Physiology

The Natural History of the Crustacea Series

SERIES EDITOR:
Martin Thiel

Editorial Advisory Board:

Geoff Boxshall, Natural History Museum, London, UK
Emmett Duffy, Virginia Institute of Marine Sciences, Gloucester, USA
Darryl Felder, University of Louisiana, Lafayette, USA
Gary Poore, Victoria Museum, Melbourne, Australia
Bernard Sainte-Marie, Fisheries and Oceans Canada, Mont-Joli, Canada
Gerhard Scholtz, Humboldt University Berlin, Berlin, Germany
Fred Schram, Friday Harbor Marine Laboratory, Seattle, USA
Les Watling, University of Hawaii, Honolulu, USA

Functional Morphology and Diversity (Volume 1)

Edited by Les Watling and Martin Thiel

Lifestyles and Feeding Biology (Volume 2)

Edited by Martin Thiel and Les Watling

Nervous Systems and Control of Behavior (Volume 3)

Edited by Charles Derby and Martin Thiel

Physiology (Volume 4)
Edited by Ernest S. Chang and Martin Thiel
 Physiology
The Natural History of the Crustacea
Volume 4

EDITED BY ERNEST S. CHANG AND MARTIN THIEL

1
1
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PREFACE

This is the fourth volume of a ten-volume series on The Natural History of the Crustacea. Our vol-
ume is on Physiology. It follows Volume 1: Functional Morphology and Diversity, Volume 2: Life Styles
and Feeding Biology, and Volume 3: Nervous Systems and Control of Behavior. The next six volumes
will focus on various aspects of the lives of crustaceans, including reproduction, development, life
history and behavioral ecology, evolution and biogeography, fisheries and aquaculture, and ecology
and conservation biology.
A comprehensive overview of the comparative physiology of crustaceans is essential to the
understanding of many aspects of their biology. General overviews of specific aspects of the physi-
ology of crustaceans can be found in some of the major invertebrate zoology textbooks. However,
due to space limitations, those textbook chapters can only present a very general introduction to
this diverse group. The last extensive overview on this topic was provided in three volumes (vols.
5, 8, and 9) of the 10-volume series on The Biology of Crustacea, which was published more than
29  years ago. Those volumes were the successors to the two-volume treatise The Physiology of
Crustacea published in 1960–1961. Since then, several reviews about particular aspects of crustacean
physiology have been published, but these are dispersed in the specialized primary literature. Thus,
we believe the time is ripe to synthesize present knowledge about the physiology of crustaceans in
a new volume where the information will be presented in a form that is attractive and accessible to
a wide general audience of comparative physiologists.
In this volume, Webster details the distinct hormones that regulate molting and metabolism
and also describes the functional overlap of these hormones in the first two chapters. McNamara
and Milograna then present a review of the physiological control of pigmentation. Muscle struc-
ture, function, and development are covered in two chapters by Medler and Mykles and Mykles
and Medler. The intriguing ability of crustaceans to drop a limb and subsequently regenerate it is
reviewed by Hopkins and Das. The various types of hearts and circulatory systems are described in
a chapter by McGaw and Reiber, and that is followed by a review of osmoregulation and excretion
by Lignot and Charmantier. Nutrition and digestion are described by Saborowski. Many of the
chapters in this volume describe not only normal physiology, but also physiological responses to
environmental stress. These include responses to varying oxygen, temperature, and pH by Whiteley
and Taylor, respiration and oxygen transport by Terwilliger, and metabolic regulation by Jimenez
and Kinsey. The cellular and molecular responses to various stressors are reviewed by Stillman and
Hurt. The volume concludes with discussions of endocrine disrupters by deFur and Williams and
various other environmental pollutants by Weis.

v
vi Preface

We hope that this volume will not only provide a synthetic overview for scholars interested in
the physiology of crustaceans and other arthropods, but also encourage further studies. As becomes
evident from many of the contributions, crustaceans are ideal model organisms to study physiologi-
cal responses to environmental factors, the understanding of which is of utmost importance in a
world that is continuously changing. Crustaceans as model organisms will become all the more
significant due to their functions as keystone members of food webs, dominance in a multitude of
habitats, and economic value as major components of world fisheries and aquaculture.
ACKNOWLEDGMENTS

Foremost, we thank our contributors for the time they have taken in synthesizing the knowledge in
their respective fields of research—their efforts and enthusiasm made this volume possible. Special
thanks to our editorial assistants, Lucas Eastman and Annie Mejaes, who provided outstanding
help in organizing, managing, and editing. The generous contribution from Universidad Católica
del Norte was essential for this project—we are grateful for the continuous support that allowed
us to focus on the task. The vision and foresight of the university authorities made this project
possible, and we hope that this and the upcoming volumes fulfill their expectations. We thank our
external referees who provided valuable comments to our authors and us about their chapters.
E.S.C. thanks Sharon A. Chang for her laboratory and personal support during this project. Finally,
we also recognize our publisher, Oxford University Press, for its commitment to the project.

Editing of this book was generously supported by Universidad Católica del Norte, Chile.

vii
CONTRIBUTORS

EDITORS
Ernest S. Chang Martin Thiel
Bodega Marine Laboratory Facultad Ciencias del Mar
University of California-Davis Universidad Católica del Norte
PO Box 247 Larrondo 1281
2099 Westshore Road Coquimbo
Bodega Bay, CA 94923 Chile
USA

AUTHORS
Guy Charmantier Penny M. Hopkins
AEO Team Department of Biology
UMR 5119, cc 92 University of Oklahoma
Université Montpellier 2 730 Van Vleet Oval, Room 314
Pl. E. Bataillon Norman, OK 73019
34095 Montpellier cedex 05 USA
France
David A. Hurt
Sunetra Das Department of Integrative Biology
Department of Biology University of California, Berkeley
University of Oklahoma 1005 Valley Life Sciences Building
730 Van Vleet Oval, Room 314 Berkeley, CA 94720
Norman, OK 73019 USA
USA
Ana Gabriela Jimenez
Peter L. deFur Department of Biology and Marine Biology
Center for Environmental Studies University of North Carolina Wilmington
Virginia Commonwealth University 601 South College Road
1000 West Cary St Wilmington, NC 28403
Richmond, VA 23284 USA
USA

ix
x Contributors

Stephen T. Kinsey Carl L. Reiber

Department of Biology and Marine Biology School of Life Sciences
University of North Carolina Wilmington University of Nevada, Las Vegas
601 South College Road 4505 Maryland Parkway
Wilmington, NC 28403 Las Vegas, NV 89154
USA USA

Jehan-Hervé Lignot Reinhard Saborowski

AEO Team Alfred Wegener Institute for Polar and Marine
UMR 5119, cc 92 Research
Université Montpellier 2 PO Box 120161
Pl. E. Bataillon 27515 Bremerhaven
34095 Montpellier cedex 05 Germany
France
Jonathon H. Stillman
Iain J. McGaw Romberg Tiburon Center and Department of
Department of Ocean Sciences Biology
Memorial University of Newfoundland San Francisco State University
0 Marine Lab Road 3150 Paradise Drive
St John’s, NL A1C 5S7 Tiburon, CA 94920
Canada USA

John Campbell McNamara Department of Integrative Biology

Departamento de Biologia University of California, Berkeley
Faculdade de Filosofia, Ciências e Letras de 5041 Valley Life Sciences Building
Ribeirão Preto Berkeley, CA 94720
Universidade de São Paulo USA
Ribeirão Preto, 14040-901 São Paulo
Brasil Edwin (Ted) W. Taylor
Emeritus Professor of Animal Physiology
Scott Medler School of Biosciences
Department of Biology University of Birmingham
State University of New York Fredonia Birmingham B15 2TT
Fredonia, NY 14063 UK
USA
Nora B. Terwilliger
Sarah Ribeiro Milograna Oregon Institute of Marine Biology
Departamento de Biologia University of Oregon
Faculdade de Filosofia, Ciências e Letras de PO Box 5389
Ribeirão Preto Charleston, OR 97420
Universidade de São Paulo USA
Ribeirão Preto 14040-901 São Paulo
Brasil Simon G. Webster
School of Biological Sciences
Donald L. Mykles Bangor University
Department of Biology Deiniol Road, Bangor
Colorado State University Gwynedd LL57 2UW
Fort Collins, CO 80523 UK
USA
 Contributors xi

Judith S. Weis Deiniol Road, Bangor

Department of Biological Sciences Gwynedd LL57 2UW
Rutgers University UK
195 University Avenue
Newark, NJ 07102 Laura E. Williams
USA Center for Environmental Studies
Virginia Commonwealth University
Nia M. Whiteley 1000 West Cary Street
School of Biological Sciences Richmond, VA 23284
Bangor University USA
CONTENTS

1. Endocrinology of Molting  •  1
Simon G. Webster

2. Endocrinology of Metabolism and Water Balance: Crustacean

Hyperglycemic Hormone  •  36
Simon G. Webster

3. Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation in

Crustacean Chromatophores  •  68
John Campbell McNamara and Sarah Ribeiro Milograna

4. Muscle Structure, Fiber Types, and Physiology  •  103

Scott Medler and Donald L. Mykles

5. Skeletal Muscle Differentiation, Growth, and Plasticity  •  134

Donald L. Mykles and Scott Medler

6. Regeneration in Crustaceans  •  168

Penny M. Hopkins and Sunetra Das

7. Circulatory Physiology  •  199

Iain J. McGaw and Carl L. Reiber

8. Osmoregulation and Excretion  •  249

Jehan-Hervé Lignot and Guy Charmantier

9. Nutrition and Digestion  •  285

Reinhard Saborowski

10. Responses to Environmental Stresses: Oxygen, Temperature, and pH  •  320

Nia M. Whiteley and Edwin (Ted) W. Taylor

xiii
xiv Contents

11. Oxygen Transport Proteins in Crustacea: Hemocyanin and Hemoglobin  •  359

Nora B. Terwilliger

12. Energetics and Metabolic Regulation  •  391

Ana Gabriela Jimenez and Stephen T. Kinsey

13. Crustacean Genomics and Functional Genomic Responses to Environmental Stress and
Infection  •  420
Jonathon H. Stillman and David A. Hurt

14. Endocrine-Disrupting Chemicals  •  461

Peter L. deFur and Laura E. Williams

15. Some Physiological Responses of Crustaceans to Toxicants  •  477

Judith S. Weis

Index  •  505
Physiology
 *
XO-CHH mRNA

* *
I II III IV
CHH-gene

PO-CHH mRNA

Fig. 2.1.
Schematic representation (not to scale) of crustacean hyperglycemic hormones (CHH) gene, transcript, and
peptide structure, illustrated by the Carcinus maenas CHH prototype. X-organ (XO)-CHH encoded by exons
I, II, IV is expressed by eyestalk neurons and gut paraneurons, whereas pericardial organ (PO)-CHH encoded
by exons I–IV is expressed by intrinsic neurons in the POs. Asterisks show positions of stop codons. CHH pre-
cursor related peptide (CPRP) is shown outlined in green, XO-CHH in blue, the PO-CHH variant C-terminal
(which differs from residues 41–72, and which is unamidated) in yellow. Modified from Dircksen et al. (2001),
with permission from The Biochemical Society.
A PLEOCYEMATA CHH
Exon
duplication Gene deletion
MIH
4 EXONS

CHH
DECAPODA CHH
MIH
DENDROBRANCHIATA

BRANCHIOPODA
Intron
insertion
ARTHROPODA 4 EXONS
ITP
3 EXONS
Exon
duplication INSECTA 5 EXONS
Drosophila
Exon
duplication
ANCESTRAL
GENE CHELICERATA
2 EXONS

B PLEOCYEMATA CHH

MIH
DECAPODA

CHH
3 EXONS
Exon Exon CHH
Exon 3 EXONS
duplication deletion
deletion MIH
4 EXONS
DENDROBRANCHIATA

BRANCHIOPODA

ITP
3 EXONS
INSECTA 5 EXONS
Drosophila
Exon
duplication

CHELICERATA

Fig. 2.2.
Evolutionary scenarios of the crustacean hyperglycemic hormones (CHH) family genes in the Arthropoda.
(A) An intron insertion in an ancestral 2 exon gene (asterisk) and two independent exon duplications resulted
in 4 exons for CHH, and a single-exon duplication gave 3 exon molt-inhibiting hormone (MIH) genes.
(B) A single exon duplication accounts for CHH, and exon deletion for MIH gene structure. Note that a fur-
ther exon duplication can account for the 5 exon ITP gene in Drosophila. The scaled schematics show exons
represented by boxes, untranslated regions (UTR) (gray), signal peptides (yellow), precursor-related peptides
(blue), ITPs (purple), CHHs (red), MIHs (green), alternatively spliced exons encoding ITP-L (violet), and
CHH (orange) C-terminus regions. Question marks indicate UTRs whose precise borders are unknown.
Figure redrawn from Montagné et al. (2010).
Fig. 2.6.
Immunohistochemical localization of crustacean hyperglycemic hormones (CHH) and molt-inhibiting hor-
mone (MIH) in neural and non-neural tissues of crustaceans. (A) Sinus gland (SG) section of Carcinus maenas
double immunostained for CHH (brown, peroxidase antiperoxidase/diamainobenzidine) and MIH (black,
silver-enhanced immunogold). Arrow points to (collapsed) hemal sinus. (B)  Double immunostained (as in
A) section of X-organ (XO) of C. maenas. Inset shows transverse section of XO-SG tract. (C) Intrinsic CHH
neurons in pericardial organ of C. maenas. (D) Endocrine cells immunoreactive (whole-mount FITC immuno-
fluorescence) to CHH surrounding the insertions of the gastric and cardiopyloric muscles at the mesocardiac
and pterocardiac ossicles of premolt (stage D2) C. maenas, dorsal view. Abbreviations: gm3c, lateral posterior
gastric muscles; gm4c, cardiopyloric muscles. Inset shows dorsal view of cells surrounding muscle insertions
of the anterior dorsal pyloric dilators (cpv1b) at the posterior mesopyloric and anterior uropyloric ossicles.
Fig. 2.6 (Continued)
(E) Structures immunoreactive to CHH (whole-mount immunofluorescence) in a recently hatched C. maenas
zoea larva. Arrows point to segmentally iterated abdominal cells (ac) close to the insertions of the abdomi-
nal flexor muscles, pericardial organs (po) and associated immunopositive cells, and XO-SG neurosecretory
system (xo). (F) Double labeled preparation showing localization of two pairs of CHH (red, Cy3) and MIH
(green, FITC) immunoreactive neurons in the brain of a C.  maenas embryo at the mid-eye stage (70–85%
development). Arrow indicates extensive arborizing dendrites. Both peptides are not colocalized, but the SG
appears yellow due to stacking of the confocal image. (G) CHH immunoreactive cells (whole-mount FITC)
in the hindgut of C. maenas, imaged from the basal (hemolymph) side, where the cells exhibit a branching
morphology. (H) CHH immunoreactive structures in the PO and adjacent areas in a C. maenas embryo just
prior to hatching; arrows point to the PO nerve trunks. Abbreviations:  ab, anterior bar; pb, posterior bar.
(I) Dual-labeled section of the retina of juvenile crayfish (Procambarus clarkii). Red structures (Texas red)
show CHH immunoreactive tapetal cells, green structures (FITC) show 5-HT immunopositive retinular cell
axons. Upper cell layer shows CHH immunoreactive tapetal cells; lower layer, 5-HT immunopositive retinular
cell axons. Scale bars: 50 µm (A, B, F, G, H), 100 µm (C), 200 µm (D) and insert (E), 20 µm (I), 25 µm insert (B).
Images: (A, B) adapted from Dircksen et al. (1988), with permission from Wiley and Sons, Inc.; (C) courtesy
of H. Dircksen; (D, E, F, H) adapted from Chung and Webster (2004), with permission from The Company of
Biologists; (G) Webster, unpublished; (I) adapted from Escamilla-Chimal et al. (2001), with permission from
The Company of Biologists.
 L-CHH cells D-CHH cells D-cells D-VIH cells L-VIH cells

preproCHH preproCHH preproCHH preproVIH preproVIH

XO +
preproVIH
Cleavage Cleavage
Signal Signal
peptide peptide

proCHH proCHH
Cleavage
Amidation a CPRP
Isomerization Amidation
Cyclization b
SG CHH D-Phe3 CHH D-Trp4 VIH VIH
(+CHH) (+VIH)

Fig. 2.7.
Diagrammatic representation of cell type specific precursor processing of crustacean hyperglycemic hor-
mones (CHH) and vitellogenesis-inhibiting hormone (VIH) isomers in the X-organ-sinus gland complex.
CPRP, CHH precursor-related peptide. Amidation (a) can occur before, during, or after cleavage of CPRP.
Cyclization of glutamate to pyroglutamate (b)  can only occur after CPRP cleavage. Some cells secrete
L-CHH, L-VIH, whereas D-CHH, D-VIH cells, while producing mainly the D-isomers, also secrete variable
amounts of L-isomers. However, a small population of cells exclusively secrete the D-isomers of both hor-
mones. Adapted from Ollivaux et al. (2009), with permission from John Wiley and Sons, Inc.
 Fig. 3.1.
The Indo-Pacific harlequin shrimp, Hymenocera elegans, exhibits one of
the most stunning chromogenic adaptations found among the Crustacea.
This gnathophyllid coral reef shrimp displays a fairly fixed, sex- and
species-specific coloration pattern that consists of irregular, bluish to
purple spots and markings, placed fairly symmetrically on a cream-white
background to form a complex pigmentary system. Here, a pair of H. ele-
gans (seen in frontal view) explores a prey species, the Indo-Pacific blue
star, Linckia laevigata. With permission from Adriano Morettin, all rights
reserved.

Fig. 3.2.
Chromatosomes consist of multicellular aggregates of highly asymmetrical, single-celled, monochromatic
chromatophores. Their intimately apposed, semispherical perikarya contain the pigment granules and form
the chromatosome center from which radiate the long chromatophore extensions through which the gran-
ules migrate. (A)  Monochromatic, red, epidermal chromatosome from the neotropical freshwater shrimp,
Macrobrachium olfersi. (B)  Monochromatic, red chromatosome, showing the nuclei of individual chromato-
phores (round clear areas within the dark red perikarya) on the dorsal surface of the fibrous capsule containing
the ovary of Macrobrachium olfersi. These particular pigmentary effectors have been employed with success
as models in deciphering the mechanisms of membrane signal transduction, intracellular second-messenger
cascades, and regulation of the molecular motors that translocate the pigment granules through the cytosol.
(C) Dichromatic, epidermal chromatosome from the New Zealand intertidal shrimp, Palaemon affinis, consist-
ing of separate monochromatic yellow (left) and monochromatic red/brown chromatophores (right) contain-
ing fully dispersed and partially dispersed pigments, respectively. Scale bars = 50 µm.
Fig. 3.3.
Chromomotor adaptation in Palaemon affinis. The nearly transparent upper shrimp was
exposed to an illuminated white background for 2 h, and the brown pigments in its epidermal
chromatosomes are fully aggregated; the white somatogastric chromatosome pigments are fully
dispersed. The lower shrimp, in which the species’ typical chromogenic pattern is clearly visible,
was placed on an illuminated black background and displays fully dispersed brown epidermal
chromatosome pigments; arrowhead indicates fully dispersed hindgut chromatosomes. Such
rapid translocations of the pigment granules through the chromatophore cytosol are regulated
by blood-borne neuropeptides called chromatophorotropins, such as red pigment concentrating
hormone, released from the eyestalk and other neurosecretory centers. Scale bar = 1 cm.
Fig. 3.4.
Epifluorescence microscopy (Leica DM5500B, λex=495  nm, λem=519  nm) showing several
red chromatosomes with fully aggregated pigments from the ovary of Macrobrachium olfersi.
Elongated microtubule bundles (green) lie along the length of the pigment-free cell extensions
that project from the perikarya of the individual constituent chromatophores among the sur-
rounding fibroblasts. Monoclonal, anti-β-tubulin primary antibody followed by an Alexa-488
conjugated goat anti-mouse IgG. Scale bar = 100 µm.
Fig. 3.5.
Sequence of pigment aggregation (A–D) in two red chromatosomes on the ovary of Macrobrachium olfersi
induced by 30 min perfusion in vitro with 25 µM Ca2+-ionophore A23187 in a 2.5 mM NaHCO3-buffered physi-
ological saline. The pigments were subsequently dispersed (E–H) by 30 min perfusion with a Ca2+-free (10−11
M Ca2+), 2 mM EDTA-chelated saline. The pigment translocation rates are not constant (see Fig. 3.6). Scale
bar = 50 µm.
Fig. 3.9.
Myosin molecular motors, intimately associated with the pigment granule membranes, play an important role
in granule translocation along the actin cytoskeleton during pigment aggregation and possibly in limiting pig-
ment dispersion brought about by opposing motors. (A) Confocal fluorescence microscopy (Leica TCS SP5,
λex= 495 nm, λem= 519 nm) showing the distribution of granule-associated, nonmuscle myosin II throughout the
cytoplasm of M. olfersi ovarian chromatophores with fully dispersed pigments, revealed using a polyclonal, non-
muscle myosin II primary antibody followed by an Alexa-488 conjugated goat anti-rabbit IgG. (B) Distribution
of a skeletal muscle pan-myosin clearly associated with the large membrane-bounded pigment granules in an
ovarian chromatophore extension, shown employing a polyclonal, anti-skeletal muscle pan-myosin primary
antibody conjugated with an Alexa-488 goat anti-rabbit IgG. Scale bars A = 20 µm, B = 1 µm.
Fig. 3.10.
Molecular motors like kinesin and dynein that respectively transport cargos to the chromatophore periphery or
perikaryon along the microtubular component of the cytoskeleton are also associated with the pigment gran-
ules. (A) Confocal fluorescence microscopy (Leica TCS SP5, λex= 495 nm, λem= 519 nm) showing the distribu-
tion of granule-associated kinesin throughout the cytoplasm of a chromatosome with fully dispersed pigments,
revealed using a monoclonal, anti-kinesin primary antibody and an Alexa-488 conjugated goat anti-mouse IgG.
(B) Dynein distribution is clearly associated with the large membrane-bound pigment granules in a chroma-
tosome with fully dispersed pigments, shown here employing a monoclonal, anti-dynein primary antibody
followed by an Alexa-488 conjugated goat anti-mouse IgG. Bright field and fluorescence images overlaid with
background subtraction, scale bars = 20 µm.
Fig. 8.3.
(A)–(D) Immunolocalization of Na+/K+-ATPase in the gills (A), epipodite (B), and branchiostegite (C) of the
epibenthic Palaemon adspersus and in the epipodite of the deep-sea hydrothermal Rimicaris exoculata (D). Scale
bars: 50 µm. (E) Transmission electron micrograph of the branchiostegite of Palaemon adspersus. Scale bar: 2.5
µm. (F) three-dimensional confocal stack showing the dorsal side of the Amphibalanus amphitrite nauplius. Black
arrow indicates areas of intense fluorescent labeling in the naupliar tissue below the dorsal shield. (G) Lateral
view of the nauplius. Black arrows denote the frontal horns where fluorescence intensity is high. Scale bars: 100
µm (F–G). Abbreviations: B, bacteria; BI, basal infoldings; BL, basal lamina; C, cuticle; CS, caudal spine (pos-
terior); E, epithelium; FH, frontal horns; GL, gill lamellae; HL, hemolymph lacuna; IC: internal cuticle; IE,
inner epithelium; M, mitochondria; N, nucleus; OC, outer cuticle; OE, outer epithelium; S, septum. Adapted
from Martinez et al. (2005), with permission from Elsevier, and from Gohad et al. (2009), with permission from
Elsevier.
 1
ENDOCRINOLOGY OF MOLTING

Simon G. Webster

Abstract
Molting processes in all arthropods are ultimately regulated by ecdysteroids produced by
Y-organs (YO) from cholesterol via a common biosynthetic pathway. Increases in ecdyster-
oid hemolymph titer initiate premolt. YO ecdysteroid synthesis is regulated by (inhibitory)
peptides produced by neurosecretory cells in the eyestalk. These hormones are defined as
molt-inhibiting hormone (MIH) and crustacean hyperglycemic hormone (CHH). Recent
research has shown that molt control (at the level of processes controlling ecdysteroid synthesis
in the YO) is complex, first by release of CHH from endocrine cells in the fore- and hindgut,
then by release of crustacean cardioactive peptide (CCAP) from the pericardial organs. Finally,
insect cuticle hardening hormone is released (together with CCAP), resulting in tanning and
sclerotization of the cuticle in immediate postmolt. In silico analyses of genomes and pepti-
domes predict the existence of other peptide hormones in crustaceans known to be centrally
important in insect molting endocrinology.

INTRODUCTION

For all arthropods, a thick, inflexible exoskeleton has inevitable consequences for almost every
aspect of their life history. In order for growth to occur, the exoskeleton must be periodically
molted. This process, called ecdysis, is followed by uptake of water (or air in the case of the
majority of insects) and redistribution of hemolymph. This results in the expansion of the
new, flexible exoskeleton to its final postmolt dimensions before the cuticle is made inflex-
ible and tough via quinone tanning and sclerotization and, for many malacostracan crusta-
ceans, subsequent calcification. The structure and function of the crustacean cuticle and its

1
2 Simon G. Webster

elaboration and mineralization is described in detail by Dillaman et al. (2013) (see Chapter 5
in volume 1).
Given the large number of processes, both somatic and behavioral, that must be precisely
coordinated during ecdysis, this process has been the subject of intense research, particu-
larly in insects, where a complex series of steroid and terpenoid hormones, neuropeptides,
and neurohormones are involved (Truman 2005, Kim et  al. 2006, Ewer 2007, Žitňan et  al.
2007). Apart from the well-studied roles of the molting hormones (ecdysteroids) in direct-
ing processes such as cell division, differentiation, and the synthesis of new cuticle in insects
(Riddiford 1989), the synthesis of which is stimulated by prothoracicotropic hormone
(PTTH; Gilbert et al. 1996, 2002), a variety of peptide hormones such as pre-ecdysis trigger-
ing hormone (PETH), ecdysis triggering hormone (ETH), kinins, diuretic hormones (DHs),
myoinhibitory peptides (MIPS), eclosion hormone (EH), crustacean cardioactive peptide
(CCAP), and bursicon are central to the insect ecdysis program (Truman 2005, Arakane
et  al. 2008). Perhaps unsurprisingly, given the rich genetic resources and tools available in
Drosophila, far more is known concerning the hormonal control of molting in insects than
(genetically intractable) crustaceans. Moreover, as discussed later, not only are there some
well-established fundamental differences in the hormonal control of molting in insects and
crustaceans, but there are also some fascinating glimpses of recognizably similar, yet rather
different, endocrine mechanisms involved in molting in crustaceans as compared to insects.
In this review, recent advances in our knowledge of the endocrine control of molting in crus-
taceans will be highlighted, and the development of the field based on early experiments and
observations will be explored.

THE CLASSICAL MODEL OF THE HORMONAL CONTROL OF MOLTING

It has long been known that bilateral eyestalk ablation often (but not invariably) leads to accel-
erated molting in malacostracan crustaceans (Zeleny 1905, Smith 1940). Such observations,
repeated many times, have led to the hypothesis that the eyestalk—or more specifically, the
X-organ sinus gland (XO-SG) neurosecretory system—is the source of a molt-inhibiting hor-
mone (MIH) that negatively regulates production of molting hormones (Passano 1953). These
observations were supported by broadly contemporaneous observations showing that ablation
of the molting glands (Y-organs, YO) prevented molting in intact and eyestalk-ablated crabs
(Gabe 1953, Echalier 1959). More recent studies have repeatedly shown that eyestalk removal
leads to increases in circulating ecdysteroids consequent upon increased ecdysteroid synthesis
by the YO (Chang et al. 1976, Keller and Schmid 1979, Jegla et al. 1983). Reciprocal experiments,
although rather infrequently reported, have clearly shown that eyestalk or sinus gland extract
injection leads to reduction in ecdysteroid titer (Hopkins 1982, Bruce and Chang 1984, Nakatsuji
and Sonobe 2004). The finding that the YO of crayfish (Orconectes limosus) taken from animals
injected with sinus gland extracts produced rather less ecdysteroids compared to saline-injected
controls (Gersch et al. 1980), as well as the finding that the production of YO cultured in vitro
could be inhibited by eyestalk and sinus gland extracts or sinus gland-conditioned media
(Pachygrapsus crassipes, Soumoff and O’Connor 1982; Cancer antennarius, Mattson and Spaziani
1985a; Carcinus maenas, Webster 1986) further supported the classical model of molt control. The
development of the YO bioassay, in which one of a pair of YO was exposed to high-performance
liquid chromatography (HPLC)-purified SG fractions followed by estimation (by radioimmu-
noassay, RIA) of repression of ecdysteroid synthesis, was instrumental in the purification and
isolation of MIH, as described later.
 Endocrinology of Molting 3

HORMONES INVOLVED IN MOLTING

Ecdysteroids

Levels During the Molt Cycle

The identification of ecdysteroids as the molting hormones of both insects and crustaceans was
achieved almost 50 years ago (Horn et al. 1966), and, with the development of sensitive immunoas-
says (e.g., RIA, Borst and O’Connor 1972, 1974, Porcheron et  al. 1976; enzyme immunoassay, EIA,
Porcheron et al. 1989), estimations of total circulating ecdysteroids have been made in many species. For
decapods, examples include crabs (Callinectes sapidus; Soumoff and Skinner 1983, Lee et al. 1998, Chung
2010), C. maenas (Lachaise et al. 1976, Styrishave et al. 2008), Emerita asiatica (Gunamali et al. 2004),
Gecarcinus lateralis (McCarthy and Skinner 1977), P. crassipes (Chang et al. 1976, Chang and O’Connor
1978), Uca pugilator (Hopkins 1983, 1986), crayfish and lobsters (Astacus leptodactylus; Durlait et al.
1988), Orconectes sanborni (Stevenson et al. 1979), Homarus americanus (Chang and Bruce 1980, Snyder
and Chang 1991a), shrimps (Palaemon serratus; Baldaia et al. 1984), Penaeus japonicus (Okumura et al.
1989), and Macrobrachium nipponense (Okumura et al. 1992). Circulating ecdysteroid levels invariably
peak during premolt (stage D2) before rapidly declining to low levels during late premolt (stages D3–4),
which is essential for ecdysis:  manipulative elevation of ecdysteroid levels by injection at this time
delays this process in H. americanus (Cheng and Chang 1991). Low ecdysteroid levels are also seen in
immediate postmolt, but in late postmolt (stages B–C1) a small but significant peak in ecdysteroid levels
is seen, and this may be correlated with increased carbonic anhydrase activity in the epidermis at this
time, which is also coincident with the large premolt peak in ecdysteroids, perhaps suggesting causal-
ity (Baldaia et al. 1984). Repeated measurement of circulating ecdysteroids from individual animals
has shown that levels can fluctuate at a very fine temporal scale as shown for H. americanus (Snyder
and Chang 1991a) or that there are well-defined peaks during premolt as determined for U. pugilator
(Hopkins 1983, 1986). For animals displaying a terminal anecdysis, the pubertal molt is followed by
reduced activity or degeneration of the YO, as for example in Sphaeroma serratum (Charmantier and
Trilles 1979) and, in consequence, low ecdysteroid levels in mature animals (e.g., in Libinia emarginata,
Laufer et  al. 2002; Chionoecetes opilio, Tamone et  al. 2005). For other malacostracans, for example,
isopods (Helleria brevicornis, Hoarau and Hirn 1978), Armadillidium vulgare (Suzuki et al. 1996), Ligia
oceanica (Girard and Maissat 1983), amphipods (Orchestia gammarellus, Blanchet et  al. 1976, 1979),
O. cavimana (Graf and Delbeque 1987), and mysids (Siriella armata, Cuzin-Roudy et al. 1989), ecdyster-
oid levels peak during premolt, decline before molting, and remain low during postmolt and intermolt.
For non-malacostracan crustaceans, measurements of ecdysteroid profiles are destructive due to their
small size. In Daphnia magna, levels of ecdysteroids peak around 40 h after molting and are associated
with the release of first- and second-clutch neonates (Martin-Creuzberg et al. 2007). In Calanus paci-
ficus, ecdysteroid levels peak in early premolt (Johnson 2003). Ecdysteroid concentrations have been
measured during the principal molt stages in the meiofaunal harpactacoid copepod Amphiascus tenui-
remis and the amphipod Leptocheirus plumulosus (Block et al. 2003). Examples of ecdysteroid profiles
during the molt cycle of diverse crustacean are shown in Fig. 1.1.
The major (free) ecdysteroids observed in the hemolymph are ecdysone, 20-hydroxyecdysone,
ponasterone A, and 3-dehydro-20-hydroxyecdysone, and ratios of these vary during the molt cycle.
For example, in C. maenas, only ecdysone and 20-hydroxyecdysone are found during intermolt, yet
the large peak of ecdysteroid observed during premolt is mainly ponasterone A, reflecting a switch
to 25-deoxyecdysone synthesis by the YO at this time (Lachaise et al. 1986, 1988); during the precipi-
tous fall in ecdysteroid levels just prior to molting, the reduction in concentration of this hormone is
relatively greater than the observed reduction in ecdysone and 20-hydroxyecdysone concentrations
4 Simon G. Webster

A B
1500 40
Homarus americanus Orconectes sanborni

Hemolymph ecdysteroids (pg/µl)

Hemolymph ecdysteroids (pg/µl)

30
1000

20

500
10

Time (days)
0 0
1.0 2.0 1 2 3 1 2 1 2 3 4 1 2 3
A B C1 C4 C4 D0 D0 D1 D1D1D2D2D3D3D3D3 A B C1 C2 C3 C4 D0 D1 D1D1 D2 D3 D4

Molt stage Molt stage

C D
300
Hemolymph ecdysteroids (pmol/100 µl)

Siriella armata Daphnia magna

250

Ecdysteroid titer (pg/individual)

300

200

200
150

100
100
50 E
E

0 0
e A 1 B C 5 D0 D1 D2 e 10 days 0 10 20 30 40 50 60 70 80

Molt stage Time (h)

Fig. 1.1.
Profiles of ecdysteroids during the molt cycle of selected crustaceans. (A) Lobster, Homarus americanus, males
(Snyder and Chang 1991a). Each point represents a mean of 3–7 determinations, error bars, ± SD. Molt stages
according to Aiken (1973), and, in late premolt, according to Cheng and Chang (1991). (B) Crayfish, Orconectes
sanborni (Stevenson et al. 1979). Each point represents a mean of 5–40 determinations, error bars, ± SD. Molt
stages according to Drach and Tchernigovtzeff (1967). (C) Mysid, Siriella armata, males (Cuzin-Roudy et al.
1989). Molt stages according to Cuzin-Roudy and Tchernigovtzeff (1985). Ecdysis (e). (D) Water flea, Daphnia
magna (Martin-Creuzberg et al. 2007). Each point represents a mean of three determinations, error bars ± SE.
Carcass ecdysteroids were determined every 4 h between two successive molts associated with release of first-
and second-clutch neonates. Redrawn from originals.

(Lachaise and Lafont 1984, Styrishave et al. 2008). Conversely, in Penaeus (Marsupenaeus) japoni-
cus, 20-hydroxyecdysone and ponasterone A are the major ecdysteroids during post- and intermolt,
whereas 20-hydroxyecdysone is the major circulating ecdysteroid during premolt (Okumura et al.
1989). Eyestalk ablation also changes the composition of ecdysteroids in the hemolymph: in C. sapi-
dus, this operation results in 20-hydroxyecdysone becoming the major ecdysteroid during premolt,
rather than ponasterone A  (Chung 2010), whereas in U.  pugilator, eyestalk removal results in a
higher ponasterone A-to-20-hydroxyecdysone ratio than for crabs in which molting is induced by
multiple limb autotomy (Hopkins 1992). Taken together, these results indicate that where eyestalk
ablation is used to induce molting, a normal endocrinological status of the animal cannot be tacitly
assumed, and the significance of eyestalk-induced perturbations of ecdysteroid synthesis relative to
completion of natural molt cycles remains to be explored.

Biosynthesis

Ecdysteroids are synthesized by the YO—small, epithelioid tissues found at the anterior margin of
the branchial chamber that were first described by Gabe (1953) in C. maenas. For a review of YO
 Endocrinology of Molting 5

structure and function, see Lachaise et al. (1993). Because arthropods cannot synthesize the precur-
sor (cholesterol) from acetate or mevalonate, this is acquired from the diet, transported in the hemo-
lymph via high-density lipoproteins, and taken up by the YO via receptor-mediated endocytosis
(Spaziani and Kater 1973, Watson and Spaziani 1985). The biosynthetic pathway of ecdysteroid syn-
thesis, which has essentially been determined using in vivo injection of 3H-25-hydroxycholesterol
(which is much more soluble than cholesterol or 7-dehydrocholesterol) and subsequent isolation
of metabolites via HPLC and mass spectrometry (MS) (Böcking et al. 1994, Wang et al. 2000) has
still not been entirely elucidated in crustaceans and seems to be rather diverse compared to insects
because a number of ecdysteroids can potentially be synthesized. However, normally only two are
released from the YO of a particular species, as shown in Table 1.1. A detailed review of our current
understanding of ecdysteroid synthesis by the crustacean YO has recently been published (Mykles
2011), but salient features are mentioned here.
The first stage of ecdysteroid biosynthesis is the conversion of cholesterol to 5β-diketol
(3-dehydro-2, 22, 25-deoxyecdysone). This occurs via conversion of cholesterol to
7-dehydrocholesterol (Rudolph and Spaziani 1992, Rudolph et  al. 1992)  via 7,8-dehydrogenase
and a series of as yet incompletely determined reactions involving 3-oxo-Δ4 intermediates,
referred to as the “black box” (Blais et al. 1996), producing Δ4-diketol, which is then reduced to
5β-diketol (2,22,25-deoxyecdysone) by 3-dehydroecdysteroid-3β-reductase (Böcking et  al. 1993).
The second stage of biosynthesis involves successive hydroxylations of 5β-diketol resulting in four
potential secretory products from the YO (3-dehydro-25-deoxyecdysone, 3-dehydroecdysone,
2-deoxyecdysone, 25-deoxyecdysone) that can undergo metabolism in peripheral tissues to pon-
asterone A  (25-deoxy-20-hydroxyecdysone) and 20-hydroxyecdysone. A  current model sum-
marizing the ecdysteroid biosynthetic pathway in crustaceans is shown in Fig. 1.2. The enzymes
involved in ecdysteroid biosynthesis from 5β-diketol have been identified in Drosophila as the
P450 mono-oxygenases (Halloween genes) Phantom (phm), Disembodied (dib), Shadow (sad), and
Shade (shd) (Rewitz et al. 2007). Although these enzymes have broad substrate specificity, it seems
that the mandatory order of hydroxylations (C25→C22→C2→C20) is maintained. Early stages

Table 1.1.  Ecdysteroids secreted by the Y-organs of decapod crustaceans

Species E 3DE 25dE 3D25dE References

Pachygrapsus crassipes • Chang and O’Connor 1977
Cancer antennarius • • Watson and Spaziani 1985,
Spaziani et al. 1989
Orconectes limosus • • Böcking et al. 1993
Procambarus clarkii • • Sonobe et al. 1991
Penaeus vannamei • • Blais et al. 1994
Macrobrachium rosenbergii • • Okumura et al. 2003
Carcinus maenas • • Lachaise et al. 1989
Callinectes sapidus • • Unpub. Cited in Wang
et al. 2000
Uca pugilator • • Hopkins 1986, 1992*
Menippe mercenaria • • Rudolph and Spaziani
1992, Wang et al. 2000

Abbreviations: E, ecdysone; 3DE, 3-dehydroecydsone; 25dE, 25-deoxyecdysone; 3D25dE, 3-dehydro-25-deoxyecdysone.

* Secretion of 25dE inferred by presence of ponasterone A in premolt.
Adapted from Mykles 2011, with permission from Elsevier.
 Cholesterol
Y-organ
7,8-dehydrogenase

7-dehydrocholesterol
Non-molting glossy (Nm-g/Shroud (Sro))
Spook (Spo)/Spookier (Spok)
Spookiest (Spot)
∆4-Diketol

5β[H]-reductase

5β-Diketol

22-hydroxylase 25-hydroxylase 3-dehydroecdysteroid 3β-reductase

3-dehydro-2,25-deoxyecdysone 3-dehydro-2, 22-deoxyecdysone 5β-Ketodiol

2-hydroxylase 22-hydroxylase 25-hydroxylase 22-hydroxylase

3-dehydro-25-deoxyecdysone 3-dehydro-2-deoxyecdysone 5β-Ketotriol 2,25-deoxyecdysone

2-hydroxylase 22-hydroxylase 2-hydroxylase

3-dehydroecdysone 2-deoxyecdysone 25-deoxyecdysone

2-hydroxylase

Ecdysone

3-dehydroecdysteroid
3β-reductase
3-dehydro-25-deoxyecdysone 3-dehydroecdysone Ecdysone 25-deoxyecdysone
3-dehydroecdysteroid
20-hydroxylase 20-hydroxylase
3β-reductase

25-deoxyecdysone 3-dehydro-20-hydroxyecdysone Ponasterone A

20-hydroxylase 20-hydroxylase 25-hydroxylase

Ponasterone A 20-hydroxyecdysone
Peripheral tissues

Fig. 1.2.
The ecdysteroid biosynthetic pathway in the crustacean Y-organ (YO). Cholesterol is first converted to
7-dehydrocholesterol by a 7,8-dehydrogenase encoded by the Neverland (nvd) gene in insects. Subsequent
conversion to Δ4-diketol occurs via a series of as yet incompletely understood reactions named the “black
box” involving 3-oxo-Δ4 intermediates, which are catalyzed by enzymes encoded by Nonmolting glossy
(nm-g)/Shroud (sro), Spook (spo)/Spookier (spok), and Spookiest (spot) genes in insects. Reduction of
Δ4-diketol by 5β[H]‌-reductase to 5β-diketol is followed by hydroxylation by enzymes encoded by Disembodied
(22-hydroxylase, Phantom (25-hydroxylase), Shadow (2-hydroxylase), and 3-dehydroecdysteroid-3β-reductase,
which potentially results in four ecdysteroid products, but only two are secreted by YO (Table 1.1). In periph-
eral tissues, further hydroxylations occur catalyzed by 20-hydroxylase, encoded by Shade, a 25-hydroxylase; and
a 3-dehydroecdysteroid-3β-reductase, resulting in a maximum of three final ecdysteroid products. Modified
from Mykles (2011), with permission from Elsevier and with information from Böcking et al. (1993), Lachaise
et al. (1993), Wang et al. (2000), Gilbert and Rewitz (2009).
 Endocrinology of Molting 7

of synthesis are controlled by Neverland (Nvd) (7,8-dehydrogenase; Yoshiyama et  al. 2006)  and
black-box intermediates are catalyzed by Nonmolting glossy (nm-g)/shroud (sro) and several
Halloween genes: Spook (spo), Spookier (spok), and Spookiest (spot) (Rewitz et al. 2007, Niwa et al.
2010). Because orthologs of all the above-mentioned genes have been identified in the Daphnia
pulex genome (Rewitz and Gilbert 2008), as well as phm in Marsupenaeus japonicus (Asazuma et al.
2009), it seems probable, indeed unsurprising, that the genes encoding the enzymes involved in
ecdysteroid biosynthesis are highly conserved across the phylum, even if the pathways involved in
species-specific ecdysteroid secretion patterns are somewhat diverse.

Metabolism

Hormonally active ecdysteroids are metabolized to more polar compounds and ecdysonoic acids,
which are excreted in the urine (the antennal gland is the principal site of excretion of ecdyster-
oids), or apolar conjugates are produced by the midgut gland, and these conjugates are excreted
in feces. A comprehensive review of metabolism of ecdysteroids is included in Mykles (2011); only
salient features are mentioned here.
Conversion of ecdysteroids to polar products and ecdysonoic acids involves conversion of
20-hydroxyecdysone to 20, 26-dihydroxyecdysone and conversion of ponasterone A to 25-deoxy-20,
26-dihydroxyecdysone. Oxidation at C26 converts 20-hydroxyecdysone to 20-hydroxyecdysonoic
acid and ponasterone A  to 25-deoxy-20-hydroxyecdysonoic acid (Lachaise and Lafont 1984,
Lafont et al. 1986, Snyder and Chang 1991b,c, 1992). In the crayfish Procambarus clarkii, epidermal
3α-reductase converts ecdysone to 3α-hydroxyecdysone and 20-hydroxyecdysone (Ikeda and Naya
1993). Excretion of ecdysteroids as highly polar conjugates in the urine is a very important inacti-
vation mechanism during the precipitous drop in ecdysteroids seen characteristic of late premolt,
for example, in H. americanus (Snyder and Chang 1991a,b), and conversion is molt-stage specific
(Snyder and Chang 1991c).
Conversion of ecdysteroids to apolar conjugates is an important route for sequestration of
dietary ecdysteroids. For example, more than 99% of ingested 3H-labelled ecdysone is excreted as
conjugated apolar metabolites in H. americanus, and, in this species, excretion of these compounds
is highest during premolt (Snyder and Chang 1992).

Molt-inhibiting Hormone

Peptide Structures, Genes, and Expression

Although the existence of an MIH had been postulated 60 years ago (Passano 1953), full chemi-
cal characterization was only realized 20 years ago (Chang et al. 1990, Webster 1991) with the full
structural identification of peptides with activities that functionally defined the hormone. These
were the ability to (i) reduce circulating ecdysteroid levels and (ii) inhibit ecdysteroid synthesis
by the YO. In the former case, a hormone with MIH activity was isolated from SG of H. america-
nus (Chang et al. 1990), which also had crustacean hyperglycemic hormone (CHH) activity and
was clearly an authentic type I (Lacombe et al. 1999) peptide—an authentic CHH (see Chapter 2
in this volume). A peptide that inhibited ecdysteroid synthesis by the YO in C. maenas (Webster
1986, Webster and Keller 1986) was fully microsequenced by Edman degradation (Webster 1991),
which, although clearly structurally related to CHH, was distinctive in that it was somewhat larger
(78 amino acids) and possessed unblocked N- and C-termini while retaining identical disulphide
bridge arrangements. Additionally, in this species, CHH also exhibited some MIH activity in the
YO bioassay, although about 10–20 times less potent than MIH (Webster and Keller 1986). This
8 Simon G. Webster

observation, when considered together with that of Homarus MIH (a CHH), clearly pointed not
only to the multifunctional nature of CHH-superfamily peptides, but also to the existence of a fam-
ily of type II peptides (Lacombe et al. 1999), with Carcinus MIH as the prototype, and also to their
often overlapping biological activities.
To date, more than 30 type II peptides have been identified (comprising MIH, mandibular
organ-inhibiting hormone [MOIH], and vitellogenesis-inhibiting hormone [VIH] peptides), of
which about 21 have been classified as authentic MIHs based on their precursor and primary struc-
tures (for accession numbers of authentic MIHs and other structurally related type II peptides,
see Table 1.2). These peptides (which have been functionally identified by their ability to repress
ecdysteroid synthesis in vitro) seem to be universal in crabs but less common in other decapod
groups (see Chapter 2 in this volume). However, it is unfortunate that many of these have not yet
been fully identified in terms of functionality by YO bioassays. Structurally, type II peptides are
always somewhat larger than type I (CHH) peptides—77–83 versus 72 amino acids. Unlike type
I peptides, the preprocessed precursor for type II peptides never includes a precursor-related pep-
tide (PRP). None has N-terminal modification (pGlu), which is commonly seen for CHHs, and
all have characteristic Gly and Val residues between the first and second Cys residues (in positions
5 and 13 relative to the first Cys). The Gly residue may be important in conferring specificity in
biological activity because insertion of this residue into the CHH of M. japonicus reduces biologi-
cal activity by at least 10-fold (Katayama and Nagasawa 2004). This residue lies within the first
predicted α-helix of MIH in this species (Katayama et al. 2003), which seems to be absent in type
I peptides. Thus, it seems possible that this structure is critical in conferring specificity between
related peptides. Although some type II peptides have C-terminal amidation (e.g., MIH: O. limo-
sus, Bulau et al. 2005; P. clarkia, Nagasawa et al. 1996; and VIH: Homarus gammarus, H. americanus,
Soyez et al. 1991, Ollivaux et al. 2006; Rimicaris kairei, Qian et al. 2009), the majority do not, in
contrast to CHHs where this modification is very common. Intriguingly, no recognizable MIH
genes have been found in D. pulex despite the identification of CHH (or more correctly ITP-like)
genes in this species (Dircksen et al. 2011).
Gene structures for MIH invariably include 3 exons (Chan et al. 1998, Udomkit et al. 2000, Chen
et al. 2005, Montagné et al. 2010), which all contain a first phase-0 intron that interrupts the signal
peptide-coding region, and a second phase-2 intron. As with CHH, the second exon always termi-
nates in the codon for the 40th or 41st amino acid of the mature MIH. Although multiple copies of
CHH genes seem to be common in all crustaceans so far examined—up to nine copies in penaeid
shrimp (Gu and Chan 1998)—maximally, only two MIH genes seem to occur in the species thus
far examined (Lu et al. 2000, Chen et al. 2007, Nakatsuji et al. 2009). Interestingly, in cancrid crabs,
these cluster with MOIH genes, which are further highly similar members of the type II peptide
family, thus clearly illustrating a gene duplication event (Lu et al. 2000). A recent review includes
details of the structures of CHH superfamily hormone genes (Webster et al. 2012). An overview of
MIH peptide, precursor, and gene structures is shown in Fig. 1.3.
Immunohistochemical studies have repeatedly shown that MIH is only present in the XO-SG
system of decapod crustaceans (crabs: Dircksen et al. 1988, Lee and Watson 2002, Hsu et al. 2006;
penaeid shrimp: Shih et al. 1998, Gu et al. 2001, 2002; palinurids: Marco and Gäde 1999). In species
where a clearly defined MOIH peptide exists, for example Cancer pagurus, MIH immunoreactive
(ir) profiles in the SG always colocalize with MIH (Webster et al. 2012). Only rarely does MIH-ir
colocalize with CHH-ir (M. japonicus, Shih et al. 1998), although another type II peptide (VIH)
often colocalizes with CHH in lobsters (De Kleijn et al. 1992, Rottlant et al. 1993, Ollivaux et al.
2009; see also Fig. 2.6. in Chapter 2 in this volume). mRNA encoding MIH has been observed in the
brain of C. pagurus (Lu et al. 2001) and in several neural tissues of Metapenaeus ensis (Gu et al. 2002),
but the presence of expressed peptide has not yet been investigated in these species. The limited
distribution of MIH peptides to the XO-SG contrasts vividly with the situation for CHH, where
Table  1.2. Molt-inhibiting hormone prepro-peptide sequences and other members of
the type II crustacean hyperglycemic hormone (CHH) superfamily. Accession numbers
(Protein) conceptually determined by cDNA, gDNA cloning and sequencing, or directly
determined by microsequencing (gray highlights indicate mature peptide sequence
information only). MIH, molt-inhibiting hormone; MOIH, mandibular organ-inhibiting
hormone; VIH (GIH), vitellogenesis (gonad)-inhibiting hormone; SGP, sinus gland
peptide.

Species Tissue source Peptide or Accession Reference

conceptual No. (Protein)
translation
Decapoda, Brachyura
Callinectes sapidus Eyestalk ganglia MIH AAA69029 Lee et al. 1995
Cancer magister Eyestalk ganglia MIH AAC38984 Umphrey et al.
1998
Cancer pagurus Sinus glands, MIH P55846, Chung et al. 1996,
X-organ, gDNA CAC39425 Lu et al. 2001
Cancer pagurus Sinus glands, MOIH1, P81034, P81025 Wainwright et al.
X-organ, gDNA MOIH2 CAB61424, 1996,
CAB61425 Tang et al. 1999,
Lu et al. 2000
Carcinus maenas Sinus glands, MIH CAA53591 Webster 1991,
X-organ Klein et al. 1993
Charybdis feriatus gDNA MIH AAC64785 Chan et al. 1998
Charybdis japonica Eyestalks gDNA MIH ACD11361, Zhu et al. 2008,
ACO90023 unpub.; Yang
et al. 2009,
unpub.
Discoplax celeste X-organ MIH AEM45616 Turner et al. 2011,
unpub.
Eriocheir sinensis Eyestalks, gDNA MIH AAQ81640, Song et al. 2003,
ABC68517 unpub; Wang
et al. 2005
unpub.
Gecarcinus lateralis Eyestalk ganglia MIH ABF06632 Lee et al. 2007
Portunus Eyestalk ganglia, MIH ABZ04547, Zhu and Shen,
trituberculatus gDNA ACF77140 unpub. 2007,
Zhu et al. 2008
unpub.
Decapoda, Astacura
Cherax Eyestalk ganglia MIH ACX55057 Pamuru et al.
quadricarinatus 2009, unpub.
Homarus americanus Sinus glands, VIH P55320 Soyez et al. 1991,
X-organ de Kleijn et al.
1994
Homarus gammarus X-organ VIH ABA42181 Ollivaux et al.
2006

(continued)
10 Simon G. Webster

Table 1.2.  (Continued)

Species Tissue source Peptide or Accession Reference

conceptual No. (Protein)
translation
Orconectes limosus X-organ MIH P83636 Bulau et al. 2005
Procambarus clarkii Sinus glands MIH P55848 Nagasawa et al.
1996
Nephrops norvegicus Eyestalk ganglia GIH AF163771 Edomi et al. 2002
Decapoda, Penaeoidea
Fenneropenaeus X-organ MIH AAL55258 Wang et al. 2003
chinensis
Litopenaeus vannamei Eyestalk ganglia, MIH1, MIH2 AAR04348, Chen et al. 2007
gDNA AAR04349
Marsupenaeus Eyestalk ganglia MIHA BAA20432 Ohira et al. 1997
japonicus (SGP-IV)
Eyestalk ganglia MIHB, BAD36757, Ohira et al. 2005
MIHC BAE78494
Metapenaeus ensis Eyestalk ganglia MIH AAC27452 Gu and Chan
(MIH-A) 1998
gDNA GIH AAL33882 Gu et al. 2002
(MIH-B)
Penaeus monodon Eyestalk ganglia, MIH1 ACS88073 Vrinda et al. 2009
gDNA AAR89516 unpublished
Yodmuang et al.
2004
gDNA MIH2 AAR89517 Yodmuang et al.
2004
X-organ GIH ABG33898 Treerattrakool
et al. 2008
Decapoda, Caridea
Rimicaris kairei Carapace tissues VIH ACS535348 Qian et al. 2009
Macrobrachium Eyestalk ganglia VIH AEJ54623 Wang et al. 2010
nipponense unpub.
Macrobrachium Eyestalk ganglia SGP-A, AAL37948, Yang and Rao
rosenbergii SGP-B AAL37949 2001
Isopoda
Armadillidium vulgare Sinus glands VIH P83627 Grève et al. 1999

expression commonly occurs in other parts of the nervous system; for example, in intrinsic neurons
of the pericardial organs of crabs (Dircksen and Heyn 1998, Dircksen et al. 2001, Chung and Zmora
2008), the second roots of the thoracic ganglia of lobsters (Chang et  al. 1999, Basu and Kravitz
2003), and in non-neural tissues such as the gut endocrine paraneurons of C. maenas (Chung et al.
1999). The distribution of CHH peptides is discussed in Chapter 2 in this volume.
During embryonic development of C. maenas, MIH-expressing neurons (two in each eye) are
seen at about 75–80% development (Chung and Webster 2004), and, during larval life, each eye-
stalk contains four MIH-expressing neurons (Webster and Dircksen 1991). Subsequently, many
 A 2015 bp e1 i10 e2 i22 e3> [1698 bp] <e3 i22 e2 i10 e1 2109 bp 8.5 kb
* *

MO-
S MIH >1314 bp 968 bp< IH-1
S 500 bp
* *

α1,2
α4

α5

α3
N

Fig. 1.3.
Gene, precursor, and primary structures of molt-inhibiting hormone (MIH) and related type II peptides. (A) Cancer pagurus mih/moih1-gene cluster (Lu et al. 2000). Light gray, UTRs; dark
gray, signal peptide; black, MIH or MOIH. Asterisks shows positions of stop codons. Figure redrawn from Webster et al. (2012). (B) Mature peptide structures for the prototype crab MIHs
from Carcinus maenas (Webster 1991) and Cancer pagurus (Chung et al. 1996) and prototype type II peptides: MOIH, from Cancer pagurus (Wainwright et al. 1996) and VIH from Homarus
americanus (Soyez et al. 1991). Cysteine residues, which are in invariant positions in all crustacean hyperglycemic hormone (CHH) family members are marked in black, and disulphide bridges
are shown. Invariant residues typical of type II peptides marked in pale gray. Other common residues are shown in dark gray. Five predicted α-helical regions are shown. (C) Ribbon model of
energy minimized average structure of Marsupenaeus japonicus recombinant MIH, showing the predicted α-helical regions, determined as a solution structure via nuclear magnetic resonance
(NMR; Katayama et al. 2003). Data from the Protein Database entry (1JOT.pdb) was remodeled using Jmol V12, which results in fusion of the first two predicted α-helices indicated in B.
12 Simon G. Webster

more neurons are recruited to give the adult complement of 28–36 MIH-ir neurons in each eyestalk
(Dircksen et al. 1988).
Measurement of circulating levels of MIH in the crayfish P.  clarkii using a highly sensitive,
time-resolved fluoroimmunoassay (TR-FIA) showed that intermolt levels of around 6 fmol/mL
decline to 1.3 fmol/mL during premolt but increase to intermolt levels during late premolt and
postmolt (Nakatsuji and Sonobe 2004). However, in C. maenas, levels of MIH measured by RIA
do not decline during premolt and are typically less than 5 fmol/mL. There is, however, evidence
of episodic release, where hemolymph levels rise to 20–40 fmol/mL, and such events seem to be
more common during darkness (Chung and Webster 2005). Steady-state transcription of MIH
mRNA (measured by Northern blotting) seems to decline during premolt in C. sapidus (Lee et al.
1998), and levels of two putative MIH transcripts measured by quantitative reverse transcription
polymerase chain reaction (RT-PCR) declined during premolt in Litopenaeus vannamei (Chen
et al. 2007). Conversely, no changes in MIH transcript abundance (Northern blotting) have been
observed during the molt cycle of P. japonicus (Ohira et al. 1997), and similar results were reported
using quantitative RT-PCR in C. maenas (Chung and Webster 2003). Clearly, these results are not
entirely in agreement with the accepted model of molt control in which a decline in MIH secretion
permits increased ecdysteroid synthesis by the YO, and because it has been repeatedly shown that
premolt YOs become entirely refractive to the inhibitory influence of MIH (Chung and Webster
2003, Nakatsuji and Sonobe 2004, Nakatsuji et al. 2006b), it seems likely that the accepted model of
molt control is rather incomplete, as discussed later.

Second Messengers and Signal Transduction Pathways

Two approaches have been used to determine the relevant second-messengers used in MIH sig-
naling in the YO:  either (i)  by a pharmacological approach using membrane permeant/slowly
hydrolyzable cyclic nucleotide analogues followed by measurement of repression of ecdysteroid
synthesis by YO or (ii) by a physiological approach; that is, measurement of intracellular cyclic
nucleotides in YO extracts following administration of MIH. Because these experiments have been
performed on a variety of decapod crustaceans, it is perhaps unsurprising that a rather disparate
literature exists (Spaziani et al. 1999, 2001, Covi et al. 2009, Nakatsuji et al. 2009). The approach
using exogenous membrane permeant analogues has implicated the involvement of both cyclic
adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in cray-
fish (Sedlmeier and Fenrich 1993, Nakatsuji et al. 2006b) and crabs (Saïdi et al. 1994, Covi et al.
2008). Exceptions include C. antennarius, where only cAMP analogues are effective (Mattson and
Spaziani 1985b, 1986a) and C. sapidus, where only cGMP analogues are effective (Nakatsuji et al.
2006a,b). Physiologically based approaches, in which cyclic nucleotides have been measured fol-
lowing addition of MIH, show that increases in cAMP are rapid and transient (1–4 min, twofold)
and are followed by impressive increases of cGMP (20- to 60-fold within 30–60 min; Baghdassarian
et al. 1996, Chung and Webster 2003, Nakatsuji et al. 2006b). Because nitric oxide (NO) donors
such as SNAP and SE175 and the guanylate cyclase agonist YC-1 can inhibit ecdysteroid synthesis
in C. maenas and G. lateralis (Covi et al. 2008, Mykles et al. 2010), and given that YO express the
catalytic (β) subunit of guanylate cyclase (GC-1) and a Ca2+/calmodulin-dependent NO synthase
in these crabs (H. W. Kim et al. 2004, Lee and Mykles 2006, Lee et al. 2007a,b), a scenario whereby
MIH activates a soluble GC-1 seems eminently feasible. Taking earlier observations concerning
transient increase in cAMP into account, an attractive and intellectually pleasing model of MIH
signaling in crab YO (Fig. 1.4) has been proposed by Chang and Mykles (2011) based on that pro-
posed for cardioacceleratory peptide 2b signaling in Drosophila Malpighian tubules (Beyenbach
et  al. 2010). An interesting peripheral feature of this model involves termination of the cGMP
 MIH Ca2+ channel
MIH-R Cell membrane

G AC
PKA

Triggering phase
ATP
cAMP

Ca2+
PDE1

PKA
AMP
CaM

NOS
Pi
CaN

Arginine NOS
dephosphorylation
Pi

Summation phase
Citrulline
NO

GC-I

PDE5
GTP cGMP GMP

PKG

Ecdysteroid synthesis
(Transcriptional/Translational Regulation)

Fig. 1.4.
Model of molt-inhibiting hormone (MIH) signaling in crab Y-organs (YOs). MIH binds to a G-protein cou-
pled receptor (MIH-R), activating adenylate cyclase (AC), resulting in cAMP production and protein kinase
A (PKA) activation. Phosphorylation of enzymes via PKA may be important in constitutive control of ecdys-
teroid synthesis but also by facultative regulation by phosphorylation of Ca2+ channels, leading to calmodu-
lin (CaM) activation of nitric oxide synthase (NOS), either directly or indirectly via calcineurin (CaN).
Calmodulin can additionally abrogate increases in cAMP via activation of phosphodiesterase 1 (PDE1). A nitric
oxide sensitive soluble guanylyl cyclase (GC-1) then increases cGMP synthesis, activating protein kinase G
(PKG), which inhibits ecdysteroid synthesis. Phosphodiesterase 5 (PDE5) can abrogate increases in cGMP,
potentially modulating this inhibitory pathway during, for example, premolt, when the YO becomes refractory
to MIH. From Chang and Mykles (2011), with permission from Elsevier.
14 Simon G. Webster

signal by phosphodiesterase 5 (PDE5) because upregulation of PDE expression/synthesis could

abrogate the MIH signal, which has in fact been observed: during premolt YOs become entirely
unresponsive to the inhibitory actions of sinus gland peptides (Sefiani et al. 1996) and notably
MIH (Chung and Webster 2003, Nakatsuji and Sonobe 2004, Nakatsuji et al. 2006b), which is
associated with a notable reduction in cGMP accumulation in premolt YO following exposure
to MIH (Chung and Webster 2003). Furthermore, addition of the nonselective PDE inhibitor
3-isobutyl-1-methylxanthine (IBMX) to premolt YO preparations restores the inhibitory activity
of MIH (Nakatsuji et al. 2006b).
Regarding long-term constitutive control of ecdysteroid synthesis (transcriptional and transla-
tional control), rather than the short-term (facultative) allosteric regulation detailed earlier, a vari-
ety of studies suggest that this is relevant.
It has long been known that eyestalk ablation dramatically increases protein and RNA synthesis
in YO (Simione and Hoffman 1975, Gersch et al. 1977) and that this also occurs during premolt
(Toullec and Dauphin-Villemant 1994). During premolt, there are dramatic changes in patterns
of gene expression (Lee and Mykles 2006). Because sinus gland extracts inhibit incorporation of
amino acids into protein (Gersch et al. 1977, Mattson and Spaziani 1986a, Dauphin-Villemant et al.
1995), and in view of the observations that cAMP, IBMX, or forskolin inhibit in vitro protein syn-
thesis by the YO, it has been proposed that constitutive action of MIH might act via cAMP signal-
ing (Mattson and Spaziani 1986a,b, Han et al. 2006). However, in the absence of critical studies
involving administration of pure MIH, this remains speculative because effects unrelated to MIH
(or indeed CHH) cannot be disregarded.
A proteomic study in which C. maenas YO protein profiles from 2D polyacrylamide electropho-
resis were compared following exposure to physiologically relevant doses of MIH showed that a
transaldolase was specifically downregulated (Lachaise et al. 1996). Because this enzyme is part of
the pentose phosphate pathway involved in nicotinamide adenine dinucleotide phosphate-oxidase
(NADPH) production, necessary for cytochrome P450 synthesis, it is entirely feasible that this
may be an important control mechanism in constitutive regulation of the YO.
cDNA-encoding P450 enzymes that may be involved in ecdysteroidogenesis have been cloned
in O.  limosus CYP4C15 (Dauphin-Villemant et  al. 1999)  and C.  maenas CYP4C39 (Rewitz et  al.
2003). The former is expressed in the YO, upregulated during premolt, and differentially down-
regulated by MIH. However, the sequence similarity of both proteins to nvd and phm is low, and,
for the latter, expression in the midgut gland would preclude a direct role in ecdysteroid synthesis.
Nevertheless, a homologue of phm with a high sequence identity to the insect homolog is expressed
in the YO of M. japonicus. Quantitative RT-PCR suggests that this transcript is upregulated during
premolt and downregulated by in vitro incubation of YOs with MIH (Asazuma et al. 2009).
Clearly, in view of the recent proteomics revolution in, for example, nanoscale identification of
proteins via in-gel digestion of Coomassie-stained 2D polyacrylamide gels followed by MS-based
sequencing and homology-based database interrogation to identify candidate proteins, there are
attractive possibilities for more fully defining constitutive mechanisms, regulated via MIH, that
control the long-term ecdysteroidogenic capacity of the YO.

Binding Sites and Receptors

Receptor binding studies using 125I-labeled MIH have shown high-affinity, saturable binding
sites (KD 1.2 × 10–10 M, BMAX 1.3 × 10–10 M) indicative of receptor binding on YO plasma mem-
brane preparations from C. maenas (Webster 1993). Interestingly, receptor binding character-
istics remain unchanged during the molt cycle (Chung and Webster 2003), thus the receptor
is presumably not involved (via changes in receptor number or affinity) in dramatic changes
 Endocrinology of Molting 15

in responsiveness of the YO to MIH during the molt cycle, as detailed earlier. Binding of
125
I-recombinant MIH to a 70 kDa YO membrane protein has been shown for M.  japonicus
(Asazuma et al. 2005) and a 51 kDa protein from C. sapidus (Zmora et al. 2009). Although these
masses are incongruent with those of a receptor guanylate cyclase (rGC) with a mass of ca.
151 kDa that is supposedly a likely candidate CHH receptor (Webster et al. 2012), some stud-
ies give evidence supporting such a scenario. An rGC has been cloned and sequenced from
C. sapidus YO cDNA (Zheng et al. 2006), and immunochemical studies using antisera raised
against the predicted extracellular domain of this protein show that it is expressed in YO plasma
membranes and that pretreatment of YOs with this antibody blunts the suppressive effect of
MIH (Zheng et al. 2008). However, because this rGC is widely distributed in many tissues and
is not specific to the YO (the only tissue that is known to bind MIH), the significance of these
interesting observations is unclear.
In view of the uncertainties regarding the nature of the MIH receptor and those involved in sug-
gested models of second-messenger signal transduction, a very important and much needed result
would be the determination of the MIH (and CHH) receptors. Ideally, candidate cDNAs should be
expressed in heterologous cells with suitable reporters. In a broader context, such an approach will
allow screening of a number of CHH superfamily peptides and would give much-needed informa-
tion on target tissues of individual peptides.

Crustacean Hyperglycemic Hormone

In many species of crustaceans, CHHs can also act as functional MIHs; indeed, the first function-
ally identified MIH, found in H. americanus, was a CHH (Chang et al. 1990). Furthermore, because
CHH is active in the YO bioassay in C. maenas (Webster and Keller 1986), although at least 10× less
potent with respect to MIH, and considering the recent report showing that a recombinant CHH
from G. lateralis inhibits ecdysteroidogenesis in C. maenas YOs (but had no hyperglycemic activity;
Zarubin et al. 2009), it seems possible that CHH might be biologically relevant in molt control,
particularly because clearly identifiable CHH binding sites are found on YO plasma membranes
in crabs (Webster 1993, Chung et al. 2010). However, because it has been shown that simultaneous
release of MIH and CHH does not occur and because both have very short half-lives in the hemo-
lymph (ca. 5 min.; Chung and Webster 2005), the functional significance of CHH in molt control
remains uncertain.
A novel action of CHH concerns its role in stimulating water uptake during ecdysis. As detailed
in Chapter 2, synthesis and release of CHH from gut paracrine cells during late premolt leads to
dipsogenesis, water absorption, and subsequent swelling of crabs to postmolt dimensions (Chung
et al. 1999; see Fig. 1.5). Further studies are now needed to see whether similar intrinsic gut cells
(Webster et al. 2000) and CHH release patterns occur in other crustaceans.

Crustacean Cardioactive Peptide and Bursicon

CCAP was first isolated from the pericardial organs of C. maenas (Stangier et al. 1987), where its
defining biological activity was in the chronotropic and inotropic acceleration of heart rate in
semi-isolated preparations. Subsequently, it has been found in a variety of crustacean and arthropod
species, and it is undoubtedly ubiquitous in its occurrence in the phylum. Notably, the structure
and distribution of CCAP immunoreactive neurons and their principal central and projection pat-
terns are remarkably similar in all arthropods, suggesting highly conserved roles (Dircksen 1998).
Transcript structure of several crustacean CCAP cDNAs has been shown to be similar to those of a
variety of insect counterparts (Chung et al. 2006).
16 Simon G. Webster

E30

E15

Molt stage injected

E5-10

E1

D4-E0

0 200 400 600 800 1000

Time to complete ecdysis (min)

Fig. 1.5.
The effect of crustacean hyperglycemic hormone (CHH) injection on ecdysis. Graph shows effect of CHH injec-
tion (100 pmol) on time to ecdysis. Filled bars, treated crabs; open bars, saline injected controls (n = 3–10); range
is shown. Vertical bars indicate means. Molt stages are shown as percentage of ecdysis. Images show the effect of a
single 300 pmol injection of CHH, upper right, 2 h after injection, compared to a saline-injected control, 2 h after
injection. Arrow indicates epimeral suture line. Lower images show radiograms of a crab injected with 100 pmol
CHH, followed by immersion in 15% barium sulphate in seawater, autoradiography 30 min later, right, and corre-
sponding saline-injected control. Arrowheads show considerable accumulation of radio-opaque barium sulphate
in stomach and hindgut as a result of drinking in the treated crab. Note trapping of barium sulphate in gills in both
crabs. Redrawn from Chung et al. (1999), with permission from the National Academy of Sciences.

In insects, CCAP is not only known to be cardioacceleratory (Tublitz and Evans 1986, Davis
et al. 1990, Dulcis et al. 2001), but is also important in a developmental context related to eclosion.
Control of blood circulation during wing inflation in Manduca sexta (Tublitz and Truman 1985), gut
contraction during embryogenesis, and larval wandering (Broadie et al. 1990, Tublitz et al. 1992) is
regulated by CCAP, and it is a central component in triggering ecdysis behavior at the larval-pupal
molt (Gammie and Truman 1997), among several other biological activities (Dircksen 1998). From
targeted ablation of CCAP-containing neurons of Drosophila, it is known that knockouts exhibit
stage-specific defects in execution and circadian gating of ecdysis behavior (Park et al. 2003).
 Endocrinology of Molting 17

In crustaceans, analogous actions of CCAP during ecdysis have been investigated in C. maenas
and O. limosus: detailed analyses of behaviors and phenotypes during ecdysis, coupled with mea-
surement of hemolymph CCAP via RIA (Stangier et al. 1988) have shown that CCAP release pat-
terns are tightly correlated with specific events during ecdysis (Phlippen et al. 2000); in particular, a
massive release of CCAP, sufficient to deplete stores in the pericardial organ, occurs during ecdysis.
Furthermore, the biphasic molting pattern of the isopod Oniscus asellus is reflected in changes in
CCAP content of the serially iterated CCAP neurons at this time ( Johnen et al. 1995). Temporally
defined ecdysis behaviors, phenotypes, and corresponding changes in circulating CCAP levels are
shown in Fig. 1.6.
The ultimate hormone in arthropod ecdysis has long been known to be bursicon, identified
as the hormone involved in cuticle tanning and melanization of the cuticle almost 50  years ago

PREMOULT ECDYSIS POSTMOULT

Passive ecdysis Active ecdysis: ≈15 min

D1 D2 D3 D4 E0E5E10 E30 E50 E60 E70 E80 E90 E100 A1 A2 B Moult stage

−72 −12 0 1 3 8 24 36 Approximate

time (h)
Morphological characteristics
Setal development
Pleural suture cracks
Decalcification of meropodite of first pereopod
Entire pleural suture opens (finally ≈ 1 mm)
Pleural suture widens (finally 2−3 mm)
Thoracoabdominal membrane bulges
Thoracoabdominal membrane ruptures
First lateralmost carapace spine out
Second lateral carapace spine out
Carapace (Cp) slimy
Cp leather-like
Cp parchment-like
Cp brittle

Overt behavioral characteristics

Body swelling (by water uptake)
Animal rolls onto its side
Lifting of carapace/cephalothorax
‘Wiggling’ (loosening of exoskeleton)
Antennule flicking stops
Rhythmic horizontal spreading movements of antennules
Shedding of head appendages
Shedding of eyestalks
Shedding of head appendages
Shedding of abdomen
Shedding of pereopods
Scaphognathite visibly beating
2–3 tailflips

CCAP hemolymph titre (Carcinus meanas)

Internal parameters
Scaphognathites beat at maximum rate
Hemolymph pressure elevated

Fig. 1.6.
Morphological and behavioral changes during the molt cycle of crab (Carcinus maenas) and crayfish (Orconectes
limosus) and cartoon of crustacean cardioactive peptide (CCAP) levels in the hemolymph. Open bars, crayfish;
filled bars, crab. Molt stages according to Drach and Tchernigovtzeff (1967). From Phlippen et al. (2000), with
permission from The Company of Biologists, Ltd.
18 Simon G. Webster

(Honegger et al. 2008). Following the identification of bursicon in Drosophila as a heterodimeric

(encoded by burs or bursα/pburs or bursβ) cystine knot protein (Luo et al. 2005, Mendive et al. 2005),
expressed sequence tag (EST) data from the D. pulex transcriptome and EST data from C. maenas
predicted the presence of burs α (Daphnia) and burs β (Carcinus), which were subsequently cloned
and sequenced in both species (Wilcockson and Webster 2008) and subsequently in H. gammarus
(Sharp et al. 2010). Contemporary techniques involving in silico data mining coupled with MS analy-
sis have confirmed these results in the D. pulex transcriptome/genome (Gard et al. 2009, Dircksen
et al. 2011) and the neuropeptidome of C. maenas (Ma et al. 2009), and similar bursicon molecules
have been discovered in L.  vannamei using this powerful approach (Ma et  al. 2010). Patterns of
expression of bursicon in the central nervous system (CNS) of C. maenas and H. gammarus using in
situ hybridization and immunohistochemistry have shown that both bursicon and CCAP are always
completely colocalized in a set of fully identified neurons in the thoracic and abdominal ganglia
(Wilcockson and Webster 2008, Sharp et al. 2010); thus, both hormones should also be co-released
from the pericardial organs. Our current studies show that this is indeed the case. Simultaneous mea-
surement of both CCAP (by RIA) and bursicon (by TR-FIA) from the same hemolymph samples
at a high temporal resolution have shown that CCAP peaks at exactly 100% ecdysis (1 pmol/mL) as
does bursicon (4 pmol/mL); hormone levels of both rapidly decline within 10 min of ecdysis and
subsequently return to basal levels within 3 h (Webster, unpublished). Thus, spatial separation of
biological actions of both peptides must occur, given co-release. Although salient biological activities
of CCAP, such as cardioacceleratory activities, are established in crustaceans, the tacit assumption
that bursicon is involved in cuticle hardening in crustaceans cannot be certain as yet. For a detailed
discussion of postecdysial changes in cuticle composition, see Chapter 5 in Volume 1.
It is noteworthy that abdominal ganglia extracts of H. americanus are active in the Sarcophaga cuti-
cle tanning bioassay (Kostron et al. 1995), as is purified Carcinus bursicon (Webster, unpublished).
Thus, a plausible scenario would be that the action of bursicon in cuticle hardening is somewhat
long-lived, compared to the rapid action of CCAP in facilitating emergence behaviors. Injection of
bursicon in Drosophila followed by gene expression analysis by microarray has shown that at least 13
genes are involved in cuticle sclerotization, and 74 diverse genes apparently unrelated to cuticular
hardening are regulated by bursicon (An et al. 2008). It would be interesting to expand these studies
to crustaceans, particularly those with well-annotated transcriptomes, to investigate downstream tar-
gets of CCAP and bursicon signaling. Current understanding of the release patterns of CHH, CCAP,
and bursicon during premolt and ecdysis is diagrammatically summarized in Fig. 1.7.

Methyl Farnesoate

Methyl farnesoate (MF) is the unepoxidated precursor of insect juvenile hormone ( JH) III, syn-
thesized and secreted from the mandibular organs (MO) of decapod crustaceans. Since its discov-
ery (Laufer et al. 1987), a large yet somewhat disparate literature has accumulated suggesting that it
is a key hormone regulating reproduction in crustaceans. Details of the role of MF in reproduction
and regulation of its secretion by neurohormones are outside the scope of this review; compre-
hensive accounts have been given by Laufer et al. (1993), Homola and Chang (1997), Borst et al.
(2001), and Nagaraju (2007). However, it is relevant here to note that MF synthesis is inhibited by
type II CHH superfamily hormones (MOIHs) in cancrid crabs (Wainwright et al. 1996, Lu et al.
2000, 2001) and by type I hormones (CHH) in C. maenas (Keller et al. 1999) and L. emarginata (Liu
et al. 1997). By analogy to well-established roles of JHs in insects (Riddiford 1996), there is evidence
that MF is involved in maintenance of juvenile morphology, inhibiting gonadal development at this
time, and, in the adult, enhancing gonadal maturation.
Regarding the influence of MF on morphogenesis in crustaceans, studies on the spider crab
L. emarginata have shown that reproductively mature males (characterized by abraded carapaces)
 Endocrinology of Molting 19

Ecdysteroid Bursicon

CHH

CHH
Hormone titer (gut)

CCAP

B C1-3 C4 D0 D1 D2 D3 D4 E A

Molt stage

Fig. 1.7.
Schematic of hormone titers during the molt cycle of Carcinus maenas. Molt stages and hormone titers not
to scale.

possess large chelae and higher levels of MF synthesis and titer than do small-clawed, unabraded,
nonreproductive males (Sagi et al. 1993, 1994, Laufer et al. 2002). Additionally, eyestalk ablation in
juvenile Libinia, which elevates levels of MF, results in maintenance of the juvenile (small chelae)
morphotype (Laufer et al. 1997). Similarly, nonreproductive male P. clarkii retain an immature mor-
photype after eyestalk ablation or dietary administration of MF (Laufer et al. 2005).
Juvenilizing effects of MF have been widely reported following exposure of crustacean larvae to
MF or dietary administration. Barnacle cyprids (Balanus amphitrite) exposed to a high concentra-
tion of MF (0.1 µM) in seawater delay metamorphosis (Smith et al. 2000). Similarly, pharmaco-
logical doses of synthetic analogues of JH III, hydroprene, and methoprene increase the number
of zoeal stages and delay megalopal development in Rhithropanopeus harrisii larvae (Christiansen
et al. 1997a,b). MF in seawater delays development of H. americanus larvae (Borst et al. 1987), and
dietary MF retards growth and development of Macrobrachium rosenbergii larvae (Abdu et al. 1998).
Exposure of D. magna to MF (Olmstead and LeBlanc 2002) and JH mimics (Tatarazako et al.
2003, Olmstead and LeBlanc 2002, Oda et al. 2005) induces parthenogenetically reproducing clones
to produce male neonates in a dose-dependent manner. Additionally, it has recently been shown
that morphological changes associated with antipredatory responses (elongation of helmet), can be
induced in Daphnia galeata by administration of MF or the JH mimic fenoxycarb (Oda et al. 2011).
It has long been known that the MOs undergo molt-cycle-related changes in structure, suggest-
ing increased secretory activity, and premolt histological and ultrastructural changes reminiscent
of those seen in YOs during premolt (Le Roux 1968, Aoto et al. 1974, Bucholz and Adelung 1980).
Injection of MO extracts accelerates molting in heterologous bioassays; for example, C.  sapidus
MO extracts promote molting in Penaeus setiferus (Yudin et  al. 1980), and P.  clarkii MO extract
accelerates molting in Caridinia denticulata (Taketomi et al. 1989). It is notable that MF (1 µM)
and co-culture with mandibular organs stimulates ecdysteroid production by YO of Cancer magis-
ter (Tamone and Chang 1993), and that MF injection can accelerate molting in several crustacean
species, for example, Cherax quadricarinatus (Abdu et  al. 2001), P.  clarkii (Laufer et  al. 2005),
and Oziotelphusa senex senex (Nagaraju 2003, Reddy et al. 2004). Because MF secretory activity
20 Simon G. Webster

increases during premolt in several species of crustaceans (Wilder et al. 1995, Ahl and Laufer 1996,
Laufer et al. 2005), it is tempting to suggest that MF forms an important part of a feed-forward loop
during the phase of rapid increase in circulating ecdysteroid so characteristic of premolt. Further
studies to elaborate possible roles of MF in modulating molting and morphogenesis using a species
which is well-studied, and, importantly, using contemporary molecular techniques, would clearly
be worthwhile.

FUTURE DIRECTIONS

During the past 20 years, there has been a remarkable resurgence of interest in the hormonal control
of molting in crustaceans. Many members of the type II CHH hormone superfamily have been
identified, and, in particular, research on authentic MIHs has been rewarding. However, in many
instances, the biological significance of a number of these peptides remains to be elucidated, par-
ticularly in the penaeid shrimps where much overlap in function seems to be common, particularly
when the large number of CHH superfamily peptides in any given species of penaeid is taken into
account. Comprehensive studies detailing the range of biological activities from the full spectrum
of CHH superfamily peptides would do much to reconcile the current situation.
Research on signaling pathways in the YO and control of ecdysteroid synthesis has been signifi-
cant and has clearly demonstrated that the accepted model of molt control is far from complete;
indeed, this may need a major revision. More studies measuring levels of MIH and CHH neuropep-
tides in the hemolymph at appropriate temporal scales are needed, and the highly sensitive assays
needed for such studies are now available for several models. In particular, the determination of the
MIH (and other CHH superfamily) hormone receptors is now urgently required, determination
of candidate receptor cDNAs expressed in heterologous cells would allow determination of ligand
specificity and affinity, and determination of expression of receptors would be invaluable in giving
much information on target tissues of respective peptides.
Downstream events related to changes in gene expression in target tissues following adminis-
tration of hormones would also seem an attractive area of future research. Given current advances
in sequencing and gene expression technologies, it is now becoming feasible to investigate global
transcriptomic changes, and coupling such studies with proteomic approaches should be an excit-
ing challenge.
Given the recent advances in our knowledge of endocrine cascades during ecdysis in insects, it is
now appropriate to investigate these in crustaceans. Genome sequencing, data mining of genomes,
transcriptomes, and EST databases are set to revolutionize crustacean neuroendocrinology, as evi-
denced by novel important studies detailing the peptidome of D. pulex (Dircksen et al. 2011, Christie
et al. 2011). Given that quite a variety of neuropeptides first identified in insects are involved in the
ecdysis cascade, a promising approach to begin corresponding peptide discovery in crustaceans
would be to identify possible transcripts and then firmly identify these as translated proteins via
proteomics. It should first be reiterated at this point that although there are peptides that seem to
be exclusively found in insects, such as PTTH, for which no homologues have yet been identified
from the Daphnia genome and, conversely, that although MIH (type II) CHH family peptides have
not yet been identified in any insect, a rigorously applied concept of “insect” or “crustacean” neu-
ropeptides is widely recognized as being somewhat naïve. Second, until relevant biological actions
of such neuropeptides have been confirmed in crustaceans, the insect hormone appellations are, at
best, putative. Peptide hormones first identified as important in insect ecdysis and that have been
identified using genomic and transcriptomic analyses in crustaceans are briefly reviewed here.
(Arg7-corazonin), pQTFQYSRGWTNamide (Veenstra 1989)  is a key neuropeptide in the
insect ecdysis cascade because, in M. sexta and Bombyx mori, it acts on Inka cells (groups of cells
 Endocrinology of Molting 21

attached to the tracheae of the prothoracic and abdominal segments of insects to elicit ETH release
(Y. J.  Kim et  al. 2004). This peptide has been predicted from analyses of the D.  pulex genome
(Christie et al. 2011), in EST databases for Daphnia carinata (Christie et al. 2010) and L. vanna-
mei (Ma et al. 2010), and the peptide has been identified by MS in D. pulex (Dircksen et al. 2011).
Furthermore, this molecule has been identified by MS of nervous system extracts (brain, thoracic
ganglia) of C. maenas (Ma et al. 2009) and L. vannamei (Ma et al. 2010).
ETH and pre-ETH (PETH) are released from Inka cells to activate specific neuronal circuits
involved in regulation of the ecdysis sequence in insects (see Zitnan et al. 2007 for review). A single
gene codes for an ETH-like precursor in D. pulex (Christie et al. 2011), which encodes two ETHs in
tandem. These are DappuETH1: DPSPEPEPFNPNYNRFRQKIPRIamide and DappuETH2: GE
GIIAEYMNSESFPHEGSLSNFFLKASKAVPRLamide (which is much longer than insect ETHs);
the presence of both of these peptides has been confirmed by MS (Dircksen et al. 2011). As yet, no
other ETH-like molecules have been predicted in crustaceans by transcriptome mining.
EH, first isolated in M. sexta and Bombyx mori (Kono et al. 1987, Katakoa et al. 1987, Marti et al.
1987) and Drosophila (Horodyski et al. 1993), acts directly on Inka cells (in Manduca and Bombyx)
to release ETH and increases cGMP in Inka cells, leading to pre-ecdysis and ecdysis burst pat-
terns in the CNS (Žitňan et al. 1996). EH-producing ventromedial (VM) cells in the brain respond
to ETH by increased activity and release of hormone (Hewes and Truman 1991, Ewer et al. 1997,
Gammie and Truman 1997, 1999), which in turn elicits cGMP production and excitability of 27/704
neurons in the CNS (Ewer et al. 1994, Ewer and Truman 1997, Fuse and Truman 2002). The 27/704
cells in turn produce CCAP and MIPs, which are released at ecdysis (Gammie and Truman 1997,
Davis et al. 2003) and are responsible for execution of the ecdysis motor program.
A single gene encoding a putative EH (dappu-eh) was identified in the D. pulex genome (Christie
et  al. 2011)  using a putative Penaeus monodon EH precursor sequence as a query (Christie et  al.
2010). This EH has a high sequence similarity with some insect EHs; for example, 71% identity with
that of the flour beetle Tribolium castaneum. A second gene, encoding a rather longer EH-like mole-
cule with lower sequence similarity to insect EH (Dappu-ehl) also occurs, next to -eh in a tail-to-tail
inverted tandem cluster (Dircksen et al. 2011). In decapods, an EST encoding an EH-like molecule
has been identified (C. sapidus, Accession No. CV224237), but this has quite a low sequence simi-
larity to insect EHs (Dircksen et al. 2011), and a very similar molecule has been identified by cDNA
cloning in C. maenas (Webster, unpublished). Additionally, EH-like molecules have been identified
by in silico data mining in Triops cancriformis (FM869498) and Marsupenaeus japonicus (CI998674,
CI998685, CI999397; Christie et al. 2010). Thus, it seems likely that EH-like molecules are widely
distributed in crustaceans, and when the presence of other neuropeptides involved in the ecdysis
program are taken into account, it seems likely that this process might be controlled in a similar
manner to that seen in insects.

CONCLUSIONS

The hormonal control of molting in crustaceans has been the subject of much interest to compara-
tive endocrinologists since early models of molt control were proposed more than 60 years ago.
Although molt cycle-related titers, biosynthesis, and metabolism of molting hormones (ecdyster-
oids) are broadly similar in all arthropods, in contrast to insects, ecdysteroid synthesis in crusta-
ceans is repressed by MIH. The first structural determinations of these hormones demonstrated
that they were members of the ever-expanding CHH family and that, in many instances, gene dupli-
cation has resulted in the evolution of specific MIH-type molecules apart from those that exhibit
hyperglycemic and molt-inhibiting activity and that can be structurally identified as CHH-type
molecules.
22 Simon G. Webster

In contrast to CHH-type molecules, which are now known to be expressed in several tis-
sues apart from the eyestalk XO-SG system, MIH-type molecules seem to be mainly expressed
in these tissues. Furthermore, MIH signal transduction pathways are associated with the only
target tissue—the YO—and notable progress has recently been made in elucidating the sig-
nal transduction cascade for MIH in crabs. This work suggests that MIH signaling involves a
membrane-bound G-protein coupled receptor that signals via a soluble guanylate cyclase and a
Ca2+/calmodulin-dependent NO synthase (NOS). However, as yet, the receptors for MIH (and
CHH) remain orphans.
The crustacean equivalent of JH ( JH-III) is MF, produced by the mandibular organ of decapod
crustaceans. The roles of this sequiterpenoid hormone in crustacean development, reproduction,
and molting are currently not well understood, yet there are tantalizing glimpses pointing to quite
fundamental functions of this hormone, and further studies, using contemporary molecular tech-
niques, are now needed to further define its role in the above-mentioned processes.
It has recently been established that CCAP and bursicon are released during the ecdysis pro-
gram of decapod crustaceans at precisely timed periods, which mirrors the situation in insect
ecdysis. However, the great complexity of the numerous hormones involved in insect ecdysis
remains to be explored in crustaceans. With recent advances in transcriptomics, it is expected
that several more “insect” neuropeptides will be discovered, and, despite the disadvantage of
genetic intractability of crustaceans (in comparison to model insects), it is expected that fun-
damental progress toward unraveling the undoubted complexity of the hormonal control of
molting in crustaceans will be made, and this will no doubt be of interest to arthropod endocri-
nologists and the aquaculture industry.

ACKNOWLEDGMENTS

The support of the Biotechnology and Biological Sciences Research Council (BBSRC), Natural
Environment Research Council (NERC), and Royal Society is gratefully acknowledged. I am par-
ticularly indebted to Dr. Sook Chung, Prof. Heiner Dircksen, Prof. Rainer Keller, and Prof. Huw
Rees for our combined research, collaboration, and friendship over more than 25 years.

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 2
ENDOCRINOLOGY OF METABOLISM AND WATER
BALANCE: CRUSTACEAN HYPERGLYCEMIC HORMONE

Simon G. Webster

Abstract
Fundamentally important regulatory processes in crustaceans, namely energy mobilization
and ionic homeostasis, are controlled by crustacean hyperglycemic hormones (CHH), which
form part of a large group of structurally related neurohormones that are also involved in con-
trol of growth and reproduction. Although it was once thought that these peptides had quite
circumscribed roles, principally related to control of hemolymph glucose levels, many more
fundamentally important processes are now known to be regulated by these hormones. Recent
research, which is highlighted in this chapter, has shown that not only do these peptides per-
form multifunctional roles, often in the same species, but they are also widely distributed in
neural and non-neural tissues in crustaceans. Furthermore, the discovery of related neuro-
hormones—the ion transport peptides (ITP) in insects—point to a long evolutionary his-
tory. This is discussed in relation to gene structure and functional diversification within the
Arthropoda.

INTRODUCTION

The most widely studied crustacean neurohormone is without doubt crustacean hyperglycemic
hormone (CHH), first described as a “diabetogenic factor” in the eyestalks of fiddler crabs,
Uca pugilator, which dramatically increases blood sugar levels when injected into blue crabs,
Callinectes sapidus (Abramowitz et al. 1944). This discovery was almost coincident with ana-
tomical studies establishing the sinus gland (SG) as a neurohemal tissue and the neural link to
the X-organ (XO; Hanström 1931, 1933, Bliss 1951, Carlisle and Passano 1953). These pioneering

36
 Endocrinology of Metabolism and Water Balance 37

studies (among others) underpinned our early understanding of the eyestalk neurosecretory
system as a major player in the endocrine orchestra of crustaceans. CHH is the most abundant
peptide in the SG. This fortuitous feature, coupled with the ease of eyestalk extirpation, stimu-
lated early studies concerned with species specificity and purification in particular. Reviews
covering the early literature are given by Kleinholz and Keller (1973), Keller et al. (1985), and
Keller and Sedlmeier (1988). In this review, recent advances in our knowledge of the structures,
functions, and mode of action of CHH are reviewed, highlighting areas where further investiga-
tions are now needed.

CHH PEPTIDES, TRANSCRIPTS, AND GENES

Following the first determination by Edman microsequencing of the amino acid sequence
of CHH from the SGs of Carcinus maenas (Kegel et  al. 1989), there has been a tremendous
upsurge of interest in determining the structures of CHHs—approximately 80 amino acid
sequences of these hormones are now known for about 40 species of decapod crustaceans
(Table 2.1), and this number is continually being added to. Additionally, after the identifica-
tion of the structurally related molt-inhibiting hormone (MIH) in C. maenas (Webster 1991),
vitellogenesis-inhibiting hormone (VIH) in Homarus americanus (Soyez et al. 1991), and that
of a functionally defined MIH in the same species, which was structurally a CHH (Chang et al.
1990), an emerging scenario became clear: the CHH neuropeptide family of crustaceans con-
tains many structurally related members that may have several overlapping biological activi-
ties. Furthermore, following the discovery of an ion transport peptide (ITP) in the locust
Schistocerca gregaria corpora cardiaca, which stimulated Cl− resorption by the ileum (Audsley
et al. 1992), it became clear that this hormone was also a member of the CHH peptide family
(Audsley et al. 1994, Meredith et al. 1996), thus CHH-like molecules seem to have a wide, if
not universal, distribution and a long evolutionary history in arthropods. These concepts are
discussed in detail later.
All CHH family members are between 72 and 83 amino acids long and possess (as an invari-
ant structural feature) six cysteyl residues arranged in three disulphide bridges. Additionally,
two arginines, one aspartate, and a phenylalanine are in identical positions. For crustaceans,
two subfamilies are recognized when amino acid sequences of the mature peptides (and their
precursors) are compared. The first, named the CHH subfamily or type I peptides (Lacombe
et al. 1999) contain 72 or occasionally 73 amino acids and are, in crabs, crayfish, and lobsters,
invariably N-terminally blocked by pyroglutamate and amidated C-terminally (for CHHs pro-
duced by the XO-SG). For the penaeid shrimps, sequences are more variable, numerous in
single species, and N-termini are often unblocked (Chen et al. 2005). For CHHs determined
by in silico database mining for the model crustacean Daphnia pulex (Gard et al. 2009) and by
cDNA cloning in Daphnia magna (Montagné et al. 2010), it is notable that these are much more
similar in a molecular phylogenetic context to insect ITPs than to their crustacean relatives
(Montagné et al. 2010).
The type II peptides (Lacombe et al. 1999) comprise a group of hormones that include mem-
bers with molt-inhibiting, vitellogenesis-inhibiting, and mandibular organ-inhibiting biological
activities named the MIH/VIH group peptides. The distinctness of this hormone group (i.e., the
mature peptides are slightly larger than CHHs, lack a precursor-related peptide, have unblocked
N- and (commonly) C-termini, have the insertion of a Gly in the N-terminal region, show circum-
scribed biological activities, and lack hyperglycemic activity) is clear cut. The structures and func-
tions of MIH/VIH peptides are discussed in detail in Chapter 1 (this volume) on molting.
Table 2.1.  Crustacean hyperglycemic hormone pre/pro-peptide sequences.

Species Tissue source Peptide or Accession Reference

conceptual No.
translation (Protein)
Branchiopoda
Daphnia magna Whole animals ITP ABO43963 Montagné and
ITP-L ABO43964 Toullec 2006
unpub.
Daphnia pulex In silico data ITP Montagné et al. 2010
mining ITP-L
Malacostraca
Isopoda
Armadillidium Sinus glands CHH P30814 Martin et al. 1993
vulgare
Eurydice pulchra Cerebral ganglia CHH JF927891 Wilcockson et al.
2011 unpub.
Decapoda
Astacus astacus Sinus glands CHH P83800 Schmitz et al. 2004
unpub.
Bythograea X-organs CHH AAK28329 Toullec et al. 2002
thermydron
Callinectes sapidus Eyestalk ganglia CHH AAS45136 Choi et al. 2006
Thoracic CHH-L ABC61678 Zheng et al. 2010
ganglia
Pericardial CHH-L DQ667141 Chung and Zmora
organs 2008
Cancer pagurus Sinus glands CHH P81032 Chung et al. 1996
Cancer productus Eyestalk ganglia CHH-I* ABQ41269 Hsu et al. 2008
CHH-IIa** ABQ41270
CHH-IIb** ABQ41271
CHH-III* ABQ41272
Carcinus maenas Sinus glands, CHH P14944 Kegel et al. 1989,
eyestalks Weidemann et al.
1989
Pericardial CHH-L AAG29432 Dircksen et al. 2001
organs
Cherax destructor Sinus glands CHH A P83485 Bulau et al. 2003
Sinus glands CHH B P83486
Chionoecetes bairdi Eyestalk ganglia CHH ACG50068 Chung et al. 2009
Discoplax celeste X-organs CHH JF894384 Turner et al. 2011
Pericardial CHH-L JF894385 unpub.
organs
Galathea strigosa X-organs CHH ABS01332 Montagné et al. 2008
Gecarcinus lateralis Eyestalk ganglia CHH ABF48652 Lee et al. 2007
Hindgut, testis CHH-L ABF58091

(continued)
Table 2.1.  (Continued)

Species Tissue source Peptide or Accession Reference

conceptual No.
translation (Protein)
Gecarcoidea natalis X-organs CHH ABL09570 Webster et al. 2006
Pericardial CHH-L ABL09571 unpub.
organs
Grapsus X-organs CHH JN048801 Webster et al. 2011
tenuicrustatus unpub.
Homarus Sinus glands, CHH A P19806 Chang et al. 1990, De
americanus eyestalks Kleijn et al. 1995
Eyestalk ganglia CHH B Q25154 De Kleijn et al. 1995
Homarus gammarus X-organs CHH A ABA42179 Ollivaux et al. 2006
CHH B ABA42180
Jasus lalandii Sinus glands CHH P56687 Marco et al. 1998
Libinia emarginata Eyestalk ganglia CHH (MOIH) AAD32706 Liu et al. 1997
Litopenaeus schmitti Sinus glands CHH P59685 Huberman et al.
2000
Litopenaeus Eyestalk ganglia CHH CAA68067 Van Wormhoudt
vannamei 1996 unpub.
Eyestalk + CHH (MIH 2) AAR11295 Lago-Lestón et al.
gDNA 2007
Eyestalk CHH-L AAN86055
+gDNA (MIH 1)
gDNA CHH, ITP-L AAN86055 Tiu et al. 2007
Macrobrachium Eyestalk ganglia CHH AAL40915 Chen et al. 2004
rosenbergii Several tissues CHH-L AAL40916
Marsupenaeus Eyestalk ganglia CHH 1 O15980 Ohira et al. 1999
japonicas CHH 2 Q9U5D2 unpub.
CHH 3 BAA13481 Ohira et al. 1997
CHH 5 O15981 Ohira et al. 1999
unpub.
Sinus glands CHH 6 P81700 Yang et al. 1997
Eyestalk ganglia CHH 7 O15982 Ohira et al. 1999
unpub.
Metapenaeus ensis Eyestalk ganglia CHH A AAD45233 Gu et al. 2000
CHH B AAF63028
Nephrops norvegicus Eyestalk ganglia CHH A* AY285782 Mettulio et al. 2004
CHH B* AY285783
Orconectes limosus Eyestalk ganglia CHH Q25589 De Kleijn et al. 1994
Pachygrapsus X-organs CHH AAO27804 Toullec et al. 2006
marmoratus Pericardial CHH-L AAO27806
organs
Pagurus bernhardus X-organs CHH ABE02191 Montagné et al. 2008

(continued)
40 Simon G. Webster

Table 2.1.  (Continued)

Species Tissue source Peptide or Accession Reference

conceptual No.
translation (Protein)
Penaeus monodon Eyestalk ganglia SGP-II AAC84143 Davey et al. 1998
unpub.
CHH 1 O97383 Davey et al. 2000
CHH 4* O97386
CHH 5 O97387
CHH* AADO3606 Chen et al. 1988
unpub.
Pontastacus Eyestalk ganglia CHH AAX09331 Ollivaux et al. 2004
leptodactylus unpub.
CHH 1 AAS45406 Mettulio et al. 2004
Portunus N/A CHH EU395808 Zhu et al. 2008
trituberculatus unpub.
Potamon ibericum X-organs CHH ABA70560 Toullec et al. 2006
CHH-L ABA70561
Ptychognathus Eyestalk ganglia CHH JN048802 Webster et al. 2011
pusillus unpub.
Procambarus Sinus glands CHH Q10987 Aguilar et al. 1996
bouvieri
Procambarus clarkii Eyestalk ganglia CHH AB027291 Yasuda-Kamatani
and Yasuda 2000
unpub.
Muscle, gDNA CHH-L AF474409 Kuepper and Jaros
2002 unpub.
Rimicaris kairei “Dorsal eyes”/ CHH FJ447499 Qian et al. 2009
carapace
“Dorsal eyes”/ CHH-L FJ447498
carapace
Scylla olivacea Eyestalk ganglia CHH AY372181 Tsai et al. 2008
Pericardial CHH-L EF530127
organs

Protein accession numbers conceptually determined from various tissue sources by cDNA, gDNA cloning and sequencing, in
silico data mining, or directly determined by microsequencing (gray highlights indicate mature peptide sequence information
only). Asterisks indicate isoforms where mature peptide sequences are identical, but where precursor peptides sequences differ.
Abbreviations: MOIH, mandibular organ-inhibiting hormone biological activity; MIH, molt-inhibiting hormone-like structure;
ITP, ion transport peptide-like structure; SGP, sinus gland peptide CHH-L; ITP-L denotes corresponding peptides derived
from alternative splicing.

A universal feature of all type I CHH prepro-hormones is the presence of a cryptic precur-
sor peptide:  CHH precursor-related peptide (CPRP). Unlike the mature CHHs, the size and
sequences of CPRPs are not well conserved, ranging from four residues in Penaeus monodon
(Davey et al. 2000) to 50 in the anomuran Pagurus bernhardus (Montagné et al. 2008). Several
CPRPs are frequently observed; for example, seven in Marsupenaeus japonicus (Yang et  al.
 Endocrinology of Metabolism and Water Balance 41

1997, Ohira et al. 1997), suggesting that there are many copies of the CHH gene, as is discussed
later. Additionally, it has been elegantly shown from nano liquid chromatography-electrospray
ionization-quadrupole time of flight mass spectrometry (LC-ESI-QTOF MS/MS) that four com-
plete and 27 truncated CPRPs (including some that are methylated at the C-terminus and that
are not extraction artifacts) occur in Cancer borealis (Fu et al. 2005). Using matrix-assisted laser
desorption ionization-Fourier transform mass spectrometry (MALDI-FTMS), it has recently
been shown that individual Cancer productus possess different inventories of CPRPs: 61% of the
examined crabs express CPRP I and II; 26% CPRP I, II, and, III; and 13% CPRP I, II, and IV
(Stemmler et  al. 2007). However, to date, no physiological role has been determined for any
CPRP. Despite stoichiometric co-release with CHH, CPRP has an extraordinarily long half-life
in circulation in Cancer pagurus, approximately 60 min, compared with 5–10 min for CHH
(Wilcockson et al. 2002), which would perhaps argue against any hormonal function. However,
it seems possible that the truncated CPRPs may actually represent the final processed, biologi-
cally active peptides (Fu et al. 2005).
Recently, an exception to the rule concerning the presence of CPRP in preprocessed CHH pre-
cursors has been observed. cDNA sequences encoding CHH-like peptides that do not contain a
CPRP have been found to be expressed in the wall of the spermatophore sac of Fenneropenaeus
chinensis (Li et al. 2010). Whether these transcripts are translated to peptide and their functionality
remain to be determined.
A notable feature of CHHs concerns the diversity of isoforms in individuals. In crab and cray-
fish SG, both N-terminally blocked and unblocked hormones are found (Chung and Webster 1996,
Bulau et al. 2003, 2004). For lobsters and crayfish, further structural diversity is generated by ste-
reo isomerization of the third amino acid, phenylalanine, which is present in L or D forms (Soyez
et al. 1994, Soyez et al. 1998, Ollivaux et al. 2009). It is thought that this diversity has biological
significance because hyperglycemia induced by injection of the D-Phe3 containing hormone in
H. americanus is prolonged compared to that seen for the corresponding L-Phe3 isoform (Soyez
et al. 1994), and only the D-Phe3 isoform exhibits MIH activity (repression of ecdysteroid synthe-
sis) in Procambarus clarkii (Yasuda et al. 1994).
Production of recombinant CHHs for several species of crustaceans has highlighted the
importance of the C-terminal amide because unamidated recombinant CHHs (rCHH) (whether
expressed in bacteria or yeast) have somewhat limited hyperglycemic activity (Katayama et  al.
2002, Trerattrakool et al. 2003), whereas correctly amidated rCHH has good biological activity,
at least 10 times that of the unamidated molecule (Mosco et al. 2008, Nagai et al. 2009, Chang
et al. 2010). Recombinant CHHs containing point or C-terminal deletions; Mettulio et al. (2004)
showed that point mutations that disrupted correct disulphide bridge formation completely
removed biological activity, as did C-terminal truncation. Similar results have been found for
C-terminal mutational analysis of recombinant ITP expressed in Drosophila Kc1 cells and assayed
(stimulation of Cl− transport) in the locust ileum (Wang et al. 2000). Clearly, these results under-
score the critical importance of C-terminal amidation. Indeed, the lack of hyperglycemic activity
observed for the (unamidated) pericardial (PO)-CHH (Dircksen et al. 2001) or biological activity
seen for the corresponding splice variant of ITP (ITP-L) in locusts (Wang et al. 2000, Phillips et al.
2001) might also reflect this phenomenon, notwithstanding the rather different C-terminal amino
acid sequences in these splice variants, as alluded to later. Interestingly, an rCHH mutant that pos-
sessed a Gly12 residue, which would mimic MIH/VIH (type II) peptides, has very limited hyper-
glycemic activity (Katayama and Nagasawa 2004). Additionally, rCHHs expressed in yeast, with
a free C-terminus or cMyc/polyhistidine tag, were effective at repressing ecdysteroidogenesis by
the Y-organ (YO; albeit at very high doses) but were almost without discernable hyperglycemic
activity (Zarubin et al. 2009).
42 Simon G. Webster

A common feature of many CHH and ITP genes concerns alternative splicing arrangements.
It was first shown in C.  maenas that tissue-specific expression of two different CHHs occurred
(Dircksen et al. 2001). The CHH mRNA expressed by XO perikarya (XO-CHH) is encoded for
by exons I, II, IV, whereas that of the intrinsic multipolar neurosecretory neurons of the pericar-
dial organ (PO-CHH) is encoded by exons I–IV. This splicing arrangement results in two pep-
tides that differ from amino acid 41 through to the C-terminus (Fig. 2.1). This arrangement of the
CHH gene (4 exons) and alternative splicing arrangements seem to be identical for the majority
of decapod CHH transcripts and genes thus far identified (e.g., Macrobrachium rosenbergii, Chen
et al. 2004; Pachygrapsus marmoratus and Potamon ibericum, Toullec et al. 2006; Rimicaris kairei,
Qian et al. 2009). However, in the penaeids Metapenaeus ensis and Litopenaeus vannamei, it has been
suggested that 3-exon CHH genes (where exon I is absent) are present (Gu et al. 2000). However,
it has recently been shown that for L. vannamei, a conventional 4-exon gene encodes CHH, and
alternative splicing mechanisms are very similar if not identical to those seen in other crustaceans
(Lago-Lestón et al. 2007, Tiu et al. 2007).
Intriguingly, for all the CHH family type II (MIH/VIH) peptides, gene arrangements consist of
3 exons, yet the splice donor/acceptor site between exons II–III corresponds exactly to that seen in
all other CHH molecules. This feature clearly points to a long evolutionary history, and scenarios of
CHH gene evolution involving either two independent exon duplications or exon duplication and
deletion, which would elegantly account for CHH gene structure and diversity across the arthro-
pods, have been recently proposed (Montagné et al. 2010), as shown in Fig. 2.2.
Regarding copy number of CHH genes, genomic Southern blot analysis has indicated at
least four CHH genes in C. maenas (Dircksen et al. 2001) and at least eight in M. ensis (Gu and
Chan 1998, Gu et al. 2000). From transcript diversity, six different CHH-like cDNAs have been
cloned and sequenced in P. monodon (Davey et al. 2000, Udomkit et al. 2000); microhetero-
geneity is frequently observed in cDNA clones (Dircksen et al. 2001) and can also be seen in
individual crabs (Hsu et al. 2008). These results all indicate that multiple copies of CHH genes
are present in crustaceans.

*
XO-CHH mRNA

* *
I II III IV
CHH-gene

PO-CHH mRNA

Fig. 2.1.
Schematic representation (not to scale) of crustacean hyperglycemic hormones (CHH) gene, transcript, and
peptide structure, illustrated by the Carcinus maenas CHH prototype. X-organ (XO)-CHH encoded by exons
I, II, IV is expressed by eyestalk neurons and gut paraneurons, whereas pericardial organ (PO)-CHH encoded
by exons I–IV is expressed by intrinsic neurons in the POs. Asterisks show positions of stop codons. CHH pre-
cursor related peptide (CPRP) is shown outlined in green, XO-CHH in blue, the PO-CHH variant C-terminal
(which differs from residues 41–72, and which is unamidated) in yellow. See color version of this figure in the
centerfold. Modified from Dircksen et al. (2001), with permission from The Biochemical Society.
 Endocrinology of Metabolism and Water Balance 43

A PLEOCYEMATA CHH
Exon
duplication Gene deletion
MIH
4 EXONS

CHH
DECAPODA CHH
MIH
DENDROBRANCHIATA

BRANCHIOPODA
Intron
insertion
ARTHROPODA 4 EXONS
ITP
3 EXONS
Exon
duplication INSECTA 5 EXONS
Drosophila
Exon
duplication
ANCESTRAL
GENE CHELICERATA
2 EXONS

B PLEOCYEMATA CHH

MIH
DECAPODA

CHH
3 EXONS
Exon Exon CHH
Exon 3 EXONS
duplication deletion
deletion MIH
4 EXONS
DENDROBRANCHIATA

BRANCHIOPODA

ITP
3 EXONS
INSECTA 5 EXONS
Drosophila
Exon
duplication

CHELICERATA

Fig. 2.2.
Evolutionary scenarios of the crustacean hyperglycemic hormones (CHH) family genes in the Arthropoda.
(A) An intron insertion in an ancestral 2 exon gene (asterisk) and two independent exon duplications resulted
in 4 exons for CHH, and a single-exon duplication gave 3 exon molt-inhibiting hormone (MIH) genes.
(B) A single exon duplication accounts for CHH, and exon deletion for MIH gene structure. Note that a fur-
ther exon duplication can account for the 5 exon ITP gene in Drosophila. The scaled schematics show exons
represented by boxes, untranslated regions (UTR) (gray), signal peptides (yellow), precursor-related peptides
(blue), ITPs (purple), CHHs (red), MIHs (green), alternatively spliced exons encoding ITP-L (violet), and
CHH (orange) C-terminus regions. Question marks indicate UTRs whose precise borders are unknown. See
color version of this figure in the centerfold. Figure redrawn from Montagné et al. (2010).

BIOLOGICAL ACTIVITIES OF CHH

Well over 60 years have elapsed since the defining role of CHH (and its corresponding appellation)
in regulation of carbohydrate metabolism was discovered and named (Chung et al. 2010, Webster
et al. 2012). However, in common with many other neurohormones, it is accepted that CHH is
truly pleiotropic and influences a variety of central physiologies such as molting, osmoregulation,
and reproduction, notwithstanding the influence on some of these processes by other structurally
related (type II) peptides in the CHH family, as alluded to earlier.
44 Simon G. Webster

Regulation of Carbohydrate and Lipid Metabolism

It has long been known that injection of eyestalk, SG extract, or purified CHH leads to rapid and sus-
tained hyperglycemia in crustaceans, almost without exception, in a group- or even species-specific
manner (Keller 1969, Leuven et  al. 1982). This response is mediated via binding to specific,
high-affinity, and saturable CHH receptors (Kummer and Keller 1993, Webster 1993) that are found
in many (perhaps all) tissues, as described later. Subsequent increases in cyclic nucleotide levels
(cyclic adenosine monophosphate [cAMP] and cyclic guanosine monophosphate [cGMP]) lead
to activation of protein kinases and thus to phosphorylase and inhibition of glycogen synthase in
a manner reminiscent to that of glucagon action (Sedlmeier and Keller 1981, Sedlmeier 1982, 1985),
although, crucially, fundamental aspects such as receptor identification and second-messenger path-
ways remain to be properly defined. The inevitable result of this cascade is hyperglycemia, yet it is
has been suggested that this phenomenon is somewhat artifactual; that is, a result of glucose leakage
from the intracellular environment, following glycogen hydrolysis (Santos and Keller 1993a,b).
The physiological significance of hyperglycemia, viz. its essential role as an adaptive hormone,
has only relatively recently been defined despite fundamental advances in our knowledge of CHH.
Circulating levels of CHH have been determined by radioimmunoassay (RIA; Keller and Orth
1990, Webster 1996, Chung and Zmora 2008) and enzyme-linked immunosorbent assay (ELISA;
Chang et  al. 1998, Lorenzon et  al. 2004). Recently, my laboratory has developed ultrasensitive
Eu3+chelate-based time-resolved fluoroimmunoassays (TR-FIA) that are 20–100-fold more sensi-
tive than current immunoassays. These offer the exciting promise of enabling repeated measure-
ments of hormone levels to be made in individuals, thus vividly illustrating remarkably rapid
minute-by-minute changes in CHH levels, viz. Gecarcoidea natalis (Morris et al. 2010), Discoplax
celeste (Turner 2010), and C. maenas (Webster unpublished).
Changes in CHH titer, which result in sustained increases in glucose 1–2 h after stressful epi-
sodes, have been shown repeatedly. Examples for various decapod crustaceans include emersion
stress and/or hypoxia (Keller and Orth 1990, Webster 1996, Chang et al. 1998, Chung and Zmora
2008), thermal stress (Zou et al. 2003, Chung and Webster 2005), parasitism (Stentiford et al. 2001),
or exposure to heavy metal pollutants (Lorenzon et  al. 2004). In particular, forced exercise (by
inducing continuous running behavior) seems to be a particularly effective and environmentally
relevant stressor. In the Christmas Island red crab, G.  natalis, this results in rapid hyperlactemia
accompanied by immediate release of CHH, which promptly declines after termination of exercise
(Morris et al. 2010). Subsequently (within an hour), the animals exhibit hyperglycemia, as sum-
marized in Fig. 2.3. CHH levels appear to be tightly controlled by positive and negative feedback
loops. Evidence for both was first provided by Santos and Keller (1993a), who demonstrated that
injection of lactate or glucose increased or reduced, respectively, circulating CHH levels in C. mae-
nas. Evidence for a negative feedback loop was recently shown in G. natalis, where glucose injec-
tion could completely inhibit exercise-induced CHH release. This was of particular significance
because this negative feedback loop only occurred during the wet season, when crabs were actively
migrating, and it is presumably of ecophysiological significance in maximizing energy efficiency
during migration. During the dry season, when these crabs are inactive and fossorial, this feedback
loop is entirely absent (Morris et al. 2010). The existence of a negative feedback mechanism has
been elegantly demonstrated in vitro, in C. borealis (Glowik et al. 1997). Dissociated CHH neu-
rons from the XO can be readily cultured in vitro and produce a recognizable phenotype in that
they produce lamelliform (veiling) growth cones and contain sufficient CHH for measurement of
the peptide content of single cells (Keller et al. 1995). Using single-cell current and voltage clamp
techniques, the glucose sensitivity of these cells can be demonstrated, whereby hyperpolarization
occurs at concentrations of D-glucose (EC50 0.25 mM) that nicely correspond to levels recorded
during hyperglycemia, thus underscoring the physiological relevance of this response (Fig. 2.4).
 70
100
60 **
80

CHH (pmol\L)
50 60

CHH (pmol/L)
40
40
20
30 0
0 20 40 60 80 100 120
20 Time (min)

10

0
0 20 40 60 80 100 120
Time (min)

10

***
8
Glucose (mmol/L)

*
2

0
0 20 40 60 80 100 120
Time (min)

25

*** ***
20 ***
***
Lactate (mol/L)

15
***
10

0
0 20 40 60 80 100 120
Time (min)

Fig. 2.3.
The effect of exercise on circulating crustacean hyperglycemic hormones (CHH), glucose, and lactate levels
in the Christmas Island red crab, Gecarcoidea natalis. Crabs were exercised for 10 min (black bar), followed by
a 110 min recovery period (white bar). Black columns, exercised animals; gray bars, controls (n = 5 for each
group). Error bars= +1 SEM. Inset shows CHH profiles from two exercised (solid lines, circles) and control
crabs (dotted lines, triangles). * P<0.05, ** P<0.01, *** P<0.001. From Morris et al. (2010), with permission from
The Company of Biologists.
 −40 mV

0.025 mmol/L

0.1 mmol/L

0.15 mmol/L

0.5 mmol/L

5 mmol/L

20 mV
10 mmol/L
40 s

1.2
4
3 3
2
0.8 5
Normalized response

1 4
4
0.4

6
2 Km = 0.25 mmol/L
6
0 1

0.01 0.1 1 10
[D-Glucose] (mmol/L)

Fig. 2.4.
Glucose dependent responses of isolated crustacean hyperglycemic hormones (CHH) immunoreactive neu-
rons of Cancer borealis to 2 min applications of glucose (0.025–10 mM). Graph shows dose–response curve
where hyperpolarizations have been normalized to the amplitude of the response at 5 mM. Numbers of cells
used are shown for each dose, curve fitted to Michaelis-Menten equation. From Glowik et  al. (1997), with
permission from The Company of Biologists.
 Endocrinology of Metabolism and Water Balance 47

Interestingly, electrical stimulation or K+ evoked exocytosis of dissected XO-SG of Cardisoma car-

nifex showed that CHH release continued for some time (tens of minutes) after the cessation of
stimulus (Keller et al. 1994).
As befits the role of CHH as an adaptive hormone, it is notable that CHH is a secretagogue and
stimulates the release of amylase from isolated midgut glands of C. maenas and Orconectes limosus
in a physiologically relevant, dose-dependent manner (Sedlmeier 1988). Additionally, in these spe-
cies, there is evidence to suggest the involvement of CHH in elevation of free fatty acids and phos-
pholipids in vitro and in vivo (Santos et al. 1997).
It has been proposed, generally on the basis of injection of biogenic amines, peptide neu-
rotransmitters, and simple measurement of hemolymph glucose, that a variety of these may
be involved in CHH release. For example, serotonin is well known to cause hyperglycemia
in a variety of crustaceans (Lüschen et al. 1993, Lorenzon et al. 2005), and this response can
be potentiated by serotonin reuptake inhibitors such as fluoxetine (Santos et  al. 2001)  and
may be mediated via 5-HT1-like receptors, since antagonists such as cyproheptadine blunt
serotonin-induced hyperglycemia (Lorenzon et  al. 2004). Nevertheless, these experi-
ments should be interpreted with caution. In some instances, eyestalk removal abolishes
serotonin-induced hyperglycemia, whereas in others this neurotransmitter seems to act inde-
pendently of CHH. The same caveats apply to experiments involving administration of dopa-
mine. This induces hyperglycemia independently of CHH, in that eyestalk-ablated animals
show no hyperglycemia following injection (Lüschen et al. 1993, Komali et al. 2005), whereas
other studies have shown that dopamine causes hypoglycemia in intact animals (Lorenzon
et al. 2004). Interestingly, retinoic acid, which appears to be an authentic signaling molecule
in crustaceans (Hopkins 2001), may mediate CHH release (as measured by hyperglycemia) in
Oziotelphusa senex senex—but only 9-cis retinoic acid and not the all-trans isomer is active in
this respect (Reddy and Sainath 2008).
Clearly, firm conclusions regarding the importance of neurotransmitters can only be reached if
CHH levels are measured in conjunction with amine administration, as have been reported (Zou
et al. 2003, Lorenzon et al. 2005). These have shown that 5-HT seems to directly stimulate CHH
release, in contrast to dopamine. However, the elegant single-cell electrophysiological approach
taken by Glowik et al. (1997) shows that 5-HT (and γ-aminobutyric acid [GABA]) hyperpolarize
CHH neurons, a result clearly at odds with other observations. Interestingly, in the lobster H. amer-
icanus, CHH immunopositive somata at the branching point of the second thoracic nerve are in
close apposition to neurosecretory terminals of serotonergic and octopaminergic neurons, and, for
octopamine, nanomolar levels of bath-applied transmitter excite spontaneous bursting activity of
these root neurons, whereas higher concentrations of both transmitters inhibit this activity (Basu
and Kravitz 2003).
Regarding the involvement of peptide neurotransmitters such as enkephalins in CHH release,
again, few firm conclusions can be drawn. On the one hand, Leu-enkephalin (L-Enk) administration
has been reported to lead to hypoglycemia in U. pugilator, C. maenas, and P. clarkii (Nagabhushanam
et al. 1995), Met-enkephalin injection has been reported to cause hyperglycemia in the freshwater
crab O. senex senex (Reddy 1999). However, more critical and sophisticated recent experiments,
whereby CHH release has been measured in isolated eyestalk ganglia of O. limosus, coupled with
immunohistochemical studies, have shown that the influence of L-Enk is likely inhibitory. Because
extensive L-Enk immunoreactive neuropils with dendrites are in close (possibly synaptic) apposi-
tion with arborizing dendrites of CHH immunoreactive XO perikarya, enkephalinergic modula-
tion of CHH release (Ollivaux et al. 2002) seems possible. However, this seems likely to be indirect
because the isolated CHH neuron preparations of Glowik et al. (1997) are entirely unresponsive to
L-Enk. Clearly, the mechanisms of modulation of CHH by relevant neurotransmitters have yet to
be satisfactorily resolved and merit further study.
48 Simon G. Webster

Inhibition of Ecdysteroid and Methyl Farnesoate Synthesis

Clearly identifiable MIHs belonging to the type II CHH family grouping that have been func-
tionally defined by their ability to repress ecdysteroid synthesis in vitro by YO bioassay (Soumoff
and O’Connor 1982, Mattson and Spaziani 1985, Webster 1986)  seem to be universal in brachy-
urans (Webster and Keller 1986, Chung and Webster 2003) and relatively common in astacurans
(Nagasawa et al. 1996, Nakatsuji and Sonobe 2004) and some penaeid shrimps (Sefiani et al. 1996,
Yang et al. 1996). However, in lobsters (Nephropidae), such MIHs have not been identified. Instead,
a CHH (type I molecule) acts as a functional MIH (CHH A in H. americanus; Chang et al. 1990),
whereas in the spiny lobster Jasus lalandii (Palinuridae), a distinct, biologically active MIH occurs
together with CHH (Marco et al. 1998, 2000).
It is notable that CHHs also exhibit some MIH activity in the YO bioassay, but at much reduced
potency (10–20 times less active) compared to MIH (Webster and Keller 1986), and it is possible
that both may act synergistically (Webster 1998). However, although high-affinity saturable recep-
tors for both CHH and MIH occur in the YO of C. maenas (Webster 1993) and C. sapidus (Chung
et al. 2010), it is as yet unclear whether CHH can exert a biologically relevant effect in control of
molting, in the context of synergistic interaction with MIH, since both peptides are rarely released
simultaneously (Chung and Webster 2005), and both have very short half-lives in the hemolymph
of between 5 and 10 min (Chung and Webster 2005, 2008). Conversely, it should also be noted that
functionally defined type II MIHs have, to date, never been shown to exhibit hyperglycemic activ-
ity, again highlighting the distinctiveness of the two peptide families.
Methyl farnesoate, the unepoxidated precursor of insect juvenile hormone III, is synthesized
by the mandibular organ (MO) of decapod crustaceans (Borst et  al. 1987, Laufer et  al. 1987a,b,
Tsukimura and Borst 1992). MF synthesis is inhibited by distinct, MIH-like (type II) neuro-
peptides from the XO in C.  pagurus:  the mandibular organ-inhibiting hormones (MOIH-I, -II;
Wainwright et al. 1996). Although similar peptides have been identified in other crabs within the
genus (Webster, unpublished), in all other decapod crustaceans so far examined, CHH fulfils the
role of a MOIH in that it profoundly inhibits in vitro MF synthesis by MO in Libinia emarginata
(Liu et al. 1997) and C. maenas (Keller et al. 1999). Clearly, further studies are now needed; in an
evolutionary context, it seems axiomatic that distinct MOIH molecules are restricted to just one
genus of decapod crustaceans.

Inhibition of Ovarian Protein Synthesis and Vitellogenesis

The inhibitory influence of SG peptides on protein and/or vitellin synthesis in decapod crusta-
ceans has long been known (Bomirski et al. 1981, Meusy and Payen 1988, Quackenbush and Keeley
1988). The hormone responsible for this process, vitellogenesis- or gonad-inhibiting hormone
(VIH/GIH), first identified in lobsters H. americanus (Soyez et al. 1991) and subsequently in wood-
lice Armadillidium vulgare (Grève et al. 1999), is a type II CHH family member. In lobsters, GIH
has no hyperglycemic activity (Soyez et al. 1987). However, by measuring semiquantitative CHH
and GIH transcript (RNase protection assay) and circulating peptide (EIA) levels, it has been
suggested from causality that GIH inhibits the start of vitellogenesis, CHH-A and -B stimulate its
onset, and CHH-B stimulates oocyte maturation in the American lobster H. americanus (De Kleijn
et al. 1998). Nevertheless, for the penaeid shrimps, an emerging scenario is one whereby CHHs
possess relevant VIH activity, and where identifiable type II VIH/GIH peptides are noticeably
absent in the SG neuropeptide inventory. For example, each of the seven CHH family SG peptides
from M. japonicus inhibited protein and mRNA synthesis in vitellogenic ovarian explants of Penaeus
semisulcatus (Khayat et al. 1998), yet a type II molecule (called Pej-SGP-IV), a putative MIH, was
entirely without inhibitory activity (Avarre et  al. 2001). Subsequently, homologous bioassay via
 Endocrinology of Metabolism and Water Balance 49

quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) analysis of vitellogenin

(Vtg) mRNA expression in this species showed that a CHH peptide (called Pej-SGP-III) pro-
foundly inhibited Vtg mRNA synthesis and, once again, that the MIH-like SG peptide SGP-IV
was ineffective in this respect (Tsutsui et al. 2005). Clearly, further research is now timely regarding
the identification of candidate VIH/GIH molecules in brachyurans and the corresponding activi-
ties of CHHs in a variety of species. Interestingly, a recent study in which Vtg mRNA levels were
measured in hepatopancreas explants of C. sapidus has shown that MIH increases Vtg mRNA levels
in mid-vitellogenesis (Zmora et al. 2009). Thus, it may be that different crustacean taxa have devel-
oped rather divergent strategies in their hormonal control of vitellogenesis, and this is a topic that
deserves further study.

Osmo- and Ionoregulation

Early work concerned with the hormonal control of osmotic and ionic regulation in mainly marine
crustaceans has been the subject of several comprehensive reviews (Mantel 1985, Muramoto 1988,
Kamemoto 1991, Morris 2001). Notwithstanding a considerable volume of literature implicating a

A −5 +G
−6
−7
Potential difference (mV)

−8
−9
−10
−11
−12
−13
−14
−G
−15
0 20 40 60 80 100 120

B 500 −G
450
Na+ influx (µEqNa+/g/h)

400
350
300
250
200
150
+G
100
0 20 40 60 80 100 120
Time (min)

Fig. 2.5.
Perfusion of posterior gills of Pachygrapsus marmoratus with high-performance liquid chromatography purified
sinus gland extract corresponding to crustacean hyperglycemic hormones (CHH) (four sinus gland equiva-
lents) showing its effect on (A) transepithelial potential difference and (B) Na+ influx. +G, −G refer to addition
and removal of sinus gland extract in gill perfusate. From Spanings-Pierrot et al. (2000) with permission from
Elsevier.
50 Simon G. Webster

variety of neurotransmitters (from almost all of the nervous system) in modulation of ion transport,
an emerging scenario from classical ablation/injection-type studies suggested that involvement of
hormones from the eyestalk neurosecretory system was relevant in such processes (Kamemoto
1976, Charmantier et  al. 1984, Freire and McNamara 1992). In particular, XO-SG peptides were
implicated: SG extracts increase hemolymph osmolarity in destalked juvenile lobsters H. america-
nus that were hyperregulating in dilute seawater (Charmantier-Daures et al. 1988), and perfusion
of posterior gills of the euryhaline crab P. marmoratus with SG extracts stimulated Na+ influx and
transepithelial potential (Pierrot et al. 1994, Eckhardt et al. 1995). Further studies on these species
have unequivocally shown CHH to be the candidate peptide involved in stimulation of Na+ uptake
(Charmantier-Daures et al. 1988, Spanings-Pierrot et al. 2000), as shown in Fig. 2.5. In the crayfish
Astacus leptodactylus, the D-Phe3 stereoisomer of CHH has been shown to be more effective than
the L-Phe3 stereoisomer in increasing hemolymph osmolarity and Na+ levels in the hemolymph of
destalked animals (Serrano et al. 2003).
In the penaeid shrimp L. vannamei, the splice variant of CHH, encoded by exons I–IV and
thus equivalent to PO-CHH (see earlier discussion), is widely expressed in several tissues,
including the gills (Tiu et al. 2007). Levels of this transcript (quantified by Northern blotting)
are highest in the posterior gills, which are considered to be of primary importance in active
transport of ions (Mantel and Farmer 1983, Towle and Weihrauch 2001). Stage-specific expres-
sion was maximal during late intermolt to early premolt, and highest levels of expression were
observed at a salinity of 15 ppt. dsRNA injection gave moderate knockdown of expression (37%
after 48 h), and injection of large quantities (0.3 μg) resulted in gill hemorrhage and complete
mortality. These results are intriguing because, as alluded to later, the equivalent of CHH in
insects (ITP) is involved in chloride resorption in the rectum of orthopteroid insects (Meredith
et al. 1996, Phillips et al. 1998a,b). However, it should be noted that only the short form of ITP
(equivalent to XO-CHH) is biologically active in insects, and the splice variant expressed in
the study by Meredith et al. (1996; ITP-L, which is equivalent to PO-CHH) seems to have no
obvious biological activity.
A novel action of CHH concerns that of water uptake during ecdysis. In C.  maenas, gut
paracrine cells in the hindgut and foregut (Fig. 2.6D,G) that are associated with muscle inser-
tions, synthesize XO-CHH only during premolt and release this as a massive but ephemeral
surge during the initiation of ecdysis. This release results in a tremendous dipsogenesis, water
absorption, and subsequent swelling of the crab, to allow it to reach its subsequent postmolt
dimensions (Chung et  al. 1999). Interestingly, this hormone surge is almost coincident with
that of crustacean cardioactive peptide, which seems to be involved with stereotyped ecdysis
behavior, thus allowing the crab to exit from its old exoskeleton well before swelling is com-
plete (Phlippen et al. 2000). Additionally, in this crab, gill plasma membrane preparations show
high-affinity saturable binding kinetics with 125 I-labeled CHH, indicative of receptor binding,
and exposure of crabs to dilute seawater (6 ppt) dramatically increased levels of cGMP in gill
tissues (Chung and Webster 2006).

Fig. 2.6 (Continued)
branching morphology. (H) CHH immunoreactive structures in the PO and adjacent areas in a C. maenas
embryo just prior to hatching; arrows point to the PO nerve trunks. Abbreviations: ab, anterior bar; pb, poste-
rior bar. (I) Dual-labeled section of the retina of juvenile crayfish (Procambarus clarkii). Red structures (Texas
red) show CHH immunoreactive tapetal cells, green structures (FITC) show 5-HT immunopositive retinu-
lar cell axons. Upper cell layer shows CHH immunoreactive tapetal cells; lower layer, 5-HT immunopositive
retinular cell axons. Scale bars: 50 µm (A, B, F, G, H), 100 µm (C), 200 µm (D) and insert (E), 20 µm (I), 25 µm
insert (B). See color version of this figure in the centerfold. Images: (A, B) adapted from Dircksen et al. (1988),
with permission from Wiley and Sons, Inc.; (C) courtesy of H. Dircksen; (D, E, F, H) adapted from Chung and
Webster (2004), with permission from The Company of Biologists; (G) Webster, unpublished; (I) adapted
from Escamilla-Chimal et al. (2001), with permission from The Company of Biologists.
Fig. 2.6.
Immunohistochemical localization of crustacean hyperglycemic hormones (CHH) and molt-inhibiting hor-
mone (MIH) in neural and non-neural tissues of crustaceans. (A) Sinus gland (SG) section of Carcinus maenas
double immunostained for CHH (brown, peroxidase antiperoxidase/diamainobenzidine) and MIH (black,
silver-enhanced immunogold). Arrow points to (collapsed) hemal sinus. (B) Double immunostained (as in
A) section of X-organ (XO) of C. maenas. Inset shows transverse section of XO-SG tract. (C) Intrinsic CHH
neurons in pericardial organ of C. maenas. (D) Endocrine cells immunoreactive (whole-mount FITC immu-
nofluorescence) to CHH surrounding the insertions of the gastric and cardiopyloric muscles at the meso-
cardiac and pterocardiac ossicles of premolt (stage D2) C. maenas, dorsal view. Abbreviations: gm3c, lateral
posterior gastric muscles; gm4c, cardiopyloric muscles. Inset shows dorsal view of cells surrounding muscle
insertions of the anterior dorsal pyloric dilators (cpv1b) at the posterior mesopyloric and anterior uropyloric
ossicles. (E) Structures immunoreactive to CHH (whole-mount immunofluorescence) in a recently hatched
C. maenas zoea larva. Arrows point to segmentally iterated abdominal cells (ac) close to the insertions of the
abdominal flexor muscles, pericardial organs (po) and associated immunopositive cells, and XO-SG neuro-
secretory system (xo). (F) Double labeled preparation showing localization of two pairs of CHH (red, Cy3)
and MIH (green, FITC) immunoreactive neurons in the brain of a C. maenas embryo at the mid-eye stage
(70–85% development). Arrow indicates extensive arborizing dendrites. Both peptides are not colocalized, but
the SG appears yellow due to stacking of the confocal image. (G) CHH immunoreactive cells (whole-mount
FITC) in the hindgut of C.  maenas, imaged from the basal (hemolymph) side, where the cells exhibit a
52 Simon G. Webster

Clearly, all these results point to a central role of CHH in osmo- and ionoregulation involv-
ing not only regulation of the internal milieu during acclimation to diminished salinity, but also
regarding one of the most all-pervading processes in crustacean life history: molting and growth.
Future directions of research to elucidate the mode of action of CHH in ionoregulation must now
concentrate on determining the action of the hormone on specific transport processes. A variety
of these Na+/K+ ATPase, V-ATPase, NKCC cotransporter, K+ and Cl− channels have been identi-
fied, cloned, and sequenced in crustacean gills, and models for branchial ionoregulation have been
proposed (Freire et al. 2008). Classical Ussing chamber-type studies, together with gene expression
profiling and CHH knockdown via RNAi are potentially powerful techniques to further define the
roles of CHH in the aforementioned processes.

DISTRIBUTION OF CHH IN THE NERVOUS SYSTEM

AND NON-NEURAL TISSUES

Many immunohistochemical studies have localized CHH expression to the XO-SG neurosecre-
tory system in the eyestalk of malacostracan crustaceans. Examples include Brachyura (Dircksen
et  al. 1988), Astacura (Soyez et  al. 1998), Penaeidae (Sithigorngul et  al. 2002), Palaemonidae
(Sithigorngul et  al. 1999), and Isopoda (Martin et  al. 1984). In general, these have shown com-
plete localization of CHH in XO perikarya and neurohemal endings in the SG (i.e., type I peptides
[CHH] are synthesized in distinct perikarya) and that overlap in cellular expression with type II
peptides (MIH, MOIH, VIH) rarely occurs. However, in a few instances, co-expression seems to be
observed between CHH and VIH in Homarus spp. (De Kleijn et al. 1992, Rottlant et al. 1993) and
CHH and MIH in M. japonicus (Shih et al. 1998). Fig. 2.6A,B,F shows representative photomicro-
graphs of CHH and MIH immunopositive structures in the XO-SG of C. maenas.
For neuropeptides, in which structural diversity due to stereo inversion occurs (Phe3 for CHH,
Trp4 for VIH) in Homarus gammarus, antisera generated against N-terminal sequences that contain
the L and D aminoacyl residues of these stereo isomers reveal fascinating and unexpected complex-
ity in expression patterns (Ollivaux et al. 2009). In total, five populations of cells can be recognized
(Fig. 2.7), including one that variably co-expresses D isoforms of both CHH and VIH, thus neatly
accounting for previously observed co-expression patterns and also raising important questions
regarding the physiological significance of this phenomenon with respect to co-release of mixtures
of neuropeptides that influence quite disparate physiological processes.
Development of CHH neurosecretory systems during embryogenesis has been studied in
C. maenas (Chung and Webster 2004). CHH mRNA expression first occurs at about 50% of embry-
onic development (eye anlagen stage) and subsequently increases dramatically. Whole-mount
immunohistochemistry revealed the presence of two pairs of CHH (and MIH) cells in the XO,
with projecting axons to the SG (Fig. 2.6F). During larval life, MIH cell number remains invariant
(i.e., four cells; Webster and Dircksen 1991), but it is not yet known when further development/
recruitment of CHH (or MIH) XO neurons occurs to increase the number to the adult comple-
ment of 62–65 in each eyestalk (Dircksen et al. 1988).
Although it was once considered that the XO-SG was the only site of CHH synthesis, it is now
known that CHH is expressed in a wide variety of neural and non-neural tissues. More than 25 years
ago, CHH was detected by RIA in the PO of C. maenas (Keller et al. 1985) and subsequently by
EIA in ventral nerve cords of H. americanus (Chang et al. 1999). The source of this material has
been shown to be from intrinsic multipolar neurons in the PO of C. maenas (Dircksen and Heyn
1998) and C. sapidus (Chung and Zmora 2008) and in the so-called second thoracic roots of the
ventral nerve cord of H. americanus (Chang et al. 1999; see Fig. 2.6C). In C. maenas embryos, CHH
immunopositive neurons are observed in the thoracic ganglion (Fig. 2.6H), which projects axons
 Endocrinology of Metabolism and Water Balance 53

L-CHH cells D-CHH cells D-cells D-VIH cells L-VIH cells

preproCHH preproCHH preproCHH preproVIH preproVIH

XO +
preproVIH
Cleavage Cleavage
Signal Signal
peptide peptide

proCHH proCHH
Cleavage
Amidation a CPRP
Isomerization Amidation
Cyclization b
SG CHH D-Phe3 CHH D-Trp4 VIH VIH
(+CHH) (+VIH)

Fig. 2.7.
Diagrammatic representation of cell type specific precursor processing of crustacean hyperglycemic hor-
mones (CHH) and vitellogenesis-inhibiting hormone (VIH) isomers in the X-organ-sinus gland complex.
CPRP, CHH precursor-related peptide. Amidation (a) can occur before, during, or after cleavage of CPRP.
Cyclization of glutamate to pyroglutamate (b) can only occur after CPRP cleavage. Some cells secrete L-CHH,
L-VIH, whereas D-CHH, D-VIH cells, while producing mainly the D-isomers, also secrete variable amounts
of L-isomers. However, a small population of cells exclusively secrete the D-isomers of both hormones. See
color version of this figure in the centerfold. Adapted from Ollivaux et al. (2009), with permission from John
Wiley and Sons, Inc.

along the segmental nerves (Chung and Webster 2004). For crabs, it is known that intrinsic neu-
rons of the PO express the splice variant of CHH encoded by exons I–IV (Dircksen et al. 2001,
Chung and Zmora 2008). Other tissues associated with neural structures now known to express
CHH include the tapetal cells of the retina in the crayfish P. clarkii (Escamilla-Chimal et al. 2001;
see Fig. 2.6I). Intriguingly, these cells, together with adjacent retinular cells that express serotonin,
undergo daily rhythms of immunoreactive content, as do cells in the XO. This is very reminiscent
of observations showing circadian rhythmicity of CHH synthesis in the XO and release from the
SG at the beginning of the scotophase and subsequent nocturnal hyperglycemia in A. leptodactylus
(Gorgels-Kallen and Voorter 1985, Kallen and Abrahamse 1989, Kallen et al. 1990). It has been sug-
gested that modulation of CHH secretion dynamics by release of serotonin from adjacent retinular
cells (Escamilla-Chimal et al. 2002) may affect circadian changes in the sensitivity of the eye. These
interesting results now need corroboration in other decapod models.
Expression of CHH in non-neural tissues now seems to be a widespread phenomenon in deca-
pod crustaceans. In C. maenas, endocrine cells (paraneurons) in the fore- and hindgut express an
XO-CHH during premolt, as described earlier (Chung et al. 1999). Detailed analysis of the distri-
bution of these cells demonstrated that they surround the muscle insertions of all extrinsic and
intrinsic muscles of the gastric and pyloric stomach, suggesting a mechanoreceptive function. In
the hindgut, they are present over the entire longitudinal musculature (Webster et al. 2000; see
Fig. 2.6D,G). During ecdysis, complete exocytosis of CHH from these cells occurs, although it is
not known whether this is associated with apoptosis. Although correlates have not been observed
in embryonic guts, serially iterated peripheral CHH cells are found at the insertions of abdominal
flexor muscles during the final stages of embryogenesis in C. maenas (Chung and Webster 2004; see
Fig. 2.6E). Because the expression and release patterns of these cells are associated with hatching of
the prezoea larva, which involves significant water uptake to rupture the eggshell, it is possible that
these cells are the functional equivalent of gut paraneurons in the adult.
54 Simon G. Webster

Transcripts that encode the exon I–IV PO-CHH seem to be present in non-neural tissues includ-
ing heart, gills, and antennal gland of M. rosenbergii (Chen et al. 2004) and epidermis, gill, and gut
of L. vannamei (Tiu et al. 2007), but, so far, there is no evidence to suggest that these are translated.
In a recent study, two rather unusual CHH encoding transcripts, transcribed from three exons that
uniquely do not code for a precursor containing a precursor-related peptide (CPRP; see the sec-
tion “CHH Peptides, Transcripts, and Genes”), were identified in an androgenic gland suppression
subtractive hybridization (SSH) library of F. chinensis (Li et al. 2010). In situ hybridization studies
showed that mRNAs encoding these peptides were restricted to the epithelial cells of the internal
wall of the spermatophore sac. This unusual and interesting finding now needs further investigation

A B NE
NE
1 Br
2 CG
CEC
on
4

CEC

COG TG

5 MdG

msc

MxG

pn

TG

Fig. 2.8.
Camera lucida drawing of CHH immunoreactive neurons in branchiopods. (A) Artemia salina. Type 1–4 cells
were seen in the brain (type 3 cells not visible in this preparation). Type 5 cells were found in the mandibular
ganglion. A strongly stained giant multipolar peripheral neuron (pn) is located on each side of the maxillary
segment that innervates unidentified muscles (msc) and bulbous tissues (arrows) in the maxillary segment.
(B) Daphnia magna. Upper figure shows brain with crustacean hyperglycemic hormones (CHH) immunoreac-
tive neurons. Lower figure shows serially homologous perikarya of bipolar neurons (arrows) within the periph-
eral nerves of the ventral body. Abbreviations:  Br, brain; CEC, circumesophageal connective; CG, cerebral
ganglion; COG, connective ganglion; MdG, mandibular ganglion, MxG, maxillary ganglion; NE, nauplius eye;
on, optic nerve; TG, thoracic ganglion. Scale bars: 50 µm. Adapted from Zhang et al. (1997), with permission
from Springer.
 Endocrinology of Metabolism and Water Balance 55

to see whether this is a common occurrence in male decapod crustaceans and to determine tissue
distribution and abundance of translated peptide.
Regarding the distribution of CHH-like peptides in nervous systems of nonmalacostracan crus-
taceans, little is as yet known. The patterns of distribution of CHH immunoreactive neurons have
been determined in the branchiopods D. magna and Artemia salina (Zhang et al. 1997), which are
shown as camera lucida reconstructions in Fig. 2.8. Distributions of CHH-ir neurons are rather
obviously different from those in the Malacostraca because none is an obvious homologue to those
seen in the XO-SG neurosecretory system; of particular interest are the peripheral neurons that
project centrally to the ventral nerve cord and to appendage muscles and maxillary somatic muscles.
They may be comparable to multipolar cells in the PO of malacostracans, and it has been suggested
that they are sensory as well as neurosecretory neurons. In silico analysis of the transcriptome of
D. pulex (Gard et al. 2009) suggests that the CHH-like peptides of D. pulex bear a somewhat higher
sequence identity to ITP than CHH (see the section “CHH Peptides, Transcripts, and Genes”).

CHH-LIKE HORMONES IN OTHER ARTHROPODS: ITP

One of the many physiological adaptations that have been central to the evolutionary success of
the insects concerns the homeostatic mechanisms involved in excretion. For insects that live in
arid environments, water conservation is of particular importance. More than 30 years ago, using
small Ussing chambers to measure short-circuit current (Isc) across ileal preparations of the locust
S. gregaria, Audsley and Phillips (1990) demonstrated that three factors in the brain, nervous lobe of
the corpora cardiaca (NCC), and ventral nerve cord (VNC) stimulated ileal Isc in a dose-dependent
manner. Fluid transport across the ileum ( Jv) was dependent on Cl− (Lechleitner et al. 1989a,b) and
likewise stimulated by similar nervous system extracts. High-performance liquid chromatography
(HPLC) analysis of the bioactive compounds revealed one from the NCC that stimulated ileal
Cl− transport; thus the ITP was partially sequenced (Audsley et al. 1992, 1994), showing it to be
very similar to CHH. cDNA library screening identified a full-length sequence of ITP (Meredith
et  al. 1996), firmly establishing this peptide as a homologous member of the CHH family (see
the section “CHH Peptides, Transcripts, and Genes”). As in crustaceans, ITPs from other insects
showed a high degree of group or even species specificity in their biological activities, and C. mae-
nas CHH was entirely inactive in the ileal bioassay (Meredith et al. 1996). Short and long isoforms
of S. gregaria ITP (SchgrITP, SchgrITPL) were identified by Meredith et al. (1996) in that species
and also in Locusta migratoria (Macins et al. 1999). Following the determination of alternative splic-
ing of pre-mRNAs for C.  maenas CHH (Dircksen et  al. 2001), it became obvious that a similar
situation occurred in locusts. The structure of itp genes and their transcripts are now known for
the lepidopterans Manduca sexta and Bombyx mori (Dai et al. 2007, Drexler et al. 2007) and for the
dipterans Aedes aegypti and Drosophila melanogaster (Dai et al. 2007, Dircksen et al. 2008). Gene,
transcript, and peptide structures of CHHs and ITPs have been discussed in more detail (see the
section “CHH Peptides, Transcripts, and Genes”), but it is of relevance to note here that the C. mae-
nas CHH gene contains 4 exons, M. sexta 3, and D. melanogaster 5 exons. To date, the biochemi-
cal identity (i.e., identification of mature processed peptide) of ITPs has only been unequivocally
demonstrated in locusts and fruitflies (Audsley et al. 1992, 2006, Dircksen et al. 2008). In a situation
analogous to that of crustaceans, where PO-CHH has no identifiable biological activities to date,
there is little information regarding the biological activities or function of the long isoforms of ITP.
Certainly, they are inactive in the ileal bioassay, thus leading to the hypothesis that the long isoforms
of ITP may antagonize the action of ITP by competing with receptor binding (Phillips et al. 1998b).
Distribution of ITP immunoreactive neurons in the CNS of insects obviously shows that prom-
inent groups of neurosecretory cells in the pars lateralis project axons to the retrocerebral complex,
56 Simon G. Webster

corpora cardiaca, and corpora allata (where release occurs). Additionally, there are also complex
arrangements of ITP-ir interneurons in the brain and subesophageal, thoracic, and abdominal gan-
glia; in efferent neurons in the abdominal ganglia; and in neurosecretory neurons associated with
the neurohemal organs of the peripheral nervous system. For a comprehensive review of the neuro-
anatomy of ITP immunoreactive structures in insect nervous systems, excellent detailed accounts
are given in a recent review (Dircksen 2009), and specific descriptions are available for M. sexta (Dai
et al. 2007) and D. melanogaster (Dircksen et al. 2008). Interestingly, in the flour beetle Tribolium
castaneum, large numbers of ITP-expressing paraneurons were detected by in situ hybridization in
the late larval midgut epithelium (Begum et al. 2009), a situation that is strikingly similar to that
seen for CHH in fore- and hindgut tissues of premolt C. maenas (Chung et al. 1999).
Regarding other arthropods, CHH-like immunoreactive structures have been observed in neu-
rohemal organs of a myriapod (centipede; Lithobius forficatus, Laverdure et al. 1994) and an arach-
nid (scorpion; Euscorpius carpathicus, Stockmann et  al. 1997), and in silico analysis of expressed
sequence tags has shown that transcripts encoding CHH/ITP-like peptides are found in the ixodid
ticks Ixodes scapularis (Christie 2008), Amblyomma variegatum, the mites Tetranychus urticae and
Suidasia medanensis, and the scorpion Mesobuthus gibbosus (Christie et  al. 2011). Thus, it seems
very likely that CHH/ITP-like peptides are widely, perhaps universally, distributed in arthropods.

TARGET TISSUES, BINDING SITES, AND SECOND-MESSENGERS

A long-held view regarding the tissues involved in CHH action was that the midgut gland and
abdominal muscle were key targets for CHH action because injection of SG extracts or puri-
fied CHH results in increases in cyclic nucleotides followed by a net decrease in glycogen
post-hyperglycemia in these tissues (Keller and Sedlmeier 1988). Traditional 125I-CHH based radio-
labeled binding experiments have shown that high-affinity, saturable binding is exhibited by plasma
membrane preparations of O. limosus and C. maenas midgut glands (Kummer and Keller 1993) and
that, in the latter species, YO membrane preparations showed similar binding characteristics to
CHH, together with clearly separate MIH binding activity (Webster 1993). Subsequently, it has
been demonstrated in C. maenas and C. sapidus that a number of other tissue membrane prepara-
tions, including those involved in ionoregulation (gills, hindgut) and respiration (heart, scaphog-
nathite) exhibit similar binding characteristics to those of midgut gland and muscle with binding
affinities (KD) 1.2–13 × 10−10 M, maximum number of binding sites (BMAX) 1-5 × 10−10 M/mg protein
(Chung and Webster 2006, Katayama and Chung 2009). However, it is not yet known whether
these receptors are involved in particular physiological processes, such as ionoregulation, or if their
presence is merely correlated with tissue-specific energy homeostasis in highly metabolically active
tissues, such as the gills.
Regarding the type of hormone receptor and second-messengers involved, there is some uncer-
tainty. At present, the balance of evidence seems to favor signaling via cGMP. Goy (1990) dem-
onstrated the presence of a membrane-bound guanylate cyclase (MGC) and increases in cGMP,
following administration of purified CHH in H. americanus tissues. A variety of tissues respond
to incubation in CHH (20 nM) with increases of up to 10-fold in cGMP levels within 30 min of
hormone administration; incubation with membrane-permeant cGMP analogues such as 8-bromo
cGMP increases tissue glucose in C.  maenas (Chung and Webster 2006). However, for ITP, it
seems likely that the transport-stimulating effects of S. gregaria ITP in the hindgut are mediated by
cAMP—that is, via a G-protein-coupled receptor (GPCR; Phillips and Audsley 1995, Phillips et al.
1998b, Lechleitner et al. 1989a). It is relevant here to mention the proposed signaling pathway of
MIH receptors (see Chapter 1 in this volume). The most attractive current model for MIH signal-
ing, based on an elegant fusion of earlier research (review by Chang and Mykles 2011) proposes that
 Endocrinology of Metabolism and Water Balance 57

the MIH receptor is a GPCR. Increases in cAMP caused by activation of adenylate cyclase activates
protein kinase A (PKA), opening Ca2+ channels; increased intracellular Ca2+ binds to calmodulin,
activating NO synthase (NOS) directly and indirectly via calcineurin. The production of NO acti-
vates a NO-sensitive guanylyl cyclase and thus stimulates cGMP production, activating a protein
kinase G that, presumably, via numerous phosphorylation steps leads to repression of ecdysteroid
synthesis via chronic (translational) and acute (transcriptional) control mechanisms. However,
given that MIH most likely evolved from a CHH-like molecule, it seems axiomatic that such closely
related peptides might have disparate receptor types and signaling mechanisms.

FUTURE DIRECTIONS

Rapid progress in identification of members of the hyperglycemic hormone family and appreciation
that they are not only pleiotropic but of widespread, if not universal occurrence in the arthropods have
perhaps been the highlights of crustacean neuroendocrinology over the past 20 years. However, the
multiplicity of CHHs in single species, as exemplified by the situation in penaeid shrimps, where there
is much confusion regarding the biologically relevant roles of a plethora of very similar CHH-like mol-
ecules, begs questions as to their biologically relevant functions. Further research, using either native
peptides or (fully) biologically active recombinant molecules is now needed. This is particularly per-
tinent when ionoregulatory roles are considered. At present, rather more is known about the physiol-
ogy of ITP-mediated ion and water transport across insect hindguts than the equivalent processes
controlled by CHH in crustaceans. Similarly, the recent findings concerning the widespread expres-
sion of mRNAs encoding the CHH-L splice variant need urgent attention. Apart from the existence of
CHH-L peptide in the intrinsic cells of the POs of crustaceans, it is not known whether tissue-specific
expression of mRNA is related to translation of peptide or, indeed, the function of these peptides
in many non-neural tissues. A significant gap in our knowledge concerns the nature of the receptors
and second-messenger signaling pathways of CHH peptides. Although notable progress has recently
been made in identifying signaling pathways for MIH (see Chapter 1 in this volume), almost nothing
is known about these processes for CHH. Similarly, for both hormones, receptors remain orphans
despite the rapid advances in insect molecular endocrinology, whereby a plethora of neuropeptide
receptors are known! Clearly, a contemporary approach to identification of the cognate CHH fam-
ily receptors will be very informative, particularly regarding identification of new target tissues, and
would of course be invaluable in identifying biologically relevant roles for these peptides.

CONCLUSIONS

This chapter has reviewed our current understanding of the nature, sites of synthesis, and biological
actions of CHH, with particular emphasis placed on demonstrating the emerging scenario of multi-
functionality of these neurohormones in the subphylum. Recent molecular studies have vividly shown
the evolutionary history and diversification of both structure and function of hyperglycemic hormones,
which have reiterated earlier observations suggesting that a wide range of functions relating to regula-
tion of a variety of quite fundamental physiologies were directly attributable to these hormones.

ACKNOWLEDGMENTS

Research performed in my laboratory has been funded by the Biotechnology and Biological
Sciences Research Council (BBSRC).
58 Simon G. Webster

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 3
ADAPTIVE COLOR CHANGE AND THE MOLECULAR
ENDOCRINOLOGY OF PIGMENT TRANSLOCATION IN
CRUSTACEAN CHROMATOPHORES

John Campbell McNamara and

Sarah Ribeiro Milograna

Abstract
This chapter focuses on the cellular underpinnings of rapid adaptive color change in crusta-
ceans. From the standpoint of molecular endocrinology, chromatic adaptation is pertinent
to the physiological, cellular, and molecular biological mechanisms that regulate intracellular
pigment granule movements. The chapter examines the distribution, organization, and ultra-
structure of pigmentary effectors—the epidermal and internal chromatophores and retinal
cells—and discusses the original characterization of pigment-aggregating and -dispersing chro-
matophorotropins that regulate pigment translocation and the sensory mechanisms that lead
to their differential release from neurosecretory nuclei. Genes present in various arthropod
groups code these peptide neurohormones, and their homology and conservation is discussed
in light of evolutionary relationships with the Crustacea. An analysis of putative mechanisms
of chromatophorotropin signal transduction is presented, focusing on membrane receptor type
and function and subsequent intracellular regulatory cascades. Structural and physiological evi-
dence concerning cytoskeletal molecular motors is investigated. Promising avenues for future
investigation are discussed.

AN INTRODUCTION TO COLOR CHANGE IN THE CRUSTACEA

Animal colors and pigments have long fascinated the astute observer, from the dazzling pelt of the
golden lion-tamarin and spectacular toco toucan’s bill to the shimmering iridescent-blue wings of

68
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 69

the Morpho butterfly and the scarlet dye in the cochineal bug. A stunning array of shades, tints,
and hues embellishes vertebrates and invertebrates alike, conferring manifest visibility or cryptic
concealment as desired.
Crustacean colors are no less spectacular, and many taxa, like the fiddler, mangrove, porcelain,
and coral crabs, and the cleaner, pistol, harlequin, hinge-beaked, and hump-backed shrimps, among
myriad others, display astonishing color patterns (Bauer 2004). Some color arrays are fixed and are
species specific, the product of convergent evolution with a particular substratum, often another
organism, whereas others are more labile, the result of rapid color adaptation to changing back-
grounds. This chapter focuses on the physiological, cellular, and molecular biological underpin-
nings of color change in crustaceans, particularly in the Caridea and Brachyura, viewed from the
standpoint of molecular endocrinology.
Chromatic adaptation in the Crustacea has long aroused scientific interest, and Kröyer (1842)
undertook the first comprehensive study on a broken-back, hippolytid shrimp almost two centu-
ries ago. Adaptive changes in pigmentary patterns may be chromogenic (morphological) in nature,
inherent to each species (Fig. 3.1), and changing only very slowly when honed by evolutionary
forces or gradually within an individual organism when the number of pigment-bearing cells or
chromatophores and their pigments increases or diminishes or when their constituent pigments
become altered in color through alterations in metabolic pathways. Such gradual changes also may
be associated with a particular developmental or molt stage or may correspond to the specific age
of an individual.
However, color changes also may be chromomotor (physiological) in character, brought about
by the rapid redistribution of pigment granules within the effector cell cytoplasm, quickly adjusting
the individual organism to its current chromatic backdrop, to ambient luminosity and temperature,
or to behavioral requirements, including circadian and tidal rhythms and reproductive cycles. It is
this process of rapid, hormonally regulated color change that we examine here, beginning with the
perception of substratum cues by the eyestalk ommatidia, through to signal transduction and the
resulting intracellular signaling cascades in the pigment cells themselves, to the activation of the
molecular motors that translocate the individual pigment granules through the cytosol and lead

Fig. 3.1.
The Indo-Pacific harlequin shrimp, Hymenocera elegans, exhibits one of the most stunning chromogenic
adaptations found among the Crustacea. This gnathophyllid coral reef shrimp displays a fairly fixed, sex- and
species-specific coloration pattern that consists of irregular, bluish to purple spots and markings, placed fairly
symmetrically on a cream-white background to form a complex pigmentary system. Here, a pair of H. elegans
(seen in frontal view) explores a prey species, the Indo-Pacific blue star, Linckia laevigata. See color version of
this figure in the centerfold. With permission from Adriano Morettin, all rights reserved.
70 John Campbell McNamara and Sarah Ribeiro Milograna

to chromatic adaptation. Our focus lies particularly on the molecular mechanisms underlying pig-
ment aggregation in the caridean shrimps, the best-studied models among the decapod Crustacea,
although information from brachyuran crabs and insect groups is included for a comparative and
evolutionary perspective.

CRUSTACEAN PIGMENTARY EFFECTORS

The Distribution, Location, and Role of Chromatophores

in Chromatic Adaptation

Crustacean pigments may be distributed freely within the exoskeleton or contained within
pigment-bearing cells or chromatophores (khrōma, “color,” phoros, “bearer”; Bauer 2004).
Chromatophores are found in nearly all crustaceans, at all stages of their life cycles, from embryos
through larvae, and, particularly, in the benthic, adult forms. The exceptions are perhaps some
cave-dwelling and abyssal species, although many of the latter do exhibit bright red pigmentation.
These uninuclear cells can contain a single pigment granule type (e.g., a monochromatic yellow
chromatophore) or several different pigment granule types (e.g., a polychromatic yellow and blue
chromatophore containing separate and distinct yellow and blue pigment granules; see Robison
and Charlton 1973, McNamara and Sesso 1983). Chromatophores are often grouped together into
multicellular effectors of a dozen or so intimately juxtaposed cells known as a chromatosome
(sōma, “body”; see Fig. 3.2A,B). These, in turn, may consist of chromatophores of a single color
(e.g., a monochromatic red chromatosome) or of several different colors (e.g., a polychromatic yel-
low and brown chromatosome composed of separate monochromatic yellow and brown chromato-
phores; see Elofsson and Kauri 1971, McNamara 1981, Bauer 2004; see Fig. 3.2C).
Chromatophores and chromatosomes are found mainly within the integumental epidermis,
often protruding into the hemolymph space lined by the basal lamina. They also occur within the
fibrous envelopes that cover the internal organs that constitute the reproductive (ovary; see Fig.
3.2B), somatogastric (hepatopancreas, stomach, hindgut; McNamara 1979), and, particularly, the
central nervous system (ganglia and ventral nerve cord; McNamara and Sesso 1982, 1983). Highly
differentiated pigment cells are also found within the crustacean visual system. Such screening pig-
ments can take the form of nonvisual accessory cells that are associated with the ommatidia. These
contain the distal retinal pigments within the elongated distal pigment cells that surround the crys-
talline cones and ommatidia and the reflecting pigments found in specialized cells that envelope
the retinula cells (Fernlund 1976). These pigments exhibit minimal translocation ability and shield
the retinula cells from extraneous photons. The proximal screening pigments, however, constitute
part of the ommatidia themselves and are located within the retinula cell cytoplasm, through which
they migrate to different degrees depending on the species. This visual pigment system is touched
on only briefly in the final part of this review.
Chromatosomes, chromatophores, and their pigments constitute the primary means by which
crustaceans attune themselves chromatically to their surroundings. This pigmentary system is
used in a variety of ways, including species-specific signaling, aposematic signaling, mate attrac-
tion, reproductive strategies, protection against ultraviolet (UV) radiation, and thermal regulation,
for example. The pigmentary system also responds to circadian and tidal rhythms in species like
the fiddler crab (Thurman 1990). A particularly characteristic function salient in the Crustacea—
blending into the background or camouflage, be it through mimicry or crypsis—is achieved by the
differential distribution of pigment granules within the chromatophore cytoplasm as the individual
organism passes from one specific chromatic background to another; that is, rapid or chromomo-
tor chromatic adaptation. Thus, when on dark-colored backgrounds, a shrimp’s dark pigments will
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 71

Fig. 3.2.
Chromatosomes consist of multicellular aggregates of highly asymmetrical, single-celled, monochromatic
chromatophores. Their intimately apposed, semispherical perikarya contain the pigment granules and form
the chromatosome center from which radiate the long chromatophore extensions through which the gran-
ules migrate. (A)  Monochromatic, red, epidermal chromatosome from the neotropical freshwater shrimp,
Macrobrachium olfersi. (B) Monochromatic, red chromatosome, showing the nuclei of individual chromato-
phores (round clear areas within the dark red perikarya) on the dorsal surface of the fibrous capsule containing
the ovary of Macrobrachium olfersi. These particular pigmentary effectors have been employed with success
as models in deciphering the mechanisms of membrane signal transduction, intracellular second-messenger
cascades, and regulation of the molecular motors that translocate the pigment granules through the cytosol.
(C) Dichromatic, epidermal chromatosome from the New Zealand intertidal shrimp, Palaemon affinis, consist-
ing of separate monochromatic yellow (left) and monochromatic red/brown chromatophores (right) contain-
ing fully dispersed and partially dispersed pigments, respectively. Scale bars = 50 µm. See color version of this
figure in the centerfold.

tend to disperse while its light pigments will tend to aggregate. Conversely, as the animal moves to
a lighter background like sand, say, from a darker encrusted rock, its light pigments will disperse
while its dark pigments may aggregate (Fig. 3.3). These events can take place within a few minutes
and are hormonally mediated because crustacean chromatophores are not innervated.
In contrast, should the individual organism be restricted to a particular background, a slower
process of chromatic adjustment ensues (Robison and Charlton 1973). To illustrate, an individual
organism’s color scheme can exhibit a considerable degree of morphological plasticity, and popula-
tions of a single species naturally residing on differently colored substrata may show corresponding
chromotypes or different chromotype frequencies (Bauer 1981). Thus, constituent chromatophores
may be lost or gained, particularly over the internal organs, whereas epidermal chromatophores
may be added during larval ontogeny and on metamorphosis to the postlarval stage, consistent
with changes in the chromatic characteristics of the new habitat. Furthermore, the color of the
pigments contained within the chromatophores may become altered as a result of catabolic and
anabolic metabolic processes, with one pigment type being synthesized while another is degraded.
These phenomena, occurring within the individual’s lifetime, are known collectively as chromogenic
adaptation because they are regulated at the level of gene expression.

Chromatophore Organization, Microanatomy, and Ultrastructure

Vertebrate chromatophores originate from the embryonic neural crest (Hall 2008), and “neural
crest-like” pigment cell precursors occur in Urochordata and Hemichordata ( Jeffery et al. 2004).
In contrast, pigment cells are of endomesodermal origin in invertebrates (Hall 2000, Bronner and
LeDouarin 2012). Findings refer mainly to the Echinodermata (Oliveri et al. 2008) whose chro-
matophore structure and physiology are not homologous with the Crustacea. The specific embry-
onic origin and migratory routes of crustacean chromatophores are yet to be elucidated.
Crustacean chromatophores are highly asymmetrical cells that contain a single nucleus and con-
sist of a spherical perikaryon of approximately 20 µm in diameter from which extend one or two
long cell processes of up to 100 µm in length. These subdivide and ramify among the surrounding
72 John Campbell McNamara and Sarah Ribeiro Milograna

Fig. 3.3.
Chromomotor adaptation in Palaemon affinis. The nearly transparent upper shrimp was exposed to an illumi-
nated white background for 2 h, and the brown pigments in its epidermal chromatosomes are fully aggregated; the
white somatogastric chromatosome pigments are fully dispersed. The lower shrimp, in which the species’ typical
chromogenic pattern is clearly visible, was placed on an illuminated black background and displays fully dispersed
brown epidermal chromatosome pigments; arrowhead indicates fully dispersed hindgut chromatosomes. Such
rapid translocations of the pigment granules through the chromatophore cytosol are regulated by blood-borne
neuropeptides called chromatophorotropins, such as red pigment concentrating hormone, released from the eye-
stalk and other neurosecretory centers. Scale bar = 1 cm. See color version of this figure in the centerfold.

epidermal cells (or fibrocytes in the case of internal organ capsules) about 50 µm after leaving the
perikaryon. Groups of 10–20 chromatophores are characteristically juxtaposed into multicellular
chromatosomes tightly linked by desmosome-like intercellular junctions in the regions of contact
between their adjacent perikarya (McNamara 1981). These multicellular pigmentary effectors can
attain up to 300 µm in diameter and are easily visible to the naked eye (Fig. 3.2).
In early chromatophores in the process of coalescing into larger chromatosomes, such as those
found in the larval stages, numerous pigment granules abound, and both the smooth and rough
endoplasmic reticulum are well developed, as are numerous microtubules and occasional mito-
chondria and polysomes (McNamara 1989). The rough endoplasmic reticulum virtually disappears
in fully developed chromatophores, whereas the smooth endoplasmic reticulum (SER) takes the
form of a tri-dimensional network that ramifies throughout the cytoplasm, becoming further differ-
entiated into a regular, transversely interconnected, scalariform conformation in the cell extensions,
much as seen in the axons of crustacean neurons (McNamara 1981, McNamara and Taylor 1987,
McNamara and Ribeiro 1999). The tri-dimensional cisternae are often encountered in structural
continuity with the pigment granules in a variety of chromatophore types (McNamara 1980).
In the planar epidermal chromatophores, bundles of 25 nm diameter microtubules abound
within the cell extensions to which they lie parallel, often occupying a central, core-like posi-
tion among the dispersed pigment granules (Elofsson and Kauri 1971, Robison and Charlton
1973, McNamara 1980). Hefty microtubule bundles are also arrayed throughout the perikaryon
(see fig.  22 in McNamara and Taylor 1987). This organization of the cytoskeleton appears to
be mainly scaffold-like in function since, curiously, microtubule-disrupting agents like col-
chicine and vinblastine do not affect pigment aggregation at pharmacological concentrations
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 73

Fig. 3.4.
Epifluorescence microscopy (Leica DM5500B, λex=495 nm, λem=519 nm) showing several red chromatosomes
with fully aggregated pigments from the ovary of Macrobrachium olfersi. Elongated microtubule bundles
(green) lie along the length of the pigment-free cell extensions that project from the perikarya of the individual
constituent chromatophores among the surrounding fibroblasts. Monoclonal, anti-β-tubulin primary antibody
followed by an Alexa-488 conjugated goat anti-mouse IgG. Scale bar = 100 µm. See color version of this figure
in the centerfold.

(Robison and Charlton 1973, Fingerman et al. 1975, Lambert and Fingerman 1976, McNamara
1980). In the chromatophores found within the fibrous capsules positioned around the
often-mobile internal organs, microtubules are far fewer and not organized into obvious bundles
(McNamara and Ribeiro 1999). However, these longitudinal arrays of tubulin polymers do bind
fluorochrome-conjugated antitubulin antibodies, revealing an unequivocal role in supporting
and delineating the long cell extensions (Fig. 3.4). In such chromatophores, an actin cytoskeleton
sensitive to cytochalasin B predominates throughout the cytoplasm, with the individual actin
filaments lying mainly parallel to the cell extensions (Robison and Charlton 1973, McNamara
and Ribeiro 1999). The actin cytoskeleton of crustacean chromatophores has been little explored
from the structural and functional points of view, although the myosin motors that employ the
actin networks to translocate the pigment granules through the cytosol have been partially char-
acterized (Boyle and McNamara 2006).

THE HORMONAL REGULATION OF PIGMENT DISTRIBUTION

WITHIN CHROMATOPHORES

The Perception of Environmental Cues

Crustacean chromatophores are not innervated. Rather, they receive neurosecretory hormonal sig-
nals derived from the transduction by the nervous system of information predominantly concern-
ing the chromatic and photic environment.
Light reaches the eyestalk ommatidia both directly, impinging on the dorsal surface, and
indirectly, reflected from the substratum to the ventral surface. The difference in light inten-
sity, detected by the visual pigments in the retinula cells, constitutes an albedo ratio; that is, a
measure of the reflectivity of the surface on which the organism is located. An elevated albedo
74 John Campbell McNamara and Sarah Ribeiro Milograna

ratio characterizes a highly reflective, light-colored or whitish substratum, whereas a low albedo
ratio signifies little reflection and thus a dark-colored surface. Integration in the optic neuropils
and cerebral ganglion thus furnishes the individual crustacean with a notion of overall substra-
tum reflection. This information is passed via afferent innervation to the perikarya of neurose-
cretory cells that constitute the various neurosecretory nuclei associated with their respective
neurohemal organs, the interfaces of distribution between the neurosecretory axon terminals
and the hemolymph. The main neurohemal organ linked to color change in the Crustacea is
the X-organ/sinus gland (XO-SG) complex located in the eyestalk medulla terminalis, although
the tritocerebral commissure/postcommissural organ and ventral nerve cord ganglia are also
important sources of chromactivating neurosecretions. The axons of adjacent neurosecretory
perikarya form short tracts like the XO-SG tract that lead to the capillary spaces on which their
axon terminals abut and replete with neurosecretory peptides.
Thus, under appropriate stimuli, originating in the perception of ambient chromatic condi-
tions, reflectivity, and luminosity, the neurosecretory perikarya receive and integrate stimulatory
and inhibitory neurotransmissions leading to membrane depolarization, action potential propaga-
tion, neurosecretory vesicle fusion with the axon terminal membrane, and the discharge of neu-
rosecretory peptide molecules into the extracellular space near the capillaries. From here, these
chromactivating substances diffuse to and are carried via the hemolymph throughout the tissues
where they are recognized by specific receptors on the chromatophores. Many neurotransmit-
ters are involved in the further fine regulation of chromactivating neuropeptide release. In the
fiddler crab Uca pugilator dopamine induces the release of pigment-aggregating hormones and
results in aggregation of the red and black epidermal chromatophore pigments (Quackenbush
and Fingerman 1984), whereas 5-hydroxytryptamine has the opposite effect (Rao and Fingerman
1983). In contrast, norepinephrine enhances the release of a pigment-dispersing hormone (PDH),
causing dispersion of the black chromatophore pigments (Rao and Fingerman 1983, Quackenbush
and Fingerman 1984).
Fish and amphibian melanophores can respond directly to UV light by melanosome disper-
sion (Hunter et al. 1979, Oshima 2001), and such nonhumorally mediated “primary” responses also
occur in crustaceans (see Thurman 1988 for review). When exposed to near UVA radiation, pig-
ment granules disperse in black chromatophores of eyestalkless crabs U. pugilator (Coohill et al.
1970) and Neohelice granulata (Gouveia et al. 2004) in situ. UVA also induces granule dispersion
in chromatophores of intact U. pugilator (Coohill and Fingerman 1975) and shrimp Palaemonetes
argentinus (Gouveia et al. 2004). Thus, like retinular cell rhabdomeric membranes (Vargas et al.
2008) and mammalian retinal ganglion cells (Peirson et al. 2009), crustacean chromatophore mem-
branes may contain a light- or UV-transducing opsin.
This primary chromatophore response to light/UV resembles photoreception by the “intrinsi-
cally photosensitive retinal ganglion cells” of the mammalian inner retina (Bellingham et al. 2006,
1334), a small subset of which expresses melanopsin (Peirson et al. 2009), an opsin/vitamin A-based
photopigment that detects light by photon absorption and triggers a G-protein-mediated photo-
transduction cascade (Bellingham et al. 2006, Peirson et al. 2009). Photosensitive amphibian mela-
nophores express melanopsin (Provencio et al. 1998), and fish, bird, and lizard genomes contain
related sequences (Bellingham et al. 2006, Peirson et al. 2009). Thus, vertebrate melanopsin-based
and crustacean primary light/UV photosensitivities may well be evolutionary linked because
opsins have been highly conserved during evolution of the Bilateria (Arendt et al. 2004).

Chromatophorotropins: Chromactivating Neurosecretory Peptides

The antagonistic neurosecretory peptides that regulate pigment aggregation and dispersion in
the Crustacea are known generically as chromatophorotropins and take the form of small peptide
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 75

chains of a dozen or so amino acids. The notion that blood-borne chromactivating substances
can regulate pigment movement has its origin in pioneering research performed some 100 years
ago (Pouchet 1872, Köller 1927, Perkins 1928), investigations that provided the first evidence for
the hormonal regulation of physiological processes in crustaceans. Although many different
physiological assays for a variety of pigmentary factors located in the neurosecretory cells of
the eyestalk medulla, tritocerebral commissure, and ventral nerve cord of many different crus-
tacean species have been developed over the years (Brown 1948, Fingerman 1969, 1970, 1985),
the final biochemical characterization and identification of a crustacean chromatophorotropin
came only much later.
Red pigment concentrating hormone (RPCH) is an octapeptide originally isolated, identified,
and sequenced (pGlu-Leu-Asn-Phe-Ser-Pro-Gly-Trp-NH2) from the eyestalks of the caridean
shrimp Pandalus borealis (Fernlund and Josefson 1968). It is considered by some to be a universal,
plesiomorphic pigment concentrating hormone (PCH) of widespread occurrence throughout the
Crustacea because this peptide consistently promotes the aggregation of the red and dark-colored
pigments, even in its synthetic form ( Josefsson 1983, Rao 1985). Indeed from the 1980s on, few
investigators extracted their own chromatophorotropins from neurosecretory tissue—a tedious
prerequisite to most studies on signal transduction and crustacean color change—relying instead
on synthetic RPCH available from commercial laboratories.
The hormone antagonistic to RPCH, that is, red PDH (RPDH) or PDH, is an octadeca-
peptide that exists as two isoforms, α-PDH (Asn-Ser-Gly-Met-Ile-Asn-Ser-Ile-Leu-Gly-Ile-Pro-
Arg-Val-Met-Thr-Glu-Ala-NH2; also isolated [Fernlund  1971] and identified and sequenced
from P.  borealis eyestalks [Fernlund 1976, Josefsson  1983]) and β-PDH (Asn-Ser-Glu-Leu-
Ile-Asn-Ser-Ile-Leu-Gly-Leu-Pro-Lys-Val-Met-Asn-Asp-Ala-NH2) isolated from the eyestalks of
the fiddler crab U. pugilator (Rao et al. 1985), shore crab Cancer magister (Kleinholz et al. 1986),
and the blue crab Callinectes sapidus (Mohrherr et al. 1990). PDH effects are only briefly men-
tioned here because this review focuses primarily on caridean shrimps in which RPCH evokes
pigment aggregation. In Uca rapax, both α- and β-PDH induce dose-dependent pigment dis-
persion, although in the isopod Ligia exotica only α-PDH triggers dose-dependent pigment
dispersion while β-PDH brings about a partial, dose-independent response. In the freshwater
shrimp Macrobrachium acanthurus α-PDH causes dose-dependent partial dispersion, but β-PDH
is without effect (Tuma et al. 1993). Both α- and β-PDH induce dose-dependent pigment dis-
persion in M. potiuna (Britto et al. 1990) whereas in M. olfersi, α-PDH triggers partial pigment
dispersion (50%) followed by pigment aggregation (Ribeiro 1998). Curiously, when the eye-
stalks are removed from otherwise intact shrimps or when caridean chromatophores are isolated
from their source of circulating RPCH in physiological preparations in vitro the red and dark
chromatophore pigments tend to disperse rapidly and spontaneously (Fingerman et  al. 1975,
McNamara and Taylor 1987, Tuma et al. 1993); in contrast, in brachyuran crabs treated similarly,
the dark pigments aggregate in the absence of PDH (Lambert and Fingerman 1976, Kulkarni
and Fingerman 1986). In crustaceans in vivo, the chromatophorotropins RPCH and PDH are
secreted to revert pigment distribution from the respective passive states of unstimulated dis-
persion and aggregation to a hormonally induced, actively aggregated or dispersed condition.
This intriguing situation has impacted widely on research in crustacean color change, and few
studies, thus, have dealt with pigment dispersion in the Caridea or with pigment aggregation in
the Brachyura. In the Caridea, at least, pigment aggregation is experimentally reversible simply
by washout of RPCH or by lowering extra- and intracellular calcium (McNamara and Ribeiro
2000; Figs. 3.5 and 3.6), and it is indeed mystifying that pigment dispersion can be brought about
experimentally without recourse to a PDH.
Kleinholz et al. (1962) demonstrated that PDH is the same substance as the distal retinal pig-
ment hormone (DRPH) mentioned later, after noting that PDH induced granule migration to the
76 John Campbell McNamara and Sarah Ribeiro Milograna

Fig. 3.5.
Sequence of pigment aggregation (A–D) in two red chromatosomes on the ovary of Macrobrachium olfersi
induced by 30 min perfusion in vitro with 25 µM Ca2+-ionophore A23187 in a 2.5 mM NaHCO3-buffered physi-
ological saline. The pigments were subsequently dispersed (E–H) by 30 min perfusion with a Ca2+-free (10−11
M Ca2+), 2 mM EDTA-chelated saline. The pigment translocation rates are not constant (see Fig. 3.6). Scale
bar = 50 µm. See color version of this figure in the centerfold.

light-adapted position in the retinal pigment cells of many arthropod species. Thus, PDH is now
also known for inducing distal pigment granule movements in the crustacean retina (Kleinholz
et al. 1986, Verde et al. 2007).
PDH or factor (PDF) also has been widely implicated in the generation, expression, and syn-
chronization of circadian activity in insects where it plays a role as a neurotransmitter and as a
neuromodulator (Verde et al. 2007). The ubiquity of PDF in the insect circadian system strongly
suggests a role similar to that of PDH in the Crustacea, where PDH is present both as a neurohor-
mone released from the SG into the circulation and as a neuromodulator and neurotransmitter
(Verde et al. 2007).
The crustacean pigment-aggregating chromatophorotropins belong to a larger class of arthro-
pod hormones known as the adipokinetic hormone (AKH)/RPCH family of structurally related,
functionally diverse peptides, and RPCH itself is also found in several insect taxa (Gäde et al.
1997, 2003, Rao 2001, Gäde 2009, Zralá et al. 2010). AKHs play an important role in regulating the
availability of metabolic substrates in insect flight muscles (Gäde and Marco 2009), a function
also attributed to RPCH in isopod muscle glucose metabolism (Zralá et al. 2010). Furthermore,
the source of both these neuropeptide hormones in the arthropods (i.e., neurosecretory cells
in the corpora cardiaca of insects and the eyestalk XO-SG complex in crustaceans) are analo-
gous to the vertebrate hypothalamus/neurohypophysis neurosecretory system (Scharrer and
Scharrer 1944). As the resemblance in signaling pathways and intracellular cascades triggered by
these peptide neurosecretions is clarified later, it will become apparent that many components
of the respective signal transduction mechanisms have been conserved, and the apparent dif-
ferences between insect and crustacean hormone actions will fade, simply constituting a likely
Pan-crustacean (Regier et al. 2010) synapomorphy.
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 77

A
+RPCH −Ca2+

Degree of pigment dispersion (%)

100 +db-cGMP +db-cGMP 100
+A23187

80 80

60 60

40 −RPCH 40
−db-cGMP
−A23187
20 −Ca2+ 20

0 0
0 10 20 30 40 50 60 70 80 90 100 110
Time (min)

B
20 20
+RPCH
Translocation velocity (µm/min)

+db-cGMP −Ca2+
15 +A23187 15
+db-cGMP

10 10
−RPCH
−db-cGMP
−A23187 −Ca2+
5 5

0 0
0 10 20 30 40 50 60 70 80 90 100 110
Time (min)

Fig. 3.6.
Remarkable similarity in pigment aggregation profiles triggered by red pigment concentrating hormone (30 nmol/L
RPCH, ●), by dibutyryl cyclic guanosine monophosphate (10 µmol/L db-cGMP, ▲) and by the Ca2+ ionophore
A23187 (25 µmol/L A23187, ◆) in Macrobrachium olfersi red ovarian chromatophore preparations perfused in vitro
with a Ca2+-containing (5.5  mmol/L) NaHCO3/HEPES-buffered physiological saline. Pigment dispersion was
induced by perfusion with saline alone (-RPCH, -cGMP) or with a comparable 2 mmol/L EDTA-chelated/Ca2+-
free saline (-Ca2+). (A) Degree of pigment dispersion. (B) Pigment translocation velocities. All effectors produce
similar although distinct kinetic phases of pigment aggregation (rapid and slow phases), suggesting crosstalk
between the different intracellular effector cascades; the cGMP effect is Ca2+-dependent. Pigment dispersion kinet-
ics on RPCH washout are intermediate between those after cGMP and Ca2+ washout. Data are the mean ± SEM,
N = 7. Redrawn from McNamara and Ribeiro (2000) and Ribeiro and McNamara (2009).

CHROMATOPHOROTROPINS AND CHROMATOPHOROTROPIN-LIKE

HORMONES

Neurosecretory Peptides in Closely Related Groups: Crustaceans and Insects

Interestingly, in addition to AKH, several other arthropod hormones produce chromatophoro-

tropic effects. Crustacean cardioactivating peptide (CCAP) and vertebrate melatonin trigger pig-
ment dispersion in M. potiuna red epidermal chromatophores, modulating the response to PDH;
in fact, CCAP produces a response threefold stronger than PDH (Nery and Castrucci 1997).
Functional substitutions among hormones also suggest their close structural relationships. AKH
78 John Campbell McNamara and Sarah Ribeiro Milograna

shares sequence homology with corazonin (CRZ), a substance to which it is probably ances-
trally related (Mercier et al. 2007). CRZ is produced by the lateral brain neurosecretory cells that
project into the corpora cardiaca of many insects, as well as in a few protocerebral brain neurons
(Mercier et al. 2007). CRZ stimulates heart rate and the contraction of hyperneural muscles, acts as
a pigmentation-controlling factor in locusts (Hua et al. 2000), and is an initiator of ecdysis behavior
(Kim et al. 2004, Žitňan et al. 2007). The multiplicity of neuropeptides found in the invertebrates
and the coexistence of diverse structurally and functionally related hormones appears to enable
fairly simple nervous systems to perform complex chemical signaling and to enhance their capabil-
ity to develop and carry out information processing (Gerarts et al. 1992, Nässel 1996).
Other neurohormones, like the crustacean hyperglycemic hormone (CHH) and the
molt-inhibiting hormone (MIH) are also produced by the XO neurosecretory perikarya and
released by the SG complex. However, these neuropeptides are structurally very distinct from the
chromatophorotropins, containing 72–78 amino acids (Mercier et al. 2007). Although they per-
form very different functions, these hormones induce their intracellular responses through very
similar signaling cascades. For example, they effect their cellular responses by increasing cytosolic
cyclic guanosine monophosphate (cGMP) concentration (Lee et al. 2007), one of the principal
intracellular second-messengers involved in pigment translocation whose role and mode of action
is discussed later. Also, in MIH signaling, cGMP is produced via the cytosolic guanylyl cyclase
(GC; Lee et al. 2007), rather than by a membrane-linked GC receptor, as is also the case in crusta-
cean chromatophores (Ribeiro and McNamara 2009).
The triggering of very similar intracellular pathways by a diversity of neuropeptide hormones
may indicate the prevalence of a single family of receptors present in different physiological systems
among the Crustacea and Insecta, thus suggesting the putative evolution of very specific agonist
recognition mechanisms and subsequent action through complex processes of signal transduction.
Arthropod neuropeptides seem to have co-evolved with their receptors, differently from the situa-
tion seen, for example, in the relationship between their biogenic amines and receptors that do not
appear to have varied much over the course of evolution in this group (Mercier et al. 2007).

Genetic Coding for the Arthropod AKH/RPCH Family of Peptides

Little is known of the biological function and the regulation of expression of the crustacean
chromatophorotropin-encoding genes, as is also true of arthropod neurohormones (Lewis et  al.
1997, Rao 2001, Lee and Park 2004). Information on arthropod neurohormone biosynthesis deals
mainly with prepro-hormones and their processing (Chin et al. 1990, O’Brien and Taghert 1994).
Although the AKH/RPCH peptide family is highly conserved, currently available sequence data
are insufficient to disclose clear arthropod evolutionary relationships (Rao 2001). Mature peptide
and prepro-hormone sequences are highly conserved not only in arthropods, but also in unrelated
species like the nematode Caenorhabditis elegans and in soybean and pea seeds. Alignment findings
for a prepro-AKH precursor reveal a significantly lower percentage identity, suggesting that, to allow
effective function in different organisms, the mature peptide must be highly conserved, whereas the
prepro-peptide may be less well conserved to play its role (Taub-Montemayor et al. 1997).
Although numerous cDNAs encoding insect AKH/RPCH precursor peptides are known
(e.g., Locusta migratoria, Schistocerca gregaria, Schistocerca nitans, Blaberus discordalis, Manduca
sexta, and Drosophila melanogaster), only three cDNAs have been described from crustaceans: the
crabs Carcinus maenas and C.  sapidus and the crayfish Cherax quadricarinatus (Martínez-Pérez
et  al. 2005). The prepro-RPCH from C.  maenas consists of a 25-amino acid signal peptide, an 8
amino-acid RPCH segment, and a 74 amino-acid RPCH-precursor-related peptide (RPRP;
Linck et  al. 1993). Compared to the prepro-hormone structure of AKH (O’Shea and Rayne
1992), this RPCH precursor shows a high percentage identity in the region corresponding to the
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 79

neuropeptides RPCH and AKH, but a low percentage identity for the precursor-related peptides
RPRP and AKH-precursor-related peptide (De Kleijn and Van Herp 1995). The same is also true
of other neuropeptides because the cDNA encoding PDH in penaeid shrimp reveals that, although
the precursor peptide structures are significantly different, the mature peptides are closely related
(Ohira et al. 2006). Thus, elevated percentage identity appears to be restricted to the peptide hor-
mone sequences while the precursor-related peptides are extremely variable, except within groups
of very closely related species (Linck et al. 1993). The fact that RPRP is not conserved suggests the
lack of an important physiological function (De Kleijn and Van Herp 1995). All AKH and RPCH
precursors, however, share a basic architectural plan: the encoding region is subdivided into a signal
peptide sequence, the hormone itself, and a precursor-related peptide (Klein et al. 1995).
The gene for the C. sapidus RPCH precursor contains an intervening element in the RPCH-PRP
coding region, an intron, not identified so far in any other crustacean species. Such introns are pres-
ent in the genes coding for insect AKH precursors, however. Although the location of this intron
is similar to the second intron in the S. nitans AKH gene, discrete differences in intron structure
between insect (D. melanogaster and S. nitans) and C. sapidus genes are present; the biological rel-
evance of such features is unknown. The C. sapidus intron, although exhibiting certain differences
with insect AKH encoding genes, shows important similarities with other regions of the RPCH
encoding gene. Nevertheless, important differences in the RPCH encoding genes do exist among
the arthropods, and the size of the introns suggests the participation of different intronic and exonic
elements during splicing to form the precursor mRNA (Martínez-Pérez et al. 2002).
Compared to noncrustacean arthropods then, the estimated amino acid identity of a 590-bp
RPCH precursor from C. quadricarinatus to insect AKHs (AKH I, II, and III) and hypertrehalos-
emic hormone (HTH) ranges from 36% to 62% (Martínez-Pérez et al. 2005; see Table 3.1). Within
the decapod Crustacea, however, the conceptual translation of a 242-bp cDNA RPCH fragment
from C. quadricarinatus is 70% identical to that of the crabs C. sapidus (Klein et al. 1995) and C. mae-
nas (Linck et al. 1993; see Table 3.1).
The isolation and characterization of the B. discoidalis HTH gene provided the first description
of an AKH/RPCH family prepro-hormone from cockroaches (Lewis et al. 1997). Thus, although
cockroaches were distinct from other arthropods by 300 mya (Kukalová-Peck 1990), their HTH
precursors have retained the simple organization of the AKH/RPCH prepro-hormones seen in

Table 3.1.  Percentage identities of various arthropod AKH/RPCH neuropeptide hormone

precursors with the amino acid sequence predicted from a 590-bp cDNA sequence for
precursor RPCH from the crab Cherax quadricarinatus. See text for references.

Peptide hormone Species Identity (%)

RPCH Callinectes sapidus 70
Carcinus maenas 70
AKH I Schistocerca gregaria 62
Locusta migratoria 41
AKH II Schistocerca nitans 48
AKH III Locusta migratoria 36
Drosophila melanogaster 36
HTH Blaberus discoidalis 50

RPCH, crustacean red pigment concentrating hormone; AKH I, II and III, insect adipokinetic hormones I, II and III; HTH,
insect hypertrehalosemic hormone.
80 John Campbell McNamara and Sarah Ribeiro Milograna

highly diverged insects and in a distantly related crustacean. Apparently, the tripartite structure
of AKH/RPCH family precursors (signal peptide, hormone plus processing site, and C-terminal
peptide of variable length and unknown function) arose early in arthropod evolution and has been
conserved across broad taxonomic groups (Lewis et al. 1997, Gäde 2004). Although functions have
been attributed to signal peptides and hormone segments within AKH/RPCH family precursors,
the significance of the C-terminal peptides is obscure. With the exception of crustacean RPCH,
C-terminal peptides from various insect species are similar in size but show only 25–35% amino acid
positional identity between orders. In comparison, the biologically active peptide segments within
the insect prepro-hormones are 44–88% identical, whereas the signal peptides show 16–45% amino
acid positional identity (Lewis et al. 1997).
The amino acid sequences of prepro-PDH from the crayfish Orconectes limosus (De Kleijn
et al. 1993) and from the crabs C. maenas (Klein et al. 1992) and C. sapidus (Klein et al. 1994) have
been deduced from cDNAs obtained employing degenerate primers based on a partial amino
acid sequence for U. pugilator PDH. The PDH peptides from O. limosus, C. maenas, and C. sapidus
(PDH I) differ by only a single amino acid (aspartic acid for glutamic acid), showing 94% identity,
whereas PDH II from C. sapidus has up to six different amino acids, corresponding to 64% identity.
The preceding RPRPs and signal peptides show 41% and 42% identity, respectively. Given over-
all prepro-hormone structure in the Crustacea, their prepro-PDHs seem to be well conserved but
show no similarity with other known peptides (De Kleijn et al. 1993, De Kleijn and Van Herp 1995).
Like many vertebrate prepro-hormones, such as insulin, post-translational modification of
prepro-neuropeptides is common in the Pan-Crustacea and extends to the AKH/RPCH and hyper-
glycemic (CHH) peptides. In insect AKHs, this usually includes two essential alterations: a Gln to
pyro-Glu exchange at the N-terminus and a carboxyamide group at the C-terminus (Gäde et al.
2007). Species-specific modifications include, for example, a conserved Leu2 for Val2 exchange
in water scorpion (Nepa cinerea) and Ser6 for Thr6 exchange in cricket (Gryllus bimaculatus; see
Nair et  al. 2001, Gäde et  al. 2007). In the crayfish O.  limosus, two distinct genes code for CHH
prepro-hormone isoforms that differ slightly in their signal and precursor-related peptides but are
identical in CHH coding region (De Kleijn et al. 1994); the two CHH isoforms expressed result
from post-translational modifications.

CELL SIGNALING AND PIGMENT MIGRATION IN CHROMATOPHORES

The Chromatophorotropin Receptor

Investigations aiming to identify the RPCH receptor type present in the plasma membrane of crus-
tacean chromatophores have been initiated only very recently. The red ovarian chromatophores
of Macrobrachium olfersi respond only partially to RPCH when previously perfused in vitro with
a generic G-protein antagonist (pGlu-Gln-D-Trp-Phe-D-Trp-D-Trp-Met-NH2; Milograna and
McNamara 2009a). This constitutes reasonable evidence that RPCH binding to a seven-domain
membrane-spanning G-protein-coupled receptor (GPCR) induces pigment aggregation. The AKHs,
structurally related to RPCH, also activate intracellular cascades after binding to GPCRs (Van der
Horst et al. 1999). AKH receptors cloned from D. melanogaster and Bombyx mori (Staubli et al. 2002) are
structurally related to the GPCR receptors for vertebrate gonadotropin-releasing hormone (Gn-RH).
The AKHs that activate glycogen phosphorylase in insect flight muscles act via membrane-located
protein Gq and phospholipase Cβ, increasing inositol trisphosphate (IP3) and leading to Ca2+ release
from IP3-gated intracellular stores and extracellular influx. In contrast, the lipase-activating AKHs
increase protein Gs and adenylyl cyclase (AC) activities, thus elevating intracellular cAMP together
with Ca2+ release from IP3-gated stores and extracellular influx (Gäde 2004).
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 81

Signal Transduction at the Chromatophore Membrane

Despite notable downstream functional diversity, the initial phase of GPCR signal transduction is
fairly uniform: the activated receptor catalyzes the exchange of guanosine diphosphate (GDP) for
guanosine triphosphate (GTP) on the stimulatory G-protein α subunit (Gsα), leading to dissocia-
tion among the Gsα-GTP and Gβγ subunits and enabling them to activate or inhibit a diversity of
effector proteins ranging from membrane-associated catalytic enzymes and ion channels to soluble
second-messenger-producing enzymes (Natochin et al. 2001, Gurevich and Gurevich 2008). The
classic enzyme system regulated by GPCRs is the membrane-located AC that catalyzes cAMP pro-
duction from ATP and its release into the cytosol. Adenylyl cyclase is activated by protein Gsα and
inhibited by protein Giα, whereas the upstream GPCRs respond to diverse external stimuli like light
and odorants and to various endogenous cues such as hormones, neurotransmitters, extracellular
Ca2+, and enzyme activity (Gurevich and Gurevich 2008).
In addition to the recent investigations on RPCH and AKH action just described (Van der
Horst et al. 1999, Staubli et al. 2002, Gäde 2004), other evidence concerning the nature of their
signaling cascades suggests that crustacean chromatophorotropins function in a classic manner via
G-protein transduction (Milograna and McNamara 2009a). Pigment translocation in crustacean
chromatophores can be induced by an increase in cytosolic Ca2+, leading to protein phosphoryla-
tion/dephosphorylation owing to downstream activation of Ca2+-regulated specific kinases and/
or phosphatases. Ca2+ signaling often occurs concomitantly but not necessarily simultaneously
with a decrease or increase in cytosolic cAMP or cGMP concentration, brought about by crosstalk
between the respective signaling cascades. This phenomenon varies widely among the crustacean
orders and depends on chromatophore type; it also varies moderately even at the species level.
These signaling cascades are discussed in detail in the following sections. The salient point here
is that most chromatophorotropin signaling cascades tend to be characteristic of GPCR signaling.

Chromatophore Membrane Ion Channels and Calcium Movements

In caridean shrimps, RPCH requires extracellular Ca2+ (Ca2+ext) to induce and sustain pig-
ment aggregation (Fingerman 1969, McNamara and Taylor 1987, Britto et  al. 1990, McNamara
and Ribeiro 1999). The Ca2+ ionophore, A23187, a Ca2+-specific mobile ion carrier that allows
concentration-dependent selective Ca2+ movements, induces complete and reversible pigment
aggregation with kinetics similar to RPCH (McNamara and Ribeiro 2000) and has been a useful
tool to investigate a role for extracellular Ca2+ (Figs. 3.5 and 3.6).
During pigment aggregation, Ca2+ derives from both extracellular and intracellular sources, fol-
lowing a sequence of intricate and finely regulated events (Ribeiro and McNamara 2007, Milograna
et al. 2010). Overall, after RPCH binds to its receptor in the plasma membrane, Ca2+ is released from
the SER (Ribeiro and McNamara 2007). Subsequent to this initial increase in intracellular Ca2+
(Ca2+int) concentration, and perhaps partly as a direct response to it, the plasma membrane resting
potential depolarizes from −76 to −34 mV (Milograna et al. 2010), leading to voltage-regulated Ca2+
influx from the extracellular fluid and an intracellular Ca2+ signaling cascade (Fig. 3.7).
The complete cellular mechanism that leads to membrane depolarization is not yet clear, but
Ba2+-sensitive plasma membrane K+ channels appear to close shortly after RPCH binding, thus
restricting K+ efflux. The resulting accumulation of positive charges below the cytosolic membrane
leaflet reduces the potential difference generated naturally in the unstimulated chromatophore
and leads to depolarization of the chromatophore membrane in which voltage-sensitive, N- and/
or P/Q-type Ca2+ channels then open, allowing Ca2+ext influx (Milograna et al. 2010; see Fig. 3.7).
An important issue here concerns the duality of the Ca2+ sources, both extra- and intracellu-
lar in nature, on which pigment aggregation depends. The velocity of pigment translocation in
82 John Campbell McNamara and Sarah Ribeiro Milograna

RPCH
A K+ receptor 1 B
channel
4
Na+/K+− a β γ α
ATPase 2 β γ

3
NCX

RyR
PMCA
ADP+Pi Pi+ADP
ATP
SER ADP+Pi
ATP
ATP 6
Ca2+
channel SERCA Pigment
−76 mV ATP
ADP+Pi granule −34 mV
5
Myosin II 7
Pigment Actin
aggregation filament

Fig. 3.7.
Model proposed for signal transduction and Ca2+-signaled pigment aggregation after red pigment concentrat-
ing hormone (RPCH) binding to its receptor in the chromatophore cell membrane. (A, left): Resting state,
fully dispersed pigment, RPCH not bound to receptor. Membrane resting voltage (−76 mV) is maintained
primarily by the Na+/K+-ATPase. K+ flows outward through open K+ channels and is recycled by the Na+/
K+-ATPase that extrudes Na+. Na+ influx (Na+ext = 140 mM) drives Ca2+ efflux through a Na+/Ca2+ exchanger
(NCX), while plasma membrane (PMCA) and sarco/endoplasmic reticulum (SERCA) Ca2+-ATPases actively
transport Ca2+ to the extracellular fluid and smooth endoplasmic reticulum (SER) lumen, respectively, main-
taining low Ca2+int (<10−9 M). The chromatophore membrane is fairly impermeable to Ca2+ influx down the
Ca2+ gradient (Ca2+ext = 5.5 mM) since Ca2+ channels are closed. (B, right): RPCH bound to putative 7-span
G-protein-coupled receptor (GPCR), pigment aggregation initiated. After RPCH binding, the activated
GPCR (1) signals a heterotrimeric G-protein, (2) leading to Ca2+ release through SER ryanodine receptors
(3) via an unknown mechanism. The increased Ca2+int (4) closes membrane K+ channels, either by a direct
Ca2+-dependent effect or through protein kinase C/Ca2+-calmodulin-mediated phosphorylation or by interac-
tion with the G-protein α- or βγ-subunits, thus (5) depolarizing the chromatophore membrane to −34 mV.
Voltage-gated N- and/or P/Q-type membrane Ca2+ channels then open (6), allowing Ca2+ influx from the
extracellular fluid down the Ca2+ gradient into the cytosol, increasing Ca2+int to ≈10−3 M. The activated intra-
cellular Ca2+ and cyclic guanosine monophosphate signaling cascades appear to stimulate Rho protein kinase
that then (7) phosphorylates a nonmuscle myosin II molecular motor, resulting in pigment aggregation. From
Milograna et al. (2010) with permission from Wiley and Sons, Inc.

palaemonid chromatophores is Ca2+ sensitive, or at least increases with increasing Ca2+int, from
<10−9 M in unstimulated chromatophores with fully dispersed pigments, to ≈10−3 M during pigment
aggregation (McNamara and Ribeiro 2000). At 5 mM Ca2+ext (equivalent to the total hemolymph
Ca2+ concentration), pigment aggregation is almost complete within 5 min of RPCH perfusion in
vitro; in ethylene glycol tetraacetic acid-buffered Ca2+-free saline, pigments previously aggregated
by RPCH or 1–5 mM Ca2+ext disperse, disclosing a process that responds differentially to variable
Ca2+ concentrations rather than a simple “on-off ” triggering mechanism. Apparently, should the
increase in cytosolic Ca2+ derive from a single source, the rate of increase in cytosolic Ca2+ nec-
essary to trigger the subsequent intracellular Ca2+ cascade is not attained as effectively as with a
simultaneous or concatenated supply from both sources. Given that Ca2+ext influx obeys a concen-
tration gradient into the cytoplasm, influx capacity may be rate limiting. Furthermore, it may be
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 83

energetically expensive for chromatophores to maintain large Ca2+ SER stocks, given the paucity
of mitochondria and the limited volume of the SER cisternae (McNamara and Ribeiro 1999). Ca2+
cycling between the cytosol and the SER thus may be limited, possibly slowing pigment aggrega-
tion and dispersion dynamics and affecting the overall efficiency of color change and the camou-
flage mechanism, for example.
A case in point, like RPCH, during the action of endogenous AHKs employed in both adipo-
kinetic and hyperprolinemic signaling in insects, neither Ca2+ext nor Ca2+ from the SER alone is
sufficient. The AKH peptide activates phospholipase Cβ, increasing IP3 levels and resulting in the
depletion of intracellular Ca2+ stores, a cue for capacitative Ca2+ entry via the plasma membrane
(Gäde and Auerswald 2003). Furthermore, extracellular Ca2+ is indispensable for AKH activity in
many insects and also may be required for hormone receptor binding (Van der Horst et al. 1999).
Caridean chromatophores are endowed with an equally rich Ca2+ transport apparatus that
enables them to reduce Ca2+int to the low levels attained via the mechanisms just described. This
system includes ATP-dependent transport proteins such as the sarco-/endoplasmic reticulum
Ca2+-ATPase (SERCA) located in the SER membranes, which actively transports free cytosolic
Ca2+ into the SER lumen. Inhibition of SERCA activity by thapsigargin and cyclopiazonic acid
alone, respectively, induces pigment aggregation of between 50% and 100% (Ribeiro and McNamara
2007). The plasma membrane Ca2+-ATPase (PMCA) actively pumps Ca2+ up its concentration
gradient to the extracellular medium. PMCA inhibition by lanthanum alone induces 12% pigment
aggregation (Milograna et  al. 2010). This spontaneous pigment aggregation seen on inhibition
of the two Ca2+-ATPases results from increased Ca2+int. The plasma membrane Na+/K+-ATPase,
linked to the Na+/Ca2+-exchanger driven by the inward Na+ gradient, is also involved in Ca2+ extru-
sion (Milograna et al. 2010; see Fig. 3.7). These proteins are also crucial to the pigment dispersion
mechanism, although whether they can by activated directly by PDHs is not known. They most
probably respond constitutively to intracellular Ca2+ levels, pumping continuously to hold Ca2+int at
the fairly constant low levels typical of pigment dispersion.
Roles for Ca2+-ATPases and Ca2+-exchangers have not been evaluated in crustacean groups other
than the palaemonid shrimps, but they likely underlie the pigment-dispersing mechanism in brachy-
uran crab chromatophores. In these crabs, Ca2+int is related to pigment movement in a manner con-
trary to that seen in carideans; that is, pigment granules are fully aggregated in unstimulated crab
chromatophores (Lambert and Fingerman 1976, Kulkarni and Fingerman 1986), exactly the reverse
of their distribution in caridean shrimp chromatophores. In fiddler crab black chromatophores,
Ca2+int, increased by the calcium ionophore A23187, induces pigment dispersion (Quackenbush 1981,
Rao and Fingerman 1983). This evidence suggests that, in crabs, PDH coupling to the plasma mem-
brane receptor induces increased Ca2+int and dispersion of the initially aggregated pigment granules,
possibly through mechanisms of signal transduction similar to that induced by RPCH in shrimps.

Intracellular Second-Messenger Cascades in Chromatophores

In addition to the increase in Ca2+int just described, RPCH binding to a putative GPCR in the plasma
membrane also leads to an increase in intracellular cGMP (McNamara and Ribeiro 2000, Ribeiro
and McNamara 2007, 2009). Dibutyryl-cGMP (db-cGMP), a lipid-soluble cGMP analogue, like-
wise induces complete, Ca2+-dependent, reversible pigment aggregation identical to RPCH- and
very similar to A23187-triggered aggregation (McNamara and Ribeiro 2000, Ribeiro and McNamara
2009; see Fig. 3.6A,B). The soluble GC activators sodium nitroprusside (SNP) and morpholinosyd-
nonimine (SIN-1) alone induce 40% pigment aggregation with granule transport kinetics similar to
that induced by RPCH (Ribeiro and McNamara 2009). Inhibition of the same cytosolic GCs by zinc
protoporphyrin IX (ZnPP-IX) and 6-anilino-5,8-quinolinedione (LY83583) reduces RPCH-triggered
pigment aggregation by 30% and also reduces the rapid phase of pigment translocation (Ribeiro and
84 John Campbell McNamara and Sarah Ribeiro Milograna

McNamara 2009). However, the type-C plasma membrane receptor GC does not seem to play a role
in cGMP synthesis during RPCH action because stimulation with Escherichia coli heat-stable entero-
toxin 1 (STa-1) does not induce pigment aggregation (Ribeiro and McNamara 2009).
Pigment aggregation in caridean chromatophores also seems to be associated with a decrease
in intracellular cyclic adenosine monophosphate (cAMP) concentration (Fingerman 1969, Nery
et  al. 1997). Furthermore, this cyclic nucleotide second-messenger induces pigment dispersion in
Palaemonetes vulgaris (Fingerman 1969)  and Macrobrachium potiuna (Nery et  al. 1998)  red epider-
mal chromatophores, although db-cAMP does not cause pigment movement in M. olfersi red ovar-
ian chromatophores (Ribeiro and McNamara 1997). The cAMP target, protein kinase A (PKA), also
plays a role in pigment dispersion in M. olfersi ovarian red chromatophores (Bell et al. 2005). PKA acti-
vation leads to a protein phosphorylation cascade that induces pigment dispersion in carideans and
brachyurans. This cascade, when deactivated, may lead to pigment aggregation in caridean shrimps
(Nery and Castrucci 1997, Nery et al. 1997). Furthermore, a decrease in cytosolic cAMP concentra-
tion, when transduced by an inhibitory G-protein (Gi)-coupled receptor, may be an alternative signal-
ing mechanism for pigment aggregation in some crustacean groups (Nery and Castrucci 1997).
Both cGMP and db-cGMP induce dose-dependent pigment dispersion in vitro in the black,
white, and red chromatophores of the crab U. pugilator, and they inhibit pigment dispersion in red
chromatophores but enhance dispersion in black and white chromatophores induced by a partially
purified eyestalk hormone in vitro (Rao and Fingerman 1983). cGMP is thus one of the important
second-messenger nucleotides that effect pigment translocation in different directions, apparently
depending on chromatophore type and taxon. This constitutes further evidence that the pigment
granules in crab and shrimp chromatophores migrate in opposite directions in response to the same
intracellular stimulus. Thus, the same cGMP signaling pathway may activate different molecular
motors that effect opposing pigment granule movements; that is, dispersion in the Brachyura and
aggregation in the Caridea (Ribeiro and McNamara 2009).
Nevertheless, pigment aggregation in caridean chromatophores is triggered by db-cGMP
only when Ca2+ is present in the extracellular medium, clearly revealing that not only does
RPCH-triggered aggregation require Ca2+, but also that the cGMP cascade is also Ca2+-dependent
(Ribeiro and McNamara 2009; Fig. 3.6A). There thus seem to be one or more points of cross-talk
between the Ca2+ and cGMP signaling cascades that together induce pigment aggregation, although
the Ca2+ cascade seems to be fairly independent of the cGMP cascade, at least in vitro. Similarly, in
the crabs that rely on different second-messenger cascades like cAMP, cGMP, and Ca2+ for black,
white, and red pigment dispersion (Quackenbush and Rao 1979, Quackenbush 1981), crosstalk and
interdependence seem to prevail. What advantage might reside in employing more than one sig-
naling cascade in a single physiological response? Crosstalk between signal transduction cascades
appears to provide cells with complex intracellular systems for the fine tuning of hormone-induced
signals (Van der Horst et al. 1999). Thus, transduction systems appear to have evolved with a certain
redundancy, thus providing functional security that may enhance the fitness of an individual.

Second-Messenger-Activated Effectors and Kinases

It is not yet clear just how cGMP and Ca2+ activate the pigment translocation mechanism in cari-
dean shrimps. Free cytosolic Ca2+ can activate many target proteins like Ca2+/calmodulin (Ca2+/
CaM), protein kinase C (PKC), and the Ca2+/calmodulin-dependent protein kinase (Ca2+/
CaMK), for example, whose activation leads to a wide variety of intracellular responses (Kheifets
and Mochly-Rosen 2007). PCH-induced pigment aggregation in M. potiuna red chromatophores
is dependent on the Ca2+/CaM complex (Nery et al. 1997), and PKC (Bell et al. 2005) and Ca2+/
CaM are involved in pigment aggregation in M. olfersi red ovarian chromatophores (Milograna et al.
2012), as revealed by N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7)
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 85

inhibition. IP3 and diacylglycerol (DAG) play a role in pigment aggregation in M. potiuna red epi-
dermal chromatophores (Nery et al. 1997) but not in M. olfersi ovarian chromatophores (Ribeiro
2002), suggesting that different elements of the same signal transduction cascade may vary among
different chromatophore types.
Ca2+/CaM can regulate or induce nitric oxide synthase (NOS) activity, a heme-protein cyto-
chrome. This enzyme generates nitric oxide (NO) from L-arginine in a reaction that requires
oxygen, nicotinamide adenine dinucleotide phosphate-oxidase (NADPH), flavines, and biopter-
ins (Moncada et al. 1991, Murad 1994). A role for NOS and NO during the RPCH signaling cas-
cade leading to pigment aggregation has been demonstrated by Nω-nitro-L-arginine methyl ester
hydrochloride (L-NAME) inhibition in perfused shrimp ovarian chromatophores (Milograna et al.
2012). cGMP can be produced by soluble cytosolic GC (GC-S) when activated by NO (Schmidt
et al. 1993, Murad 1994, Müller 1997) and a key role for GC-S in red pigment aggregation has been
demonstrated in M. olfersi ovarian chromatophores (Ribeiro and McNamara 2009; see Fig. 3.8).
The cGMP pathway appears to be Ca2+-dependent at several points, and the GC activation step

RPCH

1
Ca2+ channel β γ
α
NOS
Ca2+/CaM Guanylyl
3
cyclase
NO
5
4
6
2
Ca2+ channel
GTP cGMP
PKG
Actin filament 7

ATP
Myosin II
ADP + Pi
MLC 9
Pigment Pigment −Pi +Pi
aggregation granule
+Pi
Dynein 10
8
MLCP ROCK
Microtubule

Fig. 3.8.
Model proposed for pigment aggregation signaled by the Ca2+- and cyclic guanosine monophosphate (cGMP)
cascades after red pigment concentrating hormone (RPCH) binding to its receptor. Pre-RPCH binding,
resting state conditions are provided in Fig. 3.7 (left panel). On RPCH binding, the activated GPCR (1) sig-
nals a heterotrimeric G-protein leading to Ca2+ release through SER ryanodine receptors (2) and opening of
voltage-gated N- and/or P/Q-type membrane Ca2+ channels (3), increasing Ca2+int to ≈10−3 M (see Fig. 3.7 for
details), a concentration that (4) activates Ca2+/calmodulin (Ca2+/CaM). This intracellular effector stimulates
nitric oxide synthase (NOS) (5) to produce nitric oxide (NO) that in turn enables cytosolic guanylyl cyclase
(6) to produce cGMP from GTP. cGMP activates protein kinase G (7) that may inhibit myosin light chain
phosphatase (MLCP) activity (8), which in turn regulates myosin light chain (MLC) activity (9) through
dephosphorylation (inactive state). In contrast, Rho protein kinase (ROCK) (10), possibly activated by Ca2+ or
by cGMP, phosphorylates the MLC (active state), resulting in pigment aggregation. Thus, MLCP and ROCK
together regulate the overall activity of the myosin heavy chain motor domains (myosin II). The microtu-
bule motor, dynein, also may be activated concomitantly by this mechanism and may possibly play a role in
rapid-phase aggregation.
86 John Campbell McNamara and Sarah Ribeiro Milograna

may be particularly important in this regard (Ribeiro and McNamara 2009). In crustaceans, com-
ponents of the NO/cGMP pathway have been identified in many systems that involve sensory per-
ception, such as the neural circuits that control olfaction ( Johansson et  al. 1996, Johansson and
Mellon 1998, Scholz et al. 1998), vision (Lee et al. 2000), rhythmic motor behavior (Scholz et al.
1996, Scholz 2001), escape behavior (Aonuma et al. 2000), and neurosecretion (Lee et al. 2000).
NO induces dose-dependent pigment dispersion in the black epidermal chromatophores of
N. granulata in vitro (Vargas et al. 2008). UVA/B-induced granule dispersion in crab chromato-
phores in vivo and in cultured retinal cells also depends on NO (Filgueira et al. 2010), constituting
an important protective response against damaging UV effects. Thus, NO may act as a chroma-
tophorotropin agonist in many crustaceans (Vargas et  al. 2008), further evidence that the same
signaling pathways activate different molecular motors in the Brachyura (dispersion) and Caridea
(aggregation) (Ribeiro and McNamara 2009). Apparently, different membrane receptors, such as
UV-sensitive opsins and RPCH and PDH receptors, may converge downstream on NOS, allowing
perception of diverse environmental cues via primary and humorally mediated responses by the
same cells.
In caridean shrimps, in response to RPCH signal transduction, Ca2+ apparently occupies its
binding sites on the calmodulin molecule, forming the activated Ca2+/CaM complex that in turn
activates Ca2+/CaMK, which stimulates NOS to synthesize NO, thus activating GC-S (Ribeiro
and McNamara 2009, Milograna et al. 2012). From GTP, this cyclase produces cGMP, which is
released into the cytosol and leads to activation of protein kinase G (PKG). In addition to PKG,
cGMP can regulate ion channels and phosphodiesterases (Wong and Garbers 1992, Lincoln and
Cornwell 1993, Schmidt et al. 1993). PKG plays a crucial role in pigment aggregation in the red
ovarian chromatophores of M. olfersi (Milograna et al. 2012), as demonstrated by Rp-guanosine
cyclic 3′,5′-monophosphate triethylammonium (Rp-cGMP-triethylamine) inhibition. In the
caridean shrimps, PKG seems to be the principal kinase responsible for pigment aggregation,
although further investigation is necessary to better understand the subsequent steps in this
mechanism (Fig. 3.8). Apparently, in crustacean chromatophores, the final distributional state of
the pigments at any particular instant reflects the degree of activation of specific effectors like
protein kinases G, C, and A and the Ca2+/CaM-dependent kinase at the end points of the signaling
cascades and the subsequent phosphorylation of direction-specific molecular motors and their
specific kinase-dependent regulators (Ribeiro and McNamara 2009).
Protein kinases are second-messenger-activated enzymes that phosphorylate particular amino
acids like serine and threonine at specific sites on diverse effector proteins, to which they transfer a
high-energy phosphate group derived from the hydrolysis of ATP to ADP. Once activated, the tar-
get protein suffers a conformational change and becomes enabled to perform its functional role. In
crustacean chromatophores, protein kinases activated primarily during the second-messenger cas-
cades just described appear to activate other downstream kinases that directly regulate molecular
motor activity. For example, actin-myosin-based movements are modulated by kinases and phos-
phatases that recycle energy in the form of a phosphate group through the regulatory sites of the
myosin heavy chains, the so-called light chains. The myosin light chain (MLC) is a myosin-associated
peptide subunit that regulates contractile and other activity-related movements in nonmuscle cells.
Phosphorylation of the MLC serine 19 residue (MLCSer19) is required for the formation of the
actin-myosin complex, thus initiating the myosin motor domain ATPase activity that alters the con-
formation of the myosin dimer motor domains and leads to progressive myosin movement along
the actin filament (Fajmut and Brumen 2008). In M. olfersi red ovarian chromatophores, MLCSer19 is
apparently phosphorylated by Rho protein kinase (ROCK), a Rho GTPase effector that plays a role
in many motor responses involving the cytoskeleton (Riento and Ridley 2003). ROCK blockade
by Y-27632 and H-1152 partially inhibits pigment aggregation and accelerates pigment dispersion in
M. olfersi chromatophores (Milograna et al. 2012; Fig. 3.8).
Table 3.2.  Effects of diverse pharmacological and physiological agents on pigment movements in caridean shrimp (Macrobrachium, Palaemon, and
Palaemonetes) and brachyuran crab (Uca, Neohelice) chromatophores. These effectors, when perfused in vitro, may induce pigment translocation per
se and can inhibit or enhance RPCH-triggered pigment migration, either partially or fully. All abbreviations and references are provided in the text.

Effector Pharmacological Intracellular action Effect on shrimp Effect on crab

characteristics/activity chromatophores chromatophores
RPCH GPCR agonist Triggers Ca2+ and cGMP Complete pigment Aggregates red pigments
signaling cascades aggregation
α-, β-PDH Plasma membrane receptor Trigger Ca2+ and cAMP Partial or complete pigment Partial or complete pigment
agonists? signaling cascades dispersion dispersion
A23187 Ca2+-specific mobile ion Increases [Ca2+]int Complete pigment Complete pigment
carrier/ionophore aggregation dispersion
Ca2+ Intracellular second messenger Activates CaM, Ca2+/CaMK Complete pigment Complete pigment
and PKC aggregation dispersion
La3+ PMCA inhibitor and Ca2+ Increases [Ca2+]int; inhibits Partial pigment aggregation; No data available
channel blocker RPCH-induced Ca2+ influx partial inhibition
of RPCH-induced
aggregation
Ba2+ K+ channel blocker Depolarizes plasma membrane Partial pigment aggregation No data available
Thapsigargin SERCA inhibitor Increases [Ca2+]int Partial pigment aggregation No data available
Cyclopiazonic acid SERCA inhibitor Increases [Ca2+]int Partial pigment aggregation No data available
cGMP Intracellular second messenger Activates PKG Complete pigment Black, white, red pigment
aggregation dispersion
db-cGMP Lipid-soluble cGMP analogue Activates PKG Complete pigment Black, white, red pigment
aggregation dispersion
cAMP Intracellular second messenger Activates PKA Pigment dispersion Pigment dispersion

(continued)
Table 3.2.  (Continued)

Effector Pharmacological Intracellular action Effect on shrimp Effect on crab

characteristics/activity chromatophores chromatophores
IP3 Intracellular second messenger Releases Ca2+ from REL Pigment aggregation No data available
(M. potiuna epidermal red
chromatophores)
DAG Intracellular second messenger Activates PKC Pigment aggregation No data available
(M. potiuna epidermal red
chromatophores)
STa-1 Plasma membrane receptor Releases NO No effect No data available
guanylyl cyclase activator
SIN-1 Soluble guanylyl cyclase activator Releases NO Partial pigment aggregation Black pigment dispersion
SNP Soluble guanylyl cyclase activator Releases NO Partial pigment aggregation No data available
ZnPP-IX Soluble guanylyl cyclase inhibitor Inhibits NO release Partial inhibition of No data available
RPCH-induced aggregation
UV light Cell signaling activator Activates NOS No data available Pigment dispersion
L-NAME NOS inhibitor Inhibits NO production Partially inhibits Inhibits black pigment
RPCH-induced dispersion
aggregation
W7 Ca2+/CaM inhibitor Suppresses downstream CaM Partially inhibits No data available
cascade RPCH-induced
aggregation
Rp-cGMP-triethylamine PKG inhibitor Suppresses downstream Partially inhibits No data available
activation of RPCH-induced
PKG-phosphorylated proteins aggregation
Y-27632 Rho protein kinase inhibitor Phosphorylates MLC Partially inhibits No data available
RPCH-induced
aggregation; accelerates
pigment dispersion
H-1152 Rho protein kinase inhibitor Phosphorylates MLC Partially inhibits No data available
RPCH-induced
aggregation; accelerates
pigment dispersion
Cantharidin MLC phosphatase inhibitor Dephosphorylates MLC Accelerates pigment No data available
aggregation
BDM Myosin ATPase inhibitor Inhibits myosin/actin Hyperdispersion; reduces No data available
interaction pigment aggregation velocity
Blebbistatin Non-muscle myosin II inhibitor Impairs non-muscle myosin Partially inhibits No data available
II-based transport RPCH-induced pigment
aggregation
Pan-myosin antibody Myosin motor inhibitor Impairs myosin-based transport Partially inhibits No data available
RPCH-induced pigment
aggregation
Cytochalasin B Depolymerizes actin filaments Inhibits actin filament-based Inhibits RPCH-induced Inhibits PDH-triggered
transport pigment aggregation; no dispersion, and
effect on A23187-triggered aggregation
aggregation
Colchicine Depolymerizes microtubules Impairs microtubule-based Partial pigment No effect on PDH-triggered
transport aggregation; no effect dispersion; inhibits
on RPCH-triggered aggregation
aggregation
Taxol Microtubule stabilizer Alters cytoskeleton turn-over Partial pigment aggregation; No data available
reduces velocity of
RPCH-triggered pigment
aggregation
EHNA Dynein-ATPase inhibitor Impairs dynein-based transport Partially inhibits No data available
RPCH-induced pigment
aggregation
90 John Campbell McNamara and Sarah Ribeiro Milograna

MLC dephosphorylation is performed by the MLC phosphatase (MLCP), leading to the uncou-
pling of the myosin motor domains from the actin filaments or by simply maintaining the crossbridge
ligand sites in a dephosphorylated state (Rembold and Murphy 1990). ROCK increases MLC phos-
phorylation either by direct phosphorylation of the MLCSer19 residue (Amano et al. 1996) or by inhibi-
tion of MLCP activity (Feng et al. 1999, Chilcoat et al. 2008; Fig. 3.8). The processes underlying the
MLC phosphorylation/dephosphorylation cycle appear to depend on intracellular Ca2+ and on the
decoding of kinase signaling. When the MLC is held in a phosphorylated state by inhibition of MLCP
with cantharidin, RPCH-triggered aggregation velocity in M. olfersi chromatophores increases signifi-
cantly, indicating accelerated myosin head movement along the actin filament (Milograna et al. 2012).
This area of investigation requires detailed study because no comparative data on myosin kinases and
phosphatases are available in other crustacean pigment cells (Fig. 3.8). Table 3.2 provides summary
data for the effects of many different pharmacological and physiological agents on chromatophore
pigment movements in the main caridean and brachyuran taxa investigated.

THE MECHANICS OF PIGMENT TRANSLOCATION WITHIN

CHROMATOPHORES

Molecular Motors and the Cytoskeleton

The mechano-chemical proteins responsible for intracellular transport are known as “molecular
motors” and generally consist of two functional parts: a motor domain that reversibly binds to the
cytoskeleton and converts chemical energy into kinetic energy or movement and a tail that interacts
with a cargo, either directly or through accessory light chains (Karcher et al. 2002). Knowledge of
pigment granule translocation in invertebrate chromatophores is scanty from a biophysical stance
(McNamara and Ribeiro 1999). Traditionally, conventional myosins are the molecular motors
known to transport cargos along actin filaments, whereas kinesin and dynein interact with microtu-
bules. However, recent studies describe extremely complex interactions among molecular motors,
the cytoskeleton, and accessory proteins in cells in general (Gross et al. 2002, Ali et al. 2008), includ-
ing interactions between many kinds of molecular motors (e.g., an association between kinesin and
myosin for transporting cargos along microtubules).
It has long been suggested that more than one molecular motor is involved in pigment move-
ment, even in monochromatic chromatophores (Boyle and McNamara 2006). Pigment aggrega-
tion in M. olfersi red ovarian chromatophores clearly follows a biphasic kinetic time course: a brief 2
min pulse of rapid-phase activity at around 20 µm/min followed by a 10 min plateau of slow-phase
translocation at about 5 µm/min (Fig.  3.6A,B). Also, such chromatophores contain two mor-
phologically distinct red pigment granules that show marked differences in size (1–2 µm versus
80–100 nm diameter) and surface characteristics (membrane-bounded versus carotenoid interface;
McNamara and Sesso 1983) and that remain clearly spatially separated in the aggregated state with
no physical barrier evident between them (McNamara and Sesso 1982, 1983). This suggests that at
least two distinct molecular motors participate in pigment migration and in the spatial separation
of the pigment granules (McNamara and Ribeiro 1999).

Pigment Translocation by Actin and/or Tubulin-based Motors

We have demonstrated a prominent role for PKG and myosin in slow-phase pigment aggregation,
together with an apparent lack of function for PKG in rapid-phase translocation (Milograna et al.
2012). Slow-phase pigment aggregation velocity can be reduced by 30% by inhibiting myosin ATPase
activity with butanedione monoxime and thus appears to be underpinned by actin-myosin-based
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 91

granule transport (McNamara and Ribeiro 1999). A myosin motor also actively sustains the aggre-
gated state, regulated by Ca2+-dependent proteins activated by RPCH (McNamara and Ribeiro
1999). Myosin motor inhibition in M. olfersi red ovarian chromatophores with dispersed pigments
causes pigment hyperdispersion, suggesting that the myosin motor remains activated even in the
dispersed pigment state (Boyle and McNamara 2006).
Several different myosin types may effect pigment aggregation in crustacean chromatophores.
Myosins are currently classed into more than 20 families, each exhibiting a diversity of structural
and functional characteristics. Boyle and McNamara (2006), based on Western blotting in M. olf-
ersi ovarian chromatophores, suggest that one of the pigment-aggregating myosin motors might
be myosin II or myosin XII. Our recent findings with Alexa-conjugated anti-nonmuscle myosin II
antibodies in confocal fluorescence microscopy clearly reveal the broad distribution of nonmus-
cle myosin II in M. olfersi ovarian chromatophores (Fig. 3.9A), as well as the functional relevance
of this motor, as demonstrated by pharmacological inhibition of pigment granule aggregation
with blebbistatin, a nonmuscle myosin II inhibitor (Milograna and McNamara 2009b). Also, the
streptomycin-O mediated introduction of a pan-myosin antibody into ovarian chromatophores
causes functional inhibition of RPCH-triggered pigment aggregation (Boyle and McNamara
2006). Tetramethyl-rhodamine-isothiocyanate (TRITC)-labeled pan-myosin antibodies (Boyle
and McNamara 2006)  and Alexa-conjugated anti-pan myosin antibodies (Fig. 3.9B) also reveal
myosin to be intimately associated with the larger pigment granules, suggesting that at least two
classes of myosin participate in pigment translocation.
The role of microtubule-based transport in pigment aggregation in crustacean chromatophores
is controversial. Disruption by colchicine, vinblastine, and other tubulin depolymerizing agents
usually does not affect pigment aggregation at pharmacological concentrations (McNamara 1980,
Quackenbush 1981, Tuma et al. 1995). However, microtubule stabilization with Taxol collapses the

Fig. 3.9.
Myosin molecular motors, intimately associated with the pigment granule membranes, play an important role
in granule translocation along the actin cytoskeleton during pigment aggregation and possibly in limiting pig-
ment dispersion brought about by opposing motors. (A) Confocal fluorescence microscopy (Leica TCS SP5,
λex= 495 nm, λem= 519 nm) showing the distribution of granule-associated, nonmuscle myosin II throughout the
cytoplasm of M. olfersi ovarian chromatophores with fully dispersed pigments, revealed using a polyclonal, non-
muscle myosin II primary antibody followed by an Alexa-488 conjugated goat anti-rabbit IgG. (B) Distribution
of a skeletal muscle pan-myosin clearly associated with the large membrane-bounded pigment granules in an
ovarian chromatophore extension, shown employing a polyclonal, anti-skeletal muscle pan-myosin primary
antibody conjugated with an Alexa-488 goat anti-rabbit IgG. Scale bars A = 20 µm, B = 1 µm. See color version
of this figure in the centerfold.
92 John Campbell McNamara and Sarah Ribeiro Milograna

chromatophore structure and leads to some degree of pigment aggregation (Bell 2008), perhaps
supporting older notions (McNamara 1980, 1981) that microtubule turnover maintains the notable
chromatophore asymmetry and, particularly, the integrity of the cell extensions and finer secondary
ramifications. Taxol slightly slows RPCH-triggered pigment aggregation in the ovarian chromato-
phores of M. olfersi (Bell 2008) also suggesting that microtubule depolymerization or turnover may be
associated with aggregation. However, in the red chromatophores of M. potiuna, Taxol accelerates pig-
ment aggregation (Tuma et al. 1995), suggesting that microtubules maintained in a polymerized state
somehow sustain granule translocation. Certainly, many polymerized microtubules are present in the
cell extensions of caridean epidermal and internal organ chromatophores with aggregated pigments
(Robison and Charlton 1973, McNamara and Sesso 1983, McNamara and Taylor 1987; see Fig. 3.4).
The nature of the traditionally microtubule-associated molecular motors has not been well
investigated in crustacean chromatophores. Based on TRITC-labeled antikinesin antibody find-
ings, Boyle and McNamara (2006) have demonstrated a strong association between pigment gran-
ules and kinesin in M. olfersi ovarian chromatophores, suggesting that kinesin may be the molecular
motor responsible for pigment dispersion. Both kinesin and dynein, identified by Alexa-conjugated
antibodies in confocal fluorescence microscopy, are associated with pigment granules (Fig. 3.10A,B).
Physiological inhibition of dynein-ATPase with erythro-9-(2-hydroxy-3-nonyl) adenine hydro-
chloride also partially inhibits pigment aggregation (Ribeiro and McNamara 2004).
Given these findings, pigment aggregation in caridean shrimp chromatophores may involve
at least two active motor components. We advance two hypotheses to elucidate the dynamics of
pigment aggregation: (i) dynein may function during fast-phase aggregation via the microtubule
component of the cytoskeleton associated with the larger cell extensions; nonmuscle myosin II
may then attach to the granules, transporting them along the actin filaments in the finer secondary
ramifications; or, alternatively, (ii) granule-bound, nonmuscle myosin II may translocate over the
actin cytoskeleton during fast-phase aggregation, which would progress slowly by an association

Fig. 3.10.
Molecular motors like kinesin and dynein that respectively transport cargos to the chromatophore periphery or
perikaryon along the microtubular component of the cytoskeleton are also associated with the pigment gran-
ules. (A) Confocal fluorescence microscopy (Leica TCS SP5, λex= 495 nm, λem= 519 nm) showing the distribu-
tion of granule-associated kinesin throughout the cytoplasm of a chromatosome with fully dispersed pigments,
revealed using a monoclonal, anti-kinesin primary antibody and an Alexa-488 conjugated goat anti-mouse IgG.
(B) Dynein distribution is clearly associated with the large membrane-bound pigment granules in a chroma-
tosome with fully dispersed pigments, shown here employing a monoclonal, anti-dynein primary antibody
followed by an Alexa-488 conjugated goat anti-mouse IgG. Bright field and fluorescence images overlaid with
background subtraction, scale bars = 20 µm. See color version of this figure in the centerfold.
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 93

of dynein and myosin (Fig. 3.8). Kinesin seems to be the molecular motor responsible for pigment
dispersion, possibly through an interaction with other motors, although sustaining pharmacologi-
cal evidence is as yet unavailable. Boyle and McNamara (2008) have proposed a pigment transport
model in which kinesin and myosin mechano-chemical motors alternately stretch and compress
a structurally unified, elastic pigment matrix over a polymerized tubulin and actin cytoskeleton,
producing pigment dispersion and aggregation.

A SPECIAL CASE: THE RETINAL PIGMENTARY SYSTEM

A specialized, labile pigmentary system is also found within the cells that constitute the crustacean
compound eye, described briefly here. The photosensitive retinula cells are usually arrayed as groups
of eight cells but, depending on the taxon, are also found in groups of from five to seven cells located
beneath the cone cells. Usually four cone cells, together with the faceted, transparent, and multilay-
ered cornea, form the dioptric apparatus of the crustacean eye, which is constituted by many similar
anatomical units known as ommatidia. The retinula cells possess highly folded membrane specializa-
tions termed rhabdomeres that are the light-receptive elements of the ommatidia and that contain the
photosensitive pigments (Porter et al. 2007). Axons from the retinula cells penetrate the basement
membrane and terminate in the lamina ganglionaris, from where second-order neurons project to
other neurons located within the medulla externa. A variety of distal and proximal screening pigment
cells (Hallberg and Elofsson 1989) completes the basic structure of the crustacean compound eye
(Meyer-Rochow 2001). These various eye pigments occupy different positions within the retinula
cells, depending on ambient luminosity (Kleinholz 1966). Such pigments act as screening filters,
regulating the amount of light reaching the retina (Aréchiga et al. 1993). Photon flux to the receptors
is thus a function of the position of the pigment granules located within two distinct sets of cells in
the compound eye. In darkness, both sets of pigments are retracted, leaving most of the photorecep-
tor surface exposed to light. Under illumination, both pigment types become dispersed, depending
on light intensity, thus blocking the access of stray light to the photosensitive membranes (rhab-
domeres) in the photoreceptors (Garfias et  al. 1995). The redistribution of the pigment granules
is accompanied by changes in the SER, ranging from numerous small vesicles under conditions of
light exposure to fewer large cisternae in the dark (Frixione and Porter 1986). Photomechanically
induced changes may thus affect the position and shape of whole cells, the quantity and distribution
of organelles, and the chemical composition of membranes and photosensitive pigments, as well as
the titers of intracellular messengers (Meyer-Rochow 1999). These alterations are tuned to meet the
requirements of the crustacean eye for maximum light sensitivity and acuity while the constituent
photoreceptor cells derive maximum protection against damaging radiation (Meyer-Rochow 2001).
The mechanisms by which the conditions evoking light and dark adaptation elicit their cor-
responding intracellular pigment granule translocations are different. Proximal pigment granule
migration is a direct response of the photoreceptors themselves to light and dark (Frixione et al.
1979). The distal pigment cells, however, do not respond directly to light; they are the end effectors
of a neuroendocrine reflex. Distal pigment dispersion is triggered by light acting on extraretinal
photoreceptors (Aréchiga et al. 1985) and is mediated by the release of a light-adapting hormone
(DRPH; Kleinholz 1966). DRPH is released into the SG from neurosecretory axons whose peri-
karya are located in the protocerebrum (Hernández and Fuentes-Pardo 2001) and in the XO. In
response to this neurosecretory peptide, the distal pigment granules then migrate longitudinally,
from a distal to a more proximal position in the distal pigment cells, a change that reduces the
amount of light entering the retinula cells. The mechanisms by which retraction of the distal retinal
pigment granules is induced are yet to be disclosed (Garfias et al. 1995), but Ca2+ seems to play a
key role in regulating such movements in arthropod retinula cells (Frixione and Aréchiga 1981). In
crayfish, the transport mechanism in the retinula cells is controlled by Na+-dependent adjustments
94 John Campbell McNamara and Sarah Ribeiro Milograna

of Ca2+ concentration, dependent on uptake by SER cisternae, as is also seen in chromatophores

(Frixione and Ruiz 1988). Activation of PKC by DAG released during the phototransduction cas-
cade also may regulate the turnover of phototransductive membrane in arthropods (Blest et  al.
1993, Minke and Selinger 1992). In arthropod photoreceptors, DAG may thus constitute one of the
light-dependent second-messengers that induce transcription of rhabdomeral membrane precur-
sors (Blest et al. 1993, 1994).
A further modulatory influence on screening pigment migration is exerted by circadian rhythm.
At night, the responsiveness of the retinal photoreceptors is much higher than during the day, and
both retinal pigments are retracted at night and dispersed during the day. Such rhythmicity persists
under conditions of constant illumination (Aréchiga et al. 1993, Garfias et al. 1995) suggesting regula-
tion by a biological clock. The retinula cells also confer polarized vision or sensitivity on some crus-
tacean groups (Kleinlogel and Marshall 2009)—that is, the ability to discriminate between two light
sources of the same luminosity but of different e-vector orientation and/or degree of polarization
(Kirschfeld 1973)—and some species are also sensitive to UV light (Kleinlogel and Marshall 2006).

FUTURE DIRECTIONS

Although physiological investigations of pigment movements in crustacean chromatophores seem

to have reached their zenith toward the end of the past century, many questions still remain that now
can be examined employing novel techniques and refined methods. Knowledge of the tissues of
origin and the migration pathways of chromatophores during embryogenesis would be very useful,
for example. Perhaps the most important underlying issue will be success in culturing crustacean
chromatophores and establishing a chromatophore cell line. This will open the way for electrophys-
iological, biochemical, and molecular biological techniques that require individual cells, pure cell
suspensions, or purified chromatophore proteins and membranes. Identification of the membrane
receptors involved in chromatophorotropin signal transduction is an essential step in clarifying the
early events leading to pigment migration, and an examination of homology with other arthro-
pod hormone receptors requires molecular analysis. The use of well-established patch clamping
methodologies also will be important here to reveal voltage-dependent membrane-associated
events. Investigation of the cytoskeleton, particularly actin distribution and its associated
pigment-translocating molecular motors and their regulation, also require study, given the diverse
intracellular signaling cascades now identified. Real-time analyses of calcium movements during
pigment translocation using intracellular fluorochromes also would be very useful. Pigment dis-
persion in the caridean shrimps is still virtually unexplored, and physiological/pharmacological
investigations are necessary to reveal the receptor type, signaling cascades, and molecular motors
involved. Molecular biological methodologies will be important here to evaluate cytoskeleton and
molecular motor turnover through gene expression and to allow a comparative evaluation of how
pigment translocation systems have been conserved or have diverged among the different crusta-
cean taxa. Future molecular studies should thus contemplate a wider diversity of crustacean species
than investigated to present because most physiological and pharmacological studies have been
limited to just a few species of crab, shrimp, and crayfish, within a handful of genera.

CONCLUSIONS

We examined rapid color change in the Caridea and Brachyura from the standpoint of the physi-
ological, cellular, and molecular mechanisms that regulate pigment granule movements within
the chromatophores. We also analyzed pigmentary effector organization and revisited the char-
acterization of the pigment-aggregating and -dispersing chromatophorotropins. Genetic coding
 Adaptive Color Change and the Molecular Endocrinology of Pigment Translocation 95

for these peptide neurohormones and their homology and conservation were discussed in the
light of evolutionary relationships, notably of the Insecta, with the Crustacea. We analyzed puta-
tive mechanisms of signal transduction, focusing on receptor type and function, and the cascades
that employ both intra- and extracellular calcium and cyclic nucleotide second-messengers to
regulate the molecular motor activity that leads to pigment granule translocation. We examined
the structural and physiological evidence showing a role for cytoskeletal molecular motors like
myosin, kinesin, and dynein, putatively responsible for granule translocation, and their regula-
tion by phosphorylation-dephosphorylation mechanisms brought about by protein kinases and
phosphatases.
We conclude that necessary avenues for future investigation should include establishing a chro-
matophore cell line that will enable the use of electrophysiological, biochemical, and molecular bio-
logical techniques with individual cells, pure cell suspensions, or purified chromatophore proteins
and membranes. Greater knowledge of the tissues of origin and the migration pathways of chro-
matophores during embryogenesis is necessary. Identification of the membrane receptors involved
in chromatophorotropin signal transduction is essential to clarify early events leading to pigment
migration, and examination of homologies with other arthropod hormone receptors requires
molecular analysis. Cytoskeleton dynamics, particularly actin distribution, together with its associ-
ated pigment-translocating molecular motors, also require study. Calcium movements during pig-
ment translocation using intracellular fluorochromes should be examined. Pigment dispersion in
the caridean shrimps is little explored and physiological/pharmacological investigations are neces-
sary to reveal the receptor type, signaling cascades, and molecular motors involved. Molecular and
phylogenetic methodologies are necessary to provide a comparative evaluation of how pigment
translocation systems have been conserved or have diverged among the different crustacean taxa.

ACKNOWLEDGMENTS

We gratefully acknowledge past and ongoing financial support from the Fundação de Amparo
à Pesquisa do Estado de São Paulo (FAPESP), the Conselho Nacional de Desenvolvimento
Tecnológico e Científico (CNPq), and the Coordenadoria de Aperfeiçoamento de Pessoal de
Nível Superior (CAPES) in the form of research grants and scholarships that have financed this
line of investigation over the years. We also thank the Centro de Biologia Marinha (CEBIMAR),
Universidade de São Paulo for continued logistical support, and we are most grateful to Prof. Roy
Edward Larson and Dr.  Munira Muhammad Abdel Baqui (Departamento de Biologia Celular,
FMRP, USP) for essential support in preparing material for and performing confocal microscopy.

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 4
MUSCLE STRUCTURE, FIBER TYPES, AND PHYSIOLOGY

Scott Medler and Donald L. Mykles

Abstract
Crustacean muscles are striated muscles exhibiting a wide range of structural characteristics and
physiological capabilities. Slow fibers possess relatively wide sarcomeres and produce slow sus-
tained contractions used for diverse biological functions. Fast fibers possess narrow sarcomeres
and generate the power needed for quick movements and bursts of locomotion. Differences in
contractile rates are determined by sarcomere width and by alternate myosin heavy chain (MHC)
isoforms. Crustacean fibers exhibit thin-filament regulation of muscle contraction and possess
different isoforms of troponins and tropomyosin that influence the kinetics of muscle activation.
Crustacean muscles commonly contain fibers that do not fit neatly into one category, but lie along
a continuum of fiber types. The sarcolemma of crustacean fibers is often folded into clefts that
penetrate deeply into the fiber interior. These clefts are lined with mitochondria and presumably
facilitate diffusional exchange across the membrane in large fibers.

INTRODUCTION

Crustacean skeletal muscles have provided a rich area of research for decades, and a number of
compelling reasons exist for studying crustacean muscles. For crustacean biologists, skeletal mus-
cles are integral components of many key processes, making their biology relevant to researchers
with a wide range of interests. Growth and molting are essential features of crustacean life history,
and these complex processes must be coordinated with skeletal muscle atrophy and subsequent
growth. Many crustaceans are highly energetic and mobile animals, and any studies of locomotion
or other types of performance must incorporate an understanding of muscle function. Crustaceans,
principally decapods, are also important components of the aquaculture and commercial seafood

103
104 Scott Medler and Donald L. Mykles

industries that represent hundreds of millions of dollars of economic value annually, so an interest
in their skeletal muscles is not completely academic (Oesterling 2012).
For those interested in basic skeletal muscle structure and function, crustacean muscles have
provided a number of unique models for comparison with the more intensively studied vertebrate
skeletal muscles. Beginning in the mid-1960s, Ashley and colleagues performed a series of experi-
ments in which they injected the calcium-sensitive jellyfish fluorescent protein aqueorin into the
giant muscle fibers of barnacles (Ashley and Ridgeway 1970). When the muscles were stimulated
to contract, the aqueorin emitted light as calcium was released within the muscle to elicit contrac-
tion. These were some of the first experiments demonstrating the inextricable connection between
Ca2+ ions and muscle contraction. Not quite a decade later, Fred Lang and colleagues embarked on
a series of studies focused on the developmental changes in muscle fiber type that occur in juvenile
lobster claws (Lang et al. 1977a,b, Govind and Lang 1977). These studies showed that fibers could be
completely remodeled from fast to slow (and vice versa) and provided one of the first examples of
invertebrate muscle plasticity (see Chapter 5 in this volume). More recently, Steven Kinsey’s labora-
tory has used the exceptionally large muscle fibers of crabs to examine the diffusional limitations in
muscle fibers more generally (Kinsey et al. 2007, Hardy et al. 2009, Kinsey et al. 2011; see Chapter 12
in this volume). These studies have provided insights not only into the constraints of cell dimensions
on basic physiologic function in crustacean muscles, but also help explain how these limitations have
affected the evolution of skeletal muscle design more broadly. Collectively, these studies represent
just some of the many insights gained from studying these diverse and fascinating skeletal muscles.
In this chapter, we provide a general overview of crustacean muscle structure and physiological
function. We begin by providing some examples of the varied and complex roles crustacean mus-
cles play in an animal’s basic biology and life history. Next, we focus on crustacean muscle structure,
from the whole-muscle level down to fiber ultrastructure. We then focus on the classification of
crustacean muscles into discrete fiber types, with a particular focus on the alternate isoforms of
myofibrillar proteins that help define these fiber types. Although the current classification system
is limited to just a few species, it is clear that a significant amount of overlap exists even among these
well-defined fiber types. Finally, we conclude the chapter with a discussion of crustacean muscle
physiology. Throughout the chapter, some natural overlap in subject areas is unavoidable. For
example, it is impossible to adequately discuss muscle fiber types without covering the structural
feature of sarcomere width or physiological parameters like shortening velocity. Although we have
attempted to minimize this type of repetition, some degree of redundancy should be anticipated.

MUSCLE STRUCTURE

Overview

Crustacean muscles come in a variety of sizes, structural organizations, and even subtle colors.
Each of these characteristics can be related to the functional roles of specific muscles, and crusta-
cean muscles are highly diverse both in terms of their structural organization and function. Like all
skeletal muscles, crustacean muscles are made up of a few to hundreds of individual muscle cells
or fibers. These fibers are invariably anchored to the exoskeleton at their origin and are attached
to a moveable connection point at their insertion. In certain instances, the insertion point is a
well-defined tendon, called the apodeme, as is the case in the opener and closer muscles of claws.
In others, muscles may insert directly onto a different region of the exoskeleton, as occurs with the
extensor and flexor muscles of the tail in lobsters and crayfish. Although many crustacean muscles
are organized into bipinnate structures with a central apodeme, the precise architecture of diverse
muscles varies considerably.
 Muscle Structure, Fiber Types, and Physiology 105

The wide range in functional organization of crustacean muscles reflects their diverse special-
ized functional roles. A muscle’s functional organization is closely integrated with the exoskeleton
to which it is coupled. In some cases, the joints of crustaceans are formed by simple hinges (Warner
and Jones 1976, Schenk and Wainwright 2001), whereas others represent highly complex structures
with specialized regions of calcification in the exoskeleton that provide essential mechanical prop-
erties (Patek et al. 2004, 2007). Several well-studied musculoskeletal systems demonstrate the close
coupling between the structure of the exoskeleton and the functional organization of the associated
musculature. We discuss three of these systems here, to illustrate the diversity in muscle organiza-
tion resulting from the functional requirements of the muscles.
The claws of many crustaceans exhibit significant dimorphism in which one claw is more heav-
ily built. The closer muscles of both claws exhibit morphological and cellular adaptations that pro-
duce a slow forceful closure of the major claw and a more rapid closure of the minor claw. The major
claw, designated the crusher in clawed lobsters, has a greater mechanical advantage than its partner,
owing to the construction of the dactyl and the insertion point of the closer muscle on the apodeme
(Warner and Jones 1976, Costello and Lang 1979, Schenk and Wainwright 2001). In addition to hav-
ing an architecture that facilitates a forceful claw, all of the fibers that comprise the lobster crusher
closer muscle are long-sarcomered slow fibers (Lang et al. 1977a). The slender cutter claw in the
lobster has a mechanical advantage that favors rapid claw closure, and its closer muscle is built from
approximately 65% fast muscle fibers (Lang 1977, see Chapter 5 in this volume).
The first thoracic appendages of mantis shrimp (order Stomatopoda) are specialized into rap-
torial or hammer-like structures used for predation and defense (Patek et  al. 2004, 2007). The
velocities generated during the rapid strike from these appendages are among the highest in the
animal kingdom and exceed speeds that can be actively generated by contracting skeletal muscles
(Burrows 1969, Patek et  al. 2004). The function of the appendages has aptly been compared to
the operation of a crossbow, in which the energy input to draw back the bow is exceedingly slow
in comparison to the rapid release of energy that ensues when the trigger is activated (Patek et al.
2007). In the mantis shrimp, most of the exoskeleton of the merus is very thin and supple, but
specialized regions of calcification in the distal portion of the appendage provide structures that
function to both store energy and to effectively “cock” the structure (Patek et al. 2007). Although
the precise mechanism of energy storage is not well understood, it is clear that a series of calcified
ridges in the distal part of the merus are essential to the storage of potential energy generated by
the contracture of the extensor muscles (Patek et al. 2007, Zack et al. 2009). The primary muscles
used to generate tension for a strike are two large extensors of the carpus that insert onto two calci-
fied sclerites that function as a “click-joint” to lock the carpus in a fully flexed position (Burrows
1969, Burrows and Hoyle 1972, McNeill et al. 1972). Two smaller carpus flexors are used to lock
the “cocked” appendage in place for a short period before it is released (Burrows and Hoyle 1972).
Each of these muscles is a slow contracting muscle, with ultrastructural and physiological features
common to other slow muscles in crustaceans (McNeill et al. 1972). A similar mechanism is used
by snapping shrimp (family Alpheidae) to generate their loud snapping sounds (Ritzmann 1974,
Versluis et al. 2000; see Chapter 5 in this volume).
In crabs of the family Portunidae, the fifth pereopod has become specialized into a broad pad-
dle that is used as a swimming appendage (Hartnoll 1971, Spirito 1972). These animals are capable
of sustained swimming at speeds of up to 1 meter/s and are highly maneuverable (Spirito 1972).
The muscles used to generate power for swimming are complex, in terms of both the anatomical
arrangement of the muscles and of their fiber composition (White and Spirito 1973, Tse et al. 1983).
Swimming is powered by the interaction of four sets of muscles that receive separate innervation
and are responsible for distinct physiological activities. White muscles power short-term escape
responses, whereas pink-colored muscles drive sustained swimming for prolonged periods. Both
types of fibers have structural and physiological features characteristic of fast muscles, but the more
106 Scott Medler and Donald L. Mykles

pigmented fibers are subdivided by membrane clefts lined with high concentrations of mitochon-
dria (Tse et al. 1983, Henry et al. 2001). The specialization of these muscles into aerobic engines
that power swimming behavior has evolved in several different species of crabs (Hardy et al. 2010).
Clearly, these muscles have evolved a phenotype that is well matched to the paddle-like appendage
necessary for swimming.
The common theme illustrated by these examples is that crustacean muscles are highly sophisti-
cated organs, precisely matched to the exoskeletal structures with which they are integrated. Some
of these systems have evolved for force production, others for explosive speed, and some for sus-
tained power output. They are highly diverse in terms of their structural, metabolic, and physi-
ological properties. Although the physiological systems of invertebrates are sometimes described
as being simple, the diversity and complexity of organization are arguably greater in crustacean
muscles than in those of vertebrates.

Ultrastructural Organization

Crustacean muscles exhibit a wide variety of structural organization at the cellular level. One of
the major differences between crustacean and vertebrate skeletal muscles fibers is that crustacean
fibers display a wide range of sarcomere widths (from 3 to 20 µm), while those of vertebrates are
uniformly short (~2.5–3 µm; Hoyle 1967, 1983). Sarcomere width has commonly been used to iden-
tify different physiological fiber types in crustaceans because long-sarcomered fibers tend to be
innervated by slow motor neurons and contract slowly. Fibers with short sarcomeres are frequently
innervated by a fast motor neuron and are fast contracting, whereas intermediate fibers exhibit
intermediate sarcomere widths and are often innervated by both fast and slow motor neurons
(Atwood 1976, Govind and Atwood 1982). Sarcomere width is not simply a descriptive correlate
of contractile phenotype but is a direct determinant of contractile strength and speed (Huxley and
Niedergerke 1954, Josephson 1975). Short-sarcomered fibers have a proportionately greater number
of these contractile units in series and thereby contract with greater speed than the fibers with lon-
ger sarcomeres. Long-sarcomered fibers generally have a greater number of myosin cross-bridges
available per sarcomere and therefore produce greater forces than do fibers with short sarcomeres.
This pattern is consistent with theoretical expectations, and contractile force is directly correlated
with sarcomere width in crustacean muscles ( Jahromi and Atwood 1969, Taylor 2000).
Sarcomere width in crustacean muscles is also correlated with other common features of myofi-
brillar organization. Short-sarcomered fibers tend to have straight and well-aligned Z lines, I bands,
and A bands. Long-sarcomered fibers often possess Z lines that appear jagged or wavy in longi-
tudinal sections, and the alignment between adjacent myofibrils is often staggered ( Jahromi and
Atwood 1969, Mykles and Skinner 1981, Mellon and Stephens 1992, West et al. 1992; Fig. 4.1). In
many cases, the H zone in the middle of the A band is well-defined in short-sarcomered fibers but
is less visible or absent in the fibers with long sarcomeres. In cross-section, short-sarcomered fibers
possess thick filaments surrounded by a highly regular array of six thin filaments ( Jahromi and
Atwood 1969, West et al. 1992). The thick filaments in the long-sarcomered fibers are surrounded
by a higher number of thin filaments (~12 or more) that are scattered around the filament without
any obvious order ( Jahromi and Atwood 1969, 1971, Mykles and Skinner 1981, West et al. 1992). The
filaments themselves are narrower in fast muscles with short sarcomeres than in slow muscles. In
both muscle types, an organized array of myosin subfilaments is thought to surround a core of para-
myosin. Those of slow muscles are composed of a greater number of these myosin subfilaments and
have a thicker core of paramyosin than those of fast muscles ( Jahromi and Atwood 1969, Chapple
1982). It is generally thought that the combination of wider thick filaments and a higher number
of thin filaments associated with slow fibers results in a greater level of generated force than in fast
fibers (Mellon and Stephens 1992, Royuela et al. 2000).
 Muscle Structure, Fiber Types, and Physiology 107

Fig. 4.1.
Structural differences between short- (A, B) and long-sarcomered (C, D) fibers from the claw closer muscles
of the Australian yabby, Cherax destructor. Longitudinal section of short-sarcomered fiber (A) shows distinct
A and I bands, and these regions of adjacent myofibrils line up in register. The H zones and M lines (middle of
H zones) in the middle of each A band are distinct. In cross-section (B), each thick filament is surrounded by six
thin filaments (enclosed in circle). Longitudinal section of long-sarcomered fiber (C) show that Z lines are less
straight, do not line up in register between adjacent myofibrils, and are not always perpendicular to the fiber axis.
The H zones and M lines are not readily apparent. In cross-section (D), the long-sarcomered fibers exhibit thick
filaments surrounded by an average of 12 thin filaments (enclosed in circle). All figures are transmission electron
micrographs (TEM), scale bars = 0.2 µm. From West et al. (1992), with permission from Springer.

Each myofibril within a muscle fiber is surrounded by a collar of membranes formed by the
sarcoplasmic reticulum (SR) that is contacted by the network of tubular membranes invaginating
from the sarcolemma (Rosenbluth 1969, Franzini-Armstrong et al. 1986, Ushio and Watabe 1993;
Fig. 4.2). The extent of the SR varies greatly, depending on the speed and frequency of contrac-
tions produced by the muscle. In slow muscles, the SR and corresponding tubular system tend to
be less developed than in fast fibers (Ushio and Watabe 1993, Lagersson 2002), although in some
cases these differences are minor ( Jahromi and Atwood 1969, Jahromi and Atwood 1971). In certain
specialized high-frequency muscles, such as the antennal remoter muscle in lobsters, the relative
proportion of SR makes up the majority of the fiber volume, and myofibrils only account for about
a quarter of the total volume (Rosenbluth 1969).
Crustacean fibers possess a well-developed internal membrane system that functions to carry
depolarizations of the sarcolemma into the muscle fiber during excitation-contraction coupling
(Peachey 1967, Selverston 1967, Franzini-Armstrong et al. 1986, Ushio and Watabe 1993). In addi-
tion to the well-known T tubules that are present in vertebrate skeletal muscles, crustacean fibers
variably also possess surface membrane clefts and Z tubules (Peachey 1967, Franzini-Armstrong
et al. 1986). The clefts of crustacean fibers are often highly developed and greatly increase the sur-
face area of the sarcolemma. Various tubular systems, running not only transversely, but also in
108 Scott Medler and Donald L. Mykles

Fig. 4.2.
Membrane systems in crustacean fibers. Longitudinal- (A) and cross-sections (B) of muscle fibers demonstrate
the membrane invaginations that form networks around myofibrils (TEM). In both figures, diads are visible
where T tubules come into contact with the terminal cisternae of the SR (D in (A); T and arrows in (B)). Other
abbreviations: (A) Z tubule, ZT; M line, M; Z line, Z; (B) nonjunctional sarcoplasmic reticulum, nSR; terminal
cisternae, TC. Three-dimensional reconstruction (C) of the membrane systems in crustacean fibers demon-
strates the relationships among membrane clefts, C, T tubules (“A tubules,” TA), SR, and the terminal cisternae
(“dilated cisternae,” DR). T tubules arise as invaginations of the membrane clefts (C). Other abbreviations in
(C): Z tubules, TZ; A band, A; Z line, Z. (A) From Stokes and Josephson (1992), with permission from Springer;
(B) from Ushio and Watabe (1993), with permission from Wiley and Sons, Inc.; (C) from Peachey (1967), with
permission from Oxford University Press. Scale bars in A and B = 0.5 µm; scale bar in C = 2 µm.

longitudinal and oblique directions, arise from these clefts (Hoyle 1983). Of these, Z tubules pen-
etrate into fibers at the level of the Z line. The T tubules are defined by the fact that they form
junctions with the SR and are integral to the process of excitation-contraction coupling, although
some junctions with the SR are also formed directly with the clefts themselves. The T tubules form
flattened cisternae that are directly opposed to the SR at these positions, where they form dyads and
triads (Peachey 1967, Franzini-Armstrong et al. 1986). Fast muscles tend to have a greater density
of these connections than slow muscles. In some slow fibers, T tubules regularly penetrate into the
fiber at the level of the outer borders of the A bands, but, overall, there is no regular placement of
 Muscle Structure, Fiber Types, and Physiology 109

these tubules. Particularly in fast muscles, the T tubules may penetrate the fiber in different loca-
tions ( Jahromi and Atwood 1969, Rosenbluth 1969, Franzini-Armstrong et  al. 1986, Stokes and
Josephson 1992). Franzini-Armstrong et al. (1986) reported that the Z tubules are distinct from the
T-tubule system, although continuities between the systems do exist. The Z tubules do not appar-
ently form junctions with the SR, and their function is poorly understood ( Jahromi and Atwood
1971, Franzini-Armstrong et al. 1986).
Ultrastructural studies have provided evidence of foot processes that connect the T tubule
to the SR at dyadic and triadic junctions that are similar to those observed in vertebrate muscles
(Fig. 4.2A,B; see Mellon and Stephens 1992). We now know that these feet are ryanodine recep-
tors (RyR) responsible for coupling depolarization of the T tubule with intracellular Ca2+ release
by the SR (Franzini-Armstrong and Protasi 1997, Di Biase and Franzini-Armstrong 2005). In ver-
tebrate skeletal muscles, the ryanodine receptors are physically coupled to the dihydropyridine
receptors (DHPR) embedded in the T-tubule membranes and function together as Ca2+ release
units. In the muscles of crustaceans and other invertebrates, the ryanodine receptors serve a
similar role in excitation-contraction coupling, but there is not a direct association with the
DHPRs (Di Biase and Franzini-Armstrong 2005; and see the section “Excitation-Contraction
Coupling”).

MUSCLE FIBER TYPES

Overview

The use of specialized muscle cells to generate contraction and movement is a defining charac-
teristic of the Animal Kingdom. All animals, from creeping worms to the fastest vertebrates, rely
on the same fundamental processes of muscle contraction to power their movements. Myosin
motor proteins are organized into thick filaments that interdigitate with thin filaments of actin
and produce force when the myosin motor pulls on the actin filaments. Within this general
scheme exists a diverse array of specific levels of muscle organization (Hoyle 1967, Hoyle 1983,
Paniagua et al. 1996). In smooth muscles, thick and thin filaments interdigitate with one another,
but the spatial organization of the thick and thin filaments within the muscle cell is not well
defined. In cross-striated muscles, thick and thin filaments are organized into alternating A bands
and I bands along the fiber length, which produces the characteristic repeated banding pattern
of these muscles (Fig. 4.1). Obliquely striated muscles are similar to striated muscles in having
thick and thin filaments organized into well-defined regions, but the angle of these regions is
much less than the right angles observed in striated muscles. Broadly, different animals possess
a continuum of muscle fiber types ranging from smooth muscles, to obliquely striated, to stri-
ated fibers (Hoyle 1983, Paniagua et al. 1996). In most animal taxa, including the vertebrates and
mollusks, multiple types of muscles are present within an organism and even within the same
muscle (Paniagua et  al. 1996, Royuela et  al. 2000). Among the arthropods, all of the muscles
are cross-striated, including the muscles of the heart and other visceral organs (Mellon 1992,
Paniagua et al. 1996).
Diverse muscles within the Animal Kingdom also differ with respect to their mechanisms of
muscle activation (Lehman and Svent-Gyorgyi 1975, Svent-Gyorgyi 1975, Royuela et  al. 2000,
Hooper et  al. 2008). At rest, all muscles possess mechanisms that prohibit the myosin heads of
the thick filaments from interacting with the thin filament actins. In the muscles of all animals,
an increase in intracellular Ca2+ is required to initiate the force-producing interaction between
actin and myosin (Szent-Gyorgyi 1975, Hooper et al. 2008). In some muscles, an inhibitory state
of the myosin motor must be removed to initiate muscle contraction (thick filament regulation). In
110 Scott Medler and Donald L. Mykles

molluskan muscles and vertebrate smooth muscles, a light chain of myosin serves as the regulator
of muscle contraction (Szent-Gyorgyi 1975, Hooper et  al. 2008, Himmel et  al. 2009). In others,
troponin and tropomyosin proteins associated with the actin filaments effectively inhibit muscle
contraction by blocking the myosin binding site on the actin filaments (thin filament regulation;
Szent-Gyorgyi 1975, Royuela et al. 2000, Hooper et al. 2008). As with the structural organization
of muscles, animals broadly possess a whole range of mechanisms used for muscle activation.
Many animals possess both thick filament-regulated and thin filament-regulated muscle fiber types
(Lehman and Svent-Gyorgyi 1975, Szent-Gyorgyi 1975, Royuela et al. 2000, Hooper et al. 2008).
Arthropods, including crustaceans, rely primarily or exclusively on thin filament regulation during
muscle activation. However, there is evidence that some crustacean slow muscles possess dually
regulated systems (Lehman and Svent-Gyorgyi 1975, Szent-Gyorgyi 1975, Royuela et  al. 2000,
Hooper et al. 2008). Crustacean muscles are therefore very similar to vertebrate skeletal muscles
with respect to their cross-striated organization and in predominantly possessing thin filament
regulation of actomyosin activation.

General Classification of Muscle Fiber Types

Striated skeletal muscles are composed of populations of individual cells, or muscle fibers,
which represent the cellular basis of muscle contraction. In all animals, distinct populations
of muscle fibers are present, providing specialization of contractile function for differing
mechanical requirements (Rome et  al. 1988, Rome and Lindstedt 1997). Fast-contracting
muscles are needed for bursts of power, whereas slower muscles are used for activities that
require more prolonged periods of sustained force generation. Metabolic properties of dif-
ferent fiber types are often matched with shortening speed, with faster muscles tending to
be less aerobic and mainly relying on glycolysis and intracellular phosphagens to fuel muscle
contraction (Rome and Lindstedt 1997). Within these general parameters, specific muscle
fiber types from diverse species exhibit a wide range of contractile and metabolic proper-
ties (Rome and Lindstedt 1997). Crustacean muscle fibers are as diverse as those of any ani-
mal group. Slow fibers often control appendages and body regions where forces need to be
maintained over a period of time, but rapid contraction is not a requirement. These include
fibers of claw opener and closer muscles and many of the superficial muscles of the abdomen
( Jahromi and Atwood 1969, Ogonowski and Lang 1979, Mykles 1988, Fowler and Neil 1992,
Neil et al. 1993, Sohn et al. 2000, Medler et al. 2004). Fast fibers are important in muscles that
power rapid locomotion, such as the deep abdominal muscles in lobsters and crayfish, as well
as in the leg muscles of running crabs ( Jahromi and Atwood 1969, Ogonowski and Lang 1979,
Mykles 1985a, Li and Mykles 1990, Cotton and Mykles 1993, Medler and Mykles 2003, Perry
et al. 2009). Fast fibers are also found in the cutter claws of lobsters and the pincer claws of
snapping shrimp (Mellon and Stephens 1978, O’Connor et al. 1982, Govind 1987). Although
slow fibers tend to be more aerobic than fast fibers, this is not a strict correlation, and there
are many examples of fast aerobic fibers characterized by high mitochondrial densities (see
the section “Aerobic Capacity”). Some muscles are specialized to generate not only fast con-
tractions, but have also evolved to produce muscle twitches at high contractile frequencies
(Fahrenbach 1963, Rosenbluth 1969, Stokes and Josephson 1992, Josephson and Stokes 1994).
These muscles exhibit specializations that include high densities of T-tubule systems and SR
necessary to produce rapid increases and decreases in Ca2+ concentrations that trigger muscle
activation and relaxation, respectively.
Over the years, skeletal muscle biologists have been aware of differences in muscle fiber
types and have attempted to classify these fiber types into some logical framework. Initially,
muscles were grouped simply into “red” versus “white” fiber types based on their superficial
 Muscle Structure, Fiber Types, and Physiology 111

appearance. In other cases, physiological measurements allowed different fibers to be identi-

fied based on their contractile properties as either “fast” or “slow.” The development of histo-
chemical assays using frozen muscle sections, particularly myofibrillar ATPase histochemistry,
allowed for the identification of several different fiber types (Brooke 1970, Barnard et  al.
1971). These techniques led to the classification of mammalian fiber into three fundamen-
tal groups: slow (I), fast glycolytic (IIB), and fast oxidative (IIA). Subsequently, the myosin
heavy chain (MHC) motors responsible for generating contraction in mammalian muscles
were identified (Schiaffino and Reggiani 1996, 2011). These different isoforms are encoded by
several distinct genes, and the specific MHC isoform(s) expressed in single fibers has become
the standard for classifying muscle fiber types (Schiaffino and Reggiani 1996, 2011, Pette and
Staron 2000, 2001). Different MHC isoforms within mammalian fibers are now identified
either through labeling muscle sections with monoclonal antibodies directed against specific
MHC isoforms or through single-fiber sodium dodecyl sulfate polyacrylamide electrophoresis
(SDS-PAGE) analysis (Booth et al. 2010, Pandorf et al. 2010).
Each of these approaches has been used to distinguish crustacean muscle fiber types. In
crustaceans, fast-contracting glycolytic fibers are often large in diameter and appear either
translucent or pearly white in coloration. Fast and slow fibers with varying degrees of aero-
bic capacity range from light brown or tan in coloration, to varying shades of pink or red.
Histochemical procedures adapted from those used to identify mammalian muscle fiber
types have been used to successfully distinguish crustacean fibers (Ogonowski and Lang
1979, Ogonowski et al. 1980, Silverman and Charlton 1980, Tse et al. 1983, Maier et al. 1984,
Rathmayer and Maier 1987, Mykles 1988, Gunzel et al. 1993). These techniques stain crusta-
cean fast muscles dark brown to black but leave slow fibers relatively unstained. Preincubating
tissue sections with buffers of different pH reverses the staining reaction and reveals a range
of intermediate fiber types. This range of fiber types identified through histochemical meth-
ods can be directly correlated with the physiological properties and innervation patterns of
single fibers (Rathmayer and Maier 1987). It is reasonable to assume that these staining dif-
ferences are directly correlated with the expression of distinct MHC isoforms present within
different fibers, as is the case for mammalian fiber types (Staron and Pette 1986, Staron and
Hikida 1992). This pattern has been confirmed in at least one study of crustacean muscles
(Neil et al. 1993). Several different MHC isoforms in a limited number of species have been
identified using single-fiber SDS-PAGE analysis (LaFramboise 2000, Medler and Mykles
2003, Medler et al. 2004, Perry et al. 2009). A better understanding of the number and types
of crustacean MHC isoforms is needed to objectively classify these fiber types with a clas-
sification scheme similar to that used for mammalian fibers. Another common method used
to classify crustacean fiber types is sarcomere width, which roughly varies from 2.5 to 20
µm. This range of sarcomere dimensions is starkly different from mammalian fibers, which
have evolved to a constant width of ~2.5 µm in fast and slow fiber types alike (Hoyle 1983).
Crustacean fast fibers are constructed from narrow sarcomeres (2.5–4 µm) and slow fibers
from long sarcomeres (12–20 µm), whereas many fibers possess sarcomeres of intermediate
width (Atwood 1976, Govind and Atwood 1982).
A pattern commonly observed in different crustacean muscles is that specialized fiber types
are anatomically segregated within the same muscle. As a general rule, the more aerobic and
slower fiber types tend to be localized closer to the joint at the most proximal and distal regions
of the muscle (Mykles et  al. 2002, Medler and Mykles 2003, Perry et  al. 2009). In the closer
muscle of the cutter claw of lobsters and in the pincer of snapping shrimp, the muscle has a cen-
tral band of fast muscle fibers surrounded by slow fibers (Ogonowski et al. 1980, Govind 1987).
These different fiber types provide for a range of muscle contraction rates for distinct types of
claw movements.
112 Scott Medler and Donald L. Mykles

Identified Fiber Types in Lobster Muscles

Distinct muscle fiber types identified by specific MHC and other myofibrillar isoforms have
revealed a level of diversity and complexity that could not be detected using histochemical tech-
niques alone. The most comprehensive understanding of myofibrillar isoforms in crustacean
muscle is from the American lobster, Homarus americanus, where alternate isoforms have been
identified for MHC, myosin light chains (MLCs), paramyosin, tropomyosin, troponin T, troponin
I, troponin C, and actin (Fig. 4.3, Tables 4.1 and 4.2). Here, we discuss the current understanding
of these myofibrillar isoforms and what is known from other crustacean species for comparison.

A a b c d e f
DA CCT VCT DCT CR SF
S2 MHC
F/S1 MHC

B a b c d e f
kDa
- MHC
112 - P1
P2
81 - - P75

49.9 - ] TnT
-A
- Tm
36.2 -

29.9 - ] TnI

21.3 -

C kDa a b c d e f
112 -
81 - - P75

49.9 -
] TnT
36.2 -

29.9 -
] TnI

21.3 -
F F S1 S2 S1 S2

Fig. 4.3.
Myofibrillar protein assemblages in several muscles of the adult lobster Homarus americanus. Myosin heavy
chain isoforms are shown in (A). Silver-stained gel of multiple myofibrillar proteins are shown in (B).
Composite Western blot of P75, TnT isoforms, and TnI isoforms are shown in (C). Multiple isoforms are
expressed for many of the myofibrillar proteins, and specific fiber types are characterized by unique combina-
tions of these isoforms. Muscle fibers: deep abdominal (DA), central cutter closer (CCT), ventral cutter closer
(VCT), distal cutter closer (DCT), crusher (CR), superficial flexor (SF). These fibers can be classified as fast
(F), slow twitch (S1), or slow tonic (S2). Abbreviations: myosin heavy chain, MHC; paramyosin, P; 75 kDa
protein, P75; troponin T, TnT; actin, A; tropomyosin, Tm; troponin I, TnI. From Medler and Mykles (2003),
with permission from The Company of Biologists, Inc.
 Muscle Structure, Fiber Types, and Physiology 113

Table 4.1.  Myofibrillar protein isoforms in fiber types of the American lobster Homarus
americanus. Actin isoforms have been identified from nucleotide sequences alone.
Isoforms of MHC, Tm, and TnC have been identified at both the protein and nucleotide
levels. Isoforms of paramyosin, P75, TnT, TnI, and MLCs have been identified at the
protein level using SDS-PAGE.

Protein Fiber Type

Fast Slow Twitch (S1) Slow Tonic (S2)

MHC Fast S1 S2
Paramyosin P1>>P2 P2 P2
P75 + - -
TnT T2 T3>>T2 T1, T3
Actin SK4>5>3 (CT) SK1>2
SK8>5>>7 (DA)
Tm Fast S1 S2
TnI I1>I2>I4>I5>I3 (CT) I4>I2 (CR) I2>I4 (CR)
I1,I5>I3>I2 (DA) I3>I2>I4 (SA) I2>I3 (SA)
TnC C3 (CT) C1, C3
C2 (DA)
MLC (alpha) LC2>>LC1 LC2>>LC1>LC3 LC2>>LC1
MLC (beta) LC1 (CT) LC1 LC1
LC2 (DA)

Abbreviations: CT, cutter claw closer; CR, crusher claw closer, DA, deep abdominal muscle; SA, superficial abdominal muscle.
Table compiled from Mykles 1985a, Mykles 1985b, Mykles 1988, Li and Mykles 1990, Cotton and Mykles 1993, Mykles et al. 1998,
Medler and Mykles 2003, Koenders et al. 2004, Kim et al. 2009, Chao et al. 2010. Abbreviations for proteins: myosin heavy chain,
MHC; 75 kDa protein, P75; troponin T, TnT; tropomyosin, Tm; troponin I, TnI; troponin C, TnC; myosin light chain, MLC.

MHC exists as at least three isoforms designated fast, slow twitch (S1), and slow tonic (S2) in
lobster muscles (Li and Mykles 1990, Cotton and Mykles 1993, Medler and Mykles 2003, Medler
et al. 2004, 2007). These isoforms have been identified at the protein level using SDS-PAGE analy-
sis (Medler and Mykles 2003, Medler et al. 2004; Fig. 4.3, Table 4.1) and the 3′ terminal sequences
that encode the carboxy-terminal rod region of each isoform has been cloned (Cotton and Mykles
1993, Medler and Mykles 2003, Medler et  al. 2004; Table 4.2). The overall sequence similarity
among the identified sequences is approximately 80% within the open reading frame, and each
isoform has a distinct 3′ untranslated region (UTR), which suggests that the alternate isoforms
may be encoded by distinct genes. The fast MHC is found within several different muscles, includ-
ing the closer muscles of the cutter claw and within the deep extensor and flexor muscles of the
abdomen. The S1 MHC is expressed in various slow muscles, including those of the claw openers
and within the closer muscles of the crusher claw. This isoform is also expressed to varying levels
within the more superficial postural muscles of the abdomen. The S2 MHC isoform is expressed
in muscles that appear to correspond to the physiologically identified slow tonic fibers. These
fibers are frequently located within the proximal and distal regions of muscles near joints, and they
are likely used to maintain muscle contractions over a period of time. Within the claw closers, S2
fibers are found in a distal bundle of fibers, and the S1 isoform is frequently co-expressed to varying
degrees within single fibers (Medler and Mykles 2003). A similar pattern of co-expression at both
Table 4.2.  Myofibrillar nucleotide sequences of the American lobster Homarus america-
nus. References: a(Cotton and Mykles 1993), b(Chao et al. 2010), c(Garone et al. 1991),
d
(Kim et al. 2009), e(Koenders et al. 2002), f(Medler and Mykles 2003), g(Medler et al.
2004), h(Mykles et al. 1998).

Protein Isoform Alternative Sequence Information GenBank Ref

ID Accession #
MHC Fast Partial cds (1529 bp) C-term U03091.1 a
S1 Partial cds (1795) bp C-term AY232598.1 f
S2 Partial cds (813) bp C-term AY521626 g
Paramyosin EST (317 bp) GO271460.1
P75 Partial cds (766 bp) AY302591.1 f
TnT – – – –
Actin SK1 Complete cds (1386 bp) FJ217207 d
α actin AF399872 e
SK2 Complete cds (1395 bp) FJ217208 d
SK3 Complete cds (1224 bp) FJ217209 d
SK4 Complete cds (1248 bp) FJ217210 d
SK5 Complete cds (1295 bp) FJ217211 d
SK6 Complete cds (1243 bp) FJ217212 d
SK7 Complete cds (1276 bp) FJ217213 d
SK8 Complete cds (1245 bp) d
Tm Fast Complete cds (896 bp) AF034954.1 h
S1 Complete cds (2,223 bp) AF034953.1 h
S2 Complete cds (1526 bp) AY521627 g
TnI EST (657 bp) FD699253.1
EST (660 bp) FD467672.1
TnC TnC1 Complete cds (814 bp) FJ790218 b
aa sequence P29289 c
TnC2a Complete cds (639 bp) FJ790219 b
aa sequence P29290 c
TnC2b Complete cds (2094 bp) FJ790220 b
aa sequence P29291 c
TnC2b′′ Complete cds (2136 bp) FJ790221 b
TnC3 Complete cds (1046 bp) HM448422 b
TnC4′ Complete cds (1667 bp) FJ790223 b
TnC4′′ Complete cds (842 bp) FJ790222 b
TnC4′′′ Partial cds (563 bp) FJ790225 b
TnC6 Complete cds (2439 bp) GQ259153 b
TnC6x Complete cds (2171 bp) GQ259154 b
MLC EST (695 bp) FE044128.1
EST (194 bp) GO271581.1

Abbreviations: aa, amino acid; cds, coding sequence; C-term, C-terminal and 3′ untranslated region; EST, expressed sequence
tag; EST identified sequences are only included where published sequences are lacking. Other ESTs encoding H. americanus
MHC, actin, Tm, TnI, and TnC exist in the GenBank database.
 Muscle Structure, Fiber Types, and Physiology 115

the protein and mRNA levels is observed within the superficial extensor and flexor muscles of the
abdomen (Medler et al. 2004). In these single fibers, the S1 and S2 isoforms are expressed in vary-
ing levels, forming a continuum from the “pure” S1 and “pure” S2 fibers. Co-expression of multiple
MHCs within single fibers is common in many lobster muscles, even within fibers traditionally
classified as either fast or slow.
Six complete MHC sequences have recently been identified from the abdominal muscles of
three shrimp species, and these represent the first full-length MHC sequences from crustaceans
(Koyama et al. 2012a,b, 2013). Based on sequence comparisons, these represent two different fast
MHC isoforms (MHC1 and MHC2) expressed in deep abdominal muscles of the shrimp (Koyama
et al. 2012a,b, 2013). In addition, several partial sequences from adult pleopod muscles and from
developing shrimp muscles have also been identified (Koyama et al. 2013). One of the adult pleo-
pod MHCs exhibits sequence similarities suggesting homology with lobster S2 MHC (Koyama
et al. 2013). In crayfish muscles there are 3–4 different myosin isoforms expressed within differ-
ent muscles, but their correspondence to the lobster isoforms has not been determined (Sakurai
et  al. 1996, LaFramboise 2000). Perry et  al. (2009) identified three MHC isoforms distributed
among distinct fiber types within the carpus extensor and flexor muscle of the ghost crab Ocypode
quadrata. The 3′ terminal coding sequences and UTRs were also cloned from three distinct MHC
isoforms. The similarity among these sequences, and in comparison to those from the lobster, was
approximately 80%, but there was no clear correspondence between the crab and lobster MHCs,
thus indicating that these may not represent homologous genes. In the large anaerobic fibers from
the ghost crab muscles, two MHC isoforms (MHC1 and MHC3) were always expressed in approx-
imately 50:50 proportions. More aerobic fibers near the proximal and distal ends of the muscle
expressed a distinct isoform (MHC2), usually as a single isoform but sometimes with one of the
isoforms from the fast fibers.
MLCs exist in lobster muscles as 21–23 kDa (α) and 18–18.5 kDa (β) proteins. Three α and two β
MLC isoforms are expressed to varying degrees in different lobster fiber types (Table 4.1). A similar
pattern occurs in crayfish muscles, where each protein (α and β) is expressed as a fast and slow iso-
form. In addition, a third 31 kDa MLC is expressed in slow muscles (Sakurai et al. 1996).
Many of the other nonmyosin proteins are also present as multiple isoforms, and their expres-
sion largely mirrors that of the MHC isoforms. Many of these isoforms were identified decades
ago using SDS-PAGE gels (Costello and Govind 1984, Mykles 1985a,b, 1988). More recently, a sig-
nificant degree of progress has been made in identifying the gene sequences and tissue-specific
expression patterns of these different isoforms. Paramyosin is a large (~105–110 kDa) protein that
forms the core of thick filaments in many different invertebrate muscles, and two isoforms of this
protein have been identified at the protein level (Table 4.1; Mykles 1985a). In the lobster, fast cut-
ter and deep abdominal muscles preferentially express the larger P1 isoform, whereas slow crusher
claw and superficial abdominal muscles exclusively express the smaller P2 isoform (Mykles 1985a).
Little information is currently available about the gene sequences or mRNA distribution of these
paramyosin isoforms, although a putative sequence has been identified as an expressed sequence
tag (Table 4.2).
Tropomyosin is formed as a coiled-coil dimeric protein and is coupled to the troponin proteins
(TnI, TnT, and TnC) to form the Ca2+-sensitive “switch” in thin-filament regulation of muscle con-
traction (Hooper and Thuma 2005). In the relaxed state, tropomyosin (Tm) lies in the groove of
the actin filament and physically prevents MHC from binding to the actin and generating muscle
contraction. When activated, intracellular Ca2+ concentrations rise, the Ca2+ ions bind to troponin
C, and a conformational change takes place in the troponin/tropomyosin complex that moves the
tropomyosin away from the myosin binding sites on the actin filament. Therefore, it is anticipated
that alternate isoforms of any of these proteins might affect the steepness of the force–Ca2+ relation-
ship and thereby influence the sensitivity of muscle activation. Each of the proteins that operate as
116 Scott Medler and Donald L. Mykles

a component of this switch (tropomyosin, troponin T, troponin I, and troponin C) exists as mul-
tiple isoforms in lobster muscles (Tables 4.1 and 4.2). Tropomyosin exists as three known isoforms,
designated as fast, S1, and S2, following the MHC nomenclature (Mykles et al. 1998, Medler et al.
2004; Tables 4.1 and 4.2). Each isoform is encoded by a single gene, and specific isoforms are gener-
ated through alternative splicing (Mykles et al. 1998, Medler et al. 2004). Three skeletal muscle Tm
isoforms are also present in different muscles of the spiny lobster Panulirus japonicus and appear to
correspond to the isoforms in the American lobster (Ishimoda-Takagi et al. 1997). In addition, an
isoform specific to the heart muscle is also present (Ishimoda-Takagi et al. 1997).
Troponin T also exists as three different isoforms, designated simply as TnT1, TnT2, and TnT3 in
the order of migration on SDS-PAGE gels (T1 < T2 < T3). TnT2 is preferentially expressed within the
fast muscles, TnT3 in slow twitch (S1) muscles, and TnT1 is found specifically within the slow tonic
(S2) fibers (Table 4.1). We do not currently have information about the gene sequences encoding
the TnT isoforms in lobster muscles.
Troponin I exists as five different isoforms, with multiple isoforms frequently being expressed
within single fibers. During the juvenile stages of muscle differentiation in lobsters, single fibers
express several isoforms, but the patterns of expression become more limited as the lobsters reach
adulthood (Medler et al. 2007). In fully differentiated fast fibers of the cutter claw, the predominant
isoform is TnI1, whereas in the S1 fibers of the crusher TnI4 is the major isoform (Mykles 1985a,
Medler et al. 2007). Adult S1 fibers in the abdominal muscles predominantly express TnI3 with some
levels of TnI4; S2 fibers primarily express TnI2, whereas fast fibers express TnI1 in combination with
other isoforms (Mykles 1985a, Medler et al. 2004). Many fibers exhibiting phenotypes interme-
diate to the S1 and S2 fiber types express varying levels of the TnI isoforms (Medler et al. 2004).
Information about the genes encoding the different TnI isoforms is currently lacking, being limited
to partial sequences identified as expressed sequence tags (Table 4.2).
Troponin C is the Ca2+ binding protein that functions as the Ca2+-sensitive switch in thin
filament-regulated muscles. In lobster muscles, three isoforms of TnC have been identified at the
protein level from lobster claw and abdominal muscles using SDS-PAGE analysis (Mykles 1985a),
and three have been identified through protein purification and amino acid sequencing of isoforms
expressed in the abdominal muscles (Garone et  al. 1991; Tables 4.1 and 4.2). However, a recent
study has revealed a much greater level of complexity than has previously been anticipated. cDNA
sequences for 11 different TnC isoforms have now been identified from lobster tissues, with 6–8 of
these being predominantly or exclusively expressed within the skeletal muscles (Chao et al. 2010;
Table 4.2). The 11 different isoforms are encoded by seven different genes, with several isoforms
being generated by alternative splicing of the same gene (Chao et al. 2010). Three of the isoforms
identified by their nucleotide sequences identified by Chao et al. (2010) corresponded to those
previously identified by Garone et al. (1991).
Until recently, the protein actin that forms the backbone of the thin filament was known to exist
as a single isoform in lobster skeletal muscles. However, new data have shown that at least 12 actin
isoforms are expressed within various lobster tissues, eight of which are primarily or exclusively
expressed within the skeletal muscles (Kim et al. 2009; Table 4.2). These different isoforms are the
products of distinct genes, and the expression of specific isoforms is muscle-specific (Kim et al.
2009). In the land crab Gecarcinus lateralis, several actin isoforms are present (9–15, encoded by
7–11 genes), but their tissue-specific expression patterns are not known (Varadaraj et al. 1996). In
Artemia, 8–10 actin genes are present, and four have been cloned (Macias and Sastre 1990). Similar
patterns of actin expression are observed in crustaceans from other taxonomic groups, but the func-
tional significance of this diversity is poorly understood (Hooper and Thuma 2005). In Drosophila
spp., six different actin isoforms are expressed, four being specific to skeletal muscles (Fyrberg et al.
1998, Lovato et al. 2001, Hooper and Thuma 2005). These isoforms are selectively expressed in dif-
ferent muscles (Lovato et al. 2001) and have been shown to possess nonequivalent physiological
 Muscle Structure, Fiber Types, and Physiology 117

functions (Fyrberg et al. 1998). These patterns suggest that different actin isoforms confer subtly
different physiological properties to muscles with different functions (Fyrberg et al. 1998, Lovato
et al. 2001, Hooper and Thuma 2005).
The multiplicity of myofibrillar isoforms present in crustacean skeletal muscles suggests that
the precise contractile properties of the muscle are determined by the specific combination of
myofibrillar isoforms within a fiber. In vertebrate muscle fibers, it is well established that muscle
shortening velocity is determined directly by the MHC isoform(s) expressed. Alternate isoforms of
MHC generally provide for a range of shortening velocities, with the fastest isoforms being roughly
5–10 times greater in their velocities than the slowest (Schiaffino and Reggiani 1996, Reggiani et al.
2000). In lobster muscles, histochemical staining clearly shows that muscles possessing the fast
MHC hydrolyze adenosine triphosphate (ATP) at higher rates than the slow S1 MHC (Ogonowski
and Lang 1979, Ogonowski et al. 1980). ATPase activity measured from isolated myofibrillar pro-
teins indicates that the lobster fast MHC hydrolyzes ATP at approximately 2–5 times the rate of
the slow S1 MHC (Mykles 1985a), and ATP hydrolysis rate is directly correlated with the speed of
muscle shortening (Schiaffino and Reggiani 1996). Histochemical analysis indicates that the slow
S2 MHC is even slower than the S1 isoform (Mykles 1988, Fowler and Neil 1992, Neil et al. 1993),
and mechanical measurements from the Norway lobster Nephrops norvegicus are consistent with
this interpretation (Holmes et al. 1999). In many single fibers in lobster muscles, multiple MHC
isoforms are expressed (Medler and Mykles 2003, Medler et al. 2004). These fibers are known as
“hybrid” fibers and are often interpreted to be transitional fibers, caught in the process of switching
from one phenotype to another (Pette and Staron 2000). More recently, it has become clear that
hybrid fibers are common components of many normal muscles, in which MHC coexpression is
often the rule rather than the exception (Stephenson 2001, Caiozzo et al. 2003). In lobster muscles,
a significant level of coexpression of different MHC isoforms is present at both the mRNA and
protein levels (Medler and Mykles 2003, Medler et al. 2004, 2007). In the slow superficial muscles
of the abdomen, a continuum exists between pure S1 and S2 fibers in terms of MHC expression
and other myofibrillar isoforms as well (Medler et al. 2004). In the leg muscles of the ghost crab
O. quadrata, three different MHC isoforms are present, and single anaerobic fast fibers typically
express MHC1 and MHC3 in approximately 50:50 proportions (Perry et al. 2009). The physiologi-
cal significance of MHC co-expression is not completely understood, but in mammalian muscles,
hybrid fibers possess contractile properties intermediate to the pure fiber types (Reiser et al. 1985,
Larsson and Moss 1993, Bottinelli et al. 1996). This suggests that blending of two or more MHCs
within single fibers may provide for a continuum of contractile properties.
Alternate isoforms of myofibrillar proteins other than MHC also likely contribute to functional
differences among fibers, but their role is even less well understood than that of the MHC isoforms.
In principle, alternate isoforms of the thin filament regulatory proteins (tropomyosin, TnI, TnT, and
TnC) should affect the sensitivity of muscle activation to Ca2+ concentration. Consistent with this
expectation, alternate isoforms of TnI in two populations of fast fiber in the yabby, Cherax destruc-
tor, affect the steepness of the Ca2+–force curve (Koenders et al. 2004). The population of fibers
with the greater Ca2+-sensitivity also has slightly shorter sarcomeres, which is also consistent with
faster muscle contraction. In running ghost crabs, size-dependent differences exist in the relative
proportions of TnI and TnT isoforms that may be related to operational frequency during running
(Perry et al. 2009). In dragonfly flight muscles, alternatively spliced variants of TnT significantly
influence muscle power output and flight performance (Fitzhugh and Marden 1997, Marden et al.
1999, Marden et al. 2001, Marden and Allen 2002). Collectively, these trends suggest that the relative
proportions of the thin filament regulatory proteins may influence the kinetics of muscle activa-
tion and deactivation. However, it is possible that some of the diversity in myofibrillar isoforms
may simply represent functional redundancies or vestiges of past functional specialization. It is sur-
prising, for example, that so many distinct isoforms of actin are expressed within lobster tissues
118 Scott Medler and Donald L. Mykles

(Kim et al. 2009). Actin is generally viewed as being a passive participant in muscle contraction, and
a functional role for different actin isoforms would be an unexpected finding. Further studies link-
ing the myofibrillar isoform assemblage with the physiological properties of muscles are needed to
reveal how specific isoforms affect muscle function.

Crustacean Muscle Proteins as Allergens

One discipline that has provided unexpected insights into our knowledge of crustacean myofibril-
lar proteins is the field of seafood allergen research. Crustaceans and mollusks, collectively referred
to as shellfish, represent a major proportion of all seafood consumed worldwide (Lehrer et al. 2003,
Lopata and Lehrer 2009). A significant number of individuals within the population exhibit allergic
reactions to these foods, and, in some cases, the allergic reactions prove to be fatal. Several proteins
from crustacean muscles have been identified as allergens, including arginine kinase, SR Ca2+ bind-
ing protein, and tropomyosin (Lopata and Lehrer 2009). Of these, tropomyosin has by far been
the most consistently identified as a major allergen from multiple species of crustaceans, as well
as from mollusks (Reese et al. 1999, Ayuso et al. 2002, Lehrer et al. 2003). Tropomyosin is the only
major shrimp allergen, and more than 84% of the total IgE antibodies in shrimp-allergic patients are
directed against this protein (Lehrer et al. 2003). In addition to allergic reactions caused from con-
suming or coming in contact with crustacean muscles, tropomyosins from other arthropods (cock-
roaches and house mites) can cause allergic responses as well (Lehrer et al. 2003). The tropomyosin
amino acid sequence is highly conserved among crustaceans, thus providing several common anti-
genic sites among different tropomyosin isoforms (Motoyama et al. 2007, Suma et al. 2007).

PHYSIOLOGICAL PROPERTIES OF CRUSTACEAN MUSCLES

Nerve–Muscle Interactions

The initial identification of physiological fiber types in crustacean muscles was made independently
from the molecular and biochemical determination of muscle fiber types. Several comprehensive
reviews of the principles of neuromuscular organization and physiology of crustacean muscles have
been published (Atwood 1976, Govind and Atwood 1982, Hoyle 1983, Govind 1987, Govind 1995,
Millar and Atwood 2004). These topics are also covered in greater depth in the chapter by Atwood
(see Chapter 4 in volume 3), so they will be covered only briefly here. Crustacean muscle innerva-
tion patterns significantly complicate the relationship between the physiological properties of dif-
ferent fiber types because a single fiber may be controlled by anywhere from one to five excitatory
motor neurons. In addition, many fibers are also affected by an inhibitory motor neuron that can
modulate muscle contraction.
Single-fiber analyses of the claw closer muscle in the crab Eriphia spinifrons provide an example
of the complexities that exist between muscle phenotype and innervation patterns (Rathmayer and
Maier 1987; Fig. 4.4). Four different fiber types are present in this muscle, as identified through
histochemical, electrophysiological, and enzymatic properties. The fibers are variably innervated
by two excitatory motor neurons, one fast and one slow, as well as by a common inhibitory neuron.
Type I fibers are classified as slow oxidative. They exhibit low ATPase activities, slow contractions,
and are innervated by all three motor neurons. Type II and type III fibers are both classified as fast
oxidative glycolytic and exhibit fast contractions and high ATPase activities. The two fiber types
also possess moderate to high levels of glycolytic and oxidative enzymes. However, type II fibers
receive innervation from all three motor neurons, whereas type III fibers exclusively receive the
fast motor neuron. The largest, type IV fibers are fast glycolytic. They exhibit fast contraction, high
 Muscle Structure, Fiber Types, and Physiology 119

FCE CI SCE

7
8 6 5
9 4

3
2

200 µm

I II III IV
Fiber group

Fig. 4.4.
Schematic diagram of four fiber types present in the leg closer of the crab Eriphia spinifrons. Fiber types are
identified using combined histochemical, electrophysiological, and biochemical analyses. Three motor neu-
rons variably innervate the different fibers:  a fast excitatory motor neuron (FCE), a slow excitatory motor
neuron (SCE), and a common inhibitory neuron (CI). Type I fibers are classified as slow oxidative and are
controlled by all three neurons. Type II fibers are fast oxidative/glycolytic and are also controlled by all three
neurons. Type III fibers are fast oxidative/glycolytic and are controlled only by the FCE. Type IV fibers are fast
glycolytic and are also controlled exclusively by the FCE. From Rathmayer and Maier (1987), with permission
from Oxford University Press.

ATPase activities, and high levels of glycolytic enzymatic activity, but low oxidative capacity. Like
the type III fibers, these fibers are controlled exclusively by the fast motor neuron. These patterns
illustrate the principle that crustacean muscle physiology is not determined by fiber type alone, but
by potentially complex interactions between the activity of different motor neurons and the cellular
and molecular composition of different individual fibers. When contrasted with the organization of
mammalian skeletal muscle and motor nerves, some general differences are apparent in crustacean
neuromuscular systems. Crustacean muscles tend to have relatively few motor neurons that supply
a single muscle, but the number of contacts along a single muscle is greater than in mammalian
muscles, which only have a single synapse per fiber (Hoyle 1983, Belanger 2005).
In addition to neurotransmitters released at neuromuscular junctions, crustacean skeletal
muscles respond to a number of different neuromodulatory peptides (Kreissl et al. 1999, Mercier
et al. 2003, Weiss et al. 2003). These compounds are thought to be released into the circulation
from various sources, and they affect different physiological systems including the heart and cir-
culation, digestive system, and skeletal muscles. Their precise role in relation to skeletal muscle
function is not completely understood, but we do know that both excitatory and inhibitory pep-
tides exist. Proctolin and FMRFamide-like peptides tend to potentiate muscle contraction, whereas
120 Scott Medler and Donald L. Mykles

allatostatins act in an inhibitory capacity (Kreissl et al. 1999, Mercier et al. 2003, Weiss et al. 2003).
These effects are exerted through both presynaptic mechanisms and directly on the muscle itself
to modulate muscle contractility. There is some evidence that these compounds influence skel-
etal muscle contractile characteristics through selective phosphorylation of myofibrillar proteins
(Brüstle et al. 2001).

Excitation-Contraction Coupling

Overall, the process of excitation-contraction coupling in crustacean muscles appears to be

most similar to that in vertebrate cardiac muscles and other invertebrate muscles (Ashley
et al. 1993, Palade and Györke 1993, Lea 1996, Quinn et al. 1998, Weiss et al. 2001, Takekura and
Franzini-Armstrong 2002). A depolarization of the sarcolemma is carried along the tubular sys-
tem into the muscle fiber to the dyadic and triadic junctions between the tubule and the enlarged
cisternae of the SR. Although the threshold potential needed to initiate contraction is variable
among muscles, for most crustacean fibers, the resting potential is more negative than threshold.
In muscles that exhibit all-or-none contractions, the threshold is approximately 20–30 mV more
positive than the resting potential. In tonic fibers that produce graded contractions, the threshold
is closer to the resting potential, and the amount of tension developed is proportional to the level
of depolarization (Chapple 1982). Unlike mammalian muscle fibers, the activating depolarization
is primarily carried by the inward current of Ca2+ ions, rather than by Na+ (Ashley et  al. 1993,
Ushio et al. 1993, Weiss et al. 2001). L-type Ca2+ channels within the tubular membranes open,
and the inward flux of Ca2+ can then activate TnC to initiate contraction, but Ca2+ ions also bind
to ryanodine (RyR) receptors present in the SR membrane and initiate the release of stored Ca2+
(Weiss et al. 2001). This represents a process of Ca2+-induced Ca2+ release (CICR), which is the
principal mechanism of Ca2+ release occurring in the muscles of invertebrates and lower verte-
brates (Palade and Györke 1993, Lea 1996, Quinn et al. 1998, Weiss et al. 2001). The threshold pCa
for CICR through the RyR in the SR of lobster muscles is approximately 6.0–6.4 (Lea 1996, Quinn
et al. 1998). The degree to which Ca2+ influx from the extracellular fluid versus that released from
the SR initiates muscle contraction probably varies among different muscles, but, in most fibers,
CICR appears to play an essential role (Palade and Györke 1993, Ushio et al. 1993, Lea 1996, Quinn
et al. 1998, Weiss et al. 2001).
In most vertebrate skeletal muscles, the depolarization of the T tubule triggers a conforma-
tional change in the dihydropyridine sensitive L-type Ca2+ channels (DHPRs), and a direct
mechanical coupling between this protein and the RyR leads to opening of the RyR on the SR
membrane (Franzini-Armstrong and Protasi 1997, Endo 2009). In these muscles, the DHPR func-
tions primarily as a voltage sensor rather than a Ca2+ channel, and the process is not CICR, being
instead a direct coupling between depolarization of the T-tubule membrane and opening of the
RyR of the SR (Franzini-Armstrong and Protasi 1997). This is seen as a more advanced form of
excitation-contraction coupling and apparently evolved early in the evolution of vertebrates (Di
Biase and Franzini-Armstrong 2005). This direct coupling depends, in part, on a physical coupling
between the DHPRs within the T-tubule membrane and the RyRs on the SR (Franzini-Armstrong
and Protasi 1997, Takekura and Franzini-Armstrong 2002, Di Biase and Franzini-Armstrong 2005).
In vertebrates, each RyR is associated with four DHPRs arranged into a square pattern, where each
DHPR is attached to one of the four subunits of the RyR (Di Biase and Franzini-Armstrong 2005).
Invertebrate muscles, including those of crustaceans, lack the highly ordered arrays of DHPRs, and
there is no evidence of a close association between these Ca2+ channels and the RyRs (Loesser et al.
1992, Takekura and Franzini-Armstrong 2002, Di Biase and Franzini-Armstrong 2005). This lack
of direct coupling between these two molecules supports the view that CICR, rather than direct
coupling, provides the mechanism to link depolarization with muscle contraction.
 Muscle Structure, Fiber Types, and Physiology 121

RyRs have been isolated from crustacean muscles and studied in isolated vesicles, as well as
in intact myofibrillar bundles (Formelova et al. 1990, Seok et al. 1992, Lea 1996, Quinn et al. 1998,
Xiong et al. 1998). Each RyR is composed of four approximately 5,000 amino acid subunits, the
same as those found in vertebrate skeletal muscles (Franzini-Armstrong and Protasi 1997, Xiong
et al. 1998). Like the RyRs in vertebrates, these channels release Ca2+ in response to Ca2+ concentra-
tions in the micromolar range, but they are inhibited by Ca2+ in the millimollar range (Quinn et al.
1998, Xiong et al. 1998). Two EF-hand domains are present on each RyR subunit, but it is currently
unclear whether these Ca2+-binding sites function in channel activation or inhibition (Xiong et al.
1998). The evoked Ca2+ currents from lobster RyRs are only about half those of mammalian RyRs
(Quinn et al. 1998).
In vertebrates, multiple RyR isoforms are expressed in a tissue-specific manner. In mammals,
RyR1 is the principal isoform expressed in skeletal muscles, RyR2 in cardiac muscle, and RyR3 in the
brain (Franzini-Armstrong and Protasi 1997). In nonmammalian vertebrates, two isoforms, RyRα
and RyRβ, are expressed and are homologous to mammalian isoforms RyR1 and RyR3, respectively
(Franzini-Armstrong and Protasi 1997). Physiological studies of different muscles in the Australian
yabby are consistent with the presence of two different RyR isoforms (Launikonis and Stephenson
2000), but there has yet to be an identification made of multiple crustacean isoforms.

Mechanical Properties

Multiple parameters define the functional performance of skeletal muscles. These include the
mechanical properties of muscle stress (force/cross-sectional area) and shortening velocity (muscle
lengths per second; L/s). Maximal muscle force is proportional to the physiological cross-sectional
area of a muscle, whereas muscle stress is largely determined by sarcomere width or, more precisely,
A-band width (Huxley and Niedergerke 1954, Josephson 1975, Taylor 2000). When compared
with muscles from a wide range of animals representing different phyla, some crustacean muscles
are capable of generating the greatest forces known for any animal (Medler 2002). For example,
a variety of muscles from mammals, birds, and other vertebrates generate stresses in the range of
150–200 kN/m2. By comparison, a number of crustacean muscles produce maximal stresses ranging
from 400 to 2,000 kN/m2 or greater (Taylor 2000, Medler 2002). These trends arise from differ-
ences in the anatomical arrangement of the sarcomeres because muscle force is proportional to
the sarcomere width whereas shortening velocity is proportional to the number of sarcomeres in
series (Huxley and Niedergerke 1954, Josephson 1975, Taylor 2000). This relationship represents a
tradeoff between muscle strength and muscle speed, which has resulted in a diverse range of fiber
types in crustacean muscles adapted for different uses. In vertebrate muscles, by comparison, the
stress generated by different muscles is nearly constant, but muscles’ shortening velocities vary over
orders of magnitude.
Muscle shortening velocities (typically reported as maximum unloaded shortening velocity
or Vmax) for various crustacean muscles have not been studied as extensively as muscle forces,
but the available data suggest that these values are similar to if somewhat slower than those of
vertebrate muscles. In the broadest comparison among skeletal muscles representing different
phyla, maximal shortening velocity ranges from less than 1 to 25 muscle L/s, which appears to
represent an upper limit for shortening velocity ( Josephson 1993). Keeping in mind that some
level of variability exists among measurement parameters and approaches used in different
studies, crustacean muscles exhibit shortening velocities comparable to the muscles of other
active animals, including vertebrates. The muscles from the barnacle Balanus nubilus have very
low rates of contraction (Vmax = 0.15 L/s), but produce stresses of up to 600 kN/m2. These fibers
possess long sarcomeres (~9 µm) but also have low rates of ATP hydrolysis (Griffiths et  al.
1990). Slow fibers in the Norway lobster have maximal shortening velocities of about 0.5 L/s
122 Scott Medler and Donald L. Mykles

(Holmes et al. 1999), whereas the flagellum abductor muscle that drives rhythmically active fla-
gella in crabs has a Vmax of 7.6 L/s (Stokes and Josephson 1994). The extensor and flexor carpus
muscles of running ghost crabs were estimated to be capable of maximal shortening velocities
of approximately 5–7 L/s (Perry et al. 2009). By comparison, the limb muscles of a comparably
sized mouse have shortening velocities that range from 6 L/s (soleus) to 14 L/s (extensor digi-
torum longus); see Askew and Marsh (1997).

Aerobic Capacity

Crustacean muscles exhibit a range of aerobic capacities, from fibers that possess very few aero-
bic adaptations to those that are rich in mitochondria and have adaptations that facilitate oxy-
gen exchange (Silverman and Charlton 1980, Tse et al. 1983, Mykles 1988, Stokes and Josephson
1992, Boyle et al. 2003, Johnson et al. 2004, Perry et al. 2009, Hardy et al. 2010). Generally, slow
fibers are more aerobic than fast fibers, and slow tonic fibers are more aerobic than slow twitch
fibers (Ogonowski and Lang 1979, Lang et  al. 1980, Mykles 1988, Fowler and Neil 1992, Neil
1993). However, just as with vertebrate muscles, crustacean fast fibers also exist that possess
great aerobic capacities (Silverman and Charlton 1980, Tse et  al. 1983, Stokes and Josephson
1992, Hardy et  al. 2010). Functionally, aerobic fibers are found in muscles used for slow sus-
tained contractions (Mykles 1988, Fowler and Neil 1992, Neil 1993), those that power swim-
ming and running (Tse et al. 1983, Boyle et al. 2003, Perry et al. 2009, Hardy et al. 2010), and
those used for sustained, high-frequency contractions (Silverman and Charlton 1980, Stokes
and Josephson 1992). Within single muscles, fibers located near the proximal and distal regions
tend to be more aerobic and also tend to be composed of slower fiber types (Lang 1980, Mykles
1988, Mykles et al. 2002, Perry et al. 2009).
A common pattern observed in these crustacean aerobic fibers is one in which the mitochondria
are positioned close to the sarcolemma (Fig. 4.5). In many instances, these fibers are highly subdi-
vided by the clefts that penetrate into the fiber from the outer regions, and the subdivisions are lined
by high densities of mitochondria. The subsarcolemmal distribution of mitochondria observed in
crustacean muscles is distinct from the distribution in mammalian muscle fibers, where the mito-
chondria are more evenly scattered around the myofibrils (Boyle et al. 2003, Kinsey et al. 2007).
The subdivision of individual fibers may be to facilitate exchange of oxygen and nutrients between
the hemolymph and the muscle fibers (Hardy et al. 2009). In the aerobic fibers that power swim-
ming in portunid crabs, fibers become more divided by the membrane clefts as they get larger with
growth. The result is that the larger fibers in larger crabs are more subdivided, keeping the average
width of each subdivision relatively constant (Hardy et al. 2010). We have observed a similar trend
in the aerobic fibers of ghost crabs (Medler, unpublished observations). The function of the subsar-
colemmal distribution of mitochondria is not completely clear. Although the distribution facilitates
the exchange of oxygen between the hemolymph and mitochondria, it requires phosphagens to
diffuse across a greater distance from the mitochondria to the fiber interior (Stokes and Josephson
1992, Boyle et al. 2003, Kinsey et al. 2007).
Broadly, crustacean muscle contraction is not only dependent on the contractile properties of
the muscle fiber, but also on the motor neuron that controls contraction. Therefore, fatigue resis-
tance in crustacean muscles is not simply a consequence of muscle adaptations, but is also depen-
dent on the motor neuron(s) activating the muscle. Single fibers are often controlled by both a fast
motor neuron and a slow motor neuron. The fast motor neurons elicit a greater response from the
muscle fiber, but the neurons are quickly fatiguing, whereas the activation from the slow motor
neuron generates less force, but the muscles exhibit facilitation over time (Atwood and Cooper
1996, Millar and Atwood 2004).
 Muscle Structure, Fiber Types, and Physiology 123

Fig. 4.5.
Structural features associate with aerobic and anaerobic fibers. Aerobic fibers are demonstrated by cross-section
of the flagellum abductor (FA) muscle of Carcinus maenas (A) and in proximal fibers of the extensor carpus in
Ocypode quadrata (B). Mid-region fibers of the extensor carpus in O. quadrata (C) are not highly aerobic. In
C. maenas, mitochondria (Mc) are distributed around the fiber periphery in the FA muscle, whereas myofibrils
(MI) are located more centrally. A membrane cleft (black arrow) is visible in the figure (TEM). In O. quadrata,
NADH tetrazolium reductase staining from mitochondrial enzymes reveals a similar distribution in these
fibers. In the more proximal fibers (B), high densities of mitochondria are present, and membrane clefts (white
arrows) subdivide the fibers. In the mid-region fibers (C), mitochondria are similarly restricted primarily to the
subsarcolemmal regions, but with much lower densities. Abbreviations in (A): mitochondria, Mc; glycogen, G;
myofibrillar island, MI; nucleus, N; vacuole, Va. (A) is reprinted with permission from Stokes and Josephson
(1992), with permission from Springer. (B) and (C) are from Perry et al. (2009), with permission from The
Company of Biologists, Inc. The scale bar in A = 0.5 µm.

Physiological Differences Among Identified Fiber Types

Clear physiological differences exist among the S1, S2, and fast fiber types identified in the muscles
of lobsters and other decapod crustaceans. The fast fibers of the deep abdominal flexors and exten-
sors produce force at greater rates than the slow fibers, but the maximum force is significantly less
( Jahromi and Atwood 1969, Ogonowski and Lang 1979). Muscle fibers found in the legs and claws
of the lobster show similar differences ( Jahromi and Atwood 1971). There are also more subtle
differences between the fast muscles of the abdomen and those that compose the fast closer of
the cutter claw, although these have not been well studied. The abdominal muscle fibers appear
to be a more “pure” fast fiber type, as seen by significant differences in the expression of slow
MHC isoforms. The fast muscles of the claw co-express some level of the S1 MHC isoform, but the
expression of that isoform in the abdominal musculature is nearly zero (Medler and Mykles 2003).
Additionally, the fast fibers in cutter closer and deep abdominal muscles differ in expression of TnC
124 Scott Medler and Donald L. Mykles

isoforms (Chao et al. 2010; Table 4.1). The myosin ATP hydrolysis rate, which is directly correlated
with muscle shortening velocity, is also higher in the abdominal musculature when compared to the
fast claw fibers (Mykles 1985a). As for the slow fiber types, the S1 fibers have faster rates of contrac-
tion and relaxation than those of the S2 fibers and also exhibit higher ATPase activity than the S2
fibers in histochemical staining reactions (Mykles 1988, Galler and Neil 1994, Holmes et al. 1999).
By comparison, the S2 fibers are more sensitive to activation by Ca2+, meaning that they become
active at lower Ca2+ concentrations (Galler and Neil 1994). The S2 fibers also exhibit a greater
degree of neuromuscular facilitation, providing the capability to maintain force production even
after the S1 fibers have fatigued (Mykles et al. 2002; Fig. 4.6). The classification into these discrete
fiber types is really an oversimplification because many fibers possess a phenotype intermediate to
these extremes (Costello and Govind 1983, Medler et al. 2004).
In addition to the well-defined fiber types just discussed, there are a number of different crusta-
cean fiber types that do not clearly fit into this system. Physiological studies of several of these dif-
ferent fibers provide further insight into the functionality of different fibers. Using myosin ATPase
histochemistry, Rathmayer and colleagues (Rathmayer and Maier 1987, Galler and Rathmayer
1992) identified four different fiber types in the closer muscles of the walking legs of the crab E. spi-
nifrons (Fig. 4.4). Mechanical measurements from three of these fiber types showed a gradation in
shortening velocities, with the fastest fibers being about 2–3 times faster than the slowest. Ca2+ sen-
sitivity also differed among fiber types, but there was no direct correlation with shortening velocity.
Sarcomere width was correlated with the different fiber types, with the slowest having the longest
sarcomere width (14.6 µm), the fastest having the shortest (9.6 µm), and the intermediate fiber type
having an intermediate sarcomere width (12.3 µm). Fast muscles of crayfish abdominal extensors
and flexors possess ATPase activities that are approximately 5–7 times higher than the slow muscles
of the claw opener (Sakurai et al. 1996). West et al. (1992) found that short-sarcomered (3.25 µm)
claw closer fibers in the yabby (C. destructor) had slightly higher ATPase activities than those of
the long-sarcomered fibers (8.57 µm), but these differences were not significant. Their conclusion

A B
1 a b c d e f g h i
1
Distal
2 97 -
2
3 66 -
3 4
45 -
31 -
Central
4
5

5
Proximal
6 6 F S1 S2 proximal distal
5 mV
0.12 cm
150 ms

Fig. 4.6.
Myofibrillar protein isoform expression is correlated with synaptic efficacy. Fibers of the crayfish leg opener
exhibit regional differences in excitatory postsynaptic potential (EPSP) following stimulation of the excitatory
motor neuron (A). Proximal fibers (5 and 6) exhibit the greatest short-term facilitation, the distal fibers (1 and
2) intermediate levels, and fibers of the central region (3 and 4) show the least facilitation. Western blots of TnT
isoforms from opener fibers reveal a correlation with these physiological responses (B). The most proximal (d
and e) and distal (h and i) fibers express varying levels of TnT1 (arrow) in combination with TnT3, identifying
them as slow tonic (S2) fibers. The central fibers (f and g) express only TnT3 and are slow twitch (S1). Fibers
a–c are controls (F, fast; S1, slow twitch; S2, slow tonic). From Mykles et al. (2002), with permission from The
Company of Biologists, Inc.
 Muscle Structure, Fiber Types, and Physiology 125

was that the faster contraction of the short-sarcomered fibers was due primarily to the structural
arrangement of the sarcomeres, rather than to differences in the myosin cross-bridge kinetics.
Overall, crustacean muscles comprise highly diverse fiber types, as evident from the range of
physiological, ultrastructural, and molecular compositions among fibers within and among species.
Currently, no unified system exists that can be used to systematically classify crustacean fiber types
in an unambiguous way. In extensively studied mammalian muscles, different fibers are classified
according to the MHC isoform(s) that they express, and the genes encoding these isoforms have
been fully characterized (Schiaffino and Reggiani 1996, Schiaffino and Reggiani 2011). The most
precise classification of muscle fiber types in crustaceans is for the American lobster (H. america-
nus) in which fibers are classified as either S1, S2, or fast. This system seems to be relevant for crayfish
and at least some crabs, but currently there are not enough data for MHC isoforms in different spe-
cies to know for certain. In ghost crab leg muscles, three MHC isoforms are expressed in different
ratios, but their migration pattern on SDS-PAGE gels is different from that of the lobster (Perry
et al. 2009). Similarly, several different isoforms have been identified in crayfish muscles, but their
migration pattern is different from the lobster (LaFramboise 2000). Sequence comparisons among
orthologous decapod MHCs have failed to identify precise fiber type categories among species,
and homologies of these different isoforms remain uncertain (Cotton and Mykles 1993, Medler and
Mykles 2003, Medler et al. 2004, Perry et al. 2009). Koyama et al. (2012a,b, 2013) have recently pre-
sented phylogenetic relationships among available MHC isoforms from several different species,
but these are primarily based on the partial sequences available at this time. Partial sequences are
also available for the MHC genes of marine isopods (Holmes et al. 2002, Magnay et al. 2003), but
these are for the myosin head near the 5′ end of the molecule and offer no comparison with other
known sequences. A more comprehensive dataset consisting of both full MHC sequences and cor-
responding SDS-PAGE migration patterns would be especially helpful in defining crustacean fiber
types more objectively. This type of analysis would also be indispensable for an understanding
of the evolution of crustacean fiber type diversity. Crustaceans as a group are highly diverse and
represent several distinct evolutionary histories. It could be that several different specific fiber type
classification schemes are required to accurately classify the number of distinct fiber types and the
interrelationships among these.

FUTURE DIRECTIONS

We are currently very limited in our understanding of the relationships among the diverse fiber
types that make up crustacean muscles. These limitations exist for several reasons. First, many of
the fiber types that have been identified are still only defined by descriptive parameters like fast
versus slow, red versus white, or long- versus short-sarcomered fibers. These definitions are useful
for broadly grouping fiber types but are imprecise. A second problem is that fibers have been classi-
fied using unique systems for different species, and it is unclear how the identified fiber types cor-
respond to one another. Crab fibers defined as types I–IV by Rathmayer and colleagues (Fig. 4.4)
likely correspond in some way to the S1, S2, and fast scheme of lobster muscles (Fig. 4.3), but data are
not currently available to unravel their relationships. Finally, the identification of myofibrillar pro-
tein isoforms has been limited to just a few species, and, in most instances, only partial sequences
have been identified if molecular data are available at all.
A better understanding of precisely how many different crustacean fiber types exist and how
fiber types among different species are related to one another is clearly needed. An unambiguous
fiber type scheme would provide a foundation for other studies, such as those focused on nerve–
muscle interactions, dynamic muscle function in locomotion, skeletal muscle plasticity, and other
areas related to skeletal muscle biology. We advocate using the MHC isoforms expressed in different
126 Scott Medler and Donald L. Mykles

fibers as the definitive measure of crustacean fiber type. This has been the standard for mamma-
lian skeletal muscle classification for many years and provides an objective measure of fiber type
(Schiaffino and Reggiani 2011). Although the full-length MHC sequences from shrimp (Koyama
et al. 2012a,b, 2013) are currently the only complete crustacean MHC sequences available, several
partial sequences have been published, and we should work to obtain full sequences whenever pos-
sible. When a greater number of full-length sequences are available, we will be able to establish how
many different fiber types exist and how they are related to one another.

CONCLUSIONS

Crustacean muscles are highly diverse in both their structure and physiological function. These
muscles have evolved into specialized tissues that fulfill a variety of processes, including sustained
force generation, very rapid contractions, and sustained power output for long periods of time.
Although diverse, crustacean muscles all share several unifying features. All crustacean muscles are
striated and rely principally on thin filament regulation of muscle contraction. Fast contracting fibers
possess short sarcomeres that are roughly the width of those found in vertebrate muscles (~2.5–4
µm), whereas slow fibers have longer sarcomeres of variable width (5–20 µm). A well-developed
tubular network carries membrane depolarization into the fiber interior to come in close contact
with the SR where Ca2+ is released to trigger contraction. Crustacean fibers use DHPR proteins and
RyRs to control Ca2+ release from the SR. Unlike the process in mammalian muscles, there is not
close coupling between the DHPR and ryanodine receptor, and the process in crustacean muscles
is more similar to that in other invertebrates, lower vertebrates, and mammalian cardiac muscles.
Crustacean muscles comprise a number of distinct fiber types. These are most clearly defined in
the American lobster, where the types include fast, slow twitch (S1), and slow tonic (S2) fibers. Each
fiber type can be defined by specific assemblages of myofibrillar protein isoforms. These represent
alternate forms of MHC, paramyosin, actin, tropomyosin, troponins, and MLCs. Recent studies
have found that the number and expression patterns of these isoforms are much more complex
than previously appreciated. This diversity of proteins assembled into different fibers is presumably
responsible for the range of physiological properties. Physiological responses are also dependent on
the number and pattern of innervation from excitatory and inhibitory motor neurons.

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 5
SKELETAL MUSCLE DIFFERENTIATION, GROWTH,
AND PLASTICITY

Donald L. Mykles and Scott Medler

Abstract
During embryogenesis, muscle progenitor cells fuse and form multinucleate myotubes that dif-
ferentiate into myofibers. Fibers increase in length and diameter as animals move through larval,
juvenile, and adult stages. In large fibers, infolding of the cell membrane and the concentration
of mitochondria adjacent to the cell membrane reduce diffusional distances for energy metabo-
lism. As fibers grow, nuclei are added and occupy more central locations to maintain a relatively
constant myonuclear domain. Contractile properties and size are altered by physiological condi-
tions. Transformation requires coordinated expression of fiber type-specific isoforms of myosin,
actin, paramyosin, tropomyosin, and troponin-I and -T. Muscle load and contractile frequency
change significantly as crustaceans grow; these size-related differences require cellular remodel-
ing of muscles for locomotion. Proteasome/ubiquitin-dependent and calpain-dependent proteo-
lytic systems degrade myofibrillar proteins and are upregulated in atrophic muscle. Myostatin and
mechanistic Target of Rapamycin signaling pathways control the protein turnover required for
remodeling fiber myofibril structure.

INTRODUCTION

This chapter reviews the current state of knowledge of skeletal muscle myogenesis, growth,
and plasticity. There are few modern studies of muscle specification and differentiation during
embryogenesis, and little is known about the genes that control myogenesis. Once established
in the larval stages, the muscle fibers grow incrementally in length and width after each molt.
Because the number of muscle fibers remains relatively constant, the fibers can achieve large

134
 Skeletal Muscle Differentiation, Growth, and Plasticity 135

dimensions in adults. In the American lobster (Homarus americanus), for example, fibers in the
crusher claw closer muscle can be a centimeter in length and 1–2 mm in diameter. As in other
taxa, crustacean skeletal muscles display a remarkable plasticity, and this chapter focuses on
the best-known examples. Transformation of fibers from one phenotype to another occurs in
the American lobster, snapping shrimp, and Christmas Island red crab. A molt-induced atrophy
occurs in the closer muscles of large-clawed decapods, such as American lobster, fiddler crab,
crayfish, and blackback land crab. Autotomy of an appendage causes an unweighting atrophy in
the corresponding thoracic muscles of green shore crab, fiddler crab, crayfish, and blackback
land crab.
Skeletal muscle development begins in the embryo and is completed during the larval stages.
The early stages of myogenesis are similar to those in other taxa. Muscle progenitor cells arise
in the mesoderm and fuse to form multinucleate myotubes, which differentiate into striated
muscle fibers. Some crustaceans develop from a free-swimming nauplius larva, whereas in
others the nauplius stage takes place within the egg. In the free-swimming larva, propulsive
muscles develop to provide locomotion, and the adult muscles develop later (Kreissl et al. 2008,
Hertzler and Freas 2009). In taxa that develop without a free-swimming nauplius, the muscles
present in the adult are evident from the earliest stages of development (Harzsch and Kreissl
2010). In the lobster, early muscle fibers are recognized by the appearance of cells contain-
ing developing myofilaments and many myonuclei with diffuse chromatin (Kirk and Govind
1992, Govind 1995). Next, thick and thin myofilaments begin to form a regular latticework,
but a characteristic sarcomeric banding pattern is not yet recognizable. At this time, signs of
innervation, including neuromuscular terminals with clear synaptic vesicles, become evident.
Shortly after innervation, distinct myofibrils with recognizable sarcomeric structure become
visible (Govind 1995, Lang 1977). Myogenesis in developing crustacean muscles is asynchro-
nous because both long- and short-sarcomered fibers are already present in the larva, whereas
undifferentiated myoblasts are present in the same individual (Lang 1977, Jirikowski et al. 2010).
The close association between relatively undifferentiated myoblasts and the growing ends of
motoneurons suggests that myogenesis and motor neuron growth take place together in newly
forming muscles (Harzsch and Kreissl 2010).
Many adult decapods exhibit continual growth throughout their lives. Molting creates
additional space for tissue growth (Mykles 1980, Taylor and Kier 2006). Because muscle fibers
remain anchored to the new exoskeleton, stretching due to exoskeleton expansion stimulates
muscle growth during the postmolt period. This growth can be rapid. For example, in juvenile
lobsters, fibers grow to fill most of the available space in the claws by 3 days postecdysis (Medler
et al. 2007). In many crustacean species, an individual will increase in mass by several orders
of magnitude over its lifetime. This kind of indeterminate growth presents a number of physi-
ological challenges as the animal grows. In terms of skeletal muscles, several different aspects of
integrative muscle function are affected by organismal size. One of the most obvious of these is
the classical problem of strength-to-weight ratios. As an animal grows in size, its mass increases
in proportion to its linear dimensions3, whereas muscle strength increases in proportion to the
cross-sectional area of the muscles (approximately linear dimensions2; see Schmidt-Nielsen
1984). This reduction in relative muscle strength has varying levels of significance for a species
depending on whether it is aquatic or lives primarily a terrestrial existence. Lobsters and king
crabs can grow to sizes of several kilograms, but their relatively sedentary existence, where much
of their mass is supported by water, largely mitigates the impact of body mass. For a variety of
semiterrestrial crabs, increases in body size are expected to have a more significant impact. The
largest terrestrial arthropods are members of the infraorder Anomura, and these hermit crabs
can attain sizes of up to 3 kg (Greenaway 2003). However, the notion that increases in effective
136 Donald L. Mykles and Scott Medler

load constrain the operation of the musculoskeletal systems is not well supported. For example,
even large hermit crabs are sufficiently strong relative to their weight that they exhibit effec-
tive locomotor abilities, can climb trees, and can even open coconuts (Herreid and Full 1986a,
1986b, Greenaway 2003). Small hermit crabs are able to carry shells equaling their own body
mass or more without affecting running velocity (Herreid and Full 1986b). Exoskeletal strains
and the safety factor against limb buckling in running ghost crabs are comparable to values
for vertebrate bones (Blickhan et al. 1993). Moreover, performance parameters for these crabs
(stride frequency, speed at trot-to-gallop transition, muscle shortening velocity) are also simi-
lar to comparably sized mammals (Blickhan and Full 1987, Full 1987, Full and Weinstein 1992,
Blickhan et al. 1993). A more likely explanation for limitations on crustacean body size stems
from limitations in gas exchange rather than the perceived burden of carrying a heavy exoskel-
eton (Kaiser et al. 2007).
Skeletal muscle can alter its size and fiber type composition in response to a variety of physi-
ological conditions. Heterochely is common in decapod crustaceans (Mellon 1981, Govind 1992,
Mariappan et al. 2000), and species with dimorphic claws are well suited for the study of fiber
transformation. Some species, such as Callinectes sapidus, Carcinus maenas, and Menippe mer-
cenaria, exhibit a distinct “handedness,” in which the major claw is usually located on one side
(Mariappan et al. 2000). In the hermit crab Pagurus pollicaris, the crusher claw is always on the
right side and the cutter claw is always on the left side (Stephens et al. 1984). Other species, such
as H. americanus, Alpheus heterochaelis, and Nephrops norvegicus, exhibit an equal distribution of
left- and right-handed individuals (Mariappan et al. 2000). The claws of larvae are identical in
morphology and fiber type composition and then differentiate into major and minor claws dur-
ing the juvenile stage. Existing fibers of one type transform to a different type, which involves
coordinated expression of fiber type-specific genes, as well as remodeling of the contractile appa-
ratus. This is exemplified by H. americanus, in which fast fibers transform to slow fibers in the
presumptive major (crusher) claw and slow fibers transform to fast in the presumptive minor
(cutter) claw (Mykles 1997b). A similar process probably occurs in other species, in which the
major and minor claws differ in fiber type composition, such as the fiddler crab Uca pugilator and
the hermit crab P. pollicaris (Stephens et al. 1984, Govind et al. 1986, Ismail and Mykles 1992).
Fiber transformation can also occur in adults. Shifts in fiber type occur during claw reversal in
snapping shrimp (Alpheus sp.), in which the minor claw (pincer) transforms to the major claw
(snapper) when the snapper claw is lost. Fast fibers die, and the remaining slow fibers transform
to the snapper slow fiber phenotype over several molts. Claw reversal in the blue crab C. sapidus
does not result in changes in muscle fiber properties (Govind and Blundon 1985). Claw reversal
occurs in the stone crab M. mercenaria, but fiber transformation was not examined (Simonson
1985). In preparation for long-distance migration, the fibers in the walking legs of adult red crabs
(Gecarcoidea natalis) transform from a slow-twitch (S1) phenotype to a more fatigue-resistant
slow-tonic (S2) phenotype.
Species with large claws, such as H. americanus, freshwater yabby (Cherax destructor), and males
of Gecarcinus lateralis and U. pugilator, have proved to be excellent models for the study of muscle
growth and atrophy (Mykles 1997b). Fiber size is determined by the balance between the protein
synthetic and degradative rates. When synthesis exceeds degradation, fibers increase in diam-
eter (hypertrophy). Conversely, when degradation exceeds synthesis, fibers decrease in diameter
(atrophy). Two types of atrophy occur in crustaceans. Unweighting from claw or leg autotomy
causes a “disuse” atrophy of the corresponding thoracic musculature that operates the appendage.
A molt-induced atrophy of the claw closer muscle facilitates withdrawal of the claws from the old
exoskeleton at ecdysis. The net loss of protein results in a reduction of fiber diameter, whereas an
increased protein turnover is associated with remodeling of the contractile apparatus that results
from a preferential loss of thin filaments.
 Skeletal Muscle Differentiation, Growth, and Plasticity 137

MYOGENESIS AND EARLY MUSCLE DEVELOPMENT

Several recent studies of muscle development in crustaceans have shed some light onto these pro-
cesses, although much remains unknown (Kreissl et al. 2008, Hertzler and Freas 2009, Harzsch and
Kreissl 2010, Jirikowski et al. 2010). A common theme among these studies of early myogenesis is
that the process appears to follow the establishment of a founding muscle cell that serves as a point
of muscle development. These cells, termed pioneer cells after the myogenic cells in grasshopper
embryos (Ho et al. 1983), apparently migrate to specific anatomical locations to effectively “seed”
specific muscles throughout the body (Fig. 5.1). Following the initial muscle establishment by the
pioneer cells, some differences in the initial steps of myogenesis have been reported. In isopod
muscles, single pioneer cells appear to establish each primordial muscle cell (Kreissl et al. 2008),
whereas in lobsters and dendrobranchiate shrimp, muscles are established as a common syncytial
muscle precursor that then divides to form several muscles (Harzsch and Kreissl 2010). It is cur-
rently unclear whether the founding cells divide on their own to become polynucleate or whether
they act to recruit other cells that fuse with the founding cell.
Myogenesis in crustaceans shares a number of similarities with muscle development and dif-
ferentiation in Drosophila, a model organism that has provided one of the most detailed views
of muscle development. In Drosophila, muscle founder cells differentiate from cells within the
mesoderm and then migrate to specific locations within the developing embryo. These founder
cells then attract and fuse with fusion-competent myoblasts to form the earliest multinucleate

A Ib-5

A1

OP T8
MD
T5 T4 T3 T2 T1 MX2 MX1 A2
3 2 P1 T7 T6

B C stage 2 stage 3 stage 4

Ca
P-m Ca Pr
Pr
Da Pr1 Pr2
P-m
P-m
Da
T7 T6

Fig. 5.1.
Myogenesis in crustaceans. (A)  Muscle precursor cells in whole mount of a prehatching isopod embryo
(Idotea balthica). Myogenic cells are labeled (dark) with a monoclonal antibody directed against myosin heavy
chain (MHC). Abbreviations: A1, 2, antenna 1 and 2; MD, mandible; MX1, 2, maxilla 1 and 2; OP, operculum;
P1-3, pleopods 1 to 3; T1–7, thoracic limbs 1–7 (T1 is a maxilliped); and T8, thoracomere 8. Scale bar = 100
µm. Reprinted from Kreissl et  al. (2008), with permission from Springer. (B)  Primordial muscles (P-m) in
the endopodites of the thoracic limbs of embryonic lobsters (H.  americanus; embryonic stage E45%). The
developing muscles were labeled with a monoclonal antibody against MHC, and these muscle precursors will
develop into the closer muscle of these leg segments. Scale bar = 25 µm. Reprinted from Harzsch and Kreissl
(2010), with permission from Elsevier. (C) Schematic representation of myogenesis in the propodus of the
thoracic limbs of the isopod, I. balthica. Muscle founder cells (Pr1 and Pr2) are observed in stage 3 embryos.
In subsequent stages, the founder cells enlarge, become multinucleate, and subdivide into distinct subunits.
Abbreviations: Ca, carpus; Da, dactylus; and Pr, propodus. From Kreissl et al. (2008), with permission from
Springer.
138 Donald L. Mykles and Scott Medler

muscle precursor cell. The growing myofiber then elongates toward tendon cells with which
they subsequently fuse to form a muscle attachment site (Baylies et al. 1998, Schejter and Baylies
2010). The tendon cells secrete spatial cues that the growing myofibers use to seek out as they
elongate toward their future attachment sites (Schejter and Baylies 2010). The founder cell
then directs the ongoing differentiation of the developing fiber, with different fiber types being
determined by the original founder cell’s phenotype. Although our understanding of these
events in crustaceans is far from complete, the available data are consistent with a similar devel-
opmental process. The common processes between the crustacean muscle development and fly
development suggests that arthropod muscles may generally follow a pattern in which founder
cells (pioneer cells in crustaceans) migrate from undifferentiated mesoderm into a specific ana-
tomical position and then initiate fusion of undifferentiated cells and eventually form a specific
muscle fiber type. Further work in this area is clearly needed to understand crustacean muscle
differentiation more fully.
Myogenesis in all animals is directed, in part, by groups of transcription factors that orchestrate
the processes of muscle differentiation (Baylies et al. 1998, Wigmore and Evans 2002). In verte-
brate skeletal muscles, these include the basic helix-loop-helix proteins Myf 5, MyoD, Myf 4, and
myogenin, which play complementary roles in directing muscle development (Wigmore and Evans
2002). In Drosophila, they include comparable proteins like Twist and Nautilus (Baylies et al. 1998).
Although similar myogenic regulatory factors are presumably involved in crustacean myogenesis,
the specific proteins have not yet been identified in crustacean muscles.

MUSCLE GROWTH

Once specific muscles have been established during the early stages of development, muscles
continue to grow in both length and diameter. The available evidence suggests that increases in
overall muscle size in crustaceans are primarily accomplished by increases in the size of individual
fibers rather than through increased number of fibers. The hypertrophic growth that characterizes
most crustacean muscles means that fiber size may continue to increase over an animal’s lifetime.
This pattern is fundamentally different from that seen in mammals of vastly different size, in which
the size of individual fibers are essentially constant, being on the order of 25–75 µm in diameter
(Hoppeler and Fluck 2002, Liu et  al. 2009). The mechanism of increasing fiber length is either
through the addition of new sarcomeres at the ends of existing muscle fibers, by increasing the
length of sarcomeres throughout the fibers, or some combination of both mechanisms (Govind
et al. 1974, 1977, Bittner and Traut 1978, El Haj et al. 1984). In fully differentiated lobster muscles,
new sarcomeres are added to the ends of existing fibers, but these are the same width as existing sar-
comeres (El Haj et al. 1984). The addition of new sarcomeres to the ends of existing fibers is closely
linked to the process of molting, when the linear dimensions of the newly formed exoskeleton may
increase by approximately 15% (El Haj et al. 1984). Once the old exoskeleton has been shed and the
new one has expanded, fiber length increases with the addition of new sarcomeres. It may be that
the mechanical stretch provided as the newly formed exoskeleton expands is the physiological cue
that initiates fiber elongation (El Haj et al. 1984). In muscles of crayfish, increase in fiber length is
accomplished by lengthening of existing sarcomeres. In several different crayfish fibers, sarcomere
length increases by more than double in fast, short-sarcomered fibers and up to about fivefold in the
fibers of slow muscles (Bittner and Traut 1978). Increasing the length of sarcomeres throughout a
fiber would entail an ongoing process of fiber remodeling, whereas the addition of new sarcomeres
at the fiber ends would require more restricted remodeling. Altering sarcomere length will also
directly impact muscle shortening velocity, with increased sarcomere widths resulting in a slower
contracting fiber.
 Skeletal Muscle Differentiation, Growth, and Plasticity 139

One of the consequences of hypertrophic muscle growth is that diffusion-dependent processes

may become limiting (Kinsey et al. 2007, 2011). Large crustacean fibers are often many times larger
than mammalian skeletal muscle fibers, and the large size may restrict the diffusion of oxygen, intra-
cellular phosphagens, Ca2+, and other molecules. Kinsey and colleagues have considered this prob-
lem in detail and found that, in most cases, diffusion is not limiting to the metabolic processes but
may often be on the verge of being diffusion-limited (Kinsey et al. 2011). In the largest anaerobic
crustacean muscle fibers, the rate of arginine phosphate resynthesis following exercise is slower
than the rate that could become limited by diffusion (Kinsey et al. 2005). However, there are sig-
nificant structural and physiological adaptations evident in aerobic crustacean fibers that prevent
significant diffusion limitation. Aerobic muscle fibers in several crustacean muscles rely on a com-
mon mechanism to deal with diffusional limitations associated with hypertrophic growth. In these
fibers, the surface membrane of larger fibers becomes invaginated to form clefts that penetrate the
fiber center (see Chapter 4 in this volume). In some cases, the fibers become so highly subdivided
that it is difficult to determine whether the original fiber is still a single cell or whether it has com-
pletely separated into multiple fibers. The mitochondria in these fibers are highly concentrated near
the membrane invaginations, so that almost 90% of the mitochondria are found in this location.
These aerobic fibers are particularly evident in the muscles used for swimming in portunid crabs
(Hardy et al. 2010), as well in the proximal and distal regions of muscle used for running in ghost
crabs (Perry et al. 2009).
A related issue is the number and placement of the myonuclei within a muscle fiber. All skel-
etal muscle fibers are multinucleate, frequently with hundreds of distinct nuclei that originate from
multiple myoblasts during development (Baylies and Michelson 2001, Biressi et  al. 2007). Each
nucleus within a mature muscle fiber is thought to direct the expression of proteins within a limited
cytoplasmic space termed the myonuclear domain (Allen et al. 1999). In vertebrate muscles, myo-
nuclear domain size is relatively fixed. As vertebrate skeletal muscles grow hypertrophically, nuclei
are added to existing fibers to maintain a relatively constant myonuclear domain size (Allen et al.
1999). In mammals ranging over a 100,000-fold difference in body size, the myonuclear domain
increases with size but only on the order of three- to fivefold (Liu et al. 2009). In crustaceans, hyper-
trophic growth of existing muscle fibers also leads to the addition of new nuclei to the fibers to
maintain myonuclear domain size (Hardy et al. 2009, Jimenez et al. 2010, Kinsey et al. 2011). In
smaller fibers, the nuclei are restricted to the periphery of fibers as they are in mammalian fibers,
but, with additional growth, the new nuclei are distributed throughout the fiber, including the fiber
interior (Hardy et al. 2009, Kinsey et al. 2011). Overall, the myonuclear domain size in crustacean
muscles is comparable to sizes observed in mammalian muscles (~10,000–100,000 µm3 per nucleus;
Liu et al. 2009, Jimenez et al. 2010). In vertebrate muscles, the source of the new nuclei is the popu-
lation of undifferentiated myoblasts (satellite cells) that lie between the basement membrane and
the sarcolemma (Allen et al. 1999, Zammit et al. 2006). In crustacean muscles, putative satellite
cells have been identified morphologically, but their role in muscle growth has not been established
(Novotová and Uhrík 1992). In snapping shrimp claw muscles undergoing a cycle of degeneration
followed by regeneration of a new fiber type, the remnants of the degenerated fibers serve as scaf-
folding for myoblasts that differentiate into new fibers. The source of these myoblasts was specu-
lated to be either existing satellite cells or from undifferentiated blood cells that transformed into
myoblasts (Govind and Pearce 1994).
A different way in which skeletal muscles are impacted by body size is in the frequency of muscle
contraction during locomotion. A  universal pattern observed among diverse animal taxa is that
smaller animals move with higher frequencies in running, flying, and swimming (Hill 1950, Heglund
et al. 1974, Full 1997, Medler 2002). As a consequence, the skeletal muscles of smaller animals must
have faster intrinsic shortening velocities than in larger animals (Hill 1950, McMahon 1975, Medler
and Hulme 2009). The most well-defined example of this pattern is seen in mammalian skeletal
140 Donald L. Mykles and Scott Medler

muscles, where homologous isoforms of the myosin heavy chain (MHC) proteins have evolved
subtle differences that produce faster muscles in smaller species (Seow and Ford 1991, Reggiani
et al. 2000, Pellegrino et al. 2003, Marx et al. 2006). The relative proportion of fast fiber types is
greater in the muscles of smaller mammals than in the same muscles of larger animals (Goldspink
1977). In addition to faster muscle shortening, muscles operating at higher contractile frequencies
must be able to become activated and inactivated more quickly than in slower contracting muscles
(Rome and Lindstedt 1997, Rome 2006). It follows that one mechanism that has evolved to ensure
rapid activation and deactivation is a high density of sarcoplasmic reticulum (SR) within muscles
used at high frequencies. This ensures that the calcium ions that trigger muscle contraction can be
released and then sequestered quickly. The rates of muscle activation, deactivation, and operational
frequency are positively correlated with SR density in arthropod muscles, including those of crus-
taceans (Fahrenbach 1963, Josephson and Young 1987, Stokes and Josephson 1992, Lagersson 2002).
In vertebrate muscles, contractile frequency is correlated with concentrations of Ca2+-binding
proteins, such as parvalbumin, which buffer cytosolic Ca2+ and enhance muscle relaxation (Thys
et al. 2001, Rome 2006, Coughlin et al. 2007). Currently, there have not been any parvalbumins or
similar proteins identified in crustacean muscles, but it seems likely that they are present. Another
mechanism that influences the rate of muscle activation and relaxation is the expression of alter-
nate isoforms of the thin filament proteins that regulate muscle contraction. These could include
tropomyosin; troponin (Tn)-I, -T, and -C; or even actin itself. In two fast fiber types in the claws
of the freshwater yabby Cherax destructor, expression of alternate TnI isoforms is correlated with
Ca2+-sensitivity and muscle activation rate (Koenders et al. 2004). In fish muscles, shifts in con-
tractile frequency are correlated with changes in the expression of alternate TnT and TnI isoforms
( James et al. 1998, Thys et al. 1998, 2001). In dragonfly flight muscles, the expression of two differ-
ent TnT splice variants is correlated with contractile frequency and power output (Fitzhugh and
Marden 1997, Marden et al. 1999, 2001).
Crustaceans offer compelling examples of size-related changes in muscle function and organi-
zation, but it is currently unclear how a systematic shift in operational frequency affects their skel-
etal muscles. The most complete information comes from studies of semiterrestrial ghost crabs,
which are probably the most capable runners of all the crustaceans (Hafemann and Hubbard 1969,
Burrows and Hoyle 1973, Full and Weinstein 1992, Perry et al. 2009). These crabs are able to run
at top speeds in the range of 1–2 m/sec for short bursts, and the estimated contractile properties
are similar to a comparably sized mammal (Hafemann and Hubbard 1969, Burrows and Hoyle
1973, Full and Weinstein 1992, Perry et  al. 2009). Stride frequencies during maximal running
exhibit a significant correlation with body mass. The smallest ghost crabs reach stride frequencies
of approximately 20 Hz, but the larger animals top out at about 4 Hz (Burrows and Hoyle 1973,
Blickhan et al. 1993, Perry et al. 2009). Overall, this means that running crabs experience the same
scale-dependent shifts in operational frequency as a function of body size known for other kinds
of animals. Because these crabs increase in mass by several orders of magnitude over their life-
time, a relevant question is whether the intrinsic properties of the muscles change as the animals
grow. A recent study indicates that gradual shifts in the expression of alternate isoforms of MHC,
TnT, and TnI is correlated with changes in crab size. Single fibers from the leg extensor and flexor
carpopodite muscles express more MHC2, TnT1, and TnI1 in larger crabs than in smaller crabs
(Perry et al. 2009). Further work is needed to determine how these differences in myofibrillar
protein expression are related to functional differences in the muscles. Over the past two decades,
it has become clearer that a significant degree of complexity exists with respect to the number
and expression patterns of different myofibrillar isoforms in crustacean muscles (see Chapter 4 in
this volume). It is possible that the specific expression of unique combinations of these isoforms
provides for a degree of “fine-tuning” in response to the demands placed on different crustacean
 Skeletal Muscle Differentiation, Growth, and Plasticity 141

skeletal muscles. It remains to be determined what kinds of changes in muscle fiber type accom-
pany continued growth throughout the lifetime of crustaceans.

SKELETAL MUSCLE PLASTICITY

Fiber Transformation in Juvenile Lobsters

The dimorphic claws of the American lobster H. americanus differentiate from isomorphic claws
during the early juvenile stages (Govind 1984, 1992, Govind et al. 1987, Mykles 1997b). H. america-
nus has three planktonic larval (zoeal) stages separated by two molts. At the third molt, third-stage
larvae metamorphose into fourth-stage juveniles. By the fourth molt, fifth-stage juveniles assume
a benthic habitat (Herrick 1895). The claws of fourth-stage animals have identical fiber type com-
positions:  there is a central population of fast fibers and dorsal and ventral populations of slow
fibers (Lang et al. 1977, Govind and Lang 1978, Ogonowski et al. 1980). At each subsequent juvenile
molt, there is an incremental change in the muscle fiber composition and external morphology as
the claws differentiate into the cutter and crusher types (Fig. 5.2; Emmel 1908, Govind and Lang
1978). Fibers in the presumptive cutter claw closer muscle transform from the slow to the fast phe-
notype, whereas fibers in the presumptive crusher claw closer muscle transform from the fast to the
slow phenotype (Govind and Lang 1978, Costello and Lang 1979, Ogonowski et al. 1980). There is
no evidence of fiber degeneration and replacement during claw differentiation (Lang et al. 1977,
Costello and Lang 1979). Fiber transformation is completed in the cutter claw by the ninth stage,
whereas transformation in the crusher claw lags behind the cutter claw and it is not completed until
the 13th stage or later (Govind and Lang 1978, Lang et al. 1978, Ogonowski et al. 1980, Medler et al.
2007). Differentiation of the claws begins at the fifth stage, but it requires at least 15 molts over sev-
eral years before the final cutter and crusher claw morphologies of the adult are attained (Costello
and Lang 1979, Emmel 1908).
Changes in the innervation pattern and synaptic properties of fast and slow excitatory motor
neurons also occur during claw differentiation. In early juveniles (fourth, fifth, and sixth), most
fibers in both claws are innervated by both excitatory neurons (Costello et al. 1981). In adults,
this pattern is retained in the crusher claw closer muscle but not in the cutter. In the cutter, most
of the fast fibers receive only fast excitatory input, and the slow fibers receive either slow excit-
atory or both slow and fast excitatory input (Lang et al. 1980, Costello et al. 1981, Costello and
Govind 1983). In adults, the cutter fast closer excitatory (FCE) motoneuron fires at higher fre-
quencies for shorter bursts than the crusher FCE motoneuron (Lnenicka et al. 1988). Moreover,
there are differences in the response of the excitatory motoneuron cell bodies to sensory stimu-
lation; for both the FCE and slow closer excitatory (SCE) motoneurons, the cell bodies on
the crusher side in the thoracic ganglion have greater spike frequencies and longer burst peri-
ods than those on the cutter side with the same stimulation (Govind and Lang 1981). Changes
in the synaptic properties of the FCE motoneurons coincide with the changes in innervation
pattern during claw differentiation. Synaptic facilitation is similar in the symmetrical claws of
fourth-stage lobsters (Lnenicka et al. 1988). As the animals transition through the fifth, sixth,
and seventh stages, facilitation increases in the presumptive cutter claw but remains unchanged
in the presumptive crusher claw (Lnenicka et al. 1988). The changes in the pattern and synaptic
properties of the FCE motoneurons in the cutter claw contribute to the faster contraction times
needed to capture prey.
The determination of claw laterality is random and is restricted to fourth- and fifth-stage lob-
sters. In wild populations, the numbers of individuals with left-handed and right-handed crusher
142 Donald L. Mykles and Scott Medler

3rd. Stage
4th. Stage
5th. Stage
8th. Stage

Fig. 5.2.
Fiber transformation during claw differentiation in juvenile American lobster. Transverse sections stained
for myofibrillar ATPase activity show developmental changes in the closer muscle as the isomorphic claws of
larvae (third and fourth stages) differentiate into the cutter and crusher claws of adults. The central band of
dark-staining fast fibers expands in the presumptive cutter claw (left) by transformation of slow fibers to fast
fibers. Conversely, the dorsal and ventral bands of slow fibers expand in the presumptive crusher claw (right)
by transformation of fast fibers to slow fibers. The fast fibers in the crusher claw closer are completely replaced
by slow fibers by the 13th stage. Fiber transformation is restricted to the boundary between fast and slow fiber
populations. From Ogonowski et al. (1980), with permission from John Wiley and Sons.

claws are approximately equal (Herrick 1895, Emmel 1908), indicating that claw laterality is not
genetically fixed at hatching. Emmel (1908) first demonstrated that autotomy of one of the claws
during the fourth or fifth stage induces the remaining claw to differentiate into a crusher. This
“forces” the animal to use the intact claw until the next molt, when the contralateral claw regenerate
becomes functional (Lang et al. 1978). Once claw laterality is established in an individual, it remains
fixed in that individual for the rest of its life (Emmel 1908). Autotomy at larval stages or at sixth and
later stages has no effect on claw laterality (Emmel 1908, Lang et al. 1978). Autotomy of both claws
during the fourth and fifth stages can delay the critical period for determining claw laterality to the
sixth stage (Govind and Pearce 1989).
In an elegant series of experiments, Lang, Govind, and colleagues showed that the establish-
ment of claw laterality requires a minimal reflex activity involving sensory input and neuromuscu-
lar output (Govind et al. 1987, Govind 1992). The claw that receives the greater stimulation above
a minimum level of activity becomes the crusher claw (Lang et al. 1978, Govind and Kent 1982,
 Skeletal Muscle Differentiation, Growth, and Plasticity 143

Govind and Pearce 1986, 1992). When juveniles are raised in smooth-bottomed containers without
natural substratum or when juveniles receive equal mechanical stimulation through the fourth and
fifth stages, most of the animals develop two cutter claws with similar fiber compositions (Lang
et al. 1978, Govind and Kent 1982, Govind and Pearce 1986, 1992, Govind et al. 1991). Immobilization
has no effect on claw laterality, indicating that a complete reflex arc is needed to determine which
claw becomes the crusher (Lang et al. 1978, Govind and Kent 1982). Therefore, the cutter claw is
the “default” outcome; a crusher claw only differentiates when one of the claws is used more than
the other. The presence of a crusher claw prevents the contralateral claw from differentiating into
a crusher. However, “double-crusher” individuals occur rarely in wild populations (Emmel 1908).
The fiber compositions differ, even though the claws have the crusher morphology:  one of the
claws is a “false” crusher because it contains a mixture of fast and slow fibers resembling the com-
position of the cutter claw; the other claw is a “true” crusher that contains only slow fibers (Govind
and Lang 1979). This indicates that fiber transformation can be uncoupled from structural differ-
entiation, presumably from a genetic mutation that prevents repression of the crusher morphology
and produces a false crusher.
Changes in muscle protein gene expression occur during claw differentiation. The fast and
slow-twitch (S1) fibers in the differentiated claw of adult H. americanus express distinct assemblages
of myofibrillar protein isoforms: fast fibers in the cutter claw closer express fast MHC; actinSK3,
SK4, and SK5; paramyosin1 (P1); a 75 kDa protein (P75); troponin-T2 (TnT2) and troponin-T1
(TnI1), whereas S1 fibers in the crusher claw closer express S1 MHC, actinSK1 and SK2, P2, TnT3,
and TnI4 and no P75 (Costello and Govind 1984, Mykles 1985a, 1985b, 1988, Medler and Mykles
2003, Medler et al. 2007, Kim et al. 2009a; see Chapter 4 in this volume). The myofibrillar protein
composition of juvenile claw muscle differs from that of differentiated fast and slow fibers in the
adult (Fig. 5.3). Claw muscle from fourth-stage animals express P2, TnT3, and TnI4, but not P1, P75,
TnT2, or TnI1 (Costello and Govind 1984), which suggests that the fibers are not yet fully differen-
tiated. By the 10th stage, the myofibrillar protein isoform compositions of the cutter and crusher
claws resemble those of adults (Costello and Govind 1984), but it may take as long as 2 years for the
fast fibers to attain the TnI isoform composition of adults (Fig. 5.3; see Medler et al. 2007). This is
supported by analysis of MHC isoform and P75 expression by in situ hybridization and immunocy-
tochemistry, respectively, in juvenile lobsters (Fig. 5.4). In seventh-stage lobsters, P75 and fast and
slow MHCs are expressed in all fibers, although P75 and fast MHC are expressed at higher levels
in fast fibers and slow MHC is expressed at higher levels in slow fibers (Medler et al. 2007). By the
10th stage, the expression of MHC isoforms and P75 is more discrete between the fast and S1 fibers
(Medler et al. 2007). These data indicate that muscle fibers in juvenile claws are distinct from the
fibers in adults and that it may take months or years before the adult fiber phenotypes are achieved.
The mechanism by which the asymmetry in the nervous system directs the orderly transforma-
tion of fibers over a period of many molts is poorly understood. Transformation is restricted to the
boundary between the fast and slow fiber populations as fibers with intermediate histochemical
properties are localized to the boundary zone (Govind et al. 1987). Excitatory motoneuron activ-
ity probably plays a role. Chronic electrical stimulation induces changes in fiber properties to a
more slow-tonic-like phenotype in crayfish abdominal muscles (Cooper et  al. 1998, Gruhn and
Rathmayer 2002). In the limb opener muscle, synaptic properties of the excitatory motoneurons
may affect the contractile properties of the fibers they innervate; the slow-tonic (S2) phenotype is
correlated with larger excitatory postsynaptic potentials and greater short-term synaptic facilitation
(Mykles et al. 2002). However, the innervation pattern and synaptic properties do not strictly cor-
respond with fiber type (Costello et al. 1981, Lnenicka et al. 1988). This suggests that highly local-
ized interactions between fast and slow fibers are needed to restrict transformation to the boundary
zone. Further research is needed to determine whether fiber transformation is controlled through
direct contacts and/or by paracrine factors.
 A Silver-stained Gel
a b c d
kDa
250 MHC
150

100

75 P75

50
2/3 TnT
Actin
37 Tm

1 1
2 2
3/4 3/4 Tnl
5 5
Adult 10th Adult 10th
Cutter (Left) Crusher (Right)

B Tnl Western Blot

1 a b c d e f g h i j 1
2 2
3/4 3/4
5 5
L R L R L R L R L R
8th Stage 10th Stage 15 months 19 months 2.5 years

Fig. 5.3.
Differences in myofibrillar proteins between juvenile and adult lobster claw muscles. (A) Myofibrillar protein
composition of fibers from cutter and crusher claws from adult and 10th-stage juvenile lobsters (H. americanus).
Proteins were separated by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) and stained
with silver. Fast fibers from adult cutter claw express TnI1 as the predominant isoform. In contrast, fast fibers
from the central region of the presumptive cutter claw of 10th-stage juveniles express all five TnI isoforms at
comparable levels. Fast fibers in cutter claw from both developmental stages express P75. The slow fibers from
the adult crusher claw primarily express the TnI3/4 isoforms, whereas the juvenile fibers express less of the TnI3/4
isoforms. Both claws in juveniles express a TnT isoform with slower electrophoretic mobility than the TnT2 and
TnT3 isoforms in adult cutter and crusher claws, respectively. Abbreviations: MHC, myosin heavy chain; Tm,
tropomyosin; TnI, troponin-I; and TnT, troponin-T. Positions of molecular mass markers (kDa) indicated at
left. (B) Western blot of troponin-I isoforms in dimorphic claws during lobster development. The left claw of
fourth-stage larvae was autotomized, which induces the right claw to differentiate into the crusher claw; the left
claw regenerates and differentiates into the cutter claw (Govind and Pearce, 1989). Fibers from the central region
of the left claw (L; presumptive cutter) and the peripheral region of the right claw (R; presumptive crusher) of
juvenile lobsters at eighth stage, 10th stage, 15 months, and 19 months and adult lobster at 2.5 years were analyzed.
Proteins were separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with a lobster TnI
antibody. Positions of the TnI isoforms indicated at the sides. It may take 2 years for the presumptive cutter claw
to achieve the TnI composition of the mature adult. Reprinted with permission from Medler et al. (2007).

Fig. 5.4. (Continued)
the seventh molt stage, the developing crusher muscles have a distinct central region of fast fibers and dis-
tinct dorsal and ventral regions containing slow fibers (claw from 3 days postmolt animal). (B) Developing
seventh-stage cutter claws are primarily composed of fast fibers by this stage (claw from 7 days postmolt ani-
mal). The opener muscle and the ventral region of the claw closer are entirely slow. (C, D) 10th-stage claw
muscles possess similar staining patterns, but the fast and slow fiber regions are more distinct than the claws
from seventh-stage claw muscles (molt stage unknown in 10th-stage claws). This is especially evident in the
cutter claw, which completes fiber transformation before the crusher claw. Reprinted with permission from
Medler et al. (2007).
 A Crusher Claw (7th Stage)
fast RNA slow RNA anti-P75

fast

B Cutter Claw (7th Stage)

fast RNA slow RNA anti-P75
opener
(slow)

fast
slow

1 mm
C Crusher Claw (10th Stage)
fast RNA slow RNA

slow

fast

slow

D Cutter Claw (10th Stage)

fast RNA slow RNA

slow

fast

slow
1 mm

Fig. 5.4.
Differences in myosin heavy chain (MHC) isoform and P75 expression between developing crusher claw
muscles and cutter claw muscles in H. americanus. Serial cross-sections of muscles were labeled with fast MHC
RNA probe (left), S1 MHC RNA probe (middle), and anti-P75 antibody (seventh stage only) (right). (A) By
146 Donald L. Mykles and Scott Medler

Fiber Death and Transformation in Snapping Shrimp

Adult snapping, or pistol shrimps (Alpheus and Synalpheus spp.) possess dimorphic claws that dif-
fer greatly in morphology and function (Mellon 1981, 1999, Govind et al. 1987). The major claw,
designated the snapper, has an enlarged dactyl that fits into a corresponding socket in the propodus
(Fig. 5.5; Wilson 1903, Darby 1934, Knowlton and Moulton 1963). It is used for prey capture and
agonistic behaviors (Darby 1934, Conover and Miller 1978, Knowlton and Keller 1982, Herberholz
and Schmitz 1998, Mellon 1999). A  rapid release of the dactyl results in a loud popping sound
(Ritzmann 1973, Versluis et al. 2000), producing a continuous crackling noise in areas where large
populations occur ( Johnson et  al. 1947, Everest et  al. 1948, Knowlton and Moulton 1963, Potter
and Chitre 1999). The noise produced by snapping shrimps can serve as a cue for orientation and
settlement by invertebrate larvae on coral reefs (Stanley et al. 2010, Vermeij et al. 2010). The minor
claw, designated the pincer, is much smaller and is used for feeding and burrowing (Mellon 1999).
Right- and left-handed individuals occur in about equal numbers in wild populations (Wilson 1903,
Darby 1934). Autotomy of one of the claws in third- or fourth-stage juveniles results in a snapper
developing on the contralateral side (Young et al. 1994). These data indicate that claw laterality is
established randomly in early juveniles probably by a mechanism similar to that in lobsters.
Unlike lobsters, the laterality of the major and minor claws is not fixed in adults. If the snapper
claw is lost, the remaining pincer claw differentiates into a snapper claw over several subsequent

Fig. 5.5.
Claw transformation in snapping shrimp. Autotomy of the snapper claw causes the contralateral pincer claw to
differentiate into a snapper over several molts (upper panel), and the pincer regenerates at the location of the
original snapper. Myofibrillar ATPase histochemistry shows the dark-staining central band of fast fibers in the
pincer (A) degenerating after the first (B) and second (C) molts to the slow fiber composition after the third
molt (D). Over the next several molts, the slow fibers transform from the pincer phenotype with intermediate
sarcomere lengths (6–8 µm) and a 5–6:1 filament ratio to the snapper phenotype with longer sarcomere lengths
(10–15 µm) and a 7:1 filament ratio. Scale bar = 1 mm. Upper panel from Govind et al. (1987), with permission
from Oxford University Press; lower panel from Govind and Pearce (1994), with permission from Springer.
 Skeletal Muscle Differentiation, Growth, and Plasticity 147

molts, while the contralateral claw regenerates into a pincer (Wilson 1903, Mellon and Stephens
1978, Stephens and Mellon 1979). The transforming claw becomes a functional snapper after two
molts, but the final snapper morphology is not attained until eight molts after autotomy of the con-
tralateral snapper (Mellon and Stephens 1978, Stephens and Mellon 1979, Mellon and Greer 1987).
Claw reversal occurs when only the snapper claw is autotomized, and laterality is maintained when
only the pincer claw is autotomized or when both claws are autotomized (Wilson 1903, Darby 1934,
Mellon and Stephens 1978). This indicates that there is some “memory” of claw asymmetry but that
it can be overcome when the snapper claw is lost or is not functional. Transection of the nerve to the
pincer claw prevents or delays reversal (Wilson 1903, Young et al. 1996). Although not as effective
as pincer claw autotomy, either transection of the nerve, transection of the apodeme to the closer
muscle (tenotomy), or removal of the dactyl (dactylotomy) to the snapper triggers transformation
of the contralateral pincer, resulting in animals with two snappers; immobilization of the dactyl has
no effect (Mellon and Stephens 1978, Govind et al. 1988, Young et al. 1994, Read and Govind 1997a,
1997b). Maintaining animals in communal tanks increases the incidence of double-snapper indi-
viduals (Pearce and Govind 1987). These data indicate that the nervous system transmits the loss of
the snapper claw to the contralateral pincer and induces differentiation of the pincer to the snapper.
Differentiation of the pincer to the snapper involves a loss of fast fibers and transformation of
slow fibers to a longer sarcomere phenotype. The pincer main closer muscle has a central band
of fast fibers flanked dorsally and ventrally by slow fibers (Fig. 5.5), whereas the snapper contains
only slow fibers (O’Connor et al. 1982, Mearow and Govind 1986, Quigley and Mellon 1986, Pearce
and Govind 1987, Govind et al. 1988, Mellon and Quigley 1988). However, the sarcomere lengths
and thin-to-thick filament ratios of the slow fibers in the main closer muscles differ between the
two claws: the pincer slow fibers have intermediate sarcomere lengths (6–8 µm) and lower fila-
ment ratios (5–6:1) than snapper slow fibers (10–15 µm and 7:1, respectively; Stephens and Mellon
1979, Mellon and Stephens 1980, Stephens et al. 1983, Govind and Pearce 1994). This suggests that
the intermediate and long-sarcomere fibers in the main closer muscles of the pincer and snapper
claws, respectively, represent the two slow phenotypes. The snapper fibers express a larger TnT
isoform that is consistent with the S2 phenotype (Quigley and Mellon 1984, Govind et al. 1986,
Mykles 1988, Ismail and Mykles 1992), but this must be confirmed by a thorough analysis of myofi-
brillar protein isoforms. The fast fibers are completely gone by the second molt after snapper claw
autotomy (Fig. 5.5; Mearow and Govind 1986, Mellon and Quigley 1988, Quigley and Mellon 1986,
Govind and Pearce 1994, Young et al. 1996). Following fast fiber degeneration, there is a transfor-
mation of the slow fibers in the pincer main closer muscle to the longer sarcomere phenotype in
the snapper, which is completed within eight molts after autotomy of the snapper (Stephens and
Mellon 1979, Mellon and Stephens 1980, Govind and Pearce 1994). The remodeling of the fibers
coincides with the downregulation of pincer myofibrillar protein isoforms (e.g., pincer TnT and
fast myosin light chain-2) and the upregulation of snapper myofibrillar proteins (e.g., snapper TnT
and slow myosin light chain-2; Quigley and Mellon 1984, Mellon and Quigley 1988). Transection
of the nerve to the transforming claw within 2 days after autotomy of the contralateral snapper pre-
vents fast fiber degeneration even though claw morphological changes proceed normally (Mellon
and Quigley 1988).
Little is known about the mechanisms controlling the changes in fiber type composition dur-
ing transformation of the pincer to the snapper. The rapid degeneration of the fast fibers after the
first molt is reminiscent of the programmed cell death in the abdominal intersegmental muscles
(ISMs) of the hawkmoth Manduca sexta. At the end of pupation, contraction of the ISMs splits
the pupal case, which allows the adult to escape. After the adult emerges, the ISMs are no lon-
ger needed, and the fibers completely degenerate by 30 h after emergence (Schwartz 2008). ISM
cell death is triggered by a decline in hemolymph ecdysteroid (molting hormone) titer (Schwartz
2008). Fast fiber degeneration in the pincer claw occurs when ecdysteroid titers are low, which
148 Donald L. Mykles and Scott Medler

suggests that a decrease in hemolymph ecdysteroid also triggers programmed cell death in snapping
shrimp. Whatever the mechanism, it must explain why fast fibers die while slow fibers do not. The
transformation of slow fibers from the pincer phenotype to the snapper phenotype has not been
investigated in any detail. Gross motoneuron distribution does not appear to determine the fiber
phenotype because the innervation patterns of fast and slow excitatory motoneurons are similar
between the pincer and snapper claw closer muscles, and the patterns do not change during trans-
formation in A. californiensis (Mellon and Stephens 1979, Stephens et al. 1983). Moreover, synaptic
properties are correlated with claw type rather than with fiber type; synapses at fast and intermedi-
ate fibers in the pincer are characterized by low quantal neurotransmitter output and minimal facili-
tation, whereas synapses at long-sarcomere fibers in the snapper are characterized by large quantal
output and greater facilitation (Mellon and Stephens 1979, Stephens and Mellon 1979, Stephens
et al. 1983). This does not exclude trophic factors or other nerve properties, such as firing patterns
and peripheral branching patterns, by which the nervous system may control fiber transformation.
The cell bodies of the motoneurons in the thoracic ganglion differ in size, with the cell bodies on
the snapper side larger than the cell bodies on the pincer side (Mellon 1981, Mellon et al. 1981). Claw
reversal results in the reversal in cell body size after one molt, which is consistent with the primacy
of the nervous system in driving fiber transformation (Mellon et al. 1981).

Fiber Transformation Associated with Seasonal Migration of Red Crabs

The red crabs (G. natalis) of Christmas Island in the Indian Ocean undergo a seasonal long-distance
breeding migration. This species, as well as other members of this family, are obligate air breathers
(Adamczewska and Morris 2000). The lining of the branchial chamber is elaborated into a highly
vascularized lung-like tissue that allows high aerobic capacity. During the months of the dry season
(April–October), animals remain inactive in burrows in the interior of the island to avoid desicca-
tion. At the first monsoon rains, adult males and females begin a migration to the shore that may
require walking 1 km/day for 5–6 consecutive days (Adamczewska and Morris 2001a). Upon arrival
at the coast, males dig burrows where mating occurs. Males return inland and females remain in the
burrows while the eggs develop. After about 2 weeks, females enter the ocean, where contact with
seawater stimulates hatching and release of the zoea larvae. Over the course of 3–4 weeks, larvae
undergo several molts and metamorphose into juvenile crabs, which come ashore and migrate to
interior locations. Thus, there is a single “wave” of coastal migration, followed by three consecutive
waves of inland migration: first adult males, then adult females about 2 weeks later, and then juve-
niles about 4 weeks after that.
The transition of sedentary to migratory phases requires physiological mechanisms for sus-
tained locomotory activity. G.  natalis has a unique shunt from lungs to the gills, which allows
greater oxygenation of the hemolymph during exercise, that is lacking in other land crab species
(Adamczewska and Morris 2000, Morris 2002). Even so, dry-season G. natalis rely on anaerobic
metabolism for sustained moderate exercise. By contrast, wet-season G. natalis are completely aero-
bic and show no metabolic acidosis during migration (Adamczewska and Morris 2001a,b).
Migration causes a change in the fiber-type composition of the muscles in the walking legs to a
more fatigue-resistant phenotype. Postel et al. (2010) analyzed 2,118 expressed sequence tags (ESTs)
from muscles in the merus of walking legs from wet- and dry-season G. natalis. Differences in the
expression of several myofibrillar protein isoforms are consistent with a shift from slow-twitch (S1)
to slow-tonic (S2) fibers in preparation for migration. Histochemical studies show that S2 fibers
have a lower myofibrillar ATPase activity and higher aerobic capacity than S1 fibers (Mykles 1988).
Muscles from dry-season animals express higher levels of an ortholog of lobster actinSK1, which
is the dominant actin isoform in S1 fibers in the lobster crusher claw (Kim et  al. 2009a, Medler
et  al. 2005, Medler and Mykles 2003). Orthologs of lobster slow tropomyosin-1 (Ha-sTm1) and
 Skeletal Muscle Differentiation, Growth, and Plasticity 149

tropomyosin-2 (Ha-sTm2) are also expressed differentially between dry- and wet-season G. natalis.
In lobsters, Ha-sTm1 is preferentially expressed in S1 fibers, and Ha-sTm2 is preferentially expressed
in S2 fibers (Medler et al. 2004). In G. natalis, the S2 tropomyosin ortholog (Gn-TmS2) is expressed
at higher levels in migratory animals, whereas there is little or no change in the expression of the S1
ortholog (Gn-TmS1; Postel et al. 2010). There are also changes in 3 TnI and 2 other actin transcripts,
but assignment to specific fiber types could not be made (Postel et al. 2010). These data indicate
that a shift to slow-tonic fibers in the walking legs contribute to the ability of G. natalis to migrate
long distances without fatigue.
Transformation of S1 and S2 fibers requires remodeling the myofibrillar structure. In fiddler
crabs, S1 fibers have longer sarcomere lengths and higher thin filament-to-thick filament ratios than
do S2 fibers (Ismail and Mykles 1992). Consistent with this is the upregulation of two muscle LIM
proteins, Mlp (Contig 131) and paxillin (Contig 140), in leg muscles of migrating G. natalis (Postel
et al. 2010). Orthologs of these genes are implicated in structural remodeling of insect and mamma-
lian skeletal muscles (Postel et al. 2010). This suggests that LIM proteins facilitate the incorporation
of newly synthesized myofibrillar protein isoforms into the contractile apparatus so that animals
can initiate migration as soon as the monsoon begins.

Temperature Plasticity

Temperature is a significant abiotic factor that affects muscle contraction. Cyprinid fishes exhibit
a range of skeletal muscle plasticity in response to temperature changes to maintain muscle perfor-
mance. In carp, three temperature-specific isoforms of MHC genes are expressed (Watabe 2002).
In crustaceans, studies have examined MHC isoform diversity in the context of evolutionary adap-
tation to environmental temperature and of acclimation to temperature changes. An Antarctic iso-
pod (Glyptonotus antarcticus) expresses an MHC isoform that is not present in a temperate isopod
species (Idotea resecata) or a cold water amphipod species (Eulimnogammarus verrucosus; Holmes
et al. 2002). This isoform may have evolved to function in the cold Antarctic waters, but further
work is needed to demonstrate its functional role. In a subsequent study of seven amphipod spe-
cies, the expression of four MHC isoforms is correlated with latitudinal distribution (Rock et al.
2009). Although it was concluded that the number of expressed isoforms increased in the more
northern populations, one cannot rule out the possibility that these isoforms simply represent
differences in common fiber types (i.e., similar to fast, S1, and S2 MHC isoforms). Indeed, two
MHC isoforms were later identified as being differentially expressed in fast versus slow muscles
(Whiteley et al. 2010). Temperature-specific MHC isoforms have not been identified within any
crustacean muscles. Temperature has no effect on the expression of MHC isoforms in the abdomi-
nal muscle of lobsters (H. gammarus) reared at 10°C, 14°C, and 19°C (Whiteley and El Haj 1997,
Magnay et al. 2003). These limited data suggest that crustacean muscles do not possess the level of
temperature-dependent plasticity observed in some fish species.

Muscle Atrophy

Disuse Atrophy: Effects of Unweighting, Tenotomy, Denervation, and Immobilization

Crustacean muscles undergo a “disuse” atrophy in response to treatments that reduce the load on
the fibers, such as tenotomy and limb autotomy. Tenotomy shortens the fiber resting length 15–35%
in the crayfish (Procambarus clarkii) claw opener muscle, resulting in a 20% decrease in mean fiber
diameter by 15 days (Boone and Bittner 1974). Fiber diameter is reduced about 50% one to two
months after tenotomy (Boone and Bittner 1974). Autotomy of a walking leg in C.  maenas and
U. pugilator and a claw in crayfish (Procambarus sp.) causes an approximately 50% reduction in mass
150 Donald L. Mykles and Scott Medler

of the thoracic muscles that operate the appendage, relative to the contralateral muscle (Moffett
1987). Interestingly, the diameter of the fibers in the weighted contralateral anterior levator (AL)
muscle is reduced, but not as much as the fibers in the unweighted muscle (Schmiege et al. 1992).
Thus, there appears to be an autotomy-induced atrophy in weighted muscles. Innervation remains
intact and the muscles continue to function (Boone and Bittner 1974, Velez et al. 1981, Moffett 1987).
Atrophy occurs in intermolt animals, indicating that it does not require ecdysteroids. The atro-
phic changes are reversed when the leg regenerates and becomes functional after the animal molts
(Schmiege et al. 1992). Unlike mammalian skeletal muscle, there is no change in fiber phenotype
in unweighted P. clarkii thoracic muscle (Griffis et al. 2001). Moreover, denervation and immobili-
zation have little to no effect on crayfish claw opener muscle (Boone and Bittner 1974, Velez et al.
1981), which indicates that isometric tension per se is not essential for maintaining muscle mass.
Ultrastructural changes associated with unweighting were examined in C. maenas and crayfish
(P. clarkii and P. simulans; Velez et al. 1981, Schmiege et al. 1992). Both autotomy and tenotomy cause
disorganization of the sarcomere in C. maenas and crayfish, respectively, including fragmentation
and loss of the Z line (Velez et al. 1981). Striations are indistinct in tenotomized muscle, probably
due to hypercontraction (Velez et  al. 1981). The myofibrils have extensive areas of myofilament
erosion, and the intermyofibrillar space is increased (Velez et al. 1981, Schmiege et al. 1992). These
features are indicative of increased degradation of myofibrillar proteins. There are reductions in
mitochondria and nuclei, which are correlated with the presence of multivesicular bodies and lyso-
somes (Velez et al. 1981, Schmiege et al. 1992).

Molt-induced Atrophy of Claw Closer Muscle

In many decapod crustaceans, the first pair of pereopods develops into powerful claws that have
offensive and defensive functions. The penultimate segment (propodus) is enlarged and contains a
massive closer muscle that enables the dactyl to close with great force. Although large claws confer
a competitive advantage, it creates a mechanical challenge at ecdysis when the claws must be with-
drawn through the small basi-ischial joint that connects the claw to the body (Fig. 5.6).
Consequently, the mass of the closer muscle is reduced during the premolt period. The physi-
cal problem of withdrawing the claw was recognized by biologists in the 19th century (Salter 1860,
Herrick 1895), but it was Couch (1837, 1843) who reported a molt-induced atrophy in Cancer pagu-
rus and proposed it functioned in extricating the claws at molt. Skinner (1966) rediscovered the
phenomenon in the blackback land crab, G. lateralis, in which the claw muscle mass decreases about
40% during the premolt period. Various aspects of this molt-induced muscle atrophy have been
reviewed (Mykles and Skinner 1982a, 1985a, 1990a, 1990b, Mykles 1992, 1997b, 1999a, West 1997).
The major conclusions are:

1. Atrophy is specific to the claw closer muscle, with fiber types in the claw responding
differently to the atrophic signal. Muscles in the walking leg and cephalothorax do not atrophy
during the premolt period (Mykles and Skinner 1982a, Schmiege et al. 1992, Griffis et al.
2001). In G. lateralis, the claw closer muscle is composed mostly of large-diameter S1 fibers,
with small-diameter S2 fibers located centrally (Mykles 1988). The major claw of the fiddler
crab (U. pugnax) contains mostly S1 fibers with small numbers of S2 fibers, and the minor
claw contains only S2 fibers (Mykles 1988, Ismail and Mykles 1992). In both species, the S1
fibers undergo a greater atrophy than the smaller S2 fibers (Mykles and Skinner 1981, Ismail
and Mykles 1992). The claw closer muscle in the yabby C. destructor has equal numbers of
slow (long sarcomere) and fast (short sarcomere) fibers (West 1997, Koenders et al. 2004).
The slow fibers in late premolt animals show morphological changes associated with atrophy,
whereas fast fibers do not (West et al. 1995, West 1997). Interestingly, the major claw muscle of
 Skeletal Muscle Differentiation, Growth, and Plasticity 151

Fig. 5.6.
Dominant male blackback land crab, G. lateralis, in threat display. During premolt, the claw closer muscle atro-
phies. The reduction in muscle mass enables the animal to pull the large claws through the basi-ischial joints at
ecdysis. Photograph by D. L. Mykles.

snapping shrimp does not atrophy (Mellon 1999). These animals use a different mechanism to
extricate the claw. The exoskeleton splits open at ecdysis, providing a larger opening to remove
the appendage (Mellon 1999).
2. The magnitude of claw muscle atrophy is determined by the regeneration load. Growth of
limb regenerates occurs during the premolt period, so that a functional claw or leg is restored
at ecdysis (Hopkins 2001, Mykles 2001). A large number of limb regenerates can place a
significant demand for amino acids needed for tissue growth. In G. lateralis and U. pugnax,
there is about a 40% decrease in mass in animals regenerating no more than one walking leg
and about a 78% decrease in mass in animals regenerating 6–8 walking legs (Fig. 5.7; Skinner
1966, Mykles and Skinner 1981, Ismail and Mykles 1992). Thus, muscle protein degradation
is accentuated by regeneration of 6–8 walking legs, which would provide amino acids for
regenerate growth at a time when animals stop feeding and must rely on endogenous nutrient
stores.
3. There is no fiber degeneration. In G. lateralis, the twofold reduction in fiber diameter is
proportional to the fourfold reduction in myofibrillar cross-sectional area (Mykles and Skinner
1981), indicating that fibers retain the same number of myofibrils. The decrease in myofibrillar
diameter results from the removal and degradation of myofilaments, as indicated by areas
of myofibrillar erosion and enlarged intermyofibrillar space in atrophic muscle (Fig. 5.7;
Mykles and Skinner 1981, Ismail and Mykles 1992, West et al. 1995, West 1997). In C. destructor,
maximum Ca2+-activated tension decreases about fourfold in both fast and slow fibers in claws
of animals immediately before ecdysis (West 1999). There is also a compensatory decrease
in the SR, and organelles, such as mitochondria and nuclei, retain their normal appearance
(Mykles and Skinner 1981). Secondary lysosomes containing mitochondria and myelin figures
indicate increased breakdown of mitochondria and SR membrane as fiber volume decreases
(Mykles and Skinner 1982a).
4. There is an extensive remodeling of the sarcomere structure due to a preferential degradation
of thin filaments. In G. lateralis, 11 thin filaments are removed for each thick filament, resulting
in a decrease in the thin-to-thick filament ratio from about 9:1 to 6:1, a 31% decrease in the
actin-to-MHC ratio, and a 72% increase in thick filament packing (Fig. 5.8; Mykles and Skinner
1981, 1982b). Similar changes occur in the major claw of male fiddler crabs U. pugnax (Ismail
and Mykles 1992). In the S1 fibers, the thin-to-thick filament ratio decreases from about 9:1
152 Donald L. Mykles and Scott Medler

Fig. 5.7.
Molt-induced atrophy in the claw closer muscle of blackback land crab. Electron micrographs show the ultra-
structure of the myofibrils in fibers from intermolt (A, C) and late premolt (B, D) animals in longitudinal (A,
B) and transverse (C, D) sections. Myofibrillar cross-sectional area is reduced in late premolt fibers while the
structure of the sarcomere is retained. Arrows indicate areas of erosion within the myofibrils, and asterisks
(*) indicate enlarged interfibrillar space in the premolt fibers. Abbreviations: A, A band; D, dyad; I, I band; Mf,
myofibril; SR, sarcoplasmic reticulum; T, transverse tubule; Z, Z-line; and ZT, Z tubule. Scale bar (C, D) = 1
µm. From Mykles and Skinner (1981), with permission from Elsevier.

to 6:1, the actin-to-MHC ratio decreases 74%, and the thick filament packing increases 51%
(Ismail and Mykles 1992).

Molecular Mechanisms Regulating Protein Turnover in Crustacean Muscle

In mammalian muscle, atrophy results from a net increase in protein degradation, either from
increased proteolysis and no change in protein synthesis, decreased synthesis and no change in
proteolysis, or increased proteolysis and decreased synthesis (Schakman et al. 2009, McCarthy
and Esser 2010, Goodman et al. 2011). Molt-induced claw muscle atrophy is atypical because pro-
tein synthesis is increased as much as 13-fold in vivo and in vitro in claw muscle from late premolt
animals (Skinner 1965, El Haj et al. 1996, Covi et al. 2010). The large increase in protein synthesis
 Skeletal Muscle Differentiation, Growth, and Plasticity 153

Fig. 5.8.
Molt-induced claw muscle atrophy causes changes in myofilament ratios and packing. (A) Transverse section
of myofibril from an intermolt animal showing thick filaments surrounded by 10–15 thin filaments; the thin-to-
thick filament ratio is about 9:1. Arrowheads indicate areas where thick filaments are separated by two rows of
thin filaments. The average spacing of thick filaments is 51 nm. (B) Transverse section of myofibril from a late
premolt animal showing thick filaments surrounded by 7–11 thin filaments; the thin-to-thick filament ratio
is about 6:1. Arrowheads indicate areas lacking a row of thin filaments between thick filaments. The average
spacing of thick filaments is 45 nm. Scale bars = 0.1 µm. From Mykles and Skinner (1981), with permission from
Elsevier.

seems counterproductive because it necessitates an even greater increase in protein degradation

to produce a reduction in mass. However, an accelerated protein turnover is necessary for the
extensive remodeling of the fibers that occurs during premolt, as described earlier. Increased
protein turnover is associated with remodeling in mammalian muscle (Bassel-Duby and Olson
2006). Any modification in the filament lattice requires the exchange of proteins in the myofibril
with newly synthesized proteins in the cytoplasm (Russell et al. 2000). For example, the soluble
myofilament pool increases under catabolic conditions (Dahlmann et al. 1986). Thus, the restruc-
turing of the sarcomere depends on the flux, or turnover, of protein between the soluble and myo-
fibrillar protein pools.
Protein degradation is mediated by calpain and ubiquitin (Ub)/proteasome proteolytic systems.
Their role in molt-induced atrophy is briefly summarized here and emphasizes recent research. The
reader is referred to reviews for further details on their biochemical properties (Mykles and Skinner
1985b, 1990a, 1990b, Beyette and Mykles 1999, Mykles 1992, 1993, 1997a, 1997b, 1998, 1999a, 1999b,
2000). Calpains are cytoplasmic Ca2+-dependent proteinases (CDPs) that completely degrade
myofibrillar proteins in vitro and in situ (Mykles and Skinner 1982b, 1983, 1986, Mattson and Mykles
1993, Mykles 1990). Four calpain activities, designated CDP I, IIa, IIb, and III, occur in crustacean
154 Donald L. Mykles and Scott Medler

muscle, and cDNAs encoding three calpains, designated CalpB, CalpM, and CalpT, have been char-
acterized (Table 5.1; Mykles 2000). Total calpain activity increases twofold in atrophic claw muscle
(Mykles and Skinner 1982b).
The Ub/proteasome proteolytic system is stimulated in atrophic claw muscle. Ub is a highly
conserved protein that, when conjugated to a protein, targets that protein for degradation by the
proteasome (Mykles 1998, Glass 2010). Ubiquitin mRNA, Ub-protein conjugates, and proteasome
subunits increase five-, eight-, and twofold, respectively, in atrophic claw muscle of G. lateralis, with
greater ubiquitin expression in S1 fibers (Shean and Mykles 1995, Koenders et al. 2002). In H. ameri-
canus, Ub expression is upregulated in claw muscle during premolt (Koenders et al. 2002). During
larval development of the European lobster H. gammarus, proteasome activity in the claw muscle is
higher during the pre- and postmolt stages (Götze and Saborowski 2011). The proteasome has lim-
ited ability to degrade myofibrillar proteins (Mykles 1989, Mykles and Haire 1991, 1995). Its precise
role in muscle atrophy has yet to be determined, but its function appears secondary to calpains. As
calpains preferentially degrade the Z line (Mykles 1990), calpains initiate protein degradation by
releasing filaments from the myofibril; filaments are subsequently degraded by calpain and Ub/
proteasome systems (Mykles 1997b).
Ecdysteroids control protein metabolism in the claw closer muscle. Protein synthesis
increases during premolt, with maximum rates at late premolt when hemolymph ecdysteroid
titers are at their peak (Covi et al. 2010, Mykles, 2011). Coincident with the increase in protein
synthesis, there is a decrease in myostatin (Mstn) expression. Mstn, a member of the trans-
forming growth factor-β (TGFβ) family, is an autocrine factor that inhibits protein synthesis
and stimulates protein degradation in mammalian muscle (Schakman et al. 2009, McCarthy

Table 5.1.  Comparison of masses of deduced sequences from crustacean calpain cDNAs

with masses of lobster Ca2+-dependent proteinases (CDPs) characterized biochemically.

Type Calculated Accession CDP activity Reference

mass (cDNA) Number (subunit mass)
B Calpain:
Gl-CalpB 88.9 kDa AY639153 CDP IIb (95 kDab) (Kim et al. 2005a)
Lv-CalpB Partial cDNA GQ179742 unpublished
M Calpain:
Gl-CalpM 65.2 kDa AY639152 CDP III (59 kDaa; (Kim et al. 2005a)
Ha-CalpM 66.3 kDa AY124009 62 and 68 kDab) (Yu and Mykles
2003)
Nn-CalpM 65.9 kDa FJ666100 (Gornik et al. 2010)
T Calpain:
Gl-CalpT 74.6 kDa AY639154 CDP I or IIa (60 (Kim et al. 2005a)
kDab)

Abbreviations: Gl, Gecarcinus lateralis; Ha, Homarus americanus; Lv, Litopenaeus vannamei; Nn, Nephrops norvegicus.
Native masses of lobster CDPs I, IIa, IIb, and III are 310 kDa, 125 kDa, 195 kDa, and 59 kDa, respectively (Mykles and Skinner
1986). The putative identities of CalpB with CDP IIb and CapT with CDP I or IIa have not been established. The subunit
composition of CDP I is not known.
a
 Mass estimated by gel filtration column chromatography (Mykles and Skinner 1986).
b
 Mass estimated by SDS-polyacrylamide gel electrophoresis (Beyette et al. 1993, 1997, Beyette and Mykles 1997, Yu and Mykles
2003). A 62 kDa isoform of Ha-CalpM is expressed in claw muscle, and a 68 kDa isoform is expressed in abdominal muscle
(Yu and Mykles 2003).
Modified from Kim et al. (2005a).
 Skeletal Muscle Differentiation, Growth, and Plasticity 155

and Esser 2010). cDNAs encoding the crustacean ortholog have been cloned from G. latera-
lis, H.  americanus, C.  maenas, Eriocheir sinensis, Litopenaeus vannamei, Penaeus monodon, and
Pandalopsis japonica (Covi et al. 2008, Cho et al. 2009, Kim et al. 2009b, 2010, MacLea et al.
2010, De Santis et al. 2011, Qian et al. 2013). In G. lateralis, Gl-Mstn mRNA is reduced in both
the claw closer and thoracic muscles during premolt (Covi et al. 2010). However, the effect
of molting is greater in the claw muscle. By late premolt, Gl-Mstn mRNA in the claw mus-
cle decreases by 81% (approximately fivefold) and 94% (approximately 17-fold) in animals
induced by eyestalk ablation (ESA) and by multiple leg autotomy (MLA), respectively, and is
negatively correlated with ecdysteroids (Covi et al. 2010). Gl-Mstn mRNA in thoracic muscle
decreases by 68% (approximately threefold) and 82% (approximately fivefold) in ESA and
MLA animals, respectively, and is only weakly correlated with ecdysteroid (Covi et al. 2010).
In L.  vannamei, the effects of 20-hydroxyecdysone (20E) injection on Lv-Mstn expression
differed between muscles:  20E decreased mRNA levels in abdominal and pleopod muscles,
increased mRNA levels in pereopod muscle, and had no effect on mRNA levels in thoracic
muscle (Qian et al. 2013). However, because the control injections consisted of an equivalent
volume of saline rather than the ethanol vehicle, it is possible that the changes in Lv-Mstn
mRNA were not strictly in response to 20E (Qian et al. 2013). In H. americanus, spontaneous
molting results in a larger decrease (82%) in Ha-Mstn expression in crusher claw muscle than
in cutter claw (51%) or deep abdominal (69%) muscles (MacLea et  al. 2010). However, an
acute increase in ecdysteroids caused by ESA has no effect on Ha-Mstn mRNA levels in the
three muscles (MacLea et  al. 2010). These data suggest that ecdysteroids stimulate protein
synthesis by downregulating Mstn. The differential response of the claw and thoracic muscles
to ecdysteroid may be due to differences in the expression of the ecdysteroid receptor (Gl-EcR
and Gl-RXR isoforms; Kim et al. 2005a,b, Covi et al. 2010).
Ecdysteroids appear to activate mechanistic target of rapamycin (mTOR)-dependent protein
synthesis. mTOR is a highly conserved protein kinase that stimulates translation by phosphory-
lating ribosomal S6 kinase (S6K) and 4EF-binding protein-1 (Schakman et al. 2009, McCarthy
and Esser 2010, Goodman et  al. 2011, Frost and Lang 2012). mTOR functions as a sensor that
controls cellular growth. Its activity is regulated by nutrients, intracellular energy levels, hypoxia,
stress, and growth factors (Laplante and Sabatini 2012). The stimulation of muscle protein syn-
thesis by ecdysteroids is primarily at the translational level, as indicated by increases in ribosomal
activity and global protein synthesis during premolt (Skinner 1968, El Haj et al. 1996, Covi et al.
2010). Elevated ecdysteroids have little effect on MHC and actin mRNA levels (Whiteley and El
Haj 1997, El Haj 1999, Medler et al. 2005). Expression of Rheb (Ras homolog enriched in brain),
an activator of mTOR, is increased nearly fourfold in claw muscles from premolt G. lateralis, and
Gl-Rheb mRNA levels are positively correlated with hemolymph ecdysteroid levels (MacLea
et al. 2012). The mRNA levels of other mTOR signaling components (mTOR, Akt, and S6K)
are not correlated with hemolymph ecdysteroid titers (MacLea et al. 2012). These data indicate
that mTOR is activated by Rheb, resulting in increased synthesis of cytosolic and myofibrillar
proteins (Covi et al. 2010). Thus, Rheb expression may serve as a molecular marker for tissue
growth in crustaceans.
Mstn may have pleiotropic functions in crustaceans. Mstn is expressed in a wide variety
of tissues, which suggests its function is not restricted to controlling muscle growth (Covi
et al. 2008, Kim et al. 2009b, 2010, MacLea et al. 2010, De Santis et al. 2011, Qian et al. 2013).
Studies on shrimp (P. monodon and L. vannamei) illustrate the complex and potentially mul-
timodal actions of Mstn in crustaceans (De Santis et al. 2011, Qian et al. 2013). The expres-
sion of Pm-Mstn in abdominal muscle varies during the molting cycle, although there is not
a clear relationship between Pm-Mstn mRNA levels and periods of muscle growth. Pm-Mstn
mRNA level is elevated immediately after ecdysis (postmolt stage A), when muscles grow in
156 Donald L. Mykles and Scott Medler

response to stretching from the expansion of the new exoskeleton. Pm-Mstn expression is
low at intermolt stage when abdominal muscle growth is complete. Expression of Lv-Mstn in
“mixed muscle” samples exhibits the same pattern (Qian et al. 2013). This suggests that Mstn
is a myotropic factor that stimulates muscle protein synthesis. An apparent contradiction is
that Pm-Mstn mRNA levels are also elevated at premolt stages, when the abdominal muscle
is not growing (De Santis et al. 2011). Moreover, abdominal muscles in decapods with elon-
gated body plans (e.g., shrimp, lobsters, and crayfish) do not atrophy because the abdomen
is easily withdrawn through the large opening created at the junction between the cephalo-
thorax and abdomen at ecdysis (Chang and Mykles 2011). However, ribosomal activity and
protein synthesis are increased in lobster abdominal muscles during premolt (Whiteley and
El Haj 1997). If protein synthesis is similarly elevated in premolt shrimp abdominal muscle,
the Pm-Mstn expression pattern over the molting cycle is consistent with its function as a
stimulatory growth factor. This is supported by knockdown experiments, in which injec-
tions of Pm-Mstn ds-RNA into the abdominal muscle significantly reduced shrimp growth
over a 45-day period (De Santis et al. 2011). Interpretation of these results should be tem-
pered by possible systemic effects because the Pm-Mstn knockdown was not restricted to
the abdominal muscle. Pleopod muscle harvested at 7 days had a similar decrease (~40%) in
Pm-Mstn mRNA as abdominal muscle harvested at 45 days, indicating the effect had spread
to other regions of the abdomen. Other tissues with high levels of Pm-Mstn expression,
such as heart, gill, eyestalk, and stomach, were not included in the qPCR analysis (De Santis
et  al. 2011). An alternative explanation is that Pm-Mstn knockdown inhibited molting by
decreasing the ecdysteroidogenic activity of the molting glands (Y-organs) because Gl-Mstn
is highly expressed in the Y-organ (Mudron and Mykles, data not shown). A  reduction in
molting frequency would explain the lower growth rate of Pm-Mstn ds-RNA-injected ani-
mals. Unfortunately, the molting data were not reported, thus leaving the matter unresolved
(De Santis et al. 2011).
Little is known about how ecdysteroids regulate protein degradation in crustacean muscles. In
G. lateralis, ESA causes a transient (1–3 days) increase in Gl-CalpT and Gl-EcR mRNAs in claw
muscle, but not in thoracic muscle; ESA has no effect on Gl-CalpB and Gl-CalpM expression
(Kim et al. 2005a). Interestingly, the expression of Gl-CalpT and Gl-EcR is correlated in both claw
and thoracic muscles, suggesting the two genes are co-regulated (Kim et al. 2005a). However, the
expression of ecdysteroid receptor genes (Gl-EcR and Gl-RXR) and Gl-CalpT is not correlated
with ecdysteroid (Covi et al. 2010). In H. americanus, Ha-CalpM expression in the crusher claw is
not affected by molt stage (Yu and Mykles 2003). These data suggest that sustained activation of
calpains in atrophic muscle involves post-transcriptional mechanisms.
Atrophy in response to unweighting has a different effect on the expression of genes involved
in protein synthesis and degradation. By contrast to the effects of molt induction in G. lateralis,
Gl-Mstn mRNA increases threefold and Gl-CalpT mRNA decreases 40% in unweighted mus-
cles with respect to weighted contralateral muscle (Covi et al. 2010). In premolt animals, the
decrease in Gl-Mstn mRNA in unweighted muscle parallels the decrease in Gl-Mstn mRNA
in weighted muscle, indicating that increased ecdysteroids supersede any effect that unloading
has on Gl-Mstn expression. In other words, low ecdysteroids permit the upregulation of Mstn
in unweighted thoracic muscle. Gl-Rheb and Gl-S6K are increased 2.2-fold and 1.3-fold, respec-
tively, in unweighted thoracic muscle, indicating that mTOR-dependent protein synthesis is
stimulated (MacLea et  al. 2012). Unweighting has no effect on Gl-Akt, Gl-mTOR, Gl-EcR,
and Gl-RXR mRNA levels (Covi et al. 2010, MacLea et al. 2012). These data, which are con-
sistent with the positive relationship between Gl-Rheb and Gl-Mstn expression in weighted
thoracic muscle (MacLea et al. 2012), indicate that Mstn stimulates protein synthesis in tho-
racic muscle.
 Skeletal Muscle Differentiation, Growth, and Plasticity 157

FUTURE DIRECTIONS

Research should now turn toward elucidating the molecular control of fiber differentiation, phe-
notype, and size by neural activity and endocrine and paracrine factors. We know little about the
molecular mechanisms regulating muscle differentiation during development, in particular the
roles of transcription factors that specify skeletal muscle in other organisms (e.g., Mef2, Myf4,
Myf5, MyoD, and myogenin in vertebrates) and how neuronal activity alters myofibrillar gene
expression. The control of protein turnover is central to muscle plasticity. Fiber transformation
requires the coordinated expression of fiber type-specific myofibrillar protein isoforms, which
must be assembled and incorporated into the myofibrils while myofilaments containing protein
isoforms of the former fiber type are removed and degraded. The relative rates of protein synthesis
and degradation determine whether a muscle grows or atrophies. mTOR probably plays a critical
role because increased protein synthesis is associated with skeletal muscle growth and remodel-
ing in diverse organisms. In mammals, Mstn controls muscle growth by suppressing mTOR activ-
ity and activating Ub/proteasome-dependent degradation (Schakman et al. 2009, McCarthy and
Esser 2010, Goodman et al. 2011, Frost and Lang 2012). The function of Mstn in crustacean muscle
is not fully understood. Mstn appears to be a stimulator of protein synthesis in abdominal muscles
in P. monodon and unweighted thoracic muscle in G. lateralis (Covi et al. 2010, De Santis et al. 2011,
MacLea et al. 2012). In G. lateralis claw muscles, the downregulation of Gl-Mstn and the upregula-
tion of Gl-Rheb indicate that Mstn inhibits mTOR-mediated protein synthesis (Covi et al. 2010,
MacLea et al. 2012). Thus, muscles in the same species differ in the relationship between Gl-Mstn
and Gl-Rheb expression. In claw muscle, Gl-Rheb and Gl-Mstn mRNAs are negatively correlated in
animals induced to molt by ESA but not in animals induced to molt by MLA (MacLea et al. 2012).
By contrast, Gl-Rheb and Gl-Mstn mRNAs are positively correlated in weighted thoracic muscle
from both ESA and MLA animals (MacLea et al. 2012). The differences in the relationship between
Gl-Mstn and Gl-Rheb expression may be due to differences in the responses of the two muscles to
ecdysteroids (Covi et al. 2010). Gl-Mstn and Gl-Rheb mRNA levels in claw muscles are significantly
correlated with hemolymph ecdysteroid titers, whereas there is no correlation between Gl-Mstn
and Gl-Rheb mRNA levels and hemolymph ecdysteroid titers in thoracic muscles (MacLea et al.
2012). The next step is to gain a mechanistic understanding of the complex interactions between
endocrine/paracrine factors and signaling pathways controlling protein turnover.

CONCLUSIONS

Relatively little is known about the early stages of crustacean myogenesis, but the available data
suggest a process similar to that in insects. Muscle precursor cells migrate to specific locations
in the embryo and then differentiate into fibers. Muscle fibers with motor nerves are in place at
relatively early stages of development, and subsequent growth over several molt cycles produces
adult muscles. The genes that control muscle differentiation remain to be studied. Molting stimu-
lates the growth of muscle fibers, which increase in length by the addition of sarcomeres and/or
lengthening of existing sarcomeres and in diameter by sarcomere splitting and/or increases in myo-
fibril cross-sectional area. As in mammals, crustacean skeletal muscle is a dynamic tissue that can
undergo dramatic changes in mass and contractile properties. Molecular mechanisms controlling
the changes in fiber composition by cell death and fiber transformation are complex and probably
involve a combination of neurotrophic, endocrine, and paracrine factors, which have yet to be iden-
tified. Changes in fiber composition are coordinated with changes in claw morphology, but they
can, on rare occasions, be unlinked. In “double-crusher” lobsters, the closer muscle in the “false”
crusher has the cutter claw fiber composition. Also, fiber transformation is completed before the
158 Donald L. Mykles and Scott Medler

adult claw morphology is attained. Because both fiber transformation and molt-induced atrophy
involve extensive remodeling of the sarcomeric structure, it is likely that both processes require an
increase in protein turnover. The main difference, however, is that there is a net loss of protein in
muscle atrophy, whereas there is no net loss or net gain in protein in transforming fibers. LIM pro-
teins may play a role in both processes (Postel et al. 2010). Calpains degrade myofibrillar proteins,
but we know little about how their activities are regulated by ecdysteroids. Atrophy of unweighted
thoracic muscle is ecdysteroid-independent and is associated with modest increases in the expres-
sion of Gl-Mstn, Gl-Rheb, and Gl-S6K and decreases in the expression of Gl-CalT (Covi et al. 2010,
MacLea et al. 2012). The stimulation of muscle protein synthesis by ecdysteroids in molt-induced
claw muscle atrophy involves both Mstn and mTOR signaling pathways (Covi et al. 2010, MacLea
et al. 2012).

ACKNOWLEDGMENTS

The authors thank the many students, postdoctoral fellows, and collaborators for their contribu-
tions. The authors recognize the support of grants from the National Science Foundation and the
National Institutes of Health.

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 6
REGENERATION IN CRUSTACEANS

Penny M. Hopkins and Sunetra Das

Abstract
The ability to regenerate missing appendages is highly developed in many crustaceans.
Regeneration is often coupled with the ability to lose a limb at a preformed fracture/autotomy
plane that is part of an adaptive response to predation. Regeneration of a lost limb commences
with wound healing and blastema formation followed by development of a papilla/basal bud.
Regeneration is controlled to some extent by hormones: receptors for ecdysteroids are present in
the limb bud tissues at all stages of regeneration. Regeneration itself has profound effects on the
molt cycle, and the presence of regenerating limb buds can affect the entire cycle. Genes associ-
ated with regeneration in insect legs and imaginal discs have been identified and may play a role in
crustacean limb regeneration as well.

INTRODUCTION

Regeneration is the ability to regrow lost or damaged body parts. Frequently, regeneration con-
sists simply of the replacement of lost tissues by mitotic events. This basic regeneration is called
compensatory hyperplasia and it follows injury in many tissues (e.g., the mammalian liver). A sec-
ond more complex type of regeneration—called epimorphic regeneration—occurs in many organ-
isms. An animal that is damaged by prey or in a conspecific fight can avoid a potentially lethal
event by surrendering part of its body—usually a limb—followed by regeneration of the lost
component.
The loss of a limb—especially if it is a locomotor appendage—can present serious hardships
because such appendages are important in foraging, mating, and predator evasion. Moreover,
if a lost limb contains specialized sensory structures, there can also be a reduction in sensory
input. Epimorphic regeneration of lost appendages is also metabolically costly to the organism.

168
 Regeneration in Crustaceans 169

Regeneration of lost tissues requires reallocation of body resources, which places the organism
at a disadvantage in terms of metabolic reserves necessary for routine growth and reproduction.
Regenerated structures may also be smaller or less ornamented than the lost structure, which in
turn can interfere with social and food-seeking activities (Hopkins 1985, Uetz et al. 1996).
Evolution seems to have favored a higher degree of specialized function over regenerative
capacity. Thus, regeneration in its most spectacular manifestations is found more often in the “less
derived” more “basal” animals. In many of these animals, regeneration involves the formation of
a blastema with some degree of tissue redifferentiation linked to tissue hyperplasia. Epimorphic
regeneration occurs to variable degrees in vertebrates such as bony fish, amphibians, and reptiles
but is seen most dramatically in invertebrates such as annelids, echinoderms, mollusks, and arthro-
pods (Maginnis 2006).
A group of invertebrates in which epimorphic regeneration has been widely studied is the phy-
lum Arthropoda. Because overall body growth in the arthropods is episodic (due to the rigid exo-
skeleton), arthropod regeneration is substantially different from regeneration in other groups of
animals. Among the arthropods, regeneration has been studied most widely in the insects and crus-
taceans and has captured the interest and imagination of many scientists, beginning with Aristotle
and including such luminaries as Charles Darwin, H. Przibram, T.H. Morgan, and T.H. Huxley.
In both insects and crustaceans, molting and growth are controlled by a number of chemical
factors. A complement of neurosecretory and steroid hormones coordinate molting and growth (as
well as reproduction) in these arthropods. The arthropod steroid hormones are called ecdysteroids
and are found almost exclusively in arthropods. These multifunctional steroid hormones are able
to stimulate the physiological events leading to the shedding of the exoskeleton and thereby they
stimulate growth as well as reproductive development, especially in female arthropods. Moreover,
the same steroid hormones are also involved in the control of regeneration in both insects and crus-
taceans (Bodenstein 1955, Halme et al. 2010).
This review looks at the temporal organization of crustacean regeneration from autotomy to
wound healing, to blastema formation, and finally to hypertrophic growth. It also addresses the
roles of blood-borne factors in controlling regeneration. We do not have a complete genome for any
of the malacostracan crustaceans from whom most of the regeneration data have been accumulated,
so we know very little about the genes that are controlled by the factors that control regeneration.
We do, however, have the complete genome for some insects. We will, therefore, look at a few spe-
cific genes involved in insect limb development and regeneration with the hope that they will help
us gain insight into the control of crustacean limb regeneration.

REGENERATION IN THE PHYLUM ARTHROPODA

Regeneration in the two major groups of arthropods, the insects and the crustaceans, is some-
what different since many adult insects do not molt, and regeneration in arthropods is linked,
in part, to the molt cycle. Nevertheless, within the insects, at least 38 genera and 43 species have
been shown to undergo some degree of regeneration. Regeneration occurs in at least three of
the six classes of Crustacea, with at least 35 crustacean genera (and 45 species) able to regenerate
limbs (Maginnis 2006).
Holo- and hemimetabolous insects have life cycle stages that include egg, immature stages (such
as larva or nymphs), pupal, and adult stages. These insects molt and grow only during the imma-
ture stages. After these insects metamorphose into adults, they stop growing in order to put their
metabolic energies into reproductive activity and many die soon after reproducing. Regeneration
in these insects is limited to those times during which molting and growth occur (i.e., larval or
nymphal stages). On the other hand, the less derived apterygote insect orders (Archaeognatha and
170 Penny M. Hopkins and Sunetra Das

Thysanura) do not go through metamorphosis. These insects molt, grow, and regenerate through-
out their entire life cycle (Bodenstein 1955).
Regeneration of limbs in the less derived insects such as cockroaches, crickets, and beetles does
occur, but, for the most part, in these insects only immature tissues regenerate. Regeneration of
nymph legs, antennae, and cerci has been studied in six different species of cockroaches (order
Blattodea). Regeneration of limbs has also been studied in crickets, walking sticks, and firebrats
(orders Orthoptera, Phasmatodea, and Thysanura, respectively), as well as in true bugs. In holo-
metabolous insects, primordia of adult structures such as legs and wings are retained internally
during the larval and pupal stages. These primordia (called imaginal discs) are invaginations of the
larval epidermis that eventually give rise to the external adult structures of the head and thorax at
metamorphosis. Whereas the adult structures do not normally regenerate, these dedicated (but
undifferentiated) imaginal discs are capable of regeneration. Imaginal disc regeneration has been
described in the fruitfly Drosophila melanogaster, the moth Galleria mellonella, and the mealmoth
Ephestia kuhniella (Bergantinos et al. 2010).
Like the more basal insects, the crustaceans continue to molt, grow, and reproduce as adults.
Once these arthropods reach the adult stage, however, they must balance the physiological activities
associated with growth (and regeneration) with the physiological demands of reproduction. In the
Branchiopoda, the cladocerans Simorephalits gibbosus and Daphnia carcinata (and Daphnia magna)
can regenerate portions of the carapace and setae (Agar 1930, Anderson 1933). In the Maxillopoda,
the cirripedian Balanus balanoides can regenerate portions of shell and mantle (Tighe-Ford and
Vaile 1974). In the Malacostraca, at least four orders have been shown to regenerate: the isopods, the
amphipods, the stomatopods, and the decapods. The isopods Asellus aquaticus and Idotea baltica
can regenerate thoracic legs and antennae (Needham 1945, 1965, Varese 1960, Giordano 1961). The
isopods Porcellio dilatatus and Helleria brevicornis have been shown to regenerate legs (Hoarau 1973,
Noulin and Maissiat 1974). The amphipod Gammarus chevreuxi can regenerate a first or second
antennae (Dixey 1938), whereas Orchestia gammarella regenerates gnathopods (Charniaux-Cotton
1957). The stomatopod Squilla sp. has been reported to regenerate antennae (Giesbrecht 1910). In
some decapods, the ability to regenerate is intimately linked to the ability to undergo autotomy of a
damaged limb. Autotomy of a limb, however, does not always signify that the organism can regener-
ate the structure that is lost.

Autotomy in the Arthropoda

Autotomy is a reflexive response to injury that results in the casting off of an injured limb at a
predetermined point proximal to the injury. The advantage to an animal of an autotomy reflex
is that it allows the sacrifice of a limb at a preformed breakage site that is designed to keep blood
loss and trauma to a minimum. Injury-induced autotomy is well-documented in insects and
crustaceans. Injury can be inflicted during conspecific fights for territory or mates, as well as
during predator–prey interaction (McPeek 1997). Most “intraspecific competitive interactions”
in many crustaceans, however, are only ritualistic and do not result in autotomy ( Juanes and
Smith 1995).
The release of a limb via autotomy is a distraction to a predator. The crab Uca pugilator scav-
enges in “herds” for food on exposed intertidal flats during low tide. Shore birds swoop down
to feast on the vulnerable foragers. When a bird seizes a crab by the leg, the crab autotomizes
the leg, leaving the predator with only a small morsel to eat. This strategy enables the crab
to escape. Autotomy of limbs also can occur when an arthropod is shedding its rigid exoskel-
eton: damage to the exoskeleton during molt or physical problems in climbing out of the old
exoskeleton can cause an arthropod to shed a trapped appendage. Limbs caught in the fila-
ments of a fishing line but otherwise uninjured will also be autotomized for escape and survival
 Regeneration in Crustaceans 171

( Juanes and Smith 1995, Maginnis 2006). Autotomy is an exquisite escape mechanism that has
evolved in many groups of animals.
The term autotomy (or “self-cutting”) has recently and extensively been used to describe any act
that results in the loss of a body part irrespective of the mechanism (Maginnis 2006). In this review,
the term “autotomy” will be reserved to meet two major criteria: the loss of an appendage is a true
“autotomy event” if (i) the event occurs at a predetermined “fracture plane” and involves intrinsic
neural and muscular responses (McVean 1975), and, (ii) the event serves primarily as a defensive
function and improves the animal’s survival in an escape encounter (Wasson et al. 2002, Fleming
et al. 2007).

Extent of Autotomy in Arthropods

Examples of autotomy have been reported among many vertebrates and invertebrates.
Autotomy that meets the criteria listed here, however, is limited almost exclusively to inver-
tebrates. Loss of appendages in response to predation has been reported in many invertebrate
phyla such as Cnidaria, Annelida, Mollusca, and Echinodermata. But the phylum in which
autotomy has been more extensively studied is the Arthropoda. In addition to jettisoning
walking appendages, some arthropods are capable of autotomizing feeding structures, anten-
nae, antennules, and stingers. Examples of autotomy are found in all of the subphyla of the
Arthropoda. Spiders and centipedes lose walking legs in response to predation, but by far the
best studied examples of autotomy are in the subphyla Hexapoda (insects) and Crustacea
(Maginnis 2006, Fleming et al. 2007).
Among the crustaceans, the autotomy reflex is limited to the class  Malacostraca and most
commonly to the brachyuran crabs. No autotomy has been reported in the classes Maxillopoda,
Ostracoda, Branchiopoda, Cephalocarida, or Remipedia (although Needham stated in 1965 that
the autotomy mechanism was found in members of all classes of arthropods). Among the deca-
pods, the ability to autotomize limbs is found in both natantians and reptantians. Some natantians
have a true autotomy response, whereas other species possess a fracture plane but lack the mus-
culature for the reflex (Bliss 1956). Of the peracaridans, the amphipods and at least one species
of isopods can autotomize limbs. Some malacostracans possess a true autotomy reflex, whereas
others possess either autospasy (the pulling off of a limb at a predetermined locus of weakness) or
autotilly (the detachment of an injured limb at a predetermined point using mouth parts or other
limbs). In the Malacostraca, the macrurans and anomurans have limited autotomy responses. Some
reptantians possess the autotomy reflex in some limbs and autospasy or autotilly in remaining limbs
(Wood and Wood 1932). The degree of autotomy in a variety of arthropod populations as measured
by the number of individuals with missing or regenerating limbs is variable and can range from 10%
to 90% of the population (Fleming et al. 2007).
Some insects and crustaceans, however, can regenerate without the facilitation of autotomy, and
the ability to undergo autotomy does not always result in the ability to regenerate.

Process of Autotomy in Crustaceans

Autotomy in brachyuran crustaceans (the true crabs) has been described in detail (McVean and
Findlay 1976, McVean 1984). It is a reflexive behavior involving specialized autotomy muscles.
Specialized levator muscles insert on the proximal edge of the basioischiopodite segment of the
walking leg. The most crucial muscle to the autotomy reflex is a rotating levator muscle (rplm in
Fig. 6.1) which, when stimulated, switches the tension exerted by the remaining levators (alm1
in Fig. 6.1) so that a crucial cuticular connection between the basioischium and coxa (cp in
172 Penny M. Hopkins and Sunetra Das

rplm pplm

alm 12

th

es
cp
CSD1 Pf

bp

Fig. 6.1.
The levator muscles in the baschioischium leg segment of Carcinus maenas. In the walking legs, the anterior
levator muscle (alm) is subdivided into two parts, as is the posterior levator group (plm). The tendon head (th)
of the anterior levator muscle 1 (alm1) makes contact with the dorsal rim of the basioischium at two locations—
at a cuticular projection (es) and onto a cuticular plug (cp) that forms, at its distal point, the intact bridge across
the breakage plane (bp). This cp is bounded anteriorly by the membrane overlying the cuticular stress detector
1 (CSD1) and posteriorly by a furrow (Pf). The posterior levator muscle (pplm) helps to keep the faces of the
abutting heads closed. The main elements in the walking limbs of Carcinus that contribute to limb autotomy
are the main levator muscle alm 1, which connects to the es. The es is capable of acting as an energy store.
Functional connection is maintained to the energy store, which is tolerant of considerable distortion as long
as the catch between the abutting heads is engaged. When the catch is disconnected by rotation of the rotary
posterior levator muscle (rplm) tendon, tension accumulated in alm 1 is transferred so that it impinges directly
onto the cuticular plug (cp), which causes the breakage plane to snap open, and the basioischium is released
from the underlying coxal rim. From McVean and Findlay (1976), with permission from Springer.

Fig. 6.1) is broken, thus permitting a preformed breakage plane (bp in Fig. 6.1) to separate and
releasing the basioischiopodite from the coxal stump.
The breakage plane in both insects and crustaceans is usually an area of the cuticle with reduced
thickness that allows for easy fracture (Findlay and McVean 1977). There are species differences in
the severity of the injury stimulus needed to induce autotomy in crustaceans and insects (Maginnis
2006). Regeneration in crustaceans is always most efficient when amputation of a limb occurs near
or at the plane of autotomy (Needham 1965). In some insects, there may be more than one plane
of autotomy on each appendage (Woodruff 1937), and there is a gradient of regenerative potential
along the length of the limb. The strength of the wound stimulus to induce autotomy must increase
if other limbs are missing, and there is also some evidence of a proximo-distal sensitivity gradient
along the length of the limb: some crustaceans will not autotomize a limb if the injury is only to
the dactyl but will autotomize with increasing readiness as the injury approaches the body (Wood
and Wood 1932).
The advantage of autotomy to an animal is because the preformed “plane of autotomy” contains
a suite of structures that ensure that blood loss and injury are kept to a minimum. Immediately
proximal to the preformed fracture plane in brachyurans is a connective tissue septum called the
autotomy membrane (AM). The AM is a double membrane that extends across the entire limb base
in such a way that it divides the hemocoelic cavity (open circulatory blood space) in the coxal
 Regeneration in Crustaceans 173

segment from the cavity of the basioischiopodite (Fig. 6.2A). Following autotomy, the distal part of
the AM acts as a valve: blood pressure in the hemocoel immediately inflates the distal AM so that
it balloons into the gap left by the autotomized limb, immediately closing the hole (Hopkins 2001).
This “immediate closure” assures that there is very little blood loss and minimal bacterial invasion.
No muscle tissues cross the crustacean AM, therefore, at autotomy, the only tissue damage is to
the pedal nerve and two blood vessels that pass through a hole in the center of the AM. In insects,
some coxal muscles cross the autotomy plane and are severed at autotomy. These severed muscles,
which serve to move the femur–tibia joint, are phagocytized by blood cells within 30 h following
autotomy (Bodenstein 1957). In crustaceans, on the other hand, since there is so little tissue damage
during autotomy, there is very little phagocytotic activity by crustacean blood cells.

Fig. 6.2.
(A) Autotomy membrane, AM, in an intact limb from the crab, Uca pugilator (tissues prepared and processed as
described in Hopkins [1993] and Hopkins and Durica [1995]). The pedal nerve, PN, and blood vessels, BV, pass
through the AM as a bundle. The thickened area of the AM is the dark area in direct contact with nerve sheath.
HCC, the hemocoelic space of the coxa; HCBI, hemocoelic space of the baschioischium. (B) Light micrograph
of the autotomy membrane, AM, 4 h after autotomy. The forming scab, S, can be seen immediately below the
AM and overlying the limb bud chamber, LC. Granulocytes, G, and hyalinocytes, B, have accumulated below
the scab. The coxal wall, C, can be seen to the side. (C) An electron micrograph of the same area as shown in
B. The AM and S are seen to the right, and below them are the accumulated granulocytes, G, and hyalanocytes,
as well as a degranulated granulocyte, DG. (D) An electron micrograph of the area at the coxal wall where the
AM is attached. Granulocytes, G, are evident, and a detached epidermal cell is seen at the bottom, E, getting
ready to migrate out under the scab, S. From Hopkins (1993), with permission from Oxford University Press.
174 Penny M. Hopkins and Sunetra Das

Following autotomy or limb loss in insects and crustaceans, the process of regeneration consists
of several distinct phases: (i) wound repair, (ii) local activation and proliferation (blastema forma-
tion), (iii) pattern formation with differentiation (cell fate determination), and (iv) hypertrophic
growth of the regenerate. The similarities of leg regeneration to leg development in insects are quite
striking and have been looked at most carefully in those insects where there is genomic information
available. A closer look at developmental and regeneration cascades in insects can help us better
understand limb regeneration in crustaceans.

Wound Repair

In many arthropods, when a wound occurs to the body or walking limbs, it triggers a series of
events that eventually lead to healing and in some cases regeneration. The wound initiates a sig-
naling cascade that activates specific wound-response genes, facilitates wound repair, and recruits
blood cells (Moussian and Uv 2005). Repair of wounds by itself without subsequent regeneration
involves replacement of only small amounts of tissue and does not usually require the extensive tis-
sue redifferentiation seen in epimorphic regeneration. Wound repair is extremely important in all
animals, but especially in the arthropods that have an open circulatory system. In nature, wounds
are frequently acquired during bouts of fighting with predators or with conspecifics and occur most
often in newly molted arthropods.

Wound Healing

Wound healing occurs following various induced injuries in many insects and crustaceans.
Moreover, wound healing is the first step in any regeneration event. Wound healing includes wound
closure and epidermal repair. If the cuticle or carapace is damaged, there is also secretion of new
cuticle/exoskeleton with production of exoskeleton sculptures such as setae and spines. Although
epimorphic regeneration in insects for the most part is limited to immature forms, wound healing
has been documented in life stages that normally do not regenerate limbs. The events that culmi-
nate in wound healing in both crustaceans and insects are somewhat similar (Wu et al. 2004, Baek
et al. 2010, Kwon et al. 2010). Because a great deal more information is available on insect would
healing, it is discussed here in some detail with the underlying assumption that a number of the
molecular events involved in insect wound healing may soon be shown to be important in crusta-
cean wound healing.
Following wounding in both crustaceans and insects, the first response is an influx of blood
cells to the wound site that is thought to be in response to chemotactic signals that attract circu-
lating blood cells necessary for wound closure (Vafopoulou et al. 2007). Closure of wounds by
blood cell invasion is subsequently followed by rapid scab formation and migration of adjacent
epidermal cells.
The nucleated blood cells of crustaceans that aggregate at wound sites in response to chemical
signals are called hemocytes (Fig. 6.2B,C,D). It is generally (but not universally) agreed that there
are three types of circulating hemocytes in crustaceans: large and small granulocytes and a type of
plasmatocyte called a hyalinocyte. The different morphologies of these hemocytes are thought to
reflect different functions. The relative abundance of these cell types varies from species to species,
and fluctuating abundances may reflect differences in molt cycle, nutritional status, or disease state
(Matozzo and Marin 2010). Hemocytes function to (i) release clotting and melanization enzymes,
(ii) join with clotted blood to form a plug that further closes injury-induced breaches in the body
cavity, (iii) encapsulate invading bacteria, (iv) phagocytize necrotic tissues, and (v) possibly release
growth factors and cytokines that may help promote healing (Needham 1965).
 Regeneration in Crustaceans 175

Clotting in arthropods is unique and has very little similarity to clotting in other groups of ani-
mals. Clotting in crustaceans is part of a well-described innate immunity system (Theopold et al.
2004). Most of the components of the crustacean clotting mechanisms are stored in the hemocyte
granules and released upon activation by the lipopolysaccharide coat of invading bacteria or by
putative chemical signals released from damaged/wounded cells. In Drosophila, one chemotactic
wound signal has been identified. It is a peptide that is released by blood cells and/or by damaged
epidermis surrounding the wound (Nakatogawa et  al. 2009). The extensive clotting that occurs
upon wounding is accompanied by another chemical reaction called melanization.
The two major clotting cascades in crustaceans are (i)  the prophenoloxidase activating sys-
tem and (ii) the transglutaminase system (Theopold et al. 2004). The prophenoloxidase cascade
results in production of melanin (melanization) and other active compounds that stimulate scab
formation and increase other immune responses to kill invading bacteria. The second cascade is
the result of a transglutaminase released from granulocytes that crosslinks large blood proteins into
solid clots that are further crosslinked by the phenoloxidase products ( Jiravanichpaisal et al. 2007).
Both clotting and sclerotization reactions are essential for wound healing. Adult mutant Drosophila
lacking hemolymphatic melanization enzymes had 50% mortality when wounded, and the flies
that lived had a poor recovery from the injury (Ramet et al. 2002). Within 30 min of wounding,
two important melanization enzyme transcripts accumulate in the epidermal cells nearest to the
wound. Dopadecarboxylase (ddc) and tyrosine hydroxylase (ple) catalyze the production of cat-
echolamines that are further converted to quinones by phenol oxidase. Quinones play an important
role in clot formation and melanization of a scab (and later in cuticular protein crosslinking). It is
thought that these genes are direct targets of a wound-induced factor that appears to signal via an
extracellular signal-responsive kinase (ERK) and the transcription factor grainy head in Drosophila
(Mace et al. 2005).
In decapod crustaceans, autotomy leads to minimal wound trauma. There is immediate wound
closure by the connective tissue AM, which spans the base of all appendages that undergo autot-
omy. As described earlier, the AM is designed to close the breach left by the removal of the limb
attached to that coxa. The AM is a complex structure with a unique double membrane that is pen-
etrated by the large pedal nerves and two blood vessels (Fig. 6.2A). These structures pass through
the AM together as a bundle through a small hole in the center of the AM. There is an enlarged
space between the double membranes at the point through which the bundle of blood vessels and
pedal nerve pass. A large number of blood cells are stored in this space, which is in direct contact
with the nerve and vessels. In the crustacean coxa, the double AM separates upon autotomy and
releases the stored granulated hemocytes. The granulocytes immediately degranulate and form a
clot to seal the small hole left by the nerve bundle in the more distal AM. Within 30 min after
autotomy, the hemocoelic space immediately below the plane of autotomy is filled with granulo-
cyte cells in both insects and crustaceans (Bodenstein 1957, Adiyodi 1972). Some of these blood
cells are from the store located in the double AM membrane, but many of them are recruited from
the general circulation.
While the most distal portion of the crustacean AM balloons outward to seal the breach in
the coxa, the more proximal portion of the AM remains attached to the sheath of the pedal nerve
(Hopkins et al. 1999). This nerve is severed during autotomy at the preformed breakage plane, and
the nerve stub retracts back into the coxal stump but is held in place by the attachment of the more
proximal AM. The end of the severed nerve is thus kept in close proximity to the point at which it
was located prior to autotomy. The major site of blood loss following autotomy is the small hole
in the middle of the distal AM through which the pedal nerve and vessels pass. A clot forms very
quickly at this hole (as described earlier). Further clotting and melanization occurs along the span
of the AM. The AM together with the degranulated blood cells are incorporated into a black “scab”
176 Penny M. Hopkins and Sunetra Das

that gains its color from the chemical sclerotization described earlier. This scab seals the autotomy
plane and is visible on the outside of the coxa.
Insects have no comparable structure to the AM of crustaceans. Wounds to soft-bodied insect
larvae and the pliable larval wing discs are closed by a contraction of the wound edges. Larval ima-
ginal discs are “set aside” tissues in holometabolous insect larva that will become appendages in the
adult insect at metamorphosis. The pairs of discs within the body of the larva can become wings,
legs, genitalia, antennae, and eyes in the adult. When these discs are injured, they can regenerate.
Initial wound closure in a wounded disc is a reorganization of actin at the edges of epidermal cells
that line the injury. This actin forms a cable-like structure that is also seen in developing insect
embryos. Contraction of this cable helps to close the wound in a “purse-string” mechanism. The
wound edge contracts due to the actin purse-string, and, as the edges approach each other, the epi-
dermal cells join to anchor the two sides together and close the wound (Bosch et al. 2005).
In crustaceans, it is possible to damage only the calcified outer cuticular layers of the exoskel-
eton without injury to the underlying epidermis. Even if the epidermis is visually unharmed, hemo-
cytes will invade the area under the damaged exoskeleton. The epidermal cells that lie beneath a
damaged exoskeleton become activated in the crab Carcinus maenas and are able to recruit hemo-
cytes (Dillaman and Roer 1980). In the crayfish Procambarus clarkii, wounding of the carapace plus
the underlying epidermis results in the onset of a molt cycle (Vafopoulou et al. 2007). The induced
cycle is different in its time frame from a cycle induced by other means but, nevertheless, wounding
results in an early onset of molt compared to the controls. This suggests that the chemical signals
released at the wound site not only attract hemocytes but may also interact with the endocrine
system of the crustacean in a way that ensures that the wound will be further repaired by means of
a hastened and subsequent molt.

Epidermal Repair

When a wound breaches the cuticle and damages the underlying epidermis, the closure of the
injury gap by a blood clot and scab is followed within 6–8 hours by further activation of the epi-
dermal tissues adjacent to the wound. In crustacean penaeid shrimp, epidermal cells surrounding
a wound transform from a squamous to a columnar shape, begin to enlarge, and start to migrate
across the damaged surface below the wound (Fontaine and Lightner 1973). This same response of
the epidermal cells lining the plane of autotomy is seen following autotomy (Hopkins 1993). The
epidermal cells that line the coax—which is the most proximal segment of the lost leg that remains
attached to the animal’s body—are activated by the autotomy of the limb. At this time, these cells
detach from the coxal cuticle and change shape (Fig. 6.2C).
In insects, epidermal cells of regenerating imaginal discs have lamellipodia or filipodia that reach
toward the cells on the opposite side of the wound. They appear to migrate under the melanized
clot but are pulled together by an actin “purse string” mechanism (as described earlier). As the
filipodia from each group of cells make contact with filipodia cells from the opposite side of the
wound, they form a suture. This closure of the insect epithelium occurs within 18 hours after injury,
and the cells return to their normal shape (Ramet et al. 2002).
The initial re-epithelization of a wound in crustaceans involves only hypertrophy and migra-
tion of existing cells with no mitotic figures evident (Fontaine and Lightner 1973). The migra-
tion of epidermal cells across the plane of autotomy is accompanied by cellular changes that
include increases in chromophilia of nuclei and enlarged nucleoli. These changes suggested to
Adiyodi (1972) some degree of cellular de-differentiation in the crab Parathelphusa hydrodromus,
although Needham (1965) reported that de-differentiation was not extensive following autotomy
in most arthropods. In crustaceans, healing continues with further hypertrophy and migration of
 Regeneration in Crustaceans 177

epidermal cells under the scab. In addition to migration of epidermal cells following autotomy,
there is a marked influx of other nonepidermal, fibroblast-like cells from the coxa, the origins of
which are unclear (Needham 1965).
In Drosophilia, the JUN N-terminal kinase ( JNK) signaling pathway plays a crucial role in
re-epithelization of wounds in embryos, juveniles, and adults. Re-epithelialization of the wound
site probably does not require signals from the invading hemocytes because in Drosophila wing
discs that are injured there is no hemocyte mediation of wound healing (Wang and Xia 2010). The
upstream activating signal for JNK may be an insulin-like compound released by developing and
injured wing discs (Colombani et al. 2012, Garelli et al. 2012). The JNK pathway contains a number
of key gene products that stimulate the expression of other important genes like puckered (puc),
hemipterous (hep), basket (bsk), and kayak (Dfos or kay) that are upregulated in wound epithelia and
thus implicated in the closure and healing of wounds (Ramet et al. 2002, Xia and Karin 2004, Bosch
et al. 2005). Because puc expression is stimulated in tissues surrounding necrotic regions, it has been
suggested that there is some signal from necrotic cells that stimulates the onset of the JNK signaling
pathway in the adjacent uninjured epithelia (Mace et al. 2005, Wang and Xia 2010). The activation
of this cascade in the epidermal cells adjacent to the wound leads to the movement and fusion of the
cells during wound healing in both Drosophila adult exoskeleton and imaginal discs (Ramet et al.
2002, Bosch et al. 2005). In the embryo of Drosophila, the wound epidermis also expresses α-catenin
and E-cadherin (Garcia-Fernandez et al. 2009).

Blastema Formation

In the epimorphic regeneration that follows autotomy and wound repair, a blastema is formed.
A blastema is a mass of undifferentiated cells from which an organ or part can grow (Slack 2003).
During wound healing, the major cellular response is epidermal activation and mitosis, but during
blastema formation, in addition to epidermal activation, there is extensive cellular proliferation and
respecification. Many arthropods that lack the autotomy response are capable of blastema forma-
tion and regeneration of complete limbs. In the case of limb loss by means other than autotomy,
however, wound closure events take a considerably longer time, and these animals have longer
latency periods between limb loss and limb regeneration. In nonautomized wounds, there is tissue
damage, blood loss, and bacterial invasion. In the crab U. pugilator, a limb can be amputated distal to
the preformed breakage plane very soon after molt when the animal is still soft and unable to con-
tract its autotomy muscles. (If the limb were fully hardened, any injury along the length of the limb
would force autotomy at the plane of autotomy.) Wound healing, blastema formation, and regen-
eration can occur in these soft-shelled crabs, and only the amputated portion is regenerated (Wu
et al. 2004). Thus, positional information is found along the entire length of the limb, appears to be
inherent in the blastema itself, and does not rely on information restricted to the plane of autotomy.
The plane of autotomy, with its built-in safety valve, is the most dependable place for limb loss to
occur, but the entire length of the limb is capable of organizing a blastema and regenerating the
missing parts of the lost limb.

Cuticular Secretion

In crustaceans, following the initial wound closure after autotomy, the epidermal cells that have
migrated across the wound surface and under the scab begin to secrete a cuticle. These activated
epidermal cells secrete a thin, cuticle-like layer that helps strengthen the wound closure and fur-
ther seals the coxal gap (Needham 1965, Hopkins 2001). Seven to ten days after autotomy, the
scab drops off and the cuticular seal of the coxal surface is visible. This cuticle is quite flexible
178 Penny M. Hopkins and Sunetra Das

and forms a crenulated sac that expands as the growth of the regenerating limb bud grows. This
cuticular sac is distinct and separate from the cuticle that is secreted by the developing epidermal
shell of the regenerating segments. The cuticular sac is unique to crustaceans and has not been
reported for the insects. The cuticular sac protects the growing, regenerating crustacean limb bud.
It is shed at the time of molt when the newly regenerated but still folded limb is pulled out of the
sac and expanded with blood.

Regeneration of Limbs in Insects

As mentioned earlier, some insects can heal wounds as adults, but regeneration in the more derived
insects is limited to juvenile forms and immature tissues. The legs of hemimetabolous insect nymphs
and naiads regenerate readily, as do the imaginal discs in holometabolous insects (e.g., Drosophila
and Manduca). In the nymph of the hemimetabolous cockroach Periplaneta americana, amputation
of a leg results in regeneration of a new leg. Adult cockroaches that do not normally regenerate
lost limbs will regenerate limbs if parabiosed to a nymph, suggesting that some blood-borne factor
is necessary for regeneration competence (Bodenstein 1955). Following amputation in hemime-
tabolous and immature insects, (i) a new leg forms in the most proximal coxal segment, (ii) degen-
eration of coxal muscles creates a space in which the regenerate develops, and (iii) as regeneration
progresses, more muscles degenerate and pull away from the old exoskeleton to provide more room
for the developing regenerate. The severed leg nerve regenerates into the newly forming limb.

Insect Leg Development Compared to Leg Regeneration

The cricket leg develops from a limb bud, and adult Drosophilia legs develop from imaginal discs.
The molecular cascade that controls development in these structures is thought to be duplicated
during regeneration of these structures. It has been suggested that the regenerating leg uses the
same “molecular boundary” model for signaling during regeneration as is used during development
(Meinhardt 1983). The same signaling pathway mentioned earlier ( JNK) that triggers wound heal-
ing also stimulates epithelial movement and morphogenetic early genes involved in both leg devel-
opment and regeneration in insects. The first step in pattern formation in both development and
regeneration involves the establishment of a posterior/anterior compartmentalization. Activation
of JNK in blastema cells of the imaginal disc is one of the first responses to injury and is neces-
sary for subsequent wound healing and regeneration. Gene regulatory networks that function in
developing limbs also regulate regeneration in imaginal discs. Using RNAi, it was shown that, in
the regenerating leg of the cricket Gryllus bimaculatus, the expression of hedgehog (hh), wingless
(wg), and decapentaplegic (dpp) genes along with epidermal growth factor-related ligands set the
boundaries for posterior and anterior compartments. These compartments will eventually form
the segments of a new regenerated limb (Bergantinos et al. 2010).
In insect leg development, leg segments are sequentially formed by intercalation—starting with
the coxa/pretarsus boundary. The segments are laid down with a proximal segment intercalated
between the most proximal and most distal structures. This same pattern of proximo-distal inter-
calated segmentation is seen during limb regeneration in insects, and specific limb segments can be
identified by segment-specific gene regulatory networks. The patterns of gene expression and sig-
naling molecules can be explained by the molecular boundary model mentioned earlier (Nakamura
et al. 2008). RNAi knockdown of specific regulatory genes—such as armadillo, hedgehog, engrailed,
distaless, efgr, fat, and dachshund—results in defective development and/or leg regeneration due to
lack of positional information with the lack of distal-to-proximal respecification of the regenerate.
The ability of holometabolous insect imaginal discs to regenerate has been known for some
time (Hadorn and Buck 1962, Madhavan and Schneiderman 1969, 1977, Schubiger 1971), and access
 Regeneration in Crustaceans 179

to the Drosophila genome makes these tissues ideal to study the molecular control of regeneration
(Bergantinos et al. 2010). Damage to Drosophila wing discs results in the upregulation of several
genes, the two most important being wingless (wg) and myc (Smith-Bolton et al. 2009). Injured discs
also appear to release an insulin-like peptide that is capable of retarding growth in the rest of the
animal until disc regeneration is complete (Colombani et al. 2012, Garelli et al. 2012). Wing discs,
however, lose their regenerative capacity during the middle of the third instar (Smith-Bolton et al.
2009). The imaginal discs of some insects can actually change fates. Antenna discs that are injured
in vivo by ultraviolet radiation can be transformed so that they will grow into wing discs (McClure
and Schubiger 2007). This change in tissue identity during regeneration is called transdetermina-
tion. Transdetermination may be a widely conserved feature in insect and crustacean regeneration
and is thought to be a result of downregulation of the gene distal-less (dll), which is an important
development gene found in all arthropod limbs (Panganiban et al. 1994, Suzuki et al. 2009).

Regeneration of Limbs in Crustaceans

By 2–4 days after autotomy, most of the granulocyte cells in the area between the two AMs disap-
pear. The hypertrophied immigrant epidermal cells (the wound epidermis) have moved from the
coxal walls and completely underlie the scab in the crabs Parathelphusa and Uca (Adiyodi 1972,
Hopkins 1993). The highly activated wound epidermis of the crab secretes a thin cuticle between
the scab and itself and begins mitotic activity. Mitosis in crustacean blastemas (as measured by triti-
ated thymidine incorporation) increases 4 days after autotomy (Hopkins 1989). This first peak of
mitotic activity corresponds to the initial invagination of the epidermis to form the walls of devel-
oping segments in the crab. The formation of these epidermal foldings is the first sign of segmen-
tation and differentiation in the nascent limb bud. By 6–7 days after autotomy, the blastema has
proliferated so much that it pushes aside the scab from the face of the coxa. At this point in develop-
ment, the regenerating structure is called a papilla. The papilla is a small structure that consists of a
single segment with no internal substructure.
A second peak of mitotic activity accompanies the emergence of the papilla. This second, large
peak of mitotic activity corresponds to the formation of further limb segments and within 1–2 days
transforms the papilla into a small bud (a basal bud). Mitotic figures are first seen in the crustacean
epidermis, then in the more internal cells. The mitotic growth of the bud continues for a few more
days and then ceases (Bliss 1956; Fig. 6.3). The differentiation of the segmented exoskeleton appears
to occur solely during this early basal growth phase and is complete by the end the first 2 weeks of
basal growth.
In insects, regenerating leg segments are formed by intercalation, but differentiation of crusta-
cean segments occurs sequentially. The direction of differentiation in crustacean segments appears
to vary from group to group. In some crustaceans, regeneration of segments lacks any orientation
or they have opposite orientations, but in Uca the segments form in a proximo-distal sequence
beginning with the formation of the merus segment with further addition of more distal segments.
As the papilla emerges from the coxa and it begins to differentiate into a basal bud, there is asym-
metrical growth: one side of the developing limb bud grows faster (i.e., has more mitotic figures)
than the other (Adiyodi 1972). Asymmetrical growth causes the small basal bud to bend in upon itself
so that, as new segments are added to the regenerate, they are folded upon one another. The basal
bud is a folded structure enclosed inside the cuticular sac secreted earlier (Needham 1965; Fig. 6.4).
A comparably folded regenerating limb is also seen in a few insects. In some crustaceans such as the
macrurans, the limb buds are more elongated and less folded. There is some bending of the regen-
erating macruran limb toward the body, but the regenerate is not tightly folded within a sac as it is in
the brachyurans. The crayfish Orconectes obscurus retains the fully extended regenerating limb bud
within the coxa of the old limb and liberates the fully formed limb only upon molt.
 MOLT CYCLE
SUBSTAGES C4 D0 D1 D2 D3 D4 E

TERMINAL
25 PLATEAU
E
20
R - VALUES

15 PROECDYSIAL
BASAL GROWTH
PLATEAU
10

5
BASAL
GROWTH
0
Auto Emergence

Fig. 6.3.
Diagrammatic representation of the growth curve from a single walking leg from the crab Uca pugilator. The
large arrow at bottom left represents the point at which autotomy (Auto) occurred. The second large arrow to
the right is the point at which the regenerating bud emerged from the coxa as a papilla (Emergence). At the top
are Drach’s (1939) stages of proecdysis, with (Travis 1960) additional D0 stage, which is characteristically found
in crabs undergoing regeneration. In the center are four drawings of the limb bud at the various stages. From
left to right, the pictures depict the scab covering the coxa, a papilla emerging from the coxa, a basal bud, and a
PE bud. To the far fight is a complete walking leg. R-values are measurements of a regenerating right third (R3)
walking leg (calculated by measuring the length of the bud and dividing it by the width of the carapace then
multiplying that by 100). The solid line is a representative plot of the R3 values (y-axis) as a function of time
(x-axis) after autotomy. From Hopkins (1993), with permission from Oxford University Press.

A B
Dactylus
Propus
Cuticular
Sac Carpus

Merus
Basioischium
Coxa

Proecdysial Fully Developed

Limb Bud Limb

Fig. 6.4.
(A) Drawing of regenerating proecdysis limb bud from the crab Uca pugilator, folded and surrounded by a
cuticular sac. (B) The same newly regenerated limb after ecdysis. Individual segments are labeled. Redrawn
from Hopkins (1993), with permission from Oxford University Press.
 Regeneration in Crustaceans 181

Muscle Regeneration

The origin of new muscle tissue in the crustacean regenerating limb is unclear: the immigrant epi-
dermal cells appear to be the main source of epidermal cells in the regenerated limb bud, but there
is some question as to the origin of the other blastemal tissues (Bodenstein 1955, Skinner 1985).
There is some evidence that muscle tissues regenerate metaplastically from local epidermis, but
other reports suggest that muscle tissues arise from immigrant cells moving up from the base of the
coxa—which is the site of the large muscles that control the walking legs. Myobundles form from
cells that may be de-differentiated cells from a variety of sources, but they may also be reserve cells
located at distant points. Whatever the source, the regeneration of muscle tissue lags behind that
of segmentation by 1–2 days (Adiyodi 1972), so that the first appearance of the blastema is one of
an epidermal shell that later fills with immigrant cells that organize into myobundles (Needham
1965, Hopkins and Durica 1995). Some of the cuticular in-growths in the crustacean blastema aris-
ing during the second peak of mitotic activity form the cuticular attachment sites (apodemes) for
developing tendons of flexors and extensor muscles (Hopkins 2001). There is also a nonuniform
development of muscle fibers that is thought to be a function of variable innervation due to a vari-
able branching pattern of regenerating nerves (Lang et al. 1980).

Nerve Regeneration

As the segments develop in crustaceans, regeneration of the severed pedal nerve begins. The cut
surface of the pedal nerve is held in close proximity to the developing blastema by the attachments
of the proximal AM to the pedal nerve sheath. The regenerating nerve grows as an elongating cone
into the developing segments as they sequentially form (Hopkins and Durica 1995). It is assumed
that the pedal nerve regenerates to form the motor input to the new limb, but the source of sensory
neurons is unclear. It has been suggested that new sensory neurons differentiate from the immigrant
hypodermal cells (Skinner 1985), but the exact source of sensory nerve cells (as well as the chro-
matophore cells) is unknown.

Differentiation of the Crustacean Limb Bud

When the limb blastema emerges from the surface of the fracture plane as a papilla, it is poorly
differentiated and consists of a single segment. The length of time between autotomy and papilla
emergence depends on the same variables that determine intermolt duration. Papilla emergence
can occur at any time during the intermolt stage but is inhibited if the animal has begun prepara-
tions for molt or if exogenous molting hormones (the ecdysteroids) are administered during this
time. After papilla emergence, segmentation of the forming limb bud is evident, and the papilla
becomes a basal bud (Fig. 6.3). In the basal bud, the cuticularized segments become quite apparent.
Some organized tissues are evident within the cuticular shells, but the segments are primarily filled
with masses of what appear to be undifferentiated cells and early myocytes (Adiyodi 1972, Hopkins
et al. 1999).
During the “basal bud growth” period, organization of the tissues continues with differentia-
tion of specialized chromatophore cells, cuticular elaborations, and further muscle organization.
Proliferation (or DNA replication) also continues during basal growth, but mitosis slows down by
about 12 days after autotomy (Hopkins et al. 1999). Differentiation of the segments is pretty much
completed during the basal growth period, and the bud is encased inside the flexible cuticular sac
that expands as the bud grows. Basal growth ceases about 10–14 days after emergence and, there-
after, the small basal bud stays the same small size. This period when basal growth ceases is called
basal plateau (Fig. 6.3; Bliss 1956, Adiyodi 1972). During basal plateau, proliferation stops, epidermal
182 Penny M. Hopkins and Sunetra Das

cells secrete the secondary cuticle, and myofibrils appear within the relatively empty center of the
forming segments. Myofibrils occur in clusters attached to apodemes and are surrounded by gran-
ular sarcoplasm. Functional synapses are present in regenerating basal buds of the crab Grapsus
grapsus following basal growth, and a new AM forms. Basal buds of the crab Gecarcinus lateralis are
capable of an autotomy response (Skinner 1985). The basal bud remains small even after attaining
some degree of differentiation. This small basal bud remains folded and sequestered between the
coxa of the other, nonregenerating limbs so that the crab can participate in the foraging and repro-
ductive activities characteristic of the nonmolting periods.

Hypertrophic Growth of the Crustacean Limb Bud

As the crab that is regenerating a new limb enters preparations for molting, the small basal limb
bud begins a secondary spurt of growth that occurs only during the period just before molt called
proecdysis (PE or premolt). This growth spurt is called proecdysial growth (Fig. 6.3; Bliss 1956). This
growth is almost entirely hypertrophic, with the size of the bud reaching as much as a threefold
increase in size due primarily to muscle protein synthesis and water uptake (Hopkins et al. 1999).
During the last few days prior to ecdysis/shedding, the regenerated bud does not grow at all. This
cessation of growth is called terminal plateau of regeneration (Fig. 6.3; Bliss 1956).

Crustacean Leg Development

Hox gene expression and its correlation with appendage morphology have been well studied
in arthropods (Averof and Patel 1997, Shiga et  al. 2006). The hox genes, first discovered in
Drosophila, specify segment identities along the anteroposterior axis of both vertebrates and
invertebrates. Crustaceans have diverse body plans, with limbs developing in abdominal, tho-
racic, and head segments, whereas in insects, limb development is restricted to the thoracic and
head segments. In Drosophila, one of the earliest genes to be activated in the limb primordia is dll
(Cohen et al. 1989). This gene specifies the distal region of the limbs (Panganiban et al. 1994),
and the distal structures are not formed in absence of this gene (Cohen and Jurgen 1989). It has
been proposed that the variation in arthropod limbs may result from variation in dll regulation
(Panganiban et al. 1995).
Homeotic gene products like ubx (ultrabithorax) and abdA (abdominal A) regulate limb num-
ber. There is a significant difference in expression of ubx, abdA, and dll between insects and
crustaceans. In crustaceans like Artemia, the genes ubx/abdA and dll are co-expressed in thoracic
limb primordial, whereas in insects, ubx/abdA represses dll in abdominal segments, which in
turn inhibit limb development in that region (Pavlopoulos and Averof 2002). Studies conducted
in various crustaceans (Branchiopoda, Maxillopoda, and Malacostraca) have shown that the
hox gene ubx is usually expressed in the thoracic segments that are associated with locomotory
appendages and excluded from thoracic segments that bear maxillipeds (Averof and Patel 1997).
In Parhyale hawaiensis, a malacostracan amphipod, RNAi-mediated knockdown of ubx results in
transformation of multiple walking legs into maxilliped-like appendages (Liubicich et al. 2009).
This suggests that ubx plays a role in distinguishing posterior thoracic appendages from the more
anterior maxillipeds.
Further studies to decipher the function of hox genes in crustaceans during early limb develop-
ment will lead to identification of downstream genes necessary for this process. These genes in
turn might be activated during regeneration of limbs in adult crustaceans. The crustacean genes
mentioned here have been found in a wide variety of developing limbs from vertebrates to inverte-
brates, and the argument has been made for an “evolutionarily conserved mechanism of leg pattern
formation from protostomes to deuterostomes” (Nakamura et al. 2008, Suzuki, et al. 2009).
 Regeneration in Crustaceans 183

CONTROL OF REGENERATION IN CRUSTACEANS

Many factors have been reported to either hasten or inhibit regeneration in crustaceans and
insects: (i) wound factors, (ii) local tissues and nerve supply, plus (iii) environmental conditions
all exert some influence upon the extent and rate of regeneration. And, as mentioned earlier, (iv)
circulating ecdysteroid hormones can exert profound effects on the rate and nature of regeneration.

Wound Factors

In crustaceans, there is little evidence to indicate what (if any) factor(s) stimulate the recruitment
of blood cells into a damaged area or the activation and migration of the epidermal cells immedi-
ately following injury. A cytokine has been identified in insects that is released by wounded epi-
dermal and hemocyte cells and acts as a chemotactic agent to attract hemocytes to the wound in
the moth Pseudaleta separata (Nakatogawa et al. 2009). In crustaceans, however, there is probably
more than one message released from damaged cells because (i) hemocyte recruitment processes
are similar following limb amputation (with massive tissue damage) and autotomy (with minimal
tissue damage) and (ii) there is epidermal activation and massive influx of hemocytes into a region
where only the exoskeleton is damaged with no disruption or injury to the underlying hypodermal
cells (Vafopoulou et al. 2007). Damaged imaginal discs from the fruitfly Drosophila may release a
retinoid message into the blood that helps modulate ecdysteroid levels in the blood (Halme et al.
2010). Such a blood-borne signal could also serve as an in situ regeneration factor, as has been
recently shown in the developing chick limb bud where retinoic acid can serve as a diffusible devel-
opmental signal for proximodistal organization (Rosello-Diez et  al. 2011). Damaged tissues may
produce a signal that elevates juvenile hormones ( JH) levels (or JH sensitivity) in some insects
(Krishnakumaran 1972). In the flour beetle, Tribolium castaneum, RNAi knockdown of the dll gene
caused sufficient damage to developing limbs to produce extra larval molts, which the authors sug-
gest was due to elevated JH levels because the phenotype could be rescued with RNAi to an enzyme
that produces JH ( JH methyl transferase) and a putative JH receptor (methoprene tolerant [met];
Suzuki et al. 2009). As mentioned earlier, chemical signals released at a wound site in the crayfish
P. clarkii may serve to recruit hemocytes and may interact with the endocrine system to influence
ecdysteroid levels (Vafopoulou et al. 2007).

Local Tissue Effects

Another important control of the regeneration process is that of local tissues and positional infor-
mation. Regeneration of transplanted homoeotic legs in the crayfish P.  clarkii is affected by the
host site. Cells adjacent to a wound or regeneration site retain positional information (Mittenthal
et al. 1980). Regeneration of the uropod in the isopod A. aquaticus can be blocked by excising the
base of the limb proximal to the autotomy plane (Needham 1965). Regeneration in legs from the
crayfish P. clarkii restores the continuity of that positional information (Mittenthal and Trevarrow
1983). Claw buds transplanted from male to female Uca pugnax crabs acquire after three molts
some morphological characteristics (shape and sarcomere length) intermediate to both donor
and recipient. Also, claws regenerated from a coxa onto which a transplanted claw had been previ-
ously attached and then removed prior to the regeneration showed some donor-like characteristics
(Trinkaus-Randall 1982). Such an effect can be explained by a transfer of donor tissue with the first
transplant. Thus, when the new regenerate grows, the transferred donor tissue exerts some effect
on the morphological determination of that new regenerate. It must be stressed again, however, that
positional information is found along the entire length of the crustacean limb and is not unique to
the plane of autotomy (Wu et al. 2004).
184 Penny M. Hopkins and Sunetra Das

Local Nerve Supply

Epimorphic regeneration in all animals that produce blastemas is nerve dependent (Singer 1952,
Kumar et al. 2007). Needham (1945) demonstrated in the isopod A. aquaticus that the presence
of some intact nerve is necessary for early stages of regeneration. Bodenstein (1955) was unclear,
however, as to the importance of intact nerve input into regeneration in the cockroach. In the lob-
ster Homarus americanus, there is some indication that developing claw muscle fiber properties are
determined by innervation (Lang et al. 1980). The shrimps Alpheus heterochelis and A. armillatus
have two asymmetrical claws. One is a large snapper, and the other is a smaller pincer. If the snap-
per is removed, a nonsnapper (pincer) will regenerate in its place, and the contralateral pincer will
transform into a snapper within two molts (Mellon and Stephens 1978). Severing of the snapper
claw nerves will accomplish the same transformation. The two claws also differ in motor nerve size
as well as muscle size. The switch in development is mediated by elements within the limb nerves
(Mellon 1981). The pesticide dichlorodiphenyltrichloroethane (DDT) causes repetitive neuronal
discharges in crustacean nerves (Gordon and Welsh 1948). When DDT is placed in the holding
water of the crabs U. pugilator and U. pugnax, the growth rate of regenerating limbs is hastened
(Weis and Mantel 1976). It has been suggested in the crab U. pugilator that the pedal nerve that is
severed at autotomy releases growth factors that initiate early events in limb blastema organization
(Hopkins 2001).

Environmental Effects on Regeneration

In many autotomy-induced regenerations, environmental factors have been shown to exert an influ-
ence. Light conditions were shown to affect PE regeneration in the crab G.  lateralis (Bliss 1956,
Bliss and Boyer 1964)  and the crayfish Orconectes virilis and Procambarus clarkii (Stephens 1955,
Aiken 1969). Temperature also exerts an effect on PE regeneration in the crabs Gecarcinus, Uca, and
Pachygrapsus (Passano 1960, Rouquette and Vernet-Cornubert 1964, Bliss and Boyer 1964), and the
crayfish Faxonella clypeata (Mobberly 1963). Lack of privacy or crowding has an inhibitory effect on
regeneration rates in the crabs Ocypode macrocera, G. lateralis, and U. pugilator (Rao 1965, Skinner
and Graham 1972, Weis 1976). Low temperature slows basal growth in the crabs U.  pugnax and
O. macrocera (Passano 1960, Rao 1965). Starvation does not slow regeneration in the crabs O. mac-
rocera and U. pugilator (Rao 1965, Weis 1976) or in the cladoceran D. carcinata (Agar 1930). Addition
of inorganic phosphate to the external medium of the isopod A. aquaticus, however, results in larger
postmolt regenerates (Needham 1947). It is believed that most of these environmental effects are
mediated by the central nervous system and effected by the endocrine system.

Hormonal Control of Regeneration

Ecdysteroid hormones—20-hydroxyecdysone (20E) and ponasterone A  (PA)—are the main

effectors of molt in arthropods. As early as 1955, Bodenstein concluded that the prothoracic glands
(PGs) of insects that produce ecdysteroids and control molting also control regeneration. Early
experiments with parabiosed nymphs and adults and with reimplantation of PGs into adult cock-
roaches (P. americana) showed that the ability to regenerate (and to molt) could be reinstated by
a hormonal signal from the PGs, and the receptors for ecdysteroid hormones are present in regen-
erating tissue throughout limb regeneration in the crab U.  pugilator (Hopkins et  al. 1999). The
putative source of ecdysteroids in crustaceans are two epidermally derived structures called the
Y-organs (YOs) that are located in the anterior cephalothorax (Lachaise et al. 1993).
Exogenously administered ecdysteroids can (under certain circumstances) induce proecdysis
and ecdysis in some crustaceans, but the effects of exogenous ecdysteroids on limb regeneration
 Regeneration in Crustaceans 185

are quite variable (Krishnakumaran and Schneiderman 1969, 1970, Lowe et  al. 1968, Flint 1972,
Charmantier-Daures 1976, Bazin 1977, Gilgan et al. 1977). The effect of ecdysone and 20E on early
stages of regeneration (blastema formation through papilla emergence) is inhibitory in the crabs
U. pugilator and G. lateralis (Rao 1978, Hopkins et al. 1979). Ecdysteroids are also inhibitory to PE
limb-bud regeneration in crabs C. maenas, Pachygrapsus marmoratus, Rhithropanopeus harrisii mega-
lopa; in the crayfish Procambarus sp.; in the lobster H. americanus; and in the isopod P. dilatus. PE
growth in the crab U. pugilator and basal growth in the isopod H. brevicornis are accelerated by exog-
enous ecdysteroids (Krishnakumaran and Schneiderman 1970, Flint 1972, Hoarau 1973, Noulin and
Maissaiat 1974, Charmantier-Daures 1976, Bazin 1977, Rao 1978, McConaugha and Costlow 1980).
Furthermore, in the crab G. lateralis, PE limb-bud growth was unaffected by exogenous ecdyster-
oids (Skinner and Graham 1972, Hopkins et  al. 1979). Injected, labeled ecdysteroids are cleared
from crustacean hemolymph very quickly (McCarthy 1982). In the crab C. maenas, 80% of labeled
injected hormone was eliminated from the circulation 4 h after injection. By 24 h, however, cir-
culating levels of unlabeled steroid rose, suggesting that the crabs own source of ecdysteroids was
stimulated by the injection (Adelung 1964). Injected ecdysteroids may be quickly eliminated and
may exert their effects indirectly through feedback at the YOs. The extremely variable results of
exogenous ecdysteroid treatment may reflect variable sensitivity of the YOs and target tissues to
stimulation and/or variable rates of elimination.
In crustaceans, the last phase of regeneration (PE growth) occurs only in animals that are get-
ting ready to molt, which suggests a close relationship between the control of molting and the con-
trol of regeneration. Bliss (1956) pointed out that the rapidly growing crustacean limb bud was an
excellent external signal for the beginning of the PE stage of the molt cycle. The earliest stage of
proecdysis (stage D0 in Fig. 6.3) coincides with the onset of the PE growth phase of crustacean limb
regeneration. In the crabs C. maenas and U. pugilator, the transition from basal to PE growth coin-
cides with a small, transient peak of circulating ecdysteroids (Adelung 1971, Hopkins 1983). This
small peak marks the transition from molt stage C4 to stage D0. This temporary peak is followed
by an obligatory drop in circulating ecdysteroids. If the levels of circulating ecdysteroid does not
drop, PE growth of the bud does not occur (Hopkins 1983). It is thought that the transient rise is the
signal that the animal is getting ready to molt, but the presence of regenerating limb buds forces a
temporary reversal of that increase to allow the limb buds to complete the PE phase of regeneration.
PE growth of limb buds is rapid and can occur within 7–10 days. During PE growth, the size
of the limb bud can increase threefold. PE growth of the bud terminates with the rise in ecdyster-
oids that is necessary for apolysis of the carapace and the actual molt—during stages D1–4. The rise
in circulating ecdysteroids that is necessary to coordinate the molt event is inhibitory to further
growth of the limb bud. Bodenstein (1955) suggested that a molting hormone titer high enough to
inaugurate molting is too high to support regeneration. All phases of regeneration in crustaceans—
blastema formation, differentiation, and early and late growth—are inhibited by elevated levels of
20E (Hopkins 1983, Hopkins et al. 1979). The presence of regenerating tissue actually feeds back to
inhibit increases in circulating ecdysteroids and to maintain the low ecdysteroid levels necessary for
regeneration of limbs (Skinner 1985, Yu et al. 2002).
Wounding of the carapace of the crayfish P. clarkii results in the onset of a molt much like the
loss of a limb (Vafopoulou et al. 2007). When the carapace is wounded, there is the induction of a
new molt cycle that is similar to the cycle induced by eyestalk removal. Both groups of animals molt
by 55 and 50 days, respectively. The increase in ecdysteroids in the hemolymph in wounded ani-
mals, however, is quite different in the wounded versus the eyestalkless groups of animals. Eyestalk
removal results in a steady increase in circulating ecdysteroids until PE peak approximately 2 weeks
prior to the actual molt. In wounded animals, the final PE peak occurs only a week prior to ecdysis.
The difference in circulating ecdysteroid levels in a wounded animal is a delay in the rapid increase
of ecdysteroids. Following wounding, the circulating ecdysteroids rise, but only to basal levels, and
186 Penny M. Hopkins and Sunetra Das

then stay at those basal levels for 10–12 days. After the wound is healed, the ecdysteroid levels begin
to rise to PE levels (as in the eyestalkless animals), and the wounded animals almost catch up with
the cycles of the eyestalkless animals. This suggests that there are blood-borne signals from any
wounded epithelium that may have effects on the molting hormone production in crustaceans. The
signals allow for basal levels of ecdysteroid production that are necessary for wound healing (and
basal growth of regenerating limbs) but inhibit the surge of ecdysteroids that are necessary for the
actual ecdysis.
In crustaceans with intact eyestalks, there is a rise in circulating ecdysteroids shortly before
ecdysis (during PE): about a week prior to ecdysis, a series of large peaks of circulating ecdyster-
oids are seen (Hopkins 1983). The large peaks at the end of PE occur at (and probably induce) the
terminal plateau (or the cessation of regenerating limb-bud growth). Thus, in intact crabs (specifi-
cally, those retaining both eyestalks and missing only one walking leg), the periods of active basal
and PE limb-bud growth are correlated with periods of low circulating ecdysteroids during which
the predominant ecdysteroid varies (Hopkins 1992). The bulk of the information in the literature
suggests that injected (or infused) ecdysteroids are mostly inhibitory to all stages of regeneration
in crustaceans despite the fact that ecdysteroid receptors are present throughout these stages. This
underscores the importance of the fine control we see in the levels of ecdysteroids during regenera-
tion in both insects and crustaceans.
Insect limb regeneration is also linked to molting and ecdysteroid levels. Most adult insects,
however, do not molt because the PGs in many adult insects degenerate after the last juvenile molt.
Adult insects that do not molt may autotomize a limb for defense but very rarely regenerate the lost
structure. When adult cockroaches (Leucophaea maderae and P. americanus) that normally do not
regenerate are attached by parabiosis to juvenile nymphs and an adult appendage is removed, the
adult will regenerate the limb, but after a much longer period of time. Imaginal discs from Drosophila
will also regenerate after damage (by cutting or irradiation) but lose their ability to regenerate dur-
ing the last part of the third instar when ecdysteroid levels are rising as the larva prepares for pupari-
ation. The wg gene, which is necessary for disc regeneration, is repressed by ecdysteroids (Mitchell
et al. 2008, Smith-Bolton et al. 2009). Regeneration of imaginal discs in Drosophila also inhibits the
production of ecdysteroids by reducing the expression of the ptth gene that is responsible for the
activation of ecdysteroidogenesis by the PGs (Halme et al. 2010). This reduction of ecdysteroid
levels delays the onset of pupariation and allows for full regeneration of the discs.
Similarly, development of imaginal discs is sensitive to ecdysteroid levels. Discs will only pro-
liferate when implanted into adult abdomens, but will differentiate, metamorphose, and prolifer-
ate when implanted into third-instar larvae body cavities. These experiments suggest that there is
a blood-borne factor that allows competency and is found only in larval insects. A candidate for
such a factor may be the terpenoid JH that is the epoxidated form of retinoic acid. JH has been
reported to suppress morphogenetic growth in imaginal discs (Truman and Riddiford 1999), and
disc sensitivity to JH appears to be lost during larval development (Madhavan and Schneiderman
1977). In basal insects, JH is not present when appendages begin to grow but reappears as nymphal
differentiation occurs. In the flour beetle, T.  castaneum, RNAi knockdown of the leg patterning
genes results in loss of larval appendages and the prevention of the onset of metamorphosis (Suzuki
et al. 2009). Partial de-differentiation of appendages in insect larvae maintains high JH levels to
prevent metamorphosis. In vitro evidence with eye imaginal discs from the moth Manduca sexta
showed that the cell fate of these discs requires a “tonic presence” of ecdysteroids but at levels too
low to activate the ecdysteroid cascade that leads to metamorphosis/molt (Champlin and Truman
1998). Transplanted Drosophila imaginal discs grow more robustly in the abdomen of a female
adult than in a male abdomen. Ecdysteroid levels in adult females are low but are not detectable
in adult males (Madhavan and Schneiderman 1969). Growth in the male abdomen is enhanced
with co-transplantation of a ring gland (containing the PG) or injection of ecdysteroids. In vitro
 Regeneration in Crustaceans 187

imaginal discs from the fleshfly, Sarcophaga peregrina, in the presence of 2.5 × 10–8 M 20E discs, will
form a blastema, yet in the presence of a higher dose (1 × 10–6 M 20E) these same discs will begin
to differentiate and elongate. The concentration of ecdysteroid was critical for the correct develop-
mental response of this tissue (Kunieda et al. 1997). Wing discs in Drosophila that require ecdyster-
oid for cell determination will develop normally in the absence of steroid if the Ultraspiracle (USP)
partner for ecdysteroid receptor (EcR) is missing (Schubiger and Truman 2000). This suggests that
during normal larval development, the USP/EcR complex might inhibit development so that the
presence of ecdysteroids releases the suppression, and/or a ligand for USP may function to modu-
late the activity of EcR. Regeneration and development require low levels of ecdysteroids (as seen
in the crustaceans), and the low levels of ecdysteroids that support regeneration are insufficient to
trigger ecdysis. These data suggest that ecdysteroids subserve different functions at different con-
centrations during development. Low levels of ecdysteroid are critical for differentiation, whereas
higher levels of the same ecdysteroid are necessary for growth.
The effects of ecdysteroids are mediated by a gene activation cascade that has been known for
some time (Clever and Karlson 1960, Ashburner 1973). The genes of the ecdysteroid cascade are
ideal candidates for coordinating downstream processes of tissue patterning and morphogenesis
(Truman and Riddiford 2002). Ecdysteroids exert their effects by interacting with specific nuclear
receptors. The functional receptor for ecdysteroid hormones has been isolated and cloned for both
insects and crustaceans (Koelle et al. 1991, Guo et al. 1998, Chung et al. 1998) and is found in abun-
dance in limb bud tissues during regeneration in the crab U. pugilator (Chung et al. 1998). mRNA
and protein for the functional receptor are expressed in regenerating limb buds during early blas-
tema formation and during the PE stages (Fig. 6.5; Chung et al. 1998, Hopkins et al. 1999, Wu et al.
2004). The dimerization partner of the EcR is the retinoid X receptor (RXR), which is the homo-
logue of USP in insects. There is an 88% deduced amino acid identity (>97% similarity) between
the insect EcR and the crustacean EcR. High levels of crustacean EcR mRNA and RXR are found in
limb buds when circulating levels of ecdysteroids are lowest. Knocking down the EcR heterodimer
complex during basal growth phase in the crab U. pugilator disrupts formation and growth of the
limb buds. Simultaneous injection of EcR and RXR RNAi into early limb blastemas does not affect
migration of epidermal cells into the area but does block the proliferation of the blastema. Taken
together, these data suggest that ecdysteroid signaling plays a role in maintaining growth and sub-
sequent differentiation of regenerating limbs in this brachyuran species (Das and Durica 2013) but
only at relatively low concentrations.
The in vitro affinity for ecdysteroids of the Uca EcR/RXR complex is affected when putative
ligands for RXR are present (Hopkins et al. 2008). One such ligand is 9-cis-retinoic acid (9cRA),
which is an endogenous compound found in the developing blastema of Uca (Hopkins et  al.
2008) and has been shown to disrupt blastema organization/development in Uca. The presence
of retinoids in the developing blastema may be multifold in (i) controlling tissue respecification
(Hopkins and Durica 1995); (ii) “fine-tuning” receptor affinity so that the tissues can respond to
the obligatory low levels of circulating ecdysteroids, with lower affinity receptor conformations set
aside to control physiological responses related to molting of the carapace when circulating ecdys-
teroid levels are highest (Hopkins et al. 2008); and (iii) acting as a blood-borne signal for control-
ling other hormone levels that in turn control regeneration (Halme et al. 2010).

The Role of the YO

Ecdysteroids are thought to be produced solely in the YOs of crustaceans (see Lachaise et al. 1993).
In the isopod P. dilatatus, the YOs are necessary for both basal and PE growth (Noulin and Maissiat
1974). Likewise, it was reported for the crab C. maenas that intact YOs were necessary for both basal
and PE growth (Echalier 1956). Other work on this same crab suggests that basal growth can occur
188 Penny M. Hopkins and Sunetra Das

Total RIA-Active Material

180

in Hemolymph (pg/µl)

R3 Values of Limb Buds

C4 25
160 D0 D1-4
140 Ecdysteroids 20
120 R3 values
100 15
80 10
60
40 5
20
0 0
25 20 15 10 5 E
B Days before ecdysis (E)
0.6
0.5 *
Regenerating limb buds
EcR mRNA
abundance

0.4
0.3
0.2
0.1 nd
0.0

Fig. 6.5.
Distribution of UpEcR mRNA in limb buds from the crab Uca pugilator during late anecdysis (stage C4)
and proecdysis (stages D0 and D1–4). The solid line in the upper panel (A)  represents levels of ecdysteroid
(RIA-active material) in hemolymph during the molt cycle, matched backward from ecdysis. The dotted line
in (A) represents the same growth curve of an R3 walking leg as shown in Fig. 6.3. The bottom panel (B) rep-
resents the amounts of mRNA extracted from pooled limb buds corresponding to points in the top panel (A).
From Chung et al. (1998), with permission from Elsevier.

in the absence of YOs, but PE growth cannot (Bazin and Demeusy 1972, Demeusy 1973). The crab
Sesarma reticulatum can complete basal growth without YOs but cannot proceed into PE ( Jyssum
and Passano 1957, Passano and Jyssum 1963). The crabs P. marmoratus, Pilumnus hirtellus, and G. late-
ralis can complete both basal growth and PE growth without intact YOs (Charmantier-Daures and
Vernet 1974, Charmantier-Daures 1975, Demeusy 1982, Skinner 1985). In some YO-less crabs, the
titers of ecdysteroids rise during PE (Charmantier-Daures and DiReggi 1980). Thus, the YOs are
not indispensable to the production of ecdysteroids.

EFFECTS OF REGENERATION ON THE CRUSTACEAN

MOLT CYCLE AND GROWTH

Regeneration in larval insects delays the onset of a subsequent larval molt (O’Farrell and Stock
1953) and can accelerate subsequent molts in adult firebrats (Buck and Edwards 1990). It was suggested
that regenerating tissues were capable of reducing the concentration of molting hormone, and it has
recently been shown that regenerating imaginal discs in Drosophila can inhibit the release of a protho-
racicotropic hormone from the brain and thereby lower ecdysteroid production (Halme et al. 2010).
Yet full regeneration requires at least a basal level of ecdysteroids (Madhaven and Schneiderman 1969).
Animals undergoing regeneration of many limbs have smaller postecdysial carapace dimensions
(Bennett 1973, Kuris and Mager 1975, Hopkins 1982, Barría and González 2008) and smaller post-
ecdysial limbs (Skinner and Graham 1972, Fingerman and Fingerman 1974, Hopkins 1982). The
increase in size of the postecdysial carapace is due to (i) inflation and stretching of the new cells
of the hypodermis (Bliss et al. 1966) and (ii) increases in size and number of hypodermal cells.
Part of the increase in postecdysial carapace width is due to water uptake at molt (Bliss and Boyer
l964). One effect of regeneration on postmolt carapace size is mediated through the mechanical
distribution of this water. There is a hormonally fixed amount of water that is taken up at molt.
 Regeneration in Crustaceans 189

At molt, newly regenerated limbs that have remained folded throughout PE unfold and become
inflated. If the amount of water taken up at molt is limited, then the added volume of many legs
to be inflated will decrease the amount of water available to inflate the carapace itself. However,
Kuris and Mager (1975) were unable to demonstrate an increase in size in crabs Hemigrapsus orego-
nensis or Pachygrapsus crassipes when large limb buds were removed immediately prior to ecdysis.
Eyestalkless Uca increase in carapace width at molt much more than intact crabs (Abramowitz and
Abramowitz 1940). This effect may be due to the loss of eyestalk hormones that restrict the amount
of water taken up at molt. Regeneration of many limbs decreases this carapace growth in eyestalk-
less crabs by the same proportions that it reduces it in intact U. pugilator (Hopkins 1982), thus sug-
gesting that whatever the volume of water taken up at molt, the requirement of inflating additional
limbs reduces the mechanical aspects of growth at molt.
Regenerated limbs themselves are also smaller than unregenerated limbs from the same ani-
mals. Tchernigovtzeff (1965) reported that in the shrimp Palaemon and crab Carcinus there is a
large increase in mitotic figures in the carapace during PE. However, in the crab G. lateralis, there is
no evidence of mitotic activity during PE in integument from the branchiostegites (Skinner 1962).
In G. lateralis, a single regenerate is 75% of the size of a nonregenerated limb, whereas when sev-
eral limbs are autotomized, the postecdysial limbs are 50% of controls (Holland and Skinner 1976).
Regenerated limbs in the spider crab Chionoecetes opilio are 48% of their former size (Miller and
Watson 1976), and regenerated limbs of the large crab Paralithodes camtschatica are 35% (for juve-
niles) and 29% (for adults) of nonregenerated legs (Niwa and Kurata 1964). A second restriction on
limb growth may be stored metabolic resources.
In Uca, regenerated walking legs from crabs missing eight legs are 68% the size of controls
(Hopkins 1982). Postecdysial regenerated limbs also contain only 38% of the protein of control
limbs. Gecarcinus and Uca can regenerate only a fixed amount of protein per molt cycle (Skinner
and Graham 1972, Hopkins 1982). This protein is shared by all regenerating limbs. The more limbs
that are missing, the smaller the proportion of protein per limb. The amount of protein in a newly
regenerated limb is reduced even further if autotomy of many limbs is coupled with eyestalk abla-
tion (Hopkins 1982). When several limbs are autotomized during PE induced by eyestalk removal,
there is a prolongation of PE to allow for the organization of the limb bud and subsequent growth
prior to ecdysis. There is an incomplete growth of these buds. They grow less during PE, and the
molted limbs are smaller with much less protein.
The crab U. pugilator not only has a low threshold for molt induction, but there is also a graded
effect: the more limbs that are lost, the sooner the onset of PE (Fingerman and Fingerman 1974).
Once PE begins in Uca, it is of the same duration in crabs missing two as it is in crabs missing eight
limbs (Hopkins 1982). The effect of autotomy in Uca is then to hasten the onset of PE but not the
duration of PE itself. In the crab P. marmoratus, loss of eight walking legs reduces the actual length of
PE from 40 to 34 days. If limb losses occur during PE, there is an increase in the duration of PE. This
effect is seen whether limb loss occurs during a normal PE or during an induced PE (Fingerman
and Fingerman 1974).
In many species of brachyurans, loss of multiple limbs during intermolt hastens the onset of PE
in order that the missing limbs can be replaced as soon as possible. It was first shown in the crab
G. lateralis and later in the crab P. marmoratus that removal of several walking legs is sufficient to
induce PE in intermolt animals (Bliss et al. 1966, Vernet-Cornubert 1961). The sensitivity of animals
to autotomy-induced PE varies from group to group. In the xanthid crab P. hirtellus, loss of a single
limb is sufficient to hasten PE, whereas in U. pugilator, loss of at least two walking legs is necessary
(Fingerman and Fingerman 1974, Demeusy 1982, Hopkins 1982). In U. pugilator, loss of the large male
chelae is more effective than loss of a single walking leg in inducing the onset of PE (Hopkins 1982).
In P. marmoratus, the minimum number of limbs that must be autotomized before PE will begin
is four, and in G. lateralis five to six legs must be removed to induce PE (Bliss 1956, Rouquette and
190 Penny M. Hopkins and Sunetra Das

Vernet-Cornubert 1964). In Libinia emarginata, autotomy of as many as eight walking legs will not
induce PE (Skinner and Graham 1972). Moreover, loss of multiple limbs after the beginning of the
preparation for molt will halt the process by causing a reduction in circulating ecdysteroids (Holland
and Skinner 1976). This reduction is necessary for the early blastema to organize and grow.
The apparently contradictory effects of autotomy and regeneration on the onset and dura-
tion of PE in crustaceans can be explained by a single mechanism, if it is assumed that autotomy
and regeneration are able to reset the animal and its YO to the substage D0 of PE. Drach (1939)
developed a series of stages for the molt cycle of crustaceans. Stage D was reserved for PE, the
time during which the animal prepares for molt. Travis (1960) modified Drach’s stages to include
an additional D0 substage that encompasses the earliest stage of PE (Fig. 6.3). Substage D0 is the
period during which PE growth occurs in regenerating limbs and is a time of obligatory low levels
of circulating ecdysteroids. If autotomy and regeneration result in physiological conditions that
usually occur only at substage D0, then autotomy performed in intermolt will appear to hasten
the onset of PE. Autotomy during D0 will inhibit PE by resetting it back to the beginning of D0
(Skinner and Graham 1972, Hopkins 1982). Both of these contradictory effects suggest that there
is some crosstalk between regenerating tissue and the YOs. The feedback ensures optimal levels
of ecdysteroid for full regeneration. In P. marmoratus, multiple limb loss (and “intensive regen-
erations”) in YO-less crabs causes a shorter PE period when compared to YO-less controls. Both
groups have levels of circulating ecdysteroids comparable to intact controls (except during late
PE). Thus, in Pachygrapsus, autotomy is capable of affecting the duration of PE without affecting
the YOs. Other work by these same authors shows that, in Pachygrapsus at least, loss of many limbs
in YO-intact animals results in elevated levels of circulating ecdysteroids during PE. At this time,
it appears that autotomy in crabs stimulates the YOs but also has a stimulatory effect on the onset
and duration of PE that is independent of the YOs.
What, then, might be the mechanism responsible for the autotomy-induced resetting of early
PE? A putative factor (limb autotomy factor) has been identified in extracts of limb buds from
the crab G. lateralis. The extracts inhibit PE growth and delay molting, but the exact nature of the
putative factor, its source, and its chemical identity remain unknown (Yu et al. 2002). Imaginal
discs from Drosophila autonomously release an insulin-like peptide that can modulate normal
growth of the entire animal to ensure that disc maturation coincides with growth events in the
entire animal (Colombani et al. 2012, Garelli et al. 2012). Perhaps such a molecule is produced
and released by the developing limb buds of crustaceans. Moreover, regenerating wing discs in
Drosophila signal the central nervous system via a retinoid intermediate (Halme et al. 2009) to
keep ecdysteroid levels low enough to allow for full regeneration. Because retinoids have been
identified in the blastemas of a regenerating crustacean, retinoids may play a role in resetting PE
in crustaceans (Hopkins et al. 2008).

FUTURE DIRECTIONS

The field of crustacean regeneration control has many unanswered questions and is suitable for fur-
ther investigation. What is most needed at this point in crustacean regeneration studies (and crus-
tacean endocrinology as well) is a genome of at least one decapod crustacean. Most of the research
on crustacean regeneration control has been done on decapods, and these are the animals in which
further molecular biological investigation is absolutely essential. There are numerous expressed
sequence tag libraries for decapods, but these have limited utility in working out hormone relation-
ships and particularly in examination of control sites in hormonally triggered cascades.
One of the most important unanswered questions is to determine the factors involved in
the “resetting” effects of regeneration on the molt cycle. What does it mean exactly to reset the
 Regeneration in Crustaceans 191

animal to stage D0? What compounds are released from the regenerating tissues, and what is
their effect on the YO? It appears that the control of the YO production of ecdysteroids is much
more complicated than a mere on/off switch mediated by the molt-inhibiting hormone. Is there
a positive control of ecdysteroidogenesis from a source other than the eyestalk? Another area
that is ready for further study is the actual de-differentiation/respecification of the tissues of the
regenerating limb bud. Where do the new tissues arise, and what kinds of growth factors may be
involved? It is still unknown as to what sequence the new segments are synthesized and differen-
tiated. Moreover, the specific genes and gene cascades that control early respecification, as well
as later hypertrophic growth, are unknown in crustaceans and need to be investigated. There is a
wealth of information yet to be mined from the crustacean limb regenerating system, and it may
still provide us with new and important information about regenerative mechanisms with wide
applications and utilities.

CONCLUSIONS

This review focuses on histological, cellular, and morphological changes during limb regenera-
tion in crustaceans. The regulation of these phenomena and the similarity to leg development and
imaginal disc regeneration in insects are discussed. Studies on crustacean limb regeneration have
focused extensively on defining two distinct phases: basal growth (which includes wound healing
and blastema formation) and PE growth (which includes increases in tissue mass). The regulation
of these phases of regeneration in crustaceans appears to vary with the phase. In general, crustacean
regeneration is thought to be under the control of both growth factors and hormones, and one
of the important aspects of regeneration is how such factors will exert their influence at the cel-
lular and molecular levels. The sources of cells giving rise to different regenerating tissues within
the crustacean limb buds are not fully determined. In addition, the molecular networks that pro-
duce functional limbs from blastemas are still not completely ascertained. Limb regeneration in
crustaceans is coupled with molting and regulated by several blood-borne factors that culminate in
changes in gene expression. Because ecdysteroid receptors are expressed in regenerating crustacean
limbs throughout the regeneration and molt cycles, it is assumed that ecdysteroids play a role in
the control of downstream genes during regeneration. The gene networks that are controlled by
ecdysteroids during regeneration are, however, still unknown. These uncertainties are dictating the
direction of future limb regeneration studies in crustaceans.
Limb regeneration studies in vertebrates (especially in the salamander Ambystoma mexicanum)
have revealed that the cell potential of the cells within the blastema is restricted, and this suggests
that complete de-differentiation of blastema is not required during vertebrate limb regeneration.
In crustaceans, the degree of de-differentiation of epidermal cells in the blastema is still unknown.
The tissues arising from the blastema are quite diverse:  for example, the de-differentiated epi-
dermal cells of the blastema give rise to muscle, cuticle, nerve, chromatophores, and more.
This review also discusses the molecular networks and genes associated with regenerating
imaginal discs in Drosophila and the parallel networks that may also be involved in differentia-
tion process in crustaceans. In Drosophila, during regeneration of damaged imaginal discs, the
growth of the remaining intact disc is held up. This retardation appears to be due to release of
growth-controlling factors from the injured disc tissue. Similarly in crustaceans, the autotomy of a
limb can retard the regenerating process of previously autotomized limbs that are in basal growth,
but the growth-controlling compounds in crustaceans are still under investigation. Leg develop-
mental processes are compared to leg regenerative processes in both insects and crustaceans, and
it is generally supposed that the gene cascades and the control mechanisms in both development
and regeneration are closely related.
192 Penny M. Hopkins and Sunetra Das

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 7
CIRCULATORY PHYSIOLOGY

Iain J. McGaw and Carl L. Reiber

Abstract
The circulatory system of the subphylum Crustacea varies significantly in both anatomical and
physiological complexity. The most highly developed system occurs in decapods, where pre-
cise physiological control mechanisms rival some simple vertebrate closed systems. Changes
in heart rate have been used as an important indicator of stress and energy expenditure, and
recent technological advances allow measurement of stroke volume, cardiac output, and
hemolymph redistribution in intact animals. Decapods regulate the amount of hemolymph
delivered through each arterial system via the muscular cardioarterial valves or by changing
downstream resistance in the vessels. A number of naturally occurring peptide and amine neu-
rohormones modulate cardiac activity and differential hemolymph flow by acting directly on
the heart and cardiac nerves, or cardioarterial valves and artery walls. This chapter covers the
basic functional anatomy of the heart and vessels; developmental, neurological, and hormonal
control mechanisms; and cardiovascular responses to environmental and biotic perturbations
in decapod crustaceans.

INTRODUCTION

The cardiovascular system of the subphylum Crustacea varies significantly in both anatomical and
physiological complexity. The circulatory system is rudimentary in the class Cirripedia and consists
of a series of ill-defined sinuses. There is no heart associated with these sinuses; instead, the hemo-
lymph is circulated by way of contraction of the cirri and gut muscles. In the classes Branchiopoda
and Ostracoda, a single-chambered heart is evident, which may be globular or tubular in shape.
Most of the crustaceans within these classes lack blood vessels; hemolymph circulates directly from

199
200 Iain J. McGaw and Carl L. Reiber

the heart into the hemocoel of the body before emptying back into the heart via paired openings
or ostia (Maynard 1960, McLaughlin 1983). The circulatory system increases in complexity in the
orders Isopoda, Peracarida, and Mysidacea, consisting in these of a segmental heart and associated
vessels (Wirkner and Richter 2003, 2007a,b, 2008). The most highly developed circulatory system
is seen in the order Decapoda. A single-chambered heart pumps hemolymph into a well-defined
series of arteries that split into capillary-like vessels that ramify among the tissues. Some of the
areas of the system, as well as its physiological control mechanisms, are highly developed (Reiber
and McGaw 2009).
A number of the most highly cited articles on crustacean cardiovascular anatomy and physiol-
ogy are published in the extensive series The Biology of Crustacea (Bliss and Mantel 1983). These
have remained the standard texts for crustacean cardiovascular anatomy and physiology for
nearly three decades. McLaughlin’s (1983) chapter on internal anatomy includes reference to fine
historical articles spanning more than a century and a half. However, most of these articles were
written at the beginning of the past century or the end of the 19th century, before the advent of
modern imaging techniques allowed the detailed analysis of the circulatory system to be resolved
(Haeckel 1857, Bouvier 1891, Pearson 1908, Brody and Perkins 1930). Although a few review arti-
cles and specialized book chapters on the physiology of the cardiovascular system have been
written within the past two decades, McMahon and Wilkens (1983) chapter on ventilation, per-
fusion, and oxygen (O2) uptake has remained the gold standard for crustacean cardiorespiratory
physiology. Nonetheless, the crustacean circulatory system has received extensive attention since
this work was published. The system is now recognized as being relatively complex, with precise
physiological control mechanisms to shunt blood through specific arterial systems and pressures
that rival some of the simple vertebrate closed systems (McMahon and Burnett 1990, Wilkens
1999a, McMahon 2001, Reiber and McGaw 2009). The circulatory system of decapod crusta-
ceans may now (at least in physiologically terms) be classified as one that is partially closed,
rather than open (Reiber and McGaw 2009).
In addition to anatomical and morphological adaptations, the cardiovascular physiology
of crustaceans has also been studied extensively. For more than half a century, changes in
heart rate have been used consistently as an important measure of stress and energy expendi-
ture in crustaceans (deFur and Mangum 1979, Handy and Depledge 1999, Brown et al. 2004).
However, heart rate alone is not an accurate means of assessing the total amount of hemolymph
(cardiac output) delivered to the system. The cardiac output is also dependent on stroke vol-
ume of the heart, which can vary independently of heart rate (McGaw and McMahon 1996).
Although various methods, such as the Fick principle and thermodilution ( Johansen et  al.
1970, Burnett 1979, McMahon et al. 1979, Burnett and Bridges 1981, Bradford and Taylor 1982,
Wilkes and McMahon 1982), were used to calculate cardiac output and stroke volume of the
heart, they had their limitations (Airriess and McMahon 1994). More recently, the develop-
ment of less invasive techniques such as the pulsed-Doppler flowmeter, sonomicrometry, and
digital imaging techniques have allowed greater detail to be resolved in a wider array and size
of species (Airriess et al. 1994, Paul et al. 1997, Reiber et al. 1997, Bock et al. 2001, Guadagnoli
and Reiber 2005).
Decapod crustaceans are able to regulate the amount of hemolymph delivered through each
arterial system via the muscular cardioarterial valves at the entrance of each major artery or by
changing the downstream resistance in the vessels (Wilkens 1997, 1999a, McGaw and Reiber
2002). This is not only important for efficient delivery of nutrients and gases, but the diversion
of flow to metabolically active tissues also may enhance the ability of animals to cope with envi-
ronmental changes. Cardiac parameters and hemolymph flow rates have been found to change
in response to exercise (DeWachter and McMahon 1996a, McGaw and McMahon 1998), feeding
(McGaw and Reiber 2000, McGaw 2006a), hypoxia (Airriess and McMahon 1994, Reiber 1995b,
 Circulatory Physiology 201

Reiber and McMahon 1998), emersion (Airriess and McMahon 1996), low salinity (McGaw and
McMahon 1996, McGaw and Reiber 1998, McGaw 2006b), and temperature (DeWachter and
McMahon 1996b).
Cardiac function and blood flow are controlled by both neural and hormonal control mech-
anisms. Neural control can occur via the action of the cardioregulatory nerves (inhibitory and
acceleratory fibers) that influence both the rate and force of contraction of the heart by acting
on the cardiac ganglion. Additionally, the tonus of the cardioarterial valves can be modulated
through neuronal stimulation, which regulates hemolymph flow (Kuramoto and Kuwasawa
1980, Kuramoto and Ebara 1984). A number of naturally occurring peptide and amine neuro-
hormones modulate cardiac activity and differential hemolymph flow in crustaceans (Saver
and Wilkens 1998, Wilkens and Kuramoto 1998, Wilkens 1995, 1999a). These hormones act on
the cardioarterial valves, the artery walls, central nervous system (CNS), or the cardiac muscle
itself (Wilkens et  al. 1996, Saver et  al. 1998, Wilkens 1997, 1999a,b, McMahon 2001, Stevens
et al. 2009).
This chapter focuses primarily on articles published since the last extensive review of the topic
in The Biology of Crustacea (Bliss and Mantel 1983). Because the Malacostraca represent the bulk
of the crustaceans studied and include species with complex anatomical and physiological systems,
the physiological responses of these are covered in detail and relevant comparisons made with
other families. This chapter covers the basic anatomy and functional morphology of the heart and
vessels and their developmental, neurological, and hormonal control mechanisms, as well as cardio-
vascular responses to environmental perturbations.

FUNCTIONAL ANATOMY

The Heart

The anatomy of the crustacean heart is as diverse as the taxa and ranges from a simple tubal heart
to the complex globular hearts seen in decapods. The generalities used here illustrate primary pat-
terns observed within the decapod crustaceans, and, therefore, the cardiac anatomy outlined may
be used as a general model with some deviations between genera and species (Fig. 7.1).
The heart of the decapod crustaceans is a single-chambered box-like structure (the ventricle)
with walls consisting of bands of cardiac muscle arranged in a three-dimensional network to maxi-
mize the ability to eject hemolymph upon contraction. This trabecular network of branching and
anastomosing myocardial cells forms an avascular myocardium that acts as a functional syncytia to
facilitate coordinated contraction (Howse et al. 1971). The ventricle is suspended within the peri-
cardial cavity by elastic alary ligaments (Maynard 1960). The myocardium, alary ligaments, and
pericardial cavity together make up a functional hemolymph pump. Ventricular contraction results
in hemolymph ejection into the arterial system. Contraction is stimulated by nerves to the myocar-
dium from cardiac pacemaker cells found in the cardiac ganglion. The cardiac ganglion is located
along the dorsal surface of the heart and regulates heart rate and muscle contractile force (Wiersma
and Novitiski 1942, Maynard 1960, McMahon and Wilkens 1983). Some of the energy of contrac-
tion is stored in the elastic alary ligaments attached to the surrounding tissues. A passive expansion
of the ventricle results from this attachment, and diastolic filling begins. Venous pressure (filling
pressure) is aided by this passive expansion and allows hemolymph to fill the heart through three
paired ostia located dorsally, laterally, and ventrally on the heart. Once diastolic filling is complete,
systolic contraction begins. Initial cardiac myocardial contraction places tension on the edges of the
ostia, resulting in their closure and preventing backflow of hemolymph into the pericardial sinus
(Maynard 1960).
202 Iain J. McGaw and Carl L. Reiber

g
o v
A l.a B a
a
a
o
o
a
C

F
o a

E o
o
a
D fr o

o obl.c
o

s.l.

Fig. 7.1.
Types of crustacean hearts. (A)  Diastylis rathkei (Malacostraca, Cumacea), dorsal view, length is approxi-
mately 3 mm. (B) Daphnia magna (Branchiopoda, Cladocera) lateral view, length is approximately 200–300
µm. (C) Calanus finmarchicus (Copepoda, Calanoida), ventral (left) and dorsal views, length is approximately
330 µm. (D) Astacus astacus (Malacostraca, Decapoda), dorsal view (left) and dorsal view with dorsal heart
wall removed, length is approximately 1 cm. (E) Phronima sedentaria (Malacostraca, Amphipoda), lateral view,
length is approximately 2.5 cm. (F) Squilla mantis (Malacostraca, Stomatopoda), dorsal view, length is approxi-
mately 8 cm. Abbreviations: a, anterior aorta or opening from the heart into the anterior aorta; b, bulbus arteri-
osus; fr, musculus frontalis; g, wall of the intestine; l.a., opening from the heart into the lateral artery; o, ostium;
obl.c., musculus obliquus cordis; s.l., suspensory ligaments; v, cardioarterial valve. From Maynard (1960), with
permission from Elsevier.

The Vessels

The crustaceans comprise a group of morphologically diverse organisms making it somewhat dif-
ficult to present a general description of the circulatory system (Maynard 1960, McMahon and
Burnett 1990, McMahon 2001). Recently, a number of papers and a comprehensive review have
addressed the comparative anatomy and evolutionary relationships of the circulatory and respira-
tory systems (see Wirkner 2009, Wirkner and Richter 2013). Briefly, a wide array of systems exist,
ranging from the simple circulatory system of the cirripeds, which have no heart and simple open
sinuses, through crustaceans such as the copepods and anostracans, which possess a simple tubular
heart, but no vessels, to the most complex distribution systems seen in adult decapods (McMahon
2001). It is assumed that these variations in morphology of the circulatory system are evolution-
ary, and a complex segmental arterial system may have allowed animals to become large and active
(Wilkens 1999b). In particular, the ability of the malacostracan crustaceans to control differential
 Circulatory Physiology 203

blood flow through the tissues may have been important in their adaptive radiation and their ability
to colonize physiologically diverse environments (Wilkens 1999b).
At the top of the evolutionary chain, the circulatory system of adult decapods is the most com-
plex (Fig. 7.2), so much so that it may now be considered incompletely closed, rather than open
(Reiber and McGaw 2009); this section concentrates on this system. Hemolymph is pumped from

Fig. 7.2.
Corrosion casts showing the complexity of the circulatory system of decapod crustaceans. (A) Dorsal view
of the blue crab Callinectes sapidus, adapted from McGaw and Reiber (2002), with permission from Wiley
and Sons, Inc. (B) Dorsal view of the kelp crab Pugettia producta, adapted from McGaw and Stillman (2010),
with permission from Elsevier. (C) Ventral view of the Puget Sound king crab, adapted from McGaw and Duff
(2008), with permission from Wiley-Liss, Inc.
204 Iain J. McGaw and Carl L. Reiber

the single-chambered ventricle into seven arteries (five arterial systems); the small anterior aorta and
the paired anterolateral arteries and hepatic arteries flow anteriorly, the large sternal artery exits the
heart ventrally, and the posterior aorta complex exits from the posterior aspect of the heart. The ana-
tomical arrangement of the vessels is covered in detail elsewhere (McGaw and Reiber 2002, McGaw
2005a, Wirkner and Richter 2013). The arteries subsequently branch into smaller arteries and fine
capillary-like vessels that ramify within the tissues. Decapod crustaceans lack a complete venous
system; instead, hemolymph drains into small irregular spaces between the tissues, the interstitial
lacunae, and then into sinuses, which are large irregular spaces between the tissues (Maynard 1960).
Convention states that arterial hemolymph perfuses the tissues at the level of the lacunae where gas,
nutrient, and waste exchange takes place. However, recent work suggests that the hemolymph may
remain contained within an endothelial layer down to, or even to the level of the lacunae, which
would make lacunar exchange somewhat analogous to capillary beds of vertebrate systems (Wilkens
1997, 1999a,b, Reiber and McGaw 2009). Spent hemolymph (that has perfused the tissues) subse-
quently collects in sinuses before flowing over the gills and back into the heart. It was once thought
that sinuses were so ill defined that they almost defied successful demonstration (Pyle and Cronin
1950). However, recent work shows the sinuses to be distinct structures associated with a fine lat-
ticework of lacunae, bordered by fibrous connective tissue (McGaw and Duff 2008, McGaw and
Stillman 2010). Haeckel (1857) suggested that there were no unbound sinuses within the decapod
system. The difference between sinus and capillary therefore becomes less apparent, suggesting a
primarily histological term rather than a physiological one (Reiber and McGaw 2009). The indi-
vidual sinuses eventually drain into the large ventral thoracic sinus; from here, hemolymph col-
lects in the infrabranchial sinuses before flowing through the gills where it is reoxygenated. By the
time the hemolymph has reached the gill vessels, the pressures are only a few centimeters of water
(Blatchford 1971, McMahon and Wilkens 1983). The gill lamellae have a low branchial resistance, and
these thin-walled vessels are able to distend in response to pressure pulses (Taylor 1989). This means
that flow through the small vessels of the gill lamellae might be concerned with the redistribution of
lamellar flow, thus maximizing O2 uptake (Taylor 1989). After passing through the lamellae, hemo-
lymph drains into the pericardial sinus and back into the heart through the ostia.
Although the gills are presumed to be the major return route for venous blood, an alternative
return route has been proposed. The branchiostegal sinus is a network of lacunae and veins that
perfuses the dorsal surface of the branchial chamber and eventually drains back into the pericardial
sinus (Taylor and Taylor 1992). Although the branchiostegal network can bypass the gills, its exact
function and the partitioning of venous flow between the branchial and branchiostegal sinuses is
not fully understood (Taylor and Greenaway 1984, Greenaway and Farrelly 1990, Taylor and Taylor
1992). The branchiostegal circulation is well developed in terrestrial and amphibious crabs; in these
animals, it is used for aerial gas exchange (Taylor and Greenaway 1979, Greenaway and Farrelly
1990, Greenaway et al. 1996). However, the branchiostegal network is also well developed in many
subtidal species (McGaw and Duff 2008, McGaw and Stillman 2010). The exact function of ventila-
tory reversals has long been surmised, but they are thought to aid ventilation of dead spaces in the
branchial chamber (Hughes et al. 1969, Davidson and Taylor 1995). There is nothing to suggest that
additional O2 could not be extracted from the water in the branchial chambers via the branchioste-
gal sinus. If not, there would be a substantial mix of deoxygenated and oxygenated blood returning
to the heart, and mixing appears to be minimal (McMahon 2001; see Fig. 7.3).
Unlike mammalian systems, the majority of crustaceans do not have layers of smooth muscle
in their arterial walls; instead, the vessel walls consist of an adventitial layer of collagen fibers sur-
rounding a concentric network of fibrous elastic tissue (Shadwick et al. 1990). However, at the base
of each artery exiting the heart is a set of muscular cardioarterial valves (Alexandrowicz 1932). The
valves are innervated (Kuramoto and Ebara 1984, Kihara and Kuwasawa 1984, Tsukamoto et  al.
1992), and neural or neurohormonal stimulation of the valves causes them to contract, progressively
 Circulatory Physiology 205

Body

Branchiostegal
circulation Gills
?

Heart

Fig. 7.3.
Pictograph to show the flow of hemolymph in decapod crustaceans. Hemolymph is pumped from the
single-chambered ventricle into well-defined arteries. Most of these arteries split into smaller lacunae, which pos-
sess a well-defined structure, before emptying into sinuses or veins. In certain areas (e.g., brain, antennal gland),
the hemolymph may flow through capillaries with a closed-loop system. Spent hemolymph collects in sinuses
before it flows through well-defined gill lamellae where it is oxygenated before returning to the heart. An alterna-
tive route through the branchiostegal circulation has also been proposed. The oxygenation status is intentionally
left blank because, at this stage, the partitioning of the flow between the gills and branchiostegal circulation, and
whether the hemolymph gains oxygen or not when passing through the branchiostegites, is still unknown.

restricting hemolymph flow into the artery. Blocks of striated muscle have been reported in the
abdominal arteries of a number of crustaceans (Shadwick et  al. 1990). Because the abdominal
artery is usually the only artery that contains muscle tissue, it is postulated that this may actually be
the evolutionary remnant of a tubular heart (Wilkens 1995, Wilkens et al. 2008). The exact function
of these striated muscle blocks is unclear; however, a number of neurohormones have been shown
to cause their contraction. Whether these actually control regional blood flow as occurs in closed
systems is unclear (Martin et al. 1989, Wilkens 1997). Recent evidence suggests that the cellular
make-up of the vessel walls themselves may be important. Lobster arteries are composed of three
layers. The inner layer is an elastic connective tissue, and the outer layer is primarily composed of
collagen; the middle layer of the artery is composed of cells containing microfilaments. These cells
contain actin, myosin, and tropomyosin (Cavey and Wilkens 2000, Cavey et al. 2008, Wilkens et al.
2008). Proctolin and glutamate elicit contraction of rings of arterial tissue, possibly acting on the
actin subunits in the walls (Wilkens et al. 2008). These contractions tend to be slow (>20 min to
reach maximum) with a very slow recovery time (>2 h). However, even slight arterial contraction
will have profound effects on resistance to blood flow and may be an important component of the
control mechanisms regulating blood distribution (Wilkens et al. 2008).
Calculations of the hemolymph capacity of each arterial system have been made from corrosion
casts (McGaw and Reiber 2002, McGaw and Duff 2008, McGaw and Stillman 2010). In typical
brachyuran crabs about 40–45% of hemolymph is contained in the arteries and capillaries; as in ver-
tebrates, the venous system (sinuses, veins, heart, and gills) acts as a reservoir holding 50–65% of the
hemolymph at any one time (McGaw and Reiber 2002). A series of papers using pulsed-Doppler,
microsphere perfusion, and dye dilution techniques (Airriess et al. 1994, Guadagnoli and Reiber
2005) have shown that decapod crustaceans can partition hemolymph flow through the different
206 Iain J. McGaw and Carl L. Reiber

arterial systems, sending blood to more metabolically active tissues. Depending on the species and
the stress encountered, the sternal artery and associated branches receive most of the hemolymph,
at between 40% and 85%; the paired hepatic arteries receive between 8% and 25%, the paired antero-
lateral arteries between 8% and 40%, the anterior aorta between 0.05% and 6%, and the posterior
aorta complex receives between 0.05% and 8% of the hemolymph pumped by the heart (McGaw
et al. 1994a,b, McGaw and McMahon 1995, Reiber et al. 1997).

PHYSIOLOGICAL CONTROL

Developmental Aspects of Cardiac Function

The primary focus of research in crustacean cardiac physiology has been to gain an understanding
of the function and regulation of adult systems. This has led to major revisions in our understand-
ing of the complexity of the cardiovascular system’s regulatory capabilities in this group of animals
(McMahon and Burnett 1990, Airriess and McMahon 1992, 1994, Hill and Kuwasawa 1992, Reiber
et al. 1992, Reiber et al. 1997, Romney and Reiber 2013). Investigations by Yamagishi (1990) and
Yamagishi and Hirose (1992, 1997) have focused on the neural coupling of the CNS to the cardio-
respiratory systems in the direct developing semiterrestrial isopod (Ligia exotica) and the tadpole
shrimp (Triops longicaudatus; Yamagishi et al. 1997). The heart of the isopod was found to be myo-
genic (intrinsic cardiac pacemaker) during embryonic and juvenile stages. As cardiac development
proceeded, the more typical crustacean neurogenic pattern developed. A similar pattern of cardiac
regulation has been suggested for the chelicerate, the horseshoe crab (Limulus; Carlson and Meek
1908, Crozier and Stier 1927). The heart of both juvenile (larval) and adult tadpole shrimp was
found to be myogenic and without direct nervous innervation (Yamagishi et al. 1997). At this time,
due to the limited data available, we cannot determine if the Triops (myogenic) pattern is unique or
a more typical pattern for Crustacea.
The development of cardiac physiological function (heart rate) in the brine shrimp (Artemia
franciscana) has been shown to be dependent on cardiac differentiation early in development,
with heart rate increasing with body mass (Spicer 1994, Spicer and Morritt 1996). This pattern is
followed by a dependence on cardiac elongation with heart rate decreasing with body mass. This
relationship between differentiation and elongation is significant regarding cardiac function and
its role in developmental processes. Early cardiac activity in Artemia and in the copepod Argulus
americanus (Wilson 1904) depends more on morphological than physiological development. It is
hypothesized that there is a transition from a diffusion dependent system during early development
to an increased dependence on convective processes facilitated by the heart and gills (Spicer 1994,
Spicer and Morritt 1996). Supporting this hypothesis is the observation that the beat frequency of
natatory limbs (second antennae, used as accessory respiratory structures) decreases with devel-
opment, resulting in a decrease in gas exchange. The heart begins to take on a physiologically sig-
nificant role as natatory limb beat frequency declines. Increased cardiac and ventilatory activity
compensates for the resulting loss of respiratory function (Barlow and Sleigh 1980). This again
leads to the question of how the onset of cardiac function can be influenced by limitations in gas
exchange (environmental hypoxia).
Cardiac physiology has been investigated during development from embryonic through larval
and adult stages in a number of decapod crustaceans (Procambarus clarkii; Reiber 1997, Reiber and
Harper 2001, Harper and Reiber 2004). The embryonic crayfish tubal heart showed early signs
of contraction (although unorganized) by naupliar stage 4 (incubation day 12 at 25°C). Heart
rate increased until naupliar stage 6 then showed a progressive decrease until hatching and larval
 Circulatory Physiology 207

development was initiated. They also showed a parallel decrease in hypoxic sensitivity (Reiber
1997). Post-hatching heart rate increased through larval stages 1–3, followed by a decline as the ani-
mal gained mass (an allometric relationship; Fig. 7.4). The decrease in heart rate and hypoxic sen-
sitivity during later embryonic stages indicated a potentially diffusion-limited system as embryonic
metabolic mass increased beyond the exchange capabilities of the egg membrane (Reiber 1997).
It has also been found that embryonic heart rate increases as the partial pressure of oxygen (Po2)
in water is raised above 200 mm Hg, which also suggests the animals are O2 limited. Embryonic
and larval cardiac stroke volume was monitored during hypoxic exposure. Embryonic stroke vol-
ume and cardiac output decreased in parallel with heart rate. After hatching, larval stroke volume
increased dramatically, compensating for the lower heart rate and resulting in an increase in cardiac
output (Reiber 1995b, 1996, Harper and Reiber 2001, Harper and Reiber 2006b). It was also found
that if 10-day-old embryos were incubated under hypoxic conditions (Po2 = 75 mm Hg), hatching
occurred 4 days earlier than in control animals that were maintained under normoxic conditions
throughout development.
This abrupt termination of embryonic development was not a simple time compression of
developmental processes (as seen when incubation temperature is increased) but an abbreviation
of the ontogenic pattern. These experiments point toward a diffusion-limited embryo that can alter
its developmental pattern depending on available O2 (Reiber 1995a, 1996). If embryonic metab-
olism cannot be maintained, the animal will hatch, bringing online respiratory mechanisms into
play to facilitate gas exchange. This work complements the investigations on brine shrimp. The
ontogeny of crayfish cardiac neurohormonal regulation appears to be quite complex, with a defined
sequence of neurohormonal sensitivity developing as the animal progresses from stage to stage. It
has yet to be established if these ontogenetic changes in myocardial sensitivity to neurohormones
are due to a temporal sequence of receptor and/or ligand production or to the development of
intracellular pathways.
Injections of octopamine, serotonin, γ-aminobutyric acid (GABA), and dopamine into the peri-
cardial sinus of embryos and larvae did not elicit adult-like cardiac responses in terms of heart rate,
stroke volume, or cardiac output until late larval or early juvenile stages. Stage-3 larvae showed
a minimal response to injections; it was not until the animals reached juvenile stages (weight of
25 mg or greater) that adult-like responses were observed. Of particular interest was the embry-
onic response toward GABA injection, which caused an increase in cardiac parameters, a paradox-
ical response. An adult-like response was not observed until late larval stages had been reached
(Kuramoto and Ebara 1984, McMahon 1992, Wilkens 1995, Chapman and Reiber 1998, Reiber and
McMahon 1998, Harper and Reiber 2000). The response to GABA injection is obviously not a
simple one and may involve changes in receptor populations. More research is clearly needed to
understand this system. This insensitivity to injection of cardioactive drugs during early larval
stages would indicate that cardiac regulatory mechanisms do not mature until later in development
(Reiber 1996, Chapman and Reiber 1998, Harper and Reiber 2000).
Histological evidence supports these conclusions. Embryos stained for nerves were not found
to contain nervous innervation of the heart. The cardiac ganglion was not observed to invade the
heart until the second larval stage, which corresponds with initial sensitivity to cardioactive drug
injections. Using techniques similar to those described for crayfish, cardiovascular development
was investigated in the shrimp Metapenaeus ensis (McMahon and Chu 1994, Chu et al. 1995). In
M. ensis, the heart does not start to beat until late naupliar developmental stages. Heart rate increased
through the third protozoeal stage then fell as mysid and pelagic stages were reached (McMahon
et al. 1997). The changes in heart rate were not due to cardiac elongation, as is evident in Artemia,
but were attributed to changes in regulatory pathways. It is interesting to note that the heart begins
to beat in directly developing shrimp when comparing developmental stages (early naupliar stages
in the crayfish vs. late naupliar stages in the shrimp). The advent of internal convective processes
 A

A B C

D E F

G H I

Fig. 7.4.
(Top) Illustrations of the dorsal view of the ventricle during embryonic stages of Palaemonetes pugio (Holthuis
1949). Dimensional arrangement of the ventricle for analysis. The Y-axis runs the length of the long axis of the
 Circulatory Physiology 209

early in development increases O2 transport rates, thus allowing the crayfish to partially compen-
sate for the limitations in the diffusional surface area of the egg membrane. This compensatory
strategy may allow directly developing species to remain in embryonic stages longer and thus have
more time to develop morphologically and hatch as a well-developed larvae or juvenile.
Much of what we know regarding crustacean cardiovascular development comes from anatomi-
cal observations or from a few early studies on a limited number of species (McMahon and Chu
1994, Chu et al. 1995, McMahon and Doyle 1995, Reiber 1995b, 1996, 1997). This has resulted in
large gaps in our understanding of cardiovascular and respiratory physiological development in
Crustacea that would alone justify investigation. Added to this is the unique opportunity crusta-
cean cardiovascular development offers for the investigation of cardiac malleability and physiologi-
cal trajectories.

Neural Control of Cardiac Function and Blood Flow

Control of Cardiac Function

The neurogenic myocardium of the crustacean heart is stimulated to contract by the nerves from the
cardiac ganglion. Overlying and embedded within the cardiac muscle, the cardiac ganglion serves
as pacemaker and regulator of muscle contractile force (Maynard 1960, Taylor 1982, McMahon and
Burnett 1990). The cardiac ganglion is composed of only a small number of neurons of two cell
types, with distinct cell sizes (McMahon and Wilkens 1983, McMahon et al. 1989, Kuramoto and
Yamagishi 1990). In the crayfish, the cardiac ganglion is approximately 5 mm in length and contains
16 neurons: eight small and eight large. The two morphologically distinct cell types have functional
differences. Normally, the smaller neurons act as a pacemaker by establishing burst frequency. The
smaller cells, through chemical synapses, drive the larger neurons, which act as follower cells and
innervate the myocardium directly. Therefore, the rate of heart contraction is determined by the
burst frequency of the small cells of the cardiac ganglion. The force of the contraction is dependent
on the spike frequency within each burst.
The larger cells of the cardiac ganglion innervate the myocardium directly and show
mechanoreceptor-like sensitivity (Maynard 1960). Distention of the lobster cardiac ganglia occurs
when the heart is exposed to high filling pressure; heart frequency is then determined by the burst-
ing pattern of the large nerves (Kuramoto and Kuwasawa 1980, Kuramoto and Ebara 1985, Cooke
1988). When the heart is exposed to neurohormones such as serotonin, heart frequency is set by
the smaller neurons. The system has two layers, with the smaller cells acting as primary pacemaker
driving the larger cells; however, as just demonstrated, modulation of heart frequency can depend
on both cell types.
The neurons of the cardiac ganglion are influenced by the cardioregulatory nerves, one pair of
inhibitory fibers (SN 11) and two pairs of acceleratory fibers (SN 111; see Wiersma and Novitiski

Fig. 7.4  (Continued)

(A) Dorsal and lateral position of the heart in reference to the embryonic shrimp. (B) Development of the
ventricle during stages of embryonic development. Functional ostial pairs can be seen developing through
the stages. Solid lines signify a structure seen on the dorsal surface of the heart, and dashed lines signify a
structure seen through the heart on the ventral surface. From Romney and Reiber (2013), with permis-
sion from Brill, Inc. (Bottom) Images of embryonic stages of development of Palaemonetes pugio (Holthuis
1949), taken under a stereoscope. Stage I  and stage VIb are omitted for irrelevance of a single-shot image
properly representing characteristics for those articular stages. Stages pictured include (A)  stage II, cleav-
age; (B)  stage IIIa, gastrulation with blastopore; (C)  stage IIIb, gastrulation with V-depression; (D)  stage
IV, early nauplius; (E)  stage V, mid-nauplius; (F)  stage VIa, late nauplius with posterior segmentation;
(G)  stage VIIa, post-nauplius with eye pigmentation; (H)  stage VIIb, post-nauplius with eye condensa-
tion; (I)  stage VIII; pre-hatch embryo. Images of animals shown here are not processed together to scale.
210 Iain J. McGaw and Carl L. Reiber

1942, Maynard 1960, McMahon and Wilkens 1983). Their origins are in the anterior ventral nerve
cord and the subesophageal ganglion. The three nerves come together at the lateral pericardial
plexus and enter the pericardial sinus. Here, they separate and terminate in a variety of regions. SN
111 sends out axons that innervate the pericardial organ, the cardiac ganglion, the dorsal muscles of
the myocardium, and the alary ligaments. Cardioaccelerator and inhibitor fibers have been found
to fire tonically, with intermittent bursts of higher frequency spikes (Wiersma and Novitiski 1942,
Wilkens et al. 1974, Taylor 1982, McMahon and Wilkens 1983). If a similar burst pattern is artificially
induced electrically in the cardiac ganglion, alteration of heart rate results.
Central neural integration of heart function originates in the command interneurons found in
the circumesophageal ganglion. Interneurons have been identified that cause tachycardia, brady-
cardia, or cardiac arrest (McMahon and Wilkens 1983). The impulse frequency of cardioinhibitory
fibers has been found to vary linearly with stimulation of specific interneurons. It is interesting to
note that when these interneurons are stimulated or sensory stimulus is given, the cardioaccelerator
nerve and inhibitor nerve act in reciprocal fashion, such that when one increases firing frequency,
the other decreases its rate.
Additional nerves act on the cardiovascular system to influence cardiac output and arterial per-
fusion. The regulation of aperture size in the cardioarterial valves could be a prime means by which
decapods regulate distribution of the cardiac output (Kuramoto and Kuwasawa 1980, Kuramoto
and Ebara 1984a,b, Kuramoto and Ebara 1988). The cardioarterial valves found at the origin of each
of the major vessels as they leave the heart prevent backflow of hemolymph during diastole, as
well as regulate flow to their respective artery. This is accomplished through excitation of the two
striated muscle flaps that make up the valve. When the valve is stimulated, the free edges of the
flaps contract, resulting in reduced systolic pressure. The flap muscles contract in response to direct
stimulation by an axon of a segmental nerve from the ventral ganglion. Contraction may also result
from actions of neurohormones such as proctolin, octopamine, dopamine, noradrenalin, acetylcho-
line, and serotonin (Maynard 1960, Kuramoto and Ebara 1984, 1989).
The alary ligaments that support the heart within the pericardium are also innervated. These
ligaments are made up of elastic tissue and striated muscle. The significance of their innervation
is not known (Maynard 1960). It has been suggested that the passive expansion of the heart dur-
ing diastole may be aided by the contraction of these ligaments (McMahon and Burnett 1990).
A control of cardiac volume has also been proposed as a function for the active contraction of
the alary ligaments. This may come about through a distention of the heart by the ligaments,
resulting in a greater end diastolic volume that may increase stroke volume in a manner similar to
the Frank-Starling mechanism seen in mammalian systems. In the myogenic heart of mammals,
one of the important regulatory factors is the Starling relation (McMahon and Wilkens 1983,
Cooke 1988, Burggren et al. 1990, McMahon and Burnett 1990). When intracardiac pressure is
increased, greater stretching of the cardiac muscle occurs, resulting in greater contraction and an
increased stroke volume. In the neurogenic heart of the crustaceans, it is thought that increased
cardiac distention and/or increased intracardiac pressure will result in a distension of the car-
diac muscle, which distorts the cardiac ganglion. This distortion will cause an increased burst
frequency, which results in an increased heart rate and greater cardiac ejection (Kuramoto and
Ebara 1984a,b).
Cardiac performance in invertebrates may be influenced by the actions of neurohormones
and neuromodulators (Kuramoto and Ebara 1984a, Zatta 1987, Barthe et  al. 1989, Freschi 1989,
Freschi and Livengood 1989, Preiffer-Linn and Glantz 1989). Neuromodulation of the cardioarte-
rial valves of decapods (already discussed) plays a key role in redistribution of the cardiac output.
Neuromodulation of the heart also plays a significant role in control and regulation of heart rate and
stroke volume. The action of these cardioactive chemicals can result in changes in the output of the
cells of the cardiac ganglion and the contractile force of the myocardium itself.
 Circulatory Physiology 211

Crustacean heart rate and contraction force are determined by the summation of information
coming to the cardiac ganglion (McMahon and Wilkens 1983). Stroke volume and distribution of
flow are more complex than previously thought and involve a number of possible interactions, the
result of which alters cardiac output. Modulation of cardiac performance results from numerous
factors, including internal interactions such as neural modulation, increase hemolymph pressure,
metabolic effects, sensory modulation, circadian rhythms, and animal movement (Larimer 1962,
1964, Larimer and Tindel 1966, Freadman and Watson 1989). External factors may also play a role
in altering cardiac performance. Sensory stimuli, such as visual and mechanical, will alter heart rate.
Local environmental conditions such as water O2 and carbon dioxide (CO2) levels, osmotic regula-
tion, and temperature will all alter cardiovascular functions (Patterson and deFur 1988, Wells 1988,
Wheatly 1989, Wheatly and Toop 1989).

Control of Vascular Flow

There are four broad mechanisms by which decapods can regulate vascular hemolymph flow: car-
dioarterial valves, peripheral valves, skeletal muscle contractions, and vascular contraction, with the
last being speculative at this time. As previously discussed in this chapter, at the interface between
the heart and each arterial system is a muscular cardioarterial valve that is under neuronal and neu-
rohormonal control (Kuramoto and Kuwasawa 1980, Kuramoto and Ebara 1984a,b, Kuramoto and
Ebara 1988, Kuramoto et al. 1995, Okada et al. 1997). As the tonus of these valves changes, flow
(resistance) to each peripheral arterial system can be modulated to control bulk arterial flow. In
two species of lobsters, peripheral valves located downstream from the cardioarterial valves have
been shown to have the active ability to regulate resistance and thus hemolymph flow to individ-
ual vessels within a system (Wilkens et al. 1997, Davidson et al. 1998, Wilkens and Taylor 2003).
These two regulatory systems provide the ability to not only regulate bulk arterial flow, but to also
provide a fine level of hemolymph flow regulation to specific vascular systems within the animal.
Furthermore, fine regulatory capability may come from observations in lobster arteries of contrac-
tility and elasticity along with the identification of both actin and myosin, which may provide fine
vascular regulation of hemolymph flow (Cavey et al. 2008, Wilkens et al. 2008). Specific and refined
vascular hemolymph flow regulation in decapods accounts for the system-specific metabolic func-
tion observed in these complex animals.
The last mechanism by which decapods, specifically macrurans, modulate arterial resistance
is through contraction of major skeletal muscle groups. Although this mechanism is not inherent
to the vascular system itself, major changes in resistance and flow have been demonstrated during
activity (Reiber et al. 1997). Flow was altered substantially as a result of tail flexion in both lobsters
and crayfish. Contraction of the abdominal muscles during tail flexion increased stroke volume
substantially and caused a redistribution of cardiac output but had no effect on hart rate. Cardiac
output was redistributed during tail flexion, increasing the blood supply to the abdominal muscles
and thus serving the increased metabolic demand during activity. Blood supply also increased to
the limbs, the mouthparts, and the muscles supplying the scaphognathites. A similar shunting of
flow toward active tissues was noted during struggling in the crab Cancer magister by Bourne and
McMahon (1989).

Hormonal Control of Cardiac Function and Blood Flow

A number of neurohormones have been implicated in controlling cardiac function, primarily heart
rate, and to a lesser degree stroke volume, cardiac output, and hemolymph perfusion through indi-
vidual blood vessels. The actions of these neurohormones can vary among different species or
even within one species (McGaw et al. 1995). A number of comprehensive reviews on crustacean
212 Iain J. McGaw and Carl L. Reiber

neurohormones, including their molecular structure, control pathways, and physiological actions,
have recently appeared (Christie et al. 2010a, Christie 2011). In addition, aspects of crustacean endo-
crinology are covered in Chapters 1 to 3 of this volume. This section provides a brief review of some
of the more important neurohormones and how they affect cardiac function and regional blood
flow. The hormones may be produced in and released from the CNS and associated ganglia, or
specific neuroendocrine organs such as the pericardial organs of the heart and the X-organ com-
plex of the sinus gland (Cooke and Sullivan 1982, Christie 2011). They are released into the circula-
tion, bathing all tissues and bringing about their effects simultaneously at multiple sites (Christie
et al. 2010a). In crustaceans, they may act on the cardioregulatory nerves, cardiac ganglion, cardiac
muscle, muscle valves, or on the walls of the arteries themselves.

Crustacean Cardioactive Peptide (CCAP)

CCAP, originally isolated from the pericardial organs of the green crab Carcinus maenas, is named
for its cardioactive effects (Stangier et al. 1987). The primary site of action of CCAP is the cardiac
ganglion, where it acts to increase burst frequency and amplitude (Saver et al. 1999, Cruz-Bermudez
and Marder 2007). It is known to exert positive chronotropic and inotropic effects in a number of
crustaceans. Infusion of 10−9 M CCAP onto isolated heart preparations of C. maenas produces a pro-
nounced chronotropic and a lesser inotropic effect (Wilkens and Mercier 1993, Saver and Wilkens
1998). CCAP exerts both chronotropic and inotropic actions on the heart of the crayfish Orconectes
limosus at concentrations above 10−8 M (Stangier 1991). In the blue crab Callinectes sapidus, it causes
a dose-dependent increase in heart rate and stroke volume (Fort et al. 2007a). These chronotropic
and inotropic effects appear to be disconnected because the response level for changes in contrac-
tion occur between 10−10 and 10−9 M, whereas modulation of heart rate occurs at higher concentra-
tions of 10−8 to 10−7 M and typically lasts for less time (Fort et  al. 2007a). CCAP also produces
smaller chronotropic and inotropic increases in the giant hermit crab Aniculus aniculus at concen-
trations of 10−6 to 10−5 M (Yazawa and Kuwasawa 1992).
CCAP does not appear to affect the heart rate of Cancer pagurus, C. magister, or Homarus ameri-
canus (Stangier and Keller 1990, Stangier 1991, McGaw et al. 1994a, Wilkens et al. 1996). Although
CCAP had no effect on heart rate of C. magister, perfusion of 10−8 M CCAP causes an increase in
stroke volume. The ensuing increase in cardiac output results in an increase in hemolymph flow
delivered through the anterolateral arteries (McGaw et al. 1994a). This suggests that CCAP is either
acting on the nerves of the cardioarterial valves or directly on the valve muscle itself (McGaw et al.
1994a). CCAP is also implicated in blood flow control in the smaller arteries and arterioles: it causes
an increase in vascular resistance of lobster arteries and may be used for regulation of blood flow in
the peripheral circulation (Wilkens et al. 1997, Wilkens and Taylor 2003; Fig. 7.5).

Proctolin

Proctolin was first isolated and sequenced from the hindgut of the cockroach (Starratt and Brown
1975). It has a wide array of effects in crustaceans: its primary cardiovascular effects tend to be excit-
atory, increasing rate and force of contraction of the heart (Keller 1992). Proctolin has multiple sites
of action including the cardiac ganglion, heart muscle, CNS, the heart valves, and arteries of the
peripheral circulation (Sullivan and Miller 1984, Saver and Wilkens 1998, Saver et al. 1998, Wilkens
et al. 2005, 2008).
This peptide causes an increased heart rate in the lobster H. americanus (Wilkens et al. 1996,
Wilkens and Kuramoto 1998) and the shore crab C. maenas, with threshold effects observed at 10−8
M for both species (Wilkens et al. 1985, Wilkens and Mercier 1993, Saver and Wilkens 1998), and,
at high concentrations, the heart rate of C. maenas remains elevated for 4–5 h after infusion of the
 Circulatory Physiology 213

50 AMA

25

0
Change in resistance (%)

300 ALA 300 HA

200 200

100 100

0 0

300 SA 300 DAA

200 200

100 100

0 0
ACh
Glu
5-HT

CCAP

F1
F2

ACh
Glu
5-HT

CCAP

F1
F2
DA

DA
PR

PR
Fig. 7.5.
Relative change in resistance in flow in five arteries (mean + SEM) of the lobster Homarus americanus in
response to perfusion of acetylcholine (ACh, 10–3 mol l–1), glutamic acid (Glu, 10–3 mol l–1), serotonin (5-HT,
10–6 mol l–1), dopamine (DA, 10–6 mol l–1), crustacean cardioactive peptide (CCAP, 10–6 mol l–1), proctolin (PR,
10–8 mol l–1), and the FMRFamide-like peptides (F1, F2, 10–8 mol l–1). The actual magnitude of the change can
be estimated by multiplying the percentage change shown here by the resistance at the same flow rate, 2 mL
min–1. Abbreviations: AMA, anterior median artery; ALA, anterior lateral artery; HA, hepatic artery; SA, ster-
nal artery; DAA, dorsal abdominal artery. From Wilkens et al. (1997), with permission from The Company of
Biologists, Inc.

peptide (Wilkens et  al. 1985). Heart contractility also increases in H.  americanus (Wilkens et  al.
1996, Wilkens and Kuramoto 1998) and C. maenas (Saver and Wilkens 1998). In C. magister, a large
increase in cardiac output via an increased stroke volume leads to an increase in hemolymph flow
to the legs and mouthparts via the sternal artery (McGaw et al. 1994a).
Although proctolin appears to be cardioexcitatory in nature, it does exert inhibitory actions,
primarily related to hemodynamics. A decreased outflow through the sternal artery occurs in the
semi-isolated lobster heart (Wilkens et al. 1996). Decreases in flow rates in the abdominal artery of
the spiny lobster Panulirus japonicus (Kuramoto and Ebara 1984) and the anterolateral arteries of
C. magister also occur (McGaw et al. 1994a), presumably by the direct action of proctolin on the
cardioarterial valves (Kuramoto and Ebara 1984, Tsukamoto and Kuwasawa 2003). In addition to
controlling hemolymph flow into the major arteries, proctolin has also been implicated as a control
agent in the peripheral vasculature where it causes an increased resistance in isolated arterial rings
of the lobster (Wilkens et al. 2008).

FMRFamide-like Peptides (FLPs)

The FLPs are a superfamily of neurohormones that are important modulators of the cardiovascular
and stomatogastric systems of crustaceans (Christie et al. 2010a). The FLPs have been identified
in every invertebrate phylum, thus making them the largest and most widely distributed group of
structurally similar peptides (Price and Greenberg 1989, Greenberg and Price 1992). In crustaceans,
214 Iain J. McGaw and Carl L. Reiber

the majority of the FLPs have been isolated from the pericardial organs or the X-organ complex in
the eyestalks (Mercier et al. 2003).
Most experiments investigating hormonal actions on the cardiovascular system have been
carried out using isolated or semi-isolated heart preparations. For the most part, the FLPs
tend to be cardioexcitatory in nature when tested on such preparations. The hormones F1
(TNRNFLRFamide) and F2 (SDRNFLRFamide) consistently produce both chronotropic and
inotropic effects in a variety of lobster, crab, and crayfish species at concentrations above 10−9 M
(Mercier and Russenes 1992, Wilkens and McMahon 1992, Mercier et al. 1993, McGaw et al. 1995,
Worden et  al. 1995, Wilkens and Kuramoto 1998, Cruz-Bermudez and Marder 2007, Fort et  al.
2007b). A number of other FLPs—FMRFamide and FLRFamide (Mercier and Russenes 1992),
NRNFLRFa (Skerrett et  al. 1995, Cruz-Bermudez and Marder 2007), APSGFLGMRamide, and
TPSGFLGMRamide (Christie et  al. 2008)—produce very similar effects. In addition to exert-
ing both chronotropic and inotropic effects, application of synthetic sulfakinin (–Y (SO3H) GHM/
LRFamide) causes normalization of an irregular heart beat (Dickinson et al. 2007). The peptide
GLDLGLGRGFSGSQAAKHLMGLAAANFAGGPamide (Homam-CLDH) modulates rate and
contraction amplitude of the lobster heart at concentrations of 10−11 M, which is among the lowest
for any cardioactive peptide (Christie et al. 2010b).
Not all the FLPs have dual cardioactive effects; GYNRSFLRFamide consistently increases heart
rate but has no effect on contraction amplitude of isolated C. sapidus hearts (Krajniak 1991). In con-
trast, SYWKQCAFNAVSCFamide causes a large increase in amplitude of the heart beat of lobsters
but no clear change in rate (Dickinson et al. 2009). The insect neuropeptides SchistoFLRFamide
(PDVDHVFLRFamide) and leucomyosuppressin (PQDVDHVFLRFamide) markedly decrease
the heart rate of crayfish, suppressing the cardiac rhythm for several minutes (Mercier and Russenes
1992).
The FLPs appear to modulate cardiac activity by acting on both the heart muscle and the car-
diac ganglion (Saver et al. 1999, Fort et al. 2007a, Stevens et al. 2009). In addition to modulating the
heart, the FLPs may also regulate blood flow through the arteries, probably by their direct action
on the cardioarterial valves. F2 causes an increase in flow to anterior aorta but a decreased pres-
sure and flow into the sternal artery of lobster H.  americanus (Wilkens et  al. 1996, Wilkens and
Kuramoto 1998). Similar patterns occur in the Japanese lobster P. japonicus: flow rates into the ante-
rior aorta and anterolateral arteries increase, whereas a decrease in flow to the sternal artery and
hepatic arteries occurs (Wilkens and Kuramoto 1998). The FLPs may also influence blood flow in
arteries and sinuses more distant from the heart by increasing resistance in these vessels (Wilkens
and McMahon 1992, Wilkens 1997, Wilkens and Taylor 2003; Fig. 7.5).
Interestingly, in whole-animal preparations, a number of the FLPs appear to exert inhibitory
responses. Although F1 and F2 are cardioexcitatory in isolated C. magister hearts, both elicit a dra-
matic and long-term decrease in heart rate and stroke volume and thus cardiac output in the whole
animal at circulating concentrations between 10−9 and 10−7 M (McGaw et al. 1995). The decreases
in rate are often accompanied by periods of cardiac arrest, and a regular rhythm is not regained
for several hours (McGaw and McMahon 1995) and is accompanied by a decrease in hemolymph
flow through all arterial systems (McGaw and McMahon 1995). Cardioinhibitory actions also
occur during infusion of SchistoFLRFamide and leucomyosuppressin. Although neither hor-
mone exerts chronotropic effects, stroke volume is significantly depressed, leading to a decrease
in total cardiac output. Hemolymph flow through the sternal artery and anterolateral arteries also
decreases. Threshold for these responses occurs at circulating concentration of 10−8 M (McGaw and
McMahon 1999). Perfusion of the in vitro heart of lobsters with 10−8 M myosuppressin (pQDLD-
HVFLRFamide) causes a decrease in the frequency, with a concomitant increase in the amplitude
of heart contractions. Evidence suggests that, in addition to modulating the cardiac ganglion,
myosuppressin also acts on peripheral sites (Stevens et al. 2009). The difference between isolated
 Circulatory Physiology 215

and whole-animal preparations suggests the FLPs may have multiple action sites in whole animals
rather than a single unified effect. Multiple actions are exerted at separate locations in the cardiac
system, leading to a cascade of secondary consequences mediated by intrinsic feed-forward and
feedback coupling mechanisms (Fort et al. 2007b, Stevens et al. 2009).

Amines: Serotonin, Dopamine, Octopamine

In crustaceans, the primary source for the amines—dopamine, octopamine, and serotonin
(5-HT)—are the pericardial organs and, to a lesser degree, the subesophageal and thoracic ganglia
(Beltz 1999, Dickinson et al. 2008, Christie et al. 2010a, Christie 2011).
The effects of 5-HT are primarily cardioexcitatory in nature, causing an increase in heart rate and
contraction amplitude in lobsters, crabs, crayfish, isopods, and stomatopods, with the threshold
for these responses occurring between 10−9 and 10−7 M (Kuramoto and Ebara 1984, 1988, Wilkens
et al. 1985, Airriess and McMahon 1992, Wilkens and McMahon 1992, Wilkens et al. 1996, Saver
and Wilkens 1998, Listerman et al. 2000, Tsukamoto and Kuwasawa 2003, Ando and Kuwasawa
2004). Although primarily excitatory in nature, 5-HT causes a mild bradycardia when applied to
the myogenic heart of T. longicaudatus (Yamagishi 2003). The chronotropic effects of 5-HT suggest
that it acts directly on the cardiac ganglion, possibly increasing burst frequency, number of spikes
per burst, and spike frequency in the burst (Miller et al. 1984, Kuramoto and Ebara 1988, Berlind
1998, 2001, Saver et  al. 1999, Fort et  al. 2004, Cruz Bermudez and Marder 2007). 5-HT imparts
more variable effects on hemolymph flow. In isolated lobster hearts, it causes an increased flow
pressure into the sternal and dorsal abdominal arteries (Wilkens et al. 1996). In intact C. magister,
flow rates through the anterolateral and posterior arteries increase following perfusion of 10−7 M
5-HT (Airriess and McMahon 1992). In contrast, it constricts the cardioarterial valves of the iso-
pod Bathynomus doederleini, resulting in a decreased pressure in all the arteries (Tsukamoto and
Kuwasawa 2003). It is also known to act on the walls of arteries, constricting the lumen and increas-
ing peripheral resistance (Wilkens and Taylor 2003).
Dopamine also exerts cardioexcitatory effects, with threshold for activity ranging between
10−9 M and 10−6 M (Saver and Wilkens 1998, Fort et al. 2004, Yamagishi et al. 2004). It increases
heart rate in crayfish, lobsters, and crabs (Berlind and Cooke 1970, Florey and Rathmeyer 1978,
Wilkens et al. 1985, Wilkens and Kuramoto 1998) and also causes an increase in both rate and
amplitude of contraction of the heart of T. longicaudatus (Yamagishi 2003), C. sapidus (Fort et al.
2004), L. exotica (Yamagishi et al. 2004), and C. maenas (Saver and Wilkens 1998). Dopamine is
slow acting on the latter two species, and the effects are sustained for several hours. In contrast,
dopamine has no appreciable effect on heart rate and causes a decrease in pressure of isolated
lobster hearts (Wilkens et al. 1996); in intact C. magister, it causes a bradycardia and/or cardiac
arrest when applied in high concentrations (Airriess and McMahon 1992). Like 5-HT, dopamine
exerts its action on the neurons of the cardiac ganglion (Cooke and Sullivan 1982, Miller et al.
1984, Berlind 1998, Saver and Wilkens 1998, Yazawa and Kuwasawa 1992, 1994, Tierney et al. 2003,
Ando and Kuwasawa 2004, Cruz-Bermudez and Marder 2007). L-cell axons from the pericardial
organs and the dorsal nerve cells of the CNS also regulate heart rate via modulatory feedback on
the cardiac ganglion (Fort et al. 2004). The direct effects of the amine on the cardioarterial valves
and the myocardium may also be important in regulation of blood flow (Wilkens et  al. 1997,
Tsukamoto and Kuwasawa 2003, Yamagishi et al. 2004). Administration of dopamine causes an
increase in outflow through the sternal artery of lobsters (Wilkens et al. 1996). In C. magister, a
reduction or cessation of flow in all arteries occurs, with effects lasting for up to 15 min (Airriess
and McMahon 1992). In the isopod B. doederleini, dopamine decreases the pressure in the ante-
rior aorta and five lateral arteries but increases the pressure in the paired anterolateral arteries.
It is suggested that dopamine exerts its effects directly on the valve muscle, causing constriction
216 Iain J. McGaw and Carl L. Reiber

or relaxation of these structures (Tsukamoto and Kuwasawa 2003). Like 5-HT, dopamine causes
increased resistance in the peripheral vessels of homarid and panulirid lobsters either by con-
stricting the arterial valves or through its direct actions on the walls of the arteries and sinuses
(Wilkens et al. 1997, Wilkens and Taylor 2003).
The effects of octopamine tend to be more variable compared with 5-HT and dopamine, with
thresholds for effects occurring between 10−9 and 10−7 M. Octopamine exerts marked cardioex-
citatory effects on crayfish, crab, lobster, and Triops hearts (Florey and Rathmayer 1978, Wilkens
and Kuramoto 1998, Ando and Kuwasawa 2004, Yamagishi 2004). In isolated C. maenas hearts, it
causes a sustained increase in rate (Wilkens et al. 1985) but has little effect on the intact heart rate
of this species, suggesting that its actions are preparation-determinant (Saver and Wilkens 1998).
It exerts biphasic effects in the crabs Eriphia spinifrons and C. magister, initially causing a short
period of bradycardia followed by a more sustained and pronounced tachycardia (Florey and
Rathmayer 1978, Airriess and McMahon 1992). In the lobster P. japonicus, it is inhibitory when
perfused at minimal pressure, reducing heart rate and burst frequency of the cardiac ganglion, but
it increases heart rate and contraction when the heart is perfused at higher pressures (Kuramoto
and Ebara 1991). In other preparations, octopamine appears to produce minimal effects on the
heart or cardiac ganglion (Grega and Sherman 1975, Wilkens et  al. 1996, Cruz-Bermudez and
Marder 2007). The effects of octopamine on the cardioarterial valves appear to be dependent
on the preparation and species. Octopamine hyperpolarizes the resting membrane potential of
muscle cells in the anterior valves and hepatic arteries of P. japonicus, causing them to relax, and
it depolarizes the membrane potential in the posterior valve, causing contraction (Kuramoto and
Ebara 1984, 1991, Wilkens and Kuramoto 1998). Although octopamine produces no clear effect
on the sternal artery of P. japonicus (Wilkens and Kuramoto 1998), it does cause a decrease in
flow into the sternal artery of H. americanus (Wilkens et al. 1996). In intact C. magister, octopa-
mine infusion results in an increased flow rate through the anterolateral and posterior arteries,
presumably by causing relaxation of the arterial valves (Airriess and McMahon 1992). In contrast,
octopamine constricts the cardioarterial valves in the isopod heart, decreasing arterial pressure
in all the arteries (Tsukamoto and Kuwasawa 2003). In conjuncture with the other two amines,
octopamine causes an increased resistance in the peripheral arteries and infrabranchial sinuses
(Wilkens and Taylor 2003)

PHYSIOLOGICAL RESPONSES

Modulation of cardiac function and regional blood flow optimizes delivery of O2, nutrients, and
hormones to the tissues and the subsequent removal of wastes. As such, cardiovascular mecha-
nisms are closely coupled with respiratory and metabolic processes. These respiratory, osmoregula-
tory, and digestive processes are covered in detail elsewhere in this volume. Therefore, this section
focuses on just the cardiovascular responses to environmental stressors.

Activity Patterns

Locomotor Activity

Crustaceans display a variety of locomotory patterns; in decapod crustaceans, slow walking in an

aquatic environment (sideways or forward) is typical, interspersed with short bursts of fast run-
ning, the use of modified fifth pereopods for swimming, or the use of rapid escape mechanisms
such as tail flips (McMahon et al. 1979, Hamilton and Houlihan 1992). Animals meet the increased
metabolic demands associated with exercise by increasing O2 uptake rates, thus increasing binding
 Circulatory Physiology 217

or carrying capacity of the hemolymph and increasing tissue perfusion via the circulatory system
(DeWachter and McMahon 1996a). As such, these physiological mechanisms tend to be closely
coupled (Wilkens et al. 1985, Rose et al. 1998).
A number of articles have examined the cardiovascular responses of crustaceans to exercise;
because these measurements were made at different walking speeds and temperatures using differ-
ent experimental techniques, the relative rates and patterns of change rather than absolute values
are discussed here. Cardiovascular responses to exercise are rapid, and heart rate plateaus within a
minute or two of initiation of exercise (Gurguis and Wilkens 1995, Rose et al. 1998, O’Grady et al.
2001). In contrast, following periods of exercise, the heart rate takes considerably longer to drop
to pre-exercise levels. Resting levels in C. maenas are reached in as little as 12 min (Hamilton and
Houlihan 1992), but can take over 8 h following exhaustive exercise in C. magister (McMahon et al.
1979). These values represent repayment of an O2 debt, the time being determined by the metabolic
consequences of the exercise (Rose et al. 1998).
Cardiac output can be influenced by both heart rate and/or stroke volume of the heart. When
C. maenas is exercised at sub-burst speed (5.8 m/min), there is a 1.8-fold increase in heart rate,
coupled with a 1.5-fold increase in stroke volume leading to a 2.6-fold increase in cardiac out-
put. These circulatory changes are accompanied by concomitant changes in ventilation and O2
uptake (Hamilton and Houlihan 1992). Heart rate also influences cardiac output to a greater
degree than stroke volume during activity in the blue crab C. sapidus (Booth et al. 1982) and the
lobster Homarus vulgaris (Hamilton 1987). In contrast, when C. magister is forced to walk until
exhausted, the heart rate only increases by 20%; however, cardiac output doubles, which sug-
gests that for this species changes in cardiac output are afforded primarily by increases in stroke
volume of the heart (McMahon et al. 1979). A similar pattern occurs during moderate walking
activity in C. magister, but recovery from exercise takes less than 1 h as opposed to more than
8 h recovery from walking until exhaustion (McMahon et al. 1979, DeWachter and McMahon
1996a). In the lobster H. americanus, heart rate increases within 1 minute of exercise and remains
elevated and steady while walking (Gurguis and Wilkens 1995, Rose et al. 1998), plateauing at
30–40% above resting levels while walking at speeds of 8 m/min. However, there is no observ-
able increase in rate as a function of walking speed (Rose et al. 1998). Stroke volume, however,
does increase as a function of walking speed and accounts for the bulk of the increased cardiac
output (Rose et al. 2000). This underscores the limitation of relying on heart rate alone to inter-
pret changes in cardiac output. In contrast, O’Grady et al. (2001) reported that the heart rate of
lobsters does increase during forced walking activity; a 70% increase occurs at lower walking
speeds of 0.9 m/min. Stroke volume has a greater influence on cardiac output in the amphibious
crab Pachygrapsus marmoratus (Houlihan and Innes 1984) and the land crab Cardisoma carnifex
(Wood and Randall 1981). Interestingly, when C. carnifex is exercised for prolonged periods, a
bradycardia occurs, but this slowing of the heart likely allows increased filling time and a greater
stroke volume (Herried et al. 1979). An increased cardiac output results in an increased hemo-
lymph flow to the walking legs via the sternal artery, with smaller increases in flow through the
anterolateral arteries (DeWachter and McMahon 1996a, McGaw and McMahon 1998). Digestion
is impaired during activity (McGaw 2007) and is accompanied by a concomitant decrease in per-
fusion of the digestive gland via the hepatic arteries. Flow rates through the small posterior and
anterior aortae are maintained during exercise (DeWachter and McMahon 1996a).
Although slow walking activity is the norm for most decapod crustaceans, some species exhibit
specific behaviors such as rapid swimming or tail flips, which may be used for migration or escape
from predators (McMahon et al. 1979, Hamilton and Houlihan 1992, Rose et al. 1998). When the
portunid crab Charybdis feriatus swims, cardiac output increases by up to 60% due to an increased
heart rate, whereas stroke volume actually decreases in this species (McMahon et al. 1996). This
is accompanied by a major increase in blood flow to the leg muscles via the sternal artery, coupled
218 Iain J. McGaw and Carl L. Reiber

with lesser increases in the other major arterial systems. After prolonged swimming bouts of 2 h
it takes 5 h for full recovery (McMahon et al. 1996). In macruran crustaceans, tail flips are asso-
ciated with an increased flow through the sternal and large posterior aorta that supplies the tail
muscles; these increases in flow persist for several minutes after tail flexion (Reiber et al. 1997). In
lobsters, the heart rate remains stable during tail flexion, and so the 40% increase in cardiac output
is driven by changes in stroke volume alone. The large increases in flows are not only the result
of an increased cardiac output, but are also partially afforded by diverting hemolymph away from
the anterior arteries and into the sternal artery and posterior aorta (Reiber et al. 1997). The rapid
increase in flow through the posterior aorta supplies the O2 demand of the muscles while a con-
comitant increase in flow through the sternal arteries supplies the scaphognathite muscles of the
gill bailer (Reiber et al. 1997).

Burying

Representative species in 11 families of the Brachyura have been reported to exhibit burying behav-
ior (Bellwood 2002, McGaw 2005b). The mechanics of burying are covered in detail elsewhere
(Faulkes 2013). This mode of existence represents an unusual situation because, once buried, the
animals body is supported in the sediment and is somewhat isolated from the surrounding environ-
ment. In addition, the respiratory openings are blocked, and animals may have to employ different
ventilatory and cardiovascular mechanisms (Faulkes 2013).
Reports of cardiac function in crustaceans when they are buried are less common compared
with ventilatory or respiratory responses. The heart rate of Corystes cassivelaunus decreases
when buried (Bridges 1979), whereas in C. maenas the heart rate becomes erratic, interspersed
with periods of cardiac arrest (Cumberlidge and Uglow 1978). Cardiac output increases sig-
nificantly during the burying process in C. magister and Cancer productus (McGaw 2004). This
increase in cardiac output is afforded primarily by an increased stroke volume and, to a lesser
degree, an increase in heart rate. In both species, there is a subsequent decrease in cardiac
output once the crabs are buried (McGaw 2004). This is due to a decrease in stroke volume
in C. productus, but a decreased heart rate in C. magister (McGaw 2004). The decrease in car-
diac output represents a reduced metabolic demand when buried, when only basal metabolic
activities have to be supplied (Bridges 1979, Naylor and Taylor 1999). Spontaneous periods
of cardiac arrest are common in settled animals (McDonald et al. 1977, Burnett and Bridges
1981), and the percentage of time spent in cardiac arrest increases in C. productus once bur-
ied (McMahon and Wilkens 1977, McGaw 2004). In general, cardiac and ventilatory param-
eters are usually tightly coupled in crustaceans (Wilkens et  al. 1985, Jury et  al. 1994a, Bock
et al. 2001), with periods of cardiac arrest occurring in conjunction with ventilatory reversals
(McMahon and Wilkens 1972, Wilkens et al. 1974, McGaw et al. 1994b). However, for buried
crabs, the cardiac and scaphognathite pacemakers tend to uncouple, the reasons for which are
unclear (McGaw 2004).
Hemolymph flows through the individual arterial systems of C.  magister and C.  produc-
tus also change during digging and subsequent burial (McGaw 2004). The burying process
involves a large energy expenditure, and the greatest increase in flow rate occurs through the
sternal artery that supplies the muscles of the thoracic sterna. Flow rates reach similar levels to
those recorded during forced walking activity (DeWachter and McMahon 1996a). Once bur-
ied, hemolymph flows decrease substantially through the sternal artery, whereas hemolymph
flow to the eyestalks and antennae via the anterior aorta increase (McGaw 2004). The anten-
nae are usually the only structures protruding from the sand when the crabs are buried, and
an increased blood flow to the musculature of these structures is reflective of their increased
 Circulatory Physiology 219

flicking and sensory activity (McGaw 2005b). Hemolymph flow to the digestive organs via the
anterolateral and hepatic arteries is maintained while the crabs are buried. This suggests that, in
crabs, as in other organisms, digestion takes place during periods of inactivity (McGaw 2004).
Occasionally, hemolymph flow through the sternal artery increases for approximately 30 sec
before any movement is observed. This is followed by slight movements of the chelae, blowing
sand out from between chelae and body by way of several ventilatory reversals and maintaining
a clear pathway between the exostegal channels (Garstang 1897, McGaw 2004, 2005b). This sug-
gests that cardiac function and locomotor activity may not be tightly coupled while animals are
buried (O’Grady et al. 2001, Chabot and Webb 2008).

Feeding and Digestion

The consumption and subsequent digestion and assimilation of nutrients are associated with a gen-
eral increase in metabolic parameters most commonly termed the specific dynamic action (SDA)
of food (Secor 2009). In ectothermic invertebrates, this is represented as an increase in O2 uptake
or an increased CO2 production. These increases in O2 uptake represent the energy needed for
feeding activity, mechanical digestion, production of enzymes, and the subsequent transport of
nutrients and intracellular digestion (protein synthesis; Secor 2009). This increase in O2 uptake
and its subsequent delivery to the tissues is afforded by an increase in cardiac activity in C. sapidus
(McGaw and Reiber 2000), C.  maenas (Rovero et  al. 2000), C.  magister, and Cancer gracilis
(McGaw 2005c, 2006b,c), which occurs immediately when the food is detected and before the
animals have started feeding. The actual feeding process is associated with a sharp increase in
heart rate, stroke volume, and cardiac output, the result of increased activity during food handling
(McGaw and Reiber 2000, McGaw 2006b). This is accompanied by a doubling or tripling of flow
rates through the sternal artery, branches of which supply the mouthparts and the chelae (McGaw
and Reiber 2000). Increases in sternal artery flow rates are accompanied by increased flow through
anterolateral arteries; this is the typical pattern observed during activity (McGaw et  al. 1994b,
DeWachter and McMahon 1996a). An increased flow in the anterolateral arteries between 1 and
3 h after feeding supplies blood to the cardiac stomach muscles aiding mechanical breakdown in
the foregut (McGaw 2005c, 2006b, McGaw and Reiber 2000, 2002). Extracellular digestion by the
hepatopancreas begins within 0.5–1 h after feeding (Dall 1967, Hopkin and Nott 1980), which cor-
responds closely with the initial increase in blood flow through these arteries. The total time (8 h)
of increased perfusion of the hepatic arteries in C. sapidus correlates with the period of extracellular
digestion (McGaw and Reiber 2000).
Heart rate in blue crabs typically remains elevated for up to 24 h following feeding, and there
is a close correlation between the decline in heart rate to prefeeding levels and time for food clear-
ance from the digestive system. In C. magister and C. gracilis, heart rate remains elevated for 10 h
and 5 h, respectively (McGaw and Reiber 2000, McGaw 2005c, 2006b). In the latter two species,
the changes in cardiac parameters and flow rates are not as pronounced compared with blue crabs;
C. magister and C. gracilis were artificially fed a set meal size via a tube, whereas C. sapidus were
allowed to forage freely and tended to consume larger meals (McGaw and Reiber 2000). Oxygen
uptake usually remains elevated for longer periods (2–3 days) than cardiac parameters following
feeding in crabs. This reflects increased energetic requirements for intracellular protein synthesis,
which contributes to the bulk of the SDA response (Houlihan et al. 1990, Mente et al. 2003).
If crabs are deprived of food, a decrease in heart rate occurs between 5 and 10 days after the
last feeding, which is associated with longer term decreases in O2 uptake (Ansell 1973, Taylor and
Naylor 1999). Heart rate also becomes more stable during this period with less variation between
average day and nighttime heart rates (Ansell 1973).
220 Iain J. McGaw and Carl L. Reiber

Environmental Stressors

Temperature

Temperature is undoubtedly the most important environmental factor affecting the physiology,
behavior, and distribution of aquatic ectotherms, and, in general, metabolic rate is positively cor-
related with temperature (Willmer et al. 2005). Crustaceans routinely experience seasonal, diel, or
short-term changes in temperature (Lagerspetz and Vainio 2006) and exhibit different physiologi-
cal and behavioral reactions depending on whether the temperature change experienced is acute
or chronic (Whiteley et al. 1997, Cuculescu et al. 1998, Lagerspetz 2003). Because crustaceans are
ectotherms and cannot regulate their body temperature, it will vary directly with the environment.
The heart rate of many aquatic ectotherms is very sensitive to alterations in body temperature, and
these changes in heart rate can be an important proxy for indicating animal stress (Iftikar et  al.
2010). In crustaceans, delivery of O2 to the tissues (via the circulatory system) in response to O2
demand is essential for survival in temperature extremes, and a mismatch will determine the sur-
vival limits for many species (Frederich and Pörtner 2000). The temperature ranges within which
an individual species can function is termed the optimum range, whereas temperatures outside this
range, at which physiological mechanisms start to break down and do not recover, are referred to as
the pessimum range (Frederich and Pörtner 2000).
In crustaceans, the majority of articles on cardiac modulation in response to temperature change
concentrate on heart rate alone and report an increase in rate with increasing temperature and vice
versa (Ahsanullah and Newell 1971, Spaargaren 1973, 1974, Cumberlidge and Uglow 1977, deFur
and Mangum 1979, Burton et  al. 1980, Depledge 1984, Morris and Taylor 1984, Mercaldo-Allen
and Thurburg 1987, Reiber and Birchard 1993, Aagaard 1996, DePirro et al. 1999, DeWachter and
McMahon 1996b, DeWachter and Wilkens 1996, Stillman and Somero 1996, Goudkamp et al. 2004,
Stillman 2004, Camacho et al. 2006, Worden et al. 2006, Ungherese et al. 2008, Iftikar et al. 2010).
Increases in rate are often accompanied by a reduction or abolition of spontaneous periods of car-
diac arrest (DeWachter and McMahon 1996b, Camacho et al. 2006, Worden et al. 2006). Typical
Q 10 values for heart rate vary between 1.5 and 4; higher Q 10 values are measured outside the ther-
mal optimum range and tend to indicate a more stenothermal species (DeWachter and McMahon
1996b, Frederich and Pörtner 2000, McGaw and Whiteley 2012).
The rate and magnitude of temperature change may also affect heart rate. Lobsters respond to
a rapid change in temperature (either an increase or decrease) with a short-term bradycardia (<1
min), followed by a tachycardia ( Jury and Watson 2000). However, this response is not always
observed if the temperature is changed more slowly ( Jury and Watson 2000). In the freshwater
prawn Palaemonetes antennarius, a rapid change of up to 8°C has no effect on heart rate (Ungherese
et al. 2008). A hysteria of heart rate similar to that reported in reptiles has been observed in the
crayfish Cherax destructor, whereby the change in heart rate is significantly higher during heating
than during cooling. The increased rate during heating may act to dissipate some of the heat via the
peripheral circulatory system (Goudkamp et al. 2004). Nevertheless, heart rate does not continue
to rise indefinitely with increasing temperature. The heart rate increases up to a certain tempera-
ture, after which the rate starts to decrease and become erratic, followed by acardia at the upper
critical maxima (Fig. 7.6). This break point, referred to as the Arrhenius break temperature (Stillman
and Somero 1996) or the pejus range (Frederich and Pörtner 2000), is often used to determine the
thermotolerance of a species (Stillman and Somero 1996, Camacho et al. 2006).
Acclimation to higher temperatures (20°C vs. 4°C) raises both the Arrhenius breakpoint
temperature and the CTMax in lobsters H. americanus by approximately 3°C and 5°C, respectively
(Camacho et  al. 2006). Likewise, in different species of temperate and tropical porcelain crabs,
acclimation to high temperatures (10°C difference) increases the CTMax in all species but is more
 Circulatory Physiology 221

pronounced in the temperate species. The opposite occurs for acclimation to cooler temperatures;
the CTMin of the tropical species is extended to a greater degree (Stillman 2004). Although 2–3
weeks are usually considered the standard time for full acclimation to occur (McGaw and Whiteley
2012), in lobsters, cardiac function may be complete in as little as 3 days (Camacho et al. 2006).
Nevertheless, there may be a longer term seasonal acclimation that is not abolished even after 4
weeks acclimation to laboratory conditions. A lower basal cardiac rate has been measured for lob-
sters collected in winter ( Jury and Watson 2000). In contrast, the heart rate of winter-collected

A 2°C 12°C 22°C 29°C

0.5 Hz
0.95 Hz 1.8 Hz 0.25 Hz

B 2.5
30 Temperature
Heart Rate 2.0
25
Temperature (°C)

20

Heart Rate (Hz)

1.5
15
1.0
10

5 0.5
0
0.0
0 10 20 30 40 50 60
Time (min)

C Temperature (°C)
30 25 20 15 10 5 0
7

6
R2 = 0.94786
5 y = −10.56x + 40.7984
In Heart Rate (bpm)

3
R2 = 0.98265
2 y = 25.43x − 81.44724

0
3.30 3.35 3.40 3.45 3.50 3.55 3.60 3.65
Temperature (1000*K−1)

Fig. 7.6.
Heart rate changes in a single lobster, Homarus americanus, over a temperature range of 2–30oC. (A) Raw data
traces. (B) Changes in heart rate over an increasing temperature range; here, heart rate reaches a maximum at
22oC before declining thereafter, with a CTMax value of 29.3oC. (C) Arrhenius break plot temperature is the dis-
continuity in the slope determined by intercept of the linear fits to the rising and falling slopes. From Camacho
et al. (2006), with permission from Springer.
222 Iain J. McGaw and Carl L. Reiber

crabs, P. marmoratus, is higher at any set temperature (DePirro et al. 1999), indicating a partial meta-
bolic compensation (Hochachka and Somero 2002).
Although changes in heart rate in response to temperature change are well established, fewer
studies have measured the contractility or stroke volume of the heart of crustaceans. This is impor-
tant because it is the actual cardiac output that will determine delivery of O2 to the tissues, and
heart rate and stroke volume can vary independently to influence cardiac output (DeWachter and
McMahon 1996, McGaw and McMahon 1996). The heart rate of lobsters is low and of large ampli-
tude in cold water, becoming faster and of lower amplitude when the water is warmed (Worden
et al. 2006). The cardiac output increases up to a temperature of approximately 10°C, only decreas-
ing in strength toward the higher temperatures (22°C) tested (Worden et al. 2006). Likewise, in
Dungeness crabs, the heart rate increases as the water is warmed from 4°C to 20°C; however, the
increased rate shortens the diastolic filling period, and stroke volume decreases significantly up
to about 12°C, with a slower decline thereafter, between 12°C and 20°C. These changes in heart
rate and stroke volume result in an increase in cardiac output (DeWachter and McMahon 1996b).
This increase in cardiac output is paralleled by a general increase in flow rates through each arte-
rial system, although there is no differential hemolymph redistribution among each arterial system
(DeWachter and McMahon 1996b). Although there is a general decrease in hemolymph flows when
the spider crab Maja squinada is cooled from 12°C to 0°C, a break point occurs at 8°C, and hemo-
lymph is diverted away from the lateral arteries and toward the muscles of the thoracic sterna and
scaphognathites via the sternal artery; this may help maintain locomotor and ventilatory activity
at lower temperatures. The diversion of flow to the sternal artery is accompanied by a concomi-
tant diversion of hemolymph to the hepatopancreas via the hepatic arteries, possibly preventing
hypoxia in this homeostatic organ (Frederich and Pörtner 2000, Bock et al. 2001). Flow rates in
both the arterial and venous system decrease substantially at the lowest temperature, indicating that
the critical thermal limits have been reached (Bock et al. 2001).
Although the exact location of the thermal receptors in crustaceans is still unknown, changes in
cardiac function are thought to be mediated by the neurons rather than by the direct effects of tem-
perature on the cardiac muscle ( Jury and Watson 2000, Goudkamp et al. 2004, Worden et al. 2006).
The lobster H. americanus may be able to respond to changes in temperature by as little as 0.15°C and
can certainly detect changes of 1°C ( Jury and Watson 2000). These authors suggest that the cardio-
regulatory nerves are responsible for regulating heart rate because lobsters with severed nerves do
not respond to temperature change. In isolated lobster heart preparations, apart from a slightly lower
heart rate, changes in cardiac parameters are the same as those observed in whole-animal prepara-
tions, and so it was suggested that the temperature effects are initiated via the cardiac ganglion rather
than by peripheral regulatory nerves (Worden et al. 2006). In contrast, in isolated heart preparations
of Dungeness crabs, the heart rate was only about one-third of that of intact animals, resulting in
a decrease rather than an increase in cardiac output with increasing temperature (DeWachter and
Wilkens 1996). These workers suggested that the heart must be regulated by neurohormones and/
or the cardioregulatory nerves. In support of hormonal modulation of cardiac function, Kuramoto
and Tani (1994) found that octopamine and serotonin are released from ligamental nerves of the
pericardial organs of the spiny lobster P. japonicas during cooling. This may protect this species dur-
ing low temperature exposure because spiny lobsters do not show as pronounced a bradycardia with
decreasing temperature compared with other species (Nakamura et al. 1994, Kuramoto 1999).

Oxygen

Crustaceans encounter aquatic environments that are permanently or temporarily hypoxic,

and this has resulted in the evolution of an array of behavioral and physiological responses. The
physical characteristics of water, low O2 capacitance, high viscosity, and slow diffusion rates, all
 Circulatory Physiology 223

contribute to the stress of an animal in dealing with hypoxic exposure. Aquatic organisms must
develop compensatory mechanisms to deal with a reduction in water O2 levels because of the very
nature of water as a medium (Toulmond 1987). The diverse array of habitats that crustaceans live
in make their physiological responses toward hypoxia broad and difficult to categorize in a simple
way. Animals are classically divided into two mutually exclusive groups: O2 regulators, which main-
tain O2 consumption (Mo2) independent of environmental O2 tension (Po2), or O2 conformers,
in which Mo2 varies in proportion to water Po2. This division has been imposed on the system for
easy categorization rather than to fit observed responses (Mangum and Van Winkle 1973).
A reduction in O2 tension elicits a wide range of physiological responses, with O2 conformation
and regulation at opposite ends of a spectrum. The basic problem of maintaining O2 delivery to
metabolically active tissues with low hypoxic tolerance must remain in central focus when inter-
preting these physiological responses. Separating O2 regulation from O2 conformation is not always
readily apparent, even within species. Many animals will regulate their O2 consumption indepen-
dent of water Po2 down to some critical level below which they become O2 conformers. The inflec-
tion point is known as Pcrit and is used as the standard against which organisms are compared for
hypoxic tolerance. Pcrit for a given species is not constant. A variety of physical and physiological
parameters influence the point at which an individual will switch from O2 regulation to conforma-
tion (Maynard 1960, Taylor et al. 1977a, Herreid 1980). Pcrit must be interpreted as an integrated
homeostatic balance point that changes with internal and external conditions. The interpretation
of Pcrit remains under debate; however, conceptually, Pcrit is a useful tool in evaluating an animal’s
adaptive responses toward hypoxia. If Pcrit is considered a relative value that will be influenced by
physical (temperature, salinity, etc.) and physiological (ventilatory, cardiovascular, metabolic, etc.)
parameters, then one can make a prediction as to how a given parameter will influence this inflec-
tion point (Herreid 1980, Mangum and Van Winkle 1973).
Larger crustaceans (decapods) typically attempt to maintain O2 consumption in the face of
declining water Po2. Oxygen regulation results from a combination of physiological adjustments
leading to an increase in O2 conductance. Oxygen conductance for the whole animal is the recipro-
cal function of total resistance (the summation of resistance in a series). This implies that conduc-
tance may be increased at any point between the external environment and the utilization of O2 in
the mitochondria. It appears that the primary mechanisms used by decapods to increase conduc-
tance are the modulation of ventilatory parameters, cardiovascular functions, and hemolymph pig-
ment affinity (Dejours et al. 1970). Decapods exhibit a range of responses enabling them to regulate
Mo2 independent of water Po2; however, not all of these are fully understood.
An initial increase (short-term response) in ventilation (Vw) accompanies a reduction in water
Po2 in most actively regulating decapods. Ventilatory volume can be increased as the result of
increasing scaphognathite beat frequency (fsc), and/or by adjusting the position of the scaphogna-
thite (epipodites of the third maxillipeds) and the configuration of the pumping channel, thereby
altering stroke volume (Sv; see McMahon and Wilkens 1983). Sole reliance on fsc adjustments to
compensate for a drop in O2 availability does not appear to be a ubiquitous response in crusta-
ceans. The typical ventilatory responses of decapod crustaceans (McMahon et al. 1974, Dejours
and Beekenkamp 1977, Zanotto et al. 2005) to hypoxia is to increase both fsc and adjust scaphog-
nathite stroke volume concomitantly. This does not exclude the possibility that an increase in Vw
could result entirely from an increase in fsc. Increasing Vw brings more water over the exchange
surfaces at the energetic expense of driving the scaphognathites (Wheatly and Taylor 1981, Wheatly
1993). If Mo2 is maintained and Vw increases, then, by definition, the O2 extracted from the water
(E) will decrease, provided that Pvo2 does not decrease substantially (Dejours and Beekenkamp
1977, Herreid 1980, Wheatly and Taylor 1981, Wilkes and McMahon 1982). The compensatory respi-
ratory mechanism for short-term hypoxic exposure is to maintain O2 delivery to the gills by increas-
ing Vw, which is reflected by an increase in the convection requirement for water (Vw/Mo2). The
224 Iain J. McGaw and Carl L. Reiber

increase in Vw (hyperventilation) also results in a respiratory alkalosis that increases hemocyanin

(Hcy) O2 affinity (Bohr shift). The decrease in extraction is compensated for, as reflected by main-
tenance of the arteriovenous O2 content difference and maintenance of Mo2 (Wheatly and Taylor
1981, Wilkes and McMahon 1982).
The initial acute response toward hypoxic exposure is a transient increase in Vw, which maybe
an avoidance response (Wilkes and McMahon 1982, Wheatly 1993). Sole reliance on respiratory
adjustments does not appear to be responsible for the maintenance of Mo2 beyond short-term
exposure (6–24 h). Long-term hypoxic adaptation appears to rely in part on mechanisms other
than respiratory adjustments. Evidence for this comes from observation of a number of decapods
(McMahon et al. 1974, deFur and Mangum 1979, Wilkes and McMahon 1982, deFur and Pease 1988,
Harper and Reiber 2006a) during long-term hypoxic exposure (6–12 days). A diminution of fsc and
Sv is observed after approximately 24 h, with Vw dropping significantly compared to maximum
hypoxic exposure values. The initial increase in convection requirement for water indicates a venti-
latory compensation for the decrease in water Po2. The convection requirement for water drops as
Vw declines and Mo2 remains unchanged. Efficiency of gas exchange is increased through “other”
long-term adaptive changes, as indicated by increases in convection requirements for hemolymph
(V b/Mo2), transfer factor (To2), and hemocyanin O2 affinity, and a decrease in ventilation perfu-
sion ratio (Vw/V b; see McMahon et al. 1974, Wheatly and Taylor 1981, Wilkes and McMahon 1982).
The decline in respiratory-mediated compensation is met by an increase in O2 conductance, as
indicated earlier. The transfer factor (To2) for O2 through the gill (O2 consumption as related to
O2 uptake per kPa O2 pressure gradient across the gill) increases significantly in a number of crus-
taceans examined during prolonged hypoxic exposure (Wheatly and Taylor 1981, McMahon et al.
1974, Wilkes and McMahon 1982). The convection requirement for hemolymph (V b/Mo2) and the
ventilation perfusion ratio (Vw/V b) both increase, the latter a result of declining Vw and a mainte-
nance or increase in cardiac output (V b). These changes would indicate a cardiovascular compensa-
tory component toward increased O2 conductance (Wheatly and Taylor 1981, McMahon et al. 1974,
Wilkes and McMahon 1982, McMahon and Wilkens 1983, Reiber et al. 1992). Hemocyanin O2 affin-
ity also increases during both short- and long-term hypoxic exposure. These ventilatory-associated
and/or nonventilatory components allow the animal to maintain Mo2 during hypoxic exposure for
long periods of time.
The hypoxia-induced hyperventilation not only increases O2 delivery, but also removes CO2
from the hemolymph. The decrease in CO2 results in a rise in hemolymph pH; that is, a respi-
ratory alkalosis that causes an increase in hemocyanin O2 affinity (Redmond 1955, Dejours and
Beekenkamp 1977, Wilkes and McMahon 1982, Taylor 1982). The extreme hyperventilation
observed early during hypoxic exposure initiates the rise in hemolymph pH. The alkalosis persists
during long-term hypoxic exposure and is associated with a base deficit that is never fully compen-
sated (Wilkes and McMahon 1982, Wheatly 1993). The increase in hemocyanin O2 affinity appears
to be mediated in part by a H+ concentration-dependent increase (Bohr shift) resulting from the
respiratory alkalosis. A  second component to the increase in Hcy O2 affinity is less understood
and is H+ independent. In several crustaceans, the increased Hcy O2 binding allowed a signifi-
cantly greater saturation of postbranchial blood. This increase in Hcy O2 affinity during hypoxic
exposure allowed 60% postbranchial saturation opposed to 20% saturation under normoxic con-
ditions (Wilkes and McMahon 1982, Guadagnoli et al. 2005). The respiratory alkalosis resulting
from hyperventilation is found to be offset slightly by a metabolic acidosis (L-lactate; Wheatly and
Taylor 1981). L-lactate has been found to increase Hcy O2 affinity and may play a role in increas-
ing O2 conductance through its interactions with hemocyanin (Mangum 1983). The increase in
Hcy O2 affinity plays a major role in increasing O2 conductance maintaining the pressure gradient
across the gills by removing O2 from solution, thereby aiding maintenance of Mo2 (Booth et al.
1982, Mangum 1983, Wheatly 1993).
 Circulatory Physiology 225

The cardiovascular response toward hypoxia has seen intense investigation in recent years, yet it
is still the least understood of the compensatory mechanisms. Oxygen conductance increases with
progressive hypoxia (as stated earlier) and is in part due to alterations in cardiovascular param-
eters (McMahon et al. 1974, Wheatly and Taylor 1981, Wilkes and McMahon 1982, McMahon and
Wilkens 1983, Reiber et  al. 1992). Decapod crustaceans exposed to progressive hypoxia tend to
reduce heart rate (hypoxia induced bradycardia). This has been demonstrated in many decapods
and appears to be a consistent response to hypoxic exposure in this group (Larimer and Gold 1961,
Larimer 1962, McMahon et  al. 1974, deFur and Mangum 1979, Wheatly and Taylor 1981, Reiber
et al. 1992). As heart rate decreases, a concomitant increase in stroke volume takes place, thus main-
taining cardiac output at or near normoxic levels down to a critical O2 tension. Cardiac output in
response to hypoxic exposure in several decapod species has been quantified using a variety of
methods (Fick principle, dye dilution, and pulsed Doppler), and a hypoxia-induced bradycardia
has been observed in each instance; however, cardiac output remains unchanged due to an increase
in stroke volume (Wheatly and Taylor 1981, Wilkes and McMahon 1982, Reiber et al. 1997, Reiber
and McMahon 1998, Harper and Reiber 1999, Guadagnoli et al. 2005, Guadangoli et al. 2007). The
increase in stroke volume appears to be due to an increase in end diastolic volume resulting from
both an increased filling time and filling pressure. During hypoxic exposure, mean pericardial sinus
pressure was found to increase significantly over normoxic levels. Diastolic intracardiac pressure
did not change during hypoxic exposure. The net effect is an increase in the pressure gradient
between the pericardial sinus and the heart, which aids cardiac filling (Reiber et al. 1992, Reiber
1992, 1994, Harper and Reiber 1999, Guadangoli and Reiber 2005, Guadangoli et al. 2007).
The decrease in heart rate associated with hypoxic exposure has been of note for some decades
and seen a great deal of discussion, but, until recently, no evidence has been produced to support
the hypothesis that a benefit to the animal results from bradycardia. In 2007, Guadangoli, Tobita,
and Reiber showed in grass shrimp that cardiac work is significantly decreased during periods of
short-term hypoxic exposure and bradycardiac events (Fig. 7.7). Matched intracardiac pressures
and cardiac volumes were used to develop pressure-volume loops with the area of the loop related
to cardiac stroke work. Cardiac stroke work was seen to decrease significantly with progressive
hypoxic exposure and decreased heart rates.
Along with the hypoxia-induced bradycardia and increased stroke volume comes a redistribu-
tion of cardiac output. Arterial hemolymph flow was measured using a pulsed Doppler technique
in the anterior aorta, posterior aorta, and sternal artery of the crayfish P. clarki (Reiber et al. 1992,
Reiber et al. 1997, Reiber and Wang 1997, Reiber and McMahon 1998; Fig. 7.8). As water Po2 was
decreased, arterial flow declined in the posterior aorta and sternal artery, whereas anterior aortic
flow increased significantly. Control of arterial flow is thought to be neurohormonally modulated
via the action of the cardioarterial valves (Kuramoto and Ebara 1984, McMahon and Burnett 1990,
Reiber et al. 1992, Reiber 1994). Redistributing arterial flow toward the anterior region of the animal
may have a protective function by maintaining O2 delivery to hypoxic-sensitive nervous tissues.
Decreasing hemolymph flow to the abdominal region in combination with maintaining cardiac
output would effectively increase the minute volume of hemolymph circulating through the gills,
which would confirm the hypothesis put forward by McMahon et al. (1974), Wheatly and Taylor
(1981), and Wilkes and McMahon (1982). Altering hemolymph flow patterns through the gills to
increase the functional exchange area has also been hypothesized as a mechanism to increase O2
conductance at the gill (McMahon et  al. 1974). There is no direct evidence for this in crayfish;
however, Taylor and Taylor (1986) have found evidence of valve-like structures in the gills of other
decapod crustaceans. Functionally, these structures may play a role in regulation of gill perfusion.
The observed cardiovascular responses toward progressive hypoxia in these animals is very similar
to the diving response seen in vertebrates and may serve many of the same functions. The role the
cardiovascular responses play in increasing O2 conductance during hypoxic exposure is only now
 A B C G
25

20

Pressure at 2.2 kPa (mm Hg)

15

Area Area 10
systole diastole
5

0
Normoxia
−5 10 min
20 min
30 min
−10
0.68 0.70 0.72 0.74 0.76 0.78 0.80
D F Ventricular area at 2.2 kPa (mm2)
ROI
20
Pressure (mm Hg)

H
15
20 350
10
5 300 a
15 a
0 250

Pressure (mm Hg)

a,b a,b
−5 a a

Cardiac parameter
10 200 a,b a,b
−10 150
0 10 20 30 40 50 60 70 80 90 100 5 fH (beats min−1)
E
0.73 0 Work (area fH)
0.72 1.5 PA loop area (mm2 mm Hg)
0.68 0.69 0.70 0.71 0.72 0.73
−5
Area (mm2)

0.71 1.0 a a
0.70 Area (mm2) a,c
−10
0.69 0.5
0.68
0.67 0
0 10 20 30 40 50 60 70 80 90 100 0 5 10 15 20 25 30 35
Hypoxic exposure time at 2.2 kPa (min)

Fig. 7.7.
(A) An outline of the heart in end systole as it defines the minimal area of the heart. (B) An outline of the heart in end diastole as it defines the maximal area. (C) The difference between
the maximal (end-diastolic volume [EDV]) and minimal (end-systolic volume [ESV]) areas defines the region of interest (ROI) or stroke volume used in automated area analysis.
(D) Intraventricular pressure, as it coordinates with E. (E) Changes in cardiac area (multiple cardiac cycles are shown) calculated from ROI. (F) Eight pressure–area (P–A) loops generated
by combining the values from D and E. (G) Representative P–A loops from a single animal under normoxic conditions (20.5 kPa) and after 10, 20, and 30 min of hypoxia at 2.2 kPa. The
P–A loop areas are 1.175 for normoxia and 0.867 at 10 min, 0.854 at 20 min, and 0.848 at 30 min exposure to hypoxia (2.2 kPa). (H) Relationship between heart rate (ƒH), P–A loop area, and
minute cardiac work (CW). Measurements were made at 5, 10, 20, and 30 min after exposure to hypoxia, which was followed by exposure to normoxic water for 5 min and a final measure-
ment taken at 35 min. The letter “a” denotes a standard deviation (s.d.) from time 0 at 20.5 kPa; the letter “b” denotes s.d. from 5 min at 2.2 kPa; and the letter “c” denotes s.d. from 10 min at
2.2 kPa (P < 0.001). From Guadangoli et al. (2011), with permission from The Company of Biologists, Inc.
 Effect of tail flexion on Cardiac and respiratory
arterial hemolymph flow pause
40 Anterior aorta

Phasic velocity
30
(kHz) 20
10
0

10
(mL min−1)
Mean flow

Sternal artery
30
Phasic velocity

20
(kHz)

10
0
(mL min−1)

80
Mean flow

40
0
Posterior aorta
30
Phasic velocity

20
(kHz)

10
0

40
(mL min−1)
Mean flow

20

8
Phasic velocity

6 Lateral artery
(kHz)

4
2
0
1.5
(mL min−1)
Mean flow

1.0

0.5

0
10 min Tail flexion

Fig. 7.8.
Composite recording from two lobsters of anterior aortic, sternal arterial, posterior aortic, and right lateral arte-
rial hemolymph flow rates (both phasic velocity and mean flow rates are shown) during tail flexion (an arrow
indicates the start of tail flexion; left panel) and during a cardiac and respiratory pause (right panel). From
Reiber et al. (1997), with permission from The Company of Biologists, Inc.
228 Iain J. McGaw and Carl L. Reiber

beginning to be understood. It appears that modulation of cardiovascular parameters aids signifi-

cantly the animals’ ability to withstand prolonged hypoxic exposure.
Crustaceans exposed to environmental hypoxia are able to maintain aerobic metabolism down
to a critical Po2, below which the animal must decrease Mo2 and switch to anaerobic metabo-
lism. While exposed to water with a Po2 above Pcrit, the animal is able to mobilize a number of
mechanisms that allow the maintenance of Mo2 by increasing O2 conductance. Increasing venti-
latory volume Vw via increasing fsc and Sv brings more O2 to the gill and allows short-term com-
pensation. Modulation of Hcy O2 affinity via the Bohr effect accounts for much of the long-term
compensation. Cardiovascular parameters are also modulated, possibly increasing gill perfusion
and altering gill perfusion patterns. Cardiac output is redirected toward the anterior region of the
animal, presumably to maintain adequate O2 delivery to hypoxic-sensitive nervous tissues while
the hypoxia-induced bradycardia decreases cardiac work. The cardiovascular system plays a sig-
nificant role in increasing O2 conductance and allows the animal to survive severe hypoxic waters
for extended periods of time. A limitation in any of the above-mentioned physiological parameters
would result in an observed increase in Pcrit.

Emersion

Crustaceans exhibit three main responses to aerial exposure depending on their lifestyle (deFur
1988): animals that are obligate water breathers and are rarely emersed tend not to be able to
oxygenate the blood while exposed. Amphibious crustaceans that inhabit the intertidal zone
or ephemeral pools vary greatly in their ability to tolerate aerial exposure, ranging from those
that are essentially similar to subtidal species to the bimodal breathing crustaceans that can
respire efficiently in both water and air. Finally, a number of groups (decapods and isopods)
have made the transition to land and will drown if submerged in water for any period of time
(deFur 1988). There are a number of articles on cardiorespiratory mechanisms in a variety of
amphibious and terrestrial crustaceans. Interestingly, during emersion periods, there does not
appear to be a close coupling in ventilation and cardiac parameters (Wilkens et al. 1985, deFur
1988, Rose et al. 1998). For example, in C. productus, C. magister, and C. maenas, although heart
rate and cardiac output either remain stable or decline, there can be both short-term and longer
term increases in scaphognathite beat frequency (Taylor and Butler 1978, deFur and McMahon
1984a,b, Airriess and McMahon 1996).
In C. magister, which is rarely exposed to air, there is a slow decline in heart rate with a con-
comitant drop in stroke volume and thus cardiac output (Airriess and McMahon 1996). Likewise,
juvenile C.  productus exhibit a drop in heart rate; however, this response is size-dependent, and
in larger adults the heart rate remains unchanged when emersed (deFur and McMahon 1984a,b).
In contrast, large C. sapidus respond with an increase, whereas small crabs exhibit a decrease in
scaphognathite and heart rate during a 4 h period of emersion (deFur et al. 1988). There are sub-
stantial limitations to gas exchange during emersion in blue crabs because, like fish, the gills clump
in air. These smaller individuals may survive aerial exposure by a reduction in metabolic demand
or tolerance of the hypoxic conditions (deFur et al. 1988). A general metabolic depression occurs in
C. magister coupled with a decrease in hemolymph flow rates through all arteries except the anterior
aorta, where a substantial increase occurs. This artery serves the subesophageal ganglion (brain) of
the animals, and doubling of flow through this vessel would serve to maintain oxygenation of this
structure (Airriess and McMahon 1996).
Amphibious species, such as the green crab C. maenas, are better adapted to life in the inter-
tidal zone. They show no change in heart rate and, unlike C. productus, are able to maintain blood
oxygenation status, possibly via an increase in stroke volume (Taylor and Butler 1978). However,
heart rate is also temperature-dependent, and it will decrease if they exit into cooler air or vice versa
 Circulatory Physiology 229

(Wheatly and Taylor 1979, Taylor and Wheatly 1981, Depledge 1984). The intertidal crab P. marmo-
ratus shows a similar response—a slight but consistent bradycardia in air, which is also dependent
on the air temperature (DePirro et  al. 1999). In the amphibious purple shore crab Hemigrapsus
nudus, a tachycardia occurs when emerging, followed by a gradual but slight bradycardia in air, but
no extended periods of cardiac arrest (Greenaway et al. 1996). The higher O2 of air content and its
high diffusion rate allows H. nudus to maintain an adequate Po2 with only intermittent ventilation
(Greenaway et al. 1996). The purple shore crab Leptograpsus variegatus exhibits both elevated heart
and ventilation rates, as well as increased O2 consumption in water compared to air. Part of this
increase in rate may be due to increased activity in submerged crabs. The arterial and venous PO2
are unchanged by immersion, whereas re-emersion promotes a transient increase in venous hemo-
lymph oxygenation. Although immersion increases respiration rate, possibly via increased activity,
this does not affect gas exchange or transport, and L. variegatus is able to maintain a high Po2 in both
air and water (Morris and Edwards 1996).
In bimodally breathing crustaceans such as Cardisoma guanhumi, which can live on land or in
water, different responses are observed. These crabs have a reduced gill surface area and enlarged
branchial chambers. There is no change in heart rate when crabs move from air into water, although
after 7 days, heart rate decreases slightly. When re-emersed, a slight increase in rate occurs with
a slow decline back to preimmersion levels (Gannon et al. 2001). In contrast, Shah and Herried
(1978) report a 50% decrease in rate in this species because there is less O2 in water, and a tachycar-
dia occurs when re-emersed to repay the O2 debt. In the land crab Gecarcinus lateralis, immersion
leads to a depression of heart rate by almost 50%, with a supposed decrease in cardiac output. It
is thought that this leads to a decrease in blood delivery to the gills and results in death if the ani-
mal is immersed for extended periods. Interestingly, there is no tachycardia to repay O2 debt when
re-emersion takes place; heart rate simply returns to normal (O’Mahoney-Damon 1984). In the
terrestrial crab Holthuisana transversa, there can be a shunt of blood from gills to lungs (Taylor and
Greenaway 1984). In water, more blood is sent to the gills, whereas when the animals are emersed
the blood is then shunted to the lungs, so much so that they are capable of directing almost the
entire respiratory flow to one structure or the other depending on the medium. The switch-over
can take several hours, especially when emersed, suggesting that they retain some limited capacity
for respiration in either medium (Taylor and Greenaway 1984).

Salinity

The osmoregulatory physiology of decapod crustaceans has been studied extensively (reviewed
in Mantel and Farmer 1983, Pequeux 1995; see also Chapter 8 of this volume). The most common
physiological response to low salinity is an increase in O2 uptake (Engel et al. 1975, Taylor 1977,
Guerin and Stickle 1992, Jury et al. 1994a), which is driven by an increased scaphognathite beat
frequency (Cumberlidge and Uglow 1977, McGaw and McMahon 1996, Dufort et al. 2001). These
changes are thought to reflect an increased energy requirement for active ion uptake (Taylor 1977,
Jury et  al. 1994a). Within the field of crustacean comparative physiology, there is a growing lit-
erature on the cardiovascular responses to low salinity, and the most common cardiac response to
low salinity is a pronounced tachycardia (Hume and Berlind 1976, Cumberlidge and Uglow 1977,
Spaargaren 1982, McGaw and McMahon 1996, 2003, McGaw and Reiber 1998, Dufort et al. 2001).
Despite the fact that most crustaceans react to hyposaline exposure with an increase in cardiore-
spiratory parameters, earlier work by Kinne (1964) suggested that organisms respond to a dilu-
tion of the media by exhibiting an increase, a decrease, or no change in respiration levels. It was
hypothesized that euryhaline organisms showed an increase, and stenohaline organisms exhibited
a decrease in respiratory and cardiac parameters. However, in the past, this had been difficult to
substantiate because most studies on crustaceans were limited to those that were classed as efficient
230 Iain J. McGaw and Carl L. Reiber

hyperosmoregulators (Wheatly 1988). With the increase in respiratory and cardiovascular studies
in recent years, a general pattern is now emerging to which many decapod crustaceans conform.
In contrast to species that are able to hold the body fluid concentration above that of the medium,
osmoconforming species exhibit a bradycardia (Spaargaren 1973, Cornell 1973, 1979, McGaw 2006b,
Curtis et al. 2007) and a decrease in O2 uptake (McGaw 2006b). The osmoconforming crab C. graci-
lis may be exposed to short periods of low salinity in its natural habitat and can only survive for a
few hours in salinities below 55% seawater (Curtis et al. 2007). The decreases in cardiac function and
hemolymph flow rates of C. gracilis in low salinity are a result of behavioral adjustments (McGaw
2006b, Curtis et al. 2007). C. gracilis becomes quiescent, closing its mouthparts and retracting the
antennae as soon as the salinity starts to decrease. This closure response isolates the branchial cham-
bers from the surrounding low-salinity water (Sugarman et al. 1983); because of this rapid isolation
response, coupled with diffusive ion loss into a closed area, the water in the branchial chamber is held
at a higher osmolality than the surrounding water (Curtis et al. 2007). At the same time, the decreased
cardiac output results in a higher hemolymph residence time in the gills, thus reducing the average
exchange gradient for inward movement of water and diffusive ion loss (Cornell 1973, Hume and
Berlind 1976). Low salinity is known to cause an increased hemolymph O2 binding affinity in C. mae-
nas (Truchot 1973); therefore, a slowing of hemolymph flow at the tissue level could be beneficial
in increasing O2 extraction from the circulating hemolymph (Larimer 1964). There is a pronounced
overshoot in cardiac and ventilatory parameters when C. gracilis is returned to 100% seawater. Thus,
the short-term decrease in cardiac function and O2 uptake is probably related to decrease in activity
levels because an O2 debt suggests repayment of energetic processes (McGaw 2006b).
Weaker regulators also tend to become inactive for short periods when they experience low salinity
(Sugarman et al. 1983, McGaw et al. 1999). However, they eventually exhibit a halokinesis in an attempt
to escape (Thomas et al. 1981 McGaw and Naylor 1992, McGaw et al. 1999). The weaker regulators
tend to exhibit mixed cardiorespiratory responses to low salinity: C. magister is classified as a weak
regulator and shows no change in O2 uptake in low salinity (Brown and Terwilliger 1999, Curtis and
McGaw 2010). Although this species exhibits an increase in heart rate during low-salinity exposure,
a reduced time for filling results in a reduced stroke volume, which leads to a significant drop in car-
diac output. This results in a depression in hemolymph flow rates through all arterial systems, except
for the small posterior aorta, in which a flow increase is observed (McGaw and McMahon 1996, 2003,
McGaw 2006a). These physiological responses are thought to be due primarily to behavioral adjust-
ments, whereby the animal becomes quiescent, rather than a direct physiological response (McGaw
et al. 1999). The American lobster, H. americanus, is also a weak hyperregulator and can inhabit estu-
aries. This species exhibits a biphasic cardiac pattern, responding to the initial drop in salinity with a
tachycardia; as the salinity decreases below 22 ppt, a bradycardia ensues (Dufort et al. 2001) coupled
with a transient increase in ventilation and a subsequent apnea. When the salinity drops below 10 ppt,
O2 uptake and heart rate increase (Jury et al. 1994a). For each of these weaker regulators, it is suggested
that avoidance behavior may be the first response to low salinity; if they are forced to osmoregulate,
changes in cardiac and respiratory processes may enhance their ability to survive hyposaline exposure.
The magnitude and direction of these changes depend on the degree and duration of the low-salinity
exposure (Jury et al. 1994b, McGaw et al. 1999).
The efficient osmoregulators increase activity levels in response to low salinity; this is termed
halokinesis (Thomas et al. 1977). This behavior is accompanied by increases in respiratory and car-
diovascular functions (Engel et  al. 1975, Hume and Berlind 1976, Cumberlidge and Uglow 1977,
Taylor 1977, Spaargaren 1973, 1982, Guerin and Stickle 1992, McGaw and Reiber 1998). Two effi-
cient osmoregulators, which are essentially model organisms, are the green crab C.  maenas and
the blue crab C. sapidus. In C. maenas, there is an increase in heart and scaphognathite rate (Hume
and Berlind 1976, Taylor 1977) associated with the increased energetic demand for osmoregulation
(Taylor et al. 1978). In contrast, Taylor et al. (1977a) report that the heart rate of C. maenas does
not change in 50% seawater (16 ppt); however, this study also examined temperature interactions
 Circulatory Physiology 231

that could have affected the results. Spaargaren (1974) reports that blood flow rates are lowest at
20 ppt but increase in dilute or concentrated mediums where animals have to regulate body fluids.
The heart rate of C. sapidus also increases when the salinity decreases (Sabourin 1984, McGaw and
Reiber 1998), and, as in C. maenas, stroke volume is largely unaffected by low salinity, suggesting
that the resulting increase in cardiac output is largely driven by heart rate (deFur and Mangum
1979, McGaw and Reiber 1998). The increased cardiac output is delivered through the sternal
artery, anterior aorta, and anterolateral arteries, whereas flow rates in the hepatic arteries and pos-
terior aorta remain stable. These increases in flow likely represent the increased activity but also
an increased energetic demand for active ion uptake. Some of the flow is directed to the anterior
aorta and anterolateral arteries, which have connections with the antennal gland, which is used for
volume regulation (McGaw and Reiber 1998, 2000). The overall increase in cardiac output will
eventually be directed through the branchial vessels, ensuring increased perfusion of the active
uptake sites in the gills (McGaw and Reiber 1998). When C. sapidus is acclimated to low salinity,
the heart rate, cardiac output, and flow rates drop back down to pretreatment levels (McGaw and
Reiber 1998). This may related to an upregulation of Na+/K+-ATPase and the fact that hemolymph
osmolality has reached new stable levels (Siebers et al. 1972).
The majority of articles on crustacean cardiovascular physiology have focused on the malacos-
tracan crustaceans, probably because their large size and accessibility make them easier to study.
Recent advances in optocardiographic methods have allowed fine-scale resolution in some smaller
crustaceans (Calosi et al. 2003). Calosi et al. (2005) report an increased heart rate in the sandhopper
Talitrus saltator when exposed to salinities as low as 5.5 ppt; at salinities below this level cardiac pat-
terns tend to break down. This appears to be associated with a decrease in osmoregulatory capacity
as time progressed, and it was surmised that modulation of heart rate and O2 uptake levels in this
species is directly related to their ability to osmoregulate.
Freshwater crustaceans tend to inhabit a more stable environment but can occasionally experi-
ence periods of increased salinity. The freshwater prawn P. antennarius reacts with a short-term alarm
response and an increase in heart rate in salinities of 15 ppt and 30 ppt before rate decreases back to
normal levels. A bradycardia occurs at 20 ppt, and this salinity is close to their iso-osmotic point and
so less energy is required to maintain osmotic pressure differences within this range (Ungherese
et al. 2008). Similar patterns are reported for freshwater crayfish Astacus astacus, where an increase in
salinity depresses the normally high heart rates (Styrishave et al. 1995). In contrast, in the freshwater
crab Oziotelphusa senex, minimal heart rates are observed in crabs adapted to 50% seawater, and rates
increase in both 25% and 75% seawater (Subrahmanyam and Krishnamoorthy 1984).

FUTURE DIRECTIONS

The majority of physiological studies carried out over the past century have focused on the decapod
crustaceans, probably because of the ease of use associated with their large size and their ability to be
maintained in the laboratory environment. No doubt they will continue to be the main focus of fur-
ther physiological research. Such work could include, but would not be limited to determining their
responses to biotic and environmental stressors and the neural and hormonal control mechanisms
that elicit these responses. Although hemolymph flows through the major arteries leaving the heart
has been investigated in some detail, there is much less information on the control mechanisms used
to shunt blood through the peripheral vessels and sinuses (Reiber and McGaw 2009). The gills are
situated inside the branchial chambers and are difficult to access in intact animals; control of blood
flow through each of the individual gills, as well as the function of the associated branchiostegal
sinus, is still not understood. Thus, the determination of the exact role of the branchiostegal circula-
tion and the partitioning of venous blood between the individual gills and this sinus represents a
pressing question for crustacean physiologists (McGaw 2005a, McGaw and Stillman 2010).
232 Iain J. McGaw and Carl L. Reiber

With recent advances in technology, two areas will undoubtedly expand in the coming years.
First, a comparison with other crustacean orders is long overdue, and, at present, information on
any aspect of cardiorespiratory physiology of nondecapodan orders is very limited compared to the
plethora of information available for decapods. This is important given the wide variation in ana-
tomical and morphological complexity among crustacean cardiovascular systems, and this should
be a fruitful area of research (Wirkner and Richter 2013). There is also a push toward carrying out
experiments in a more natural setting or even in the field. The improvement and miniaturization
of remote archival data tagging methods has accelerated so much in the past decade that tags can
now be affixed to and used to gather data from relatively small animals (Curtis and McGaw 2008).
As this technology continues to expand, it will open up an exciting new area for crustacean cardio-
respiratory physiologists, one in which multiple stressors can be investigated in the field and com-
pared with data derived from “controlled” laboratory experiments (Styrishave et al. 2003).

CONCLUSIONS

Crustaceans display a wide anatomical diversity of circulatory systems, ranging from simple tubular
hearts with no vessels through to the highly developed system of the decapods. The majority of the
work on crustaceans has focused on the decapods, probably because their large size, and the fact
they are readily available and survive well in the laboratory setting makes them an easy subject to
use. Historically, the cardiovascular system was regarded as a simple, low-pressure system with very
little control over regional blood flow; however, during the past three decades, precise physiologi-
cal control mechanisms have been shown to modulate cardiac output and partition regional blood
flow, so much so that these mechanisms rival those of some of the simple vertebrate closed systems.
Cardiac function and blood flow are modulated by both neural and hormonal control mechanisms.
Neural control can occur via the action of the cardioregulatory nerves that influence both the rate
and force of contraction of the heart by acting on the cardiac ganglion. Additionally, the tonus of the
cardioarterial valves can be modulated through neuronal stimulation, which regulates hemolymph
flow. A number of naturally occurring peptide and amine neurohormones modulate cardiac activity
and differential hemolymph flow in crustaceans. These hormones act on the cardioarterial valves,
artery walls, CNS, or the cardiac muscle itself. In addition, cardiac parameters and hemolymph flow
rates have been found to change during development and in response to exercise, feeding, hypoxia,
emersion, low salinity, and temperature change. These changes are not only important for efficient
delivery of nutrients and gases, but the diversion of flow to metabolically active tissues also may
enhance the ability of animals to cope with environmental changes. Future work will undoubt-
edly reveal further physiological control mechanisms, not only in the decapods, but also across the
diverse array of organisms that comprise the subphylum Crustacea.

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 8
OSMOREGULATION AND EXCRETION

Jehan-Hervé Lignot and Guy Charmantier

Abstract
Most extant crustaceans live in aquatic habitats with diverse salinity conditions (stable or predict-
ably or unpredictably variable). Therefore, some crustaceans are stenohaline osmoconformers
(marine species), whereas others are steno- or euryhaline regulators (some marine and all freshwa-
ter species). The capacity to inhabit these various habitats can also differ through ontogeny. A great
deal of information is currently available concerning the main optimized physiological cues, such as
hemolymph composition, the efficiency of the different osmoregulatory strategies, hormonal con-
trol, and functional organization at tissue, cellular, and molecular levels of the key osmoeffectors.
The effects of internal cues (gender, molting, nutrition, and ontogeny), as well as external factors
(parasitism, season, temperature, dissolved oxygen, pollutants) are also key elements to consider.
This broad review discusses these elements, focusing on different physiological strategies that allow
osmoregulating species to maintain internal solute concentration at a level different from that of the
external environment.

INTRODUCTION

Most of the extant crustaceans are aquatic (about 90%), living in the water column or as part of the
benthic community. The crustaceans inhabit various habitats: coastal areas where salinity condi-
tions can vary abruptly and unpredictably, the abysses (very stable environment), and hydrother-
mal vents (unstable) (Wirkner and Richter 2013). Crustaceans in different developmental stages
may also have to move and adapt to very distinct haline media (parasites, ontogenetic migrators).
This versatility makes crustaceans the most diverse aquatic invertebrates (Ruppert and Barnes
1994). Tolerance to changes in environmental salinity widely varies, and all types of osmoregu-
latory strategies are displayed within crustaceans. Euryhaline species can tolerate wide variations

249
250 Jehan-Hervé Lignot and Guy Charmantier

in salinity, whereas others are intolerant to changes in salinity and are stenohaline. Some crusta-
ceans are stenohaline osmoconformers (marine species), and others are stenohaline or euryhaline
regulators (some marine and all freshwater species). A species living in a very stable environment
with no salinity fluctuations (i.e., crayfish in freshwater) does not experience any change in salin-
ity in the wild but can, nevertheless, tolerate salinity changes (up to 50% seawater) in experimen-
tal conditions. The freshwater prawns from the genus Macrobrachium, like crayfish, maintain high
hemolymph osmolalities in freshwater and tolerate a range of salinities and are therefore recent
colonizers of the freshwater habitat (Freire et al. 2003, Ordiano et al. 2005).
In osmoconformers, intracellular fluid is isosmotic to the extracellular fluid (Fig. 8.1). This lim-
its the individuals to water bodies that do not vary in their ionic composition (strength). Although
cell volume (water balance) is easily maintained in this situation, intracellular ionic homeostasis
still requires finely tuned adjustments of the intracellular osmotic effectors (e.g., de novo synthesis
and degradation of free amino acids [FAAs] and phosphoric compounds) in order to retain mini-
mal osmotic gradients at the cell membrane surface (Schoffeniels and Gilles 1970, Péqueux 1995,
Wehner et al. 2003). This type of regulation, which is presumed to be an ancestral trait, is relatively
slow and thus is inefficient when rapid salinity changes occur (Anger 2001). Despite this, in some
euryhaline crustaceans, the intracellular concentration of these FAAs may be 10 times higher than
that observed in mammals (Gilles and Delpire 1997).
Crustacean regulators maintain their extracellular hemolymph osmolality with reduced varia-
tions, regardless of the salinity of the surrounding medium (Fig. 8.1). This anisosmotic extracellular
regulation is based on several mechanisms, thus implying various permeability and salt transport
properties within different ion-transporting epithelia. For example, in dilute seawater, crustaceans
maintain their hemolymph at a higher solute concentration than their environment and thereby
minimize osmotic water influx and diffusive salt loss across body surfaces by reducing drinking,
epithelial water permeability, or urine production and by actively pumping in salts from their envi-
ronment. These mechanisms are detailed later.
iso

1500
Hemolymph osmolality (mOsm/kg)

1000
3
2 1

500 2’

500 1000 1500

Medium osmolality (mOsm/kg)

Fig. 8.1.
Patterns of osmoregulation in crustaceans:  relation between medium and hemolymph solute concentra-
tion (mOsm/kg). 1: osmoconformers; 2: hyperisoregulators; 2′: hyperisoregulators in freshwater; 3: hyper-/
hyporegulators. Iso: isosmotic line. Adapted from Charmantier et al. (2009).
 Osmoregulation and Excretion 251

A CENTURY OF RESEARCH

The pioneering work on osmoregulation was done by Hecht (1914), and, since then, many other
studies have been published, culminating in the recent reviews by Péqueux (1995) and Charmantier
et al. (2009). Most studies have dealt with the composition of hemolymph, the efficiency of osmo-
regulatory strategies, the hormonal control and functional organization of osmoregulatory effec-
tors (at tissue, cellular, and molecular levels), and the effects of internal cues (molting, nutrition,
ontogeny), as well as with external factors (temperature, dissolved oxygen, season, pollutants).
Species of the order Decapoda have been the most studied because of their size and prevalence in
coastal and freshwater environments. An estimated 32% of these decapods occupy freshwater habi-
tats (De Grave et al. 2009, Coelho de Faria et al. 2011). Some only spend one or a few developmental
stage(s) in this milieu, whereas others have become fully adapted to freshwater and they spend their
entire life cycle there (several hololimnic caridean shrimps, trichodactylid, potamoid, and grapsid
crabs). Therefore, comparative studies in these decapods contributed to a better understanding of
the physiological mechanisms sustaining the invasion of the freshwater environment and enabled a
clearer view of the ecological implications involved.
The first studies addressed the ionic and water hemolymph compositions in osmoconform-
ers and regulators. The studies indicated that there are always (yet sometimes small) differences
between the hemolymph and the external milieu and between the composition of the intra- and
extracellular fluids. Intracellular isosmotic regulation has been particularly well studied by Gilles,
Péqueux, and colleagues. In parallel, the structure and function of gills and other extrabranchial
organs were explored using biochemistry, chemical blockers, isolated and perfused organs, elec-
trophysiology, microscopy, and molecular techniques. One of the key and most-studied players is
Na+/K+-ATPase, a membrane channel involved in the active pumping of sodium out of the cell
in exchange for K+ or NH4+. Among the numerous studies that focused on this enzyme, those by
David Towle must be highlighted. From the early 1980s onward, he and his colleagues studied the
physiological role of this pump and located it, as previously assumed, along the basolateral side
of the ionocytes present in osmoregulating organs such as the posterior gills of brachyuran crabs.
They then cloned and sequenced the α-subunit for the first time. David Towle was a true compara-
tive physiologist who used molecular techniques and genomics as additional tools for the ecophysi-
ological study of invertebrates.
Excretory physiology was poorly explored until the 1960s. After this date, the structure of anten-
nal and maxillary glands, urine formation (ultrafiltration), and volume regulation were described
(Péqueux 1995, Freire et  al. 2008, Charmantier et  al. 2009). Furthermore, the specific functional
adaptations of these excretory organs when crustacean species are exposed to dilute seawater or con-
centrated seawater, and for terrestrial life have also been documented. It appears that the contribu-
tion of the excretory organs is geared toward the control of water balance (urine production) while
total ionic balance remains minimal for most species. Therefore, the urine produced is usually isos-
motic and shows similar sodium and chloride concentrations to the hemolymph. However, for some
freshwater hyperregulators (crayfish) and freshwater and brackish water gammarids, compensatory
ionic reabsorption occurs. Similarly, in terrestrial species, ionic reabsorption from the produced
urine occurs in the gills and in the gut. As far as the digestive tract and its connected organs (hepato-
pancreas and midgut ceaca) are concerned, a direct role in osmoregulation was first demonstrated in
Artemia, with careful measurements of the osmotic pressure of the gut fluid indicating that it is less
concentrated than the medium but considerably more concentrated than the hemolymph (Croghan
1958a,b). The hepatopancreas has since been clearly identified as the main site for nutrient absorp-
tion (see studies by Ahearn and collaborators; e.g., Ahearn 1996), with some sodium-dependent
transporters seen to be at work at least during nutrient assimilation. The midgut region has also been
identified as a key player for water absorption at ecdysis (McNamara et al. 2005).
252 Jehan-Hervé Lignot and Guy Charmantier

The neurohormonal control of hydromineral regulation took off with early studies on molting
(McWhinnie 1962) and involved physiological experiments with organ ablation, extract injections,
organ perfusion, and transepithelial ion transport. The organs involved are the antennal gland in
isopods, and the brain, the “eyestalk complex,” the thoracic ganglionic centers, and the pericardial
organ in decapods. Various hormones, second-messengers like cyclic adenosine monophosphate
(cAMP), and regulatory factors have also been detected.
Finally, advances during the past 20 years produced a deeper understanding of the cellular and
molecular mechanisms and adaptive role of osmoregulation in crustaceans. Several membrane pro-
teins (exchangers, cotransporters, other ion pumps or ion channels) involved in ionic and water
transport have been revealed, along with their gene regulations, not only in the gills but also in
other osmoregulating tissues such as the gut lining and hepatopancreas, as well as in the excretory
organ. A more integrated understanding is now available at the gene and phenotypic level.

INTRINSIC FACTORS INFLUENCING THE HYDROMINERAL

BALANCE IN CRUSTACEANS

Different internal factors such as gender, body size, nutritional status, and ontogenetic and non-
ontogenetic migratory patterns, as well as the growth steps induced by molting, can directly or
indirectly influence the osmo- and ionoregulatory capacity of a crustacean.

Gender

In Gammarus roeseli, females exposed to high salinity show the lowest tolerance and the highest
and lowest hemolymph Na+ and Cl− concentrations, respectively (Sornom et al. 2010). This inter-
sexual difference of sensitivity may strongly impair the population structure and could partly be
explained by the increased energetic cost during oogenesis and egg incubation and the following
increase of lipidic synthesis and mobilization, in comparison to spermatogenesis processes that are
less demanding in terms of energy use (Buikema and Benfield 1979).

Age (Body Size/Weight)

A positive allometry has been documented for the freshwater crayfish Procambarus clarkii, with a
net ion uptake of Ca2+, Na+, Cl–, and NH4+ that increases with body mass (Zanotto et al. 2004). In
this species, between 72% and 97% of variation in ionic regulation is related to body mass. However,
in G.  roeseli, interage difference has no impact on salinity stress tolerance (Sornom et  al. 2010),
although, with a larger age range, several studies have demonstrated a higher sensitivity of juvenile
gammarids in comparison to adults (Naylor et al. 1995, Alonso et al. 2009).
The direct size effect may be due to different physiological factors, as well as to different
surface-to-volume ratios and weight-to-gill surface area ratios. For example, compared to later life
stages, juveniles could regulate their hemolymph at a lower osmolality, and the maximum salin-
ity beyond which they cannot regulate their hemolymph osmolality could be lower (i.e., juveniles
become osmoconformers at a lower salinity). Also, the maximum hemolymph osmolality that they
can tolerate is lower than in adults (Kefford et al. 2007). Furthermore, for a hyperregulating spe-
cies, juveniles with a proportionally greater gill surface area for their weights than adults experi-
ence larger water gain and salt loss and a higher metabolic rate per weight unit (Schmidt-Nielsen
1984). This has been demonstrated for postmetamorphic Callinectes sapidus hyperregulating in
diluted seawater (Kinsey et al. 2003, Li et al. 2006). Small blue crabs experience a bigger challenge
 Osmoregulation and Excretion 253

than larger crabs, one that is incompletely compensated by lower integument permeability and
higher levels of gill Na+/K+-ATPase compared to larger crabs. Finally, growth rates are higher in
younger individuals than older ones with more frequent critical molting steps and shorter intermolt
durations.

Nutrition

Feeding induces increased activity of the digestive tract (digestion, nutrient absorption, ion trans-
port and secretion), and a large postprandial increase in oxygen uptake and in cardiovascular vari-
ables that correspond to the well-described specific dynamic action (SDA; Carefoot 1990, McGaw
2005). Cellular protein synthesis then follows (Houlihan et al. 1990, Mente et al. 2003). Therefore,
the simultaneous demands of nutrition and osmoregulation can imbalance these physiological sys-
tems (prioritization or additive effects). For example, the osmoconforming Cancer gracilis can slow
food processing in the gut and can even regurgitate food from the foregut at low salinity (McGaw
2006a). Increased mortality has also been reported for postprandial crabs (Carcinus maenas, Cancer
magister) maintained at low salinity (Legeay and Massabuau 2000, McGaw 2006a,b). A  strongly
osmoregulating crab such as C. sapidus fed in seawater and then exposed to low salinity can coordi-
nate the responses to both of these physiological challenges, whereas C. magister, a weak osmoregu-
lator, under the same conditions prioritizes the osmoregulatory demand over digestion (Curtis and
McGaw 2010). In the osmoconforming C. gracilis, exposure to low salinity immediately after feeding
induces a temporary decrease in oxygen uptake, bradycardia, and reduced hemolymph flow rates.
However, the crab soon recovers and prioritizes digestion over the metabolic response to low salinity.

Molting

Crustaceans present an incremental increase in size when molting but usually spend most of their
time in intermolt, a period during which they feed and reproduce. Before shedding their confining
exoskeleton (premolt), crustaceans stop feeding, and the exoskeletal calcium is solubilized and then
transferred to the blood. It is temporarily stored in specialized organs such as the gastrolith (lobster,
crayfish), anterior integumental sterites (isopods), or posterior midgut caeca (amphipods). It is
then redeposited at postmolt into the new exoskeleton in order to increase its strength and rigidity.
Therefore, large amounts of calcium are transferred through different epithelial cell layers during
the molt cycle without altering the intracellular calcium activity (the cell signaling role of low intra-
cellular calcium concentrations). To accomplish this massive transcellular movement of calcium in
different directions through the cells, specific calcium transport proteins of the plasma membranes,
the endoplasmic reticulum (ER) calcium ATPase (SERCA), the apical ryanoride receptor (RyR),
and Na/Ca exchangers that use the transmembrane sodium gradient to drive calcium out of the cell
can, together, regulate calcium flow. Furthermore, down- and upregulation of 26 different cuticle
genes involved in the synthesis, breakdown, and resorption of chitin have been identified recently
across the molt cycle (Seear et al. 2010).
During intermolt, transcellular calcium fluxes are minimal, and the plasma membrane
Ca-ATPase largely controls cytoplasmic calcium activities by acting as a “housekeeping” protein.
As a result of the transepithelial calcium transporting processes and ion compartmentalization
occurring during premolt and postmolt, the ER may contain calcium activities greatly in excess to
those in the bulk cytoplasm, thus allowing the cell to retain its cell signaling functions throughout
these phases of the molt cycle.
Furthermore, before hardening by mineralization, the new exoskeleton is rapidly expanded
through water uptake. This massive water absorption (that is equivalent to 60–80% of body
254 Jehan-Hervé Lignot and Guy Charmantier

volume) occurs through the digestive tract, mostly. It accounts for 60–70% of the total water uptake
(Chung et al. 1999), whereas the gills and the newly soft cuticle may be responsible for the rest
(Neufeld and Cameron 1994).

Ontogenetic and Nonontogenetic Migrations

Some crustaceans spend their entire life cycle in the same environment, whereas others dis-
play complex migratory life history patterns, during which successive developmental stages are
exposed to different osmotic conditions. For instance, salinity can affect egg production and
development, as well as the development of post-hatch stages. In some cases, the salinity of the
surrounding milieu during egg incubation can later impair the reproductive capacities of these
individuals and can even induce sterility. Ontogenetic migrating crustaceans can have early life
stages living in shallow coastal waters, lagoons, or freshwater environments while the adults live
at sea, deeper in the water column and under more stable salinity conditions. The opposite onto-
genetic migrating pattern also exists: semiterrestrial crabs for which hatching in water is com-
pulsory, and freshwater palaemonid shrimps (Macrobrachium spp.) and brachyuran crabs (e.g.,
Eriocheir sinensis) migrate and release the larvae in saline waters. Salinity tolerance is usually
higher in adults than in larvae, which generally show weaker regulatory capabilities than juveniles
or adults (see the section “Ontogeny of Osmoregulatory Capacity”). Among the 200 extant spe-
cies of the genus Macrobrachium, M. amazonicum, distributed along the northern and northeast-
ern coasts of South America, typically displays ontogenetic migrations: freshwater-living berried
females migrate downstream, and hatching occurs in brackish water of estuaries where larvae
develop before recruitment to limnic populations through an upstream migration of juveniles to
freshwater habitats. Adults are strong hyper-/hypo-osmoregulators. However, isolated popula-
tions, currently assigned to the same species, are found in freshwater habitats of the Pantanal in
Brazil, where they have evolved a hololimnetic life cycle. Compared to the diadromous popula-
tions, the Pantanal shrimps have acquired an increased ability to hyperosmoregulate in freshwa-
ter, and, most strikingly, they show a complete loss of the ability to hypo-osmoregulate at high
salinity (Charmantier and Anger 2011).
Nonontogenetic migration also occurs for species like spiny lobsters and homarid lobsters
that move over large distances but in relatively stable environments. As weak regulators, they
can adapt to only small salinity changes. Salinity fluctuation therefore may not be the major
driving force for these migrations. For the brown crab Cancer pagurus, long-distance migrations
of more than 260 km have also been identified from the European coastline, along specific direc-
tions and usually against the main oceanic currents (Bennett and Brown 1983). Although juve-
niles and adult males are more sedentary, inhabiting the coastal waters, females present a more
migratory behavior. This may be linked to the stable salinity conditions found deeper offshore
that allows better hatching success and larval survival. Vertical salinity gradients (haloclines)
also affect the distribution and/or migration of planktonic crustaceans such as free-living cope-
pods (Lougee et al. 2002). Both phototactic and geotactic behaviors can be altered by salinity.
Indeed, various pelagic larvae of marine and estuarine crustaceans present downward move-
ment when exposed to lower salinity seawater. This has been observed for copepods, branchio-
pods, barnacles and cirriped nauplii, and multiple decapod larvae (Grindley 1964, Hughes and
Richard 1973). A change in sodium chloride concentration is the main environmental cue for
detection of salinity variation (Forward 1989, 2009). In the supralittoral sandhopper amphi-
pods, the salt concentration of the water influences their directional choice (Terracini-De
Benedetti 1963, Scapini 2006). More specifically, Ca2+ concentration of seawater appears to be
an important cue for the correct functioning of the sun compass mechanism to orientate along
the sea–land axis (Ugolini et al. 2009).
 Osmoregulation and Excretion 255

OSMOREGULATORY STRATEGIES

Osmoconformers do not widely regulate their body fluid ionic composition and osmotic strength,
although they have to face some fluctuations in salinity (Fig. 8.1). They allow body fluid osmolarity
to vary directly with that of the environment. Osmoconformity is usually the result of a high perme-
ability for monovalent ions and water through the integument (cuticle and underlying epithelium)
therefore inducing an entirely passive influx or efflux of ions and water and lack of ion selectivity.
Some species can reversibly switch from osmoconformation to osmoregulation depending on the
salinity of their surrounding milieu and the time of exposure. Among regulating crustaceans, some
hyperosmoregulate when exposed to a dilute environment, but they remain isosmotic when con-
fronted with highly concentrated media (“weak” and “strong” hyperisoregulators). These individu-
als maintain their hemolymph at a higher solute concentration than the environment (i.e., more
concentrated than the surrounding media). Strong hyperosmoregulators like E. sinensis are capable
of maintaining their extracellular fluids at hyperosmotic levels even in freshwater (Péqueux and
Gilles 1978, Cieluch et al. 2005). Crayfish, for instance, can maintain their hemolymph osmolal-
ity up to 400 times higher than their environment. Along with other freshwater decapods (e.g.,
Pseudothelphusa jouyi and Metopaulias depressus), crayfish are fully adapted to freshwater, have a
high capacity for salt absorption in the gills and a very low permeability coefficient for the integu-
ment, and can tolerate increased salinity but are incapable of returning to full-strength seawater
(Charmantier et  al. 2009). Weak hyperosmoregulators (e.g., Homarus gammarus; Charmantier
et al. 1984) are also able to cope with a hypo-osmotic environment but only for a restricted time
and are not able to migrate to freshwater (Felder 1978).
Other regulators have the ability to maintain a constant extracellular fluid in a wide salinity
range from concentrated seawater (>35‰) to freshwater (e.g., Armases roberti, A. ricordi, A. miersii;
Schubart and Diesel 1998, Charmantier et al. 1998). They hyperosmoregulate in dilute seawater,
but hyporegulate in seawater. Beachfleas (e.g., talitrid amphipods; Fig. 8.2) and terrestrial crabs are
also very good osmoregulators and can even face desiccation. Upon return to seawater, they remain
hypo-osmotic to the marine environment.
In light of these different osmoregulating strategies, complex mechanisms involving different
organs, active uptake or active excretion of ions, water transport, and regulation of intracellular
amino acid concentration have evolved. Water is not actively transported and can pass directly by
osmosis across cell membranes following the ion concentration gradient. It is therefore indirectly
controlled by the cellular response to changes in ion concentration. These strategies involve the
“pumps and leaks” system through limiting and compensatory processes (Péqueux 1995). Limiting
processes are mostly used by osmoconformers, which act on the permeability properties of the
cell membrane in order to minimize the diffusive movements of osmotic effectors (Schoffeniels
and Gilles 1970), whereas compensatory processes are mostly used by regulators and involve active
movements of solutes to compensate for diffusive fluxes between spaces inside and outside the cell.
The metabolic demands associated with this type of osmoregulation directly impact the animal’s
cardiac vascular system, oxygen uptake rate, and protein synthesis (Taylor et al. 1977, McGaw and
Reiber 1998). For example, lobsters at low salinity increase their metabolism and heart and ventila-
tion rates to help fuel the Na+/K+-ATPase necessary to keep their blood osmolarity higher than the
ambient water (Charmantier et al. 2001). Also, in Macrobrachium rosenbergii, salinity change has a
direct effect on protein synthesis in juveniles but not in postlarvae (Intanai et al. 2009). Osmotic
stress also influences lipid synthesis and gluconeogenic activity in the gills, muscles, and hepato-
pancreas (Schein et al. 2004, Martins et al. 2011). For example, total lipid concentrations in gills
and muscle of the crab Neohelice granulata (previously known as Chasmagnathus granulata and
C. granulatus) are lower in crabs exposed to hypo-osmotic media (Luvizotto-Santos et al. 2003).
A hyperosmotic stress also induces a decrease of the total lipid concentration in the posterior gills,
256 Jehan-Hervé Lignot and Guy Charmantier

1200
1100

Hemolymph osmolality (mOsmol/kg)

1000
900
800
700
600
500
400 Traskorchestia traskiana
300 Orchestia scutigerula
Orchestia gammarellus
200 Orchestia cavimana
100
0
100 200 300 400 500 600 700 800 900 1000 1100 1200 1300
Medium osmolality (mOsmol/kg)

Fig. 8.2.
Hemolymph osmolality plotted against medium osmolality for different talitrid amphipods. Note the hyper-/
hyporegulatory patterns displayed by these semiterrestrial beachfleas, except for Orchestia cavimana, which
maintains its hemolymph hyperosmotic between external concentrations of 50 and 900 mOsmol/kg. Note
also the regulation of O. scutilerula at ~635 mOsmol/kg during short-term exposure to a wide range of envi-
ronmental salinities (0–900 mOsmol/kg). It is hypothesized that the potential wide range of osmotic stresses
experienced on the high shore has selected the same osmoregulatory pattern. For O. cavimana, which is also
the only one to produce hypo-osmotic urine from the antennary gland (all the other species examined having
their urine isomotic with the hemolymph), the loss of hyporegulatory ability is explained by the fact that this
species is unlikely to experience hypersaline stress at the freshwater end of temperate estuaries. The relatively
high hemolymph concentration of ~350 mOsmol/kg for O. cavimana in freshwater and its euryhalinity may
be conservative traits, indicating evolution from brackish water ancestors. Adapted from Morritt and Spicer
(1998), with permission from NRC Research Press.

hepatopancreas, and muscles in this species (Chittó et al. 2009). Furthermore, during hypo-osmotic
stress, the hepatopancreatic phosphoenolpyruvate carboxykinase (PEPCK) activity of the shrimp
Litopenaeus vannamei (Rosas et  al. 2001)  and the PEPCK and glucose 6-phosphatase (G6Pase)
activities in posterior gills of N. granulata are decreased.

IONIC HEMOLYMPH COMPOSITION

The presence or absence of ions in the bathing medium and their relative importance in the hemo-
lymph can play key roles depending on the habitats used by the different developmental stages of
a species. These ions are the main osmotic effectors (Na+, Cl− being the dominant ions, along with
other major ions such as K+, Ca2+, and Mg2+) accounting for up to 90% of the hemolymph osmolal-
ity, and the rest being due to organic compounds such as FAAs and carbohydrates. Na+ and Cl−
concentrations account for 75–85% of the hemolymph osmotic pressure, as, for example, in penaeid
shrimps (Castille and Lawrence 1981, Lin et al. 2000) and in the freshwater shrimp Macrobrachium
olfersii when salinity of the external medium is more than 21 ppt (Lima et al. 1997).
The Mg2+ concentration is inversely related to the level of activity in different species of decapod
crustaceans (Robertson 1953, 1960, Walters and Uglow 1981) and in the amphipod Talitrus saltator
(Spicer et al. 1994). It has an anesthetic effect on marine invertebrates (Pantin 1946) and inhibits syn-
aptic transmitter release at the neuromuscular junction (Mantel and Farmer 1983). Therefore, low
magnesium concentrations along with low sulphate and calcium levels should positively influence
 Osmoregulation and Excretion 257

swimming activity (increase buoyancy) of active planktonic larval stages (Newton and Potts 1993).
For instance, unlike Brachyura, Anomura, Palinura, and Astacidea (Reptantia), caridean shrimps
(Natantia) have a high capacity for magnesium excretion and therefore maintain hemolymph Mg2+
concentrations far below that of seawater (Walters and Uglow 1981, Tentori and Lockwood 1990).
This can have a direct consequence on species distribution. It may explain the presence of fewer
decapods in the Antarctic continental shelf (caridean shrimps are present, but not brachyuran or
anomuran crustaceans) compared to subantarctic regions (Wittmann et al. 2010, 2011).
Crustaceans also regulate their serum K+ concentration over a wide range of salinities
(Robertson 1960). However, a low K+ concentration in inland saline waters that are abundant
in Australia (with an otherwise similar inorganic ion concentration compared to the open sea)
has been identified as the main cause for high mortalities in penaeid shrimp post-larvae (Tantulo
and Fotedar 2006). Although the contribution of K+ to the total hemolymph osmolality is small
in these post-larvae, its role in maintaining Na+/K+-ATPase activity is crucial, and an imbalance
between K+ and Na+ concentrations in the hemolymph can be the cause of the high mortality
rate observed in these inland saline waters (Zhu et al. 2004, Sowers et al. 2005). Because K+ con-
centration has a positive correlation with intracellular Na+ (Burton 1995), low K+ concentration
may result in disproportionate Na+ and K+ concentrations in the hemolymph, which may in turn
disturb the Na+/K+-ATPase activity.
The hemolymph Ca2+ concentration is significantly affected by molt stages and is usually
strongly regulated over a wide range of salinities (Ferraris et  al. 1986, Parado-Estepa et  al. 1989,
Neufeld and Cameron 1993). In freshwater with a low calcium concentration, for example, adapted
crustaceans have a reduced integumental permeability, a high calcium affinity, and a net Ca2+ influx
that occurs through active transport.

HORMONAL CONTROL

The Neuroendocrine System

The neuroendocrine control of water and ionic balance was recently reviewed (Chung et al. 2010,
Mykles et al. 2010). It has been studied in a few isopods but mostly using decapod crustaceans.
It involves the brain, the “eyestalk complex,” the thoracic ganglionic centers, and the pericardial
organ. In decapods, the brain is located between the two eyestalks and joins the thoracic ganglion
(coalesced paired ganglia of all thoracic and abdominal segments) by two circumesophageal con-
nectives, whereas the eyestalk complex is composed of the medulla terminalis X-organ (MTXO)
and the sinus gland (SG). Initial studies focusing on animals with ablated eyestalks and injections
of extracts from the different neuroendocrine centers indicated the presence of regulatory factors
within the eyestalks (Zeleny 1905); their ablation impaired adaptation to different salinities (Mantel
and Farmer 1983). One of the biogenic molecules is the crustacean hyperglycemic hormone neu-
ropeptide (CHH; see Chapter 2 in this volume) produced by the MTXO (Charmantier-Daures
et al. 1994). In C. maenas, a release of CHH in the hemolymph initiates molting and water uptake
(Chung et al. 1999). A long-term acclimation to dilute seawater also shows elevation of circulating
CHH (Chung and Webster 2005). Therefore, CHH and associated signaling pathways must have
a central role in the overall response to hypo-osmotic conditions. Structurally identical CHH and
CHH-like peptides have also been reported for non-eyestalk tissues, as, for example, in the gut
and pericardial organ of the shore crab C. maenas and for estuarine and freshwater species (Freire
and McNamara 1992, Tiu et al. 2007). Furthermore, in various decapod crustaceans, other biogenic
molecules including serotonin (5-HT), dopamine, octopamine, and norepinephrine have been
pinpointed as involved in the control of osmoregulation (Zatta 1987, Kuo et al. 1995). Finally, the
258 Jehan-Hervé Lignot and Guy Charmantier

SG has been identified as a storage and release center for the different crustacean neurohormones
synthesized in the brain and the MTXO complex.

Target Organs

The neuroendocrine extracts affect osmoregulation through increased circulation rate by the heart
and movement of salt and water in the gills, stomach, intestine, and excretory organs (the antennal
glands; Kamemoto 1991). CHH and ion transport peptide (ITP) are members of the same arthro-
pod neuropeptide family. In insects, ITP regulates Cl− transport and is involved in the gut water
reabsorption (Phillips et al. 1998). In estuarine and freshwater decapods, CHH is involved in bran-
chial ion transport and increases the transepithelial voltage and Na+ influx (Serrano et al. 2003).
Animals acclimated to low salinity also present high levels of CHH. Therefore, CHH has multiple
functions and is associated with supplying the energy required for iono-/osmoregulation (glucose
mobilization), molting, and reproduction.

OSMOSENSING AND OSMOREGULATORY EFFECTORS

The perception of salinity change and ionic composition of the external milieu, along with the reg-
ulation of water and ion concentrations in the body, relies on several osmoregulatory effectors and
involves different epithelia (integument, gills, extrabranchial organs, gut lining, and excretory organs).

Osmosensing

Adult crustaceans and their larval stages can perceive salinity changes. The osmoreceptors are
located in different areas: the walking legs, the branchial chamber, or near the excurrent opening of
the branchial chamber (Larimer 1964, Davenport and Wankowski 1973, Schmidt 1989). Depending
on the species, the antennae and antennules can also play an important role (Gleeson et al. 1996,
1997, Dufort et al. 2001). Finally, the system involved in salinity detection can be sensitive to the
concentration of certain ions only, rather than to the overall osmolality. For example, the American
lobster’s receptors located in or near the branchial chamber are primarily sensitive to chloride ions
(Dufort et al. 2001). Similarly, decreased concentration of sodium and cations of equivalent size
allows the perception of a salinity decrease, whereas cation size is not an important parameter for
detecting increased salinity (Harges and Forward 1982).

Integument

The permeability of the cuticle and underlying epithelium, because of its surface-to-body ratio,
can represent the main exchanging site for water, ions, and other elements. For instance, direct
integumental absorption of the exogenous amino acid glycine as a nutrient has recently been dem-
onstrated in phyllosoma larvae of the Japanese spiny lobster Panulirus japonicus (Souza et al. 2010).
Lowering integumental permeability limits water and ion fluxes. This is one of the key adapta-
tions to freshwater and terrestrial life. Therefore, salt and water permeabilities are higher in marine
and lower in freshwater species (Subramanian 1975), and acclimation to low salinity may induce
a rapid decrease in water permeability. Furthermore, osmoregulators are less permeable than
osmoconformers.
For brachyuran and anomuran terrestrial crustaceans, water and ion balance is related to their
habitat and osmoregulatory pattern. Some regularly return to seawater for water uptake, excretion,
 Osmoregulation and Excretion 259

and reproduction, whereas others completely avoid immersion but live in moist environments or
use inland freshwater, dew, and rainwater. These terrestrial crustaceans are capable of conserving
salts by postrenal modification of the urine. For instance, they all retain gills and other osmoreg-
ulating tissues that are implicated in ion reabsorption (Taylor and Greenaway 2002). Therefore,
although most marine and brackish water crustaceans generally produce urine isosmotic to their
hemolymph, terrestrial crabs are able to modify the final composition of their urine in their bran-
chial chambers. In some species, urine can also be reingested. Furthermore, evaporative water loss
is strongly reduced. As a comparison, in intertidal hermit crabs (Clibanarius), the evaporative water
loss rate is high and can be increased three times more if the animals are removed from their shells
(Herreid 1969). However, in coenobitids such as the terrestrial Coenobita scaevola, removal from the
shell increases evaporative loss by only 11% (Achituv and Ziskind 1985). For these terrestrial hermit
crabs, which only migrate back to the sea for reproduction, producing urine that is only slightly
hyperosmotic to the hemolymph generates increased drinking rates when drinking seawater (De
Wilde 1973, Greenaway et al. 1990). Despite this increased drinking in seawater in order to compen-
sate for the large quantity of urine produced, the net water gain still remains limited. Thus, when
saline water is provided for drinking, intake must be considerably enhanced because much of the
volume gained is needed to excrete the salt load, and the net gain of pure water, required to replace
evaporative loss, is small.

Gills

The gills (pleurobranchs, arthrobranchs, and podobranchs, depending on the location of gill
attachment) are the main organs for respiratory gas exchange and for homeostasis of the extracel-
lular fluid (Wirkner and Richter 2013). A description of the structure, ventilation, microvascula-
ture, histology, and ultrastructure of gills in various crustacean groups has recently been updated
(Freire et al. 2008, Charmantier et al. 2009). They are designated as phyllobranchiate (brachyurans,
carideans, and some anomurans), trichobranchiate (crayfish and lobsters), and dendrobranchiate
gills (penaeid and sergestoid shrimps) depending on their degree of surface amplification (Freire
et al. 2008). In the gills, osmoregulating sites can coexist with respiratory epithelial cells or can be
restricted to the posterior ones, as observed in several estuarine crabs (Charmantier et al. 2009).
In some species, gills are restricted to gas exchange, whereas epipodites and branchiostegites par-
ticipate in osmoregulation (i.e., lobsters, see next paragraph). Furthermore, although gas diffusion
is accomplished through thin and poorly differentiated cells, ion transport requires larger cells
with well-characterized structures. These “ionocytes” possess apical infoldings and basolateral
infoldings associated with numerous mitochondria. Adjacent ionocytes can be tightly linked with
septate junctions and lateral infoldings, thus providing structural strength and permeability regula-
tion through the intercellular space. A detailed review about the physiological and molecular data
obtained for the combined cytosolic and membrane-bound proteins at work in these ionocytes
is now provided in the most recent review (Charmantier et al. 2009). Therefore, a brief outline is
detailed in the last paragraph of this review detailing new challenges along with the current working
models for ion transport across the different osmoregulating tissues in crustaceans.

Extrabranchial Organs

Ionocytes and ion-permeable areas other than the gills have been described in several seawater,
estuarine, and freshwater crustaceans (Figs. 8.3 and 8.4). Some are located in the branchial cham-
bers: the pleurites, the inner epithelia of the branchiostegites and the epipodites (Bouaricha et al.
1994, Kikuchi and Matsumasa 1995, Lignot et  al. 1999). A  few others are situated outside these
Fig. 8.3.
(A)–(D) Immunolocalization of Na+/K+-ATPase in the gills (A), epipodite (B), and branchiostegite (C) of
the epibenthic Palaemon adspersus and in the epipodite of the deep-sea hydrothermal Rimicaris exoculata (D).
Scale bars: 50 µm. (E) Transmission electron micrograph of the branchiostegite of Palaemon adspersus. Scale
bar: 2.5 µm. (F) three-dimensional confocal stack showing the dorsal side of the Amphibalanus amphitrite nau-
plius. Black arrow indicates areas of intense fluorescent labeling in the naupliar tissue below the dorsal shield.
(G) Lateral view of the nauplius. Black arrows denote the frontal horns where fluorescence intensity is high.
Scale bars: 100 µm (F–G). Abbreviations: B, bacteria; BI, basal infoldings; BL, basal lamina; C, cuticle; CS, cau-
dal spine (posterior); E, epithelium; FH, frontal horns; GL, gill lamellae; HL, hemolymph lacuna; IC: internal
cuticle; IE, inner epithelium; M, mitochondria; N, nucleus; OC, outer cuticle; OE, outer epithelium; S, sep-
tum. See color version of this figure in the centerfold. Adapted from Martinez et al. (2005), with permission
from Elsevier, and from Gohad et al. (2009), with permission from Elsevier.
 Osmoregulation and Excretion 261

A B

1st

NB

2nd He

3rd
Pereopods 1-7

4th

5th
6th

7th

C
C

SM
M I
N

BL

Fig. 8.4.
Pereopodal disks of the estuarine amphipod Melita setiflagella. (A) Medial view of the right pereopods treated
by the silver nitrate nitric acid method. The arrows indicate the pereopodal disks as darkly stained discoid organ
located on the medial surface of each basipodite, except the third and fourth pereopods. Scale bar: 500 µm.
(B) Longitudinal section of a basipodite through the pereopodal disk. The disk (asterisk) is composed of a spe-
cialized thick epithelium (white block arrows) covered with a cuticle layer (arrowhead) thinner than that of the
ordinary epithelium. Scale bar: 25 µm. (C) Ultrastructure of the pereopodal disk (cross-section of an epithelial
cell). Note the presence of numerous mitochondria, deep basolateral infoldings (arrows), complicated inter-
digitations between the adjacent epithelial cells, and very shallow and sparse apical infoldings (arrowheads).
Scale bar: 2.5 µm. Abbreviations: BL, basal lamina; C, cuticle layers; He, hemocoel; I, internal infoldings; M,
mitochondria; N, nucleus; NB, nerve bundle; SM, striated muscle. From Kikuchi and Matsumasa (1995), with
permission from Elsevier.

cavities in nondecapod crustaceans: the neck organ of the nauplii, the metepipodites of the phyl-
lopodia, gut of the brine shrimp Artemia salina, the gut of terrestrial isopods, and the pereopodal
disk on the medial surface of the basipodite in amphipods, as well as the sterna, sternal gills, and
patch-like areas of the afferent blood vessels of the coxal gills in estuarine amphipods (Wägele 1992,
Aladin and Potts 1995, Hosfeld and Schminke 1997, Kikuchi and Matsumasa 1997; see Fig. 8.4).

Digestive Tract

In arthropods, the gut is divided into three distinct parts: the fore-, mid-, and hindgut (see Chapter 9
in this volume). In some species, diverticula and anterior and posterior caeca showing considerable
morphological diversity are also present (Icely and Nott 1985, Brunet et al. 1994). For example, in
decapods, the hepatopancreas or midgut gland forms a large branching gut diverticulum and is the
major site for nutrient absorption (Lockwood 1968).
262 Jehan-Hervé Lignot and Guy Charmantier

Whether the gut of crustaceans plays a role in osmoregulation has been questioned in very
early studies (Olmsted and Baumberger 1923, Drach 1939)  and, since then, has been regularly
addressed, taking into account the different parts of the gut, the hepatopancreas, and the midgut
caeca, and using different osmoconforming and osmoregulating species as well as terrestrial species
(for a review, see Charmantier et al. 2009). It appears that continuous or rhythmic antiperistaltic
water uptake can occur through the mouth and anus, and this drinking acts as an enema, main-
tains intestinal antiperistalsis, and stretches the gut wall muscles (Fox 1952). Some of this water
passes through the gut wall into the hemolymph and out of the body by way of the excretory organs
(Greco et al. 1986). The water absorption through the gut varies according to the external salinity
(De Wilde 1973, Greenaway et al. 1990) but is minor compared to the total water influx realized
through the gills and cuticle. It is solute-dependent, and Na+, Cl− and K+ are the key ions that drive
this water uptake (Mantel and Farmer 1983). Furthermore, several ion and ionic-mediated nutrient
transporters have been identified along the gut. For example, Na+/K+-ATPase has been detected
in several parts of the gut. In the hepatopancreas, the Na+/Cl− exchanger, different isoforms of the
electrogenic 2Na+/1H+ antiporter, and the proton-gradient-dependent Cl–/HCO3– have also been
identified (Ahearn et al. 1987). In the Chinese mitten crab (E. sinensis), fatty acid-binding proteins
(FABPs) have been identified in the hepatopancreas, intestine, and gills but were not detected in
the stomach. Leptin receptor has also been identified in the intestine but is less expressed in the
hepatopancreas and gills ( Jiang et al. 2010, Gong et al. 2010). Whether this is related to osmoregula-
tion remains to be investigated. Sodium-dependent glucose transport has also been reported in the
hepatopancreas of the American lobster, Homarus americanus (Ahearn et al. 1985). The presence of
glucose transporters (GLUT2-like, GLUT5-like, and SGLT1-like transporters) was later reported
in this species (Sterling et al. 2009). With the sodium-dependent hexose SGLT1-like transporter,
fructose is preferentially transported over glucose. It is potentially located in either the apical brush
border or basolateral side of the hepatopancreatic cells, or possibly both.
Therefore, water and ion transport by the gut lining is related to physiological processes such
as drinking, nutrient absorption, and molting. It is used by some marine hypo-osmoregulators and
terrestrial species for limiting dehydration through active salt extrusion (Dall 1967a,b) and urine
reingestion (Ahearn et  al. 1999). For example, in the terrestrial crab Gecarcinus lateralis, perme-
ability to water and ions by the foregut from lumen to hemolymph and in the reverse direction
is hormonally driven and occurs during the intermolt period and immediately after ecdysis. It is
therefore an important adaptation to conserve water (Mantel 1968). Similarly, in C. maenas, the
massive and rapid release of CHH from the fore- and hindgut endocrine cells only during premolt
induces water uptake by these tissues and, thus, size increase just after ecdysis (Chung et al. 1999).
The midgut and the midgut caeca have also been reported as ion and water transporting sites in
penaeids and various crab species exposed to dilute seawater (Dall 1967a,b, Mantel and Farmer
1983). Nevertheless, despite these data and the acute role of the digestive system for water intake
during molting, differentiating the osmoregulatory function from digestive processes is still diffi-
cult, and additional research is necessary, particularly in conditions of hypo-osmoregulation.

Excretory Organs

Data available concerning the crustacean excretory organs (antennal, antennary, maxillary, and
green or renal glands) indicate that they are functionally and structurally equipped for regulating
volume and ionic composition of their hemolymph through urine production (Henry and Wheatly
1992, Péqueux 1995). These morphological and functional data have been reviewed recently and
are briefly described (Freire et al. 2008, Charmantier et al. 2009). Among these different organs,
the antennal glands have been the most studied (Webb 1940, Behnke et al. 1990, Gao and Wheatly
2004). Antennal glands possess the same basic structure as the maxillary glands and consist of an
 Osmoregulation and Excretion 263

end sac, a convoluted duct (or tubule) that can be divided into proximal and distal portions, and
an exit duct that may differentiate as a bladder. In some species, the proximal part can be partially
differentiated as a labyrinth. This structure can appear as a very convoluted, coiled tubule, a maze of
anastomosing tubules, or a spongy tissue (Freire et al. 2008). Furthermore, different epithelial cell
types can be found along the tubule: podocytes filled with small and large vacuoles, lysosomes and
residual bodies in the end sac, typical transporting cells with an apical brushborder and basal infold-
ings associated with numerous mitochondria in the labyrinth and proximal tubule, and, last, epi-
thelial cells with no or less dense apical microvilli in the distal tubule. Finally, the bladder not only
functions as a storage compartment, but also possesses ionocytes. It is thus involved in absorptive
and secretory processes and further modification of the urine (Freire et al. 2008, Charmantier et al.
2009). Functionally, different key ion-transporting enzymes can be found in the excretory organs,
such as carbonic anhydrase and Na+/K+-ATPase, as well as Ca2+-ATPase (Peterson and Loizzi 1974,
Sarver et al. 1994, Khodabandeh et al. 2005a,b). Thus, the hemolymph filtrate in the end sac (Riegel
1961, Kirschner 1967) is modified by the reabsorption of some substances (i.e., organic molecules
such as glucose and amino acids) and secretion of others into the fluid (Riegel and Kirschner 1960,
Lin et al. 2000). As a result, most crustaceans living in seawater and/or subjected to dilute seawater
produce urine that is isosmotic to their hemolymph. Only in a few freshwater species, including
crayfish and some amphipods, are ions reabsorbed through the excretory glands, thus producing
a hypotonic urine (review in Charmantier et al. 2009). For brachyuran and anomuran terrestrial
crabs, specific adaptations allow them to conserve salts by postrenal modification of the urine. For
crustaceans exposed to higher salinities than seawater, the urine produced is isosmotic.

ONTOGENY OF OSMOREGULATORY CAPACITY

Different postembryonic developmental strategies can be found in crustaceans (Anger 2001),

with some species having numerous larval, postlarval, and juvenile stages (e.g., penaeid shrimps),
whereas others possess only a few post-hatch juvenile stages (freshwater decapods). Physiological
studies have clearly demonstrated that significant changes occur during the ontogenesis of osmo-
regulation in various crustaceans, including cladocerans (daphnids), isopods, amphipods, and
decapods (crabs, lobsters, shrimps, and crayfish; Cieluch et al. 2004, Khodabandeh et al. 2005a,b;
see Figs. 8.5 and 8.6).
Crustacean larvae are planktonic and swim actively in the water column. The larvae possess
a very thin cuticle that is permeable to water and ions. Furthermore, they do not have gills, and
salinity tolerance must therefore be achieved by actively regulating their internal concentration.
This isosmotic intracellular regulation has been shown in the megalopa of the blue crab C. sapidus
(Burton 1992) and in all the developmental stages of H. gammarus (Haond et al. 1999). Anisosmotic
extracellular regulation is also possible in larvae (Kalber and Costlow 1968, Charmantier et  al.
2009). Three patterns of ontogeny of osmoregulation have been recognized (Charmantier 1998,
Charmantier et al. 2009). In osmoconformers, osmoregulation does not vary throughout devel-
opment. The adult type of osmoregulation is established in the first postembryonic stage, thus
osmoregulation occurs, usually late, during embryonic development. In addition, freshwater deca-
pods display life cycles exhibiting varying degrees of abbreviated larval development, produce leci-
thotrophic eggs, and maintain low hemolymph osmolalities and diminished intracellular FAA titers
(Lee and Bell 1999, Augusto et al. 2007). In a third pattern, the early stages of development, usually
larvae, osmoconform or slightly osmoregulate; following metamorphosis, the juveniles display the
adult type and capacity of osmoregulation, become more euryhaline, and are able to change their
habitat (Fig. 8.7). These changes accompany ontogenetic migrations between sea and estuaries, as
seen for example for the grapsid crab C. granulata (Charmantier et al. 2002).
 A B
3 3
Zoea II Zoea II
metecdysis diecdysis
E

2 2 N

∆FPi °C

∆FPi °C
1 1

e
li n
ic
ot 1 hour after molt
sm
Iso

12 hours after ecdysis

1 2 3 1 2 3
∆FP0 °C ∆FP0 °C

C 3
Zoea III
metecdysis E

N
2
∆FPi °C

1 2 3
∆FP0 °C

Fig. 8.5.
Osmoregulation curves for zoea II and III of Rhithropanopeus harisii. Adapted from Kalber and Costlow 1966, with
permission from Oxford University Press. (A) Comparison of osmoregulation in normal metecdysial zoea II at 1 h
following molt and at 12 h ecdysis. (B) Osmoregulatory responses of normal zoea II and the eyestalkless animals of
the same age. (C) The effects of eyestalk removal on zoea III metecdysis at 12 h after molt. Abbreviations: E, eye-
stalkless; N, normal; ΔFPi, ΔFP0, hemolymph, medium freezing point depression. Osmotic pressure (mOsmol/
kg) = (|ΔFP|/1.858) × 1,000 (1.858 is the freezing point constant for water). These results point out the effects of
molting and of the sinus gland on the larval osmoregulatory capacity. Premolt (data not shown) and postmolt larvae
(here, zoea II) immediately after molt are hyperosmotic above 33‰, most probably to ensure inflow of water and to
establish a greater body volume during hardening of the new exoskeleton (A). Also, zoea II larvae do not hyperos-
moregulate during diecdysis, but eyestalkless individuals show a significant gain to osmoregulate at all salinities (B).
Normal zoea III hyperregulate at salinities below 30‰ and show a slight hyporegulation near 40‰ seawater (C).
Eyestalkless larvae, however, hyporegulate at all salinities below 33‰ seawater and hyperosmoregulate against 40‰
seawater. Therefore, the sinus gland located in the eyestalk influences water uptake during molting.

Fig. 8.6. (Continued)
lings follow a hyperisosmotic regulation pattern. The established osmotic gradient required for the uptake of
water results in the swelling of these embryos and occurs before the appearance of the coxal gills and possibly
via the vitelline membrane and/or the dorsal organ. Stage 5, 6, and 7 embryos, however, exhibit a distinct
hyper-/hypo-osmotic pattern of regulation. For all these developmental stages, the isosmotic point is similar
(between 500 and 550 mOsmol/kg). It is also worth noting that for this species, the changes in osmoregula-
tory pattern are not associated with a profound metamorphosis. Adapted from Morritt and Spicer (1995), with
permission from John Wiley and Sons.
 A
Stage 2/3 embryos Cephalothorax Dorsal organ
1250

1000 Limb bud

750
Vitelline
500
membrane
250 Peri-embryonic space

0
0 250 500 750 1000 1250
Peri-embryonic fluid osmolality (mOsmol/kg)

B
Dorsal organ
1250 Stage 5 embryos Non-beating heart
1000

750 Developing
coxal gill
500 Embryonic
eye
250
Peri-embryonic space Vitelline membrane
0
0 250 500 750 1000 1250

C
Dorsal
1250 Stage 6/7 embryos organ
Vitelline Beating heart
1000 membrane

750

500
Embryonic
250
eye
0 Developing coxal gill
0 250 500 750 1000 1250 Peri-embryonic space

1500 Hatchlings
Hemolymph osmolality

1250
(mOsmol/kg)

1000
750
500
250
0
0 250 500 750 1000 1250
Medium osmolality (mOsmol/kg)

Fig. 8.6.
Changes in the pattern of osmoregulation in the embryonic development of the brackish water amphipod
Gammarus duebeni. (A) Stage 2/3 embryos, reddening cephalothorax with prominent dorsal organ and limb
bud development; (B) stage 5 embryos, appearance of eye pigmentation and nonbeating heart; (C) stage 7
embryos, heart beat present, initially irregular becoming regular; (D) hatchlings, immediately posteclosion.
Periembryonic fluid (fluid from the space between the embryo and the vitelline membrane) or hemolymph
osmotic pressure is plotted against the corresponding medium concentration. Stage 2 and 3 embryos and hatch
266 Jehan-Hervé Lignot and Guy Charmantier

A B
1600 Zoea I Zoea II
1600
1400 1400

1200 1200

1000 1000

800 800

600 600

e
e

lin
400
lin
400

ic
ic

ot
ot

sm
sm
Hemolymph osmolality (mOsmol/kg)

Iso
200 200
Iso

0 0
0 200 400 600 800 1000 1200 1400 1600 1800 0 200 400 600 800 1000 1200 1400 1600 1800
C D
Megalopa Crab 1
2000 2000

1800 1800

1600 1600

1400 1400

1200 1200

1000 1000

800 800

600 600
e
lin

e
lin
ic

400 400
ot

ic
sm

ot
sm
Iso

200 200 Iso

0 0
0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200 0 200 400 600 800 1000 1200 1400 1600 1800 2000 2200
Medium osmolality (mOsmol/kg)

Fig. 8.7.
Osmoregulation curves for larvae and early postlarvae of Uca subcylindrica. This species living in semiarid
habitats and facing regular and unpredictable salinity extremes present condensed larval development that is
completed with only two brief zoeal stages followed by a postmetamorphic megalopa (2–3 days after hatch-
ing). The first crabs appear within 4.5–8 days after hatch. The ability to hyperosmoregulate is already present at
hatching time (A), and tolerance to higher salinities increases with successive stages (B, C, and D) as the ability
to hypo-osmoregulate improves (C, D). Adapted from Rabalais and Cameron (1985).

EXTERNAL CUES

Parasitism

Parasite-induced changes in physiology and behavior of the host appear adaptive for the parasite
and can directly affect the osmoregulatory capacity (OC) of the host. For instance, hemolymph
osmolality of hyperosmoregulating estuarine species, such as the blue crabs Callinectes rathbu-
nae and C.  sapidus, is lowered when parasitized respectively with the rhizocephalan barnacle
Loxothylacus texanus (Alvarez et al. 2002) and microsporidian Ameson michaelis (Findley et al. 1981).
In C. sapidus, hemolymph osmolality and sodium and chloride concentrations were reduced while
hemolymph K+ and amino acid concentrations increased. For C. rathbunae, which lives in tropical
estuaries and is regularly subjected to sudden salinity changes, the OC of the parasitized crabs is
significantly affected with only decreasing salinity, therefore limiting this crab to high-salinity areas.
Protist parasites of the dinoflagellate-like genus Hematodinium also affect the OC of the osmoregu-
lating C. maenas. No such alteration was detected in the osmoconforming C. pagurus and Pagurus
bernhardus, although acid–base balance is affected (Hamilton et  al. 2010). Supralittoral talitrid
 Osmoregulation and Excretion 267

sandhopper amphipods such as Talorchestia quoyana infected with the mermithid nematode para-
site Thaumamermis zealandica burrow more deeply into sand layers than uninfected amphipods in
order to reach the interstitial seawater level. This water-seeking behavior is induced by an increase
in osmolality of body fluids and allows the parasite that is almost completely filling the body cavity
of its host to emerge into moist sand, a necessary requisite for the free-living adult stage (Poinar
et  al. 2002, Williams et  al. 2004). The presence of acanthocephalan parasites (e.g., Polymorphus
minutus, in the cystacanth stage) inside the hemocoel of gammarid amphipods (G. roeseli, G. pulex)
also changes hemolymph osmolality (Bentley and Hurd 1993), solute concentration (carotenoids,
proteins, and glycogen; Plaistow et al. 2001), and salinity tolerance of the host (Piscart et al. 2007).
Although the sodium pump activity is not affected in parasitized gammarids, reduced oxygen
consumption, increased intra- and extracellular FAAs, and hemolymph hemocyanin concentra-
tion may explain the observed increased salinity tolerance (Patrick and Bradley 2000, Piscart et al.
2007). Devoting more resources to tolerate increased salinity may thus induce the death of the
parasites and, subsequently, must decrease the parasitic-induced stress.

Season and Temperature

Temperature variations have been reported repeatedly to affect crustacean osmoregulation, with
high and low temperatures generally decreasing the capacity to osmoregulate in temperate species.
As observed for crab larvae and juvenile shrimp, the range of tolerance of salinity narrows when
temperature deviates from the optimum and vice versa (Charmantier-Daures et  al. 1988, Anger
2001). Temperature and salinity are therefore two extrinsic factors that can jointly evolve (Allan
et al. 2006). Protein synthesis, amino acid concentration, and the position of the isosmotic point
can also be modified according to the water temperature (Bückle et al. 2006, Intanai et al. 2009).
Also, in some species, the ionic composition of the hemolymph varies annually, and this may
have direct physiological and behavioral consequences. For example, in the supralittoral amphi-
pods, burrowing activity is seasonal and is related to the Mg2+ concentration in their hemolymph
(Spicer et al. 1990). Mg2+ has a narcotizing effect on many marine invertebrates, and there is a gen-
eral negative relationship between extracellular Mg2+ concentrations and activity in crustaceans
(Morritt and Spicer 1993). In talitrid amphipods, Mg2+ is maintained at very low levels during the
summer months, while in winter, when the amphipods have reduced activity and stay in deep bur-
rows, the concentration of Mg2+ is significantly higher (Spicer et al. 1990).

Food Availability and Quality of the Diet

Diet quality also has a direct consequence on osmoregulation. For example, dietary n-3 highly
unsaturated fatty acid (HUFA) has positive effects on the ability of penaeid shrimp to resist an
osmotic shock (Rees et al. 1994, Palácios et al. 2004). It results in better tolerance to salinity stress
in larval stages of E. sinensis and higher larval development (Sui et al. 2007). For that species, the
ability to endure salinity stress (e.g., sudden transfer from 60% seawater to seawater and hypersaline
seawater) for zoeal and megalopa stages is also improved when animals are fed diets with higher
ratios of docosahexaenoic acid/eicosapentaenoic acid (DHA/EPA; Sui et al. 2007). However, in
juvenile L. vannamei, although fatty acid composition in gill membranes is modified in response to
their proportion in the diet, their osmotic pressure, FAA content, and gill Na+/K+-ATPase activity
do not appear affected by HUFA supplementation (Hurtado et al. 2007). Marine crustaceans have a
limited ability to elongate C18 n-3 poly-unsaturated fatty acids (n-3 PUFA) to n-3 HUFA (Kanazawa
et al. 1977, 1979, Suprayudi et al. 2004), which therefore must be obtained from their food. In con-
trast to marine species, freshwater species have a lower requirement for n-3 HUFA and a higher
capacity to elongate and desaturate these from shorter chain fatty acids. Therefore, the benefit of
268 Jehan-Hervé Lignot and Guy Charmantier

HUFA complementation from the diet, although not completely identified, could be related to a
modification of the fatty acid composition within cell membranes. Fatty acid composition of the
cell membranes is modified by salinity in amphipods (Morris et al. 1982). It can change permeability
(Haines et al. 1994) and can modulate the activity of Na+/K+-ATPase and other membrane-bound
enzymes (Turner et al. 2003). The effects of a prebiotic used as a dietary supplement for growth
and health management have also been tested on tolerance to low-salinity conditions in the Pacific
white shrimp L. vannamei and proved to enhance survival (Li et al. 2009).

Environmental Stressors

A wide range of stressors affect crustacean osmotic and ion regulation (see Lignot et  al. 2000,
Charmantier et al. 2009), and, in most cases, exposure to stress results in a disruption of ionic regu-
lation, mostly of Na+ and Cl− regulation, and therefore of osmotic regulation in osmoregulating
crustaceans. The most studied stressors are pollutants such as metals (i.e., aluminium, cadmium,
copper, lead, and zinc), oil, phenols, pesticides, and polychlorinated biphenyls (PCBs). Other
stressing parameters, such as turbidity (Lin et al. 1992), low levels of dissolved ammonia and oxy-
gen (Hagerman and Uglow 1982, 1983, Henry and Wheatly 1992), pathogenic agents (Souheil et al.
1999, Vinagre et al. 2002), acid stress (Felten et al. 2008), and the presence of antibiotic residues in
the water (Tu et al. 2010) also disrupt ionic regulation.
Usually, the observed effects are size- and dose-dependent. The OC measured as hyper-OC in
freshwater and diluted seawater or hypo-OC in seawater and concentrated seawater is markedly
affected. Ion-transporting organs are also impacted (hemocytic congestion, necrotic blackening of
the gills, intracellular vacuolization, nuclear pycnosis, enlarged mitochondria, and disruption of the
basolateral infoldings of the ionocytes). Although most pollutants have a decreasing adverse effect
on crustaceans as they develop, others, such as organophosphorus insecticides, act the opposite
way by specifically inhibiting the activity of the acetylcholinesterase (AchE) that becomes fully
functional in postmetamorphic stages and, then, by disrupting the osmotic capacity and the ionic
hemolymph composition (Kobayashi et al. 1990, Lignot et al. 1997).

EVOLUTION FROM SEAWATER TO FRESHWATER

AND TERRESTRIAL HABITATS

Invasions across environmental realms might have occurred directly (sea to freshwater and sea
to land) or indirectly (sea to land via freshwater and sea to freshwater via land) and at different
times since the Cambrian. Adaptive shifts in osmoregulatory strategies might have occurred, as is
demonstrated, for example, by Dilocarcinus pagei, a neotropical hololimnetic crab (Augusto et al.
2007). Embryos, juveniles, and adults increase their total FAA content in muscles by 100% when
acclimated from freshwater to 25 ppt saltwater. These authors therefore argue that anisosmotic
extracellular regulation is replaced by isosmotic intracellular regulation in this species and that it
can represent one of the main shifts that occurred in the first brachyurans that invaded freshwater.
Species that have invaded the freshwater environment in the recent past appear less efficient at
osmoregulation than ancient freshwater species (Lee and Bell 1999), but this can vary according
to species (Lee et  al. 2012). For example, Corophium curvispinum requires more energy to regu-
late sodium in freshwater than do freshwater amphipods (Taylor and Harris 1986). Also, in order
to be fully adapted to freshwater, crustacean species must have evolved different ontogenetic pat-
terns because development in freshwater is associated with great physiological constraints (osmo-
regulation, but also tolerance to high pH, oxygen, and calcium concentrations; Powers and Bliss
1983). Therefore, for crustacean species that invaded freshwaters early in their history (i.e., crayfish,
 Osmoregulation and Excretion 269

palaemonids, and potamid crabs but also some Sesarminae species), exporting the pelagic larvae
to marine waters, reducing the larval phase (with, e.g., two zoeal stages and one megalopa, as seen
in freshwater Sesarma species), or laying only a few large eggs (i.e., reduced fecundity) from which
fully developed, adult-like juveniles hatch, are the effective strategies (Anger 1995). Furthermore,
brooding (as mostly seen in distantly related freshwater families such as gammarid and talitrid
amphipods, crayfish, and potamid crabs) has been considered as another prerequisite for success-
ful invasion of freshwater (Fig. 8.8), although it also occurs in some seawater species living at high
latitudes or with extreme habitat specialization (Anger 1995, 2013, Vogt 2013). This parental care can
limit predation in an environment with more limited feeding resources than in seawater. Reduction
of the geographic range (endemism) also appears as a consequence of this shift to freshwater habi-
tats. Some populations that have invaded freshwater appear to have lost their high salinity tolerance,
a pattern found in more ancient freshwater inhabitants. This pattern suggests tradeoffs in osmo-
regulatory capability.
For terrestrial and semiterrestrial crustaceans, desiccation is overcome by different behavioral
and physiological traits (Hoese 1981, Edney 1977). Among these species, the oniscoid isopods are
the most successful terrestrial crustaceans today (Carefoot and Taylor 1995). The oniscoid isopods
have developed key adaptations to minimize body evaporation in order to survive from the supra-
littoral zone up to arid deserts (Edney 1977). Some are endogenous and live in cool and humid
areas, and those living in the most arid environments are nocturnal. Along the seashore, the semi-
terrestrial oniscoid Ligia exotica shows poor resistance to desiccation and cannot live without water
(Tsai et al. 1997). They use closely apposed pereopods 6 and 7 (walking legs) to take water up from
standing water (Horiguchi et al. 2007). The water moves upward by capillary action along grooves
formed between the apposed legs. L. exotica also possess a water-conducting system that is coordi-
nated with respiration and thermoregulation.

1250
Hemolymph osmolality (mOsmol/kg)

1000

750
24 h
48 h
500
72 h
96 h
250
168 h

0
100 200 300 400 500 600 700 800 900 1000 1100 1200
Medium osmolality (mOsmol/kg)

Fig. 8.8.
Relationship between the hemolymph osmolality for hatchling O. gammarellus at different ages post-hatching
and medium osmolality. In Talitrid amphipods, hatchlings remain within a ventral groove that serves as a brood
pouch or marsupium. These Talitrids are the only amphipod to have successfully invaded supralittoral habitats
and colonized land, and the fluid resident in the ventral groove is isolated from the aquatic environment. In the
experiment, the osmotic capacity (OC; osmotic difference between the hemolymph and external medium)
is tested for hatchlings cultured in vitro at different salinities. Note the improvement of the OC (as indicated
by the arrows) with time of development. These results support the hypothesis that hatchlings must attain a
level of physiological competency in osmoregulation before leaving the marsupium. Adapted from Morritt and
Spicer (1999), with permission from Elsevier.
270 Jehan-Hervé Lignot and Guy Charmantier

NEW CHALLENGES: FROM MOLECULES TO MACROEVOLUTION AND

BIOGEOGRAPHY, REDUCTIONIST AND HOLISTIC APPROACHES

The physiological, cellular, and molecular information obtained over the past 20 years or so on crus-
taceans’ osmoregulating tissues and controlling sites allows the creation of hypothetical working
models, such as those developed, for example, for NaCl uptake across the gills of hyperosmoregulat-
ing aquatic crustaceans. In these models, extracellular anisosmotic regulation appears controlled by
specific morphological features (as already detailed for branchial and extrabranchial osmoregulating
epithelia), but also by key apical, basolateral, and cytosolic proteins such as carbonic anhydrase, Na+/
K+-ATPase, V-type H+-ATPase, the 2Na+/1H+ antiporter, the Na+/K+/2Cl− co-transporter, the Na/K
exchanger, and the proton-gradient-dependent Cl–/HCO3– antiport (Ahearn et  al. 1987). Among
these key proteins, a few are detailed here to highlight the most recent findings.
Cytosolic carbonic anhydrase is used to catalyze the formation of H+ and HCO3–, and these
counterions support Na+ and Cl− uptake by the gills (Na+/H+ and Cl–/HCO3− exchange). Therefore,
this pool of carbonic anhydrase provides support for the overall transport mechanisms and is spe-
cifically involved in the physiological and biochemical adaptation to low salinity, as seen by its tran-
scriptional regulation and downstream translational and/or post-translational induction (Serrano
and Henry 2008). This has been evaluated in the posterior gills of euryhaline crustaceans (C. mae-
nas, C. sapidus) exposed to diluted seawater (Henry 1988, 2001, Serrano et al. 2007) and in the gills
and epipodites of penaeid shrimps (L. vannamei, Penaeus monodon; Roy et al. 2007, Pongsomboon
et al. 2009). Furthermore, carbonic anhydrase is under inhibitory regulation by a putative repressor
present in the eyestalk (Henry and Campoverde 2006). Finally, branchial carbonic anhydrase activ-
ity is not modified by salinity transfer in Cancer irroratus, a stenohaline osmoconforming species
(Henry and Campoverde 2006). Therefore, carbonic anhydrase may only function in hyperosmo-
regulating species acclimating to dilute media, such as crabs and the European lobster H. gammarus
(Olsowski et al. 1995, Pavicic-Hamer et al. 2003).
Na+/K+-ATPase is the enzyme responsible for the exclusion of three sodium ions from the
cell in exchange for two K+ ions at the expense of the hydrolysis of one adenosine triphosphate
(ATP) molecule. Its activity maintains cell-membrane potential, mediates transmembrane trans-
port, and thus maintains osmotic equilibrium and cell volume (Lucu and Towle 2003). In crus-
taceans, it has been detected in the different organs containing ionocytes, such as the gills (e.g.,
in brachyuran crabs, caridean shrimp, crayfish), the epipodites (the European lobster, penaeid
shrimps), the inner side of the branchiostegite (the European lobster), the Artemia salt gland, and
the pleopod endopodites in isopods, as well as in the calcium-transporting sternal epithelium of
the terrestrial isopod Porcellio scaber. For example, in brine shrimp living in hypersaline waters,
metepipodites of the phyllopodia have very high activities of Na+/K+-ATPase, and chloride ions
must be actively pumped out of the body against large concentration and charge gradients. Sodium
ions also leave the shrimp against a large concentration gradient but reduce the net electrochemical
(concentration and charge) gradient across the shrimp’s body wall (Holliday et al. 1990). Also, in
hyperosmoregulating species, when subjected to a hyposomotic stress (low salinity), the activity of
Na+/K+-ATPase located along the basolateral membrane of the crustacean ionocytes is increased,
but this response is less pronounced in osmoconformers and weak regulators (Spencer et al. 1979,
Lucu et al. 2000). Therefore, Na+/K+-ATPase is largely involved in both hyperosmoregulation in
dilute seawater and hypo-osmoregulation in concentrated seawater. It creates polarized cells with
an intracellular Na+ gradient that drives secondary active transport.
The molecular characteristics of Na+/K+-ATPase have been well detailed in crustaceans,
including the brine shrimp Artemia franciscana, the American lobster H.  americanus, and the
crabs C.  sapidus, Scylla paramamosain, and Pachygrapsus marmoratus (Chung and Lin 2006,
Charmantier et al. 2009).
 Osmoregulation and Excretion 271

The vacuolar type H+-ATPase (V-type H+-ATPase) is responsible for acid–base balance and
nitrogen excretion (Weihrauch et al. 2001), and it plays a crucial role in the adaptation to freshwater,
as seen in euryhaline crabs (Tsai and Lin 2007). It is also considered, with Na+/K+-ATPase, as a key
enzyme during the transition from a marine environment to freshwater (Lee et al. 2011) and to land
(Morris 2001, Weihrauch et al. 2004). It can be cytosolic or located along the apical or basolateral
side of the ionocytes (Weihrauch et al. 2001, Ziegler et al. 2004, Tsai and Lin 2007). Also, in the
calcium-transporting sternal epithelial cells of the terrestrial isopod P. scaber, a basolateral to apical
shift occurs during the transition from calcium deposition to calcium resorption of intermittent
CaCO3 deposits (Ziegler et al. 2004).
The amiloride-sensitive 2Na+/1H+ antiporter (or exchanger) is associated with pH regulation
and appears present in crustaceans not only in the gills (Strauss and Graszynski 1992, Towle et al.
1997, Kimura et al. 1994), but also in the hepatopancreas and antennal glands (Ahearn et al. 1990).
A role in osmoregulation has been demonstrated in Daphnia magna (Bianchini and Wood 2008),
crayfish (Kirschner 2002), and crabs (Cameron 1979, Martinez et al. 1998, Onken and McNamara
2002, Siebers et al. 1987, Zeiske et al. 1992).
Despite the working models that are available in the most recent literature, a clear functional
view of all the mechanisms at play is still incomplete, especially concerning the mechanisms of
ion excretion in species that hypo-osmoregulate. Also, some transporters have been identified at
the molecular level but still need to be precisely located. Large-scale gene expression studies using
microarrays can also discriminate between transcriptional and post-transcriptional responses.
As an example, one can take advantage of the recent study focusing on gene expression of pos-
terior gills of C. maenas maintained in seawater and diluted seawater and analyzed by microarray
(Towle et al. 2011). Expression ratios of selected transcripts of transporters and other membrane
proteins revealed upregulation in diluted seawater-acclimated crabs of Na+/K+-ATPase and cyto-
plasmic carbonic anhydrase, both previously shown by quantitative polymerase chain reaction to
respond positively to salinity reduction in C. maenas and other euryhaline crabs (Henry et al. 2002,
Luquet et al. 2005, Serrano and Henry 2008). Other upregulated transcripts concerned an organic
cation transporter, the sodium/glucose cotransporter, an endomembrane protein, a voltage-gated
calcium channel, an anion exchanger, and an octopamine receptor that doubled in less than 2 h
after transfer from seawater to low salinity. Many other transcripts encoding other transport-related
proteins implicated in osmoregulatory ion transport in crustacean gill remained unaffected by the
salinity challenge. These include the Na+/H+ exchanger, Na+/K+/2Cl− cotransporter, the V-type
H+-ATPase, the K+/Cl− symporter, the Rhesus-like ammonium transporter, the CFTR-like ABC
transporter, and aquaporin. Therefore, for these proteins, gene regulation is not part of the adaptive
process (Towle et al. 2011). The study also indicates no transcriptional response among the selected
stress-related proteins but a significant upregulation of the transcript level for mitochondrial pro-
teins. Although only a small number of the microarray positive hits have been analyzed, they clearly
indicate that the integrated molecular and physiological response of the crab in front of an acute
salinity transfer (from 100% to 50% and 32% seawater) does not represent a stressful challenge.
Finally, functional models should also integrate ontogeny and the molting cycle, although the
molecular events occurring before, during, and after ecdysis have been tackled recently (Shechter
et al. 2007, Shi et al. 2009, Seear et al. 2010).
Wide adaptive radiation occurred during crustacean evolutionary history that enabled them to
inhabit different marine, brackish, freshwater, and terrestrial habitats. Some of these ecosystems
correspond to transitory habitats (estuaries, sandy or rocky shores) with low species diversity due
to large fluctuations of salinity and temperature. Therefore, being able to regulate internal solute
concentration at a level different from that of the external environment using a suite of molecular,
morphological, and physiological traits allowed some crustaceans to compensate for unpredictable
fluctuations of the external environment. Evolutionary trends leading to increased independence
272 Jehan-Hervé Lignot and Guy Charmantier

from fluctuations in the external environment include decreased permeability of the body surface
to water and solutes, restrictions in the rate of water influx and ion loss, and enhanced ion uptake
through modulation of transporter function.
The capacity to osmoregulate is also a key function for invasive species. The role of salinity tol-
erance of amphipod invaders was recently highlighted by Devin and Beisel (2007) who studied the
biological and ecological traits of gammarid amphipods from Western Europe and North America
that may explain their successful invasion. Their study has revealed a particular ecological profile
for invaders, with a strong influence of salinity tolerance. This is also illustrated by the European
green crab C. maenas, a strong hyper-/hyporegulator and one of the world’s most successful aquatic
invaders (Darling et al. 2008).

FUTURE DIRECTIONS

Since their early developments, investigations on crustacean ion and water regulation have been
conducted in species differing in their ecology, thus establishing osmoregulation as one of the main
adaptations to various habitats throughout development. However, future research is still necessary
and concerns different levels of integration.
First, relatively few crustaceans are able to both hyper- and hypo-osmoregulate, and most stud-
ies on crustacean osmoregulation have been conducted under conditions of salinity decrease.
This stems from the usually high isosmotic salinity that is close to seawater; that is, 1,000 mOsm
kg–1 in marine crustaceans. As a result, most of what is known about the mechanisms of crustacean
osmoregulation concerns hyperosmoregulation. It is thus necessary to focus on the mechanisms of
hypo-osmoregulation in those species that are able to do so when salinity increases above the values
of seawater. Also, as in marine fish, the gut is probably involved in water uptake, as are the gills for
ion excretion. Therefore, the corresponding cells and molecular mechanisms should be studied
along with Cl− transport at the basal and apical sides of the cells.
As part of developmental biology in crustaceans, the origin of the osmoregulatory function
should be addressed. The occurrence of ionocytes and their molecular functionality should be fol-
lowed at different sites during embryonic and early postembryonic development in relation to the
role of osmoregulation in ecophysiological strategies.
Finally, global climate change and environmental stressors should promote further investiga-
tions on their multilevel effects on osmoregulation.

CONCLUSIONS

This broad overview concerning mostly osmoregulating crustaceans clearly indicates that these
species can maintain internal solute concentration at a different level from that of the external envi-
ronment mostly because of a reduced permeability of their body surface to water and solutes, lim-
ited water influx and ion loss, and an enhanced ion uptake through modulation of the transporter
function. Furthermore, ion and water regulation in these species is influenced by many internal and
external factors (e.g., the effects of gender, molting, nutrition, ontogeny, parasitism, season, temper-
ature, dissolved oxygen, pollutants, etc.). The way these factors influence the OC was discussed, as
was the efficiency of the different osmoregulatory strategies; the functional organization at tissue,
cellular, and molecular levels of the key osmoeffectors; and the hormonal system that controls the
osmoregulatory function, each according to different ecological constraints. It appears therefore
that these mechanisms are highly adaptive and at play in crustaceans living in environments with
fluctuating salinities, for invasive crustaceans, and in populations colonizing new habitats.
 Osmoregulation and Excretion 273

ACKNOWLEDGMENTS

This chapter is dedicated to David W. Towle, who inspired several generations of carcinologists.

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 9
NUTRITION AND DIGESTION

Reinhard Saborowski

Abstract
The crustaceans colonized all aquatic habitats. In the oceans they are present from the coasts down
to the deep sea basins. They are also frequent in freshwater systems and some species even live on
land. Accordingly, they adapted to utilize a variety of food items. These items comprise organic
matter from dead or alive animals, vascular plants, detritus, macro- and microalgae, microbial
films, and organic sediment compounds. This food is utilized in the digestive organs with the aid
of highly active enzymes. The enzymes are synthesized in specific cells along the midgut lining or,
in the higher crustaceans, in the complex midgut diverticula. From there they are released into the
stomach where they initiate food digestion. According to the biochemical nature of the major food
compounds the crustaceans possess a variety of endo- and exopeptidases to hydrolyze proteins
and peptides, amylases to cleave glycogen, and lipases and esterases to utilize lipids. The liberated
nutrients are resorbed by specific cells of the midgut or the midgut diverticula. Besides, crustaceans
can also digest organic materials which are known for their chemical stability, namely chitin, cel-
lulose, and hemicelluloses. The acting enzymes are for the most part of endogenous origin. Bacteria
may be present in the gut in quite high numbers but their contribution to the digestive processes is
not mandatory. The digestive enzymes of Crustacea show extraordinary catalytic properties which
form the basis to efficiently utilize a variety of food items and, thus, to meet the nutritive demands
of the organisms.

INTRODUCTION

Feeding and assimilation of food are cardinal physiological processes in heterotrophic organ-
isms. They comprise the uptake as well as the chemical degradation of organic material. The
purpose is to gain nutrients and energy for maintaining homeostasis and facilitating growth

285
286 Reinhard Saborowski

Proteins Carbohydrates Lipids

DIGESTIVE ENZYMES

GENERATION OF METABOLIC ENERGY

Peptides Oligosaccharides Fatty acids
Amino acids Glucose Glycerol

Pyruvate

Acetyl CoA

Krebs Cycle

NH4 H2O CO2 ATP

Fig. 9.1.
Simplified diagram of the catabolic pathways of major storage products.

and reproduction (Fig. 9.1). The digestive organs of crustaceans and their function have been
researched for more than 100 years. The first detailed anatomical studies on the stomach of
crayfish and on the basic enzymatic processes within the digestive organs were published in
the 19th century.
During the past 30–40 years, research on crustacean digestive physiology increased consider-
ably. This interest is based on both emerging knowledge about the ecological significance of crus-
taceans in various habitats and also on their relevance in food production. The ecological aspects
deal with the trophic interactions of crustaceans within their habitats and their ecophysiological
adaptations and specializations. The economic aspects mostly target the optimization of feeds to
increase yield and thus earnings.
This chapter is aimed at providing an overview of the feeding and utilization of food in
crustaceans from the physiological and biochemical point of view. More details about crusta-
cean feeding biology can be found in extended reviews from the past decades. The anatomy
of the crustacean digestive system is presented by Watling (2013). The decapod hepatopan-
creas and its function in digestion and food storage were thoroughly described by Gibson
and Barker (1979). The gut structure and the detailed cellular processes of digestion in
Malacostraca and other crustaceans were presented by Brunet et al. (1994). Dall and Moriarty
(1983) contributed a chapter entitled “Functional Aspects of Nutrition and Digestion” to the
series The Biology of Crustacea. Metabolism and transport processes were reviewed by Chang
and O’Connor (1983). A comprehensive overview on crustacean nutrition was edited and pub-
lished by D’Abramo et  al. (1997). A  recent review on the digestive tract of crustaceans was
contributed by Ceccaldi (2006).
 Nutrition and Digestion 287

CRUSTACEAN NUTRITION

Crustaceans occupy a variety of habitats. To utilize a variety of food from these habitats, crustaceans
show specific morphological and anatomical adaptations to handle and to process their food (Watling
2013). Moreover, they also developed physiological and biochemical properties to utilize the food.

Food Sources and Feeding Habits

The various morphotypes of crustaceans are generally capable of utilizing almost all kinds of
organic material. Food may originate from live or dead animals, higher plants and their remains,
macro- and microalgae, and organic surface layers or the organic fraction of sediments (Fig. 9.2).
Some species evolved to extract nutrients from other living organisms, thus acting as exo- or endo-
parasites. Other species process hardly digestible food items such as cellulose and lignocellulose
with the aid of endogenous enzymes (Linton et al. 2006, King et al. 2010).
Feeding frequencies, gut passage times, and assimilation efficiencies differ widely between spe-
cies and also depend on the quantity and the chemical composition of the food (Waddington 2008).

A B

C D

Fig. 9.2.
Examples of crustaceans feeding on different food items. (A) Herbivorous isopods Idotea emarginata feeding
on seaweed. Photo by Reinhard Saborowski. (B) Christmas Island land crab Discoplax hirtipes feeding on a leaf.
Photo by Reinhard Saborowski. (C) Parasitic fish louse Argulus japonicus. The digestive system is filled with
the blood of the host. Abbreviations: AM, anterior midgut; ED, central diverticulum; F, foregut; H, hindgut;
PM, posterior midgut ventral to reproductive system (RS). The length of the animal is 4 mm. From Tam and
Avenant-Oldewage (2009), with permission from Elsevier. (D) Hyas araneus and Carcinus maenas feeding on a
dead flatfish. Photo by Uwe Nettelmann.
288 Reinhard Saborowski

The gut passage may last for several minutes or up to hours and days. It depends on the size of the
animal, the feeding strategy, and on the anatomy of the digestive tract. For example, the spiny lob-
ster Jasus edwardsii showed poor growth on formulated diets due to limitations in food uptake and
low gut transition times (Simon and Jeffs 2008). Feeding experiments showed that food uptake
was rapid, but satiation occurred quickly due to low foregut capacity. The filling time of the foregut
amounted to 1–2 h, whereas the clearance time lasted for 10 h. The gut throughput was slow (34–42 h),
and appetite revival was evident after 18 h (Simon and Jeffs 2008). The scavenging Antarctic isopod
Natatolana obtusata has a very plastic mode of food uptake. The mouth opening is very flexible, and
the anterior hindgut is extremely variable in its lumen and very distensible. The gut is perfectly suited
to store huge amounts of food and to overcome long periods of food deficiency (Storch et al. 2002).

Nutritional Requirements

The nutritional requirements of crustaceans were most intensively studied and are best docu-
mented for species with high economic value and relevance in aquaculture. In this respect, the
nutritional requirements for Litopenaeus vannamei were reviewed by Cuzon et al. (2004). A general
and comprehensive overview on crustacean nutrition was presented by D’Abramo et al. (1997).
The chemical gross compositions of food were reported for a range of different crustaceans and
their ontogenetic stages. The amounts of protein, lipids, carbohydrates, and inorganic compounds
vary widely, depending on the ability of the species to accumulate storage products and on the
degree of calcification of the shell. As a rough overview, the following numbers may be given. The
amount of water in the food may range from 75% to 85% of the total fresh mass. The inorganic frac-
tion (ash) may amount to 10–20% of dry weight and may increase significantly when species are
strongly calcified. The protein content can account for up to 70% of ash-free dry mass and total
lipids for less than 10% to up to 60% of ash-free dry weight.

Water

The uptake of water happens predominantly through the epithelia of the digestive system and, to
a lesser extent, through the gills. In some species, highly specialized adaptations may have evolved
that allow those animals to cope with exceptional environmental conditions. The most common
method of water uptake is to swallow it through the mouth and esophagus into the stomach. Land
crabs have been observed drinking water from a small creek. They dipped the tips of their claws
into the water, moved the claws toward the mouth, and allowed the adhering water to drip onto
and behind the maxillipeds that cover the mouth. In addition to oral water uptake, uptake through
the anus into the hindgut was reported (Fox 1952). Other groups, such as some isopods, developed
highly specialized cuticular microstructures on their legs for water uptake (Horiguchi et al. 2007).

Proteins and Essential Amino Acids

The requirements for protein vary greatly between species and also depend on feeding habits.
Carnivorous and omnivorous species will easily meet their demand with their normal diet, whereas
herbivorous species need to optimize their ability to assimilate proteins. In addition to protein, chi-
tin, which is present in arthropod shells, provides another valuable source for nitrogen. Herbivorous
crustaceans adapted to low-nitrogen diets by elevating their feeding rates, keeping gut retention times
short, and increasing the assimilation rates for soluble cellular materials (Linton and Greenaway
2007). In aquaculture, fish meal is a common source for proteins. The dietary amounts of proteins
should range between 30% and 50%. Supplements of carbohydrate can reduce the demand of proteins.
 Nutrition and Digestion 289

Table 9.1.  Essential amino acid ratio (percentage of all essential amino acids) of whole
animals at different stages of growth and tail muscle (*).

Amino acid Litopenaeus Penaeus Penaeus Macrobrachium

vannamei monodon japonicus rosenbergii *
Arginine 13.7 15.3 15.2 20.6
Histidine 3.8 4.7 4.5 4.5
Isoleucine 10.8 8.5 8.6 7.2
Leucine 16.3 14.6 15.0 14.8
Lysine 14.4 14.5 15.8 17.3
Methionine 1.4 7.4a 7.5a 6.5
Phenylalanine 11.3 15.5 16.8b 7.4b
Threonine 9.1 7.6 8.2 7.6
Tryptophan 2.1 2.1 – –
Valine 10.6 9.9 8.3 7.3

 Methionine and cysteine.

a

 Phenylalanine and tyrosine.

b

After Mente et al. (2002).

The dietary amino acid composition should be correlated with the amino acid profile of the
whole body. Ten amino acids cannot be synthesized by crustaceans. These essential amino acids and
their relative amount are listed in Table 9.1. Shrimp tail muscle is rich in arginine. Therefore, arginine
was often the first limiting factor when experimental diets were administered (Cuzon et al. 2004).

Lipids and Essential Fatty Acids

Lipids are both important structural compounds and valuable energy stores in crustaceans.
Phospholipids (PL) represent the majority of polar lipids and are essential elements of cellular mem-
branes. Storage lipids are predominantly deposited as neutral lipids in the form of wax esters or triacyl
glycerols, with both of these helping to overcome periods of food deprivation. The preference for
either of these storage lipids varies between species (Kreibich et al. 2010; Fig. 9.3). Wax esters serve as
long-term lipid stores and may remain in the body for several months, as, for example, in high-latitude
species. Triacyl glycerols, in contrast, are considered short-term depot lipids. They are used to meet
metabolic energy requirements and are subjected to continuous turnover. Lipids are predominantly
stored in the midgut gland. Some species of euphausids deposit lipids in a connective tissue forming
the so-called fat body. High-latitude species of copepods may store lipids within oil sacs.
Fatty acids, the principal compounds of most of the crustacean storage lipids, are synthesized
by elongation of acetyl CoA primers. Crustaceans are capable of synthesizing saturated fatty acids
and monounsaturated fatty acids. However, they cannot synthesize significant amounts of n-3 and
n-6 polyunsaturated fatty acids. Accordingly, essential fatty acids like eicosapentaenoic acid (EPA,
20:5(n-3)) and docosahexaenoic acid (DHA, 22:6(n-3)) need to be extracted from the diet (Dalsgaard
et al. 2003). Elevated dietary levels of linoleic acid (18:2n-6) and n-3 highly unsaturated fatty acids
increased fecundity, egg hatching efficiency, and larval quality of Macrobrachium rosenbergii (Cavalli
et al. 1999). Supplements of sterols and polyunsaturated fatty acids had beneficial effects on Daphnia
magna (Martin-Creuzburg and von Elert 2009). Sterols seem to be important for unconstrained
290 Reinhard Saborowski

Meganyctiphanes norvegica
A
30
WE
25 TAG
PL
20 y = 0.92x − 9.66

Lipid class (% DM)

r2 = 0.923
15 n = 10

10

0
0 10 20 30 40
Total lipids (% DM)

B Hymenodora glacialis
60
WE
50 TAG
PL
Lipid class (% DM)

40 y = 0.83x + 1.82
r2 = 0.827
30 n = 18

20

10

0
0 10 20 30 40 50 60
Total lipids (% DM)

Fig. 9.3.
Correlation between the amount of wax esters (WE), triacylglycerols (TAG), phospholipids (PL), and
the amount of total lipids in % of the dry mass for (A)  Meganyctiphanes norvegica (Euphausiacea) and
(B) Hymenodora glacialis (Caridea). Note the different scaling of the axes. From Kreibich et al. (2010), with
permission from Inter-Research.

somatic growth, whereas polyunsaturated fatty acids support egg production. Cholesterol is essential
and needs to be administered with the diets to meet demands (Cuzon et al. 2004).

Carbohydrates

Crustaceans possess a variety of digestive glucanases. They are able to digest polysaccharides such
as starch, cellulose, or laminarin. Polysaccharides are sources for sugars that can be directly con-
verted into energy, stored as glycogen, or used for the synthesis of chitin, fatty acids, or sterols, for
example. Dietary requirements for carbohydrates are not defined. Glucose is only poorly utilized
by crustaceans. Glucose-supplemented diets yield lower growth rates in shrimps than do diets with
polyglucans like starch or glycogen (Shiau 1998). Rock lobsters (J. edwardsii) were fed diets with
 Nutrition and Digestion 291

different carbohydrate-to-lipid ratios ( Johnston et al. 2003). Maximum growth and highest levels
of lipid and dry matter in midgut glands and whole body were present at a diet of 27% carbohydrate
and 13.5% lipid (2:1 ratio). Balanced supplements of carbohydrates may improve the growth perfor-
mance of crustaceans because they provide immediate energy for metabolic processes.

Vitamins

Vitamin requirements were studied in several, predominantly commercial species. The recom-
mended doses vary distinctly between species but also between ontogenetic stages within a spe-
cies. Many vitamins are cofactors for enzymes or important metabolites. Others act as antioxidants,
growth factors, or structural components (Table 9.2).

Antioxidants
L-Ascorbic acid (vitamin C) and tocopherol (vitamin E) are potent antioxidants. Both vitamins
are traditionally used in shrimp feeds to improve the animals’ health. Vitamin C deficiency caused
“black death disease” in penaeid shrimps (Magarelli Jr. et  al. 1979). Dietary levels of 20–130 mg
vitamin C per kg diet supplied as a stable and bioavailable source allow a normal growth. Elevated

Table 9.2.  The physiological functions of vitamins and their recommended feed levels.

Vitamin Physiological function Recommended

feed level
(mg·kg−1 diet)
Thiamin Co-factor for metabolic enzymes 60
Riboflavin FMN and FAD are intermediates in electron transfer 25
in biological redox reactions
Niacin Precursor for NAD+/NADH and NADP+/NADPH 40
Vitamin B6 Co-factor for metabolic enzyme 50
Pantothenic acid Required for CoA synthesis 75
Biotin Prosthetic group of enzymes, epigenetic regulation 1
of gene function
Folate Deficiency causes poor growth 10
Vitamin B12 Cobalt-containing cofactors of enzymes involved in 0.2
nucleic acid and protein synthesis
Choline Compound of cell membranes 600
(phosphatidylcholine)
myo-inositol Basis for signaling and second messenger molecules 400
Vitamin C Antioxidant, synthesis possible in some species 200
Vitamin A Many physiological functions, compound of eye 250
pigments
Vitamin E Antioxidant, protection of membrane-bound 100
PUFAs
Vitamin D Mediator in calcium metabolism 0.1
Vitamin K Blood coagulation, calcium transport, co-factor for 5
enzymes

After Conklin (1997).

292 Reinhard Saborowski

levels of 1,500 mg/kg or more enhance the resistance to stress and diseases. Because ascorbic acid
is quite vulnerable to oxidation, more stable derivatives with sulfate and phosphate moieties show
beneficial effects when used in feeds.
Vitamin E in the diet can provide immediate protection against free radicals. Although the
hepatopancreatic tissue has a strong intrinsic antioxidant capability, addition of vitamin E distinctly
increased the radical scavenging ability (Díaz et al. 2004).

Carotenoids
Carotenoids fulfill various physiological functions in crustaceans (Meyers and Latscha 1997). In
aquaculture, they were administered to improve growth, reproduction, oxidative defense, pigmen-
tation, and cellular protection from light damage (Liñán-Cabello et al. 2002). However, the effects
reported were often not consistent. Dietary carotenoids are the sole biological precursors of reti-
noids and thus pro-vitamin A in crustaceans. Retinoids, in turn, are involved in the activation of
nuclear hormone receptors. They play important roles in many developmental processes, including
embryogenesis and cell differentiation. Administration of retinol palmitate caused induction of the
primary vitellogenic phase in crayfish females (Cherax quadricarinatus). In the shrimp L. vannamei,
the concentrations of carotenoids as well as vitamin A were higher in the digestive glands of wild
catches than in captive animals (Liñán-Cabello et al. 2003). The authors suggested that supple-
mentation of carotenoids is important for captive shrimps to improve cellular protection at oocyte
differentiation, for example.

Minerals

Minerals fulfill a variety of biochemical functions in cell homeostasis and signaling, as cofactors for
enzymes, and in skeletal and tissue formation. The minerals representing the highest proportions
are denoted macrominerals and comprise calcium, phosphorus, potassium, sodium, and magne-
sium. Microminerals are represented by copper, iron, manganese, selenium, and zinc (Table 9.3).
Calcium carbonate (CaCO3) is the most important inorganic compound of the mineral phase
of crustacean shells. Depending on the degree of calcification, animals lose a significant amount of
calcium with the molt. Marine species are capable of extracting calcium from seawater, predomi-
nantly via the gill epithelia. The net influx amounts to 2 mmol·kg−1·h−1 (Zanotto and Wheatly 2003).
Unlike marine crustaceans, animals from freshwater and terrestrial habitats are more restricted in
their calcium availability. In these species, the uptake of calcium occurs predominantly via the diet,
and, accordingly, calcium influx is facilitated through the digestive organs. The ingestion of calcium
may range between 10 and 20 mmol·kg−1·h−1 over a calcification period of 72 h. The physiological
processes of calcium transport in crustacean digestive organs are reviewed in detail by Zanotto and
Wheatly (2003). In addition to calcium uptake from water or food, most species also developed
mechanisms to retain calcium in the body over ecdysis. Crayfishes and some crabs retain most of
the calcium as calcified concretions (gastroliths) in the cardiac stomach (Fig. 9.4). After ecdysis,
the gastroliths are released into the digestive tract, and the calcium is rapidly reabsorbed. Other
storage sites are the midgut gland that, however, predominantly accumulates calcium phosphate
and sulfate rather than calcium carbonate. The calcium concentrations in the R and E cells of the
midgut gland were higher during premolt than in the intermolt stage (Chavez-Crooker et al. 2003).

Probiotics, Prebiotics, and Other Supplements

Probiotics and prebiotics are increasingly used in fish and crustacean aquaculture to improve the
health and survival of the hosts (Yousefian and Amiri 2009, Ganguly et al. 2010). For example, the
 Table 9.3.  Minerals in crustaceans and their function.

Mineral Physiological function

Macrominerals
Calcium Component of enzymes, signaling, support of skeletal
structures
Phosphorus Component of membranes, nucleic acids, and various other
biomolecules; structural support
Potassium Maintenance of membrane potential, osmoregulation, acid–
base regulation
Sodium Same as potassium
Magnesium Muscle function
Microminerals
Copper Integral part of the respiratory pigment hemocyanin (most
Crustacea)
Iron Component of hemoglobin (e.g., Daphnia), cytochrome, and
enzymes
Manganese Component of some enzymes
Selenium Component of antioxidative enzyme glutathione peroxidase
Zinc Component of some enzymes (zinc endopeptidases)

After Mente et al. (2002).

A B

C D

Fig. 9.4.
Localization, shape, and structure of gastroliths from crayfishes. (A)  Radiography of the cephalothorax of
Cherax quadricarinatus showing the X-ray dense gastroliths. (B) Dissected gastroliths from Orconectes limosus
and Cherax quadricarinatus. The size is proportional to the size of the animals. (C) Broken gastrolith showing
its internal aspects. (D) Cross-section showing the dense and striated structure of the gastrolith. From Luquet
et al. (2013), with permission from MDPI AG.
294 Reinhard Saborowski

supplement of probiotics (photosynthetic bacteria and Bacillus sp.) to basal shrimp diets led to sig-
nificantly better growth than did unsupplemented feed (Wang 2007). Supplement of Lactobacillus
plantarum modified the bacterial microbiota in shrimp digestive tracts and increased resistance to
Vibrio harveyi infection (Vieira et al. 2010). In addition to dietary supplement of bacteria, adminis-
tration of soybean isoflavones showed positive effects on the growth of shrimps L. vannamei as well
(Chen et al. 2011). Immunological parameters like total hemocyte counts, activities of antioxidant
enzymes, and resistance against Vibrio alginolyticus also increased. The defense mechanisms of crus-
taceans depend completely on the innate immune system. It is activated when pathogen-associated
molecular patterns are recognized by soluble or cell surface host proteins (Vazquez et al. 2009). In
order to understand the effect of probiotics in crustaceans, more intensive studies are needed on
the function of their immune reactions.

Reactions to Starvation

Crustaceans experience starvation during periods of food scarcity. However, they also starve during
and after molt when the exoskeleton, and, thus, the feeding appendages and masticatory structures
of the foregut have to harden before the animals can continue feeding. Starvation affects the organ-
ism on different organizational levels ranging from behavioral changes to alterations of subcellular
structures and molecular properties (Sánchez-Paz et al. 2006).
The reactions to prolonged starvation depend, on one hand, on the ability of the animal to
reduce its metabolic rates. On the other hand, it depends on the amounts and availability of nutri-
ent reserves in the body. Small planktonic copepods without significant lipid reserves (e.g., Temora
longicornis) show first reactions to starvation very rapidly, within 24 h. They successively decrease
digestive and metabolic enzyme activities, reduce egg production rates, and catabolize remaining
body lipids (Kreibich et al. 2008).
Species that accumulate higher amounts of storage products can consequently better cope with
starvation. Short-term starvation of up to 5 days in whiteleg shrimp (L. vannamei) caused a rapid
reduction of plasma and hepatopancreas glucose and a decrease in hepatopancreatic glycogen levels
(Sánchez-Paz et al. 2007). Triacylglycerol levels decreased first at the end of the starvation period
(5 days), whereas protein levels remained unchanged.
The life cycle of Antarctic krill Euphausia superba is characterized by a strong seasonality of
biological production. Krill may face severe starvation during the low-productive Antarctic winter.
To withstand these conditions, E. superba is capable of reducing its metabolic activity. Laboratory
experiments revealed a reduction of metabolic rates by 30% after 10 days of starvation (Auerswald
et al. 2009). Moreover, E. superba is capable of catabolizing muscle proteins, which entails shrinkage
of the animal (McGaffin et al. 2002).
Cytological changes appeared in the midgut gland of larval spider crabs Hyas aranaeus and post-
larval prawns Penaeus monodon after starvation for a few days (Storch and Anger 1983, Storch et al.
1984). The R cells were most sensitive and showed reduction in size, depletion of lipid stores, and
pronounced swelling of mitochondria. In contrast to R cells, B cells showed only slight reactions,
and F and E cells remained almost unchanged (Vogt et  al. 1985). However, after a starvation of
13 days, all cell types disintegrated, and after 15 days the postlarvae died.
Effects of starvation on the immune response and oxidative stress were studied in Carcinus
aestuarii (Matozzo et al. 2011). Crabs that starved for 7 days showed elevated numbers of hemo-
cytes. Glucose concentrations increased in the cell-free hemolymph, but protein values declined.
Phenoloxidase activity was elevated in hemocyte lysates. Matozzo et al. (2011) concluded that star-
vation influenced immune parameters in crabs and that the animals can modulate their cellular and
biochemical parameters to cope with starvation.
 Nutrition and Digestion 295

Natural and Anthropogenic Toxins and Their Effects

Many micro- and macroalgae, bacteria, fungi, and protists are capable of producing and releasing
adverse metabolites. Exposure of crustaceans to harmful chemicals can have various effects on all
organizational levels (Brouwer and Lee 2008).
Tannins and phlorotannins, for example, are polyphenolic compounds from terrestrial
plants and kelp, respectively. They appear predominantly in coastal areas and are nutritionally
adverse because they can precipitate proteins, inhibit digestive enzymes, and impair the utili-
zation and uptake of vitamins and minerals (Chung et al. 1998, Sotka and Whalen 2008). On
the other hand, it was reported that tannins may be beneficial by reducing mutagenicity, show-
ing anticarcinogenic activity, and inhibiting growth of fungi, bacteria, and viruses. Exposure
of freshwater crustaceans (Cladocera, Copepoda, and Ostracoda) to tannic acids showed a
sequential degradation of the midgut epithelium. The strength of the effect differed between
species and depended on the duration of exposure and the concentration assayed (Pautou et al.
2000). The resistance to tannic acid of the crustacean was correlated with qualitative and quan-
titative features of detoxification enzymes, cytochrome P450 enzymes, esterases, and glutathi-
one S-transferase (Rey et al. 2000).
During blooms of cyanobacteria, elevated amounts of biologically active secondary metabolites
are released. Among these metabolites, proteinase inhibitors are present that are capable of inhibit-
ing the digestive proteinases of the cladoceran D. magna (Schwarzenberger et al. 2010). The cladoc-
erans react to the presence of cyanobacterial proteinase inhibitors by upregulation of proteinase
expression. They increase their capacity for protein digestion and thus may compensate for the
adverse effects of the inhibitors (Schwarzenberger et al. 2010).
In addition to natural toxins, numerous anthropogenic xenobiotics pollute aquatic and terres-
trial habitats. In coastal regions, industrial activities, ship traffic, or oil drilling activities liberate, for
example, petroleum components (hydrocarbons), heavy metals, or antifouling agents. Exposure to
water-soluble petroleum constituents significantly impairs the reception of food in European lob-
sters Homarus gammarus (Walter et al. 2008). Moreover, Lavarías et al. (2006) observed an increase
in the activities of lipid-catabolizing enzymes in shrimps Macrobrachium borellii after exposure to
sublethal concentrations of water-soluble petroleum compounds. Ultimately, exposure to these
pollutants adversely affects the shrimp by raising its energy demand.
Some crustaceans, however, may also make use of environmental toxins. For example, the
hydrothermal vent crabs Xenograpsus testudinatus are adapted to feed on poisoned planktonic
organisms. The crabs live in crevices very close to shallow water hydrothermal vents off of
Taiwan ( Jeng et al. 2004). The discharges of the vents are highly acidic and rich in sulfur. The
crabs can resist elevated temperatures, low pH, and high concentrations of dissolved toxic gases.
Moreover, they feed on planktonic organisms that were killed by the toxic plumes and fell to
the bottom (Hu et al. 2011). During slack water, huge numbers of crabs leave their crevices and
pick up fallen dead organisms. With the onset of the tidal currents, they seek shelter again.
Accordingly, these hydrothermal vent crabs are exposed to toxins in two ways: the surrounding
water and the ingested food. Apparently, the crabs are capable of coping with toxins and high
metal ion loads and thus might serve as suitable models in crustacean toxicology and, particu-
larly, in toxin resistance studies.

Dietary Shifts During Ontogenesis

Ontogenetic shifts in feeding habits and nutrition may appear in different ways. The animals may
simply grow and, consequently, change the size spectrum of their prey. As they grow, they change
296 Reinhard Saborowski

their morphology or anatomy. During development, the ontogenetic stages may change their habi-
tat and thus food sources. They may also change between lecithotrophy and planktotrophy, as dem-
onstrated for the larvae of some species.
Size gain and habitat shift often appear concomitantly. For example, lobster larvae feed
on small zooplankton or phytoplankton. As the lobsters metamorphose, the food originates
from the benthic environment but still includes small organisms that can be handled by the
early post-larvae. When the post-larvae mature and grow, they develop strong claws and
start hunting for larger prey organisms. They are able to crack other crabs and mollusks with
their strong claws and can even catch bottom-living fishes. Stomach content analysis of early
benthic stages of American lobsters revealed a progressive dietary shift with increasing size
(Sainte-Marie and Chabot 2002). Small lobsters fed on soft or readily available food such as
fish carrion, small bivalves, macroalgae, and meiobenthic crustaceans. Larger ones preferred
more mobile and more nutritious prey including other crustaceans and fish (Sainte-Marie
and Chabot 2002).
Significant morphological and anatomical changes are frequent in lecithotrophic larvae from
higher latitudes. These larvae do not start feeding after hatching but utilize yolk reserves that they
maintained from the embryonic stages. In this respect, the larvae of the king crab Lithodes santolla
do not feed during the first larval stage but catabolize their yolk (Calcagno et al. 2004). These larvae
also lack a functional digestive tract and do not express digestive enzymes such as endopeptidases,
which are required for the digestion of external food (Saborowski et al. 2006). In the subsequent
stages, however, they start feeding and develop a functional digestive system to utilize external food
(Fig. 9.5).
Most crustacean species undergo a more continuous larval development through a sequence
of predetermined larval stages. Between these stages, however, distinct differences in enzyme
expression and thus capacity of food utilization may also occur. For example, larval development
of the pink shrimp Farfantepenaeus paulensis passes through five to six nonfeeding naupliar stages,
followed by three protozoeal stages and three mysid stages prior to the postlarval stages. Lemos
et al. (1999) found a significant increase of endopeptidase activities in the protozoea stages, activity
that declined again in the subsequent mysis and postlarval stages. The authors conclude that the
elevated enzyme activities may be related to higher energy demands due to increased swimming
behavior and feeding activity.

DIGESTIVE ENZYMES

Studies on digestive enzymes of crustaceans can be traced back to the 1870s. Hoppe-Seiler (1877)
and Krukenberg (1878) were the first to report about digestive processes and the proteolytic
properties of the gastric fluid of the crayfish Astacus astacus. Thereupon, in the early 20th cen-
tury, a number of excellent physiologists and biochemists established the cytological and bio-
chemical fundamentals of crustacean digestive physiology and biochemistry which, for the most
part, are still valid. However, the detailed factors that determine the properties and composition
of the expressed enzymes are poorly understood and thus still under debate. A certain genetic
disposition seems apparent that determines the basic expression patterns of enzymes, such as
the predominance for cysteine- or serine-proteinases (Teschke and Saborowski 2005, Navarrete
del Toro et al. 2006). But, beyond that, the composition of enzymes seems also to favor specific
feeding behavior and may indicate trophic resource utilization, as shown in brachyuran crabs
( Johnston and Freeman 2005).
 Nutrition and Digestion 297

70 A B

Ratio (Ala-AP/Chymotrypsin) 60

50

40

30

20

10

1
E ZI ZII ZIII/M Cl Juv GF
1

10

Ratio (Chymotrypsin/Ala-AP)
20

30

40

50

60

70

Fig. 9.5.
Activity ratios of alanine-aminopeptidase (Ala-AP) and chymotrypsin in different larval stages, juveniles, and
the gastric fluid of adult Lithodes santolla. (A)  Eggs (E), zoea I  (ZI), zoea II (ZII), zoea III, and megalopa
(ZIII/M), crab I (CI). (B) Juvenile midgut gland (juv), gastric fluid of adults (GF). From Saborowski et al.
(2006), with permission from Springer.

Sites of Enzyme Synthesis and Enzyme Action

In all crustaceans studied so far, enzyme synthesis and enzyme secretion take place in specialized
cells in the midgut region or in the midgut caeca (Fig. 9.6). The digestive cellular processes in
higher and in lower Crustacea were reviewed in detail by Brunet et al. (1994).
In those crustaceans that do not possess distinct midgut diverticula, the midgut lining forms
specialized cells that may produce and secrete enzymes. Cladocera, for example, show a quite sim-
ple cellular organization of their gut. However, they also show variations in cell structure along the
midgut that can be related to particular functions, such as enzyme secretion and nutrient resorption
(Schultz and Kennedy 1976). Digestive enzymes are secreted in a holocrine fashion, preferably by
the cells in the mid-regions of the gut. Posteriorly, the midgut cells become smaller and seem to be
more responsible for the resorption of nutrients.
In copepods, the epithelium of the midgut regions consists of different cell types called R, F, and
B cells, according to those in the higher crustaceans. The F cells show structures such as abundant
rER and active dictyosomes, which indicate enzyme synthesis and secretion.
 R F
Transition
zone

Lumen
B-Cell
zone

R gol
B F
R
vac

HEM

Fig. 9.6.
Schematic drawing of the different mature cell types (R, F, and B cells) within the hepatopancreatic tubules of a
decapod. Abbreviations: Golgi body, gol; hemolymph surrounding tubule, HEM; VAC, vacuoles. From Loizzi
(1971), with permission from Springer.

Fig. 9.7.
Immunohistochemical localization of protease in the hepatopancreatic tubules of the crayfish Astacus astacus.
(A) Intracellular labeling of protease in F cells and predominant appearance in the apical part (arrows) of the
cells and around the cell nucleus, ×240. (B) Strong fluorescence is located in the lumen of the midgut tubule
hours after induction of enzyme production and release of enzymes into the lumen, ×400. B cells (B), R cells
(R), F cells (F), lumen (L), hemolymph space (H). From Vogt et al. (1989), with permission from Springer.
 Nutrition and Digestion 299

In the higher crustaceans, the midgut gland forms the principal site of digestive enzyme synthe-
sis and enzyme secretion. The tubules of the midgut gland contain three cell types that are directly
involved in the digestion and assimilation of nutrients: the R, F, and B cells. The R cells perform the
resorption of nutrients, whereas the F cells are reported as the sites of enzyme synthesis. Vogt et al.
(1989) confirmed the production of digestive proteases in the F cells of crayfish by immunohisto-
chemistry and incorporation of radiolabeled amino acids (Fig. 9.7). Toullec et al. (1992) separated
the different cell types of the midgut gland of the caridean shrimp Palaemon serratus and found
maximum amylase activities within the fractions also containing the F cells. Lehnert and Johnson
(2002), as well as Hu and Leung (2007), detected the mRNA for cathepsin L and other digestive
enzymes solely within the cytoplasm of the F cells in the shrimps P. monodon and Metapenaeus ensis.
No enzyme synthesis was detected in the cytoplasm of B cells. However, several studies revealed
the presence of the mature digestive enzymes within the B cells and also in the lumen of the mid-
gut gland tubules. A plausible model for the function of the midgut gland and the interplay of the
different cell types was given by Vogt (1994). In brief, the mRNAs, as well as the enzymes, are
synthesized within the F cell. The enzymes are released through subapical vesicles into the tubule
lumen of the midgut gland, seep through the tubules and the ducts, and finally accumulate in the
stomach. After ingestion and mechanical processing of food within the stomach, the chyme consist-
ing of enzymes and predigested foods is pressed through the pyloric filter and back into the midgut
gland tubules. Part of the nutrients is resorbed by the R cells. B cells incorporate the chyme by
endocytosis and continue to process the nutrients intracellularly in the huge vacuoles of the cells
(Lehnert and Johnson 2002). This process is terminated by apocrine or holocrine extrusion of the
B cells into the lumen of the tubules. Accordingly, it can be considered that the B cells fulfill two
functions: intracellular digestion of nutrients and elimination of waste material (Vogt 1994).
Recent comparative studies gave evidence that the composition of digestive enzymes var-
ies considerably between species and, probably, may show specific patterns for certain groups or
taxa. Most distinctly, group-specific separation becomes evident in the expression of proteolytic
enzymes (endopeptidases). Most of the endopeptidases in the crustacean digestive tract belong to
a group of serine-, cysteine-, aspartate-, or metallo-endopetidases. The serine-endopeptidases com-
prise trypsin, chymotrypsin, and the recently established group of brachyurins. These enzymes
form the major share of gastric endopeptidases in many of the decapod taxa such as the Anomura,
Brachyura, Palinura, and Penaeidea.
The extracellular degradation of food items within the lumen of the digestive tract is the initial
step of enzymatic degradation. It releases oligomers, monomers, or other fragments of biological
molecules that, in the second step of enzymatic degradation, can be incorporated by the resorp-
tive cells of the midgut gland. The resorbed nutrients are finally processed by the digestive cells.
They can be catabolized to provide metabolic energy, used for somatic growth or reproduction, or
deposited as storage material.
For a long time, it was not clear whether crustacean digestive enzymes and, particularly, pro-
teinases, are synthesized and released by the secretory cells in an already active form or as an inac-
tive pro-enzyme. The first description of a putative zymogen sequence was provided by Klein
et  al. (1998) for trypsin from the shrimp L.  vannamei. The inactive precursor, trypsinogen, was
detected in the cells and the tubule lumen of the midgut gland of L. vannamei (Sainz et al. 2004).
Cathepsin L, a cysteine endopeptidase, was also shown to be synthesized as an inactive pro-enzyme
(Le Boulay et al. 1998). Further studies were carried out on astacin, a novel metalloprotease from
the crayfish A. astacus. Astacin is produced as a zymogen (Geier at al. 1997). The pro-enzyme has
an extension of 49 amino acids, which is removed during the maturation process of the enzyme.
Pro-astacin was detected within the F cells. After release, the pro-enzyme was also present within
the tubules of the midgut gland but not in the cardiac stomach. The activation of astacin happens by
stepwise autoproteolytic degradation of the 49 amino acid extension (Möhrlen et al. 2001, Guevara
300 Reinhard Saborowski

et al. 2010). It seems likely that many, if not all, proteolytic enzymes are synthesized as pro-enzymes
but are rapidly activated after secretion.

Extracellular and Intracellular Digestion

The digestive enzymes secreted by the hepatopancreatic cells accumulate in the stomach. Upon
starvation, the gastric fluid contains almost no food items but consists solely of an aqueous solution
of digestive enzymes, some emulsifiers, and some inorganic compounds. The amount of protein
comprises about 5%. As soon as food is ingested and further minced with the aid of the gastric mill,
the enzymes start to degrade the food compounds.
The enzymatic cocktail in the stomach consists predominantly of endo-hydrolases. These
enzymes cleave internal bounds of their targets, which are dietary macromolecules such as pro-
teins, polysaccharides, or nucleic acids. The resulting mono- and oligomers are absorbed by the
resorptive cells of the midgut region. In the higher crustaceans, the entire chyme including the pre-
digested nutrients is pressed through the pyloric filter into the tubules of the midgut gland. There,
the nutrients are incorporated by the R cells and subjected to further metabolic processing.
Studies on membrane transport within the gastrointestinal tract of crustaceans were most
intensively carried out on isolated brush border membrane vesicles (BBMV; Ahearn 1987). BBMV
from American lobster (Homarus americanus) midgut glands possess at least seven distinct trans-
port proteins for nutrient absorption (Ahearn et  al. 1992). Four of them are sodium dependent
(carrier systems for glucose, inositol, L-leucine, L-glutamate) and three are sodium independent
(L-alanine, L-proline, and a second system for L-leucine; Berra et al. (2006). Reduction of the pH
(down to pH 4) stimulated the uptake rates of both Na+-dependent and Na+-independent systems.
Glucose transport mechanisms in crustaceans were reviewed by Verri et al. (2001). The hepa-
topancreatic brush border membranes of shrimps (L. vannamei) and lobsters (H. americanus) have
sodium-dependent D-glucose transporters of the GLUT-family (Zhao and Keating 2007) and, at
least in lobsters, also for fructose. The transporters showed immuno-crossreactivity with antibod-
ies raised against mammalian GLUT transporters and high sequence similarity with mammalian
and insect transporters (Ahearn et al. 1985, Verri et al. 2001, Sterling et al. 2010).
Uptake and transport of other dietary requirements were studied for only a few substances. The
transport of myo-inositol was shown to be Na+- but not K+-dependent. However, it was sensitive
to pH, showing optimal influx at neutral pH inside and outside the vesicles (Siu and Ahearn 1988).
The transport system of folic acid (Pte-Glu) was studied in BBMV isolated from the midgut gland
of the prawn Marsupenaeus japonicus. The transport into the vesicles appeared to be carrier medi-
ated. It also was stimulated by an inward proton gradient (pH 5.5 outside, 7.4 inside) and was unaf-
fected by a sodium gradient (Blaya et al. 1998).

The Major Digestive Enzymes

Digestive enzymes can be studied from midgut gland extracts or directly from the gastric fluid,
which can be simply and repeatedly obtained from living animals (Fig. 9.8). Most of the digestive
enzymes belong to the enzyme class of hydrolases (EC 3). They are capable of cleaving various
molecular bonds such as ester bonds, glycosidic bonds, or peptide bonds by reactions involving the
addition of water.
From the beginning, research on digestive enzymes of Crustacea was focused on the proteo-
lytic enzymes and, particularly, on trypsin. The reasons for this fascination may be the fact that
trypsin was one of the first proteases identified, and it also plays an important role in digestion, as
well as in the regulation of various physiological processes (Neurath 2001). The proteinases that
have been characterized so far belong predominantly to four major groups: the serine-, cysteine-,
 Nutrition and Digestion 301

A B C 150
Gastric Proteins 100
Water
fluid 75
Salts 50
Emulsifiers
35

25

15
kDa

Fig. 9.8.
Gastric fluid from lobster Homarus americanus. (A) Sampling of gastric fluid from live lobsters using a syringe
and a flexible Teflon tube. The sampling does not harm the animal and can be repeated after a few days. Photo
by Kristine Reuter. (B)  The gastric fluid contains about 5–6% protein, 2–3% inorganic salts, and a similar
amount of emulsifiers. The majority (>90%) of the gastric contents consists of water. (C) Electrophoretic sepa-
ration of gastric proteins. When the animals have starved for 2–3 days before sampling, these proteins represent
solely the digestive enzymes but no alimentary proteins.

and metallo-endopeptidase families, and, recently, aspartic proteases were isolated from the gastric
fluid of lobsters (Rojo et al. 2010).

Trypsin

Trypsin shows a distinct catalytic specificity, preferably hydrolyzing proteins and peptides at the
carboxylic site of arginine and lysine residues. Trypsin-like enzymes were considered by many
authors to be the most important proteolytic enzymes in crustaceans. In fact, these enzymes were
identified in a large number of species including ancient forms such as the “living fossil” Triops sp.
(Maeda-Martínez et al. 2000) or the brine shrimp Artemia salina (Pan et al. 1991). Other crusta-
ceans, such as the cladoceran D. magna (von Elert et al. 2004), several copepod species (Hallberg
and Hirche 1980, Mayzaud et al. 1998, Lischka et al. 2007, França et al. 2010), and the nauplius larvae
of the cirriped Elminius modestus (Le Vay et al. 2001) possess high trypsin activities as well. Tryptic
enzymes were studied most intensively in Malacostraca and, particularly, in those species with sig-
nificant commercial importance. For example, Klein et al. (1998) obtained the coding sequences
of three trypsin genes from the whiteleg shrimp L. vannamei. Two of them were expressed in the
midgut gland. The iso-enzymes were isolated and genetically characterized by Sainz et al. (2004,
2005). A detailed review of invertebrate trypsins and some of their properties was recently provided
by Muhlia-Almazan et al. (2008).

Chymotrypsin

Chymotrypsin, another serine endopeptidase in crustaceans, appears in lower numbers of isoforms

but it often exhibits a higher catalytic activity than does trypsin. Chymotrypsin is less specific,
preferentially cleaving peptide bonds at the carboxyl side of the aromatic amino acids tyrosine,
tryptophan, and phenylalanine. Probably due to analytical difficulties, it was long considered to
be absent in crustacean digestive organs. However, Kimoto et al. (1985) reported high activities of
302 Reinhard Saborowski

chymotrypsin in the Antarctic krill E. superba. Using a new and specific substrate, Tsai et al. (1986,
1991) detected and isolated chymotrypsin-like enzymes in three penaeid species. However, only
marginal activity was present in the caridean shrimp M. rosenbergii, which, in turn, indicated that
chymotrypsin may not be ubiquitous among Crustacea. Subsequently, van Wormhoudt et al. (1992)
isolated chymotrypsin isoforms from L. vannamei and cloned the cDNAs encoding these enzymes
(Sellos and van Wormhoudt 1992). To date, chymotrypsin-like enzymes were identified in a variety
of crustacean species such as the squat lobster Pleuroncodes planipes and the crayfish Pacifastacus
astacus (García-Carreño et al. 1994), the edible crab Cancer pagurus (Saborowski et al. 2004), and
the branchiopod D. magna (von Ehlert et al. 2004). Chymotrypsin-like enzymes are, however, not
ubiquitous in crustaceans. Comparative studies among some eucarid crustaceans revealed that
crustaceans belonging to the Brachyura, Anomura, and Euphausiacea express high chymotrypsin
activities. In contrast, representatives of the Astacura and Caridea often express only low activities
(Saborowski, unpublished). It seems as if the appearance of chymotrypsin-like enzymes might be a
group-specific characteristic within the Crustacea.

Collagenases, Brachyurins

Since the 1960s and 1970s, proteolytic enzymes were isolated from crustacean midgut glands and
were reported to possess unique catalytic properties. In addition to showing trypsin and chymo-
trypsin activities, they were highly active in degrading native triple-helix collagen under physiologi-
cal conditions. The prototype of these collagenolytic enzymes was isolated from the midgut gland
of the fiddler crab Uca pugilator (Eisen and Jeffrey 1969, Eisen et al. 1973). The amino acid sequence
revealed its nature as a serine endopeptidase and, consequently, its divergence from mammalian
collagenases, which are zinc-metalloenzymes (Grant et al. 1980). The enzyme from U. pugilator,
denoted as serine collagenase 1, was recognized as a novel member of the chymotrypsin protease
family (Tsu and Craik 1996, Perona et al. 1997). Soon, proteases with similar collagenolytic properties
were identified in a shrimp (L. vannamei), the shore crab Carcinus maenas, and in the Kamtschatka
crab Paralithodes camtschaticus (Sellos and van Wormhoudt 1992, Roy et  al. 1996, Rudenskaya
et al. 2004). A cold-adapted analogue from Antarctic krill E. superba, named euphaulysin, shares
many of these characteristics (Gudmundsdóttir 2002). Since 1992, the new term “brachyurins”
(EC 3.4.21.32) was recommended by the Nomenclature Committee of the International Union of
Biochemistry and Molecular Biology (NC-IUBMB) for these collagenolytic serine endopeptidases
(Rudenskaya 2003). The brachyurins are of interest for studies of structure–function relationships
and for understanding of the evolution of serine proteases. Due to their catalytic properties, they
appear interesting for dermatological applications in terms of wound care.

Astacin

Astacins are another example of a new group of digestive enzymes that were first discovered and
studied in crustaceans. These enzymes are zinc endopetidases (Stöcker et al. 1988) that were first
isolated from the gastric fluid of the crayfish Astacus fluviatilis. Although already described by
Pfleiderer et al. (1967), the detailed analyses of the enzyme started with the identification of the
amino acid sequence by Titani et  al. (1987). The endopeptidase, with a molecular mass of 22.6
kDa, showed unusual specificities and was not affected by the hitherto known inhibitor for the
established families of proteases. An enzyme from the crayfish A. astacus served as the prototype
for the new astacin family. The preferred cleavage sites of astacin are peptide bonds in front of small
aliphatic residues. The crystal structure of the enzyme was discovered by Bode et al. (1992), and
the catalytic mechanism was investigated by Stöcker et  al. (1993) and Stöcker and Bode (1995).
 Nutrition and Digestion 303

Today, the astacin family of metalloproteases comprise a versatile and diverse group of enzymes
with respect to function and evolutionary history. They are present in bacteria and animals but
were not found in plants and fungi. Their physiological roles span a variety of key functions beyond
digestion (Becker-Pauly et al. 2009).

Cathepsin L

The term cathepsin arose from the ancient Greek meaning “digestion,” and cathepsins comprise a
number of proteolytic enzymes with different structural and catalytic characteristics. Cathepsins
are involved in a variety of intracellular metabolic reactions. About a dozen cathepsins are known
today, which, however, belong to the different groups of serine-, cysteine-, and aspartate-proteinases.
Laycock et al. (1989, 1991) isolated a cysteine protease that accounted for 80% of the proteolytic activ-
ity in the gastric fluid of the American lobster. It showed high similarity to papain and, particularly, to
the L cathepsins of vertebrates. Le Boulay et al. (1996, 1998) cloned the cathepsin L from L. vannamei
and analyzed its gene organization, which is homologous to that of rat cathepsin L. Accordingly,
the shrimp cathepsin L was categorized as a member of the cysteine proteinase of the papain super-
family. Other digestive enzymes of this family were identified and studied in the penaeid shrimp
M. ensis (Hu and Leung 2004, 2007), and the caridean shrimps Pandalus borealis (Aoki et al. 2003,
2004), Crangon crangon, and C. allmani (Teschke and Saborowski 2005). Cathepsin L is known as
an intracellular lysosomal enzyme in many tissues. Thus, its role in extracellular digestion within the
crustacean stomach is unusual and remains to be investigated in more detail.

Exopeptidases

From a functional point of view, endopeptidases should be secreted by the midgut gland and
should perform protein digestion in the stomach, whereas exopeptidases are supposed to be highly
active in the midgut gland tubules and the brush border membranes of the resorbing cells. Early
studies on exopeptidases, including carboxypeptidase A  and B, arylamidases, and dipeptidases,
are summarized by Dall and Moriarty (1983). Recent studies addressed various physiological and
biochemical aspects with respect to digestive exopeptidases. Carboxypeptidases were observed in
the Atlantic blue crab Callinectes sapidus (Dendinger 1987) and leucine-aminopeptidases were iso-
lated and characterized from the midgut gland of the crayfish Procambarus clarkii (de la Ruelle et al.
1992). Fernandez et al. (1997) showed variation in the activities of amino- and carboxypeptidases
in Farfantepenaeus notialis during the reproduction and molting cycles, and Ezquerra et al. (1999)
showed that different diets can influence the activities of a set of exopeptidases in the whiteleg
shrimp L. vannamei. Sakharov and Prieto (2000) characterized carboxypeptidases from the midgut
gland of the king crab P. camtschatica, which is the ingredient of a commercial enzyme preparation.
The concerted interplay, however, between endo- and exopeptidases in crustaceans is still poorly
understood. Recent comparative investigation showed that high activities of exopeptidases may
also occur in the gastric fluid of the crayfish Astacus leptodactylus but not in the clawed lobster
H. gammarus and the crab C. pagurus (Weber and Saborowski, unpublished). The physiological
function of the enzyme in the gastric fluid, as well as the cellular origin and expression pattern of
the enzyme, demands deeper investigation.

Lipases/Esterases

Digestive lipases catalyze the hydrolysis of triacylglycerols and thus the liberation of fatty acids
and glycerol. Lipases known to date vary considerably in size and in primary structure. However,
304 Reinhard Saborowski

they all belong to the α/β-hydrolase superfamily and they have in common a catalytic triad of
His, Ser, and Asp (Rivera-Pérez et  al. 2011). Crustacean lipases do not depend on colipase or
bile salts as mammalian lipases do. Cherif et  al. (2007) and Cherif and Gargouri (2009) iso-
lated a thermostable lipase with a molecular mass of 65 kDa from the midgut gland of the shore
crab C. maenas. The larval stages of the shrimp L. vannamei show lipase activity already in the
first stages, indicating that these stages are capable of degrading dietary lipids or storage lipids
(Rivera-Pérez et al. 2010).

Chitinases

The process of biochemical chitin degradation demands a set of enzymes that first produce oligo-
mers of the chitin chain by an endochitinase, commonly referred to as chitinase. The oligomers, in
turn, serve as substrates for an exochitinase that, in concert with unspecific β-glucosidases, liberates
the monomers, acetylated amino sugars (Saborowski et al. 1993). The exochitinase is commonly
termed N-acetyl-β-D-glucosaminidase (NAGase). Chitinolytic enzymes are involved in different
physiological processes such as molting, pathogen suppression, and digestion. In the euphausiids
E. superba and Meganyctiphanes norvegica, Peters et al. (1998, 1999) distinguished enzymes that are
involved in molting and those that are digestive enzymes. Digestive chitinases are not only suitable
in degrading and utilizing chitinous arthropod shells; they can also help to utilize crystalline chitin
from the spines of phytoplankton, as shown in Antarctic krill (Saborowski and Buchholz 1999).
Several studies in various species identified genes encoding for digestive chitinase (Watanabe et al.
1998, Tan et al. 2000, Proespraiwong et al. 2010) suggesting that the utilization of dietary chitin is an
ancient intrinsic characteristic of crustaceans.

Amylases

Amylases hydrolyze the α(1,4)-glycosidic bonds of starch and glycogen and liberate glucose oligo-
mers and monomers. The distribution and activities of amylases from crustaceans were studied
in detail by van Wormhoudt et al. (1995). Among 40 species of decapods, highest activities were
present in shrimps and crabs. The molecular mass of the enzymes ranged between 30 and 55 kDa,
and a high degree of polymorphism was apparent in many species. The sequences of amylase genes
in the shrimp L. vannamei were studied by van Wormhoudt and Sellos (2003). It is not fully under-
stood yet whether the polymorphic amylase genes are of advantage for the animal. However, this
knowledge might help in the formulation of carbohydrate-enriched feeds for shrimp aquaculture.
The activities of amylase and, particularly, the ratios between amylase and protease activities have
often been used to interpret dietary preferences of shrimps and their ontogenetic stages, respec-
tively (Ribeiro and Jones 2000, Johnston et al. 2004).

Cellulases and Laminarinases

One of the most remarkable findings of the past decade’s research is the verification of endog-
enous cellulolytic enzymes in many crustaceans. Cellulolytic activity was reported earlier in
several species, but the activity could not be unambiguously assigned to the crustaceans and
was possibly contributed by symbiotic bacteria. Byrne et al. (1999) obtained a complete cDNA
open reading frame for an endo-β-1,4-glucanase of 469 amino acids from a hepatopancreas
cDNA library of the crayfish C.  quadricarinatus. The endogenous origin of the enzyme was
confirmed by amplification of a polymerase chain reaction (PCR)-product from genomic
DNA. The gene, belonging to the glycosyl hydrolase family 9 (GHF9), showed conserved
 Nutrition and Digestion 305

regions, which supports the ancestral nature of this gene (Crawford et  al. 2004, Crawford
2005). Cellulase fragments were also sequenced from two other crayfishes (Cherax destructor,
Euastacus sp.) and the terrestrial brachyuran Austrothelphusa transversa (Linton et al. 2006).
The high cellulolytic activities in terrestrial anomurans also support the presence of endog-
enous enzymes (Greenaway 2003, Wilde et al. 2004). Recent studies on marine crustaceans
confirmed the presence of the cellulase genes in various species of branchiopods, isopods,
amphipods, euphausiids, and decapods (King et al. 2010, Saborowski unpublished). The eco-
logical significance of cellulose digestion in marine crustaceans remains to be investigated in
more detail. In coastal areas, detritivorous species may benefit from endogenous cellulases
when feeding on terrestrial plant material washed into the sea or on kelp. In terrestrial crusta-
ceans like the woodlouse Porcellio scaber, the benefits of endogenous cellulose digestion are
obvious because these animals are important decomposers of plant material (Kostanjšek et al.
2010). Cellulose degradation in land crabs differs from the hitherto existing model (Fig. 9.9).
It depends on a two-enzyme catalysis (endo-β-1,4-glucanase and glucohydrolase) rather than
the generally proposed three-enzyme process (Allardyce et al. 2010). Studies on the digestion
of storage products from kelp, such as laminarin or mannitol, are rare. Laminarinase enzymes
from the freshwater crayfish C. destructor and land crab Gecarcoidea natalis were isolated and
characterized by Allardyce and Linton (2008).

Properties of Enzymes

Proteolysis Resistance

The digestive enzymes that are released into the stomach are exposed to various proteinases and
esterases that were simultaneously synthesized and secreted. Although it was shown that some pro-
teolytic enzymes appear within the cell as inactive zymogenes after synthesis, they mature to fully

A B

Short oligomer
Cellobiose
(e.g. cellopentaose)

Glucose Glucose

Cellobiohydrolase Endo-β-1, 4-glucanase β-glucosidase Glucohydrolase

Fig. 9.9.
Scheme of cellulose degradation in (A)  fungi and in (B)  crustaceans. The conventional reactions involved in
the degradation of cellulose comprise the catalytic action of an endo-β-1,4-glucanase, a cellobiohydrolase, and a
β-glucosidase. In crustaceans, the glucohydrolase is capable of hydrolyzing glucose from both cellobiose (dimer)
and short oligomers. From Allardyce et al. (2010), with permission from The Company of Biologists, Inc.
306 Reinhard Saborowski

active enzymes after secretion and release into the stomach. Many of these extracellular enzymes
show a remarkable stability and may resist degradation for extended periods (Saborowski et  al.
2004). The enhanced stability may be related to structural properties such as the location of hydro-
lytic cleavage sites and stabilization through Ca2+-incorporation and electrostatic interactions. For
example, a trypsin isoform from the edible crab C. pagurus has a lower number of autolytic cleav-
age sites at the surface of the molecule compared to vertebrate trypsin (Fig. 9.10). And although a
C-terminal fragment of about 11 amino acids was removed, the enzyme retained almost full activity
(Hehemann et al. 2008).
Enhanced stabilities were also reported for other digestive enzymes, such as an endochitinase
from the Antarctic krill E. superba (Saborowski et al. 1993) and a chymotrypsin from C. pagurus
(Saborowski et al. 2004). These properties of the enzymes may help to avoid rapid degradation and
thus may contribute to saving metabolic energy for enzyme synthesis and secretion. Depending on
the feeding habits of the species or on the trophic environment (i.e., the supply of food), specimens
may starve for extended periods. However, as soon as food appears, they must be able to digest it
efficiently. An example for such a strategy of food utilization may be the Antarctic krill E. superba,
which shows high digestive enzyme activities also on starvation and successfully exploits patchy
food environments (Saborowski and Buchholz 1999).

Effects of pH

The gastric fluids at the extracellular sites of the digestive systems of crustaceans may range from
slightly acidic to slightly alkaline. In the gut of the copepod Calanus helgolandicus, the pH was deter-
mined within the foregut and in the hindgut with a microinjection technique (Pond et al. 1995).
The pH ranged between 6.11 and 7.25 in starved animals and up to 9.35 in animals that were fed with
phytoplankton (Fig. 9.11). In the stomachs of Malacostraca, the pH ranges between approximately
5 and 6. The lowest pH values were measured in lobsters, at around 4.7 (Navarrete del Toro et al.
2006). Upon feeding, the pH rises 0.5 to almost 1 unit. The shift in pH favors the catalytic action of

A K87 N-terminal domain B R62 N-terminal domain

K87 K107
C
K61 R67
K82 C
R235 K90 K113
R96
K126
K127 K97 R117
K153
K230
N N
K157
K175

K188

K188
R221
R184
C-terminal domain C-terminal domain

Fig. 9.10.
Computer-generated model of the three-dimensional structure of a trypsin isoform of the marine crab Cancer
pagurus (A) compared with a trypsin from rat (B). Arginine (R) and lysine (K) residues are indicated and
labeled according to the common chymotrypsin numbering. From Hehemann et al. (2008), with permission
from Elsevier.
 Nutrition and Digestion 307

A B C

Fore

6.8

Micropipette

Hind

8.5

Fig. 9.11.
Calanus helgolandicus. (A)  Restrained female with a micropipette inserted into the hindgut via the anus to
facilitate the injection of the pH-sensitive dye BCECF into the gut. (B and C) Gray-shaded images of the guts
of individual females indicating the pH after starvation (B) and after feeding (C). The gray scale represents the
pH from 6.8 to 8.5. From Pond et al. (1995), with permission from Springer.

proteolytic enzymes that show their maximum activity at neutral and slightly alkaline conditions.
This passive pH alteration may act as an efficient mechanism to control proteolytic activity. During
periods of starvation, the pH in the stomach drops to lower values that, in turn, cause a reduction
of proteolytic activity. Lowered proteolytic activity also prevents hydrolysis and thus destruction of
other enzymes that accumulate simultaneously with the proteolytic enzymes in the stomach. After
feeding, the pH rises again, and the proteolytic enzymes gain activity and capability of digesting
alimentary proteins.

Thermal Properties

The thermodynamic properties of crustacean digestive enzymes and their catalyzed reactions,
respectively, were predominantly studied in view of ecophysiological adaptations (Somero 2004).
Most enzymes exhibit a distinct thermal stability and remain fully active up to temperatures of
40–50°C or even higher. The increase of activity with rising temperature from 0° to about 30°C
generally follows an exponential relationship according to van’t Hoff ’s law. At higher temperatures,
the enzymes become progressively degraded. The optimum curve reaches its maximum before the
activity drastically declines and drops to zero. The “thermal optimum” by itself has no physiological
meaning but mirrors the physicochemical properties of the protein. However, the initial activity at
low temperatures may provide some indication about the cold tolerance of animals, and the course
of the exponential increase can be used to calculate the activation energy of the reaction according
to the Arrhenius equation.

Polymorphism of Digestive Enzymes

Polymorphism of digestive enzymes has been reported in many crustacean species and for
a number of enzymes. Van Wormhoudt and Sellos (2003) found eight electromorphs of
α-amylase in the whiteleg shrimp L. vannamei. The authors characterized three genes encod-
ing for mature proteins of 495 amino acids. All of the three genes were expressed in the midgut
gland of the shrimps. The sequences of the genes suggested an ancient duplication event and
a long evolutionary development between the present and ancestral genes. Similarly, several
308 Reinhard Saborowski

isoforms for amylase, but also for endopeptidases and unspecific esterases, were reported in
the spiny lobster Panulirus argus by Perera et al. (2008). The ecological benefit of polymorphic
enzyme expression may be seen as an adaptation to better cope with variable food supply.
According to Nelson and Hedgecock (1980), species with a broad food spectrum showed a
higher degree of heterozygosity than did feeding specialists. Moreover, among the special-
ists, carnivorous species were more often heterozygous than herbivorous species. Perera et al.
(2010) studied the in vitro digestibility of food by spiny lobsters with three different major
endopeptidase isoenzyme patterns. It was apparent that the different phenotypes released dif-
ferent amounts of amino acids from various protein sources. The different activities of trypsin
phenotypes in L. vannamei are also related to different kinetic properties of isoenzyme (Sainz
Hernández and Cordova Murueta 2009). Accordingly, enzyme polymorphism contributes a
surplus of catalytic potential due to slight structural and thus kinetic variations of the iso-
forms. These slight differences improve the concerted degradation of the natural target mol-
ecules and the yield of bioavailable products.

Dietary Effects on Enzyme Expression and Activities

During the past decades, a huge number of studies were published dealing with the effects of
natural or formulated diets or nutrients on the expression of digestive enzymes. The general
aims were, on one hand, to improve formulated diets for aquaculture and, on the other hand,
to define physiological proxies for the trophic preferences of a species or a population. The
studies revealed a huge variety of results, of which only a few examples can be presented here.
Le Moullac et al. (1996) reported a significant dose–response effect between the amount of
dietary casein supplement and activity and, particularly, the amount of trypsin in L. vanna-
mei. Chymotrypsin and amylase levels showed high scatter but no distinct trends. In contrast
to casein, gelatin-supplemented diet inhibited trypsin activity. Muhlia-Almazán et al. (2003)
showed that the same species of shrimp (L. vannamei) fed with a diet containing 30% of pro-
teins exhibited higher trypsin and chymotrypsin activities than did animals fed with lower
or higher protein contents. Moreover, differential expression of trypsin mRNA was observed
in the midgut gland of L.  vannamei under starvation (Sánchez-Paz et  al. 2003). In another
species, L.  stylirostris, saturation levels for midgut gland glucogenesis and amylase activity
were apparent at a diet containing 21% of carbohydrates (Rosas et al. 2000). Simon (2009)
fed juvenile spiny lobsters J. edwardsii with natural and formulated diet. The latter caused a
decrease of digestive enzyme activities, nutritional conditions, and changes of midgut cytol-
ogy. To reduce the amount of protein from fish meal, diets with soybean meal and blood meal
were tested. Both showed low in vitro digestibility due to high inhibitory effects (Lemos et al.
2004). However, feeding shrimps (L. vannamei) with diets containing different amounts of
soybean trypsin inhibitor over the course of 10 weeks had no effect on overall weight gain
(Sessa and Lim 1992).
Amphipods from different habitats and with different food preferences were analyzed for their
digestive enzyme activities ( Johnston et  al. 2005). According to their habitats, the amphipods
showed high laminarinase and lipase activities (supralittoral kelp-feeder); high α- and β-glucosidase,
cellobiase, and xylanase activities (low shore intertidal feeder); or high activities of all of these
enzymes (forest litter feeder). These results support a relationship between dietary preferences and
digestive enzyme complements ( Johnston et al. 2005). Very rapid responses to starvation or change
of diet were exhibited by small copepods (T. longicornis). Starvation caused significant reductions
in digestive and metabolic enzyme activities within 24 h. Change in food altered rapidly the fatty
acid composition and the expression pattern of digestive enzymes (Kreibich et al. 2008, 2011). A fast
 Nutrition and Digestion 309

response of enzyme expression may reflect an adaptive strategy to cope with a variable and chang-
ing food supply.

Effect of Hormones

Vertebrate gastrointestinal hormones were found to stimulate the release of enzymes from
midgut gland tissue of the crayfish Orconectes limosus (Resch-Sedlmeier and Sedlmeier 1999).
The authors concluded that crustaceans possess endogenous factors similar to vertebrate hor-
mones or, at least, receptors that accept these hormones. Putative endocrine cells were already
described by Mykles (1979) in the midgut epithelium of the crab Cancer magister. However,
only recently was their hormone content investigated and SIFamide- and tachykinin-related
peptide (TRP)-like immunopositive cells were identified (Christie et al. 2007). SIFamide-like
labeling prevailed in the anterior part of the midgut and the paired anterior midgut caeca. The
TRP-like immunoreactivity predominated in the posterior midgut and posterior midgut cae-
cum. Moreover, TRPs were detected in the hemolymph of starved animals but not in fed ani-
mals. Christie et al. (2007) suggested that midgut-derived TRP in Cancer spp. may function in
the paracrine/hormonal control of feeding behavior.

Symbiotic Bacteria and Other Microbes

The variety of potential food items of Crustacea also include materials that are hard to digest.
Such materials are chitin from the shell of crustaceans or other invertebrates, cellulose and hemi-
celluloses from plants, and other structural compounds such as lignin. The biochemical ability
to degrade these materials was often attributed to symbiotic bacteria or other microorganisms.
However, limnorid wood-boring isopods do not rely on symbiotic microbes for lignocellulose
digestion but are capable of producing the relevant enzymes by themselves (King et al. 2010).
Nevertheless, the presence of external and internal bacteria was reported for various taxa and
species of crustaceans such as copepods, decapods, or isopods. Terrestrial isopods show bacte-
ria within their midgut glands that seem beneficial in the digestion of plant material. In contrast,
marine species showed no significant microbiota in their midgut glands (Zimmer et al. 2001).
Low numbers of bacteria in the midgut gland are not surprising because the midgut divertic-
ula are separated from the stomach by a fine pyloric filter system. Unlike the midgut gland,
microbes have direct access to the hindgut. The physiological conditions in the gut lumen of
terrestrial isopods would allow for the coexistence of aerobic and anaerobic microorganisms
with fermentative activities (Zimmer and Brune 2005). In addition to direct contribution to
digestion, high bacterial proliferation rates in the posterior hindgut promote dense colonization
of feces with bacteria. After egestion, these feces may serve as partly fermented and high-quality
supplements to leaf litter. However, even if high numbers of bacteria are present in the animal’s
intestines, their contribution to digestion is not mandatory. Nordic krill M.  norvegica, which
omnivorously feeds on microalgae and zooplankton, was studied for bacterial contribution in
chitin degradation (Donachie et al. 1995). Chitinolytic bacteria were successfully isolated from
the digestive organs of krill. However, the enzymes of these bacteria showed much lower activi-
ties and different chromatographic properties than the putative endogenous enzymes of the
krill. It is self-evident that bacteria that are somehow associated with the foods are ingested by
crustaceans and accumulate and proliferate in their guts. However, rapid gut transit times, as
well as the formation of peritrophic membranes around the undigested food, are detrimental in
the establishment of a persistent and potent microbial community. Nevertheless, the microbes
present in the gut may certainly produce or liberate important micronutrients.
310 Reinhard Saborowski

FUTURE DIRECTIONS

During the past decades, significant progress was achieved in the research on digestive processes in
crustaceans. Morphological and anatomical studies made use of groundbreaking inventions such
as raster electron microscopy or laser scanning microscopy. This progress in analytical techniques
allows for biochemical measurements in very small crustaceans, larvae, and isolated organs or tis-
sues (Kreibich et al. 2011), and the results provide new and exciting insights into the biology and
digestive physiology of crustaceans. Now the “omics” are entering the field of crustacean digestive
physiology and may open new dimensions in the understanding of these cardinal living processes
(Ward et al. 2010).
Despite progress in research, today, the entirety of the gathered knowledge still does not pro-
vide a clear and comprehensive picture of the digestive physiology of Crustacea. There are still
open questions, apparently contradicting findings, and unsolved regulatory pathways. For example,
there is no consistent view on the genesis and development of the R, F, and B cells in the midgut
gland. A general and often presented view is that B cells develop from F cells. However, there is
also evidence that all of the three cell types directly arise from the embryonic E cells and develop
independently. Concerted cytological and biochemical studies are necessary to answer this ques-
tion. Basic research is also important to reveal the background for the different preferences of taxa
in expressing proteolytic enzymes. Certainly, this knowledge will also be helpful for understanding
the cytological processes within the midgut gland (Hu and Leung 2007). The transfer of nutrients
through the membranes of the midgut gland cells, as well as hormonal control of feeding activity
and digestion, are further important targets of basic research.
The major branches of applied crustacean biology and digestive physiology are aquaculture
and, to a lesser extent, biotechnology. Aquaculture of crustaceans increased significantly in the past
decades. In order to improve assimilation of the diets and, in turn, to reduce expenses for feeds, a
line of crustacean digestive research evolved to study the effects of food additives on the perfor-
mance of shrimp stocks, for example. Moreover, optimized food can help to lower excretion and
thus reduce pollution in and around aquaculture facilities. Another promising field in aquaculture
is the improvement of larval rearing. Optimized feeds and supplements are suitable to improve
growth, health, and survival of larvae and early juvenile stages (Simon 2009).
Biotechnological approaches make use of different products derived from crustaceans such as
chitin, chitosan, or astaxanthin. In addition, the versatile enzymes from the digestive tract of crus-
taceans often show exceptional catalytic properties. Such exceptional enzymes are suitable tools
for biotechnological, analytical, or clinical applications. For example, shrimp alkaline phospha-
tase, which is isolated from the northern shrimp P. borealis, became an important tool in molecu-
lar biology and genetics. There is evidence that crustacean enzymes may have a high potential for
applications either as byproducts directly obtained from crustaceans or as recombinant proteins
(Saborowski et al. 2006, Hehemann et al. 2008).
Crustacea represent one of the most important and versatile group of invertebrates on Earth.
Their relevance in both human alimentation as well as a source for new biotechnological products
is rising. Accordingly, nutrition of Crustacea, along with digestive physiology, biochemistry, and
molecular biology, will provide challenging and exciting fields of future research.

CONCLUSIONS

Crustacean fossil records can be back-dated to the Middle Cambrian. During the 500 million years
that have passed since then, the crustaceans evolved to occupy almost all aquatic and also some ter-
restrial habitats. Due to their wide geographic distribution, but also due to their huge span in size,
 Nutrition and Digestion 311

they are capable of feeding on a variety of food items ranging from microscopic plankton organisms
to fish and huge vertebrate carrion. Food uptake is facilitated by the aid of the primary feeding
appendages, the mandible and maxillae. Higher species developed additional feeding appendages
such as maxillipeds, gnathopods, or filter baskets that derived from the thoracopods. They use vari-
ous feeding modes such as filter feeding, deposit feeding, or scavenging. Several taxa also developed
parasitic forms that feed on the body fluids of their hosts.
The extraction and utilization of the nutrients occur in the digestive organs. In primitive taxa,
these organs can have the apparently simple appearance of a tube-like gut pervading the body from
the esophagus to the rectum. The gut is separated into different sections that bear epithelial cells
for enzyme secretion and nutrient resorption. Advanced taxa show a progressive septation of the
ectodermal foregut and hindgut and an entodermal midgut. The foregut forms the esophagus and
the complex chitinous stomach, whereas the diverticula of the midgut are the principal sites of
digestive enzyme synthesis and nutrient resorption. In order to cover the nutritive demands for
growth and reproduction, crustaceans express a variety of digestive enzymes. These enzymes ful-
fill the general tasks of hydrolyzing the common biopolymers proteins, carbohydrates, and lipids.
However, recent studies revealed a unique variety of structural characteristics and thus functional
properties. In particular, many of these enzymes are very resistant to proteolysis as an adaption to
their concomitant appearance with active peptidases in the lumen of the stomach. Crustaceans are
even capable of hydrolyzing quite inert biological substances like cellulose or chitin by the aid of
their endogenous enzymes.
In conclusion, the Crustacea developed a complex but efficient digestive system. The functional
match between anatomy and biochemistry is unique among the animal kingdom. It forms the key for
the successful utilization of a large and diverse range of food items to meet their nutritional demands.

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 10
RESPONSES TO ENVIRONMENTAL STRESSES: OXYGEN,
TEMPERATURE, AND pH

Nia M. Whiteley and Edwin (Ted) W. Taylor

Abstract
Although crustaceans are primarily a marine group, they have diversified into and adapted
to a wide range of environments, including the deep sea, where oxygen and sulfite levels may
vary; the littoral zone, with its daily fluctuations in water levels, temperature, and respiratory
gas concentrations; and freshwater and terrestrial habitats, either as facultative air breathers or
as fully terrestrial species resisting desiccation. The ability of crustaceans to compensate for
anthropogenic factors such as global warming and acidification of aquatic habitats is poorly
understood. Early studies on the effects of ocean acidification imply that adult crustaceans’
survival is affected by their ability to compensate for acid–base disturbances. Future studies
should concentrate on integrated crustacean responses to complex environmental changes,
such as the relationship between physiological plasticity and the transcriptome, as well as
their ability to undergo genetic adaptations to progressive anthropogenic factors such as those
resulting from climate change.

INTRODUCTION

The subphylum Crustacea comprises primarily aquatic animals inhabiting marine environ-
ments. Although the conditions in oceanic waters have changed over geological time, they can
be characterized within recorded time as having relatively stable temperatures well within the
biological temperature range, a relatively alkaline pH of around 8, and sufficient oxygen (O2)
to support aerobic metabolism. This generalization does not apply to isolated arms of the sea
(such as the Baltic Sea) and is currently being challenged by the increase in atmospheric car-
bon dioxide generated by progressive industrialization, with its associated reduction in the pH

320
 Responses to Environmental Stresses 321

and salinity of surface waters, and increase in temperature due to global warming. Relatively
small changes in these variables are likely to have disproportionate effects on the distribution
and survival of animal populations, including crustaceans, over the long term. A large num-
ber of crustacean species are found in shallow seas where many are important constituents of
plankton, either as larvae or adults, and where they may show diel vertical migrations through
the thermocline, thus experiencing rapid changes in temperature and sometimes in O2 levels.
Others form part of the benthos where O2 levels may be very low, and, in extremely anaerobic
conditions, sulfides accumulate. Many species have left the oceans to invade freshwater and
even terrestrial habitats where they are likely to encounter various combinations of highly
variable temperatures, areas or periods of hypoxia, and, in some circumstances, changes in pH.
For example, in small pools of salt- or freshwater, daytime temperatures can rise and plant pho-
tosynthesis can generate very high levels of dissolved O2 accompanied by high pH levels due
to consumption of carbon dioxide. By contrast, at night, the same pools can cool and become
hypoxic and acidotic due to plant and animal respiration (Taylor and Butler 1973, Truchot and
Duhamel-Jouve 1980).
Any consideration of the effects of temperature, O2 availability, and pH on animals has to take
account of the fact that these environmental variables are interrelated. In water, an increase in tem-
perature will reduce the solubility coefficient for O2, effectively reducing its availability, and will
result in a reduction in the pH at neutrality. Similar effects will be experienced by the body fluids
and tissues of an animal exposed to temperature change, although they can actively combat these
changes. As well as the physical solubility of O2 in body fluids being reduced by increased tem-
peratures, most crustaceans show a reduction in the O2 affinity of the respiratory blood pigment
hemocyanin with temperature. This is in part related to the direct allosteric effect of temperature
on O2 binding, but it also relates to the reduction in the pH of body fluids as temperature increases,
which typically reduces O2 affinity. So, as O2 demand increases with temperature, the supply of O2
to the metabolizing tissues may be compromised.
Much of our current understanding of respiratory physiology in crustaceans, including acid–
base balance, comes from studies on decapodan crustaceans because of their convenient size
for sampling and their value as a food source. More recently microtechniques have been devel-
oped that enable the study of respiratory responses in smaller crustaceans such as the bran-
chiopods Daphnia spp. and Triops cancriformis (Paul et al. 2004, Weber and Pirow 2009). The
transparency of Daphnia, for instance, has allowed optical techniques to be used to determine
respiratory variables such as the O2-binding properties of respiratory pigments, the use of fluo-
rescent microspheres for investigating ventilatory flows, and O2-sensitive phosphorescent dyes
to examine the distribution of partial pressure of O2 (Po2) within the hemolymph (Paul et al.
2004). Microspectral fluorometry, along with pH-sensitive dyes, has been used to determine
acid–base balance from microliter hemolymph samples, whereas fluorescence techniques have
also been used as highly sensitive methods for measuring small changes in O2 levels in water
samples in order to determine rates of O2 uptake of individual crustaceans weighing less than
100 mg (Rastrick and Whiteley 2011).
The following account features the integrated metabolic, physiological, and behavioral responses
shown by a range of crustacean species to aquatic hypoxia (which can include the facultative use
of air as a source of O2) and to environmental temperatures ranging from near freezing in polar
environments to the relatively high temperatures found in the tropics via the variable temperatures
encountered with changes in latitude and height on the seashore. The combination of relatively
high temperatures and aquatic hypoxia is considered to have been associated with the evolution of
air breathing, and the adaptations necessary for survival in air are discussed. Finally, we consider
the responses of crustaceans to anthropogenic effects such as global warming and environmental
acidification.
322 Nia M. Whiteley and Edwin (Ted) W. Taylor

RESPIRATORY GAS EXCHANGE WITH WATER

Oxygen Exchange

The gills in typical aquatic decapod crustaceans are contained within branchial chambers on
either side of the body and are ventilated with a forwardly directed stream of water driven by
a pair of balers or scaphognathites (Taylor 1982). These create subambient, oscillating hydro-
static pressures around the gills. When an animal is active, it ventilates large volumes of water
in order to maintain a high O2 partial pressure of 15–20 kPa in the water contained in the
branchial chambers (P WO2). This in turn can generate high O2 levels in the arterialized hemo-
lymph (Pao2) of 10–13 kPa. However, despite the potential for countercurrent or cross-current
exchange, Po2 levels in arterialized hemolymph rarely exceed those in expired water. This was
long considered to be due to a reduced diffusion coefficient for O2 over crustacean gills caused
by the presence of a relatively impermeable layer of chitin covering the gill lamellae, although
this has been questioned (Innes and Taylor 1986). In settled, inactive crustaceans, the O2 levels
in the hemolymph can apparently be far lower than have been previously reported (Massabuau
and Forgue 1996), with recorded values for arterialized hemolymph of less than 1 kPa that
are well below the partial pressures for saturation of crab hemocyanin described by Truchot
(1975). In the prebranchial (venous) hemolymph, Pvo2 levels in routinely active animals vary
between 0.67 and 2.67 kPa. This ensures a relatively high diffusion gradient for O2 transfer
across the gills from normoxic water, giving a partial pressure difference (Δ Pgo2) of up to 12
kPa. Despite their limited O2 carrying capacity due to the respiratory pigment hemocyanin
being carried in colloidal suspension in the hemolymph, decapods are able to maintain a sub-
stantial venous reservoir of O2 by virtue of their large circulating hemolymph volume and the
relatively high affinity for O2 of hemocyanin. Values for these variables that govern respiratory
gas exchange and transport were given for several crustacean species in a review by Taylor and
Innes (1988).
In the cladoceran Daphnia, O2 exchange takes place across the entire body surface because
the carapace is relatively permeable. Gas exchange also occurs between the hemolymph and the
ambient medium at specific sites: the carapace lacuna and the posterior part of the head (Pirow
et al. 1999). Ventilatory currents are maintained to the inner walls of the carapace by the thoracic
appendages that are also responsible for filtering food particles from the water stream. Hemolymph
Po2 immediately after the carapace lacuna is 0.4–0.5 kPa below ambient and ranges from 0.9 to 6.1
kPa (Pirow et al. 2004). The respiratory pigment in Daphnia, as in other cladocerans, is hemoglo-
bin (Hb; Kobayashi and Hoshi 1982). Both ventilation and heart rate vary with body size and in
response to algal concentrations and O2 levels in the ambient medium (Paul et al. 2004).

Carbon Dioxide Exchange

Crustaceans, like all other organisms, maintain pH levels in the body fluids within set limits.
This occurs against a continuous production of CO2 by cell metabolism and, in some species,
against fluctuations in CO2 in the surrounding water. The high ventilation rates of routinely
active aquatic gill-breathers relate to the relatively low availability of O2 in water, which is lim-
ited by its solubility, and is affected by temperature or salinity. Consequently, there is an effec-
tive hyperventilation with respect to the elimination of carbon dioxide so that partial pressure
of carbon dioxide (Paco2) levels in the hemolymph are very low (<0.67 kPa; Cameron 1986).
Hemolymph buffer base levels are correspondingly low and range between 4 and 10  mmol l–1
(Wheatly and Henry 1992), with the exception of daphnids, in which hemolymph [HCO3–]
reaches 21  mmol l–1 (see summary table of acid–base status in Crustacea by Weber and Pirow
 Responses to Environmental Stresses 323

2009). Elevated HCO3− levels in Daphnia hemolymph are associated with an alkaline pH despite
elevated Paco2 levels due to relatively high metabolic rates. In all crustaceans, hemolymph pH
regulation involves the passive buffering of CO2 changes by the carbonate system and other non-
bicarbonate buffers (e.g., proteins) and the electroneutral exchange of acid–base equivalents
(Fig. 10.1). The most dominant mechanism is electroneutral exchange of H+ and HCO3– ions across
ion transporting epithelia of the gills in decapods (Wheatly and Henry 1992) and the epipodites
in daphnids (Glover and Wood 2005). In decapods, it is generally accepted that electroneutral ion
exchange involves branchial Cl–/HCO3− and Na+/NH4+ or H+ exchangers, active ion transporters,
and the hydration/dehydration of CO2 by the enzyme carbonic anhydrase (Wheatly and Henry
1992, Whiteley 1999, Freire et al. 2008). Although the interrelationships among the various com-
ponents of this exchange system are still unclear, it appears that inward HCO3− fluxes are depen-
dent on apical Cl–/HCO3– exchange driven by an apical V-type H+-pump following the hydration
of CO2 into H+ and HCO3− by carbonic anhydrase (Onken and Putzenlechner 1995, Henry 2001,
Freire et al. 2008). The outward flux of acid equivalents depends on apical Na+/H+ exchangers
driven by a basolateral Na+/K+ ATPase (Taylor and Taylor 1992, Towle and Weihrauch 2001). As
a result, acid–base regulation in decapods is closely associated with ion regulation because both
homeostatic processes share the same mechanisms (Whiteley et al. 2001b). These relationships

GILL HEMOLYMPH CARAPACE

Non-calcified Extracellular and Intracellular Calcified

exoskeleton Epithelium compartments Epithelium exoskeleton

Protein buffers - weak acids

H 2O ? 2lactate+
HCO–3 + H+ CO2 CO2 CO2 H2O
CO2 HCO–3 + H+
CA CA CO32–
TISSUE Lactic ?
HCO–3 HCO–3 acid
Cl– + + CaLactate
Protein
+ + + Phosphate
H H H Bicarbonate pH = 7.4
+ –
Na Cl
NH+4 H+ + NH3 NH3 NH3 + H+ Metabolism CaCO3
Aerobic CO2 +
+ ?
H+(NH+4 ) NH+4 Anaerobic (lactic acid) H H+
Na+ Na+ Na+ H + H+ Cl– Na+/H+
K+ K+
?
(NH+4 ) (NH+4 ) Ca2+
Na+, K+ 2Na+ Na+ HCO–3 +
?
2Cl– H+ + HCO–3 HCO–3
H+
CO2
pH = 8.1 pH = 7.7 pH = 8.2

Fig. 10.1.
Schematic diagram of the proposed mechanisms involved in regulating acid–base status in the extra- and intra-
cellular compartments of a typical aquatic crustacean. Acid–base regulation takes place against a continuous
production of carbon dioxide by the cells and is dominated by passive buffering due to the presence of bicar-
bonate and nonbicarbonate buffers and by the exchange of acidic/basic equivalents (H+/HCO3–) between
body compartments and the surrounding water. All of the mechanisms responsible for branchial acid–base
exchange are also involved in ion regulation. See text for further details. The broken lines represent passive
diffusion. CA, carbonic anhydrase. Circles with crosses represent electrogenic ion pumps, and open circles
represent electroneutral ion exchange. Mechanisms labeled with a question mark are speculative. Modified
from Whiteley (1999).
324 Nia M. Whiteley and Edwin (Ted) W. Taylor

are summarized in Fig. 10.1. Although the ion exchange mechanisms responsible for hemolymph
pH regulation in Daphnia appear to be similar to those in decapods, the involvement of carbonic
anhydrase (CA) has not been established. However, recent molecular investigations have iden-
tified 31 genes with CA-like coding regions in the D.  pulex genome (Weber and Pirow 2009).
Twenty-five of the CA sequences had sequence similarities to secretory proteins, indicating that
CA is secreted into the hemolymph, unlike in decapods, which lack circulating CA (Cameron
1986). The presence of CA activity in the hemolymph of Daphnia could help to explain the trans-
port of CO2 in the hemolymph as HCO3− rather than as dissolved CO2 as modeled by Weber and
Pirow (2009). Overall, resting aquatic crustaceans are either in acid–base balance or they show net
base excretion (acid uptake) due to a largely herbivorous diet (Wheatly and Henry 1992).
As described for hemolymph, pH regulation in the intracellular compartment (designated as
pHi) is primarily regulated by electroneutral ion exchange (Taylor et al. 1991, Wheatly and Henry
1992). Early experiments reveal that intracellular pH in crustaceans is regulated by a Na+-dependent
Cl–/HCO3− exchanger and an electrogenic antiporter that exchanges Na+ for H+ (Wheatly and
Henry 1992, Towle and Weihrauch 2001). Closer examination of the sodium/proton antiporter
using brush border membrane vesicles from the hepatopancreas in marine (Homarus americanus)
and freshwater (Macrobrachium rosenbergii) crustaceans has revealed that two Na+ ions are trans-
ferred in exchange for one H+ (Ahearn et al. 1990 and see Fig. 10.1). This uneven stoichiometry
results in considerable Na+ uptake across a relatively small transmembrane pH gradient and is
responsible for a wide array of functions in addition to pHi regulation (Ahearn et al. 2001). Within
the intracellular compartment, buffering capacities are higher than in the hemolymph due to the
higher concentrations of proteins and phosphates (Wheatly and Henry 1992). Intracellular bicar-
bonate levels are relatively low at around 2 mmol l–1, whereas Pco2 levels match those in the venous
hemolymph. Intracellular pH values are typically 0.2–0.6 pH units lower than the pH values in the
hemolymph, apart from the exoskeletal compartment where pH levels are 0.3 pH units higher in
order to maintain the integrity of its calcium carbonate (Wheatly et al. 1991, Wheatly and Henry
1992). This compartment has an important role in acid–base regulation because it constitutes a
source of buffer base and is the site for the sequestration of protons and lactate ions during anaero-
bic metabolism ( Jackson et al. 2001) (Fig. 10.1).

AQUATIC HYPOXIA

Aquatic crustaceans can encounter environmental hypoxia and even anoxia in their natural envi-
ronment. In the marine environment, hypoxia occurs naturally in O2-minimum zones, deep
basins, upwelling areas of eastern boundary currents, and fjords (Helly and Levin 2004, Rabalais
et al. 2010). Over the past 50 years, the number of reported hypoxic sites in the global ocean has
increased, mainly in coastal regions but also in the open ocean due to anthropogenic activities such
as increased nutrient pollution leading to eutrophication (Diaz and Rosenberg 1995, 2008, Gilbert
et al. 2010, Rabalais et al. 2010). Climate-related changes are also involved because the rise in sur-
face water temperatures is increasing the likelihood of strengthened stratification, and increased
precipitation is likely to increase freshwater discharge and associated influx of nutrients (Rabalais
et al. 2010). The sensitivity of estuarine systems to hypoxia is also on the rise due to climate-driven
changes in ocean circulation patterns that have driven the upwelling of O2-poor but nutrient-rich
water from the deep ocean into coastal habitats (Howarth et al. 2011). The interstitial water in tidal
mudflats and the fluid seeping from hydrothermal vents are both anoxic and contain elevated levels
of hydrogen sulfide due to the activity of anaerobic bacteria. Finally, in freshwater ecosystems, O2
deprivation is characteristic of eutrophic lakes and streams, especially where water flow is low or
nonexistent and subject to eutrophication.
 Responses to Environmental Stresses 325

Crustaceans can respond to variations in O2 supply by changing their behavior and/or their
physiology. Behavioral changes are summarized here and usually involve movement away from
hypoxia in order to find more oxygenated water or emersion into air. Physiological responses range
from changes in ventilation and heart rate to variations in the factors determining the functional
range of respiratory pigments and metabolic responses. Particular attention is given to the provi-
sion of O2 to developing eggs and the adaptations shown by burrowing crustaceans and those living
at great depths. Finally, the facultative or obligate use of air as a source of O2 is considered.

Behavioral Responses to Hypoxia

Mobile aquatic crustaceans avoid chronic hypoxia by seeking out normoxic waters. In general,
crustaceans increase their activity rates up to threefold as Po2 levels start to decline both in the
laboratory and in the field (Hagerman and Uglow 1985, deFur et al. 1990, Bell et al. 2003, 2009,
Haselmair et al. 2010). At Po2 levels down to 2 kPa, benthic species show avoidance behavior as they
abandon their secluded benthic habitats and move vertically to find oxygenated water (Hagerman
and Uglow 1985, McMahon 2001, Haselmair et al. 2010). Increased activity occurs regardless of the
threat of exposure to predation and the existence of entrained diurnal activity patterns. During
severe hypoxia (0.04–2.0 kPa), benthic crustacean species become less active, presumably to reduce
O2 demand when O2 supply is limiting ( Johansson 1997), and can demonstrate a number of atypical
behaviors. For example, territorial species are no longer able to avoid each other but will aggregate
together in areas where Po2 levels are higher (Haselmair et al. 2010). Predator–prey interactions
are also reduced, as well as feeding rates, as shown in the isopod Saduria entomon, which feeds
on the benthic amphipod Monoporeia affinis. During moderate hypoxia, the interactions between
S. entomon and M. affinis decline mainly due to a decrease in activity rates of the latter (Sandberg
and Bonsdorff 1996). During anoxia, most species become immobile and moribund, although tol-
erances do vary because those species with a burrowing lifestyle are more active under such con-
ditions (Haselmair et al. 2010). In addition, crayfish have been shown to actively seek out colder
environments during hypoxia to reduce metabolic rates and consequently O2 demand (Morris
2004). This behavior is known as hypoxia-induced behavioral hypothermia. Other species, such
as the northern krill Meganyctiphanes norvegica, use cooler temperatures to enable migration down
into severely hypoxic waters during the day (Spicer and Saborowski 2010). Migrations to the surface
during the night are essential to pay off the O2 debt. Other extensive diel migrations are observed in
the zooplankton. In the coastal upwelling regions off the coast of Chile, for example, the copepod
Eucalanus inermis and the euphausiid Euphausia mucronata are closely associated with the O2 mini-
mum zone (Escribano et al. 2009). E. mucronata in particular performs extensive diel migrations
between the surface waters and the core of the O2 minimum zone. If the temperatures increase as
expected due to global warming, then the depth of vertical migrations in these crustacean species
could be limited, subjecting them to greater predation risk.
Despite collective behavioral responses to hypoxia, crustacean species demonstrate different
threshold levels. Those species with higher tolerances will remain active down to lower Po2 values.
To date, it has been shown that species-specific thresholds for reduced activity are lower in situ
than they are in the laboratory (Haselmair et al. 2010). They are also lower in adults compared with
juveniles (Eriksson and Baden 1997). It has also been demonstrated that activity thresholds can
vary within species (Bell et al. 2009). For example, nutritional state can influence an individual’s
response to hypoxia because recently fed individuals will behave differently to unfed individuals.
In Cancer magister, declining Po2 levels (21–1.5 kPa) reduced both the amount of food eaten and the
amount of time spent feeding (Bernatis et al. 2007). In the laboratory, it was observed that feeding
had no effect on the behavior of the crabs because both fed and unfed individuals preferred the
highest Po2 levels when placed into an O2 gradient. Out in the field, however, unfed crabs remained
326 Nia M. Whiteley and Edwin (Ted) W. Taylor

mobile and migrated up to 1.4 km in 6 h. Crabs that had recently fed settled in well-oxygenated areas
to digest their meal for at least 48 h (Bernatis et al. 2007). Hypoxia also affects foraging behavior in
Cancer setosus because hypoxic crabs consume less prey of smaller body sizes than normoxic crabs
(Cisterna et al. 2008).

Physiological Responses to Hypoxia

The differences just described between species in behavioral thresholds to hypoxia can be
accounted for by differences in their respiratory physiology. Generally speaking, crustaceans either
regulate rates of O2 uptake down to a critical value of ambient O2 partial pressure (Pcrit) or conform
to declining O2 levels in their aquatic environment. In the former case, rates of O2 uptake are main-
tained during moderate hypoxia by changes in cardiorespiratory responses and in the ability of the
circulatory system to transport O2 to the metabolizing tissues. Both act to improve O2 uptake and
delivery at a time when ambient O2 levels are declining. In general, crustaceans maintain O2 uptake
rates down to Pcrit by increasing ventilation rates (Wheatly and Taylor 1981, Taylor 1982, Airriess and
McMahon 1994, McMahon 2001). This effective hyperventilation serves to “blow-off ” CO2 and
results in a respiratory alkalosis (Fig. 10.2 B,D). In contrast, in the cladoceran Daphnia, which uses
its appendages to filter feed, ventilation rates increase but only when the animals are maintained
in nutrient-free conditions. When nutrients are available to Daphnia, ventilation by the thoracic
appendages remains unchanged or even decreases (Paul et al. 1997, Pirow and Buchen 2004). Heart
rate either increases, as observed in Daphnia (Paul et al. 1997), or declines, as observed in crayfish,
lobsters, and crabs (Wheatly and Taylor 1981, Airriess and McMahon 1994, Reiber and McMahon
1998). During hypoxia, cardiac output is typically maintained by increased cardiac stroke volume
so that perfusion of the respiratory gas exchange surfaces remains unchanged or may even increase
(Taylor 1982, Airriess and McMahon 1994, Reiber and McMahon 1998). Hemolymph flow may also
be adjusted during hypoxia, resulting in its redistribution to tissues with higher energy demands. In
the crab C. magister, for example, hemolymph flow is redistributed from the anterodorsal regions
to the posterior and anteroventral regions by the differential operation of valves on the arterial out-
flow from the heart (Airriess and McMahon 1994). Below Pcrit, ventilation and heart rate decrease
and the O2 content of the hemolymph decreases (Fig. 10.2 C). At this stage, crustaceans resort to
anaerobic metabolism, resulting in the accumulation of lactic acid as the main metabolic end prod-
uct (McMahon 2001) (Fig. 10.2 E).
The values for Pcrit in crustaceans vary with aquatic habitat. Table 10.1 summarizes Pcrit values
obtained from crustacean species inhabiting a range of freshwater and marine environments.
Generally speaking, those species more likely to encounter hypoxia and anoxia in their natural
environment have lower Pcrit values. In mid-water crustacean species, for instance, it appears that
Pcrit values of less than 3 kPa are associated with specific adaptations for hypoxia in the O2 minimum
layer (Childress and Seibel 1998). Species with the highest Pcrit values are mid-water species living
at high environmental O2 levels or those living in burrows. Intertidal crustaceans have intermediate
Pcrit values (Table 10.1; for squat lobsters see Lovrich and Thiel 2011). The responses to hypoxia in
water breathing crustaceans are affected by temperature because, in the short term at least, it affects
rates of aerobic metabolism and consequently O2 demand. In the shore crab Carcinus maenas, Pcrit
for the reduction in both rate of O2 uptake and heart rate during progressive hypoxia varied from
around 8 kPa at 20°C to 5.3 kPa at 10°C (Taylor et al. 1977, Taylor 1981). In the crayfish Pacifastacus
leniusculus, Pcrit of O2 uptake was reduced from 5 kPa at 20°C to 2.5 kPa at 5°C (Gorr et al. 2010).
At the lower acclimation temperature, crayfish accumulated less lactic acid, suggesting that aero-
bic adenosine triphosphate (ATP) production was unaffected by hypoxia at 5°C. In northern krill
M. norvegica, Pcrit decreased from 8–11 kPa at 15°C to 4–6 kPa at 10°C (Strömberg and Spicer 2000).
This shift in Pcrit has important implications because M. norvegica has a moderate ability to regulate
 Responses to Environmental Stresses 327

A 6 B 8.10

5 8.05
*
4 8.00
Pao2 (kPa)

pHa
3 7.95
*
2 7.90
*
1 7.85
0 7.80
0 5 10 15 20 25 0 5 10 15 20 25
C 0.5 D 0.5

0.4
0.4
Cao2 (mmol L–1)

Paco2 (kPa) 0.3 *

0.3 *
0.2
*
0.2
0.1

0.1 0.0
0 5 10 15 20 25 0 5 10 15 20 25

E F 8
1.4
1.2 *
[HCO–3] (mmol L–1)

6
Lactate (mmol L–1)

1.0 *
*
0.8 4
0.6
0.4 2
0.2
0.0 0
0 5 10 15 20 25 0 5 10 15 20 25
Plo2 (kPa) Plo2 (kPa)

Fig. 10.2.
Changes in postbranchial hemolymph oxygen and acid–base variables during exposure to hypoxia in the fresh-
water crayfish Austropotamobius pallipes. Aquatic hypoxia caused a proportional reduction in (A) hemolymph
oxygen partial pressure (Pao2), (B) an increase in hemolymph pH (pHa) due to a respiratory alkalosis, (C) the
maintenance of hemolymph oxygen content (Cao2) in moderate hypoxia, (D) a reduction in hemolymph CO2
partial pressure (Paco2), (E) an accumulation of lactate during severe hypoxia, and (F) a reduction in hemo-
lymph bicarbonate concentration ([HCO3–]). Values taken from Wheatly and Taylor (1981).

rates of O2 uptake in the face of environmental hypoxia and rapidly accumulates large concentra-
tions of L-lactate during anoxia (Spicer 1999). Temperature changes during migration into hypoxic
layers during diel vertical migration of this mesopelagic species are hypothesized to be crucial for
survival (Spicer and Saborowski 2010).

The Role of Respiratory Pigments

The ability of crustaceans to tolerate hypoxia relies to a large extent on the functional properties
of their respiratory pigment. This is hemocyanin in most crustaceans, although Hb is present in
branchiopod crustaceans such as Daphnia, Artemia, and Chirocephalus (Terwilliger and Ryan 2001).
Table 10.1.  Critical oxygen partial pressures (Pcrit) for rates of oxygen uptake in crus-
tacean taxa occupying a range of aquatic habitats. Amphipods classified to subor-
der, decapods to family (apart from thalassinidean shrimps, which are classified to
infraorder).

Pcrit (kPa) Temp (°C) Taxa Source

Mid-water (Mesopelagic)
Polar (Antarctic) 3.9–5.6 0.5 Gammarid and Torres et al.
hyperiid (1994)
amphipods,
euphausiids,
ostracod
Temperate 4.0–6.0 10 Euphausiid Strömberg and
Spicer (2000)
8.0–11.0 15 Euphausiid Strömberg and
Spicer (2000)
Subtropical 2.7–4.0 7 Sergestid and Donnelly and
oplophorid Torres (1988)
shrimps,
penaeid prawns
3.3–4.7 14 Sergestid and Donnelly and
oplophorid Torres (1988)
shrimps,
penaeid prawns
4.0–6.7 20 Sergestid and Donnelly and
oplophorid Torres (1988)
shrimps,
penaeid prawns
Oxygen minimum layer 1.2–1.5 4 Mysid and Childress (1975)
oplophorid
shrimps,
copepod,
ostracod
1.0–1.1 5 Mysid, oplophorid Childress (1975)
and pandalid
shrimps,
copepod,
gammarid
amphipod
1.3–2.4 7 Pandalid shrimp, Childress (1975)
copepod
2.1–2.7 10 Sergestid shrimp, Childress (1975)
hyperiid
amphipod,
euphausiid

(continued)
 Responses to Environmental Stresses 329

Table 10.1. (Continued)
Pcrit (kPa) Temp (°C) Taxa Source
Deep-sea hydrothermal 1.7 5 Bythograeid crab Mickel and
vents (vent crab) Childress
(1982)
Burrow environments 1.3–6.7 10–25 Thalassinidean James et al.
shrimps (2005)
Intertidal 5.3 10 Portunid crab Taylor (1981)
~8.0 20 Portunid crab Taylor et al.
(1977)
~2.0 ? Palaemonid Morris and
shrimp Taylor (1985)
Freshwater 2.5 5 Astacid crayfish Gorr et al. (2010)
4.0 15 Astacid crayfish Wheatly and
Taylor (1981)
6.0 25 Cambarid crayfish Reiber and
McMahon
(1998)
Hypogeal 3.3–3.9 22 Cambarid crayfish Gannon et al.
(1999)

During hypoxia, alterations in both carrying capacity and O2 affinity can occur. Above Pcrit, the
rise in ventilation rate in moderate hypoxia results in a hyperventilation with respect to CO2 and
causes a respiratory alkalosis that left shifts the O2 equilibrium curve for hemocyanin (Wheatly
and Taylor 1981, McMahon 2001). This maintains O2 loading at the respiratory surfaces, conserv-
ing a high O2 content in postbranchial hemolymph that sustains O2 supply to the tissues despite
reductions in hemolymph Po2 (Fig. 10.2). Below Pcrit, it is possible that other modulators increase
in importance, such as increases in hemolymph [Ca2+] and [Mg2+] associated with the mobilization
of HCO3− from internal stores (Taylor and Whiteley 1989), increases in hemolymph [L-lactate]
(Truchot 1980, Mangum 1997), and increases in hemolymph urate (Lallier and Truchot 1989). All
of these modulators increase O2 affinity of crustacean hemocyanins. Further details of the modula-
tion of the binding properties of respiratory pigments and how these relate to hypoxia are covered
in Chapter 11 of this volume. Increases in the O2 carrying capacity of hemolymph due to increased
hemocyanin content have been observed in Callinectes sapidus but only after prolonged exposure
to hypoxia (deFur et  al. 1990), and in C.  maenas on exposure to hypoxia after feeding (Legeay
and Massabuau 2000). Hb concentrations rise in response to declining O2 levels in Daphnia and
Artemia (Kobayashi and Hoshi 1982, Mangum 1997, Paul et al. 2004). The induction of Hb is a
standard response, and the regulation of Hb gene expression is central to the medium-term survival
of Daphnia during hypoxia because it leads to the synthesis of Hb subunits with high O2 affinities
(Paul et al. 2004). Between-individual variation in Pcrit is related to the properties of the respiratory
pigments, with increases in the O2 affinity of hemocyanin associated with lower Pcrit in C. sapidus
(Bell et al. 2009) and an increase in both Hb concentration and O2 affinity being responsible for
lower Pcrit in Daphnia (Kobayashi and Hoshi 1984). In the branchiopod crustacean Chirocephalus
diaphanus, an inhabitant of temporary ponds in which temperatures and respiratory gas concentra-
tions are highly variable, this response is gender-specific because females show the induction of Hb
during chronic hypoxia whereas the males do not. Poisoning with carbon monoxide increased the
330 Nia M. Whiteley and Edwin (Ted) W. Taylor

Pcrit in females to the level shown by normal males (Taylor 1965). This difference between the sexes
may relate to the high cost of egg production and to behavioral differences because the females were
observed to swim close to the bottom of temporary pools, where they may avoid predation but be
exposed to low O2 levels, whereas the males swam close to or even at the surface, except for periods
when they swam in tandem with the females during insemination.

Metabolic Responses to Hypoxia

A number of metabolic adjustments at the cellular and molecular level also take place during
hypoxia, either to protect the tissues from O2 deprivation or to provide alternative pathways for
ATP production. During hypoxia, animals can show several defense mechanisms against O2 depri-
vation, including a reduction in energy (measured as ATP) turnover. Studies on vertebrate tissues,
mainly hepatocytes and brain tissue, have shown that the defense system in hypoxia-tolerant ani-
mals involves an O2 sensor and a signal transduction pathway, including protein kinase cascades
that regulate preferential expression of several proteins (Hochachka 1997). Prolonged hypoxia or
anoxia leads to downregulation of ATP turnover rates to achieve a new hypometabolic steady state.
Extremely low levels of ATP turnover are achieved via the downregulation of protein turnover, urea
synthesis, glucose synthesis, and the maintenance of electrochemical gradients (Wu 2002). The
main molecular responses to hypoxia are regulated by the transcription factor hypoxia-inducible
factor 1 (HIF-1), which leads to differential gene expression under hypoxia and a series of biochemi-
cal and physiological responses (Wu 2002). To date, it has been shown that the synthesis of Hb in
Daphnia magna during hypoxia is dependent on HIF-1 (Gorr et al. 2004). It has also been suggested
that HIF-1α is involved in the regulation of gene expression in crustaceans during the molt and in
response to infection (Gorr et al. 2010). In mammals, HIF-1α is even upregulated during normoxia
and is associated with cell proliferation, apoptosis, vascularization, and a strong immune response.

Provision of Oxygen for Developing Eggs

Female decapod crustaceans typically hold a mass of fertile eggs on the underside of their
abdomens to be released into the plankton as motile larvae following a period of development.
Conditions within the egg mass may become markedly hypoxic, especially in brachyuran crabs
where the egg mass is relatively large in proportion to body size (Naylor et  al. 1999, Fernández
et al. 2000, Baeza and Fernández 2002). There is evidence that the female edible crab Cancer pagu-
rus may induce development and synchronized release of the eggs after an overwintering dormant
period by actively ventilating the egg mass with water. Female crabs attach a mass of up to 3 million
fertile eggs to their abdominal pleopods in October/November, then move offshore into deeper,
calmer water where they stay half buried in the sand or silt, neither moving nor feeding for about
6–9 months (Naylor et al. 1997). During this inactive period, they show reduced rates of O2 uptake
and decreased heart rates compared to their nonovigerous counterparts (Naylor and Taylor 1999).
They also do not appear to aerate or agitate the egg mass. In the spring, these ovigerous female
crabs adopt a characteristic stance, which takes the form of a raised posture and increased pumping
actions by the abdomen and pleopods, which serves to clean and aerate the eggs (Naylor and Taylor
1999). This was confirmed by measurements of O2 levels within the egg mass that ranged from 4.5
kPa during early developmental stages to 13.1 kPa at stages close to hatching (Naylor et al. 1999).
These increased Po2 levels within the egg mass coincided with a rise in O2 demand of the eggs and
the O2 sensitivity of the egg masses; Pcrit increased from 8.0 to 11 kPa with development (Naylor
et al. 1999). Preliminary experiments on ovigerous C. pagurus revealed that an injection of potas-
sium cyanide in the vicinity of the egg mass increased ventilatory activity by the ovigerous female,
indicating that her O2 receptors were sampling water collected from around the eggs (Naylor et al.
 Responses to Environmental Stresses 331

1999) so that she is able to measure the effectiveness of her ventilation of the egg mass. The final
stages of egg development in C. pagurus can be fairly rapid, taking only 2–3 weeks, and is associated
with the active ventilation of the egg mass by the female. Accordingly, the behavioral change lead-
ing to the onset of active irrigation of the egg mass in spring enables its synchronized release by the
ovigerous female.
The success of in vitro cultivation of decapod eggs has been shown to rely on the degree of
agitation of the eggs (Helluy and Beltz 1991). Continuous agitation of eggs isolated from C. mae-
nas resulted in higher survival rates and faster rates of embryonic development compared to eggs
still attached to adults under similar conditions (Hartnoll and Paul 1982). These observations sug-
gest that circulation of well-aerated water around the eggs is important for development. Oxygen
uptake rates in C. pagurus eggs were shown to drop by 76% between stirred and stationary con-
ditions, thus indicating that the availability of O2 is limiting to aerobic metabolism (Naylor et al.
1997). Because the eggs are extremely small, O2 uptake of individual eggs is unlikely to be diffusion
limited. However, they are closely packed within the egg mass so that their O2 supply is strongly
convection limited, with Po2 in the vicinity of the eggs determined by the brooding behavior
of the ovigerous female. Brooding behaviors in brachyuran crabs range from movements of the
abdomen to grooming of the egg mass with the chelae (Naylor and Taylor 1999, Naylor et al. 1999,
Fernández et  al. 2000, Baeza and Fernández 2002, McCleary 2006). Active brood behavior can
represent a substantial parental investment due to the costs associated with ventilating the large
egg mass (Fernández et  al. 2000). Costs increase during development as the O2 demand of the
embryos increases. Movements of the abdomen, for example, increase in frequency during devel-
opment accompanied by an increase in rates of O2 consumption of the brooding females (Baeza
and Fernández 2002). Furthermore, abdominal movements are continuous during later stages of
development and are not subject to diurnal activity rhythms (Ruiz-Tagle et al. 2002). Active brood
care has also been observed in freshwater (Crangonyx pseudogracilis) and intertidal (Apherusa
jurinei) amphipods that normally encounter wide variations in O2 levels in their natural environ-
ment (Dick et al. 1998, 2002). Ovigerous females ventilate the embryos by using a flexing motion
to flush water through the brood pouch. The time spent by the female ventilating the brood pouch
increases when the O2 levels in the surrounding water are reduced. Brooding behaviors, however,
decline at more advanced stages of development when the embryos have functional cardiovascular
and respiratory systems.

Hypoxia and Larval Development

Hypoxia has been shown to affect hatching in copepods. Hatching success was suppressed by up
to 50% when the eggs were exposed to hypoxia for 12 days, although newly spawned eggs survived
exposure to anoxia longer than fully developed eggs (Marcus and Lutz 1994). Experiments on spi-
der crab eggs showed that the eggs are resistant to periods of anoxia and can tolerate low O2 levels
for much of the embryonic development until 1–2 months before hatching (Peterson and Anger
1997). This latter phase is likely to coincide with increases in protein synthesis rates, growth, and
differentiation as observed in C. pagurus embryos (McCleary 2006). An extreme case is shown by
Artemia eggs, which can tolerate anoxia during desiccation, surviving as dehydrated cysts for up
to 4 years. When water supply is limited, they undergo profound metabolic depression, accom-
panied by a dramatic drop in intracellular pH (Hand 1998). Both O2 depletion and a drop in pH
depress protein synthesis due to the presence of a molecular O2 receptor and proton sensitive
translational components (Kwast and Hand 1996). The biochemical and molecular adjustments
needed to sustain extended metabolic depression are considered elsewhere (see Chapter 12 in this
volume). In developing larvae exposed to progressive hypoxia, maintenance of metabolic rate with
hypoxia is less well developed than in either post-larvae or adults. Regulation of O2 uptake rates in
332 Nia M. Whiteley and Edwin (Ted) W. Taylor

N. norvegicus improves in post-larvae as gas exchange shifts from the extrabranchial surfaces (telson
and uropods) to the highly specialized gills (Spicer and Eriksson 2003). This transition occurs as
the general exoskeleton surface calcifies and becomes less permeable to O2 and CO2. Larvae, how-
ever, can improve tolerances to hypoxia by increasing hemocyanin O2 affinity and possibly circulat-
ing hemocyanin concentrations (Spicer and Eriksson 2003).

Living at Depth

Hypoxia and anoxia are features of the open ocean where low ambient O2 levels occur at intermedi-
ate depths as O2 minimum layers (400–1,000 m) and in the deep ocean at depths below 1,000 m at
hydrothermal vents and cold-seeps. In the absence of physical turnover of large masses of seawater,
as happens during autumnal cooling in temperate regions, reaeration from the atmosphere of water
in the deep ocean is inhibited, and oceanic O2 minimum layers develop (Childress and Seibel 1998).
Knowledge of the ability of crustaceans to live in O2 minimum layers comes from studies on the
bathypelagic mysid Gnathophausia ingens. Morphological changes to the gills involve an increase in
surface area and a reduction in diffusion distances (Childress 1971). G. ingens is also highly effective
at extracting O2 from the ventilatory stream due to relatively high ventilation rates and increased
circulatory capacity (Belman and Childress 1976). In addition, hemocyanin from G. ingens has a
high affinity for O2, a high cooperativity, and a large Bohr effect (Sanders and Childress 1990).
Marine species living within the O2 minimum layer tend to have poor anaerobic abilities, suggest-
ing that changes in aerobic capacity are sufficient for survival in conditions where Po2 levels are
consistently low at only a fraction of 1 kPa (Childress and Seibel 1998).
Deep-sea habitats present further challenges to marine life due to increases in barometric pres-
sure, decreases in temperature, and the lack of light apart from bioluminescence. Conditions can
be even more unfavorable at hydrothermal vents and cold-seeps where ambient seawater at 2°C
mixes with anoxic and acidic (pH 3.0) vent fluid at 350°C; this fluid contains high concentrations
of heavy metals, CO2, and sulfide (Hourdez and Lallier 2007). Cold-seeps occur where hypersaline
brine containing reduced sulphur, methane, and often mineral oils emerge from the sediments on
the seafloor without an appreciable increase in temperature (Levin 2005). Despite these harsh and
variable conditions, deep-sea habitats support thriving marine communities characterized by low
species diversity but high species abundance. Crustaceans are well represented in both communi-
ties, with 125 species of decapod crustaceans from more than 33 families being reported in 2005
(Martin and Haney 2005). Amphipods, isopods, and copepods have also been reported to occur in
cold-seeps (Levin 2005). Although these deep-sea sites are largely inaccessible, studies on animals
successfully brought to the surface and kept under high pressure are beginning to explore the respi-
ratory adaptations of deep-sea crustaceans. Currently most of our knowledge comes from work on
hydrothermal vent species as opposed to cold-seep species and those that occupy O2 minimum
zones where hypoxic conditions are more stable.
In general, rates of O2 uptake in deep-sea species at low Po2 levels are similar to those of related
shallow water species at higher Po2 values (Seibel and Drazen 2007). A  reduction in metabolic
rate with depth in pelagic species is thought to be associated with a reduction in the use of visual
predation and the requirements for high activity rates (Childress and Seibel 1998). Metabolic rates
in benthic decapods and amphipods do not change with depth when values were standardized for
temperature and body size. Therefore, it appears that neither pressure nor hypoxia has an effect on
metabolic rates in deep-sea benthic crustaceans (Childress and Seibel 1998). To date, physiological
studies have shown that metabolic rates are maintained during hypoxia in deep-sea crustaceans
via a series of morphological and respiratory adaptations. It appears that only those species that
frequently encounter hot, anoxic conditions show morphological differences in their branchial
chambers. Both the vent shrimp Rimicaris exoculata and the vent crab Bythograea thermydron show
 Responses to Environmental Stresses 333

a significant increase in scaphognathite surface area when compared with related littoral species
(Decelle et  al. 2010). Presumably, this morphological adaptation serves to improve water flow
through the branchial chambers in order to increase O2 supply to the gills. Gill surface areas in both
vent species are also significantly higher due to a doubling of the number of gill lamellae. Despite
the increase in gill surface area observed in R. exoculata, the branchial chambers are enlarged to
contain chemoautotrophic bacteria that oxidize sulfur from the hydrothermal vents to fix carbon
and provide a direct food source for the shrimps. This is a highly successful arrangement because
R. exoculata can live in very large swarms at densities of 1,000–3,000 individuals m–2 (Cottin et al.
2010). Impairment of respiratory gas exchange by the bacteria is avoided because they are restricted
to distinct sites within the branchial chambers. Each branchial chamber is divided into three parts,
with the bacteria housed in two areas, one with and one without mineral deposits of iron oxides
(Schmidt et al. 2008). The gills occupy the third part of the branchial chamber and remain free of
both bacteria and mineral formations. In contrast, scaphognathite and gill surface areas in those
species with access to colder more oxygenated waters in the vicinity of hydrothermal vents are
similar to those in littoral species (Decelle et al. 2010). None of the deep-sea species examined by
Decelle et al. (2010) showed a reduction in diffusion distances across the gills. Overall deep-sea
crustaceans have a good capacity for anaerobic respiration that enables them to survive bouts of
severe hypoxia and/or anoxia (Hourdez and Lallier 2007).
The respiratory pigments, hemocyanin or Hb, of deep-sea crustaceans are characterized by
a high affinity for O2 and a large Bohr effect (Hourdez and Lallier 2007). These characteristics
facilitate O2 loading at the gill and O2 release at the tissues. Lactate increases the O2 affinity of
hemocyanin in some species but not others, such as the vent-chimney crab Cyanagraea praedator
(Chausson et al. 2001), although thiosulphate increases hemocyanin O2 affinity in the vent crab
B. thermydron (Vetter et al. 1987). Interestingly, temperature has no or little effect on the O2 affin-
ity of hemocyanins in the vent crustaceans sampled to date (Lallier and Truchot 1997, Chausson
et al. 2001). This insensitivity to temperature may represent an adaptation to living in the highly
variable temperatures of hydrothermal vent systems. Hb concentrations in the two deep-sea vent
copepods studied to date indicate that concentrations are relatively low. It appears that Hb serves
to facilitate O2 uptake during hypoxia because its O2 affinity is so high, as discussed by Hourdez
and Lallier (2007).

Burrowing Crustaceans

A number of aquatic crustaceans burrow into the substratum in order to shelter from extreme
environmental perturbations and to protect themselves from predators (Taylor and Atkinson 1991,
James et al. 2005). Species either bury themselves into the sediment for short-term concealment or
construct semipermanent burrows within the sediment that they rarely leave unless they have to
move to feed and to breed. Burying behavior occurs in many Brachyura but also in anomurans and
has been observed in reptant decapods such as Astacidea and Thalassinidea, as well as in natant
decapods such as the Alpheidae (Taylor and Atkinson 1991, James et al. 2005).
Burrowing aquatic decapods can encounter very severe hypoxia or even anoxia in their burrows
on a regular basis, especially species that inhabit tidal mud flats where levels of hydrogen sulphide
may be elevated. In burrows inhabited by two species of upogebiid mud-shrimp (Thalassinidea),
Po2 values varied between 10.7 and 13.3 kPa at the burrow mouth but dropped to 1.3–6 kPa in the
deeper, more poorly irrigated parts (Astall et al. 1997). The first response of many burrowing spe-
cies is to increase the rates of beating of their pleopods that are used to irrigate the burrows and
to increase the time spent irrigating (Taylor and Atkinson 1991). However, if Po2 of the burrow
water falls below Pcrit (Table 10.1), pleopod beating will cease and the animal will either leave the
burrow to find oxygenated water, as observed in N. norvegicus and burrowing crayfish (Hagerman
334 Nia M. Whiteley and Edwin (Ted) W. Taylor

and Uglow 1985, McMahon 2001), or it will tolerate anoxia by resorting to anaerobic metabolism
( James et al. 2005, Holman and Hand 2009).
In general, burrowing aquatic decapod crustaceans are highly tolerant of hypoxia, although
juveniles and larger adults are more sensitive (Taylor and Atkinson 1991, Eriksson and Baden 1997,
Holman and Hand 2009). Burrowing crustaceans show the same hypoxic response as nonburrow-
ing species: ventilation rates increase and heart rate remains unchanged or decreases but fails to
show bradycardia during severe hypoxia/anoxia (Astall et al. 1997, James et al. 2005). Burrowing
decapods such as callianassid species, Upogebia mud-shrimps and N. norvegicus are able to main-
tain rates of O2 uptake down to relatively low critical Po2 levels during progressive hypoxia (Table
10.1; see, e.g., Hagerman and Uglow 1985, Eriksson and Baden 1997). They achieve this by possess-
ing respiratory pigments with higher O2 affinities than those found in other aquatic decapod spe-
cies and, in burrowing crayfish at least, by having higher O2 carrying capacities (Taylor et al. 2000,
McMahon 2001). An increase in hemocyanin O2 affinity facilitates O2 uptake at the gills despite the
presence of relatively low circulating Po2 levels.
In those species that can tolerate anoxia, such as the mud-shrimp Calocaris macandreae (lethal
time for 50% mortality, LT50 = 43 h at 10°C) and the ghost shrimp Lepidophthalmus louisianensis
(LT50 = 64–113 h at 25°C), animals accumulated L-lactate as the metabolic end product (Anderson
et al. 1994, Holman and Hand 2009). Recovery from anoxia was slow in C. macandreae, and anaer-
obic metabolism was only utilized in extreme cases when burrow Po2 levels dropped below 0.9
kPa (Anderson et al. 1994). Moreover, it appears that metabolic depression, a strategy employed
to reduce O2 demand during deficiencies in O2 supply, is delayed in the ghost shrimp for up to 48
h of anoxia (Holman and Hand 2009). ATP production between 6 and 12 h anoxia was sustained
up to 55–77% of the aerobic rate from lactate and arginine phosphate metabolism, which could
help to explain why this species is so tolerant of anoxia. Alternatively, those species with access to
the air–water interface at the burrow openings, such as species occupying intertidal mud flats, will
utilize air as a source of O2. A similar response has been observed in burrowing crayfish, which can
undergo total emergence into air for approximately 75% of the hypoxic period (McMahon 2001).
Access to O2 increases circulating Po2 levels, but a concomitant accumulation of hemolymph Pco2
results in a small Bohr shift and subsequent reduction in hemocyanin O2 affinity. This allows O2
delivery at the tissues despite the increase in circulating Po2 (McMahon 2001).

Facultative Air Breathing

Some species of crustaceans live routinely at the air–water interface. The shore crab C.  maenas
inhabits the littoral zone along the Atlantic coast of Europe, as well as the Mediterranean and
increasingly the Pacific coast of North America. It can often be found in rock pools that can become
markedly hypoxic at night. Under these circumstances, the crab raises its exhalent openings above
the water surface and, by reversing the normal direction of ventilation, bubbles air through the
water surrounding the gills in the branchial chambers, a behavior termed the emersion response
(Taylor and Butler 1973, Taylor et al. 1973). The O2 partial pressure at which this behavior is elicited
varies with metabolic rate, which is affected by temperature, salinity (Taylor et al. 1977), and the
consumption of food (Robertson et al. 2002). The behavior serves to aerate the water surround-
ing the gills and to raise the O2 content of the arterialized hemolymph (Taylor et al. 1973) while
retaining the aquatic route for the excretion of carbon dioxide. Female crabs carrying eggs direct
the stream of bubbles backward over the egg mass, and they show this specific emersion behavior,
which seemingly relates to the O2 requirements of the developing eggs, at higher O2 levels than do
nonovigerous females (Wheatly 1981). This response is highly temperature dependent because an
increase in acclimation temperature from 15°C to 25°C increased the percentage time spent aerat-
ing the egg mass by the ovigerous females. Over the same temperature range, the Pcrit values for O2
 Responses to Environmental Stresses 335

uptake of the eggs increased from 6.7 to 10.7 kPa (Wheatly 1981). Collectively, these observations
imply that the ovigerous female responds to the O2 requirements of her eggs despite them being in
a mass external to her body.
Many primarily aquatic crustaceans are able to survive relatively long periods out of water. This
ability is exploited by marine littoral species that can feed on the shore between tides and by some
freshwater species that can migrate onto land to feed at night and to migrate between bodies of
water during drought. This behavior can be elicited by increased temperature. When the shore crab
was exposed to a progressive increase in ambient temperature, it moved into air at a mean tempera-
ture of 28°C. In air, it is able to maintain a similar rate of O2 consumption as when it is submerged in
normoxic water despite being reliant on gas exchange over chitin-covered gills (Taylor and Butler
1978, Taylor and Wheatly 1979). The freshwater crayfish can survive in air for up to 72 hours and
routinely leaves water to forage for food or to change habitats. Under experimental conditions, it
can be driven into air from shallow water by progressive hypoxia or increasing temperature (Taylor
and Wheatly 1980). When in air, its gills collapse, resulting in a pronounced reduction in the effec-
tive surface area for gas exchange. As a result, the animal becomes markedly hypoxic and hypercap-
nic (Fig. 10.3A,E). However, an initial respiratory and metabolic acidosis is countered by elevation
of buffer base in the form of bicarbonate ions (Fig. 10.3D). Evidence that these are mobilized from
the calcified exoskeleton was provided by the simultaneous elevation of calcium levels in the hemo-
lymph (not shown in Fig. 10.3). Oxygen supply to the tissues falls to zero, then is restored during
aerial exposure, initially by the reversal of a Bohr shift due to compensation for the acidosis (Taylor
and Wheatly 1981) and subsequently by an increase in the affinity of hemocyanin for O2 resulting
from the accumulation of lactate and calcium ions in the hemolymph (Morris et al. 1986a,b) (Fig.
10.3F). Both protons and lactate are then effectively buffered/sequestered in the calcified exoskel-
eton ( Jackson et al. 2001). Following resubmersion there is a lactate washout into the hemolymph,
likely from the exoskeleton (Fig. 10.3F). So, the exoskeleton buffers protons, releases bicarbonate
and calcium, and sequesters lactate during aerial exposure (Fig. 10.1).
These adaptations to survival out of water are exploited by the commercial fishing industry that
transports live crustaceans long distances in air. The adaptations enabling survival have been stud-
ied in some detail in the lobster Homarus gammarus and are described by means of a pH-bicarbonate
or Davenport diagram (Fig. 10.4; Taylor and Whiteley 1989, Whiteley and Taylor 1989, 1992). The
effects of aerial exposure and recovery have also been described in other commercial species such as
the Norway lobster N. norvegicus (Lund et al. 2009, Albalat et al. 2010) and the South African Cape
lobster Jasus lalandii (Haupt et al. 2006).
The ability of aquatic gill-breathing decapods to survive aerial exposure appears to be related to
the differences in habitat and to the degree of exposure that species normally encounter (Whiteley
1999). Subtidal species such as C.  sapidus (Cameron and Batterton 1978)  and the red rock crab
Cancer productus (deFur et  al. 1983)  bury into the substratum if exposed on rare occasions and
remain quiescent. Species distributed further up the shore can function as facultative air breathers,
as mentioned earlier for C. maenas, and can survive longer periods of aerial exposure.

Terrestrial, Air-breathing Crustaceans

The combination of relatively high temperatures and frequent periods of hypoxia in tropical
habitats such as mangrove swamps likely presented the environmental pressures that led to the
evolution of air breathing in crustaceans (Innes and Taylor 1986). Regular exposure in air is often
associated with a reduction in gill area although some air-breathing crabs may continue to use the
gill surfaces for gas exchange and in particular for the excretion of CO2. They achieve this by hold-
ing a small volume of water in the branchial chambers while in air, and they flick this water over
the gill surfaces. This reservoir of water is used as a sink for CO2 and ammonia (Wood and Randall
336 Nia M. Whiteley and Edwin (Ted) W. Taylor

A 10 B 0.7
0.6
8
0.5

Cao2 (mmol L–1)

Pao2 (kPa) 6 0.4

4 0.3
0.2
2
0.1
0 0.0
0 10 20 30 40 0 10 20 30 40

C 8.0 D 16

7.9
14

[HCO–3 ] (mmol L–1)

7.8
12
7.7
pHa

7.6 10
7.5
8
7.4
7.3 6
0 10 20 30 40 0 10 20 30 40
E 1.8 F 10
1.6
8
1.4
Lactate (mmol L–1)
Paco2 (kPa)

1.2 6
1.0
0.8 4
0.6
2
0.4
0.2 0
0 10 20 30 40 0 10 20 30 40
Time (h) Time (h)

Fig. 10.3.
Changes in postbranchial hemolymph oxygen and acid–base variables during 24 h of exposure to air (open
circles) and subsequent recovery in aerated water (filled circles) in the freshwater crayfish Austropotamobius
pallipes. Values are (A) oxygen partial pressure (Pao2), (B) oxygen content (Cao2), (C) pH (pHa), (D) bicar-
bonate concentration ([HCO3–]), (E)  CO2 partial pressure (Paco2), and (F)  lactate concentration. Values
taken from Taylor and Wheatly (1981).

1981). This adaptation ties them to a local supply of water to refresh their reservoir when it becomes
saturated with CO2 or ammonia, so that they cannot stray far from the margins of the sea or from
their water-filled burrows. Many of these land crabs continue to release larvae into the plankton,
which is an additional factor tying them to the seashore. Oxygen is typically obtained by diffu-
sion over a lung-like structure elaborated from the wall of the branchial chamber; this structure is
characterized by an increased surface area and a thin cuticle, resulting in short diffusion distances
(Greenaway and Taylor 1976, Taylor and Taylor 1992). Thus, these structures satisfy the require-
ments of Krogh’s law of diffusion, and land crabs can be characterized as maintaining O2 levels of
between 6.66 and 13.33 kPa and CO2 levels below 1.33 kPa by the combined use of gills and lungs
(Fig. 10.5).
 Responses to Environmental Stresses 337

18
14hA
16

14
[HCO–3] (mmol L–1)

12 3hAD
3hA 0.5hR

10 S

3hR
6

7.3 7.5 7.7 7.9

pH

Fig. 10.4.
A pH-bicarbonate (Davenport) diagram of the changes in acid-base variables in the postbranchial hemolymph
during 14 h of exposure in air and subsequent recovery in aerated seawater in the lobster Homarus gammarus.
On exposure in air for 3 h, the submerged animal (S) showed an increase in Paco2 along the nonbicarbonate
buffer line, accompanied by a mild acidosis when undisturbed (3hA), but a much greater acidosis when dis-
turbed in air (3hAD). After 14 h in air (14hA), pH was restored to submerged levels by elevation of bicarbonate
at constant Paco2. Resubmergence resulted in a recovery alkalosis (0.5hR and 3hR). The ✰ symbol denotes
animals sampled on arrival at market following transport in air. Values taken from Taylor and Whiteley (1989)
and Whiteley and Taylor (1992).

A few species are more independent of water, living for periods remote from a source of stand-
ing water. Preeminent among this group is the Trinidadian mountain crab Pseudothelphusa germani.
This South American crab lives in a tropical rainforest and never returns to the sea to breed. During
the dry season, the female aestivates in a burrow, holding a small batch of eggs that she releases
as fully formed crabs into freshwater streams at the onset of the rainy season. She then molts and
joins the males in a period of active feeding and mating. During this period, both sexes are active
air breathers that can, however, utilize freshwater as a sink for CO2 and ammonia by irrigating the
gills, using the scaphognathites. At the onset of the dry season, both sexes retreat back into their
burrows on the banks of streams that dry up and deprive them of access to free water. The animals
then rely on respiratory gas exchange with air over their elaborately formed lungs. These are dif-
fuse structures that are invaginated into the walls of the branchial chambers and present a large
surface area of thin epithelium to a stream of deoxygenated hemolymph that perfuses large sinuses
(Innes and Taylor 1986, Taylor and Innes 1988, Taylor and Taylor 1992). Use of these invaginated
lungs can generate very high O2 partial pressures (up to 20 kPa) in arterialized hemolymph (Fig.
10.5). These O2 levels seem disproportionate in relation to the binding properties of hemocyanin
because the respiratory pigment will be saturated at lower O2 levels. The answer to this apparent
paradox lies in the function of the lungs in excreting CO2 in the absence of an aquatic route. When
the respiratory gas exchange properties of the gills and lungs of a range of animals are plotted as
a CO2/O2 diagram (Fig. 10.5), it shows that the mountain crab is effectively blowing off CO2 by
 20
18
8
16
X
14 11 9 16
13 15
12 14
1 6
10

Po2 (kPa)
8
6 4 12
10
4 7
3 2
2
5
0
0 Y 1 2 3 4 5 6

12 15
16
10
Cao2 (mmol L–1)

8
14
6
13
4
11 12
9 8
2
3 4 7
6
0 1 10 2,5

0 1 2 3 4 5 6

35
14
30 13
16
[HCO–3] (m mol L–1)

25 15
20 2 8
12
6
15 5
7
10 11 4
9,10
5 1 3
0
0 1 2 3 4 5 6
Pco2(kPa)

Fig. 10.5.
Comparison of the relationships between crustacean hemolymph and vertebrate blood values of: oxygen par-
tial pressure (Po2), oxygen content (Cao2), and bicarbonate concentration ([HCO3–]), plotted against carbon
dioxide partial pressure. The two oblique lines on the plot of Po2 against Pco2 labeled X and Y are the respira-
tory gas exchange (R) lines for a ratio of 1 in air breathers and water breathers, respectively (see Rahn 1966).
All of the crustaceans, whether in water or air (numbered 1–10), have CO2 partial pressures below 1.33 kPa
(10 mm Hg), indicated by the vertical broken line. This relates to the low nonbicarbonate buffering capacity
of their hemolymph that reflects its low protein content, which in turn determines its low oxygen content.
Air-breathing vertebrates (numbered 12–16) have elevated CO2 partial pressures and oxygen contents in the
blood that relate to their increased blood protein levels, with hemoglobin packaged into red cells. Bicarbonate
concentrations are elevated in air-breathing crustaceans to about the level shown by lungfish but not as high
as the committed air breathers among the vertebrates. These relationships are further discussed in the text.
The numbered points represent shore crab, Carcinus maenas: 1, submerged in seawater; 2, in air; 3, in shallow
seawater with access to air; crayfish, Austropotamobius pallipes: 4, submerged in freshwater; 5, in air; land crab,
 Responses to Environmental Stresses 339

apparent hyperventilation at a respiratory exchange ratio of unity (R = 1). In fact, its rate of ventila-
tion is very low, being generated by small, slow changes in pressure in the branchial chambers, in
order to reduce respiratory water loss. This ventilation rate, however, is high relative to the flow of
hemolymph, and this, coupled with a high diffusion coefficient over the lung, results in this highly
effective exchange of respiratory gases. The primary role of the lung under these circumstances is
clearly to reduce levels of CO2 in the hemolymph below a critical level of 1.33 kPa in order to main-
tain acid–base balance (Innes and Taylor 1986, Taylor and Innes 1988). In comparison to vertebrate
blood with its red cells, the hemocyanin level in crustacean hemolymph is limited by the colloid
osmotic pressure it exerts (Mangum and Johansen 1975). This represents a potential limit on its O2
carrying capacity and also on its buffering capacity against the accumulation of CO2 that, in most
land crabs, is countered by retaining the aquatic route for CO2 excretion but in the mountain crab is
achieved by exchange of respiratory gases over its highly effective lung (Fig. 10.5).

TEMPERATURE

Animals in general live within a biological temperature range set by the freezing point of water and
the upper thermal limits for protein stability, but, within these limits, species vary according to their
thermal histories. The body temperatures of ectotherms including crustaceans vary with those of
their environment. However, thermal ranges differ considerably and reflect evolutionary adaption
to particular thermal niches. Those species that inhabit relatively constant temperatures, such as
polar and tropical marine species as well as deep-sea and cave-dwelling species, are stenothermal and
sensitive to temperature change. In general, those that inhabit more variable thermal environments,
such as freshwater and shallow coastal species, are eurythermal and are less sensitive to temperature
change. Furthermore, the latter can vary within genetically fixed limits over temporal and spatial
scales (Hochachka and Somero 2002). Temperature has a profound effect on ectotherms at all levels
of biological organization from influencing biological rate functions to influencing the biogeographi-
cal distribution patterns of a species. Although crustaceans are unable to control body temperatures
independently of the external environment, they can show a number of behavioral responses to
avoid temperature extremes. Many can also acclimate to temperature change in the laboratory and
acclimatize in the natural environment to simultaneous changes in multiple environmental variables.
Ecophysiological responses to temperature change have been studied in crustaceans since the early
1950s, and there is a current interest in the physiological characteristics of marine species distrib-
uted along natural thermal gradients, such as those that occur with latitude and vertical distance up
the shore, because of the growing concern over the effects of increasing sea surface temperatures
occurring as a result of climate change. Physiological responses can indicate thermal tolerances, thus
enabling us to understand factors affecting the survival, distribution, and abundance of marine inver-
tebrates (including crustaceans) and predict how they will respond to increasing temperatures and
be able to adjust their thermal tolerances to survive climate change (Stillman 2003, Somero 2010).

Thermal Limits

The degree of eurythermy shown by ectotherms is ultimately affected by physiological responses

at the level of the whole organism (Pörtner 2002). Early experiments on the spiny spider crab Maja
squinado showed that thermal tolerances were influenced by limitations in O2 supply (Federich and

Fig. 10.5. (Continued)
Cardisoma carnifex: 6, in air; 7, in water; Trinidad mountain crab, Pseudothelphusa germani: 8, in air; 9, in shal-
low water with access to air; 10, submerged in freshwater; 11, fish, Scyliorhinus canicula; 12, lungfish, Protopterus;
13, amphibian, Bufo marinus; 14, reptile, Pseudemys; 15, bird, Gallus; 16, mammal, Homo sapiens.
340 Nia M. Whiteley and Edwin (Ted) W. Taylor

Pörtner 2000). Measurements of heart and ventilation rate between 0°C and 40°C showed that
both rate functions increased with temperature to compensate for the rise in O2 demand. Above
the “pejus temperature,” which is the early limit to temperature tolerance, both heart and ventila-
tion rate stabilized, and arterial Po2 started to decrease even though the crabs were in fully aerated
seawater. As temperatures reached the upper critical limit, systemic hypoxia preceded anaerobic
metabolism as O2 demand outstripped O2 supply. At the lower limit of the temperature range, both
rate processes decreased to such an extent that O2 uptake and transport was insufficient. This con-
cept of O2 and capacity limitation demonstrates that optimal aerobic performance in M. squinado is
only maintained between 6°C and 16°C (Federich and Pörtner 2000). Above and below these ther-
mal limits, performance decreases to unsustainable levels. Thermal limits differ between species
and populations and can be influenced by concomitant changes in another environmental variable.
The simultaneous exposure of two crab species, C. pagurus and Hyas araneus, to hypercapnia plus
an increase in temperature reduced the upper thermal limits and increased mortality rates in both
species (Metzger et al. 2007, Walther et al. 2009).

Physiological Responses to Thermal Gradients in the Littoral Zone

Differences in thermal sensitivities of crustacean species distributed along a vertical environmental

gradient in the intertidal zone have been attributed to physiological variation with height on the
shore (Somero 2002). Thermal adaptations on the shore are necessary because, during emersion,
organisms are no longer under the influence of the buffering effects of the seawater but are exposed
to higher air temperatures, solar insolation (radiation), and changes in humidity and wind speed. As
a result, there is considerable spatial and temporal variation in temperatures on the shore, further
influenced by the timing of low tide, climatic conditions, aspect, cloud cover, and occurrence of
microclimates (reviewed by Helmuth et al. 2006). This can result in rapidly fluctuating and often
extreme temperatures, particularly in sessile intertidal crustaceans such as barnacles, and especially
in those species distributed higher up on the shore. Whereas sessile species are unable to avoid
thermal extremes, mobile intertidal crustaceans, such as intertidal decapods, isopods, and amphi-
pods, can escape to favorable microclimates by hiding in crevices or under stones and seaweed
where temperatures are more moderate. Some also rely on behavioral thermoregulation, such as
the semiterrestrial isopod Ligia oceanica, which uses evaporative cooling to keep body temperatures
several degrees lower than ground temperatures (Edney 1953). This behavioral response, however,
is limited because L. oceanica needs to return to humid conditions at intervals to prevent death by
desiccation.
Physiological adaptation to increased thermal stress is of key importance to the survival of crus-
tacean species on the shore. To date, studies are limited to observations on barnacles and porcelain
crabs. High shore barnacles are able to keep their filter-feeding cirri beating at markedly higher
temperatures than are individuals from further down the beach. Thermal limits for the circulatory
system also vary. Two sympatric species of the porcelain crab (Petrolisthes) living in different verti-
cal zones of the intertidal were examined by Stillman and Somero (1996). Upper and lower thermal
limits were compared over the range of temperatures normally encountered in the field. They varied
with the thermal conditions in the intertidal habitats of crabs from different biogeographic regions
(Stillman and Somero 1996). These thermal tolerances are relatively plastic, although adjustments
in LT50 were lower in the upper intertidal species compared with low intertidal and subtidal spe-
cies (Stillman 2002). There was a strong positive correlation between LT50 and maximum habitat
temperature that varied with vertical position on the shore. Arrhenius break temperatures (ABT)
of heart rate varied with the upper shore species, Petrolisthes cinctipes, characterized by an ABT
of 31.5°C and the low intertidal/subtidal species, Petrolisthes eriomerus, characterized by an ABT
of 26.6°C. The upper shore species, which normally experiences a maximum habitat temperature
 Responses to Environmental Stresses 341

of 33°C, is more eurythermal and is remarkably tolerant to a wide range of temperatures. P.  eri-
omerus could not survive at the temperatures experienced by the upper shore species. The upper
thermal limits of heart function (CTmax) after temperature acclimation showed a similar response
because CTmax decreased linearly among species in inverse proportion to maximal habitat tempera-
ture (Stillman 2003). In P. cinctipes, heart failure was one of the proximate causes of heat death in its
native habitat (Stillman and Somero 1996). It is possible that thermal limits were set by differences
in the properties of the cardiac muscle or differing sensitivities in the nerves innervating the heart.
It was concluded that upper shore species with the greatest tolerance to high temperatures are the
most threatened by increases in habitat temperature because they are already living at temperatures
close to their LT50s and so have reduced ability to adjust their thermal limits (Stillman 2002).
Cold temperatures can also be a problem in the intertidal zone, but the physiological effects are
rarely mentioned. Coastal areas at high latitudes are subject to freezing conditions in the winter
months for part of the tidal cycle. Intertidal invertebrates in such areas are freeze tolerant because
they cannot avoid the numerous ice crystals present in the environment and have little time to pro-
tect themselves from ice formation. The barnacle Semibalanus balanoides can tolerate freezing and
thawing in the extracellular compartment on a regular basis (Murphy 1983). Freeze tolerance and
lower lethal temperatures also vary with season because summer S. balanoides are unable to survive
at −6°C when about 40–45% body water freezes. In winter, death in barnacles occurs at −18.3°C
when more than 80% of the body water freezes. Other species, such as the gammarid amphi-
pod Gammarus duebeni, can avoid freezing by inhabiting hypersaline rock pools where sea water
remains unfrozen even when temperatures reach −8°C (Davenport 1992). Physiological responses
to cold acclimatization involve classic strategies, such as changes in lipid composition in the mem-
branes and changes in enzyme activities. For example, crabs undergo increases in membrane fluid-
ity due to a decrease in cholesterol levels within the membrane, with the more stenothermal crab
C. pagurus having slightly more fluid membranes and lower cholesterol/phospholipid ratios than
the eurythermal species C. maenas (Cuculescu et al. 1995).

Physiological Responses to Latitudinal Thermal Gradients

Metabolic Rate

Temperature typically has a marked effect on physiological and biochemical rate processes in
ectothermic animals, causing a two- to threefold increase in rate over a 10°C rise in temperature.
Nevertheless, responses to temperature vary tremendously because many species are able to com-
pensate biological rate processes such as metabolic rate. Comparisons within species demonstrate
that crustaceans from latitudinally separate populations usually compensate for environmental
temperature gradients. The overall effect is the maintenance of similar rates of O2 uptake when
measured at the habitat temperatures experienced by the different populations (e.g., metabolic rates
in species such as the fiddler crab Uca rapax and the prawn Pandalus montagui are higher in north-
ern versus southern populations, when measured at the same temperature; Fox and Wingfield 1937,
Vernberg 1962). A similar response has been observed in northern krill M. norvegica from three geo-
graphically separate populations (Saborowski et al. 2002) and in boreal/temperate and temperate
gammarid amphipod species from different latitudes (Rastrick and Whiteley 2011). However, lack
of any metabolic compensation with latitude has been observed in the porcelain crab Petrolisthes
granulosus sampled from three sites along the Chilean coast (20–36°S; Monaco et al. 2010) and in the
subarctic/boreal gammarid species at high polar latitudes (Rastrick and Whiteley 2011). Metabolic
compensation was confined to the more eurythermal species that may benefit from the mainte-
nance of a consistency in biological rate processes across latitudes. In those species where meta-
bolic compensation does take place with latitude, there is some evidence of local adaptation. In Uca
342 Nia M. Whiteley and Edwin (Ted) W. Taylor

pugilator, for instance, individuals from three latitudinal populations were hatched and reared under
identical laboratory conditions (Vernberg and Costlow 1966). Metabolic rates were higher in cold
water populations from North Carolina and Massachusetts compared with warm water populations
from Florida. The persistence of differences in metabolic rate across generations suggests they are
genetically fixed, resulting in differences in acclimatory ability with latitude. Moreover, a southern
population of the copepod Euterpina acutifrons from Brazil (24°S) reduced metabolic rate at high
acclimation temperatures more effectively than did a population from South Carolina (35°N), thus
reflecting better adaptation to mean habitat temperatures (Vernberg and Moreira 1974).
Species-related comparisons of standard metabolic rate indicate that marine crustaceans do not
compensate metabolic rates in order to live in the extreme cold, as previously suggested by the
concept of metabolic cold adaptation. Antarctic isopod crustaceans do not upregulate their meta-
bolic rates in the cold and stable conditions of the Southern Ocean (Luxmoore 1984, Chapelle and
Peck 1995, Whiteley et al. 1996). Instead, they maintain relatively low rates of O2 uptake resulting
in low maintenance costs, which is a significant advantage in the cold because food resources may
be limiting (Clarke 1993, Pörtner et al. 2005). Arctic amphipods also maintain relatively low rates of
O2 consumption because rates were lower in the subarctic/boreal species Gammarus oceanicus and
Gammarus setosus compared with the temperate species G. locusta (Rastrick and Whiteley 2011). It
has been suggested that lower metabolic rates and therefore O2 demand, as well as the increase in O2
solubility at low temperatures, will allow for larger body sizes (a feature of many Antarctic marine
ectotherms) because sufficient O2 uptake from O2-rich waters is possible despite the reductions in
O2 gradients and diffusion distances that accompany an increase in body size (Chapelle and Peck
1999, Pörtner et al. 2007). This suggestion ignores the role of the circulatory system.

Protein Synthesis Rates

Protein synthesis rates are reported to be closely related to rates of O2 uptake mainly because protein
synthesis is energetically costly and can account for up to 42% of basal metabolic rate (Houlihan
et al. 1995, Whiteley and Fraser 2009). Rates of protein synthesis generally increase with tempera-
ture, but values are influenced by the rate of feeding, which also increases with temperature. Under
these conditions, whole-animal rates of protein synthesis in a range of crustacean species collected
from different thermal regimes and acclimated to a constant temperature for 4 weeks showed an
exponential increase with temperature (Fig. 10.6). In all cases, animals were fed ad libitum so that
protein synthesis rates were not limited by lack of food. All values were also standardized for differ-
ences in body mass, which is also known to influence protein synthesis rates (Whiteley and Fraser
2009). Figure 10.6 shows that the lowest rates of protein synthesis were observed in the Antarctic
isopod Glyptonotus antarcticus. The relatively low rate of protein synthesis plus the observation
that Antarctic invertebrates are characterized by a low protein retention efficiency, helps to explain
the lower than average growth rates observed in Antarctic versus temperate and tropical species
(Pörtner et al. 2007).
Despite these broad species-related differences in protein synthesis rates and presumably
growth potential, there are also examples of compensatory responses in which protein synthesis
rates vary independently from temperature. The first example of this comes from a study on the
semiterrestrial, intertidal isopod L.  oceanica (Whiteley and Faulkner 2005). L.  oceanica inhabits
rocky shores at the high water mark of spring tides where it experiences a wide range of tempera-
tures. In summer, the surface of rocks facing direct sunlight, even in temperate regions, can rise
to over 40°C, but L. oceanica inhabits crevices and the undersurface of loose rock, which provide
shade and increased humidity (Edney 1953). In winter, temperatures drop to around 5°C. In accli-
matized animals straight from the shore, whole-animal rates of protein synthesis are maintained at a
relatively low but constant level despite acute changes in temperature. Summer and winter animals
 Responses to Environmental Stresses 343

Loge fractional rates of protein synthesis (% day–1) 13

2

11 12

1 6,8

9
7
0 10
3
1

4,5
–1
2

–2
0 5 10 15 20 25 30 35
Temperature (°C)

Fig. 10.6.
Relationship between fractional whole-animal rates of protein synthesis (ks) and acclimation temperature in
crustaceans from different thermal habitats. All protein synthesis values scaled to a standard body mass of 1g
wet mass using a weight exponent of −0.2. Numbers represent the giant Antarctic isopod Gyptonotus antarcticus
(1–3), the temperate isopod Idotea rescata (4 & 10), the Baltic isopod Saduria entomon (5 & 9), boreal/tem-
perate amphipod Gammarus duebeni from two different populations (6 & 7), the subarctic/boreal amphipod
Gammarus oceanicus (8), and the tropical prawn Macrobrachium rosenbergii (11–13). In all cases, the animals
were acclimated to their respective experimental temperatures for 1 month and fed ab libitum. The fitted regres-
sion line is described by the exponential function: Log ks = log 0.415 + 0.085.log temp, r2= 0.874. Data taken
from Whiteley et al. (1996) for G. antarcticus and I. rescata; Robertson et al. (2001a, 2001b) for G. antarcticus and
S. entomon; Rastrick and Whiteley (2013) for the gammarid amphipods and for M. rosenbergii.

showed a similar response. A relatively low and constant rate of protein synthesis was also observed
in the boreal/temperate amphipod species Gammarus duebeni (Rastrick and Whiteley 2013). This
high shore species occupies areas under the influence of freshwater runoff and is highly tolerant
of environmental change (Bulnheim 1979). Whole-animal rates of protein synthesis did not dif-
fer between acclimatized individuals from populations at different latitudes experiencing different
thermal regimes. The abilities of both L. oceanica and G. duebeni to maintain relatively low rates of
protein synthesis could be an adaptation for life in a highly changeable environment. It is possible
that this response represents a strategy for enhancing survival by reducing the energetic costs asso-
ciated with the replacement, repair, and synthesis of proteins.

Specific Dynamic Action

In addition to its effects on rates of metabolism and protein synthesis in crustaceans, temperature
can also influence duration and timing of the rise in O2 uptake rate after feeding or the specific
dynamic action (SDA). Comparisons between species from different thermal habitats revealed that
the SDA response in the Antarctic isopod G. antarcticus is considerably slower than in temperate
isopod species, lasting for 8 days instead of 10 h and taking 3 days instead of several hours to reach
peak values (Whiteley et al. 2001a). Differences in SDA responses are largely explained by differ-
ences in temperature, as demonstrated by acclimating the shore crab C. maenas to a range of tem-
peratures. A reduction in acclimation temperature from 22°C to 7°C decreased the magnitude of
the SDA response in C. maenas by increasing SDA duration but decreasing the SDA factorial scope
344 Nia M. Whiteley and Edwin (Ted) W. Taylor

(Robertson et al. 2002). Further comparisons between cold water isopod species such as G. ant-
arcticus and the Baltic isopod Saduria entomon, which inhabits the Baltic and Bothnia Seas as well as
the Arctic Ocean, revealed marked differences in SDA response. G. antarcticus had a lower absolute
rate of O2 uptake at peak SDA, but the SDA duration was extended when compared to S.  ento-
mon at the same acclimation temperature (Robertson et al. 2001a,b). Whole-animal rates of protein
synthesis increased in proportion to the postprandial increase in O2 uptake rate in both species.
Estimates on the costs of protein synthesis after a meal gave similar values in both species, despite
their differences in SDA response and thermal habitat (Whiteley et al. 2001a). The extended SDA
duration observed in the Antarctic isopod species when compared to either S. entomon or C. maenas
held at the same temperature is thought to be caused by the extreme stenothermy of the Antarctic
environment, where the longer SDA duration reflects lower aerobic scopes and a marked sensitivity
to temperature change (Whiteley et al. 2001a). Overall, the rates at which Antarctic species process
their food is much slower, further contributing to the observed reductions in growth rate.

Seasonal Temperature Change

A long-term study of the effects of seasonal temperature variation between 1°C and 21oC on a popula-
tion of the freshwater crayfish revealed that both water and hemolymph pH varied with temperature
during the spring warming. However, following the summer molt, the hemolymph pH increased
and remained virtually unchanged as water temperatures cooled toward winter. This relative inde-
pendence of pH with variation in temperature was explained by a complex of seasonal changes in
CO2 solubility, bicarbonate concentrations, and Pco2, as well as by measured values for the carbonic
acid dissociation constant, pK1 (Whiteley and Taylor 1993). This apparent regulation of hemolymph
pH was accompanied by active regulation of intracellular pH (pHi; Fig. 10.7). Tissues of functional
importance in the winter months, such as the abdominal muscles responsible for the escape response,
had a pHi that varied inversely with body temperature in order to maintain the integrity of protein
function (alphastat hypothesis; Reeves 1972). The tissues that are less active in the winter, when
the crayfish do not feed, such as claw muscle and the hepatopancreas, maintained a constant pHi as
temperature fell. As a result, these tissues became relatively acidotic, which may serve to depress the
activity of metabolic enzymes during the cold winter months (Whiteley et al. 1995). Under labora-
tory conditions, pHi normally varies with temperature according to the alphastat hypothesis, but
different tissues can maintain very different pH levels (Wheatly and Henry 1992).

ACIDIFICATION OF AQUATIC ENVIRONMENTS

Acidification of Freshwater Environments

Acidification of freshwater ecosystems can occur naturally or as a result of anthropogenic activi-

ties (Dangles et al. 2004, Weber and Pirow 2009). In contrast to naturally occurring acidification,
the anthropogenic input of acidified material into freshwater lakes and rivers is a recent event that
began in Europe and North America in the middle of the past century and peaked in the 1970s and
1980s. The major causes of anthropogenic acidification include “acid rain” and the effect of acid
mine drainage. Sulfuric and nitric acids can reduce the pH of uncontaminated rainwater in equi-
librium with atmospheric CO2 from pH 5.6 to less than 4.3, whereas freshwater areas influenced by
acid mine drainage can have pH values of less than 3.0 (Deneke 2000). Their impact is dependent
on the capacity of the surrounding geology and soils to neutralize acids, with granite and gneiss
regions being the most vulnerable.
 Responses to Environmental Stresses 345

8.4

8.0

7.6
Tissue intracellar pH

7.2

6.8

6.4

6.0
0 2 4 6 8 10 12 14
Temperature (°C)

Fig. 10.7.
Intracellular pH (pHi) in the heart (▲), claw muscle (■), abdominal muscle (□), and hepatopancreas (♦)
of the freshwater crayfish Austropotamobius pallipes caught in the winter and acclimated to 1°C, 5°C, and 12°C.
Values are means ± SE. Equivalent changes in hemolymph pH (∆) and the changes in pH of pure water or pN
(● and broken line) are also given. There was little change in pHi in the claw muscles and hepatopancreas with
temperature, but heart pHi changed in parallel to the changes in the hemolymph at −0.010 pH units °C–1. The
largest temperature-related change in pHi occurred in the abdominal muscles at −0.025 pH units °C–1. From
Whiteley et al. (1995).

Survival rates of freshwater crayfish declined as pH levels dropped below pH 6.0, but crayfish
showed a remarkable ability to tolerate extremely low pH levels for short periods. For example, 50%
of Procambarus clarkii survived for 4 days at pH 3.0 and for 60 days at pH 4.0, although increased
mortality was observed in Astacus astacus kept for extended periods at pH 5.6 (McMahon and
Stuart 1989). Initial exposure of A. astacus and P. clarkii to freshwater acidification caused a drop
in hemolymph pH and a substantial loss of Na+ and Cl− ions from both the hemolymph and the
tissues due to the large uptake of acid equivalents from the water (McMahon and Stuart 1989,
Jensen and Malte 1990). However, both species were able to recover by increasing ventilation rates
to reduce hemolymph Pco2 levels rather than depending on ion regulation. Nevertheless, crayfish
exposed to acid conditions in the medium term reached a new ionic equilibrium (McMahon and
Stuart 1989). Other species, such as the amphipod Gammarus pulex, were more sensitive to pH
levels of less than 6.0 because they experienced a reduction in hemolymph osmolarity, alterations in
Na+ regulation, and reductions in ventilatory and locomotory activity (Felten et al. 2008). Survival
in Daphnia, which is an important member of freshwater zooplankton, was ensured by improved
tolerance to acid stress despite alterations in acid–base balance, ventilation, and energy metabo-
lism (Weber and Pirow 2009). More specifically, chronic exposure of Daphnia to acidic conditions
(ΔpH of 0.16–0.23 pH units) resulted in a slight hemolymph acidosis and a decrease in [HCO3–]
346 Nia M. Whiteley and Edwin (Ted) W. Taylor

by up to 65%. When external pH was reduced to 6.0, the hemolymph acidosis was accompanied by
an increase in nonbicarbonate buffering capacity, as well as by tachycardia, hyperventilation, and
hypermetabolism (Weber and Pirow 2009). The highest tolerance to acid stress was observed at an
external pH of 5.5, which was attributed to the activation of defense mechanisms that occurred at
the level of transcription.
Alterations in freshwater pH have also been demonstrated to affect larval survival and devel-
opment. An interesting example is provided by the crab Metopaulias depressus, which occupies
the water retained in the leaf axils of large bromeliads (Diesel and Schuh 1993). Although the axil
water provides stable and reliable breeding sites, water pH ranges from 3.8 to 6.8, and it can be
both hypoxic and hypercapnic (Diesel 1992). Maternal manipulation of the environment helps to
improve larval survival by increasing median water pH from 4.8 to 6.8 (Diesel and Schuh 1993).
This is achieved by the removal of dead leaf litter and other organic material, along with the supply
of small shells to increase buffering capacities of the axil water. The exposure of freshwater crusta-
ceans to long-term reductions in water pH of less than 6.0 is known to alter species distribution and
abundance (e.g., France 1993), species richness (Walseng et al. 2003), and community structure
and function (e.g., Dangles et al. 2004). As a result, planktonic crustacean species (cladocerans and
copepods) are currently being used to gauge recovery of freshwater lakes from past anthropogenic
acidification events and to assess the evolutionary reversal of acid tolerance (Walseng et al. 2003).
Freshwater acidification effects remain valid examples of the negative impacts of acidification on
aquatic communities because of the relatively long periods of time required for recovery once pol-
lution controls have been put into place and anthropogenic emissions have been reduced. However,
freshwater communities exposed to natural acidification processes appear to be more tolerant of
reductions in external pH, indicating that communities can adapt to protracted low pH if given
sufficient time (Dangles et al. 2004).

Ocean Acidification

Since preindustrial times, more than a third of the CO2 released into the atmosphere has been
absorbed by the oceans, thus reducing both pH and carbonate ion concentrations in the surface
waters (Feely et al. 2004, Sabine et al. 2004, Fabry et al. 2008). These changes in carbonate chemis-
try are causing some concern, especially as the saturation of the oceans with respect to aragonite, a
soluble form of calcium carbonate required by marine calcifiers for their shells and exoskeletons, is
also decreasing (Caldeira and Wickett 2003, Fabry et al. 2008). If atmospheric Pco2 continues to rise
according to the “business as usual” CO2 emission scenario (Houghton et al. 2001), then the pH of
the oceans is predicted to fall from current pH levels of 8.1 by a further 0.3–0.4 pH units by the end of
the century and by 0.7 pH units by 2300 (Caldeira and Wickett 2003, Feely et al. 2004). The effects of
CO2 emissions in the 21st century may also be strongly delayed. For example, if CO2 emissions were
reduced or stopped by 2100, their effects would still be experienced in 2500 when changes in ocean
pH are predicted to extend from the surface down to a depth of 2,000 m (Frölicher and Joos 2010).
The ability to compensate for the effects of ocean acidification (OA) varies between crustacean
species and is linked to the ability to buffer the resulting changes in Pco2 and pH in the hemolymph
(Whiteley 2011). Strongly iono- and osmoregulating crabs are able to fully compensate for expo-
sure to extremely high CO2 levels (hypercapnia) by elevating hemolymph HCO3− levels to reach
a new acid–base equilibrium (Cameron and Iwama 1987). The majority of the HCO3− ions (93%)
originate from the seawater, but a small percentage comes from internal stores (7%; Cameron 1985).
Species that maintain a higher hemolymph HCO3− are more tolerant of hypercapnia (Pörtner et al.
2004, Melzner et al. 2009), although it appears that [HCO3–] elevation is limited to a threshold
value of approximately 50 mmol l–1 (Cameron and Iwama 1987, Spicer et al. 2007). The inability
to increase [HCO3–] beyond this value is thought to be a compromise between acid–base and ion
 Responses to Environmental Stresses 347

regulation (Cameron and Iwama 1987), in which ion regulation takes priority over acid–base bal-
ance to maintain cell volume control (Whiteley et al. 2001b). When exposed to lower external CO2
levels (i.e., 0.10–0.20 kPa) for 24 h, even poor ion regulators such as the subtidal crabs Necora puber
(Spicer et al. 2007) and C. magister (Pane and Barry 2007) are able to buffer the resulting hemo-
lymph acidosis by elevating [HCO3–]. However, HCO3− buffering was inadequate in N. puber after
16 and 30 days exposure at a Pco2 of 0.20 kPa and after 4–5 days exposure at a Pco2 of 2.0 kPa (Spicer
et al. 2007, Small et al. 2010). In all three cases, the elevation of hemolymph [HCO3–] was limited
below 55 mmol l–1 and led to the development of an internal acidosis. The inability to compensate
an internal acidosis can lead to hypoxemia and death from asphyxiation because the subsequent
increase in [H+] suppresses the O2 affinity of hemocyanin to such an extent that O2 delivery to the
tissues is severely disrupted (Taylor and Whiteley 1989, Whiteley and Taylor 1992). In contrast, the
strongly iono- and osmoregulating prawn species Palaemon elegans and P. serratus fully compen-
sated for the effects of 30 days exposure to a Pco2 of 0.30 kPa (Dissanayake and Ishimatsu 2011).
The ability to compensate for acid–base disturbances during hypercapnia is also related to cir-
culating levels of the nonbicarbonate buffer, hemocyanin. The level of this respiratory pigment
varies among crabs according to routine levels of activity (Watt et al. 1999). Active crabs tend to
have higher hemocyanin levels when compared with slow-moving species. Higher hemocyanin
levels increase nonbicarbonate buffering and O2 carrying capacities to match the higher produc-
tion of metabolic CO2 and to satisfy the higher aerobic requirements. Slow-moving crustaceans
with low hemocyanin levels are therefore less likely to be able to buffer an acidosis resulting from
elevated seawater Pco2. The deep-sea tanner crab Chionoecetes tanneri is unable to compensate for
exposure to a Pco2 of 1.28 kPa for 24 h (Pane and Barry 2007). Its relatively low hemolymph protein
levels, which are mainly hemocyanin, plus the fact that this species is unable to elevate hemolymph
HCO3− levels beyond 3 mmol l–1 (Pane and Barry 2007), means that this species is poorly adapted
for acid–base compensation and is likely to be more sensitive to OA. In contrast, the deep-sea steno-
thermal prawn Pandalus borealis is able to partially compensate for the effects of exposure to a Pco2
of 0.90 kPa by increasing [HCO3–] levels threefold within the first day of exposure up to 15 mmol
l–1 (Hammer 2012). The ability to partially compensate for the resulting extracellular acidosis was
attributed to the higher rates of activity characteristic of this species because it undergoes diel
migrations in order to feed. The diversity of acid–base responses in deep-sea crustaceans to hyper-
capnia further confirms the importance of routine activity levels in determining the compensatory
capacities of crustaceans to OA. Low activity rates are generally characteristic of Antarctic benthic
marine species because of the low and stable temperatures and a lack of resources (Whiteley 2011).
From the few studies carried out to date on the giant Antarctic isopod G. antarcticus, it appears that
polar crustaceans also have very poor hemolymph buffering capacities (Whiteley et al. 1997). The
circulating protein levels in G. antarcticus are up to 7.5 times lower than the values characteristic of
temperate crustaceans. In addition, hemocyanin O2 affinity in G. antarcticus is highly sensitive to
a reduction in pH ( Jokumsen et al. 1981). Both characteristics suggest that OA could potentially
disrupt both acid–base balance and O2 transport in the hemolymph of G. antarcticus and lead to
death by asphyxiation.

FUTURE DIRECTIONS

Many of the experiments described in this chapter have adopted the historical approach to physi-
ological experimentation whereby crustacean species have been exposed to acute changes in single
environmental variables under controlled conditions for relatively short periods of time; their
responses are averaged then subjected to routine statistical analysis. The purpose of these experi-
ments was to explore the physiological mechanisms by which species adjust to environmental
348 Nia M. Whiteley and Edwin (Ted) W. Taylor

variation. More recently, attention has shifted from the mechanisms themselves to the metabolic
costs of short- and long-term adjustments. This approach is of ecological importance because costs
can affect tolerances and therefore the abundance and distribution patterns of populations or spe-
cies (Monaco and Helmuth 2011). In addition, there has been a growing interest in the study of mul-
tiple environmental variables on the ability of individuals to adjust to change. This has come about
in part in response to the increasing interest in climate change, in which environmental variables are
changing simultaneously. Researchers in the field of OA for instance, have realized the importance
of studying the effects of more than one variable, such as OA and either temperature, O2 levels,
or salinity (Whiteley 2011). To date, it has been shown that OA and temperature change have a
synergistic effect (Melzner et al. 2009). There is also a growing interest in exposing crustaceans to
longer term changes in environmental variables (months to years), which raises interesting ques-
tions about the experimental regime and whether experimental temperatures, for example, should
change naturally with the seasons or remain constant. The effect of variable temperatures has also
attracted attention in recent years with early experiments showing that an increase in the ampli-
tude of daily temperature variation can decrease thermal sensitivity and increase metabolic rate in
the terrestrial isopod Porcellio laevis (Folguera et al. 2011). However, it is still unknown how crus-
taceans will be able to cope physiologically with the changes in temperature variability expected
from global climate change. Finally, there is a great deal of interest in the role of adaptation at the
molecular level in determining physiological responses to environmental change. As present, cli-
mate changes are occurring at rates that make it difficult for marine species to adapt; future survival
of populations and their constituent individuals will depend to a large extent on the plasticity avail-
able within the existing genotype (Somero 2010). Physiological experiments are needed to define
the degree of plasticity, whereas multiple generational studies are required to determine the role of
genetic adaptation. Other molecular approaches involve the use of genomic technologies and the
growing availability of DNA sequence data (both genomic and expressed sequence tag) to improve
our understanding of the effects of environmental change on the transcriptome of crustaceans
(Stillman et al. 2008). Such an approach has yielded novel genes and identified genes that were pre-
viously unknown to be involved in temperature acclimatization responses (Tagmount et al. 2010).
Overall, contemporary crustacean physiology needs to consider the responses of a range of organ-
isms living in a range of habitats to more clearly define the processes that constrain physiological
adaption to change. Crustaceans remain a rich source of material for such studies because they
demonstrate the full spectrum of responses, from those that are unable to respond to environmen-
tal change to those that are highly adaptable, as demonstrated in this chapter.

CONCLUSIONS

Crustaceans occupy a range of aquatic and terrestrial habitats where they encounter variable tem-
perature, O2, and pH levels. They manage to survive by changing their behavior and by undergo-
ing physiological and metabolic adjustments. During environmental hypoxia, aquatic crustaceans
move away to find more oxygenated water and, in the case of berried females, spend more time
ventilating their eggs. Adults typically maintain their ventilation rates and cardiac output down to a
critical Po2 value, and they maintain O2 transport in the hemolymph via increases in carrying capac-
ity and O2 affinity of the blood pigment. Early stages of development tend to be more resistant to
hypoxia and anoxia, with eggs tolerating hypoxia for most of embryonic development. Crustaceans
living at depth and in burrows survive hypoxia by increasing aerobic capacity. Burrowing crusta-
ceans can also tolerate anoxia by resorting to anaerobic metabolism. Several species can resort to air
breathing by raising the exhalent openings above the surface of the water and reversing the normal
direction of ventilation to bubble air through the branchial chambers. Some species can also live
 Responses to Environmental Stresses 349

for several days out of water, despite becoming markedly hypoxic, by buffering the accumulating
hemolymph acidosis with HCO3− ions from the exoskeleton and improving O2 transport to the
tissues by increasing hemocyanin O2 affinity. Hypoxia combined with warmer temperatures in the
tropics has probably led to the evolution of air breathing in crustaceans. Terrestrial crabs are charac-
terized by reduced gills that are responsible for the excretion of CO2, and elaborately formed lungs
that exchange O2.
The thermal ranges experienced by crustaceans differ considerably. Thermal limits are deter-
mined at the level of the whole organism by O2 and capacity limitation and are associated with
physiological variability. Along natural thermal gradients, there is evidence of metabolic com-
pensation but only in more eurythermal species. Protein synthesis rates in crustaceans generally
increase with acclimation temperature, but in those species that occupy the high intertidal, pro-
tein synthesis rates can be independent of temperature, which is beneficial in a highly fluctuating
environment. Temperature can also affect the duration and magnitude of the SDA response in
crustaceans, and the relationship between pHi and temperature can be influenced by season.
Freshwater crayfish can survive for 4 days at an environmental pH of 3.0 but suffer from a sub-
stantial loss of Na+ and Cl− ions. Improved tolerance to acid stress is possible. The ability of
crustaceans to survive OA depends on their compensatory capacities. Strong iono- and osmo-
regulators are more likely to survive, although the added effects of elevated temperatures and/or
reduced salinities are unknown.
In summary, aquatic crustaceans respond to environmental change in a number of ways. In
order to avoid the most immediate effects, individuals move away from the problem and show a
range of unusual behaviors, such as the aggregation of both predators and prey to avoid bouts of
hypoxia. Physiological responses further enhance the ability of individuals to survive relatively
short-term changes in either O2, temperature, or pH levels in the surrounding water. Compensatory
responses are observed in a range of crustacean species and are focused on adjustments in respira-
tory physiology, including acid–base homeostasis and ion regulation. Survival depends on the abil-
ity to maintain the most appropriate conditions for protein function. The physiological responses
and consequent survival of aquatic crustaceans to simultaneous changes in environmental pH and
either temperature or O2 over the longer term remain to be studied.

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a burrowing mode of life. Pages 211–234 in A.J. Woakes, M.K. Grieshaber, and C.R. Bridges, editors.
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Series 41. Cambridge University Press, Cambridge.
Taylor, A.C., C M. Astall, and R.J.A. Atkinson. 2000. A comparative study of the oxygen transporting
properties of the haemocyanin of five species of thalassinidean mud-shrimps. Journal of Experimental
Marine Biology and Ecology 244:265–283.
Taylor, E.W. 1965. Respiration in species inhabiting temporary freshwater habitats. Ph.D. thesis, University of
Southampton.
Taylor, E.W. 1981. Some effects of temperature on respiration in decapodan crustaceans. Journal of Thermal
Biology 6:239–248.
Taylor, E.W. 1982. Control and co-ordination of ventilation and circulation in crustaceans: responses to
hypoxia and exercise. Journal of Experimental Biology 100:289–319.
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Taylor, E.W., and P.J. Butler. 1978. Aquatic and aerial respiration in the shore crab, Carcinus maenas L. at 15oC.
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Taylor, E.W., and A.J. Innes. 1988. A functional analysis of the shift from gill to lung breathing during the
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Taylor, E.W., and M.G. Wheatly. 1980. Ventilation, heart rate and respiratory gas-exchange in the crayfish
Austropotamobius pallipes (Lereboullet) submerged in normoxic water and following 3 h exposure in air
at 15°C. Journal of Comparative Physiology 138:67–78.
Taylor, E.W., and M.G. Wheatly. 1981. The effect of long-term aerial exposure on heart rate, ventilation,
respiratory gas exchange and acid-base status in the crayfish, Austropotamobius pallipes. Journal of
Experimental Biology 92:109–124.
Taylor, E.W., and N.M. Whiteley. 1989. Oxygen transport and acid-base balance in the haemolymph of the
lobster, Homarus gammarus, during aerial exposure and resubmersion. Journal of Experimental Biology
144:417–436.
Taylor, E.W., P.J. Butler, and P.J. Sherlock. 1973. The respiratory and cardiovascular changes associated with
the emersion response of Carcinus maenas (L.) during environmental hypoxia, at three different
temperatures. Journal of Comparative Physiology 86:95–115.
Taylor, E.W., P.J. Butler, and A. Al-Wassia. 1977. Some responses of the shore crab, Carcinus maenas L. to
progressive hypoxia at different acclimation temperatures and salinities. Journal of Comparative
Physiology 122:391–402.
Taylor, E.W., N.M. Whiteley, and M.G. Wheatly. 1991. Respiratory gas exchange and the regulation of
acid-base status in decapod crustaceans. Pages 79–106 in A.J. Woakes, M.K. Grieshaber, and C.R.
Bridges, editors. Physiological strategies for gas exchange and metabolism. Society for Experimental
Biology, Seminar Series 41. Cambridge University Press, Cambridge.
Taylor, H.H., and E.W. Taylor. 1992. Gills and lungs: the exchange of gases and ions. Pages 203–293 in
F.W. Harrison and A.G. Humes, editors. Decapod Crustacea. Vol. 10 of Microscopic Anatomy of
Invertebrates. Wiley-Liss, New York.
Terwilliger, N.B., and M. Ryan. 2001. Ontogeny of Crustacean respiratory proteins. Integrative and
Comparative Biology 41:1057–1067.
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Antarctic micronektonic Crustacea as a function of depth of occurrence and season. Marine Ecology
Progress Series 113:207–219.
Towle, D.W., and D. Weihrauch. 2001. Osmoregulation by gills of euryhaline crabs: molecular analysis of
transporters. American Zoologist 41:770–780.
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Carcinus maenas (L.). Respiration Physiology 223:351–360.
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45:35–45.
11
OXYGEN TRANSPORT PROTEINS IN CRUSTACEA:
HEMOCYANIN AND HEMOGLOBIN

Nora B. Terwilliger

Abstract
In crustaceans, oxygen transport proteins hemocyanin and hemoglobin increase oxygen transport
capacity of hemolymph and sustain metabolic needs. Hemocyanins are large, multi-subunit mol-
ecules containing highly conserved copper-based oxygen binding sites; their structure and sub-
unit heterogeneity have been explored using techniques that enhance our understanding of their
evolution and the role of specific amino acid residues in their assembly and function. Hemocyanin
also functions as a phenoloxidase, a carrier protein, and in immune response. Increasing evidence
implicates hemocyanin in sclerotization of new exoskeleton at ecdysis. Additional gene family
members are cryptocyanin, a copperless hexamer expressed during premolt that functions in
exoskeleton formation, and a hemocyte phenoloxidase involved in wound repair, sclerotization,
melanin synthesis, and immune system. Future directions call for understanding the regulation
and coordination of expression of multiple hemocyanin or hemoglobin genes in crustaceans and
the possibility of transgenerational epigenetic inheritance in response to development, molting,
and environmental change.

INTRODUCTION

Challenges of Obtaining Oxygen

Small organisms have high surface-to-volume ratios that allow them to obtain O 2 through
simple diffusion. As an animal grows, metabolic needs tend to increase cubically, while the
surface of the animal, the window by which an organism obtains O 2, only increases as the
square. Thus, the surface-to-volume ratio decreases with increasing size. Organisms also

359
360 Nora B. Terwilliger

must contend with the diffusion rate of gases, which is equal to the cross-sectional area
exposed to the gas multiplied by the diffusion properties of the gas (in either air or water)
multiplied by the concentration gradient of the gas. Only the area and the concentration
gradients are subject to modification by the organism. Anatomical modifications to sat-
isfy O 2 needs through increasing the surface area are seen in many soft-bodied animals. In
Annelida, for example, certain oligochaetes and leeches take up O2 directly across the thin
body wall to underlying capillaries; capitellids assume flattened string-like shapes; nereid
and glycerid polychaetes extend thin-walled flaps or cirri from their segmentally arrayed
parapodia, and tube-dwelling terebellids sport corkscrew gills that protrude into the oxy-
genated seawater from the most anterior segments and contribute up to one-third of the
total body surface. These thin-walled “gills” all function to increase the general surface area
and minimize the diffusion path for O 2 from outside to inside the organism, the first step
in aerobic respiration.
Crustaceans and other Arthropoda, whose bodies are encased in exoskeletons, have lim-
ited opportunities to increase their gas exchange areas and must rely on the gills and epithe-
lial lining of the branchial chambers where the exoskeleton coverings are markedly thinner.
Crustaceans do possess numerous behavioral and biochemical strategies to improve the O2
flux and steepen the O2 gradient between the outside milieu and inside the chitin-enclosed
gill. Externally, ventilatory movements by appendages like the scaphognathite or gill bailer,
that push O2-depleted water out of the gill chambers and allow well-oxygenated water to flow
in and around the gills, enhance the O2 gradient (McMahon 2001). Internally, the highly reg-
ulated circulatory system of crustaceans effectively removes O2 from inside the thin barrier
gill, both steepening the diffusion gradient and delivering O2 elsewhere (see Chapter 7 in this
volume). Finally, O2 transport proteins that are specialized to bind O2 in regions of high con-
centration and release it in regions of lower concentration circulate in the hemolymph, thus
providing a molecular means to sustain the metabolic needs of the crustaceans. The proteins
markedly increase the O2 transport capacity of the hemolymph in comparison to the very low
solubility of dissolved O2. Like ventilation and circulation, O2 transport proteins effectively
improve the O2 flux and enhance the O2 gradient.
Arthropods abound in the Middle Cambrian fossils from the Burgess Shale. This site in
the Canadian Rockies, renowned for its excellent preservation of soft-bodied animals that
are more than 500 million years old, also has exceptional diversity of early arthropod species.
The exoskeletons covering these mobile, multicellular bilaterians suggest that the O2 needs of
these arthropods could not have been met through simple diffusion; they would have required
proteins that could bind the O2 molecules and transport them via a circulatory system to the
respiring tissues. Phylogenetic studies estimate the O2 transport proteins evolved prior to the
Cambrian, perhaps between 600 and 700  million years ago, and preceded the appearance of
fossils with hard exoskeletons and shells (Decker and van Holde 2011). Marrella splendens, a
basal arthropod frequently found in the Burgess Shale, is often preserved with a dark stain
under or around the fossil (Fig. 11.1). This stain probably represents the fossilized traces of a
copper-based hemocyanin that circulated in the hemolymph. Elevated concentrations of cop-
per identified specifically in the dark stain, but also in other regions of the fossilized Marrella
using synchrotron X-ray fluorescence imaging, provide intriguing evidence for a most ancient
hemocyanin (Pratt et al. 2010).
Today’s crustaceans are among the most sensitive taxa to O2 levels, showing the highest LC50
and the shortest LT50 when compared with fish, mollusks, annelids, echinoderms, cnidarians, and
priapulids in a survey of 872 published experiments (Vaquer-Sunyer and Duarte 2008). Obtaining
O2 is a critical task for a crustacean. This chapter focuses on recent findings on the structures, func-
tional properties, and phylogenetic relationships of the crustacean respiratory proteins, hemocya-
nins and hemoglobins.
 Oxygen Transport Proteins in Crustacea 361

Fig. 11.1.
Marrella splendens Walcott, a Middle Cambrian basal arthropod from the Burgess Shale. The dark stain visible
toward the posterior of the ~500-million year old fossil is rich in copper and probably represents an ancient
hemocyanin. Scale bar = 1 cm. Copyright Peabody Museum of Natural History, Yale University, New Haven,
CT. YPM catalog no. 5861. Photography by W.K. Sacco.

Oxygen Transport Proteins: Hemocyanin, Hemoglobin, Hemerythrin

Three types of O2 binding proteins reversibly transport O2 within an organism. First, hemocyanins,
found exclusively in Arthropoda and Mollusca, are large, multi-subunit molecules composed of
individual polypeptide chains or subunits containing highly conserved copper-based O2-binding
sites (Van Holde and Miller 1995, Decker et al. 2007a, Decker and van Holde 2011). Each of the two
copper atoms in the active site is covalently bound by three histidine residues (Fig. 11.2). Upon oxy-
genation, a di-O2 is bound as a peroxide in a side-on (μ-η2:η2) coordination between the two copper
atoms (type III copper binding site). When O2 is bound, the coppers oxidize from Cu(I) to Cu(II)
and the hemocyanin becomes blue; this is reversed when O2 is released in the deoxygenated state.
Hemocyanins are extracellular and circulate dissolved in the hemolymph, in contrast to vertebrate
hemoglobins in erythrocytes. Arthropod hemocyanins are hexameric arrays of individual approxi-
mately 75 kDa subunits, and each subunit has one O2-binding site. The hexamers assemble into
multiples of hexamers depending on the arthropod species, as described below. The largest, the
8-hexamer oligomer of horseshoe crab hemocyanin, has 48 O2-binding sites. Molluskan hemocya-
nins are cylindrical structures whose 10 or 20 subunits are much larger than arthropod hemocya-
nin subunits and assemble into decamers, didecamers, or multidecamers. A molluskan subunit of
350–400 kDa contains 7–8 paralogous functional units (FU). Each 50 kDa FU contains one active
site, so that intact hemocyanin of a mollusk may have 160 O2-binding sites (Cuff et al. 1998, Miller
362 Nora B. Terwilliger

Cu Cu

O
Cu
Cu
O

Fig. 11.2.
Diagram of active site of oxy and deoxy hemocyanin showing each copper atom complexed by a set of three
histidines, which form the connection to the protein backbone. (A) Deoxygenated state without any ligands.
(B) Oxygenated state with oxygen bound as peroxide in a side-on coordination. From Panzer et al. (2010), with
permission from the American Chemical Society.

et al. 1998, Perbandt et al. 2003). The protein sequences of arthropod and molluskan hemocya-
nins appear to be phylogenetically unrelated except perhaps at the functional site (Van Holde et al.
2001, Burmester 2002), yet overall structural aspects and functional properties show hints of shared
history (Durstewitz and Terwilliger 1997, Jaenicke et al. 2010). Whether these circulating copper
proteins have evolved independently or in fact share a common ancestor is an ongoing topic of
investigation and conjecture (see later discussion).
The other two O2 transport proteins, hemoglobins and hemerythrins, both have iron-based
O2-binding sites. Hemoglobins are nearly ubiquitous, being widely distributed in animals, plants, pro-
tists, and bacteria, and are expressed in multiple tissues, although they are not all involved in revers-
ible O2 transport (Terwilliger 1992, Terwilliger 1998, Weber and Vinogradov 2001, Royer et al. 2005,
Brunori and Vallone 2007, Burmester and Hankeln 2007). The single iron in the heme of a hemoglo-
bin subunit is coordinated on its proximal side to a histidine of the globin protein. On its distal side,
the heme iron reversibly binds a di-O2 molecule within the protective globin framework. Vertebrates
contain the well-known α2β2 tetrameric hemoglobin, each subunit with a single heme, in a circulating
red blood cell or erythrocyte. Across the phyla, however, circulating hemoglobins range from intracel-
lular to extracellular, single to multiple hemes per subunit, and monomeric to multimeric subunits per
molecule, and they self-assemble into a wide range of beautiful three-dimensional arrays.
The third O2 transport protein, hemerythrin, has been functionally described in only four pro-
tostome groups: Sipuncula, Annelida, Brachiopoda, and Priapulida (Kurtz 1992, Vanin et al. 2006).
In contrast to hemoglobin, two iron atoms in the O2 binding site of a hemerythrin subunit are
coordinated directly to the hemerythrin protein itself, there is no heme group, and the protein
sequence has no resemblance to a globin (Vanin et al. 2006, Meyer and Lieb 2010). Oxygenated
hemerythrin is deep pink-purple, whereas deoxyhemerythrin is colorless. Hemerythrins are
intracellular in circulating hemerythrocytes as trimeric or octomeric assemblages that may have
 Oxygen Transport Proteins in Crustacea 363

evolved from a monomeric ancestor, and hemerythrins are also present in muscle and nervous tis-
sue. Hemerythrin-related sequences have been determined in Cnidaria and Bacteria, but not in
Arthropoda—nor in any deuterostomes (Xiong et al. 2000, Bailly et al. 2008, French et al. 2008,
Traverso et al. 2010). Although future genomic analyses may reveal a more extensive distribution of
hemerythrins and a cosmopolitan occurrence of hemocyanins and hemoglobins, at the moment,
expression of circulating O2 transport proteins in the Crustacea is limited to hemocyanin and
hemoglobin.

CRUSTACEAN OXYGEN BINDING PROTEINS: HEMOCYANIN

Hemocyanins have been identified in all four subphyla of Arthropoda, Crustacea, and Chelicerata
(Markl and Decker 1992, Van Holde and Miller 1995, Rehm et  al. 2012), Myriapoda (Mangum
et al. 1985), and Hexapoda (Hagner-Holler et al. 2004, Pick et al. 2008). They are also found in the
related ecdysozoan, Onychophora (Kusche et al. 2002). Within the Crustacea, hemocyanin occurs
in Remipedia (Ertas et al. 2009) and is expressed extensively in Malacostraca, from the most basal
Phyllocarida, Nebalia (Vierthaler et al. 2003), to the shrimps, lobsters, and crabs, but it has not been
described in other Crustacea.

Hemocyanin Structure

Detailed knowledge of the subunit structure of a crustacean hemocyanin is the basis for under-
standing the complex quaternary assemblages and functional properties of the circulating proteins,
as well as the evolution of the arthropod hemocyanin gene family. Crystal structure analyses of
single subunits from hemocyanins of a crustacean Panulirus interruptus (spiny lobster) and a che-
licerate Limulus polyphemus (horseshoe crab) have shown that the arthropod hemocyanin subunit
is folded into three distinct regions or domains (Gaykema et al. 1984, Volbeda and Hol 1989, Hazes
et al. 1993, Magnus et al. 1994). The N-terminal domain 1, predominantly α-helical, is thought to
form a narrow channel for the O2 to enter and exit the molecule. Upon oxygenation, an 8-degree tilt
of domain 1 occurs, which may block the channel. Thus, domain 1 shields the entrance to the active
site. The central domain 2, a four-α-helix bundle, contains the O2-binding site, where each of the
two copper atoms is complexed to three histidines. The C-terminal domain 3 has a seven-stranded
β-barrel folding motif. Six of these approximately 75 kDa subunits assemble into a sandwich of two
trimers. The resulting approximately 450 kDa hexamer is the smallest physiologically active circu-
lating hemocyanin.
Hemocyanin hexamers and multiples of hexamers are composed of species-specific combina-
tions of unique subunits, rather than multiple copies of a single protein. This diversity, referred to
as subunit heterogeneity, enhances the assembly and functional properties of the oligomers. Subunit
heterogeneity, as well as phylogenetic relationships among the hemocyanin subunits and among
the Arthropoda, have been established through numerous studies at the protein, mRNA, and gene
levels (Markl et al. 1979, Markl and Decker 1992, Burmester 2001, 2002, Terwilliger and Ryan 2006).
Determination of the complete cDNA sequences of a species’ array of hemocyanin subunits, as in
the seven subunits of the tarantula Eurypelma californicum and the six subunits of the brachyuran
crab Cancer (Metacarcinus) magister, confirms each subunit’s identity as a unique gene product
(Voit et al. 2000, Terwilliger et al. 2006).
Within the malacostracan crustaceans, hemocyanin subunits appeared to group into several
types, α, β, and γ, based on immunogenicity and roles in aggregation (Markl 1986). As hemocyanin
sequences representing more malacostracans have become available, additional subunit types and
support for new interpretations of crustacean phylogenetic relationships are emerging (Terwilliger
364 Nora B. Terwilliger

and Ryan 2006, Ertas et al. 2009, Scherbaum et al. 2010). For example, hemocyanin is expressed
in Nebalia kensleyi, a leptostracan in the subclass Phyllocarida, considered the most basal of the
Malacostraca (Fig. 11.3). Nebalia hemocyanin, present only as hexamers, combines reversibly with
O2, indicating that it functions as an O2 transporter (Vierthaler et al. 2003). Its sequence clusters with
other malacostracan hemocyanins, but separate from the α, β, and γ types, consistent with the basal
position of the Phyllocarida (Fig. 11.4). In contrast, four hemocyanin subunits of the stomatopod
Odontodactylus scyllarus (Malacostraca, subclass Hoplocarida) all belong to the branch of β-type
subunits (Scherbaum et  al. 2010). Eucaridan decapods (Malacostraca, subclass Eumalacostraca)
also express β subunits; in the brachyuran C. magister, two of the six hemocyanin subunits are β
whereas the other four are γ, with no α representatives (Terwilliger and Ryan 2006), and hemo-
cyanin in the anomuran Petrolisthes cinctipes is composed of β and γ subunits (Scherbaum et al.
2010). Thus, an early hemocyanin gene duplication seems to have given rise to the β subunit lineage
as early as 520–480 mya, near the time when the Eumalacostraca and Phyllocarida diverged, fol-
lowed by the Hoplocarida at approximately 404 mya (Fig. 11.5; Scherbaum et al. 2010). Hemocyanin
sequences from amphipod and isopod species (all eumalacostracan Peracarida) cluster within the
malacostracan hemocyanins and cryptocyanins but in a separate branch distinct from the α and γ
type hemocyanin sequences of the eumalacostracan Eucarida (Figs. 11.4 and 11.5; Hagner-Holler
et al. 2005, Terwilliger and Ryan 2006, Jaenicke et al. 2009). This may reflect a gene duplication,
separation, and subsequent evolution of hemocyanin types as eumalacostracan crustaceans evolved
into separate superorders of Eucarida and Peracarida, and this theory is substantiated by functional

Fig. 11.3.
Electron micrograph of Nebalia kensleyi hemocyanin shows the presence of single hexamers. Hemolymph
negatively stained with 1% uranyl acetate. Microscopy by E. Schabtach.
 Oxygen Transport Proteins in Crustacea 365

PmaHc1
StuHc1 Insecta & Remipedia
0.81 StuHc3
/38 0.97 PmaHc2
/43 */64 StuHc2
OscHc1
*/* OscHc3
*/* OscHc2 Malacostraca
*/* OscHc4
CmaHc1 β-type
*/* hemocyanins
*/* CmaHc2
*/* PleHc
0.77/63 PciHc1
*/* PciHc2
*/* NkeHc phyllocarid hemocyanin
EpuHc1
0.96
/66 */* EpuHc2
peracarid
CcsHc hemocyanins
*/* GpuHc1
0.92/54
*/* GroHc1
HamHcA
*/* PleHc2
PinHcA
*/92 */* α-type
*/* PinHcB
hemocyanins
*/* PelHc1
*/* PelHc4
0.53/43 PelHc2
*/69 PelHc3
*/94 HamPHc1
*/* HamPHc2
cryptocyanins
*/* CmaCC1
*/* CmaCC2
PvaHc
*/* */* MjaHcy
*/* FchHc1
*/* PvaHc1
0.97/84 */94 MjaHcL
PinHcC γ-type
PciHc3 hemocyanins
*/96
CmaHc3
*/*
CsaHc
0.1 */*
CmaHc4
*/*
*/99 CmaHc5
*/73 CmaHc6

Fig. 11.4.
Phylogeny of crustacean hemocyanins, including remipedian and malacostracan (phyllocaridan, hoplo-
caridan, eumalacostracan eucaridan, and eumalacostracan peracaridan) hemocyanin and eumalacostracan
eucaridan cryptocyanin (pseudohemocyanin) sequences. Sister-group relation of hexapodan and remi-
pedian hemocyanins to malacostracans is included. See Table 11.1 for abbreviations. The numbers at the
nodes are Bayesian posterior probabilities (first number) and ML nonparametric bootstrap support values
(second number). Asterisks indicate 1.0 Bayesian posterior probability and 100% ML bootstrap support,
respectively. The bar represents 0.1 PAM distance. From Scherbaum et al. (2010), with permission from
Springer Science.

differences between eucaridan and peracaridan hemocyanins (phenoloxidase function). Further

gene duplications and subunit evolution into α and γ type subunits, including the cryptocyanins,
occurred as the eucaridans diversified (Hagner-Holler et al. 2005, Scherbaum et al. 2010). Thus, the
hemocyanin molecular phylogenies inform the crustacean phylogenies and vice versa.
The discovery of hemocyanin in the Remipedia Speleonectes tulumensis is the first unambigu-
ous evidence for crustacean hemocyanin expression outside the Malacostraca (van der Ham and
Felgenhauer 2007, Ertas et  al. 2009). The Remipedia hemocyanin is most similar to Hexapoda
hemocyanins, which places both in a sister-group relation to the hemocyanins of malacostracan
crustaceans (Fig. 11.4; Ertas et al. 2009). Evidence indicates that the Remipedia hemocyanin sub-
units are orthologs of hexapod hemocyanin subunits; therefore, the hemocyanin lineages would
have split before the Remipedia and Hexapoda groups diverged. A  hexapod-crustacean clade
has support from both morphological and molecular analyses, although there have been several
366 Nora B. Terwilliger

O S D C P TR J K E N
hoplocaridan hoplocarid
β-type Hc
β-type Hc
eumalacostracan: eucaridan eucarid
β-type Hc
phyllocaridan phyllocarid Hc

isopod Hc
eumalacostracan: peracaridan
amphipod Hc
astacid
α-type Hc
α-type Hc achelate
α-type Hc
eumalacostracan: eucaridan
Cryptocyanins
penaeid
γ-type Hc
γ-type Hc
achelate
γ-type Hc
brachyuran
γ-type Hc

500 400 300 200 100 0 MYA

Fig. 11.5.
Molecular clock analysis of malacostracan hemocyanin subunits based on Fig. 11.4. The geological periods are
indicated at the top. Є, Cambrian; O, Ordovician; S, Silurian; D, Devonian; C, Carboniferous; P, Permian; TR ,
Triassic; J, Jurassic; K, Cretaceous; E, Paleogene; N, Neogene. From Scherbaum et al. (2010), with permission
from Springer Science.

candidates for the crustacean sister group, including Malacostraca, Branchiopoda, Copepoda,
Remipedia, and Cephalocarida, and the debates on arthropod phylogeny still continue (Kusche
et al. 2003, Dunn et al. 2008, Edgecombe 2010, von Reumont et al. 2012).
A major consequence of subunit heterogeneity in arthropod hemocyanin is the ability of hex-
amers to assemble into larger oligomers (Giomi and Beltramini 2007). Hemocyanin hexamers
self-assemble into 2-, 4-, 6-, and 8-hexameric aggregates in a species-specific distribution, presum-
ably based on availability of amino acid residues providing intra- and interhexameric contacts. Of
the malacostracan crustaceans, for example, phyllocaridans, caridean shrimp, spiny lobsters, and
amphipods have 1-hexamer hemocyanins, whereas crabs, clawed lobsters, and isopods usually have
both 1- and 2-hexamer forms. The 2-hexamer hemocyanins typically require an additional subunit
type, a linker, not present in the 1-hexamer form, and the two oligomer populations are not in a
simple equilibrium mixture (Markl et al. 1979, Terwilliger 1982, Terwilliger and Terwilliger 1982).
Hemocyanins of thalassinid crustaceans circulate predominantly as aggregates of 4-hexamers,
along with a low percentage of 1-hexamers and 2-hexamers (Fig. 11.6). Within the Chelicerata, 1-,
2-, 4-, and 8-hexamer hemocyanins have been described. However, the 4-hexamer hemocyanins of
scorpions and tarantulas differ from the 4-hexamer hemocyanin of thalassinid crustaceans in the
three-dimensional arrangement of the hexamers (Hartmann and Decker 2002, Paoli et  al. 2007,
Micetic et al. 2010). Unique 6-hexamer aggregates occur in the Myriapoda, and the largest hemocy-
anins, 8-hexamers, are found in horseshoe crabs (Chelicerata). Hemocyanin structures have been
studied intensively for many years as models for protein-protein interaction and subunit assem-
bly using electron microscopy, ultracentrifugation, and electrophoresis (Van Holde and Miller
1982, Salvato and Beltramini 1990, Markl and Decker 1992, Van Holde and Miller 1995). Numerous
sequence comparisons have shown that important secondary structure features of arthropod
hemocyanin subunits are highly conserved, and this has led to homology-based molecular mod-
eling of the giant hexameric aggregates. Integration of models with high-resolution cryoelectron
microscopy images and small-angle X-ray scattering (SAXS) provides new ways of understanding
the role of specific amino acid residues in the intra- and interhexameric contacts that promote both
Table 11.1.  Hemocyanin (Hc), cryptocyanin (Cc), and pseudohemocyanin (PH) sequences used in Fig. 11.4.

Abbr Species Subclass Superorder Order Infraorder Acc. No. Protein

Subphylum Hexapoda, Class Insecta
PmaHc1 Perla marginata Plecoptera AJ555403 Hc1
PmaHc2 Perla marginata Plecoptera AJ555404 Hc2
Subphylum Crustacea, Class Remipedia
StuHc1 Speleonectes tulumensis Nectiopoda FM863709 Hc1
StuHc2 Speleonectes tulumensis Nectiopoda FM863710 Hc2
StuHc3 Speleonectes tulumensis Nectiopoda FM863711 Hc3
Subphylum Crustacea, Class Malacostraca
NkeHc Nebalia kensleyi Phyllocarida Leptostraca GQ279108 Hc
OscHc1 Odontodactylus Hoplocarida Stomatopoda FM99928 Hc1
scyllarus
OscHc2 Odontodactylus Hoplocarida Stomatopoda FM99929 Hc2
scyllarus
OscHc3 Odontodactylus Hoplocarida Stomatopoda FM99930 Hc3
scyllarus
OscHc4 Odontodactylus Hoplocarida Stomatopoda FM99931 Hc4
scyllarus
GroHc1 Gammarus roeseli Eumalacostraca Peracarida Amphipoda AJ937836 Hc1
GpuHc1 Gammarus pulex Eumalacostraca Peracarida Amphipoda EST data Hc 2
CcsHc Cyamus scammoni Eumalacostraca Peracarida Amphipoda DQ230983 Hc
EpuHc1 Euridice pulcra Eumalacostraca Peracarida Isopoda GQ153951 Hc1
EpuHc2 Euridice pulcra Eumalacostraca Peracarida Isopoda GQ153952 Hc2
PleHc Pacifastacus leniusculus Eumalacostraca Eucarida Decapoda Astacidea AF522504 Hc1
PleHc2 Pacifastacus leniusculus Eumalacostraca Eucarida Decapoda Astacidea AY193781 Hc2

(continued)
Table 11.1. (Continued)

Abbr Species Subclass Superorder Order Infraorder Acc. No. Protein

HamHcA Homarus americanus Eumalacostraca Eucarida Decapoda Astacidea AJ272095 HcA
PinHcB Panulirus interruptus Eumalacostraca Eucarida Decapoda Palinura P10787 Hcb
PinHcA Panulirus interruptus Eumalacostraca Eucarida Decapoda Palinura P04254 Hca
PinHcC Panulirus interruptus Eumalacostraca Eucarida Decapoda Palinura S21221 Hcc
PvuHc Palinurus vulgaris Eumalacostraca Eucarida Decapoda Palinura P80888 Hc
PelHc1 Palinurus elephas Eumalacostraca Eucarida Decapoda Palinura AJ344361 Hc1
PelHc2 Palinurus elephas Eumalacostraca Eucarida Decapoda Palinura AJ344362 Hc2
PelHc3 Palinurus elephas Eumalacostraca Eucarida Decapoda Palinura AJ344363 Hc3
PelHc4 Palinurus elephas Eumalacostraca Eucarida Decapoda Palinura AJ516004 Hc4
CmaHc6 Cancer magistera Eumalacostraca Eucarida Decapoda Brachyura U48881 Hc6
CmaHc1 Cancer magíster Eumalacostraca Eucarida Decapoda Brachyura AY861676 Hc1
CmaHc2 Cancer magíster Eumalacostraca Eucarida Decapoda Brachyura AY861677 Hc2
CmaHc3 Cancer magíster Eumalacostraca Eucarida Decapoda Brachyura AY861678 Hc3
CmaHc4 Cancer magíster Eumalacostraca Eucarida Decapoda Brachyura AY861679 Hc4
CmaHc5 Cancer magíster Eumalacostraca Eucarida Decapoda Brachyura AY861680 Hc5
CsaHc Callinectes sapidus Eumalacostraca Eucarida Decapoda Brachyura AF249297 Hc
PciHc1 Petrolisthes cinctipes Eumalacostraca Eucarida Decapoda Anomura EST data Hc1
PciHc2 Petrolisthes cinctipes Eumalacostraca Eucarida Decapoda Anomura EST data Hc2
PciHc3 Petrolisthes cinctipes Eumalacostraca Eucarida Decapoda Anomura EST data Hc3
PvaHc1 Penaeus vannameib Eumalacostraca Eucarida Decapoda Penaeoidea AJ250830 Hc1
PvaHc Penaeus vannamei Eumalacostraca Eucarida Decapoda Penaeoidea X82502 Hc
FchHc Fenneropenaeus Eumalacostraca Eucarida Decapoda Penaeoidea FJ594414 Hc
chinensis
MjaHcL Marsupenaeus Eumalacostraca Eucarida Decapoda Penaeoidea EF375711 HcL
japonicus
MjaHcY Marsupenaeus Eumalacostraca Eucarida Decapoda Penaeoidea EF375712 HcY
japonicus
HamPHc1 Homarus americanus Eumalacostraca Eucarida Decapoda Astacidea AJ132141 PH
HamPHc2 Homarus americanus Eumalacostraca Eucarida Decapoda Astacidea AJ132142 PH
CmaCC1 Cancer magíster Eumalacostraca Eucarida Decapoda Brachyura AF091261 Cc
CmaCC2 Cancer magíster Eumalacostraca Eucarida Decapoda Brachyura DQ230982 Cc

 Cancer (Metacarcinus) magister.

a

 Penaeus (Litopenaeus) vannamei.

b
370 Nora B. Terwilliger

Fig. 11.6.
Comparison of oligomers of three hemocyanins using small angle X-ray scattering (SAXS) refinement of struc-
tural models derived by negative-stain electron microscopy. (A) Brachyuran crab Carcinus estuarii 2-hexamer
hemocyanin is typical of most crustacean 2-hexamer hemocyanins. (B)  Peculiar stacking of stomatopod
Squilla mantis 2-hexamer hemocyanin resembles 2-hexameric substructure of larger cheliceratan hemocyanins.
(C) Thalassinid Upogebia pusilla 4-hexamer hemocyanin has a unique tetrahedral packing in contrast to square
planar shape of 4-hexamer hemocyanins of chelicerates. Structures of final, best-fit models. The trimeric axes
of the hexameric units are oriented differently with respect to one another in the three hemocyanin oligomers.
See ­figure 6 in Micetic et al. for structural parameters and geometries. From Micetic et al. (2010), ­figures 2, 3, 4
with permission from Elsevier.

the assembly and the complex functional properties of the hemocyanins. Thus, the structures of
the 1-hexamer hemocyanin of the spiny lobster Palinurus elephas (Meissner et  al. 2003)  and the
2-hexamer hemocyanins of the clawed lobster Homarus americanus (Hartmann et  al. 2001), the
brachyuran crab Carcinus aestuarii, and the stomatopod Squilla mantis (Micetic et al. 2010) have
been clarified. The structure of C. aestuarii hemocyanin is similar to those of most other crustacean
2-hexamer hemocyanins, with the two hexamers aligned along their short rough sides, but it differs
from the arrangement of the two uniquely linked hexamers of S. mantis and O. scyllarus, stacked on
their long flat sides, which more closely resemble a 2-hexameric substructure from a cheliceran 4-
or 8-hexamer hemocyanin (Fig. 11.6; Bijlholt and van Bruggen 1986, Micetic et al. 2010, Scherbaum
et  al. 2010). Myriapod hemocyanin, with its unique 6-hexamer structure, was first described in
centipedes (Mangum et al. 1985) and diplopods ( Jaenicke et al. 1999a). A high-resolution analy-
sis modeling 6-hexamer hemocyanins with strikingly different functional properties, one from a
slow-moving diplopod Spirostreptus (h ~1.3, P50 ~5 torr) and the other from a speedy scutigero-
morph centipede Scutigera coleoptrata (h ~10; P50 ~50 torr) has led the authors to suggest how
 Oxygen Transport Proteins in Crustacea 371

detailed differences in amino acids at the interhexameric interfaces explain the striking divergence
in functional allostery within the 6-hexamer framework of these myriapod hemocyanins (Markl
et  al. 2009). The giant structures of 4-hexamer hemocyanin in the scorpion Pandinus imperator
(Cong et al. 2009) and 8-hexamer hemocyanin in the horseshoe crab L. polyphemus (Martin et al.
2007) have also been finely resolved. These intricate multi-subunit hemocyanins provide excellent
opportunities to explore the effects of gene duplications and subsequent independent evolutionary
trajectories of heterogeneous subunits within a given structural framework.

Hemocyanin Gene Family

Gene duplications and mutations have resulted not only in hemocyanin subunit heterogeneity,
but also in the evolution of several phylogenetically related hexameric proteins. The arthropod
hemocyanin gene family includes hemocyanins, cryptocyanins, phenoloxidases, and hexamerins
(Terwilliger 1998, Burmester 2001). The four proteins show strong conservation of primary and
quaternary structure, but each has unique features that have led to diverse functions. Cryptocyanin
(pseudohemocyanin), like hemocyanin, is synthesized in the hepatopancreas of crabs and lobsters
and circulates in the hemolymph. Its hexamers are so similar to hemocyanin hexamers that early
researchers were unaware of its presence, and often it was inadvertently co-purified with hemocya-
nin from the hemolymph. Its active site has a reduced number of the six histidine residues critical
for copper binding, and therefore it lacks copper and has no O2 transport or oxidase functions
(Terwilliger et  al. 1999, Burmester 1999). It is produced in high concentrations during premolt
and helps form the new exoskeleton (Terwilliger et al. 2005, Kuballa et al. 2007, Terwilliger 2011).
The possibility that there are tissue-specific cryptocyanins with unique functions in some species
should be studied further. Phenoloxidase is a hexameric enzyme synthesized in hemocytes that cir-
culate in the hemolymph of some crustaceans and insects (Aspán et al. 1995, Terwilliger and Towle
2007). The active site of the phenoloxidase subunit contains the two copper atoms and six histi-
dine residues similar to hemocyanin, and therefore it binds O2 (Lerch and German 1988, Decker
and Terwilliger 2000, Terwilliger and Ryan 2006, Cong et al. 2009). Rather than transporting O2
reversibly, however, phenoloxidase functions in the arthropod immune response (Söderhäll and
Cerenius 1998) and participates in crosslinking the new exoskeleton after molting, in exoskeleton
repair, and in encapsulating foreign material (Sugumaran 1998). Hemocyanin can be converted
to enzymatically active phenoloxidase (see the section “Hemocyanin Function:  Phenoloxidase
Activity”). Hexamerins, the fourth member of the gene family, are extracellular hexameric proteins
found in insect hemolymph. A hexamerin subunit, like cryptocyanin, lacks one or more of the six
histidines in the highly conserved active site and binds neither copper nor O2 (Telfer and Massey
1987, Beintema et al. 1994, Pick et al. 2008, Pick and Burmester 2009). Although a number of func-
tions have been demonstrated for different hexamerins, they are frequently referred to as storage
proteins.

Hemocyanin Function

Oxygen Binding and Transport

Arthropod hemocyanins demonstrate variable O2 affinities and high cooperativities. The dramati-
cally wide range of allosteric functional properties is another major consequence of subunit hetero-
geneity and assembly into multiple oligomers. Hemocyanins have high O2 transport capacities due
to their copious O2-binding sites and high concentrations in the hemolymph. Numerous biochemi-
cal and physiological studies have enhanced our understanding of how crustaceans are able to live
372 Nora B. Terwilliger

in widely different habitats and obtain sufficient O2 under potentially stressful conditions (Morris
1990, Truchot 1992, Mangum 1997a, Morris and Airriess 1998, Terwilliger 1998, Bridges 2001, Giomi
and Beltramini 2007). Crustacean hemocyanins, like other respiratory proteins, have the ability
to adopt different conformations that have different affinities for O2. The conformational changes
are regulated by allosteric effectors, including inorganic ions, such as H+, Na+, Ca2+, Mg2+, Cl–, the
organic molecules lactate and urate, sulfide and thiosulfate, and neurohormones (Truchot 1980,
Morris et al. 1985, Burnett 1992, Truchot 1992, Sanders and Childress 1992, Van Holde and Miller
1995, Hagerman and Vismann 1999). Hemocyanin O2 affinities are affected by external environ-
mental factors as well, such as temperature and salinity. However, O2 binding of hemocyanin from
crustaceans regularly exposed to highly variable temperatures, including deep-sea hydrothermal
vent species and certain shallow-water species, show little or no temperature sensitivity (Hourdez
and Lallier 2007).
Conformational changes in the tertiary and quaternary structure of hemocyanins affect both
subunit interactions and the active site of the subunit. Most hemocyanins exhibit highly coopera-
tive O2 binding, and this extraordinary cooperativity often is stronger in the more complex multi-
hexamer structures with higher molecular masses. Cooperativity in the higher aggregation forms,
from 2-hexamer to 8-hexamer, has been described as hierarchies of the equilibrium between differ-
ent types of allosteric units, a nesting model, in which one level, a 1-hexamer for example, is embed-
ded in the next, a 2- or 4-hexamer unit (Robert et al. 1987, Decker and Sterner 1990, Decker et al.
2007a). Although hemocyanins with increased degrees of subunit heterogeneity and oligomers
tend to exhibit a broader range of functional properties, this is not always true. Recent work on
hemocyanin of Upogebia pusilla, a burrowing mud shrimp, has shown that, despite the 4-hexamer
structure, the functional properties in this species are based on the hexamer as the allosteric unit
without additional interactions between the hexamers (Hellmann et al. 2010). Furthermore, unlike
4-hexamer hemocyanins from several other thalassinid shrimps, the O2 affinity in this species
increases in the presence of L-lactate. The authors suggest that possibly the lactate compensates
even at resting conditions for a low intrinsic O2 affinity in U. pusilla. This exception is a reminder
that although model organisms can provide critical templates for understanding biological patterns,
evolution occurs at the species level. Studies characterizing structure and function of an unusual
female-specific hemocyanin in the crab Scylla olivacea (Chen et  al. 2007)  and previously unde-
scribed hemocyanins in the crab Calappa granulata (Olianas et al. 2006) and spiny lobster Palinurus
gilchristi (Olianas et al. 2009) continue to enhance our knowledge base of both typical and unusual
systems.
Some of the parameters that affect the binding of O2 to the copper atoms at the active site and
the transition from deoxy Cu(I) to oxy Cu(II)-O2-Cu(II) include the geometries of the Cu-Cu
coordination and access to them by the binding ligands, as well as the distance between the two
coppers. The Bohr effect, the well-known sensitivity of O2 binding in respiratory proteins to fluctu-
ations in pH, permits the proteins to vary their affinities in response to changes in the physiological
demands of the organism. An increase in proton concentration generally decreases hemocyanin O2
affinity and cooperativity (Truchot 1992). Studies on the molecular basis of the effect of hydrogen
ions on hemocyanin, comparing three well-characterized hemocyanins from P. interruptus, C. aes-
tuarii, and L. polyphemus, used flash photolysis and X-ray edge measurements to focus on the coor-
dination geometry of the copper at the active site (Hirota et al. 2008). Findings indicate that the
Bohr effect results from changes in the constant kon , the rate of O2 binding to the protein, and not
from changes in the koff . Thus, there is a structural rearrangement in the deoxygenated protein that
produces a change in the rate of binding O2. This structural effect of pH is on the coordination
geometry of the individual Cu(I) sites of the binuclear center, which may in turn affect the redox
potential of the copper ions, as well as on a change in the Cu-Cu distance, as others have suggested.
 Oxygen Transport Proteins in Crustacea 373

In contrast to the Bohr effect, the binding of lactate, a metabolic byproduct of glycolysis under
anaerobic conditions, usually increases O2 affinity and cooperativity of hemocyanin (Truchot 1980,
Graham et al. 1983). The lactate effect appears to counterbalance the potential decrease in O2 affin-
ity caused by the Bohr effect under conditions of acidosis. In the portunid crab Carcinus maenas,
lactate increases O2 affinity of hemocyanin by raising the association equilibrium constant of the
low-affinity Tense state without affecting that of the high-affinity Relaxed state (Weber et al. 2008).
The same study shows the important dual roles for lactate and proton ions in affecting the tem-
perature sensitivity of hemocyanin O2 binding in crustaceans. The structural basis of the lactate
effect on O2 binding in the closely related C. estuarii is a modulation of the global conformation of
the protein, but lactate binding does not affect the active site, in contrast to the Bohr effect (Hirota
et al. 2010). Based on SAXS measurements, at pH 8.3, L-lactate had little effect, but at pH 6.5, lactate
caused a concentration-dependent shift in the interhexameric distances. Changes in quaternary
structure of both hexamers and 2-hexamers were affected by lactate binding, supporting the bind-
ing of lactate at an intersubunit site rather than an exterior site. The dual functional roles of a circu-
lating 6-hexamer hemocyanin and a tracheal system in Myriapoda have been explored in the huge
African diplopod Archispirostreptus gigas (Damsgaard et al. 2013). The authors compare the mecha-
nisms of allosteric control, including effects of lactate, Ca2+, and pH on this diplopod hemocyanin
with other O2 transport proteins.
Another study, using L-edge X-ray absorption spectroscopy (XAS) under physiological condi-
tions to detail the changes in the active copper sites in hemocyanin of H.  americanus, has dem-
onstrated that O2 binding does not simply switch the copper valence state between Cu I and Cu
II (Panzer et al. 2010). Rather, the authors show that, upon deoxygenation, water can replace the
di-O2. When O2 is added, the water molecules keep the copper atoms partially oxidized. This new
finding promises to stimulate further studies on water-O2-protein functions.

Hemocyanin Function: Phenoloxidase Activity

In addition to binding O2 reversibly and functioning as O2 transport molecules, hemocyanins

of arthropods and mollusks can be activated to function as phenoloxidases under certain in
vitro conditions (Zlateva et  al. 1996, Salvato et  al. 1998, Decker et  al. 2001, Siddiqui et  al. 2006,
Terwilliger and Ryan 2006, Decker et  al. 2007b; and see also the section “Hemocyanin Gene
Family”). Phenoloxidases, found in many organisms, are enzymes that catalyze the hydroxylation
of monophenols, such as tyrosine, to o-diphenols, and their subsequent oxidation to highly reac-
tive o-quinones. The quinones are on the pathway to melanin synthesis, a compound critical in
the arthropod immune response (Sánchez-Ferrer et al. 1995). “Phenoloxidase” is a generic term
that includes tyrosinases, capable of catalyzing both reactions, and catecholoxidases, able to cata-
lyze the second reaction only. The enzymes are responsible for the browning of fruits, coloring of
mammalian skin and hair, and wound healing, immune defense, and sclerotization in arthropods.
Because melanin has antimicrobial, antifungal, and antiviral properties, phenoloxidases are impor-
tant components of the innate immune system. In many crustaceans and insects, phenoloxidases
occur as prophenoloxidases in circulating hemocytes, and their activation is regulated by a tight sys-
tem of enzymatic reactions (the prophenoloxidase cascade; Ashida and Yamazaki 1990, Söderhäll
and Cerenius 1998). Hemocyanin is closely related to phenoloxidase phylogenetically, and the two
share the binuclear copper active site structure of type 3 copper proteins. That hemocyanin can
function as both an O2 transporter and phenoloxidase raises intriguing questions.
First, dramatic evidence for the structural mechanism of activation of arthropod hemocyanin and
phenoloxidase has been presented by Decker and colleagues (Cong et al. 2009). Sodium dodecyl
sulfate (SDS) has been commonly used in lieu of natural activators to convert prophenoloxidase
374 Nora B. Terwilliger

and hemocyanin into enzymatically active phenoloxidases and measure phenoloxidase activity, but
how SDS worked was unknown. Analysis of electron cryomicroscopy and pseudoatomic models
of the 4-hexameric hemocyanin from the scorpion P. imperator in resting and SDS-activated states
validates an activation model proposed earlier (Decker and Tuczek 2000). At the individual sub-
unit level, SDS induces a conformational change in which the flexible domain I twists away from
domains II and III, exposing the entrance to the two coppers in the subunit’s active site and allow-
ing access by bulky phenol or diphenol substrates (see the section “Hemocyanin Structure”). The
shape change in individual subunits results in a cooperative structural rearrangement of the entire
4-hexamer oligomer. SDS, instead of acting as a denaturant, functions as an allosteric modulator of
the hemocyanin megamolecule. The results provide a new example of how arthropod hemocya-
nin hexamers interact and behave cooperatively during oxygenation or phenoloxidase activation.
Because the active site of type 3 copper proteins is well conserved, these results may explain the
mechanism of action of other systems as well.
Second, the relative contributions of hemocyte phenoloxidase versus activated hemocyanin
have been investigated in a number of crustaceans (Decker et al. 2001, Terwilliger and Ryan 2006,
García-Carreno et al. 2008, Nillius et al. 2008, Jaenicke et al. 2009). At the physiological ratio at
which they occur in the hemolymph, hemocyte phenoloxidase in the crab C.  magister has high
activity, but hemocyanin is present in a much higher concentration (Terwilliger and Ryan 2006).
Hemocyte phenoloxidase is able to catalyze both reactions—the conversion of monophenols to
diphenols and the oxidation to o-quinones—but the activated hemocyanin of a chelicerate or crus-
tacean often functions only as a catecholase, depending on the species and/or experimental condi-
tions (Decker et al. 2001, Nillius et al. 2008).
A third important question is whether hemocyanins function as phenoloxidases in vivo. Two
groups of arthropods, chelicerates and peracarid crustaceans, have the same needs for immune
defense and hardening of the exoskeleton after molting as do other arthropods. Surprisingly, spe-
cies of horseshoe crabs, tarantulas, isopods, and amphipods examined to date lack a hemocyte
phenoloxidase, but their hemocyanins do function as phenoloxidases in vitro (Pless et  al. 2003,
Arellano and Terwilliger 2004, Terwilliger 2007, Jaenicke et al. 2009). Anionic phospholipids such
as phosphatidylserine, as well as coagulation factors and antimicrobial peptides, have been impli-
cated as natural activators of horseshoe crab hemocyanin in vivo (Nagai et al. 2001, Coates et al.
2011). Hemocyanins in chelicerates and peracarid crustaceans, then, must be able to switch back
and forth between O2 transport and phenoloxidase, although there is no information on how the
two functions are regulated or how circulating hemocyanin molecules are allocated to a particular
function. Whether all chelicerates and peracarid crustaceans, including the tanaids, cumaceans, and
mysids, share a reliance on hemocyanin as the sole source of phenoloxidase activity remains to be
explored.
In those eucaridan malacostracans expressing both hemocyte phenoloxidase and hemocya-
nin, regulation of hemocyanin function in vivo between reversible O2 transport and oxidative
enzyme also needs to be addressed (Terwilliger and Ryan 2006, Cerenius et al. 2008). It has been
reported that crab hemocyanin may be activated into a functional phenoloxidase by hemocyte
components (Adachi et al. 2003, Fan et al. 2009). Perhaps hemocyte phenoloxidases of crabs,
shrimps, and lobsters function as rapid responders to microbial or viral challenge. Hemocyanin
may be activated to phenoloxidase activity only under extreme stress, as in prolonged hypoxia
when hemocyte phenoloxidase activity is suppressed (Tanner et al. 2006) or immediately post-
molt when there is a need for concerted, global sclerotization (Terwilliger 2007). Support for
this hypothesis comes from work on the crayfish Cherax quadricarinatus. Hemocyanin with phe-
noloxidase activity has been localized in the chitin matrix of the crayfish gastrolith, a transient
calcium deposit secreted similarly to the crayfish exoskeleton by an epithelium continuous with
that forming the exoskeleton (Glazer et al. 2013). Hemocyanin transcript expression was specific
 Oxygen Transport Proteins in Crustacea 375

to the hepatopancreas during premolt, consistent with other studies on hemocyanin synthesis in
crayfish and crabs. The authors suggest the hemocyanin may indeed be functioning as a sclero-
tizing agent in hardening the chitinous layers of the gastrolith as calcium is deposited during
premolt. Problems resulting from a crustacean immune response with overactive or unregulated
hemocyanin and/or hemocyte phenoloxidase activity are manifested in the extreme melaniza-
tion of lobster and crab shells in response to bacterial infection, “black shell disease,” as well as
postmortem darkening of whiteleg shrimp Penaeus vannamei, both of which reduce marketability
(Vogan et al. 2002, Terwilliger 2007, García-Carreno et al. 2008). Although uncommon, albino
C. magister and H. americanus do occur, and these may be due in part to alterations in the path-
way of melanin synthesis (Protas et al. 2011). In vertebrates, defects in melanin regulation range
from albinism to melanomas. Contamination of the marine environment with alkylphenols that
competitively interact with the phenoloxidase-dependent incorporation of tyrosine into the
exoskeleton may exacerbate problems associated with crustacean molting, metamorphosis, and
survival (Laufer et al. 2005) and provide added impetus for understanding the balance between
O2 transport and phenoloxidase activity in hemocyanin.

Additional Functions of Hemocyanin

Circulating hemocyanin oligomers may function as specific or nonspecific carrier proteins for
a variety of molecules in a role similar to that played by serum albumin in vertebrate blood
(Carter and Ho 1994, Terwilliger 2011). High hemolymph concentrations and approximately 75
Kda subunits of hemocyanin, as well as cryptocyanin and insect hexamerins, potentially offer
multiple binding sites for steroids, peptide hormones, fatty acids, and other ligands. The ste-
roid hormone ecdysone is bound with low affinity by hemocyanin from the tarantula E.  cal-
ifornica ( Jaenicke et  al. 1999b), similar to the ecdysone binding affinity seen in some insect
hexamerins (Enderle et  al. 1983). Furthermore, antimicrobial, antifungal, and antiviral prop-
erties have been reported for several arthropod hemocyanins and their peptides (Bachere
2000, Destoumieux-Garzon et al. 2001, Lee et al. 2003, Lei et al. 2008). Because the first and
third domains of the hemocyanin subunit, rather than the copper-containing second domain,
appear to be responsible for these protective functions, cryptocyanin might also have these
immune functions. In the shrimp Litopenaeus vannamei, hemocyanin showed hemolytic activity
(Zhang et al. 2009), and in the shrimp Penaeus japonicus, differences in antiviral activity among
its hemocyanin subunits have been reported (Lei et al. 2008), which offers more evidence for
the adaptive advantage of subunit heterogeneity. Other functions proposed for hemocyanins
include cation transport and exoskeleton formation ( Jaenicke et al. 1999b, Jaenicke and Decker
2004, Terwilliger 2011).

Hemocyanin Synthesis and Regulation

Site of Synthesis

Hemocyanin subunits of brachyuran crabs are synthesized in R cells of the hepatopancreas,

as shown by in situ hybridization, Northern blots, and immunohistology of the crab C. magis-
ter (Terwilliger et  al. 1999, 2005, Terwilliger 2011). Cryptocyanin is also synthesized in the
hepatopancreas R cells. Both proteins are secreted basally into the blood vessels surround-
ing the hepatopancreas tubules, consistent with the ultrastructure of the hepatopancreas R
cells in Penaeus semisulcatus (Al-Mohanna and Nott 1989). Self-assembly of subunits into
hexameric quaternary structures probably occurs either in the hepatopancreas cells prior to
secretion or in the hemolymph immediately after secretion. Monomers of each protein have
376 Nora B. Terwilliger

not been identified in the circulating hemolymph. Earlier studies had proposed that reserve
cells (Cuenot 1893, Johnson 1980)  in the connective tissue were the site of hemocyanin
synthesis, based in part on electron micrographs of large crystalline inclusions in the cells
(Ghiretti-Magaldi et al. 1973, 1977). Our studies showed, however, that reserve cells express
neither hemocyanin nor cryptocyanin mRNA. The reserve cells accumulate and increase in
size in the connective tissue during premolt. They become strongly immunoreactive against
cryptocyanin-specific monoclonal antibodies immediately prior to ecdysis and then decrease
in size and disappear in early postmolt. Instead of synthesizing hemocyanin or cryptocyanin,
it appears that reserve cells endocytose and metabolize excess cryptocyanin from the hemo-
lymph at ecdysis. Hemocyanin synthesis has been reported to take place in the hepatopancreas
of the shrimps Penaeus monodon and P. japonicus (Lehnert and Johnson 2001, Lei et al. 2008),
and both hemocyanin and cryptocyanin mRNA have been amplified in the hepatopancreas
of many brachyuran and anomuran crabs and lobsters (Terwilliger and Ryan, unpublished).
We and others have also found hemocyanin mRNA in the hepatopancreas of the cheliceran
horseshoe crab L.  polyphemus. Cyanocytes of L.  polyphemus, like the reserve cells of crabs,
probably are also involved in hemocyanin catabolism and not its synthesis, contrary to previ-
ous interpretation (Fahrenbach 1970).

Phenotypic Plasticity: Regulation of Hemocyanin Subunit Expression

Hemocyanin subunit heterogeneity is responsive to developmental trajectories and environ-

mental challenges. Ontogenic regulation of expression of the six genes coding for the six hemo-
cyanin subunits of the crab C.  magister occurs during development from megalopa to adult
(Terwilliger and Brown 1993, Terwilliger and Ryan 2001, Terwilliger et al. 2006). Hemocyanin
hexamers circulating in the early megalopa include three different subunits, whereas the
2-hexamer hemocyanins include the same three plus a linker subunit. Shortly after metamor-
phosis from megalopa to juvenile crab, a fifth unique subunit (Hc subunit 4) appears in each
oligomer, and, after several more juvenile crab molts, the sixth subunit type is present. These
structural changes in expression of hemocyanin subunits are accompanied by increases in intrin-
sic O2 affinity (Brown and Terwilliger 1992, 1998). In synchrony with these shifts is an increased
capacity for ion regulation, especially Mg2+, an ion that allosterically raises the hemocyanin O2
affinity. Developmentally correlated decreases of hemolymph Mg2+ to adult levels counterbal-
ance the increased intrinsic O2 affinity of the hemocyanin, effectively maintaining a constant
O2 affinity of whole hemolymph from megalopa to adult. Ontogenic changes in hemocyanin
subunits and changes in hexamer/2-hexamer ratios have also been reported in the American
lobster H. americanus (Olson 1991).
Changes in expression of specific hemocyanin subunits, with a concomitant shift in O2 affin-
ity, occur in response to nondevelopmental, external stimuli as well and have been studied at the
protein level. In the amphipod Chaetogammarus marinus, an increase in one and maybe more of the
eight hemocyanin subunits in response to environmental salinity caused an increase in O2 affinity
(Spicer and Hodgson 2003). Hemocyanin of the blue crab Callinectes sapidus showed a decrease in
three of its six subunit types and a resulting increase in the ratio of 1-hexamers to 2-hexamers after
exposure to environmental hypoxia (deFur et al. 1990, Mangum et al. 1991, Mangum 1997b). The
1-hexamers had a higher O2 affinity and lower cooperativity than did the 2-hexamers. Molecular
studies on C. sapidus have confirmed the effect of environmental hypoxia as a stimulus of hemo-
cyanin subunit expression (Brouwer et al. 2004, Brown-Peterson et al. 2005). Environmental tem-
perature may be another effector of hemocyanin subunit expression, as evidenced by a shift in
expression of the linker subunit in hemocyanin of the crayfish Astacus leptodactylus (Decker and
 Oxygen Transport Proteins in Crustacea 377

Foll 2000). Animals acclimatized to higher temperatures showed a decrease in both linker subunit
and 2-hexamer hemocyanin. When juvenile C. magister were raised in varying regimes of tempera-
ture and food level, those raised in warm water had higher 1-hexamer hemocyanin concentrations
than did those in cold water (Terwilliger and Dumler 2001). Levels of the 2-hexamer hemocyanin
were relatively unresponsive to differences in food and temperature. The instar timing of the devel-
opmental switch from juvenile to adult hemocyanin subunit composition in C. magister was altered
by both nutritional state and water temperature, indicating that ontogeny of gene expression can
be altered by environmental conditions. External parameters clearly can affect hemocyanin subunit
expression, even though identifying the precise stimulus or combination of factors—O2 level, pH,
organic or inorganic ions, temperature—can be difficult. The epigenetic mechanisms of pheno-
typic plasticity in crustacean hemocyanins are unknown, although in Drosophila (another arthro-
pod), piRNAs have been shown to shut down genes, and modifier proteins add methyl groups
to histones (Huang et al. 2013, Saey 2013). Whether environmentally induced epigenetic changes
in hemocyanin expression can be transgenerationally inherited is also unknown. Global climate
change and ocean acidification will prove challenging to crustaceans trying to obtain O2 and main-
tain sufficient calcium in their exoskeletons. Both genetic and epigenetic processes may participate
in the evolution of hemocyanin structure and function.

Hemocyanin Concentration

Many measurements of changes in total hemocyanin concentration have been reported (Giomi
and Beltramini 2007). Major determinants of hemocyanin concentration in the hemolymph are
molt stage and nutrition, and both factors must be accommodated into the experimental design
for meaningful interpretation of results. In addition, valid concentration measurements must dis-
tinguish between hemocyanin and cryptocyanin hexamers. During the molt cycle, circulating
levels of hemocyanin and cryptocyanin vary greatly from a gradual increase in premolt to their
highest levels just before ecdysis, followed by almost undetectable levels immediately after ecdysis
in both juvenile and adult crabs (Terwilliger et al. 2005, Terwilliger 2011). In the adult crab, hemo-
lymph cryptocyanin disappears after ecdysis until the following premolt, months later, whereas
hemocyanin levels resume almost immediately after molting. These changes in hemocyanin and
cryptocyanin expression as a function of the molt cycle have been confirmed at the subunit level
in C. magister by M.R. Phillips using a cDNA microarray and characterizing global transcription
patterns (Phillips 2007, Stillman et al. 2008), as have gene expression studies in Portunus pelagicus
(Kuballa et al. 2011). The potent influence of the molt cycle on hemocyanin concentration is wide-
spread across crustaceans, including krill Meganyctiphanes norvegica (Spicer and Stromberg 2002).
Adaptive changes in hemocyanin levels are usually attributed toward maintaining O2 transport
needs. In this context, the sudden drop in hemocyanin levels just before ecdysis seems counterpro-
ductive. Perhaps some of the high premolt levels of hemocyanin are destined for epidermal seques-
tration. There, the hemocyanin molecules could be mobilized to provide phenoloxidase-related
functions for the new postmolt exoskeleton. Localization of a prophenoloxidase-activating
enzyme in the epidermis of the blue crab C. sapidus (Buda and Shafer 2005) is consistent with
this provocative model, as is the discovery of a phenoloxidase-active hemocyanin in the crayfish
gastrolith (Glazer et al. 2013, see the section “Hemocyanin Function”). Alternatively, the decrease
in synthesis and concentration of hemocyanin at ecdysis might protect against excess O2 uptake
and reactive O2 species production, since O2 could more readily diffuse across the unsclerotized
new exoskeleton. In addition to molt stage and food availability, other factors invoked as modify-
ing hemocyanin concentration include gender, collecting site, hypoxic exposure, low salinity, and
temperature (Giomi and Beltramini 2007).
378 Nora B. Terwilliger

CRUSTACEAN OXYGEN TRANSPORT PROTEINS: HEMOGLOBINS

Hemoglobin Distribution

Hemoglobin expression is limited in the Arthropoda. Neither Chelicerata nor Myriapoda are
known to express circulating hemoglobin. Hemoglobins are present in Hexapoda, but they occur
mostly in noncirculating cells and rarely as O2 transporters (Burmester and Hankeln 2007). In
the Crustacea, hemoglobins are extensively distributed in Branchiopoda, and they are present in
Copepoda, Ostracoda, some parasitic Cirrepedia, and several Malacostraca (Terwilliger 1992, 1998,
Hourdez et al. 2000, Weber and Vinogradov 2001, Zeis et al. 2003).
The predominance of hemocyanins in Malacostraca versus hemoglobins in Branchiopoda has long
intrigued biologists. A  resurgence of interest in crustacean hemoglobins since 2001, especially with
publication of the draft genome of Daphnia pulex and related studies (Colbourne et al. 2011), warrants
a full review. Here, space allows only a summary of some of the more tantalizing findings. Like hemo-
cyanins, crustacean hemoglobins that function in O2 transport are large, multi-subunit molecules that
circulate in the hemolymph. No crustacean erythrocytes have been described. The hemoglobin com-
plexes exhibit much variability in O2 affinities, cooperativities, Bohr effects, and responses to allosteric
modulators including divalent cations. The only Cirripedia with a significant amount of circulating O2
transport protein are the parasitic rhizocephalan barnacles, primarily Briarosaccus callosus, whose host
is the king crab (Shirley et al. 1986, Terwilliger et al. 1986). Both the externa and interna of this large rhi-
zocephalan are filled with a bright red hemoglobin. The giant hemoglobin molecules circulate between
the externa that protrudes outward from the ventral thoracic-abdominal junction of the crab, and the
interna, whose thin-walled rootlets are bathed in crab hemocyanin inside the crab (Terwilliger 1998).
Hemoglobin is also present in smaller rhizocephalans Peltogaster pagurus and Peltogasterella gracilis,
whose host, the hermit crab Pagurus samuelis, like the king crab, has a circulating hemocyanin (Torchin
1994). Oxygen affinities of the rhizocephalan hemoglobins are higher than those of their hosts’ hemo-
cyanins, indicating an O2 transfer system from host to parasite that ensures an ample supply of O2 for
the rhizocephalan embryos developing in the externa.

Hemoglobin Phenotypic Plasticity, Hypoxia, and HIF

Crustacean hemoglobins have been studied most thoroughly in the Branchiopoda, one of the
classes touted as a potential sister group to the Hexapoda, along with Remipedia and Malacostraca.
Branchiopods and their hemoglobins are models for phenotypic responses to abiotic environmental
fluctuations, especially hypoxia (Kimura et al. 1999, Guadagnoli et al. 2005, Zeis et al. 2009), and, in
Daphnia, the rise and fall of hemoglobin levels in response to O2 availability has been shown to be
hypoxia-inducible factor (HIF) dependent (Gorr et al. 2004, Hoogewijs et al. 2007). Genomic analysis
of the 11 hemoglobin genes in D. pulex and seven in D. magna teases apart the tandem gene duplica-
tion and subsequent differentiation, in concert with proteome analysis of the acclimatory responses of
D. pulex to environmental changes (Colbourne et al. 2011). Demonstration of the eco-responsiveness
of these magnificently coordinated multi-subunit hemoglobins and other expanded gene families in
Daphnia promise to fuel more comparative physiological genomics studies in Crustacea and enhance
our comprehension of how and why subunit heterogeneity and phenotypic plasticity are maintained.

CO-EXPRESSION OF HEMOCYANIN AND HEMOGLOBIN

The majority of crustaceans express either hemocyanin or hemoglobin, but there are excep-
tions. First, although all Branchiopods have an extracellular hemoglobin, Artemia franciscana
and Triops longicaudatus (and probably others) also express phenoloxidase, evidence they have
 Oxygen Transport Proteins in Crustacea 379

retained the hemocyanin gene family in addition to hemoglobin (Terwilliger and Ryan 2006).
Second, the cyamid amphipod Cyamus scammoni (Malacostraca, subclass Eumalacostraca,
Peracarida), commensal on the gray whale, has both circulating hemoglobin and hemocyanin
(Terwilliger 1991, 2008b). This is the first example of hemoglobin in a Malacostraca. Most
amphipods have only hemocyanin. Similar to other peracaridans, the “whale rider” lacks a
separate hemocyte prophenoloxidase, so the hemocyanin probably also functions as a phe-
noloxidase. Third, a membrane-bound hemoglobin has been described in the gills of the
green crab C. maenas (Ertas et al. 2011). Just like other brachyuran crabs, C. maenas has 1- and
2-hexamer hemocyanin circulating in its hemolymph. The hemoglobin does not function in
O2 transport, but the authors propose that it may protect cell membrane lipids from reactive
O2 species, similar to bacterial membrane-bound hemoglobins. Phylogenetic analysis indi-
cates that the C. maenas membrane-bound hemoglobin does not cluster with the extracellular
circulating hemoglobins of the Branchiopoda, insect globins, or expression sequence tagged
(EST) tick (Chelicerata) globins. Its location in the gills may be analogous to hemoglobins in
insect tracheal cells. This surprising finding should stimulate further database searches and
experiments that may reveal the widespread presence of similar membrane-bound hemoglo-
bins among the crustaceans, insects, and chelicerates and lead to functional analyses. Today,
multiple functions in addition to O2 transport are recognized for both hemoglobins and hemo-
cyanins (Decker and van Holde 2011). Genes for both families of O2-binding proteins in one
species are probably present throughout the Arthropoda, including the crustaceans, and will
be identified along with potential expression patterns in synchrony with the development of
emerging technologies.

FUTURE DIRECTIONS

New insights about the phylogenetic relatedness of hemocyanin between arthropods and
mollusks stem from recognition of structural similarities among an arthropod hemocyanin
subunit (Limulus hemocyanin subunit II), an unusual C-terminal extension of one func-
tional unit of a molluskan hemocyanin, KLH-h (Megathura crenulata, keyhole limpet), and
a type 1 copper protein from a cucumber ( Jaenicke et al. 2010). The cucumber basic protein
(CBC) has one copper-binding site and is a member of the cupredoxin family, a phytocyanin
or plantacyanin (Guss et al. 1996). Another cupredoxin, the bacterial CopC protein, has two
monocopper-binding sites (Arnesano et al. 2002) and is structurally correlated with domain
3 of arthropod hemocyanin. Jaenicke and colleagues propose that both arthropod and mol-
luskan hemocyanins evolved from ancestral tyrosinases, each with two copper-binding sites,
to which the cupredoxin-like domains were added to enhance copper loading onto the active
sites (Fig. 11.7). The ancestral tryosinases were considered to have evolved from a single last
common ancestor. An additional domain (domain 1 in arthropod hemocyanin) was added
for specificity of O2 binding. As the two hemocyanins continued to evolve in the two phyla,
acquiring multiple subunits and functional properties, the cupredoxin-like domain acquired
new functions and lost its monocopper binding site. Each arthropod hemocyanin subunit still
maintains the cupredoxin-like domain 3, whereas the molluskan hemocyanin subunit adds it
only to the final functional unit of the long subunit. This co-evolutionary pattern of addition
and subsequent functional loss of a copper-loading domain is remarkable because it is based
on the concept of the two hemocyanins evolving congruently from ancestral tyrosinases that
had themselves evolved from a last common ancestor. The model is consistent with the pat-
terns of gene duplication and preservation by entrainment that describe the Daphnia genome
(Colbourne et al. 2011) and the concept of protein evolution by domain rearrangements, the
emergence of new domains, and the loss of old ones (Moore and Bornberg-Bauer 2012).
380 Nora B. Terwilliger

Primordial
tyrosinase

Ancestral
tyrosinases

Shielding Cupredoxin-like Shielding

domain domains domain

Molluskan
Arthropod hemocyanin
hemocyanin
D1
#2 C
C
#3 D1
D3 D2
#1 N
N
FU-h C D2
N
FU-a to FU-g

Fig. 11.7.
Tracing the evolution of arthropod and molluskan hemocyanins from their putative common ancestor, a pri-
mordial tyrosinase. The oxygen-binding domain (white) of arthropod and molluskan hemocyanin presumably
evolved independently from ancestral tyrosinases. To enhance efficient copper loading to the active site in
the oxygen-binding domain, a cupredoxin-like domain (dark gray) with an additional monocopper center was
added. A high specificity for oxygen was established by an additional domain (light gray), which shields the
binuclear copper site from regular tyrosinase substrates but still allows entry and exit of oxygen molecules.
Slight tilting movements of this shielding domain, which opens and closes the narrow oxygen diffusion chan-
nel, added allosteric regulation. The cupredoxin-like domain eventually assumed new functions in assembly
and allosterism of the hemocyanin multimers, and its monocopper center was lost. Today, the cupredoxin-like
domain is still ubiquitous in the 75 kDa arthropod hemocyanin subunits, whereas in molluskan hemocyanin
it is restricted to FU-h, the tail of the 400 kDa subunit. Black dots, copper-binding units. Reproduced from
Jaenicke et al. (2010), with permission from The Biochemical Society.

Integrating the new tools of contemporary biology, genomics, proteomics, metabolomics,

microarrays, and modern technologies with ecologically relevant studies that address multiple envi-
ronmental factors promises a treasure box full of potential answers about how animals adapt and
evolve, as the Daphnia genome study has revealed. We anticipate similar studies in more crustaceans,
recognizing that relying on only a few model organisms in the past has been both beneficial and lim-
iting. Explanations about regulation of these processes are tantalizingly close as well. Regulation of
phenotypic plasticity in crustacean hemoglobin expression by HIF has been described, and we are
moving toward a better understanding of a regulatory role of HIF in hemocyanin expression and
 Oxygen Transport Proteins in Crustacea 381

function (Li and Brouwer 2007, Hoogewijs et al. 2007, Terwilliger 2008a, Soñanez-Organis et al.
2009, Gorr et al. 2010).

CONCLUSIONS

Oxygen transport proteins assist in the challenge of obtaining sufficient O2, especially in an arthro-
pod whose body, including the gills, is covered by a chitinous exoskeleton. Hemocyanins and
hemoglobins, specialized to bind O2 in regions of high concentration and release it in regions of
lower concentration, circulate in the hemolymph of crustaceans and provide a molecular means to
sustain the metabolic needs of the organism. In Crustacea, both the copper-based hemocyanin and
the iron-heme-based hemoglobin are large extracellular molecules composed of individual poly-
peptide chains that self-assemble into species-specific aggregates. Creative studies using technolog-
ical advances have enhanced our understanding of key structural features in both the assembly and
function of these molecules. High degrees of subunit heterogeneity in the two types of O2 trans-
port proteins provide opportunities for highly cooperative O2 binding, a wide range of O2 affinities,
and phenotypic plasticity. New models have resulted from studies that integrate conformational
changes in structure with functional properties. Other roles for hemocyanin and hemoglobin are
surfacing as well.
Although crustacean O2 transport proteins provide excellent examples of gene duplication and
diversification, we await precise descriptions of how the functional expression of multiple hemo-
cyanin or hemoglobin genes is coordinated during the molt cycle, in response to temporary “dead
zones” of hypoxic waters, or under immune challenge. As we search to understand the roles of
phenotypic plasticity, as well as the initiation of novel phenotypes in response to issues such as
climate change, ocean acidification, global nutritional needs, and health issues, hemocyanin and
hemoglobin will continue to provide experimental model systems.

ACKNOWLEDGMENTS

I acknowledge Charlotte Mangum, Steve Morris, Bob Terwilliger, and David Towle for their inspir-
ation and appreciation of balancing form and function, molecule and organism. I am grateful to
students and colleagues around the world who have contributed to these studies on invertebrate
hemocyanins and hemoglobins through stimulating discussions, collaborative research, and valued
friendships. I thank the editors, Ernie Chang and Martin Thiel, for their invitation to participate in
this project.

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12
ENERGETICS AND METABOLIC REGULATION

Ana Gabriela Jimenez and Stephen T. Kinsey

Abstract
Crustaceans exploit diverse habitats and face numerous environmental challenges. This chapter
reviews mechanisms by which crustaceans maintain energy homeostasis in the face of physical and
physiological stresses. The basic metabolic pathways of energy metabolism present in higher ver-
tebrates also function in crustaceans, with some important differences. Crustaceans store major
metabolic fuels in the form of glycogen, protein, and several types of lipids, and muscle and hepato-
pancreas are their most important depots. Biological energetic and environmental challenges cause
an increase in ATP demand. Although maintaining homeostasis under variable environmental con-
ditions may lead to increased energy expenditure, lack of tolerance to environmental changes is not
necessarily due to energetic constraints. Current understanding of crustacean energy homeostasis
provides a framework for future work addressing the likely effects of climate change on species
distributions and invasions and novel approaches for enhancing aquaculture production of com-
mercially viable species.

INTRODUCTION

The maintenance of energetic homeostasis is a prerequisite for all organisms. Crustaceans, like
other animals, use carbohydrates, lipids, and proteins as principal fuel molecules, as well as building
blocks for intra- and extracellular structural components. These molecules are either synthesized
or acquired by ingestion, and some can be oxidized to produce adenosine triphosphate (ATP).
Storage forms of carbohydrates, lipids, and proteins play an important role in maintaining energy
balance, particularly during periods of reduced ATP supply or increased ATP demand. Changes in
synthesis or mobilization of these fuels can therefore occur during changes in physiological state,
such as during locomotion, reproduction, or molting, which increase ATP demand, or during

391
392 Ana Gabriela Jimenez and Stephen T. Kinsey

starvation, which limits fuel supply. Similarly, the cellular and whole-body energy state can be
altered by changing environmental conditions, including fluctuations in temperature, O2 availabil-
ity, or salinity.
Many of the mechanisms by which crustaceans respond to the specific physiological states and
environmental conditions just mentioned are covered in detail in other chapters of this volume.
Therefore, this chapter considers only aspects of energy homeostasis, with an emphasis on the con-
sequences and metabolic costs associated with physiological challenges, and how fuel reserves are
allocated to maintain or restore energy balance at the cellular, tissue, and whole-animal level. We
focus largely, but not exclusively, on decapod crustaceans and avoid substantial discussion of model
crustaceans such as Artemia or Daphnia, for which there is an abundance of literature. To limit the
scope of this chapter, we also concentrate on more recent literature. Older studies are discussed in
detailed reviews elsewhere (Cameron and Mangum 1983, Chang and O’Connor 1983, Claybrook
1983, Gilles and Pequeux 1983).

ENERGY METABOLISM

Early work on crustacean biochemistry centered on characterizing metabolic pathways, gener-

ally by determining whether enzymes and regulatory mechanisms identified in mammals were
also present in crustaceans, and many of these studies focused on the oxidation and synthesis of
metabolic fuel molecules. Chang and O’Connor (1983) reviewed the synthesis and breakdown of
carbohydrates and lipids, while Claybrook (1983) evaluated early studies on protein and amino
acid metabolism. Although there are some obvious differences in crustacean biochemistry com-
pared to that in mammals (e.g., crustacean cuticle formation), the major enzymatic pathways of
energy metabolism and biosynthesis of fuel molecules found in mammals are present in crusta-
ceans (Fig. 12.1).

Major ATP-Producing and Consuming Pathways

Fuel Molecules

Carbohydrates
As in most vertebrates, D-glucose is the principal monosaccharide present in the hemolymph of
crustaceans, and it serves several purposes, including the synthesis of mucopolysaccharides, chitin,
nicotinamide adenine dinucleotide phosphate (NADPH), and glycogen, as well as the formation
of pyruvate (Fig. 12.1). Glucose in hemolymph comes from the direct absorption of dietary glucose
through hepatopancreatic and intestinal epithelial cells or from peripheral tissues such as the hepa-
topancreas and muscle, where it is stored as glycogen or synthesized by the gluconeogenic pathway.
Glucose levels in hemolymph are tightly controlled, particularly by the crustacean hyperglycemic
hormone (CHH), a neuropeptide produced by the sinus gland of eyestalks (Verri et  al. 2001).
Relatively little free glucose occurs in cells because it is rapidly converted to glucose-6-phosphate
by hexokinase (HK). Then, glucose-6-phosphate follows one of three primary fates: glycogene-
sis, glycolysis, or the pentose-phosphate pathway (Chang and O’Connor 1983, Santos and Keller
1993, Oliveira et al. 2004). Glucose in crustaceans is stored in the form of glycogen, and the larg-
est depots are in the hepatopancreas and muscle (Vinagre and Da Silva 1992, Oliveira et al. 2003,
Buckup et al. 2008). The net storage mobilization of glucose depends on a variety of biotic and
abiotic factors, including molt stage, diet, nutritional state, season, salinity, and dissolved O2, which
are discussed in later sections (Vinagre and Da Silva 1992, Buckup et al. 2008). However, in general,
 Energetics and Metabolic Regulation 393

Glycogen

Glycogen synthesis Glycogenolysis

Glucose Triacylglycerol
Gluconeogenesis Glycolysis
Serine, Glycine Lipid synthesis Lipolysis
ATP, e–
Cysteine

Alanine Pyruvate Fatty acid

Fatty acid
e– synthesis
Lactate B-oxidation
of fatty acids
Acetyl-CoA

e–
Citrate
Electron transport
Aspartate e– and ATP synthase
Oxaloacetate
Asparagine
GTP ATP
Citric Acid Cycle CO2
α-Ketoglutarate

Protein
Glutamate
Glutamine
Proline
Essential Amino Acids
Arginine Methionine
Histidine Phenylalanine Tyrosine
Isoleucine Threonine
Leucine Tryptophan Ariginine kinase
Lysine Valine Arginine phosphate + ADP Arginine + ATP

Fig. 12.1.
Overview of energy metabolism in crustaceans. Storage forms of major fuel molecules are in large type, metabolic
pathways are in italics, and major metabolites are also shown. Pathways associated with adenosine triphosphate
(ATP) production are designated with solid lines. These pathways include glycolysis and fatty acid oxidation,
both of which partially oxidize carbon substrates and feed acetyl-CoA into the citric acid cycle, where the acetyl
groups are fully oxidized to CO2. These pathways yield the direct production of ATP as well as electrons (e–) that
are carried to the electron transport system by nicotinamide adenine dinucleotide (NADH) and flavin adenine
dinucleotide (FADH2). Electron transport in the mitochondria leads to a proton motive force, which is used
to make ATP via ATP synthase. The dashed lines show biosynthetic pathways, as well as other pathways that
can interconvert molecules in crustaceans. Amino acids are oxidized to produce ATP by being converted to an
intermediate of glycolysis or the citric acid cycle. In addition to these pathways, the arginine kinase reaction can
provide ATP by transferring a phosphate from the phosphagen arginine phosphate to ADP.

when hemolymph glucose levels are low, glycogen stored in tissues is broken down into glucose and
mobilized into the hemolymph, whereas when glucose levels are high, glycogen synthesis occurs
via gluconeogenesis (Santos and Keller 1993).
Under aerobic conditions, glucose is fully oxidized to CO2 through glycolysis and the citric acid
cycle, and ATP is produced via oxidative phosphorylation (Chang and O’Connor 1983). Under
anaerobic conditions, lactate is the exclusive metabolic end-product in crustaceans, as in vertebrates
394 Ana Gabriela Jimenez and Stephen T. Kinsey

(Gade and Grieshaber 1986). Lactate accumulates when ATP demand exceeds the supply available
by oxidative metabolism, either because of increased demand, such as occurs in muscles during
intensive exercise (England and Baldwin 1983, Morris and Adamczewska 2002, Johnson et al. 2004,
Kinsey et al. 2005, Hardy et al. 2006, Jimenez et al. 2008), or due to reduced supply, such as during
anoxia (Oliveira et al. 2001, Abe et al. 2007, Holman and Hand 2009).

Lipids
Lipids play a major role in crustacean metabolism and in cellular and subcellular membrane struc-
ture. The principal site of lipid storage in crustaceans appears to be the hepatopancreas (Chang
and O’Connor 1983, Kucharski and Da Silva 1991a, Muriana et  al. 1993, Garcia et  al. 2002). The
catabolism of fatty acids appears to occur via β-oxidation, whereas the synthesis of fatty acids, tria-
cylglycerols, and phospholipids also occurs via the same pathways found in mammals (Fig. 12.1;
Chang and O’Connor 1983). The principal circulating lipids in these animals are phospholipids, in
contrast with vertebrates, and the hemolymph also contains triglycerides and sterols, all of which
are packaged in high-density lipoproteins or very high-density lipoproteins, although there is only
a small fraction of free fatty acids (Yepiz-Plascencia et al. 2000, 2002). Additionally, the principal
storage form of lipids in some crustaceans is wax esters, although in most crustaceans, including
decapods, it is triacylglycerols (Chang and O’Connor 1983, Chang 1995). Lipid synthesis follows
the same pathways as in vertebrates, and lipid droplets accumulate in specific tissues to serve as
energy stores, particularly during reproduction.

Amino Acids
Biosynthetic and degradation pathways for amino acids in crustaceans are largely the same as those
of mammals (Fig. 12.1; Claybrook 1983). Although most amino acids are in proteins, the concen-
tration of free amino acids in most crustaceans is several-fold higher than in vertebrate tissues.
There are 10 essential amino acids for crustaceans, nine of which are the same as in humans (in
crustaceans, arginine is also considered essential). Glucose can supply carbon skeletons for nine of
the nonessential amino acids via intermediates of glycolysis and the citric acid cycle. In glycolysis,
3-phosphoglycerate and pyruvate are precursors to amino acids, whereas in the citric acid cycle,
α-ketoglutarate and oxaloacetate are substrates for amino acid synthesis. Tyrosine is derived from
phenylalanine, which is an essential amino acid. The pathways by which amino acids are degraded
also are thought to be closely related to those found in vertebrates, where glucogenic amino acid
carbon skeletons are converted to pyruvate or a citric acid cycle intermediate, while ketogenic
amino acid carbon skeletons are converted to acetyl-CoA or acetoacetyl-CoA, thus facilitating their
oxidation for ATP production or conversion to glucose or lipids. Nitrogen from amino acids is
excreted as ammonia, uric acid, or urea, although there does not appear to be a true urea cycle in
crustaceans (Claybrook 1983).

Tissue Specificity

Hepatopancreas and muscle are the major storage depots for glycogen owing to the relatively
large mass of these tissues, whereas gills and gonads are smaller reservoirs (although gonad gly-
cogen reserves are dependent on the reproductive stage; Fig. 12.2; Chang and O’Connor 1983,
Vinagre and Da Silva 1992, 2002, Oliveira and Da Silva 1997, Oliveira el al. 2003, Antunes et al.
2010). In the absence of adipose tissue, the hepatopancreas is the principal storage site for lipids
(Chang and O’Connor 1983, Schmitt and Santos 1993), whereas muscle is the main protein stor-
age location in crustaceans (Claybrook 1983, Buckup et al. 2008). Although most of the metabolic
pathways associated with energy metabolism are similar to those in vertebrates, the metabolic
 Energetics and Metabolic Regulation 395

Muscle
Glucose Gills
Glucose
Glycogen Glycogen
Lipid Lipid
Protein
Protein
Hemolymph
Glucose Ovaries (during
Hepatopancreas
Lipid reproduction)
Glucose
Protein Glucose
Glycogen Glycogen
Lipid Lipid
Protein Protein

Fig. 12.2.
Generalized schematic of the relative importance of major tissues in fuel storage. The size of the box indicates
the relative size of the tissue, and the size of the type indicates the importance of that tissue for each type of
fuel molecule. These relationships are highly species specific, and this diagram is not meant to be quantitative.

capacity for these processes is sometimes more evenly distributed among the major tissues in
crustaceans. For instance, gluconeogenesis appears to occur in hepatopancreas, gills, muscle, and
hemocytes, rather than being confined to specific tissues (Lallier and Walsh 1991, Oliveira et al.
2004). However, the hepatopancreas does appear to be the principal site of lipid and lipoprotein
synthesis (Walker et al. 2003).

Mechanisms of Regulation

Energy metabolism responds to environmental challenges in part through changes in gene expres-
sion. Hyperglycemia is a response to various kinds of stress, such as changes in temperature and
pH, and is linked to increased expression of CHH (Chang et al. 1999). Changes in temperature, O2,
or metal ion concentrations can lead to elevation in the expression of heat shock proteins (HSPs),
which help preserve the function of other proteins (Ryan and Hightower 1994), as well as elevate
the expression of other stress proteins (Willsie and Clegg 2001, Gorr et al. 2004). Hypoxia leads to
significant decreases in transcription of superoxide dismutase, hemocyanin, and ribosomal genes in
blue crabs (Callinectes sapidus) after 5 days of exposure. The decrease in two of the three ribosomal
cDNAs analyzed suggests that protein synthesis may have been slowing down in these animals
during prolonged hypoxia (Brouwer et al. 2004). In the grass shrimp, Palaemonetes pugio, severe
chronic hypoxia leads to an initial upregulation of the mitochondrial genes associated with electron
transport, whereas long-term exposure leads to a downregulation of both transcript and protein lev-
els (Brouwer et al. 2008). Hyposmotic stress causes an increased expression of the enzyme carbonic
anhydrase, which plays a role in osmoregulation in gills of the green crab Carcinus maenas (Serrano
and Henry 2008) and the tiger shrimp Penaeus monodon (Pongsomboon et al. 2009), as well as the
activity of the citric acid cycle enzyme citrate synthase in gills of C. sapidus (Kinsey et al. 2003).
Changes in physiological state also alter the expression of genes involved in the energy metabo-
lism of crustaceans. In the shrimp Metapenaeus ensis, oocyte maturation is associated with increased
expression of glyceraldehyde-3-phosphate dehydrogenase and arginine kinase (AK) transcripts,
which are involved with ATP production via glycolysis and phosphagen hydrolysis, respectively
(Sze Lo et al. 2007). During molting, there is a substantial increase in expression of transcripts for
cuticular proteins in the swimming crab Portunus pelagicus (Kuballa et al. 2011), and in the Antarctic
krill Euphausia superba (Seear et al. 2010) and P. pelagicus, the increased demand for ATP during
396 Ana Gabriela Jimenez and Stephen T. Kinsey

cuticle formation is associated with an increased expression of mitochondrial genes (Kuballa et al.
2011). Jiang et al. (2009) examined the relationship between the expression of genes that are associ-
ated with nutritional status and those that are associated with reproduction in hepatopancreas and
testis of the mitten crab, Eriocheir sinensis. With respect to energy metabolism, these authors found
that nutritional state altered the expression of AK, which is an ATP buffer important during periods
of increasing ATP demand.
In addition to changes in gene expression, energy metabolism is also controlled hormonally
and by allosteric regulation of enzyme activity, the latter usually in the same manner as in mam-
mals (Claybrook 1983, Chang and O’Connor 1983). For instance, CHH plays a key role in regulat-
ing tissue synthesis and release of glucose and lipids from hepatopancreas and muscle (Chang and
O’Connor 1983, Santos et al. 1997). Glycogen breakdown in crustaceans is regulated by the enzyme
glycogen phosphorylase, and activity of this enzyme is controlled by phosphorylation state and
by adenosine monophosphate (AMP; Kamp 1989). The glycolytic enzymes, phosphofructokinase
and pyruvate kinase, are allosterically regulated by the adenylates, AMP and adenosine diphosphate
(ADP) (activators) and ATP (inhibitor) (England and Baldwin 1985). The citric acid cycle enzyme,
citrate synthase, is allosterically inhibited by ATP in crustaceans, providing a negative feedback
that slows metabolism when energy needs are met (Vetter 1995). Glutamate dehydrogenase, which
regulates the production of ammonia during amino acid breakdown, is inhibited by nicotinamide
adenine dinucleotide (NADH), the citric acid cycle intermediate α-ketoglutarate, and guanosine
triphosphate (GTP), but is activated by ADP (Claybrook 1983). These and other examples of allo-
steric regulation are essentially the same as in mammals, indicative of the highly conserved nature
of pathways of energy metabolism.

Conditional Responses of Metabolism

Responses to Physiological State

Locomotion/Activity
Locomotion modes for crustaceans include walking (aquatic and terrestrial), swimming, and
tail-flipping (in lobsters and shrimp). For crustaceans, walking and swimming have been divided
into short-term high-speed bursts leading to rapid fatigue, prolonged high- to moderate-speed
movement leading to eventual fatigue, and long-term low-speed activity that can be sustained for
extended periods (Wood and Randall 1981). Tail-flipping entails one or more abdominal flexions
that are used as an escape response and therefore always are rapid, high-speed movements for short
durations. As in other taxa, burst locomotion is primarily fueled by anaerobic metabolism and
powered by muscle fibers with few mitochondria (usually <3% of cell volume is mitochondria).
Prolonged locomotion in crustaceans is usually characterized by an increase in O2 consumption
and some accumulation of lactate and is powered by aerobic muscle fibers that may have more
than 25% of their cell volume devoted to mitochondria (Houlihan and Innes 1984, Boyle et al. 2003,
Johnson et al. 2004, Hardy et al. 2009, Kinsey et al. 2011).
Most species of crustaceans have a diversity of muscle fiber types that reflect functional demand
(Tse et  al. 1983, Silverman et  al. 1987, Stokes and Josephson 1992, Hardy et  al. 2009, 2010). The
metabolic range of muscle function has been well characterized in the blue crab C. sapidus, which is
a fast and efficient swimmer that exhibits both burst-escape locomotion to avoid predators and sus-
tainable aerobic locomotion (Booth and McMahon 1992). Both types of locomotion are powered
by a large mass of “backfin” muscles attached to the paddle-like fifth pereopods, which move in a
sculling-type motion to propel the animal sideways while swimming (White and Spirito 1973). The
swimming muscle responsible for these distinct forms of locomotion are classified into two major
 Energetics and Metabolic Regulation 397

metabolic categories: so-called “dark” aerobic fibers that power sustained swimming and a large
mass of anaerobic “light” fibers that are used for burst swimming (Tse et al. 1983; see Chapter 4
in this volume). The aerobic fibers are noticeably yellow-brown in color due to the presence of a
dense population of mitochondria and are highly perfused ( Johnson et al. 2004, Hardy et al. 2009).
The dark and light fibers in crustaceans are functionally analogous to the red and white fibers of
fishes, but the aerobic fibers are not red in color since crustaceans lack myoglobin. The dark fibers
are highly subdivided and appear to have arisen from light fiber precursors during the evolution of
sustained exercise in the swimming crabs (Tse et al. 1983, Hardy et al. 2009, 2010). To accommodate
the demands of aerobic swimming, the fiber sarcolemmal membrane became highly invaginated,
permitting intrafiber perfusion and forming small, isolated metabolic functional units (fiber sub-
divisions) that have high mitochondrial density and short O2 diffusion distances. However, the
contractile functional unit remains the fiber as a whole because innervation patterns are identical in
the dark and light fibers. This separation of the metabolic (fiber subdivision) and contractile (fiber)
functional units in dark muscle reflect the importance of O2 diffusion in governing fiber structure,
whereas contractile function is not constrained by diffusion (Hardy et al. 2009, 2010). Similarly,
subdivided fibers are present in many crustaceans, and the extent of subdivision and mitochondrial
density reflects the demand for sustained contraction.
Burst contraction in crustacean muscles relies initially on AK, which catalyzes the transfer of a
phosphate from the phosphagen arginine phosphate (AP) to ADP, thus forming ATP (Fig. 12.1).
The AK/AP system is analogous to the creatine kinase/creatine phosphate system in vertebrates
and some other invertebrates (Ellington 2001). There is typically 30–40 mM AP in crustacean
light muscle, and, during successive burst contractions, the AP pools are nearly depleted (Fig. 12.3;
England and Baldwin 1983, Baldwin et al. 1999, Kinsey et al. 2005, Hardy et al. 2006, Jimenez et al.
2008). For additional contractions, ATP is supplied by anaerobic glycogenolysis, which is reflected
by the accumulation of lactate and depletion of glycogen, as well as by a reduced contractile speed
(Fig. 12.1; Booth and McMahon 1992, Milligan et al. 1989, Morris and Adamczeskwa 2002, Johnson
et al. 2004, Kinsey et al. 2005). The ATP, AMP, and ADP concentrations in decapod crustacean light
muscle are higher than in insects, and AP concentration is generally higher than is creatine phos-
phate in vertebrates, perhaps indicative of the highly anaerobic poise of these muscles (Beis and
Newsholme 1975, England and Baldwin 1983, Hill et al. 1991, Speed et al. 2001). Previous work has
demonstrated that factors limiting anaerobic capacity include the size of the phosphagen and gly-
cogen stores and sensitivity to anaerobic end-products (Baldwin et al. 1999). The glycogen content
of leg muscle in the terrestrial Christmas Island red crab Gecarcoidea natalis and other land crabs,
can become extraordinarily high when adequate food is available (Henry et al. 1994, Adamczewska
and Morris 1994, 2000) and appears to be crucial in supporting the elevated level of anaerobiosis
in G. natalis and several other species of crab (Henry et al. 1994). The large depletion of glyco-
gen and the ensuing glycolysis ultimately require the large flux of lactate from the muscles into the
hemolymph, as is also the case in other exercising decapods (Fig. 12.3; Henry et al. 1994). On the
other hand, large amounts of glucose mobilized from glycogen remained as glucose-6-phosphate to
directly fuel muscle glycolysis during exercise (Morris and Adamczewska 2002).
Although the metabolic processes that power burst contraction in crustaceans are similar to
those in vertebrates, metabolic recovery following contraction in crustaceans does not always fol-
low the vertebrate paradigm. Vertebrates rely exclusively on aerobic metabolism to power resyn-
thesis of creatine phosphate, and lactate does not accumulate following contractions (Kushmerick
1983, Meyer 1988), even in highly anaerobic fish white muscle (Curtin et al. 1997). In contrast, post-
contractile restoration of AP pools in crustaceans is largely powered by anaerobic glycogenolysis,
leading to glycogen depletion and lactate accumulation after contraction (Fig. 12.3; England and
Baldwin 1983, Boyle et  al. 2003, Johnson et  al. 2004, Kinsey et  al. 2005). Postcontractile lactate
accumulation in crustacean muscle appears to be a mechanism for accelerating certain phases of
398 Ana Gabriela Jimenez and Stephen T. Kinsey

35
40

Glycogen (µmol glucosyl/g)

30

Arginine Phosphate (mM)

30 25

20
20
15
10
10

0 5
0 20 40 60 0 200 400 600 800
16
30 Muscle
12 Hemolymph

Lactate (mM)
20 8
Pi (mM)

4
10
0

0
0 20 40 60 0 200 400 600 800
Time (min) Time (min)

Fig. 12.3.
Changes in locomotor muscle and hemolymph metabolites following a short-burst contraction in the blue
crab, Callinectes sapidus. Note the rapid initial change in arginine phosphate and inorganic phosphate (Pi)
due to high adenosine triphosphate (ATP) demand during the burst contractile period and then the slower
recovery. Glycogen is depleted, and lactate accumulates over a longer time course primarily after contraction,
indicating that anaerobic glycogenolysis is largely powering the recovery of arginine phosphate and Pi, as well
as other processes such as the restoration of intracellular pH (not shown). Aerobic metabolism is ultimately
responsible for the slow restoration of glycogen and lactate levels. Data from Boyle et al. (2003), Johnson et al.
(2004), and Kinsey et al. (2005).

the recovery process to facilitate additional high-force contractions because aerobic capacity is so
low in light muscle (Boyle et al. 2003, Johnson et al. 2004, Kinsey et al. 2005, Jimenez et al. 2008).
Furthermore, postcontractile lactate accumulation is much greater in the very large muscle fibers
of adults compared to the small fibers of juveniles, again indicating the increasing importance of O2
diffusion in limiting aerobic metabolic flux as fibers grow and diffusion distances increase (Boyle
et al. 2003, Johnson et al. 2004, Hardy et al. 2006, Kinsey et al. 2011).
Despite their reliance on anaerobic metabolism to power specific recovery processes, complete
recovery ultimately must depend on aerobic pathways. Furthermore, crustacean muscle fibers do
not appear to express a lactate transporter (Kinsey and Ellington 1996), and lactate efflux from
crustacean muscle is extremely slow (Milligan et al. 1989, Kinsey and Ellington 1996). The subse-
quent resynthesis of muscle glycogen, presumably from lactate (Milligan et al. 1989), and the res-
toration of intracellular pH (pHi) occurs in situ over a protracted time course (several hours) and
constitutes the aerobic phase of recovery (Kamp 1989, Milligan et al. 1989, Henry et al. 1994). The
restoration of high-energy phosphates and depleted O2 stores generally occurs more rapidly than
the removal of lactate (Morris and Adamczewska 2002). Although the fate of lactate in crustaceans
is still unclear, some possibilities for metabolic regulation of lactate accumulation after exercise
include its reincorporation into glycogen or oxidation in a variety of tissues (Ellington 1983, Hill
 Energetics and Metabolic Regulation 399

et al. 1991, Lallier and Walsh 1992, Oliveira and da Silva 2000, Hervant et al. 1999), excretion into
the environment (Head and Baldwin 1986, Hervant et al. 1999a), or endogenous glyconeogenesis
within muscles (Henry et al. 1994, Hervant et al. 1999).
Exercise at low rates can be powered almost exclusively by aerobic metabolism in crustaceans,
and, as intensity increases, the contribution of anaerobic glycolysis increases. Terrestrial crabs gen-
erally respond to exercise with a combination of elevated aerobic metabolism, further accompanied
by anaerobic lactate accumulation (Herreid and Full 1988, Full and Weinstein 1992). For example,
air-breathing crabs like the ghost crabs of the genus Ocypode exhibit large and rapid elevations in
aerobic metabolism matched to high endurance, whereas fiddler crabs of the genus Uca fatigue more
quickly and are unable to elevate O2 uptake to the extent of ghost crabs (Herreid and Full 1988).
Thus, as a general rule in crustaceans, the intensity of exercise is largely governed by the ability to
accelerate aerobic metabolism and glycolysis and to tolerate accumulated anaerobic end-products
(Full and Weinstein 1992, Adamczewska and Morris 2000, Weinstein 2001). Disruption of aerobic
metabolism directly influences exercise capacity. For example, C. sapidus injected with a sublethal
dose of Vibrio campbellii had a reduced aerobic capacity that led to a lower rate of O2 consumption
and higher lactate accumulation during exercise (Thibodeaux et al. 2009).
An interesting feature of crustacean muscles is that they are typically composed of very large
fibers (cells), and because this leads to large O2 diffusion distances that may constrain aerobic
metabolism, this property has long puzzled crustacean biologists (Kinsey et al. 2007, 2011). Jimenez
et  al. (2011) recently showed that large fibers provide a means of reducing basal metabolic cost
due to a lower surface area-to-volume ratio (SA:V) and therefore less membrane over which to
maintain the membrane potential via Na+/K+ -ATPase (NAK) transporter. In the American lobster,
Homarus americanus, adults have abdominal muscle fibers that have a twofold larger diameter (and
twofold higher SA:V) than juveniles, and this leads to a proportional twofold lower NAK activity
and cost of operating the NAK in resting muscle (Fig. 12.4). We have recently found that NAK cost
is proportional to fiber SA:V in muscle from a broad range of crustaceans and fishes, suggesting that
fibers are large to reduce whole-animal metabolic rate ( Jimenez et al. 2013).

Reproduction
The cost of reproduction can be direct, such as that associated with gamete production, or indi-
rect, such as that associated with courtship and mating behaviors or parental care/brooding. The
energetic expenditure associated with reproduction in female decapods is reflected by an increased
metabolic demand (Guadagnoli et  al. 2005), a doubling of food intake (Teshima et  al. 1986),
decreased growth rate, and increased susceptibility to predators (Berglund and Rosenqvist 1986).
Lipids and proteins appear to be the principal metabolic reserves mobilized during reproduction,
whereas glycogen is more important in fueling activity (Harrison 1990, Quackenbush 1994, Lee
and Walker 1995, Rosa and Nunes 2003, Vinagre et al. 2007, Antunes et al. 2010). For instance, in
the blue crab C. sapidus, lipid droplets, which form a minor component in immature ovaries, con-
stitute nearly a third of the total lipids in mature ovaries (Lee and Walker 1995). The origin of lipids
reaching the ovary is not fully understood. Lipids stored in the hepatopancreas have been shown to
be transported to the ovary during vitellogenesis (Harrison 1990). However, the amount of lipids
accumulated within the ovaries is greater than that stored in the hepatopancreas, suggesting some
lipid synthesis likely occurs within the ovaries and developing oocytes (Millamena and Pascual
1990, Khayat et al. 1994, Palacios et al. 2000). In addition, some lipid requirements of the develop-
ing ovary seem to be more dependent on the ingestion of dietary lipids than on hepatopancreatic
reserves.
Synthesis of several proteins, including enzymes and egg yolk proteins, are also important in
maturation and reproduction, and vitellogenesis is accompanied by yolk protein synthesis largely in
the form of high-density lipoproteins (HDL) and glycoproteins (Yehezkel et al. 2000). In decapods,
400 Ana Gabriela Jimenez and Stephen T. Kinsey

NAK activity (µmol/min/g)

0.016 30
0.014 25

Fiber SA:V (µm–1)

0.012
0.010 20
0.008 * 15 *
0.006 10
0.004
0.002 5
0.000 0
Juvenile Adult Juvenile Adult
Animal size class Animal size class

0.14 Na+/K+-ATPase
SR Ca 2+-ATPase
0.12 Transcription/Translation
ATP consumption rate (mM/min)

0.10

0.08

0.06

0.04
*
0.02

0.00
Juvenile Adult
Animal size class

Fig. 12.4.
The effect of fiber size on metabolic maintenance costs in abdominal muscle from the American lobster,
Homarus americanus. Fiber surface area-to-volume ratio (SA:V) was twofold lower in adults, and this resulted in
a twofold lower Na+/K+-ATPase (NAK) activity, and a twofold lower adenosine triphosphate (ATP) cost asso-
ciated with NAK function. In the lower panel, the black horizontal lines represent the total basal ATP demand
in the muscle, and three processes—transcription/translation, the sarcoplasmic reticulum Ca2+-ATPase,
and the NAK—account for nearly all of this. Of these processes, only the NAK cost is significantly different
between the size classes and has dependence on the SA:V because it is a sarcolemmal membrane protein. The
difference in the NAK cost accounts for most of the difference in cost between the juveniles and adults, indi-
cating the savings associated with the larger fibers in adults. The * indicate significant differences between size
classes. Data are from Jimenez et al. (2011).

the dominant HDLs are LP-I and LP-II (also known as lipovitellin), which are important in trans-
porting lipids from the hepatopancreas to peripheral tissues (Harrison 1990, Lee and Walker 1995,
Walker et al. 2003). In C. sapidus, LP-I has been shown to be synthesized in the hepatopancreas in
both larvae and adults. Free sterols also are abundant in developing ovaries, where they contrib-
ute to membrane structure and are precursors of hormones and steroids (Rosa and Nunes 2003,
Antunes et al. 2010).
However, in addition to the biosynthetic costs associated with reproduction, there may be
costs associated with parental care. Crustaceans may release eggs into the environment, brood the
embryos until hatching, or carry offspring in brood pouches after hatching. The cost of brood-
ing embryos has been examined because, among brooders, crustaceans are large animals and must
expend energy in maintaining an adequate O2 supply to their relatively large egg masses. In some
brachyuran crabs, the rate of O2 consumption can nearly double in brooding females, suggesting
 Energetics and Metabolic Regulation 401

60

Cardiac output (mL min–1)

50 * *

40 * *

30 *
Non-gravid
Gravid
Ovig/gravid
20
0 25 50 75 100 125 150
mm Hg

Fig. 12.5.
The effect of reproductive state and oxygen partial pressure (Po2) on cardiac output in the grass shrimp,
Palaemonetes pugio. Ovigerous/gravid females had a higher cardiac output than gravid females, which had in
turn higher rates than nongravid females, indicating the high aerobic cost of egg production and brooding.
Cardiac output also decreased with declining Po2, and the higher demand of egg production and brooding
reduces hypoxia tolerance because ovigerous/gravid females could maintain cardiac output at a Po2 as low as
75 mm Hg, gravid females as low as 50 mm Hg, and nongravid females as low as 15 mm Hg. The § indicates
significant differences between reproductive groups, and the * indicates significant differences from normoxia
(150 mm Hg). Data from Guandagnoli et al. (2005), with permission from John Wiley and Sons.

that the cost of brooding can be high, whereas in other species the costs are negligible (Fernández
et al. 2000, Taylor and Leelapiyanart 2001). Guadagnoli et al. (2005) showed that in the grass shrimp
P. pugio cardiac output and pleopod fanning frequency was highest in ovigerous females, indicating
the elevated cost of egg production and brooding. Ovigerous females were intermediate in cardiac
output, whereas nonovigerous females had the lowest metabolic demand (Fig. 12.5).

Molting
Molting is controlled hormonally, and the high energetic cost of molting is reflected by changes
in fuel storage and mobilization, as well as by changes in metabolic rate prior to and during the
molt. Oxygen consumption and heart rate increases during premolt and declines following ecdysis
(Penkoff and Thurberg 1982, Cockcroft and Wooldridge 1985, Kuramoto 1993), although metabo-
lism may remain elevated in early postmolt (Mangum et al. 1985). The increased metabolic costs
associated with molting are largely related to the net synthesis of protein, new cuticle formation,
and remodeling of tissues. For instance, in the swimming crab P. pelagicus, an analysis of transcript
expression revealed that genes associated with energy metabolism, such as NADH dehydrogenase,
cytochrome c oxidase, and ATP synthase, were upregulated during the premolt period, presumably
to meet the energy demands associated with molting. This is consistent with the increased expres-
sion of cuticular protein transcripts during molting (Fig. 12.6; Kuballa et al. 2011). A similar large
increase in transcript expression of 23 cuticular proteins was observed during molting in Antarctic
krill, E. superba, indicating the extensive metabolic cost associated with protein synthesis (Seear
et al. 2010).
Changes in protein, lipid, and carbohydrate content of the hepatopancreas have been seen
during the course of the molt cycle in several different species. Most notable is the pre-ecdysial
increase in lipid content and shifts in lipid classes in the hepatopancreas that have been recorded
in a number of species (Chang and O’Connor 1983, Jeckel et al. 1990, Chang 1995). This increase in
402 Ana Gabriela Jimenez and Stephen T. Kinsey

Fig. 12.6.
Changes in transcript expression during the molt cycle (M, molt; P, postmolt; I, intermolt; E, early premolt; L,
late premolt). Transcripts for mitochondrial proteins involved with energy metabolism, such as the adenosine
triphosphate (ATP) synthase nicotinamide adenine dinucleotide (NADH) dehydrogenase and cytochrome c
oxidase increase during premolt, presumably to provide the ATP needed for molting, whereas cuticle protein
transcripts are upregulated largely during the molt and in postmolt. Data from Kuballa et al. (2011), with per-
mission from BioMed Central.

premolt lipid content is likely due to an increased rate of fatty acid synthesis and an increased capac-
ity for esterification of ingested fatty acids forming triacylglycerols. For example, Bollenbacher et al.
(1972) demonstrated several decades ago that the premolt increase in lipid storage in the hepato-
pancreas paralleled an increase in enzyme activity associated with fatty acid synthesis. There is also
an increase in hemolymph glucose and hepatopancreas glycogen during premolt, which may reflect
both the energy needs associated with ecdysis as well as the usage of glucose as a precursor to chitin
(Chang 1995, Galindo et al. 2009). Large changes in protein expression and free amino acid levels
have also been observed in the premolt hepatopancreas (Chang 1995). The amount of DNA in the
hepatopancreas also increased during premolt and may be a mechanism to increase the capacity for
transcription as needed for the molt (Chang 1995).
In addition to the synthesis of new cuticle proteins, there is also extensive tissue degradation
in some muscles of decapod crustaceans during premolt, which allows the animal to escape from
the confines of its previous exoskeleton at ecdysis (Mykles 1999). This is consistent with reduced
muscle proteins during molting in the Pacific white shrimp Litopenaeus vannamei (de Oliveira
Cesar et al. 2006). This process likely contributes to the increase in protein turnover that occurs
during ecdysis in the American lobster, H. americanus, as reflected by higher protein synthesis rates
in muscle during the molt cycle (El Haj et al. 1996).

Starvation
Unlike the conditions just described, which increase ATP demand, starvation constitutes a limita-
tion of oxidizable fuels used to make ATP. Fuel reserves used during starvation in crustaceans have
been reviewed by Sanchez-Paz et al. (2006). Several studies of crustacean metabolism have shown
high variability of energy reserve mobilization during starvation, making it difficult to outline a
general metabolic profile. Experimental results have led to the long-standing view that the primary
source of energy in crustaceans is protein (Neiland and Scheer 1953, Claybrook 1983, Anger 2001),
contrasting with the paradigm for mammals and birds, which utilize mainly carbohydrates and lip-
ids as energy sources while sparing protein (Cherel et al. 1992). However, some crustaceans may
also minimize protein depletion during starvation. For example, the glycogen and lipid stores in the
hepatopancreas and muscle were depleted during starvation in the shrimp species Penaeus japonicus
(Cuzon et al. 1980), P. duorarum (Schafer 1968), and Crangon crangon (Cuzon and Ceccaldi 1973).
 Energetics and Metabolic Regulation 403

In the Pacific white shrimp, L. vannamei, a strict reliance on carbohydrate metabolism was observed
for short bouts of starvation, followed by use of plasma protein (Fig. 12.7; Sanchez-Paz et al. 2007).
Similarly, for the copepod Calanus finmarchicus (Helland et al. 2003) and in the isopod Stenasellus
virei (Hervant and Renault 2002), carbohydrate and lipid reserves were used early in starvation, and
protein was used in the later stages. Once the animals were allowed to feed again, energy reserves
were fully recovered after a period of 7–15 days of refeeding (Hervant and Renault 2002).
Experiments that reduced dietary protein by replacing it with carbohydrates suggested that
the ability of shrimp to utilize carbohydrates is limited as a consequence of both the low stor-
age capacity and the low capability of enzymatic processing (Rosas et al. 2000). However, poly-
saccharides may be more useful dietary sources of energy than simple sugars because they may
have protein- and lipid-sparing effects. For instance, P.  monodon shrimp fed starch or dextrin
had significantly higher weight gain and survival than those fed glucose (Shiau and Peng 1992).
These authors suggested that a diet high in glucose may lead to a rapid elevation of plasma glu-
cose, leading to excretion of glucose. The ability of certain crustaceans to utilize protein for fuel
may further reduce the need for simple sugars (Sanchez-Paz et al. 2006). For example, crabs fed
a high-protein diet had a lower hemolymph glucose level and lower hepatopancreas and mus-
cle glycogen levels than animals fed a high-carbohydrate diet (Oliveira et  al. 2004, Pellegrino
et  al. 2008). During starvation, hemolymph glucose was maintained largely by hydrolysis of
hepatopancreatic glycogen stores in the high-carbohydrate diet group, whereas gluconeogenic

0.6
Glucose (mg/mL)

0.4

0.2 *
* * * * * *
0.0
2 4 8 12 18 24 48 72 96 120
Time (h)
*
Glycogen (mg/g)

8.0

4.0
* *
*
* *

0.0
2 24 48 72 96 120
Time (h)

Fig. 12.7.
Hemolymph glucose and hepatopancreas glycogen during short-term starvation and refeeding in the Pacific
white shrimp, Litopenaeus vannamei. Both glucose and glycogen decrease indicate the critical role of carbohy-
drates in supporting short-term starvation. Empty bars, control; gray bars, starved; black bar, refed. The * indi-
cates significant difference from control. Data from Sanchez-Paz et al. (2007), with permission from Elsevier.
404 Ana Gabriela Jimenez and Stephen T. Kinsey

conversion of alanine to glucose and muscle glycogen synthesis from lactate appears to be the
major mechanism for maintaining hemolymph glucose (and lactate) in the high-protein diet
group (Oliveira et al. 2004, Pellegrino et al. 2008).
It is generally thought that the activities of digestive enzymes parallel food availability and that
these enzymes are inactive until secretion, although there is limited supporting data (Sanchez-Paz
et  al. 2006). The crustacean hepatopancreas produces and secretes several digestive enzymes,
including proteases like trypsin and chymotrypsin, lipases, and carbohydrate-degrading enzymes
(Dall et al. 1990). Trypsin and chymotrypsin activities in L. vannamei hepatopancreas were 40–60%
lower after 120 h of starvation (Muhlia-Almazan and Garcıa-Carreno 2002), whereas trypsin mRNA
was 30% lower (Sanchez-Paz et  al. 2003). Lipase activity has been found in crustaceans such as
L. vannamei (Gamboa-Delgado et al. 2003), red claw crayfish Cherax quadricarinatus (Lopez-Lopez
et al. 2003), the shrimp Macrobrachium borellii (Gonzalez-Baro et al. 2000), and terrestrial isopods
(Zimmer 2002). However, the role and regulation of these enzymes under starvation periods is
unknown. As dietary fuel sources are reduced and intracellular stores are mobilized, there is some
evidence of upregulation of glycolytic enzymes. In the hepatopancreas of L. vannamei, the glyco-
lytic enzyme phosphofructokinase mRNA levels increased 120-fold after 96 h of starvation, sug-
gesting an increased reliance on cellular carbohydrate stores to maintain ATP supply in this tissue
(Sanchez-Paz et al. 2007).

Responses to Environment

Temperature
Reduced temperatures make it less likely that enzymatic reactions will exceed their activation ener-
gies, thus reducing the metabolic rate. The aggregate effect of temperature on many reactions is
often encapsulated by the operational term Q  10, where a 10°C change in temperature is associated
with about a twofold change in metabolic rate, thus producing a Q  10 of 2 (Schmidt-Nielsen 1997).
During thermal stress, crustaceans must either address the temperature change by metabolically
compensating, if the animal needs to remain active, or avoid the thermal stressor by drastically
reducing the metabolic rate, a process called estivation or brumation (Vernberg and Vernberg 1968).
Thus, there are clear energetic consequences associated with temperature acclimation associated
either with compensatory remodeling of tissues or a need to cease activity.
Colson-Proch et al. (2009) found in the subterranean aquatic amphipod Niphargus rhenorhoda-
nensis that cold stress caused a large reduction in ventilatory rate and locomotion, although there
was no increase in lactate production, suggesting that aerobic metabolism was able to meet energy
demand. This is consistent with the view that anaerobic metabolism is only invoked when tempera-
tures drop below a critical level at which aerobic metabolism cannot keep pace with resting ATP
demand (Pörtner et al. 2006). However, the glucose and glycerol pools were reduced (glycerol can
be used as a precursor for antifreeze compounds or as a precursor to glucose to maintain stable
hemolymph levels), and there were significant increases in the free amino acid pool, largely due to
changes in concentrations of alanine, glutamine, lysine, and arginine, which all seem to be impor-
tant in conferring cryoprotection (Issartel et al. 2005, Colson-Proch et al. 2009). Seasonal changes
in temperature appear to alter the fuel utilization in decapods, and there can be large changes in
glycogen and lipid storage in the hepatopancreas and muscle, although the seasonal patterns are
variable and species-specific (Kucharski and Da Silva 1991b, Oliveira et al. 2004, Vinagre et al. 2007,
Buckup et al. 2008, Pellegrino et al. 2008). These shifts in fuel storage likely reflect changes not only
due to temperature, but also to seasonal differences in other factors such as activity level, reproduc-
tive status, and food intake.
When the temperature drops below a threshold level, some species can undergo brumation,
which is a dramatic decrease in metabolic rate associated with a cessation in activity. For instance,
 Energetics and Metabolic Regulation 405

Temperature Acclimated
0.1 Acute Temperature Transfer

Corrected O2 Consumption (mg/h STP)

Brumation

0.01

∆ = 0.0097 mg/h

0.001

5 10 15 20 25
Temperature (°C)

Fig. 12.8.
Oxygen consumption rates as a function of temperature in the fiddler crab, Uca pugilator (STP, standard tem-
perature and pressure). The acute temperature transfer groups (circles) were acclimated to 20°C and then
transferred to 14°C or 26°C. The temperature acclimated groups (squares) were acclimated to 5°C, 10°C, or
20°C. At 5°C, there was a 90-fold reduction of metabolic rate compared to crabs acclimated to 20°C, indicative
of brumation, during which crabs were inactive and nonresponsive to stimuli. Data from Jimenez and Bennett
(2007), with permission from Elsevier.

brumation is common in the fiddler crab Uca pugilator ( Jimenez and Bennett 2007) and the beach
flea Talochestia megalopthalma (Edwards and Irving 1943). This compensatory change in meta-
bolic rate allows the animal to survive unfavorable thermal conditions in an energetically efficient
manner. For example, when acclimated to 5°C, metabolic demand in brumating U. pugilator pop-
ulations decreased 90-fold compared to active crabs acclimated to 20°C (Fig. 12.8; Jimenez and
Bennett 2007).
Thermal tolerance range is particularly important in dictating vertical and latitudinal zonation
patterns in the intertidal zone (Somero 2002). For example, among porcelain crabs in the genus
Petrolisthes, tolerance to high habitat temperatures is associated with a capacity to maintain heart
rate, O2 consumption rates, and nerve action potentials and to limit lactate production during a
high-temperature challenge (Stillman and Somero 1996, Somero 2002). At the cell level, some of
these thermal adaptations likely reflect tradeoffs between protein flexibility and substrate binding
affinity and alterations in membrane fluidity that preserve enzyme and membrane function under
different temperature regimes (Hochachka and Somero 2002). For instance, there is some evidence
that citrate synthase and pyruvate kinase substrate affinities are adjusted in response to acclimation
temperature in the krill Meganyctiphanes norvegica and in the isopod genus Idotea over a seasonal
temperature range (Vetter and Buchholz 1997, Buchholz and Saborowski 2000).

Hypoxia and Anoxia

Tolerance of hypoxia or anoxia in crustaceans is highly species-specific, and although some repre-
sentatives are O2 conformers, most complex species have the ability to regulate O2 consumption
even at low partial pressures by increasing physiological parameters such as heart rate and venti-
lation rate (McMahon 2001). However, when O2 levels fall below the O2 regulating range, three
types of adaptation appear to permit survival: maintenance of large stores of glycogen and AP in tis-
sues, utilization of anaerobic pathways to produce ATP and to maintain redox balance in anaerobic
406 Ana Gabriela Jimenez and Stephen T. Kinsey

conditions, and the reduction of metabolic rate (Oliveira et al. 2001). Unlike other invertebrates
that may generate a variety of anaerobic end-products (Hochachaka and Somero 2002), crusta-
ceans employ only classical anaerobic glycolysis, leading to lactate as the sole end-product (Oliveira
et al. 2001, Abe et al. 2007). For example, the ghost shrimp Lepidophthalmus louisianensis maintains
an ATP production rate at near-aerobic levels during the first 12 h of anoxia, and between 12 h and
48 h of anoxia still supports about 50% of its ATP demand via aerobic metabolism. However, after
48 h, there is a large accumulation of lactate, as well as a major depression of metabolism (Holman
and Hand 2009).
The phosphagen AP provides an immediate but fairly rapidly depleted source of ATP during
anaerobiosis and, in addition, helps limit acidification because AP hydrolysis is a proton-consuming
process (Ellington 2001). Hypoxia or anoxia in muscles of the shrimp species Marsupenaeus japoni-
cus and L. louisianensis led to a near depletion of AP during the early stages of hypoxia, whereas ATP
levels remained fairly constant. As AP was depleted, glycogenolysis became the dominant source
of ATP, and lactate gradually accumulated (Abe et al. 2007, Holman and Hand 2009). However,
in M. japonicus muscle, the glycolytic enzyme fructose bisphosphate aldolase was downregulated
after 6 h of hypoxia, suggesting a suppression of glycolysis perhaps due to cellular acidification or
a whole-organism level metabolic downregulation (Abe et al. 2007). In fact, metabolic rate depres-
sion is probably essential for extended bouts of severe hypoxia or anoxia (Hill et al. 1991, Holman
and Hand 2009). It has even been proposed that crustaceans intentionally maintain low arterial
blood O2 (even when environmental O2 is high), which limits metabolic rate somewhat but also
prevents excessive reactive O2 species production associated with high cellular O2 (Massabuau
2001, Corbari et al. 2004).
Recovery from hypoxia or anoxia entails replenishment of ATP, AP, and glycogen stores, as well
as a restoration of pH. These processes may be powered exclusively by aerobic metabolism without
the accumulation of additional lactate (Oliveira et al. 2001, Abe et al. 2007), or there may be addi-
tional glycogen depletion and lactate accumulation during recovery, similar to the case for burst
contraction (Hill et al. 1991). The latter case likely reflects a mechanism to speed up key phases of
recovery in tissues such as muscle, which in crustaceans often has a very low aerobic capacity as
well as possible diffusion constraints on aerobic metabolism (Kinsey et al. 2007, 2011). Three path-
ways have been proposed for the clearance of lactate: complete oxidation, conversion into products
such as glycogen, and excretion (Ellington 1983), and it appears that crustaceans rely on all three
processes to eliminate lactate (Gade and Grieshaber 1986, Hill et al. 1991, Henry et al. 1994, Hervant
et al. 1999, Oliveira et al. 2001, 2004, Marqueze et al. 2006, Maciel et al. 2008).
Although transient exposure to hypoxia is common in some coastal ecosystems, midwater
crustaceans that reside in the O2 minimum zones of the oceanic water column may be chronically
exposed to hypoxia. The mysid shrimp Gnathophausia ingens resides in the O2 minimum zone and
has been the subject of considerable study (Childress and Seibel 1998). This species has a number
of traits that allow it to maintain aerobic function in the O2 minimum layer, including a high ven-
tilatory rate and circulatory capacity; a high gill surface area; short O2 diffusion distances across
the gills; and hemocyanin with a high affinity for O2, high cooperativity, a large Bohr effect to aid
delivery of O2 to tissues, and a low concentration, which presumably lowers the circulatory costs.
These traits lead to O2 extraction from the water that may be as great as 90%, thus allowing O2
consumption rates to be regulated even at the lowest O2 pressures encountered in the environment
(Childress and Seibel 1998). However, as O2 falls below critical levels, a reduction in metabolic rate
or reliance on anaerobic metabolism is necessary to maintain energy homeostasis (Seibel 2011).
Oxygen minimum zones also intercept the benthos at continental margins and can create vast
regions of essentially permanent, severely hypoxic benthic habitats (Levin 2003). Squat lobsters are
highly tolerant of hypoxia and are one of the relatively few large invertebrates that can exploit these
habitats (Zainal et al. 1992, Matabos et al. 2012). Adult squat lobsters in the genus Munida have been
 Energetics and Metabolic Regulation 407

shown to use the same increased ventilator response just described to regulate O2 consumption
over a broad range of O2 tensions (Zainal et al. 1992). The larvae of the squat lobster Pleuroncodes
monodon, which are often released into the O2 minimum zone, are also highly tolerant to hypoxia,
although less so than the adult. As the larvae develop from zoeal stages to megalopae, the capacity
to regulate O2 consumption increases, which corresponds to a shift toward lower O2 environments
(Yannicelli et al. 2013, Yannicelli and Castro 2013). The expected increase in hypoxic zones on con-
tinental shelves associated with global climate change will favor the relatively few hypoxia-tolerant
species, leading to a reduction in community diversity (Matabos et al. 2012).
The molecular basis of the hypoxic response in crustaceans is not well resolved. Crustaceans
express a hypoxia inducible factor-1 (HIF-1) that has homology with vertebrate HIF (Hoogewijs
et  al. 2007), but the regulation of expression may not follow the vertebrate paradigm (Li and
Brouwer 2007, Soñanez-Organis et al. 2009, Head 2010). For example, in the Pacific white shrimp
L. vannamei, HIF-1α has a decreased transcript level during hypoxia in gill, muscle, and hepatopan-
creas (Soñanez-Organis et al. 2009). In mammals, HIF-1α is typically regulated post-translationally,
and the protein levels increase during hypoxia, so the surprising downregulation of transcript
expression led these authors to suggest that elevated HIF-1α protein may invoke a decrease in
mRNA levels. A follow-up study on L. vannamei, Soñanez-Organis et al. (2011) found that activity
of the glycolytic enzyme HK was upregulated during hypoxia, and this effect was blocked in gills
and reduced in muscle, when the HIF-1α or -β expression was silenced (Fig. 12.9). Head (2010)
further suggested that HIF-1 may regulate the increase in blood hemocyanin content and changes
in isoform expression that accompany hypoxia in the crab Cancer magister.

Salinity
The energetic challenge associated with osmoregulation in euryhaline crustaceans appears to be
largely associated with (i) anisosmotic regulation of the extracellular fluid, where the osmolality
of the hemolymph is independent of the osmolality of the external medium, and (ii) isosmotic
regulation of the intracellular fluid (Gilles and Delphire 1997). Hemolymph osmolality is regu-
lated during hyper- or hyposmotic regulation in crustaceans primarily by a suite of ion transport-
ers, which generally occur in specialized tissues such as the gills (McNamara and Faria 2012). For
instance, in decapods such as the blue crab C. sapidus, which has been a principal subject in studies
of osmoregulation, the posterior gills are specialized for ion transport whereas the anterior gills
are largely respiratory (Mantel and Farmer 1983, Towle and Weihrauch 2001). The energetic cost
associated with ion transport in C. sapidus is reflected by the greater capacity of the posterior gills
to produce ATP (Piller et al. 1995, Kinsey et al. 2003) and to upregulate ion transport proteins and
increase gill O2 consumption under hypo-osmotic stress (Péqueux 1995). These responses lead, in
part, to increases in whole-animal respiration in response to reduced salinity, although the effects
may be relatively small in some species (Guerin and Stickle 1997, McGaw and McMahon 2003). For
instance, the small increase in cardiac output during low salinity exposure in C. sapidus was associ-
ated with increased blood flow to the legs and mouthparts and behavioral changes, rather than to
ion pumping per se (Fig. 12.10; McGaw and Reiber 1998), whereas blood flow rates and cardiac
dynamics were only modestly influenced by salinity in the Dungeness crab, C. magister (McGaw
and McMahon 2003). This suggests that ion transport associated with osmoregulation may incur a
relatively small energetic cost, particularly in highly euryhaline species because it is often confined
to the posterior gills, which represent a very small fraction of body mass. Nevertheless, crustaceans
maintained in suboptimal salinities do often show reduced growth rates and feed conversion rates,
and the view that this effect is due to energetic stress cannot be ruled out (Romano and Zeng 2012).
Although ion pumps in the gills play a central role in osmoregulation, there can still be signifi-
cant variation in hemolymph osmolarity, and the intracellular fluid must remain isosmotic with the
hemolymph to minimize cell volume changes. This is accomplished by increasing the concentration
408 Ana Gabriela Jimenez and Stephen T. Kinsey

A 0.0024 * Control
dsRNA α
0.0020 dsRNA β

(Units/min/mg protein)
0.0016

HK activity
0.0012

0.0008

0.0004
* *
0.0000

B 0.0015
*
0.0012
(Units/min/mg protein)

*
0.0009
HK activity

0.0006 *

*
0.0003

0.0000
ia

ox h

ia

ox h

ia

ox h

h
a1

24

24

24
ox

ox

ox
a

ia
rm

rm

rm
i

i
ia

ia

ia
ox

ox

ox
No

No

No
yp

yp

yp
yp

yp

yp
H

H
H

Treatment/Condition

Fig. 12.9.
Hexokinase (HK) activity during hypoxia in (A)  gills and (B)  muscle from the shrimp Litopenaeus vanna-
mei, as a function of silencing of hypoxia inducible factor-1 (HIF-1α and HIF-1β) with double stranded RNA
(dsRNA). HK activity increases under hypoxia in controls, whereas silencing of either HIF-1 subunit elimi-
nates or reduces the response, indicating control of HK activity by HIF-1. The * indicates a significant differ-
ence from the normoxia group. Data from Soñanez-Organis et al. (2011).

of certain free amino acids and other compatible or counteracting solutes that, unlike some inor-
ganic ions, do not interfere with protein or DNA structure (Gilles and Delpire 1997, Yancey 2001).
For instance, Holt and Kinsey (2002) showed that in isolated superfused muscle from C. sapidus,
extracellular osmolarity increases caused a reduction in AK function, presumably due to the influx
of inorganic ions. However, in a follow-up study, changes in salinity of a similar magnitude did not
alter AK function in vivo, suggesting that the ion pumping capacity of the gills moderated changes
in blood osmolarity (Kinsey and Lee 2003). Thus, the integrated responses—immediate increases
in gill ion pumping followed by a slower accumulation of compatible solutes—allowed C. sapidus
to preserve enzyme function in vivo despite large acute changes in salinity.
Much of our understanding of the sources of amino acids and other compatible solutes and
the mechanisms of removal from both the intracellular fluid and hemolymph is derived from the
 Energetics and Metabolic Regulation 409

100% seawater 25% seawater

160 A

f H (min–1)
70
0.6 B
Vs (ml.beat–1)

0.1
55 C
Vb (ml.min–1)

15
0 10 20 30 40 50 60 70 80
Time (h)

Fig. 12.10.
The effect of reduced salinity on (A) heart rate, (B) cardiac stroke volume, and (C) cardiac output in the blue
crab, Callinectes sapidus. Note the elevation in heart rate and cardiac output that occurs due to hyposmotic
regulation, suggesting a modest energetic cost that gradually decreases during 2–3 days of acclimation. Data
from McGaw and Reiber (1998), with permission from Elsevier.

numerous metabolic studies that were conducted several decades ago (Claybrook 1983). In the crab
Neohelice granulata, hyperosmotic stress leads to a decrease in lipid concentrations in the hepato-
pancreas, muscle, and posterior gills suggesting that increased lipid oxidation is associated with
hyper- and hypo-osmotic regulation (Luvizzotto-Santos et  al. 2003, Chitto et  al. 2001). In addi-
tion, hepatopancreatic and muscular gluconeogenesis is involved in osmoregulatory adjustments
(Oliveira and Da Silva 2000, Schein et al. 2005), suggesting that free amino acids released from
different organs during hypo-osmotic stress are deaminated in the hepatopancreas, and the car-
bon chains are used as a substrate for the gluconeogenic pathway (Oliveira and Da Silva 2000).
Consistent with this view, Martins et al. (2011) also found an upregulation of gluconeogenic enzyme
activities in the gills and hepatopancreas of N. granulata during changes in salinity.

COMPARISON WITH OTHER TAXA

The basic metabolic pathways and mechanisms of regulation of energy metabolism in crustaceans
are largely the same as in other invertebrates and vertebrates, although there are some notable
differences. For instance, the first line of defense in defending ATP levels in response to an ener-
getic challenge in most animals is phosphagen hydrolysis. However, crustaceans rely exclusively
on the phosphagen AP, whereas other invertebrate groups have a variety of phosphagens, and ver-
tebrates have only creatine phosphate (Ellington 2001). Similarly, crustaceans produce only lac-
tate as an anaerobic end-product, as do vertebrates, whereas other invertebrates produce a wide
range of metabolic end-products in order to maintain cellular redox balance during anaerobiosis
(Hochachka and Somero 2002). Crustaceans produce urea, but lack a formal urea cycle analogous
to that in mammals, and they also excrete uric acid and ammonia (Claybrook 1983). The fact that
crustaceans, like other arthropods, grow by molting means that growth is highly pulsatile. This
410 Ana Gabriela Jimenez and Stephen T. Kinsey

leads to major swings in energy metabolism and fuel storage/utilization associated with the molt
cycle, which is different from vertebrates and most other invertebrates that do not grow incremen-
tally. There is also substantial energy devoted to the synthesis of the cuticle in crustaceans (Chang
1995). Many crustaceans also undergo an increase in body mass during growth and development
that encompasses several orders of magnitude. In addition, the transition from planktonic larvae
to benthic organisms represents a major reorganization of the body plan. Among the vertebrates,
only fishes experience such a broad size range within a species. Thus, many crustaceans experience
very different respiratory challenges during growth as cellular and egg mass O2 transport distances
increase with animal size (Fernández et al. 2000, Taylor and Leelapiyanart 2001, Kinsey et al. 2011).

FUTURE DIRECTIONS

Prior work summarized here and in earlier reviews has provided a sound basis for our understand-
ing of crustacean energetics and metabolic regulation and has given us a platform for extending
this work to areas that are likely to be increasingly important. In our view, there are three major
areas where advancements are needed. First, climate change is likely to alter species distributions
in a dramatic fashion. This will lead to species composition changes that are likely to alter entire
ecosystems. Understanding the manner in which climate change impacts individual species will
aid our understanding of which populations are likely to extend their range, perhaps leading to a
wide range of new invasive species, and which species are likely to have their range reduced or to
even face extinction. Second, population growth and further development of coastal and terres-
trial habitats are likely to lead to an increased frequency and duration of deleterious events such as
hypoxic episodes, harmful algal blooms, or toxin loading. A better understanding of the metabolic
consequences of these challenges will aid our ability to manage ecosystems, and key species may
be useful indicators of impending deleterious conditions. Finally, much of the current and future
work is devoted to understanding the energetic and nutritional conditions that maximize aqua-
culture production. As coastal crustacean populations are increasingly under duress and natural
stocks face increasing harvesting pressure, the need for efficient aquaculture operations will likely
continue to grow.

CONCLUSIONS

Much of the early work on crustacean energetics and metabolic regulation centered on basic
aspects of biochemistry and physiology. These studies demonstrated that most of the biochemis-
try of crustaceans was similar to that of higher vertebrates, although there are notable departures.
Most recent work has probed mechanisms by which crustaceans respond energetically to different
physiological states, as well as to abiotic environmental challenges. We have attempted to sum-
marize some of the work conducted in the past two to three decades in an effort to point out both
consistent patterns among groups as well as species specific responses. Much of the future work
on crustacean energetics will likely focus on issues such as the effects of climate change on species
distributions and invasions, as well as means of enhancing the productivity of marketable species
in an aquaculture setting.

ACKNOWLEDGMENTS

This work was supported by a National Science Foundation grant to S.T.K. (IOS-0719123).
 Energetics and Metabolic Regulation 411

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 13
CRUSTACEAN GENOMICS AND FUNCTIONAL GENOMIC
RESPONSES TO ENVIRONMENTAL STRESS AND INFECTION

Jonathon H. Stillman and David A. Hurt

Abstract
High-throughput DNA sequencing has facilitated genome-scale studies of many organisms and
issues, including how crustaceans respond to environmental stresses, including temperature, viral
and bacterial infection, metal and organic toxicants, hypoxia, and salinity. The work has been uneven;
whereas economic drivers have spurred studies across a wide array of experimental conditions in
shrimp and bioindicator taxa (e.g., Cladoceran crustaceans in the genus Daphnia) dominate studies
of environmental pollutants, ecologically important crustaceans (isopods, amphipods, euphausids)
have been neglected. From these studies have emerged genes that encode mitochondrial proteins,
chaperonins, structural proteins, reproductive proteins, and extracellular receptor proteins and are
responsive to multiple types of environmental stressors across a range of crustaceans. This work
highlights aspects of crustacean cellular biology that elucidate how these diverse organisms respond
to a small set of changing environmental stressors across many of Earth’s habitats.

INTRODUCTION

Functional genomic, proteomic, and metabolomic analyses are powerful tools for examin-
ing the comprehensive set of responses that organisms make in response to environmental
variation. Increasingly, these tools are applied to nonmodel organisms as advances in genomics
and high-throughput screening technologies have lowered both the time and cost investment.
Genomic-scale data have been generated for a small number of crustaceans (Fig. 13.1, Table 13.1);
in this chapter, we review the literature on the application of functional genomic (transcriptomic),
proteomic, and metabolomic approaches that have been applied to investigate how crustaceans
respond to changes in their physical environment, to environmental toxicant exposure, and to
infection by viral and bacterial pathogens (Table 13.2). Responses to those abiotic and biotic factors

420
 Anostraca
Laevicaudata
Notostraca
Spinicaudata
Cyclestherida
Cladocera*
Remipedia
Cephalocarida
Tantulocarida
Cirripedia
Branchiura
Mormonilloida
Harpacticoida
Poecilostomatoida
Siphonostomatoida

Monstrilloida

Misophrioida
Cyclopoida
Gelyelloida
Calanoida
Platycopioida
Mystacocarida
Ostracoda
Stomatopoda
Euphausicea
Mysida
Syncarida
Decapoda
Thermosbanacea
Spelaeogriphacea
Amphipoda

Lophogastrida
Mictacea

Tanaidacea
Cumacea

Isopoda

Leptostraca
Hexapoda

Fig. 13.1.
Phylogenetic distribution of GenBank nucleotide data for the Crustacea. Branch weight corresponds to the
number of nucleotide entries as of May 2011. Dashed lines indicate no entries. Due to an unresolved crustacean
phylogeny, this tree is a composite and is for illustrative purposes only (not evolutionary inference).* Genome
has been sequenced for Daphnia pulex, and there are currently 14,177 entries in GenBank for this species.
Table 13.1.  Genomic data available in public databases for the Crustacea.

Class (Subclass, Infraclass) Order Genus species Genomic Archived Data (GenBank)
Data Types1
Nucleotide ESTs GEO Data
Series
Branchiopoda (Sarsostraca) Anostraca Artemia franciscana M,E 1,673 37,590 0
Artemia parthenogenetica E 695 0
Artemia sinica E 2 0
Branchiopoda (Phyllopoda) Notostraca Triops cancriformis M,E 1,046 3,981 0
Triops longicaudatus M 0 0
Branchiopoda (Phyllopoda, Diplostraca) Laevicaudata 252
Spinicaudata 875
Cyclestherida 14
Cladocera Daphnia carinata E 12,742 6,364 0
Daphnia magna G,E 13,517 11
Daphnia pulex G,M,E 200 Mbp 15,2659 11
Maxillopoda (Copepoda) Calanoida Calanus finmarchicus E 4,697 11,461 0
Cyclopoida Paracyclopina nana M 1,018 0 0
Gelyelloida 0
Harpacticoida Tigriopus californicus M,E 1,380 4,801 0
Tigriopus japonicus M 0 0
Misophrioida 0
Monstrilloida 15
Mormonilloida 0
Platycopioida 0
Poecilostomatoida 310
Siphonostomatoida Caligus clemensi E 7,674 14,806 0
 Caligus rogercresseyi E 32,037 0
Lepeophtheirus salmonis M,E 129, 4
250
Lernaeocera branchialis E 14,927 0
Maxillopoda (Thecostraca, Cirripedia) Pygophora 7
Apygophora 14
Kentrogonida Sacculina carcini G 220 0 0
Akentrogonida 16
Pedunculata Capitulum mitella M 1,212 0 0
Pollicipes pollicipes E 4,191 0
Sessilia Balanus amphitrite E 7,141 905 0
Megabalanus volcano M 0 0
Tetraclita japonica M 0 0
Facetotecta 21
Ascothoracida 23
Maxillopoda (Branchiura) Arguloida Argulus americanus M 92 0 0
Cyclida 0
Maxillopoda (Pentastomida) Cephalobaenida 8
Porocephalida Armillifer armillatus M 54 0 0
Maxillopoda (Mystacocarida) Mystacocaridida 44
Maxillopoda (Tantulocarida) Basipodellidae 0
Deoterthridae 0
Doryphallophoridae 0
Microdajidae 0
Ostracoda (Myodocopa) Myodocopida Vargula hilgendorfii M 630 0 0
Halocyprida 95
Ostracoda (Podocopa) Platycopida 5

(continued)
Table 13.1.  (Continued)

Class (Subclass, Infraclass) Order Genus species Genomic Archived Data (GenBank)
Data Types1
Nucleotide ESTs GEO Data
Series
Podocopida 2,563
Malacostraca (Eumalacostraca) Bathynellacea 39
Anaspidacea 28
Spelaeogriphacea 1
Thermosbaenacea 4
Lophogastrida 17
Mysida 1,195
Mictacea 2
Amphipoda Gammarus pulex E 8,776 12,345 0
Caprella mutica M 0 0
Caprella scaura M 0 0
Metacrangonyx longipes M 0 0
Onisimus nanseni M 0 0
Parhyale hawaiensis E 55,663
Isopoda Eurydice pulchra E 5,301 1,026 0
Eophreatoicus sp. M 0 0
Ligia oceanica M 0 0
Tanaidacea 98
Cumacea 153
Euphausiacea Euphausia superba G,E 1,193 6,142 0
Amphionidacea 0
Decapoda Alpheus distinguendus M 39,985 0 0
Bythograea thermydron G 0 0
Callinectes sapidus M,E 10,563 0
Carcinus maenas E 15,558 0
Celuca pugilator E 3,646 0
Charybdis japonica M 0 0
Cherax destructor M 0 0
Cherax quadricarinatus E 120 2
Eriocheir hepuensis M 0 0
Eriocheir japonica M 0 0
Eriocheir sinensis M,E 70,985 16,987 0
Exopalaemon carinicauda M 0 0
Farfantepenaeus M 0 0
californiensis
Fenneropenaeus chinensis G,M,E 10,446 1
Fenneropenaeus indicus E 714 0
Fenneropenaeus merguiensis E 11 0
Gandalfus yunohana M,E 310 0
Gecarcoidea natalis E 2,118 0
Geothelphusa dehaani M 0 0
Halocaridina rubra M 0 0
Homarus americanus E 29,957 2
Ilyoplax pusilla E 438 0
Litopenaeus setiferus E 1,042 0
Litopenaeus stylirostris M,E 416 0
Litopenaeus vannamei G,M,E 75,329 16,1248 4
Macrobrachium lanchesteri M 0 0

(continued)
Table 13.1.  (Continued)

Class (Subclass, Infraclass) Order Genus species Genomic Archived Data (GenBank)
Data Types1
Nucleotide ESTs GEO Data
Series
Macrobrachium nipponense M,E 557 8,458 0
Macrobrachium rosenbergii M,E 602 4,427 0
Marsupenaeus japonicus G,M,E 3,156 0
Metacarcinus magister E 1,137 0
Metapenaeus ensis E 13 0
Neocaridina denticulata E 132 0
Pacifastacus leniusculus E 802 0
Pagurus longicarpus M 0 0
Palaemonetes pugio E 42 1
Panulirus argus E 16 0
Panulirus japonicus M,E 2,673 0
Panulirus ornatus M 0 0
Panulirus stimpsoni M 0 0
Penaeus monodon G,M,E 39,397 3
Petrolisthes cinctipes E 97,806 2
Portunus trituberculatus M,E 9,552 0
Procambarus clarkii E 42 0
Pseudocarcinus gigas M 0 0
Scylla olivacea M 0 0
Scylla paramamosain M 0 0
Scylla serrata M 0 0
 Scylla tranquebarica M 0 0
Shinkaia crosnieri M 0 0
Upogebia major M 0 0
Xenograpsus testudinatus M 0 0
Malacostraca (Hoplocarida) Stomatopoda Gonodactylus chiragra M 607 0 0
Harpiosquilla harpax M 0 0
Lysiosquillina maculata M 0 0
Oratosquilla oratoria M 0 0
Squilla empusa M 0 0
Malacostraca (Phyllocarida) Leptostraca Squilla mantis M 242 0 0
Remipedia Nectiopoda Speleonectes tulumensis M,E 102 981 0
Cephalocarida Brachypoda Hutchinsoniella M 145 0 0
macracantha

1 . Sequence data types: G, genomic; M, mitochondrial; E, gene expression (cDNA / EST).
Table 13.2.  Synopsis of genome-wide studies in the Crustacea.

Species Taxonomy1 Tissue ‘Omic Type2 Method Experiment Citation Reviewed

here
Temperature
Petrolisthes cinctipes Malacostraca Hepatopancreas T cDNA microarray Kinetics of heat stress Teranishi and Yes
Decapoda, response Stillman 2007
Anomura: Porcellanidae
Petrolisthes cinctipes Malacostraca Heart T cDNA microarray Acclimatization of Stillman and Yes
Decapoda, heat and cold Tagmount
Anomura: Porcellanidae stress response 2009
Petrolisthes cinctipes Malacostraca Heart T cDNA microarray Thermal acclimation Ronges et al. No
Decapoda, of extreme cold 2012
Anomura: Porcellanidae tolerance
Rimicaris exoculata Malacostraca Abdomen with T SSH2 Heat stress Cottin et al. 2010 Yes
Decapoda, Caridea: Bresiliidae cuticle
Penaeus monodon Malacostraca Hemocytes T cDNA microarray Heat stress de la Vega et al. Yes
Decapoda, 2007
Penaeoidea: Penaeidae
Temperature + ecotoxicant mixed effects
Calanus finmarchius Maxillopoda Whole organism T SSH Heat stress, MEA, Hansen et al. Yes
Calanoida: Calanidae WSFs, Cu 2007
Oxygen
Penaeus monodon Malacostraca Hemocytes T cDNA microarray Hypoxia de la Vega et al. Yes
Decapoda, 2007
Penaeoidea: Penaeidae
Fenneropenaeus Malacostraca Hepatopancreas P 2DE3 + LC Hypoxia Jiang et al. 2009 Yes
chinensis Decapoda, ESI-MS/MS4
Penaeoidea: Penaeidae
Palaemonetes pugio Malacostraca Hepatopancreas T cDNA microarray Hypoxia Li and Brouwer Yes
Decapoda, 2009b
Caridea: Palaemoninae
Palaemonetes pugio Malacostraca Hepatopancreas T SSH Hypoxia Li and Brouwer Yes
Decapoda, 2009a
Caridea: Palaemoninae
Infection
Penaeus (Litopenaeus) Malacostraca Hemocytes P 2DE + LC Taura Syndrome Chongsatja et al. Yes
vannamei Decapoda, ESI-MS/MS Virus (TSV) 2007
Penaeoidea: Penaeidae
Litopenaeus vannamei Malacostraca Gills, Hemocytes, T SSH, cDNA White spot syndrome Robalino et al. Yes
Decapoda, Hepatopancreas, microarray virus (WSSV) 2007
Penaeoidea: Penaeidae Muscle
Procambarus clarkii Malacostraca Hemocytes T SSH, cDNA WSSV Zeng and Lu Yes
Decapoda, microarray 2009
Astacidea: Cambarinae
Litopenaeus stylirostris Malacostraca Hemocytes T SSH Vibrio penaeicida de Lorgeril et al. Yes
Decapoda, 2005
Penaeoidea: Penaeidae
Penaeus monodon Malacostraca Hemocytes P 2DE + LC Vibrio harveyi Somboonwiwat Yes
Decapoda, ESI-MS/MS et al. 2010
Penaeoidea: Penaeidae
Homarus americanus Malacostraca Muscle, gill, heart, T SSH Epizootic shell Tarrant et al. Yes
Decapoda, hepatopancreas, disease 2010
Astacidea: Nephropidae brain,
branchiostegite,
gonad

(continued)
Table 13.2.  (Continued)

Species Taxonomy1 Tissue ‘Omic Method Experiment Citation Reviewed

Type2 here
Penaeus (Litopenaeus) Malacostraca hepatopancreas T cDNA microarray WSSV Dhar et al. 2003 Yes
stylirostris Decapoda,
Penaeoidea: Penaeidae
Litopenaeus vannamei Malacostraca Gill (proteome) T (review) SSH, cDNA WSSV Robalino et al. Yes
Decapoda, + P (new microarray, 2009
Penaeoidea: Penaeidae data) dsRNA (RNAi),
2D LC-MS-MS
Marsupenaeus Malacostraca Hemocytes T cDNA Microarray Peptidoglycan Fagutao et al. Yes
japonicus Decapoda, 2008
Penaeoidea: Penaeidae
Marsupenaeus Malacostraca Hemocytes T EST library WSSV Rojtinnakorn Yes
japonicus Decapoda, et al. 2002
Penaeoidea: Penaeidae
Armadillidium vulgare Malacostraca Hemocytes P 2DE Wolbachia Herbiniere et al. Yes
Isopoda, Ligiamorpha, 2008
Armadillididae
Armadillidium vulgare Malacostraca Hemocytes T cDNA library Wolbachia Chevalier et al. No
Isopoda, Ligiamorpha, 2012
Armadillididae
Ecotoxicant: Organics
Daphnia magna Branchiopoda Whole animal T cDNA microarray Estrogens Iguchi et al. 2006 Yes
Diplostraca,
Cladocera: Daphniidae
Palaemonetes pugio Malacostraca Hepatopancreas T SSH EST library Pyrene Li and Brouwer Yes
Decapoda, 2009a
Caridea: Palaemoninae
Diporeia spp. Malacostraca Whole animal M GCxGC/ Atrazine Ralston-Hooper Yes
Amphipoda, TOF-MS6 et al. 2008
Gammaridea: Pontoporelidaae
Penaeus monodon Malacostraca Hemolymph P 2D-DIGE7 Enrofloxacin and Silvestre et al. Yes
Decapoda, furazolidone 2010
Penaeoidea: Penaeidae
Neocaridina denticulate Decapoda, Caridea, Atyidae Whole animal T SSH Nonylphenol Liu and Sung No
2011
Daphnia magna Branchiopoda Whole animal M FT-ICR MS8 Fenvalerate, Taylor et al. 2010 Yes
Diplostraca, dinitrophenol, and
Cladocera: Daphniidae propranolol
Daphnia magna Branchiopoda Whole animal T cDNA microarray Pyrene and Vandenbrouck Yes
Diplostraca, fluoranthene et al. 2010
Cladocera: Daphniidae
Daphnia magna Branchiopoda Whole animal T cDNA microarray Ibuprofen Heckmann et al. Yes
Diplostraca, 2008
Cladocera: Daphniidae
Daphnia magna Branchiopoda Whole animal T SSH, cDNA Fenarimol Soetaert et al. Yes
Diplostraca, microarray 2007
Cladocera: Daphniidae
Daphnia magna Branchiopoda Whole animal T cDNA microarray bNF Watanabe et al. Yes
Diplostraca, 2008
Cladocera: Daphniidae
Procambarus clarkii Malacostraca Nervous tissue, P 2DE Chlorpyrifos and Vioque- Yes
Decapoda, digestive gland carbaryl Fernandez
Astacidea: Cambarinae et al. 2009

(continued)
Table 13.2.  (Continued)

Species Taxonomy1 Tissue ‘Omic Type2 Method Experiment Citation Reviewed

here

Ecotoxicant: Metals
Litopenaeus vannamei Malacostraca Whole animal T EST library Cadmium (Cd) Keating et al. Yes
Decapoda, (larvae) 2007
Penaeoidea: Penaeidae
Tigriopus japonicus Branchiopoda Whole animal T EST library, Copper (Cu) Ki et al. 2009 Yes
Maxillopoda, Oligonucleotide
Copepoda: Harpacticidae microarray
Palaemonetes pugio Malacostraca Hepatopancreas T SSH EST library Copper (Cu) Li and Brouwer Yes
Decapoda, 2009a
Caridea: Palaemoninae
Daphnia magna Branchiopoda Whole animal T cDNA microarray Cu, Cd, Zn Poynton et al. Yes
Diplostraca, 2008
Cladocera: Daphniidae
Daphnia magna Branchiopoda Whole animal T cDNA microarray AgNO3, Ag Poynton et al. Yes
Diplostraca, nanoparticles 2012
Cladocera: Daphniidae
Daphnia pulex Branchiopoda Whole animal T cDNA microarray Cd Shaw et al. 2007 Yes
Diplostraca,
Cladocera: Daphniidae
Eriocheir sinensis Malacostraca Gills P 2DE Cd Silvestre et al. Yes
Decapoda, Brachyura: Varunidae 2006
Daphnia magna Branchiopoda Whole animal M FT-ICR MS Cd Taylor et al. 2010 Yes
Diplostraca,
Cladocera: Daphniidae
Daphnia magna Branchiopoda Whole animal M FT-ICR MS Cu Taylor et al. 2009 Yes
Diplostraca, (adult and
Cladocera: Daphniidae larvae)
Artemia sinica Branchiopoda Whole animal P 2DE Cu Zhou et al. 2010 Yes
Anostraca, (larvae)
Artenmiina: Artemiidae
Daphnia magna Branchiopoda Hemolymph M, T FT-ICR MS, Cd Poynton et al. Yes
Diplostraca, NMR, 2011
Cladocera: Daphniidae Oligonucleotide
microarray
Daphnia magna Branchiopoda Whole animal T cDNA microarray Cu, Cd, Zn Poynton et al. Yes
Diplostraca, 2007
Cladocera: Daphniidae
Daphnia magna Branchiopoda Whole animal T cDNA microarray Ni, Vandenbrouck Yes
Diplostraca, Ni+Pb. et al. 2009
Cladocera: Daphniidae Ni+Cd
Salinity
Penaeus monodon Malacostraca Hemocytes T cDNA microarray Hypo-osmotic de la Vega et al. Yes
Decapoda, salinity 2007
Penaeoidea: Penaeidae
Developmental Regulation
Artemia franciscana Branchiopoda Embryos: T EST library Dehydrated and Chen et al. 2009 No
Anostraca, rehydrated cysts
Artenmiina: Artemiidae
Balanus amphitrite Maxillopoda Whole animals P 2DE Nauplius, the Thiyagarajan No
Sessilia, Balanoidea: Balanidae swimming cyprid, and Qian
the attached 2008
cyprid, and the
metamorphosed
cyprid

(continued)
Table 13.2.  (Continued)

Species Taxonomy1 Tissue ‘Omic Method Experiment Citation Reviewed

Type2 here

Balanus amphitrite Maxillopoda Whole animal P 2DE Cyprid larvae Zhang et al. 2010 No
Sessilia, Balanoidea: Balanidae
Balanus amphitrite Maxillopoda Whole animals P, PhosphoP 2DE Larvae Thiyagarajan No
Sessilia, Balanoidea: Balanidae et al. 2009
Artemia franciscana Branchiopoda Embryos: P 2DE Post-diapaused cysts Wang et al. 2007 No
Anostraca,
Artenmiina: Artemiidae
Lepeophtheirus Maxillopoda Post molting T EST library Pre-adult and adult Eichner et al. No
salmonis Copepoda, maturation and 2008
Siphonostomatoida: Caligidae egg production
Endocrine/Exocrine
Daphnia pulex Branchiopoda Whole Pre- T cDNA microarray Methyl farnesoate Eads et al. 2008 No
Diplostraca, vitellogenic (MF)
Cladocera: Daphniidae females
Balanus amphitrite Maxillopoda Cyprid larvae P 2DE Biofilm, conspecific Thiyagarajan No
Sessilia, Balanoidea: Balanidae settlement factor 2010
Cherax quadricarinatus Malacostraca Hepatopancreas T cDNA microarray Ecdysteroid Shechter et al. No
Decapoda, 2007
Astacidea: Parastacidae
Cherax quadricarinatus Malacostraca Hypodermis, T cDNA microarray Ecdysone Yudkovski et al. No
Decapoda, gastrolith disk 2010
Astacidea: Parastacidae
Daphnia magna Branchiopoda Whole animal T SSH, cDNA 20-hydroxyecdysone Soetaert et al. No
Diplostraca, microarray 2007
Cladocera: Daphniidae
 Sexual Differentiation
Echinogammarus Malacostraca Whole animals T cDNA microarray Normal and intersex Ford and Thain No
marinus Amphipoda, populations 2008
Gammaridae: Gammaridae
Scylla serrata Malacostraca Gonad? T EST Gonad Zou et al. 2009 No
Decapoda, Brachyura: Portunidae
Fenneropenaeus Malacostraca Ovary T SSH Diploid vs. Triploid Xie et al. 2010 No
chinensis Decapoda,
Penaeoidea: Penaeidae
Eriocheir sinensis Malacostraca Testes T EST Testes Zhang et al. 2011 No
Decapoda, Brachyura: Varunidae
Molt Cycle/Exoskeleton Formation
Portunus pelagicus Malacostraca Various tissues and T EST, cDNA Molt, post-molt, Kuballa et al. No
Decapoda, Brachyura: Portunidae whole animals microarray intermolt, 2007
pre-molt
Portunus pelagicus Malacostraca Various tissues and T EST, cDNA Molt, post-molt, Kuballa and No
Decapoda, Brachyura: Portunidae whole animals microarray intermolt, Elizur 2008
pre-molt
Euphausia superba Malacostraca Heads T EST, cDNA 8 molt stages (A/B, C Seear et al. 2010 No
Euphausiacea: Euphausidae microarray early, C, C late, D0,
D1, D2, D3)
Portunus pelagicus Malacostraca Various tissues and T EST, cDNA Molt, post-molt, Kuballa et al. No
Decapoda, Brachyura: Portunidae whole animals microarray intermolt, 2011
pre-molt
Callinectes sapidus Malacostraca Gills, hypodermis T EST Pre-molt D2 and Shafer et al. 2006 No
Decapoda, Brachyura: Portunidae early post-molt

(continued)
Table 13.2.  (Continued)

Species Taxonomy1 Tissue ‘Omic Method Experiment Citation Reviewed

Type2 here

Gecarcinus lateralis Malacostraca Y-organs P 2DE Intermolt Lee and Mykles No

Decapoda, 2006
Brachyura: Gecarcinidae
Olfaction
Homarus americanus Malacostraca Olfactory organ T EST library, cDNA Olfactory genes McClintock No
Decapoda, mature zone microarray et al. 2006
Astacidea: Nephropidae

1. Taxonomy: Class, Order, Infraorder or Superfamily: Family.

2. Omic Type: T, Transcriptome; P, Proteome; M, Metabolome.
3. SSH: EST libraries constructed from suppressive subtractive hybridization.
4. 2DE: Two-dimensional gel electrophoresis.
5. LC ESI-MS/MS: Liquid chromatography electrospray ionization tandem mass spectroscopy.
6. GCxGC/TOF-MS: Two-dimensional gas chromatography coupled with time of flight mass spectroscopy.
7. 2D-DIGE: Two-dimensional differential in gel electrophoresis.
8. FT-ICR MS: Fourier transform ion cyclotron resonance mass spectrometry.
 Crustacean Genomics and Functional Genomic Responses 437

vary across factor and taxonomic group, and interpreting the responses of commonly observed dif-
ferentially expressed genes (Table 13.3) may lead to new fundamental understanding of crustacean
physiology. Additional studies using these tools in crustaceans and involving other processes such
as growth, development, and metamorphosis are listed in Table 13.2, but are not reviewed here.
Because this field is one of rapid advancement and discovery fueled by growing access to advanced
technology and increased expertise in the crustacean community, we fully expect future advance-
ments in the field to eclipse what has been learned to date (Ou et al. 2012). Nevertheless, this review
may provide a useful foundational resource.

TEMPERATURE

Crustaceans are found in just about every thermal habitat on Earth, from icy Antarctic waters, to
highly variable marine intertidal zone habitats, to constantly warm tropical reefs, and extremely
hot hydrothermal vent habitats. The literature is rich in examples of candidate gene approaches to
studying thermal adaptation, acclimation/acclimatization, and thermal stress responses. In con-
trast, crustacean biologists are at the dawn of the genomics era with respect to thermal biology,
and relatively few studies have specifically examined genome-wide responses to thermal habitat or
thermal stress (Fig. 13.2).
Crustacean responses to thermal stress have been studied extensively in the porcelain crab
Petrolisthes cinctipes, for which one of the largest collections of expressed sequence tags (ESTs) of
any crustacean species presently exists (Table 13.1), comprising a large fraction of genomic data
for the Decapoda (Fig. 13.1; see Stillman et al. 2006, Stillman et al. 2008, Tagmount et al. 2010).
Transcriptomic analysis of responses to thermal stress have been conducted in hepatopancreas and
cardiac tissues of P. cinctipes (Table 13.2) using 5 K unigene cDNA microarrays (5 K unigene is equal
to ~5,000 spots, each representing a different gene; see Stillman et al. 2006, Teranishi and Stillman

Shrimp # of ‘omics
studies
Lobster
Isopod 1
Euphausid 2
Crayfish
Organism

3
Crab
4
Copepod
5
Cladoceran
Barnacle 6
Artemia 7
Amphipod
Organotoxicants
Molt Cycle
Bacterial Infection
Cold Stress
Development
Endocrine/Exocrine
Heat Stress
Hypoxia
Metals

Salinity
Temperature
Viral Infection
Olfaction

Reproduction

Experimental Condition

Fig. 13.2.
Graphical representation of the number of genomic and proteomic studies that have been conducted on taxo-
nomic groups of crustaceans across a range of experimental conditions. Data obtained from Table 13.2.
Table 13.3.  Genes and proteins that were observed as differentially expressed in response to multiple types of environmental stressors. Response to
stressors indicated as up (upregulation), down (downregulation), and up & down (different expression patterns were observed in multiple studies).

Gene or Protein Warm Cold Heat Shock Viral Bacterial Organic Heavy Hypoxia
Acclimation Acclimation Infection Infection Ecotoxicants Metals
ATP synthase Up Down Down Up
Myosin Up Down Down Up
Actin Up Down Up Up
β-tubulin down Up Up & Down Up
Chaperonin Up Down Up
Thioredoxin Up Up Down
Hsp70 Up Up Down
Lysozyme Up & Down Up Up
14-3-3-γ Up Down Up
cytochrome c oxidase Up Down Up
Crustin Up Up Up
Hsp90 Up Down Up
Extensin-like down Up Up & Down
Ribin down Up Up & Down
Vitellogenin Down Down Up & Down
α-tubulin Up Down
Glutathione-S-transferase Down Down
Mannose receptor I Up Up
Ubiquitin Up Up
Serine protease Up Up & Down
Ankyrin Up Down
Cuticular proteins Up Down
Cytochrome b Up Down
Cytochrome c Up Down
Isocitrate dehydrogenase Up Down
NADH dehydrogenase Up Down
Succinate dehydrogenase Up Down
Titin Up Down
Tropomyosin Up Down
Troponin Up Down
Hemocyanin Up & Down Up & Down
440 Jonathon H. Stillman and David A. Hurt

2007, Stillman and Tagmount 2009). In hepatopancreatic tissue, gene expression was monitored at
nine time points from 30 min to 30 h of recovery following thermal stress (Teranishi and Stillman
2007). Genes involved with protein chaperone activity, including heat shock proteins (hsp), were
strongly induced following heat stress (Teranishi and Stillman 2007). Heat shock protein (hsp) 20
and hsp70 genes had equally high induction at all time-points, whereas induction of hsp90 genes
was strongest during the first 2 h of recovery and undetectable following 18 h of recovery (Teranishi
and Stillman 2007). Ubiquitin, a tag for protein denaturation, was only strongly induced during the
first 2 h following heat stress (Teranishi and Stillman 2007). Hsp20 and hsp70 induction beyond
2 h of recovery from heat stress suggests chaperone activity for newly synthesized proteins rather
than the refolding of extant proteins. Ribosomal proteins were strongly induced for the first 2 h
following heat stress but not at longer recovery times, thus suggesting that hepatopancreatic tis-
sue responds to thermal stress by increasing protein synthesis. Relatively few genes were repressed
throughout the recovery period. The mitochondrial ATP synthase was the most strongly repressed
gene and was repressed for up to 24 h following heat stress. A number of other genes involved in
oxidative metabolism were repressed immediately following heat stress. Hemocyanin expression
was repressed between 12 and 24 h, but not during the first 12 h following heat stress (Teranishi and
Stillman 2007).
In cardiac tissue, transcriptome profiles were monitored following both heat and cold stress in
porcelain crabs acclimatized to winter or summer conditions at one latitude ( June and December
in Cape Arago, Oregon) or across latitude within one season ( June in Monterey, California; Cape
Arago, Oregon; and Bamfield, British Columbia; Stillman and Tagmount 2009; see Table 13.2).
In both sets of comparisons, there was variation in both average temperature (summer warmer
than winter) as well as the degree of thermal variability (summer more thermally variable than
winter, high latitudes more thermally variable than low latitude due to difference in the timing of
low tide; Stillman and Tagmount 2009). Specimens were given a heat stress, cold stress, or held
at the collection temperature as a control, and the stress effect was calculated as the difference in
gene expression between the heat- or cold-stressed individuals and the control individuals for each
gene that significantly differed in expression (Stillman and Tagmount 2009). Overall, there were
much stronger transcriptome responses to heat stress than to cold stress in all specimens, and only
heat-stress responses varied among porcelain crabs acclimatized to different temperatures. Heat
stress induced the expression of hsp20, hsp70, and some other proteins regardless of thermal his-
tory, but induction was much stronger (up to 32-fold induction) in crabs acclimatized to warmer
and more thermally variable habitats (e.g., summer, high latitudes). In contrast, hsp90 was weakly
induced following heat stress in summer or high-latitude acclimatized crabs and was not induced at
all in winter or low-latitude acclimatized crabs.
Observed gene expression profiles, taken in aggregate, proved to be useful to discriminate across
stress type as well as thermal acclimatization state. Principal components analysis of genes that were
commonly differentially expressed in crabs from different latitudes and seasons that were exposed
to heat stress (Fig. 13.3) or cold stress (Fig. 13.4) show that heat stress has a stronger impact on
genome activity and that acclimatization is stronger with respect to heat stress than cold stress. The
data also indicate that thermal acclimatization presents a stronger overall effect on transcriptome
profiles than does heat stress because the first principal component largely separated seasonal and
latitudinal groupings, whereas the second principal component separated control and heat-stressed
crabs (Fig. 13.3). Crabs from cool, thermally constant habitats (December in Oregon, June in
Monterey California) had similar shifts across the principal component that were distinct from
warm, thermally variable habitats ( June in Cape Arago, Oregon and Bamfield, British Columbia;
Fig. 13.3). Although such groupings were also present in cold-stressed crabs (e.g., clustering of
June in Monterey, California and December in Cape Arago, Oregon cold stress and stress effect
crabs away from other groups), the patterns were not as strong (Fig. 13.4). These results prove that
 Crustacean Genomics and Functional Genomic Responses 441

Monterey_StressEffect

0.4
essEffect
Bamfield_Str
Cape_Arago_StressEffect
December_StressEffect

Monterey_HS June_StressEffect
0.2 Bamfield_HS
Decemb Cape_Arago_HS
er_HS June_HS
PC2

0.0

June_Cont

December_cont
Bamfield_cont
Cape_Arago_cont
–0.2 Monterey_cont

–0.5 0.0 0.5 1.0

PC1

Fig. 13.3.
Graphical representation of the first two principal components from a reanalysis of data on the effect of heat
stress across samples collected in two different seasons (December, June) at Cape Arago, Oregon, and at
three different latitudes during summer (Monterey, California; Cape Arago, Oregon; and Bamfield, British
Columbia). Microarray features used for principal components analysis were only those that commonly signifi-
cantly differed in the seasonal and latitudinal study (total n = 40 features). Data from Stillman and Tagmount
(2009).

transcriptome profiles are a reliable molecular-level biomarker for thermal acclimatization state and
prove their utility in broader scale ecological studies.
The largest variation in cardiac transcriptome response to heat stress across crabs acclimatized
to different conditions was in a small set of mostly uncharacterized genes that included β-tubulin,
a 28s rRNA-encoded gene known as ribin, and genes whose only homology is to extensin-like pro-
teins (Stillman and Tagmount 2009). These transcripts were constitutively repressed in crabs accli-
matized to cool, low-variability temperatures but were strongly induced following heat stress (up
to 16-fold induction). In contrast, these transcripts were constitutively induced in crabs acclima-
tized to warm, thermally variable habitats but strongly repressed following heat stress (up to 16-fold
repression). Thus, heat stress caused 256-fold variation in the expression of these transcripts relative
to control specimens, depending on thermal acclimatization state (Stillman and Tagmount 2009).
The functional roles of the genes within this cluster are not known, although because α-tubulin had
a very different expression profile from β-tubulin, microtubule formation is not likely one of those
functions (Stillman and Tagmount 2009). β-tubulin, in addition to being a component of microtu-
bules, has been shown to have chaperone activity in refolding heat denatured proteins (Guha et al.
1998) and restoring activity to unfolded enzymes (Manna et al. 2001), and thus it may be acting as a
chaperonin in porcelain crab cardiac tissue.
Cardiac tissue of porcelain crabs acclimatized to cool, thermally constant habitats showed
induction of a large number of genes involved in oxidative energy metabolism, including electron
442 Jonathon H. Stillman and David A. Hurt

Monterey_Control
June_StressEffect
0.25
June_CS

Cape_Arago_StressEffect

Cape_Arago_Cold_Stress
0.00 C o ntrol Bamfield_Control
_
mbe
r ffect Control
Dece StressE Cape_Arago_
ld_ June_Control
fie Bamfield_Cold_Stress
Bam
PC2

–0.25 December_StressEffect
December_CS
Monterey_Cold_Stress

–0.50

Monterey_StressEffect

–0.5 0.0 0.5 1.0

PC1

Fig. 13.4.
Graphical representation of the first two principal components from a reanalysis of data on the effect of cold
stress across samples collected in two different seasons (December, June) at Cape Arago, Oregon, and at three
different latitudes during summer (Monterey, California; Cape Arago, Oregon; and Bamfield, British Columbia).
Microarray features used for principal components analysis were those that commonly significantly differed in
the seasonal and latitudinal study (total n = 20 features). Data from Stillman and Tagmount (2009).

transport and citric acid cycle, and these genes were repressed following heat stress (Stillman
and Tagmount 2009), similar to that observed in hepatopancreatic tissue (Teranishi and Stillman
2007). Cold acclimatization may induce greater expression of these genes to overcome Q10 effects
and/or lower efficiency of mitochondrial function. Genes encoding structural and muscle pro-
teins had acclimatization-dependent responses to heat stress, with induction following heat stress
in warm acclimatized specimens and repression following heat stress in cool acclimatized speci-
mens (Stillman and Tagmount 2009). Why thermal acclimatization should alter the thermal stress
responses of these proteins is unknown, but ultrastructural analyses of muscle could be informative.
Immune responsive genes including antimicrobial peptides and complement were induced in
porcelain crabs acclimatized to cooler temperatures but were not further induced by thermal stress
(Stillman et al. 2006). Expression of the antimicrobial peptide carcinin has been shown to be more
responsive to thermal stress than to infection load in Carcinus maenas (Brockton and Smith 2008),
suggesting that immune-responsive genes may have temperature-related regulatory mechanisms.
Additional efforts to dissect the role that temperature versus pathogen load has in the expression of
immune responsive genes in crustaceans are warranted.
Variation in the transcriptome response to heat stress has been investigated in the hydrothermal vent
shrimp Rimicaris exoculata (Cottin et al. 2010). These shrimp swarm near black smoker chimneys and
may experience large variation in habitat temperature, including periods that exceed their long-term
thermal maxima (Cottin et al. 2010). Thus, it was of interest as to whether they exhibited similar gene
expression responses to heat stress as organisms living in more mesic and constant thermal environ-
ments. Shrimp exposed to a transient 20°C temperature spike, from 10°C to 30°C in 1 h and back to 10°C
 Crustacean Genomics and Functional Genomic Responses 443

for 2 h were used to identify differentially expressed genes using suppression subtractive hybridization
cDNA libraries (SSH; Cottin et al. 2010). Of the 192 cloned differentially expressed cDNAs, approxi-
mately 47% of the induced clones and 62.5% of the repressed clones following heat stress matched known
genes (Cottin et al. 2010). Genes identified as differentially expressed between control and heat-stressed
R. exoculata specimens represented a wide range of functional processes: cellular stress response; anti-
oxidant defense; immune response; energetics and metabolism; structural and cytoskeletal; ribosomal,
transcription, and protein regulation; and calcium homeostasis (Cottin et al. 2010).
Based on the number of redundant clones sampled, the most abundant repressed transcripts
encoded sarcoplasmic calcium-binding protein (SCP)-1 genes and structural and cytoskeletal
genes, suggesting that processes involving muscle function and cellular organization were nega-
tively impacted by heat stress (Cottin et al. 2010). The most abundant induced transcripts were
hsp70, antioxidants, and ribosomal proteins. Quantitative real-time PCR (qPCR) was used to
verify the differential expression of a select number of the transcripts identified by SSH. Of those,
only two hsp70 genes and one hsp90 gene were observed to shift in expression greater than twofold
(either induced or repressed) in heat-stressed versus control shrimp (Cottin et al. 2010).
In hemocytes of the black tiger shrimp Penaeus monodon, SSH and non-subtracted libraries were
used to construct a 4 K cDNA microarray that was used to profile responses to multiple environmen-
tal stressors, including temperature (de la Vega et al. 2007). P. monodon were exposed to temperature
increases of 6°C, from 29°C to 35.5°C (at a rate of 2°C/h), maintenance at 35.5°C for 24 h, and decrease
to 29°C (at a rate of 1°C/h). Hemolymph samples were withdrawn for transcriptome profiling at four
time points: (i) before the initial thermal ramp, (ii) after reaching 35.5°C, (iii) 24 h after returning to
29°C, and (iv) several days following return to 29°C (de la Vega et al. 2007). Sixty-eight features were
significantly differentially expressed across the four sampling time points, and those did not include
hsps. The fact that this study did not observe induction of hsps following thermal stress and that
relatively few differentially expressed genes were observed could be due to the relatively low sample
sizes (n = 2 for each time point) leading to low statistical power. Alternatively, the transcriptional
responses of hemocytes to heat stress may be limited to just the few genes observed here and do not
include induction of the cellular stress response. Finally, it is possible that 35.5°C is not a stressful
temperature for these tropical shrimp and that there was little physiological requirement to induce
any sort of response following a 1 day exposure to that temperature.
In the copepod Calanus finmarchicus, SSH was used to capture genes that were responsive to a
mixed array of environmental stressors, including elevated temperature (Hansen et al. 2007). However,
since there were no stressor-specific SSH libraries constructed, results can only be used to generate a
generalized stress-responsive transcriptome profile. Genes observed to be induced in response to stress
included common elements of the cellular stress response, β-tubulin, but not α-tubulin (Hansen et al.
2007), similar to what was observed in porcelain crabs (Stillman and Tagmount 2009).

INFECTION

The economic importance of crustaceans has motivated genomics-level studies of response to viral
and bacterial pathogens commonly encountered in marine aquaculture and fisheries. These studies
aim for enhanced detection of disease, as well as an understanding of disease mechanisms in order
to allow the development of preventative measures.

Viral Infection

Penaeid shrimp are an important aquaculture species and are known to be highly susceptible to
viral infections for which there are no drug treatments available. Genomic and proteomic screens
thus afford the ability to identify target cellular responses that are repressed following infection
444 Jonathon H. Stillman and David A. Hurt

and present possible candidates for genetic engineering of shrimp variants that possess greater
immunity. The response of the pacific whiteleg shrimp Litopenaeus vannamei hemocytes to Taura
Syndrome Virus (TSV), a virus that can cause large-scale mortality in shrimp aquaculture, was
monitored using a proteomic approach (Chongsatja et al. 2007). Using two-dimensional gel elec-
trophoresis of infected and control specimens, a total of 32 protein spots were found to be differ-
entially expressed between control and infected shrimp, with eight proteins strongly upregulated
and five proteins strongly downregulated. Proteins strongly upregulated following infection func-
tioned in signal transduction, carbohydrate metabolism, detoxification, and cellular structure and
protein modifications potentially involved with stress responses. Proteins strongly downregulated
following infection have functional roles in cellular defenses against oxidative stress. Hemocyanins
that did not have infection-dependent expression were among the most highly abundant proteins.
Specific TSV responses of hemocyanins were inferred from the fact that differentially expressed
hemocyanins upregulated following TSV infection matched mainly the acidic C-terminus of
the protein (average pI  =  4.98), whereas those downregulated following infection matched the
N-terminus (average pI = 5.23; Chongsatja et al. 2007).
Response to White Spot Syndrome Virus (WSSV) has been investigated in multiple studies
utilizing genomics approaches (Fig. 13.4, Table 13.2), including transcriptomic studies in L.  vanna-
mei hepatopancreas, gill, hemocyte, and muscle tissues (Robalino et al. 2007, Robalino et al. 2009),
transcriptomic studies of Litopenaeus stylirostris (Dhar et  al. 2003), transcriptomic responses in
Procambarus clarkii hemocytes (Zeng and Lu 2009), and proteomic responses of L. vannamei gill tis-
sue (Robalino et al. 2009). L. vannamei were injected with active virus at two temperatures (27°C and
32°C; the higher of which inhibits virulence), and, as controls for general response to stimuli, shrimp
were also injected with heat-killed bacteria and fungal spores (Robalino et al. 2007). ESTs (n = 7,021)
captured in cDNA and SSH libraries assembled into 3 K unigenes, approximately 900 of which were
represented by multiple ESTs. Immune response-related cellular responses including antimicrobial,
antiviral, cell adhesion, cell death, oxidative stress, proteases, protease inhibitors, RNA interference,
signal transduction, and transcriptional control were represented by 98 unigenes, many of which were
differentially regulated with respect to infection. Among these, ESTs with high biological significance
in response to viral infection included IκB kinase, a positive regulator of the NF-κB pathway, and sig-
nal transducer and activator of transcription (STAT), a core component in the interferon response in
vertebrates and antiviral responses in arthropods (Robalino et al. 2007). Additional potential antiviral
genes identified included three components of the RNAi pathway (Robalino et al. 2007). Following
WSSV infection, a 2.5 K unigene cDNA microarray was used to examine differential expression in
hepatopancreas tissues of L. vannamei (Robalino et al. 2007). WSSV induced genes involved in anti-
microbial response, protein degradation, chromatin remodeling and energy metabolism. WSSV
repressed genes involved xenobiotic response, transmembrane proteins and cell signaling, protein
synthesis, and oxidative stress (Robalino et al. 2007). To what extent antimicrobial genes induced
by viral infection represent secondary microbial infection versus general immune response pathways
requires further study. Many of the putative immune-responsive genes identified in the SSH librar-
ies were not observed to be differentially expressed following WSSV infection in hepatopancreas by
cDNA microarray, a result that could be due to tissue-specific responses to WSSV.
Gill proteome response to WSSV infection in L. vannamei was examined using 2-dimensional liquid
chromatography together with tandem mass spectrometry (2D LC-MS-MS) at 0 and 12 h post infection
(Robalino et al. 2009). Only at 12 h post infection were any peptides determined to be upregulated by
the infection, although this could be a result of endogenous rhythmicity of protein expression. Those
peptides indicate that changes in chromatin structure, cytoskeleton, protein turnover, pigments, and pos-
sibly energy metabolism may result from WSSV infection in gill tissue (Robalino et al. 2009).
Transcriptomic responses of L. stylirostris hepatopancreas tissue to WSSV infection, monitored
on a very small (n = 100 features) microarray made from cDNAs cloned from infected shrimp and
 Crustacean Genomics and Functional Genomic Responses 445

in differential display libraries, found strong induction of a serine protease and genes involved
within cellular surface recognition proteins, thus suggesting strong antimicrobial response (Dhar
et al. 2003). Repressed genes were difficult to functionally characterize (Dhar et al. 2003). Some
of the unknown genes were cloned by differential display and thus may represent 3′ untranslated
regions (UTRs) for the genes, requiring further DNA sequencing into the protein coding region of
the gene before assessing gene homology (Dhar et al. 2003). These results indicate that antibacte-
rial genes are induced following viral infection, similar to that observed by (Robalino et al. 2007).
In the crayfish P. clarkii, hemocytes of WSSV infected and noninfected specimens were used
to make a cDNA microarray from cDNAs captured using SSH (Zeng and Lu 2009). A total of 255
cDNAs were induced, and 23 cDNAs were repressed following WSSV infection. Of the induced
cDNAs, the strongest response (four- to fivefold induction) was an inhibitor of apoptosis (Zeng
and Lu 2009). Additional genes induced by WSSV encoded proteins involved in protein homeo-
stasis, the cytoskeleton, and energy metabolism (Zeng and Lu 2009). There was little overlap in
the most strongly differentially expressed genes in shrimp and crayfish following WSSV infection,
although potentially a different set of genes was examined.

Bacterial Infection

Transcriptomic and proteomic responses to bacterial infection have been examined in crusta-
ceans infected with Vibrio (gram negative γ-proteobacteria), Wolbachia (α-proteobacteria), and
unknown bacteria or bacterial assemblages that cause lesions in crustacean shells known as epi-
zootic shell disease.
Response to Vibrio penaeicida infection was examined using SSH in hemocytes of L. stylirostris
that survived to 96 h postinfection (de Lorgeril et al. 2005). Macroarrays were constructed from
320 randomly selected cloned cDNAs that assembled into 52 clusters and 158 singletons, for a total
of 210 unigenes (de Lorgeril et al. 2005). Those cDNAs with homology to functionally annotated
genes were involved with immune function (21% of identified cDNAs); cell proliferation (20%);
metabolism (17%); DNA modification, gene expression, and protein synthesis (15%); cell signaling
(12%); and cell structure (5%; de Lorgeril et al. 2005). There was relatively little differential expres-
sion of any of the SSH identified genes between infected and uninfected shrimp, but qPCR analysis
indicated a greater than twofold upregulation of several immune function genes and a gene product
involved in cell division and proliferation following infection (de Lorgeril et al. 2005).
Proteomic analysis of black tiger shrimp, P. monodon, hemocytes following Vibrio harveyi infec-
tion identified 27 protein spots by two-dimensional gel electrophoresis that differed between con-
trol and infected specimens (Somboonwiwat et al. 2010). Proteins upregulated by infection included
prophenoloxidase 2 and actin 2, suggesting that those proteins have a specific role in response to
infection (Somboonwiwat et al. 2010). Proteins upregulated in infected shrimp but also present
in control specimens include hemocyanin, arginine kinase, twinstar, tubulin, serine protease, and
transaldolase (Somboonwiwat et al. 2010). Proteins downregulated by infection included multiple
immune-responsive proteins, including α2 macroglobulin, prophenoloxidase-1 and -2, serine pro-
teases, hsp90, 14-3-3 protein epsilon, calmodulin, karyopherin, and ATP synthase (Somboonwiwat
et al. 2010). It is likely that the differences in protein pools between infected and uninfected shrimp
are reflections of post-translational modification rather than protein expression, and thus the pro-
phenoloxidase observed in control shrimp may be the same gene product as the one observed in
infected shrimp but with a different set of post-translational modifications involved with regulation
of this immune-responsive enzyme (Somboonwiwat et al. 2010).
Cell wall peptidoglycans prepared from Bifidobacterium thermopilum were fed to the kuruma
shrimp Marsupenaeus japonicus to examine hemocyte transcriptome responses to noninfec-
tious bacterial components (Fagutao et  al. 2008). cDNA microarrays containing 2 K unigenes
446 Jonathon H. Stillman and David A. Hurt

identified from WSSV-infected and -uninfected EST library construction projects in M. japonicus
(Rojtinnakorn et al. 2002) and P. monodon (Supungul et al. 2002) were used to examine responses
at 1, 7, and 14 days following peptidoglycan injections (Fagutao et al. 2008). The strongest gene
expression response in transcripts induced or repressed by peptidoglycans occurred at 1  day
postingestion. Induction of known antimicrobial, clotting, healing, and melanization genes was
observed (Fagutao et al. 2008). Transcripts strongly induced for up to 2 weeks following peptido-
glycan exposure were involved in protein synthesis, immune response, and other functions, sug-
gesting that a strong and prolonged immune response can be initiated by noninfectious elements of
bacterial cell walls (Fagutao et al. 2008).
Terrestrial arthropods are commonly infected by Wolbachia bacteria, and these infections
can be passed to subsequent generations in the germ line (Herbiniere et al. 2008). To investi-
gate the immune response of terrestrial isopod crustaceans to Wolbachia infection, proteomic
analysis was performed on hemolymph samples using two-dimensional gel electrophoresis
(Herbiniere et al. 2008). Of the 300 protein spots that were identified in all three of the repli-
cate gels, about one-third were excised and characterized using Q-TOF-MS analysis, resulting
in 56 identified proteins (Herbiniere et al. 2008). Of those proteins, a number were found to be
involved with immune functioning, including non-self recognition and melanization of xeno-
biotics coagulation, detoxification, cell adhesion, cell communication, protease defense, stress
response, and the cytoskeleton (Herbiniere et al. 2008). Identification and characterization of
these proteins in a terrestrial isopod form a valuable tool for examination of infection in nonin-
sect terrestrial arthropods.
Epizootic shell disease is manifested by characteristic lesions on the external surface of crusta-
ceans. Although the bacteria responsible for causing these lesions are not well characterized, one
hypothesis is that they are normal members of the marine microbial assemblage that cause disease
when the infected animals are suffering from poor physiological condition. Variation in multiple
tissues of American lobsters, Homarus americanus, that were either suffering from epizootic shell
disease or were asymptomatic were used to determine whether general physiological condition
plays a role in the susceptibility of lobsters to the bacteria that cause shell erosion. Differentially
expressed transcripts were captured by SSH using two sets of symptomatic and asymptomatic
lobsters (Tarrant et  al. 2010). Approximately equal numbers of genes were induced (n  =  73) as
repressed (n = 66) in symptomatic lobsters. There was little concordance in the specific ESTs that
were identified as induced or repressed in symptomatic lobsters between the two sets of SSH librar-
ies. For example, in one SSH library, myosin, actin, and a mannose-binding protein were induced;
whereas in the second SSH library, arginine kinase, cysteine protease, cytochrome b, cytochrome
c, and keratinocyte-associated and mannose-binding proteins were induced, with the only overlap
between the two libraries being the mannose-binding protein (Tarrant et al. 2010). However, to
confuse matters, mannose-binding protein was also observed in both SSH libraries to be repressed
in symptomatic lobsters, as were a number of the other genes identified as induced in the second
SSH library in symptomatic lobsters (Tarrant et al. 2010). Muscle arginine kinase expression, quan-
tified by qPCR, was repressed in symptomatic lobsters, a result that was interpreted as represent-
ing an energetic drain resulting from or enabling the infection (Tarrant et al. 2010). Hemocyanin
expression in hepatopancreatic tissue was also repressed in symptomatic lobsters, suggesting that
lower respiratory capacity is involved in epizootic shell disease (Tarrant et al. 2010). Ovarian levels
of α2 macroglobulin were induced in symptomatic lobsters, suggesting that these lobsters are cop-
ing with higher levels of proteolytic activity, potentially due to infection, in ovarian tissues (Tarrant
et al. 2010). Although this initial study of transcriptional response to epizootic shell disease was by
no means comprehensive, nor did it identify a smoking gun, the study does lend some evidence
that the shell disease is at least associated with changes across the entire organism and not just at
the sites where lesions are located.
 Crustacean Genomics and Functional Genomic Responses 447

ENVIRONMENTAL TOXICANTS

Much of the “omics-based” work on crustacean responses to environmental stress has been in
the area of environmental toxicology. Ecotoxicogenomics is a rapidly growing field wherein
researchers are realizing the potential of genomic and other approaches for characterizing
stressor-specific response profiles (Snape et  al. 2004). The applied potential of ecotoxicoge-
nomics for identifying biomarkers is great, and this approach has identified rapid biomarkers
for reproductive effects, such as vitellogenin mRNA (Soetaert et al. 2006), and it has character-
ized novel metallothioneins (Shaw et al. 2007) in cladocerans, which are efficacious as markers
of trace metal exposure (Amiard et al. 2006). Although there are many overlapping, common
molecular stress response pathways induced by ecotoxicants in general, evidence is compelling
for stressor-specific responses. These specific response profiles may be grouped broadly into
two categories based on the chemical characteristics of toxicants: those induced by heavy met-
als and those induced by organic compounds.
The bulk of published work on ecotoxicogenomics in crustaceans has thus far focused on met-
als, specifically the trace metal cadmium, likely due to its effects on human health (Nordberg 2009).
Other work has focused on responses to copper and organic compounds such as atrazine, mono-
ethyl amine, propiconazole, pyrene, water-soluble fractions of oil, and organophosphates. For the
most part, studies have focused on responses at the level of the transcriptome, but some proteomic
(Silvestre et al. 2010) and metabolomics (Ralston-Hooper et al. 2008) approaches have been taken
as well. Daphnids have been the main focus of ecotoxicogenomics efforts, due to the history of these
species in toxicity testing. However, work has also been done on other crustacean taxa, including
other groups within the Branchiopoda, Maxillopoda, and Malacostraca.

Organic Compounds

Crustacean sensitivity to insecticidal organics (e.g., organochlorines, organophosphates, and pyre-

throids) is likely due to their shared ancestral lineage with the insect arthropods that those toxins
target. Thus, establishing genomic response profiles of crustaceans to organic toxicants is of great
utility to ecotoxicology. For these reasons, there is a growing body of work aimed at characterizing
genome-wide induction by organics in crustaceans to develop biomarkers and elucidate basic cel-
lular response mechanisms.
In Daphnia magna, functional genomics approaches have been employed to investigate
molecular responses to the triazole fungicide propiconazole (Soetaert et al. 2006) and to poly-
cyclic aromatic hydrocarbons (PAHs) fluoranthene and pyrene (Vandenbrouck et  al. 2010).
SSH was used to generate a cDNA library of differentially expressed genes in D. magna between
adults and juveniles, enriched for reproduction-specific transcripts (Soetaert et al. 2006). This
cDNA library has been utilized in multiple studies (Soetaert et al. 2006, Soetaert et al. 2007,
Vandenbrouck et al. 2010), which illustrates the utility to the scientific community of construct-
ing “omics-scale” resources.
The 1,189 unique reproduction-specific cDNA fragments generated for D. magna corresponded
to transcripts involved in ribosomal RNA/proteins, cell cycle, molting, embryonic development,
energy metabolism, nervous system development, and other cellular processes (Soetaert et al. 2006).
The effect of propiconazole, a compound known to have detrimental effects on daphnid embryos,
was measured via a cDNA microarray generated from the reproduction-specific library (Soetaert
et  al. 2006). Far more transcripts were repressed than induced, and these displayed a time- and
concentration-dependent expression pattern because the majority of transcripts were most strongly
repressed immediately following exposure (Soetaert et al. 2006). The most strongly repressed tran-
scripts were vitellogenin, larval-specific gene, stromal cell-derived factor, and chaperonin, and the
448 Jonathon H. Stillman and David A. Hurt

most strongly induced transcripts were hsp90 and ATP synthase (Soetaert et al. 2006). Although
multiple genes involved in cellular energy metabolism were represented by the array, ATP synthase
was the only differentially expressed metabolic gene in response to propiconazole.
The same cDNA library was used to investigate the effects of the PAHs fluoranthene and pyrene
on D. magna in an integrated approach incorporating metabolomics and energetics (Vandenbrouck
et al. 2010). To investigate the effect of various mixtures of the similarly acting compounds and to
potentially parse out the molecular differences in response, mixtures of varying toxic units, as well
as just one compound or the other, were used as treatments (Vandenbrouck et al. 2010). Along with
cDNA microarray experiments, validations were performed using qPCR, and metabolomics analy-
ses were conducted using nuclear magnetic resonance spectroscopy (1H NMR) and gas chroma-
tography mass spectrometry (GC-MS). Similar to the response observed to propiconazole, more
genes were repressed than induced: 34 in response to solely fluoranthene and 27 to solely pyrene
(Vandenbrouck et  al. 2010). Unlike propiconazole, gene expression changes elicited by fluoran-
thene and pyrene did not change in a concentration-dependent manner, and, based on a hierarchi-
cal clustering analysis on all differentially expressed genes, there was no difference in response to
the two compounds (Vandenbrouck et al. 2010).
Vitellogenin was repressed in response to fluoranthene and pyrene in D.  magna, as was also
observed in response to propiconazole; however, there was no reported induction of hsp90 or any
other canonical stress response proteins or significant induction of energy metabolism-related tran-
scripts (Vandenbrouck et al. 2010). Although all differentially expressed genes were not significantly
different between the two PAHs, there were observed differences between them at the single tran-
script level. For example, in response to pyrene, two carboxypeptidases and a chymotrypsin-like
protease were induced, whereas fluoranthene elicited a repression of cuticular protein-related tran-
scripts (Vandenbrouck et al. 2010), which appears to be a common response to metal contaminants,
as discussed in upcoming paragraphs.
D.  magna also responded differently to binary mixtures of fluoranthene and pyrene, exhibit-
ing greater differential expression than the expected sum of the two parts when exposed to both
compounds (Vandenbrouck et al. 2010). This finding supports a potential synergistic rather than
additive interaction effect on gene expression. Thus, although commonalities exist among daphnid
responses to the organics propiconazole, fluoranthene, and pyrene, there are also stressor-specific
responses that can be valuable for establishing unique response profiles.
SSH libraries were generated for the copepod C. finmarchicus exposed to a sublethal mixture of
environmental stressors (Hansen et al. 2007) and to diethanolamine (DEA; Hansen et al. 2010).
The sublethal mixture was composed of monoethanolamine (MEA), water-soluble fractions of oil,
copper, and elevated temperature, and it induced differential expression of genes such as hsp, anti-
oxidants, and cytochrome P450 (Hansen et al. 2007). Although exposing organisms to a mixture of
compounds may provide some qualitative insight into the inducible transcriptome, it is difficult to
determine which changes are attributable to which compounds, and one mixture represents a min-
ute fraction of the possible combinations of toxicants to which an organism may be exposed. Further,
interactions of specific compounds make it difficult to characterize a response because a pair of toxi-
cants may share an antagonistic interaction on gene expression. Pairing investigations of exposure
to mixtures with single-compound exposures as a contrast will help to provide insight into potential
antagonistic, additive, or synergistic effects on the expression of genes or proteins in the future.
A concentration of DEA corresponding to 25% of the lethal concentration for C. finmarchicus
caused differential expression of 865 clones that were identifiable by basic local alignment search
tool (BLAST; Hansen et al. 2010). Of the 865 clones, gene ontology (GO) terms could be used to
annotate 194 of the induced transcripts and 511 of the repressed transcripts, the majority of which
corresponded to molecular functions of binding, catalytic activity, structural molecule activity, and
transcription regulator activity (Hansen et  al. 2010). Interestingly, the proportion of enzymatic
 Crustacean Genomics and Functional Genomic Responses 449

transcripts that were repressed was 60% higher than the proportion induced and approximately
four times in magnitude (202 transcripts repressed vs. 48 transcripts induced; Hansen et al. 2010),
much like the aforementioned effects of propiconazole, fluoranthene, and pyrene on gene expres-
sion in D. magna.
Metabolomic analysis of C. finmarchicus exposed to DEA using a high-resolution magic angle
spinning NMR (HR-MAS NMR) indicated choline deficiency (Hansen et al. 2010), which could
have an effect on lipid metabolism (Barbee and Hartung 1979). Indeed, changes in transcripts for
enzymes involved in lipid metabolism were found in the SSH library and confirmed using qPCR
(Hansen et al. 2010).
Antibiotics and their effect on crustacean physiology is a growing concern due to their
increasing inclusion as a common practice in aquaculture. The effect of the antibiotics enroflox-
acin and furazolidone on P. monodon has been evaluated using a differential proteomic approach
(Silvestre et al. 2010). P. monodon did not exhibit significantly different protein expression pro-
files from controls in response to the antibiotics except for one unidentified protein, which
was significantly decreased in abundance in response to enrofloxacin (Silvestre et  al. 2010).
However, this study was considerably limited by statistical power due to experimental design
(three replicates per treatment of six pooled individuals). The type of culture system used had
a more significant effect on protein expression than did antibiotic treatment. Two systems
were used: intensive culturing ponds and improved extensive culturing ponds (Silvestre et al.
2010). Intensive culture systems are characterized as being small and relatively deep, with high
stocking densities and water exchange regulated by pumps and aeration systems, whereas the
improved extensive systems are large, shallow, with low stocking densities and a tidal water
exchange (Silvestre et al. 2010).
Nine proteins were found to be differentially expressed between P. monodon reared in intensive or
improved extensive culturing systems, of which three were identified as hemocyanins and were highly
expressed in the intensive system (Silvestre et al. 2010). Two proteins were identified as SCPs and were
highly downregulated in the intensive system. These results suggest that perhaps hypoxic conditions
in the intensive system led to increased hemocyanin production to bind oxygen more effectively and
that these individuals were experiencing suppressed immune capacity because SCPs are involved in
Ca2+ homeostasis and linked to immune function in Drosophila (Engstrom et al. 2004).
Crustacean responses to organic compounds at the omic level, although still quite under-
studied, are beginning to show some commonalities. For example, whether at the transcript- or
protein-level, it remains consistent among studies, compounds, and organisms that repression far
outweighs induction of gene expression, perhaps due to an energetics tradeoff necessary for cellular
repair mechanisms or sustaining metabolic performance under stress. Evidence thus far is sugges-
tive of a dose-dependent magnitude of expression change for some compounds. More studies tar-
geted at gaining a quantitative understanding of expression responses to toxicants will be necessary
to establish reliable indicators of exposure to organic ecotoxicants.

Metals

Almost without exception, omic studies of crustacean responses to metals have focused on cad-
mium (Cd), copper (Cu), or zinc (Zn). Many studies have focused on daphnids: eight involving
D. magna and one D. pulex. Other taxa studied include the Chinese mitten crab Eriocheir sinensis,
shrimp L. vannamei, brine shrimp Artemia sinica, copepod Tigriopus japonicas, and the grass shrimp
Palaemonetes pugio. Within the daphnids, most published work is transcriptomic, although there
have been some metabolomics studies. The D. pulex genome sequencing effort (Colbourne et al.
2011) will undoubtedly increase the output of toxicogenomics studies, and productivity in years to
come will be prolific for daphnids and other crustacean taxa as a result.
450 Jonathon H. Stillman and David A. Hurt

The same cDNA libraries enriched for energy metabolism, molting, and life stage-specific
processes (Soetaert et al. 2006) mentioned in the previous section on organics were used to estab-
lish a cDNA microarray for D.  magna to measure transcriptome responses to 0, 10, 50, and 100
μg/L of Cd at 48 and 96 h exposures (Soetaert et al. 2007). Cd exposure resulted in differential
expression (≥1.8-fold) of 112 nonredundant gene fragments that exhibited a very clear time- and
concentration-dependent relationship (Soetaert et al. 2007). The differentially expressed fragments
corresponded to genes involved in digestion, oxygen transport, acid–base balance, and immune
response, based on GO annotations (Soetaert et al. 2007). A first-generation cDNA microarray was
also established for D. pulex to measure expression changes resulting from Cd exposure, leading to
the identification of novel metallothioneins (Shaw et al. 2007). Since these initial studies, a number
of efforts have been made to further investigate the effects of Cd and other metals on daphnids—
although mostly in D. magna, likely due to its history as a sentinel for aquatic ecosystem health.
D.  magna exposed to nickel (96 h at either 0.125, 0.5, 1, or 2 mg/L), exhibited repression of
hemoglobin-associated genes but induced genes potentially involved in early heme biosynthesis
(Vandenbrouck et  al. 2009). These conflicting results suggest that hemoglobin effects observed
in daphnids exposed to metals could be due to disruption of heme biosynthesis and not hypoxia
inducible factor-related transcriptional regulation of the hemoglobin protein (Vandenbrouck et al.
2009). By pairing microarray results with a time-course of available energy reserves, it was con-
cluded that vitellogenin repression under heavy-metal stress might be due simply to a lack of energy
reserves, which could preclude the use of vitellogenin as a biomarker for juvenile hormone expo-
sure (Vandenbrouck et al. 2009) although targeted expression studies will be needed in the future.
A cDNA microarray consisting of 5 K randomly selected cDNA clones from the Daphnia
Genome Consortium was used to generate distinct expression profiles for D. magna exposed to
Cu, Cd, and Zn at 5% of the lethal concentration for 24 h (Poynton et al. 2007). Animals exposed
to Cd exhibited the greatest differential expression, with the majority of transcripts being repressed
(Poynton et al. 2007). In D. magna exposed to Zn and Cu, the proportion of repressed and induced
transcripts was roughly equal, whereas Cd-exposed individuals had fourfold greater repressed
genes (Poynton et al. 2007). There were only four differentially expressed genes common to all
three metal exposures; all four were repressed and corresponded to digestion and nutrient absorp-
tion (Poynton et al. 2007). A larger 15 K oligonucleotide was used to demonstrate that in D. magna
exposure to silver (Ag) complexed with nanoparticles caused physiological responses that were dif-
ferent from exposure to Ag alone (Poynton et al. 2012). Metal responsive and DNA damage repair
genes were induced by Ag in nanoparticle complexes, but not AgNO3, suggesting that we need to
re-evaluate toxicity of metals when they are complexed in nanoparticles.
To establish biomarkers for metal exposure that extend in utility beyond a qualitative indi-
cator, concentration- and metal-dependent gene expression profiles have been generated and
field-validated for D. magna (Poynton et al. 2008). When exposed to concentrations of 5% of the
effective concentration, 5% of the lethal concentration, and a nonobservable effect concentration of
Cu, Cd, and Zn, D. magna gene expression, quantified using a previously developed cDNA micro-
array (Poynton et al. 2007), exhibited very specific profiles dependent on the concentration and
type of metal (Poynton et al. 2008). Exposure to lower concentrations resulted in a more resolved
and metal-specific expression profile for all three metals, whereas exposures of near-acute toxic-
ity resulted in many more common stress response genes being expressed (Poynton et al. 2008).
Thus, the utility of gene expression profiles for distinguishing between metal types may be lim-
ited to chronic sublethal exposures in daphnids, although a general inference of metal exposure
may be gained under acutely toxic conditions. The feasibility of using microarray technology to
identify a specific metal in a natural setting has been validated for D. magna. Animals exposed to
field samples collected from Cu mines in California were used to create gene expression profiles
and compared to previously generated profiles for Cu, Cd, and Zn (Poynton et al. 2008). Cu was
 Crustacean Genomics and Functional Genomic Responses 451

successfully identified as the primary pollutant based on clustering analysis and class prediction
algorithms (Poynton et  al. 2008), thus demonstrating the applied potential of daphnid ecotoxi-
cogenomic assessments.
Although a number of studies have demonstrated that gene expression profiles are metal- and
concentration-specific in daphnids, expression data without any indication of ecological signifi-
cance are of little importance if one aims to evaluate ecosystem health. In D. magna, transcriptome
profiles generated using a 14 K feature cDNA microarray were compared to population growth
rate (Connon et al. 2008). As population growth rates declined with increasing Cd concentrations,
increased differential expression of transcripts associated with cellular processes such as growth
and molting, ion transport, and general stress responses was observed (Connon et  al. 2008).
Similarly, D.  magna fed a Zn-enriched diet suffered decreases in population and delays in molt
cycle relative to controls, and differential expression of transcripts involved in growth and molting
was also observed (De Schamphelaere et  al. 2008). The pairing of transcriptomics studies with
higher level observations, such as decreased population growth rates and depleting energy reserves
(Vandenbrouck et al. 2009), suggests that chronic effects observed in daphnids due to metal expo-
sure may be due in part to a reduction in the bioavailability of resources to the organism and thus
the ability to assimilate energy that would otherwise drive somatic growth.
There are limited metabolomics studies involving crustacean responses to metals. When com-
bined with expression data from a 44 K oligonucleotide microarray, results from Fourier trans-
form ion cyclotron resonance mass spectrometry and NMR spectroscopy analyses indicate that
fatty acid metabolism and nutrient absorption may be primary avenues through which Cd reduces
energy reserves in D. magna (Poynton et al. 2011). Four fatty acids decreased in D. magna hemo-
lymph resulting from Cd exposure: lauric acid, myristic acid, palmitic acid, and steric acid, which
are all part of the fatty acid biosynthesis pathway (Poynton et al. 2011). Furthermore, six of the
seven amino acids that decreased in D. magna hemolymph after Cd exposure were essential, thus
indicating the perturbation of nutrient absorption, a result supported by transcriptome changes
of genes involved in protein and carbohydrate metabolism (Poynton et  al. 2011). Metabolomic
analysis has been used in D. magna to establish signatures of specific compounds with differing
modes of action, such as Cd and various nonmetal compounds (Taylor et al. 2009, Taylor et al.
2010). Initial studies provide compelling evidence that metabolomics, especially when paired
with transcriptomics and other approaches, will prove a valuable tool in elucidating mechanisms
underlying toxicity in crustaceans.
Outside of the daphnids, omic studies of crustacean responses to metals are taxonomically
limited. Most nondaphnid studies are on the decapods or copepods. Within the Decapoda, there
have been two EST studies and one proteomic study. ESTs have been generated in response to
Cd in postlarvae of the shrimp L. vannamei (Keating et al. 2007) and in Cu-exposed P. pugio (Li
and Brouwer 2009a). A proteomic analysis of the anterior gill of the mitten crab E. sinensis under
exposure to Cd has also been performed (Silvestre et al. 2006). In copepods, there has been one
oligochip microarray study in T. japonicas exposed to Cu and an SSH library (Ki et al. 2009) pre-
pared from C. finmarchicus exposed to a mixture of MEA, Cu, and water-soluble fractions of oil at
elevated temperature (Hansen et al. 2007). One other proteomic study on the response of A. sinica
larvae to Cu-sulfate has been performed (Zhou et al. 2010). There is a great necessity for future
efforts to include functional genomics approaches to metal exposure in nondaphnid taxa because
such studies will allow comparative approaches between these taxa and the well-studied D. magna.
In E.  sinensis, acute and chronic Cd-exposure regimes express a very different gill proteome
(Silvestre et al. 2006). E. sinensis expressed six two-dimensional gel electrophoresis protein spots
differentially after acute (500 μg/L for 3 days) exposure and 31 spots after chronic (50 μg/L for
30 days) Cd exposure, which resulted in acclimation or increased resistance to subsequent acute
exposure (Silvestre et al. 2006). The upregulation of several antioxidant proteins and chaperones
452 Jonathon H. Stillman and David A. Hurt

during chronic acclimation suggests that Cd induces toxicity primarily through oxidative stress and
sulfhydryl-binding in E. sinensis (Silvestre et al. 2006). Similarly, in response to Cu-sulfate, A. sinica
larvae upregulated chaperones as well as peroxiredoxin (Zhou et al. 2010). Although these results
are highly suggestive of oxidative stress as a primary route of toxicity for these metals in both mala-
costracan and maxillopodan crustaceans, many more proteomic analyses in multiple species will be
necessary to offer a better-resolved mechanism.
T. japonicas consistently induced and repressed 138 and 375 genes, respectively, when exposed to
10 μg/L Cu for 6, 12, and 24 h (Ki et al. 2009). Similar to the effects of propiconazole on daphnids
(Soetaert et al. 2006), the greatest number of differentially expressed transcripts occurred at the
earliest time point, indicating an acute effect on gene expression, and the majority of differentially
expressed genes were repressed (Ki et al. 2009), an observed commonality in crustacean responses
to both organics and metals. The majority of repressed genes were involved in growth and develop-
ment, again suggesting an energetics tradeoff in transcription during stress. Contrary, however, to
the common finding of repressed mRNA as well as protein-level responses to environmental toxi-
cants, the copepod C. finmarchicus exhibited more induction (127 ESTs) than repression (54 ESTs)
of identifiable transcripts in response to a combination of stressors (Hansen et al. 2007). Increased
induction may be a product of limited annotation power, but it could also be a true result indicating
an emergent property of gene expression under multiple stressors, and it highlights the confound-
ing effects toxicant mixtures may have on crustacean biology.

HYPOXIA

Crustaceans are commonly found in hypoxic habitats, and induction of hemoglobins is well known
in the Branchiopoda following hypoxic or anoxic exposure. However, relatively few studies have
employed omics approaches to examine the responses of crustaceans to hypoxia.
Grass shrimp, P. pugio, exposed for 3 to 5 days to severe (1.5 mg/L O2) and moderate (2.5 mg/L O2)
chronic hypoxia and to variable oxygen tensions between severe hypoxia and normoxia (1.5→7 mg/L)
were used to construct hepatopancreas tissue SSH libraries in order to identify genes involved with
response to environmental oxygen (Li and Brouwer 2009a). For each oxygen exposure, induced and
repressed cDNAs were identified and sequenced. The three oxygen exposures resulted in three dis-
tinct sets of differentially expressed transcripts for which functional homology was ascribed, with only
one transcript differing specifically between oxygen treatments (Li and Brouwer 2009a). Translation
elongation factor 2 was induced in both chronic oxygen exposures, and cytochrome c oxidase sub-
unit III was induced in both chronic and variable exposures where severe hypoxia was reached (Li
and Brouwer 2009a). Cytochrome c oxidase subunit I was observed to be upregulated across all oxy-
gen exposures (Li and Brouwer 2009a). Chronic severe hypoxia resulted in six induced transcripts,
three of which were not observed in any of the other oxygen treatments (Li and Brouwer 2009a). In
contrast, moderate chronic hypoxia and cyclical hypoxia resulted in 15 and 22 differentially induced
genes, respectively. Both moderate and cyclical hypoxia induced genes involved with oxygen transport
and gluconeogenesis when mean oxygen tensions were low but not severe (Li and Brouwer 2009a).
Genes induced in chronic moderate hypoxia included several lipid-binding/modification genes and
oxygen-binding proteins (Li and Brouwer 2009a). Genes expressed only in cyclical hypoxia included
potential DNA modification proteins, as well as oxidative metabolism (Li and Brouwer 2009a). There
was a larger magnitude response of genes repressed by chronic and cyclical hypoxia, and, like induced
genes, most differentially repressed genes were expressed only in one of the three hypoxia treatments.
Chronic severe hypoxia resulted in 21 repressed genes, one of which, vitellogenin, was also observed
as repressed in the other two treatments. Moderate chronic hypoxia and cyclical hypoxia resulted in
47 and 58 repressed genes, respectively, with five transcripts repressed in both of those treatments
(Li and Brouwer 2009a). Under chronic severe hypoxia, the repressed genes encoded some immune
 Crustacean Genomics and Functional Genomic Responses 453

responsive proteins, fermentative metabolism, lipid binding, and cell–cell communication (Li and
Brouwer 2009a). Moderate chronic hypoxia repressed some structural protein genes, immune respon-
sive genes, protein synthesis, and polyamine synthesis inhibition. Genes involved in sulfur redox and
(homo)cysteine metabolism were all repressed in response to cyclic hypoxia. Additionally, cyclical
hypoxia repressed genes that may be involved in vitamin metabolism and, potentially, pH regulation
(Li and Brouwer 2009a).
The SSH libraries generated by Li and Brouwer (2009a) were used to construct a cDNA microar-
ray to determine a higher resolution response to hypoxia in thorax and hepatopancreatic tissues of
P. pugio at between 0 and 240 h after severe chronic hypoxia (Li and Brouwer 2009b). The first 5 days
of hypoxia caused induction of genes that were involved in oxygen transport, cell surface recognition,
and iron homeostasis but little in the way of gene repression. At 10 days of hypoxia, there was a shift in
gene expression patterns, with induction of only a few genes but repression of a large number of genes
involved in metabolism and, potentially, cell cycle regulation (Li and Brouwer 2009b). Overall, the
results suggested that a limited set of transcripts were modulated by hypoxia but that these transcripts
were both induced and repressed by hypoxia, depending on the hypoxia duration. Genes induced at
6, 24, and 120 h hypoxia tended to be repressed at 12, 48, and 240 h hypoxia (Li and Brouwer 2009b),
potentially because of confounding effects of endogenous rhythms of gene expression.
Proteomic two-dimensional electrophoretic analysis of hepatopancreas response to hypoxia in
the fleshy prawn Fenneropenaeus chinensis identified 67 protein spots that changed following 3.5 h
at 45% oxygen ( Jiang et al. 2009). Fifty-one of the 67 differentially expressed protein spots had
homology to known proteins. Fifteen spots representing 11 proteins were upregulated, and 36 spots
representing 23 proteins were downregulated during hypoxia. Proteins upregulated by hypoxia
were involved in carbohydrate metabolism, immune response, chaperone proteins, and exoskel-
etal pigmentation ( Jiang et al. 2009). Proteins downregulated by hypoxia were involved in energy
metabolism, antioxidant response, chaperones, and cytoskeletal proteins ( Jiang et al. 2009). The
most strongly upregulated and among the most strongly downregulated proteins were the protein
disulfide isomerase chaperonins (PDIs). Because PDIs were observed in spots both up- and down-
regulated following hypoxia, these spots likely represent different post-translational modifications
that play a role in regulation of activity ( Jiang et al. 2009).
In hemocytes of P. monodon, SSH and nonsubtracted libraries were used to construct a 4 K feature
cDNA microarray that was used to profile responses to multiple environmental stressors, including
hypoxia (de la Vega et al. 2007). P. monodon held at 29°C and salinity 35 were exposed to 1 part per mil-
lion (ppm) oxygen saturation for 8 h and then returned to normoxia. Control specimens were main-
tained under normoxic conditions for the duration of the experiment. Hemolymph samples were
withdrawn for transcriptome profiling at four time points: (i) before the hypoxia, (ii) after reaching
1 ppm O2, (iii) 1 day after returning to normoxia, and (iv) several days following return to normoxia
(de la Vega, Hall et al. 2007). Seventy-five features were significantly differentially expressed across
the four sampling time points within the hypoxia-treated shrimp, only a few of which had putative
functional homology. The strongest repressed genes during hypoxia encoded proteins involved in
oxygen transport and the immune response (de la Vega et al. 2007). At time point (ii), comparison
of hypoxia-treated and control specimens yielded 26 differentially expressed genes, including hemo-
cyanin and crustin, both of which were repressed in response to hypoxia (de la Vega et al. 2007). This
result suggests that hypoxia exposure could result in increased susceptibility to infection.

OSMOTIC STRESS

There exists little published work utilizing omics-based investigations of crustacean responses to
osmotic stress. Many targeted expression and candidate gene approaches have been taken, and uti-
lizing the power of omics technologies may increase the mechanistic implications of these studies.
454 Jonathon H. Stillman and David A. Hurt

de la Vega et al. (2007) constructed a cDNA microarray from both SSH and normal cDNA librar-
ies from hemocytes of P. monodon (as previously mentioned) to evaluate transcriptomic responses
to multiple environmental stressors. Genome-wide expression profile responses to hypo-osmotic
conditions in P. monodon indicated differential expression of a number of cDNA clones; however,
many did not correspond to sequences of known biological function (de la Vega et al. 2007). Genes
induced following hyposmotic stress encoded respiratory and immune proteins. Repression of ret-
rotransposons was observed immediately following exposure to hypo-osmotic conditions, which
warrants further investigation into the role of these elements in the stress response because ret-
rotransposons were also observed to be differentially expressed in response to temperature stress
(de la Vega et al. 2007). When compared to controls, differentially expressed genes were almost all
repressed immediately following osmotic stress except for hemocyanin, which was induced.
Although de la Vega et  al. (2007) were able to identify few cDNA clones with putative bio-
logical function, there were 69 clones that exhibited temporal variation in expression through the
course of exposure to osmotic stress and 45 clones that were differentially expressed relative to con-
trols immediately following exposure, indicating that perhaps the largest magnitude of expression
change occurs relatively quickly. However, the lack of experiments in this area makes it difficult to
infer any general omic-scale responses in crustaceans to osmotic stress. Because of this, it is crucial
that future studies be targeted at this area to better elucidate effects of salinity on crustacean gene
and protein expression.

ACIDIFICATION

Carbon dioxide-driven acidification of seawater, a process termed ocean acidification (OA), has
recently been the focus of much research in environmental physiology. Compared to studies on
mollusks and echinoderms, relatively few studies have examined the effects of OA on crustaceans,
and, of those, there have been only a small number involving genome-scale approaches. Studies to
date suggest that there are moderate to small changes at the transcriptome or proteome level fol-
lowing exposure to OA conditions in crustaceans. For example, in a two-dimensional gel analysis
of the barnacle Balanus amphitrite cyprid, only nine of 566 protein spots changed in response to
acidification, four of which were upregulated and five of which were downregulated in individuals
exposed to pH 7.6 versus the control pH 8.1 (Wong et al. 2011). Although ascribing cellular function
shifts following such a small change in the proteome is tenuous at best, upregulated proteins were
involved in protein synthesis, respiratory gas transport, and energy metabolism, whereas down-
regulated proteins encoded endocytosis and proteolytic proteins (Wong et al. 2011).

FUTURE DIRECTIONS

Genome-scale studies of crustacean responses to environmental stress that have been done to date have
been focused on relatively few taxa (Fig. 13.1) and with unequal representation of stressors examined
across taxa (Fig. 13.4). Future studies, aided by the reduced costs and ease of next-generation DNA
sequencing for genomic and transcriptomic (i.e., RNA-seq) data collection and new approaches in
LC-MS/MS-based proteomics that have hugely more resolving power than two-dimensional gel elec-
trophoresis, are likely to be more numerous in crustaceans and have the potential to be conducted on
a greater array of taxa, hence presenting a better balance of studies and taxa than seen now (Fig. 13.4).
Data collected to date have begun to reveal some common cellular response pathways to an array of
stressors (Table 13.3), and it is entirely likely that new fundamental understanding of crustacean physi-
ology will be aided by comparative functional genomic and proteomic analyses in the years to come.
 Crustacean Genomics and Functional Genomic Responses 455

Of significance in understanding the ecological responses of crustaceans using a molecular

physiology approach is the examination of responses to interactive or synergistic stressors (e.g.,
temperature and salinity; temperature and infection) in comparison to the responses to those
stressors in isolation. The natural world is changing in a multifactorial and complex manner, and
whereas molecular biomarkers such as transcriptome fingerprints have great potential for unrav-
eling physiological differences in field-acclimatized specimens (Fig. 13.3), realizing that potential
requires characterization of those molecular fingerprints under controlled conditions where envi-
ronmental stressor combinations reflect the natural habitat conditions. Although such experiments
are difficult to conduct and can be expensive, it is those studies that are likely to be highly infor-
mative in understanding the biology of crustaceans under future biotic and abiotic scenarios. Of
particular interest should be resolving physiological responses under combinations of temperature,
pH, hypoxia, infectious organisms, and ecotoxicants because all of those stressors are predicted to
change in the future, and crustacean responses to those variables have not been studied in a multi-
factorial context.

CONCLUSIONS

We hope that this chapter provides a valuable benchmark for the state of studies employing genomic
approaches in the analysis of crustacean responses to environmental stress and infection. We have
aimed to provide a comprehensive review of the literature, inclusive of all germane publications,
and apologize for any studies we neglected to include. An abundance of genomic, proteomic, and
metabolomic studies in crustaceans also exist for studies of the endocrine system, developmental
regulation, and the molt cycle, which were not included here due to space limitations.
Taxonomic breadth of coverage of crustacean omic-scale responses to environmental stress
and infection is quite limited. However, the relatively few studies that have been conducted to date
allow some early inferences to made into shared stress responses. For example, we begin to see that
there are many genes that have expression responses, whether up or down, to multiple types of
environmental stressors (Table 13.3). Based on the literature reviewed here, some common mecha-
nisms shared between taxa can be gleaned. For example, it appears that in crustaceans there is a
specific time course of hsp transcript expression in response to a stressor, be it temperature or a
heavy-metal toxicant. Generally, it appears that hsp90 is expressed rapidly in response to an acute
exposure, whereas onset of other chaperones of this family is slower and more drawn out (e.g.,
hsp70, hsp20). Due to the differential expression of oxidative stress genes by various stressors (e.g.,
infection, heavy metal; Table 13.3) it appears that this may be a common pathway through which a
number of stressors operate to cause detrimental cellular and physiological effects across the crusta-
cean phylogeny. However, more comparative work on the role of oxidative stress and the molecular
constituents involved will be necessary to elucidate this process on a mechanistic level because
oxidative stress genes are repressed under pathogen infection and induced under heavy-metal expo-
sure. Vitellogenin is another gene that has been observed to respond to environmental stressors
of numerous types across crustacean taxa. Although there is much mechanistic work targeted at
the role of vitellogenin in various endocrine and other cellular processes, future efforts should be
directed at reconciling the differences in expression observed in response to different stressors in
different crustacean taxa (Table 13.3).
Overall, the use of transcriptomic and proteomic approaches to understand physiological
processes occurring across environmental gradients, as well as in laboratory studies investigating
physiological responses to abiotic and biotic stressors, is just beginning in crustaceans. The devel-
opment of genomics information in crabs (Tagmount et al. 2010), shrimp ( Jung et al. 2011, Ma et al.
2012), amphipods (Zeng et al. 2011), and Daphnia (Colbourne et al. 2011, Orsini et al. 2011), among
456 Jonathon H. Stillman and David A. Hurt

other crustaceans (Table 13.1), as well as the spectacular reduction in both cost and effort of new
genomic data acquisition afforded by next-generation DNA sequencing technologies will likely
accelerate the pace of such studies in the near future.

ACKNOWLEDGMENTS

This material is based on work supported by the National Science Foundation (grant no. 1041225 to
J.H.S.; Graduate Research Fellowship to D.A.H.).

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14
ENDOCRINE-DISRUPTING CHEMICALS

Peter L. deFur and Laura E. Williams

Abstract
Chemicals that disrupt animal endocrine systems have been the topic of scientific investigations
for some years and of public interest in recent years. Much of the attention has been focused on
vertebrates, including the recent phenomena of sex reversal in male fish. However, two of the
best examples of environmental endocrine disruption are from invertebrates: gastropod mollusks
affected by tributyltin and insects affected by pesticides. Crustacean endocrine systems are known
to be sensitive to several types of endocrine-disrupting chemicals, largely because of the similarity
between insect and crustacean hormones. Crustacean endocrine systems, although not as com-
prehensively examined as vertebrate endocrine systems, utilize a range of compounds including
steroids, terpenoids, amines, and peptides as neurohormones. Early investigations into crustacean
endocrine disruption is derived from research on a few experimental models and field studies.
Subsequent research extends these observations on endocrine disruption in crustaceans to ter-
restrial species and several other orders.

INTRODUCTION

The topic of endocrine disruption entered the lexicon of modern biological researchers in the 1990s
based on laboratory and field investigations on vertebrates, although earlier work included a poly-
phyletic background (Gorbman and Davey 1991, deFur et al. 1999). Soto et al. (1991) reported that
chemicals leaching from plastic culture dishes exhibited estrogenic properties in the MCF7 breast
cancer cell line that is responsive only to estrogen. Soto et al. (1991) then screened other commonly
used chemicals and found that many exhibited estrogenic activity. Based on the in vitro assay, a
number of commonly used chemicals have the potential to activate a hormone pathway known to

461
462 Peter L. deFur and Laura E. Williams

be a cancer risk factor. The significance of these results is their applicability to other receptor-based
hormones. A broader perspective and more complete picture of endocrine disruption resulted from
a focused workshop comparing their effects across vertebrate species (including humans), physi-
ological systems, and animal models (workshop proceedings published by Colborn and Clement
1992). It is clear from the results presented and reviewed in Colborn and Clement (1992) and in
the subsequent review paper (Colborn et al. 1993) that basic physiological mechanisms could be
affected by anthropogenic chemicals. The physiological systems of greatest interest in these reviews
were endocrine and developmental.
The review by Colborn et  al. (1993) across laboratories and species indicated that man-
made compounds could interact with endocrine systems, including those of crustaceans, but
no review of endocrine disruption had taken a truly multiphyletic approach. Indeed, reviews
of the topic by the National Academy of Sciences (NRC 1999), Kendall et al. (1998), and the
Environmental Protection Agency (US EPA 1997)  only included a passing reference to inver-
tebrates. deFur et al. (1999) was the first volume (proceedings of a workshop organized by the
Society for Environmental Toxicology and Chemistry [SETAC]) to address invertebrate endo-
crine disruptors across phyla and end points. Among the conclusions by deFur et al. (1999) are
that endocrine-disrupting chemicals can influence growth, reproduction, and sexual differen-
tiation in various species, notably arthropods. Oberdorster and Cheek (2000) and more recent
publications (summarized in this chapter) reached a similar conclusion in reviewing the phe-
nomenon in marine crustaceans.
Invertebrates constitute about 95% of the known species on our planet and play a pivotal role
in ecosystem dynamics (deFur et al. 1999), in addition to acting as biological indicators for other
biological systems. Of the invertebrates, crustaceans are critical components of many ecosys-
tems: zooplankton abound throughout the world’s oceans, larger species are scavengers and pred-
ators in shallow marine and estuarine habitats, and many species are a food source for the human
population. The evaluation of the effects of endocrine-disrupting chemicals on crustaceans is nec-
essary to understanding the broader impacts of endocrine-disrupting chemicals.
Aspects of the crustacean endocrine system are described in detail in Webster’s chapters (see
Chapters 1 and 2 in this volume), and the reader is referred to them for a comprehensive treatment
of the systems and hormones. Some important features need to be appreciated in a consideration
of crustacean endocrine disruptors. Typically, endocrine systems function via cascades that are
initiated by environmental or physiological cues and result in a terminal hormone and its action
on a target organ. The cascade acts as a line of communication between the nervous system and
the endocrine system and has been appreciably conserved over evolutionary time. The activity of
all hormones is mediated by specific hormone receptors. Common across all hormone action is
(i) the presence of specific, high-affinity protein receptors found within or on the target cell for a
particular hormone, and (ii) the complex formed from binding of the particular hormone and its
receptor results in biochemical activity in the target tissue. Invertebrate hormone systems generally,
and crustacean systems specifically, rely to a great extent on neurohormones that are not steroidal;
the ecdysteroids are more an exception to this generalization as an important steroid hormone class
in Arthropoda.
A key feature of endocrine systems is the multistep aspect of the cascading response system.
Such systems begin with hormone synthesis and proceed with intracellular transport, release
into a body fluid, transport (possibly in a bound form) in the body, and recognition at the tar-
get tissue/cell site, followed by the response at the cellular level. Cellular responses include
receptor binding, additional protein binding, nuclear transport (in the case of steroids), gene
activation, and any subsequent metabolic synthesis, transformations, and secretion. The cascad-
ing sequence of events means that anthropogenic chemicals may interfere at any one of a large
 Endocrine-Disrupting Chemicals 463

number of steps in the long series of biochemical events of normal endocrine functioning that
are present in all systems.

ENDOCRINE DISRUPTION

Endocrine disruption is a form of toxicological effect that targets endocrine systems and
frequently causes changes that are not acutely lethal or even chronically debilitating.
Definitions of endocrine-disrupting compounds (EDCs) abound in the open literature and
in regulatory documents (US EPA 1997, US EPA 1998, deFur et al. 1999, NRC 1999); most
of these sources describe an EDC as a chemical that interacts with a hormonal pathway
and elicits an abnormal or harmful response, a generalized definition we adopt here. Weis’s
chapter (see Chapter 15 in this volume) discusses toxicology in the Crustacea, and the pres-
ent chapter does not attempt to duplicate the basic toxicological information. Endocrine
disruption deals with one specific mode of action in toxic chemicals, and, for this reason,
the definition may provoke considerable controversy. Classifying a chemical as an EDC,
with all the associated connotation and regulatory implications, would seem to require that
the compound functionally interact with the endocrine system and not simply affect an
endpoint under endocrine control. The subject itself deals with endocrinology, toxicology,
physiology, and chemical regulatory policy.
The recent scientific and regulatory interest over endocrine-disrupting chemicals began with a
focus on the specific mechanism of action of the receptor binding step in hormone function; some
chemicals can bind to hormone receptors and modulate the activity of these receptors (McLachlan
et al. 1992, Kelce et al. 1995). The estrogen system was an early focus of endocrine disruptor research.
Some xenobiotics exhibit agonistic behavior by binding to a hormone receptor and functioning like
that hormone (Arnold et al. 1996). Other xenobiotics exhibit antagonist behavior by binding to the
hormone receptor without stimulating the activity of the receptor. This binding blocks the receptor
from accepting the endogenous hormone for that receptor, thus competitively inhibiting its activ-
ity. The endocrine system, however, consists of much more than receptor binding, as noted earlier,
and xenobiotics can also act as endocrine disruptors without interacting with hormone receptors.
Instead, some chemicals can induce increases or decreases in levels of an endogenous hormone. For
example, the fungicide ketoconazole can lower serum testosterone levels by inhibiting testosterone
synthesis (Arnold et al. 1996).
A critically important result in the early investigation of endocrine disruption was the obser-
vation of altered secondary sexual characteristics in fish, first reported in the United Kingdom
(Purdom et al. 1994, Sumpter and Jobling 1995). Several investigations in John Sumpter’s lab
revealed that male fish in waters downstream from sewage treatment facilities produced egg
protein, vitellogenin, in response to estrogenic chemicals in the discharges. These results dem-
onstrated that external exposure to at least estrogens and perhaps other endocrine disruptors
could effectively initiate responses other than the expected responses, whether lethal or not.
The significance for crustaceans is that many are aquatic and all are oviparous, as are fish, raising
the possibility that aquatic crustaceans may be susceptible to similar exposures and effects as
reported by Sumpter and Jobling (1995) in fish. Subsequent research supports the applicability
to aquatic crustaceans (deFur et al. 1999), as described later (see Lye et al. 2005, Lye et al. 2008).
Recent developments regarding the susceptibility of freshwater crustaceans to estrogenic sub-
stances provide evidence that gammaridian amphipods are likely susceptible to wastewater
effluents (Schirling et al. 2005).
464 Peter L. deFur and Laura E. Williams

Another outcome of the work reported by Sumpter and co-workers in Great Britain was
completion of similar investigations in the United States, reported first by Folmar et  al.
(1996). As a result, the US Geological Survey (USGS) initiated a more comprehensive sur-
vey of contaminants in surface waters of the United States to include chemicals that had
not been previously assessed. The USGS termed these chemicals “emerging contaminants”
and published the results of a survey of 139 locations across the continental United States
(Kolpin et al. 2002). The results indicated the widespread occurrence of a range of chemicals
in rivers, streams, lakes, and ponds. The chemicals include pharmaceuticals (ethinyl estra-
diol) and industrial chemicals (polychlorinated biphenyls) known to be hormonally active
in cell-based or whole-animal bioassays (see Colborn et al. 1993 for early review on the clas-
sification). The significance of these results for crustaceans is that aquatic crustaceans in all
types of habitats are likely exposed to some range of chemicals known to be hormonally
active in animal systems.
Early indications of the sensitivity of aquatic crustaceans to EDCs is found in the investigations
of such chemicals as chlordecone (trade name Kepone), spilled into the James River, Virginia, in
the 1970s (Schimmel et al. 1979) and interfering with normal molting of blue crabs. Chlordecone
is an estrogenic insecticide that was removed from the market following the Virginia incident. In
addition, the early evidence that farnesol mimics juvenile hormone ( JH) activity was accompanied
by evidence that insect JH mimics also altered normal molting patterns in crustaceans (Laufer et al.
1998, Tuberty and McKenney 2005, Zou 2005).

VERTEBRATE RESEARCH INFORMS THE FIELD

We gain valuable perspectives and insights from understanding the research on EDCs in ver-
tebrates, where the topic has been more extensively studied as a general and more public matter
(NRC 1999). Colborn and Clement (1992), Colborn et al. (1993), and the NRC (1999) reviewed a
number of cellular and biochemical mechanisms by which EDCs may act, but the most frequently
investigated mechanism was estrogen receptor function. EDCs interfere with steroid hormones,
such as estrogens, and have been associated with altered reproductive function in all vertebrate
classes (Tyler et al. 1998). The vertebrate hormone receptor system is highly conserved and is bet-
ter understood than that of the invertebrates. Receptor-binding activity of xenobiotics has been
largely documented with steroid hormone receptors in vertebrate systems (deFur et al. 1999). One
of the first indications of vertebrate exposure to EDCs was the presence of vitellogenin (an egg yolk
protein) in male fish (Purdom et al. 1994). A decreased population of juvenile alligators in Lake
Apopka, Florida ( Jennings et  al. 1988)  led Guillette et  al. (1994) to determine that the juvenile
males had poorly organized testes and small phalli, and the females had multinucleated oocytes.
Dicofol, DDT, and agricultural runoff were the active agents with hormonal activity reported by
Guillette et al. (1994).

INVERTEBRATES

Endocrine disruption was documented in the invertebrates (deFur et  al. 1999)  long before
the phenomenon was recognized in vertebrates in the past two decades. A  number of insec-
ticidal agents were intentionally formulated to interfere with growth and/or metamorpho-
sis, and some exhibited serious effects on nontarget species of aquatic crustaceans (Touart
1989). Furthermore, the boat paint biocidal agent tributyltin has been known to alter normal
 Endocrine-Disrupting Chemicals 465

reproductive organ growth and development in marine snails since at least 1981 (LeBlanc and
Bain 1997). Scientific understanding of endocrine disruption in the invertebrates generally has
developed along quite a different pathway than in the vertebrates, as summarized earlier and as
described in numerous volumes. Both the basic endocrinology and life history of those verte-
brates investigated were reasonably well described, even if imperfectly in some species. But the
same is not true for the vast majority of invertebrates (Fingerman 1997, deFur et al. 1999, Chang
and Mykles 2011). The state of biological knowledge of most invertebrate endocrine systems is
not sufficient to explore mechanistic aspects of EDCs.
Compared to the vertebrates, invertebrates have evolved a multitude of varying strategies
to complete growth, development, and reproduction. Due to the high degree of evolutionary
divergence found among the invertebrate phyla, regulation of invertebrate neuroendocrine
systems is much more diverse than in vertebrates. Furthermore, regulation of invertebrate
neuroendocrine systems is based on steroid, terpenoid, and peptide hormones, where pep-
tide hormones are by far the most common (Gorbman and Davey 1991). One major differ-
ence among the invertebrate phyla is between the deuterostomes that use vertebrate-type
sex steroids as terminal hormones and the protostomes that use neuropeptides much more
commonly. Insects and crustaceans, however, challenge a simple dichotomous distinction and
utilize both ecdysteroid (steroid) and terpenoids as terminal hormones within a neuroendo-
crine cascade. Invertebrate secretory structures more often consist of neurosecretory cells or
organs instead of true glands, maintaining a close connection between nervous and endocrine
tissues (deFur et al. 1999).

Arthropods

Insects as a Basis for Understanding Crustaceans

The insect endocrine system is the most well known of the invertebrates generally and the
arthropods more specifically, owing to the importance of insects in agriculture and the manip-
ulation of insect physiology to control pests; this system serves as the prototype for the other
arthropods, including the crustaceans. Several of the hormones, notably molting hormones
and JHs, are structurally similar or identical among arthropods. The insect system consists of
neurosecretory cells found mostly in the central nervous system, three endocrine glands, the
corpora allata, the corpora cardiaca and prothoracic gland, and the gonads. Neurosecretory
cells of the insect release neuropeptides into the hemolymph, controlling growth, molting,
and reproduction in response to external cues. Growth and development is accomplished
through a series of molts that produce ever larger exoskeletons revealed after the shedding of
the old cuticle (Nijhout 1994). An important neuropeptide in the regulation of molting is pro-
thoracicotropic hormone (PTTH), which is released from neurosecretory cells in the brain
and stimulates the prothoracic glands to secrete ecdysone, a steroid hormone also known as
the molting hormone (generically named ecdysteroids). Ecdysone travels via hemolymph to
other tissues that convert ecdysone to 20-hydroxyecdysone (20E), which acts on certain tar-
get cells. Ecdysone is involved in cell proliferation (Champlin and Truman 1998), and 20E is
involved in cell differentiation important in the production of the new cuticle. Production of
the new cuticle usually does not begin until ecdysone starts to decline. JH, a terpenoid hor-
mone, is the secretory product of another neuroendocrine cascade and plays a role in regulat-
ing the action of 20E. JH is important for the maturation of an animal that molts to transition
from juvenile to adult stages.
466 Peter L. deFur and Laura E. Williams

Ecdysteroids

Ecdysone was the first steroid hormone shown to have an action directly on DNA, summarized
in Nijhout (1994). Clever and Karlson (1960) demonstrated that ecdysone applied to the salivary
glands of the midge Chironomus tentans caused synthesis of mRNA and proteins (Clever 1964).
Similar studies in Drosophila led to a model for ecdysteroid action (Ashburner et al. 1974) in which
the biologically active 20E, coupled to the ecdysone receptor (EcR), differentially regulates several
classes of target genes.

Metamorphosis and Development

The presence or absence of the sequiterpenoid hormone JH determines whether the

end-product of the molt is a juvenile or an adult. In the absence of JH at the time of ecdy-
sone release, metamorphosis will occur. Exposure of a pupa to JH prevents development to
an adult and causes a second pupal molt. Timing of the JH release is critical. The presence of
JH during the final larval stage produces excess larval molting, and the absence of JH from
earlier larval instars can cause premature transformation to adulthood or precocious meta-
morphosis (Riddiford 1994). In contrast, the higher flies, such as Drosophila melanogaster,
have a fixed number of instars, and most of the adult is formed from precursor cells that pro-
liferate during larval life (Riddiford 1993). However, the presence of JH at the critical final
instar can prevent metamorphosis of the nervous system (Restifo and Wilson 1998) and of
the adult abdomen (Postlethwait 1974, Riddiford and Ashburner 1990). Exposure to JH dur-
ing the JH-free stages of embryonic development can disrupt later embryonic development
(Staal 1975, Riddiford 1994).

Reproduction

In adult female insects, JH regulates egg maturation (Wyatt and Davey 1996) and, in many insects,
JH stimulates the production of vitellogenin, an egg protein. Vitellogenin is secreted from the fat
body into the hemolymph, transported to the gonad, and taken up by specific receptors on the
oocyte membrane. In D. melanogaster, both the fat body and the ovarian follicle cells make vitel-
logenin, and both JH and 20E are needed for yolk production and deposition (Riddiford 1993).
In most insects, the female also produces a sex pheromone that attracts a mate. In some cock-
roaches and houseflies, pheromone biosynthesis is regulated by JH and by ecdysone, respectively
(Blomquist et al. 1993). Male spermatogenesis is under the control of hormones responsible for
metamorphosis and therefore begins when ecdysone is released in the absence of JH (Happ 1992).
In some insects, ecdysteroids are produced in the testis and play a role in genital duct development
(Adams 1997). Male accessory glands are often also under the control of JH (Happ 1992, Wyatt and
Davey 1996, Wolfner 1997).

Endogenous Regulators of JH Production

The activity of the corpora allata appears to be under the control of the regulatory neuropep-
tides that either stimulate (allatotropins) or inhibit (allatostatins) the production of JH. The first
allatotropin to be characterized came from Manduca sexta and stimulates the adult corpora allata
(Kataoka et al. 1989), whereas allatostatins were first characterized from the brains of cockroaches
Diploptera punctata (Woodhead et al. 1989). The distribution of allatostatin-like immunoreactivity
within the nervous system of a range of parasitic worms, or helminths, has led to speculation that
 Endocrine-Disrupting Chemicals 467

A O
C N N C

H O

B H3C OH
HO CH3

CH3 OH
CH3
CH3
HO
H OH

HO
H
O

Fig. 14.1.
Structure of insecticide tebufenozide (A) compared with the molting hormone 20-hydroxyecdysone (B).

they may have neurotransmitter/neuromodulation functions with a role in locomotion, feeding,

reproduction, and sensory perception (Smart et al. 1995).

Endocrine Disruption via Ecdysteroid-mediated Processes

Effects of the insect ecdysteroids can be interrupted by inhibition of synthesis and/or release from
the prothoracic glands, interference with the peripheral metabolism of 20E, or by competition at
its receptor level. The interaction of a compound at the ecdysone receptor can be either agonistic,
causing persistent 20E, or antagonistic, causing low levels/absence of 20E. Because of differences
in receptors, the activity of a chemical in one order of insects or other arthropods does not mean it
will act similarly in another. There are few ecdysteroid-mimicking pesticides; tebufenozide is one
that has been developed and marketed in recent years (Fig. 14.1).
Tebufenozide is formulated as a lepidopteran-specific ecdysteroid pesticide that acts as an ago-
nist at the receptor level, increasing the rate of expression of upregulated genes. Retnakaran et al.
(2001) found that ingestion of tebufenozide impacts the molting process of spruce budworm larvae
and thereby causes premature death. Because of its persistence and ecdysteroid activity, tebufeno-
zide also inhibits the expression of genes that are “downregulated” and “normally expressed in the
absence of 20E” (Retnakaran et al. 2001).

Juvenile Hormone

The great majority of identified EDCs in insects are JH agonists or antagonists; the most commonly
used commercial EDCs are agonists (Fig. 14.2). Methoprene is one such JH agonist or mimic that
is used commonly to control insect pests, and a number of other JH agonists with similar chemical
structures are registered for use in the United States.
Methoprene is a JH analog ( JHA) that mimics the action of naturally occurring JHs. These
analogs can be applied at specific life stages of an insect life cycle to complicate the molting process
and ultimately cause death. The strategic application of JHAs as a pesticide introduces JHs during
468 Peter L. deFur and Laura E. Williams

A Ecdysteroid Titer
400
Diflubenzuron
Control

Picograms/microliter 300

200

100

quantification
0 limit
AB C D0 D1 D2 D3–4 E
Molt Stage

B Ecdysteroid Titer
400
Fenoxycarb
Control
300
Picograms/microliter

*
200

100

quantification
0 limit
A B C D0 D1 D2 D3–4 E
Molt Stage

Fig. 14.2.
Circulating ecdysteroid levels in grass shrimp Palaemonetes pugio exposed to (A) diflubenzuron or (B) fenoxy-
carb compared with unexposed control shrimp. With permission from Touart (1989).

a period of growth when JH should be absent (Dhadialla et al. 1998). JHAs such as methoprene are
commonly used in flea and/or tick control growth regulators (Young et al. 2004).
A range of commercial formulations of JHAs are in production and have been the subject of
some research on the effects of JHAs on aquatic crustaceans (Touart 1989). As Touart (1989)
reported, several JHAs alter normal growth and molting in grass shrimp Palaemonetes pugio at con-
centrations that may occur in surface waters.

Crustacea

The current understanding of EDCs in crustaceans is based on limited direct research prior to 2000
and a subsequent growing literature in the fields of endocrinology, toxicology, and environmen-
tal biology in recent years. Crustacean endocrinology has been investigated principally in larger
 Endocrine-Disrupting Chemicals 469

animals, crabs, lobster, shrimp, and crayfish, for various logistical and practical reasons (availability,
blood volumes, economic importance). Modern methods have enabled measurements on smaller
samples and thus smaller species, providing an important understanding of key processes and
phenomena (i.e., the androgenic hormone of amphipods). Research on EDCs in crustaceans has
employed both laboratory- and field-based approaches, combining the ability to manipulate and
control experimental conditions with the knowledge of conditions in the animals’ natural habi-
tat. EDC research in crustaceans, as is the case with all invertebrates, has been hampered by a less
than complete understanding of the underlying endocrinology and life history in some species,
limited long-term funding, and difficulty of obtaining data from natural habitats. Notwithstanding
these challenges, recent research is filling in some of these gaps and providing information on EDC
effects in crustaceans, with some mechanistic insights.
A wide range of physiological processes and functions are known to be under endocrine control
and often cued or influenced by external environmental stimuli (briefly reviewed in Fingerman
1997), including molting, sexual maturation, reproduction, sexual differentiation, limb regenera-
tion, secondary limb regeneration, pigmentation, hemolymph glucose levels, water balance, ion
transport, heart rate, blood pressure, and cardiovascular function. Indeed, continued research into
crustacean endocrine function suggests that additional systems, such as the pigment control hor-
mones, likely play a role in reproductive function, thus opening the possibility for other control
pathways and the modulation thereof (Sarojini et al. 1995).
Several lines of investigation and monitoring have added greatly to the understanding of EDCs in
crustaceans: (i) field investigations conducted on populations of crustaceans (deFur et al. 1999), (ii)
laboratory toxicity studies using methods ranging from whole animals to isolated cell fractions and
molecular identification (see LeBlanc 2007), and (iii) endocrinology research at multiple levels of orga-
nization (see Chapters 1 and 2 in this volume). These research efforts have added greatly to the current
understanding of how anthropogenic chemicals alter endocrine function and the physiological process.
The limiting factor in the current research is that specific mechanisms of action are known for only a few
chemicals and endocrine pathways. Nonetheless, endocrine disruption in crustaceans has been dem-
onstrated for three processes or pathways: ecdysteroid-regulated molting, juvenoid-controlled devel-
opmental maturation, and sexual maturation/determination; these are examined in the next sections.

Ecdysteroids and Molting

The crustacean ecdysteroids, isolated and characterized in the lobster Jasus lalandei (Hampshire and
Horn 1966, Horn et al. 1966) are responsible for the principal biological events of molting (Chang
and Mykles 2011). Ecdysteroids are, in turn, regulated or under the influence of molt-inhibiting
hormone (MIH) and methyl farnesoate (MF; the equivalent of crustacean JH). Crustacean ecdys-
teroids function in a similar fashion as originally described in insects, the details of which are
described in Chapter 1 of this volume.
Little experimental information exists on endocrine-disrupting artificial or synthetic ecdysteroids
because pesticides targeting the molting hormone have not reached the market until quite recently.
The insecticide tebufenozide, described earlier, is one of the few of these developed for the market, and
research has yet to identify any consequences for nontarget crustaceans, either terrestrial or aquatic.

Feminization/Masculinization

Imposex, or feminization and masculinization, can occur as a result of exposure to several different
contaminants, and the most studied case of imposex is in gastropod mollusks exposed to tributyltin
(TBT; reviewed in deFur et al. 1999). Female snails grow penises and vas deferens after exposure to
470 Peter L. deFur and Laura E. Williams

just 1 ng/L of TBT (Matthiessen and Gibbs 1998). LeBlanc et al. (2005) offer evidence that TBT acts
on testosterone esterification in snails, yet Oberdorster et al. (2005) suggest that TBT increases levels
of the neurotransmitter APWGamide, a gastropod penile growth stimulator. Neither of these mecha-
nisms would appear to be operative in crustaceans. The morphological phenomenon of feminization/
masculinization has, however, been reported in crustaceans in several developmental contexts. Males

O Methyl farnesoate

O
JH-I
O
O

O JH-II
O

O JH-III
O

O JH-III bis epoxide

O O

O Hydroprene

O
O
Methoprene
O

NH O Fenoxycarb
O
O

O N
O
Pyriproxyfen

Fig. 14.3.
Structures of several commercially formulated juvenile hormone agonists that are used as insecticides, shown
with methyl farnesoate and insect juvenile hormone for comparison.
 Endocrine-Disrupting Chemicals 471

100

90

Percentage Survival 80 *

70
Control

60 10 µg/L pyriproxyfen *
50 µg/L fenoxycarb

50
0(1st instar) 6(4th instar) 11(5th instar)
Day and Stage of Development

Fig. 14.4.
Mean survivorship of Palaemonetes pugio from day 0 to 11 of development. Asterisk (*) denotes significant
(P < 0.05) difference from control. From Tuberty and McKenny (2005), with permission from Oxford
University Press.

of the freshwater crab Geothelphusa dehaani in Japan have undergone feminization, demonstrating
female genital (gonopore-like) openings at a proportion of 8–32%. This value increased with male
growth and was absent in males collected from unpolluted waters (Ayaki et al. 2005). Takahashi et al.
(2000) also found the presence of penis-like appendages on the female crab of this species in Japan.
The mechanisms of action suspected to cause crustacean imposex are several, including inhibition of
cytochrome P450, alteration of reductase activities, and conversion of testosterone to 17β-estradiol.

Juvenile Hormone and MF

The crustacean JH MF was first identified in crustaceans by Laufer et al. (1987); the same labora-
tory later reported the stimulatory effect of MF on ovarian maturation. In several reviews (deFur
et al. 1999, Oberdorster and Cheek 2000), MF is described as the crustacean equivalent of insect JH,
and this point is now accepted by an increasing number of investigators (LeBlanc 2007, Rodríguez
et al. 2007). The several JH agonists being marketed as insect growth regulators (e.g., methoprene,
pyriproxyfen, hydroprene; the structures shown in Fig. 14.3) are able to act on aquatic crustaceans
as well as insects, impairing growth, metamorphosis, and survival (Touart 1989, McKenney 2005,
Tuberty and McKenney 2005, LeBlanc 2007, Rodríguez et al. 2007).
Touart (1989) found that several different insecticides formulated as growth regulators and
acting on the molting process in insects—largely through interfering with maturation at the
molt—also impair normal molting in grass shrimp P. pugio (Fig. 14.2). Diflubenzuron and metho-
prene (not shown in Fig. 14.2) both depress circulating ecdysteroid titers in grass shrimp at the
molt stage when ecdysteroid peaks (Fig. 14.2A). Fenoxycarb both depresses ecdysteroid titers and
causes ecdysteroid levels to peak early, at stage D1 instead of D2 (Fig. 14.2B). These JH agonists alter
the normal pattern of molting hormone and can result in increased larval mortality (Fig. 14.4).
The adverse effects of JH or JHA have also been reported in a number of other aquatic crusta-
ceans (McKenny 2005)  including daphnids (Olmstead and LeBlanc 2003, LeBlanc 2007), crabs
(Takahashi et al. 2000, Lye et al. 2008), grass shrimp (Touart 1989, Tuberty and McKenny 2005),
and lobsters (Walker et al. 2005). Recent investigations have turned to other crustacean groups,
472 Peter L. deFur and Laura E. Williams

including gammarid amphipods and isopods, thus extending the observations of endocrine disrup-
tion to these two crustacean orders.

FUTURE DIRECTIONS

The current trends in research on crustacean endocrinology and endocrine disruption are encour-
aging with respect to identifying the occurrence of EDCs and elucidating their underlying mecha-
nisms. Given the importance of crustaceans in aquatic ecosystems (e.g., amphipods in freshwaters,
copepods in estuaries and coastal waters), in commercial open-water fishing (shrimp, crabs, lob-
ster) where chemical pollution is a known risk factor, and in fish farming (shrimp, lobster), there is
every reason to anticipate a continued need for further understanding of the topic. One example of
research that extends scientific information on endocrine disruption in a new area is the ongoing
work on terrestrial isopods in the lab of Lemos (Lemos et al. 2010a,b,c). Their work examines how
two known endocrine disruptors, vinclozolin (antiandrogenic) and bisphenol A (estrogenic), may
interfere with normal function via protein expression. The important ecological role of terrestrial
isopods is clearly threatened by widespread and continued exposures to EDCs.
We expect to see elaboration of all aspects of the endocrine disruption in crustaceans, notably
(i) the endocrine mechanisms in crustaceans generally; (ii) specific mechanistic information about
MF ( JH) and ecdysteroids, as well as crustacean hyperglycemic hormone (CHH) and the CHH
complex of neuropeptides; (iii) the mechanism of action of various types of EDCs, specifically
JHAs, ecdysteroids, and signaling pathway inhibitors; (iv) the occurrence of endocrine disruption
in crustacean populations in nature (as predicted by the multiple authors in deFur et  al. 1999);
(v) the development of alternative research designs for assessing field-level effects that are not read-
ily observable; (vi) investigations into the consequences of EDC effects at the population level,
similar in scope and focus to the work on gastropod mollusks exposed to TBT; and (vii) interac-
tions between or among known and suspected EDCs and other environmental perturbations, such
as increased temperatures, hypoxia, altered water chemistry, and the like.
A few of these research areas warrant some additional discussion. First, the understanding of
basic endocrinology is absolutely essential, especially for the crustaceans that utilize neuroendo-
crine control to a far greater extent than the vertebrates and in a fashion not found in the insects
(the two groups that have traditionally been the focus of much endocrinological research). As
noted by several recent investigators (Zou 2005, Rodríguez et al. 2007, Chang and Mykles 2011), the
control of molting is under the direct or indirect influence of both environmental factors (deFur
et al. 1999) and multiple hormones. Ecdysteroids directly influence the target tissues, but ecdyster-
oid synthesis and release is in turn regulated by other (neuro)hormones, and even those are or may
be regulated by neuroendocrine secretions.
Field methods for detecting endocrine disruption in invertebrates are now extending to caged
amphipods ( Jubeaux et  al. 2012, Besse et  al. 2013), although the methods and confirmation of
consistency with lab results are in process. We note that caged-animal studies by Sumpter and
Jobling (1995) were instrumental in the early investigations of in situ endocrine disruption in fish
(Bundschuh et al. 2011).
New biochemical methods of investigating crustacean endocrinology and toxicology have the
capability of detecting and identifying changes in gene expression and protein regulation (Stillman
et al. 2008). As these new approaches and methods provide information regarding changes in gene
expression, scientists will be better able to inform managers about the types of biological responses
to anthropogenic chemicals. These new methods will surely change the way in which ecotoxicology
is practiced around the world.
 Endocrine-Disrupting Chemicals 473

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15
SOME PHYSIOLOGICAL RESPONSES OF
CRUSTACEANS TO TOXICANTS

Judith S. Weis

Abstract
Investigating effects of pollutants on various aspects of crustacean physiology has a long history
and an enormous literature. This chapter focuses on work on decapods, mysids, and amphipods
and covers effects of metals, oil, and other organic contaminants on physiological functions includ-
ing feeding and digestion, respiration, osmoregulation, excretion, regeneration, and molting.
Physiological processes generally tend to be reduced or inhibited by contaminants, but in each case
some chemicals or species are exceptions to this general tendency. Mechanisms of uptake, stor-
age, and metabolism of metals and organic contaminants are also reviewed. Although most studies
have been laboratory bioassays in which organisms are exposed to particular chemicals for a rela-
tively short time, a number of studies compared animals collected from contaminated field sites
with those from cleaner areas. These studies give greater insight into ecological effects resulting
from physiological changes and compensatory mechanisms that may take place after long-term
exposures.

INTRODUCTION

The other chapters in this volume review recent work on various aspects of crustacean physiol-
ogy. It is unfortunate that many crustaceans today live in contaminated environments, and toxic
contaminants may alter their physiology. Unless an entire book is devoted to this topic, it is impos-
sible to do a thorough review of such a comprehensive topic, which has been the focus of intensive
study for more than 40 years. Review articles have generally focused on a type of effect, a group
of crustaceans, or a type of pollutant, for example, the recent review of Kouba et  al. (2010) of
crayfish responses to metals. Consequently, this chapter is only a general survey that touches on

477
478 Judith S. Weis

the different types of responses (omitting reproduction and endocrine disruption) and focuses
primarily on decapods. Also, there is an enormous literature on daphnids and copepods that is
not covered.
Among the contaminants of greatest concern are those that are persistent in the environment, are
toxic, and bioaccumulate in organisms; among the types of chemicals that have received the most
attention are metals and organic chemicals including oil, pesticides, and industrial chemicals like
polychlorinated biphenyls (PCBs). Some of these chemicals are no longer in use, but they remain in
the environment because they are stable and persistent, especially in aquatic sediments where they
can continue to affect crustaceans and other organisms. In contaminated environments, crustaceans
at higher trophic levels may be exposed via their food, as well as through the water or sediments.
Generally, when an organism is exposed to contaminants, rates of physiological processes tend
to be reduced, although there are cases in which rates are increased. Most of the research has uti-
lized a laboratory bioassay approach in which organisms are exposed (generally through the water,
but sometimes via food) to different concentrations of particular chemicals and the responses
quantified. Initial work in environmental toxicology sought to establish concentrations that caused
mortality (the LC50 approach), which is most useful in ranking chemicals in terms of their toxicity.
Studies subsequently examined physiological responses, which are sublethal responses to lower
concentrations of chemicals and are more likely to occur in nature. Fundamental work in this field
was presented in a series of symposia on pollution and physiology of marine organisms in the
1970s and 1980s organized by John and Winona Vernberg, Fred Thurberg, and Anthony Calabrese.
A number of studies from these symposia, although “old,” are discussed in this review because they
are basic to the field and might otherwise be lost to future researchers. Early studies generally used
high concentrations; more recently, lower concentrations have been used that are closer to environ-
mental levels. There have also been some studies in which organisms from polluted areas have been
compared to ones from reference sites. These studies are ecologically more realistic, but because
contaminated sites usually have multiple pollutants, it is difficult to attribute responses to any par-
ticular contaminant.

FEEDING AND DIGESTION

Reduced feeding and digestion are commonly observed responses to pollutants. Because crusta-
ceans play a major role in the cycling of nutrients in estuarine and freshwater food webs, alterations
in their feeding rates, nutrient assimilation, and energetics could impact not only their own popula-
tion dynamics, but also could have community-wide repercussions.

Feeding

Reduced food consumption is an almost universal response in a wide variety of taxa to a wide vari-
ety of toxicants (Taylor et al. 1993, Maltby and Crane 1994, Blockwell and Taylor 1998, Wallace et al.
2000). However, increased feeding rates have occasionally been found, for example, in amphipods
exposed to lindane (Blockwell and Taylor 1998). Pollutant-induced decreased feeding is not only
a general response to contaminants, but also can result in a “positive feedback” situation, in that
poor nutrition resulting from decreased feeding can in turn make animals more susceptible to con-
taminants (Dissanayake et al. 2008b). These authors advised that “ecotoxicological studies need
to take into account the nutritional state of the test organism to achieve the full assessment of con-
taminant impact” (p. 40). However, it is also likely that reduced feeding will reduce further uptake
of contaminants. This is particularly true for organisms that acquire much of their body burden of
contaminants from their food.
 Some Physiological Responses of Crustaceans to Toxicants 479

Metals

Chronic exposure to copper (Cu; 85 and 212 μg/L) and zinc (Zn; 106, 212, and 525 μg/L) reduced
growth of shrimp larvae (Farfantepenaeus paulensis) due to reduced feeding. Both metals reduced
the number of Artemia captured by the shrimp larvae during 30 min. Oxygen consumption was
reduced by about 30% in all concentrations (Santos et  al. 2000). Similarly, gut fullness of the
shrimp Metapenaeus ensis larvae feeding on Chaetoceros gracilis was reduced by a 2 h exposure to
Cu at 0.25 mg/L. In contrast, gut fullness was not affected even after 24 h exposure to chromium
(Cr), Cu, or nickel (Ni) at concentrations close to the 48 h LC50. However, postlarval shrimp
exposed for 24 h to those concentrations of Cr, Cu, or Ni consumed fewer Artemia nauplii (Wong
et al. 1993).

Organic Contaminants

Reduced feeding activity of juvenile amphipod Gammarus pulex was a sensitive response to lindane
and 3,4-dichloroaniline (3,4-DCA). Reduced feeding was detected after 96 h exposure at 8.4 μg/L
lindane and 240 hours exposure at 918 μg/L 3,4-DCA (Blockwell and Taylor 1998). However, a sig-
nificant increase was seen in feeding of those exposed for 240 h to 0.09 μg/L lindane. This may be
evidence of a hormetic response. Jensen and Carroll (2010) examined feeding of copepods exposed
to the water-soluble fraction (WSF) of crude oil. Feeding was inhibited in Calanus finmarchicus
exposed to 0.4 µg/L of the WSF, showing that adults are sensitive to exposure to crude oil.

Impacts in Polluted Sites

A number of studies have assessed feeding rates or prey capture in animals living in contaminated
sites. Perez and Wallace (2004) found that grass shrimp (Palaemonetes pugio) from a clean reference
site captured brine shrimp about twice as fast as shrimp from more contaminated sites. Shrimp
from the clean site that were maintained in the laboratory for 8 weeks with sediment and water
from the contaminated site showed reduced prey capture ability, comparable to that of grass shrimp
inhabiting that site, thus demonstrating that the difference was due to the environment. Videotape
analysis indicated that the reduced prey capture was due to shrimp using a less efficient grab type
of capture, rather than a lunge or pursuit type of attack. Khoury et al. (2009) compared feeding
rates (number of scoops) of fiddler crabs (Uca pugnax) from a contaminated site and a reference
site. Crabs from the reference site had twice the number of scoops (on the same sediment) as those
from the contaminated site. Blue crabs (Callinectes sapidus) from a contaminated site captured
fewer active prey (killifish or juvenile blue crabs) compared with crabs from cleaner environments,
but ate comparable amounts of less active prey (fiddler crabs and mussels) suggesting that coor-
dination was affected, rather than appetite (Reichmuth et al. 2009). Gut content analysis showed
that crabs from the contaminated site ate much less fish or crab but much more detritus, algae, and
sediment than did crabs from the clean site. These are not typical food items for this predatory
species. Transplanting polluted crabs to the clean site or keeping them in the laboratory on food
from the clean site allowed them to become better predators on juvenile blue crabs. Transplanting
clean crabs to the polluted site or maintaining them in the lab on food from the polluted site caused
them to become poor predators on juvenile blue crabs; this was correlated with the accumulation
of mercury (Hg).
Cellular activity, immune function, cardiac activity, and foraging behavior were studied in green
crabs, Carcinus maenas, collected from a polycyclic aromatic hydrocarbon (PAH)-contaminated
site and two comparatively clean field sites and compared with responses of crabs exposed in the
laboratory to the PAH pyrene (200 μg/L) for 28  days (Dissanayake et  al. 2010). Impacts at the
480 Judith S. Weis

cellular level were evaluated in hemocytes by assessing membrane integrity and immune func-
tion (phagocytosis), which were decreased by contaminant exposure in the laboratory. In the
field study, no significant impacts were observed at the cellular or physiological level in crabs from
the contaminated site, but, when brought into the laboratory, foraging behavior was significantly
reduced, thus demonstrating that feeding behavior is a more sensitive response. Crabs from the
contaminated estuary took significantly longer than other field-collected and laboratory-exposed
crabs to approach a cockle and break into the shell, resulting in differences in overall prey handling
time, with PAH-contaminated groups showing significantly longer handling times.

Digestion

Ingested pollutants can alter digestive physiology even before they are assimilated; when in the gut
fluids, they can affect gut motility, enzyme activities, or absorption (De La Ruelle et al. 1992). This
is termed “pre-assimilatory toxicity.” Post-assimilatory toxicity occurs after the pollutant has been
incorporated into tissues; this may damage gut tissues, interfere with enzyme synthesis or release,
and interfere with absorption, transport, and assimilation of nutrients and thus impact energy
reserves (Seebaugh 2010). Most studies, however, do not attempt to distinguish between pre- and
post-assimilatory toxicity.

Metals

In general, digestive enzymes are inhibited by metal contaminants. Cadmium (Cd) exposure
reduced amylase activity in gastric juice of the crayfish Procambarus clarkii (Reddy and Fingerman
1994). Activities of tryptase, pepsin, cellulase, amylase, and the metabolic enzymes, alkaline phos-
phatase, acid phosphatase, superoxide dismutase, and glutathione-S-transferase in the hepatopan-
creas of the prawn Macrobrachium rosenbergii were reduced after exposure to Cu2+ concentrations
ranging from 0.01 to 0.5 mg/L (Li et al. 2008). Inhibition of digestive enzymes was observed, with
the maximum inhibition in amylase. Acid and alkaline phosphatase were decreased, correlated with
increased Cu2+ concentrations. Decreased glutathione-S-transferase activity was observed after
exposure to 0.01 mg/L Cu2+.
Digestive enzymes (cellulase, amylase, β-galactosidase, trypsin, and esterase) were studied in
Daphnia magna after exposure to various metals (De Coen and Janssen 2007). Both Cd (0.8 μg/L)
and Hg (1.8 μg/L) inhibited enzyme activities after 48 h exposure. However, after 96 hours, no
inhibition was produced by Hg, and increased enzyme activity was seen with Cd. This increased
enzyme activity was considered to reflect altered food assimilation efficiency to cope with reduced
food uptake. Gaudy et al. (1991) also found that Cd exposure (0.05 mg/L) reduced assimilation
efficiency and fecal pellet production in Leptomysis. The decreases in fecal pellet production and
assimilation efficiency reflected a significant decrease in energy (about 43%), which authors felt
would lead to an unbalanced energy budget and lower reproductive potential. Hydrolase activi-
ties increased initially in the presence of 0.2 mg/L Cd, but declined after 48 h, reaching very low
values at 72 h. The unbalanced energy budget was considered a consequence of the inability to
utilize food.
Cd body burdens in prey also altered assimilation efficiency of Cd in the grass shrimp P. pugio
(Seebaugh and Wallace 2004). Cadmium assimilation was positively correlated with gut residence
time in shrimp collected along a pollution gradient. Increased gut residence time can, in turn,
influence (increase) pollutant assimilation. Ingestion of a pulse of Cd reduced protease activi-
ties and fecal elimination rate (Seebaugh 2010). Protease activities could have been influenced
by pre-assimilatory interactions between Cd in the gut and enzyme-secreting cells, or they could
have resulted from impacts on stored or circulating enzymes. Previous exposure to dietary metals
 Some Physiological Responses of Crustaceans to Toxicants 481

can induce changes in digestive physiology and affect digestive enzymes that may influence future
digestion and assimilation.

Organic Contaminants

There have been few studies on effects of organic pollutants on digestion. Horst et  al. (2007)
found that exposure to the juvenile hormone analog methoprene (50 μg/L) caused upregulation
of some genes in the hepatopancreas of the lobster Homarus americanus, including the enzymes
betaine-homocysteine S-methyltransferase (BHMT) and other enzymes of the methionine cycle.
Increased levels of enzymes associated with protein turnover, including trypsin, ubiquitin conjugat-
ing enzyme, and ubiquitin carboxyl terminal hydrolase were also observed.

Animals from Polluted Sites

Grass shrimp (P. pugio) from polluted sites had reduced digestive protease activity compared to
shrimp from a reference site. Casein hydrolysis rates were negatively correlated with gut residence
time and inversely related to assimilation efficiency of Cd (Seebaugh 2010), which would affect
future assimilation of pollutants. However, carbon assimilation was not affected in these shrimp,
suggesting that they can compensate for metal-induced post-assimilatory toxicity to maintain
assimilation of nutrients (Seebaugh 2010). There was a trend of increasing gut residence time
with increasing dietary Cd but not with Hg or C. Increased gut residence time can compensate
for reduced digestive enzyme activities. Fecal elimination rate was not affected by field exposure,
which also may be a compensatory response to impacts of pollutants. It appears that gut plasticity
allows shrimp in contaminated sites to maintain adequate assimilation of essential nutrients but
may increase the risk of dietary exposure to specific pollutants.

RESPIRATION/METABOLIC RATE

Most toxicants have been found to reduce metabolic rate and respiration of crustaceans and other
organisms. Many studies have relied primarily on oxygen consumption to determine changes in
metabolic rates. In some cases, lowered oxygen consumption can be attributed to reduced gill ven-
tilation, and, in other cases, the mechanism is disruption of the enzymes of cellular respiration.
Few studies have related effects on respiration to total carbon assimilation through measures of
feeding and excretion or have examined effects on the total carbon, nitrogen, or energy budget of
a crustacean.

Metals

Cadmium

Early studies showed that Cd reduced O2 consumption in both larval and adult fiddler crabs,
Uca pugilator (Vernberg et al. 1974) and grass shrimp (0.1 and 0.5 mg/L) (Hutcheson et al. 1985).
Similarly, Barbieri (2007) found that exposure of pink shrimp (F. paulensis) to Zn (0.31 mg/L) or
to Cd (0.18 mg/L) inhibited oxygen consumption. However, lobsters exposed to much lower con-
centrations of Cd (3 μg/L) had elevated gill oxygen consumption and increased ATPase activity
(Thurberg et al. 1977). This may be an example of hormesis (the tendency for low doses of contam-
inants to have “positive” results, whereas at high doses processes are inhibited). Gaudy et al. (1991)
482 Judith S. Weis

found that temperature affected the responses of the mysid Leptomysis lingvura to Cd. At 18°C,
the respiration rate was affected only by concentrations greater than 0.05 mg/L Cd. Exposure to
0.1 mg/L Cd depressed the respiration rate more significantly at 20°C than at 10°C (Fig. 15.1).
Because most studies are performed at only one temperature, this may help explain some of the
differences observed in the literature in the direction of change of respiration.

Mercury

Oxygen consumption in adult fiddler crabs, U. pugilator, was reduced by exposure to 0.18 mg/L
Hg (Vernberg and Vernberg 1972). St-Amand et  al. (1999) exposed zoea larvae of the shrimp
Pandalus borealis to inorganic Hg (0–160 µg/L) for 27 h and measured oxygen consumption,
potential respiration (determined by respiratory electron transfer system activity [ETSA]), and
swimming activity. ETSA was constant after 27 h exposure to 160 µg/L Hg, whereas both oxygen
consumption and swimming decreased, showing that Hg disturbed part of the respiration pro-
cess but not activity of the enzymes involved in the ETSA assay. In the crayfish Astacus astacus,

A C

10 10

5 5
Respiration rate (mg O2 g−1d−1)

0 0
10 20 10 20

B D

5 5

0 0
10 20 10 20°C
Temperature

Fig. 15.1.
Respiration rate of Leptomysis lingvura at 10oC and 20oC in four experiments (A,B,C,D). Open circles are con-
trols, closed circles are Cd-exposed at 0.1 mg/L, stars are Cd-exposed at 0.01 mg/L. Mean + SD. From Gaudy
et al. (1991), with permission from Springer.
 Some Physiological Responses of Crustaceans to Toxicants 483

exposure to HgCl2 (0.1 mg/L Hg) produced cardiac arrhythmia (Styrishave and Depledge 1996,
Kouba et al. 2010), which may be related to metabolic disturbances.

Copper

Spicer and Weber (1991) found impairment of respiratory function of Cancer pagurus after 7 days
of exposure to sublethal concentrations of Cu and Zn (0.4 mg/L), but only during hypoxic expo-
sures. The Cu and Zn did not cause significant changes in ventilation or perfusion rates, although
there was some indication that cardiac output may increase in respiratory-impaired individuals.
The authors thought that respiratory impairment was due to an increase in diffusion barrier thick-
ness at the gills and that this was reversible even during continued exposure. In a review, Spicer and
Weber (1992) concluded that the essential metals Cu and Zn act on the respiratory system primar-
ily by disrupting gill function, causing development of internal hypoxia, whereas the more toxic
Hg and Cd interfere with the respiratory system at every level of organization, including cellular
respiration itself.

Lead

Lead (Pb) decreased the respiration rate in crayfish P.  clarkii (Torreblanca et  al. 1987). Oxygen
uptake of whole animals generally decreased with increasing Pb concentration, but was not statisti-
cally significant, whereas that of excised gills decreased significantly. Histology of the gill filaments
of crayfish treated with 200 mg/L Pb indicated a general disorganization. Ahern and Morris (1999)
found that exposure of the crayfish Cherax destructor to 100 or 0.5 mg/L Pb reduced oxygen con-
sumption significantly, along with a decrease in heart rate, although ventilation rate was unchanged.
There was also reduction of the oxygen transfer factor across the gills after 21 days. Despite reduced
oxygen consumption and oxygen transfer factor, there was almost no change in acid–base status.
Although part of the typical response of crustaceans to hypoxia is hyperventilation, Pb did not elicit
an increase in ventilation and therefore no attempt was made to maintain normal oxygen levels.
Metabolism was not supplemented by anaerobiosis, and thus overall energetic demand would have
been lowered.

Organic Contaminants

Oil

Effects of oil and its constituent hydrocarbons vary considerably among crustaceans, with a
number of studies showing increased metabolic rates in response to exposure. Studies have gen-
erally been done with individual hydrocarbons or with the WSF of oil, which lowered respira-
tion in the shrimp Crangon (Edwards 1978). However, in adult shrimp P. borealis, energy balance
(scope for growth [SFG]) declined after exposure to WSF (20–36 μg/L) due to reduced food
intake, but remained positive at all oil concentrations (Stickle et  al. 1987). The energy bud-
get developed by these investigators measured costs of respiration and ammonia excretion and
found that costs did not change significantly in response to differing levels of fuel oil, whereas
the reduction in feeding rate was concentration dependent. In fact, at low concentrations, feed-
ing rates increased, a possible case of hormesis, wherein low levels of contaminants have “ben-
eficial” results, whereas at high doses feeding decreased. Because the metabolic costs remained
relatively constant at all exposures, consumption alone determined energy production in this
study. Nitrogen excretion accounted for only 10–20% of metabolic costs, whereas oxygen
484 Judith S. Weis

metabolism accounted for 80–90% of the costs. Conversely, blue crabs (C. sapidus) exposed to
WSF increased their energy expenditure and decreased their SFG in a dose-dependent man-
ner, due primarily to reduced feeding. They reduced their energy intake without a reduction
in maintenance costs, and thus had reduced growth and longer intermolt periods at 800 μg/L
(Wang and Stickle 1987). In both these studies, metabolic costs such as respiration remained
relatively constant while feeding rate changed in response to contamination. Lobster larvae
(H. americanus) exposed to 0.25 mg/L South Louisiana crude oil showed a reduction in respira-
tion rate and O:N ratio (Capuzzo and Lancaster 1981). Low O:N ratios suggest the organism is
deriving energy from protein catabolism rather than carbohydrates or lipids. Energy metabo-
lism did not return to control values after a week in clean water. Cancer irroratus incubated
in 14C-naphthalene-labeled oiled sea water accumulated the isotope into their hemolymph
(Vandermeulen et al. 1980). Respiration was lowered in 11.0 mg/L and returned to control lev-
els when crabs were returned to clean sea water. The O2 binding potential and structural integ-
rity of hemocyanin were unaltered, suggesting that disruption of hemocyanin-O2 binding is
not a mechanism of hydrocarbon toxicity. Whereas oxygen consumption increased in 8 mg/L
naphthalene-treated mud crabs Scylla serrata (Vijayavel and Balasubramanian 2006), activity
of the respiratory enzymes lactate dehydrogenase, isocitrate dehydrogenase, succinate dehy-
drogenase, malate dehydrogenase, and α-ketoglutarate dehydrogenase decreased in the hepa-
topancreas, ovary, and gills for all the tested concentrations. Naphthalene at the much lower
concentration of 0.2 mg/L also increased oxygen consumption in adult Neomysis americana
(Smith and Hargreaves 1985). In another example of increased metabolic rate, Laughlin and
Linden (1983) found that exposure to high levels of WSF of crude oil (200 and 1,000 μg/L)
increased metabolic rate and ammonia excretion in the mysid Neomysis integer. Effects were
influenced by temperature, with the greatest effect at 21.5°C, the highest temperature tested.
This again suggests that temperature may be a partial explanation for the opposite results seen
among the various studies.
Differences have been found in sensitivity of juvenile versus adult shore crabs (C. maenas) to oil
(Dissanayake et al. 2008a). Seven days of exposure to 200 μg/L of pyrene reduced immunocom-
petence, elevated basal heart rate, and decreased respiration of juveniles but had no overall impact
on adults. Juveniles were more susceptible than adults using various endpoints. Thus, basing “safe”
concentrations on the tolerances of adults fails to protect more sensitive life stages, and all life stages
need to be studied.

Pesticides and PCBs

Fenvalerate, a pyrethroid insecticide, reduced weight gain in P.  pugio larvae and juveniles at 10
μg/kg due to altered energy metabolism. Affected larvae contained significantly less N than con-
trols, whereas exposed postlarvae contained significantly less carbon and less energy (McKenney
et al. 1998; Figs. 15.2 and 15.3). Thiobencarb, a carbamate insecticide, at 100 μg/L also stimulated
the respiratory rate in Mysidopsis bahia, thus reducing the amount of energy available for growth.
Higher O:N ratios suggested a greater reliance on energy-rich lipid substrates resulting in less
lipid being available for gamete production (McKenney 1985). Energy metabolism of M. bahia
was also altered by the pesticide fenthion. Juveniles responded with elevated respiration, which
reduced the amount of energy available for growth and resulted in reduced growth (McKenney
and Matthews 1990).
Verslycke et al. (2004) studied cellular respiratory responses of N. integer, including SFG and
cellular energy allocation (CEA). Both assays are based on the concept that energy in excess
of that required for normal maintenance will be available for growth and reproduction. Mysids
were exposed to environmentally realistic concentrations of the organophosphate chlorpyrifos,
 Premetamorphic
A 36 80
32 Day 7 76 Day 14
*
N (µg/shrimp)

28 72 *
*
24 68
20 64
16 60
0 1 10 100 0 1 10 100

Postmetamorphic
B 175
330
Day 21 Day 28
310
N (µg/shrimp)

155
290
*
135 270 *
250
115 230
0 1 10 100 0 1 10 100
µg fenvalerate/kg sediment µg fenvalerate/kg sediment

Fig. 15.2.
Nitrogen content (mean + SE) of Palaemonetes pugio larvae and postlarvae in different concentrations of fen-
valerate in sediment. Asterisks indicate significant differences (p <0.05) from control. From McKenney et al.
(1998), with permission from Springer.

Premetamorphic
A 5 11
Day 7 Day 14
J (× 103/shrimp)

10
4
8

3 8
0 1 10 100 0 1 10 100

Postmetamorphic
B 24 50
Day 21 Day 28
J (× 103/shrimp)

21 45

18 * 40

15 35 *

12 30
0 1 10 100 0 1 10 100
µg fenvalerate/kg sediment µg fenvalerate/kg sediment

Fig. 15.3.
Energy content (in joules) (+ SE) of Palaemonetes pugio larvae and postlarvae in difference concentrations of
fenvalerate in sediment. Asterisks denote significant differences (p <0.05) from control. From McKenney et al.
(1998), with permission from Springer.
486 Judith S. Weis

1.0
48 h
96 h
0.9

Oxygen consumption (µl O2 mg−1 wet wt h−1)

168 h

0.8

0.7

0.6

0.5

0.4

0.3
0
Control 0.038 0.056 0.072 0.100
(100 µl acetone L−1)
Exposure concentration (µg chlorpyrifos L−1)

Fig. 15.4.
Oxygen consumption (+ SD) by Neomysis integer following exposure to chlorpyrifos at three different time
periods. From Verslycke et al. (2004), with permission from Elsevier.

and assays were conducted. Results of both assays were correlated, and both were significantly
affected (Fig. 15.4). CEA was more sensitive and reduced at lower concentrations (0.038 and
0.056 μg/L) than SFG. Effects of pentachlorophenol, a pesticide and wood preservative, on met-
abolic rate of grass shrimp depended on the molt cycle stage of the animal, with molting-stage
animals much more sensitive than intermolt animals (Cantelmo et  al. 1978). This study also
found inhibition of several respiratory enzymes in blue crabs, including fumarase, succinate
dehydrogenase, malate dehydrogenase, glucose-6-phosphate dehydrogenase, pyruvate kinase,
and lactic dehydrogenase.
Effects of PCBs on U.  pugilator respiration were variable:  at some temperatures, exposure
to 50 μg/L increased metabolic rate, and at other temperatures, it decreased the metabolic rate
(Vernberg et al. 1978). Again, temperature may be responsible for some of the disparate results
obtained in different studies.

OSMOREGULATION

The ability to maintain salt concentrations in the body regardless of the salt concentration of
the environment is particularly important in animals living in estuaries, which may be exposed
to variable salinity and pollution stress. Two enzymes play a major role in osmoregulation: Na+/
K+-ATPase and carbonic anhydrase (CA). Na+/K+-ATPase in intestines and gills maintains gradi-
ents needed for salt movement and is related to Na+ and Cl− exchanges across tissues. CA is involved
in the hydration of CO2 to produce H+ and HCO3−, playing a role in osmoregulation, as well as in
gas exchange and acid–base balance (Lionetto et al. 2000). It should be noted that impaired osmo-
regulation, like feeding, may result in altered uptake of toxicants (by altered rates of pumping),
which could then modify toxic effects.
 Some Physiological Responses of Crustaceans to Toxicants 487

Metals

The toxicity of many trace metals is higher in less saline water, in part because more of the metal
is in free ion form and more bioavailable, but also because of physiological responses of organ-
isms. Metal uptake may be reduced as salinity approaches the isosmotic point of a species because
of reduced activity of ion exchange pumps. Effects of metals on estuarine animals were reviewed
by Monserrat et al. (2007). A key mechanism of acute metal toxicity in many organisms has been
reported to be osmoregulatory impairment associated with gill Na+/K+-ATPase inhibition. Copper,
Ag, Cd, Pb, Zn, and Hg have all been found to impair osmoregulation related to inhibition of Na+/
K+-ATPase in freshwater, brackish, and marine animals (Péqueux et al. 1996, Bianchini and Castilho
1999). Inhibitory effects of Ag (0.05–0.5 μM), Cd (0.05–0.5 μM or 1.25 mg/L), Cu (0.05–0.5 μM),
and Zn (2–6 μM) have been also reported on CA in euryhaline crabs (Vitale et al. 1999, Skaggs and
Henry 2002; Fig. 15.5).

Copper

Exposure of C. maenas to 1 mg/L Cu altered hemolymph osmolality and ion balance (Bjerregaard
and Vislie 1986); disruption of Na+/K+-ATPase was considered the cause of the disturbance.
Hansen et al. (1992) found that exposure to 10 mg/L Cu for 1 week reduced this enzyme by 50–60%
and resulted in a major reduction in hemolymph Na+ concentration. However, osmoregula-
tion was less sensitive to Cu than respiration (Hebel et al. 1999). In this species, the anterior gills
(numbers 1–6) are primarily respiratory in function, whereas the posterior gills (numbers 7–9)
play an osmoregulatory role. Following exposure to sublethal concentrations of Cu, damage (epi-
thelial hyperplasia and necrosis) initially occurred in respiratory gills at 100 μg/L Cu. No dam-
age was seen in osmoregulatory gills at levels up to 300 μg/L Cu. In an investigation of Cu (0.78
µM) effects on C. sapidus at low (2 ppt) and high (30 ppt) salinity, Martins et al. (2011) found that
crabs acclimated to dilute seawater showed inhibition of expression of mRNA of the genes for the
Na+/K+-ATPase and the Na+/K+/2Cl– co-transporter, but Na+/K+-ATPase activity itself was not

3000
CA Activity (µmol CO2 mL−1 min−1)

2500

2000

1500

1000

500

0.0 2.0 4.0 6.0 8.0 10.0 12.0 14.0 16.0 18.0
Io (µmol L−1)

Fig. 15.5.
Inhibition of branchial carbonic anhydrase activity in Callinectes sapidus by Cu 2+. Open circles are 1 mM and
solid circles are 2.5 mM CO2 substrate. Modified from Skaggs and Henry (2002), with permission from Elsevier.
488 Judith S. Weis

affected, indicating that the gene transcription is downregulated before significant inhibition of
enzyme activity occurs. No effects were seen at high salinity, possibly because of lower bioavailabil-
ity of the free ion. Bambang et al. (1995) found that concentrations of 500 (low), 1,000 (medium),
and 1,500 (high) μg/L Cu altered both hypo- and hyper-osmoregulation in larval shrimp Penaeus
japonicus. Hypo-osmoregulation was reduced after 4 days at low Cu and was suppressed at medium
and high levels. Hyperosmoregulatory capacity was significantly reduced after 4 days exposure to
low and medium concentrations. Only the shrimps exposed to low and medium concentrations of
Cu recovered their hypo-osmoregulatory capacity after 7 days back in control seawater. Tolerance
to Cu increased when nauplii became juveniles.

Mercury

Mercury (0.04 μM) inhibited Na and Ca influx in the freshwater isopod Asellus aquaticus
(Wright and Welbourn 1991). Péqueux et al. (1996) investigated the possibility that toxicity
results from interference with osmoregulatory mechanisms and that impairment of osmoregu-
lation is greater in lower salinity. They examined effects on three crab species with various
degrees of osmoregulatory ability: the strong regulator Eriocheir sinensis, the weak regulator
C.  maenas (both euryhaline), and the stenohaline osmoconformer C.  pagurus. They found
synergistic effects between salinity and HgCl2 (0.1 mg/L) toxicity in the euryhaline species
that are hyperregulators in dilute media (E.  sinensis and C.  maenas). In E.  sinensis, Na+ and
Cl− permeability of the gill epithelium was affected, as well as Na+ and Cl− active transport
processes. They showed that Hg drastically disturbs the Na+/K+ pump and the Cl− channels
in the posterior gills.

Cadmium

Sublethal Cd (7.5 and 15 μg/L) exposure of the amphipod G. pulex caused a significant decrease of
osmolality and hemolymph Ca2+ but not hemolymph Na+ and Cl− concentrations. However, Na+/
K+-ATPase activity was significantly increased (Felten et al. 2008), contrary to the typical response.
On the other hand, feeding rate and locomotor and ventilatory activities were significantly reduced
in Cd-exposed organisms.

Organic Contaminants

There has been less work on effects of organic chemicals on osmoregulation.

Oil

Palaemon adspersus, a hyper- and hypo-osmoregulating shallow-water shrimp, was exposed

to 20, 70, 100, and 200 µg/L WSF of North Sea crude oil (Baden 1982). The ability to maintain
hyper-osmolality decreased after 1, 2, and 3 weeks exposure to 200, 70, and 100 µg/L, respectively,
but no effect was observed at 20 µg/L.

Pesticides and PCBs

Neufeld and Pritchard (1979a) found that gill Na+/K+-ATPase was inhibited by both in vitro and
in vivo exposure to DDT (1 µg/L) in C. irroratus. However, in C. sapidus, the in vivo response
was transient and disappeared after return to clean water (Neufeld and Pritchard 1979b). The
 Some Physiological Responses of Crustaceans to Toxicants 489

authors felt it was likely that induction of new Na+/K+-ATPase in response to osmoregulatory
stress protected the crabs from osmotic failure in response to DDT. Sublethal levels of lindane
altered the ionic and osmoregulatory ability of the mud crab Eurypanopeus depressus. Chloride
(Cl) ion regulation was disrupted at 0.70 µg/L, whereas hemolymph osmotic concentration was
reduced at 1.45 µg/L (Shirley and McKenney 1987). In juvenile P. japonicus in seawater or diluted
seawater, the insecticide fenitrothion decreased osmoregulatory capacity at both lethal and sub-
lethal (low μg/L) concentrations (Lignot et al. 1997). The effect was time- and dose-dependent.
In seawater, shrimp could recover in less than 48 when transferred to clean water. In dilute sea-
water, recovery at 48 h was possible only after exposure to the lowest concentration (Fig. 15.6).
The PCB mixture Aroclor 1254 at 7.5 or 29 μg/L did not significantly alter hemolymph Cl and
osmotic concentrations or Cl-exchange kinetics in adult P. pugio (Roesijadi et al. 1976). However,
disruption of hemolymph Cl regulation was seen in juveniles and was associated with mortalities
not seen in adults.
Shrimp (P. japonicus) tolerance to tributyltin increased with development from larvae to juve-
niles. Acute exposures (0.88 μg/L for nauplii to 708 μg/L for juveniles) decreased osmoregulatory
capacity (difference between the hemolymph osmolality and the osmolality of the medium) of ani-
mals exposed to lethal and sublethal concentrations (Lignot et al. 1998). Pathology in gills increased
with the concentration and was considered the cause of impaired osmoregulation. However, the
ability to osmoregulate recovered after shrimp were put in water without TBTO for 48–120 h.

Animals from Polluted Sites

Animals from polluted sites have been studied in terms of their tolerance to contaminants. Effects of
Zn were studied in two populations of the freshwater amphipod G. pulex. Animals from a relatively

A B C
−100 −100 0 µg L−1
0.50 µg L−1
0.75 µg L−1
−80 −80
* * 1.00 µg L−1
Hypo-OC (mosM kg−1)

*
−60 −60

−40 −40

−20 −20

n = 8 n = 12 n = 4 n = 10 n = 11 n = 9 n=9 n = 8 n = 11
0 0
96 h: SW + Ft 96 h: SW + Ft 144 h: SW
Exposure time (h)

Fig. 15.6.
Hypo-osmoregulatory capacity of juvenile Penaeus japonicus after 96 h exposure to (A) control, 0.5 and 1 μg/L
fenitrothion in seawater; (B) controls, 0.75 and 1 μg/L in seawater, and (C) controls, 0.75 and 1 μg/L fenitro-
thion followed by 48 h in clean water. Asterisks indicate significant (p <0.05) differences. From Lignot et al.
(1997), with permission from Springer.
490 Judith S. Weis

clean site showed a marked hemoconcentration after 4 days at 37 μmol/L Zn or 5 days at 18.2 μmol/L
Zn as shown by an increase in hemolymph osmotic pressure (OPh) and [Na+] and [K+]. However,
after 5 days at 37 μmol/L Zn, hemolymph exhibited an OPh significantly less than controls. Animals
from a contaminated site showed a reduction in OPh (but not ions) only after 5 days at 76.2 μmol/L
Zn, indicating they had a higher tolerance to Zn than the reference site animals (Spicer et al. 1998).
This paper did not examine differences between controls from the different sites, however. The man-
grove crabs Ucides cordatus and Callinectes danae were sampled from “polluted” mangrove areas and
a reference site (Harris and Santos 2000). Individuals of both species from the polluted site showed
greater ability to regulate blood osmotic concentrations at low salinity (9 ppt). However, U. cordatus
showed reduced hyporegulatory ability in 34 ppt. C. danae from the polluted site had significantly
higher Na+/K+-ATPase levels in posterior gills than did “unpolluted” crabs. These differences may
reflect adaptive changes following long-term exposure to contamination.

EXCRETION

Although there has been much research on the excretion of metals and other contaminants, there
have been relatively few studies on the physiological effects of contaminants on the process of
excretion itself. There have been reports of both increases and decreases in ammonia excretion in
different species, sometimes in response to the same toxicants. Reduced excretion rate can contrib-
ute to an increased body burden of contaminants.

Metals

Gaudy et al. (1991) found that Cd (0.05 mg/L) reduced ammonia excretion in Leptomysis. Exposed
mysids had reduced ability to utilize food (see the section “Digestion”), so this could be a result of
reduced protein. Ammonia excretion rate decreased with increasing concentrations of Zn (<0.2
mg/L) and Cu (<0.1 mg/L) in the freshwater shrimp Macrobrachium carcinus (Correa 1987). After
exposure to Cd or Zn (1 mg/L of each), ammonium excretion in the white shrimp Litopenaeus van-
namei was higher than in controls (Wu and Chen 2004)—the opposite effect from that observed by
Correa (1987) and Gaudy et al. (1991) with much lower concentrations. Barbieri (2007) found simi-
lar results in Litopenaeus schmitti: 0.18–0.98 mg/L Cd and 0.31–1.64 mg/L Zn increased ammonium
excretion (Fig. 15.7). Barbieri et al. (2005) found that Hg (0.045 mg/L) reduced oxygen consump-
tion but increased ammonia excretion in larvae of F. brasiliensis, a commercial shrimp. However,
postlarvae of Penaeus indicus showed a decrease in ammonia excretion with increasing concentra-
tions of Pb up to 7 mg/L (Chinni et al. 2002).

Organic Contaminants

Laughlin and Linden (1983) found that exposure of the Baltic mysid N. integer to WSF at concentra-
tions of 200–1,000 pg/L produced decreases in ammonia excretion that were strongly influenced
by temperature, with the greatest effect at the highest temperature tested. Cypermethrin (0.01
and 0.1 μg/L) increased ammonia excretion in the freshwater crab Trichodactylus borellianus in a
dose-dependent manner (Verónica and Collins 2003), suggesting increasing catabolism of amino
acids. Montagna and Collins (2008) studied effects of chlorpyrifos and endosulfan on ammonia
excretion of the freshwater crab T. borellianus, and found a significant increase of excretion at 150
and 300 μg/L of chlorpyrifos. The O:N ratio decreased with chlorpyrifos and with 2,500 μg/L
endosulfan, indicating a shift toward protein metabolism.
 Some Physiological Responses of Crustaceans to Toxicants 491

0.36

Ammonium-N excretion (µg g−1 min−1)

0.32

0.28

0.24

0.20

0.16
0.0 0.5 1.0 1.5 2.0 2.5 3.0
Concentration of Zn (mg L−1)

Fig. 15.7.
Shrimp (Litopenaeus schmitti) ammonium excretion in different Zn concentrations. From Barbieri (2007),
reprinted with permission from Water Environment Research. Copyright © 2007 Water Environment
Federation, Alexandria, Virginia.

REGENERATION AND MOLTING

Decapods can autotomize injured limbs—that is, break them off at a preformed breakage plane where
there is a membrane, thus minimizing tissue damage. Autotomy is an effective antipredator response,
leaving the predator with only a limb or claw while the intended prey escapes and can regenerate the
lost appendage. Regeneration begins after a period of tissue reorganization and is first noticeable as
a small bump at the autotomy plane. The limb buds grow within a thin covering of cuticle. In crabs,
they grow folded and will unfold when the animal molts. In shrimp, they are not folded, but have joints
nestled within one another so they also cannot expand until the animal molts. Thus, regeneration is
closely tied with the molt cycle. Regeneration can be divided into two stages: basal growth, when
tissue differentiation occurs and that is independent of the molt cycle, and proecdysial growth, in
which rapid growth occurs and that is dependent on the presence of molting hormones. There may be
a plateau phase when basal growth is completed and proecdysial growth has not yet started. Multiple
autotomy, removal of many limbs at once, results in accelerated regeneration and molting.
Many chemicals have been found to alter the rate of limb regeneration and molting. In some
cases, regeneration and molting may be affected independently, but in many studies it is not pos-
sible to distinguish effects on regeneration per se from those on the molt cycle because they are
usually coupled, and both processes are simultaneously retarded. A number of toxicants produce
morphological alterations in the regenerated limbs. These may be relatively minor, such as reduced
number of pigment cells, setae, or tubercles in the regenerated limbs (these tubercles function in
stridulatory sound production during courtship), or there may be more major deformities, such as
abnormal bending in the limb or claw or defects in chitin formation in the exoskeleton.

Metals

The most common effect of metals is retardation of regeneration accompanied by a delay in ecdy-
sis; in some cases, regeneration is affected without altering the timing of molting. A series of studies
492 Judith S. Weis

on fiddler crabs was conducted in the 1970s and 1980s by Weis and colleagues; these are reviewed
in Weis et al. (1992). Delayed regeneration and molting were observed in U. pugilator after exposure
to HgCl2, methyl mercury (MeHg), Cd, and Zn at concentrations of 0.5–1.0 mg/L. Retardation
of regeneration was accompanied with a delay in ecdysis so that, at molt, limbs were fully formed.
MeHg at 0.1 mg/L inhibited the development of melanin pigment in the regenerated limbs and
reduced the number of tubercles on regenerated first walking legs of males. Although Hg and
Cd individually retarded limb regeneration, the presence of MeHg reduced the particularly toxic
effects of the Cd at low salinity. Zinc and Hg together were additive, whereas Zn and Cd interacted
in an antagonistic manner. Pre-exposure to a lower concentration of Cd enhanced Cd tolerance;
these crabs regenerated more rapidly in Cd than those that had not been pre-exposed, but molting
still was delayed. U. pugnax from a contaminated site were less affected by MeHg than those from
a relatively clean site (limb regeneration was less retarded) indicating that they had acquired toler-
ance to MeHg. However, short-term pre-exposure to low concentrations of MeHg did not enhance
tolerance to higher concentrations. In U. pugnax, MeHg did not retard ecdysis, but did retard regen-
eration. Juvenile tiger shrimp, Penaeus monodon, experienced shortened time to the first molt and
decreased molting frequency after exposure to 0.9 mg/L Cr (Chen and Lin 2001).

Organic Contaminants

Oil and Its Constituents

Exposure of juvenile blue crabs (C. sapidus) to 1 mg/L benzene or dimethylnaphthalene increased

the length of the intermolt cycle, decreased the increment per molt, and retarded limb regeneration
(Cantelmo et al. 1982). Affected crabs showed a longer plateau stage and a longer time for regener-
ated limbs to develop pigmentation. Wang and Stickle (1987) found that the WSF (1.5 or 2.5 mg/L)
of South Louisiana crude oil inhibited growth and molting in blue crabs, reduced the increment at
molt, and prolonged the intermolt period.

Pesticides, PCBs, and Dioxins

The PCB mixture Aroclor 1242 (8 mg/L) was found by Fingerman and Fingerman (1978) to inhibit
limb regeneration in U. pugilator, with greater inhibition at high and low salinities than at inter-
mediate salinities. PCBs retarded molting also in crabs that were not undergoing regeneration.
Chlorophenols and dithiocarbamates at 0.1–1.0 mg/L retarded regeneration in P.  pugio without
affecting the timing of molting. Early stages of regeneration were more sensitive to pentachloro-
phenol than later phases (Rao et  al. 1979). Other chlorophenols (2,3,4,5-tetrachlorophenol and
2,3,4,6-tetrachlorophenol) inhibited limb regeneration at 0.3 mg/L and 0.7 mg/L respectively) but
did not alter the molt cycle, suggesting an effect on limb growth rather than on molting. (Rao et al.
1981). However, DDT (10 μg/L) accelerated limb regeneration in fiddler crabs (Weis and Mantel
1976). In the case of crabs with multiple autotomy, the time to ecdysis was shortened as well. These
responses may have been due to heightened excitation of the nervous system and secretion of
neuroendocrine factors promoting molting. Exposure to the antifouling agent tributyltin (TBT)
retarded limb regeneration and molting in U. pugilator at concentrations of 0.5 µg/L and produced
anatomical abnormalities in regenerated chelae of males, in which the regenerated dactyl curved
upward, away from the pollex, instead of downward toward it (Weis et al. 1987a).
Current insect growth-regulating pesticides are very toxic to growth processes in crustaceans.
The chitin synthesis inhibitor diflubenzuron (Dimilin) at levels of 0.5, 5.0, and 50 μg/L produced a
dose-dependent retardation of regeneration in U. pugilator; crabs that molted in higher concentra-
tions showed high mortality (Weis et al. 1987b). Regenerated limbs had blackened areas in which
 Some Physiological Responses of Crustaceans to Toxicants 493

the cuticle had not developed properly. Diflubenzuron at 0.11 μg/L also retarded the molt cycle of
grass shrimp and caused dose-related inhibition of limb regeneration (Touart and Rao 1987). This
suggests that, in addition to its effects on cuticle synthesis, it affects molting and inhibits mito-
sis and differentiation of limb buds. Stuekle et al. (2008) found that methoprene, a juvenile hor-
mone mimic, retarded regeneration in U. pugnax, with greater effects on males, which took longer
than females at 0.1 µg/L and exhibited greater frequency of abnormal limb formation at 1.0 µg/L.
Abnormal limbs failed to regenerate; had bent or bulging merus, carpus, or propodus; or had a
hook-shaped dactyl.

BIOACCUMULATION, STORAGE, DETOXIFICATION, METABOLISM

OF CONTAMINANTS

Uptake of contaminants occurs via the skin, respiratory system, or food. For crustaceans with a
chitinous exoskeleton, uptake is mostly via the gills or food, except perhaps directly after ecdysis
when the exoskeleton is paper-thin.

Metals

Bioaccumulation

Crustaceans can take up metals from the water, the sediments, or their food. The accumulation
pattern (distribution among tissues) of different metals varies considerably among taxa depend-
ing on the uptake rate, degree to which excretion plays a role, and where the metal is stored. The
ionic form of most metals is the most bioavailable form, thus is generally taken up the most. The
subsequent fate of the metal depends on the animal’s physiology and whether or not the metal is
essential. Essential metals tend to be regulated at optimum concentrations, above which excre-
tory mechanisms come into play. Potentially toxic metals must either be excreted or sequestered
in a nonavailable form if they are not to cause damage. Toxicity occurs when the concentration
exceeds the amount that can be stored in nontoxic form or excreted. Metals tend to be stored in
specific tissues such as the midgut gland (hepatopancreas; Rainbow 1988), which is generally the
site with the highest accumulation of Cd, Zn, Cu, Pb, and Cr. However, significant concentra-
tions may be found in muscle, which has implications for human consumption of edible species.
For example, in many crustaceans including the edible crayfish and blue crab, Hg (the most toxic
metal and one that has caused neurotoxic effects in humans from consuming contaminated fish)
is accumulated largely in the muscle (Simon et al. 2000, Kouba et al. 2010, Reichmuth et al. 2010).
However, crustaceans do not feed so high on the food chain as to accumulate high levels like those
found in large predatory fishes.
Cadmium is taken up and accumulated from the water and food (Devi et al. 1996), and it is
stored and detoxified mainly in the hepatopancreas (White and Rainbow 1986). The order of Zn
accumulation in crayfish tissue was found by Bagatto and Alikhan (1987) to be hepatopancreas>ex
oskeleton>digestive tract>abdominal muscle. Marine crustaceans appear to have similar Zn accu-
mulation patterns. Copper in decapod crustaceans is essential as part of the structure of the hemo-
cyanin molecule. It is regulated to an approximately constant level until it exceeds a threshold and
net accumulation begins (White and Rainbow 1982, Rainbow and White 1989). Excess levels of Cu
and Zn, both of which are essential metals, can be rapidly depurated when the animals are returned
to clean water (Kouba et al. 2010). Some species, such as Palaemon elegans, can match excretion to
uptake of certain metals such as Zn (Rainbow 1993). Martins et al. (2010) showed that Cu influx
into the gills of blue crabs was higher than into other tissues and that gills were important sites of
494 Judith S. Weis

Cu accumulation at both high and low salinities. In vitro experiments with isolated perfused gills
showed a positive relationship between Cu accumulation in both anterior and posterior gills and
the metal’s concentration in the incubation media.
Lead is neither essential nor beneficial and generally accumulates to the greatest extent in the
hepatopancreas. After 10 weeks of exposure of A. astacus to 0.02 mg/L Pb, it accumulated to the
highest concentration in the hepatopancreas, carapace, and gills (Meyer et al. 1991, cited in Kouba
et al. 2010). However, the freshwater crab Potamonautes perlatus had the lowest concentration in the
hepatopancreas and the highest concentration in the gonads (Reinecke et al. 2003).
Because crustaceans periodically molt their exoskeleton, depositing metals in the exoskeleton
prior to molting is a potential way to depurate contaminants. However, when P. pugio were exposed
to Cu, Zn, and Cd and then placed in clean water to molt, relatively low percentages of metals were
actually depurated via ecdysis (<26% for Cd). It appeared that some of the Cu in the exoskeleton
was reabsorbed prior to molting (Keteles and Fleeger 2001). In contrast, U. pugnax, particularly
specimens from a contaminated site, moved considerable amounts of Pb and Hg from their soft
tissues into their exoskeleton prior to molting while moving the essential Cu and Zn from their
exoskeleton into the soft tissues (Bergey and Weis 2007). This was an effective method to depurate
the more toxic metals and may be a mechanism of increased tolerance for the population in the
contaminated site.

Subcellular Disposition/Detoxification

The cellular localization of metals can be critical to their toxicity. Metals associated with
metal-sensitive intracellular components (e.g., organelles and enzymes) may impact cell func-
tioning. Metals tend to bind to proteins and may prevent them from functioning normally. For
example, metals can bind to active sites of enzymes, changing their configuration and inhibiting
their activity. Khoury et al. (2009) found that fiddler crabs (U. pugnax) from a contaminated
site had elevated metals in the heat-denatured proteins (HDP, enzymes) fraction. Other types
of proteins can bind to and then detoxify the metals (to a degree). Thus, the metal may be toxic
and available or may be detoxified, depending on what kind of protein it is bound to. In environ-
ments with high levels of available metals, animals tend to have evolved mechanisms to enhance
detoxification.

Metallothioneins
Metallothioneins are low-molecular-weight heat-stable proteins that are rich in cysteine and capa-
ble of binding high amounts of Ag, Cu, Zn, Cd, and Hg. They normally play a role in regulating Cu
and Zn. Blue crabs that had not been exposed to metals were examined for partitioning of Zn and
Cu into different fractions during different phases of the molt cycle (Engel 1987). Copper and Zn in
the hemolymph and digestive gland decreased during molt. Metallothioneins were highest during
intermolt and premolt and lowest just after ecdysis, suggesting that they are naturally occurring and
are involved in Cu regulation (perhaps hemocyanin synthesis) and Zn regulation. However, these
proteins are also involved in detoxification of nonessential metals. Their synthesis, primarily in
hepatopancreas and gills, is stimulated by metal exposure, and, by binding metals, they limit their
toxicity (Engel and Brouwer 1989). Most of the Cd taken up by P. elegans binds to metallothioneins
without being excreted (Rainbow and White 1989). Recently, it has been found that different iso-
forms of metallothionein are involved in Cu metabolism versus metal detoxification (Schlenk and
Brouwer 1991, Monserrat et al. 2007).
Mechanisms involved in tolerance can be linked to trophic transfer of contaminants. Metals
bound to metallothioneins tend to be available for trophic transfer and are therefore more avail-
able to predators than are metals associated with insoluble cellular constituents. Seebaugh et al.
 Some Physiological Responses of Crustaceans to Toxicants 495

(2005) fed P. pugio brine shrimp that had been exposed to Cd. Metals associated with metallothio-
neins in the brine shrimp were associated with enhanced trophic transfer of Cd to the grass shrimp.
Trophically available metal (TAM) consists of the metals associated with the subcellular fractions
containing metallothioneins, enzymes, and organelles. The overall amount of TAM may be a way
to predict transfer to predators (Seebaugh and Wallace 2004). A direct relationship was observed
between the partitioning of Cd and Zn to the TAM compartment of brine shrimp and the absorp-
tion of these metals by the grass shrimp. Similarly, when Cd-exposed amphipods (Gammarus law-
rencianus exposed to 0.01–0.51 mg/L Cd) were fed to grass shrimp, the amount of TAM was related
to the assimilation efficiency of grass shrimp for Cd. The TAM was considered to be the maximum
bioavailable Cd in the prey (Seebaugh et al. 2006). Although the partitioning of metals to TAM can,
in some cases, be a predictor of metal trophic transfer, it is clear from recent studies that ultimate
absorption of TAM metals by predators is also linked to digestive processes in the predator (Goto
and Wallace 2009).

Lysosomes
Lysosomes can sequester metals in many invertebrates and thereby play an important role in detox-
ification. Lysosomes may accumulate metals and trap them in lipofuscin granules, making them
unavailable to the cell; this is a mechanism that leads to elimination of the metals through the kid-
neys. Nassiri et al. (2000) investigated the amphipod Orchestia, which uses lysosomes in the ven-
tral caeca as a mechanism of metal detoxification. Animals from contaminated and reference sites
were investigated. There was no difference in Zn uptake by the different populations when they
were exposed to the same concentration of Zn. However, the contaminated population took up
significantly less Cd than the reference population after exposure. Following laboratory exposure
to Cu, Zn, and Cd, the lysosomes usually contained both Cu and Zn but not Cd. Although Cd was
mostly associated with metallothioneins, the lysosomes in cells of the ventral caeca appeared to be
a major detoxification pathway for Cu and Zn. Ahearn et al. (2010) summarized recent investiga-
tions of lysosomal function in lobster hepatopancreatic epithelia and described carrier-mediated
transport processes on lysosomal membranes that accumulate metals from the cytoplasm. They
discussed how metal transporters are linked with the uptake of anions that may then precipitate
concretions within the lysosomes. The potential role of the organic anion transporter (OAT) in
transporting glutathione with its associated metals from the cytoplasm into the lysosomal interior
was described.

Insoluble Granules
Trace metals can be detoxified in the form of insoluble concretions or deposits, which prevents
their toxicity because the metals are trapped in an insoluble form. Juvenile prawns, P. monodon,
were exposed to Cu and Pb to investigate the formation and accumulation of metal granules as well
as their excretion (Vogt and Quinitio 1994). Copper-containing granules accumulated primarily
in the hepatopancreatic tubules. The amount and size of the granules increased along the tubules
in accordance with the cell age; the granules were released by discharge of senescent cells into the
intestine and were added to the feces. Lead-containing granules were found in the thoracic exten-
sions of the antennal gland (kidney), where they were discharged into the gland lumen by secretion
and then excreted with the urine. Although metals associated with metallothioneins are available
to predators, metals in granules are less available for trophic transfer (Wallace and Lopez 1997) and
can pass, unaltered, through the gut of a predator.

Stress Proteins
Stresses can bring about changes in protein conformation, and it is important to have mechanisms
to maintain proteins in their functional conformation. Heat-shock proteins, originally found
496 Judith S. Weis

in various organisms after exposure to elevated temperature, play a role in protein folding and
assembly and can protect damaged cells from further damage. These proteins are also induced
by contaminants and have general protective functions, so are more accurately termed “stress
proteins.” They confer increased tolerance to toxicants (Sanders 1993). The rate of survival and
stress protein response were investigated in the amphipod Gammarus fossarum during a stress and
recovery experiment by Schill et al. (2003). Lower Cd2+ concentrations led to induction of stress
proteins whereas higher Cd2+ concentrations resulted in a reduced response, most likely due to
pathological damage. Those amphipods that survived the exposure retained the ability to produce
stress proteins during the recovery period. Eckwert et al. (1997) found that the expression of stress
proteins in the terrestrial woodlouse Oniscus asellus was marginally increased by low concentra-
tions of a variety of metals; at intermediate concentrations, a strong induction occurred, but high
concentrations caused a decline of stress protein levels, which may reflect pathological damage.
Combinations of metals increased the induction more intensely than comparable concentrations
of individual metals.

Organic Contaminants

The uptake of foreign hydrocarbons presents organisms with the need to metabolize, store, and
excrete them. Organic contaminants tend to be accumulated primarily in the hepatopancreas,
where they may be transformed into metabolites that can be excreted via the gills and kidneys.
Sometimes a metabolite may be more toxic than the original chemical. Synthesis of detoxification
enzymes can be induced by chemical exposure.

Metabolism

Pathways of organic contaminant metabolism involve two phases. Phase I reactions typically reduce
the contaminant’s activity and render the molecule susceptible to phase II reactions, often by hydro-
lyzing or oxidizing the molecule to make it more polar or water soluble. Phase II reactions involve
conjugation of the product of the phase I reaction with a substance that makes it less bioactive and
more readily excreted. (Some compounds are directly conjugated without a phase I metabolism.)
The most common phase I reaction is carbon oxidation. The enzymes responsible for oxidation
of foreign compounds are termed mixed function oxidases (MFOs), and these include the highly
studied cytochrome P-450 (CYP) system. CYPs, found in many tissues, are involved in oxidative
metabolism of a wide range of organic compounds including PAHs, PCBs, pesticides, and other
chemicals. The MFO system requires nicotinamide adenine dinucleotide phosphate (NADP or
NADPH) plus molecular oxygen to convert nonpolar PAHs into polar hydroxy derivatives and
arene oxides (some of which may be more toxic than the parent compound). In phase II reactions,
another molecule (such as acetate, glucuronic acid, sulfate, glycine, or glutathione) is conjugated to
a group on the xenobiotic, making it more readily excreted.
Rates of detoxification in crustaceans were initially found to be slow, which partially explains
their sensitivity to oil pollution (Burns 1976). More recent studies demonstrate that crustaceans
do have CYPs to metabolize organic compounds, and the activity of these systems varies among
species. The shore crab C.  maenas has a high capacity to metabolize PAHs with CYP enzymes.
Expression of CYP2- and CYP3-like genes fluctuated over the molt cycle, with low expression dur-
ing premolt and maximum expression during late postmolt, and expression was predominant in the
hepatopancreas, whereas expression of CYP4-like genes was predominant in gills and epidermis
(Dam et al. 2008). In addition, CYP2- and CYP3-related genes respond to ecdysteroid and xeno-
biotic treatment. The data suggest that premolt crabs with low gene expression would be more
susceptible to organic pollutants than postmolt crabs.
 Some Physiological Responses of Crustaceans to Toxicants 497

PAHs can be conjugated and then excreted in the urine. C. maenas individuals were exposed to
phenanthrene and pyrene (separately) at 0–200 μg/L. After 48 h, urine samples were taken and ana-
lyzed. Urinary levels were dose dependent for both compounds (Fillmann et al. 2004). In another
study, C. maenas was exposed to waterborne pyrene for 48 h, and depuration was monitored. No
unchanged pyrene was detected in samples from exposed crabs in this study, which had biotrans-
formed it into conjugates that were then excreted in the urine (Watson et al. 2004). Urinary levels
reached a maximum 2–4 days after exposure and then decreased.

FUTURE DIRECTIONS AND CONCLUSIONS

Viewing these physiological responses individually, they are, for the most part, deleterious.
Although there are exceptions, there is a general response of reducing the rates of digestion, metab-
olism, excretion, osmoregulation, and molting. However, when looking at the “big picture,” these
effects collectively could be viewed as adaptive responses to conserve homeostasis. The cluster
of effects of reduced respiration, digestion, osmoregulation (which requires energy expenditure),
excretion, regeneration, and molting may all be associated with the almost-universal response of
decreased food intake. With reduced food consumption and more energy devoted to detoxify-
ing the contaminants, slowing down physiological processes is a way to compensate for reduced
energy intake. Although productivity (growth and reproduction) would be expected to be lower in
contaminated habitats, reduced productivity may not always be the case. Reduced respiration and
excretion may in some cases be able to compensate for reduced food intake.
There are, however, cases in which physiological processes are increased after contaminant
exposure in the laboratory. One would predict that when physiological functions are increased
while energy intake is reduced, the most deleterious effects would occur because a negative energy
balance could not be sustained over more than a short time. Future research should examine in
greater detail those cases in which physiological processes are increased while food/energy intake is
reduced to see whether these effects are transient and if the rate subsequently decreases to compen-
sate for reduced energy intake. It may be that some of the observed cases of increased physiological
function were really examples of hormesis, and higher concentrations of the toxicant would have
the opposite effect. The role of temperature and other aspects of seasonality in modifying or even
changing the direction of responses to toxicants is also in need of further investigation.
Future work is likely to continue in more biochemical, molecular, genomic, and proteomic
directions in order to better understand mechanisms underlying physiological responses, and such
work will also need to consider a suite of “emerging” contaminants such as flame retardants, phar-
maceuticals and personal care products, nanoparticles, and the ubiquitous reduction of the ocean’s
pH (ocean acidification). For example, elevated Pco2 levels in seawater, as predicted for the year
2300, reduce calcification, growth, and molting frequency in marine crustaceans. At these levels,
embryonic development is impaired, and larvae and juveniles are affected when changes in Pco2
are accompanied by rising temperatures (Whiteley 2011). Regarding nanoparticles, the nano form
of metals is generally far more toxic than the ionic forms that have been studied previously (Karu
and Dubourguier 2010).
There is also a great need for connecting effects observed in laboratory bioassays to the lives of
animals living in the “real world.” Additional studies on contaminated populations will be needed
in order to understand ecological consequences of altered physiology, as well as the compensa-
tory mechanisms that are used to survive in stressful environments. The various mechanisms that
increase tolerance to contaminants (MTs, stress proteins, CYPs, etc.) all take energy. Environmental
stress can reduce the assimilated energy available for maintenance and reproduction, but com-
pensatory partitioning could be a common phenomenon to counteract deleterious effects of
498 Judith S. Weis

pollutants. For example, effects on growth do not necessarily result in altered population dynamics
if the effects are compensated for by increased reproduction. A reduction in resources allocated to
respiration, for example, could allow additional resources to be allocated to growth or reproduction
in order to maintain a population. More studies are needed of compensatory partitioning of energy
resources in contaminated populations, which could be an important strategy to deal with anthro-
pogenic stressors. Such populations may have been subjected to selection pressure and may have
evolved enhanced tolerance to the contaminants. They may be more tolerant because of reduced
uptake, altered inducibility of cytochrome P-450 systems, enhanced binding to molecules such as
metallothioneins, or increased ability to depurate or store contaminants in intracellular structures
that prevent damage. The inherent or induced tolerance of these populations enables them to per-
sist. However, tolerance comes with energy and fitness costs, and it has frequently been noted that
populations adapted to one set of environmental stressors have increased susceptibility to other
stressors and may show tradeoffs among life history traits.

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INDEX

20-hydroxyecdysone (20E),╇ 3–7, 155, 184, 434, Aorta,╇ 202, 204, 206, 214, 215, 217, 218, 225, 227, 228,
465, 467 230, 231
Abdomen,╇ 17, 110, 113, 115, 117, 123, 156, 186, 330, 331, Apherusa,╇331
428, 466 Apodeme,╇ 104, 105, 147, 181, 182
Acid rain,╇ 344 Arginine kinase (AK),╇ 118, 393, 395, 445, 446
Actin,╇ 73, 82, 85, 86, 89–95, 109, 110, 112–118, 126, 134, Arginine phosphate (AP),╇ 139, 334, 393, 397, 398
140, 144, 148, 149, 151, 152, 155, 176, 205, 211, 438, Argulus,╇ 206, 287, 423
445, 446 Armadillidium,╇ 3, 10, 38, 48, 430
Activity rate,╇ 325, 332, 347 Armases,╇255
Adenosine triphosphate (ATP),╇ 117, 270, 326, 391, Artemia,╇ 54, 55, 116, 182, 206, 207, 251, 261, 270, 301,
393, 398, 400 327, 329, 331, 378, 392, 422, 433, 434, 437,
Aedes, 55 449, 479
Aerobic capacity,╇ 110, 122, 148, 332, 348, 398, 399, 406 Artery,╇ 199–202, 204–206, 210, 213, 214–219, 222, 225,
Agonist,╇ 12, 78, 86, 87, 146, 463, 467, 470, 471 227, 228, 231, 232
Allergen,╇118 Asellus,╇ 1, 70, 488
Alpheus, 136, 146, 184, 425 Astacidea,╇ 257, 329, 333, 366, 369, 429, 431, 434, 436
Amino acid,╇ 7, 8, 14, 37, 41, 42, 75, 78–80, 86, 114, Astacus,╇ 3, 38, 50, 202, 231, 296, 298, 302, 303, 344,
116, 118, 121, 151, 187, 250, 255, 258, 263, 266, 267, 376, 482
286, 288, 289, 299, 301, 302, 304, 306, 308, 359, Atrophy,╇ 103, 134–136, 149–154, 156–158
366, 371, 392–394, 396, 402, 404, 408, 409, Austrothelphusa,╇305
451, 490 Autospasy,╇171
Amphiascus, 3 Autotilly,╇171
Amphipoda,╇ 3, 149, 170, 171, 182, 202, 253–256, 261, Autotomy,╇ 4, 135, 136, 142, 144, 146, 147, 149, 150, 155,
263, 265, 267–269, 272, 305, 308, 325, 328, 331, 168–177, 179–184, 186, 189–191, 491, 492
332, 340–343, 345, 364, 366, 367, 374, 376, 379,
404, 420, 421, 424, 431, 435, 437, 455, 463, 469, Balanus,╇ 19, 121, 170, 423, 433, 434, 454
472, 477, 479, 488, 489, 495, 496 Barnacle,╇ 19, 104, 121, 254, 266, 340, 341, 378, 437, 454
Aniculus, 212 Basal growth,╇ 179–182, 184–188, 191, 491
Anomura,╇ 40, 135, 171, 257–259, 263, 299, 302, 305, Bathynomus,╇215
333, 364, 368, 376, 428 Benthic,╇ 70, 141, 249, 296, 325, 332, 347, 406, 410
Anoxia,╇ 324–327, 330–334, 348, 394, 405, 406 Benzene,╇492
Antagonist,╇ 47, 74, 75, 80, 448, 463, 467, 492 Bicarbonate,╇ 323, 327, 335, 337, 358, 344, 346, 347
Antenna,╇ 7, 54, 107, 137, 170, 176, 179, 205, 206, 218, Binding site,╇ 14, 15, 56, 86, 110, 115, 121, 359, 361, 362,
230, 231, 252, 256, 258, 262, 271, 495 363, 371, 375, 379
Antennule,╇ 17, 171, 258 Bioaccumulation,╇493

505
506 Index

Biphenyl,  268, 464, 478 Cardioarterial valve,  199–202, 204, 210–216, 225, 232

Blaberus, 78, 79 Cardisoma,  47, 217, 229, 339
Blastema,  168, 169, 174, 177–179, 181, 184, 185, 187, Caridea,  10, 69, 70, 75, 81, 83, 84, 86, 87, 90, 92, 94,
190, 191 95, 251, 257, 259, 270, 290, 299, 302, 303, 366,
Bohr effect,  228, 332, 333, 372, 373, 378, 406 428–432
Bombyx,  20, 21, 55, 80 Caridinia, 19
Brachyura,  9, 49, 52, 69, 70, 75, 84, 86, 87, 90, 94, Cell membrane,  13, 82, 134, 250, 255, 268, 291, 379
171, 172, 179, 187, 189, 205, 218, 251, 254, 257–259, Cellulose,  285, 287, 290, 305, 309, 311
263, 268, 270, 296, 299, 305, 330, 331, 363, 366, Cephalothorax,  17, 150, 156, 184, 265, 291
368–370, 375, 376, 379, 400, 432, 435, 436 Cerebral ganglion,  38, 54, 74
Bradycardia,  215–217, 220, 222, 225, 228–230, 231, Chaetogammarus, 376
253, 334 Charybdis,  9, 217, 425
Branchial chamber,  4, 148, 204, 229–231, 258, 322, Chasmagnathus,  2, 53, 498
333–337, 339, 348, 360 Chelae,  19, 189, 219, 331, 492
Branchiopoda,  38, 54, 55, 170, 171, 182, 199, 202, 302, Cherax,  9, 19, 38, 78, 79, 107, 117, 136, 140, 220, 292,
305, 327, 329, 366, 378, 379, 422, 430–434, 293, 305, 374, 404, 425, 434, 483
447, 452 Chionoecetes,  3, 38, 189, 347
Branchiostegal circulation,  204, 205 Chirocephalus,  3, 25, 329
Brooding,  269, 331, 399–401 Chitin,  253, 285, 288, 290, 304, 309, 310, 311, 322, 335,
Buffering capacity,  338, 339, 346 360, 374, 375, 381, 392, 402, 491–493
Burrow,  146, 148, 267, 325, 326, 329, 333, 334, 336, 337, Chlordecone, 464
348, 372 Chlorophenol, 492
Bythograea,  38, 332, 425 Chlorpyrifos,  431, 484, 486, 490
Bythograeidae, 329 Cholesterol,  1, 5, 6, 290, 341
Chromatic adaptation,  68–70
Cadmium (Cd),  268, 432, 447, 480, 481, 488, 493 Chromatophore,  68–75, 77, 78, 80–88, 90–92, 94,
Calanus,  3, 202, 306, 307, 403, 422, 428, 443, 479 95, 181, 191
Calappa, 372 Chromatophorotropin,  68, 72, 74–78, 80, 81, 94, 95
Calcium (Ca),  75, 81, 83, 94, 95, 104, 140, 251, 256, Chromium (Cr),  477
257, 268, 270, 271, 291, 292, 293, 324, 335, 346, Circulation,  2–4, 7, 12, 16, 17, 20, 41, 44, 45, 48, 75, 76,
374, 375, 377, 443 119, 174, 175, 183, 185–187, 190, 199, 204, 205, 212,
Callinectes,  3, 5, 9, 36, 38, 75, 79, 136, 203, 212, 252, 266, 214, 225, 230, 231, 257, 258, 322, 324, 331, 332, 334,
303, 329, 368, 376, 395, 398, 409, 425, 435, 479, 347, 360–363, 366, 371, 373, 374–379, 381, 394,
487, 490 468, 471, 480
Calmodulin,  12, 13, 22, 57, 82, 84–86, 445 Cladocera,  170, 184, 202, 263, 295, 297, 301, 322, 326,
Calocaris, 334 346, 420, 421, 422, 430–434, 437, 447
Cambaridae, 329 Claw,  104, 105, 107, 110, 111, 113, 115, 116, 118, 123, 124,
Cancer,  2, 5, 8, 9, 11, 19, 38, 41, 46, 75, 150, 211, 212, 218, 135, 136, 139–158, 183, 184, 288, 296, 344, 345,
219, 253, 254, 270, 302, 306, 309, 325, 326, 330, 404, 491
335, 363, 368, 369, 407, 483, 484 Clibanarius, 259
Cancridae, 8, 18 Climate change,  272, 320, 339, 348, 381, 391, 407, 410
Carapace,  10, 17, 18, 40, 170, 174, 176, 180, 185, 187–189, Coastal,  148, 249, 251, 254, 285, 295, 305, 324, 325, 339,
322, 494 341, 406, 410, 472
Carbohydrate,  43, 44, 256, 286, 288, 290, 291, 304, Coenobita, 259
308, 311, 401, 404, 444, 451, 453, 484 Cold-seep, 332
Carbon dioxide,  211, 321–323, 334, 454 Compensatory hyperplasia,  168
Carbonic anhydrase,  3, 263, 270, 323, 486, 487 Copepoda,  3, 202, 206, 254, 289, 294, 295, 297, 301,
Carcinus,  2, 5, 8, 9, 11, 17–19, 37, 38, 42, 51, 78, 79, 123, 306, 308, 309, 325, 328, 331–333, 342, 346, 366,
136, 172, 176, 189, 212, 232, 253, 287, 294, 302, 326, 378, 403, 422, 432, 434, 437, 443, 448, 449, 452,
338, 370, 373, 395, 425, 442, 479 472, 478, 479
Cardiac ganglion,  201, 207, 209–212, 214–216, Copper (Cu),  268, 292, 293, 359–363, 371–375,
222, 232 379–381, 432, 447–449, 479, 483, 487, 493–495
Cardiac output,  207, 211, 217, 218, 225, 401 Corophium, 268
 Index 507

Corystes, 218 Dendrobranchiata,  43, 137, 259

Crab Detoxification,  295, 443, 444, 493–497
blackback land,  135, 150, 151 Dicofol, 464
blue,  36, 75, 136, 203, 212, 217, 219, 228, 230, 252, Diecdysis, 264
263, 266, 303, 376, 377, 395, 396, 398, 399, 407, Diflubenzuron,  468, 471, 492, 493
409, 464, 468, 484, 486, 492–494 Digestion,  14, 119, 216, 217, 219, 251–254, 261,
brown, 254 262, 285–288, 290, 292, 294–297, 299–311,
Chinese mitten,  262, 396, 449, 451 326, 404, 431, 450, 477, 478, 480, 481, 490,
Christmas Island land,  287 493–495, 497
Dungeness,  2, 22, 407 Digestive tract,  251, 253, 254, 261, 286, 287, 292, 294,
fiddler,  36, 69, 70, 74, 75, 83, 135, 136, 149–151, 302, 296, 299, 310, 493
341, 399, 405, 479, 481, 482, 492, 494 Dilocarcinus, 268
ghost,  115, 117, 122, 125, 136, 139, 140, 399 Dioxin, 492
green,  212, 228, 230, 272, 379, 395, 479 Discoplax,  9, 38, 44, 287
hermit,  135, 136, 212, 259, 378 Dithiocarbamate, 492
horseshoe,  206, 361, 363, 366, 371, 374, 376 DNA sequence,  41, 79, 116, 348, 363
Kamtschatka, 302 Dopamine,  47, 74, 207, 210, 213, 215, 216, 257
kelp, 203 Dynein,  85, 89, 90, 92, 93, 95
king,  135, 203, 296, 303, 378
mangrove, 69, 490 Ecdysis,  1–4, 15–18, 20–22, 50, 53, 78, 136, 150, 151, 155,
mud,  4, 82, 489 156, 180, 182, 184–189, 251, 262, 264, 271, 292, 359,
porcelain,  69, 220, 340, 341, 405, 437, 440–443 376, 377, 401, 402, 491–494
purple shore,  229 Ecdysone receptor,  4, 64, 467
red,  44, 45, 135, 136, 148, 397 Ecdysteroid,  1–8, 12–15, 19–21, 41, 48, 57, 147, 148, 150,
red rock,  335 154–158, 168, 169, 181, 183–188, 190, 191, 434, 462,
shore,  75, 135, 212, 229, 257, 302, 304, 326, 334, 335, 465–469, 471, 472, 496
338, 343, 484, 496 Egg mass,  330, 331, 334, 400, 410
spider,  18, 189, 222, 294, 331, 339 Elminius, 299
stone, 136 Embyro,  10, 16, 50–53, 70, 71, 94, 95, 134, 135, 137, 157,
swimming,  3, 93, 401 176, 177, 206–209, 263–265, 268, 272, 292, 296,
tanner, 347 310, 331, 348, 378, 400, 433, 434, 447, 497
Trinidadian mountain,  3, 35, 339 Emerita, 3
vent,  295, 329, 332, 333 Emersion response,  334
Crangon,  303, 402, 483 Endocrine disruption,  461–465, 467, 469, 472, 478
Crangonyx, 331 Energetics,  410, 443, 448, 449, 452, 478
Crustacean cardioactive peptide (CCAP),  1, 2, 15, 17, Environmental stress,  320, 420, 447, 454, 455, 497
50, 77, 212, 213 Enzyme,  3, 5–7, 13, 14, 44, 81, 85, 86, 118, 119, 123, 174,
Crustacean hyperglycemic hormone (CHH),  1, 7, 9, 175, 183, 219, 251, 263, 268, 270, 271, 285–287,
11, 15, 16, 36, 38, 42, 43, 45, 46, 49, 51, 53, 54, 78, 291–311, 323, 341, 344, 371, 373, 374, 377, 392, 395,
257, 392, 472 396, 399, 402–409, 441, 445, 448, 449, 480–482,
Cryptocyanin,  359, 364–367, 371, 375–377 484, 486–488, 494–496
Cyamus,  3, 65, 379 Eriocheir,  9, 155, 254, 396, 425, 432, 435, 449, 488
Cyanagraea, 333 Eriphia,  118, 119, 216
Cyclic adenosine monophosphate,  44, 84, 250 Escherichia, 84
Cyclic guanosine monophosphate,  12, 44, 77, 78, Estrogen,  430, 461, 463, 464, 472
82, 85, 86 Estuary,  230, 254, 256–259, 261, 263, 266, 271, 324,
Cytoskeleton,  68, 72, 73, 86, 89–95, 443–446, 453 462, 472, 478, 480, 486, 487
Euastacus, 305
Daphnia,  3, 4, 7, 18–21, 37, 38, 54, 170, 202, 271, 289, Eucalanus, 325
293, 321–324, 326, 327, 329, 330, 345, 378–380, Eucarida,  302 364, 365, 367, 368, 369, 374
390, 420–422, 430–434, 447, 450, 455, 480 Eulimnogammarus, 149
DDT,  184, 464, 488, 492 Eumalacostraca,  364–369, 379, 424
Deep sea,  260, 285, 320, 329, 332, 333, 339, 347, 372 Euphausia,  294, 325, 395, 424, 435
508 Index

Euryhaline,  50, 229, 249, 250, 256, 263, 270, 271, 407, Glucose,  36, 44–47, 56, 76, 256, 258, 262, 263, 271,
487, 488 286, 290, 294, 300, 304, 305, 330, 392–397,
Eurypanopeus, 489 402–404, 469, 486
Eurythermal,  339, 341, 349 Gluconeogenesis,  255, 392, 393, 395, 403, 409, 452
Euterpina, 342 Glycerol,  286, 289, 303, 360, 404
Eutrophication, 324 Glycogen,  44, 56, 80, 123, 267, 285, 290, 294, 304,
Excretory organ,  252, 258, 262, 263 391–399, 402–406
Exoskeleton,  1, 17, 50, 70, 104–106, 135, 136, 138, 151, Glycolysis,  110, 373, 392–395, 397, 399, 406
156, 169, 170, 174, 176–179, 183, 253, 264, 294, 323, Glyptonotus,  1, 49, 342
324, 332, 335, 346, 349, 359, 360, 371, 374, 375, 377, Gnathophausia,  3, 30, 406
381, 402, 435, 453, 465, 491, 493, 494 Gnathophyllidae, 69
Eyestalk,  1, 2, 4, 9, 10, 12, 14, 17, 19, 22, 36–40, 42, 44, Gnathopod,  1, 70, 311
47, 50, 52, 69, 72–76, 84, 155, 156, 185, 186, 189, Gonad,  9, 18, 48, 394, 429, 435, 465, 494
191, 214, 218, 252, 257, 264, 270, 392 Grapsidae,  2, 49, 263

Facultative air-breather,  320, 334, 335 Hatching,  50, 51, 53, 142, 148, 206, 207, 209, 254, 265,
Farfantepenaeus,  296, 303, 425, 479 266, 269, 289, 296, 330, 331, 342, 400
Fatty acid,  47, 262, 267, 268, 289, 290, 303, 308, 375, Heart,  15, 54, 56, 78, 109, 116, 119, 156, 199, 200–202,
393, 394, 402, 451 204–207, 209–222, 225, 226, 228–232, 255, 258,
Faxonella, 184 265, 322, 325, 326, 330, 334, 340, 341, 345, 401,
Fenitrothion, 489 405, 409, 428, 429, 469, 483, 484
Fenneropenaeus,  10, 41, 369, 425, 428, 435, 453 Helleria, 3, 170
Fenoxycarb,  19, 468, 470, 471 Hemerythrin,  361, 362, 363
Fenvalerate,  431, 484, 485 Hemigrapsus,  1, 89, 229
Fiber Hemocyanin,  224, 267, 293, 321, 322, 327, 329,
acceleratory, 201, 209 332–335, 337, 339, 347, 349, 359–367, 370–381,
aerobic,  110, 111, 115, 122, 123, 139, 396, 397 395, 406, 407, 439, 440, 444–446, 449, 453,
fast,  103, 106, 107, 110–113, 115–117, 122–124, 136, 454, 484, 493, 494
140–144, 146–148, 150 Hemocyte,  174–177, 183, 268, 294, 359, 371, 373–375,
inhibitory, 209, 210 379, 395, 428–430, 433, 443–445, 453, 454, 480
slow tonic,  112, 113, 116, 122, 124, 126, 136, 143, Hemoglobin,  301, 322, 338, 359–363, 378–381,
148, 149 450, 452,
slow twitch,  112, 113, 116, 122, 124, 126, 136, 143, 148 Hepatopancreas,  49, 70, 219, 222, 251, 252, 255, 256,
Foregut,  1, 50, 53, 56, 219, 253, 261, 262, 287, 294, 306, 261, 262, 271, 286, 292, 294, 298, 300, 304, 324,
307, 311 344, 345, 371, 375, 376, 391, 392, 394–396, 399,
400–404, 407, 409, 428–430, 432, 434, 437,
Gammarus,  170, 252, 265, 341, 342, 345, 367, 424, 479, 440, 442, 444, 446, 452, 453, 481, 484, 493–496
495, 496 Hindgut,  1, 16, 38, 50, 51, 53, 56, 57, 70, 72, 212, 261,
Gecarcinus,  3, 9, 38, 116, 136, 154, 182, 184, 189, 229, 262 262, 288, 306, 307, 309, 311
Gecarcoidea,  39, 44, 45, 136, 305, 397, 425 Hippolytidae, 69
Gene expression,  14, 18, 20, 52, 71, 94, 143, 157, 178, Holthuisana, 229
182, 191, 271, 329, 330, 377, 395, 396, 427, 440, Homarus,  3, 4, 8, 9, 11, 22, 37, 39, 52, 112–114, 135, 154,
442, 445, 446, 448–453, 472, 496 184, 212, 213, 217, 221, 255, 262, 295, 300, 301,
Genome,  1, 7, 18, 20, 21, 42, 74, 169, 174, 179, 190, 324, 335, 337, 368–370, 399, 400, 425, 429, 436,
251, 304, 324, 348, 363, 378, 380, 420, 422, 424, 446, 481
426–428, 437, 440, 443 Homeostasis,  36, 55, 56, 222, 223, 250, 259, 285, 292,
Geothelphusa,  4, 23, 471 323, 349, 391, 392, 406, 443, 445, 449, 453, 497
Gills,  16, 49, 50, 52, 54, 56, 148, 156, 204–206, Hoplocarida, 364–367, 427
218, 223–225, 228–231, 251–256, 258–265, 267, Hyas,  287, 294, 340
268, 270–272, 288, 292, 322, 323, 332–337, 349, Hydroprene,  19, 470, 471
360, 379, 381, 394, 395, 406–409, 429, 430, 432, Hydrothermal vent,  249, 295, 324, 329, 332, 333, 372,
435, 444, 451, 481, 483, 484, 486–490, 493, 437, 442
494, 496 Hymenodora, 290
 Index 509

Hypercapnia,  335, 340, 346, 347 Lipid,  44, 83, 87, 252, 255, 285, 286, 288–291, 294, 295,
Hyperglycemia,  15, 21, 37, 41, 44, 47–49, 53, 57, 80, 304, 311, 341, 379, 391–396, 399–404, 409, 449,
395, 472 452, 453, 484
Hypersaline,  256, 267, 270, 332, 341 Lithodes,  2, 94, 297
Hyposaline,  2, 29, 230 Litopenaeus,  10, 12, 39, 42, 154, 155, 256, 288, 289, 369,
Hypoxia,  44, 155, 200, 206, 207, 222–226, 228, 232, 375, 402, 403, 408, 425, 429, 430, 432, 444,
321, 324–327, 329–335, 340, 346, 348, 349, 374, 490, 491
376–378, 381, 395, 401, 405–408, 410, 420, 428, Lobster
429, 437, 438, 449, 450, 452, 453, 472, 483 American,  48, 112–114, 116, 125, 126, 135, 141, 142,
230, 258, 262, 270, 296, 300, 303, 376, 399, 400,
Idotea,  137, 149, 170, 287, 343, 405 402, 406
Imposex,  4, 67, 471 clawed,  105, 303, 366, 370
Infection,  267, 294, 330, 375, 420, 429, 437, 438, European,  154, 270, 295
442–446, 453, 455 Norway,  117, 121, 335
Ingestion,  7, 292, 295, 299, 300, 309, 391, 399, 402, rock, 290
467, 480 spiny,  48, 116, 213, 222, 254, 258, 287, 308, 363, 366,
Intertidal,  71, 170, 228, 229, 259, 308, 326, 329, 331, 334, 370, 372
340–342, 349, 405, 437 squat,  303, 326, 406
Ion exchange,  323, 324, 487 Locusta, 55, 78, 79
Ion transport peptide (ITP),  36, 37, 40, 256 Lysosome,  150, 151, 263, 303, 495
Ionoregulation,  49, 52, 56, 57, 252, 258, 346, 347, 349
Isopoda,  3, 10, 17, 38, 52, 75, 76, 125, 137, 149, 170, 171, Macrobrachium,  3, 5, 10, 19, 39, 42, 71, 73, 75–77, 80,
183–185, 187, 200, 206, 215, 216, 228, 252, 253, 84, 87, 250, 254, 256, 289, 295, 324, 343, 404, 425,
257, 261, 263, 269–271, 287, 288, 305, 309, 325, 426, 480, 490
332, 340, 342–344, 347, 348, 364, 366, 367, 374, Maja,  2, 22, 339
403–405, 420, 421, 424, 430, 437, 446, 472, 488 Malacostraca,  1–3, 38, 52, 55, 169–171, 182, 201, 202,
231, 286, 301, 306, 363–367, 374, 378, 379, 424,
Jasus,  39, 48, 287, 335, 469 427–436, 447, 452
Juvenile hormone,  18, 48, 183, 450, 464, 467, 470, Mandible,  8, 9, 18, 19, 22, 37, 40, 48, 54, 137, 311
471, 481, 493 Marrella,  3, 58, 361
Marsupenaeus,  4, 7, 10, 11, 21, 39, 40, 300, 369, 406,
Kepone, 464 426, 430, 445
Ketoconazole, 463 Maxilla,  54, 55, 137, 251, 262, 311
Kinesin,  90, 92, 93, 95 Maxilliped,  137, 182, 223, 288, 311
Maxillopoda,  170, 171, 182, 422, 423, 428, 432–434,
Lactate,  44, 45, 323, 324, 327, 333–336, 372, 373, 393, 447, 452
394, 396–399, 404–406, 409, 484 Medulla externa,  93
Lactic acid,  3, 21, 326 Medulla terminalis,  74, 257
Lamina ganglionaris,  93 Megalopa,  19, 185, 263, 266, 267, 269, 297, 376, 407
Latitude,  149, 269, 296, 321, 339, 341–343, 405, Meganyctiphanes,  290, 304, 324, 377, 405
440–442 Membrane receptor,  68, 83, 84, 86–88, 94, 95
Lead (Pb),  69, 268, 483, 494, 498 Menippe, 5, 136
Lepidophthalmus,  3, 32, 406 Mercury (Hg),  479, 482, 488, 492
Leptocheirus, 3 Meropodite, 17
Leptograpsus, 229 Metabolism
Leptomysis,  480, 482, 490 aerobic,  228, 320, 326, 331, 397–399, 404, 406
Libinia,  3, 19, 39, 48, 190 anaerobic,  326, 334, 348, 396, 398, 404, 406
Ligia,  3, 75, 206, 269, 340, 424 Metal,  44, 268, 295, 332, 395, 420, 432, 437, 438,
Limb bud,  168, 173, 178–183, 185–187, 189, 191, 265 447–452, 455, 477–481, 483, 487, 490, 491,
Limb regeneration,  151, 168, 169, 174, 177, 178, 493–497
184–186, 191, 469, 491–493 Metallothionein,  447, 450, 494, 495, 498
Limulus,  206, 363, 379 Metamorphosis,  19, 71, 141, 148, 169, 170, 176, 186,
Lindane,  478, 479, 489 263, 264, 296, 375, 376, 433, 464, 466 376, 471
510 Index

Metapenaeus,  8, 10, 39, 42, 207, 299, 395, 426, 479 Nephrops,  10, 39, 117, 136, 154, 334, 349
Metecdysis, 264 Neuroendocrine,  20, 57, 93, 212, 257, 258, 465, 472,
Methoprene,  19, 183, 467, 468, 470, 471, 481, 493 492
Methyl farnesoate,  18, 48, 434, 469, 470 Neurohormone,  2, 18, 36, 43, 57, 68, 76, 78, 95, 204,
Metopaulias, 346 205, 207, 209–213, 222, 225, 232, 252, 258, 372,
Microarray,  18, 271, 377, 380, 428–437, 441, 442–445, 461, 462
447, 448, 450, 451, 453, 454 Neuropeptide,  2, 18, 20–22, 37, 48, 52, 57, 72, 74, 76,
Microtubule,  72, 73, 85, 89–92, 441 78, 79, 214, 255, 258, 392, 465, 466, 472
Midgut,  7, 14, 47, 56, 251, 253, 261, 262, 287, 289, 291, Neurosecretion, 74, 76, 86
292, 294, 295, 297–304, 307–311, 493 Nickel (Ni),  4, 48, 479
Migration,  44, 70, 71, 75, 80, 84, 87, 90, 93–95, 116, Niphargus, 404
125, 136–138, 148, 149, 157, 173, 174, 176, 177, 183, Norepinephrine, 74, 257
187, 217, 249, 252, 254, 255, 259, 263, 321, 325–327, Normoxia,  207, 224–226, 322, 325, 326, 330, 335, 401,
335, 347 408, 452, 453
Mitochondria,  72, 83, 103, 106, 110, 122, 123, 134, 139, Nutrition,  174, 249, 251–253, 272, 285, 286, 288, 295,
150, 151, 223, 259–261, 263, 268, 271, 294, 393, 308, 310, 311, 325, 377, 381, 392, 396, 410, 478
395–397, 402, 420, 427, 440, 442
Molecular motor,  68, 69, 71, 82, 86, 90–95 Ocean acidification,  320, 346, 377, 381, 454, 497
Molting hormone,  2, 3, 21, 147, 181, 185, 186, 188, 465, Octopamine,  47, 207, 210, 215, 216, 222, 257, 271
467, 469, 471, 491 Ocypode,  115, 123, 184, 399
Molt-inhibiting hormone (MIH),  1, 2, 7, 9, 11, 13, 37, Odontodactylus,  3, 62, 367
40, 43, 51, 78, 191, 469 Ommatidia,  69, 70, 73, 93
Monoporeia, 325 Oniscus, 17, 496
Motor neuron,  106, 118, 119, 122, 124, 126, 135, 141, Ontogeny,  71, 207, 249, 251, 252, 254, 263, 268, 271,
143, 148 272, 288, 291, 295, 296, 304, 376, 377
Munida, 406 Orchestia,  3, 170, 256, 495
Muscle Orconectes,  2–5, 10, 17, 39, 47, 80, 179, 184, 212,
closer,  104, 105, 107, 110, 111, 113, 118, 124, 135–137, 293, 309
141, 142, 147, 148, 150–152, 154, 157 Organic contaminant,  477, 479, 481, 483, 488, 490,
extensor,  104, 105, 113, 115, 122–124, 140, 181 492, 496
fast,  105, 106, 108, 109, 111, 116, 123, 124 Osmoconformation,  230, 249–253, 255, 258, 262, 263,
flexor,  51, 53, 104, 105, 112, 113, 115, 122–124, 140, 181 266, 270, 488
levator,  150, 171, 172 Osmolality,  230, 231, 250, 252, 255–258, 263, 265–267,
opener,  143, 144, 149, 150 269, 407, 487–489
slow,  105–108, 110, 113, 115, 124, 138, 149 Osmoregulation,  43, 216, 249–259, 261–272, 293, 346,
Myofiber,  104, 106–108, 111–114, 117, 118, 120–126, 134, 347, 395, 407, 409, 477, 486–489, 497
135, 138, 140, 142–144, 146–155, 157, 158, 182 Ovary,  48, 70, 71, 73, 76, 77, 80, 84–86, 90–92, 395,
Myogenesis,  134, 135, 137, 138, 157, 206, 210, 215 399, 400, 435, 446, 466, 471, 484
Myosin,  73, 82, 85, 86, 89–93, 95, 103, 106, 109–113, Oxygen minimum zone,  3, 26, 406
115, 124, 125, 134, 137, 140, 144, 145, 147, 205, 211, Oxygen transport,  359, 361, 378, 381, 450, 452, 453
438, 446 Oziotelphusa,  19, 47, 231
Myosin heavy chain (MHC),  85, 86, 103, 111–113, 137,
140, 144, 145 Pachygrapsus,  2, 5, 39, 42, 49, 184, 185, 189, 190,
Myosin light chain (MLC),  85, 86, 112, 113, 147 217, 270
Mysidacea,  3, 4, 200, 207, 296, 328, 332, 374, 406, 477, Pacifastacus,  302, 326, 367, 426
482, 484, 490 Pagurus,  39, 40, 136, 266, 378, 426
Mysidopsis, 484 Palaemon,  3, 22, 71, 72, 87, 189, 260, 299, 347, 488,
493, 498
Natatolana, 288 Palaemonetes,  74, 84, 87, 208, 209, 220, 395, 401, 426,
Nebalia,  363, 364, 367 429, 430, 432, 449, 468, 471, 479, 485
Necora, 347 Palaemonidae,  52, 82, 83, 254, 269, 329
Neohelice,  74, 87, 255, 409 Palinurus,  368, 370, 372
Neomysis,  4, 82, 486 Pandalopsis, 155
 Index 511

Pandalus,  75, 303, 311, 482 Protein

Panulirus,  116, 213, 258, 308, 363, 368, 426 kinase,  13, 44, 57, 82, 84–86, 88, 89, 95, 155, 330
Paralithodes,  1, 89, 302 synthesis,  14, 48, 152, 154–158, 182, 219, 253, 255,
Parasitism,  44, 249, 266, 267, 272, 287, 311, 378, 466 267, 291, 331, 342–344, 349, 395, 399, 401, 402,
Parathelphusa,  1, 76, 179 440, 444–446, 453, 454
Parhyale,  1, 82, 424 Pseudothelphusa,  255, 337, 339
Penaeus,  3–5, 10, 19, 21, 40, 48, 155, 270, 289, 294, 368, Pyriproxyfen,  4, 68, 471
369, 375, 376, 395, 402, 426, 428, 429, 431, 433, Pyruvate,  286, 392–394, 396, 405, 486
443, 488–490, 492
Peracarida,  171, 200, 364–367, 374, 379 Red pigment concentrating hormone (RPCH),  72,
Pereopod,  17, 105, 150, 155, 216, 261, 269, 396 75, 77, 79, 82, 85
Pericardial organ,  1, 10, 15, 17, 18, 38–42, 51, 201, 204, Regeneration,  39, 142, 144, 146, 147, 150, 151, 168–191,
207, 210, 212, 214, 215, 222, 225, 252, 257 469, 477, 491–493, 497
Peripheral tissue,  5, 6, 392, 400 epimorphic,  168, 169, 174, 177, 184
Pesticide,  184, 268, 461, 467, 469, 478, 484, 486, 488, Remipedia,  171, 363, 365–367, 378, 421, 427
492, 496 Reproduction,  18, 19, 22, 36, 43, 69, 70, 169, 170, 182,
Petrolisthes,  340, 341, 364, 368, 374, 405, 426, 428 253, 254, 258, 259, 285, 287, 292, 299, 303, 311,
pH,  111, 224, 268, 271, 295, 300, 306, 307, 320–324, 327, 391, 394–396, 399–401, 404, 420, 437, 447, 462,
331, 332, 335–337, 344–349, 367, 369, 372, 373, 464–467, 469, 478, 480, 484, 497, 498
377, 395, 398, 406, 453–455, 497 Resistance,  122, 199, 200, 204, 205, 211–216, 223, 267,
Phenoloxidase,  175, 294, 359, 365, 371, 373–375, 269, 292, 294, 295, 305, 306, 311, 320, 331, 348, 451
377–379 Respiration,  56, 202, 206, 207, 209, 216, 218, 223, 224,
Phyllocarida,  363–367, 437 227–230, 259, 269, 293, 320–322, 325–327, 329,
Pigment cell,  69–71, 76, 90, 93, 491 331, 332–335, 337–339, 347, 349, 360, 372, 407, 410,
Pigment dispersion,  75, 77, 83, 84, 86–89, 91–95 446, 454, 477, 481–484, 486, 487, 493, 497, 498
Pigment translocation,  68, 76–78, 81, 83, 84, 87, 90, Respiratory gas,  259, 322, 326, 329, 333, 337–339, 454
91, 94, 95 Respiratory pigment,  293, 321, 322, 325, 327, 329, 333,
Pigmentary effector,  68, 70–72, 94 334, 337, 347
Pilumnus, 188 Rhithropanopeus,  19, 185, 264
Pleocyemata, 43 Rimicaris,  8, 10, 40, 42, 260, 332, 428, 442
Pleopod,  115, 137, 155, 156, 270, 330, 333, 401 RNA,  8, 12, 14, 42, 48, 49, 52, 54, 57, 79, 115, 117, 145,
Pleuroncodes, 302, 407 154–157, 178, 182, 183, 186–188, 299, 308, 363, 376,
Pollutant,  44, 247, 251, 268, 272, 295, 420, 451, 477, 404, 407, 408, 430, 444, 447, 452, 454, 466, 487
478, 480, 481, 496, 498 Ryanodine receptor,  82, 85, 109, 120, 126
Polychlorinated biphenyl (PCB),  268, 464, 478
Polycyclic aromatic hydrocarbon (PAH),  4, 45, 479 Saduria,  3, 23, 343
Ponasterone A (PA),  3–7, 184 Salinity,  50, 52, 201, 223, 229–232, 249, 250, 252–258,
Porcellio,  170, 270, 305, 348 262, 263, 264, 266–272, 321, 322, 334, 348, 39, 372,
Portunidae,  105, 122, 139, 217, 329, 373, 435 376, 377, 392, 407–409, 420, 433, 437, 453–455,
Portunus,  9, 40, 395, 426, 435 486–488, 490, 492, 494
Postmetamorphic,  252, 266, 268, 485 Sarcomere,  103, 104, 106, 111, 117, 121, 124–126, 135, 138,
Potamidae, 269 146, 147, 149–153, 157, 158, 183
Potamon, 40, 42 Scaphognathite,  17, 56, 211, 218, 222, 223, 228–230,
Potamonautes, 494 322, 333, 337, 360
Predation,  105, 168, 170, 171, 174, 217, 269, 325, 330, Schistocerca, 37, 78, 79
332, 333, 349, 396, 399, 462, 479, 491, 493–495 Sclerotization,  1, 18, 175, 176, 359, 373–375
Premetamorphic, 485 Scylla,  40, 270, 426, 427, 435, 484
Premolt,  1, 3, 4, 5, 7, 12–15, 17–20, 50, 51, 53, 56, Second-messenger cascade,  71, 83, 84, 86
150–156, 182, 253, 262, 264, 92, 359, 371, 375–377, Serotonin,  47, 53, 207, 209, 210, 213, 215, 222, 257
401, 402, 435, 494, 496 Sesarma,  1, 88, 269
Procambarus,  5, 7, 10, 40, 41, 50, 149, 176, 184, 185, Shrimp
206, 252, 303, 345, 426, 429, 431, 444, 480 brine,  206, 207, 261, 270, 301, 449, 479, 495
Proctolin,  119, 205, 210, 212, 213 broken-back, 69
512 Index

Shrimp (cont.) Tebufenozide,  4, 65, 469

ghost,  3, 32, 406 Temora, 294
grass,  225, 395, 401, 449, 452, 468, 471, 479–481, Temperature,  69, 149, 184, 201, 207, 211, 217, 220–223,
486, 493, 495 228–230, 232, 249, 251, 267, 271, 272, 295, 307,
harlequin, 69 320–322, 324, 325, 326, 329, 332–335, 339–345,
kuruma, 445 347–349, 372, 373, 376, 377, 392, 395, 404, 407,
mantis, 105 420, 437, 440–444, 448, 454–456, 472, 482,
pink,  2, 94, 481 484, 486, 490, 496, 497
pistol, 69, 146 Terpenoid hormone,  2, 186, 461, 465
snapping,  105, 110, 111, 135, 136, 139, 146, 149, 151 Thalassinidea,  328, 329, 333, 366, 370, 372
tadpole, 206 Thermal sensitivity,  3, 38, 348
tiger,  395, 443, 445, 492 Thermal tolerance,  339, 340, 405
vent,  3, 30, 442 Thermoregulation,  2, 67, 340
white,  268, 294, 301, 303, 307, 375, 402, 403, 407, 444, 490 Thoracic ganglion,  10, 21, 38, 52, 54, 141, 148, 215, 252, 257
Signal peptide,  8, 11, 43, 53, 78–80 Tigriopus,  422, 432, 449
Signal transduction,  12, 15, 22, 68, 69, 71, 75, 76, 78, Toxicant,  420, 447–449, 452, 455, 477, 478, 481, 486,
81–86, 94, 95, 330, 444 490, 491, 496, 497
Simorephalits, 170 Toxin,  295, 410, 447
Sinus gland,  2, 9, 10, 14, 36, 38–40, 49, 51, 53, 74, 212, Traskorchestia, 256
257, 264, 392 Triacylglycerol,  289, 290, 303, 393, 394, 402
Siriella, 3, 4 Tributyltin (TBT),  461, 464, 469, 489, 492
Species distribution,  257, 346, 391, 410 Trichodactylidae, 251
Species diversity,  2, 69, 332 Trichodactylus, 490
Species richness,  344 Triops,  21, 206, 216, 301, 321, 378, 422
Specific dynamic action(SDA),  219, 253, 343 Tropomyosin,  103, 110, 112, 113, 115–118, 126, 134, 140,
Speleonectes,  365, 367, 427 144, 148, 149, 205
Sphaeroma, 3 Troponin,  103, 110, 112, 113, 115, 116, 126, 134, 140, 143, 144
Squilla,  170, 202, 370, 427 Tyrosinase,  373, 379, 380
Stenasellus, 403
Stenohaline,  229, 249, 250, 270, 488 Uca,  3, 5, 36, 74, 75, 87, 136, 170, 173, 179, 180, 183, 184,
Stenothermal,  220, 339, 341, 347 187–189, 266, 302, 341, 399, 405, 479, 481
Stomach,  16, 53, 70, 156, 219, 258, 262, 285, 288, 292, Ucides, 490
296, 299, 300, 303, 305–307, 309, 311 Upogebia,  334, 370, 372, 427
Stomatopoda,  105, 170, 202, 215, 364, 367, 370, 421, 427
Stress,  44, 121, 155, 172, 199, 200, 206, 220, 223, 252, Vein,  2, 04, 205
255, 256, 267, 268, 270, 292, 294, 340, 345, 346, Ventilation,  200, 204, 217, 223, 224, 228–230, 255, 259,
349, 374, 395, 404, 407, 409, 420, 428, 437, 322, 325, 326, 329–332, 334, 339, 340, 345, 348,
440–444, 446–455, 486, 489, 495–497 360, 405, 481, 483
Stress protein,  395, 495–497 Ventral nerve cord (VNC),  52, 55, 70, 74, 75, 210
Subtropical, 328 Vitelline membrane,  2, 62, 265
Synalpheus, 146 Vitellogenin,  8, 9, 37, 48, 49, 53, 292, 399, 438, 447,
System 448, 450, 452, 455, 463, 464, 466
arterial,  199–202, 204–206, 211, 214, 218, 222, 230
cardiovascular,  199, 200, 206, 210, 214, 228, 232 Xanthidae, 189
circulatory,  174, 199, 200, 202, 203, 217, 220, 232, Xenobiotic,  295, 444, 446, 463, 464, 496
326, 340, 342, 360 Xenograpsus,  2, 93, 427
digestive,  119, 219, 262, 286–288, 296, 306, 311 X-organ,  2, 9, 10, 36, 38–40, 42, 51, 53, 74, 212,
endocrine,  176, 183, 184, 257, 455, 461–463, 465 214, 257
nervous,  10, 18, 21, 50, 52, 55, 56, 70, 73, 78, 143, 147,
148, 184, 190, 201, 447, 462, 465, 466, 492 Y-organ,  1, 2, 5, 6, 13, 41, 156, 184, 436

Talitrus,  2, 31, 256 Zinc (Zn),  83, 268, 292, 293, 302, 449, 479, 492
Talorchestia, 267 Zoea,  19, 51, 141, 148, 264, 266, 267, 269, 297, 407, 482