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Plant Nematology
 DEDICATION

To Clare and Monique with thanks for their patience and support during the preparation
of this book.
Plant Nematology

Edited by

Roland N. Perry
Plant Pathogen Interaction Division, Rothamsted Research,
Harpenden, Hertfordshire AL5 2JQ, UK and Biology Department,
Ghent University, K.L. Ledeganckstraat 35, B9000 Ghent, Belgium
and

Maurice Moens
Agricultural Research Centre, Crop Protection Department, Burg.
Van Gansberghelaan 96, B9280 Merelbeke, Belgium and
Department of Crop Protection, Ghent University, Coupure links
653, B9000 Ghent, Belgium
 CABI is a trading name of CAB International

CABI Head Office CABI North American Office

Nosworthy Way 875 Massachusetts Avenue
Wallingford 7th Floor
Oxfordshire OX10 8DE Cambridge, MA 02139
UK USA
Tel: +44 (0)1491 832111 Tel: +1 617 395 4056
Fax: +44 (0)1491 833508 Fax: +1 617 354 6875
E-mail: [email protected] E-mail: [email protected]
Website: www.cabi.org

© CABI 2006. All rights reserved. No part of this publication may be repro-
duced in any form or by any means, electronically, mechanically, by photo-
copying, recording or otherwise, without the prior permission of the copyright
owners.

A catalogue record for this book is available from the British Library, London,
UK.

Library of Congress Cataloging-in-Publication Data

Plant nematology / edited by Roland N. Perry and Maurice Moens.

p. cm.
Includes bibliographical references.
ISBN-13: 978-1-84593-056-1 (alk. paper)
ISBN-10: 1-84593-056-8 (alk. paper)
1. Plant nematodes. 2. Nematode diseases of plants. I. Perry, R. N. (Roland
N.) II. Moens, Maurice. III. Title.

SB998.N4P56 2006
632’.6257--dc22
2005020480

ISBN-10: 0-85199-027-4
ISBN-13: 978-0-85199-027-9

Typeset by SPi, Pondicherry, India.

Printed and bound in the UK by Biddles Ltd, King’s Lynn.
 Contents

The Editors xi
Contributors xiii
Preface xv

PART I. TAXONOMY AND PRINCIPAL GENERA 1

1 Structure and Classification 3

Wilfrida Decraemer and David J. Hunt
1.1. Introduction 4
1.2. General Morphology 5
1.3. Life Cycle Stages 23
1.4. Feeding Groups 26
1.5. Classification of Plant-parasitic Nematodes 27
1.6. Common Morphometric Abbreviations 32

2 Molecular Taxonomy and Phylogeny 33

Sergei A. Subbotin and Maurice Moens
2.1. Taxonomy and Phylogeny 34
2.2. Species Concepts and Delimiting Species in Nematology 34
2.3. Phylogeny and Classification 35
2.4. Molecular Techniques 36
2.5. Genes Used for Molecular Systematics 45
2.6. Microsatellites 47
2.7. Phylogenetic Inference 47
2.8. Reconstruction of Historical Associations 50
2.9. Databases 51
2.10. Examples of Molecular Phylogenies 51

v
vi Contents

3 Root-knot Nematodes 59
Gerrit Karssen and Maurice Moens
3.1. Introduction to Root-knot Nematodes 60
3.2. Life Cycle and Behaviour 60
3.3. Host Response to Parasitism 63
3.4. Post-infection Biology 66
3.5. Effect on Plant Growth and Yield 68
3.6. Survival 68
3.7. Cytogenetics 69
3.8. General Morphology 71
3.9. Principal Species 73
3.10. Biochemical and Molecular Identification 85
3.11. Interactions with Other Plant Pathogens 86
3.12. Management and Control 88

4 Cyst Nematodes 91
Susan J. Turner and Janet A. Rowe
4.1. Introduction to Cyst Nematodes 92
4.2. Life Cycle and Behaviour 92
4.3. The Hatching Process 98
4.4. General Morphology of the Subfamily Heteroderinae 99
4.5. Genera and Principal Species 103
4.6. Pathotypes and Races 113
4.7. Biochemical and Molecular Diagnosis 115
4.8. Interactions with Other Plant Pathogens 118
4.9. Management and Control 120

5 Migratory Endoparasitic Nematodes 123

Larry W. Duncan and Maurice Moens
5.1. Introduction to Migratory Endoparasitic Nematodes 124
5.2. The Pratylenchids: Lesion, Burrowing and Rice Root
Nematodes 125
5.3. Anguinids and the Stem and Bulb Nematode, Ditylenchus
dipsaci 138
5.4. Plant-parasitic Aphelenchs 142

6 Ectoparasitic Nematodes 153

Wilfrida Decraemer and Etienne Geraert
6.1. Introduction to Ectoparasitic Nematodes 154
6.2. Definition of Ectoparasites/Ectoparasitism 154
6.3. Classification 155
6.4. Tylenchina (Chromadorea, Chromadoria) 155
6.5. Enoplea 166
6.6. Biochemical and Molecular Diagnostics 179
6.7. Ectoparasitic Nematodes as Vectors of Plant Viruses 181
6.8. Management 182
Contents vii

PART II. NEMATODE BIOLOGY AND PLANT RESPONSES 185

7 Reproduction, Physiology and Biochemistry 187

Denis J. Wright and Roland N. Perry
7.1. Reproduction and Development 188
7.2. Musculature and Neurobiology 192
7.3. Biosynthesis 194
7.4. Respiration and Intermediary Metabolism 198
7.5. Osmotic and Ionic Regulation and Excretion 200
7.6. Survival Strategies 202

8 Behaviour and Sensory Perception 210

A. Forest Robinson and Roland N. Perry
8.1. Sense Organs or Sensilla 211
8.2. Undulatory Propulsion 213
8.3. Random Movement 218
8.4. Movement in Response to Stimuli 222
8.5. Other Types of Movement and Behaviour 233

9 Molecular Aspects of Plant–Nematode Interactions 234

Godelieve Gheysen and John T. Jones
9.1. Nematode Parasitism of Plants 234
9.2. Invasion and Migration 237
9.3. Wound and Defence Responses of the Plant 240
9.4. Protection from Host Responses 241
9.5. Molecular and Cellular Aspects of Nematode Feeding Cells 242
9.6. Nematode Signals for Feeding Site Induction 249
9.7. Comparison Between Cyst and Root-knot Nematodes 252
9.8. Resistance and Avirulence Genes 252

10 Genetic Engineering for Resistance 255

Chris Thomas and Amanda Cottage
10.1. Targets in the Nematode–Plant Interaction for Engineered
Resistance 256
10.2. Past and Present Engineered Resistance Work 257
10.3. The Need for Promoters 265
10.4. The Role of Reporter Genes 266
10.5. Stacked Defences 267
10.6. The Research Approach to Engineer Resistance 267
10.7. The Future 272

PART III. QUANTITATIVE NEMATOLOGY AND MANAGEMENT 273

11 Plant Growth and Population Dynamics 275

Corrie H. Schomaker and Thomas H. Been
11.1. Introduction 276
11.2. Relationships of Nematodes with Plants 276
viii Contents

11.3. Predictors of Yield Reduction 276

11.4. Different Response Variables of Nematodes 278
11.5. Stem Nematodes (Ditylenchus dipsaci) 279
11.6. Root-invading Nematodes 280
11.7. Effect of Nematicides 293
11.8. Validation of the Model 294
11.9. Population Dynamics 295

12 Distribution Patterns and Sampling 302

Thomas H. Been and Corrie H. Schomaker
12.1. Introduction 302
12.2. Practical Application 303
12.3. Horizontal Distribution 304
12.4. Vertical Distribution 324

13 International Plant Health – Putting Legislation into Practice 327

Sue Hockland, Renato N. Inserra, Leah Millar and Paul S. Lehman
13.1. Introduction and Terminology 328
13.2. Historical Considerations 329
13.3. International Phytosanitary Frameworks 331
13.4. Early Legislation Enacted against Plant-parasitic Nematodes 333
13.5. International Phytosanitary Initiatives against Plant-parasitic
Nematodes 334
13.6. Phytosanitary Problems Posed by Plant-parasitic Nematodes 336
13.7. Determining the Risk Posed by Plant-parasitic Nematodes Using
Pest Risk Analysis 336
13.8. Phytosanitary Measures for Plant-parasitic Nematodes 339
13.9. Phytosanitary Measures and their Associated Cost:Benefits 340
13.10. Future Challenges for the Control of Regulated Nematodes 343
13.11. Challenges Facing Scientific Advisers and Researchers 343

14 Biological and Cultural Management 346

Nicole Viaene, Danny L. Coyne and Brian R. Kerry
14.1. Biological Control 347
14.2. Cultural Management 359

15 Resistant Cultivars 370

Roger Cook and James L. Starr
15.1. Introduction 370
15.2. Definitions 371
15.3. Variation and Specificity 373
15.4. Origins and Functions 378
15.5. Exploitation 379
15.6. Successes and Opportunities 389

16 Chemical Control of Nematodes 392

Patrick P.J. Haydock, Simon R. Woods, Ivan G. Grove and Martin C. Hare
16.1. History and Development of Nematicides 392
16.2. Active Substances: Chemical Groups and Modes of Action 396
Contents ix

16.3. Formulation and Application 397

16.4. Nematicides in the Environment 403
16.5. Human Safety 405
16.6. Naturally Occurring Nematicides 408

References 411
Glossary 432
Index 440
This page intentionally left blank
 The Editors

Roland Perry
Professor Roland Perry is based at Rothamsted Research. He graduated with a BSc (Hons)
in Zoology from the University of Newcastle upon Tyne, where he also obtained a PhD in
Zoology on physiological aspects of desiccation survival of Ditylenchus spp. After a
year’s postdoctoral research at Newcastle, he moved to Keele University, UK, for 3 years
where he taught Parasitology. He was appointed to Rothamsted Research in 1976. His
research interests have centred primarily on plant-parasitic nematodes, especially focus-
ing on nematode hatching, sensory perception, behaviour and survival physiology, and
several of his past PhD and postdoctoral students are currently involved in nematology
research. He co-edited The Physiology and Biochemistry of Free-living and Plant-para-
sitic Nematodes and is author or co-author of over 40 book chapters and refereed

xi
xii The Editors

reviews and over 100 refereed research papers. He is Editor-in-Chief of Nematology and
Chief Editor of the Russian Journal of Nematology. In 2001, he was elected Fellow of the
Society of Nematologists (USA) in recognition of his research achievements. He is a
Visiting Professor at the University of Ghent, Belgium, where he lectures on nematode
biology, and also gives regular lectures to MSc courses at Imperial College, London and
Reading University.

Maurice Moens
Professor Maurice Moens is head of the Crop Protection Department of the Agricultural
Research Centre at Merelbeke, Belgium and part-time professor at Ghent University
where he gives a lecture course on Agronematology at the Faculty of Bioscience
Engineering. He is also director of the Postgraduate International Nematology Course,
which is organized in the Faculty of Sciences of the same University. He graduated as an
agricultural engineer from Ghent University and obtained a PhD at the same University
on the spread of plant-parasitic nematodes and their management in hydroponic crop-
ping systems. Within the framework of the Belgian Cooperation, he worked from 1972 to
1985 as a researcher in crop protection, including nematology, at two research stations in
Tunisia. Upon his return to Belgium, he was appointed as senior nematologist at the
Agricultural Research Centre, Merelbeke. There, he gradually expanded the research in
plant nematology over various areas covering molecular characterization, biology of
host–parasite relationships, biological control, resistance and other forms of non-chemi-
cal control. He supervised ten PhD students, who now are active in nematology all over
the world. He is a partner in research projects being executed in Europe and several
developing countries. He is co-author of over 70 refereed research papers and a member
of the editorial board of Nematology and the Russian Journal of Nematology.
 Contributors’ Contact Information

Thomas H. Been, Wageningen University and Research Centre, Plant Research

International BV, Agrosystems Research, PO Box 16, 6700A Wageningen, The
Netherlands (e-mail: [email protected])
Amanda Cottage, Department of Plant Sciences, University of Cambridge, Downing
Street, Cambridge CB2 3EA, UK (e-mail: [email protected])
Roger Cook, Institute of Grassland and Environmental Research, Aberystwyth,
Ceredigion SY23 3EB, UK (e-mail: [email protected])
Danny L. Coyne, International Institute of Tropical Agriculture (IITA), c/o Lambourn &
Co., Carolyn House, 26 Dingwall Road, Croydon CR9 3EE, UK (e-mail:
[email protected])
Wilfrida Decraemer, Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B1000
Brussels, Belgium (e-mail: [email protected])
Larry W. Duncan, University of Florida, Institute of Food and Agricultural Sciences,
Citrus Research and Education Center, 700 Experiment Station Road, Lake Alfred, FL
33850, USA (e-mail: [email protected])
Etienne Geraert, Department of Biology, Ghent University, Ledeganckstraat 35, B9000
Ghent, Belgium (e-mail: [email protected])
Godelieve Gheysen, Department of Molecular Biotechnology, Ghent University,
Coupure links 653, B9000 Ghent, Belgium (e-mail: [email protected])
Ivan G. Grove, Crop and Environment Research Centre, Harper Adams University
College, Newport, Shropshire TF10 8NB, UK (e-mail: [email protected])
Martin C. Hare, Crop and Environment Research Centre, Harper Adams University
College, Newport, Shropshire TF10 8NB, UK (e-mail: [email protected])
Patrick P.J. Haydock, Crop and Environment Research Centre, Harper Adams University
College, Newport, Shropshire TF10 8NB, UK (e-mail: [email protected])
Sue Hockland, Pest and Disease Identification Team, Plant Health Group, Defra, Central
Science Laboratory, Sand Hutton, York YO41 1LZ, UK (e-mail: [email protected])
David J. Hunt, CABI Bioscience, Bakeham Lane, Egham, Surrey TW20 9TY, UK (e-mail:
[email protected])

xiii
xiv Contributors’ Contact Information

Renato N. Inserra, Florida Department of Agriculture and Consumer Services, Division

of Plant Industry, PO Box 147100, Gainesville, FL 32614-7100, USA (e-mail:
[email protected])
John T. Jones, Plant–Pathogen Interactions Programme, Scottish Crop Research Institute,
Invergowrie, Dundee DD2 5DA, UK (e-mail: [email protected])
Gerrit Karssen, Plant Protection Service, PO Box 9102, 6700 HC Wageningen, The
Netherlands (e-mail: [email protected])
Brian R. Kerry, Nematode Interactions Unit, Rothamsted Research, Harpenden,
Hertfordshire AL5 2JQ, UK (e-mail: [email protected])
Paul S. Lehman, Nematologist Emeritus, Florida Department of Agriculture and
Consumer Services, 721 Spiegle Road, Boswell, PA 15531-1902, USA (e-mail: paul-
[email protected])
Leah Millar, US Department of Agriculture, Center for Plant Health Science and
Technology, 1730 Varsity Drive, Suite 300, Raleigh, NC 27606-2194, USA (e-mail:
[email protected])
Maurice Moens, Agricultural Research Centre, Crop Protection Department, Burg. Van
Gansberghelaan 96, B9820 Merelbeke, Belgium (e-mail: [email protected])
Roland N. Perry, Plant Pathogen Interaction Division, Rothamsted Research, Harpenden,
Hertfordshire AL5 2JQ, UK (e-mail: [email protected])
A. Forest Robinson, USDA-ARS, Southern Crops Research Laboratory, 2765 F&B Road,
College Station, TX 77845, USA (e-mail: [email protected])
Janet A. Rowe, Nematode Interactions Unit, Plant Pathogen Interactions Division,
Rothamsted Research, Harpenden, Hertfordshire AL5 2JQ, UK (e-mail:
[email protected])
Corrie H. Schomaker, Wageningen University and Research Centre, Plant Research
International BV, Biometris, PO Box 16, 6700 AA, Wageningen, The Netherlands
(e-mail: [email protected])
James L. Starr, Department of Plant Pathology and Microbiology, Texas A&M University,
College Station, TX 77843-2132, USA (e-mail: [email protected])
Sergei A. Subbotin, University of California, Riverside, CA 92521, USA (e-mail: sergei.sub-
[email protected])
Chris Thomas, Milton Contact Limited, 3 Hall End, Milton, Cambridge CB4 6AQ, UK (e-
mail: [email protected])
Susan J. Turner, Department of Agriculture for Northern Ireland, Applied Plant Science
Division, Agriculture and Food Science Centre, Newforge Lane, Belfast BT9 5PX, UK
(e-mail: [email protected])
Nicole Viaene, Crop Protection Department, Agricultural Research Centre, Burg. van
Gansberghelaan 96, B9820 Merelbeke, Belgium (e-mail: [email protected])
Simon R. Woods, Crop and Environment Research Centre, Harper Adams University
College, Newport, Shropshire TF10 8NB, UK (e-mail: [email protected])
Denis J. Wright, Division of Biology, Faculty of Life Sciences, Imperial College London,
Silwood Park Campus, Ascot, Berkshire SL5 7PY, UK (e-mail: d.wright@
imperial.ac.uk)
 Preface

Plant-parasitic nematodes are of considerable importance worldwide and their devastat-

ing effects on crops have major economic and social impacts. Control of these plant pests
is imperative and with the banning or limitation of the use of many nematicides, alterna-
tive control strategies are required that, in turn, will have to be based on a sound knowl-
edge of nematode taxonomy and biology. Such information is also a basic necessity for
effective formulation and implementation of quarantine regulations. Molecular
approaches to all aspects of nematology have already made a major contribution to tax-
onomy and to our understanding of the host–parasite interactions, and will undoubtedly
become increasingly important.
There have been several excellent specialized texts on plant-parasitic nematodes,
aimed primarily at research scientists. However, there is no book on plant-parasitic
nematodes aimed at a broader readership, especially one including students specializing
in the subject at undergraduate and postgraduate levels. The driving force for this book
was the need for a text to support the MSc course in Nematology run by the University of
Ghent, Belgium. The students on this course come from a wide spectrum of scientific
backgrounds and from many different countries and, after obtaining their degree, will
return to their own country to undertake various jobs, including advisory work, statutory
and quarantine work, PhD degrees and teaching posts. Many of these students will return
to countries where facilities for plant nematology work are basic. Thus, the book needed
to provide a wide range of information on plant-parasitic nematodes and needed to be
inclusive, appealing to workers from developing and developed countries. An excellent
book, edited by John Southey entitled Plant Nematology, provided this type of informa-
tion but is now very dated and has long been out of print. We have used the general for-
mat of Southey’s book as a template for the present volume. We hope that, as well as
being informative, this book will stimulate interest in plant-parasitic nematodes.
Research on taxonomy, biology, plant–nematode interactions and control has gen-
erated an extensive volume of literature. In this book, we have deliberately limited the
number of references, although key research papers have been included where these are
of major significance. Important book chapters and reviews are cited so that a reader
interested in a specific aspect can access these to obtain source references.
We are grateful to all the authors of the chapters for their time and effort in compiling
their contributions. In addition, we wish to thank David Hunt (CABI, UK), John Jones

xv
xvi Preface

(SCRI, UK) and Brian Kerry (Rothamsted Research, UK) for their advice and comments on
various chapters, and Bram Moens (Wetteren, Belgium) for preparing some of the figures.
This book is primarily for students and the impetus for it came from students; we would
like to thank all those whose enthusiasm and interest in plant nematology made this book
possible.

Roland Perry and Maurice Moens

May 2005
I Taxonomy and Principal
Genera

Nematode identification underpins all aspects of research, advisory work, implementa-

tion of quarantine legislation and selection of control strategies. Classical taxonomy is
now supplemented to a large extent by molecular diagnostics. In some regions of the
world, molecular techniques and bar-coding may even replace traditional microscopic
identification, and this may be accelerated by the undoubted decline in the numbers of
expert classical taxonomists. However, from a worldwide perspective, both classical and
molecular techniques are required and this is reflected in the chapters in Part I. The basic
structure and classification of nematodes is followed by a chapter on molecular taxon-
omy and phylogeny and then four chapters on major groups of plant-parasitic nema-
todes. Throughout this book, the systematic scheme follows the higher classification (i.e.
at family level and above) of De Ley and Blaxter (2002).
This page intentionally left blank
1 Structure and Classification
WILFRIDA DECRAEMER1 AND DAVID J. HUNT2
1
Royal Belgian Institute of Natural Sciences, Vautierstraat 29, B1000
Brussels, Belgium and Department of Biology, University Ghent,
Ledeganckstraat 35, B9000 Ghent, Belgium; 2CABI Bioscience, Bakeham
Lane, Egham, Surrey TW20 9TY, UK

1.1. Introduction 4
1.2. General Morphology 5
1.2.1. Body cuticle 5
1.2.1.1. Outer cuticle structure and ornamentation 5
1.2.1.2. Cuticle ultrastructure 7
1.2.2. Epidermis 10
1.2.3. Somatic musculature 10
1.2.4. Pseudocoelom 12
1.2.5. Head region, sense organs and nervous system 12
1.2.5.1. Head region and anterior sensilla 12
1.2.5.2. Central nervous system 15
1.2.5.3. Peripheral and pharyngeal nervous system 15
1.2.6. Digestive system 16
1.2.6.1. Stomodeum 16
1.2.6.2. Mesenteron 18
1.2.6.3. Proctodeum 19
1.2.7. Secretory–excretory system 19
1.2.8. Type of reproduction and reproductive system 19
1.2.8.1. Female reproductive system 20
1.2.8.2. Male reproductive system 23
1.3. Life Cycle Stages 23
1.4. Feeding Groups 26
1.5. Classification of Plant-parasitic Nematodes 27
1.6. Common Morphometric Abbreviations 32

© CABI 2006. Plant Nematology (eds R.N. Perry and M. Moens) 3

4 W. Decraemer and D.J. Hunt

1.1. Introduction

Nematodes are pseudocoelomate, unsegmented worm-like animals, commonly

described as filiform or thread-like, a characteristic reflected by the taxon name nema
(Greek, nema = thread) and its nominative plural nemata. Zoologically speaking, the ver-
nacular word ‘nematode’ is a corruption for the order name Nematoidea, one of the five
historical orders of the class Helminthia (Rudolphi, 1808), which embraced all thread-
like forms or roundworms (gordians and nematodes). At present, nematodes are gener-
ally regarded as a separate phylum, the Nematoda or Nemata (De Ley and Blaxter, 2002).
The systematic scheme presented follows the higher classification proposed by De Ley
and Blaxter (2002).
Nematodes are the most numerous Metazoa on earth. They are either free-living or
parasites of plants and animals. Although they occur in almost every habitat (Cobb,
1915), they are essentially aquatic animals. Nematodes depend on moisture for their
locomotion and active life and therefore soil moisture, relative humidity and other envi-
ronmental factors directly affect nematode survival. However, many nematodes can sur-
vive in an anhydrobiotic state (see Chapter 7). Soil structure is influential as pore size
affects the ease with which nematodes can move through the soil interstices (see Chapter
8). In general, sandy soils provide the best environment for terrestrial nematodes but sat-
urated clay soils can be colonized successfully by certain specialized nematodes, includ-
ing Hirschmanniella and some Paralongidorus. Soil pH may affect nematodes, but soil
temperature is rarely a particularly important factor.
This review of the anatomy of plant-parasitic nematodes will also include mention
of some free-living and animal-parasitic species for comparative purposes. Although
currently only about 4100 species of plant-parasitic nematodes have been described
(i.e. 15% of the total number of nematode species known), their impact on humans by
inflicting heavy losses in agriculture is substantial. The maxim that ‘where a plant is able
to live, a nematode is able to attack it’ is a good one. Nematodes are even able to attack
the aerial parts of plants provided that the humidity is high enough to facilitate move-
ment. Such conditions are provided in flooded rice fields where foliar species, such as
Aphelenchoides besseyi Christie, 1942 and Ditylenchus angustus (Butler, 1913) Filipjev,
1936, can be devastating. Some Bursaphelenchus species, vectored by wood-boring
insects, directly attack the trunk of coconut palm or pines. Other nematodes, such as
some Hirschmanniella and Halenchus spp., attack algae and can live in sea water. It has
been estimated that a single acre of soil from arable land may contain as many as
3,000,000,000 nematodes and a single wheat gall formed by Anguina tritici (Steinbuch,
1799) Filipjev, 1936 typically contains approximately 11,000–18,000 nematodes,
although as many as 90,000 have been recorded.
In order to constrain or even banish this limiting factor in agricultural production,
it is vital to identify accurately the nematode pests and to understand their biology. At
present, most nematode identification relies upon morphological characters,
although this is complemented by other approaches, including DNA sequences (see
Chapter 2).
Despite their great diversity in lifestyle, nematodes display a relatively conserved
body plan. The body consists of an external cylinder (the body wall) and an internal cylin-
der (the digestive system) separated by a pseudocoelomic cavity filled with fluid under
pressure and containing a number of cells and other organs such as the reproductive
tracts. About 99% of all known nematodes have a long, thin cylindrical body shape,
which is round in cross-section and tapered towards both ends, although usually more
towards the posterior or tail end. The tail may be short or long and varies in form from
broadly rounded to filiform. The tail may also differ in shape between developmental
stages or between sexes.
Structure and Classification 5

Nematodes usually crawl or swim with undulating movements in a dorsoventral

plane (see Chapter 8). Aberrant body shapes, for example, a swollen female body, may
either indicate a loss of locomotion, as in cyst nematodes, or be related to an aberrant
locomotory pattern, as in the free-living marine families Draconematidae and
Epsilonematidae, which move on their ventral side. In plant-parasitic nematodes, all
migratory ectoparasites of roots, including all Trichodoridae and Longidoridae and many
Tylenchomorpha, are vermiform throughout their life cycle. Other, more highly evolved
Tylenchomorpha have a sedentary endoparasitic lifestyle, one or more stages inciting
specific feeding cells or feeding structures within the root tissue and becoming obese.
This type of life cycle is seen in the root-knot and cyst nematodes where the mature
female becomes pyriform to globose in form.
The body shape assumed by nematodes on relaxation varies from straight through
ventrally curved to spiral. Free-living and plant-parasitic nematodes are mostly smaller
than 1 mm in length, although some species may greatly exceed this (e.g. the 12 mm long
Paralongidorus epimikis Dalmasso, 1969). Animal-parasitic nematodes can be substan-
tially longer and may achieve lengths of many centimetres. Externally, the body shows lit-
tle differentiation into sections apart from the tail region. The nematode body can be
divided into a dorsal, a ventral and two lateral sectors. The secretory–excretory (S–E)
pore, vulva and anus in the female or the cloacal opening in the male are all located ven-
trally whereas the lateral regions may be identified by the apertures of the amphids (few
exceptions), deirids and phasmids, when present. The mouth opening is usually located
terminally at the anterior end. The body displays a bilateral symmetry although the ante-
rior end also shows a radial symmetry. The body wall consists of the body cuticle, epider-
mis and somatic musculature.

1.2. General Morphology

1.2.1. Body cuticle

Although some nematodes lack a cuticle in the adult (e.g. the insect-parasitic female of
Fergusobia), most nematodes possess a cuticle, the structure of which may be extremely
variable (Figs 1.1 and 1.2; Box 1.1), not only between different taxa, but also intraspecifi-
cally between sexes and developmental stages or between different body regions of an
individual (Decraemer et al., 2003). The nematode cuticles vary from simple and thin
(Fig. 1.2G) to highly complex, multilayered structures, as in some marine species
(Fig. 1.2A). The body cuticle invaginates at the mouth opening, amphids, phasmids, S–E
pore, vulva, anus or cloacal opening and forms the lining to the cheilostome, amphidial
fovea (canal), terminal canal, part of the terminal duct of the S–E system, pars distalis vagi-
nae, cloaca and rectum. As nematodes lack both a skeleton and a circular muscle system,
the cuticle functions as an antagonistic system that prevents radial deformation of the body
when the longitudinal muscles contract during undulatory locomotion. Initially, the cuticle
plays a role in maintaining body shape after elongation of the embryo. The cuticle together
with the epidermis also functions as a barrier to harmful elements in the environment and,
being semipermeable, plays a role in secretion–excretion or uptake of substances.

1.2.1.1. Outer cuticle structure and ornamentation

The body cuticle is often marked by transverse and/or longitudinal striae (Fig. 1.1). The
transverse striae range from being very fine, superficial (i.e. restricted to the cortical zone)
and close together, to being deeper and wider apart, in which case they delimit the
6 W. Decraemer and D.J. Hunt

Fig. 1.1. Surface structure of the body cuticle. A. Transverse striae, SEM (Trichodoridae).
B. Longitudinal ridges (Actinolaimidae, Dorylaimida) (Vinciguerra and Claus, 2000; courtesy
Universidad de Jaén). C and D. Criconema paradoxiger. C, External cuticular layer in female;
D, scales in juvenile (adapted from Decraemer and Geraert, 1992); E, lateral field with
longitudinal ridges and areolation (arrow) in Scutellonema; F, perineal pattern in Meloidogyne
(Siddiqi, 1986; courtesy CAB International); G, caudal alae or bursa in Scutellonema male.

annuli. Transverse striae are present in Longidoridae and Trichodoridae (Fig. 1.1A) where
they are indistinct or difficult to observe by light microscopy. When both transverse and
longitudinal striae are present all over the body, the cuticle has a tessellate appearance.
Apart from striae, longitudinal elevations or ridges may also be present (with or without
an internal support). Alae are thickened wing-like extensions of cuticle, which are often
found laterally or sub-laterally on the body, or localized in the caudal region of the male
when they form the bursa (Fig. 1.1G). Other cuticular outgrowths, such as transverse or
longitudinal flaps, occur at the female genital opening where they cover the vulva. More
elaborate cuticular ornamentation may also occur (spines, setae, papillae, tubercles,
warts, bands, plates, rugae and pores). In plant-parasitic nematodes, cuticular ornamen-
tations are important diagnostic features, especially in Criconematidae; in Criconema
(Amphisbaenema) and Nothocriconema, an extra cuticular layer may be found
(Decraemer et al., 1996) (Fig.1.1D; Box 1.1).
In many nematodes, the lateral body cuticle is modified to form the lateral fields. In
Tylenchomorpha, the lateral fields are marked by longitudinal incisures and may rise
above the body contour to form longitudinal ridges or bands. These ridges may be inter-
rupted by the transverse striae, in which case the lateral field is described as being areo-
lated (Fig. 1.1E). The number of longitudinal lines or incisures is of taxonomic
importance but, as their number decreases towards the extremities, the number of lines
should be counted at the mid-body. It is important to differentiate between longitudinal
ridges and lines or incisures. Sometimes, the lateral field shows anastomoses and in some
cases lateral cuticle differentiation may be absent, as in obese females of Heterodera or
Structure and Classification 7

A B
1
2 1
2
3
3
4

4 195 nm
1 C
2
4
1
2
3
1 µm
4
D
1
2
3
1 µm
4 0.2 µm
E F
0.5 µm H
1
2
3
1
2
3 4
21
4 3
4
G
0.2 µm 0.5 µm 0.5 µm

Fig. 1.2. Diverse ultrastructure of body cuticle. Free-living nematodes. A. Enoplus, adult
(Enoplida). Plant-parasitic nematodes. B. Xiphidorus, adult (Longidoridae). C. Trichodorus,
adult (Trichodoridae). D–H. Tylenchomorpha, females. D, Heterodera; E, Helicotylenchus;
F, Rotylenchus; G, Pratylenchus; H, Tylenchorhynchus, lateral field (Decraemer et al., 2003).
Courtesy Cambridge Philosophical Society (Biological Reviews).

all Longidoridae and Trichodoridae. Cuticular differentiations may also occur at or

around the vulva and anus, as in the perineal patterns of mature females of root-knot
nematodes (Fig. 1.1F).

1.2.1.2. Cuticle ultrastructure

The cuticle is secreted in layers and essentially consists of four parts: (i) a thin epicuticle
at the external surface, which is provided with a surface coat of glycoproteins and other
surface-associated proteins or, more rarely, with an additional sheath formed from cuti-
cle or extra cuticular particles; (ii) a cortical zone; (iii) a median zone; and (iv) a basal
zone. Certain zones may be absent. For example, in Tylenchomorpha, the body cuticle
changes in structure at the base of the head, i.e. the median and radially striated basal
zones disappear (Fig. 1.5B). The latter appears to continue as the electron-dense zone of
the cephalic capsule.
8 W. Decraemer and D.J. Hunt

Box 1.1. Ultrastructure of nematode body cuticle.

b a
1

Scheme ultrastructure. 1, Epicuticle; 2, cortical zone; 3, median zone; 4, basal zone; a, sur-
face coat; b, extracuticular or non-cuticular material in criconematids; c, basal lamina; d,
epidermis (based on Bird and Bird, 1991).
Epicuticle: Trilaminar outermost part of the cuticle, first layer to be laid down during
moulting.
Cortical zone: Zone beneath the epicuticle, may be more or less uniform in structure,
amorphous or with radial striae, or may show a subdivision into an outer amorphous part
and an inner, radially striated layer, or be multilayered in criconematids. Cortical radial
striae are unknown from plant parasites.
Median zone: Internal to the cortical zone, variable in structure: homogeneous or layered,
with or without granules, globules, struts, striated material or fibres. The median zone
may be absent.
Basal zone: Innermost zone of the cuticle, usually with the most complex structure of the
three main zones; comprises outer sub-layers of spiral fibres and an inner layer with or
without other fibres, laminae or radial striae.
Radial striae: Either cortical or basal in position, consist of longitudinal and transverse cir-
cumferential interwoven laminae which, at high magnification, appear as osmiophillic
rods separated by electron-light material; the spacing and periodicity of these rods may
vary between species, but in transverse sections of tylenchids it is about 19 nm.
Radial elements: Superficially resembling radial striae (in Longidoridae).
Basal radial striae: In basal cuticle zone, always interrupted at level of lateral chords and
usually replaced by oblique fibre layers; mainly found in free-living terrestrial stages and
most plant-parasitic Tylenchina.
Cortical radial striae: In cortical zone, not interrupted at level of lateral chords; character-
istic of a free-living aquatic lifestyle.
Basal spiral fibre layers: Helically arranged fibre layers (angle 54º44′ or more) play a role in
maintaining the internal turgor pressure.
Struts: Column-like supporting elements in the median zone (common in free-living
aquatic nematodes and some animal parasites).
Structure and Classification 9

The most important structural elements of the cuticle morphology are the presence
or absence of (i) cortical radial striae; (ii) basal radial striae; (iii) spiral fibre layers in the
basal zone; and (iv) supporting elements, e.g. struts, in the fluid matrix of the median
zone (Fig. 1.2; Box 1.1). All these features are thought to be responsible for the radial
strength of the cuticle. At the level of the lateral chords, the cuticle may not only show an
external differentiation in ornamentation or cuticular outgrowths, such as lateral alae,
but also show ultrastructural differences when compared to the dorsal and ventral sides
of the body. Intracuticular canals have been observed in many species (e.g.
Trichodoridae, Hoplolaimidae) and may be involved in transport of material from the
epidermis to the other layers of the body cuticle.
In Longidoridae, adults and juveniles have the same cuticle structure. The cuticle
consists of three main zones: (i) the cortical zone with radial filaments and radial ele-
ments at the inner base; (ii) the median zone with a layer of median thick longitudinal
fibres; and (iii) the basal zone with two spiral fibre layers and either a layered or a homo-
geneous inner region (Fig. 1.2B).
In Trichodoridae, the cortical zone has no radial striae and consists of two sub-lay-
ers, the outer electron-lucent and the inner electron-dense; the median zone is a rather
thick homogenous layer and the basal zone is subdivided into a thin to thick outer
electron-dense sub-layer and an inner layer with concentric striation in transverse as
well as in longitudinal section (Fig. 1.2C). The structure of the basal zone with concentric
layers is characteristic of this family. The absence of radial striae in the cortical or the
basal zone as well as the absence of spiral fibre layers may be related to the low inter-
nal pressure in trichodorids (trichodorids do not ‘explode’ when punctured) as well as
to their slow locomotion.
In Tylenchomorpha, the cuticle structure is more diverse but cortical radial striae
are always absent (Fig. 1.2D–H). The cortical zone is rarely subdivided but rather
appears homogeneous, finely granular, fibrillar or amorphous, although in females of
the family Criconematidae, the cortical zone is multilayered except in the head
region. The median zone in Tylenchomorpha may be absent and the cortical zone
abuts the radially striated basal zone (e.g. Aphelenchus avenae). Sometimes, the limits
of the median zone are difficult to define. The majority of Tylenchomorpha have a vac-
uolated median zone with or without granular material or ovoid to globular structures
whereas in criconematids it is loose and fibrillar. In the majority of Tylenchomorpha,
all stages have the basal zone characterized by a radially striated layer. In
Hoplolaimidae and Heteroderidae (Fig. 1.2D), the basal zone of females possesses
additional sub-layers internal to the radially striated layer, initially referred to as endo-
cuticle. In agreement with Maggenti (1979), these sub-layers are considered part of
the basal zone; their differences in numbers, appearance and thickness are reliable
taxonomic characters in Heteroderidae (Cliff and Baldwin, 1985). In globose females
of this family, the radially striated layer is discontinuous (Fig. 1.2D (Heterodera
schachtii A. Schmidt, 1871)), but this is not the case in sausage-shaped females of the
genus Verutus. The cuticle at the level of the lateral field is differently structured com-
pared to the rest of the body in Tylenchomorpha. Laterally, the striated layer is inter-
rupted and often appears as forked where it is replaced by two to four obliquely
orientated, fine, fibrillar layers (Fig. 1.2H).
Most juveniles of plant-parasitic Tylenchomorpha have a similar cuticle structure to
the adults. Cuticular changes other than during the moulting process occur when chang-
ing lifestyles and in sedentary stages. Upon invasion of plant roots, the conspicuous radi-
ally striated basal zone of the cuticle of the pre-parasitic second-stage juvenile (J2) of
Meloidogyne, is modified in the parasitic J2 into a thicker cuticle lacking basal radial
striae (Jones et al., 1993).
10 W. Decraemer and D.J. Hunt

Basal radial striae appear to induce some physical constraints, e.g. to growth, which
may explain their absence under certain conditions. In obese endoparasitic females of the
Heteroderinae, the thick basal zone retains only patches of radial striae. Further, the lateral
differentiation of the cuticle, with replacement of the basal radial striae by fibre layers,
appears to be a functional requirement to accommodate small changes in the diameter of
the nematode. Basal radial striae also appear to be involved in locomotion as they disap-
pear in J2 of Meloidogyne shortly after the juvenile becomes a sedentary endoparasite.
The surface-coat protein components show stage-specific differences and play an
important role in the resistance of an invading nematode to the host reaction (e.g.
Meloidogyne). All stages of the plant-parasite Hemicycliophora arenaria Raski, 1958
possess an additional sheath covering the normal cuticle (Johnson et al., 1970). This
sheath is composed of a trilaminate outer layer and either four (female) or two (male)
inner layers of cuticle.
Most nematodes moult four times (exceptionally three times, e.g. as in some
Longidoridae) during their development. At each moult the cuticle is reconstructed.
During moulting, the cuticle can either be shed completely (Trichodoridae) or partially
resorbed (Meloidogyne). New cuticle formation is characterized by the occurrence of
epidermal folds or plicae over which the new cuticle becomes highly convoluted (Bird
and Bird, 1991). The epicuticle is the first layer laid down. The body cuticle is connected
to the epidermis by hemidesmosomes.
In addition to the anterior cuticular sense organs such as labial and cephalic sensilla
and the amphids, there are also somatic sense organs that terminate in setae or in pores,
phasmids or deirids (see section 1.2.5.3).

1.2.2. Epidermis

The epidermis secretes the cuticle and is responsible for the overall architecture, including
elongation of the embryonic tadpole stage (Costa et al., 1997). The epidermis is probably
the limiting structure in homeostatic regulation. The epidermis consists of a thin layer and
four main internal bulges that form the longitudinal chords, one dorsal, one ventral and
two laterals, dividing the somatic muscles into four fields (Box 1.2.). Anteriorly, it pervades
the region of the cephalic framework and is responsible for its formation. The epidermis
can be cellular, partly cellular or syncytial (Tylenchomorpha). The cellular condition is a
primitive one occurring in free-living species and some parasitic species plus juveniles of
parasites that possess a syncytial epidermis in adults. In some species there are no cell
boundaries between the chords but cell walls exist within the chords, especially the lateral
chords. The cell nuclei are usually located in the chords, although the dorsal chord only
has nuclei in the pharyngeal region. The structure of the epidermis may show pronounced
changes during development. For example, in the insect parasitic stage of Fergusobia, the
cuticle and feeding apparatus are degenerate and the epidermis is convoluted into the
numerous microvilli responsible for uptake of nutrients (Giblin-Davis et al., 2001). The
epidermis contains various specialized structures such as epidermal glands, caudal glands
and ventral gland(s) of the S–E system. In some aquatic nematodes, such as
Geomonhystera disjuncta (Bastian, 1865), vacuoles in the epidermal chord may act as a
compartmentalized hydrostatic skeleton (Van De Velde and Coomans, 1989).

1.2.3. Somatic musculature

Only a single layer of obliquely orientated and longitudinally aligned somatic muscle
cells lies beneath the epidermis. The number of rows per quadrant between the chords
Structure and Classification 11

Box 1.2. Body wall and pseudocoel.

6
5
c 2
3
b
4

a
Diagram of transverse section depicting the epidermal chords, the somatic musculature
with detail of muscle cell and pseudocoel. Internal organs have been omitted. 1, Dorsal
epidermal chord; 2, ventral chord with ventral nerve; 3, muscle cell; 4, basal lamina; 5, lat-
eral alae; 6, pseudocoel; a, contractile part of muscle cell; b, non-contractile part; c,
process of muscle cell (based on Bird and Bird, 1991).
Somatic muscle cell: Mainly spindle-shaped, consists of: (a) a contractile portion of the
cell towards the epidermis, (b) a non-contractile portion towards the body cavity and (c)
an arm or process that extends from the non-contractile portion of the cell towards the
dorsal or the ventral nerve; muscle cells anterior to the nerve ring send processes
directly into the nerve ring.
Platymyarian muscle cell: The whole contractile part of the muscle cell is flat and broad
and borders the epidermis; common in small species.
Coelomyarian muscle cell: Spindle-shaped muscle cell; laterally flattened so that the
contractile elements are arranged not only along the epidermis, but along the sides of
the flattened spindle as well; coelomyarian muscle cells bulge into the pseudocoel;
common in large species.
Circomyarian muscle cell: Muscle cell in which the sarcoplasm is completely surrounded
by contractile elements.
Meromyarian musculature: Few (up to 5–6) rows of muscle cells are present per quadrant.
Polymyarian musculature: More than six rows of muscle cells present per quadrant; spin-
dle-shaped muscle cells laterally flattened.

varies from a few (up to five cells), the meromyarian condition, to many rows, the poly-
myarian condition. The general sinusoidal movement of nematodes is brought about by
alternate contraction of the ventral and dorsal musculature, thereby giving rise to waves
in a dorsoventral plane (see Chapter 8). In the Criconematidae with strongly developed
transverse cuticular annuli, contraction of the somatic muscles shortens and relaxation
extends the body, resulting in a movement comparable to that of earthworms. A typical
characteristic of a nematode muscle cell, a feature found in only a few other invertebrate
taxa (e.g. some Gastrotricha), is that instead of the nerve process running towards the
muscle, a process of the non-contractile portion of the muscle cell extends towards the
dorsal or ventral nerve in the corresponding epidermal chord. The arrangement of the
contractile portion groups the muscle cells into three types: (i) platymyarian; (ii) coelo-
12 W. Decraemer and D.J. Hunt

myarian; and (iii) circomyarian (Box 1.2). The platy-meromyarian type is more common
in small species such as plant-parasitic nematodes. Specialized muscle cells are associ-
ated with the digestive system and male and female reproductive systems.

1.2.4. Pseudocoelom

The pseudocoelom or body cavity is a secondary structure lacking mesentery and is lined
by the somatic muscles and the basal lamina that covers the epidermal chords. This fluid-
filled cavity bathes the internal organs and contains some large amoeboid cells called
pseudocoelomocytes. These vary in number, size and shape and their function includes
osmoregulation, secretion and transport of material. The pseudocoelomic fluid acts as
part of the turgor-pressure system, but also has some circulatory function.

1.2.5. Head region, sense organs and nervous system

1.2.5.1. Head region and anterior sensilla

The basic pattern in nematodes is the presence of six lips around the mouth opening
(two sub-dorsal, two sub-ventral and two lateral lips) (Fig. 1.3A). The lips can be fused,
for example two by two resulting in three lips, one dorsal and two ventrosublateral
lips (Ascaris), or the lateral lips may be reduced or absent (Pseudoacrobeles (Bonobus)
pulcher (Loof, 1964) De Ley, Siddiqi & Boström, 1993). The lips are either clearly sep-
arated or partially to completely fused (Longidoridae, Trichodoridae) (Fig. 1.3C). In
Tylenchomorpha, the anterior head end shows an amalgamated, usually hexagonal,
lip region and so lip-like differentiations, when present, are better referred to as lip
sectors or lip areas (Fig. 1.3B), e.g. six lip sectors in aphelenchs, four lip sectors upon
reduction of the lateral ones in Belonolaimus. Loof and De Grisse (1974) introduced
the term pseudolips for the six areas around the oral opening of some Criconematidae
(Fig. 1.3E). The area around the oral opening may be differentiated into an oral and/or
labial disc. In many groups of Tylenchomorpha there are two consecutive openings
due to bending in of the head cuticle, the outermost is called the pre-stomatal open-
ing, the innermost, the stomatal opening. The region between the openings or anterior
to the stoma opening when the pre-stoma opening is wide, is referred to as the pre-
stoma (Fig. 1.5B).
In Tylenchomorpha, the cephalic region is internally supported by a variously devel-
oped cephalic cuticular framework that may be well developed and heavily sclerotized.
The lip region can be continuous with the body contour or more or less offset from the
rest of the body, either by a depression or constriction, or be broader than the adjoining
body and therefore expanded. In Tylenchomorpha, the true head height is not always
easy to establish as the head may be offset at a different level to the basal cephalic frame-
work. The head region may be smooth or bear transverse striae.
The lip region carries a concentration of anterior sensilla, each composed of a neu-
ronal and non-neuronal section formed by two epidermal cells, the socket cell and the
sheath cell (Coomans, 1979). In nematodes, there are primitively twelve labial sensilla
and four cephalic sensilla arranged in three circlets to form six inner labial sensilla, six
outer labial sensilla and four cephalic sensilla. This is described as the 6 + 6 + 4 pattern.
Two chemoreceptor sense organs, the amphids, are primitively clearly posterior to the
three circlets of anterior sensilla, but in more derived forms, such as in Tylenchomorpha,
they have migrated forward on to the lip region. Each lip bears an inner and an outer sen-
Structure and Classification 13

A B C

ols

amp
ils
cs

D
cs

amp ils

ld Or.o
p. p.
ols
p.

F G H I
Fig. 1.3. Head region and anterior sensilla. A. Basic scheme (de Coninck, 1965).
B. Scutellonema with six lip areas (arrows), en face view. C. Paratrichodorus in en face
view with outer labial and cephalic papillae in a single circlet (arrow). D. Arrangement in
Aphelenchoides: amp, amphid; cs, cephalic sensilla; ils, inner labial sensilla; ld, labial disc;
ols, outer labial sensilla; Or.o, oral opening. E. Criconemoides (= Criconemella), en face
view showing pseudolips (arrows) (Van den Berg and De Waele, 1989). Amphid structure.
F and G. Spiral fovea, lateral and ventral view (p, pore). H. Pocket-like amphidial fovea in
lateral (left) and ventral (right) view. I. Xiphidorus, pouch-like amphidial fovea with
pore-like opening.

sillum on its radial axis, this hexaradial pattern being maintained when lip number is sec-
ondarily reduced. The four cephalic sensilla are bilaterally arranged (two laterodorsal,
two lateroventral) and represent the anteriormost somatic sensilla. In the plant-parasitic
families Longidoridae and Trichodoridae, the cephalic sensilla have migrated on to the
lip region and are close to the outer labial sensilla, thereby forming a single circlet or 6 +
(6 + 4) pattern. In Tylenchomorpha, the anterior sensilla are arranged in three circlets but,
because of the small size of the head, the two posterior circlets are located close together.
In general, the six inner labial sensilla protrude from the surrounding head cuticle
via a terminal pore on top of a papilla. In a number of plant- and animal-parasitic nema-
todes, the inner labial sensilla either have pore-like openings around the oral opening or
inside the pre-stoma (Pratylenchus), or pores may be lacking entirely and the receptors
end blind in the cuticle of the oral disc (Hemicycliophora). Inner labial sensilla in open
connection with the environment are chemoreceptive; those covered or embedded in
the cuticle are mechanoreceptive. In most tylenchs, the inner labial sensilla possess two
ciliary receptors and show a combined chemo- and mechanoreceptive function. In
Longidorus, four such receptors can be found, while there are two or three in
Trichodorus.
The outer labial sensilla may protrude via papillae or setae, but in many plant- and
animal-parasitic nematodes they end in simple pores or are embedded in the head cuti-
14 W. Decraemer and D.J. Hunt

cle; the cuticle above each termination may show a slight depression. Their nerve end-
ings may be displaced from a sub-dorsal or sub-ventral position to a nearly sub-median
one (= position of cephalic nerves) before they return to their original position. The lateral
outer labial sensilla are often reduced, a reduction that may be related to the develop-
ment of the amphids (Meloidogyne).
The cephalic sensilla are sub-median in position and usually protrude from the sur-
rounding cuticle as setae or papillae with a terminal pore. In many parasitic nematodes,
they are embedded in the head cuticle.
The main constituent parts of an amphid are the aperture, the fovea, the canalis and
the fusus or sensillar pouch (Fig. 1.4). The distal part of the amphid, the fovea, is either an
external excavation of the head or body cuticle (as in many free-living Chromadorida
species; Fig. 1.3F) or an invagination of the cuticle, thus forming a pocket connected with
the exterior through an aperture (as in plant-parasitic nematodes; Fig. 1.3H). The

cu.

ep.

fov.

s.r. a.d.
s.c

g.c

g.c

m.d

Fig. 1.4. Ultrastructure of amphid: a.d., amphidial duct; cu., cuticle; ep., epidermis; fov.,
fovea; g.c., gland or sheath cell; m.d., multivillous dendrite; s.c., socket cell; s.r., sheath
receptor. (After Coomans, 1979.)
Structure and Classification 15

amphidial aperture is typically located laterally, but may be shifted dorsad. The amphidial
opening varies in form: in Trichodoridae (Fig. 1.3C) it is a post-labial transverse slit,
whereas in Longidoridae it is of variable shape and size, being either a post-labial trans-
verse slit or a pore (Fig. 1.3I). In Tylenchomorpha, the amphid openings have migrated on
to the lip region and are usually close to the oral opening, their openings being greatly
reduced in size and slit-like to oval in form (Fig. 1.3B). The fovea is the most variable part
of the amphid, varying in size and shape according to taxa and even within a taxon
between sexes and may be completely or partially filled with a gelatinous substance (cor-
pus gelatum) secreted by the amphidial gland and may protrude from the body. In nema-
todes with an internal fovea, variation in form is less. In Longidoridae, the fovea varies
from stirrup- or goblet-shaped to a pouch, which may be bilobed. In tylenchs, there is no
sharp demarcation between fovea and canalis and the sensilla pouch may be located as
far posterior as the stylet knobs. The amphids are the largest of the chemoreceptors and
possess a much greater number of receptors than other sensilla. In Tylenchomorpha, lip
patterns and arrangement of the anterior sensilla and amphids are considered important
diagnostic features in identification and for the analysis of relationships.

1.2.5.2. Central nervous system

The nerve ring usually encircles the isthmus of the pharynx, rarely the intestine. The nerve
ring is connected to several ganglia, longitudinal nerves running anteriorly towards the
anterior sensilla in the head region, while posteriorly four large nerves, the largest being
the ventral nerve, run through each of the four epidermal chords, with four smaller
nerves, two laterodorsal and two lateroventral, running adjacent. The cell bodies of the
nerves of the anterior sensilla are located in six separate ganglia anterior to the nerve ring
or in a single ganglion (Caenorhabditis elegans). The amphidial nerves have an indirect
connection with the nerve ring, their cell bodies being located in the paired lateral gan-
glia. Of the posterior longitudinal nerves, the lateral nerve contains a few ganglia, includ-
ing the lumbar ganglia in the tail region. The ventral nerve has a chain of ganglia, the
anteriormost being the retrovesicular ganglion whereas the dorsal and sub-median
nerves lack posterior ganglia. Posterior to the nerve ring, the longitudinal nerves are con-
nected to each other by commissures. The presence and position of some of these com-
missures are of taxonomic importance. The most important commissure is the
lateroventral commissure, which is also known as the hemizonid. This is visible as a
refractive body near the S–E pore. At a short distance from the hemizonid lies the hemi-
zonion. Caudalids in the tail region correspond to the paired anal–lumbar commissure,
which links the pre-anal ganglion to the lumbar ganglion.

1.2.5.3. Peripheral and pharyngeal nervous system

The peripheral nervous system consists of a lattice-like network of nerves that connect
setae and papillae. It coordinates impulses from these somatic sense organs, both with
each other and with the central nervous system. This system has mainly been described
from free-living marine taxa. In Rhabditia we do not usually find a complex system of sur-
face sensory nerves innervating the cervical setae. The innervation of deirids (cervical
papillae common in Tylenchomorpha) is from the lateral nerves from the lateral ganglia.
Other somatic sense organs common in Tylenchomorpha (although they may be lacking,
as in criconematids) are the postdeirids and phasmids, which are usually situated in the
tail region. Phasmids have the basic structure of a sensillum, the ciliary receptor being in
open connection with the environment via a pore. The pore may be provided with a plug,
which is probably secreted by the sheath cell, and which may be small, as in many
Tylenchomorpha, or large and scutellum-like (e.g. in Scutellonema; Fig. 1.1E).
16 W. Decraemer and D.J. Hunt

The pharynx possesses its own visceral nervous system with its own neurons,
processes and receptors, but is also connected to the nerve ring. The detailed structure of
this system is mainly known from work on C. elegans.

1.2.6. Digestive system

The wide diversity of food sources and methods of ingestion is reflected in the diversity of
the structure of the digestive system. In general, this system consists of three regions: (i)
stomodeum; (ii) mesenteron; and (iii) proctodeum. Only the mesenteron or midgut is of
endodermal origin, the stomodeum being of mixed ecto-mesodermal origin and the
proctodeum or rectum formed from the ectoderm. The stomodeum region is referred to
as the pharyngeal, neck or cervical region.

1.2.6.1. Stomodeum
The stomodeum comprises the mouth opening, stoma sensu lato (composed of
cheilostome and pharyngostome), pharynx and cardia. The oral aperture is mostly situ-
ated terminally. The cheilostome or lip cavity has a hexa- or triradial symmetry and is
lined by body cuticle. The cuticle lining may be more or less sclerotized and is supported
by simple rods (cheilorhabdia, a synapomorphy of the Chromadorida). Originally, the
cheilostome was delimited by the lips but in derived conditions may extend further pos-
teriorly, as in Longidoridae where the cheilostome represents the stoma sensu stricto, and
extends from the oral opening to the guiding ring (Fig. 1.5A). In Tylenchomorpha, the
cheilostome largely lines the cavity of the cephalic framework through which the stylet
moves. The pharyngostome is triradially symmetrical, as is the pharynx, of which it is a
specialized part. Its structure reflects the method of feeding and food source. In plant-
parasitic nematodes, some entomophilic and some predatory nematodes, the pharyn-
gostome possesses a protrusible stylet; in Trichodoridae, the stylet is a protrusible dorsal
tooth. The feeding apparatus or stylet in the majority of plant-parasitic nematodes
is either a hollow stomatostyle (Tylenchomorpha) or odontostyle (Dorylaimida,
Longidoridae). The stomatostyle consists of three parts: (i) a conus with a ventral aperture;
(ii) a shaft; and (iii) a posterior region that may enlarge to form three basal knobs acting as
attachments for the stylet protractor muscles. Tylenchids have no stylet retractor muscles;
the resulting tension in the alimentary tract causes retraction of the stylet when the pro-
tractor muscles are relaxed (Fig. 1.5B). The dorylaimid stylet is made up of two parts: (i)
an anterior odontostyle with a dorsal aperture anteriorly and with its posterior end fur-
cate or simple and (ii) a posterior stylet extension or odontophore, a modification of the
anterior pharyngeal region, which acts as a supporting structure for the odontostyle. The
base of the odontophore may be enlarged to form three flanges for enhanced insertion of
the stylet protractor muscles (Fig. 1.5.A). Longidorids possess eight stylet protractor mus-
cles (typical of dorylaims) but true stylet retractor muscles are poorly developed or
absent. Pharyngeal retractor muscles at the level of the narrow part of the pharynx assist
in retracting the odontostyle. Because of the extreme elongation of the cheilostome,
Xiphinema species possess a special set of dilatores buccae muscles, which counteract
compression of the cheilostome wall on stylet protraction. In Longidoridae, the odon-
tostyle is long and needle-like. When it is protruded, the anterior stomatal lining folds
along the odontostyle, thereby forming a guiding sheath, the inner cuticular lining being
separated from the rest of the wall (except at the level of the guiding ring and at its poste-
rior end) by a fluid-filled cavity, which acts as a hydrostatic skeleton. In Longidorus, for
example, the hydrostatic tissue forms four compensation sacs to regulate the pressure on
Structure and Classification 17

A B C
7 6
9
1 8
12
1 14
2
13 10

16

3
15

4 4

11 4

Fig. 1.5. Stoma region and types of feeding apparatus in plant-parasitic nematodes.
A. Odontostyle and odontophore (Longidoridae). B. Stomatostylet with detail of body cuticle
(inset) at base of cephalic framework (Tylenchomorpha). C. Onchiostyle (Trichodoridae).
1, Cheilostome; 2, odontostyle; 3, somatic muscles; 4, stylet protractor muscles; 5,
odontophore with flanges; 6, pre-stoma; 7, thickening of cuticle around pre-stoma; 8, stylet
opening; 9, stoma; 10, stylet conus; 11, stylet shaft and knobs; 12, basal cephalic frame-
work; 13, body cuticle in detail, showing disappearance of median and striated basal zone in
head region; 14 and 15, onchiostyle with onchium (14) and onchiophore (15); 16, dilatores
buccae. A, From Coomans (1985); B, adapted from Endo (1980); C, from Maafi and
Decraemer (2002).

stylet protraction. The odontostyle is secreted by a cell in one of the ventrosublateral

sectors of the pharynx; the odontophore, together with the guiding sheath and the pha-
ryngeal lining, is derived from pharyngeal tissue. The ventrally curved onchiostyle in
Trichodoridae consists of an anterior stylet-like tooth or onchium with a solid tip and a
posterior support, the onchiophore, which is formed by the thickened and sclerotized
dorsal lumen wall of the pharyngostome (Fig. 1.5C). The protrusion of the onchiostyle
occurs upon contraction of protractor muscles of the pharyngostom; there are no retrac-
tor muscles.
Posterior to the pharyngostome, the structure of the pharynx itself is related to feeding
mode. In Tylenchomorpha, the anterior part of the pharynx (or corpus) is subdivided into
an anterior, muscular procorpus and a muscular, larger diameter and more robust, meta-
corpus located posteriorly. The corpus is followed by a non-muscular isthmus (may be
very short or absent) and three pharyngeal glands arranged either in a terminal bulb (post-
corpus) or in a lobe(s) overlapping the intestine (Fig. 1.6C and D). The arrangement of pha-
ryngeal glands is of taxonomic and phylogenetic importance. In the Sphaerularioidea, a
group of taxa combining fungus feeding and insect parasitism, the pharynx is simple and
lacks differentiation into a procorpus. Species consuming liquid food, such as plant-
parasitic tylenchs, have a median bulb or metacorpus with a cuticularized triradiate val-
vate apparatus attached to strong musculature, thereby allowing a stronger pumping
action. Within the Tylenchomorpha, the position of the outlet of the dorsal gland is of tax-
onomic importance and differentiates the Aphelenchoidea (where the outlet is in the
metacorpus) from the other taxa of the infraorder (outlet in the procorpus and often close
18 W. Decraemer and D.J. Hunt

A B C D

1 7 7
1
17
2
8 8

17
9 9
3
6 5
6 15

3
16
12 11 13
4 11
10 12
11 11
5
14 11
17 5 12
12 13 13
13
4 11

13 5

Fig. 1.6. Digestive system of plant-parasitic nematode taxa. A. Paraxiphidorus

(Longidoridae). B. Paratrichodorus (Trichodoridae). C. Pratylenchoides (Pratylenchidae). D.
Aphelenchoides (Aphelenchoididae). 1, Cheilostome; 2, pharyngostome; 3, narrow anterior
region of pharynx; 4, pharyngeal bulb; 5, pharyngeal–intestinal junction; 6, isthmus; 7,
stomatostylet; 8, procorpus; 9, metacorpus; 10, post-corpus; 11, ventrosublateral pharyngeal
gland nuclei; 12, dorsal pharyngeal gland nucleus; 13, intestine; 14, intestine dorsally
overlapping pharynx; 15, pharyngeal–intestinal junction valve cell; 16, pharyngeal gland lobe;
17, dorsal pharyngeal gland orifice. A, From Decraemer et al. (1998); B, from Decraemer and
De Waele (1981); C, from Siddiqi (1986); D, from Shepherd et al. (1980).

to the stylet base) (Fig. 1.6C and D). In Longidoridae, the pharynx is flask-shaped and pos-
terior to the pharyngostom continues as a narrow flexible tube followed by an offset, mus-
cular and glandular bulb that is often cylindroid in form (Fig. 1.6A). This terminal bulb
usually contains five pharyngeal glands and their respective orifi, the cuticular lining of
the lumen being reinforced by six platelets. In Trichodoridae, the pharynx consists of a
narrow isthmus, which gradually expands into a largely glandular basal bulb with five
gland nuclei and gland orifi (Fig. 1.6B). The pharyngo-intestinal junction may be directly
abutting or characterized by various types of overlap (either a ventral overlap by the pha-
ryngeal glands or a dorsal overlap by the intestine over the pharynx, or both types of over-
lap together). In most virus vector trichodorid species, the entire lining of the pharynx acts
as a retention site for tobraviruses. The pharyngo-intestinal junction or cardia exists in
many different types. In Tylenchomorpha, the cardia usually consists of two cells, which
differ in size and position in relation to the anterior intestine, whereas in Trichodoridae,
the cardia is only weakly developed (Fig. 1.6B and D).

1.2.6.2. Mesenteron
The intestine is entirely of endodermal origin. It is a simple, single layered tube, either
cellular or syncytial (criconematids), with or without a clear lumen lined with microvilli.
Structure and Classification 19

The function of the intestine is mainly absorption (its lumen being bordered with
microvilli), storage and secretion of proteins and enzymes. In advanced root parasites,
such as Meloidogyne, the intestine becomes a storage organ; the thin lumen wall lacking
microvilli loses connection with the anus. Anteriorly, the intestine may be differentiated
into a ventricular part that may overlap the pharynx (Trichodoridae) or become modified
to house symbiotic bacteria (entomopathogenic Steinernema species). Posteriorly, the
intestine may be differentiated into a pre-rectum, which is separated from the intestine by
a valve-like structure formed by columnar cells (Longidoridae).

1.2.6.3. Proctodeum
The rectum is of ectodermal origin and is lined with body cuticle. It is a very simple short
tube, which is apparently formed by a few cells. The junction between the rectum and
pre-rectum is guarded by an H-shaped sphincter muscle. Defecation is mediated by the
H-shaped dilator ani. In criconematids, both rectum and anus are poorly developed and
probably non-functional. In Meloidogyne, six large rectal glands produce the gelatinous
matrix in which the eggs are deposited. In Mononchida and Dorylaimida, males possess
rectal glands consisting of three to five pairs of cells located dorsolaterally or laterally
from the posterior intestine (pre-rectum). Caudally or dorsocaudally from the spicules the
ducts of these rectal glands form loops that run anteriorly between the spicules and open
via the dorsal wall of the cloaca.

1.2.7. Secretory–excretory system

The S–E system is a system of variable complexity. It is often called the ‘excretory system’
on morphological grounds, but physiologically the evidence supports more of a secre-
tory and osmoregulatory function than an excretory one. The S–E-system is either: (i)
glandular, in general consisting of a renette cell or ventral gland connected to a ventral
pore by a duct, the terminal part of which is lined by cuticle or (ii) tubuloglandular, the
most complex system consisting of an H-shaped cell with longitudinal canals running in
the lateral chords and joined by a transverse duct connected to the S–E pore by a median
duct, associated with a S–E sinus and an A-shaped binucleated gland cell (C. elegans;
Fig. 1.7B). In Tylenchomorpha, the S–E system is of the tubuloglandular type and is asym-
metrical with a single renette cell situated laterally or lateroventrally, usually posterior to
the pharynx (Fig. 1.7C). The S–E duct leads to the S–E pore, which is usually located pos-
terior to the nerve ring, although exceptionally it may be close to the head region or the
vulva region (Tylenchulus). In Tylenchulus, the renette cell is enlarged and produces a
gelatinous matrix in which eggs are deposited. In Trichodoridae, the S–E system is not
developed and only a ventral pore and a very short duct are present. In Longidoridae it is
not yet clear if a S–E system exists, a glandular structure with two cells being described
only from Longidorus macrosoma Hooper, 1961 (Fig. 1.7A).

1.2.8. Type of reproduction and reproductive system

Most nematodes are dioecious (having the sexes separate) and gonochoristic (meaning
they are either male or female) but bisexual individuals or protandrous hermaphrodites
(e.g. C. elegans) exist; the latter usually have the appearance of females. Gonochoristic
species reproduce by amphimixis or cross-fertilization. Uniparental reproduction or
autotoky takes the form of either parthenogenesis, where development occurs through
females producing female offspring (i.e. without fertilization), or automixis or self-
fertilization in hermaphrodites (where male and female gametes are produced in the
20 W. Decraemer and D.J. Hunt

Fig. 1.7. Secretory–excretory (S–E) system. A. Glandular system in Longidorus macrosoma

(Longidoridae). B. Complex tubular system in Caenorhabditis elegans (Rhabditoidea).
C. Simple tubular system in tylenchid nematodes. 1, S–E gland cell; 2, S–E duct; 3, tubular
cell; 4, transverse duct; 5, S–E pore. A, Based on Aboul-Eid (1969); B, from Nelson et al.
(1983) in Bird and Bird (1991); C, from de Coninck (1965).

same individual). Parthenogenesis may be either meiotic (following meiosis the diploid chro-
mosome number is restored by fusion with a polar body or by first doubling of the chromo-
somes) or mitotic (i.e. without meiosis). In both types of autotoky, males may show up
sporadically (i.e. upon environmental stress) and amphimixis may then be possible in some
species. Autotoky has arisen independently in several taxa of the phylum. Intersexes are
found in some species and should not be confused with hermaphrodites as only one set of
reproductive organs is functional, the other being vestigial. Pseudogamy, a way of reproduc-
tion intermediate between amphimixis and automixis/parthenogenesis, where development
of the egg is activated by a spermatozoon, which then plays no further role, is less common.
Sex is mostly determined genotypically, mostly XX in female and hermaphrodites and, XO or
XY in male, but in some cases the genotype is changed under epigenetic influences.
The reproductive system is quite similar in both sexes and generally comprises one
or two (rarely multiple) tubular genital branches. Apart from sexual characters, sexual
dimorphism is not a common feature among nematodes, but when it occurs it is most
evident among parasitic groups, e.g. a swollen saccate female contrasting with the vermi-
form male (Verutus, Tylenchomorpha).

1.2.8.1. Female reproductive system

The basic system is didelphic (i.e. composed of two uteri), amphidelphic (referring to the
uteri extending in opposite directions) and connected to a single vagina opening to the
Structure and Classification 21

outside via a mid-ventral vulva (Fig. 1.8A). A derived system with a single uterus is called
monodelphic. The monodelphic system with only the anterior branch developed is
described as prodelphic; the opposite being opisthodelphic. The terms monogonic and
digonic refer, respectively, to the presence of one or two ovaries. In didelphic systems,
the vulva is located at about 50% of the entire body length from the anterior end,
although it may be more anterior in monodelphic conditions or virtually subterminal in
position as in some monodelphic species or certain obese females. Each genital branch
consists of an ovary (= gonad) and a gonoduct. The gonoduct consists of oviduct and
uterus and may have one or two sphincters (valves) and a spermatheca. The spermatheca
is a specialized part of either the oviduct or the uterus. Reduction of one of the genital
branches is not uncommon, ranging from partial reduction in various degrees (the
pseudomonodelphic condition, as in some Xiphinematinae) or complete loss, apart from
the possible retention of a small post-uterine sac (monodelphic system as in
Criconematidae). In the didelphic condition, both branches may, in nematodes where
the vulva is virtually subterminal, be anteriorly outstretched, i.e. prodelphic.
The ovary is usually a tubular structure, either outstretched or reflexed, and consists
of three main zones. The blind end usually functions as a germinal zone, the telogonic
condition, (although primary oocytes may be formed before the last moult), and is fol-
lowed by the growth zone and ripening zone. In some taxa the oocytes are connected to
a central protoplasmic core or rachis. In species without a rachis, the oocytes are inter-
connected by protoplasmic bridges.
The oviduct may show great variation in structure and development between taxa,
and is considered to be of fundamental importance in nematode systematics (Geraert,
1983). In Tylenchomorpha, the oviduct is generally formed by two rows of a few cells
(e.g. three or four cells in Tylenchoidea, Criconematoidea and two cells in
Aphelenchoididae); the number of cells corresponds to major taxa and is of diagnostic
and phylogenetic importance; the tightly packed oviduct cells form a tiny canal when a
ripe oocyte is squeezed through. In Trichodoridae, the oviduct consists of two cells, while
in Longidoridae, the oviduct consists largely of flattened disc-like cells with a collapsed
lumen and a wider part that acts as a spermatheca. The presence or absence of a sper-
matheca(e) is of diagnostic importance (Trichodoridae). In Tylenchomorpha, the number
of cells of the spermatheca, their shape and spatial arrangement are of taxonomic impor-
tance (Bert et al., 2003).
The uterus may be a simple tube (as in Paratrichodorus), but is usually more complex,
being subdivided, for example, into a glandular, a muscular and a non-muscular portion
(Fig. 1.8B). In Tylenchomorpha, the uterus sensu stricto is restricted to the eggshell-
producing region of the gonoduct, the crustaformeria, of which the number and arrange-
ment of cells (tricolumella, tetracolumella, quadricolumella) are of taxonomic
importance (Fig. 1.8C and D). In Longidoridae the uterus may vary from very short and
simple to very long and complex local uterine differentiations such as the Z-organ
(Xiphinema). The Z-organ probably slows down the descent of the eggs towards the vulva,
although its function is not entirely clear. Part of the uterus or uteri opposite the vagina may
be differentiated into a muscular ovejector to assist with egg ejection. The uterine structure
is of taxonomic importance in Longidoridae. Various sets of muscles attach to the vagina,
some serving to dilate the lumen while others constrict the vagina or suspend it in the
body, thereby preventing it from prolapsing during egg laying. Egg laying is also mediated
by the vulva dilators, which connect the vulva to the lateral body wall. Eggs may be laid
singly or stuck together in masses in a gelatinous matrix secreted by the female. Such egg
masses are associated with species where the females swell and become sedentary,
although some obese genera retain all the eggs within the body, the female cuticle tanning
on death to form a tough cyst. Egg sacs and cysts serve to protect the vulnerable eggs. Most
nematode eggs are morphologically very similar, i.e. ellipsoidal in shape with a transpar-
22 W. Decraemer and D.J. Hunt

Fig. 1.8. Female reproductive system in plant-parasitic taxa. A. Didelphic–amphidelphic

system (Trichodorus, Trichodoridae). B. Pseudomonodelphic system (Xiphinema surinamense,
Longidoridae). C–E. Monodelphic system. C. Entire female reproductive system of
Pratylenchus coffeae (Pratylenchidae); D. Detail of oviduct–uterus region in Rotylenchus
goodeyi (Tylenchoidea). E. Posterior body region (Hemicriconemoides minor,
Criconematoidea). 1, Ovary; 2, oviduct; 3, spermatheca; 4, sphincter/valve; 5, ovarial sac; 6,
uterus; 7, vagina; 8, vulva; 9, crustaformeria uterus; 10, uterine sac; 11, post-vulval uterine
sac; 12, uterus pars dilata; 13, reduced anterior branch; 14, sperm. A, From Decraemer
(1991); B, from Decraemer et al. (1998); C, from Roman and Hirschmann (1969); D, courtesy
W. Bert; E, from Decraemer and Geraert (1992).

ent shell, except for some animal-parasitic nematodes, and are of similar dimension, irre-
spective of the size of the adult. Different sized eggs may occur within a species between
free-living forms and parasitic forms. The eggshell consists of three main layers: the outer
vitelline layer derived from the oolemma and first layer formed after sperm penetration,
the middle chitinous layer and the inner lipid layer. The lipid layer is largely responsible for
Structure and Classification 23

the impermeability of the eggshell, which is only permeable to chemicals before the lipid
layer is formed during the passage of the egg down the uterus; the permeability of the lipid
layer also alters before hatching (see Chapter 7).

1.2.8.2. Male reproductive system

Nematodes may have a single gonad or testis (monorchic system) or two testes (diorchic
system) (Fig. 1.9). In Longidoridae, the male reproductive system comprises two testes,
the posterior one being reflexed; the gonoduct consists of a single vas deferens, which
may be differentiated into a strongly muscular ductus ejaculatorus (Enoplida) opening to
the cloaca, a common cavity with the outlet of the digestive system. The posterior part of
the testis(-es) and/or the anterior part of the vas deferens may form a vesicular seminalis or
sperm storage zone. Ejaculatory glands may be associated with the vas deferens.
Typically four cells are present along each side of the intestine at pre-rectum level
in dorylaims and the ducts merge posteriorly with the vas deferens. Such glands are
absent in Trichodoridae. Trichodoridae males are monorchic as are males of the
Tylenchomorpha, except for some Meloidogyne males, which occasionally have two
testes due to sex reversal during development. Sperm are continuously produced in most
plant-parasitic taxa but in genera of the Criconematoidea and some Sphaerularioidea
they are produced before the final moult. In such forms the testis appears degenerate
although well-developed sperm fill the gonoduct.
The copulatory apparatus generally consists of two equal spicules, cuticularized,
tubular structures. The spicules are rarely absent (e.g. marine Monoposthia), may be
dimorphic, partly fused (in rhabditids) or reduced to a single spicule (as in some
mermithids). Spicules are formed by gradual invagination of the posterior wall of the
spicular pouches, which originate from the spicular primordium, specialized cells of the
dorsal wall of the cloaca. Each spicule contains sensilla with one or two dendrites and
dendritic process or receptor enclosed in a channel leading to a pore(s) near the tip of the
spicule. In several taxa, the spicules are differentiated and show ornamentations, such as
striae, bristles, a ventral velum or sub-ventral vela (flanges), which are of taxonomic
importance. The gubernaculum is a cuticular thickening of the dorsal wall of the spicular
pouch and acts as a guide during spicule protrusion. The gubernaculum can be very sim-
ple to complex; in Longidoridae only the crura remain (Fig. 1.9B). The copulatory appa-
ratus functions by means of sets of protractor and retractor muscles. There are protractor
and retractor muscles for each spicule as well as protractor and retractor muscles related
to the gubernaculum. In Triplonchida (Trichodoridae) the spicule protractor muscles
form a capsule of suspensor muscles not directly attached to the spicules. In the caudal
region, accessory genital structures such as caudal alae, pre- and post-cloacal supple-
ments, genital papillae or rays on the bursa, setae or suckers may be present. The arrange-
ment of the pre- and post-cloacal supplements (see Longidoridae, Trichodoridae) or
genital papillae (Rhabditomorpha) are of taxonomic importance. Paired genital papillae
may also be present on the post-cloacal lip (= hypoptygma) as in Merliniinae
(Tylenchomorpha). Nematode sperm cells are non-flagellate, non-ciliate and show
amoeboid motility. They are diverse in size and shape and possess a major sperm protein
associated with their unusual motility and correlated with a fibrillar skeleton in mature
spermatozoa. Except for Enoplida, mature sperm lack a nuclear envelope.

1.3. Life Cycle Stages

Nematodes typically have an egg stage, four juvenile stages and the adult male
and female (Fig. 1.10). The egg is usually cylindrical with a chorion/eggshell of varying
24 W. Decraemer and D.J. Hunt

Fig. 1.9. Male reproductive system. A. Posterior body region (Paraxiphidorus). B. Spicule and
guiding piece or crura (arrow). C. Monorchic system (Trichodorus). D. Spicule with velum
(arrow). E. Diorchic system (Xiphinema). F. Gubernaculum. G. Muscles and copulatory
apparatus of Hoplolaimus. 1, Ejaculatory gland; 2, mid-ventral supplement; 3, adanal
supplement; 4, corpus; 5, cuneus; 6, capitulum; 7, apophysis or apodeme; 8, crura;
9, anterior spicule protractor; 10, gubernaculum protractor; 11, posterior spicule protractor;
12, spicule retractor muscles; 13, germinal zone of testis; 14, vesicular seminalis; 15, vas
deferens. A and B, From Decraemer et al. (1998); C–D, from Decraemer (1991); E, based on
Luc and Coomans (1992); F, from Maggenti (1979); G, from Coomans (1962).

thickness. Eggs may be deposited singly or in masses. In the latter case they may be held
within a gelatinous matrix (root-knot nematodes) or protected within a tough cyst formed
from the body of the dead female. Most nematodes moult four times before becoming
adult, although there are nematodes, such as certain Xiphinema species, that have life
cycles shortened to only three moults, probably due to epogenesis (maturation of
gametes before completion of body growth), a phenomenon not uncommon in partheno-
genetic organisms. In the Longidoridae and Trichodoridae, the juvenile that hatches from
the egg is the J1, whereas in Tylenchomorpha it is the J2, the first moult occurring within
the egg. In some animal-parasitic nematodes, two moults occur within the egg.
Structure and Classification 25

Egg Egg

Hatches from egg in J1 J1

Longidoridae/Trichodoridae

Hatches from egg in Resistant infective stage,

J2 J2
Tylenchomorpha e.g. Anguina

(Ensheathed in Dauer stage, e.g.

animal parasites: J3 Caenorhabditis elegans J3
infective stage)

J4 Resistant infective stage,

e.g. Ditylenchus

Adult Adult, e.g.

in some
Longidoridae
Fig. 1.10. Basic life cycles of nematodes.

Before reaching maturity, the juveniles usually resemble the adult female in mor-
phology, differing in the absence of a mature reproductive system and in certain meas-
urements and proportions. In some groups, one juvenile stage (usually the J3, but may be
the J2 or the J4) is more resistant to environmental stress than the other stages. This juve-
nile stage is therefore specialized for dispersal and for surviving inhospitable conditions,
and usually represents the infective stage of most animal-parasitic forms. This infective
stage is non-feeding and retains the cuticle from the previous stage as a protective sheath
around its body. In free-living rhabditid nematodes, this modified stage is often called a
dauer (from the German for endurance) larva, dauer juvenile or dauer, and may represent
an alternative pathway to the normal development process. Dauers possess a thicker
cuticle, which is often hydrophobic, and are non-feeding, the oral aperture being closed,
the non-functional pharynx and intestine reduced in development and the anterior sen-
silla modified. Many free-living species with dauers are dispersed by insects or other
arthropods, the dauers either attaching themselves to the vector or congregating beneath
the elytra or within the intersegmental folds. This is known as phoresy; dauers attached to
the outside of the insect vectors being ectophoretic, whilst those inside the vectors (e.g.
in the Malpighian tubes, intestine, tracheal system or bursa copulatrix) are endophoretic.
When conditions are suitable, the dauer stages can reactivate by absorbing water; the life
cycle then continues. The term dauer has also been used for resistant stages of some
plant-parasitic nematodes (e.g. J2 of anguinids) although here it does not seem to repre-
sent an alternative pathway to the formation of a normal J2.
26 W. Decraemer and D.J. Hunt

Nematodes are typically amphimictic and have separate males and females (see
Chapter 7). Many species, however, lack males and reproduce either by parthenogene-
sis (the usual case) or, more rarely, by hermaphrodism. The generation time of nema-
todes can, depending on the species concerned, vary from a few days to a year or more.
Females are usually oviparous, but in some groups the eggs can hatch inside the body of
the female (ovoviviparity), usually resulting in her death (this is known as endotokia
matricida). Ovoviviparity can be induced in normally reproducing species by pollu-
tants, e.g. sulphur dioxide (Walker and Tsui, 1968). In some specialized plant parasites,
the female body swells and becomes greatly enlarged. The cuticle in some species thick-
ens and tans/darkens on the death of the female to form a tough cyst that surrounds the
retained eggs and protects them from deleterious factors such as drought or extremes of
temperature.

1.4. Feeding Groups

Nematodes display a wide range of feeding habits or trophisms. Some species of nema-
todes are microphagous/microbotrophic, feeding on small microorganisms, while others
are saprophagous, feeding on dead and decaying organic matter. Many species of nema-
todes are phytophagous, obtaining nourishment directly from plants, whilst others are
omnivorous or predatory. Parasitism of invertebrates or vertebrates is also common. There
are three main types of plant parasitism: (i) ectoparasitic; (ii) endoparasitic; and (iii) semi-
endoparasitic.

1. Ectoparasitic: The nematode remains in the soil and does not enter the plant tissues. It
feeds by using the stylet to puncture plant cells – the longer the stylet the deeper it can
feed within the plant tissues. The majority of ectoparasitic species remain motile whereas
others, e.g. Cacopaurus, become permanently attached to the root by the deeply embed-
ded stylet.
2. Endoparasitic: In this type of parasitism the entire nematode penetrates the root tissue.
Migratory endoparasites, such as Pratylenchus and Radopholus, retain their mobility and
have no fixed feeding site within the plant tissue, whereas the more advanced sedentary
endoparasites have a fixed feeding site and induce a sophisticated trophic system of
nurse cells or syncytia (see Chapter 9). Establishment of a specialized feeding site
enhances the flow of nutrients from the host, thereby allowing the females to become
sedentary and obese in form and highly fecund. Sedentary endoparasites also have a
migratory phase before the feeding site is established. In root-knot and cyst nematodes it
is only the J2 and adult male that are migratory but in Nacobbus, for example, all juvenile
stages, the male and the immature vermiform female are migratory, only the mature
female being sedentary.
3. Semi-endoparasitic: Only the anterior part of the nematode penetrates the root, with
the posterior part remaining in the soil phase.

The above categories are not mutually exclusive as some genera may, depending on the
host, be either semi-endoparasitic or migratory ecto-endoparasitic, e.g. Hoplolaimus
(Tytgat et al., 2000) or Helicotylenchus, whilst some sedentary parasites have only
the anterior body embedded in the root (= sedentary semi-endoparasites), e.g.
Rotylenchulus, Tylenchulus.
In ectoparasites and most migratory endoparasites, any vermiform stage may feed
on, or penetrate, the root, but in those plant-parasitic nematodes where the female
becomes obese and sedentary, the infective stage is usually the vermiform J2. This is
Structure and Classification 27

true for Heterodera/Globodera and Meloidogyne species, for example, although in

Rotylenchulus it is the immature female that is the infective stage, the non-feeding
juveniles and males remaining in the soil. Tylenchulus has a similar life cycle, although
here the juvenile females browse on epidermal cells, the immature females penetrating
deeper into the root cortex. In Nacobbus, perhaps a less specialized parasite in some
ways, all stages, including the vermiform immature female are infective. In a number of
Tylenchomorpha the males have a degenerate pharynx and do not feed. Such males are
found in Radopholus, for example, and occur throughout the Criconematoidea. In
Paratylenchus, the J4 female lacks a stylet and does not feed on plant cells.

1.5. Classification of Plant-parasitic Nematodes

Nematode classification is currently in a state of flux as molecular phylogenies

become increasingly pertinent. For this reason, the higher classification is particularly
fluid with proposals for Infraorders, etc., and the bringing together of groups which
previously, under classical systematic systems, had been regarded as distantly related
(De Ley and Blaxter, 2002). For plant-parasitic nematodes, the most recent classical
schemes are those of Siddiqi (2000) for the Tylenchina; Hunt (1993) for the
Aphelenchoidea and Longidoridae; and Hunt (1993) and Decraemer (1995) for the
Trichodoridae.
In the following scheme, the higher systematic categories (i.e. at family level and
above) are as proposed by De Ley and Blaxter (2002) whilst at subfamily and generic
level, a simplification of Siddiqi (2000) and Hunt (1993) is followed. The most important
plant-parasitic genera are indicated in ‘bold’ .
It is useful to note that the various systematic ranks have different suffixes attached to
the stem:

Class -ea
Subclass -ia
Order -ida
Infraorder -omorpha
Suborder -ina
Superfamily -oidea
Family -idae
Subfamily -inae
Tribe -ini

PHYLUM NEMATODA POTTS, 1932

CLASS CHROMADOREA INGLIS, 1983
Subclass Chromadoria Pearse, 1942
Order Rhabditida Chitwood, 1933
Suborder Tylenchina Thorne, 1949
Infraorder Tylenchomorpha De Ley & Blaxter, 2002
Superfamily Tylenchoidea Örley, 1880
Family Tylenchidae Örley, 1880
Subfamily Tylenchinae Örley, 1880
Tylenchus Bastian, 1865
Aglenchus Andrássy, 1954
Coslenchus Siddiqi, 1978
Filenchus Andrássy, 1954
28 W. Decraemer and D.J. Hunt

Subfamily Boleodorinae Khan, 1964

Boleodorus Thorne, 1941
Basiria Siddiqi, 1959
Subfamily Thadinae Siddiqi, 1986
Thada Thorne, 1941
Subfamily Duosulciinae Siddiqi, 1979
Duosulcius Siddiqi, 1979
Malenchus Andrássy, 1968
Miculenchus Andrássy, 1959
Ottolenchus Husain & Khan, 1967
Subfamily Tanzaniinae Siddiqi, 2000
Tanzanius Siddiqi, 1991
Subfamily Ecphyadophorinae Skarbilovich, 1959
Ecphyadophora de Man, 1921
Subfamily Ecphyadophoroidinae Siddiqi, 1986
Ecphyadophoroides Corbett, 1964
Subfamily Atylenchinae Skarbilovich, 1959
Atylenchus Cobb, 1913
Subfamily Eutylenchinae Siddiqi, 1986
Eutylenchus Cobb, 1913
Subfamily Tylodorinae Paramonov, 1967
Tylodorus Meagher, 1964
Subfamily Pleurotylenchinae Andrássy, 1976
Cephalenchus Goodey, 1962
Pleurotylenchus Szczygiel, 1969
Subfamily Epicharinematinae Maqbool & Shahina, 1985
Epicharinema Raski, Maggenti, Koshy & Sosamma, 1980
Gracilancea Siddiqi, 1976
Family Dolichodoridae Chitwood in Chitwood & Chitwood, 1950
Subfamily Dolichodorinae Chitwood in Chitwood & Chitwood,
1950
Dolichodorus Cobb, 1914
Neodolichodorus Andrássy, 1976
Subfamily Brachydorinae Siddiqi, 2000
Brachydorus de Guiran & Germani, 1968
Subfamily Belonolaiminae Whitehead, 1960
Belonolaimus Steiner, 1949
Carphodorus Colbran, 1965
Ibipora Monteiro & Lordello, 1977
Morulaimus Sauer, 1966
Subfamily Telotylenchinae Siddiqi, 1960
Telotylenchus Siddiqi, 1960
Bitylenchus Filipjev, 1934
Histotylenchus Siddiqi, 1971
Neodolichorhynchus Jairajpuri & Hunt, 1984
Paratrophurus Arias, 1970
Quinisulcius Siddiqi, 1971
Sauertylenchus Sher, 1974
Telotylenchoides Siddiqi, 1971
Trichotylenchus Whitehead, 1960
Trophurus Loof, 1956
Tylenchorhynchus Cobb, 1913
Structure and Classification 29

Uliginotylenchus Siddiqi, 1971

Subfamily Meiodorinae Siddiqi, 1976
Meiodorus Siddiqi, 1976
Subfamily Macrotrophurinae Fotedar & Handoo, 1978
Macrotrophurus Loof, 1958
Subfamily Merliniinae Siddiqi, 1971
Merlinius Siddiqi, 1970
Amplimerlinius Siddiqi, 1976
Geocenamus Thorne & Malek, 1968
Nagelus Thorne & Malek, 1968
Scutylenchus Jairajpuri, 1971
Family Hoplolaimidae Filipjev, 1934
Subfamily Hoplolaiminae Filipjev, 1934
Hoplolaimus Daday, 1905
Aorolaimus Sher, 1963
Peltamigratus Sher, 1964
Scutellonema Andrássy, 1958
Subfamily Rotylenchoidinae Whitehead, 1958
Helicotylenchus Steiner, 1945
Rotylenchus Filipjev, 1936
Subfamily Aphasmatylenchinae Sher, 1965
Aphasmatylenchus Sher, 1965
Subfamily Rotylenchulinae Husain & Khan, 1967
Rotylenchulus Linford & Oliveira, 1940
Senegalonema Germani, Luc & Baldwin, 1984
Subfamily Verutinae Esser, 1981
Verutus Esser, 1981
Bilobodera Sharma & Siddiqi, 1992
Subfamily Acontylinae Fotedar & Handoo, 1978
Acontylus Meagher, 1968
Subfamily Heteroderinae Filipjev & Schuurmans Stekhoven, 1941
Heterodera Schmidt, 1871
Afenestrata Baldwin & Bell, 1985
Betulodera Sturhan, 2002
Cactodera Krall & Krall, 1978
Dolichodera Mulvey & Ebsary, 1980
Globodera Skarbilovich, 1959
Punctodera Mulvey & Stone, 1976
Subfamily Meloidoderinae Golden, 1971
Meloidodera Chitwood, Hannon & Esser, 1956
Subfamily Ataloderinae Wouts, 1973
Atalodera Wouts & Sher, 1971
Bellodera Wouts, 1985
Cameolodera Krall, Shagalina & Ivanova, 1988
Cryphodera Colbran, 1966
Ekphymatodera Bernard & Mundo-Ocampo, 1989
Hylonema Luc, Taylor & Cadet, 1978
Rhizonema Cid del Prado, Lownsbery & Maggenti, 1983
Sarisodera Wouts & Sher, 1971
Family Pratylenchidae Thorne, 1949
Subfamily Pratylenchinae Thorne, 1949
Pratylenchus Filipjev, 1936
30 W. Decraemer and D.J. Hunt

Zygotylenchus Siddiqi, 1963

Subfamily Hirschmanniellinae Fotedar & Handoo, 1978
Hirschmanniella Luc & Goodey, 1964
Subfamily Radopholinae Allen & Sher, 1967
Radopholus Thorne, 1949
Achlysiella Hunt, Bridge & Machon, 1989
Apratylenchoides Sher, 1973
Hoplotylus s’Jacob, 1960
Pratylenchoides Winslow, 1958
Radopholoides De Guiran, 1967
Zygradus Siddiqi, 1991
Subfamily Nacobbinae Chitwood in Chitwood & Chitwood, 1950
Nacobbus Thorne & Allen, 1944
Family Meloidogynidae Skarbilovich, 1959
Subfamily Meloidogyninae Skarbilovich, 1959
Meloidogyne Goeldi, 1892
Subfamily Nacobboderinae Golden & Jensen, 1974
Meloinema Choi & Geraert, 1974
Bursadera Ivanova & Krall, 1985
Superfamily Criconematoidea Taylor, 1936 (1914)
Family Criconematidae Taylor, 1936
Subfamily Criconematinae Taylor, 1936
Criconema Hofmänner & Menzel, 1914
Bakernema Wu, 1964
Lobocriconema De Grisse & Loof, 1965
Neolobocriconema Mehta & Raski, 1971
Ogma Southern, 1914
Subfamily Macroposthoniinae Skarbilovich, 1959
Criconemoides Taylor, 19361
Discocriconemella De Grisse & Loof, 1965
Xenocriconemella De Grisse & Loof, 1965
Subfamily Hemicriconemoidinae Andrássy, 1979
Hemicriconemoides Chitwood & Birchfield, 1957
Family Hemicycliophoridae Skarbilovich, 1959
Subfamily Hemicycliophorinae Skarbilovich, 1959
Hemicycliophora de Man, 1921
Colbranium Andrássy, 1979
Subfamily Caloosiinae Siddiqi, 1980
Caloosia Siddiqi & Goodey, 1964
Hemicaloosia Ray & Das, 1978
Family Tylenchulidae Skarbilovich, 1947
Subfamily Tylenchulinae Skarbilovich, 1947
Tylenchulus Cobb, 1913
Trophotylenchulus Raski, 1957
Subfamily Sphaeronematinae Raski & Sher, 1952
Sphaeronema Raski & Sher, 1952
Subfamily Meloidoderitinae Kirjanova & Poghossian, 1973
Meloidoderita Poghossian, 1966
Subfamily Paratylenchinae Thorne, 1949

1
= Criconemella, Macroposthonia and Mesocriconema
Structure and Classification 31

Paratylenchus Micoletzky, 1922

Cacopaurus Thorne, 1943
Tylenchocriconema Raski & Siddiqui, 1975
Superfamily Sphaerularioidea Lubbock, 1861
Family Anguinidae Nicoll, 1935
Subfamily Anguininae Nicoll, 1935 (1926)
Anguina Scopoli, 1777
Ditylenchus Filipjev, 1936
Orrina Brzeski, 1981
Pterotylenchus Siddiqi & Lenné, 1984
Subanguina Paramonov, 1967
Superfamily Aphelenchoidea Fuchs, 1937
Family Aphelenchidae Fuchs, 1937
Subfamily Aphelenchinae
Aphelenchus Bastian, 1865
Subfamily Paraphelenchinae
Paraphelenchus Micoletzky, 1922
Family Aphelenchoididae Skarbilovich, 1947
Subfamily Aphelenchoidinae
Aphelenchoides Fischer, 1894
Schistonchus Cobb, 1927
Subfamily Parasitaphelenchinae
Bursaphelenchus Fuchs, 1937
CLASS ENOPLEA INGLIS, 1983
Subclass Dorylaimia Inglis, 1983
Order Dorylaimida Pearse, 1942
Suborder Dorylaimina Pearse, 1942
Superfamily Dorylaimoidea Thorne, 1935
Family Longidoridae Thorne, 1935
Subfamily Longidorinae
Longidorus Micoletzky, 1922
Australodorus Coomans, Olmos, Casella & Chaves, 2004
Longidoroides Khan, Chawla & Saha, 1978
Paralongidorus Siddiqi, Hooper & Khan, 1963
Paraxiphidorus Coomans & Chaves, 1995
Xiphidorus Monteiro, 1976
Subfamily Xiphinematinae
Xiphinema Cobb, 1913
Subclass Enoplia Pearse, 1942
Order Triplonchida Cobb, 1920
Suborder Diphtherophorina Coomans & Loof, 1970
Superfamily Diphtherophoroidea Micoletzky, 1922
Family Trichodoridae Thorne, 1935
Trichodorus Cobb, 1913
Ecuadorus Siddiqi, 2002
Paratrichodorus Siddiqi, 1974
Allotrichodorus Rodriguez-M., Sher & Siddiqi, 1978
Monotrichodorus Andrássy, 1976
32 W. Decraemer and D.J. Hunt

1.6. Common Morphometric Abbreviations

Nematodes are characterized by a combination of measurements and ratios derived from

the various body parts (see Hooper, 1986). Such morphometric characters are usually
abbreviated, the most common being listed in Box 1.3. Measurements of, for example,
the body, pharynx and tail are taken along the mid-line of the relevant structure.
Measurements of the spicule, a curved structure, are usually taken along the median line
(a genuine indicator of actual length), although occasionally (and particularly in aquatic
nematodes and in older descriptions) the chord, a straight line joining the two extremities
of the spicule, is used. Body diameter should be measured perpendicular to the longitu-
dinal body axis and care should also be taken that the nematode being quantified is not
squashed (as a result, for example, of the coverslip not being properly supported with
glass rods or beads) as this will produce a higher value than would otherwise be the case.
Nematodes that have a long tail, often fragile that may be readily broken, are often meas-
ured from the head to the anal or cloacal aperture instead of to the end of the tail. By
removing the error or variable element caused by a long and/or broken tail, ratios result-
ing from this modified measurement of body length, such as female vulval position, are
more consistent and are therefore of greater utility in diagnostics.

Box 1.3. A list of the most commonly used abbreviations.

L = Total body length (head to tail tip).

L′ = Body length from head to anal or cloacal aperture (used when tail is very long and/or
frequently in a damaged state).
a = Total body length divided by maximum body diameter.
b = Total body length divided by pharyngeal length (the pharynx is defined as head end
to the pharyngo-intestinal junction, i.e. not to the posterior tip of the overlapping
gland lobes).
b′ = Total body length divided by distance from anterior end of body to posterior end of
pharyngeal glands.
c = Total body length divided by tail length.
c′ = Tail length divided by body diameter at the anal/cloacal aperture.
V = Position of vulva from anterior end expressed as percentage of body length. Superior
figures to the left and right refer to the extent of anterior and/or posterior gonad or
uterine sac, respectively, and are also expressed as a percentage of body length.
V′ = Position of vulva from anterior end expressed as percentage of distance from head to
anal aperture.
T = Distance between cloacal aperture and anteriormost part of testis expressed as per-
centage of body length.
m = Length of conical part of tylenchid stylet as percentage of whole stylet length.
o = Distance of dorsal pharyngeal gland opening behind stylet knobs expressed as a
percentage of stylet length.
MB = Distance of median bulb from anterior end expressed as a percentage of total pha-
ryngeal length.
Caudal ratio A = Length of hyaline tail divided by its proximal diameter.
Caudal ratio B = Length of hyaline tail divided by its diameter at a point 5 µm from its
terminus.
µm = One thousandth of a millimetre (micron).
2 Molecular Taxonomy and
Phylogeny
SERGEI A. SUBBOTIN1 AND MAURICE MOENS2
1
University of California, Riverside, CA 92521, USA and Biology
Department, Ghent University, K.L. Ledeganckstraat 35, B9000 Ghent,
Belgium; 2Agricultural Research Centre, Burg. Van Gansberghelaan 96,
B9820 Merelbeke, Belgium and Laboratory for Agrozoology, Ghent
University, Coupure links 653, B9000 Ghent, Belgium

2.1. Taxonomy and Phylogeny 34

2.2. Species Concepts and Delimiting Species in Nematology 34
2.3. Phylogeny and Classification 35
2.4. Molecular Techniques 36
2.4.1. Protein electrophoresis 36
2.4.2. Polymerase chain reaction (PCR) 37
2.4.3. PCR–restriction fragment length polymorphism (PCR–RFLP) 38
2.4.4. Multiplex PCR 39
2.4.5. Random amplified polymorphic DNA (RAPD) 39
2.4.6. Amplified fragment length polymorphism (AFLP) 41
2.4.7. Reverse dot blot hybridization 42
2.4.8. Sequencing of DNA 42
2.4.9. DNA bar-coding 44
2.4.10. Real-time PCR 44
2.4.11. Perspectives of molecular diagnostics of nematodes 44
2.5. Genes used for Molecular Systematics 45
2.5.1. Nuclear ribosomal RNA genes 45
2.5.2. Nuclear protein-coding genes 46
2.5.3. Mitochondrial DNA 46
2.6. Microsatellites 47
2.7. Phylogenetic Inference 47
2.7.1. Alignment 47
2.7.2. Methods for inferring phylogenetic trees 48
2.7.2.1. Minimum evolution method 48
2.7.2.2. Maximum parsimony 48
2.7.2.3. Maximum likelihood 49
2.7.2.4. Bayesian inference 49

© CABI 2006. Plant Nematology (eds R.N. Perry and M. Moens) 33

34 S.A. Subbotin and M. Moens

2.7.3. Phylogenetic tree and tree terminology 49

2.7.4. Evaluation of the reliability of inferred trees 50
2.7.5. Testing of hypothesis 50
2.8. Reconstruction of Historical Associations 50
2.9. Databases 51
2.10. Examples of Molecular Phylogenies 51
2.10.1. Position of Nematoda within metazoans 51
2.10.2. The phylum Nematoda 52
2.10.3. Root-knot nematodes of the family Meloidogynidae 53
2.10.4. Cyst nematodes of the subfamily Heteroderinae 55
2.10.5. Stem and gall-forming nematodes of the family Anguinidae 55
2.10.6. Needle nematodes of the family Longidoridae 55
2.10.7. Pinewood nematode and other Bursaphelenchus species 58

2.1. Taxonomy and Phylogeny

The tasks of systematics are (i) to name, identify and catalogue organisms (taxonomy); (ii)
to discover the ancestral relationships among organisms (phylogeny); and (iii) to organize
information about the diversity of organisms into a hierarchical system (classification).
Molecular systematics is the application of knowledge of genome information, espe-
cially sequence and structure of DNA, RNA molecules and amino acid chains, for
addressing problems of phylogeny and taxonomy of organisms.
There are several reasons why molecular data are more suitable for phylogenetic
studies than morphological ones. First, DNA and protein sequences are strictly heritable
entities, whereas morphological characters can be influenced by various environmental
factors. Second, the interpretation of molecular characters, such as the assessment of
homologies, is generally easier than that of morphological characters. Third, molecular
characteristics generally evolve much more regularly than morphological ones and,
therefore, can provide a clearer picture of relationships. Fourth, molecular characters are
much more abundant than morphological features, and many can be generated in a rela-
tively short period of time. Various preserved, deformed and partly degraded materials
can often be used for molecular studies. Using standard protocols and commercial kits,
sequence information of certain genes or DNA fragments can be obtained from a single
nematode or even only a part of one. Using species-specific primers, nematode DNA can
be amplified from soil or plant extracts. Moreover, specialized methods enable the
extraction of short DNA fragments from long-time preserved, formalin-fixed and glycer-
ine-embedded specimens. Recent achievements in molecular biology and the wide
application of molecular techniques have revolutionized our knowledge in taxonomy
and phylogeny of nematodes. The use of such techniques is becoming routine in nema-
tology ( Jones et al., 1997; Powers, 2004; Subbotin et al., 2004b; Blok, 2005).

2.2. Species Concepts and Delimiting Species in Nematology

There has been considerable debate concerning the definition of a species. Species were
at first merely taxonomic units, i.e. the named categories to which Linnaeus and other
taxonomists of the 18th century assigned organisms, largely on the basis of appearance.
Later the typological species concept was favoured, in which the species is considered a
community of specimens described by characteristic features of its type specimen.
Another random document with
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“The wagon? I have a wagon, yes. One bought for me by my
servant.”
“In Brussels?”
“As it happens, in Brussels.” The paleness of Gordon’s face was
accentuated now, and his eyes held cores of dangerous flame. “And
because I am an English milord, and bring a wagon from Brussels,
you assume that I am a robber?”
“You were driven from your own country,” menaced the other. “Do
you think we hear nothing, we Swiss? This canton knows you well
enough! Stop those horses!” he snarled, for the great coach, ready
for its trip to the town, was rolling down the driveway. The syndic
sprang to the horses’ heads.
At the same instant the two strangers who had been in the
overturned boat, now with clothing partially dried, came from the
house.
“There!” The syndic pointed to the ornate vehicle. “Do you deny this
is the wagon described in that newspaper, and that you absconded
with it from Brussels?”
The older of the two strangers turned quick eyes on Gordon, then on
the wagon. Before Gordon could reply, he spoke in nervous French:
“I beg pardon. I was the owner of that conveyance, and the one
who sold it.”
“Maybe,” said the functionary, “but you did not sell it to this person,
I have reason to believe.”
“No, yonder is the purchaser.” He pointed to a prosaic figure at the
steps.
“His valet!” Shelley thrust in explosively.
“I told you so,” grunted the man of law, and stared with the surprise
of recognition, as the syndic, ruffling with anger, turned on the
strangers with sarcasm: “Friends of the English milord, no doubt!”
The counsellor laid a hasty hand on his sleeve:
“Stop!” he said. “I think I have had the honor of meeting these
gentlemen in Geneva. Allow me to present you, monsieur, to Prince
Mavrocordato, minister of foreign affairs of Wallachia, and”—he
turned to the latter’s younger companion—“his secretary, Count
Pietro Gamba, of Ravenna.”
The sour-faced official drew back. These were names whose owners
had been public guests of the canton. This Englishman, evil and
outcast as he might be, he had no legal hold upon. He could
scarcely frame a grudging apology, for the resentment of self-
righteousness that was on his tongue, and stalked off up the terrace
in sullen chagrin not consoled by the chuckles of the attorney beside
him.
Gordon saw them go, his hands trembling. He replied mechanically
to the grateful farewells of the two strangers as they entered the
coach, and watched it roll swiftly down the darkening shore road, a
quivering blur before his eyes. A fierce struggle was within him, the
peace which the tranquil poise of Shelley’s creed had lent him,
warring against a clamant rage.
Not only in England was he maligned. Here, on the edge of this
mountain barrier, defamation had followed him. The pair riding in his
own carriage knew who he was; the older had spoken his name and
title. And they had not elected to stay beyond necessity. Yet for their
momentary presence, indeed, he should be grateful. But for this trick
of coincidence he should now be haled before a bungling Genevan
tribunal, his name and person a mark for the sparring of pettifogging
Swiss officials!
These thoughts were clashing through his mind as he turned and
walked slowly down to the bank where Shelley’s Swiss servant had
moored the stranger’s rescued boat, bailed out and with sail
stretched to dry. The sunset, as he stood, flamed redly across the
lake, its ray glinting from the rim of a bright object whose broken
chain had caught beneath the boat’s gunwale. He leaned and drew it
out.
It was an oval miniature backed with silver—the portrait of a young
girl, a face frail and delicately hued, with fine line of chin and slender
neck, with wistful eyes the deep color of the Adriatic, hair a gush of
tawny gold, skin like warm Arum lilies, and a string of pearls about
her neck. Evidently it had belonged to one of the two men with
whom the craft had capsized. It was too late now to overtake the
coach; he would send it after them that evening.
He turned the miniature over. On the back was engraved a name:
“Teresa Gamba.” Gamba? It had been one of the names spoken by
the attorney, that of the young count for whose rescue he had swum
so hard.
He looked again at the ivory. His wife? No, no; innocence of life,
ignorance of its passions and parades were there. His sister? Yes.
The fair hair and blue eyes were alike. And now he caught a subtle
resemblance of feature. She was dear to this brother, no doubt—
dear as was his own half-sister to him, well-nigh the only being left
in England who believed in him and loved him.
He looked up at a hail from the lake. A boat was approaching,
bearing a single feminine rower. As he gazed, she looked over her
shoulder to wave something white at the porch.
“It is Jane. She has been to the post,” cried Shelley from the terrace,
and hastened down the bank.
Gordon thrust the ivory into his pocket as the skiff darted in to the
landing.
 CHAPTER XXIII
THE DEVIL’S DEAL

As he took the two missives the girl handed him Gordon caught his
breath, for one he saw was directed in Annabel’s hand. For a
moment a hope that overleaped all his suffering rose in his brain.
Had those months wrought a change in her? Had she, too, thought
of their child? Had the cry he voiced on the packet that bore him
from England struck an answering chord in her? He opened its cover.
An inclosure dropped out.
He picked it up blankly. It was the note he had pencilled on the
channel, returned unopened.
The sudden revulsion chilled him. He broke the seal of the second
letter and read—read while a look of utter sick whiteness crept
across his face, a look of rage and suffering that marked every
feature.
It was from his sister, a letter written with fingers that soiled and
creased it in their agony, blotted and stained with tears. For the
thing it told of was a dreadful thing, a whispered charge against him
so damning, so satanic in its cruelty, that though lip might murmur it
to a gloating ear, yet pen refused to word it. The whole world turned
black before him, and the dusk seemed shot through with barbed
and flaming javelins of agony.
He crushed the letter in his hand, and, with a gesture like a
madman’s, thrust it into Shelley’s, turning to him a countenance
distorted with passion, gauche, malignant, repulsive.
“Read it, Shelley,” he said in a strangled voice. “Read it and know
London, the most ineffable centaur ever begotten of hypocrisy and a
nightmare! Read what its wretched lepers are saying! There is a
place in Michael Angelo’s ‘Last Judgment’ in the Sistine Chapel that
was made for their kind, and may the like await them in that of our
Lord and Saviour Jesus Christ—Amen!”
With this fearful imprecation he flung away from their startled faces
along the winding vineyarded lane, on into the dusk, lost to a sense
of direction, to everything save the blackness in his own soul.
The night fell, odorous with grape-scents, and the moon stained the
terraces to amber. It shone on Gordon as he sat by the little wharf
where the skiff rocked in the ripples, his eyes viewless, looking
straight before him across the lake.
For him there was no sanctuary in time or in distance. The passage
he had read at Newstead Abbey in his mother’s open Bible, beside
her body, flashed through his mind: And among these nations shall
thou find no ease, neither shall the sole of thy foot have rest.... In
the morning thou shalt say, Would God it were even! and at even
thou shalt say, Would God it were morning! He had found—should
find—no ease nor rest! The captive of Chillon had been bound only
with fetters of iron to stone pillars. He was chained with fiery links of
hate to the freezing walls of the world’s contumely!
Footsteps went by along the shadowy lane. Shelley’s voice spoke:
“He will come back soon, and we must comfort him if we can.”
The words came distinctly as the footsteps died away.
Something clutched tangibly at Gordon’s soul. In that instant his
gaze, lifted, rested on a white square in the moonlight. It was a
familiar enough object, but now it appeared odd and outré. He rose
and approached it. It had been a sign-post bearing an arrow and the
words “Villa Diodati.” Now malice had painted out the name and
replaced it with new and staring characters.
“Atheist and Fool.” It glared level at him with a baleful malevolence
that chilled the moment’s warmer softening into ice. Atheist! Without
God. What need, then, had he for man? Let the moralists have it so,
since they stickled so lustily for endless brimstone. Fool? He would
be so, then! His brain should lie fallow and untilled—he would write
no more!
With a quick gesture he drew from his pocket his commonplace-
book. He laid it against the disfigured sign-board, pencilled a few
words on its cover and, turning, hurled it far from him into the
shrubbery.
A twig snapped. He looked around. Jane Clermont stood near him,
her eyes smiling into his, fringed with intoxication and daring.
“I know,” she said; “they are hounding you still. They hated me,
too!” She came quite close to him. “What need we care? What are
they all to us?”
It was the Jane of the Drury Lane greenroom he saw now—the Jane
whose brilliance and wit had held him then; but there was
something deeper in her look that he had never seen before: a
recklessness, an invitation and an assent.
“Jane!” he exclaimed.
She touched his hand. “Why should we stay here? Let us go away
from them all—where they cannot follow us to sting!”
Gordon stared at her, his eyes holding hers. To go away—with her?
To slip the leash of all that was pagan in him? What matter? He was
damned anyway—a social Pariah; why strive to undeserve the
reputation? His thought was swirling through savage undercurrents
of vindictive wrath, circling, circling like a Maelstrom, about this one
dead center: Civilization had cast him off. Henceforth his life was his
own, to live to himself, for his own ends, as the savage, as the beast
of the field. To live and to die, knowing that no greater agony than
was meted to him now could await him, even in that nethermost
reach where the lost are driven at the end.
“We must comfort him if we can!” The words Shelley had spoken
seemed to vibrate in the stillness like the caught key of an organ. He
turned to where Villa Diodati above them slept in the long arms of
the night shadows, listening to the contending voices within him.
Comfort? The placid comfort of philosophy for him whose flesh was
fever and his blood quicksilver? In this girl life and action beckoned
to him—life full and abundant—forgetfulness, wandering, and
pleasure, fleeting surely, but still his while it should last! And yet—
The girl’s hand was on the skiff. On a sudden a cry of fear burst from
her lips and she shrank back as a disordered figure broke from the
darkness and clutched Gordon’s arm fiercely.
“Where are you taking her now?”
Gordon’s thought veered. In his numbness of feeling there scarce
seemed strangeness in the apparition. As he looked at the oriental,
mustachioed face, haggard and haunted, his lips rather than his
mind replied:
“Who knows?”
“You lie! You ruined her career and stole her away from London and
from me! Now you want to take her from these last friends of hers—
for yourself! But you cannot go where I will not find you! And where
you go the world shall know you and despise you!”
Jane’s eyes flashed upon the speaker. “You!” she cried in
contemptuous anger. “You hated him even in London; now you have
followed him here. It is you who have set the peasants to spy upon
us! It is you who have spread tales through Geneva! You whose lies
sent the syndic to-day!”
Gordon had been staring at the Moorish, theatric face with a gaze of
singular inquiry, his brain searching, searching for a lost clue. All at
once the haze lightened. His thought leaped across a chasm of time.
He saw a reckless youth, a deserter from the navy, whom he had
befriended in Greece—a youth who had vanished suddenly from
Missolonghi during the feast of Ramazan. He saw a shambling,
cactus-bordered road to the seashore—a file of Turkish soldiers, the
foremost in a purple coat, and carrying a long wand—a beast of
burden bearing a brown sack—
“Trevanion!” he said. “Trevanion—by the Lord!”
He burst into a laugh, reëchoing, sardonic, a laugh now of absolute,
remorseless unconcern, of crude recklessness flaunting at last
supreme over crumbled resolve—the laugh of a zealot flagellant
beneath the lash, a derisive Villon on the scaffold.
“So I stole her from you! You, even you, dare to accuse me. Out of
my sight!” he said, and flung him roughly from the path.
Gordon held out his hand to Jane Clermont, lifted her into the skiff,
and springing in, sent the slim cockle-shell shooting out into the still
expanse like an arrow on the air.
Then he took up the oars and turned its prow down the lake to
where the streaming lights of the careless city wavered through the
mists, pale green under the moonbeams.

The journal which Gordon had hurled from him lay in the vine-rows
next morning when Shelley, with a face of trouble and foreboding,
passed along the dewy lane. He read the words written on its cover:
“And all our yesterdays have lighted fools the way to dusty death. I
will keep no record of that same hesternal torch-light; and to
prevent me from returning, like a dog, to the vomit of memory, I
throw away this volume, and write in Ipecacuanha: Hang up justice!
Let morality go beg! To be sure, I have long despised myself and
man, but I never spat in the face of my species before—‘O fool! I
shall go mad!’”
 CHAPTER XXIV
THE MARK OF THE BEAST

“Your coffee, my lord?”

It was Fletcher’s usual inquiry, repeated night and morning—the
same words that on the Ostend packet had told his master that his
wanderings were shared. After these many months in Venice, where
George Gordon had shut upon his retreat the floodgates of the
world, the old servant’s tone had the same wistful cadence of
solicitude.
Time for Gordon had passed like wreckage running with the tide.
The few fevered weeks of wandering through Switzerland with Jane
Clermont—he scarcely knew where or how they had ended—had left
in his mind only a series of phantom impressions: woods of withered
pine, Alpine glaciers shining like truth, Wengen torrents like tails of
white horses and distant thunder of avalanches, as if God were
pelting the devil down from Heaven with snowballs. And neither the
piping of the shepherd, nor the rumble of the storm; not the torrent,
the mountain, the glacier, the forest or the cloud, had lightened the
darkness of his heart or enabled him to lose his wretched identity in
the Power and the Glory above and beneath him.
In that night at Geneva the tidal wave of execration which had rolled
over his emerging manhood had left as it ebbed only a bare reef
across which blew cool, infuriate winds of avid recklessness; and
through these insensate blasts he moved in a kind of waking
somnambulism, in which his acts seemed to him those of another
individual, and he, the real actor, poised aloft, watching with a
sardonic speculation.
At Rome his numbed senses awakened, and he found himself alone,
and around him his human kind which he hated, spying tourists and
scribblers, who sharpened their scavenger pencils to record his
vagaries. He fled from them to Venice, where, thanks to report,
Fletcher had found his master.
But it was a changed Gordon who had ensconced himself here, a
Gordon to whom social convention had become a sneer, and the
praise or blame of his fellows idle chaff cast in the wind. He ate and
drank and slept—not as other men, but as a gormand and
débauché. Such letters as he wrote—to his sister, to Tom Moore, to
Hobhouse—were flippant mockeries. Rarely was he seen at opera, at
ridotto, at conversazione. When he went abroad it was most often
by night, as though he shunned the daylight. More than one cabaret
in the shadow of the Palace of the Doges knew the white satiric face
that stared out from its terrace over the waterways, where covered
gondolas crept like black spiders, till the clock of St. Mark’s struck
the third hour of the morning. And more than one black and red-
sashed boatman whispered tales of the Palazzo Mocenigo on the
Grand Canal and the “Giovannotto Inglese who spent great sums.”
The gondolieri turned their heads to gaze as they sculled past the
carved gateway. Did not the priests call him “the wicked milord”?
And did not all Venice know of Marianna, the linen-draper’s wife of
the street Spezieria, and of Margarita Cogni, the black-eyed
Fornarina, who came and went as she pleased in the milord’s
household? They themselves had gained many a coin by telling
these tales to the tourists from the milord’s own country, who came
to watch from across the canal with opera-glasses, as if he were a
ravenous beast or a raree-show; who lay in wait at nightfall to see
his gondola pass to the wide outlying lagoon, haunted the sand-spit
of the Lido where he rode horseback, and offered bribes to his
servants to see the bed wherein he slept. They took the tourists’
soldi shamefacedly, however, for they knew other tales, too: how he
had furnished money to send Beppo, the son of the fruit peddler, to
the art school at Naples; how he had given fifty louis d’or to rebuild
the burned shop of the printer of San Samuele.
“Your coffee, my lord?” Fletcher repeated the inquiry, for his master
had not heard.
“No; bring some cognac, Fletcher.”
The valet obeyed, though with covert concern. He had seen the
inroads that year had made; they showed in the lines on the pallid
face, in the brown hair now just flecked with gray, in the increasing
fire in the deep eyes. The brandy sat habitually at his master’s elbow
in these days.
It was two hours past midnight, for to Gordon day and night were
one, and sleep only a neutral inertness, worse with its dreams than
the garish day he dreaded. On the hearth a fire blazed, whose flame
bred crimson marionettes that danced over the noble carved ceiling
panels, the tall Venetian mirrors supported by gilt lions, the faded
furnishings and the mildew-marked canvases whose portraits looked
stonily from the walls.
A gust of voices and the sound of virginals, flung up from the canal,
came faintly through the closed casement. He moved his shoulders
wearily. Yesterday had been Christmas Day. To-night was the eve of
St. Stephen, the opening of the carnival season, with every corner
osteria a symphony of fiddles, when Venice went mad in all her
seventy islands. What were holidays, what was Christmas to him?
Even in the warm blaze Gordon shivered. Ghosts had troubled him
this day. Ghosts that stalked through the confused mist and rose
before him in the throngs that passed and repassed before his
mind’s eye. Ghosts whose diverse countenances resolved
themselves, like phantasmagoria, into a single one—the pained
eager face of Shelley. The recurring sensation had brought a sick
sense of awakening, as of something buried that stirred in its
submerged chrysalis, protesting against the silt settling upon it.
But brandy had lost its power to lay those ghosts. He went to the
desk which held the black phial, the tiny glass comforter to which he
resorted more and more often. Once with its surcease it had brought
a splendor and plenitude of power; of late its relief had been lent at
the price of distorted visions. As he drew out the thin-walled drawer,
its worm-eaten bottom collapsed and its jumble of contents poured
down on the mahogany.
He paused, his hand outstretched. Atop of the mélange lay a silver-
set miniature. He picked it up, holding it nearer the light. A girl’s
face, hued like a hyacinth, looked out of his palm, painted on ivory.
A string of pearls was about her neck.
For an instant he regarded the miniature fixedly, his recollection
travelling far. The pearls aided. It was the one he had found in the
capsized boat at Villa Diodati! He had purposed sending it after the
two strangers. The events of that wild night had effaced the incident
from his mind, as a wet sponge wipes off a slate. Fletcher, finding
the oval long ago in a pocket lining, had put it in the desk-drawer for
safe-keeping, where until this moment it had not met his master’s
eye.
“Teresa.” Gordon suddenly remembered the name perfectly. With the
memory mixed a sardonic reflection: the man who had lost the
miniature that day in Switzerland had hastened away with clothing
scarce dried. Well, if that brother had deemed himself too good to
linger with the outcast, the balance had been squared. The sister,
perforce, had made a longer stay!
He put down the miniature, found the phial of laudanum and
uncorked it, but the face drew him back. It was not the external
similitude now, but something beneath, unobserved the day he had
found it—the pure sensibility, shining unsullied through the
transparent media. A delicate convent slip, she seemed, not yet
transplanted to the unsifted soil of the world! A strange portrait for
him to gaze upon here in this palace of ribaldry—him, the moral
Caliban, the dweller in Golgotha on whose forehead was written the
hic jacet of a dead soul!
The antithesis of the picture, bold, Medea-like, tall as a Pythoness,
with hair of night, black flashing eyes and passion blent with ferocity,
projected itself, like a materialization in a séance, from the air. He
turned his head with a sensation of bodily presence, though he knew
the one of whom he thought was then in Naples. If she should enter
and find him with that ivory in his hand, what a rare sirocco would
be let loose!
He tried to smile, but the old arrant raillery would not come. The
miniature blotted out the figure of the Fornarina. Against his will, it
suggested all the pure things that he had ever known—his youthful
romance, his dreams, Ada, his child!
Holding it, he walked to a folded mirror in a corner of the wall and
opened its panels. There had been a time when he had said no
appetite should ever rule him; the face he saw reflected now wore
the lines of incorrigible self-indulgence, animalism, the sinister badge
of the bacchanal.
“Is that you, George Gordon?” he asked.
“IS THAT YOU, GEORGE GORDON?” HE ASKED. p. 178.
The ghosts drew nearer. They peered over his shoulders. He felt
their fingers grasping at him. He cursed them. By what right did they
follow him? By what damnable chance, ruled by what infernal
jugglery, came this painted semblance to open old tombs?
Something had awakened in him—it was the side that recollected,
remorseless and impenitent but no longer benumbed, writhing with
smarting vitality. Awake, it recoiled abashed from the voiceless vade
retro of that symbol. What part had he in that purity whose visible
emblem mocked and derided him? What comradeship did life hold
for him save the hideous Gorgon of memory, the Cerberus of ill
fame, spirits of the dark, garish fellows of the half-world—“they
whose steps go down to hell!”
A fury, demoniac, terrible, fell on him. He seized the miniature,
dashed it on the floor, stamped it with his heel and crushed and
ground it into indistinguishable fragments.
Then he sprang up, and with an oath whose note was echoed by the
tame raven croaking on the landing, rushed down the stairway and
threw himself into his gondola.
The moon rose red as a house afire. Before it paled, he had passed
the lagoon. In the dim light that presaged the sodden dawn he
leaped ashore on the mainland, pierced the damp laurel thickets that
skirted the river Brenta and plunged into the forest.
 CHAPTER XXV
TERESA MEETS A STRANGER

Through the twittering dawn, with its multitudinous damp scents, its
stubble-fields of maize glimpsed through the stripped ilex trees,
whose twigs scrawled black hieroglyphics on the hueless sky, Gordon
strode sharply, heedless of direction.
The convulsion of rage with which he had destroyed the miniature
had finished the work the latter’s advent had begun. The nerve,
stirring from its opiate sleep to a consciousness of dull pain, had
jarred itself to agony. His mind was awake, but the wind had swept
saltly through the coverts of his passion, and their denizens
crouched shivering.
The sight of a dove-tinted villa guarded by cypress spears—a gray
gathering of cupolas—told him he had walked about two miles. This
was La Mira, one of the estates of the Contessa Albrizzi, a great
name in Venice. He turned aside into the deserted olive grove above
the river. A slim walk meandered here, thick with dead leaves, with a
cleared slope stretching down to where the deep-dyed Brenta
twisted like a drenched ribbon on its way to the salt marshes.
Fronting this breach, Gordon came abruptly upon a wooden shrine,
with a weather-fretted prayer bench.
He stopped, regarding it half-absently, his surcharged thought
rearranging disused images out of some dusty speculative
storehouse. A more magnificent shrine rose on every campo of
Venice. They stood for a priestly hierarchy, an elaborate clericalism—
the mullioned worship that to his life seemed only the variform
expression of the futile earth-want, the satiric hallucination of finite
and mortal brain that grasped at immortality and the infinite. This,
set in the isolation of the place, seemed a symbol of more primitive
faith and prayer, of religion rough-hewn, shorn of its formal
accessories.
He went a step nearer, seeing a small book lying beside the prayer
bench. He picked it up. It was a reprint in English of his own
“Prisoner of Chillon,” from a local press in Padua.
A sense of incongruity smote him. It was the poem he had
composed in Geneva. He readily surmised that it was through
Shelley the verses had reached his publisher in England, to meet his
eye a year afterward, in a foreign dress, in an Italian forest.
He turned the pages curiously, conning the scarce remembered
stanzas. Could he himself have created them? The instant wonder
passed, blotted out by lines he saw penned in Italian on the fly-leaf
—lines that he read with a tightening at his heart and an electric-like
rush of strange sensations such as he had never felt. For what was
written there, in the delicate tracery of a feminine hand, and in
phrases simple and pure as only the secret heart of a girl could have
framed them, was a prayer:
“Oh, my God! Graciously hear me. I take encouragement
from the assurance of Thy word to pray to Thee in behalf
of the author of this book which has so pleased me. Thou
desirest not the death of a sinner—save, therefore, him
whom Venice calls ‘the wicked milord.’ Thou who by sin art
offended and by penance satisfied, give to him the desire
to return to the good and to glorify the talents Thou hast
so richly bestowed upon him. And grant that the
punishment his evil behavior has already brought him be
more than sufficient to cover his guilt from Thine eyes.
“Oh blessed Virgin, Queen of the most holy Rosary!
Intercede and obtain for me of thy Son our Lord this
grace! Amen.”
A step fell behind him. He turned half-dazed, his mind full of conflict.
A girl stood near him, delicate and alert and wand-like as a golden
willow, her curling amber hair loosely caught, her sea-blue eyes wide
and a little startled. She wore a Venetian hood, out of whose green
sheath her face looked, like lilies under leaves.
Gordon’s mind came back to the present of time and space across
an illimitable distance.
He stared, half believing himself in some automatic hallucination.
There had been no time to speculate upon what manner of hand
had written those words, what manner of woman’s soul had so
weirdly touched his own out of the void. Knowledge came
staggeringly. Hers was the face of the miniature that his heel had
crushed to powder.
He noted that her eyes had fallen to the book in his hand, as
mechanically he asked, in Italian:
“This book is yours, Signorina?”
“Yes.” There was a faint flush of color in her cheek, for she saw the
volume was open at the written page.
Gordon was looking at her palely, seeing her face set in a silver oval.
Eyes, hair and lips; there in lifeless pigments, here in flesh and
blood! The same yet more, for here were unnunned youth,
slumbrous, glorious womanhood unawaked, stirring rosily in every
vein, giving a passionate human tint to the spiritual impression. And
underneath all, the same unsullied something he had raged at that
black night, even while her prayer for him lay here dumb at the feet
of Our Lady of Sorrows!
His voice sounded unreal to his own ears as he spoke, his mind
feeling its way through tumbled predispositions to an unfamiliar
goal. “If apology be owed,” he said, “for reading what was intended
for purer eyes than this world’s, I most humbly offer it, Signorina! I
did so quite inadvertently.”
He held out the book as he spoke, and her fingers closed over it, the
gesture betraying confusion. Who was this stranger, with face of
such wan luster and gray-blue eyes so sadly brilliant? Some sense in
her discerned a deeper, unguessed suffering that made her heart
throb painfully.
“If there be an ear which is open to human appeal,” he added
gently, “that prayer was registered, I know!”
He spoke calmly enough, but a hundred thoughts were ricochetting
through his mind. Pulpits had fulminated against him, priest and laic
had thundered him down, but when—in London, in Geneva, in
Venice—had a single disinterested voice been lifted in a prayer for
him before? And this girl had never seen him.
“If there be!” Her thought stirred protestingly. “Ah, Signore, surely
there is Some one who hears! How could one live and pray
otherwise?”
How indeed? To such a one as she, to pray and to live were one and
the same thing. Prayer to her was not a mental process—it was as
instinctive and unconscious as breathing. For such as she, shrines
like this were erected; not for him! So, across the riot in his breast,
Gordon’s waked habit paused to smile—a satire-smile, at itself, at the
new sweet flower that was lifting head there amidst desert ruins.
The girl caught the mixed feeling in his face. He was not Italian—his
accent had told her that. He was an Englishman, too, perhaps. “Do
you know him, Signore?”
His head turned quickly toward her. In truth, had he ever really
known himself? “Yes,” he answered after a pause. “I know him,
Signorina—far better than most of the world.”
She was gazing with varied feelings, her heart beating strangely,
curiosity and wonder merging. In her few short weeks at La Mira,
fresh from the convent, the Englishman of whom all Venice told tales
had been but a dim and unsubstantial figure. She had thought of the
grim Palazzo Mocenigo with a kind of awe, as a child regards a
mysterious cavern bat-haunted and shunned. Into her poetic world
of dreams had fallen the little book, and thereafter the shadowy
figure that roamed nightly Venice had taken on the brilliant and
piteous outline of a fallen angel. Here, wonderfully, was a man who
knew him, whose speech could visualize the figure that had grown
to possess such fascination. Questions were on her tongue, but she
could not frame them. She hesitated, opening and closing the book
in her hands.
“Is he all they say of him?”
“Who knows, Signorina?”
It was an involuntary exclamation that sounded like acquiescence.
The girl’s face fell. In her thought, the man of her dreaming, lacking
an open advocate, had gained the secret one of sympathy. Was it all
true then? Her voice faltered a little.
“I have not believed, Signore, that with a heart all evil one could
write—so!”
Into the raw blend of tangent emotions which were enwrapping
Gordon, had entered, as she spoke, another well-defined. Never in
his life, for his own sake, had he cared whether one or many
believed truth or lie of him. But now there thrilled in him, new-born,
a desire that this slight girl should not judge him as did the world.
The feeling lent his words a curious energy:
“Many tales are told, Signorina, that are true—some that are false. If
he were here—and I speak from certain knowledge of him—he
would not wish me to extenuate; least of all to you who have written
what is on that leaf. Perhaps that has been one of his faults, that he
has never justified himself. By common report he has committed all
crimes, Signorina. He has thought it useless to deny, since slander is
not guilt, nor is denial innocence, and since neither good nor bad
report could lighten or add to his wretchedness.”
The tint of her clear eyes deepened. “I knew he was wretched,
Signore! It was for that reason I left the prayer here overnight
before Our Lady of Sorrows—because I have heard he is an outcast
from his own country and his own people. And then, because of
this.” She touched the volume. “Ah, I have read little of all he has
written—this is the only poem—for I read his English tongue so
poorly; but in this his heart speaks, Signore. It speaks of pain and
suffering and bondage. It was not only the long-ago prisoner he
sang of; it was himself! himself! I felt it—here, like a hurt.”
She had spoken rapidly, stumblingly, and ended with a hand pressed
on her heart. Her own feeling, as she suddenly became aware of her
vehemence, startled her, and she half turned away, her lips
trembling.
A sentiment at variance with his whole character was fighting in
Gordon. The Babel he had builded of curses was being smitten into
confusion. Something granite-like, mural and sealed by time, was
breaking and melting unaccountably away. His face was turned from
hers—toward the slope below, where the river bubbled and sparkled.
When he spoke it was in words choked and impeded:
“I think if he were here—this wicked milord—he would bless you for
that, Signorina. He has suffered, no doubt. Perhaps if there had
been more who felt what he wrote—as you have felt,—if there had
been more to impute good of him rather than evil—I am quite sure if
this could have been, Signorina, he would not now be in Venice the
man for whom you have written that prayer. I know him well enough
to say this. It is through his wretchedness that I have come to know
him—because, like him, I am a wanderer.”
A softer light suffused her cheek. The words smote her strangely. His
pain-engraved face brought a mist to her eyes. She was a child of
the sun, with blood leaping to quick response, and a heart a well of
undiscovered impulses. The wicked milord’s form lost distinction and
faded. Here was a being mysterious, wretched, too, and alone—not
intangible as was he of the Palazzo Mocenigo, but beside her,
speaking with a voice which thrilled every nerve of her sensitive
nature. Unconsciously she drew closer to him.
At that moment a call came under the bare boughs: “Teresa!
Teresa!”
She drew back. “It is la Contessa,” she said; “I must hasten,” and
started quickly through the trees.
His voice overtook her. “Signorina!” The word vibrated. “Will you give
me the prayer?” He had come toward her as she stopped. “There is
a charm in such things, perhaps.”
The voice called again, and more impatiently: “Teresa!”
She opened the book and tore out the leaf with uncertain fingers. As
he took it his hand met hers. He bent his head and touched it with
his lips. She flushed deeply, then turned and ran through the naked
trees toward the villa shielded in its cypress rows.
The girl ran breathlessly to the terrace, where a lady leaned from a
window with a gently chiding tongue:
“Do they teach you to do wholly without sleep in convents?” she
cried. “Do you not know your father and Count Guiccioli, your lord
and master to be, are to arrive to-day from Ravenna? You will be
wilted before the evening.”
The girl entered the house.
Under the olive wood a man, strangely moved, a rustling paper still
in his hand, walked back with quick strides to his gondola, striving to
exorcise a chuckling fiend within him, who, with mocking and
malignant emphasis, kept repeating:
“Oh blessed Virgin, Queen of the most holy Rosary! Intercede and
obtain for me, of Thy Son our Lord, this grace!”
 CHAPTER XXVI
A WOMAN OF FIRE AND DREAMS

From the moment those lips touched her hand in that meeting at the
wood shrine Teresa Gamba felt her life unfold to rose-veined visions.
Her unmothered childhood and the placid convent school years at
Bagnacavallo, near Ravenna, had known no mystery other than her
day-dreams had fashioned. She had dreamed much: of the time
when she should marry and redeem the fortunes of her house,
which, despite untainted blood and ancient provincial name, was
impoverished; of the freedom of Italy, the sole topic, aside from his
endless chemical experiments, of which her father, now growing
feeble, never tired; of her elder brother, away in Wallachia, secretary
to the Greek Prince Mavrocordato; of the few books she read, and
the fewer people she met. But these dreams had not possessed the
charm of novelty. Even when, at eighteen, through family friendship,
she became a member of the Albrizzi household and exchanged the
dull convent walls for the garlanded La Mira—even with those rare
days when she saw the gay splendor of Venice from a curtained
gondola—even then her mental life suffered small change.
The marriage arranged for her with Count Guiccioli, the oldest and
richest nobleman of Ravenna, a miser and twice a widower, had
aroused an interest in her mind scarce greater than had the tales of
the Englishman of the Palazzo Mocenigo. Such marriages were of
common occurrence in the life she knew: the “wicked milord” was a
stranger thing—one to speculate more endlessly upon.
It was Tita, the gigantic black-bearded gondolier and door-porter, a
servant in the Gamba family since she was born, whom she had
brought with her as her own attendant—one who worshiped her
devoutly, and in whose care her father entrusted her more
confidently than to any duenna—who had first pointed out to her the
gloomy building which shielded that mysterious occupant, and had
piqued her interest with weird tales: how in his loneliness for human
kind the outcast surrounded himself with tamed ravens and
paroquets, and used for a wine cup a human skull, that of a woman
he had once loved. With her rapt eyes on the palazzo front, Teresa
had wondered and shuddered in never ending surmise.
The little volume from the Paduan press had deepened her curiosity
and given it virgin fields in which to wander. The English books in
her father’s library were prose and for the most part concerned his
pet hobby, chemistry. This volume, given her on a saint’s day by the
Contessa Albrizzi, who took pride in her protégée’s scholarship, was
her first glimpse of English poetry, and her pulses had leaped at the
new charm. Thereafter the personality of the contradictory being
who had written it had lived in her daily thought. She retained the
faiths of her childhood unshattered, and the prayer she had left at
the shrine of Our Lady of Sorrows sprang from an impulse as natural
as it was significant.
But that meeting in the wood had turned the course of her
imaginings. “A wanderer—like him”; the words had bridged the
chasm between the dreaming and the real. The secret thought given
to the “wicked milord” found itself absorbed by a nearer object. The
palazzo on the Grand Canal grew more remote, and the stranger she
had seen stepped at a single stride into a place her mind had
already prepared.
The blush with which she had taken the book from Gordon’s hand
was one of mere self-consciousness; the vivid, burning color which
overspread her face as she ran back through the trees was
something very different. It was a part of her throbbing heart, of the
tremulous confusion that overran her whole body, called into life by
the touch of those palely carved lips upon her fingers. His colorless
face—a face with the outline of the Apollo Belvedere—the gray
magnetic eyes, the words he had said and their accent of sadness,
all were full of suggestive mystery. Why was he a wanderer—like
that other? Not for a kindred reason, surely! He could not be evil
also! Rather it must have been because of some loss, some hurt of
love which time might remedy.
Her agile fancy constructed more than one hypothesis, spun more
than one romance, all of like ending. A new love would heal his
heart. Sometime he would look into a woman’s eyes—not as he had
looked into hers; some one would feel his lips—not as he had kissed
her hand. She in the meantime would be no longer a girl; she would
be the Contessa Guiccioli, with a palazzo of her own in Ravenna, and
—a husband.
But, somehow, this reflection brought no satisfaction. The old count
she had seen more than once driving by in state when she played as
a child in the convent woods; and that he with his riches should
desire her, had given her father great pride, which was reflected in
her. Her suitor had brought his age and ailments to La Mira on the
very day she had met the stranger at the shrine—the day her heart
had beat so oddly—and with his arrival, her marriage had projected
itself out of the hazy future and become a dire thing of the present.
She felt a fresh distaste of his sharp yellow eyes, his cracked laugh.
His eighty wiry years seemed as many centuries. She became
moody, put her father off and took refuge in whims. The contessa
advised the city, and the week’s end saw the Albrizzi palazzo thrown
open.
In Venice, Teresa’s spirits rose. She loved to watch the bright little
shops opening like morning-glories, the sky-faring pigeons a silver
quiver of wings; to lie in the gondola waiting while her father drank
his brandy at the piazzetta caffè; to buy figs from little lame
Pasquale, who watched for her at a shop-door in a narrow calle near
at hand; to see the gaudy flotillas of the carnival, and the wedding
processions, fresh from the church, crossing the lagoon to leave
their gifts at the various island-convents; or, propelled by Tita’s
swinging oar, to glide slowly in the purpling sunset shadows, by the
Piazza San Marco, around red-towered San Giorgio, and so home
again on color-soaked canals in the gleaming ruby of the afterglow,
through a city bubbling with ivory domes and glistening like an opal’s
heart under its tiara of towers.
She scarcely told the secret to her own heart—that it was one face
she looked to see, one mysterious stranger whose image haunted
every campo, every balcony and every bridge. She flushed whenever
she thought of that kiss on her fingers; in the daytime she felt it
there like a sentient thing; at night when she woke, her hand burned
her cheek.
Who was he? Why had he asked her for the prayer? What had he
done with it? Was he still in Venice? Should she see him again? She
wondered, as, parting the gondola tenda, she watched her father
cross the pave for his cognac.
“Are there many English in Venice, Tita?”
The gondolier, lounging like a brilliant-hued lizard, shrugged his
shoulders. “Bellissima, there are hundreds in the season. They come
and go. They are all lasagnoni, these Englishmen!”
Teresa’s sigh checked itself. Tita suddenly turned his head. Across
the piazzetta a crowd was gathering. It centered before the shop at
whose front the five-year-old fig-seller was used to watch for her.
“He fell from the scaffolding!” said a voice.
“If it should be little Pasquale!” cried Teresa, and springing out, ran
quickly forward. Tita waited to secure the gondola before he
followed her.
A sad accident had happened. Before the calle a platform had been
erected from which spectators might watch the flotillas of the
carnival. Little Pasquale’s delight was a tame sparrow, whose home
was a wicker cage, and climbing to sun his pet when he had been
left to tend the empty shop, the child had slipped and fallen to the
pavement.
Teresa broke through the circle of bystanders and knelt by the
tumbled little body, looking at the tiny face now so waxen. The
neighbors thronged about, stupefied and hindering. A woman ran to
fetch the mother, gossiping with a neighbor. Another called loudly for
a priest.
The girl, looking up, was bewildered by the tumult. “He must be got
in,” she murmured, half helplessly, for the people ringed them round.
A voice answered close beside her: “I will carry him, Signorina”—and
a form she knew bent beside her, and very gently lifted the small
bundle in his arms.
Teresa’s heart bounded. Through these days she had longed to hear
that voice again how vainly! Now, in this moment, she was brought
suddenly close to him. She ceased to hear the sounds about her—
saw only him. She sprang up and led the way through the press,
down the close damp calle and to the shop where the child lived.
“Dog of the Virgin! He need touch no finger to child of mine!” swore
a carpenter from the adjoining campo.
“Nor mine!”
“Why didn’t you carry him in yourself, then?” growled Giuseppe, the
fruit-vender. “Standing there like a bronze pig! What have you
against the Englishman? Didn’t he buy your brother-in-law a new
gondola when the piling smashed it?”
“Scellerato!” sneered the carpenter. “Why is his face so white? Like a
potato sprout in a cellar! He is so evil he fears the sun!”
The fruit-vender turned away disdainfully. His foot kicked a shapeless
wicker object—it was little Pasquale’s cage smashed flat. The
sparrow inside was gasping. He picked up the cage and carried it to
the shop.
In the inner, ill-lighted room, Gordon laid the child on a couch. He
had spoken no further word to Teresa. At the first sight of her,
kneeling in the street, he had started visibly as he had done in the
forest of La Mira when he recognized her face as that of the
miniature. Now he was feeling her presence beside him with a
curious thrill not unlike her own—a pleasure deeply mixed with pain
that was almost a physical pang.
Since that dawn walk above the plane-treed Brenta he had been
treading strange ways. In the hours that followed, remorse had been
born in him. And as the first indrawn breath racks the half-drowned
body with agony greater than that of the death it has already tasted,
so the man had suffered. During a fortnight, words written on a
sheet of paper that he carried in his pocket had rung through his
brain. Day after day, as he sat in his gloomy palazzo, he had heard
them; night after night they had floated with him as his gondola
bore him through the waterways ringing with the estro of the
carnival. To escape them he had fled again and again to the black
phial, but when he awoke the pain was still with him, instinct and
unrenounceable. It was more acute at this moment than it had ever
been.
Teresa scarcely noted the fruit-vender as he put the battered cage
into her hand just before its feathered occupant breathed its last.
Her look, fixed on Gordon, was still eloquent with the surprise. She
saw the same pale face, the same deep eyes, the same chiselled
curve of lips. His voice, too, as he despatched the kind-hearted
Giuseppe for a surgeon on the Riva, had the same cadence of
sadness. She had noticed that his step halted as he walked, as
though from weakness. And surely there was illness in his face, too!
Had there been any tender hands near him—as tender as those with
which he now examined the unconscious child?
As Gordon bent above him, little Pasquale opened his eyes. His gaze
fell first not on the man or on Teresa, but on the broken cage beside
him, where the bird lay still, one claw standing stiffly upright. He
tried to lift his head, and called the sparrow’s name.
There was no answering chirp. The claw was very still.
Then little Pasquale saw the faces about him and knew what had
happened.
“He’s dead!” he shrilled, and burst into tears.
 CHAPTER XXVII
THE EVIL EYE

Tears, too, had rushed to Teresa’s eyes, with a sweet, glad sense of
something not akin to grief. Her hand on the couch in the semi-
darkness touched another and she drew it away, trembling.
Suddenly a wail came from the calle, a hurried step crossed the shop
floor, and the slattern mother burst into the room. Close behind
followed Tita, who, seeing his mistress, blocked the inner door with
his huge frame against the curious, with whom the place now
overflowed.
The weeping woman had thrown herself beside the couch where the
child lay, his eyes closed again. All at once she saw the man who
stood above her, and to Teresa’s astonishment sprang up and spat
out coarse imprecations.
“The evil eye!” she screamed. “Take the Inglese away and fetch
some holy water! He has the evil eye!”
Teresa saw the spasm of pain that crossed the colorless face. “No,
no!” she cried.
“What did I say!” sneered the carpenter.
Tita’s great hand took him by the throat. “Silence, devout jellyfish!”
he said, “or I crack your skull. Didn’t you hear the signorina?”
“The evil eye!” wailed the woman, flinging back inky hair from her
brows. “He looked at the heart-of-my-life or he would not have
fallen!”
“For shame!” protested Teresa indignantly. “He who carried him in
his own arms! Ah, do not listen!” She turned to Gordon appealingly.
“She is mad to say such things! Let us go,” she added hastily, as
murmurs swelled from the shop. “We can do no more!”
“Go, son of the Black One!” screamed the woman. “Go before my
child dies!”
Gordon had distinguished in the girl’s voice a note of pity and of fear
for his safety, and a flash of smile softened the bitterness of his lips.
“You are right, Signorina,” he answered, and preceded her. The
people parted as they passed, some peering maliciously, some
shamefaced. Tita, bringing up the rear, glared about him, his fist
clenched like a hammer. He knew well enough who the stranger
was, but his signorina walked with him and that was sufficient. Tita
knew what was expected of him.
It was growing dusky as they emerged. The group before the shop
had run to watch the great surgeon alighting at the water-stairs. The
dozen steps that brought them to the open piazzetta they walked in
silence.
There Teresa paused, wishing to say she knew not what, burning
with sympathy, yet timid with confusion. The street seemed to wear
an unwonted, un-everyday luster, yet she knew that around the
corner lay little Pasquale woefully hurt, in full view Tita was
unlashing the gondola, and across the piazzetta she could see the
entrance of the caffè where her father was sipping his cognac. A
fear lest the latter should appear and find her absent from the
gondola mixed with the wave of feeling with which she held out her
hand to the man beside her.
“Poor little fig-merchant!” she said—the scene with the mother was
too painfully recent to touch upon at once. “He watched for my
gondola every day. I hope he is not badly hurt. What do you think,
Signore?”
“No bones were broken,” he rejoined. “But as to internal injury, I
could not tell. I shall hope doubly for him,” he added, “since you love
him.”