Biochem. Cell. Arch. Vol. 15, No. 1, pp.

259-264, 2015 ISSN 0972-5075

DETERMINATION OF FEEDING STIMULANTS IN SHRIMP USING A SOLID

MATRIX BIOASSAY
Cheryl Antony1, P S B R James2 and B. Sundaramoorthy3
1
Professor, Institute of Fisheries Technology, Tamil Nadu Fisheries University, Ponneri-601 204.Thiruvallur, India.
2
Director (Retd.) Central Marine Fisheries Research Institute, ICAR, Kochi, India.
3
Professor, Fisheries College and Research Institute, Thoothukkudi, India.
(Accepted 23 February 2015)

ABSTRACT : Chemoreception plays an important role in the processes of feeding, mating, and defense against predators.
Studies have identified amino acid complexes and marine-origin compounds as effective attractants that can be included in the
feed of farmed marine shrimp. Marine shrimp have developed mechanisms to compensate for their poor visual sense, by using
chemoreceptors located in both locomotion appendices and buccal parts, as effective tactile, gustatory and olfactory senses. A
nutritionally adequate and organoleptically pleasing diet is essential to achieve satisfactory intake and growth in shrimps.To
improve the palatability of diets, various substances have been investigated for their effectiveness in aqua-feed including natural
feed ingredients and synthetic flavor substances. Diet palatability and attractiveness would help to reduce the time that shrimp
spend approaching the feed and it would limit nutrient leaching and feed loss which in turn reduce deterioration of rearing
pondenvironments from overloaded nutrient input. A feeding bioassay system which uses agar discs was used for evaluating
chemosensory stimuli influencing ingestive behaviour in Fenneropenaeus indicus and in Metapenaeus dobsonii.Various
extract fractions were assayed to determine the tissue component that is responsible for eliciting ingestion activity in the
shrimps.
Key words : Chemoreception, feeding response, extract fractions, tissue components, Fenneropenaues indicus, Metapenaues
dobsoni, bioassay system, agar matrix bioassay, test stimuli, ingestion.

INTRODUCTION an optimal feeding regime.

Careful attention of the basic crustacean behavioural l rapid and positive uptake lessens residual time in
patterns is critical in order to provide an accurate system the aqueous system.
for evaluation of single and mixed chemical signals by l study the factors that influence feeding behaviour
the crustacean, resulting in more efficient utilization of and response of cultured animals to a particular
the feed. The shrimp’s feeding habits change from diet like water quality and nature of the test diet.
continuous to nocturnal as size increases. Growth rates
A feeding bioassay system which uses agar discs
in shrimp pond can be improved by decreasing feeding
was developed by Holland and Borski (1993) for
response time. A shortened response time means nutrients
evaluating chemosensory stimuli influencing ingestive
will have less opportunity to leach from the food pellet.
behaviour in Penaeus vannamei. This system was found
In addition, less energy is expended by the shrimp in
suitable for rapid screening of a wide variety of
search of food.
compounds. The agar matrix bioassay served as a cheap
Attractiveness and palatability are critical factors in and efficient method to screen a wide variety of
formulation of aquatic diets, especially in weaning of attractants and stimulants.
young ones from natural foods to dry pelleted diets. The
1. Standard experimental conditions followed in
need for attractants and stimulants were best studied,
the trial
l By identification of feeding stimuli often needed
1. Shrimps were acclimated to the laboratory
to obtain the full sequence of feeding behaviour.
condition at least for 5 days, before the trial.
l response varies among aquatic species and is
2. Animals were deprived of food for 24 hrs prior
related to different chemosensory sites and their
to the trial.
interaction with a specific chemical or group of
chemicals. 3. Observation times were standardised occurring
before 10.00 hrs and after 17.00 hrs.
l understanding the feeding behaviour of the
aquaculture species necessary in order to obtain 4. Uniform water flow was maintained throughout
260 Cheryl Antony et al
the experimental tank. pieces from the agar disc and pass them into the mouth
5. Aeration was not provided during the trial. directly for ingestion, avoiding wastage. The positions of
the disc were randomized for every test. The discs were
6. Stimulus was assigned to the centre of the
weighed before and after the test period to determine
experimental chamber.
the consumption and preference. A set of control discs
7. Rearing of shrimps and study was conducted at were also placed in the water without the animal for four
20 ppt salinity. hours to determine the weight gain or loss due to hydration
8. Animals used for the study were healthy with or dehydration of the disc. The blank agar disc prepared
complete appendages. without stimulus served as the reference.
9. Test was declared negative if no response Test Stimuli : 1) Whole extracts (at 0.5, 1.0, 1.5,
occurred within 10 minutes. 2.0 and 2.5% levels of 15 tissue sources. 2) Whole
10. Experimental tank was drained, and cleaned extract, protein free extract, lipid free extract, free amino
after each trial. acid fraction, synthetic aminoacid prepared at the same
11. Animals once used for a test were discarded and level as that of the free amino acid composition of the
for every new trial, fresh sets of animals were selected sources (Table 1) 3) Amino acids.
used. RESULTS
METHOD OF EVALUATION Ingestive property of natural and chemical stimuli
Palatability bioassay Tissue extracts were assayed for their feeding
Shrimps of 5.0 - 6.0 g were used for the solid matrix stimulus activity in shrimps, the results of which are
ingestion bioassay. They were maintained and tested presented in Tables 2 and 3. Unflavoured agar gel elicits
individually in glass aquaria of 10 l capacity. To prevent little feeding activity by itself, while agar flavoured with
any erroneous preconditioning to a particular flavour, the tissue extracts had higher palatability as indicated by the
shrimp were fed in excess with diets containing the test higher consumption rates in both the species. Agar
materials used in the bioassay and the tanks were consumption varied significantly for different tissue types
siphoned clean before the experiment. (P<0.0l) indicating variation in their stimulatory efficacy.
F. indicus and M. dobsoni varied in their affinity to
The standard procedure of the bioassay was to
different test samples. In all trials, F. indicus preferred
present the test stimulus to the shrimp in an agar disc as
agar gel flavoured with squid extract followed by crab,
described by Adams and Johnsen (1986). The blank agar
clam, oyster and shrimp extracts. M. dobsoni preferred
disc elicits little feeding activity by itself as demonstrated
agar gel flavoured with squid, shrimp, clam, crab and oyster
by very poor consumption in two-choice blank vs. blank
extracts in the order of preference. F. indicus showed
feeding tests. To reduce any possible effects of previous
least preference to gel flavoured with seaweed extract
trials, tests were always conducted as two -choice
and M. dobsoni to the gel flavoured with earthworm
experiments which allow direct comparison of
extract. In both the species, agar gel consumption
consumption and preference between the two discs. To
increased significantly with the test sample concentration
maximise the amounts ingested and for sufficient hardness
(P<0.05).Incorporation of squid ink in the gel decreased
so as to withstand the handling by shrimps 2.0% (w/v)
its palatability. The agar consumption decreased with the
agar was used for the block. The stimulus prepared was
increase in the concentration of squid ink in the gel and
added to melted agar at 55-60°C temperature and the
at higher concentration, the gel was totally avoided.
agar master block cast into a petridish in which 3.5 cm.
long plastic rods with a base were suspended. After the Various extract fractions were assayed to determine
agar gets solidified they were cut into 3 cm dia. and 1.5 the tissue component that is responsible for eliciting
cm thick discs fixedwith the plastic rod. These rods were ingestion activity in shrimps. Details of agar consumption
fixed to a mounting PVC pipe and this assembly lowered flavoured with different extract fractions and their
carefully into the experimental tank and fixed with a clamp relative potencies in Table 4. The gel flavoured with the
and allowed to be fed for 4 hours. The agar discs were whole extract showed the same pattern of preference as
placed in a corner against the floor and wall of the aquaria stated above. But the preference towards extract
so that the shrimp’s rostrum prevented it from getting its fractions varied from that of whole extracts in both
mouthparts directly onto the discs, which would result in species. Maximum agar consumption occurred when
rendering the amounts actually consumed immeasurable. flavoured with whole extracts.
In this case the shrimps used only the dactyls to pick off Removal of soluble protein from the extract led to a
 Determination of feeding stimulants in shrimp using a solid matrix bioassay 261
Table 1 : Percentage composting of free amino acids in different tissue extracts.
Amino acids Crab Shrimp Squid Clam Fish Oyster Mussel
Alanine 7.304 11.250 7.589 8.525 8.870 13.300 8.380
Arginine 7.817 4.680 6.324 6.164 0.940 1.130 3.830
Asparagine 0.668 0.531 - - 1.140 0.420 -
Aspartic acid 0.126 0.440 0.506 1.377 1.140 1.780 2.590
Cystine 0.411 - - 0.328 - 0.240 0.410
Glutamic Acid 0.970 0.876 1.467 6.754 1.410 1.830 8.690
Glutamine 10.100 3.343 - - 1.710 1.400 -
Glycine 38.804 49.721 24.790 21.574 10.240 8.700 6.620
Histidine 0.474 0.170 0.430 0.590 22.57 0.182 1.140
Isoleueine 0.365 0.562 0.784 0.721 0.470 0.073 0.830
Leucine 0.839 1.008 1.594 1.311 0.870 0.150 1.340
Lysine 0.776 0.260 0.810 1.639 3.960 0.600 5.170
Methionine 1.341 0.551 0.987 0.721 0.240 0.040 1.650
Phenylalanine 0.320 0.287 - 1.311 0.270 0.018 0.830
Proline 19.06 6.049 40.730 1.049 1.580 4.450 2.280
Serine 0.907 0.711 0.936 1.574 1.650 1.200 2.480
Taurine 6.448 17.510 9.36 43.54 38.280 60.130 47.160
Threonine 1.381 0.340 1.240 0.852 1.610 0.200 2.690
Tryptophan 0.451 - - - - - -
Tryrosine 0.348 0.488 0.632 1.049 0.130 0.090 1.240
Valine 1.090 1.220 1.037 0.918 0.940 0.150 2.690
Ornithine - - - - 0.910 0.350 -
Quartenary ammonium compound
Betaine 48.995 45.280 42.540 67.900 51.240 91.070 97.090

Table 2 : Agar gel consumption (g/100 g. body weight of shrimp) by Fenneropenaeus indicus in a two choice feeding preference test.
Stimulus concentration (% W/V)
Test stimulus 0 0.5 1.0 1.5 2.0 2.5
References
Shark 0.321 0.032 0.336 0.396 0.529 0.788
Mussel 0.035 0.233 0.438 0.521 0.645 0.817
Cuttle Fish 0.032 0.268 0.373 0.483 0.612 0.825
Squila 0.031 0.126 0.337 0.473 0.613 0.753
Fish 0.035 0.218 0.402 0.495 0.537 0.794
Crab 0.034 0.340 0.426 0.605 0.739 0.988
Shrimp 0.036 0.266 0.440 0.505 0.696 0.842
Black Clam 0.032 0.298 0.422 0.639 0.718 0.948
Squid 0.034 0.336 0.439 0.607 0.779 0.998
Earthworm 0.032 0.169 0.219 0.393 0.448 0.680
Mysid 0.033 0.149 0.313 0.424 0.565 0.603
White Clam 0.032 0.128 0.352 0.467 0.545 0.784
Seaweed 0.034 0.102 0.263 0.310 0.465 0.564
Oyster 0.036 0.274 0.418 0.593 0.712 0.883
Squid (with ink sac) 0.030 0.180 0.116 0.076 0.037 0.023

much higher reduction in agar consumption and higher When the agar gel was flavoured with free amino
loss in potency than the lipid free fraction. In F. indicus, acid fraction (FAA) alone, the potency came down to
minimum loss in potency was recorded for protein free 47.5 to 68.6% in F. indicus and 45.5 to 57.2% in M.
fraction of crab extract and maximum for the clam extract, dobsoni for various extract types. In both the species
whereas, in M. dobsoni it was for protein free fraction maximum consumption occurred when the gel was
of shrimp and squilla extracts respectively. flavoured with free amino acid fraction of crab, followed
262 Cheryl Antony et al
Table 3 : Agar gel consumption (g/100 g. body weight of shrimp) by Metapenaeus dobsoni in a two choice feeding preference test.
Stimulus concentration (% W/V)
Test stimulus 0 0.5 1.0 1.5 2.0 2.5
References
Shark 0.061 0.295 0.351 0.395 0.404 0.523
Mussel 0.059 0.269 0.418 0.533 0.667 0.734
Cuttle Fish 0.062 0.259 0.414 0.503 0.600 0.698
Squila 0.062 0.234 0.364 0.428 0.553 0.628
Fish 0.058 0.227 0.367 0.431 0.567 0.634
Black Clam 0.059 0.390 0.442 0.596 0.744 0.882
Oyster 0.064 0.314 0.437 0.534 0.683 0.786
Shrimp 0.061 0.382 0.492 0.603 0.783 0.886
Squid 0.063 0.483 0.534 0.687 0.723 0.899
Earthworm 0.062 0.218 0.310 0.346 0.436 0.505
Mysid 0.065 0.254 0.321 0.353 0.439 0.517
White Clam 0.065 0.296 0.316 0.413 0.529 0.593
Seaweed 0.065 0.263 0.308 0.352 0.416 0.511
Squid (with ink sac) 0.061 0.114 0.103 0.083 0.023 0.000

Table 4 : Ingestive potency of tissue extract fractions and synthetic fractions on Fenneropenaeus indicus and Metapenaeus dobsoni.
Extract source TE LFE PFE FAA SAA SN
a. F. indicus
Fish 1.0 0.950 0.807 0.506 0.273 0.215
Clam 1.0 0.877 0.705 0.529 0.316 0.127
Shrimp 1.0 0.927 0.848 0.455 0.319 0.156
Crab 1.0 0.987 0.923 0.572 0.390 0.174
Oyster 1.0 0.931 0.844 0.489 0.312 0.151
Squid 1.0 0.942 0.858 0.522 0.335 0.158
Mussel 1.0 0.958 0.734 0.508 0.320 0.166
Squilla 1.0 0.896 0.798 0.527 - -
b. M. dobsoni
Fish 1.0 0.923 0.920 0.580 0.495 0.192
Clam 1.0 0.917 0.834 0.559 0.386 0.217
Shrimp 1.0 0.981 0.903 0.520 0.421 0.261
Crab 1.0 0.973 0.874 0.566 0.434 0.247
Oyster 1.0 0.928 0.843 0.475 0.385 0.182
Squid 1.0 0.894 0.826 0.501 0.357 0.217
Mussel 1.0 0.964 0.863 0.526 0.349 0.168
Squilla 1.0 0.913 0.790 0.86 - -

by squid, clam and shrimp and minimum for FAA of fish differ significantly in their ability to induce ingestion
extract. Synthetic amino acid fraction (SAA) produced activity in shrimps (P<0.05). Species also differ
a potency of 34.9 to 49.5% in M. dobsoni and 27.3 to significantly in their specificity to different amino acids
39.0% in F. indicus. The SAA fraction based on the amino studied (P<0.05). Methionine produced maximum agar
acid profile of clam produced maximum agar consumption consumption and higher relative activity in F.indicus
and of fish the minimum consumption in both the species. followed by lysine, proline, arginine, aspartic acid, taurine,
The potency of synthetic amino acid fraction compared alanine and histidine in the order. Maximum relative
to the free amino acid fraction was low in both species activity was recorded for methionine (83.12%) in F.
and is of the order of 54% to 86% in F. indicus and 66 to indicus and minimum for tyrosine (11.32%). In M.
86% in M. dobsoni. dobsoniaspartic acid produced maximum agar
The stimulatory efficacy of different amino acids on consumption with a relative activity of 82.52% and
agar consumption is depicted in Table 5. Amino acids minimum by alanine (4.87%). The order of preference
 Determination of feeding stimulants in shrimp using a solid matrix bioassay 263
Table 5 : Agar gel consumption (g/100 g body weight of shrimp) and the relative ingestive activity of the test samples (amino acids) for F.
indicus and M. dobsoni in a two choice feeding preference test.
F. indicus M. dobsoni
Stimuli
Agar Gel Relative Agar Gel Relative
Consumption (g) Activity Consumption(g) Activity
Amino acid
Synthetic Amino acid Fraction of Shrimp 0.751 100 0.698 100
Alanine 0.366 48.79 0.232 33.24
Arginine 0.449 59.79 0.242 34.67
Aspartic acid 0.418 55.66 0.597 82.52
Glutamic acid 0.327 43.54 0.422 60.46
Glycine 0.325 43.28 0.238 34.10
Histidine 0.339 45.14 0.240 34.38
Isoleucine 0.274 36.48 0.486 69.63
Leucine 0.242 32.20 0.514 73.64
Lysine 0.624 83.09 0.278 39.83
Methionine 0.629 83.12 0.538 77.08
Ornithine 0.209 27.83 0.181 25.93
Phenylalanine 0.239 31.82 0.207 29.60
Proline 0.484 64.45 0.305 43.70
Serine 0.242 32.22 0.218 31.23
Taurine 0.410 54.59 0.401 51.45
Threonine 0.241 32.09 0.271 38.83
Tryptophan 0.277 36.88 0.505 72.35
Tyrosine 0.085 11.32 0.175 25.07
Valine 0.181 24.10 0.034 4.87

of M. dobsoni towards amino acids was aspartic acid, stimuli and gives good indication of relative rank and
methionine, leucine, tryptophan, isoleucine, glutamic acid, general preference trends. For the tissue extracts tested
taurine and proline. Hence, it is observed that the order equal weights with F. indicus, the palatability ranking
of preferences in the amino acid fractions showed was Squid>Crab>Clam>Oyster>Shrimp extract and for
differences between the species. These results show that M. dobsoni it was Squid>Shrimp>Clam>Crab>Oyster
the observations can be utilized in the formulating of feeds extracts (Tables 2 and 3). Nakamura (1987) reported
based on the amino acid profiles in the ingredients.It is that specific amino acids elevate only orientation and
also observed that when the test stimulus squid extract searching behaviour in P. japonicus, but for ingestion
with ink was tested, there was a significant reduction in response, some proteins such as casein and gelatin as
the consumption when compared to the other ingredient well as saccharides, glucose and starch were more
sources. stimulative than amino acids. These present study
DISCUSSION indicated that free amino acids form the major flavour
component of tissue extracts eliciting ingestion activity
A feeding bioassay which uses agar discs was in bothspecies. Betaine was found to have limited role as
developed for evaluating chemosensory stimuli influencing an ingestant in F. indicus and M. dobsoni (Table 5).
ingestive behaviour in F. indicus and M. dobsoni. The Both of these components also produced very poor
palatability assay required only small amounts of stimuli response in behavioural evaluation indicating that they
and is suitable for rapid and inexpensive screening of a have no role in the feeding activity of this shrimps, when
wide variety of compounds. The added advantage of the tested alone. But several workers have reported that
system is that only quite small amounts of stimuli can be betaine acted is a stimulant in various aspects of feeding
used to test large number of individuals. Unlike feed tests behaviour in diverse crustaceans when tested alone
which incorporate stimuli into feed pellets, this assay is (Hodgson, 1958; Laverack, 1963; Hashimoto, 1967; Carr,
independent of confounding factors such as pellet size, 1978; Harpaz et al, 1987a, b; Harpaz, 1988). Betaine
texture, or hardness, and the results are not influenced HCl was found to be one of the most potent chemo-
by other compounds extent in the pellets. This test allows attractant for M. rosenbergii in behavioural observations
direct comparison of the relative efficacy of the paired (Harpaz et al, 1987 a, b) as well as in electro physiological
264 Cheryl Antony et al
studies (Derby and Harpaze, 1988) and in one of the Palaemonetespugio: comparative studies of stimulatory
major components in extracts of fish, crab and shrimp substances in human serum. Biol. Bull. 148, 380-392
which are readily fed upon by M. rosenbergii likewise Case J (1964a) Adequate stimulus of certain Cancer dactyl
chemoreceptors. Amer. Zool. 4, 37.
Hodgson (1988) and Laverack (1963) reported that
Case J (1964b) Properties of the dactyl chemoreceptors of Canceran
betaineis a feeding incitant in crayfish, crabs and lobsters.
tennarius and C. productus. Biol. Bull. 127, 428-446.
Arginine is one of the most potent amino acid inducing
Derby C D and Harpaz S (1988) Physiology of chemoreceptor cells
ingestion in F. indicus, but have only a less significant in the legs of the freshwater prawn Macrobrachium rosenbergii.
role in inducing feeding response in P. japonicus. Comp. Biochem. Physiol. 90A, 85-91.
Similarly, glycine elicited feeding behaviour in both F. Harpaz S, Kahan D and Galum R (1987a) Variability in feeding
indicus and M. dobsoni, but had only poor ingestant behaviour of the Malaysian prawn Macrobrachium rosenbergii
activity. But glycine was considered as the most potent (De Mann) during the moult cycle (Decapoda Caridea).
Crustaceana 52, 53-60.
stimulant for P. japonicus (Nakamura, 1987) and P.
Harpaz S, Kahan D, Galun R and Moore I (1987b) Responses of
monodon (Murai et al, 1981). In lobsters, Levandowsky
freshwater prawn, Macrobrachium rosenbergii, to chemical
and Hodgson (1958) found glycine as less stimulative. attractants. J. Chem. Ecol. 13, 1957-1965.
Most of the workers reported taurine as the major extract Hollander M and Wolffe D A (1973) Nonparametric Statistical
component of prey organisms inducing feeding activities Methods Wiley series in probability and mathematical statistics
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Carr, 1976; 1978; Carr et al, Johson and Ache, 1978; Harpaz S and Steiner J E (1987) Quantitative analysis of feeding
Trott and Robertson, 1984). Taurine was also found as a behaviour stereotypes and the rejection of over satiating food in
the freshwater prawn. Macrobrachium rosenbergii. Taste and
potent feed ingestant for P. indicus and M. dobsoni in Olfaction. In the Annual Meeting of the New York Academy of
the present study, but had a very poor role in eliciting the Science, pp. 347-348.
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Holland K N and Borski R J (1993) A palatability bioassay for
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