Journal of Chemical Ecology, Vol. 13, No. 9, 1987
RESPONSES OF FRESHWATER PRAWN, Macrobrachium rosenbergii, TO CHEMICAL ATTRACTANTS
HARPAZ, 1 DAVID KAHAN, 1 RACHEL ITZHAK MOORE 3
GALUN, 2 and
IDepartment of Zoology ZDepartment of Parasitology, Hadassah Medical School The Hebrew University of Jerusalem Jerusalem 91904, Israel 3Department of Entomology, A.R. O. The Volcani Center, Bet Dagan, Israel (Received September 19, 1986; accepted November 17, 1986) Abstract--Chemoreception in the adults of the freshwater prawn Macrobrachium rosenbergii was investigated under controlled laboratory conditions, using behavioral assays. Tests were carried out on groups, as well as on individuals, all at their intermolt stages of the molt cycle, and prestarved for three to four days. Of 28 different substances tested, the amino acids taurine, glycine, arginine, and betaine, as well as trimethyIamine, elicited a positive behavioral response in at least 50 % of the test animals when applied at a threshold concentration of 10-5-10 -8 M. A positive response comprises enhanced antennular flicking and food search motion. Of the various nucleotides tested for chemoattraction, only adenosine monophosphate elicited a response similar to that of the above amino acids, although at a concentration of 10 4 M, whereas adenosine diphosphate required a dosage of 10 ~ M. Key Words--Macrobrachium rosenbergii, Crustacea, chemoreception, taufine, glycine, trimethylamine, adenosine monophosphate, feeding stimulants.
INTRODUCTION C h e m o r e c e p t i o n is a n i m p o r t a n t m e c h a n i s m g o v e r n i n g t h e p r o c e s s o f f o o d s e a r c h i n g in m a n y a q u a t i c a n i m a l s . E a r l i e r w o r k o n t h e a t t r a c t i o n o f Macro-
brachium rosenbergii to foodstuffs h a s s h o w n t h a t w h i l e l a r v a e o f t h i s p r a w n do n o t s e e m to r e s p o n d to w a t e r b o r n e s t i m u l i , p o s t l a r v a e a c t i v e l y s w i m t o w a r d s the a p p a r e n t s o u r c e o f c h e m i c a l a t t r a c t a n t s ( M o l l e r , 1978). M o l l e r ' s w o r k , 1957 0098-0331/87/0900-1957505.00/0 9 1987 Plenum Publishing Corporation
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however, was of a preliminary and qualitative nature, performed mainly in Petri dishes on crude materials, and did not include the adult stage. In a recent preliminary study by Holland (1985), in which M. rosenbergii prawns were individually tested, a fish extract and some chemically defined materials were identified as inducing two grades of reaction: food detection and search. A similar characterization of the behavioral response pattern of the prawn has been observed by Harpaz et al. (1987), who also demonstrated the importance of the prawn's stage in the molt cycle during chemotactic examinations. The present work reports the results of quantitative studies on a number of chemically identified crustacean attractants, including certain nucleotides. The latter were included because they serve as potent attractants for some marine decapods (Carr and Thompson, 1983; Derby et al., 1984).
METHODS AND MATERIALS
The effects of chemoattractants on M. rosenbergii were tested on groups of prawns and on individuals. Tests on groups served only as a preliminary screening of various chemicals. Those found attractive were then tried on individuals to obtain dose-response data. Animals were kept in glass aquaria at a constant temperature of 27 _+ 2 ~ They were fed commercial pelleted fish feed containing 25 % protein supplemented occasionally with 1-g pieces of frozen fish and fresh leaves (see Harpaz and Schmalbach, 1986). Prior to every test, the animals were starved for three to four days. The weight range of both sexes of adult prawns employed in the tests was 6-20 g, although within each test group prawns were selected for more uniform weight. M. rosenbergii are nocturnal, thus the tests were conducted under dimmed light conditions; when necessary, only red light illumination (brightness of 0.9 lamberts) was used, since crustaceans are not sensitive to it (Kennedy and Bruno, 1961). These conditions minimized the interfering effects of shyness due to the presence and movements of the human experimenter. The test attractants were introduced into the aquaria by means of a 10-ml pipet containing 7 ml of the stimulant, with an enlarged tip aperture. The pH of aquarium water fluctuated between 6.5 and 7.5 and, when necessary, the stimulant's pH was adjusted accordingly. A similar pipet, containing aquarium water only, was introduced simultaneously at another comer of the aquarium in order to distinguish between chemical, as opposed to visual and/or mechanical response. For this particular purpose, water circulation (see below) had to be stopped, and time allowed for the animals to readjust. Under such almost still conditions, diffusion of the attractants from the pipet would be very slow were it not for the differences between the level of liquid in the pipet and that of the water in the aquarium. This provided an initial hydrostatic pressure for pushing
P R A WN RESPONSES T O A T T R A C T A N T S
the solution out of the pipet so as to elicit a response within 60 sec. The respective concentration reaching the prawn's chemoreceptors might be up to 1000-fold lower depending on the position of the prawn and the slight water turbulence caused by the gill bailers, as well as prawn's movements. In dye simulation, the cloud covered close to half the aquarium volume within 1 rain. In the solitary aquaria the attractant was introduced at a point most distant from the prawn's head. All test chemicals were purchased from Sigma Chemical Company, St. Louis, Missouri. A response was considered positive only when a specific sequence of events relating to feeding behavior took place. These include increased antennular flicking as described by Schmitt and Ache (1979), followed by food searching motions carried out by the first pereiopods. The latter search the substratum in front of the animal in a sweeping movement with their chelae, frequently bringing them towards the mouthparts. This sequence usually ends with the animal's eventual arrival at the stimulant release point. For a detailed description, see Harpaz et al. (1987). In a given assay, each prawn was scored only once unless stated otherwise. Each stimulant was tested at a minimum of five concentrations with at least 10 replicates per concentration. A total of 50 prawns took part in these experiments. Where appropriate, the significance of differences between compounds was analyzed using ANOVA followed by the Student-Newman-Keuls test (Sokal and Rohlf, 1969), as well as by log probit analysis according to Daum (1970) for determination of EDs0. Tests on Groups. Ten groups of six adult prawns each were kept in 50 liters of water contained in glass aquaria measuring 50 x 40 x 30 cm. A 5-cm layer of gravel, placed at the bottom of each aquarium, served as a biological filter, from which water was constantly circulated through the entire volume of the aquarium by an electrical air pump. Sections of plastic tubing (5 cm diameter, 10 cm long) were placed on the gravel layer as hiding sites to reduce cannibalism. The group trials served as a first approximation in which the same concentration (0.1 M) was applied for every one of the 23 test chemicals. Attractiveness was expressed in terms of the time required for 50% of the group to respond positively. A ceiling limit of 6 min was set for a positive response, any later arrivals at the target were considered as accidental and nonsignificant under these experimental conditions. When cannibalism occurred (rare and normally following molting), the whole group was disqualified. Tests on Individuals, The individual tests were carried out only with prawns that were at responding stages of the molt cycle. Prawns at the nonfeeding stages (D 3 and A) were not tested. Designation of the different stages of the molt cycle was according to Peebles (1977). This was based on the recognized
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phenomenon that many crustaceans, including M. rosenbergii, do not feed during the periods immediately before and after ecdysis. Moreover, during these particular nonfeeding stages of the molt cycle, the animals do not respond in the typical manner to chemical attractants (Harpaz et HI., 1987). The aquaria for individual prawns measured 30 • 15 • 20 cm and contained 7 liters of water each. A 300-ml cup filled with gravel and placed on the bottom of the aquarium was used as above for constant water filtration. Each of these aquaria was optically insulated from its neighbor to avoid visual-response interference arising from prawns learning and memorizing as reported by Singer et al. (1979). The molt cycle was recorded for each individual in this type of aquarium. The light-dark regime in these aquaria was 12L: 12D to simulate the conditions of this prawn's tropical origin. Based on preliminary tests, the time limit in this series for reaching the target in a positive response had to be raised to 10 min. In the control assays (pipets containing aquarium water only) included in each chemoattractant test, no more than 2 % of the animals actually reached the pipet tip. However, practically all these "positive" responses should be regarded as chance arrivals since very rarely did any of these prawns go through the entire repertoire constituting a complete positive response. On the other hand, at very low concentrations (varying according to the attractants), the animal performed the behavioral repertoire, but finally failed to reach the target. Nevertheless, such cases were still recorded as positive response. RESULTS
Test on Groups. Table 1 lists the chemicals eliciting a positive response in M. rosenbergii at an initial concentration of 0.1 M at the point of introduction. TABLE 1. MEAN TIME REQUIREDFOR 50% OF GROuP-TESTEDPRAWNSTO DETECT AND RESPONDTO 10 -1 M CONCENTRATIONOF VARIOUSCHEMOATTRACTANTS(N ~ 10 GROUPSFOR EACH ATTRACTANT) Attractant Glycine L-Arginine Trimethylamine (TMA) Casein hydrolysate Betaine L-Isoleucine L-Proline
Time (sec, X + SD) 125.5 _+ 36.3aa 135.0 +_41.9a 142.2 + 41.9a 143.5 +_ 38.4a 193.0 +_+_75.2 206.0 _+ 79.0b 245.0 _+ 84.2b
aFigures with the same letter do not differ significantly (P > 0.05).
PRAWN RESPONSES TO ATTRACTANTS
Results are presented in terms o f the time (in seconds) required for 50 % o f the prawns to detect and perform the food searching behavioral pattern while approaching the attractant source. The following chemicals either did not elicit any response, or the reaction was nonsignificantly positive at this relatively high concentration: adenosine, adenosine 5'-triphosphate (ATP), alanine, aspartic acid, cysteine, glutamic acid, glutathione, histidine, leucine, lysine, rnethionine, phenylalanine, serine, threonine, tryptophan, tyrosine. All amino acids were L forms. Tests on Individuals. Except for casein hydrolysate and proline, the attractants eliciting a positive response in the group tests are indicated in Table 2, where the minimum molar concentrations required for these chemicals to attract at least 50% o f the test prawns are presented. Taurine and nucleotides, which have been reported as powerful chemoattractants to other decapods (Case, 1964; Ache, 1972; Shepheard, 1974; Fuzessery et al., 1978; Heinen, 1980; C a r t and Thompson, 1983; Derby et al., 1984), were tested as well. A m o n g the amino acids, the most attractive were taurine, glycine, and isoleucine. Table 3 presents the percentages o f prawns positively responding to various nucleotides at ascending log concentrations. The most potent was A M P . However, at concentrations higher than 10 - z M, the response to this nucleofide decreased, indicating an inhibitory effect o f A M P at higher concentrations. As shown in Table 3 (for 10 -1 M), the potencies o f G M P , A D P , and I M P were low and decreased in the indicated order. A T P and adenosine were inactive even at the highest concentration tested. The results obtained with individual prawns actually confirmed the conclu-
TABLE 2. EDs0a VALUES
OF VARIOUS CHEMOSTIMULANTS ELICITING POSITIVE
BEHAVIORAL RESPONSES IN INDIVIDUALLY TESTED
PRAWNS Test chemical TMA taurine glycine L-isoleucine betaine L-arginine AMP
EDso 3.03 4.55 2.78 2.34 4.98 1.14 3.29
x 10-8 x 10-8 • 10 -7 X 10 -6
x 10 6 x 10 5 • 10 4
Slope ___SE 0.204 0.273 0.246 0.281 0.274 0.268 0.337
+ 0.032 + 0.040 _ 0.031 + 0.033 + 0.042 _+ 0.041 _ 0.057
aEDs0 = mean molar concentration required for eliciting a positive response in 50 % of the tested prawns, calculated from log probit regression according to Daum (1970). Statistical differences exist only between concentrations which are one log step apart. Saturation levels are similar except for AMP (see text).
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TABLE 3. RESPONSE a OF INDIVIDUALLY TESTED Macrobrachium
CONCENTRATIONS OF VARIOUS NUCLEOTIDES
GMP ADP IMP ATP Adenosine
+ + + + + + no response no response
~Since the response did not exceed 50%, rating was according to the following scale: + + + _< 50%; + + _ 40%; + < 30%.
sions drawn from the group trials in the case of glycine, arginine, TMA, betaine, and isoleucine. Also, the pattern of behavior towards the attractant's release point was observed to be identical in the two series of trials. At a high concentration of attractant, the entire sequence of events, as mentioned above, is performed with remarkable intensity and, upon reaching the target area, the prawns vigorously shake the pipet with their chelae. As the concentration decreases, the intensity of food searching movements diminishes, and the prawn at times gives up the search before reaching the target area, thus indicating that the mechanism involved is much more complex than a mere "all-or-none" response.
In this study, chemoattractants for M. rosenbergii were identified and their dose-response relations determined through the use of behavioral methods commonly employed in such studies: tests on prawn groups and individuals. Many studies on feeding behavior of decapod crustaceans have been carried out on groups rather than individual animals (Carr, 1978; Deshimaru and Yone, 1978; Cart and Thompson, 1983; Carr et al., 1984). This is because, besides experimental convenience, results of group trials can at times be better related to field conditions, thereby making them more directly applicable to aquaculture. However, group experiments have some potential disadvantages; one of them is that all prawns are often not at the same stage of the molt cycle (see Methods and Materials). Another drawback is that at the beginning of each experiment animals are not equally distant from the pipet tip, so that the concentration gradient between the tip of the pipet and the site of the animal about to respond can influence the results. Furthermore, one cannot rule out the possibility of a responding animal stimulating other members of the group into
P RA WN R E S P ON S E S T O A T T R A C T A N T S
motion. Another source for possible error is the prawn's territorial behavior. This is displayed by its tendency, upon discovering a food site, to deter other prawns from approaching the site (Harpaz, 1986). Tests carried out on individuals offer a better chance of controlling experimental conditions, validating group test results, and avpiding most of their drawbacks. Most of the chemicals tested in the present study have been reported as stimulants with various degrees of potency for other crustaceans (Case, 1964; Ache, 1972; Shepheard, 1974; Carr and Gurin, 1975; Carr, 1978; Fuzessery et al., 1978; Johnson and Ache, 1978; Heinen, 1980; Derby and Atema, 1982; Carr and Thompson, 1983; Santos-Filho, 1983; Derby et al., 1984). Some of these chemicals were also tested on M. rosenbergii, although only preliminarily (Holland, 1985). It is difficult to compare results obtained in different studies since the methods, experimental set-ups, and criteria used are not the same. Nevertheless, as regards M. rosenbergii, the ranking of the different chemoattractants is similar. The most potent were TMA, glycine, and taurine. In addition, we have found that betaine and, to a lesser extent, nucleotides are also active. The most potent nucleotide was AMP followed by GMP, ADP, and IMP in the listed order, while ATP and adenosine were not active. These results are similar to those reported by Carr and Thompson (1983) for Palaemonetes pugio, and by Derby et al. (1984) for the spiny lobster Panulirus argus. Yet, even the most potent nucleotide AMP was two to three orders of magnitude less effective than the amino acids for M. rosenbergii. For other arthropods, e.g., hematophagous insects, the trend of nucleotide potency ranking was found to be reversed (Galun et al., 1985). In the present study, no attempt was made to discriminate between chemoreceptors associated with the senses of smell and taste. Following antennular removal, M. rosenbergii, like Procambarus clarkii (Ameyaw-Akumfi, 1977), regains (after a short recovery period) its ability to home in on several attractants. This points toward the possibility that other parts of the body (presumably the first pereiopods and maxillipeds) possess chemoreceptors which are able to substitute for those located on the antennules (Harpaz, unpublished). It seems that some of the amino acids and nucleotides, identified as chemoattractants for M. rosenbergii, have a dual function. All the amino acids eliciting a powerful response in M. rosenbergii stimulated (with varying degrees of activity) the chemoreceptors of the walking legs of the lobster H. americanus (Derby and Atema, 1982). Since, in Crustacea, leg and mouthparts contain the sensory structures involved in the sense of "taste" (gustation) (Atema, 1980), it seems that these amino acids may be powerful phagostimulants. Thus, incorporation of trimethylamine hydrochloride to a low-grade feed of adult M. rosenbergii brought about a 30-38 % increase in food consumption (Costa-Pierce and Laws, 1985). In an attempt to improve the nutritional value of commercial pellets fed to adult M. rosenbergii, Farmanfarmaian and Lauterio (1979) supplemented
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t h e s e p e l l e t s w i t h v a r i o u s a m i n o a c i d s r e p o r t e d as e s s e n t i a l f o r C r u s t a c e a . Among these amino acids, arginine appreciably stimulated growth. This beneficial effect m a y h a v e b e e n c o n t r i b u t e d also b y t h e p o w e r f u l a t t r a c t a n c y o f arg i n i n e to M. r o s e n b e r g i i as s h o w n in t h e p r e s e n t study.
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