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Olfactory and gustatory responses to amino acids in two marine teleosts—Red sea bream and mullet
Comp. Biochem. PhysioL, Vol. 66C, pp. 217 to 224
0306-4492/80/0701-0217502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
OLFACTORY A N D GUSTATORY RESPONSES TO A M I N O ACIDS IN TWO MARINE TELEOSTS---RED SEA BREAM A N D MULLET YASUMASAGOH and TAMOTSUTAMURA Fisheries Laboratory, Faculty of Agriculture, Nagoya University, Nagoya, Japan
(Received 19 November 1979) Abstraet--l. The olfactory and gustatory responses to amino acids were recorded electrophysiologically in two stocks of red sea bream (Chrysophyrys major) and mullet (Mugil cephalus). 2. The olfactory spectra of amino acids determined from the bulbar response in the two stocks of red sea bream were more or less same, and the spectrum in mullet was also similar to that in the red sea bream. 3. A comparison of the spectra revealed high similarity among intra- and inter-species. 4. The gustatory spectra of amino acids were similar between the different two stocks of red sea bream, but the spectra were different from those obtained in other fishes. 5. The gustatory spectrum of amino acids in mullet was different from that in other fishes. 6. A comparison of the gustatory spectra revealed a species specificity. 7. From the view of the species non-specificity of the olfactory spectrum of amino acids and the species specificityof the gustatory one, the functional difference between the olfactory and the gustatory response to amino acids in the feeding behaviour was discussed.
INTRODUCTION Many behavioural studies have shown that amino acids and their compounds are effective in inducing feeding behaviours in Japanese eel (Hashimoto et al., 1968; Konosu et al., 1968), silverside, flounder, kiUfish (Sutterlin, 1975), pinfish (Carr et al., 1976; Carr & Chancy, 1976) pigfish (Carr, 1976), whiting, cod (Pawson, 1977), herring larvae (Dempsey, 1978)~ rainbow trout (Adron & Mackie, 1978) and puffer (Hidaka et al., 1978; Ohsugi et al., 1978). However, few of the behavioural studies could identify the sense organ through which the amino acids were perceived. Electrophysiological studies have shown that amino acids are effective not only on the taste system in catfish (Caprio, 1975), bullhead (Bardach et al., 1967a), Atlantic salmon (Sutterlin & Sutterlin, 1970), puffer (I-Iidaka et al., 1975) and goby (Yoshii & Yamashita, 1975), but also on the olfactory system in white catfish (Suzuki & Tucker, 1971), Atlantic salmon (Sutterlin & Sutterlin, 1971), rainbow trout (Hara, 1973), brook trout, whitefish (Hara et al., 1973), char (Berghaug & D~ving, 1977), carp (Goh & Tamura, 1978), channel catfish (Caprio, 1978), red sea bream and conger eel (Goh et al., 1979). In olfaction, it was found that a sequence of the stimulatory effectiveness of amino acids (spectrum of amino acids) resulted from electrophysiological studies is similar among Atlantic salmon, white catfish, rainbow trout, whitefish, brook trout, carp, red sea bream and char (Goh et al., 1979). On the other hand, the gustatory spectrum of amino acids seems to differ from species to species. The most typical example is found in the comparison of the spectrum between two species of the same genus Ictarulus, i.e. catfish and bullhead: the taste system of the former (Caprio, 1975) was far more sensitive to L-glutamine and to L-serine than that of the
latter which was more sensitive to L-leucine and L-glutamic acid (Fujiya & Bardach, 1966) than the taste system of the former. In the present experiment, we attempted firstly to add further evidences to the similarity of the olfactory spectrum of amino acids among intra- and interspecies, secondly to show the species specificity of the taste spectrum of amino acids, and lastly to discuss the relative importance in the feeding behaviour between the olfaction and the gustation. MATERIALS AND METHODS Two stocks of the red sea bream (Chrysophyrys major) were used: one was obtained from a fish farmer in Mie Prefecture and consisted of 24 individuals (Fork Length, 85-112 mm) and the other was from the Fisheries Laboratory of University of Tokyo in Shizuoka Prefecture and consisted of 14 individuals (F.L, 120-138 mm). 14 mullets (Mu#il cephalus, F.L., 125-235mm) were also obtained from the laboratory. All the fishes were kept in out-door ponds with continuous flowing of the seawater, and fed with artificial diets. The temperature of the pond water was 13.5-21.5°C. In the electrophysiological recording of the taste and olfactory responses, the fish was immobilized with the intramuscular injection of Flaxedil (gallamine triethiodide; 0.1--0.15mg/100 g body weight in red sea bream, 0.2-0.3 mg/100 g in mullet). The fish was then wrapped in a tissue paper and fixed in a lead (Pb) plate. For the recording of the olfactory response, the skull of the fish was opened, and the forepart of the brain was exposed. Ag-AgCI type bipolar electrode (0.15mm dia, 1-1.5 mm apart) insulated with lacquer except the tips was placed on the surface of the olfactory bulb. The electrical activity from the bulb was amplified and displayed on a pen recorder. The stimulating solution was introduced to the olfactory mucosa by adding the solution (0.1 ml) to a continuously flowing seawater (5-8 ml/min) to the olfactory sac. The diluting factor of the stimulating solution
217
218
YASUMASAGOH and TAMOTSUTAMURA
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IO-5M mt,~.,""a . . ~ , . , Fig. 1. Schematic diagram showing a recording system of the taste response. S, stimulus solution; CS, continuously flowing seawater to the responsive area; OSC, oscilloscope; E, electrode; GS, seawater for gill irrigation; SN, stimulating nozzle.
added to the continuously flowing seawater was about 1/20 (Goh et al., 1979). In the recording of the taste response, the eyeball of the fish was removed, and a branch of the facial and trigeminal nerve complex innervating the lower lip region or a branch of the facial nerve innervating the anterior palate region (Herrick, 1899, 1901) was exposed, and isolated from the connective tissue and from the central connection. The nerve branch was then hooked on a bipolar platinum electrode. The electrical activity from the nerve bundle was amplified, passed through an integrating circuit and displayed on a pen recorder. A continuous flow of the seawater (40 ml/min) was directed to the lower lip or anterior palate region, and the stimulating solution (0.2ml) was injected into the continuously flowing seawater (diluting factor about 1/2) (Fig. 1). Throughout the experiment, the gills were perfused with seawater. The water used in the experiment was filtrated natural seawater. The pH value of the seawater was about 8.1, and the stimulating solution of each chemical was adjusted to pH 7.5-8.5 with 1/10 N HCI and 0.4% NaOH.
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Fig. 2. A series of olfactory responses to L-glutamine 10-2-10 -8 M in red sea bream of MS.
RESULTS Olfactory response to amino acids R e d sea bream. In order to test the similarity of the olfactory spectrum of a m i n o acids a m o n g different strains, we recorded the olfactory response from the olfactory bulbs of two stocks of red sea b r e a m ; Mie
01 m
rr
41
41
Fig. 3. Olfactory spectra of amino acids in three stocks of red sea bream. Lower bar, YS (Goh et al., 1979); middle bar, SS; upper bar, MS; * not tested; each bar represents a relative response magnitude (+SD) as a percentage of the response to standard L-glutamine 10 -3 M.
Gustatory responses by fish to amino acids I0-2H
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Fig. 4. A series of olfactory responses to L-glutamine 10-2-10-7 M in mullet.
stock (MS) and Shizuoka stock (SS), and the results were compared to those obtained from Yamaguchi stock of rod sea bream (YS) by Gob et al. in 1979. Figure 2 shows an example of the bulbar response to L-glutamine 1 0 - 2 - 1 0 - S M in MS. The response magnitude increased with the increase of the stimulus concentration, and the application of seawater, as a control, induced no response. The threshold concentration for L-glutamine was about 10 - s M under the condition of the present experiment. Figure 3 shows the three spectra of 15 amino acids and betaine in 11 fishes of MS, 6 fishes of S.S, and fishes of YS. All the chemicals were tested at the same concentration of 10- 3 M. The response magnitude of each chemical is the mean amplitude of the induced
219
waves in the initial 5 sec, and expressed as a percentage of that obtained by the stimulation with the standard 1 0 - 3 M L-glutamine. From this figure, it is reasonably concluded that three stocks have the almost same spectrum. In order to compare the three spectra more quantitatively, we computed the regression lines and their correlation coefficients by the method of least squares in each pair of the spectra of the three stocks. The equations of the regression lines and their correlation coefficients were y = 0.97x + 3.71 (r = 0.97) between YS and MS, y = 1.03x - 0.26 (r = 0.98) between MS and SS and y = 1.01x - 4.06 (r = 0.99) between SS and YS. Mullet. Figure 4 shows an example of the olfactory response to L-glutamine 10- 2-10-" M obtained from the olfactory bulb of the mullet. The response magnitude increased with the increase of the stimulus strength, and the threshold concentration for L-glutamine was about 10-TM in this fish under the present experimental condition. The olfactory spectrum of amino acids in 6 fishes of mullet is shown in Fig. 5. All chemicals were tested at the same concentration of l0 -3 M. The spectrum in the mullet was compared to that in carp (Goh & Tamura, 1978), red sea bream (YS) (Gob et al., 1979), rainbow trout (Hara, 1973) and catfish (Suzuki & Tucker, 1971) by computing the regression line and its correlation coefficient. The equations of the regression fines and their correlation coefficients were y = 1.04x + 4.31 (r -- 0.98) between red sea bream and mullet, y = 0.83x + 20.09 (r--0.91) between catfish and mullet, y - - 0 . 5 8 x + 30.89 (r = 0.72) between rainbow trout and mullet and y = 1.30x - 15.80 (r = 0.84) between carp and mullet. Taste response to amino acids Red sea bream. In the recording of the taste responses, we stimulated different regions of the gustatory system between large fish (Shizuoka stock of red sea bream and mullet) and small fish (Mie stock of red sea bream), because of the technical convenience.
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Fig. 5. Olfactory spectrum of amino acids in mullet. Other notation of this figure is same as in Fig. 3.
220
YASU~,SAGOH and TAMOTSUTAMURA !llllllllml
Cont,rol
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Fig. 6. A series of gustatory responses to L-alanine 10-z-10 -7 M in red sea bream of MS. Note that there is a small response even at 10 -6 M. In the former fishes, the anterior palate region was stimulated and from the nerve running to this region the responses were recorded. In the latter fishes, the lower lip region was stimulated and the responses were recorded from the nerve running to this region. Figure 6 shows an example of the integrated taste response to L-alanine 10-2-10 -6 M in MS. The response in this experiment always consisted of an apparent phasic phase and a small tonic phase probably because of the short stimulating duration. The response magnitude was, therefore, represented by the height of the phasic response. The response to L-alanine increased with the increase of the stimulus concentration, and the threshold concentration was about 10 -6 M. The gustatory spectra of amino acids (the sequence of the stimulatory effectiveness of amino acids in taste) were determined in 12 fishes of MS and 7 fishes of SS as shown in Fig. 7. All chemicals were tested at the same concentration of 10 -z M. The spectra were more or less same between the two stocks of red sea bream, although the responses to some amino acids such as g-lysine, L-glutamine, L-proline and betaine were slightly larger in SS than
in MS. However, the difference in the responses between the two groups may partly be attributed to the different gustatory systems mentioned above, since even in the same stock (MS) the responses to these amino acids were different between the two gustatory systems (Fig. 8), and the afferent trigeminal component of the anterior nerve bundle of facialtrigeminal complex was reported not to respond to taste stimuli (Kiyohara et al., 1975). Because of the small number of the available fish, each response obtained from anterior palate region is represented as a dot in Fig. 8. In each spectrum of MS and SS, L-alanine was the most effective amino acid, and glycine, L-arginine and L-serine were relatively effective. L-alanine + betaine and giycine + betaine were very effective because of the synergistic interaction (Hidaka et al., 1976). L-tyrosine and L-cystine were almost non-stimulative. In addition to the test of amino acids, we tested seven nucleotides (adenosine, AMP, ADP, ATP, IMP, U M P and GMP), but all the nudeotides were nonstimulative in the fishes of both the stocks, when the test solution was 10 -2 M. Mullet. In mullet, the taste response was recorded
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Fig. 7. Gustatory spectra of amino acids in two stocks of red sea bream. Upper bar, MS; lower bar, SS, each bar represents a relative response magnitude (+ SD) as a percentage of the response to standard L-alanine 10-2 M. The concentration of 1/2 (alanine + betaine) and 1/2 (glycine + betaine) is adjusted to l 0 - 2 M of total of both the chemicals.
Gustatory responses by fish to amino acids
221
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Fig. 8. A comparison of the taste spectra of amino acids in red sea bream of MS between two taste systems; lower lip region and anterior palate. Each dot represents a response obtained from anterior palate. Each bar represents a relative response magnitude (___SD) obtained from lower lip region. from a branch of the facial nerve running to the anterior palate. Figure 9 shows a series of responses to L-alanine 10-2-10 - 7 M. The threshold concentration to this amino acid was about 10 -6 M. Figure 10 shows the taste spectrum of amino acids in 7 mullet, when the amino acids were tested at the same concentration of 10-ZM. The most effective amino acid was L-arginine, and L-lysine, L-alanine and L-serine were the next effective amino acids. L-cystine, L-tyrosine and L-isoleucine were not effective. Seven nucleotides tested in red sea bream were also tested in mullet, but none of the nucleotides was effective in inducing any response. DISCUSSION
Olfaction Since the olfactory epithelium of Atlantic salmon (Sutterlin & Sutterlin, 1971) and catfish (Suzuki &
Tucker, 1971) was found to respond well to amino acids, the electrical response to amino acids has been investigated in the olfactory system of many fish species. From the experiments, it is concluded that the olfactory system of fishes has high sensitivity to amino acids, and the threshold concentration of the olfactory organ is ranging from 10 - 9 to 10-6M for some effective amino acids. In the present experiment, the threshold concentration for L-glutamine in red sea bream of Mie stock and for mullet was about 10- s M and about 10-TM respectively, and the high sensitivity to amino acids was also demonstrated in the two species. Comparing the olfactory spectrum among the three different stocks of red sea bream: Yamaguchi stock (Goh et al., 1979), Mie stock and Shizuoka stock, high similarity was found in each pair of the spectra (r = 0.97 between YS and MS, r = 0.98 between MS
LLIiiLL$1i I
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Fig. 9. A series of the taste responses to L-alanine 10-z-10-7 M in mullet. Note that there is a small response at 10-4 M.
222
YASUMASAGOH and TAMOTSUTAMURA
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Fig. 10. Gustatory spectrum of amino acids in mullet. Each bar represents a relative response magnitude (± SD) as a percentage of the response to the standard L-arginine 10-2 M. Other notation of this figure is same as in Fig. 7. and SS and r = 0.99 between SS and YS). The fact that the threshold concentration for L-glutamine is similar between YS and MS, and that the olfactory spectrum of amino acids is very close in the three stocks of red sea bream suggests that the olfactory sensation to amino acids may be more or less same in any stock of red sea bream. In our previous work ((;oh & Tamura, 1978; Goh et al., 1979), we pointed out that the olfactory spectrum of amino acids is similar among catfish, Atlantic salmon, rainbow trout, brook trout, whitefish, char, carp and red sea bream, and we assumed that the olfactory spectrum may be similar in all teleosts. In order to confirm the assumption, the olfactory spectrum of amino acids in mullet was obtained and compared to that in catfish (Suzuki & Tucker, 1971), rainbow trout (Hara, 1973), red sea bream (Gob et al., 1979) and carp (Goh & Tamura, 1978). In each pair of the spectra the similarity was high (r = 0.98 between red sea bream and mullet, r = 0.91 between catfish and mullet, r = 0.72 between rainbow trout and mullet and r = 0.84 between carp and mullet). These results support the assumption that the olfactory spectrum of amino acids is similar in all teleosts. Taste The gustatory response to amino acids has been reported in several fishes, and the taste spectrum of amino acids is different from species to species. For instance, the taste system of hake was reported to be sensitive to L-aspartic acid, L-glutamic acid, L-phenylalanine and glycine (Bardach & Case, 1965), while other "fishes were sensitive to other amino acids, i.e. bullhead was sensitive to L-cysteic acid, L-cysteine and L-alanine, tomcod to L-cysteine (Bardach et al., 1967a), Atlantic salmon to L-proline (Sutterlin & Sut-
terlin, 1970), catfish to L-alanine, L-arginine, L-serine and L-glutamine (Caprio, 1975), puffer to L-proline, glycine and L-alanine (Hidaka et al., 1975), Japanese eel to L-arginine, glycine, L-alanine, L-proline, L-lysine (Yoshii et al., 1979). The taste spectrum obtained in this study in red sea bream and mullet was also different from that in any other fishes reported earlier, though the spectrum in Japanese eel was considerably similar to that in mullet. Comparing the two spectra in mullet and red sea bream, the response magnitude to 12 amino acids and alanine + betaine was considerably similar, but that to L-arginine, L-lysine, glycine and L-glutamine was much different. Thus, the species specificity of the gustatory spectrum was also confirmed in red sea bream and mullet. We would like to assume that the species specificity of the taste spectrum of amino acids could be due to different feeding substances among different species. If this is true, the taste spectrum may be different even in the same species of different stocks. In the comparison of the spectrum between Mie stock and Shizuoka stock of red sea bream, however, we could not find a large difference in the spectrum as expected. But the taste spectrum of amino acids in the two stocks differed clearly from that in the same species investigated by Hidaka (personal communication), where the responses to L-arginine and L-histidine were much larger than the responses to the same two amino acids obtained in our study. This difference is very interesting, but further studies are needed to make any conclusion on the problem. Functional difference between olfaction and taste Since amino acids were found to be good stimulants not only for the taste system but also for the olfactory system of fish, it has become an important
Gustatory responses by fish to amino acids problem what is the functional difference between the responses to amino acids in olfaction and in taste. The solution to this problem may be given by comparing the threshold between the two sensory systems. The threshold concentration for amino acids seems to be lower in olfaction than iff taste except for a case in catfish (Caprio, 1975, 1978). In the present experiment, the threshold concentration in olfaction of the two species was also lower than that in taste. If the threshold decided from the electrical responses represents an actual sensitivity in the daily behaviour of fish, the higher sensitivity to amino acids in olfaction than in taste may support the general recognition that the olfaction is a distant sense and the taste is a contact or near sense. An interspecies comparison of the spectra of amino acids in olfaction and taste may give some insight to this problem. As discussed above, the olfactory spectrum of amino acids seems to be similar through all the teleosts, but the taste spectrum of amino acids seems to be similar through all the teleosts, but the taste spectrum of amino acids is species specific. We presume that the species specificity of the gustatory spectrum of amino acids in fishes may be a reflection of the difference of their foods, and the taste responses to amino acids may have more intimate relationship to the feeding behaviour than the olfactory responses. Many studies on the role of amino acids in the feeding behaviour were performed in fishes, and amino acids were found to be effective in inducing the feeding urge in fishes. Few of the studies, however, identified the sense organ through which the amino acids were recognized. In yellow bullhead (Ictalurus natalis), the sense of taste was demonstrated to be able to function as a distant receptor, and the destroy of the olfactory epithelium caused no effect on the feeding behaviour (Bardach et al., 1967b; Atema, 1971). But this may be considered to be a special case, because the taste sensitivity to amino acids in channel catfish (I. punctutus) was revealed to be higher than the olfactory sensitivity, to amino acids (Caprio, 1975, 1978). In conclusion, we would like to state that the taste sensation to amino acids have far more intimate relationships to the feeding behaviour of the fish than the olfactory one.
Acknowledgements--We wish to express our thanks to Professor C. Shimizu of Fisheries Laboratory of University of Tokyo for permitting use of the facilities. Thanks are also due to the staff of the Laboratories for providing the fishes, and to Mrs K. Koga for preparing the manuscript. This work was supported in part by research grant from the Ministry of Education of Japan. REFERENCES ADRONJ. W. & MACKIEA. M. (1978) Studies on the chemical nature of feeding stimulants for rainbow trout, Salmo gairdneri Richardson. J. Fish. Biol. 12, 303-310. ArEMA J. (1971) Structures and functions of the sense of taste in the catfish (lctalf~rus natalis). Brain. Behav. Evol. 4, 273-294. BAaD^CH J. E. & CASEJ: (1965) Sensory capabilities of the modified fins of squirrel hake (Urophycis chuss) and searobins (Prionotus carolinus and P. evolans). Copeia 2, 194-206.
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BARDACHJ. E., FUJIYA M. & HOLL A. (1967a) Investigations of external chemoreceptors of fishes. In Olfaction and Taste, VoL 2 (Edited by HAYASHIT.), pp. 647-665. Pergamon Press, New York. BARDACHJ. E., TODD J. H. & CRICKMERR. (1967b) Orientation by taste in fish of the genus Ictalurus. Science, N. ¥ 155, 1276-1278. BELGHAUG R. & DOVING K. B. (1977) Odour threshold determined by studies of the induced waves in the olfactory bulb of the char (Salmo alpinus L.). Comp. Biochem. Physiol. 57A, 327-330. CAPRIO J. (1975) High sensitivity of catfish taste receptors to amino acids. Comp. Biochem. Physiol. 52A, 247-251. CAPRIOJ. (1978) Olfaction and taste in the channel catfish: An electrophysiologicai study of the responses to amino acids and derivatives. J. comp. Physiol. 123, 357-371. CARR W. E. S. (1976) Chemoreception and feeding behavior in the pigfish, Orthopristis chrysopterus: characterization and identification of stimulatory substances in a shrimp extract. Comp. Biochem. Physiol. 55A, 153-157. CARR W. E. S. & CHAtqEV T. B. (1976) Chemical stimulation of feeding behavior in the pinfish, Lacjodon rhomboides: characterization and identification of stimulatory substances extracted from shrimp. Comp. Biochem. Physiol. 54A, 437-441. CARR W. E. S., GONDECK A. R. & DELANOYR. L. (1976) Chemical stimulation of feeding behavior in the pinfish, LacJodon rhomboides: A new approach to an old problem. Comp. Biochem. Physiol. 54A, 161-166. DEMPSEYC. H. (1978) Chemical stimuli as a factor in feeding and intraspecific behaviour of herring larvae. J. mar. biol. Ass. U.K. 58, 739-747. FUJIYAM. & BARDACHJ. E. (1966) A comparison between the external taste sense of marine and freshwater fishes. Bull. Jap. Soc. scient. Fish. 32, 45--56. Gon Y. & TAMURAT. (1978) The electrical responses of the olfactory tract to amino acids in carp. Bull. Jap. Soc. scient. Fish. 44, 341-344. GOH Y., TAMURAT. & KOBAYASH!H. (1979) Olfactory responses to amino acids in marine teleosts. Comp. Biochem. Physiol. 62A, 863--868. I-IAgA T. J. (1973) Olfactory responses to amino acids in rainbow trout, Salmo gairdneri. Comp. Biochem. Physiol. 44A, 407-416. HARA T. J., LAW Y. M. C. & HOSDEN B. R. (1973) Comparison of the olfactory response to amino acids in rainbow trout, brook trout and whitefish. Comp. Biochem. Physiol. 45A, 969-977. HASHIMOTOY., KONOSUS., FUSETANIN. & NOSE T. (1968) Attractants for eels in the extracts of short-necked clam--I. Survey of constituents eliciting feeding behavior by the omission test. Bull. Jap. Soc. scient. Fish. 34, 78-83. HERRICK C. J. (1899) The cranial and first spinal nerves of Menidia; a contribution upon the nerve components of the bony fishes. J. comp. Neurol. 9, 153-544. HERRICK C. J. (1901) The cranial nerves and cutaneous sense organs of the North American Siluroid fishes. J. comp. Neurol. II, 177-249. HIDAKA I., KIYOHARAS. & TABATAM. (1975) Gustatory responses in the puffer. Bull. Jap. Soc. seient. Fish. 41, 275-281. HIDAKA I., NVU N. & KIYOHARAS. (1976) Gustatory response in the puffer--IV. Effects of mixtures of amino acids and betaine. Bull. Fac. Fish. Mie Univ. 3, 17-28. HIDAKAI., OHSUGIT. & KUnOMATSUT. (1978) Taste receptor stimulation and feeding behaviour in the puffer, Fugu paradalis--I. Effect of single chemicals. Chem. Senses Flay. 3, 341-354. KIYOHARAS., HIDAKAI. & TAMURAT. (1975) The anterior cranial gustatory pathway in fish. Experientia 31, 1051-1053. KONOSU S., FUSETANIN., NOSET. & HASHIMOTOY. (1968)
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Attractants for eels in the extracts of short-necked clam--II. Survey of constituents eliciting feeding behavior by fractionation of the extracts. Bull. Jap. Soc. scient. Fish. 34, 84-87. OHSUGi T., HID^K^ I. & IKEDA M. (1978) Taste receptor stimulation and feeding behaviour in the puffer, Fuou paradalis--II. Effects produced by mixture of constituent of clam extract. Chem. Senses Flay. 3, 355-368. P^WSON M. G. (1977) Analysis of a natural chemical ~ttractant for whiting Merlangius merlangus L. and cod Gadus morhua L. using a behavioural bioassay. Cutup. Biochem. Physiol. 56A, 129-135. SUTTERLIN A. (1975) Chemical attraction of some marine fish. In Olfaction and Taste, Vol. 5 (Edited by DENTON D. A. & CtmnL~N J. P.), pp. 153-156. Academic Press, New York.
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0306-4492/80/0701-0217502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
OLFACTORY A N D GUSTATORY RESPONSES TO A M I N O ACIDS IN TWO MARINE TELEOSTS---RED SEA BREAM A N D MULLET YASUMASAGOH and TAMOTSUTAMURA Fisheries Laboratory, Faculty of Agriculture, Nagoya University, Nagoya, Japan
(Received 19 November 1979) Abstraet--l. The olfactory and gustatory responses to amino acids were recorded electrophysiologically in two stocks of red sea bream (Chrysophyrys major) and mullet (Mugil cephalus). 2. The olfactory spectra of amino acids determined from the bulbar response in the two stocks of red sea bream were more or less same, and the spectrum in mullet was also similar to that in the red sea bream. 3. A comparison of the spectra revealed high similarity among intra- and inter-species. 4. The gustatory spectra of amino acids were similar between the different two stocks of red sea bream, but the spectra were different from those obtained in other fishes. 5. The gustatory spectrum of amino acids in mullet was different from that in other fishes. 6. A comparison of the gustatory spectra revealed a species specificity. 7. From the view of the species non-specificity of the olfactory spectrum of amino acids and the species specificityof the gustatory one, the functional difference between the olfactory and the gustatory response to amino acids in the feeding behaviour was discussed.
INTRODUCTION Many behavioural studies have shown that amino acids and their compounds are effective in inducing feeding behaviours in Japanese eel (Hashimoto et al., 1968; Konosu et al., 1968), silverside, flounder, kiUfish (Sutterlin, 1975), pinfish (Carr et al., 1976; Carr & Chancy, 1976) pigfish (Carr, 1976), whiting, cod (Pawson, 1977), herring larvae (Dempsey, 1978)~ rainbow trout (Adron & Mackie, 1978) and puffer (Hidaka et al., 1978; Ohsugi et al., 1978). However, few of the behavioural studies could identify the sense organ through which the amino acids were perceived. Electrophysiological studies have shown that amino acids are effective not only on the taste system in catfish (Caprio, 1975), bullhead (Bardach et al., 1967a), Atlantic salmon (Sutterlin & Sutterlin, 1970), puffer (I-Iidaka et al., 1975) and goby (Yoshii & Yamashita, 1975), but also on the olfactory system in white catfish (Suzuki & Tucker, 1971), Atlantic salmon (Sutterlin & Sutterlin, 1971), rainbow trout (Hara, 1973), brook trout, whitefish (Hara et al., 1973), char (Berghaug & D~ving, 1977), carp (Goh & Tamura, 1978), channel catfish (Caprio, 1978), red sea bream and conger eel (Goh et al., 1979). In olfaction, it was found that a sequence of the stimulatory effectiveness of amino acids (spectrum of amino acids) resulted from electrophysiological studies is similar among Atlantic salmon, white catfish, rainbow trout, whitefish, brook trout, carp, red sea bream and char (Goh et al., 1979). On the other hand, the gustatory spectrum of amino acids seems to differ from species to species. The most typical example is found in the comparison of the spectrum between two species of the same genus Ictarulus, i.e. catfish and bullhead: the taste system of the former (Caprio, 1975) was far more sensitive to L-glutamine and to L-serine than that of the
latter which was more sensitive to L-leucine and L-glutamic acid (Fujiya & Bardach, 1966) than the taste system of the former. In the present experiment, we attempted firstly to add further evidences to the similarity of the olfactory spectrum of amino acids among intra- and interspecies, secondly to show the species specificity of the taste spectrum of amino acids, and lastly to discuss the relative importance in the feeding behaviour between the olfaction and the gustation. MATERIALS AND METHODS Two stocks of the red sea bream (Chrysophyrys major) were used: one was obtained from a fish farmer in Mie Prefecture and consisted of 24 individuals (Fork Length, 85-112 mm) and the other was from the Fisheries Laboratory of University of Tokyo in Shizuoka Prefecture and consisted of 14 individuals (F.L, 120-138 mm). 14 mullets (Mu#il cephalus, F.L., 125-235mm) were also obtained from the laboratory. All the fishes were kept in out-door ponds with continuous flowing of the seawater, and fed with artificial diets. The temperature of the pond water was 13.5-21.5°C. In the electrophysiological recording of the taste and olfactory responses, the fish was immobilized with the intramuscular injection of Flaxedil (gallamine triethiodide; 0.1--0.15mg/100 g body weight in red sea bream, 0.2-0.3 mg/100 g in mullet). The fish was then wrapped in a tissue paper and fixed in a lead (Pb) plate. For the recording of the olfactory response, the skull of the fish was opened, and the forepart of the brain was exposed. Ag-AgCI type bipolar electrode (0.15mm dia, 1-1.5 mm apart) insulated with lacquer except the tips was placed on the surface of the olfactory bulb. The electrical activity from the bulb was amplified and displayed on a pen recorder. The stimulating solution was introduced to the olfactory mucosa by adding the solution (0.1 ml) to a continuously flowing seawater (5-8 ml/min) to the olfactory sac. The diluting factor of the stimulating solution
217
218
YASUMASAGOH and TAMOTSUTAMURA
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IO-5M mt,~.,""a . . ~ , . , Fig. 1. Schematic diagram showing a recording system of the taste response. S, stimulus solution; CS, continuously flowing seawater to the responsive area; OSC, oscilloscope; E, electrode; GS, seawater for gill irrigation; SN, stimulating nozzle.
added to the continuously flowing seawater was about 1/20 (Goh et al., 1979). In the recording of the taste response, the eyeball of the fish was removed, and a branch of the facial and trigeminal nerve complex innervating the lower lip region or a branch of the facial nerve innervating the anterior palate region (Herrick, 1899, 1901) was exposed, and isolated from the connective tissue and from the central connection. The nerve branch was then hooked on a bipolar platinum electrode. The electrical activity from the nerve bundle was amplified, passed through an integrating circuit and displayed on a pen recorder. A continuous flow of the seawater (40 ml/min) was directed to the lower lip or anterior palate region, and the stimulating solution (0.2ml) was injected into the continuously flowing seawater (diluting factor about 1/2) (Fig. 1). Throughout the experiment, the gills were perfused with seawater. The water used in the experiment was filtrated natural seawater. The pH value of the seawater was about 8.1, and the stimulating solution of each chemical was adjusted to pH 7.5-8.5 with 1/10 N HCI and 0.4% NaOH.
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Fig. 2. A series of olfactory responses to L-glutamine 10-2-10 -8 M in red sea bream of MS.
RESULTS Olfactory response to amino acids R e d sea bream. In order to test the similarity of the olfactory spectrum of a m i n o acids a m o n g different strains, we recorded the olfactory response from the olfactory bulbs of two stocks of red sea b r e a m ; Mie
01 m
rr
41
41
Fig. 3. Olfactory spectra of amino acids in three stocks of red sea bream. Lower bar, YS (Goh et al., 1979); middle bar, SS; upper bar, MS; * not tested; each bar represents a relative response magnitude (+SD) as a percentage of the response to standard L-glutamine 10 -3 M.
Gustatory responses by fish to amino acids I0-2H
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Fig. 4. A series of olfactory responses to L-glutamine 10-2-10-7 M in mullet.
stock (MS) and Shizuoka stock (SS), and the results were compared to those obtained from Yamaguchi stock of rod sea bream (YS) by Gob et al. in 1979. Figure 2 shows an example of the bulbar response to L-glutamine 1 0 - 2 - 1 0 - S M in MS. The response magnitude increased with the increase of the stimulus concentration, and the application of seawater, as a control, induced no response. The threshold concentration for L-glutamine was about 10 - s M under the condition of the present experiment. Figure 3 shows the three spectra of 15 amino acids and betaine in 11 fishes of MS, 6 fishes of S.S, and fishes of YS. All the chemicals were tested at the same concentration of 10- 3 M. The response magnitude of each chemical is the mean amplitude of the induced
219
waves in the initial 5 sec, and expressed as a percentage of that obtained by the stimulation with the standard 1 0 - 3 M L-glutamine. From this figure, it is reasonably concluded that three stocks have the almost same spectrum. In order to compare the three spectra more quantitatively, we computed the regression lines and their correlation coefficients by the method of least squares in each pair of the spectra of the three stocks. The equations of the regression lines and their correlation coefficients were y = 0.97x + 3.71 (r = 0.97) between YS and MS, y = 1.03x - 0.26 (r = 0.98) between MS and SS and y = 1.01x - 4.06 (r = 0.99) between SS and YS. Mullet. Figure 4 shows an example of the olfactory response to L-glutamine 10- 2-10-" M obtained from the olfactory bulb of the mullet. The response magnitude increased with the increase of the stimulus strength, and the threshold concentration for L-glutamine was about 10-TM in this fish under the present experimental condition. The olfactory spectrum of amino acids in 6 fishes of mullet is shown in Fig. 5. All chemicals were tested at the same concentration of l0 -3 M. The spectrum in the mullet was compared to that in carp (Goh & Tamura, 1978), red sea bream (YS) (Gob et al., 1979), rainbow trout (Hara, 1973) and catfish (Suzuki & Tucker, 1971) by computing the regression line and its correlation coefficient. The equations of the regression fines and their correlation coefficients were y = 1.04x + 4.31 (r -- 0.98) between red sea bream and mullet, y = 0.83x + 20.09 (r--0.91) between catfish and mullet, y - - 0 . 5 8 x + 30.89 (r = 0.72) between rainbow trout and mullet and y = 1.30x - 15.80 (r = 0.84) between carp and mullet. Taste response to amino acids Red sea bream. In the recording of the taste responses, we stimulated different regions of the gustatory system between large fish (Shizuoka stock of red sea bream and mullet) and small fish (Mie stock of red sea bream), because of the technical convenience.
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Fig. 5. Olfactory spectrum of amino acids in mullet. Other notation of this figure is same as in Fig. 3.
220
YASU~,SAGOH and TAMOTSUTAMURA !llllllllml
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Fig. 6. A series of gustatory responses to L-alanine 10-z-10 -7 M in red sea bream of MS. Note that there is a small response even at 10 -6 M. In the former fishes, the anterior palate region was stimulated and from the nerve running to this region the responses were recorded. In the latter fishes, the lower lip region was stimulated and the responses were recorded from the nerve running to this region. Figure 6 shows an example of the integrated taste response to L-alanine 10-2-10 -6 M in MS. The response in this experiment always consisted of an apparent phasic phase and a small tonic phase probably because of the short stimulating duration. The response magnitude was, therefore, represented by the height of the phasic response. The response to L-alanine increased with the increase of the stimulus concentration, and the threshold concentration was about 10 -6 M. The gustatory spectra of amino acids (the sequence of the stimulatory effectiveness of amino acids in taste) were determined in 12 fishes of MS and 7 fishes of SS as shown in Fig. 7. All chemicals were tested at the same concentration of 10 -z M. The spectra were more or less same between the two stocks of red sea bream, although the responses to some amino acids such as g-lysine, L-glutamine, L-proline and betaine were slightly larger in SS than
in MS. However, the difference in the responses between the two groups may partly be attributed to the different gustatory systems mentioned above, since even in the same stock (MS) the responses to these amino acids were different between the two gustatory systems (Fig. 8), and the afferent trigeminal component of the anterior nerve bundle of facialtrigeminal complex was reported not to respond to taste stimuli (Kiyohara et al., 1975). Because of the small number of the available fish, each response obtained from anterior palate region is represented as a dot in Fig. 8. In each spectrum of MS and SS, L-alanine was the most effective amino acid, and glycine, L-arginine and L-serine were relatively effective. L-alanine + betaine and giycine + betaine were very effective because of the synergistic interaction (Hidaka et al., 1976). L-tyrosine and L-cystine were almost non-stimulative. In addition to the test of amino acids, we tested seven nucleotides (adenosine, AMP, ADP, ATP, IMP, U M P and GMP), but all the nudeotides were nonstimulative in the fishes of both the stocks, when the test solution was 10 -2 M. Mullet. In mullet, the taste response was recorded
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Fig. 7. Gustatory spectra of amino acids in two stocks of red sea bream. Upper bar, MS; lower bar, SS, each bar represents a relative response magnitude (+ SD) as a percentage of the response to standard L-alanine 10-2 M. The concentration of 1/2 (alanine + betaine) and 1/2 (glycine + betaine) is adjusted to l 0 - 2 M of total of both the chemicals.
Gustatory responses by fish to amino acids
221
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Fig. 8. A comparison of the taste spectra of amino acids in red sea bream of MS between two taste systems; lower lip region and anterior palate. Each dot represents a response obtained from anterior palate. Each bar represents a relative response magnitude (___SD) obtained from lower lip region. from a branch of the facial nerve running to the anterior palate. Figure 9 shows a series of responses to L-alanine 10-2-10 - 7 M. The threshold concentration to this amino acid was about 10 -6 M. Figure 10 shows the taste spectrum of amino acids in 7 mullet, when the amino acids were tested at the same concentration of 10-ZM. The most effective amino acid was L-arginine, and L-lysine, L-alanine and L-serine were the next effective amino acids. L-cystine, L-tyrosine and L-isoleucine were not effective. Seven nucleotides tested in red sea bream were also tested in mullet, but none of the nucleotides was effective in inducing any response. DISCUSSION
Olfaction Since the olfactory epithelium of Atlantic salmon (Sutterlin & Sutterlin, 1971) and catfish (Suzuki &
Tucker, 1971) was found to respond well to amino acids, the electrical response to amino acids has been investigated in the olfactory system of many fish species. From the experiments, it is concluded that the olfactory system of fishes has high sensitivity to amino acids, and the threshold concentration of the olfactory organ is ranging from 10 - 9 to 10-6M for some effective amino acids. In the present experiment, the threshold concentration for L-glutamine in red sea bream of Mie stock and for mullet was about 10- s M and about 10-TM respectively, and the high sensitivity to amino acids was also demonstrated in the two species. Comparing the olfactory spectrum among the three different stocks of red sea bream: Yamaguchi stock (Goh et al., 1979), Mie stock and Shizuoka stock, high similarity was found in each pair of the spectra (r = 0.97 between YS and MS, r = 0.98 between MS
LLIiiLL$1i I
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Fig. 9. A series of the taste responses to L-alanine 10-z-10-7 M in mullet. Note that there is a small response at 10-4 M.
222
YASUMASAGOH and TAMOTSUTAMURA
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Fig. 10. Gustatory spectrum of amino acids in mullet. Each bar represents a relative response magnitude (± SD) as a percentage of the response to the standard L-arginine 10-2 M. Other notation of this figure is same as in Fig. 7. and SS and r = 0.99 between SS and YS). The fact that the threshold concentration for L-glutamine is similar between YS and MS, and that the olfactory spectrum of amino acids is very close in the three stocks of red sea bream suggests that the olfactory sensation to amino acids may be more or less same in any stock of red sea bream. In our previous work ((;oh & Tamura, 1978; Goh et al., 1979), we pointed out that the olfactory spectrum of amino acids is similar among catfish, Atlantic salmon, rainbow trout, brook trout, whitefish, char, carp and red sea bream, and we assumed that the olfactory spectrum may be similar in all teleosts. In order to confirm the assumption, the olfactory spectrum of amino acids in mullet was obtained and compared to that in catfish (Suzuki & Tucker, 1971), rainbow trout (Hara, 1973), red sea bream (Gob et al., 1979) and carp (Goh & Tamura, 1978). In each pair of the spectra the similarity was high (r = 0.98 between red sea bream and mullet, r = 0.91 between catfish and mullet, r = 0.72 between rainbow trout and mullet and r = 0.84 between carp and mullet). These results support the assumption that the olfactory spectrum of amino acids is similar in all teleosts. Taste The gustatory response to amino acids has been reported in several fishes, and the taste spectrum of amino acids is different from species to species. For instance, the taste system of hake was reported to be sensitive to L-aspartic acid, L-glutamic acid, L-phenylalanine and glycine (Bardach & Case, 1965), while other "fishes were sensitive to other amino acids, i.e. bullhead was sensitive to L-cysteic acid, L-cysteine and L-alanine, tomcod to L-cysteine (Bardach et al., 1967a), Atlantic salmon to L-proline (Sutterlin & Sut-
terlin, 1970), catfish to L-alanine, L-arginine, L-serine and L-glutamine (Caprio, 1975), puffer to L-proline, glycine and L-alanine (Hidaka et al., 1975), Japanese eel to L-arginine, glycine, L-alanine, L-proline, L-lysine (Yoshii et al., 1979). The taste spectrum obtained in this study in red sea bream and mullet was also different from that in any other fishes reported earlier, though the spectrum in Japanese eel was considerably similar to that in mullet. Comparing the two spectra in mullet and red sea bream, the response magnitude to 12 amino acids and alanine + betaine was considerably similar, but that to L-arginine, L-lysine, glycine and L-glutamine was much different. Thus, the species specificity of the gustatory spectrum was also confirmed in red sea bream and mullet. We would like to assume that the species specificity of the taste spectrum of amino acids could be due to different feeding substances among different species. If this is true, the taste spectrum may be different even in the same species of different stocks. In the comparison of the spectrum between Mie stock and Shizuoka stock of red sea bream, however, we could not find a large difference in the spectrum as expected. But the taste spectrum of amino acids in the two stocks differed clearly from that in the same species investigated by Hidaka (personal communication), where the responses to L-arginine and L-histidine were much larger than the responses to the same two amino acids obtained in our study. This difference is very interesting, but further studies are needed to make any conclusion on the problem. Functional difference between olfaction and taste Since amino acids were found to be good stimulants not only for the taste system but also for the olfactory system of fish, it has become an important
Gustatory responses by fish to amino acids problem what is the functional difference between the responses to amino acids in olfaction and in taste. The solution to this problem may be given by comparing the threshold between the two sensory systems. The threshold concentration for amino acids seems to be lower in olfaction than iff taste except for a case in catfish (Caprio, 1975, 1978). In the present experiment, the threshold concentration in olfaction of the two species was also lower than that in taste. If the threshold decided from the electrical responses represents an actual sensitivity in the daily behaviour of fish, the higher sensitivity to amino acids in olfaction than in taste may support the general recognition that the olfaction is a distant sense and the taste is a contact or near sense. An interspecies comparison of the spectra of amino acids in olfaction and taste may give some insight to this problem. As discussed above, the olfactory spectrum of amino acids seems to be similar through all the teleosts, but the taste spectrum of amino acids seems to be similar through all the teleosts, but the taste spectrum of amino acids is species specific. We presume that the species specificity of the gustatory spectrum of amino acids in fishes may be a reflection of the difference of their foods, and the taste responses to amino acids may have more intimate relationship to the feeding behaviour than the olfactory responses. Many studies on the role of amino acids in the feeding behaviour were performed in fishes, and amino acids were found to be effective in inducing the feeding urge in fishes. Few of the studies, however, identified the sense organ through which the amino acids were recognized. In yellow bullhead (Ictalurus natalis), the sense of taste was demonstrated to be able to function as a distant receptor, and the destroy of the olfactory epithelium caused no effect on the feeding behaviour (Bardach et al., 1967b; Atema, 1971). But this may be considered to be a special case, because the taste sensitivity to amino acids in channel catfish (I. punctutus) was revealed to be higher than the olfactory sensitivity, to amino acids (Caprio, 1975, 1978). In conclusion, we would like to state that the taste sensation to amino acids have far more intimate relationships to the feeding behaviour of the fish than the olfactory one.
Acknowledgements--We wish to express our thanks to Professor C. Shimizu of Fisheries Laboratory of University of Tokyo for permitting use of the facilities. Thanks are also due to the staff of the Laboratories for providing the fishes, and to Mrs K. Koga for preparing the manuscript. This work was supported in part by research grant from the Ministry of Education of Japan. REFERENCES ADRONJ. W. & MACKIEA. M. (1978) Studies on the chemical nature of feeding stimulants for rainbow trout, Salmo gairdneri Richardson. J. Fish. Biol. 12, 303-310. ArEMA J. (1971) Structures and functions of the sense of taste in the catfish (lctalf~rus natalis). Brain. Behav. Evol. 4, 273-294. BAaD^CH J. E. & CASEJ: (1965) Sensory capabilities of the modified fins of squirrel hake (Urophycis chuss) and searobins (Prionotus carolinus and P. evolans). Copeia 2, 194-206.
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Attractants for eels in the extracts of short-necked clam--II. Survey of constituents eliciting feeding behavior by fractionation of the extracts. Bull. Jap. Soc. scient. Fish. 34, 84-87. OHSUGi T., HID^K^ I. & IKEDA M. (1978) Taste receptor stimulation and feeding behaviour in the puffer, Fuou paradalis--II. Effects produced by mixture of constituent of clam extract. Chem. Senses Flay. 3, 355-368. P^WSON M. G. (1977) Analysis of a natural chemical ~ttractant for whiting Merlangius merlangus L. and cod Gadus morhua L. using a behavioural bioassay. Cutup. Biochem. Physiol. 56A, 129-135. SUTTERLIN A. (1975) Chemical attraction of some marine fish. In Olfaction and Taste, Vol. 5 (Edited by DENTON D. A. & CtmnL~N J. P.), pp. 153-156. Academic Press, New York.
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