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Effect of amino acids on the feeding behaviour in red sea bream
Comp. Biochem. Physiol., Vol. 6612, pp. 225 to 229
0306-4492/80/0701-0225502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
EFFECT OF AMINO ACIDS ON THE FEEDING BEHAVIOUR IN RED SEA BREAM YASUMASAGOH and T~OTSU TAMURA Fisheries Laboratory, Faculty of Agriculture, Nagoya University, Nagoya, Japan
(Received 19 November 1979)
Abstract--1. The effects of amino acids and betaine on the feeding behaviour of red sea bream (Chrysophyrys major) were studied by adding the chemicals to a casein-base purified diet. 2. Among the 15 chemicals tested, 1/2 (alanine + betaine), 1/2 (glycine + betaioe), L-alanine, L-valine, glycine, L-serine, L-arginine and L-glutamine were found to be effective in activating the feeding behaviour. 3. The behavioural experiments were concluded to harmonize well with the electrical activities of the gustatory system rather than those of the olfactory system.
INTRODUCTION
MATERIALS AND METHODS
Since amino acids were found to be good stimulants not only for the taste but also for the olfaction in fish, it has become very important problem what is the functional difference of the response to amino acids between the olfaction and the taste. In our previous electrophysiological study (Goh et al., 1979), we pointed out that the responses to amino acids in taste may have an intimate relation to the feeding behaviour of fish but those in olfaction may have no direct relationship to the behaviour. Many behavioural studies show that amino acids are effective on the feeding behaviour of fishes, and some of the studies suggest that the taste plays a more important role than the olfaction in the behaviour. For instance, betaine which was electrophysiologically known to enhance the responses to several amino acids when it was combined with certain amino acids (Hidaka et al., 1976; G o h & Tamura, 1980), was also known to enhance the feeding urge to amino acids mixtures (Ohsugi et al., 1978; Carr & Chaney, 1976). However, these previous works except a series of studies by Hidaka and his colleagues (Hidaka et al., 1975, 1976, 1978; Kiyohara et al., 1975; Ohsugi et al., 1978), were limited to the electrophysiological study or the behavioural study only in the different species, and even the studies by Hidaka and his colleagues offered no information concerning the sense of olfaction. Thus, there are, to our knowledge, no data showing the relationship between results obtained electrophysiologically from the olfactory system and behavioural responses to amino acids. In our previous work (Gob & Tamura, 1980), we obtained electrophysiologically the olfactory and gustatory responses to amino acids in red sea bream. The present study was designed to compare the electrophysiological results to the feeding behavioural response to amino acids by using the same stock of fishes as used in our previous work. 225
Fish maintenance Red sea bream (Chrysophyrys major; T.L. 75-115mm) used in this study were the same as used in the previous work ((/oh & Tamura, 1980). The fish were kept in an out-door tank (2 x l x 1 m depth) and fed on the same diet as used in the experiment mentioned below.
Experimental tank Eight tanks (24 x 45 x 29 cm depth) used in this experiment were made of glass, and were supplied with a continuous in-flow of natural seawater (about 4 l/min). The water level of the tank was kept consistently of 25 cm depth by regulating the out-flow of the water with a siphon. The tanks were visually isolated from each other by a sheet to avoid the visual effects of the neighbouring fishes and other disturbances. For keeping a uniform lighting condition of any part in a tank, a fluorescent lamp was hung above the tank and switched on during day time hours. The temperature of the tank water was 22-28.5°C.
Preparation of the food-ball The basic diet for food-balls was made of purified components after Yone & Fujii (1975). The composition of the basic diet is shown in Table 1. The diet was made once Table 1. The composition of the basic diet Casein from milk Gelatin Dextrin Pollack residual oil Mineral mixture* Vitamin mixturet u-Cellulose Water
54 12 8 9 8 3 6 200
Total
300
* McCollum salts mixture (Nakarai Chemicals Ltd, Kyoto). t Panvitan powder (Takeda Chemical Industries Ltd, Osaka).
226
YASUMASA GOH and TAMOTSU TAMURA To Recorder
spiral wire of stainless steel which kept the ball tolerable against repeated attacks of the fishes. er I
In-Flow
Fig. 1. Schematic diagram showing the experimental apparatus. The tank was covered with a sheet (not shown in this figure) to avoid visual effects. every two or three days and stored at the temperature of 4°C. The test chemicals were added to the diet just before each test. The concentration of added amino acids was represented as mole/liter of water contained in the diet. The pH of the diets of both amino acid flavoured and non-flavoured was adjusted to 6.0-6.5 by ½N NaOH and ½N HCI. In this manner the test diet was produced without changing the colour and the texture. The diet was made into a ball of about 1.2 cm in diameter with a core of a
Experimental procedure The fish accustomed to the basic diet for several months in the out-door pond was transferred individually to the eight tanks a week prior to the experiment. During the week, the fish was trained once a day to attack a pair of the non-flavoured food-balls. For testing the effect of amino acids, a pair of the foodballs, one of which was flavoured with the amino acid and the other non-flavoured, was hung from a pair of special mechano-electric transducers with 10cm apart, and put into the water at the depth of 10 cm. The attacks of the fish to the food-balls were translated to electrical signs by the transducer, and the signs were displayed on a pen recorder (Fig. 1). The position of the paired food-balls of flavoured and non-flavoured was changed alternatively. The test was mostly performed twice a day (lst 10:00-11:00a.m., 2nd 4:00-5:00p.m.); only one test was, however, done when the temperature of the tank water was lower than 24°C.
Data analysis The number of the attacks of the fish to the paired foodballs in the initial 150 see was counted from the recordings. The preference or repellence of the fish to the tested amino acids was determined from the numbers of attacks to the paired food-balls with t-test.
illl;[[lll A
B
C
D
E
A
E
~- .....
Time in Sec II jill
Fig. 2. Taste (upper traces) and olfactory (lower traces) responses to the purified diet. A: response to the purified diet without vitamin mixture and mineral mixture; B: response to purified diet without mineral mixture; C: response to full purified diet: D, response to L-alanin¢ 10 -z M in taste and to L-glutamine 10-3M in olfaction: E, control.
227
Effect of amino acids on fish feeding ! 40
30
"~ 20
-I0
I
2
3
4
5
6
7
Number of Experience
Fig. 3. A relationship between the response magnitude (Rv) to L-alanine 10- t M and increase of experience. A relative response value (Rv) was computed with the following equation. Rv = (Nt - Nc)/(Nt + Nc) x 100 where Nt = Number of attacks to flavoured food-ball, Nc = Number of attacks to non-flavoured food-ball.
E lectr ophysiolog y For testing the olfactory and gustatory quantity of the diet, electrophysiological experiments were performed. The procedure of the experiments were the same as described elsewhere (Goh & Tamura, 1980). RESULTS
In order to test the stimulatory effects of the basic diet, 1 g of the m a s h e d diet was suspended in 100ml of the seawater a n d the s u p e r n a t a n t solution was used as a stimulant for the electrophysiologieal experiments. The results showed that the s u p e r n a t a n t solution h a d n o effects o n the olfaction a n d taste of the fish (Fig. 2), suggesting that the diet m a y be odourless a n d tasteless to the fish. At the same time, Table 2. Behavioural responses to 17 chemicals at the same concentration of 10- t M Chemicals ½(ala + bet) ½(gly + bet) L-alanine L-valine glycine L-serine L-proline L-arginine L-glutamine L-lysine betaine L-tyrosine L-threonine L-leucine L-histidine L-cystine L-methionine control
n 10 10 32 24 20 10 lO 13 12 8 16 14 8 lO lO 20 6 37
Rv ___SE 45.8 40.6 33.9 33.6 32.8 32.8 26.4 22.6 21.3 14.0 4.0 -- 1.3 --3.0 --4.6 - 8.8 --9.4 -- 14.0 -3.7
+ 8.72 + 9.87 + 3.44 _ 5.44 _+ 6.60 _ 9.08 _+ 7.22 _ 10.11 _ 9.47 -t- 9.27 _+ 10.30 _ 12.20 _+ 3.54 -t- 8.91 + 11.20 _ 8.97 _ 12.18 + 6.99
§ P < 0.00l, ~/P < 0.005, i" P < 0.01, *P < 0.05.
alanine + betaine a n d glycine + betaine were tested to the olfactory system at the concentration of 1 0 - 3 M of the total of b o t h the chemicals. However, n o synergistic effect was observed in the olfactory response, t h o u g h the effect was very clear in the taste response as shown by G o h & T a m u r a (1980). In the initial trials, the fish preferably attacked one side of the paired food-balls regardless of flavoured or non-flavoured. The choice of the one side of the paired food-balls appeared to be decided by chance. However, after the repeated experiences, the fish tended to attack more frequently the a m i n o acid flavoured food-ball than non-flavoured one when the a m i n o acid was effective. Consequently, Rv to the flavoured one increased. Figure 3 shows the increase of Rv to L-alanine flavoured ball with the increase of the experience of trials. Rv to this a m i n o acid was saturated at a b o u t 30 after the 4th trial. Even the fish which h a d once gained high Rv to L-alanine, attacked the non-flavoured food-ball at the beginning of succeeding trials as frequently as the flavoured one, b u t soon the attacks were tended to concentrate o n the latter. This was one of the reasons why Rv did not become more t h a n the values a r o u n d 50 (Table 2). The increase of Rv with the increase of experience was also observed in L-valine, a n d the saturated Rv to this a m i n o acid was a b o u t 30. The fish which h a d once gained saturated Rv to L-alanine, responded well also to L-valine with the saturated Rv of a b o u t 30 even at the first trial. Therefore, in the following experiment, we used the Rv to L-alanine 10-1 M as an indicator to check the normality of the fish behaviour. Figure 4 shows the relation of Rv to L-alanine 1 0 - 3 - - 5 x 10 -1 M. The response increased with the logarithmic increase of the stimulus concentration of the range between 10 -3 a n d 10 -2 M. The response to 10- 3 M was doubtful (n = 32, t = 1.60, P < 0.2), b u t 2 x 10 -3 M L-alanine was effective to induce the preference response (n = 15, t = 2.16, P < 0.05). Rv to L-alanine was saturated at 1 0 - 2 M (n = 30, t = 7.47, P < 0.001), a n d it was a b o u t 30. The control test was performed with a pair of the food-balls of n o chemical additive, resulting Rv of the control test was a b o u t zero (Fig. 3, Fig. 4 a n d Table 2).
t 5.25§ 4.11~/ 7.45§ 6.18§ 4.97§ 3.61t 3.66t 2.24* 2.25* 1.52 0.39 0.11 0.85 0.52 0.79 1.05 1.t4 0.53
50
"~ 25 E
~
1 I
J
I
I
2x 5x Control
I0 -3
I
I
2x 10-2
I
1
5x I0 - I
I0 0
Fig. 4. A relationship between the response magnitude (Rv) and the molar concentration of L-alanine.
228
YASUMASAGOH and TAMOTSUTAMURA
In order to compare the behavioural results to the electrophysiological results of olfaction and taste (Goh & Tamura, 1980), 14 amino acids, betaine, alanine + betaine and glycine + betaine were tested at the same concentration of 10-1 M. Table 2 shows the feeding behavioural spectrum of these chemicals. In the series of this experiment, Rv to L-alanine was often checked to be about 30. The effectiveness of the chemicals to induce the preference response was in the following order; ½ (alanine + betaine) > ½ (glycine + betaine) > L-alanine > L-valine > glycine -- Lserine > L-proline > L-arginine > L-glutamine. Other amino acids were not recognized to be effective by the statistic analysis, though some amino acids such as L-lysine and L-methionine might be preferred or repelled by the fish. DISCUSSION The present experiment was designed to investigate the role of amino acids as a stimulus to activate the fish to attack and to swallow the food. The stimulus due to the amino acids is supposed to be received by two chemical sensory systems--the olfaction and the taste. In this experiment, several amino acids, namely ½ (alanine + betaine), ½ (glycine + betaine), alanine, glycine, arginine, L-serine, L-proline, L-valine and L-glutamine which were effective in inducing the electrical taste response (Goh & Tamura, 1980) were also effective in activating the food-ball attacking behaviour, although L-threonine, betaine, L-leucine, L-histidine and L-methionine were not effective in the attacking behaviour in spite of their effectiveness on the taste receptors. However, all amino acids which induced the preference response were always effective on the taste receptors. Furthermore, betaine which was known to enhance the neural responses to L-alanine and glycine in taste (Goh & Tamura, 1980) were also proved to enhance the food-ball attacking behaviour when it was combined with each of the two amino acids. On the other hand, the order of the effectiveness of amino acids in the behaviour was much different from the olfactory spectrum of amino acids. For the most typical example, L-proline was hardly effective on the olfactory bulbar response (Goh & Tamura, 1980), but it was effective on the behaviour. Furthermore, no synergistic interaction between betaine and L-alanine or glycine was observed in the olfactory response, while the interaction was obtained in the feeding behavioural response. Thus, the comparison of the spectra of amino acids between the food-ball attacking behaviour and the taste or the olfaction revealed a high similarity between the behaviour and the taste and no similarity between the behaviour and the olfaction. To evaluate quantitatively the similarity or dissimilarity of the behavioural response to the neural responses of the taste and the olfaction, correlation coefficients were calculated by the method of least squares, resulting 0.78 between the behaviour and the taste and 0.01 between the behaviour and the olfaction. Similar results suggesting the strong relationship between the feeding behaviour and the sense of taste
were reported in puffer (Hidaka et al., 1978; Ohsugi et al., 1978). In the experiments, L-alanine, glycine, L-proline, L-serine and betaine which were found to be effective to the tip chemoreceptor of the puffer have potency for the fish to accept a starch pellet when they were added singly to the pellet. Furthermore, a mixture of these amino acids plus betaine were much more effective than a mixture of the amino acids only. Betaine was also known in the neural response of the lip chemoreceptor of the same fish to enhance the response to L-alanine, glycine and L-serine (Hidaka et al., 1976). The experiments by Carr and his colleagues (Carr, 1976; Carr & C h a n e y , 1976; Carr et al., 1976) also suggest the intimate relation between the taste and the behaviour of fish. In the experiments, with a cue of pure chemical stimulus only, the fishes attacked a solution-delivering rubber, suggesting that amino acids may be received by the sense of taste rather than olfaction, since betaine enhanced the stimulatory effectiveness of a mixture of amino acids when it was mixed. Higher effectiveness of amino acids mixtures than any single amino acids was reported in neural taste responses in puffer and Japanese eel (Hidaka et al., 1976; Yoshii et al., 1979). Similar effect of amino acids mixtures were also demonstrated behaviourally in cod and whiting (Pawson, 1977) and Japanese eel (Hashimoto et al., 1968; Konosu et al., 1968). Thus, a strong relationship between the taste response to amino acids and the feeding behaviour of fish was demonstrated in many works. It is said that yellow bullhead (lctarulus natalis) can orientate the food only by the sense of taste (Bardach et al., 1967; Atema, 1971). Indeed, the taste sensitivity to amino acids in channel catfish (I. punctatus) is known to be higher than the olfactory one (Caprio, 1975, 1978). In some fishes except a case of this channel catfish, electrophysiological studies showed that the sensitivity to amino acids was higher in olfaction than in taste (Sutterlin & Sutterlin, 1970, 1971; Goh & Tamura, 1980), and seems to support the general recognition that taste is a contact or near sense and olfaction is a distant sense. However, when we think that olfactory response to amino acids is species non-specific while taste response to amino acids is species specific (Goh et al., 1979; Goh & Tamura, 1980) and that many behavioural works demonstrate the strong relation of the taste to the behaviour, the role played by the taste organ in feeding behaviour as a distant receptor to amino acids has to be studied in various species of fish. Acknowledoements--We wish to express our thanks to Professor C. Shimizu of Fisheries Laboratory of University of Tokyo for permitting use of the facilities. This work was supported in part by research grant from the Ministry of Education of Japan.
REFERENCES
AT~MAJ. (1971) Structures and functions of the sense of taste in the catfish (Ictalurus natalis). Brain. Behav. Evol. 4, 273-294. BARDACHJ. E., TODDJ. H. & CRICKMERR. (1967) Orienta-
Effect of amino acids on fish feeding tion by taste in fish of the genus lctalurus. Science, N. Y 155, 1276--1278. CAPRIO J. (1975) High sensitivity of catfish taste receptors to amino acids. Comp. Bioehem. Physiol. 52A, 247-251. CAPRIO J. (1978) Olfaction and taste in the channel catfish: An electrophysiological 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. & CHANEY T. B. (1976) Chemical stimulation of feeding behavior in the pinfish, La#odon rhomboides: Characterization and identification of stimulatory substances extracted from shrimp. Comp. Biochem. Physiol. 54A, 437--441. CARR W. E. S., GONDECK A. R. & DELANOY R. L. (1976) Chemical stimulation of feeding behavior in the pinfish, Lagodon rhomboides: A new approach to an old problem. Comp. B iochem. Physiol..r~4A, 161-166. GOHY. & TAMUR^ T. (1980) Olfactory and gustatory responses to amino acids in two marine teleosts---red sea bream and mullet. Comp. Biochem. Physiol. 66, 217-224. GOH Y., TAMURAT. & KOBAYASHI H. (1979) Olfactory responses to amino acids in marine teleosts. Comp. Biochem. Physiol. 62A, 863-868. HASHIMOTO Y., KONOSU S., FUSETANI N. & 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. HIO^KA I., KIeOHARA S. & TABATA M. (1975) Gustatory responses in the puffer. Bull. Jap. Soc. scient. Fish. 41, 275-281. HIDAKA I., NVU N. & KIVOHARA S. (1976) Gustatory re-
229
sponse in the puffer--IV. Effects of mixtures of amino acids and betaine. Bull. Fac. Fish. Mie Univ. 3, 17-28. HIDAKA I., OHSUGI T. & KOBOMATSUT. (1978) Taste receptor stimulation and feeding behaviour in the puffer, Fugu paradalis--I. Effect of single chemicals. Chem. senses Flavour 3, 341-354. KIYOHARA S., HIDAKA I. & TAMURA T. (1975) Gustatory response in puffer--II. Single fiber analyses. Bull. Jap. Soc. scient. Fish. 41, 383-391. KONOSU S., FUSETANI N., NOSE T. & HASHIMOTO Y. (1968) 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., HIDAKA I. & IKEDA M. (1978) Taste receptor stimulation and feeding behaviour in the puffer, Fugu paradalis--II. Effects produced by mixture of constituent of clam extract. Chem. senses Flavour 3, 355-368. PAWSON M. G. (1977) Analysis (ff a natural chemical attractant for whiting Merlan#ius merlan#us L. and cod Gadus morhua L. using a behavioural bioassay. Comp. Biochem. Physiol. $6A, 129-135. SUTTERLIN A. M. & SUTIERUN N. (1970) Taste responses in Atlantic salmon (Salmo salar) parr. J. Fish. Res. Bd Can. 27, 1927-1942. SUTTERLIN A. M. & SUTTERLIN N. (1971) Electrical responses of the olfactory epithelium of Atlantic salmon (Salmo salar). J. Fish. Res. Bd Can. 28, 565-572. YONE Y. & FuJn M. (1975) Studies on nutrition of red sea bream--XI. Effect of to3 fatty acid supplement in a corn oil diet on growth rate and feed efficiency. Bull. Jap. Soc. scient. Fish. 41, 73-77. YOSHU K., KAMO N., KURIHARA K. & KOBATAKEY. (1979) Gustatory responses of eel palatine receptors to amino acids and carboxylic acids. J. gen. Physiol. 74, 301-317.
0306-4492/80/0701-0225502.00/0
© Pergamon Press Ltd 1980. Printed in Great Britain
EFFECT OF AMINO ACIDS ON THE FEEDING BEHAVIOUR IN RED SEA BREAM YASUMASAGOH and T~OTSU TAMURA Fisheries Laboratory, Faculty of Agriculture, Nagoya University, Nagoya, Japan
(Received 19 November 1979)
Abstract--1. The effects of amino acids and betaine on the feeding behaviour of red sea bream (Chrysophyrys major) were studied by adding the chemicals to a casein-base purified diet. 2. Among the 15 chemicals tested, 1/2 (alanine + betaine), 1/2 (glycine + betaioe), L-alanine, L-valine, glycine, L-serine, L-arginine and L-glutamine were found to be effective in activating the feeding behaviour. 3. The behavioural experiments were concluded to harmonize well with the electrical activities of the gustatory system rather than those of the olfactory system.
INTRODUCTION
MATERIALS AND METHODS
Since amino acids were found to be good stimulants not only for the taste but also for the olfaction in fish, it has become very important problem what is the functional difference of the response to amino acids between the olfaction and the taste. In our previous electrophysiological study (Goh et al., 1979), we pointed out that the responses to amino acids in taste may have an intimate relation to the feeding behaviour of fish but those in olfaction may have no direct relationship to the behaviour. Many behavioural studies show that amino acids are effective on the feeding behaviour of fishes, and some of the studies suggest that the taste plays a more important role than the olfaction in the behaviour. For instance, betaine which was electrophysiologically known to enhance the responses to several amino acids when it was combined with certain amino acids (Hidaka et al., 1976; G o h & Tamura, 1980), was also known to enhance the feeding urge to amino acids mixtures (Ohsugi et al., 1978; Carr & Chaney, 1976). However, these previous works except a series of studies by Hidaka and his colleagues (Hidaka et al., 1975, 1976, 1978; Kiyohara et al., 1975; Ohsugi et al., 1978), were limited to the electrophysiological study or the behavioural study only in the different species, and even the studies by Hidaka and his colleagues offered no information concerning the sense of olfaction. Thus, there are, to our knowledge, no data showing the relationship between results obtained electrophysiologically from the olfactory system and behavioural responses to amino acids. In our previous work (Gob & Tamura, 1980), we obtained electrophysiologically the olfactory and gustatory responses to amino acids in red sea bream. The present study was designed to compare the electrophysiological results to the feeding behavioural response to amino acids by using the same stock of fishes as used in our previous work. 225
Fish maintenance Red sea bream (Chrysophyrys major; T.L. 75-115mm) used in this study were the same as used in the previous work ((/oh & Tamura, 1980). The fish were kept in an out-door tank (2 x l x 1 m depth) and fed on the same diet as used in the experiment mentioned below.
Experimental tank Eight tanks (24 x 45 x 29 cm depth) used in this experiment were made of glass, and were supplied with a continuous in-flow of natural seawater (about 4 l/min). The water level of the tank was kept consistently of 25 cm depth by regulating the out-flow of the water with a siphon. The tanks were visually isolated from each other by a sheet to avoid the visual effects of the neighbouring fishes and other disturbances. For keeping a uniform lighting condition of any part in a tank, a fluorescent lamp was hung above the tank and switched on during day time hours. The temperature of the tank water was 22-28.5°C.
Preparation of the food-ball The basic diet for food-balls was made of purified components after Yone & Fujii (1975). The composition of the basic diet is shown in Table 1. The diet was made once Table 1. The composition of the basic diet Casein from milk Gelatin Dextrin Pollack residual oil Mineral mixture* Vitamin mixturet u-Cellulose Water
54 12 8 9 8 3 6 200
Total
300
* McCollum salts mixture (Nakarai Chemicals Ltd, Kyoto). t Panvitan powder (Takeda Chemical Industries Ltd, Osaka).
226
YASUMASA GOH and TAMOTSU TAMURA To Recorder
spiral wire of stainless steel which kept the ball tolerable against repeated attacks of the fishes. er I
In-Flow
Fig. 1. Schematic diagram showing the experimental apparatus. The tank was covered with a sheet (not shown in this figure) to avoid visual effects. every two or three days and stored at the temperature of 4°C. The test chemicals were added to the diet just before each test. The concentration of added amino acids was represented as mole/liter of water contained in the diet. The pH of the diets of both amino acid flavoured and non-flavoured was adjusted to 6.0-6.5 by ½N NaOH and ½N HCI. In this manner the test diet was produced without changing the colour and the texture. The diet was made into a ball of about 1.2 cm in diameter with a core of a
Experimental procedure The fish accustomed to the basic diet for several months in the out-door pond was transferred individually to the eight tanks a week prior to the experiment. During the week, the fish was trained once a day to attack a pair of the non-flavoured food-balls. For testing the effect of amino acids, a pair of the foodballs, one of which was flavoured with the amino acid and the other non-flavoured, was hung from a pair of special mechano-electric transducers with 10cm apart, and put into the water at the depth of 10 cm. The attacks of the fish to the food-balls were translated to electrical signs by the transducer, and the signs were displayed on a pen recorder (Fig. 1). The position of the paired food-balls of flavoured and non-flavoured was changed alternatively. The test was mostly performed twice a day (lst 10:00-11:00a.m., 2nd 4:00-5:00p.m.); only one test was, however, done when the temperature of the tank water was lower than 24°C.
Data analysis The number of the attacks of the fish to the paired foodballs in the initial 150 see was counted from the recordings. The preference or repellence of the fish to the tested amino acids was determined from the numbers of attacks to the paired food-balls with t-test.
illl;[[lll A
B
C
D
E
A
E
~- .....
Time in Sec II jill
Fig. 2. Taste (upper traces) and olfactory (lower traces) responses to the purified diet. A: response to the purified diet without vitamin mixture and mineral mixture; B: response to purified diet without mineral mixture; C: response to full purified diet: D, response to L-alanin¢ 10 -z M in taste and to L-glutamine 10-3M in olfaction: E, control.
227
Effect of amino acids on fish feeding ! 40
30
"~ 20
-I0
I
2
3
4
5
6
7
Number of Experience
Fig. 3. A relationship between the response magnitude (Rv) to L-alanine 10- t M and increase of experience. A relative response value (Rv) was computed with the following equation. Rv = (Nt - Nc)/(Nt + Nc) x 100 where Nt = Number of attacks to flavoured food-ball, Nc = Number of attacks to non-flavoured food-ball.
E lectr ophysiolog y For testing the olfactory and gustatory quantity of the diet, electrophysiological experiments were performed. The procedure of the experiments were the same as described elsewhere (Goh & Tamura, 1980). RESULTS
In order to test the stimulatory effects of the basic diet, 1 g of the m a s h e d diet was suspended in 100ml of the seawater a n d the s u p e r n a t a n t solution was used as a stimulant for the electrophysiologieal experiments. The results showed that the s u p e r n a t a n t solution h a d n o effects o n the olfaction a n d taste of the fish (Fig. 2), suggesting that the diet m a y be odourless a n d tasteless to the fish. At the same time, Table 2. Behavioural responses to 17 chemicals at the same concentration of 10- t M Chemicals ½(ala + bet) ½(gly + bet) L-alanine L-valine glycine L-serine L-proline L-arginine L-glutamine L-lysine betaine L-tyrosine L-threonine L-leucine L-histidine L-cystine L-methionine control
n 10 10 32 24 20 10 lO 13 12 8 16 14 8 lO lO 20 6 37
Rv ___SE 45.8 40.6 33.9 33.6 32.8 32.8 26.4 22.6 21.3 14.0 4.0 -- 1.3 --3.0 --4.6 - 8.8 --9.4 -- 14.0 -3.7
+ 8.72 + 9.87 + 3.44 _ 5.44 _+ 6.60 _ 9.08 _+ 7.22 _ 10.11 _ 9.47 -t- 9.27 _+ 10.30 _ 12.20 _+ 3.54 -t- 8.91 + 11.20 _ 8.97 _ 12.18 + 6.99
§ P < 0.00l, ~/P < 0.005, i" P < 0.01, *P < 0.05.
alanine + betaine a n d glycine + betaine were tested to the olfactory system at the concentration of 1 0 - 3 M of the total of b o t h the chemicals. However, n o synergistic effect was observed in the olfactory response, t h o u g h the effect was very clear in the taste response as shown by G o h & T a m u r a (1980). In the initial trials, the fish preferably attacked one side of the paired food-balls regardless of flavoured or non-flavoured. The choice of the one side of the paired food-balls appeared to be decided by chance. However, after the repeated experiences, the fish tended to attack more frequently the a m i n o acid flavoured food-ball than non-flavoured one when the a m i n o acid was effective. Consequently, Rv to the flavoured one increased. Figure 3 shows the increase of Rv to L-alanine flavoured ball with the increase of the experience of trials. Rv to this a m i n o acid was saturated at a b o u t 30 after the 4th trial. Even the fish which h a d once gained high Rv to L-alanine, attacked the non-flavoured food-ball at the beginning of succeeding trials as frequently as the flavoured one, b u t soon the attacks were tended to concentrate o n the latter. This was one of the reasons why Rv did not become more t h a n the values a r o u n d 50 (Table 2). The increase of Rv with the increase of experience was also observed in L-valine, a n d the saturated Rv to this a m i n o acid was a b o u t 30. The fish which h a d once gained saturated Rv to L-alanine, responded well also to L-valine with the saturated Rv of a b o u t 30 even at the first trial. Therefore, in the following experiment, we used the Rv to L-alanine 10-1 M as an indicator to check the normality of the fish behaviour. Figure 4 shows the relation of Rv to L-alanine 1 0 - 3 - - 5 x 10 -1 M. The response increased with the logarithmic increase of the stimulus concentration of the range between 10 -3 a n d 10 -2 M. The response to 10- 3 M was doubtful (n = 32, t = 1.60, P < 0.2), b u t 2 x 10 -3 M L-alanine was effective to induce the preference response (n = 15, t = 2.16, P < 0.05). Rv to L-alanine was saturated at 1 0 - 2 M (n = 30, t = 7.47, P < 0.001), a n d it was a b o u t 30. The control test was performed with a pair of the food-balls of n o chemical additive, resulting Rv of the control test was a b o u t zero (Fig. 3, Fig. 4 a n d Table 2).
t 5.25§ 4.11~/ 7.45§ 6.18§ 4.97§ 3.61t 3.66t 2.24* 2.25* 1.52 0.39 0.11 0.85 0.52 0.79 1.05 1.t4 0.53
50
"~ 25 E
~
1 I
J
I
I
2x 5x Control
I0 -3
I
I
2x 10-2
I
1
5x I0 - I
I0 0
Fig. 4. A relationship between the response magnitude (Rv) and the molar concentration of L-alanine.
228
YASUMASAGOH and TAMOTSUTAMURA
In order to compare the behavioural results to the electrophysiological results of olfaction and taste (Goh & Tamura, 1980), 14 amino acids, betaine, alanine + betaine and glycine + betaine were tested at the same concentration of 10-1 M. Table 2 shows the feeding behavioural spectrum of these chemicals. In the series of this experiment, Rv to L-alanine was often checked to be about 30. The effectiveness of the chemicals to induce the preference response was in the following order; ½ (alanine + betaine) > ½ (glycine + betaine) > L-alanine > L-valine > glycine -- Lserine > L-proline > L-arginine > L-glutamine. Other amino acids were not recognized to be effective by the statistic analysis, though some amino acids such as L-lysine and L-methionine might be preferred or repelled by the fish. DISCUSSION The present experiment was designed to investigate the role of amino acids as a stimulus to activate the fish to attack and to swallow the food. The stimulus due to the amino acids is supposed to be received by two chemical sensory systems--the olfaction and the taste. In this experiment, several amino acids, namely ½ (alanine + betaine), ½ (glycine + betaine), alanine, glycine, arginine, L-serine, L-proline, L-valine and L-glutamine which were effective in inducing the electrical taste response (Goh & Tamura, 1980) were also effective in activating the food-ball attacking behaviour, although L-threonine, betaine, L-leucine, L-histidine and L-methionine were not effective in the attacking behaviour in spite of their effectiveness on the taste receptors. However, all amino acids which induced the preference response were always effective on the taste receptors. Furthermore, betaine which was known to enhance the neural responses to L-alanine and glycine in taste (Goh & Tamura, 1980) were also proved to enhance the food-ball attacking behaviour when it was combined with each of the two amino acids. On the other hand, the order of the effectiveness of amino acids in the behaviour was much different from the olfactory spectrum of amino acids. For the most typical example, L-proline was hardly effective on the olfactory bulbar response (Goh & Tamura, 1980), but it was effective on the behaviour. Furthermore, no synergistic interaction between betaine and L-alanine or glycine was observed in the olfactory response, while the interaction was obtained in the feeding behavioural response. Thus, the comparison of the spectra of amino acids between the food-ball attacking behaviour and the taste or the olfaction revealed a high similarity between the behaviour and the taste and no similarity between the behaviour and the olfaction. To evaluate quantitatively the similarity or dissimilarity of the behavioural response to the neural responses of the taste and the olfaction, correlation coefficients were calculated by the method of least squares, resulting 0.78 between the behaviour and the taste and 0.01 between the behaviour and the olfaction. Similar results suggesting the strong relationship between the feeding behaviour and the sense of taste
were reported in puffer (Hidaka et al., 1978; Ohsugi et al., 1978). In the experiments, L-alanine, glycine, L-proline, L-serine and betaine which were found to be effective to the tip chemoreceptor of the puffer have potency for the fish to accept a starch pellet when they were added singly to the pellet. Furthermore, a mixture of these amino acids plus betaine were much more effective than a mixture of the amino acids only. Betaine was also known in the neural response of the lip chemoreceptor of the same fish to enhance the response to L-alanine, glycine and L-serine (Hidaka et al., 1976). The experiments by Carr and his colleagues (Carr, 1976; Carr & C h a n e y , 1976; Carr et al., 1976) also suggest the intimate relation between the taste and the behaviour of fish. In the experiments, with a cue of pure chemical stimulus only, the fishes attacked a solution-delivering rubber, suggesting that amino acids may be received by the sense of taste rather than olfaction, since betaine enhanced the stimulatory effectiveness of a mixture of amino acids when it was mixed. Higher effectiveness of amino acids mixtures than any single amino acids was reported in neural taste responses in puffer and Japanese eel (Hidaka et al., 1976; Yoshii et al., 1979). Similar effect of amino acids mixtures were also demonstrated behaviourally in cod and whiting (Pawson, 1977) and Japanese eel (Hashimoto et al., 1968; Konosu et al., 1968). Thus, a strong relationship between the taste response to amino acids and the feeding behaviour of fish was demonstrated in many works. It is said that yellow bullhead (lctarulus natalis) can orientate the food only by the sense of taste (Bardach et al., 1967; Atema, 1971). Indeed, the taste sensitivity to amino acids in channel catfish (I. punctatus) is known to be higher than the olfactory one (Caprio, 1975, 1978). In some fishes except a case of this channel catfish, electrophysiological studies showed that the sensitivity to amino acids was higher in olfaction than in taste (Sutterlin & Sutterlin, 1970, 1971; Goh & Tamura, 1980), and seems to support the general recognition that taste is a contact or near sense and olfaction is a distant sense. However, when we think that olfactory response to amino acids is species non-specific while taste response to amino acids is species specific (Goh et al., 1979; Goh & Tamura, 1980) and that many behavioural works demonstrate the strong relation of the taste to the behaviour, the role played by the taste organ in feeding behaviour as a distant receptor to amino acids has to be studied in various species of fish. Acknowledoements--We wish to express our thanks to Professor C. Shimizu of Fisheries Laboratory of University of Tokyo for permitting use of the facilities. This work was supported in part by research grant from the Ministry of Education of Japan.
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