Feeding Attraction Activities of Amino Acids and Lipids for Juvenile Yellowtail*1, 2

Transcription

1 Bulletin of the Japanese Society of Scientific Fisheries 51(3), (1985) Feeding Attraction Activities of Amino Acids and Lipids for Juvenile Yellowtail*1, 2 Katsuhiko HARADA*3 (Accepted July 21, 1984) Fractions of basic, neutral and acidic amino acids, and of neutral, phospho- and glyco-lipids were prepared from the amino acid- and the lipid-extracts obtained from the mid-gut gland of squid Todarodes pacificus. The basic amino acid- and the phospholipid-fractions induced a high behavioral feeding attraction activity for juvenile yellowtail Seriola quinqueradiata and the neutral amino acid- and the glycolipid-fractions did bring about a low activity. Of twenty-six authentic L- and DL-amino acids, the activities were found in histidine, arginine, lysine and ornithine among the basic amino acids. Especially, histidine and arginine strikingly induced an attraction behavior in the fish. In the neutral amino acids, glycine, valine, threonine, methionine and cystine were involved in the attraction, specially glycine and threonine showing high activity. In the acidic amino acids, asparatic acid and glutamic acid didn't show an activity, while their amides did. Both L-histidine and L-threonine elicited the attraction in contrast to D-types which showed little effects. Of thirty authentic lipids, the activities were high in many kinds of phospholipids. Among the other lipids, however, only palmitic acid and stearic acid, monostearin, and ceramide were effective. The threshold values were estimated to be 5 ~10-7 M and 6 ~10-8 M for L-histidine and egg lecithin, respectively. It has been demonstrated that the amino acid and the lipid-extracts from the mid-gut gland of squid Todarodes pacificus evoke remarkable ex ploratory and feeding behaviors for juvenile yellowtail Seriola quinqueradiata.1) This enabled the author to separate both the amino acid and the lipid into three fractions each: basic, neutral and acidic amino acid-ones, and neutral, phospho and glyco-lipid-ones, respectively, and to in vestigate whether they are effective in the attrac tion or not. The characterization and the identification of attractants in the fractions are, further - more, of practical importance. Various amino acids and lipids were therefore examined for the attraction behaviors of the fish. This paper deals with the results. Materials and Methods Experimental Juvenile Yellowtail One hundred and sixteen specimens of juvenile yellowtail of 5.0cm and 2.0g in average fork length and body weight were obtained from the Senzaki Station of Aquaculture, on June 30, The yellowtails were maintained in each compartment of an experimental aquarium2) into which sea water was continuously run at a velocity of 1000ml/min. The rearing was done in a similar fashion described previously2) At the end of the experiments, on October 31, 1983, the yellowtails grew to 18cm and 112g in average. Separation of Acidic, Neutral and Basic Amino Acids, and Neutral, Phospho- and Glyco-lipids The amino acids1) obtained from the midgut gland of the squid were separated into three frac tions of basic, neutral and acidic ones in a similar manner described in the preceding paper.3) The fractions were concentrated below 60 Ž in vacuo and the concentrates were dissolved in water to prepare test solutions containing 35mg/ml of amino acids as leucine. The solutions were adjusted to ph with sodium hydroxide. The crude lipids1) obtained from the midgut gland were separated into three fractions of neutral, phospho- and glyco-lipids by the following *1 Contribution from Shimonoseki University of Fisheries No *2 Studies on the Feeding Attractants for Fishes and Shells-X. *3 Laboratory of Biochemistry, Department of Food Science and Technology, Shimonoseki University of Fisheries, Shimonoseki , Japan (Œ c Ÿ F: ŽY åšw Z» Šw È»Šw ³Žº).

2 454 HARADA method: One volume of the crude lipids dissolved in a small amount of chloroform was added with ten volumes of acetone. The mixture was centri fuged for 20 min at 12,000 ~g, after incubating 1h at room temperature. The precipitate ob tained was further added with five volumes of acetone. The mixture was treated as above. Both supernatants (acetone soluble fraction, neutral lipids) were combined. The second pre cipitate was added with ten volumes of ether. The mixture was centrifuged to separate into super natant (ether soluble fraction, phospholipids) and precipitate (ether insoluble fraction, glycolipids). All the fractions were concentrated below 60 Ž in vacuo to remove solvents. The concentrates obtained were suspended in an appropriate amount of water and homogenated by Ultra-Turrax apparatus to prepare homogenates containing 12 mg/ml of lipids. The homogenates were adjusted to ph with sodium hydroxide. Authentic Chemicals Amino acids (guaranteed), purchased from Nakarai Chemical Co. Ltd., were as follows: L and D-histidines, L-arginine, L-lysine, DL-hydroxylysine hydrochloride and L-ornithine hydro chloride (thus far, basic amino acids), glycine, L-ƒ - alanine, L-valine, L-leucine, L-isoleucine, D- and L-threonines, L-serine, DL-phosphoserine, L- methionine, L-cystine hydrochloride, L-cysteine, DL-homocysteine, L-phenylalanine, L-tyrosine and L-tryptphan (thus far, neutral amino acids), and L-asparatic acid, L-asparagine, L-glutamic acid, L- glutamine, L-proline and L-hydroxyproline (thus far, amides, acidic amino and imino acids). Each amino acid-solution was prepared to a concentration of 225 mm of amino acid. The solutions were, if necessary, adjusted to ph by the addition of sodium hydroxide or hydrochloric acid. Fatty acids (extra pure), glycerine (extra pure), neutral lipids (extra pure), and phospholipids of lecithin and cephalin (extra pure) were purchased from Tokyo Kasei Chemical Co. Ltd.. Other phospholipids (analytical grade), and glycolipids (analytical grade) were bought from Sigma Che mical Co.. Fatty acids and lipids used were as follows: butyric, caproic, caprylic, capric, lauric, myristic, palmitic, stearic, arachidic, behenic and lignoceric acids (thus far, fatty acids), mono palmitin, monostearin, monoolein, tripalmitin, tristearin and triolein (thus far, neutral lipids), egg- and bean-lecithins, animal-cephalin,pho sphatidyl-l-serine, phosphatidyl-inositol, sphingo sine and cardiolipin (thus far, phospholipids), and cerobroside, ceramide and ganglioside (thus far, glycolipids). The chemicals were suspended in 10% polyvinyl alcohol with polymerization grade 1100, adjusted to ph by the addition of sodium hydroxide, and homogenized by Ultra- Turrax apparatus. The concentrations of fatty acids, neutral lipids, lecithin, and cephalin in the homogenates were 12 mg/ml, while those of the other phospholipids, and glycolipids were 1.2 mg/ml. Estimation of Activity by Attraction Index The attraction index gr (referred to as A.I. gr hereon) was obtained on the basis of applying the time-course obtained from the behavioral experi ments to a logistic curve y=g/(l+exp [-r(j-a)]}, as described previously.2) The gauze (25 ~25cm) which soaked 7.5ml of the test solution or homo genate was used as the attractant sample for es timating the A.I. gr. Results and Discussion Attraction Activities of Amino Acid- and Lipid - Fractions Table 1 shows the attraction activities of re spective three fractions of amino acids and lipids. Of the amino acid-fractions (No. 1), the basic one Table 1. Attraction activities of amino acid- and lipid-fractions from the mid-gut gland of squid *1 The gr of attraction index gr (A.I. gr) is the product of coefficients g and r of a logistic curve y=g/{1+exp [-r(j-a)]} in the entering leaving time-course.2) or *2,8,4 The eluates of ph N, ph N and ph N sodium citrate buffer solutions, respectively.8) *5,6,7 Acetone soluble, ether soluble and ether insoluble, respectively.? Pr(x2>x20.100)>0.100 and slightly less than Pr=0.100, respectively.2)

3 Attraction of Amino Acid and Lipid for Yellowtail 455 Table 2. Attraction activities of various kinds of L- and DL-amino acids was most effective and the neutral one was slightly effective, but the acidic one was not because the A.I. gr of the acidic one was lower than that of the dummy as control. Of the lipid-fractions (No. 2), the phospholipid-one was most effective in the attraction and glycolipid-one was somewhat effective, but neutral lipid-one was not. The results indicate that some compounds in the basic amino acid- and phospholipid-fractions played important roles inducing attracting, exploratory and feeding behaviors for the yellowtails. Further, some of the compounds in the neutral amino acid - and glycolipid-fractions may be involved in the attraction. The non-effective fractions, the acidic amino acid- and neutral lipid-ones, appear to be irrelevant in the attraction, but there is a possi bility that they contain also effective compounds because their A.I. gr's were the sum of attraction and non-attraction or repellance activities and because the latter could be stronger than the former. Attraction Activities of Authentic Amino Acids Table 2 shows the attraction activities of twenty -six kinds of L- and DL-amino acids. Of the basic amino acids (Nos. 3 and 4), four compounds were effective in the attraction, i.e., the activities were highest in histidine and lowest in lysine and orni thine, being intermediate in arginine. Of the neutral amino acids (Nos. 5-9), at least four com pounds were effective, i.e., the activities were highest in glycine and threonine, lowest in valine, being intermediate in methionine. Of the amides, acidic amino and imino acids (Nos. 10 and 11), two amide compounds, asparagine and glutamine were effective in the attraction, but their activities were moderate. Among histidine, glycine and threonine (No. 12), which showed high attraction indexes, the activities were high in histidine and threonine and low in glycine. In this context, it was ascertained that the majority of basic amino acids elicited remarkably the feeding attraction behavior from the fish.

4 There are many papers on the attraction ac tivities of amino acids for fishes by behavioral experiments. Most of them dealt with the ac tivity of each amino acid by omission or combina tion test, but a few by single test. The results have been well documented in recent reviews.4-10) It may be said that the attractant common to a variety of fishes could not be found out yet, al though at least a part of the difference in the results might be due to the difference in the method of experiment adopted. The attraction of basic amino acids was found in Japanese eel Anguilla japonica,7,11) killifish Fundulus heteroclitus,12) rainbow trout Salmo gairdneri 13) and red sea bream Chrysophrys major.14) But these groups of the compounds didn't show any attraction to the Table 3. Attraction activities of L- and n-amino acids Table 4. Attraction activities of various kinds of lipids

5 Attraction of Amino Acid and Lipid for Yellowtail 457 following species: winter flounder Pseudopleuronectes americanus,12) silverside Menidia menidia,12) tomate Bathystoma rimator,15) puffer Fugu par dalis,16) red sea bream,17) dover sole Solea solea,18) European eel Anguilla anguilla19) and salmon Salmo salar.20) In the behavioral experiments using juvenile yellowtails, the present results didn't accord with those of HOSOKAWA et al.* Using the method of different experiment, they observed that the attractants were not basic but neutral amino acids. The reason of this dis crepancy is not clear at present. Table 3 shows the attraction activities of L- and D-types of histidine and threonine which were found most effective. L-Histidine was effective while D-histidine was non-effective (No. 13). This coincided well with the results obtained on L- and D-threonines (No. 14). These accord with the results obtained from behavioral experiments on red sea breams5) and rainbow trout.10) On the other hand, in the electrophysiological experi ments using rainbow trout21) it was reported that D-amino acids also responded to olfactory bulbar of the fish above the concentration of about 10-7 M. The attraction activities of D-amino acids, however, were low, being about a half of those of L-amino acids even at the same concentrations. It may be, therefore, necessary to investigate behaviorally on the activities of various kinds of D-amino acids over wide range of concentrations. Attraction Activities of Authentic Lipids Table 4 shows the attraction activities of thirty kinds of lipids and their related compounds. Of the fatty acids (Nos ), two compounds were effective in the attraction, i.e., the activities were moderate in palmitic and stearic acids. Of the neutral lipids (Nos. 19 and 20), the activity was high only in monostearin. Of the phospholipids (Nos ), seven compounds were effective in the attraction, i.e., the activities were highest in cardiolipin, lowest in bean-lecithin, animal -cephalin, sphingomyelin and phosphatidic acids, being intermediate in egg-lecithin and phos phatidyl-inositol. Of the glycolipids (No. 24), only ceramide was effective in the attraction. In comparison with the activities of egg-lecithin, cardiolipin and ceramide (No. 25), the activities were high in egg-lecithin and cardiolipin and low in ceramide. In this context, it was ascertained that the majority of phospholipids evoked re markable feeding attraction behavior for the yellowtails. To my knowledge, there is no available paper on the attraction activities of lipids for fish in a behavioral experiment. However, KANEDA et al.22) reported a possibility that the lipids may be involved in the attraction behavior for yellowtail. On the other hand, there are several papers on the attraction activities of lipids for the following species in electrophysiological experiments: carp Cyprinus carpio,23) catfish Parasilurus asotus,24) Atlantic salmon25) and eel A. japonica.26) As most of lipids are water-insoluble, they should reached to experimental fish as colloidal particles. In the case of phospholipids, however, they are ampholytic similar to amino acids,27) and it may be the reason why phospholipids are effective attractants for fishes, because HARA21) suggested that an attraction depends on the ionized amino and carboxyl groups. Difference in Attraction Activities and Threshold Values of Histidine and Lecithin In order to elucidate difference in activities be tween histidine and lecithin, their attraction ac tivities were tested, as indicated in Table 5. In single test (No. 26), the activity of lecithin was higher than that of histidine. In combination test (Nos. 27 and 28), lecithin was also more effective in the attraction than histidine, because the ac tivity of histidine increased with the addition of Table 5. Attraction activities of L-histidine, egg -lecithin and their combination *8 Extracted with water from the mid-gut gland of squid.1) * H. HOSOKAWA, K. TAKII, T. KIKUCHI and M. TAKEDA; Presented at the Annual Meeting of the Japanese Society of Scientific Fisheries on April 2, 1977.

6 lecithin, but that of lecithin didn't with histidine. Judging from the results above and the fact that the concentrations used were 0.1 mmol in lecithin (apparent molecular formula, C42H80O8NP, M.W. 757) and 1.7 mmol in histidine, lecithin is more potent attractant for yellowtail. In estimating the threshold values of histidine and lecithin, the attraction activities, A.I. gr in six concentrations of histidine and lecithin were investigated behaviorally. Figs. 1 and 2 show the semi-logarithmic plots of the dose-attraction ac tivity relationships in histidine and lecithin, re spectively. The ratio of gr at each concentration to gr of dummy was taken as ordinate (=y) and the logarithmic ratio of each concentration to 225 mm in histidine and 12mg/ml in lecithin as abscissa (=x). In these figures, two plots at the concentration ratio, 0.25 in histidine and one plot, 0.25 in lecithin were excluded, because they showed the relationships quite different from those in dicated by the other plots. It was obvious that the attraction activities depended on the concen trations. From the results of a regression ana lysis,28) it was estimated that the regression lines in histidine and lecithin were y= x and y= x with the significance at 0.05 and 0.01 level, respectively. When the ab solute minimum concentration in the attraction behaviors for the fish is set to be x at y=1 in the equations, the concentrations in histidine and lecithin were 13.3ƒÊmol and 1.6ƒÊmol, respectively. Assuming that the concentrations were diffused Fig. 1. The correlation between the ratio of attrac tion activity (A. I. gr) and the logarithmic ratio of concentration in L-histidine. Abscissa; The logarithmic ratio of each con centration to 225 mm. Ordinate; The ratio of gr at each concentra tion to that of dummy. Regression line estimated; y= x (n=l0, F=9.560*, one asterisk: significant at 0.05 level). into the test compartment of experimental aquarium2) (28.5l) and the diffusates reached to the fish, the threshold values could be calculated as 5 ~10-7 M in histidine and 6 ~10-8 M in lecithin. This coincided roughly with the finding that when 10-3 M methylene blue was used the dye solution of ca M reached to the fish in a minute. In addition, the threshold value in histidine accorded roughly with those of amino acids obtained elec trophysiologically.21) Accordingly these findings showed that lecithin induced more effective at traction than histidine. Acknowledgements The author is grateful to Dr. H. MAEDA for his help in the statistical analysis and Mr. K. NAKAGAWA for kind supply of yellowtails. References Fig. 2. The correlation between the ratio of attrac tion activity (A. I. gr) and the logarithmic ratio of concentration in egg-lecithin. Abscissa; The logarithmic ratio of each con centration to 12mg/ml. Ordinate; The ratio of gr at each concentra tion to that of dummy. Regression line estimated; y= x (n=11, F=14.899**, two asterisks: significant at 0.01 level). 1) K. HARADA and H. MATSUDA: Bull. Japan. Soc. Sci. Fish., 50, (1984). 2) K. HARADA: Bull. Japan. Soc. Sic. Fish., 48, (1982). 3) K. HARADA and I. IKEDA: Bull. Japan. Soc. Sci. Fish., 50, (1984). 4) I. HIDAKA: in "Chemical Sense of Fish and Feeding Stimulants" (ed. by Japan. Soc. Sci. Fish.), Vol. 37, Koseisha Koseikaku Co., Tokyo, 1981, pp ) K. INA: in "Chemical Sense of Fish and Feeding

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