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Identification of feeding stimulants from a jack mackerel muscle extract for young yellowtail Seriola quinqueradiata
Aquaculture 181 Ž2000. 115–126 www.elsevier.nlrlocateraqua-online
Identification of feeding stimulants from a jack mackerel muscle extract for young yellowtail Seriola quinqueradiata Iwao Hidaka a,) , Jun Kohbara a , Toshiyoshi Araki b, Tatsuo Morishita b, Toshiaki Miyajima a , Shigeki Shimizu a , Isao Kuriyama a a
b
Laboratory of Fish Physiology, Faculty of Bioresources, Mie UniÕersity, Tsu, Mie 514-8507, Japan Laboratory of Utilization of Marine Products, Faculty of Bioresources, Mie UniÕersity, Tsu, Mie 514-8507, Japan Accepted 25 May 1999
Abstract The feeding-stimulatory components of an aqueous extract of jack mackerel white muscle for young yellowtail were identified. The extract was fractionated using anion-exchange chromatography, and the feeding-stimulatory effectiveness of fractionated components examined by adding them to starch pellets and feeding to yellowtail. The filtrate ŽFE. of the muscle extract filtered through a membrane having a mol. wt. cut-off of 10,000 Da was found to have a high feeding-stimulatory effectiveness on the yellowtail. FE was fractionated using DEAE-Sephadex A-25 at pH 5.5. The DEAE-Sephadex adsorbate ŽA A . showed an effectiveness close to but slightly weaker than that of FE. The DEAE-Sephadex non-adsorbate ŽNA . had no appreciable effect. Subdividing A A components by stepwise elutions with NaCl solutions suggested that inosine-5X-monophosphate and lactic acid were largely responsible for the stimulatory effectiveness of A A. Some components in NA might also be synergistic with A A components to elicit the full effectiveness of FE. q 2000 Elsevier Science B.V. All rights reserved. X
Keywords: Fish; Yellowtail; Feeding stimulant; Muscle extract; Inosine-5 -monophosphate; Lactic acid; Behavior
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Corresponding author. Tel.: q81-59-231-9533; fax: q81-59-231-9523; E-mail: [email protected]
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 2 2 1 - 5
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1. Introduction Considerable work has been reported on feeding stimulants for fish, and diverse compounds are thought to be attractants or feeding stimulants ŽHarada, 1985; Mackie and Mitchell, 1985; Adams and Johnsen, 1986; Takeda and Takii, 1992; Carr et al., 1996.. Carr et al. found that amino acids and betaine were potent stimulants for the pinfish Lagodon rhomboides ŽCarr and Chaney, 1976. and pigfish Orthopristis chryropterus ŽCarr et al., 1977.. Betaine was also shown to have an enhancing effect when added to amino acids ŽCarr and Chaney, 1976; Carr et al., 1977.. Similar collaboratory effects on feed-enhancing capabilities of betaine are known for several coastal fishes ŽOhsugi et al., 1978; Goh and Tamura, 1980; Mackie, 1982; Mackie and Mitchell, 1983. and invertebrates ŽCarr, 1967; Harpaz, 1997.. Mackie et al. reported species differences in feeding-stimulants contained in a squid muscle extract: amino acids were effective for rainbow trout ŽAdron and Mackie, 1978., inosine and inosine-5X-monophosphate ŽIMP. for turbot ŽMackie and Adron, 1978., mixtures of amino acids, and others for plaice ŽMackie, 1982.. Different foods may have different sets of stimulants for an omnivorous fish. Thus, Takeda et al. studying the feeding-enhancing effects of extracts of jack mackerel muscle and krill on yellowtail and jack mackerel, found that IMP and amino acids plus nucleotides were major stimulants in the jack mackerel muscle extract and in the krill extract, respectively, for both species of fish ŽTakeda, 1980; Ikeda et al., 1988a,b.. We previously studied the effects of squid and jack mackerel muscle extracts on yellowtail and found that, in the squid extract, amino acids were major stimulants, and nucleotides had enhancing effects when added to amino acids ŽFukuda et al., 1989., while in the jack mackerel muscle, a synthetic mixture containing amino acids, ATP-related compounds, quaternary ammonium and others, based on the analysis by Konosu et al. Ž1974., was inferior to the natural extract, suggesting that some effective components were absent in the synthetic mixture ŽKohbara et al., 1989.. Later we found that lactic acid enhanced the synthetic mixture when added to it ŽKohbara et al., 1993., but it was still less effective than the natural extract. In the present study, we attempted to identify the effective components in the natural extract by fractionating it using ion-exchange chromatography. We also studied the stimulatory effects of the fractionated components on the gustatory and olfactory receptors of the same fish, the results of which will be reported separately ŽKohbara et al., 1999..
2. Materials and methods 2.1. Experimental periods, animals and their maintenance The feeding experiments were performed from July to September at the Fisheries Research Laboratory, Mie University, Zagashima Island, Ago Bay, Mie Prefecture. The experimental animals were wild juvenile yellowtail Ž Seriola quinqueradiata. caught by local fishermen. After being kept in a floating net pen set off from the shore and fed a
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mixture of minced raw fish and an artificial feed ŽMarubeni Shiryo., they were moved to indoor tanks and kept there for another two weeks. Then they were anesthetized with ethyl m-aminobenzoate methanesulfonate ŽMS222, Sankyo. and distributed to six round tanks Ž200 l each. with 20 fish per tank. The tanks were supplied with filtered seawater at 6–9 mlrmin. The fish were fed the same feed every day at 1600 at about 10% of body wt. and kept for another week before the experiments. A 20-W fluorescent light was hung 2 m above each tank. The light was on 12 hrday from 0800 to 2000. Fish size at the start of the experiments ranged 12–67 g in body wt. Ž23.0–31.0; 12.2–23.5; 32.4–67.0. and 10–18 cm in fork length Ž11.6–13.5; 10.2–12.5; 13.7–17.4.. The water temperature ranged 21–298C Ž21.7–26.0; 23.5–26.6; 25.2–28.5.. 2.2. Preparation of the jack mackerel white muscle extracts Freshly caught jack mackerels weighing 9–26 g were transported on ice to the Faculty of Bioresources at Tsu taking 2 h. Upon arrival, the white muscle was carefully isolated from the dorsal trunk in a cold room at 48C. Fifty grams of the muscle was homogenized in 500 ml of water. The homogenate was centrifuged at 10,000 rpm Ž8720 = g . for 10 min. The supernatant obtained was divided, freeze-dried, and kept at y308C before use. The dried crude extracts ŽCE. were dissolved in water for further use. The ‘‘original concentration’’ was used as the unit of a solution containing the extracts in amounts corresponding to 50 g wet wt. of the muscle in 50 ml of water. The CE was filtered through a UK-10 membrane ŽAdvantec. having a mol. wt. cut-off of 10,000, and the filtrate of CE obtained ŽFE. was used as the starting extract for further fractionations. All chromatographic treatments were done in a cold room Ž48C.. 2.3. Ion exchange chromatography FE was loaded onto an anion-exchange column Ž5 = 10 cm, Pharmacia. of DEAESephadex A-25 equilibrated with 0.05 M NaCl adjusted to pH 5.5 with 0.1 M NaOH and 0.1 M HCl; we did not use any buffer to avoid contaminating the samples to be used for behavioral assay. The flow rate was 60 mlrh and fraction size was 10 ml. The column was eluted with the same aqueous solution, and the unadsorbed fractions ŽNA . were collected. The DEAE-Sephadex adsorbate was eluted by one-step elution with 0.6 M NaCl or by two-step elution with 0.2 and 0.6 M NaCl. The fractions eluted by one-step elution were referred to as A A . Those eluted stepwise with 0.2 and 0.6 M NaCl solutions were termed A A0.2 and A A0.2 – 0.6 , respectively ŽFig. 1.. The eluates were desalted, if necessary, by Sephadex G-10 gel chromatography. 2.4. Analytical treatments 2.4.1. Gross monitoring of the fractionated extract components The components in fractions produced by ion-exchange chromatography were scanned by ultraviolet absorption spectra ŽUV-1200, Shimadzu., ninhydrin reactions and enzymatic measurements of lactic acid ŽMarbach and Weil, 1967..
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Fig. 1. Flow chart showing the procedures for fractionating the ultrafiltrate of the muscle extract ŽFE. using an ion-exchanger.
2.4.2. High performance liquid chromatography (HPLC) Amino acids were analyzed by a Shimadzu LC-6A system equipped with a Simpack Amino-Li column Ž6 = 100 mm, Shimadzu. and a fluorescence detector ŽShimadzu RF550; excitation wavelength 348 nm, fluorescence wavelength 450 nm.. The column was eluted with 0.15 N ŽA. and 0.3 N ŽB. lithium citrate solutions at 398C. The flow rate was 0.6 ml miny1 . ATP-related compounds were analyzed by a Waters 625 LC System equipped with a reverse phase column ŽSTR ODS-II, 4.6 = 150 mm, Shimadzu. and UV monitor Ž250 nm.. The column was eluted with a mobile phase containing 18 mM diethylaminoethanol and 12 mM citric acid at 408C. The flow rate was 1.0 ml miny1 . Organic acids were analyzed by a Waters 625 LC System equipped with an organic acid column Ž7.8 = 300 mm, Waters. and a UV monitor Ž214 nm.. The column was eluted with 0.1% phosphoric acid at 408C. The flow rate was 1.0 ml miny1 . 2.5. Preparation of test pellets Wheat starch ŽNacalai Tesque, C.P. grade. was treated with 0.08 M NaOH to remove dissolved proteins and polyphenols from the insoluble starch layer and then freeze-dried. Pellets of test samples were made as follows: 4.7 g of starch was mixed with 4 ml of a
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test sample solution at original concentration and 0.3 g of carboxymethylcellulose ŽCMC, as a binding agent., and the mixture was then kneaded and extruded into pellets of size 25 mg each Ž3 = 4 mm.. Test samples were dissolved in 100% or diluted artificial seawater to get a salt concentration equivalent to seawater to avoid stimulating the taste receptors of yellowtail which has a high sensitivity to dilute seawater ŽHidaka and Ohsugi, 1979.. 2.6. Application of test pellets and statistical treatments Six groups of fish were used for each behavioral assay. The test pellets were dispensed through an opaque pipe Ž10 cm diameter= 50 cm length. set vertical in the middle of the tank ŽFukuda et al., 1989.. The fish were fed starch pellets containing CE or FE for two or three days to adapt to eating pellets. The experiment was performed once a day at 1400. One hundred pellets were offered simultaneously to each group of fish, and the number of pellets eaten within the first min was estimated by collecting and counting the uneaten pellets. When two Žor three. kinds of test samples were tested in
Fig. 2. The feeding-stimulatory effectiveness of the adsorbate ŽA A . and non-adsorbate ŽNA . fractions of FE from DEAE-Sephadex A-25 chromatography. Ordinate shows the average percent of pellets eaten per group of fish: 100 test pellets were offered to each of 6 groups of 20 fish and the number of pellets eaten in the first minute was estimated by counting the uneaten pellets. See also text.
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combination, three Žor two. tanks were allotted to each sample per day and tested in all six tanks taking two Žor three. days. To compare the stimulatory effectiveness among extract samples, Wilcoxon signedrank test for two samples was used ŽLehmann, 1975.. For samples containing data with a score of 100, which means all of the pellets offered were eaten and therefore the data were censored at the point of 100, a modified Wilcoxon test ŽGehan, 1965. was used.
3. Results 3.1. The feeding-stimulatory effects of FE, A A and NA Fig. 2 shows the results of a feeding experiment with A A and NA together with FE as a control. In this experiment, FE was completely eaten in all six groups of fish; A A was completely eaten in two groups but not in the others; NA was hardly eaten in all six groups, thus the average number of pellets eaten per group of fish being 100, 87.7 and 4.7, respectively. FE was usually completely eaten Žin 35 out of 36 groups tested w6 trials with 12 groups of fishx., while A A was completely eaten in 8 groups out of 24 groups Ž4
Fig. 3. The feeding-stimulatory effectiveness of A A and its two subdivided fractions, A A0.2 and A A0.2 – 0.6 . Ordinate shows the average percent of pellets eaten per group of fish as in Fig. 2. See also text.
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trials with 12 groups., but partially in 16. The modified Wilcoxon test ŽGehan, 1965. showed that A A was less effective at 5% level of significance. 3.2. The feeding-stimulatory effects of A A0 .2 and A A0 .2 – 0 .6 Fig. 3 compares the consumption A A and its two subdivided fractions, A A0.2 and A A0.2 – 0.6 . In this experiment, CE was used as a between trial control. In Fig. 3, the average number of pellets eaten per group of fish for A A , A A0.2 and A A0.2 – 0.6 were 89.7, 27.0 and 65.2, while CE was completely eaten in all six groups. The Wilcoxon test indicated A A0.2 - A A0.2 – 0.6 and A A0.2 - A A at the 5% level of significance, while A A0.2 – 0.6 and A A were not significantly different. In another experiment with the same groups as in Fig. 3, A A0.2 ) NA at the 5% level of significance. 3.3. Substances contained in A A , A A0 .2 and A A0 .2 – 0 .6 HPLC revealed that A A contained mainly lactic acid, IMP and glutamic acid ŽTables 1 and 2.. By stepwise elution of A A components from the DEAE-Sephadex column with 0.2 M and 0.6 M NaCl, glutamic acid and lactic acid were eluted separately from IMP. Analyses of A A , A A0.2 and A A0.2 – 0.6 for lactic acid by the enzyme method, for IMP by ultraviolet absorption and for amino acids by HPLC ŽTable 2., showed their presence at mM Žor mmolrkg of muscle. as follows: lactic acid in A A 34.8, in A A0.2 32.7 and in A A0.2 – 0.6 not detectable; IMP in A A 6.0, in A A0.2 – 0.6 6.1, and in A A0.2 no detectable amount; and glutamic acid in A A 1.15, in A A0.2 0.96 and in A A0.2 – 0.6 0.05 and minute quantities of phosphoserine and other amino acids in A A0.2 and A A0.2 – 0.6 ŽTable 2..
Table 1 ATP related substances and organic acids ŽmM a . detected by HPLC in FE and A A and NA from DEAE-Sephadex A-25 chromatography Žsee also text.. Ž – ., No detectable absorption was observed at 250 nm for ATP-related substances and at 214 nm for organic acids at the corresponding retention time Substances
AA
NA
FE
AMP ADP ATP IMP Inosine Hypoxanthine Ascorbic acid Citric acid Fumaric acid Lactic acid Malic acid Succinic acid Tartaric acid
– – – 6.61 0.04 0.04 – – 0.05 51.71 – – –
– – – 0.07 0.79 0.19 – – – – – – –
– – – 6.93 0.96 0.18 – – 0.10 61.53 – – –
a
Concentration of the compounds extracted from 50 g weight of the muscle in 50 ml solution.
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Table 2 Amino acids ŽmM a . detected by HPLC in A A , A A0.2 and A A0.2 – 0.6 fractions of FE from DEAE-Sephadex chromatography. Ž – ., Not detected or error; Žtr. trace Amino acids
AA
A A0.2
A A0.2 – 0.6
Phosphoserine Taurine Aspartic acid Hydroxy proline Threonine Serine Asparagine Glutamic acid Glutamine Sarcosine a-Amino-n-adipic acid Proline Glycine Alanine Citrulline a-Amino-n-butyric acid Valine Cystine Methionine Cystathionine Isoleucine Leucine Tyrosine Phenylalanine b-Alanine Histidine Ethanolamine Ornithine Lysine Arginine Total amino acids
0.01 0.00 0.02 – 0.00 0.00 – 0.96 – 0.00 0.01 0.00 0.00 0.00 tr 0.00 0.01 – 0.00 0.00 0.00 0.00 0.00 0.00 – 0.00 – 0.00 0.00 0.00 1.01
0.06 0.02 0.00 – 0.00 0.02 – 0.05 – 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00 – 0.00 0.00 0.00 0.00 0.00 0.00 – 0.01 – 0.00 0.00 0.00 0.16
0.07 0.02 0.01 – 0.00 0.00 – 1.15 – 0.00 0.03 0.00 0.02 0.00 tr 0.00 0.00 – 0.00 tr 0.00 tr 0.00 0.00 – 0.02 – 0.00 0.00 0.00 1.32
a
The same as in Table 1.
These results indicated that both lactic acid and glutamic acid were eluted in A A0.2 , and IMP was almost exclusively eluted in A A0.2 – 0.6 .
4. Discussion In the present experiment, pellets containing plain sea water were hardly eaten, while those containing either CE or FE were as well eaten as previously ŽFukuda et al., 1989.. Thus, it appeared that neither starch nor CMC had any stimulatory or inhibitory effects on the feeding behavior of yellowtail. We also examined the stimulatory effects of the starch and CMC on the taste receptors of yellowtail by applying a 10% starch suspension and a 1% CMC solution to the palate and recording the taste nerve responses
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ŽFukuda et al., 1989.. We observed no appreciable response to the starch, but observed a tiny but continuing impulses to CMC which were considered to be a kind of mechanical response ŽKiyohara et al., 1985.. The treatment of FE on the DEAE-Sephadex column showed that A A contained the main stimulants. HPLC showed that A A mainly contained lactic acid, IMP and glutamic acid. Of these, lactic acid and glutamic acid were eluted in A A0.2 and IMP in A A0.2 – 0.6 . The results in Fig. 3 show that A A0.2 was less effective than A A and A A0.2 – 0.6 , while A A0.2 – 0.6 was no less effective than A A . However, it was 25% less effective in the average percentage of pellets eaten than A A . This suggests that either glutamic acid and lactic acid or both enhance IMP to elicit the effectiveness of A A , although they were effective rather weakly as A A0.2 alone. However, glutamic acid did not seem to stimulate the taste receptors ŽHidaka et al., 1985.. Whereas lactic acid is a potent stimulant for the taste receptors with a threshold of 10y6 –10y5 M ŽHidaka et al., 1992. and also enhances feeding-stimulatory effectiveness of a synthetic jack mackerel muscle extract ŽKohbara et al., 1993.. We previously observed that a synthetic mixture of jack mackerel muscle extracts ŽKonosu et al., 1974. was less effective than the natural extract ŽCE. ŽKohbara et al., 1989., even after lactic acid was added to it ŽKohbara et al., 1993.. In the latter mixture, IMP and lactic acid were 7.5 mM and 50 mM, respectively. The amounts are quite comparable to those found for FE in Table 1. Therefore, it might be that components of NA also have an enhancing effect on A A components although NA had no appreciable effect when applied alone. Takeda et al. studied the feeding-enhancing effects of jack mackerel extracts on yellowtail by adding test stimulants to a casein-based diet ŽTakeda, 1980.. They observed the daily feeding rate and found that IMP was a major stimulant. Whereas in a similar study on the krill extract, they found that the amino acid fraction plus the nucleotide fraction were effective ŽTakeda, 1980.. The amino acids involved were alanine, methionine and proline. These amino acids were contained in the krill extract at 40–261 mgr93 ml Ž3–30 mM., while in the jack mackerel extract at 1–21 mgr93 ml Ž70 mM–2 mM.. On the other hand, IMP was 2.4 mgr93 ml Ž70 mM. in the former and 308 mgr93 ml Ž9 mM. in the latter. Thus, the difference in the main stimulatory substances between the two extracts appears to be the abundance of stimulatory substances. Takaoka et al. Ž1990. reported that for marbled rockfish, inosine was the most effective stimulant in a krill extract. In this extract inosine was 137 mgr93 ml Ž5.5 mM., whereas, IMP and other nucleotides were present in much smaller quantities. They also reported that IMP and other nucleotides were as effective as inosine at high concentrations, whereas inosine in the krill extract was not a potent stimulant for yellowtail and jack mackerel ŽTakeda, 1980; Ikeda et al., 1988b.. In our previous study with the synthetic mixture of squid muscle extracts ŽFukuda et al., 1989., we found that the amino acid fraction was the most effective, and the nucleotide fraction somewhat enhanced the amino acid fraction. We also found that amino acids which were stimulatory for the taste receptors were involved in the feeding-stimulatory effect. In the squid muscle extract also, amino acids are abundantly found ŽSuyama and Kobayashi, 1980..
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The high feeding-stimulatory potency of IMP and inosine has also been reported in the turbot ŽMackie and Adron, 1978.. Components of a squid muscle extract were tested by adding them to a casein-based diet, and inosine was identified as a specific stimulant: removal of inosine from the synthetic mixture effectively abolished feeding stimulation and inosine alone was almost as effective as the complete mixture. IMP was also as or more effective than inosine when compared at equal molesrg diet ŽMackie and Adron, 1978.. In the largemouth bass also, IMP had a high feeding-enhancing effect when added to a soybean based diet while inosine did not ŽKubitza et al., 1997.. In puffer Takifugu rubripes ŽTakaoka et al., 1995. and T. pardalis Žformerly Fugu pardalis, Ohsugi et al., 1978., marked synergistic effects between amino acids and betaine were found in clam Tapes japonica extract. Similar synergism was also reported for juvenile Dover sole ŽMackie et al., 1980.. Mixtures of several amino acids have been reported to be more effective than single or combinations of a few amino acids in rainbow trout ŽAdron and Mackie, 1978., red sea bream ŽFuke et al., 1981. and eel ŽMackie and Mitchell, 1983; Takeda et al., 1984.. Also, some amino acids and other constituents of tissue extracts have been reported to behave as a feeding incitant or feeding deterrent in mixtures with other constituents ŽMackie, 1982; Mearns et al., 1987; Ikeda et al., 1988b..
Acknowledgements We thank Mr. Hiromu Nakamura, the Fisheries Research Laboratory, Faculty of Bioresources, for help in transporting the experimental animals. We also thank Dr. Seishi Kimura for help during our stay at the Laboratory. This work was supported by grants from the Ministry of Education, Science and Culture of Japan ŽNos. 0366190, 0766024..
References Adams, M.A., Johnsen, P.B., 1986. Chemical control of feeding in herbivorous and carnivorous fish. In: Duvall, D., Muller-Schwarze, D., Silverstein, R.M. ŽEds.., Chemical Signals in Vertebrates IV. Plenum, New York, pp. 45–61. Adron, J.W., Mackie, A.M., 1978. Studies on the chemical nature of feeding stimulants for rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 12, 303–310. Carr, W.E.S., 1967. Chemoreception in the mud snail, Nassarius obsoletus: II. Identification of stimulatory substances. Biol. Bull. 133, 106–127. Carr, W.E.S., Chaney, T.B., 1976. Chemical stimulation of feeding behavior in the pinfish, Lagodon rhomboides: characterization and identification of stimulatory substances extracted from shrimp. Comp. Biochem. Physiol. A 54, 437–441. Carr, W.E.S., Blumenthal, K.M., Netherton, J.C. III, 1977. Chemoreception in the pigfish, Orthopristis chrysopterus: the contribution of amino acids and betaine to stimulation of feeding behavior by various extracts. Comp. Biochem. Physiol. A 58, 69–73. Carr, W.E.S., Netherton, J.C. III, Gleeson, R.A., Derby, C.D., 1996. Stimulants of feeding behavior in fish: analyses of tissues of diverse marine organisms. Biol. Bull. 190, 149–160. Fuke, S., Konosu, S., Ina, K., 1981. Identification of feeding stimulants for red sea bream in the extract of marine worm Perinereis breÕicirrus. Bull. Jpn. Soc. Sci. Fish. 47, 1631–1635.
I. Hidaka et al.r Aquaculture 181 (2000) 115–126
125
Fukuda, K., Kohbara, J., Zeng, C., Hidaka, I., 1989. The feeding-stimulatory effects of squid muscle extracts on the young yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi 55, 791–797, Žin Japanese, with English abstract.. Gehan, E.A., 1965. A generalized Wilcoxon test for comparing arbitrarily singly-censored samples. Biometrika 52, 203–223. Goh, Y., Tamura, T., 1980. Effects of amino acids on the feeding behaviour in red sea bream. Comp. Biochem. Physiol. 66C, 225–229. Harada, K., 1985. Feeding attraction activities of amino acids and lipids for juvenile yellowtail. Bull. Jpn. Soc. Sci. Fish. 51, 453–459. Harpaz, S., 1997. Enhancement of growth in juvenile freshwater prawns, Macrobrachium rosenbergii, through the use of a chemoattractant. Aquaculture 156, 221–227. Hidaka, I., Ohsugi, T., 1979. High sensitivity of the palatal chemoreceptors of the yellowtail to dilution of sea water. Bull. Jpn. Soc. Sci. Fish. 45, 643. Hidaka, I., Ohsugi, T., Yamamoto, Y., 1985. Gustatory response in the young yellowtail Seriola quinqueradiata. Bull. Jpn. Soc. Sci. Fish. 51, 21–24. Hidaka, I., Zeng, C., Kohbara, J., 1992. Gustatory responses to organic acids in the yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi 58, 1179–1187. Ikeda, I., Hosokawa, H., Shimeno, S., Takeda, M., 1988a. Identification of feeding stimulant for jack mackerel in its muscle extract. Nippon Suisan Gakkaishi 54, 229–233, Žin Japanese, with English abstract.. Ikeda, I., Hosokawa, H., Shimeno, S., Takeda, M., 1988b. Identification of feeding stimulant in the krill extract for jack mackerel. Nippon Suisan Gakkaishi 54, 235–238, Žin Japanese, with English abstract.. Kiyohara, S., Hidaka, I., Kitoh, J., Yamashita, S., 1985. Mechanical sensitivity of the facial nerve fibers innervating the anterior palate of the puffer, Fugu pardalis, and their central projection to the primary taste center. J. Comp. Physiol. A 157, 705–716. Kohbara, J., Fukuda, K., Hidaka, I., 1989. The feeding-stimulatory effects of the jack mackerel white muscle extracts on the young yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi 55, 1343–1347, Žin Japanese, with English abstract.. Kohbara, J., Hidaka, I., Morishita, T., Miyajima, T., 1993. The feeding-stimulatory effectiveness of L-lactic acid on the young yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi 59, 183. Kohbara, J., Hidaka, I., Morishita, T., Miyajima, T., 1999. Gustatory and olfactory sensitivity to extracts of jack mackerel muscle in young yellowtail Seriola quinqueradiata. Aquaculture, submitted. Konosu, S., Watanabe, K., Shimizu, T., 1974. Distribution of nitrogenous constituents in the muscle extracts of eight species of fish. Bull. Jpn. Soc. Sci. Fish. 40, 909–915. Kubitza, F., Lovshin, L.L., Lovell, R.T., 1997. Identification of feed enhancers for juvenile largemouth bass Micropterus salmoides. Aquaculture 148, 191–200. Lehmann, E.L., 1975. Nonparametrics: Statistical Methods Based on Ranks Žtranslated into Japanese by Nabeya, S., Kriya, T., Miura, R., 1978.. Morikita Shuppan, Tokyo, 484 pp. Mackie, A.M., 1982. Identification of the gustatory feeding stimulants. In: Hara, T.J. ŽEd.., Chemoreception in Fishes. Elsevier, Amsterdam, pp. 275–291. X Mackie, A.M., Adron, J.W., 1978. Identification of inosine and inosine 5 -monophosphate as the gustatory feeding stimulants for the turbot, Scophthalmus maximus. Comp. Biochem. Physiol. A 60, 79–83. Mackie, A.M., Mitchell, A.I., 1983. Studies on the chemical nature of feeding stimulants for the juvenile European eel, Anguilla anguilla ŽL... J. Fish Biol. 22, 425–430. Mackie, A.M., Mitchell, A.I., 1985. Identification of gustatory feeding stimulants for fish-applications in aquaculture. In: Cowey, C.B., Mackie, A.M., Bell, J.G. ŽEds.., Nutrition and Feeding in Fish. Academic Press, London, pp. 177–189. Mackie, A.M., Adron, J.W., Grant, P.T., 1980. Chemical nature of feeding stimulants for the juvenile Dover sole, Solea solea ŽL... J. Fish Biol. 16, 701–708. Marbach, E.P., Weil, M.H., 1967. Rapid enzymatic measurement of blood lactate and pyruvate. Use and significance of metaphosphoric acid as a common precipitant. Clin. Chem. 13, 314–325. Mearns, K.J., Ellingsen, O.F., Døving, K.B., Helmer, S., 1987. Feeding behaviour in adult rainbow trout and Atlantic salmon parr, elicited by chemical fractions and mixtures of compounds identified in shrimp extract. Aquaculture 64, 47–63.
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Ohsugi, T., Hidaka, I., Ikeda, M., 1978. Taste receptor stimulation and feeding behaviour in the puffer, Fugu pardalis: II. Effects produced by mixtures of constituents of clam extracts. Chem. Senses Flavour 3, 355–368. Suyama, M., Kobayashi, H., 1980. Free amino acids and quaternary ammonium bases in mantle muscle of squids. Bull. Jpn. Soc. Sci. Fish. 46, 1261–1264, Žin Japanese, with English abstract.. Takaoka, O., Takii, K., Nakamura, M., Kumai, H., Takeda, M., 1990. Identification of feeding stimulants for marbled rockfish. Nippon Suisan Gakkaishi 56, 345–351, Žin Japanese, with English abstract.. Takaoka, O., Takii, K., Nakamura, M., Kumai, H., Takeda, M., 1995. Identification of feeding stimulants for tiger puffer. Fisheries Sci. 61, 833–836. Takeda, M., 1980. Feeding stimulants for fish. Iden ŽHeredity. 34, 45–51, Žin Japanese.. Takeda, M., Takii, K., 1992. Gustation and nutrition in fishes: application to aquaculture. In: Hara, T.J. ŽEd.., Fish Chemoreception. Chapman & Hall, London, pp. 271–287. Takeda, M., Takii, K., Matsui, K., 1984. Identification of feeding stimulants for juvenile eel. Bull. Jpn. Soc. Sci. Fish. 50, 645–651.
Identification of feeding stimulants from a jack mackerel muscle extract for young yellowtail Seriola quinqueradiata Iwao Hidaka a,) , Jun Kohbara a , Toshiyoshi Araki b, Tatsuo Morishita b, Toshiaki Miyajima a , Shigeki Shimizu a , Isao Kuriyama a a
b
Laboratory of Fish Physiology, Faculty of Bioresources, Mie UniÕersity, Tsu, Mie 514-8507, Japan Laboratory of Utilization of Marine Products, Faculty of Bioresources, Mie UniÕersity, Tsu, Mie 514-8507, Japan Accepted 25 May 1999
Abstract The feeding-stimulatory components of an aqueous extract of jack mackerel white muscle for young yellowtail were identified. The extract was fractionated using anion-exchange chromatography, and the feeding-stimulatory effectiveness of fractionated components examined by adding them to starch pellets and feeding to yellowtail. The filtrate ŽFE. of the muscle extract filtered through a membrane having a mol. wt. cut-off of 10,000 Da was found to have a high feeding-stimulatory effectiveness on the yellowtail. FE was fractionated using DEAE-Sephadex A-25 at pH 5.5. The DEAE-Sephadex adsorbate ŽA A . showed an effectiveness close to but slightly weaker than that of FE. The DEAE-Sephadex non-adsorbate ŽNA . had no appreciable effect. Subdividing A A components by stepwise elutions with NaCl solutions suggested that inosine-5X-monophosphate and lactic acid were largely responsible for the stimulatory effectiveness of A A. Some components in NA might also be synergistic with A A components to elicit the full effectiveness of FE. q 2000 Elsevier Science B.V. All rights reserved. X
Keywords: Fish; Yellowtail; Feeding stimulant; Muscle extract; Inosine-5 -monophosphate; Lactic acid; Behavior
)
Corresponding author. Tel.: q81-59-231-9533; fax: q81-59-231-9523; E-mail: [email protected]
0044-8486r00r$ - see front matter q 2000 Elsevier Science B.V. All rights reserved. PII: S 0 0 4 4 - 8 4 8 6 Ž 9 9 . 0 0 2 2 1 - 5
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1. Introduction Considerable work has been reported on feeding stimulants for fish, and diverse compounds are thought to be attractants or feeding stimulants ŽHarada, 1985; Mackie and Mitchell, 1985; Adams and Johnsen, 1986; Takeda and Takii, 1992; Carr et al., 1996.. Carr et al. found that amino acids and betaine were potent stimulants for the pinfish Lagodon rhomboides ŽCarr and Chaney, 1976. and pigfish Orthopristis chryropterus ŽCarr et al., 1977.. Betaine was also shown to have an enhancing effect when added to amino acids ŽCarr and Chaney, 1976; Carr et al., 1977.. Similar collaboratory effects on feed-enhancing capabilities of betaine are known for several coastal fishes ŽOhsugi et al., 1978; Goh and Tamura, 1980; Mackie, 1982; Mackie and Mitchell, 1983. and invertebrates ŽCarr, 1967; Harpaz, 1997.. Mackie et al. reported species differences in feeding-stimulants contained in a squid muscle extract: amino acids were effective for rainbow trout ŽAdron and Mackie, 1978., inosine and inosine-5X-monophosphate ŽIMP. for turbot ŽMackie and Adron, 1978., mixtures of amino acids, and others for plaice ŽMackie, 1982.. Different foods may have different sets of stimulants for an omnivorous fish. Thus, Takeda et al. studying the feeding-enhancing effects of extracts of jack mackerel muscle and krill on yellowtail and jack mackerel, found that IMP and amino acids plus nucleotides were major stimulants in the jack mackerel muscle extract and in the krill extract, respectively, for both species of fish ŽTakeda, 1980; Ikeda et al., 1988a,b.. We previously studied the effects of squid and jack mackerel muscle extracts on yellowtail and found that, in the squid extract, amino acids were major stimulants, and nucleotides had enhancing effects when added to amino acids ŽFukuda et al., 1989., while in the jack mackerel muscle, a synthetic mixture containing amino acids, ATP-related compounds, quaternary ammonium and others, based on the analysis by Konosu et al. Ž1974., was inferior to the natural extract, suggesting that some effective components were absent in the synthetic mixture ŽKohbara et al., 1989.. Later we found that lactic acid enhanced the synthetic mixture when added to it ŽKohbara et al., 1993., but it was still less effective than the natural extract. In the present study, we attempted to identify the effective components in the natural extract by fractionating it using ion-exchange chromatography. We also studied the stimulatory effects of the fractionated components on the gustatory and olfactory receptors of the same fish, the results of which will be reported separately ŽKohbara et al., 1999..
2. Materials and methods 2.1. Experimental periods, animals and their maintenance The feeding experiments were performed from July to September at the Fisheries Research Laboratory, Mie University, Zagashima Island, Ago Bay, Mie Prefecture. The experimental animals were wild juvenile yellowtail Ž Seriola quinqueradiata. caught by local fishermen. After being kept in a floating net pen set off from the shore and fed a
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mixture of minced raw fish and an artificial feed ŽMarubeni Shiryo., they were moved to indoor tanks and kept there for another two weeks. Then they were anesthetized with ethyl m-aminobenzoate methanesulfonate ŽMS222, Sankyo. and distributed to six round tanks Ž200 l each. with 20 fish per tank. The tanks were supplied with filtered seawater at 6–9 mlrmin. The fish were fed the same feed every day at 1600 at about 10% of body wt. and kept for another week before the experiments. A 20-W fluorescent light was hung 2 m above each tank. The light was on 12 hrday from 0800 to 2000. Fish size at the start of the experiments ranged 12–67 g in body wt. Ž23.0–31.0; 12.2–23.5; 32.4–67.0. and 10–18 cm in fork length Ž11.6–13.5; 10.2–12.5; 13.7–17.4.. The water temperature ranged 21–298C Ž21.7–26.0; 23.5–26.6; 25.2–28.5.. 2.2. Preparation of the jack mackerel white muscle extracts Freshly caught jack mackerels weighing 9–26 g were transported on ice to the Faculty of Bioresources at Tsu taking 2 h. Upon arrival, the white muscle was carefully isolated from the dorsal trunk in a cold room at 48C. Fifty grams of the muscle was homogenized in 500 ml of water. The homogenate was centrifuged at 10,000 rpm Ž8720 = g . for 10 min. The supernatant obtained was divided, freeze-dried, and kept at y308C before use. The dried crude extracts ŽCE. were dissolved in water for further use. The ‘‘original concentration’’ was used as the unit of a solution containing the extracts in amounts corresponding to 50 g wet wt. of the muscle in 50 ml of water. The CE was filtered through a UK-10 membrane ŽAdvantec. having a mol. wt. cut-off of 10,000, and the filtrate of CE obtained ŽFE. was used as the starting extract for further fractionations. All chromatographic treatments were done in a cold room Ž48C.. 2.3. Ion exchange chromatography FE was loaded onto an anion-exchange column Ž5 = 10 cm, Pharmacia. of DEAESephadex A-25 equilibrated with 0.05 M NaCl adjusted to pH 5.5 with 0.1 M NaOH and 0.1 M HCl; we did not use any buffer to avoid contaminating the samples to be used for behavioral assay. The flow rate was 60 mlrh and fraction size was 10 ml. The column was eluted with the same aqueous solution, and the unadsorbed fractions ŽNA . were collected. The DEAE-Sephadex adsorbate was eluted by one-step elution with 0.6 M NaCl or by two-step elution with 0.2 and 0.6 M NaCl. The fractions eluted by one-step elution were referred to as A A . Those eluted stepwise with 0.2 and 0.6 M NaCl solutions were termed A A0.2 and A A0.2 – 0.6 , respectively ŽFig. 1.. The eluates were desalted, if necessary, by Sephadex G-10 gel chromatography. 2.4. Analytical treatments 2.4.1. Gross monitoring of the fractionated extract components The components in fractions produced by ion-exchange chromatography were scanned by ultraviolet absorption spectra ŽUV-1200, Shimadzu., ninhydrin reactions and enzymatic measurements of lactic acid ŽMarbach and Weil, 1967..
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Fig. 1. Flow chart showing the procedures for fractionating the ultrafiltrate of the muscle extract ŽFE. using an ion-exchanger.
2.4.2. High performance liquid chromatography (HPLC) Amino acids were analyzed by a Shimadzu LC-6A system equipped with a Simpack Amino-Li column Ž6 = 100 mm, Shimadzu. and a fluorescence detector ŽShimadzu RF550; excitation wavelength 348 nm, fluorescence wavelength 450 nm.. The column was eluted with 0.15 N ŽA. and 0.3 N ŽB. lithium citrate solutions at 398C. The flow rate was 0.6 ml miny1 . ATP-related compounds were analyzed by a Waters 625 LC System equipped with a reverse phase column ŽSTR ODS-II, 4.6 = 150 mm, Shimadzu. and UV monitor Ž250 nm.. The column was eluted with a mobile phase containing 18 mM diethylaminoethanol and 12 mM citric acid at 408C. The flow rate was 1.0 ml miny1 . Organic acids were analyzed by a Waters 625 LC System equipped with an organic acid column Ž7.8 = 300 mm, Waters. and a UV monitor Ž214 nm.. The column was eluted with 0.1% phosphoric acid at 408C. The flow rate was 1.0 ml miny1 . 2.5. Preparation of test pellets Wheat starch ŽNacalai Tesque, C.P. grade. was treated with 0.08 M NaOH to remove dissolved proteins and polyphenols from the insoluble starch layer and then freeze-dried. Pellets of test samples were made as follows: 4.7 g of starch was mixed with 4 ml of a
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test sample solution at original concentration and 0.3 g of carboxymethylcellulose ŽCMC, as a binding agent., and the mixture was then kneaded and extruded into pellets of size 25 mg each Ž3 = 4 mm.. Test samples were dissolved in 100% or diluted artificial seawater to get a salt concentration equivalent to seawater to avoid stimulating the taste receptors of yellowtail which has a high sensitivity to dilute seawater ŽHidaka and Ohsugi, 1979.. 2.6. Application of test pellets and statistical treatments Six groups of fish were used for each behavioral assay. The test pellets were dispensed through an opaque pipe Ž10 cm diameter= 50 cm length. set vertical in the middle of the tank ŽFukuda et al., 1989.. The fish were fed starch pellets containing CE or FE for two or three days to adapt to eating pellets. The experiment was performed once a day at 1400. One hundred pellets were offered simultaneously to each group of fish, and the number of pellets eaten within the first min was estimated by collecting and counting the uneaten pellets. When two Žor three. kinds of test samples were tested in
Fig. 2. The feeding-stimulatory effectiveness of the adsorbate ŽA A . and non-adsorbate ŽNA . fractions of FE from DEAE-Sephadex A-25 chromatography. Ordinate shows the average percent of pellets eaten per group of fish: 100 test pellets were offered to each of 6 groups of 20 fish and the number of pellets eaten in the first minute was estimated by counting the uneaten pellets. See also text.
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combination, three Žor two. tanks were allotted to each sample per day and tested in all six tanks taking two Žor three. days. To compare the stimulatory effectiveness among extract samples, Wilcoxon signedrank test for two samples was used ŽLehmann, 1975.. For samples containing data with a score of 100, which means all of the pellets offered were eaten and therefore the data were censored at the point of 100, a modified Wilcoxon test ŽGehan, 1965. was used.
3. Results 3.1. The feeding-stimulatory effects of FE, A A and NA Fig. 2 shows the results of a feeding experiment with A A and NA together with FE as a control. In this experiment, FE was completely eaten in all six groups of fish; A A was completely eaten in two groups but not in the others; NA was hardly eaten in all six groups, thus the average number of pellets eaten per group of fish being 100, 87.7 and 4.7, respectively. FE was usually completely eaten Žin 35 out of 36 groups tested w6 trials with 12 groups of fishx., while A A was completely eaten in 8 groups out of 24 groups Ž4
Fig. 3. The feeding-stimulatory effectiveness of A A and its two subdivided fractions, A A0.2 and A A0.2 – 0.6 . Ordinate shows the average percent of pellets eaten per group of fish as in Fig. 2. See also text.
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trials with 12 groups., but partially in 16. The modified Wilcoxon test ŽGehan, 1965. showed that A A was less effective at 5% level of significance. 3.2. The feeding-stimulatory effects of A A0 .2 and A A0 .2 – 0 .6 Fig. 3 compares the consumption A A and its two subdivided fractions, A A0.2 and A A0.2 – 0.6 . In this experiment, CE was used as a between trial control. In Fig. 3, the average number of pellets eaten per group of fish for A A , A A0.2 and A A0.2 – 0.6 were 89.7, 27.0 and 65.2, while CE was completely eaten in all six groups. The Wilcoxon test indicated A A0.2 - A A0.2 – 0.6 and A A0.2 - A A at the 5% level of significance, while A A0.2 – 0.6 and A A were not significantly different. In another experiment with the same groups as in Fig. 3, A A0.2 ) NA at the 5% level of significance. 3.3. Substances contained in A A , A A0 .2 and A A0 .2 – 0 .6 HPLC revealed that A A contained mainly lactic acid, IMP and glutamic acid ŽTables 1 and 2.. By stepwise elution of A A components from the DEAE-Sephadex column with 0.2 M and 0.6 M NaCl, glutamic acid and lactic acid were eluted separately from IMP. Analyses of A A , A A0.2 and A A0.2 – 0.6 for lactic acid by the enzyme method, for IMP by ultraviolet absorption and for amino acids by HPLC ŽTable 2., showed their presence at mM Žor mmolrkg of muscle. as follows: lactic acid in A A 34.8, in A A0.2 32.7 and in A A0.2 – 0.6 not detectable; IMP in A A 6.0, in A A0.2 – 0.6 6.1, and in A A0.2 no detectable amount; and glutamic acid in A A 1.15, in A A0.2 0.96 and in A A0.2 – 0.6 0.05 and minute quantities of phosphoserine and other amino acids in A A0.2 and A A0.2 – 0.6 ŽTable 2..
Table 1 ATP related substances and organic acids ŽmM a . detected by HPLC in FE and A A and NA from DEAE-Sephadex A-25 chromatography Žsee also text.. Ž – ., No detectable absorption was observed at 250 nm for ATP-related substances and at 214 nm for organic acids at the corresponding retention time Substances
AA
NA
FE
AMP ADP ATP IMP Inosine Hypoxanthine Ascorbic acid Citric acid Fumaric acid Lactic acid Malic acid Succinic acid Tartaric acid
– – – 6.61 0.04 0.04 – – 0.05 51.71 – – –
– – – 0.07 0.79 0.19 – – – – – – –
– – – 6.93 0.96 0.18 – – 0.10 61.53 – – –
a
Concentration of the compounds extracted from 50 g weight of the muscle in 50 ml solution.
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Table 2 Amino acids ŽmM a . detected by HPLC in A A , A A0.2 and A A0.2 – 0.6 fractions of FE from DEAE-Sephadex chromatography. Ž – ., Not detected or error; Žtr. trace Amino acids
AA
A A0.2
A A0.2 – 0.6
Phosphoserine Taurine Aspartic acid Hydroxy proline Threonine Serine Asparagine Glutamic acid Glutamine Sarcosine a-Amino-n-adipic acid Proline Glycine Alanine Citrulline a-Amino-n-butyric acid Valine Cystine Methionine Cystathionine Isoleucine Leucine Tyrosine Phenylalanine b-Alanine Histidine Ethanolamine Ornithine Lysine Arginine Total amino acids
0.01 0.00 0.02 – 0.00 0.00 – 0.96 – 0.00 0.01 0.00 0.00 0.00 tr 0.00 0.01 – 0.00 0.00 0.00 0.00 0.00 0.00 – 0.00 – 0.00 0.00 0.00 1.01
0.06 0.02 0.00 – 0.00 0.02 – 0.05 – 0.00 0.00 0.00 0.02 0.00 0.00 0.00 0.00 – 0.00 0.00 0.00 0.00 0.00 0.00 – 0.01 – 0.00 0.00 0.00 0.16
0.07 0.02 0.01 – 0.00 0.00 – 1.15 – 0.00 0.03 0.00 0.02 0.00 tr 0.00 0.00 – 0.00 tr 0.00 tr 0.00 0.00 – 0.02 – 0.00 0.00 0.00 1.32
a
The same as in Table 1.
These results indicated that both lactic acid and glutamic acid were eluted in A A0.2 , and IMP was almost exclusively eluted in A A0.2 – 0.6 .
4. Discussion In the present experiment, pellets containing plain sea water were hardly eaten, while those containing either CE or FE were as well eaten as previously ŽFukuda et al., 1989.. Thus, it appeared that neither starch nor CMC had any stimulatory or inhibitory effects on the feeding behavior of yellowtail. We also examined the stimulatory effects of the starch and CMC on the taste receptors of yellowtail by applying a 10% starch suspension and a 1% CMC solution to the palate and recording the taste nerve responses
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ŽFukuda et al., 1989.. We observed no appreciable response to the starch, but observed a tiny but continuing impulses to CMC which were considered to be a kind of mechanical response ŽKiyohara et al., 1985.. The treatment of FE on the DEAE-Sephadex column showed that A A contained the main stimulants. HPLC showed that A A mainly contained lactic acid, IMP and glutamic acid. Of these, lactic acid and glutamic acid were eluted in A A0.2 and IMP in A A0.2 – 0.6 . The results in Fig. 3 show that A A0.2 was less effective than A A and A A0.2 – 0.6 , while A A0.2 – 0.6 was no less effective than A A . However, it was 25% less effective in the average percentage of pellets eaten than A A . This suggests that either glutamic acid and lactic acid or both enhance IMP to elicit the effectiveness of A A , although they were effective rather weakly as A A0.2 alone. However, glutamic acid did not seem to stimulate the taste receptors ŽHidaka et al., 1985.. Whereas lactic acid is a potent stimulant for the taste receptors with a threshold of 10y6 –10y5 M ŽHidaka et al., 1992. and also enhances feeding-stimulatory effectiveness of a synthetic jack mackerel muscle extract ŽKohbara et al., 1993.. We previously observed that a synthetic mixture of jack mackerel muscle extracts ŽKonosu et al., 1974. was less effective than the natural extract ŽCE. ŽKohbara et al., 1989., even after lactic acid was added to it ŽKohbara et al., 1993.. In the latter mixture, IMP and lactic acid were 7.5 mM and 50 mM, respectively. The amounts are quite comparable to those found for FE in Table 1. Therefore, it might be that components of NA also have an enhancing effect on A A components although NA had no appreciable effect when applied alone. Takeda et al. studied the feeding-enhancing effects of jack mackerel extracts on yellowtail by adding test stimulants to a casein-based diet ŽTakeda, 1980.. They observed the daily feeding rate and found that IMP was a major stimulant. Whereas in a similar study on the krill extract, they found that the amino acid fraction plus the nucleotide fraction were effective ŽTakeda, 1980.. The amino acids involved were alanine, methionine and proline. These amino acids were contained in the krill extract at 40–261 mgr93 ml Ž3–30 mM., while in the jack mackerel extract at 1–21 mgr93 ml Ž70 mM–2 mM.. On the other hand, IMP was 2.4 mgr93 ml Ž70 mM. in the former and 308 mgr93 ml Ž9 mM. in the latter. Thus, the difference in the main stimulatory substances between the two extracts appears to be the abundance of stimulatory substances. Takaoka et al. Ž1990. reported that for marbled rockfish, inosine was the most effective stimulant in a krill extract. In this extract inosine was 137 mgr93 ml Ž5.5 mM., whereas, IMP and other nucleotides were present in much smaller quantities. They also reported that IMP and other nucleotides were as effective as inosine at high concentrations, whereas inosine in the krill extract was not a potent stimulant for yellowtail and jack mackerel ŽTakeda, 1980; Ikeda et al., 1988b.. In our previous study with the synthetic mixture of squid muscle extracts ŽFukuda et al., 1989., we found that the amino acid fraction was the most effective, and the nucleotide fraction somewhat enhanced the amino acid fraction. We also found that amino acids which were stimulatory for the taste receptors were involved in the feeding-stimulatory effect. In the squid muscle extract also, amino acids are abundantly found ŽSuyama and Kobayashi, 1980..
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The high feeding-stimulatory potency of IMP and inosine has also been reported in the turbot ŽMackie and Adron, 1978.. Components of a squid muscle extract were tested by adding them to a casein-based diet, and inosine was identified as a specific stimulant: removal of inosine from the synthetic mixture effectively abolished feeding stimulation and inosine alone was almost as effective as the complete mixture. IMP was also as or more effective than inosine when compared at equal molesrg diet ŽMackie and Adron, 1978.. In the largemouth bass also, IMP had a high feeding-enhancing effect when added to a soybean based diet while inosine did not ŽKubitza et al., 1997.. In puffer Takifugu rubripes ŽTakaoka et al., 1995. and T. pardalis Žformerly Fugu pardalis, Ohsugi et al., 1978., marked synergistic effects between amino acids and betaine were found in clam Tapes japonica extract. Similar synergism was also reported for juvenile Dover sole ŽMackie et al., 1980.. Mixtures of several amino acids have been reported to be more effective than single or combinations of a few amino acids in rainbow trout ŽAdron and Mackie, 1978., red sea bream ŽFuke et al., 1981. and eel ŽMackie and Mitchell, 1983; Takeda et al., 1984.. Also, some amino acids and other constituents of tissue extracts have been reported to behave as a feeding incitant or feeding deterrent in mixtures with other constituents ŽMackie, 1982; Mearns et al., 1987; Ikeda et al., 1988b..
Acknowledgements We thank Mr. Hiromu Nakamura, the Fisheries Research Laboratory, Faculty of Bioresources, for help in transporting the experimental animals. We also thank Dr. Seishi Kimura for help during our stay at the Laboratory. This work was supported by grants from the Ministry of Education, Science and Culture of Japan ŽNos. 0366190, 0766024..
References Adams, M.A., Johnsen, P.B., 1986. Chemical control of feeding in herbivorous and carnivorous fish. In: Duvall, D., Muller-Schwarze, D., Silverstein, R.M. ŽEds.., Chemical Signals in Vertebrates IV. Plenum, New York, pp. 45–61. Adron, J.W., Mackie, A.M., 1978. Studies on the chemical nature of feeding stimulants for rainbow trout, Salmo gairdneri Richardson. J. Fish Biol. 12, 303–310. Carr, W.E.S., 1967. Chemoreception in the mud snail, Nassarius obsoletus: II. Identification of stimulatory substances. Biol. Bull. 133, 106–127. Carr, W.E.S., Chaney, T.B., 1976. Chemical stimulation of feeding behavior in the pinfish, Lagodon rhomboides: characterization and identification of stimulatory substances extracted from shrimp. Comp. Biochem. Physiol. A 54, 437–441. Carr, W.E.S., Blumenthal, K.M., Netherton, J.C. III, 1977. Chemoreception in the pigfish, Orthopristis chrysopterus: the contribution of amino acids and betaine to stimulation of feeding behavior by various extracts. Comp. Biochem. Physiol. A 58, 69–73. Carr, W.E.S., Netherton, J.C. III, Gleeson, R.A., Derby, C.D., 1996. Stimulants of feeding behavior in fish: analyses of tissues of diverse marine organisms. Biol. Bull. 190, 149–160. Fuke, S., Konosu, S., Ina, K., 1981. Identification of feeding stimulants for red sea bream in the extract of marine worm Perinereis breÕicirrus. Bull. Jpn. Soc. Sci. Fish. 47, 1631–1635.
I. Hidaka et al.r Aquaculture 181 (2000) 115–126
125
Fukuda, K., Kohbara, J., Zeng, C., Hidaka, I., 1989. The feeding-stimulatory effects of squid muscle extracts on the young yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi 55, 791–797, Žin Japanese, with English abstract.. Gehan, E.A., 1965. A generalized Wilcoxon test for comparing arbitrarily singly-censored samples. Biometrika 52, 203–223. Goh, Y., Tamura, T., 1980. Effects of amino acids on the feeding behaviour in red sea bream. Comp. Biochem. Physiol. 66C, 225–229. Harada, K., 1985. Feeding attraction activities of amino acids and lipids for juvenile yellowtail. Bull. Jpn. Soc. Sci. Fish. 51, 453–459. Harpaz, S., 1997. Enhancement of growth in juvenile freshwater prawns, Macrobrachium rosenbergii, through the use of a chemoattractant. Aquaculture 156, 221–227. Hidaka, I., Ohsugi, T., 1979. High sensitivity of the palatal chemoreceptors of the yellowtail to dilution of sea water. Bull. Jpn. Soc. Sci. Fish. 45, 643. Hidaka, I., Ohsugi, T., Yamamoto, Y., 1985. Gustatory response in the young yellowtail Seriola quinqueradiata. Bull. Jpn. Soc. Sci. Fish. 51, 21–24. Hidaka, I., Zeng, C., Kohbara, J., 1992. Gustatory responses to organic acids in the yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi 58, 1179–1187. Ikeda, I., Hosokawa, H., Shimeno, S., Takeda, M., 1988a. Identification of feeding stimulant for jack mackerel in its muscle extract. Nippon Suisan Gakkaishi 54, 229–233, Žin Japanese, with English abstract.. Ikeda, I., Hosokawa, H., Shimeno, S., Takeda, M., 1988b. Identification of feeding stimulant in the krill extract for jack mackerel. Nippon Suisan Gakkaishi 54, 235–238, Žin Japanese, with English abstract.. Kiyohara, S., Hidaka, I., Kitoh, J., Yamashita, S., 1985. Mechanical sensitivity of the facial nerve fibers innervating the anterior palate of the puffer, Fugu pardalis, and their central projection to the primary taste center. J. Comp. Physiol. A 157, 705–716. Kohbara, J., Fukuda, K., Hidaka, I., 1989. The feeding-stimulatory effects of the jack mackerel white muscle extracts on the young yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi 55, 1343–1347, Žin Japanese, with English abstract.. Kohbara, J., Hidaka, I., Morishita, T., Miyajima, T., 1993. The feeding-stimulatory effectiveness of L-lactic acid on the young yellowtail Seriola quinqueradiata. Nippon Suisan Gakkaishi 59, 183. Kohbara, J., Hidaka, I., Morishita, T., Miyajima, T., 1999. Gustatory and olfactory sensitivity to extracts of jack mackerel muscle in young yellowtail Seriola quinqueradiata. Aquaculture, submitted. Konosu, S., Watanabe, K., Shimizu, T., 1974. Distribution of nitrogenous constituents in the muscle extracts of eight species of fish. Bull. Jpn. Soc. Sci. Fish. 40, 909–915. Kubitza, F., Lovshin, L.L., Lovell, R.T., 1997. Identification of feed enhancers for juvenile largemouth bass Micropterus salmoides. Aquaculture 148, 191–200. Lehmann, E.L., 1975. Nonparametrics: Statistical Methods Based on Ranks Žtranslated into Japanese by Nabeya, S., Kriya, T., Miura, R., 1978.. Morikita Shuppan, Tokyo, 484 pp. Mackie, A.M., 1982. Identification of the gustatory feeding stimulants. In: Hara, T.J. ŽEd.., Chemoreception in Fishes. Elsevier, Amsterdam, pp. 275–291. X Mackie, A.M., Adron, J.W., 1978. Identification of inosine and inosine 5 -monophosphate as the gustatory feeding stimulants for the turbot, Scophthalmus maximus. Comp. Biochem. Physiol. A 60, 79–83. Mackie, A.M., Mitchell, A.I., 1983. Studies on the chemical nature of feeding stimulants for the juvenile European eel, Anguilla anguilla ŽL... J. Fish Biol. 22, 425–430. Mackie, A.M., Mitchell, A.I., 1985. Identification of gustatory feeding stimulants for fish-applications in aquaculture. In: Cowey, C.B., Mackie, A.M., Bell, J.G. ŽEds.., Nutrition and Feeding in Fish. Academic Press, London, pp. 177–189. Mackie, A.M., Adron, J.W., Grant, P.T., 1980. Chemical nature of feeding stimulants for the juvenile Dover sole, Solea solea ŽL... J. Fish Biol. 16, 701–708. Marbach, E.P., Weil, M.H., 1967. Rapid enzymatic measurement of blood lactate and pyruvate. Use and significance of metaphosphoric acid as a common precipitant. Clin. Chem. 13, 314–325. Mearns, K.J., Ellingsen, O.F., Døving, K.B., Helmer, S., 1987. Feeding behaviour in adult rainbow trout and Atlantic salmon parr, elicited by chemical fractions and mixtures of compounds identified in shrimp extract. Aquaculture 64, 47–63.
126
I. Hidaka et al.r Aquaculture 181 (2000) 115–126
Ohsugi, T., Hidaka, I., Ikeda, M., 1978. Taste receptor stimulation and feeding behaviour in the puffer, Fugu pardalis: II. Effects produced by mixtures of constituents of clam extracts. Chem. Senses Flavour 3, 355–368. Suyama, M., Kobayashi, H., 1980. Free amino acids and quaternary ammonium bases in mantle muscle of squids. Bull. Jpn. Soc. Sci. Fish. 46, 1261–1264, Žin Japanese, with English abstract.. Takaoka, O., Takii, K., Nakamura, M., Kumai, H., Takeda, M., 1990. Identification of feeding stimulants for marbled rockfish. Nippon Suisan Gakkaishi 56, 345–351, Žin Japanese, with English abstract.. Takaoka, O., Takii, K., Nakamura, M., Kumai, H., Takeda, M., 1995. Identification of feeding stimulants for tiger puffer. Fisheries Sci. 61, 833–836. Takeda, M., 1980. Feeding stimulants for fish. Iden ŽHeredity. 34, 45–51, Žin Japanese.. Takeda, M., Takii, K., 1992. Gustation and nutrition in fishes: application to aquaculture. In: Hara, T.J. ŽEd.., Fish Chemoreception. Chapman & Hall, London, pp. 271–287. Takeda, M., Takii, K., Matsui, K., 1984. Identification of feeding stimulants for juvenile eel. Bull. Jpn. Soc. Sci. Fish. 50, 645–651.