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Utilization of synthetic carotenoids by the prawn Penaeus japonicus reared under laboratory conditions
Aquaculture, 110 (1993) 151-159 Elsevier Science Publishers B.V., Amsterdam
151
AQUA 50046
Utilization of synthetic carotenoids by the prawn Penaeus japonicus reared under laboratory conditions Genevieve N&e-Sadargues”, Rem5 Castillo”, Helene Petit”, Sophie San&, Ramon Gomez Martinezb, Jose-Carlos G. Milicua”, Georges Choubertd and Jean-Paul Trilles” Wwertebrate Ecophysiology Laboratory, Languedoc Sciences and Technics University,Montpellier, France bTecnologia de Los Alimentos, Facultad de Farmacia, Vitoria, Spain ‘Department of Biochemistry, Faculty of Sciences, Universityof Basque Country, Bilbao, Spain ‘Fish Nutrition Laboratory, I.N.R.A. Hydrobiological Station, Saint-P&e-sur-Nivelle, France (Accepted 8 June 1992 )
ABSTRACT Negre-Sadargues, G., Castillo, R., Petit, H., San&, S., Gomez Martinez, R., Milicua, J-C.G., Choubert, G. and Trilles, J-P., 1993. Utilization of synthetic carotenoids by the prawn Penaeus japonicus reared under laboratory conditions. Aquaculture, 110: 15 1- 159. Four groups of prawns fed three different pigmented diets (astaxanthin 100 mg/kg, canthaxanthin 100 mg/kg, astaxanthin/canthaxanthin SO/SO mg/kg) and a pigment-free diet were maintained under laboratory rearing conditions during one moulting cycle. Dietary astaxanthin was found to be stored in the integument (carapace and epidermis) and hepatopancreas. Individuals fed the astaxanthin/canthaxanthin mixture showed an accumulation of carotenoids in the epidermis and exhibited the highest survival rate. There was no experimental evidence supporting a possible influence of these pigments on growth under the conditions used in this study.
INTRODUCTION
Carotenoids are widely distributed in living organisms and represent the main pigments of most aquatic species like fishes and crustaceans. Astaxanthin (3,3’-dihydroxy-4,4’-diketo+,/?-carotene) is considered to be the major carotenoid of crustacea, comprising about 90% of the total pigments in Penaeusjuponicus (Ishikawa et al., 1966) and can be recovered from the carapace and the internal organs of the prawn (Katayama et al., 1972). Correspondence to: G. Negre-Sadargues, Invertebrate Ecophysiology Laboratory, Languedoc Sciences and Technics University, Place Eugene Bataillon, 34095 Montpellier, Cedex 5, France.
0044-8486/93/$06.00
0 1993 Elsevier Science Publishers
B.V. All rights reserved.
G. NBGRE-SADARGUES
152
ET AL.
The metabolic scheme for oxidative transformation of B-carotene to astaxanthin (Katayama et al., 1971, 1972) has been demonstrated using pure carotenoids or pigmented products like alfalfa and spirulina (Tanaka et al., 1976). Astaxanthin may also be formed in the animal from zeaxanthin (3,3’dihydroxy-/I&carotene) (Tanaka et al., 1976). Several studies on carotenoid metabolism (Negre-Sadargues, 1978; Goodwin, 1984) of different crustaceans have demonstrated that most decapods are able to absorb and metabolize P-carotene. Certain species can convert canthaxanthin (4,4’-diketo-/I&carotene) into astaxanthin (Maugle et al., 1980; Yamada et al., 1990) whereas no transformation has been found to occur in dietary astaxanthin; on the other hand, the functions of these pigments in animals, crustaceans in particular, remain hypothetical. The purpose of this work was to investigate the effects of dietary carotenoids on zootechnical parameters like survival, growth and moulting frequency of the prawn Penaeus juponicus reared in the laboratory. A comparison of the tissue distribution of the pigments in shrimp fed carotenoid supplemented and carotenoid-free diets was also conducted. MATERIALS
AND METHODS
Experimental animals Juvenile prawns, Penaeusjaponicus, (about 12 g ) were purchased from SCA Mari-Aude, Port Leucate, France, aquacultural center. The moult stage was determined immediately after the arrival of the animals in the laboratory according to the method of Drach and Tchernigovtzeff ( 1967) as modified for penaeids (Cognie, 1970). Only shrimp in moult stage C were maintained for experimental purposes. Ten fresh-caught individuals were dissected and constituted the 0 group. Four groups of 48 shrimp per treatment were reared during the period between two successive C moult stages. All animals were sacrificed at the same time in order to minimize possible time effects on carotenoid distribution (Otazu-Abrill and Ceccaldi, 1978; Valin et al., 1987). Fresh weight determinations were made at the beginning ( W, ) and end of the experiment ( W, ) . Carapace and epidermis, dissected from the cephalothoracic region, and hepatopancreas were sampled and frozen at - 15 ‘C for subsequent pigment extraction. Results correspond to individual sample analysis. Rearing conditions The rearing system consisted of 60-l white PVC tanks each with a sand bottom. The sea water, changed every 2 days, was aerated by a permanent bubbling device and passed through an activated charcoal filter (400 l/h). The initial density was 12 individuals per tank. Water temperature was con-
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stant at 20’ C and artificial light was used to maintain a diurnal 12 h light : dark cycle. It should be noted that even under these conditions, some mortality was observed during the first week. Experimental diets and feeding The composition of our standard carotenoid-free diet (STD ) is presented in Table 1. The three experimental diets were prepared by supplementing the standard carotenoid-free diet as follows: diet C 100, 100 mg of canthaxanthin (Carophyll red) /kg of diet; diet Al 00, 100 mg of astaxanthin (Carophyll pink)/kg of diet; and diet A50/C50,50 mg of astaxanthin plus 50 mg of canthaxanthin/kg of diet. Astaxanthin and canthaxanthin were obtained from Hoffman-La Roche and Co., Basel, Switzerland in the form of water-dispersible gelatin beadlets. The carotenoid content of the four diets was analysed at the beginning and end of the experiment and showed similar results. The STD diet was found to contain detectable amounts of an unidentified yellow pigment which apparently resulted from some dietary ingredient. Mean duplicate analyses of the carotenoid concentration of the experimental diets were 88.9, 12 1.1 and 97.9 mg/kg dry weight for diets C 100, A 100 and A50/C50, respectively. The diets were prepared by adding the carotenoids to the standard diet; the mixture was transformed into pellets, dried and stored at - 18 ‘C. Shrimps were fed daily at 17.00 h according to a feeding rate of 0.5 g diet/ shrimp day- I. The experiment lasted 4 weeks. Analyticalprocedure Carotenoid analyses were con ucted according to previously outlined methods (Castillo and Lenel, 197 8; Castillo et al., 1988 ). Petroleum ether solutions of carotenoids were measured at 470 nm (absorption peak of astaxanthin ) using a Uvikon 8 10 scan-recorder spectrophotometer. TABLE 1 Composition
of standard diet
Ingredient
% dry weight
Ingredient
I dry weight
Fish meal Precooked starch Mineral mix’ Vitamin mix2 Saccharose
55 19 5 5 3.5
Cholesterol Cod liver oil Cellulose Gelatin Agar
0.5 5 1 3 3
‘Luquet (1971). ‘EIFAC ( 197 1).
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154 RESULTS
Rearing parameters Survival. Under these rearing conditions, the mortality rate was found to be very similar for shrimp fed the STD (48.77%), Al00 (46.15%) and Cl00 diets (53.06%), but lower for shrimps fed the A5O/C50 diet (23.07%). The loss recorded at ecdysis may be ascribed to a physiological weakness of the animals during this period. Frequency of moulting. The shrimp were in moult stage C at the beginning of the experiment. Exuviae were collected daily. The number of moults, which appeared to be very low during the second week except for the C loo-fed group, was higher during the third week, We noted a shortening of the moult cycle in the group fed the Cl00 diet; in fact, all of these animals had undergone a moult before the fourth week. Growth rate. Shrimp fed the standard diet had a lower growth rate than that observed for the animals fed the carotenoid-supplemented diets (Table 2). The shrimp fed canthaxanthin had a growth rate higher than that of individuals receiving the other three diets. Carotenoid content The carotenoid concentrations in the different tissues of the shrimp are summarized in Table 3. The values obtained for the digestive gland and epidermis of the 0 group of shrimp were quite scattered. In fact, the carotenoid content of the hepatopancreas of animals depends directly on the availability of food in their natural environment. Consequently, analyses of the results for the different groups (Table 3 ) did not allow statistical comparison of these values. However, the data obtained for the carapace (Table 3) did permit TABLE 2 Growth rates of shrimp fed the experimental Diet
wo (g)
STD Cl00 Al00 ASO/CSO
12.10f0.23 10.71 kO.19 11.65kO.20 11.23f0.27
diets’,2.3 Growth rate (Oh)
W, (9) (48)” (48)b (48)” (48)b
13.02t0.46 12.61 f0.47 12.76f0.42 12.54k0.39
(13)’ (19)’ (12)’ (20)’
1.60 17.74 9.52 11.66
’ W, and W,: initial and final wet body weight. ‘Means* s.e.m. of (x) shrimp per group. ‘Means with different superscripts in each column are significantly different (PC 0.05).
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TABLE 3 Carotenoid concentration Dietary group
in different tissues of the shrimp’,’
Tissue (concentration Hepatopancreas
0
STD Cl00 Al00 ASO/CSO
34.77f 106.97+ 112.97f 232.16+ 117.83+
5.24 10.22 14.01 19.51 12.52
expressed as ,ug/g wet weight ) Epidermis
(8) (12)” (16)” ( 10)b (16)”
149.58+ 17.49 118.10t17.87 167.76+21.51 277.66k31.17 381.86k29.77
Carapace (10) (12)” (15)’ (ll)b (18)’
28.31 k 3.36 15.71 f2.30 10.77+ 1.84 24.56f2.64 10.44+ 1.76
( 9)b (12)” (12)” ( 10)b (17)”
‘Values are means f s.e.m. of (x) samples per group. *Means with different superscripts in each column are significantly different (PC 0.05).
such analyses. All tested tissues were found to contain astaxanthin. Free and esteritied forms occurred in the carapace, epidermis and hepatopancreas. The hepatopancreas of shrimp fed the STD, Cl00 and ASO/CSO diets had a quite similar low carotenoid content, significantly lower than that observed for the AlOO-fed group. Therefore, it appears that only astaxanthin is stored in the digestive gland. The carotenoid concentration of the epidermis was very similar for the STD and C loo-fed groups. Significantly higher concentrations were observed in shrimp fed the Al00 and ASO/CSO diets. Only the carapace from the A loo-fed group contained a carotenoid concentration similar to that of freshly collected shrimp (group 0). The remaining animals showed a decrease in pigment content in this tissue at the end of the experiment. All the analyses provided evidence for astaxanthin absorption by the hepatopancreas prior to its distribution in the integument. DISCUSSION
Survival
A higher survival rate was observed for the animals fed the ASO/CSO supplemented diet. This tendency for enhanced survival must, however, be confirmed with a larger number of individuals over a longer experimental period. The influence of carotenoid pigments on survival remains unclear. Different experiments have, nevertheless, shown a significant loss for adult shrimp fed on a carotenoid-free diet (unpublished results) in comparison with individuals receiving carotenoid-supplemented diets. Yamada et al. ( 1990) reported a survival rate of 9 1.4% for individuals fed on either an astaxanthin ( 100 mg/ kg) or pcarotene ( 100 mg/kg )-supplemented diet. This value was higher than that observed for their control (85.7%) group and for shrimp fed a cantha-
156
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ET AL.
xanthin-supplemented diet ( 100 mg/kg) (80%) during the first 4 weeks of their study. Moreover, their data indicated important variations beyond the fourth week, i.e., a higher rate of survival (9 1.3%) was observed for the astaxanthin-fed shrimp, whereas a value of 57.1% survival was observed for the control group. Moulting It should be pointed out that although the different groups of shrimp were selected randomly, an evaluation of the initial fresh weights indicates a mean value of 10.7 1 If:0.19 g for the animals fed the C 100 diet, whereas values for the three other groups (A 100, ASO/CSO and STD) were 11 to 12 g (see Table 2). The moulting cycle in shrimp of about 12 g fresh weight lasts about 3 weeks. A slight shortening of this period was observed for animals fed the C 100 diet. This may have been due to the smaller average size of this group, since it is well known that younger individuals show a higher moulting frequency. However, recent data concerning adult individuals as well as post-larval stages fed on carotenoid-supplemented diets (unpublished results) corroborate these observations. An evaluation of the ecdysteroids in the haemolymph of adult shrimps fed on carotenoid-supplemented diets is needed to provide more information on the possible physiological interaction between pigment and moulting hormones. Growth rate It is well known that in crustacea the growth rate decreases concomitantly with increasing age and consequently with increasing body weight (Choe, 1970). Thus, the growth rate ( 17.74% and 11.66%) observed for the prawn fed the C 100 and the ASO/CSO diets may ascribed to the fact that at the starting point ( IV,), their weight was slightly less than that of the other two groups (Table 2), suggesting that carotenoids may not have been involved in improving growth rate, in agreement with the findings of Yamada et al. ( 1990). Analogous growth rates have also been obtained for fish (rainbow trout ) fed diets with or without supplemented astaxanthin or canthaxanthin for several months (Foss et al., 1984). Consequences of different pigmen ted diets on tissue pigmen tation Canthaxanthin did not seem to be stored by the shrimp under our experimental conditions i.e., the carotenoid content of the hepatopancreas, epidermis and carapace of shrimp fed the C 100 diet were comparable to those observed for individuals fed the carotenoid-free diet. Similar results have been observed in previous experiments with synthetic canthaxanthin (unpublished results). It has, however, been reported that the prawn can transform dietary synthetic canthaxanthin into astaxanthin (Tanaka et al., 1976).
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On the contrary, dietary astaxanthin was found to be stored in the three different tissues analyzed (Table 3 ). Recent studies (Yamada et al., 1990) have likewise indicated that the integuments of Penaeus juponicus fed canthaxanthin and astaxanthin diets for 4 weeks showed comparable carotenoid concentration; however, an improved storage was observed in the shrimp fed the A 100 diet over a period of 8 weeks. In fish, on the other hand, astaxanthin and canthaxanthin are deposited unchanged, e.g. in salmonid flesh (Hata and Hata, 1973; Foss et al., 1984; Torrissen, 1986; Choubert and Storebakken, 1989); however, astaxanthin is better retained (Storebakken et al., 1987; Bjerkeng et al., 1990) due to a higher digestive retention rate. Hata and Hata ( 1972) have suggested that the occurrence of hydroxyl groups within the molecular configuration of the carotenoid enhances its absorption by the digestive epithelium. Thus astaxanthin (dihydroxycanthaxanthin ) may be more easily absorbed than canthaxanthin. The hepatopancreas as well as the carapace from shrimp fed the ASO/CSO diet had carotenoid concentration values comparable to those found in prawns fed the STD and C 100 diets, whereas the epidermis contained a higher quantity of these pigments. Astaxanthin and canthaxanthin seemed to be readily absorbed by the epidermis, provided that equal amounts of these pigments occur simultaneously in the food (A5O/C50). However, the accumulation of carotenoids in the digestive gland was not observed in this case. Furthermore, the pigment content of the carapace of animals fed the ASO/CSO diet was quite low. Few studies have been successful concerning the use of dietary astaxanthin-canthaxanthin mixtures in fish. Results vary (Foss et al., 1987; Torrissen, 1989) and are probably due to the relative concentration of these pigments in the diet. Rainbow trout fed a mixture of astaxanthin (72%) and canthaxanthin (28%) showed a flesh carotenoid content higher than that of individuals fed diets containing 100% astaxanthin and 50% astaxanthin/50% canthaxanthin as the supplemental carotenoids (Torrissen, 1989). It seems likely that these two xanthophylls act synergistically, probably being absorbed in different parts of the digestive tract. On the other hand, the nature of pigment carriers in fish blood, recently established in the chum salmon Oncorhynchus keta (Nakamura et al., 1985; Ando et al., 1986) as well as in the trout Oncorhynchus mykiss (Choubert et al., 1992), must likewise be determined in crustacea in order to improve knowledge on the consequences of the use of synthetic carotenoids for shrimp. ACKNOWLEDGEMENTS
This study is supported by a France-Spain Technical and Scientific Cooperation Programme (Action Integree no. 263). Carophyll red and Carophyll
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pink were kindly supplied by Hoffmann-La Roche (Basle, Switzerland). The authors thank Mrs. E. Grousset for technical assistance.
REFERENCES Ando, S., Takeyama, T. and Hatano, M., 1986. Transport associated with serum vitellogenin of carotenoid in chum salmon Oncorhynchus keta. Agric. Biol. Chem., 50: 557-563. Bjerkeng, B., Storebakken, T. and Liaaen-Jensen, S., 1990. Response to carotenoids by rainbow trout in the sea: resorption and metabolism of dietary astaxanthin and canthaxanthin. Aquaculture, 9 1: 153- 162. Castillo, R. and Lenel, R., 1978. Determination and metabolism of carotenoid pigments in the hermit crab Clibanarius erythropus Latreille ( 18 18) [ Syn. Cl. misanthropus Heller ( 1863) 1. Comp. Biochem. Physiol., 59B: 67-73. Castillo, R., Ntgre-Sadargues, G. and Lenel, R., 1988. The carotenoids of the hermit crab Clibanarius erythropus Latreille ( 18 18 ) (Crustacea, Decapoda, Anomoura) during the moulting cycle. I. Quantitative aspects. Comp. Biochem. Physiol., 89B: 239-243. Choe, S., 1970. Study on feeding and growth of the oriental brown shrimp Penaeus japonicus Bate. Bull. Korean Fish. Sot., 3: 16 1- 17 1. Choubert, G. and Storebakken, T., 1989. Dose response to astaxanthin and canthaxanthin pigmentation of rainbow trout fed various dietary carotenoid concentrations. Aquaculture, 8 1: 67-77. Choubert, G., Milicua, J.-C.G., Gomez Martinez, G., Sante, S., Petit, H., Negre-Sadargues, G., Castillo, R. and Trilles, J.P., 1992. Transport of canthaxanthin in serum of rainbow trout Oncorhynchus mykiss Walb. Comp. Biochem. Physiol., 103A: 403-406. Cognie, D., 1970. Contribution a l’etude de la biologie de Penaeus kerathurus (Forsklll, 1775), Crustace Decapode, en fonction du cycle d’intermue. These de Doct. Specialite, Universite Aix-Marseille, France, 83 pp. Drach, P. and Tchernigovtzeff, C., 1967. Sur la methode de determination des stades d’intermue et son application g&t&ale aux crustacts. Vie Milieu, Ser. A, 18: 595-6 10. European Inland Fisheries Advisory Commission (EIFAC), 197 1. Salmon and trout feeds and feeding. FAO, Rome, Tech.. Pap. 12: 29 pp. Foss, P., Storebakken, T., Schiedt, K., Liaaen-Jensen, S., Austreng, E. and Streiff, K., 1984. Carotenoids in diets for salmonids. I. Pigmentation of rainbow trout with the individual optical isomers of astaxanthin in comparison with canthaxanthin. Aquaculture, 41: 213226. Foss, P., Storebakken, T., Austreng, E. and Liaaen-Jensen, S., 1987. Carotenoids in diets for salmonids. V. Pigmentation of rainbow trout and sea trout with astaxanthin and astaxanthin dipalmitate in comparison with canthaxanthin. Aquaculture, 65: 293-305. Goodwin, T.W., 1984. The Biochemistry of the Carotenoids, Vol. II. Animals. Chapman and Hall, London, 224 pp. Hata, M. and Hata, M., 1972. Carotenoid pigments in goldfish. IV. Carotenoid metabolism. Bull. Jpn. Sot. Sci. Fish., 38: 331-338. Hata, M. and Hata, M., 1973. Studies on astaxanthin formation in some freshwater fishes. Tohoku J. Agric. Res., 24: 192- 196. Ishikawa, Y., Miyake, Y. and Yasuie, S., 1966. Chromatophores and carotenoids in cultured kuruma-prawn, Penaeus japonicus Bate, of different body colours. Bull. Fish. Exp. Stn. Okayoma Pref., 5: 18-24 (in Japanese). Katayama, T., Hirata, K. and Chichester, C.O., 1971. The biosynthesis of astaxanthin. IV. The
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carotenoids in the prawn, Penaeus juponicus Bate (Part 1). Bull. Jpn. Sot. Sci. Fish., 37: 614-620. Katayama, T., Kamata, T., Shimaya, M., Deshimaru, 0. and Chichester, C.O., 1972. The biosynthesis of the carotenoids. VIII, The conversion of labelled p-carotene-l 5,l 5’-3Hz into astaxanthin in prawn, Penaeus juponicus Bate. Bull. Jpn. Sot. Sci. Fish., 38: 117 l-l 175. Luquet, P., 197 1. Efficacite des proteines en relation avec leur taux d’incorporation dans l’alimentation de la truite arc-en-ciel. Ann. Hydrobiol., 2: 175- 186. Maugle, P., Kamata, T., McLean, S., Simpson, K.L. and Katayama, T., 1980. The influence of eyestalk ablation on the carotenoid composition of juvenile Mucrobruchium rosenbergii. Bull. Jpn. Sot. Sci. Fish., 46: 90 l-904. Nakamura, K., Hata, M. and Hata, M., 1985. A study on astaxanthin in salmon Oncorhynchus ketu serum. Bull. Jpn. Sot. Sci. Fish., 5 1: 979-983. N&e-Sadargues, G., 1978. Sur les transformations metaboliques des pigments carotenoides chez les crustads: synthbe bibliographique. Ann. Biol., 17: 415-454. Otzu-Abrill, M. and Ceccaldi, H.J., 1978. Variations circadiennes des pigments carotenoides dans les yeux et l’hepatopancreas de Penueus juponicus (Crustace, Decapode). CR. Seanc. Sot. Biol., 172: 684-690. Storebakken, T., Foss, P., Schiedt, K., Austreng, E., Liaaen-Jensen, S. and Manz, U., 1987. Carotenoids in diets for salmonids. IV. Pigmentation of Atlantic salmon with astaxanthin, astaxanthin dipalmitate and canthaxanthin. Aquaculture, 65: 279-292. Tanaka, Y., Matsuguchi, H., Katayama, T., Simpson, K.L. and Chichester, C.O., 1976. The biosynthesis of astaxanthin. XVIII. The metabolism of the carotenoids in the prawn. Penueus juponicus Bate. Bull. Jpn. Sot. Sci. Fish., 42: 197-202. Torrissen, O.J., 1986. Pigmentation of salmonids. A comparison of astaxanthin and canthaxanthin as pigment sources for rainbow trout. Aquaculture, 53: 27 l-278. Torrissen, O.J., 1989. Pigmentation of salmonids: interactions of astaxanthin and canthaxanthin on pigment deposition in rainbow trout. Aquaculture, 79: 363-374. Valin, A., Castillo, R., Negre-Sadargues, G. and Lenel, R., 1987. Quantitative aspects of carotenoid pigment circadian variations in the crayfish Astucus leptoductylus. Biochem. Syst. Ecol., 15: 607-6 10. Yamada, S., Tanaka, Y., Sameshima, M. and Ito, Y., 1990. Pigmentation of prawn (Penueus juponicus) with carotenoids. I. Effect of dietary astaxanthin, p-carotene and canthaxanthin on pigmentation. Aquaculture, 87: 323-330.
151
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Utilization of synthetic carotenoids by the prawn Penaeus japonicus reared under laboratory conditions Genevieve N&e-Sadargues”, Rem5 Castillo”, Helene Petit”, Sophie San&, Ramon Gomez Martinezb, Jose-Carlos G. Milicua”, Georges Choubertd and Jean-Paul Trilles” Wwertebrate Ecophysiology Laboratory, Languedoc Sciences and Technics University,Montpellier, France bTecnologia de Los Alimentos, Facultad de Farmacia, Vitoria, Spain ‘Department of Biochemistry, Faculty of Sciences, Universityof Basque Country, Bilbao, Spain ‘Fish Nutrition Laboratory, I.N.R.A. Hydrobiological Station, Saint-P&e-sur-Nivelle, France (Accepted 8 June 1992 )
ABSTRACT Negre-Sadargues, G., Castillo, R., Petit, H., San&, S., Gomez Martinez, R., Milicua, J-C.G., Choubert, G. and Trilles, J-P., 1993. Utilization of synthetic carotenoids by the prawn Penaeus japonicus reared under laboratory conditions. Aquaculture, 110: 15 1- 159. Four groups of prawns fed three different pigmented diets (astaxanthin 100 mg/kg, canthaxanthin 100 mg/kg, astaxanthin/canthaxanthin SO/SO mg/kg) and a pigment-free diet were maintained under laboratory rearing conditions during one moulting cycle. Dietary astaxanthin was found to be stored in the integument (carapace and epidermis) and hepatopancreas. Individuals fed the astaxanthin/canthaxanthin mixture showed an accumulation of carotenoids in the epidermis and exhibited the highest survival rate. There was no experimental evidence supporting a possible influence of these pigments on growth under the conditions used in this study.
INTRODUCTION
Carotenoids are widely distributed in living organisms and represent the main pigments of most aquatic species like fishes and crustaceans. Astaxanthin (3,3’-dihydroxy-4,4’-diketo+,/?-carotene) is considered to be the major carotenoid of crustacea, comprising about 90% of the total pigments in Penaeusjuponicus (Ishikawa et al., 1966) and can be recovered from the carapace and the internal organs of the prawn (Katayama et al., 1972). Correspondence to: G. Negre-Sadargues, Invertebrate Ecophysiology Laboratory, Languedoc Sciences and Technics University, Place Eugene Bataillon, 34095 Montpellier, Cedex 5, France.
0044-8486/93/$06.00
0 1993 Elsevier Science Publishers
B.V. All rights reserved.
G. NBGRE-SADARGUES
152
ET AL.
The metabolic scheme for oxidative transformation of B-carotene to astaxanthin (Katayama et al., 1971, 1972) has been demonstrated using pure carotenoids or pigmented products like alfalfa and spirulina (Tanaka et al., 1976). Astaxanthin may also be formed in the animal from zeaxanthin (3,3’dihydroxy-/I&carotene) (Tanaka et al., 1976). Several studies on carotenoid metabolism (Negre-Sadargues, 1978; Goodwin, 1984) of different crustaceans have demonstrated that most decapods are able to absorb and metabolize P-carotene. Certain species can convert canthaxanthin (4,4’-diketo-/I&carotene) into astaxanthin (Maugle et al., 1980; Yamada et al., 1990) whereas no transformation has been found to occur in dietary astaxanthin; on the other hand, the functions of these pigments in animals, crustaceans in particular, remain hypothetical. The purpose of this work was to investigate the effects of dietary carotenoids on zootechnical parameters like survival, growth and moulting frequency of the prawn Penaeus juponicus reared in the laboratory. A comparison of the tissue distribution of the pigments in shrimp fed carotenoid supplemented and carotenoid-free diets was also conducted. MATERIALS
AND METHODS
Experimental animals Juvenile prawns, Penaeusjaponicus, (about 12 g ) were purchased from SCA Mari-Aude, Port Leucate, France, aquacultural center. The moult stage was determined immediately after the arrival of the animals in the laboratory according to the method of Drach and Tchernigovtzeff ( 1967) as modified for penaeids (Cognie, 1970). Only shrimp in moult stage C were maintained for experimental purposes. Ten fresh-caught individuals were dissected and constituted the 0 group. Four groups of 48 shrimp per treatment were reared during the period between two successive C moult stages. All animals were sacrificed at the same time in order to minimize possible time effects on carotenoid distribution (Otazu-Abrill and Ceccaldi, 1978; Valin et al., 1987). Fresh weight determinations were made at the beginning ( W, ) and end of the experiment ( W, ) . Carapace and epidermis, dissected from the cephalothoracic region, and hepatopancreas were sampled and frozen at - 15 ‘C for subsequent pigment extraction. Results correspond to individual sample analysis. Rearing conditions The rearing system consisted of 60-l white PVC tanks each with a sand bottom. The sea water, changed every 2 days, was aerated by a permanent bubbling device and passed through an activated charcoal filter (400 l/h). The initial density was 12 individuals per tank. Water temperature was con-
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stant at 20’ C and artificial light was used to maintain a diurnal 12 h light : dark cycle. It should be noted that even under these conditions, some mortality was observed during the first week. Experimental diets and feeding The composition of our standard carotenoid-free diet (STD ) is presented in Table 1. The three experimental diets were prepared by supplementing the standard carotenoid-free diet as follows: diet C 100, 100 mg of canthaxanthin (Carophyll red) /kg of diet; diet Al 00, 100 mg of astaxanthin (Carophyll pink)/kg of diet; and diet A50/C50,50 mg of astaxanthin plus 50 mg of canthaxanthin/kg of diet. Astaxanthin and canthaxanthin were obtained from Hoffman-La Roche and Co., Basel, Switzerland in the form of water-dispersible gelatin beadlets. The carotenoid content of the four diets was analysed at the beginning and end of the experiment and showed similar results. The STD diet was found to contain detectable amounts of an unidentified yellow pigment which apparently resulted from some dietary ingredient. Mean duplicate analyses of the carotenoid concentration of the experimental diets were 88.9, 12 1.1 and 97.9 mg/kg dry weight for diets C 100, A 100 and A50/C50, respectively. The diets were prepared by adding the carotenoids to the standard diet; the mixture was transformed into pellets, dried and stored at - 18 ‘C. Shrimps were fed daily at 17.00 h according to a feeding rate of 0.5 g diet/ shrimp day- I. The experiment lasted 4 weeks. Analyticalprocedure Carotenoid analyses were con ucted according to previously outlined methods (Castillo and Lenel, 197 8; Castillo et al., 1988 ). Petroleum ether solutions of carotenoids were measured at 470 nm (absorption peak of astaxanthin ) using a Uvikon 8 10 scan-recorder spectrophotometer. TABLE 1 Composition
of standard diet
Ingredient
% dry weight
Ingredient
I dry weight
Fish meal Precooked starch Mineral mix’ Vitamin mix2 Saccharose
55 19 5 5 3.5
Cholesterol Cod liver oil Cellulose Gelatin Agar
0.5 5 1 3 3
‘Luquet (1971). ‘EIFAC ( 197 1).
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154 RESULTS
Rearing parameters Survival. Under these rearing conditions, the mortality rate was found to be very similar for shrimp fed the STD (48.77%), Al00 (46.15%) and Cl00 diets (53.06%), but lower for shrimps fed the A5O/C50 diet (23.07%). The loss recorded at ecdysis may be ascribed to a physiological weakness of the animals during this period. Frequency of moulting. The shrimp were in moult stage C at the beginning of the experiment. Exuviae were collected daily. The number of moults, which appeared to be very low during the second week except for the C loo-fed group, was higher during the third week, We noted a shortening of the moult cycle in the group fed the Cl00 diet; in fact, all of these animals had undergone a moult before the fourth week. Growth rate. Shrimp fed the standard diet had a lower growth rate than that observed for the animals fed the carotenoid-supplemented diets (Table 2). The shrimp fed canthaxanthin had a growth rate higher than that of individuals receiving the other three diets. Carotenoid content The carotenoid concentrations in the different tissues of the shrimp are summarized in Table 3. The values obtained for the digestive gland and epidermis of the 0 group of shrimp were quite scattered. In fact, the carotenoid content of the hepatopancreas of animals depends directly on the availability of food in their natural environment. Consequently, analyses of the results for the different groups (Table 3 ) did not allow statistical comparison of these values. However, the data obtained for the carapace (Table 3) did permit TABLE 2 Growth rates of shrimp fed the experimental Diet
wo (g)
STD Cl00 Al00 ASO/CSO
12.10f0.23 10.71 kO.19 11.65kO.20 11.23f0.27
diets’,2.3 Growth rate (Oh)
W, (9) (48)” (48)b (48)” (48)b
13.02t0.46 12.61 f0.47 12.76f0.42 12.54k0.39
(13)’ (19)’ (12)’ (20)’
1.60 17.74 9.52 11.66
’ W, and W,: initial and final wet body weight. ‘Means* s.e.m. of (x) shrimp per group. ‘Means with different superscripts in each column are significantly different (PC 0.05).
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TABLE 3 Carotenoid concentration Dietary group
in different tissues of the shrimp’,’
Tissue (concentration Hepatopancreas
0
STD Cl00 Al00 ASO/CSO
34.77f 106.97+ 112.97f 232.16+ 117.83+
5.24 10.22 14.01 19.51 12.52
expressed as ,ug/g wet weight ) Epidermis
(8) (12)” (16)” ( 10)b (16)”
149.58+ 17.49 118.10t17.87 167.76+21.51 277.66k31.17 381.86k29.77
Carapace (10) (12)” (15)’ (ll)b (18)’
28.31 k 3.36 15.71 f2.30 10.77+ 1.84 24.56f2.64 10.44+ 1.76
( 9)b (12)” (12)” ( 10)b (17)”
‘Values are means f s.e.m. of (x) samples per group. *Means with different superscripts in each column are significantly different (PC 0.05).
such analyses. All tested tissues were found to contain astaxanthin. Free and esteritied forms occurred in the carapace, epidermis and hepatopancreas. The hepatopancreas of shrimp fed the STD, Cl00 and ASO/CSO diets had a quite similar low carotenoid content, significantly lower than that observed for the AlOO-fed group. Therefore, it appears that only astaxanthin is stored in the digestive gland. The carotenoid concentration of the epidermis was very similar for the STD and C loo-fed groups. Significantly higher concentrations were observed in shrimp fed the Al00 and ASO/CSO diets. Only the carapace from the A loo-fed group contained a carotenoid concentration similar to that of freshly collected shrimp (group 0). The remaining animals showed a decrease in pigment content in this tissue at the end of the experiment. All the analyses provided evidence for astaxanthin absorption by the hepatopancreas prior to its distribution in the integument. DISCUSSION
Survival
A higher survival rate was observed for the animals fed the ASO/CSO supplemented diet. This tendency for enhanced survival must, however, be confirmed with a larger number of individuals over a longer experimental period. The influence of carotenoid pigments on survival remains unclear. Different experiments have, nevertheless, shown a significant loss for adult shrimp fed on a carotenoid-free diet (unpublished results) in comparison with individuals receiving carotenoid-supplemented diets. Yamada et al. ( 1990) reported a survival rate of 9 1.4% for individuals fed on either an astaxanthin ( 100 mg/ kg) or pcarotene ( 100 mg/kg )-supplemented diet. This value was higher than that observed for their control (85.7%) group and for shrimp fed a cantha-
156
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xanthin-supplemented diet ( 100 mg/kg) (80%) during the first 4 weeks of their study. Moreover, their data indicated important variations beyond the fourth week, i.e., a higher rate of survival (9 1.3%) was observed for the astaxanthin-fed shrimp, whereas a value of 57.1% survival was observed for the control group. Moulting It should be pointed out that although the different groups of shrimp were selected randomly, an evaluation of the initial fresh weights indicates a mean value of 10.7 1 If:0.19 g for the animals fed the C 100 diet, whereas values for the three other groups (A 100, ASO/CSO and STD) were 11 to 12 g (see Table 2). The moulting cycle in shrimp of about 12 g fresh weight lasts about 3 weeks. A slight shortening of this period was observed for animals fed the C 100 diet. This may have been due to the smaller average size of this group, since it is well known that younger individuals show a higher moulting frequency. However, recent data concerning adult individuals as well as post-larval stages fed on carotenoid-supplemented diets (unpublished results) corroborate these observations. An evaluation of the ecdysteroids in the haemolymph of adult shrimps fed on carotenoid-supplemented diets is needed to provide more information on the possible physiological interaction between pigment and moulting hormones. Growth rate It is well known that in crustacea the growth rate decreases concomitantly with increasing age and consequently with increasing body weight (Choe, 1970). Thus, the growth rate ( 17.74% and 11.66%) observed for the prawn fed the C 100 and the ASO/CSO diets may ascribed to the fact that at the starting point ( IV,), their weight was slightly less than that of the other two groups (Table 2), suggesting that carotenoids may not have been involved in improving growth rate, in agreement with the findings of Yamada et al. ( 1990). Analogous growth rates have also been obtained for fish (rainbow trout ) fed diets with or without supplemented astaxanthin or canthaxanthin for several months (Foss et al., 1984). Consequences of different pigmen ted diets on tissue pigmen tation Canthaxanthin did not seem to be stored by the shrimp under our experimental conditions i.e., the carotenoid content of the hepatopancreas, epidermis and carapace of shrimp fed the C 100 diet were comparable to those observed for individuals fed the carotenoid-free diet. Similar results have been observed in previous experiments with synthetic canthaxanthin (unpublished results). It has, however, been reported that the prawn can transform dietary synthetic canthaxanthin into astaxanthin (Tanaka et al., 1976).
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On the contrary, dietary astaxanthin was found to be stored in the three different tissues analyzed (Table 3 ). Recent studies (Yamada et al., 1990) have likewise indicated that the integuments of Penaeus juponicus fed canthaxanthin and astaxanthin diets for 4 weeks showed comparable carotenoid concentration; however, an improved storage was observed in the shrimp fed the A 100 diet over a period of 8 weeks. In fish, on the other hand, astaxanthin and canthaxanthin are deposited unchanged, e.g. in salmonid flesh (Hata and Hata, 1973; Foss et al., 1984; Torrissen, 1986; Choubert and Storebakken, 1989); however, astaxanthin is better retained (Storebakken et al., 1987; Bjerkeng et al., 1990) due to a higher digestive retention rate. Hata and Hata ( 1972) have suggested that the occurrence of hydroxyl groups within the molecular configuration of the carotenoid enhances its absorption by the digestive epithelium. Thus astaxanthin (dihydroxycanthaxanthin ) may be more easily absorbed than canthaxanthin. The hepatopancreas as well as the carapace from shrimp fed the ASO/CSO diet had carotenoid concentration values comparable to those found in prawns fed the STD and C 100 diets, whereas the epidermis contained a higher quantity of these pigments. Astaxanthin and canthaxanthin seemed to be readily absorbed by the epidermis, provided that equal amounts of these pigments occur simultaneously in the food (A5O/C50). However, the accumulation of carotenoids in the digestive gland was not observed in this case. Furthermore, the pigment content of the carapace of animals fed the ASO/CSO diet was quite low. Few studies have been successful concerning the use of dietary astaxanthin-canthaxanthin mixtures in fish. Results vary (Foss et al., 1987; Torrissen, 1989) and are probably due to the relative concentration of these pigments in the diet. Rainbow trout fed a mixture of astaxanthin (72%) and canthaxanthin (28%) showed a flesh carotenoid content higher than that of individuals fed diets containing 100% astaxanthin and 50% astaxanthin/50% canthaxanthin as the supplemental carotenoids (Torrissen, 1989). It seems likely that these two xanthophylls act synergistically, probably being absorbed in different parts of the digestive tract. On the other hand, the nature of pigment carriers in fish blood, recently established in the chum salmon Oncorhynchus keta (Nakamura et al., 1985; Ando et al., 1986) as well as in the trout Oncorhynchus mykiss (Choubert et al., 1992), must likewise be determined in crustacea in order to improve knowledge on the consequences of the use of synthetic carotenoids for shrimp. ACKNOWLEDGEMENTS
This study is supported by a France-Spain Technical and Scientific Cooperation Programme (Action Integree no. 263). Carophyll red and Carophyll
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G. NkGRE-SADARGUES ET AL.
pink were kindly supplied by Hoffmann-La Roche (Basle, Switzerland). The authors thank Mrs. E. Grousset for technical assistance.
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