Review of some aspects of marine fish larviculture

V. Culture ICES mar. Sei. Symp., 201: 138-142. 1995 Review of some aspects of marine fish larviculture Patrick Sorgeloos, Marleen Dehasque, Philippe Dhert, and Patrick Lavens Sorgeloos, P., Dehasque, M., Dhert, P., and Lavens, P. 1995. Review of some aspects of marine fish larviculture. - ICES mar. Sei. Symp., 201: 138-142. Dependable availability of quality fry to stock grow-out production systems has been one of the most critical factors in the commercial success of industrial production of fish and shellfish. Large-scale production of marine fish fry was realized only from the 1980s onwards. Although Japan was the pioneer with the red sea bream (Pagrus major), the most competitive hatchery methods were eventually developed in Europe for the sea bass (Dicentrarchus labrax) and the gilthead sea bream (Sparus aurata). Improved knowledge of larval dietary requirements, not the least with regard to (n-3) highly unsaturated fatty acids (HUFAs), combined with the adoption of live-food enrichment protocols allowed the successful transition from pilot to commercial-scale larviculture. Initially based on an empirical approach, larviculture nutrition research today is of a multidisciplinary nature. There are good indications that the more fundamental approach will lead to significant progress in hatchery outputs. The larval dietary regimes will eventually be adjusted as a function of the cultured species and/or specific developmental stages, e.g., changes in the enrichment protocols for Brachionus and A nem ia, the selected formulation of substitution diets, and/or the adoption of co-feeding techniques. The area that has been most neglected so far, but might provoke the biggest impact in future hatchery technology is microbiology. Also the prophylactic and therapeutic use of antibiotics and other chemotherapeutics is expected to undergo significant improvements in the near future. In addition, it is very likely that better broodstock conditioning and feeding can ensure improved and constant larval qualities. Finally, improved zoo techniques will make fish larviculture more predictable and more cost-effective, e.g., adoption of modular hatchery systems, selection and use of new materials, reduction of the so-called human factors by increased automation, etc. P. Sorgeloos, M. Dehasque, P. Dhert, and P. Lavens: Laboratory o f Aquaculture and Artemia Reference Center, University o f Ghent, Rozier44, B-9000 Ghent, Belgium [tel: (+32)92643754, fax: (+32) 92644193] Introduction Large-scale production of marine finfish was pioneered by the Japanese, with the red sea bream (Pagrus major). Since the 1960s the Japanese Prefectural Institutes of Fisheries have been involved in sea bream mass production (Hirano and Oshima, 1963). In Europe and the USA research in marine fish larviculture started at the same time, be it on a more modest scale (Morris, 1956; Riley, 1966). The expertise developed at British and French research institutes eventually led to the settingup of the first commercial hatcheries of the European sea bass (Dicentrarchus labrax), the gilthead sea bream (Sparus aurata) and the turbot (Scophthalmus maximus) in the early 1980s. Today, Japan is still the biggest producer with about 200 million fry produced per year; i.e., sea bream (Pagrus major) and Japanese flounder (Paralichthys olivaceus) making up 70% of the total production, the rest consisting of puffer (Takifugu rubripes), rockfish (Sebastes schlegeli), mud dab (Limanda yokohamae) and several other species (Fukusho, 1993). Europe is in second place, but with much more competitive hatchery outputs. In 1993, hatcheries in the Mediterranean were expected to produce more than 100 million fry of bass and bream (Stephanis and Divanach, 1993). In this review, we overview what we consider to have been, and to some extent still are, important bottlenecks in the successful larviculture of marine fish.

ICES mar. Sei. Symp., 201 (1995) Marine fish larviculture 139 Nutritional requirements Without doubt, the most significant progress achieved during the last decade has been the result of improved nutrition, more particularly through the manipulation of the fatty acid profile of the live prey, Brachionus and Artemia (reviews in Sorgeloos and Léger, 1992; Lavens et al., 1994). However, we have been misled by the most widely used food item, the brine shrimp Artemia, of which more than 2000 metric tonnes of cysts are currently marketed annually for feeding in fish and shellfish hatcheries. As the studies of Watanabe et al. (1983) and Léger et al. (1985) revealed, the presence of the fatty acid 20:5(n-3) (eicosapentaenoic acid or EPA) determined the suitability of a given Artemia source for successful production of marine fish larvae. Because of these findings, major emphasis was placed on increasing the EPA levels in the live prey Brachionus and Artemia by using algae or, emulsified and particulate enrichment products (review in Léger et al., 1986). However, more attention should have been paid to the levels of 22:6(n-3) (docosahexaenoic acid or DHA) when evaluating the results of feeding tests with different Artemia preparations on European sea bass larvae (Lisac et al., 1986): good survival appeared to be correlated with high EPA levels in the diet; however, the best growth was achieved in the diets with the highest DHA levels. Recent studies with various species have revealed the importance of DHA and more particularly the requirement for high DHA/EPA ratios in promoting growth, stress resistance, and pigmentation (Lavens et al., 1995; Kraul et al., 1992; Reitan et al., 1994; and Mourente et al., 1993). Whereas in the past satisfactory results were achieved with DHA/EPA ratios of less than 1, the emphasis today is on attaining levels of 4 and higher in the live prey Brachionus and Artemia (Dhert et a i, 1993). As pointed out by Sargent et al. (1993), neural tissues contain elevated concentrations of DHA and it is likely that the DHA-deficient live prey that has been offered to date has resulted in fish with poor performance with regard to visual perception of the prey and other behavioural aspects. The requirement in the larval diet for (n-3) HUFAs might also vary as a function of larval quality. This is illustrated with turbot larvae at first-feeding. In early spawning adults, when egg and larval quality are generally at their best, the requirement for DHA-enriched rotifers is limited to two days only, whereas late in the season, when poor-quality larvae prevail, the turbot larvae need to be offered DHA-rich rotifers for a longer period (Dhert et al., 1995). Furthermore, Harel et al. (1994) documented a clear effect of the fatty acid composition of the maternal diet on the egg quality of Sparus aurata. We recently found that, in their early larval development, turbot can turn black as a result of stressed conditions, e.g., unsuitable light conditions or elevated larval densities (Lavens et al., 1995). Recovery to a normal colour appears to be a function of the (n-3) HUFA composition of the diet, i.e., slow when offered a coconut-enriched Brachionus, fast when feeding EPA and DHA enriched diets. It is clear that in the area of larval nutrition most attention has been paid to the qualitative and quantitative (n-3) HUFA requirements; further work is required to verify ratios of n-3/n-6/n-9 fatty acids, and especially to evaluate the effects of other lipid classes, not the least the phospholipids which might alter the requirements for (n-3) HUFAs. Aside from the lipids it is clear that many other dietary components deserve more attention in future larviculture nutrition research, e.g., different vitamins (recent work by Merchie et al. (1995a, b, c) illustrates significant effects in growth and/or stress resistance with vitamin C in different species), free amino acids, pigments, etc. In our opinion, the challenge remains of analytically and biologically unravelling those components in wild copepods which are responsible for their excellent dietary value for marine larval fish. The aim of this would be to eventually allow their incorporation in Brachionus and/or Artemia. Similarly, the use of green water in the commercial rearing of most marine fish species requires further study. Various hypotheses are worth pursuing, e.g. a source of micro-nutrients, source of immunostimulants, water quality conditioner, microbiological conditioner. Formulated diets The ultimate goal in commercial marine fish larviculture is to be able to rely on formulated dry diets right from start-feeding; however, for most species of marine fish this remains wishful thinking. Nonetheless, good progress has been reported at the experimental level, with artificial diets for start-feeding of some Japanese fish species (Kanazawa, 1993) and in advancing early weaning to 400 day degrees and less in the European sea bass (Person-Le Ruyet, 1993; Devresse et al., 1991). We are convinced that application of the co-feeding principle, i.e., supplementing live food with formulated compounds, in the early larval stages might provide opportunities for better identification of qualitative/quantitative dietary requirements (e.g., with components such as phospholipids that cannot be manipulated easily in the live-food fraction). This might also allow for a possible reduction of live-food requirements, eventualy resulting in improved outputs as compared to a diet consisting of live food only (Lavens et a i, 1995).

140 P. Sorgeloos et al. ICES mar. Sei. Symp., 201 (1995) Microbial control An area that urgently deserves more attention is the microbial flora of marine fish hatcheries. With upscaling and expansion of commercial fish larviculture, hatcheries have been plagued by an increased incidence of microbial diseases, often claimed to be caused by Vibrio spp. (several pers. comm, with marine fish hatchery operators in the Mediterranean). Similar to practices in marine shrimp hatcheries, the indiscriminate use of antibiotics in prophylactic treatment of the fish tanks has resulted in the development of resistant strains and the need to switch to other antibiotics, a practice which is doomed to fail (Brown, 1989). Verdonck et al. (1994) and Verdonck and Swings (1995) performed an extensive sampling campaign in three marine fish hatcheries in the Mediterranean during the first 30 days of rearing of sea bass (Dicentrarchus labrax) and sea bream (Sparus aurata) larvae; i.e., the microbial diversity and quantitative presence were recorded, as well as the relationship between the microflora of the culture water, the live food, and the colonization of the larval intestine during development. The microbial diversity appeared to be enormous (over 1200 bacterial strains were characterized) and varied from season to season as well as from hatchery to hatchery. Quantitative data revealed the presence of 100-10000 bacteria per rotifer and per brine shrimp nauplius. The bacterial numbers found in the gut of larvae increased with age and feeding regime from 1000 to 100000 per fish larva. They also noted that the numbers found on a Vibrio-specific TCBS-medium were always 10 to 100- fold lower than the total counts on a marine agar culture medium, except in cases of mortality outbreaks, when the values were similar. The fact that the composition of the microflora in the intestine of healthy fish was not an exact copy of the microflora found in the culture water or the live food might be an indication of selective colonization of the gut or the existence of so-called probiotic bacteria. The major surprise of the microbiological study was the important input of bacteria (and potentially fish pathogens) via the live-food chain. New measures should be considered to reduce bacterial loads as well as to selectively manipulate the microflora both in the live feeds produced in the hatchery and in the culture water prior to stocking of the fish larvae. Selected bacterial inoculation of the culture tank prior to stocking with the larvae would not only reduce the chances of opportunistic bacteria becoming dominant, but eventually have a beneficial effect on first colonization of the fish larva s gut. In general, strict hygienic measures should be taken in hatcheries. Furthermore, regular disinfection and dryout of the complete culture circuit (including all piping) between production cycles should be implemented. In this respect new hatcheries should consider the use of modular systems rather than having all culture units in one building. When antibiotic treatment is justified for therapeutic reasons, the drugs should not be added to the culture tank but rather oral biomedication applied using Brachionus and/or Artemia as a carrier for the antibiotics ingested from an emulsified or particulate preparation. Doses ranging from 20 to 100 ppm sulfadrugs can be incorporated in sea bass and turbot larval tissue respectively within less than 4h after feeding Artemia metanauplii boosted with the antibiotics (Chair et al., 1991; Chair et al., submitted). The therapeutic efficacy of this oral delivery system has been documented by Chair et al. (1995) and Dhert et al. (1995) with turbot larvae challenged with a Vibrio anguillarum pathogen. Conclusion The intensification of hatchery activities brought about new problems not experienced on the experimental scale, e.g., in relation to upscaling of life-food production, washing and cleaning of live food, intensity of manual labour, etc. To date, limited attention has been paid to improved zoo techniques, which might make marine fish larviculture more predictable and more costeffective, e.g., selection and use of new materials (i.e., stainless-steel welded-wedge filters instead of woven filters for Artemia washing) (Léger and Sorgeloos, 1992), increased automation to reduce the so-called human factor often responsible for reduced performances after several months of operation, etc. In the past decade, marine fish hatcheries have evolved from hit and miss ventures into profitable enterprises. In Europe, market saturation of bass, bream, and even turbot fry has been reached faster than was anticipated (Sorgeloos and Léger, 1992); i.e., fry prices have dropped to US$ 0.75 and less per larva (Stephanis and Divanach, 1993). Although there is still room for further improvement, especially with regard to cost-effectiveness, present-day hatchery technology for marine fish species has proven to be widely applicable, in terms of both geographic location and fish species. The list of commercially cultivated species is growing rapidly (e.g., various bass and bream species, turbot, Japanese flounder, fugu, milkfish, mullet) and many candidate species will hopefully soon join this list, e.g., dolphin fish mahi-mahi, various grouper species, Atlantic halibut, red snapper, cod, wolffish. Acknowledgements This research was sponsored by the Belgian National Science Foundation, the Belgian Ministry for Science

ICES mar. Sei. Symp., 201 (1995) Marine fish larviculture 141 Policy, the Belgian Administration for Development Cooperation, the European Community, SINTEF, and IN VE Aquaculture S.A. References Brown, J. 1989. Antibiotics: their use and abuse in aquaculture. World Aquacult. 20(2): 34-43. Chair, M., Dehasque, M., Sorgeloos, P., Nelis, H., and De Leenheer, A. 1995. Live-food mediated drug delivery as a tool for disease treatment in larviculture: a case study with turbot (Scophthalmus maximus). J. World Aquacult. Soc., 26(2): 217-219. Chair, M., Nelis, H. J., Léger, P., Sorgeloos, P., and De Leenheer, A. Accumulation of trimethoprim, sulfamethoxazole and N-acetylsulfamethoxazole in fish and shrimp fed on medicated Artemia. (Submitted). Chair, M., Romdhane, M., Dehasque, M., Nelis, H., De Leenheer, A., and Sorgeloos, P. 1991. Live-food mediated drug delivery as a tool for disease treatment in larviculture. II. A case study with European seabass. In Larvi 91 - Fish and Crustacean Larviculture Symposium, pp. 412-414. Ed. by P. Lavens, P. Sorgeloos, E. Jaspers, and F. Ollevier. Eur. Aquacult. Soc., Spec. Publ. No. 15, Ghent. 427 pp. Devresse, B., Candreva, P., Léger, Ph., and Sorgeloos, P. 1991. A new artificial diet for the early weaning of seabass (Dicentrarchus labrax) larvae. In Larvi 91 - Fish and Crustacean Larviculture Symposium, pp. 178-182. Ed. by P. Lavens, P. Sorgeloos, E. Jaspers, and F. Ollevier. Eur. Aquacult. Soc., Spec. Publ. No. 15, Ghent. 427 pp. Dhert, P., Lavens, P., Dehasque, M., and Sorgeloos, P. 1995. Improvements in the larviculture of turbot Scophthalmus maximus: zootechnical and nutritional aspects, possibility for disease control. In Turbot culture: problems and prospects, pp. 32-46. Ed. by P. Lavens and R. A. M. Remmerswaal. Eur. Aquacult. Soc., Spec. Publ. No. 22, Ghent. 358 pp. Dhert, P., Sorgeloos, P., and Devresse, B. 1993. Contributions towards a specific DHA enrichment in the live food Brachionus plicatilis and Artemia sp. In Fish farming technology, pp. 109-115. Ed. by H. Reinertsen, L. A. Dahle, L. Jørgensen and K. Tvinnereim. A. A. Balkema, Rotterdam. 482 pp. Fukusho, K., 1993. Mass larval rearing technology of marine finfish in Japan: 9. In Book of Abstracts, National Seminar - Workshop on Breeding and Seed Production of Cultured Finfishes in the Philippines, 4-5 May 1993, SEAFDEC/ AQD, Tigbauan, Philippines. Harel, M., Tandler, A., and Kissil, G. Wm. 1994. The kinetics of nutrient incorporation into body tissues of gilthead seabream (Sparus aurata) females and the subsequent effects on egg composition and egg quality. Br. J. Nutr. 72: 45-58. Hirano, R., and Oshima, Y. 1963. On the rearing of larvae of marine animals with special reference to their food organisms. Bull. Japan. Soc. Sei. Fish., 29(3): 282-297. Kanazawa, A. 1993. Importance of dietary docosahexanenoic acid on growth and survival of fish larvae. In Finfish hatchery in Asia: Proceedings of finfish hatchery in Asia 91, pp. 87-95. Ed. by C. S. Lee, M. S. Su, and I. C. Liao. TML Conference Proceedings 3. Tungkang Marine Laboratory, Tungkang. 252 pp. Kraul, S., Nelson, A., Brittain, K., Ako, H., and Ogasawara, A. 1992. Evaluation of live feeds for larval and postlarval mahimahi Coryphaena hippurus. J. World Aquacult. Soc. 23(4), 299-306. Lavens, P., Sorgeloos, P., Dhert, P., and Devresse, B. 1995. Larval Foods. In Broodstock management and egg and larval quality, pp. 373-397. Ed. by N. R. Bromage and R. J. Roberts. Blackwell, Oxford. 424 pp. Léger, P., Bengtson, D. A., Simpson, K. L., and Sorgeloos, P. 1986. The use and nutritional value of Artemia as a food source. Oceanogr. Mar. Biol. ann. Rev., 24: 521-623. Léger, P., and Sorgeloos, P. 1992. Optimized feeding regimes in shrimp hatcheries. In Marine shrimp culture: principles and practices, pp. 225-244. Ed. by A. W. Fast and L. J. Lester. Elsevier, New York. 862 pp. Léger, P., Sorgeloos, P., Millamena, O. M., and Simpson, K. L. 1985. International study on Artemia. XXV. Factors determining the nutritional effectiveness of Artem ia: The relative impact of chlorinated hydrocarbons and essential fatty acids in San Francisco Bay and San Pablo Bay Artemia. J. exp. mar. Biol. Ecol. 93: 71-82. Lisac, D., Franicevic, V., Vejmelka, Z., Buble, J., Léger, P., and Sorgeloos, P. 1986. International study on Artemia. XLIII. The effect of life food fatty acid content on growth and survival of sea bream (Sparus aurata) larvae. Paper presented at the conference Ichthyopathology in Aquaculture, 21-24 October, Inter-University Center, Dubrovnik. Merchie, G., Lavens, P., Dhert, P., Pector, R., Mai Soni, A. F., Abbes, M., Nelis, H., Ollevier, F., De Leenheer, A., and Sorgeloos, P. 1995a. Live food mediated vitamin C transfer to Dicentrarchus labrax and Clarias gariepinus. In Proceedings of the EIFAC Workshop on Methodology for Determination of Nutrient Requirements in Fish, Eichenau, Germany, 29 June to 1 July 1993. J. Appl. Ichthyol. (In press). Merchie, G., Lavens, P., Verreth, J., Ollevier, F., Nelis, H., De Leenheer, A., Storch, V., Sorgeloos, P. 1995b. The effect of supplemental ascorbic acid in enriched live food for Clarias gariepinus larvae at startfeeding. Aquaculture. (In press). Merchie, G., Lavens, P.. Verreth, J., Ollevier, F., Pector, R., Nelis, H., De Leenheer, A., Storch, V., and Sorgeloos, P. 1995c. The effect of graded levels of supplemental ascorbic acid in enriched live food fed to Clarias gariepinus larvae. In Proceedings of the 6th International Symposium on Fish Nutrition and Feeding, Hobart, Australia, 4-7 October 1993. Aquaculture. (In press). Morris, R. W. 1956. Some aspects of the problem of rearing marine fish. Bull. Inst. Oceanogr., 1082: 1-61. Mourente, G., Rodriguez, A., Tocher, D. R., and Sargent, J. R. 1993. Effects of dietary docosahexaenoic acid (DHA; 22:6n-3) on lipid and fatty acid composition and growth in gilthead sea bream (Sparus aurata L.) larvae during firstfeeding. Aquaculture, 112: 79-98. Person Le Ruyet, J., Fischer, C., and Thébaud, L. 1993. Sea bass (Dicentrarchus labrax) weaning and ongrowing onto sevbar. In Proceedings of IV International Symposium on Fish Nutrition and Feeding, Fish Nutrition in Practice, pp. 623-628. Ed. by S. J. Kaushik and P. Luquet. INRA editions, Paris. 972 pp. Reitan, K. I., Rainuzzo, J. R., and Olsen, Y. 1994. Infiuenceof lipid composition of live feed on growth, survival and pigmentation of turbot larvae. Aquacult. Int., 2: 1-16. Riley, J. D. 1966. Marine fish culture in Britain, VII. Plaice (Pleuronectes platessa L.) post-larval feeding on Artemia salina L. nauplii and the effects of varying feeding levels. J. Cons. perm. int. Explor. Mer, 30: 204-221. Sargent, J. R., Bell, J. G., Bell, M. V., Henderson, R. J., and Tocher, D. R. 1993. The metabolism of phospholipids and polyunsaturated fatty acids in fish. In Aquaculture: Fundamental and Applied Research, pp. 103-124. Ed. by B. Lahlou and P. Vitiello. Coastal Marine Studies No. 43, American Geophysical Union, Washington, DC. Sorgeloos, P., and Léger, P. 1992. Improved larviculture out

142 P. Sorgeloos et al. ICES mar. Sei. Symp., 201 (1995) puts of marine fish, shrimp and prawn. J. World Aquacult. Soc., 23(4): 251-264. Stephanis, J., and Divanach, P. 1993. Farming of Mediterranean finfish species, present status and potentials. In Book of Abstracts World Aquaculture 93, pp. 290-291. Ed. by M. Carillo, L. Dahle, J. Morales, P. Sorgeloos, N. Svennevig, and J. Wyban. Eur. Aquacult. Soc., Spec. Publ. No. 19, Ghent. 632 pp. Verdonck, L., and Swings, J. 1995. Microbial problems related to marine fish hatcheries in southern Europe: a case study. In Memorias II Congreso Ecuatoriano de Acuicultura. pp. 197 199. Ed. by J. Calderon and P. Sorgeloos. CENAIM, Escuela Superior Politecnica del Litoral, Guayaquil. 306 pp. Verdonck, L., Swings, J., Kersters, K., Dehasque, M., Sorgeloos, P., and Léger, P. 1994. Variability of the microbial environment of rotifer (Brachionus plicatilis) and Artemia production systems. J. World Aquacult. Soc., 25(1): 55-59. Watanabe, T., Kitajima, C., and Fujita, S. 1983. Nutritional values of live organisms used in Japan for mass propagation of fish: a review. Aquaculture, 34: 115-143.