FISH LIVE. Organizado

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1 LARVITA FISH LARVAE TRAINING SCHOOL LIVE FEEDS Pedro Pousão Ferreira a INRB,, I.P. IPIMAR Faro/Olhão, Portugal de Novembro de 2010 Organizado pelo Centro de Ciências do Mar (CCMAR) e pelo Instituto Nacional de Recursos Biológicos (INRB, I.P. I IPIMAR).

2 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR Live Feeds Pedro Pousão- Ferreira INRB, I.P. IPIMAR Av. 5 de Outubro s/n, Olhão, Portugal ppousao@ipimar.pt Introduction The most important groups that we produce in marine aquaculture - molluscs, shrimps and fish present small larval stages that need to be fed with live organisms during the first period of live. This period vary from different groups and different species inside each group. In marine fish the larval period is considered the most crucial for survival; starvation and/or predation are considered to be major impediments to successful recruitment into juvenile/adult. The visual range over which zooplankton can be detected by most fish larvae, more particularly in the start-feeding period, is very important for the detection of plankton, which usually are distributed in groups. Zooplankton organisms usually have continuous movement that generally assures a good distribution in the water column and a much better contrast than artificial feeds, which attracts the larvae facilitating their detection by the feeding larva. The quantity of plankton in the water column is also important because can promote more frequent encounters between plankton and the larvae, which in most cases have a low mobility, and it s usually a key between starvation or feeding. The first stages of bass, bream, meager, sole and many other marine fish have small larvae (Table 1) with very limited yolk reserves at hatching which are consumed in two or three days at 20ºC. They have also very small mouths (Fig. 1) which restrict the size of the food particles that can be ingested. In shrimp, the size of the larval food is also a problem but not so restrict because they have appendices that helps to catch and break the food. Some cultivated groups like penaeid

3 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR shrimpss pass through different larval stages changing from a herbivorous filter-feeding eating microalgae to carnivorous feeder eating Artemia. Table 1 - Eggs size and larval length at hatching in different species of fish (after Jones and Houde 1981). Species Samon (Salmo salar) Trout (Onchorhynchus mikiss) Carp (Cyprinus carpio) Milkfish (Chanos chanos) European sea bass (Dicentrarchus labrax) Gilthead seabream (Sparuss aurata) Turbot (Scophtalmus maximus) Senegalensis sole (Solea senegalensis) ) Grouper (Epinephelus marginatus) Meager (Argyrosomus regius) Egg diameter (mm) Length of larvae (mm) Figure 1 Seabream larvae mouth and live preys size. The mollusk bivalve like oysters, clams, mussels and the gastropod likee abalone (Haliotis( spp.) are filter-feeding eating microalgae during all their life cycle.

4 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR In the nature the diet of the referred marine fish larvae, marine crustacean larvae and mollusks consists of a wide diversity of phytoplankton species with different sizes and biochemical composition (like diatoms, flagellates and green algae) and zooplankton organisms (like copepods and small larvae from other crustacean groups). The different sizes and biochemical composition of the plankton are important for meeting all the nutritional requirements for these larvae but collecting natural plankton for use as aquaculture feed isn t viable in industrial production. Three groups of live diets are widely applied in larviculture of marine mollusks, fish and crustaceans (Table 2). Table 2 Groups of plankton and their use for different species feeding. Live Feeds Dimension Species Microalgae 2 to 20 µm Bivalves Penaeid shrimps Rotifers Copepods Artemia Rotifers 50 to 220 µm Penaeid shrimps Marine fish Artemia spp. 400 to 800 µm Mollusks Penaeid shrimps Marine fish The use of plankton for aquaculture feeds are selected according to a number of criteria (Fig.2) that are a compromise between the practical viewpoint of the culturist and the behavior (size and nutritional needs) of different species that we want to feed. The live feeds must be in one hand available, cost-effective, simple as well as versatile in application and in another must have nutritional value and be easily captured and digested by the larvae.

5 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR In recent years different formulations of supplementation and substitution products have been developed and use to improve the qualityy of live feeds (and for supplement specific nutrients) and to advance the transition to t inert feeds. Figure 2 - Selection criteria for larval food sources from the viewpoint v of the culturist and the cultured larva (from Léger et al., 1987). The microalgae Phytoplankton comprises the basis of essentially all marine and freshwater aquatic food chains. Microalgae are indispensable as a food source for all growthh stages of bivalve molluscs, larval stages of some s crustacean species and to produce mass quantities of zooplankton (rotifers, copepods, Artemia) which serve in turnn as food for larval and earlyuseful in: juvenilee stages of crustaceans and fish. Microalgae are also used in larval fish tanks (green water technique) wheree they are i) stabilizing the water quality, ii) nutrition of the zooplankton and larvae, iii) better pray contrastt and light dispersion and microbial control. Diatoms (eg. Skeletonema costatum, Thalassiosiraa pseudonana, Chaeotoceros calcitrans), flagellates (eg. Isochrysis galbana, Isochrysis aff. galbana) and green algaee (eg. Nannochloris

6 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR gaditana, Dunaliella salina, Tetraselmis suecica) are the dominant groupss of cultured micro- culture algae for the different groups. They have been selected on the basis of f their masss potential, cell size, digestibility and nutritional value. Diatoms are rich in silicates, which constitute their cell walls (frustules) and are usually given to bivalve molluscs and crustacean larvae for the formation of rigid structures (Conceição et al, 2010). Various techniques have been developed to grow microalgae on a large scale but the commonly in hatcheries they are progressively cultured in a multi-stage back-up system (Fig. 3). Starting from sterile Petri boxes and/ /or test-tubes the algaee are inoculated in larger glass containers and carboys, then to bigger plastic bags (Fig. 4) and a finally to large, indoor or outdoor tanks. Cell concentration in phytoplankton cultures are high and therefore must be enriched with nutrients. Macronutrients include nitrate, phosphate and silicate (for diatoms) and micronutrients consist of various trace metals and vitamins. There are different enrichment media butt the most widespread are the Walne mediumm and the Guillard's F/2 medium. Figure 3 - Schematic outline of steps involved in the mass production of microalgae.

7 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR Figure 4 Mass production off microalgaee Nutritional value of microalgae Microalgae vary appreciably in their biochemical composition, even when grown under standard conditions (Brown et al, 1997). The nutritional composition of specific microalgae can vary considerably according to the culture conditions and growth phase/age of the culture. The nutritional value of any algal species for a particular organism depends on its cell size, digestibility, production of toxic compounds, and biochemical l composition. Although there are marked differences in the compositions of the microalgal classes andd species, protein is always the major organic constituent, followed usually by lipid and then by carbohydrate (Lavens and Sorgeloos, 1996). A mixture of two or several algal species is often used in order to supply the adequatee amount of nutrients. Particular microalgae may lack a nutrient that is present p in another. In molluscs culture is normally used a mixture of several microalgae and in i crustacean and fish larvae a mix of two.

8 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR The content of highly unsaturated fatty acids (HUFA), in particular eicosapentaenoic acid (20:5n-3, EPA), arachidonic acid (20:4n-6, ARA), and docosahexaenoic acid (22:6n-3, DHA), is of major importance in the evaluation of the nutritional composition of an algal species to be used as food for marine organisms (Lavens and Sorgeloos, 1996). The fatty acid composition of 4 species of microalgae is presented in Figures 5 and 6. The alternative to on-site algal culture is the use of preserved microalgae. There are in the market specialized companies that sells paste of specific microalgae (by centrifugation of microalgae to concentrate it into a paste), frozen microalgae concentrate and dried specific or mixed microalgae (also dried extracts of microalgaes and other substance for green water). Other method is the preparation of microalgal concentrates based on chemical flocculation (Brown and Robert, 2002). Other techniques have been developed for the large scale production of marine microalgae under heterotrophic growth conditions, by utilizing organic carbon instead of light as an energy source. The productions of this technology are not yet important but they can have an important role in a next future as alternative sources of ARA, EPA and DHA (Harel et al., 2002, Harel & Place, 2004). 25% N. oculata N. galbana 20% 15% % Dw PS 10% 5% 0% dias Days 18:2n-6 18:2n-6 18:3n-3 18:3n-3 20:4n-6 20:4n-6 20:5n-3 20:5n-3 22:6n-3 22:6n-3 Figure 5 - Fatty acids profile of Nannochloropsis oculata e Nannochloropsis gaditana.

9 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR 16% 14% 12% 10% I. galbana T iso % Dw 8% 6% 4% 2% 0% Days 18:2n-6 18:2n-6 18:3n-33 18:3n-3 20:4n-6 20:4n-6 20:5n-3 20:5n-3 22:6n-3 22: 6n-3 Figure 6 - Fatty acids profile of Isochrysis galbana e Isochrysis aff. galbana (T-Iso) The rotifer Brachionus spp. This brackishwater Brachionus plicatilis (Fig. 7) was identifiedd by Japanese aquaculturists as a suitable starter diet in marine fish larviculture (Fukusho, 1989). Figure 7 Brachionus plicatilis, female and male (diagram by Schach, S Y., after Koste 1980, Pourriot 1990).

10 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR In function of the larval mouth size of thee fish larvae which is variable v withh species and age, a small and super small Brachionus strain S or SS ( µm length), l medium SM ( ( µm) or a large one L ( µm) is used. Hatcheries are currently using rotifer body size to distinguish their marine Brachionus cultures into Large (L) andd Small (S) and use them alone and mixed. This classification by sizes is still used in the industry but is inaccurate to define the Brachionus speciess complex that is currently dividedd in new species or biotypes (Gomez et al, 2002). Nowadays inside each of this groups L, SM, and S or SS there are divisions in different species and biotypes with possible species status and different d Brachionus species or biotypes may have different optima withh respect to culture conditions (Gomez et al, 2002; Papakostas et al, 2007; Baer et al, 2008). Different species or biotype of this Brachionus plicatilis species complex inside each group (L, SM or S/SS) can be adapted and growth well in a hatchery and can be a failure in another. Under optimal culture conditions, Brachionus spp. has a high fecundity and reproduces by parthenogenesis asexual reproduction were each female produces several eggs at a time. After hatching the rotifer reach reproductive stage in a few days only, this allows population duplication in one or two days. In the industrial production the t most common is the batch culture c system with 4 to 6 cylinder-conical tanks (Fig.8), using microalgae often Nannochloropsis sp and or yeast based product or dried algae formulated products as food. Figure 8 - Schematic outline of steps involved in the mass production of rotifers.

11 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR Tanks are inoculated at a start density of approximately rotifers per milliliter at 26-29ºC and 20 ppt seawater and the production collected after 3 to 5 days (Fig. 9). The quality, quantity and number of meals of food and the temperature plays a very important role in growth rate. Rotifers ingest almost all kind of particles, of adequate dimension (1-20µm), in suspension in the water column by filtration. Usually temperature about 25-27ºC are used for L type and 28-30ºC for S type rotifer. Rot/ml Rot/ml Rot/ml Day Growth % Total Growth % Day ,0% Day ,0% Day ,0% Day ,0% 311,1% Figure 9 Growth rate of rotifers with different initial population In Figure 10 is presented a correlation between temperature and growth (BernAqua, 2009). Correlation between temperature and growth y = 0,7595x - 18,09 R² = 0, % 450% 400% 350% 300% 250% 200% 150% 100% 50% 0% Temperature ( C) Figure 10 Correlation between temperature and growth. Rotifer fed with ω3algae. Data and product from BernAqua. The microalgae and commercial products used in rotifer production may usually have enough nutritional value for the normal production but not enough for fish larvae mainly in essential fatty acids. The enrichment with specific products with high profile in ARA, EPA and DHA prior to feed the larvae is critical in optimizing the food value of the rotifers.

12 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR The HUFA enrichment of rotifers is performed either in the culture vessel or mostly in a separate tank, during 6 to 12 hours, according with product manufacturer instructions, before they are used as feed. In Figure 11 we present the essential fatty acids profile of rotifers feed with different products µg/ml DW :2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 Rot. levedura Rot. Protein Selco Rot. DHA P. Selco Rot. Rich Rot. N. oculata Rot. N oculatal/tiso Rot. Cod liver oil Rot. Seabream eggs Figure 11 - Fatty acids profile of rotifers feed with different products. The enrichment profile of the rotifers reflects not only the profile of the products used but also the period of enrichment (Fig. 12). µg/mg DW Baker's yeast BrPS 4hEnr BrPS 6hEnr BrPS 8hEnr 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 DHA/EPA 1,04 1,03 1,02 1,01 1,00 0,99 0,98 0,97 Figure 12 Rotifers profile in baker s yeast and after different hours of enrichment with Protein Selco.

13 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR The amino acid profile of rotifer can be also unbalanced for fish larvae affecting larval performance and quality, see Saavedra et al 2006; Saavedra et al a 2007; Conceição et al The brine shrimp Artemia spp. Brine shrimp Artemia nauplii constitute the most widely used species s of the live food used in larval rearing of fish and crustaceans. Artemia cystss are easy too keep in dormant stage. After 24 hours of immersion in saltwater at 26-28ºC, the cysts (embryos) hatch and releasee nauplii instar I (0,4-0,5mm) that swim in the water (Fig. 13). The different techniques for decapsulating (decapsulation treatment - cyst hydration, exposure to hypochlorite solution, washing, chlorine deactivation in hydrochloric acid, washing and/or brine dehydration (Sorgeloos et al., 1986; Léger and Sorgeloos, 1992)) and hatching Artemia are welll documented in literature. Figure 13 Hatching of Artemia cysts. These nauplii are used immediately before they growth too much to fit the fish larval mouth and their nutritional value decrease. One of the biggest disadvantages of Artemia is their deficiency in essential fatty acids for marine fish larvae. They have low levels of EPA, less in continental strains, and no DHA and the fatty acids profile can t be modified because the naupliii still don tt have a functional digestive system and don t feed. Therefore is necessary to choose on the market the cyst which gives the smallest nauplii and withh better fatty acids profile (Fig. 14) ).

14 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR 0,7 30 0,6 25 0,5 20 mm 0,4 0,3 µg/mg Dw ,2 5 0,1 0 M 1 C 0 18:2n-6 M C 18:3n-3 20:4n-6 20:5n-3 22:6n-3 Figure 14 Size at hatching and fatty acids profile of marine and continental cysts. Other parameters should be considered. The hatching synchrony (i.e. hatching time between the first 10% and 90% of hatching cysts) is important to have most m of the cysts with same size and nutritional value. The hatching efficiency (i.e. the number of o nauplii that can be produced under standard conditions from one gram cyst product) reflects on the pricee per nauplii. After 6-8hours the metanauplius I (Instar II) start filter- feeding and can bee fed / enriched with specificc products with high profile in ARA, EPA and DHA prior to feed the larvae. This method called bioencapsulation, with a mixture of microalgae, home maid emulsions or commercial products, is widely applied att marine fish and crustacean hatcheries for enhancing the nutritional value of Artemia with essential fatty acids. This T enrichment is critical for optimizing the food value of the Artemia (Fig. 15) and avoid sub nutrition and the consequent mass mortality on the larvae. Figure 15 Enriched Artemia.

15 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR The Artemia enrichment profile tends to reflect the enrichment product (Fig. 16 and Fig. 17) that must be chosen in agreement with the species that we are feeding µg/mg Dw h 9h 24h 33h 48h 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 Figure 16 Fatty acids profile of Artemia enriched during different periods with Cod liver oil. µg/mg Dw h 9h 24h 33h 48h 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 Figure 17 Fatty acids profile of Artemia enriched during different periods with Sunflower oil. As we can see in the Figures 16 and 17 the profile changes with much higher quantity in EPA (22:5n3) when we use Cod liver oil to enrich Artemia and no DHA (22:6n3) and higher Linoleic acid (18:2n6) with Sunflower oil. Nevertheless, even with best product profile in fatty acids, an important fraction of the

16 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR lipids are digested, assimilated into the Artemia body and metabolized and in addition the differential metabolism of certain fatty acids, incorporated fatty acids redistribute themselves among lipid classes with high unpredictability, both during enrichment and particularly under starving conditions, after being added to the larval rearing tanks (Conceição et al, 2010). Despite a lot of work that has been done on fish and crustacean nutrition and the progress on products for Artemia enrichment, however there still remain difficult to enrich Artemia with high levels of DHA and a correct balance between DHA and EPA, given the natural tendency of Artemia to retroconvert DHA into EPA (Navarro et al, 1999), leading to a product with a low DHA:EPA ratio that is advisable to be 2 or higher for marine fish larvae (Sargent, Bell et al. 1999). DHA nutritional deficiencies have major effects on larval growth and development of neural and visual systems (with major impacts on the ability of larvae to capture their prey) (Sargent, et al,1999a; Sargent, et al,1999b). Even with the same products differences are found and considerable variability occur in the essential fatty acid content, inside the same hatchery or between hatcheries using the same products and protocol. Other problem concerning Artemia and the metabolism of lipids is metanauplius starvation. If the Artemia metanauplius stays too long in the fish/crustaceans rearing tanks before being eaten by the larvae, begins starving, the HUFA profile will be much lower (Fig. 18). µg/mg Dw ,6 0,5 0,4 0,3 0,2 0,1 0 24h S.Selco 24h S.Selco+3h starvation 24h S.Selco+6h starvation 48h S.Selco 48h S.Selco+3h starvation 48hS.Selco+6h starvation 0,0 18:2n-6 18:3n-3 20:4n-6 20:5n-3 22:6n-3 DHA/EPA Figure 18 Fatty acids profile of Artemia after enrichment and with 3 and 6 hours of starvation.

17 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR The fish and crustacean larvae shouldd be fed several times during the day to ensure thee quantity (preys/ml) and quality of preys. To avoid Artemia from growth and Artemia and rotifers from lose their enrichment profile they can be conserved in a freezer along the day at 6±1ºC (Fig. 19), and distributed to fish/crustacean tanks when necessary. Rotifers and Artemia metanauplius are continuous non-selective filter-feeding on particles of adequate size, which enables enrichment with nutrients (ie. fatty acids and vitamins) and other substances like hormones antibiotics, prophylactic products and vaccines (Fig. 20). Figure 19 Freezer too keep rotifers and Artemia during the day. Figure 20 Artemia bioencapsulation with different products, Agh A & Sorgeloos, The copepods Copepods are an important component of marine larvae diet in nature. In aquaculture they also were used as live feeds for marine fish larvae because of their nutritional value. They naturally have a much higher content in EPA and DHA and superior nutritional value for fish larvae when compared with rotifer and Artemia. Also, in absolute values, amino acid concentrations were generally lower in rotifers and Artemia, compared to the copepods (Van der Meeren et al, 2008). However due too the relative ease andd low cost of culturing rotifers

18 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR and Artemia in high densities it is unlikely that copepod cultures will replace them as an economically viable alternative in the near future. The main copepod groups used in aquaculture (Fig. 21), are Calanoids, C mainly Acartia spp., and Harpacticoids like Tisbe spp. Besides their superior nutritional value copepodss have a wide range of body sizes both within andd between species. Thee early nauplii and copepodites can be extremely useful as initial prey for species with small mouth gapes at first feeding. However copepod rearing require spacee and is very laborious. Their density in culture is usually very low when compared with rotifers and Artemia being a constrain for industrial production (Støttrup, 2003). In aquaculture they are mostly used in semi intensive larval rearing systems for a initial short period of time during the larval stage to ensure normal development. Theyy may be cultured in large quantities in outdoor mesocosms, collected in the wild orr reared indoor tanks to t obtain resting eggs that will later be hatched for nauplii according to fish larval requirements. Intensive-rearing systems for copepods pass by exploiting the capacity of some copepod species to produce resting eggs. Figure 21 Calanoid copepodid (Acartiaa grani) and Harpacticoid Tisbes graciloides females. (Photos: M.E. Cunha).

19 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR Literature - Agh, N. & Sorgeloos, P. (Eds), Handbook of Protocols and Guidelines for Culture and Enrichment of Live Food for Use in Larviculture. Laboratory of Aquaculture and Artemia Reference Center University of Ghent, Ghent, Belgium. 60p. - Baer A.; Langdon C.; Mills S.; Schulz C. & Hamre K., Particle size preference, gut filling and evacuation rates of the rotifer Brachionus Cayman using polystyrene latex beads. Aquaculture 282, Bernaqua, New rotifer feed from BernAqua. Hatchery International. Jan/Feb Brown, M.R.; Jeffrey, S.W.; Volkman, J.K. & Dunstan, G.A., Nutritional properties of microalgae for mariculture. Aquaculture, 151(1-4): Brown, M.R. & Robert, R., Preparation and assessment of microalgal concentrates as feeds for larval and juvenile Pacific oyster (Crassostrea gigas). Aquaculture 207: Conceição, L.E.C.; Yúfera, M.; Makridis, P.; Morais, S. & Dinis, M.T., 2010, Live feeds for early stages of fish rearing. Aquaculture Research, 41: Fukusho, K Biology and mass production of the rotifer Brachionus plicatilis. Int. J. Aq. Fish. Technol., 1: Gómez, A.; Serra, M.; Carvalho, G. R.& Lunt, D. H., Speciation in ancient cryptic species complexes: evidence from the molecular phylogeny of Brachionus plicatilis (rotifera). Evolution, 56; Harel, M.; Koven, W.; Lein, I.; Behrens, Y. B.; Stubblefield, J.; Zohar, Y. & Place, A. R., Advanced DHA, EPA and ARA enrichment materials for marine aquaculture using single cell heterotrophs. Aquaculture 213: Harel, M. & Place, A. R., Heterotrophic production of marine algae for aquaculture. In Handbook of Microalgal Culture - Biotechonology and Applied Phycology, (Amos Richmond ed.), pp Blackwell Science Ltd, Iowa, USA. - Lavens, P; Sorgeloos, P. (eds.). Manual on the production and use of live food for aquaculture. FAO Fisheries Technical Paper. No Rome, FAO p - Jones A.; Houde, E.D Mass rearing of fish fry for aquaculture. p In: Realism in Aquaculture: Achievements, Constraints, Respectives. Bilio, M., Rosenthal, H. and Sinderman G.J. (Eds). European Aquaculture Society, Bredene, Belgium, 585 pp.

20 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR - Léger, Ph.; Chamorro, R.; Sorgeloos, P Improved hatchery production of postlarval Penaeus vannamei through application of innovative feeding strategies with an algal substitute and enriched Artemia. Paper presented at the 18th Ann. Meeting of the WAS, Guayaquil (Ecuador), January 18-23, Léger, Ph.; Sorgeloos, P Optimized feeding regimes in shrimp hatcheries. In: Culture of Marine Shrimp: Principles and Practices. A.W. Fast, L.J. Lester (Eds). Elsevier, p Navarro, J.C.; Henderson, R.J.; McEvoy, L.A.; Bell, M.V. & Amat, F.,1999. Lipid conversions during enrichment of Artemia. Aquaculture174: Papakostas, S.; Wolf, T.; Triantafyllidis, A.; Vasileiadou, K.; Kanellis, D.; Cecconi, P.; Kappas, I. & Abatzopoulos, T.J., Follow-up of hatchery rotifer cultures with regard to their genetic identity. Journal of Biological Research 7: Pourriot, R., Rotifers Biology. In: Aquaculture vol.1, (Barnabe, G. ed.) : Ellis Horwood, England. - Saavedra, M.; Conceição, L.E.C.; Pousão-Ferreira, P. & Dinis, M.T., Amino acid profiles of Diplodus sargus (L., 1758) larvae: Implications for feed formulation. Aquaculture, 261: Saavedra,M., Beltran,M., Pousão-Ferreira,P., Dinis,M.T., Blasco,J. & Conceição, L.E.C., Evaluation of bioavailability of individual amino acids in Diplodus puntazzo larvae: Towards the ideal dietary amino acid profile. Aquaculture 263: Sargent, J.; Bell, G.; McEvoy, L.;Tocher, D. & Estevez, A., 1999a. Recent developments in the essential fatty acid nutrition of fish. Aquaculture,177: Sargent, J.; McEvoy, L.; Estevez, A.; Bell, G.; Bell, M.; Henderson, J. & Tocher, D., 1999b. Lipid nutrition of marine fish during early development: current status and future directions. Aquaculture,179; Sorgeloos, P.; Lavens, P.; Léger, P.; Tackaert, W. & Versichele, D., Manual for the culture and use of brine shrimp Artemia in aquaculture. State University of Ghent, Faculty of Agriculture, 319pp. - Støttrup. J.G., Production and nutrition value of copepods. in Live Feeds in MarineAquaculture (ed. by J.G. Støttrup & L.A. McEvoy), pp Blackwell Publishing, Oxford, UK.

21 LARVITA Fish Larvae Training School 2010 Live feeds Pedro Pousão Ferreira, INRB, I.P. IPIMAR - Van der Meeren, T., Olsen, R.E., Hamre K. & Fyhn, H.J., Biochemical composition of copepods for evaluation of feed quality in production of juvenile marine fish. Aquaculture. 274: Other literature Andersen, R. (Ed), 2005.Algal Culturing Techniques, Elsevier Academic Press. Amsterdam. 578p. Lavens, P; Sorgeloos, P. (eds.) Manual on the production and use of live food for aquaculture. FAO Fisheries Technical Paper. No Rome, FAO. 295p. - Moretti, A.; Fernandez-Criado, M.P.; Cittolin, G. & Guidastri, R. (EDs) Manual on Hatchery Production of Seabass and Gilthead Seabream. Volume 1. FAO, Rome. 194p. - Moretti, A.; Fernandez-Criado, M.P. & Vetillart, R. (Eds) Manual on Hatchery Production of Seabass and Gilthead Seabream. Volume 2. FAO, Rome. 152p. Pousão-Ferreira, P Manual de cultivo e bioencapsulação da cadeia alimentar para a larvicultura de peixes marinhos. INRB I.P. IPIMAR. Lisboa. 239p. (ISBN: ). Richmond, A. (Ed) Handbook of Microalgal Culture: Biotechnology and Applied Phycology. Blackwell Publishing, Oxford, UK. 566p. Stottrup, J.G. and McEvoy, L.A., (eds) Live Feeds in Marine Aquaculture. Blackwell Publishing, Oxford, UK. 318p.