1. CIMAR LA - CCMAR Centro de Ciências do Mar, Universidade do Algarve, Campus de Gambelas, Edifício 7 8005-139 Faro, Portugal ADVANCES IN FISH LARVAE NUTRITION Aquaculture Research Group Encontro Nacional de Ciência - CIÊNCIA 2010 Sessão: Aquacultura e culturas offshore, 5 de Julho de 2010

2. Larvae vs. larger fish: Poorer capacity to digest and/or absorb nutrients higher growth rates (10-20%/day; up to 50%/day) => higher requirements in terms of HUFAs, phospholipids, amino acids, vitamins and other nutrients Background

3. Methodological difficulties: small size of the animals most species do not perform well on inert diets nutritional composition of live food is not easily manipulated assessing food intake and digestibility of diets is a major challenge dose-response studies very difficult to perform Background

4. Review new tools in fish larvae nutrition Examples of their present use, as well as potential future applications ? This presentation

5. Rust et al. 1993; Rønnestad et al. 2000, 2001 14 C-labelled nutrients, as amino acids or fatty acids Tracer studies: Tube-feeding Methodology

6. Tracer studies: Tube-feeding Methodology

7. Pinto et al. 2009. Amino Acids 36: 177-183 Tyrosine retention increases during Senegalese sole metamorphosis Tracer studies: Tube-feeding Example: Tyrosine utilisation during metamorphosis

8. Stress affects tyrosine metabolism in Senegalese sole post-larvae Tracer studies: Tube-feeding Example: Tyrosine and stress response Aragão et al. 2010. Aquaculture Europe 2010

9. Dietary tyrosine supplementation avoids disturbance in protein metabolism during stress Tracer studies: Tube-feeding Example: Tyrosine and stress response

10. Air suplly 14 CO 2 trap 14 C-enriched Artemia 14 C-enriched fish Metabolic chambers Methodology Tracer studies: Diet labelling

11. Digestibility ArtR (+25% of inert diet) ST: Live feed b b a a Example Protein utilisation by Senegalese sole larvae Engrola et al. 2009. Aquaculture 287: 185-190 Tracer studies: Diet labelling

12. 81% 100% Feed intake 19% Faeces 15% Catabolised 66% Retained 100% Feed intake 11% Catabolised 27% Faeces 62% Retained 73% 22 DAH Sole postlarvae Tracer studies: Diet labelling Live feed treatmentArtemia replacement treatment Engrola et al. 2009. Aquaculture 287: 185-190 Example: Protein utilisation by Senegalese sole larvae

13. Proteomics Methodology: Two-dimensional electrophoresis 1 st dimension: isoelectric focusing Before focusing After focusing Separation according to pI 2 nd dimension: SDS-PAGE Separation according to MW

14. MS: separation and detection of peptide ions according to their ratio mass/charge (m/z) Protein Trypsin digestion Peptides Mass spectrum Protein sequence Theoretical mass spectrum Theoretical peptides obtained after trypsin digestion Proteomics Methodology: Protein spot identification by MS

15. Proteomics Example: Vitamin K1 diet enrichment  proteome 487 protein spots detected (pI : 4-7) Vit K1 supplementation: 47 proteins over- expressed (ratio >1.5) 29 under-expressed (ratio < 0.6) Richard et al. 2009. Interdisciplinary Approaches in Fish Skeletal Biology

16. 16 spots analyzed by LC-MS/MS (ESI-Ion Trap) Collagen VI: - Expressed in most tissues - Involvement in matrix structural integrity - Bind other collagen fibrils - Role in bone formation?  Under-expressed with Vit K supplementation Proteomics Example: Vitamin K1 diet enrichment  proteome Richard et al. 2009. Interdisciplinary Approaches in Fish Skeletal Biology

17. Prenatal or early postnatal events exerted at critical developmental windows, result in lifelong contributions to postnatal growth potential and health status Concept Nutritional programming in early life stages Possible biological mechanisms for “imprinting” the nutritional programming stimulus until adulthood include: adaptive changes in gene expression preferential clonal selection of adapted cells in programmed tissues programmed differential proliferation of tissue cell types Knowledge in fish is extremely scarce Egg Larvae Juvenile Adult Growth potential & health status Heterogeneity

18. These methodologies have made decisive contributions, and are expected to do even more in the near future, to build a knowledge basis for development of optimised diets and feeding regimes for different species of larval fish. Conclusions

19. Coordinator: Maria Teresa Dinis CIMAR LA Researchers: Luís Conceição Jorge Dias Post-Docs: Cláudia Aragão Sofia Engrola Nadège Richard Dulce Martins Catarina Martins Sonia Martínez-Páramo Catarina Oliveira PhD Students: Benjamín Costas Catarina Campos Elisabete Matos Mahaut Vaireles Patrícia Silva Tomé Silva Wilson Pinto Research assistants: Ana Ramalho André Santos Filipa Castanheira Filipa Rocha Helena Teixeira Odete Cordeiro Patrícia Diogo Rita Colen Vera Rodrigues Aquaculture Research Group

20. Obrigado !!!