CIMAR LA - CCMAR Centro de Ciências do Mar, Universidade do Algarve,

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

2. Background 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

3. Background 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

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

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

6. Tracer studies: Tube-feeding Methodology

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

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

9. Tracer studies: Tube-feeding Example: Tyrosine and stress response Dietary tyrosine supplementation avoids disturbance in protein metabolism during stress Aragão et al. 2010. Aquaculture Europe 2010

10. Tracer studies: Diet labelling Methodology 14C-enriched Artemia 14C-enriched fish Air suplly 14CO2 trap Metabolic chambers

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

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

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

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

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. Proteomics Example: Vitamin K1 diet enrichment  proteome 16 spots analyzed by LC-MS/MS (ESI-Ion Trap) Bold numbers: spots over-expressed in larvae fed with diet 250 relative to control larvae Regular numbers: spots under-expressed in larvae fed with diet 250relative to control larvae • Collagen VI: • Under-expressed with Vit K supplementation 8802 1809 Myosin heavy chain 1801 4829 4727 5732 4729 5822 Collagen VI 8807 4733 4732 5719 6704 4726 MW (KDa) 8808 5702 6702 6711 4735 - Expressed in most tissues 4611 64 Chaperonin 5712 Fibrinogen β 1601 4609 6612 ATP synthase β polypeptide Myosin binding protein 3624 4602 8506 Enolase 5606 6534 6506 3518 2620 51 - Involvement in matrix structural integrity 5603 6522 0514 4606 Keratin 3519 1511 7414 5510 2402 7428 Keratin I isoform 1 39 6417 Keratin I isoform 2 Creatine kinase 1301 7215 6302 0211 8204 • Bind other collagen fibrils • Role in bone formation? 1207 28 Myosin light chain 1 5205 0115 8221 2215 Muscle creatine kinase 3123 Muscle creatine kinase 19 1112 14 8106 6108 8118 8117 Linear pH (4-7) Muscle creatine kinase 4 Richard et al. 2009. Interdisciplinary Approaches in Fish Skeletal Biology 7

17. Adult Growth potential & health status Juvenile Larvae Egg Heterogeneity Nutritional programming in early life stages Concept Prenatal or early postnatal events exerted at critical developmental windows, result in lifelong contributions to postnatal growth potential and health status 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

18. Conclusions 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.

19. Aquaculture Research Group 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

20. Obrigado !!!