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- Programming the metabolic gene expression in zebrafish juveniles (Danio rerio) by supra-physiological glucose level during embryogenesisPublication . Rocha, F.; Dias, J.; Engrola, S.; Gavaia, Paulo J.; Geurden, Inge; Dinis, Maria Teresa; Panserat, S.Using zebrafish as a model organism, assess the effect of high glucose levels during early embryonic stages as a lifelong moderator of metabolic pathways.
- Nutrition, quality and performance in marine fish larvaePublication . Conceição, L. E. C.; Alves-Martins, D.; Engrola, S.; Gavaia, Paulo J.; Richard, Nadège; Aragão, C.O cultivo de larvas de peixes marinhos é caracterizado por altas mortalidades e por problemas de qualidade, nomeadamente malformações esqueléticas. A solução de parte destes problemas passa por uma melhor compreensão dos requisitos nutricionais. Este trabalho pretende fazer uma revisão de trabalhos recentes que demonstram como os aminoácidos, os ácidos gordos e a vitamina K, mas também o regime alimentar, podem influenciar a qualidade e a performance das larvas de peixes marinhos.
- Tolerance of zebrafish embryos to supra-physiologic yolk levels of glucosePublication . Rocha, F.; Dias, J.; Engrola, S.; Gavaia, Paulo J.; Dinis, Maria Teresa; Panserat, S.Once spawned and fertilized the oviparous fish eggs operate as closed systems; only respiratory gases, heat and negligible amounts of solutes and water are exchanged freely, as a result of an extremely low permeability of the egg surface membranes. Currently, the opportunities to exert a nutritional stimulus during a stage of high metabolic plasticity, such as fish embryogenesis, are restricted to maternal transfer and the onset of exogenous feeding.
- What determines growth potential and juvenile quality of farmed fish species?Publication . Valente, L. M. P.; Moutou, K. A.; Conceição, L. E. C.; Engrola, S.; Fernandes, J. M. O.; Johnston, Ian A.Enhanced production of high quality and healthy fry is a key target for a successful and competitive expansion of the aquaculture industry. Although large quantities of fish larvae are produced, survival rates are often low or highly variable and growth potential is in most cases not fully exploited, indicating significant gaps in our knowledge concerning optimal nutritional and culture conditions. Understanding the mechanisms that control early development and muscle growth are critical for the identification of time windows in development that introduce growth variation, and improve the viability and quality of juveniles. This literature review of the current state of knowledge aims to provide a framework for a better understanding of fish skeletal muscle ontogeny, and its impact on larval and juvenile quality as broadly defined. It focuses on fundamental biological knowledge relevant to larval phenotype and quality and, in particular, on the factors affecting the development of skeletal muscle. It also discusses the available methodologies to assess growth and larvae/juvenile quality, identifies gaps in knowledge and suggests future research directions. The focus is primarily on the major farmed non-salmonid fish species in Europe that include gilthead sea bream, European sea bass, turbot, Atlantic cod, Senegalese sole and Atlantic halibut.
- Novel methodologies in marine fish larval nutritionPublication . Conceição, L. E. C.; Aragão, C.; Richard, Nadège; Engrola, S.; Gavaia, Paulo J.; Mira, Sara; Dias, J.Major gaps in knowledge on fish larval nutritional requirements still remain. Small larval size, and difficulties in acceptance of inert microdiets, makes progress slow and cumbersome. This lack of knowledge in fish larval nutritional requirements is one of the causes of high mortalities and quality problems commonly observed in marine larviculture. In recent years, several novel methodologies have contributed to significant progress in fish larval nutrition. Others are emerging and are likely to bring further insight into larval nutritional physiology and requirements. This paper reviews a range of new tools and some examples of their present use, as well as potential future applications in the study of fish larvae nutrition. Tube-feeding and incorporation into Artemia of 14C-amino acids and lipids allowed studying Artemia intake, digestion and absorption and utilisation of these nutrients. Diet selection by fish larvae has been studied with diets containing different natural stable isotope signatures or diets where different rare metal oxides were added. Mechanistic modelling has been used as a tool to integrate existing knowledge and reveal gaps, and also to better understand results obtained in tracer studies. Population genomics may assist in assessing genotype effects on nutritional requirements, by using progeny testing in fish reared in the same tanks, and also in identifying QTLs for larval stages. Functional genomics and proteomics enable the study of gene and protein expression under various dietary conditions, and thereby identify the metabolic pathways which are affected by a given nutrient. Promising results were obtained using the metabolic programming concept in early life to facilitate utilisation of certain nutrients at later stages. All together, 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.
- Optimization of monoclonal production of the glass anemone Aiptasia pallida (Agassiz in Verrill, 1864)Publication . Leal, M. C.; Nunes, Carla; Engrola, S.; Dinis, Maria Teresa; Calado, RicardoSea anemones of genus Aiptasia are commonly used as biologicalmodels for biotechnological and molecular research. They are also employed to study the symbiotic interactions between cnidarians and zooxanthellae. In addition, Aiptasia is an important prey for the culture of the highly priced ornamental nudibranch Aeolidiella stephanieae. The purpose of this study was to determine the best culture conditions for establishing large monoclonal populations of this anemone. This study analyzed the effect of the following factors on Aiptasia pallida propagation and biomass increase throughout 60 days: initial anemone stocking density, light regimes, water temperature and different live diets. The best results were achieved at a higher water temperature (26 °C) and in darkness. Artemia nauplii were a better live prey than Artemia metanauplii to maximize biomass production, with lower initial anemone stocking densities maximizing propagation ratios. This research provides initial data that enables a large-scale production of monoclonal A. pallida, either to be used as a biological model, for the screening of new natural products or in the aquaculture of ornamental sea slugs.
- Glucose overload in yolk has little effect on the long-term modulation of carbohydrate metabolic genes in zebrafish (Danio rerio)Publication . Rocha, Filipa; Dias, J.; Engrola, S.; Gavaia, Paulo J.; Geurden, Inge; Dinis, Maria Teresa; Panserat, S.The use of early nutritional stimuli to program metabolic pathways in fish is ill defined. Therefore, studies were undertaken with zebrafish to assess the effect of high glucose levels during the embryonic stage as a lifelong modulator of genes involved in carbohydrate metabolism. Genes related to carbohydrate metabolism were expressed at low levels at 0.2 and 1 day post-fertilization (dpf). However, from 4 dpf onwards there was a significant increase on expression of all genes, suggesting that all analysed pathways were active. By microinjection, we successfully enriched zebrafish egg yolk with glucose (a 43-fold increase of basal levels). Acute effects of glucose injection on gene expression were assessed in larvae up to 10 dpf, and the programming concept was evaluated in juveniles (41 dpf) challenged with a hyperglucidic diet. At 4 dpf, larvae from glucose-enriched eggs showed a downregulation of several genes related to glycolysis, glycogenolysis, lipogenesis and carbohydrate digestion in comparison with control (saline-injected) embryos. This inhibitory regulation was suppressed after 10 dpf. At the juvenile stage, and upon switching from a low to a high digestible carbohydrate diet, early glucose enrichment had no significant effect on most analysed genes. However, these same fish showed altered expression of the genes for cytosolic phosphoenolpyruvate carboxykinase, sodium-dependent glucose cotransporter 1 and glycogen synthase, suggesting changes to the glucose storage capacity in muscle and glucose production and transport in viscera. Overall, supplementation of egg yolk with high glucose levels had little effect on the long-term modulation of carbohydrate metabolic genes in zebrafish.
- Individual differences in cortisol levels and behaviour of Senegalese sole (Solea senegalensis) juveniles: evidence for coping stylesPublication . Silva, P. I. M.; Martins, C. I. M.; Engrola, S.; Marino, G.; Øverli, Ø.; Conceição, L. E. C.Individual variation in stress physiology and behaviour has been previously reported in several fish species. As seen in other vertebrates, existence of stress coping styles seemsto be reflected by the presence of individual variation. Aggressive behaviour, amongst others, is one of the most commonly used parameters to characterize coping styles. However, not all fish species exhibit aggressive behaviour, such as the flatfish Senegalese sole, Solea senegalensis (Kaup, 1858). Therefore, the goal of this study was to determine the magnitude of individual variation in behavioural parameters other than aggression (feeding motivation and activity during stress) as well as in growth and stress response in Senegalese sole. The relationship between these variables was investigated to determine whether they could be used as indicators of coping styles. Thirty-six juvenile fish (9.9 2.2 g) were individually housed for 73 days. Feedingmotivation,measured as the time(in s) taken by each fish to react to feed,was determined on days 10, 17, 24 and 31. Bloodsamples for plasmacortisol were collected on days 51 and 71 for determination of undisturbed and stress levels, respectively. Thestress test consisted of holding each fish individually in a net, outside the water, for 3min. Duration of escape attempts, i.e. the time takenby each fish to stop struggling (in an attempt to escape) in the net, was quantified. The results showed a pronounced individual variation in both control (CV = 54%) and acute stress (CV = 71%) cortisol levels. Senegalese sole also exhibited high coefficient of variationinthebehavioural parameters:75%in feeding latency and96%induration of escape attempts. Growth (RGR = 1.17 0.38) showed to be the parameter with lower variation of only 32% and was not correlated with any of the measured parameters. A significant correlation between undisturbed cortisol levels and duration of escape attempts was found. Undisturbed cortisol levels (8.08 4.36 ng/ml) were negatively correlated with duration of escape attempts (P = 0.009, rs = 0.503). Correlations between plasma cortisol levels after stress (398.45 282.67 ng/ml) and the behavioural parameters were not found. The observed individual variation in behaviour and stress physiology as well as their relationship suggests the existence of coping styles in Senegalese sole where proactive individuals exhibit shorter feeding latency, higher duration of escape attempts and lower undisturbed cortisol levels than passive individuals.
- The role of dietary methionine concentrations on growth, metabolism and N-retention in cobia (Rachycentron canadum) at elevated water temperaturesPublication . Nguyen, Minh Van; Espe, Marit; Conceição, Louis E. C.; Le, Hoang Minh; Yúfera, Manuel; Engrola, Sofia; Jordal, A. E. O.; Rønnestad, IvarThis study determined impacts of dietary methionine concentrations at two temperatures on growth, feeding efficiency and N-metabolites in juvenile cobia. Methionine concentrations of the experimental diets were deficient (M9; 9 g/kg), sufficient (M12; 12 g/kg) and surplus (M16, 16 g/kg). Water temperature was normal (30°C) or elevated (34°C). Twenty cobia in triplicate tanks were fed the experimental diets for 6 weeks. Both methionine and temperature affected cobia's growth and feeding efficiency. Cobia fed M9 performed lower than the fish fed M12 and M16 diets. Additionally, cobia reared at 34°C performed poorer than at 30°C, probably due to lower voluntary feed intake in the fish reared at 34°C. Protein efficiency ratio and protein productive value in cobia fed M9 diet were less than M12 or M16 diets. This was confirmed with the improved retentions of indispensable amino acids (AAs). No interactions between methionine and temperature were observed in growth and protein accretion. At 30°C, CF improved, while HSI and VSI declined upon methionine supplementation levels. Of which an interaction between temperature and methionine was present. Plasma, muscle and liver free AA and N-metabolites were affected by methionine and temperature. Furthermore, temperature affected cobia's lipid class composition, resulting in increased phospholipids and cholesterol at 34°C.
- Dietary tryptophan supplementation induces a transient immune enhancement of gilthead seabream (Sparus aurata) juveniles fed fishmeal-free dietsPublication . Ramos-Pinto, Lourenco; Martos-Sitcha, Juan A.; Reis, Bruno; Azeredo, Rita; Fernandez-Boo, Sergio; Perez-Sanchez, Jaume; Alvar Calduch-Giner, Josep; Engrola, Sofia; Conceicao, Luis E. C.; Dias, Jorge; Silva, Tome S.; Costas, BenjaminEuropean aquaculture is an industry with a high sustainability profile contributing to the supply of safe seafood. However, several diseases can affect farmed fish and it is imperative to find alternatives for chemotherapeutic treatments when disease outbreaks occur. Maintenance of health through nutrition is well-establish in modern animal farming, and amino acids (AA) are promising candidates as functional additives to improve fish health. Therefore, the goal of this research is to provide a better understanding of the influence of tryptophan supplementation on nutritional condition and immune mechanisms in fish. Triplicate groups of fish (13.3 +/- 0.3g) previously fed with a fishmeal-based diet were either fed a control diet with an extreme formulation (0% fishmeal) but meeting the AA requirements (CTRL), or the SUP diet, formulated as the CTRL with an increase in tryptophan (TRP) content. After 2 and 13 weeks of feeding, head-kidney (HK), liver (L) and white skeletal muscle (WSM) were collected for gene expression, whereas plasma was suited for humoral immune parameters. A holistic approach using transcriptomic, humoral and zootechnical parameters was undertaken. The expression of 29-31 genes for WSM, L or HK confirms an effect due to the treatment across time. A two-way ANOVA analysis revealed that 15-24 genes varied significantly depending on the tissue, and the multivariate analysis by means of PLS-DA explained (R-2) and predicted (Q(2)) with four components up to 93% and 78% of total variance, respectively. Component 1 (R-2 = 50.06%) represented the time effects, whereas components 2 (24.36%) and 3 (13.89%) grouped fish on the basis of dietary treatment, at early sampling. The HK results in particular suggest that fish fed SUP diet displayed an immunostimulated state at 2 weeks. No major differences were observed in plasma humoral parameters, despite an increase in antiprotease and peroxidase activities after 13 weeks regardless of dietary treatment. These results suggest that tryptophan supplementation may improve the seabream immune status after 2 weeks. Hence, the use of functional feeds is especially relevant during a short-term feeding period before a predictable stressful event or disease outbreak, considering that these putative advantageous effects seem to disappear after a 13 weeks feeding period.