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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.
- Recent advances in nutrition of fish larval | Avanços recentes em nutrição de larvas de peixesPublication . Conceição, L. E. C.; Aragão, C.; Richard, Nadège; Engrola, S.; Gavaia, Paulo J.; Mira, Sara; Dias, J.Os requisitos nutricionais de larvas de peixes são ainda mal compreendidos, o que leva a altas mortalidades e problemas de qualidade no seu cultivo. Este trabalho pretende fazer uma revisão de novas metodologias de investigação, tais como estudos com marcadores, genómica populacional, programação nutricional, génomica e proteómica funcionais, e fornecer ainda alguns exemplos das utilizações presentes e perspectivas futuras em estudos de nutrição de larvas de peixes.
- Improving weaning strategies for Senegalese sole: effects of body weight and digestive capacityPublication . Engrola, S.; Conceição, L. E. C.; Dias, L.; Pereira, R.; Ribeiro, Laura; Dinis, Maria TeresaTo optimize Senegalese sole-weaning strategies, three experiments were performed.The first trial tested four weaning strategies with a10mg sole. Artemia-fed sole grew threefold less than fish fed an inert diet. Sudden weaning (abrupt change from Artemia to inert diet) and weaning with co-feeding produced larger sole than did a late weaning treatment; delayed weaning negatively affected ¢sh growth. In the second experiment, the digestive capacity of early-weaned 1, 2 and 4mg sole was investigated. The highest growth was observed in sole weaned at 4mg. Digestive enzyme pro¢les suggest that sole have an adaptation period to inert diets, with reduced feed intake. This adaptation period is inversely proportional to post-larvae weight. The third experiment examined weaning with cofeeding at di¡erent weights (2,5 and11mg).These studies demonstrate that sole of 5^10mg can be weaned, with high survival rates. On the basis of the digestive enzyme pro¢les, the early introductionof inert diets in co-feeding with Artemia seems to affect intestinal processes in smaller postlarvae. This study also suggests that trypsin and alkaline phosphatase may be used as indicators of nutritional status in sole ofo5mg.