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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.
- 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.
- 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.
- 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.
- Co-feeding of inert diet from mouth opening does not impair protein utilization by Senegalese sole (Solea senegalensis) larvaePublication . Engrola, S.; Mai, M.; Dinis, Maria Teresa; Conceição, L. E. C.In most marine species inert diets alone have a poor ability to sustain fish larvae growth and development. Furthermore, results of co-feeding inert diets and live prey are variable, which may be related to the effect of inert diets on digestive maturation and subsequently protein utilization. The aim of the present work was to investigate how different feeding regimes, live feed alone or co-fed with an inert diet, influence protein utilization in Senegalese sole larvae. Feed intake, protein absorption, protein retention and protein catabolism were estimated in sole from 8 to 35 days after hatching (DAH), using 14C-labelled Artemia protein and posterior incubation in metabolic chambers. Postlarvae that were co-fed with an inert diet from mouth opening ate more than postlarvae fed Artemia alone at most sampling ages. Sole Artemia protein digestibility ranged from 56.97 (16 DAH) to 81.32% (22 DAH). Sole larvae that were fed a second meal had a slightly, though significant, higher digestibility than sole fed a single meal. Digestibility was lower in co-fed sole during metamorphosis climax, and similar between treatments at other developmental stages. Retention efficiency remained almost constant during early development, and was not affected by feeding regime. In short, co-feeding of an inert diet from mouth opening does not impair protein utilization by Senegalese sole larvae.
- Skeletal evaluation and technical improvements to decrease the incidence of skeletal deformities in Solea senegalensis, KaupPublication . Gavaia, Paulo J.; Richard, Nadège; Dâmaso, L.; Dinis, Maria Teresa; Pousão-Ferreira, P.; Engrola, S.; Conceição, L. E. C.; Cancela, LeonorThe appearance of skeletal deformities is a major problem associated to intensive, large-scale culture of fish species, affecting growth, development and ultimately, survival of produced fish. Although the mechanisms working behind it are still unclear, it is known that environmental, genetic and nutritional factors play fundamental roles. Given its importance as a vertebrate model to study skeleton-related diseases, the selection of an appropriate dietary protocol is a critical step in order to diminish the nutritional effect on bone and cartilage development. To better understand the effect of diet on skeletal development and deformities, as well as on larvae and juvenile global performance, we have tested four distinct dietary regimes, composed of rotifers Brachionus sp., Artemia nauplii and dry, formulated food.
- Comparing skeletal development of wild and hatchery-reared Senegalese sole (Solea senegalensis, Kaup 1858): Evaluation in larval and postlarval stagesPublication . Gavaia, Paulo J.; Domingues, S.; Engrola, S.; Drake, P.; Sarasquete, C.; Dinis, Maria Teresa; Cancela, LeonorThe Senegalese sole is a marine pleuronectiform that naturally occurs in Southern Europe and Mediterranean region where it is being produced in aquaculture, in particular in Portugal and Spain. The aim of this study was to assess the quality of hatchery-reared larvae in comparison with those reared in the wild, and determine to which extension wild growing larvae are also affected by skeletal deformities. The main structures affected included those forming the axial skeleton, the caudal fin complex and both anal and dorsal fins, with the most prevalent anomalies affecting caudal vertebrae and arches. Hatchery-reared fish presented a higher incidence of deformities (79%) compared with the 19% observed in wild specimens. In wild postlarvae collected in Autumn no deformities were observed. This work clearly shows that wild Senegalese sole present less skeletal deformities than those hatchery-reared during larval stages, indicating a selective mortality of wild deformed fish and/or an effect of aquaculture-related rearing conditions in the development of skeletal deformities in sole.
- Hormonal induction of Atlantic dusky grouper (Epinephelus marginatus) broodstockPublication . Conceição, L. E. C.; Cabrita, Elsa; Engrola, S.; Lacuisse, M.; Pousão-Ferreira, P.; Dinis, Maria TeresaImmature fish and young females were sex reversed using hormonal induction in two consecutive years. In the first year one permanent male was obtained. Collected sperm of both years ranges from 30 μl to 300 μl and cell concentration varies from 3 to 6.5 x109 spermatozoa/ml. Atlantic dusky grouper can successfully be induced to functional males with 17α-methyltestosterone implants.
- Nutritional physiology during development of Senegalese sole (Solea senegalensis)Publication . Conceição, L. E. C.; Ribeiro, Laura; Engrola, S.; Aragão, C.; Morais, S.; Lacuisse, M.; Soares, Florbela; Dinis, Maria TeresaThe Senegalese sole, a species with a complex metamorphosis, difficulties in weaning and with occasional problems of malpigmentation and skeletal deformities, is a good model species to study larval nutritional physiology. In addition, the early metamorphosis and acquisition of a peculiar non-proactive bottom-feeding behaviour make early weaning an important issue in sole hatcheries. The present work reviews recent findings in different aspects of nutritional physiology during the development of Senegalese sole, in an attempt to optimize the composition of sole diets and to understand what are the limiting factors for weaning sole. Both digestive enzymes activity and tracer studies using 14C-Artemia show that sole larvae, even at young stages, have a high capacity for digesting live preys. This is reflected in a high growth potential and low mortality rates for this species during the larval stage compared to other marine fish species. Based on the observation of the digestive enzymes profile, early introduction of inert microdiets in co-feeding with Artemia does not seem to affect intestinal function. However, when co-feeding is not provided, intestinal activity may be depressed. Furthermore, early introduction of microdiets in co-feeding with Artemia may have a positive effect on survival rates, but at the expense of lower growth rates and higher size dispersal. This may reflect variation in the adaptation capacity of individual larvae to inert diets. High dietary neutral lipid (soybean oil) content results in reduced growth and accumulation of lipid droplets in the enterocytes and affects the capacity of Senegalese sole larvae to absorb and metabolise dietary fatty acids (FA) and amino acids (AA). Through tube feeding of different 14C-lipids and free FA it has been shown that FA absorption efficiency increases with unsaturation and that sole larvae spare DHA from catabolism. In addition, it was demonstrated that absorption efficiency varies according to molecular form, being highest for free FA, lowest for triacylglycerols and intermediate for phospholipids. Live preys commonly used in larviculture do not seem to have a balanced AA profile for sole larvae. Furthermore, the ideal dietary AA composition probably changes during development. Rotifers and Artemia metanauplii are apparently deficient in one or more of the following AA depending on the larval development stage: histidine, sulphur AA, lysine, aromatic AA, threonine and arginine. It has also been demonstrated that balancing the dietary AA profile with dipeptides inArtemia-fed larvae increases AA retention and reduces AA catabolism. When supplementing larval diets with limiting AA it should also be considered that sole larvae have different absorption, and retention efficiencies for individual AA, and that they have the capacity to spare indispensable AA. In addition, the absorption of free AA is faster and more efficient than that of complex proteins. Improvements in biochemical composition of inert microdiets for sole are likely to contribute to the reproducible weaning success of Senegalese sole.
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