Cláudia Aragão

Stress response and changes in amino acid requirements in Senegalese sole (Solea senegalensis Kaup 1858).

Summary.Fish in aquaculture are often exposed to various stressors that may change their ability to survive or limit growth. Amino acids are used for processes other than growth, including stress response. This study intended to analyse how repeated acute handling stress can affect growth and amino acid requirements in fish. Senegalese sole juveniles were weekly held in the air during 3 min (Handling) for 9 weeks; Control groups were left undisturbed. Growth and plasma levels of stress indicators and of free amino acids were assessed at the end of the experiment. Plasma cortisol and osmolality levels showed that fish in the Handling treatment were stressed, but growth was unaffected. Plasma amino acid concentrations indicate that their requirements in stressed fish were altered, which probably reflects the synthesis of proteins or other specific compounds related to stress response.

Assimilation and catabolism of dispensable and indispensable free amino acids in post-larval Senegal sole (Solea senegalensis).

The postprandial metabolism of dietary free amino acids (AA) was studied in post-larval Senegal sole, Solea senegalensis, aged 32 days after hatching (DAH). The diet was administered as a single pulse (36 nl, 43.1 mmol/l) using a micro tube-feeding technique and a dissolved mixture of crystalline AA. In four separate treatments the diet contained L [U-(14)C] tracer for two indispensable AA (IAA), lysine and arginine or two dispensable amino acids (DAA), glutamate and alanine. The post-larva absorbed all tested AA from the diet with similar efficiency (97.5%). A small fraction of the IAA was catabolised (11.5+/-1.1 and 15.1+/-3.3%, for lysine and arginine, respectively) and a high proportion was retained in the body (86.7+/-1.3 and 81.6+/-4.1%). For the DAA more were catabolised (64.9+/-5.3% and 41.4+/-7.2% for glutamate and alanine, respectively) and less were retained (32.9+/-5.1% and 56.3+/-7.2%). On this basis, it appears that post-larval Senegal sole use DAA in preference to IAA as energy substrates while the retention (assimilation efficiency) is better for the IAA. These results support other recent studies that early stages of fish have a better capacity to regulate AA catabolism than previously believed and that indispensable AA are saved for body growth.

Novel methodologies in marine fish larval nutrition

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.

Cloning, tissue and ontogenetic expression of the taurine transporter in the flatfish Senegalese sole (Solea senegalensis)

Flatfish species seem to require dietary taurine for normal growth and development. Although dietary taurine supplementation has been recommended for flatfish, little is known about the mechanisms of taurine absorption in the digestive tract of flatfish throughout ontogeny. This study described the cloning and ontogenetic expression of the taurine transporter (TauT) in the flatfish Senegalese sole (Solea senegalensis). Results showed a high similarity between TauT in Senegalese sole and other vertebrates, but a change in TauT amino acid sequences indicates that taurine transport may differ between mammals and fish, reptiles or birds. Moreover, results showed that Senegalese sole metamorphosis is an important developmental trigger to promote taurine transport in larvae, especially in muscle tissues, which may be important for larval growth. Results also indicated that the capacity to uptake dietary taurine in the digestive tract is already established in larvae at the onset of metamorphosis. In Senegalese sole juveniles, TauT expression was highest in brain, heart and eye. These are organs where taurine is usually found in high concentrations and is believed to play important biological roles. In the digestive tract of juveniles, TauT was more expressed in stomach and hindgut, indicating that dietary taurine is quickly absorbed when digestion begins and taurine endogenously used for bile salt conjugation may be recycled at the posterior end of the digestive tract. Therefore, these results suggest an enterohepatic recycling pathway for taurine in Senegalese sole, a process that may be important for maintenance of the taurine body levels in flatfish species.

Effects of dietary amino acids and repeated handling on stress response and brain monoaminergic neurotransmitters in Senegalese sole (Solea senegalensis) juveniles

The present study aimed to assess the effects of increased availability of dietary amino acids (AA) on brain monoamine neurotransmitters and the metabolic processes resulting from stressful situations in fish. Senegalese sole (Solea senegalensis) juveniles (24.2±0.4g wet mass) were weekly subjected to an acute handling stressor (HDLG) or remained undisturbed (CTL). Additionally, both treatments were fed a control or a high protein (HP) diet (CTL, CTL HP, HDLG and HDLG HP). The HP diet slightly increased the levels of digestible indispensable AA, together with tyrosine and cysteine. Repeated handling induced a stress response after 14 and 28 days in fish held at both HDLG and HDLG HP treatments. While dietary treatment and handling stress activated the serotonergic system at 14 days, these effects were not observed after 28 days. In addition, the HP diet minimized the decrease in plasma indispensable AA due to repeated handling stress after 28 days. It was concluded that HP diet decreased post-stress plasma glucose and lactate levels in HDLG HP specimens only at 14 days of treatment. Moreover, dietary treatment was also effective in stimulating DA synthesis and release, thus dietary phenylalanine supplementation can increase DA biosynthesis in fish.

Dietary nitrogen and fish welfare

Little research has been done in optimizing the nitrogenous fraction of the fish diets in order to minimize welfare problems. The purpose of this review is to give an overview on how amino acid (AA) metabolism may be affected when fish are under stress and the possible effects on fish welfare when sub-optimal dietary nitrogen formulations are used to feed fish. In addition, it intends to evaluate the current possibilities, and future prospects, of using improved dietary nitrogen formulations to help fish coping with predictable stressful periods. Both metabolomic and genomic evidence show that stressful husbandry conditions affect AA metabolism in fish and may bring an increase in the requirement of indispensable AA. Supplementation in arginine and leucine, but also eventually in lysine, methionine, threonine and glutamine, may have an important role in enhancing the innate immune system. Tryptophan, as precursor for serotonin, modulates aggressive behaviour and feed intake in fish. Bioactive peptides may bring important advances in immunocompetence, disease control and other aspects of welfare of cultured fish. Fishmeal replacement may reduce immune competence, and the full nutritional potential of plant-protein ingredients is attained only after the removal or inactivation of some antinutritional factors. This review shows that AA metabolism is affected when fish are under stress, and this together with sub-optimal dietary nitrogen formulations may affect fish welfare. Furthermore, improved dietary nitrogen formulations may help fish coping with predictable stressful events.

How does fish metamorphosis affect aromatic amino acid metabolism

Aromatic amino acids (AAs, phenylalanine and tyrosine) may be specifically required during fish metamorphosis, since they are the precursors of thyroid hormones which regulate this process. This project attempted to evaluate aromatic AA metabolism during the ontogenesis of fish species with a marked (Senegalese sole; Solea senegalensis) and a less accentuated metamorphosis (gilthead seabream; Sparus aurata). Fish were tube-fed with three l-[U-14C] AA solutions at pre-metamorphic, metamorphic and post-metamorphic stages of development: controlled AA mixture (Mix), phenylalanine (Phe) and tyrosine (Tyr). Results showed a preferential aromatic AA retention during the metamorphosis of Senegalese sole, rather than in gilthead seabream. Senegalese sole’s highly accentuated metamorphosis seems to increase aromatic AA physiological requirements, possibly for thyroid hormone production. Thus, Senegalese sole seems to be especially susceptible to dietary aromatic AA deficiencies during the metamorphosis period, and these findings may be important for physiologists, fish nutritionists and the flatfish aquaculture industry.

European Sea Bass (Dicentrarchus labrax) Immune Status and Disease Resistance Are Impaired by Arginine Dietary Supplementation.

Infectious diseases and fish feeds management are probably the major expenses in the aquaculture business. Hence, it is a priority to define sustainable strategies which simultaneously avoid therapeutic procedures and reinforce fish immunity. Currently, one preferred approach is the use of immunostimulants which can be supplemented to the fish diets. Arginine is a versatile amino acid with important mechanisms closely related to the immune response. Aiming at finding out how arginine affects the innate immune status or improve disease resistance of European seabass (Dicentrarchus labrax) against vibriosis, fish were fed two arginine-supplemented diets (1% and 2% arginine supplementation). A third diet meeting arginine requirement level for seabass served as control diet. Following 15 or 29 days of feeding, fish were sampled for blood, spleen and gut to assess cell-mediated immune parameters and immune-related gene expression. At the same time, fish from each dietary group were challenged against Vibrio anguillarum and survival was monitored. Cell-mediated immune parameters such as the extracellular superoxide and nitric oxide decreased in fish fed arginine-supplemented diets. Interleukins and immune-cell marker transcripts were down-regulated by the highest supplementation level. Disease resistance data were in accordance with a generally depressed immune status, with increased susceptibility to vibriosis in fish fed arginine supplemented diets. Altogether, these results suggest a general inhibitory effect of arginine on the immune defences and disease resistance of European seabass. Still, further research will certainly clarify arginine immunomodulation pathways thereby allowing the validation of its potential as a prophylactic strategy.

Taurine and Fish Development: Insights for the Aquaculture Industry

Expansion of the aquaculture industry is limited by incomplete knowledge on fish larval nutritional requirements. Nevertheless, it is believed that dietary taurine deficiencies may be particularly critical for fish larvae. The reasons include the high taurine levels found during egg and yolk-sac stages of fish, suggesting that taurine may be of pivotal importance for larval development. Moreover, unlike aquaculture feeds, natural preys of fish larvae contain high taurine levels, and dietary taurine supplementation has been shown to increase larval growth in several fish species. This study aimed to further explore the physiological role of taurine during fish development. Firstly, the effect of dietary taurine supplementation was assessed on growth of gilthead sea bream (Sparus aurata) larvae and growth, metamorphosis success and amino acid metabolism of Senegalese sole (Solea senegalensis) larvae. Secondly, the expression of taurine transporter (TauT) was characterised by qPCR in sole larvae and juveniles. Results showed that dietary taurine supplementation did not increase sea bream growth. However, dietary taurine supplementation significantly increased sole larval growth, metamorphosis success and amino acid retention. Metamorphosis was also shown to be an important developmental trigger to promote taurine transport in sole tissues, while evidence for an enterohepatic recycling pathway for taurine was found in sole at least from juvenile stage. Taken together, our studies showed that the dependence of dietary taurine supplementation differs among fish species and that taurine has a vital role during the ontogenetic development of flatfish, an extremely valuable group targeted for aquaculture production.

Avanços recentes em nutrição de larvas de peixes

Major gaps in knowledge on fish larval nutritional requirements still remain, what leads to high mortalities and quality problems in marine larviculture. This paper reviews a range of new tools, such as tracer studies, population genomics, nutritional programming, functional genomics and proteomics, as well as some examples of their present use, and potential future applications in the study of fish larvae nutrition.Major gaps in knowledge on fish larval nutritional requirements still remain, what leads to high mortalities and quality problems in marine larviculture. This paper reviews a range of new tools, such as tracer studies, population genomics, nutritional programming, functional genomics and proteomics, as well as some examples of their present use, and potential future applications in the study of fish larvae nutrition. Recent advances in nutrition of fish larval