Effect of Dietary Incorporation of Yellow Mealworm as a Partial Fishmeal Replacer on Growth, Metabolism, and Intestinal Histomorphology in Juvenile Meagre (Argyrosomus regius).

Efforts have been made to find alternatives to fish meal (FM), as the sustainability of aquaculture depends on it. Insect meal (IM) is a potential candidate to partially replace FM, being more sustainable and economically viable. In this experimental trial, three diets were tested with different yellow mealworm incorporation: a control diet with no IM, a diet with an inclusion of 10% IM (Ins10), and a diet with an incorporation of 20% IM (Ins20). The diets were tested on 10.5 g meagre for 47 days. The results showed that an IM inclusion higher than 10% affected both growth (2.6 vs. 2.2) and FCR (1.5 vs. 1.9) of meagre juveniles. However, this reduction in growth did not result from lower protein retention or changes in muscle fibre area or density. Little differences were observed in the activity of pancreatic and intestinal enzymes except for aminopeptidase total activity which was higher in the control and Ins10 compared to Ins20 (3847 vs. 3540 mU/mg protein), suggesting no limitations in protein synthesis. Also, the alkaline phosphatase intestinal maturation index was higher in the control group compared to the IM groups (437 vs. 296). On the contrary, several differences were also found in the proteolytic activity in the hepatic and muscle tissues of meagre juveniles fed the Ins10 diet. The inclusion of IM had no impact on intestine histomorphology but changes were detected in the enterocytes of fish from control and Ins10 which showed hypervacuolization and nucleus misplacement compared to the Ins20 treatment. Nevertheless, a higher percentage of Vibrionaceae was recorded for meagre fed on the Ins20 diet. Since no signs of inflammation were observed in the distal intestine, this suggests IM incorporation could have had an important impact on intestinal health due to its antimicrobial properties. This is supported by an increase in the haematocrit in the treatments where IM was added (20 to 25%). In conclusion, incorporations of IM at percentages up to 10% do not seem to have a negative impact on meagre performance at this age but can enhance the fish immune system and protection against intestinal inflammation.

Short-Term Immune Responses of Gilthead Seabream (Sparus aurata) Juveniles against Photobacterium damselae subsp. piscicida.

Photobacteriosis is a septicaemic bacterial disease affecting several marine species around the globe, resulting in significant economic losses. Although many studies have been performed related to the pathogen virulence and resistance factors, information regarding the host defence mechanisms activated once an infection takes place is still scarce. The present study was designed to understand innate immune responses of farmed juvenile gilthead seabream (Sparus aurata) after Photobacterium damselae subsp. piscicida (Phdp) infection. Therefore, two groups of seabream juveniles were intraperitoneally injected with 100 µL of PBS (placebo) or 100 µL of exponentially growing Phdp (1 × 106 CFU/mL; infected). The blood, plasma, liver, and head kidney of six fish from each treatment were sampled immediately before infection and 3, 6, 9, 24 and 48 h after infection for the broad screening of fish immune and oxidative stress responses. Infected animals presented marked anaemia, neutrophilia and monocytosis, conditions that are correlated with an increased expression of genes related to inflammation and phagocytic activity. Similar studies with different fish species and bacteria can be useful for the definition of health biomarkers that might help fish farmers to prevent the occurrence of such diseases.

Plant-based diets fed to juvenile meagre Argyrosomus regius with low methionine and taurine supplementation led to an overall reduction in fish performance and to an increase in muscle fibre recruitment.

Methionine and taurine are amino acids (AA) that are usually deficient when fish meal is replaced by plant proteins. In this study, three diets were tested in juvenile meagre (initial weight: 13.4 g) for 8 weeks. The D1 diet had 0.2% methionine and 1% taurine supplementation; the D2 and D3 diets had 0.6% methionine and 1% and 2% taurine supplementation, respectively. The results showed that meagre fed the D1 diet had lower specific growth rate (2.2 to 2.5), lower feed efficiency (0.9 to 1.2) and higher food conversion rate (FCR, 1.1 to 0.8) as well as a lower activity of the alanine aminotransferase (ALAT) enzyme. Furthermore, a higher recruitment of muscle fibres (46% compared to 36%) as well as a higher fibre density was observed (1019 compared to 870 fibres mm-2 ). This study shows that meagre requires a sufficient quantity of methionine in plant-based diets to avoid a reduction in fish performance. Furthermore, taurine supplementation in the D1 diet was not able to mitigate the effects of methionine deficiency. A higher taurine supplementation did not improve meagre performance.

Living in a multi-stressors environment: An integrated biomarker approach to assess the ecotoxicological response of meagre (Argyrosomus regius) to venlafaxine, warming and acidification.

Pharmaceuticals, such as the antidepressant venlafaxine (VFX), have been frequently detected in coastal waters and marine biota, and there is a growing body of evidence that these pollutants can be toxic to non-target marine biota, even at low concentrations. Alongside, climate change effects (e.g. warming and acidification) can also affect marine species' physiological fitness and, consequently, compromising their ability to cope with the presence of pollutants. Yet, information regarding interactive effects between pollutants and climate change-related stressors is still scarce. Within this context, the present study aims to assess the differential ecotoxicological responses (antioxidant activity, heat shock response, protein degradation, endocrine disruption and neurotoxicity) of juvenile fish (Argyrosomus regius) tissues (muscle, gills, liver and brain) exposed to VFX (via water or feed), as well as to the interactive effects of warming (ΔT °C = +5 °C) and acidification (ΔpCO2 ~ +1000 µatm, equivalent to ΔpH = -0.4 units), using an integrated multi-biomarker response (IBR) approach. Overall, results showed that VFX toxicity was strongly influenced by the uptake pathway, as well as by warming and acidification. More significant changes (e.g. increases surpassing 100% in lipid peroxidation, LPO, heat shock response protein content, HSP70/HSC70, and total ubiquitin content, Ub,) and higher IBR index values were observed when VFX exposure occurred via water (i.e. average IBR = 19, against 17 in VFX-feed treatment). The co-exposure to climate change-related stressors either enhanced (e.g. glutathione S-transferases activity (GST) in fish muscle was further increased by warming) or attenuated the changes elicited by VFX (e.g. vitellogenin, VTG, liver content increased with VFX feed exposure acting alone, but not when co-exposed with acidification). Yet, increased stress severity was observed when the three stressors acted simultaneously, particularly in fish exposed to VFX via water (i.e. average IBR = 21). Hence, the distinct fish tissues responses elicited by the different scenarios emphasized the relevance of performing multi-stressors ecotoxicological studies, as such approach enables a better estimation of the environmental hazards posed by pollutants in a changing ocean and, consequently, the development of strategies to mitigate them.

Effect of copper exposure and recovery period in reared Diplodus sargus.

The aquaculture growth can be followed by the occurrence of more and new pathogenic agents, since the production leads to higher fish densities in confined areas more appropriate to the appearance and propagation of pathologies. Copper sulfate has been widely used in preventing and controlling fish parasites. The objective of this study is to investigate the effects of copper treatments in the fish tissues (bioaccumulation and histological changes in different organs), mortality and evaluate what happens during the recovery period. White sea bream (Diplodus sargus) were exposed to copper sulfate (0.25 and 0.5 mg L-1) during 60 days followed with a 75-day recovery period. The results showed that the concentration of copper in fish liver was significantly higher in the 0.5 mg L-1 treatment than in the 0.25 mg L-1 treatment. Conversely, copper load in the muscle did not differ significantly between treatments and control. Copper levels in muscle, and especially in liver, increased during copper exposure (up to 60 days). In summary, at higher concentrations copper sulfate treatment (0.5 mg L-1) might be toxic to fish, which showed histological alterations and copper accumulation in their tissues, mainly in the liver. Nevertheless, individuals returned to their original state after a 75-day recovery period and the tested copper concentrations does not represents risk for food safety.

Humoral and mucosal immune responses in meagre (Argyrosomus regius) juveniles fed diets with varying inclusion levels of carob seed germ meal.

Many studies have assessed the effects of incorporation of plant feedstuffs in fish diets on growth performance, whereas few studies have addressed the effects of fish meal replacement by plant protein sources on fish immune parameters. Thus, the aim of this study was to evaluate the effects on immune response of different inclusion levels of carob seed germ meal (CSGM) as partial replacement for fish meal in diets for meagre (Argyrosomus regius) juveniles. Fish were fed four experimental diets with increased CSGM inclusion levels [0% (control), 7.5% (CSGM7.5), 15% (CSGM15) and 22.5% (CSGM22.5)]. After 1, 2, and 8 weeks of feeding fish were sampled to determine haematological profile and several humoral parameters in plasma and intestine. Results showed that dietary inclusion of CSGM did not negatively affect the immune parameters of meagre. In addition, total numbers of red and white blood cells, as well as thrombocytes, lymphocytes, monocytes, and neutrophils counts were not affected by dietary treatments. All parameters evaluated in plasma were unaffected by dietary CSGM inclusion after 1 and 2 weeks of feeding, with only the haemolytic complement activity showing an increase in fish fed diets with CSGM after 1 week and in fish fed CSGM22.5 diet after 2 weeks. Regarding the innate immune parameters analysed in the intestine, it could be highlighted the increase in alkaline phosphatase and antiprotease activities in fish fed the diet with the higher inclusion of CSGM at 8 weeks. Overall, results suggest that high dietary CSGM inclusion do not compromise immune status or induce an inflammatory response in meagre juveniles.

Fish energy budget under ocean warming and flame retardant exposure.

Climate change and chemical contamination are global environmental threats of growing concern for the scientific community and regulatory authorities. Yet, the impacts and interactions of both stressors (particularly ocean warming and emerging chemical contaminants) on physiological responses of marine organisms remain unclear and still require further understanding. Within this context, the main goal of this study was to assess, for the first time, the effects of warming (+ 5 °C) and accumulation of a polybrominated diphenyl ether congener (BDE-209, brominated flame retardant) through dietary exposure on energy budget of the juvenile white seabream (Diplodus sargus). Specifically, growth (G), routine metabolism (R), excretion (faecal, F and nitrogenous losses, U) and food consumption (C) were calculated to obtain the energy budget. The results demonstrated that the energy proportion spent for G dominated the mode of the energy allocation of juvenile white seabream (56.0-67.8%), especially under the combined effect of warming plus BDE-209 exposure. Under all treatments, the energy channelled for R varied around 26% and a much smaller percentage was channelled for excretion (F: 4.3-16.0% and U: 2.3-3.3%). An opposite trend to G was observed to F, where the highest percentage (16.0 ±â€¯0.9%) was found under control temperature and BDE-209 exposure via diet. In general, the parameters were significantly affected by increased temperature and flame retardant exposure, where higher levels occurred for: i) wet weight, relative growth rate, protein and ash contents under warming conditions, ii) only for O:N ratio under BDE-209 exposure via diet, and iii) for feed efficiency, ammonia excretion rate, routine metabolic rate and assimilation efficiency under the combination of both stressors. On the other hand, decreased viscerosomatic index was observed under warming and lower fat content was observed under the combined effect of both stressors. Overall, under future warming and chemical contamination conditions, fish energy budget was greatly affected, which may dictate negative cascading impacts at population and community levels. Further research combining other climate change stressors (e.g. acidification and hypoxia) and emerging chemical contaminants are needed to better understand and forecast such biological effects in a changing ocean.

Stress effects of amyloodiniosis in gilthead sea bream Sparus aurata.

Amyloodiniosis is a parasitological disease caused by one of the most common and important parasitic dinoflagellates in fish, Amyloodinium ocellatum (Brown), that represents a major bottleneck for gilthead seabream semi-intensive aquaculture in Southern Europe. In this experiment, we analyzed some metabolic, osmoregulatory and stress indicators to elucidate some of the physiological responses of gilthead sea bream when exposed to an A. ocellatum outbreak. We observed significant differences between Control and Infection groups in the cortisol, lactate and gill Na+/K+-ATPase (NKA) activity levels but that glucose, osmolarity, pH and total protein did not present such differences. This could indicate that the presence of the parasite induced a stress response, possibly enhancing the metabolization of glucose and subsequently lactate to cope with the higher energy requirements of the organism. There was also a decrease in gill NKA activity possibly due to severe epithelial damage and increased mucus production caused by the parasite A. ocellatum, which could induce anoxia and osmoregulatory impairment in the organism. However, further works must be performed to fully understand the physiological reactions of fish for A. ocellatum outbreaks.

Preliminary studies on haematological and plasmatic parameters in gilthead sea bream (Sparus aurata) held under day/night temperature variations.

This study aimed to evaluate to what extent diel water temperature oscillations over a short period of time (41 h) affected haematologic and plasmatic parameters of gilthead sea bream. Sea bream juveniles (160 fish; 177.2 ± 20.9 g) were divided in two homogenous groups, experimental and control. The experimental group was exposed to water at 22.3 ± 0.6 °C between 5 a.m. and 5 p.m. and to colder water from 5 p.m. to 5 a.m. (temperature variation of 8.6 ± 0.3 °C) during 41h period, whereas for the control group, water temperature was maintained constant (22.4 ± 0.7 °C) for the same period. Fish were fed three times per day (10:30 a.m., 2 p.m., 4 p.m.). Blood was collected from fish caudal veins (n = 6) at different time points (10 a.m., 1 p.m., 5 p.m., 6:30 p.m., 8 p.m. and 10 a.m. of the following day) from each group, for haematologic (haematocrit and haemoglobin) and plasmatic (glucose, cortisol, triglycerides and total protein) determinations. All parameters studied tended to vary throughout the study period, both for experimental and control groups. Significant differences were observed between the two groups for all the studied parameters at different sampling points. These differences were observed near the influence of the cold-water period or during the cooling-water period. Results suggested that colder water temperature cycles depressed fish metabolic activity and affected the levels of the analysed blood-circulating metabolites. Overall, results suggested that stress response was similar for experimental and control groups and that gilthead sea bream were able to cope with diel water temperature variations.

Prebiotics effect on immune and hepatic oxidative status and gut morphology of white sea bream (Diplodus sargus).

The aim of this study was to evaluate the effects of short-chain fructooligosaccharides (scFOS), xylooligosaccharides (XOS) and galactooligosaccharides (GOS) on immune and hepatic oxidative status, and gut morphology of white sea bream juveniles. Four diets were formulated: a control diet with fish meal (FM) and plant feedstuffs (PF) (30FM:70PF) and three test diets similar to the control but supplemented with 1% of scFOS, XOS or GOS. Dietary prebiotic incorporation did not affect total blood cell counts, hematocrit, hemoglobin, red blood indices or differential white blood cell counts. Fish fed GOS had lower ACH50 and nitric oxide than fish fed control diet. XOS enhanced immune status through the increase in alternative complement pathway (ACH50), lysozyme and total immunoglobulin. The higher activity of glucose 6-phosphate dehydrogenase in fish fed FOS compared to the other dietary groups was the only related antioxidant enzyme affected by prebiotics in the liver. GOS ameliorated the precocious adverse effects of PF based diet on gut histomorphology, as denoted by the lower incidence of histological alterations in fish fed GOS for 15 days. In conclusion, XOS and GOS at 1% might have potential to be used as prebiotics in white sea bream juveniles.

Metabolic responses to dietary protein/carbohydrate ratios in zebra sea bream (Diplodus cervinus, Lowe, 1838) juveniles.

This study aims to evaluate the effect of diets with different protein to carbohydrate ratios (P:C) on the omnivorous zebra sea bream (Diplodus cervinus) juveniles growth performance, feed efficiency, N excretion and metabolic response of intermediary metabolism enzymes. Four isoenergetic and isolipidic diets were formulated to contain increasing protein levels (25, 35, 45 and 55%) at the expense of carbohydrates (43, 32, 21 and 9%): diets P25C43, P35C32, P45C21 and P55C9. Growth performance, feed efficiency (FE), N intake [(g kg(-1) average body weight (ABW) day(-1))], N retention (g kg(-1) ABW day(-1)) and energy retention (kJ kg(-1) ABW day(-1)) increased with the increase of P:C ratio. The best growth performance and FE were achieved with diet P45C21. Ammonia excretion (mg NH4­N kg(-1) ABW day(-1)) increased as dietary protein level increased. Alanine aminotransferase and glutamate dehydrogenase activities increased with the increase of dietary P:C ratio. The opposite was observed for malic enzyme activity. Aspartate aminotransferase, hexokinase, glucokinase, fructose-1, 6-bisphosphatase and fatty acid synthetase activities were unaffected by dietary treatments. Response of key amino acid catabolic enzymes and N excretion levels to dietary P:C ratio supports the metabolic adaptability of this species to dietary protein inclusion levels. Overall, zebra sea bream seems capable of better utilize dietary protein rather than dietary carbohydrates as energy source which may be an obstacle for using more economically diets and thus for reducing environmental N loads in semi-intensive aquaculture of this species.

Comparative analysis on natural spawning of F1 meagre, Argyrosomus regius, with wild broodstock spawns in Portugal.

The purpose of this study was to describe and compare the reproductive success and egg and larvae quality between wild and first-generation (F1) breeders of Argyrosomus regius. Wild broodstock were adapted to captivity, and good-quality spawns were obtained in 2009-2010, after GnRH treatment. In 2012, the F1 meagre (3 years old) spawned naturally at IPMA's Aquaculture Research Station facilities. From each spawning event, the following parameters were determined: number of floating and non-floating eggs, egg size, hatching success and larval total length. Eggs size and percentage of hatching obtained from F1 breeders (1.04 ± 0.10 mm and 90.5 ± 6.4%) were significantly higher when compared with wild breeders (0.97 ± 0.13 mm and 17.0 ± 12.7%). Although wild breeder spawns exhibited 2.7 ± 0.2 mm for larval total length, F1 breeder spawns presented 2.6 ± 0.2 mm. The wild and F1 breeder spawns exhibit a good egg and larval quality, indicating a promising starting point for a successful meagre hatchery production.

Defective skeletogenesis and oversized otoliths in fish early stages in a changing ocean.

Early life stages of many marine organisms are being challenged by rising seawater temperature and CO2 concentrations, but their physiological responses to these environmental changes still remain unclear. In the present study, we show that future predictions of ocean warming (+4°C) and acidification (ΔpH=0.5 units) may compromise the development of early life stages of a highly commercial teleost fish, Solea senegalensis. Exposure to future conditions caused a decline in hatching success and larval survival. Growth, metabolic rates and thermal tolerance increased with temperature but decreased under acidified conditions. Hypercapnia and warming amplified the incidence of deformities by 31.5% (including severe deformities such as lordosis, scoliosis and kyphosis), while promoting the occurrence of oversized otoliths (109.3% increase). Smaller larvae with greater skeletal deformities and larger otoliths may face major ecophysiological challenges, which might potentiate substantial declines in adult fish populations, putting in jeopardy the species' fitness under a changing ocean.

Oleic acid as modulator of oxidative stress in European sea bass (Dicentrarchus labrax) juveniles fed high dietary lipid levels.

Although the benefits of oleic acid (OA) have been established in mammals, its effects on fish remain understudied. The aim of this study was to evaluate the antioxidant potential of OA in the liver, intestine, and muscle of European sea bass juveniles fed diets containing different lipid levels. For that purpose, six diets with 16 or 22% lipids and 0, 1, and 2% OA were formulated and triplicate groups of European sea bass juveniles (21.4 g) were fed with these experimental diets for 9 weeks. Increasing dietary lipid levels or OA supplementation did not affect antioxidant enzyme activity in the liver and muscle. Superoxide dismutase (SOD) activity in the intestine increased with both the dietary lipid and OA levels, while glucose 6 phosphate dehydrogenase (G6PDH) activity increased only with dietary OA supplementation. Reduced glutathione (GSH) and total glutathione (tGSH) content were higher in the liver and intestine of fish fed the low-lipid diets, while in the high-lipid diets it was lower in the muscle than in the liver and intestine. Present findings suggest that OA plays a role in the antioxidant defense mechanisms of European sea bass, particularly at the intestine level, but additional research is required to further assess the potential benefits of incorporating OA into the diets.

Early life stage mechanisms of an active fish species to cope with ocean warming and hypoxia as interacting stressors.

Ocean's characteristics are rapidly changing, modifying environmental suitability for early life stages of fish. We assessed whether the chronic effects of warming (24 °C) and hypoxia (<2-2.5 mg L-1) will be amplified by the combination of these stressors on mortality, growth, behaviour, metabolism and oxidative stress of early stages of the white seabream Diplodus sargus. Combined warming and hypoxia synergistically increased larval mortality by >51%. Warming induced faster growth in length and slower gains in weight when compared to other treatments. Boldness and exploration were not directly affected, but swimming activity increased under all test treatments. Under the combination of warming and hypoxia, routine metabolic rate (RMR) significantly decreases when compared to other treatments and shows a negative thermal dependence. Superoxide dismutase and catalase activities increased under warming and were maintained similar to control levels under hypoxia or under combined stressors. Under hypoxia, the enzymatic activities were not enough to prevent oxidative damages as lipid peroxidation and DNA damage increased above control levels. Hypoxia reduced electron transport system activity (cellular respiration) and isocitrate dehydrogenase activity (aerobic metabolism) below control levels. However, lactate dehydrogenase activity (anaerobic metabolism) did not differ among treatments. A Redundancy Analysis showed that ∼99% of the variability in mortality, growth, behaviour and RMR among treatments can be explained by molecular responses. Mortality and growth are highly influenced by oxidative stress and energy metabolism, exhibiting a positive relationship with reactive oxygen species and a negative relationship with aerobic metabolism, regardless of treatment. Under hypoxic condition, RMR, boldness and swimming activity have a positive relationship with anaerobic metabolism regardless of temperature. Thus, seabreams may use anaerobic reliance to counterbalance the effects of the stressors on RMR, activity and growth. The outcomes suggests that early life stages of white seabream overcame the single and combined effects of hypoxia and warming.

Effect of dietary l-glutamine supplementation on the intestinal physiology and growth during Solea senegalensis larval development.

The maturation of the intestinal digestive and absorptive functions might limit the amount of absorbed nutrients to fulfil the high requirements of the fast-growing marine fish larva. Glutamine (Gln) has been described to improve intestinal epithelium functions, due to its involvement in energy metabolism and protein synthesis. The purpose of this study was to evaluate dietary 0.2% Gln supplementation on aspects of intestinal physiology, protein metabolism and growth-related genes expression in Senegalese sole larvae. Experiment was carried out between 12 and 33 days post hatching (DPH) and fish were divided into two experimental groups, one fed Artemia spp. (CTRL) and the other fed Artemia spp. supplemented with Gln (GLN). GLN diet had two times more Gln than the CTRL diet. Samples were collected at 15, 19, 26 and 33 DPH for biometry, histology, and digestive enzymes activity, and at 33 DPH for gene expression, protein metabolism and AA content determination. Growth was significantly higher for Senegalese sole fed GLN diet, supported by differences on protein metabolism and growth-related gene expression. Slight differences were observed between treatments regarding the intestinal physiology. Overall, GLN diet seems to be directed to enhance protein metabolism leading to higher larval growth.

Ecotoxicological responses of juvenile Sparus aurata to BDE-99 and BPA exposure: A multi-biomarker approach integrating immune, endocrine and oxidative endpoints.

Pentabromodiphenyl ether (BDE-99) and bisphenol A (BPA) are synthetic organic compounds present in several daily use products. Due to their physicochemical properties, they are ubiquitously present in aquatic ecosystems and considered highly persistent. Recent evidence has confirmed that both emerging compounds are toxic to humans and terrestrial mammals eliciting a wide range of detrimental effects at endocrine and immune levels. However, the ecotoxicological responses that they can trigger in vertebrate marine species have not yet been established. Hence, this study aimed to investigate the ecotoxicological responses of juvenile Sparus aurata upon chronic (28 days) dietary exposure to BDE-99 and BPA (alone and combined) following an integrated multi-biomarker approach that combined fitness indicators (Fulton's K and splenosomatic indexes) with endocrine [cortisol, 17ß-estradiol (E2), 11-ketotestosterone (11-KT) concentrations] and immune (peroxidase and antiprotease activities) endpoints in fish plasma, and oxidative stress [superoxide dismutase (SOD), catalase (CAT) and glutathione S-transferase (GST) activities, and lipid peroxidation (LPO)] endpoints in the fish spleen. The mixture of BDE-99 and BPA yielded the highest IBR index value in both plasma and spleen biomarkers, therefore, suggesting that the effects of these compounds are more severe when they act together. Endocrine biomarkers were the most responsive in the three contaminated treatments. Fitness indicators were not affected by the individual nor the interactive effects of BDE-99 and BPA. These findings highlight the relevance of accounting for the interactive effects of emerging chemical contaminants and integrating responses associated with distinct biological pathways when investigating their impacts on marine life, as such a multi-biomarker approach provides a broader, more realistic and adequate perspective of challenges faced by fish in a contaminated environment.

Solea senegalensis vasa transcripts: molecular characterisation, tissue distribution and developmental expression profiles.

The Vasa protein is an RNA helicase belonging the DEAD (Asp-Glu-Ala-Asp)-box family. The crucial role played by the vasa gene in the germ-cell lineage of both vertebrates and invertebrates has made this gene a useful molecular marker for germinal cells and a useful tool in surrogate broodstock production using primordial germ cell transplantation. With the aim of establishing a novel approach to improving Solea senegalensis broodstock management, the vasa gene in this species was characterised. Four S. senegalensis vasa transcripts were isolated: Ssvasa1, Ssvasa2, Ssvasa3 and Ssvasa4. Their phylogenetic relationship with other vasa homologues was determined confirming the high degree of conservation of this helicase throughout evolution. Our qPCR results showed that S. senegalensis vasa transcripts are prevalently expressed in gonads, with ovary-specific expression for Ssvasa3 and Ssvasa4. During embryonic and larval development, a switch between the longest and the shortest transcripts was observed. While Ssvasa1 and Ssvasa2 were maternally supplied, Ssvasa3 and Ssvasa4 depended on the de novo expression program of the growing juveniles, suggesting that vasa mRNA could be involved in Senegalese sole gonad differentiation. In situ hybridisation and immunohistochemical analysis performed in 150-days after hatching (DAH) larvae showed vasa product expression in the germinal region of early gonads. In our work we demonstrated the usefulness of Ssvasa mRNAs as molecular markers for primordial germ cells and germinal cells during embryonic development, larval ontogenesis and gonad differentiation. Furthermore, our results confirmed the potential of vasa to help investigate germinal cell biotechnology for Senegalese sole reproduction.

Effects of dietary arachidonic acid on cortisol production and gene expression in stress response in Senegalese sole (Solea senegalensis) post-larvae.

Dietary fatty acids, particularly arachidonic acid (ARA), affect cortisol and may influence the expression of genes involved in stress response in fish. The involvement of ARA on stress, lipid, and eicosanoid metabolism genes, in Senegalese sole, was tested. Post-larvae were fed Artemia presenting graded ARA levels (0.1, 0.4, 0.8, 1.7, and 2.3%, dry matter basis), from 22 to 35 days after hatch. Whole-body cortisol levels were determined, before and 3 h after a 2 min air exposure, as well as the expression of phospholipase A2 (PLA 2 ), cyclooxygenase-2 (COX-2), steroidogenic acute regulatory protein (StAR), glucocorticoid receptors (GRs), phosphoenolpyruvate carboxykinase (PEPCK), and peroxisome proliferator-activated receptor alpha (PPARα). Relative growth rate (6.0-7.8% day(-1)) and survival at the end of the experiment (91-96%) and after stress (100%) were unaffected. Fish reflected dietary ARA content and post-stress cortisol increased with ARA supply up to 1.7%, whereas 2.3% ARA seemed to enhance basal cortisol slightly and alter the response to stress. Results suggested that elevating StAR transcription might not be necessary for a short-term response to acute stress. Basal cortisol and PLA 2 expression were strongly correlated, indicating a potential role for this enzyme in steroidogenesis. Under basal conditions, larval ARA was associated with GR1 expression, whereas the glucocorticoid responsive gene PEPCK was strongly related with cortisol but not GR1 mRNA levels, suggesting the latter might not reflect the amount of GR1 protein in sole. Furthermore, a possible role for PPARα in the expression of PEPCK following acute stress is proposed.

Postprandial pattern of digestive enzymes and protein turnover in meagre (Argyrosomus regius) juveniles.

After a meal, a sequence of physiological changes occurs in animals in response to digestion, absorption and assimilation of the ingested nutrients. These processes are very important for the aquaculture sector since they will define the efficiency by which food is converted into growth, thus contributing to reduce the production costs and also undigested food in the effluents. Here we investigated the activity of digestive enzymes in the midgut and the protein degradation systems following a single meal to define postprandial patterns of action in hepatic and muscle tissues of meagre juveniles. Fish were fed with a single meal followed by a period of 24 h without feed. The activity of several digestive enzymes (α-amylase, trypsin, aminopeptidase, alkaline and acid phosphatases) plus the analysis of key players of the ubiquitin-proteasome (UPS) and autophagy-lysosomal (ALS) systems were examined just before feeding (0 h, basal point) and 2, 4, 6, 8 and 24 h after food ingestion. Digestion was activated around 4 h after food ingestion and nutrients available for protein degradation 2 h later. This work provided information about the short-term physiological effects induced by a single meal to support scientists' decision when planning a specific study involving digestion and protein degradation, and also to fish farmers on how to better manage feeding protocols when producing A. regius juveniles. Our results suggested that meagre juveniles, under the experimental conditions used, can be fed every 4 h, time when digestion starts, however further studies should be addressed to find the optimal feeding regime for this juvenile species.