Evaluation of the Inclusion of the Green Seaweed Ulva ohnoi as an Ingredient in Feeds for Gilthead Sea Bream (Sparus aurata) and European Sea Bass (Dicentrarchus labrax).

This study evaluated the use of Ulva ohnoi as an ingredient in feeds for aquaculture in three different experiments. Experiment 1 was oriented to confirm the negative effect of U. ohnoi on fish digestion. Experiment 2 assessed the effect on growth, feed efficiency, and immune status of juvenile sea bass (Dicentrarchus labrax) fed on diets including U. ohnoi, previously treated or not with carbohydrases used to partially hydrolyze indigestible polysaccharides. Experiment 3 was aimed to evaluate the potential protective effect of U. ohnoi on the oxidative status of sea bream (Sparus aurata) challenged by the consumption of a feed formulated with the oil fraction completely oxidized. Results show a negligible effect of U. ohnoi meal on protein digestion when included in feeds at levels of 10% or less. Moreover, results of growth and feed use evidenced the possibility of using up to 5% inclusion of algal meal in feeds without adverse effects on the zootechnical parameters, while the enzyme pretreatment was ineffective to improve its nutritional use. Finally, the inclusion of U. onhoi in feeds determined both an immunostimulatory effect, evidenced by an increase in skin mucus lysozyme in the two mentioned fish species, and a positive influence on the oxidative metabolism of seabream when fed on a diet including rancid oil.

Targeting the Mild-Hypoxia Driving Force for Metabolic and Muscle Transcriptional Reprogramming of Gilthead Sea Bream (Sparus aurata) Juveniles.

On-growing juveniles of gilthead sea bream were acclimated for 45 days to mild-hypoxia (M-HYP, 40-60% O2 saturation), whereas normoxic fish (85-90% O2 saturation) constituted two different groups, depending on if they were fed to visual satiety (control fish) or pair-fed to M-HYP fish. Following the hypoxia conditioning period, all fish were maintained in normoxia and continued to be fed until visual satiation for 3 weeks. The time course of hypoxia-induced changes was assessed by changes in blood metabolic landmarks and muscle transcriptomics before and after exhaustive exercise in a swim tunnel respirometer. In M-HYP fish, our results highlighted a higher contribution of aerobic metabolism to whole energy supply, shifting towards a higher anaerobic fitness following normoxia restoration. Despite these changes in substrate preference, M-HYP fish shared a persistent improvement in swimming performance with a higher critical speed at exercise exhaustion. The machinery of muscle contraction and protein synthesis and breakdown was also largely altered by mild-hypoxia conditioning, contributing this metabolic re-adjustment to the positive regulation of locomotion and to the catch-up growth response during the normoxia recovery period. Altogether, these results reinforce the presence of large phenotypic plasticity in gilthead sea bream, and highlights mild-hypoxia as a promising prophylactic measure to prepare these fish for predictable stressful events.

Dietary tryptophan supplementation induces a transient immune enhancement of gilthead seabream (Sparus aurata) juveniles fed fishmeal-free diets.

European 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 ±â€¯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 (R2) and predicted (Q2) with four components up to 93% and 78% of total variance, respectively. Component 1 (R2 = 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.

Metabolic and Stress Responses in Senegalese Soles (Solea senegalensis Kaup) Fed Tryptophan Supplements: Effects of Concentration and Feeding Period.

: The objective of this study was to assess the impact of different dietary Trp concentrations on the stress and metabolism response of juvenile Senegalese soles (Solea senegalensis). Fish (38.1 ± 1.9 g) were fed different Trp-enriched feeds (0%, 1% and 2% Trp added) for two and eight days, and later exposed to air stress for three min. Samples were taken pre- and 1 h post-stress (condition). Plasma cortisol, lactate, glucose and proteins were significantly affected by the sampling time, showing higher values at 1 h post-stress. Trp concentration in food also had significant effects on lactate and glucose levels. However, the feeding period did not affect these parameters. Post-stress values were higher than in the pre-stress condition for every plasma parameter, except for lactate in two days and 1% Trp treatment. Nevertheless, cortisol, glucose and lactate did not vary significantly between pre- and post-stress samplings in fish fed the 1% Trp-enriched diet for two days. The lack of variability in cortisol response was also due to the high pre-stress value, significantly superior to pre-stress control. The exposure time to Trp feeding did not significantly affect any enzyme activity; however, Trp added and condition influenced protein-related enzyme activities. In spite of decreasing stress markers, Trp-enriched diets altered the protein metabolism.

Dietary Tryptophan Induces Opposite Health-Related Responses in the Senegalese Sole (Solea senegalensis) Reared at Low or High Stocking Densities With Implications in Disease Resistance.

High rearing densities are typical conditions of both inland and onshore intensive aquaculture units. Despite obvious drawbacks, this strategy is nonetheless used to increase production profits. Such conditions inflict stress on fish, reducing their ability to cope with disease, bringing producers to adopt therapeutic strategies. In an attempt to overcome deleterious effects of chronic stress, Senegalese sole, Solea senegalensis, held at low (LD) or high density (HD) were fed tryptophan-supplemented diets with final tryptophan content at two (TRP2) or four times (TRP4) the requirement level, as well as a control and non-supplemented diet (CTRL) for 38 days. Fish were sampled at the end of the feeding trial for evaluation of their immune status, and mortalities were recorded following intra-peritoneal infection with Photobacterium damselae subsp. piscicida. Blood was collected for analysis of the hematological profile and innate immune parameters in plasma. Pituitary and hypothalamus were sampled for the assessment of neuro-endocrine-related gene expression. During the feeding trial, fish fed TRP4 and held at LD conditions presented higher mortalities, whereas fish kept at HD seemed to benefit from this dietary treatment, as disease resistance increased over that of CTRL-fed fish. In accordance, cortisol level tended to be higher in fish fed both supplemented diets at LD compared to fish fed CTRL, but was lower in fish fed TRP4 than in those fed TRP2 under HD condition. Together with lower mRNA levels of proopiomelanocortin observed with both supplementation levels, these results suggest that higher levels of tryptophan might counteract stress-induced cortisol production, thereby rendering fish better prepared to cope with disease. Data regarding sole immune status showed no clear effects of tryptophan on leucocyte numbers, but TRP4-fed fish displayed inhibited alternative complement activity (ACH50) when held at LD, as opposed to their HD counterparts whose ACH50 was higher than that of CTRL-fed fish. In conclusion, while dietary tryptophan supplementation might have harmful effects in control fish, it might prove to be a promising strategy to overcome chronic stress-induced disease susceptibility in farmed Senegalese sole.

Impact of Air Exposure on Vasotocinergic and Isotocinergic Systems in Gilthead Sea Bream (Sparus aurata): New Insights on Fish Stress Response.

The hypothalamus-pituitary-interrenal (HPI) and hypothalamus-sympathetic-chromaffin cell (HSC) axes are involved in the regulation of the stress response in teleost. In this regard, the activation of a complex network of endocrine players is needed, including corticotrophin-releasing hormone (Crh), Crh binding protein (Crhbp), proopiomelanocortin (Pomc), thyrotropin-releasing hormone (Trh), arginine vasotocin (Avt), and isotocin (It) to finally produce pleiotropic functions. We aimed to investigate, using the gilthead sea bream (Sparus aurata) as a biological model, the transcriptomic response of different endocrine factors (crh, crhbp, pomcs, trh), neuropeptides (avt and it), and their specific receptors (avtrv1a, avtrv2, and itr) in four important target tissues (hypothalamus, pituitary, kidney and liver), after an acute stress situation. We also investigated several stress hormones (catecholamines and cortisol). The stress condition was induced by air exposure for 3 min, and hormonal, metabolic and transcriptomic parameters were analyzed in a time course response (15 and 30 min, and 1, 2, 4, and 8 h post-stress) in a total of 64 fish (n = 8 fish per experimental group; p = 0.05; statistical power = 95%). Our results showed that plasma noradrenaline, adrenaline and cortisol values increased few minutes after stress exposure. At hypothalamic and hypophyseal levels, acute stress affected mRNA expression of all measured precursors and hormonal factors, as well as their receptors (avtrs and itr), showing the activation, at central level, of HPI, HSC, and Avt/It axes in the acute stress response. In addition, stress response also affected mRNA levels of avtrs and itr in the head kidney, as well as the steroidogenic acute regulatory protein (star) and tyrosine hydroxylase (th) expression, suggesting their participation in the HPI and HSC axes activation. Moreover, the pattern of changes in hepatic avtrs and itr gene expression also highlights an important role of vasotocinergic and isotocinergic pathways in liver metabolic organization after acute stress events. Our results demonstrate, both at transcriptional and circulating levels of several hormones, the existence of a complex activation of different endocrine pathways in S. aurata related to the stress pathways, where vasotocinergic and isotocinergic systems can also be considered key players of the acute stress response orchestration.

Myrcia sylvatica essential oil mitigates molecular, biochemical and physiological alterations in Rhamdia quelen under different stress events associated to transport.

The effects of pre-transport handling and addition of essential oil of Myrcia sylvatica (EOMS) during transport on stress pathways activation in Rhamdia quelen were investigated. Fish (n=400, 25.2±2.9g) were captured in production ponds and transferred to 100-L tank (density 100g L-1). After 24h, 10 fish were sampled (before transport group). The remaining fish were placed in plastic bags (n=30 or 32 fish per bag, density 150g L-1) containing 5L of water (control), ethanol (315µLL-1, vehicle) or EOMS (25 or 35µLL-1), in triplicate, transported for 6h and sampled (n=10 animals per group). Indicators of stress and metabolism, as well as mRNA expression of brain hormones were evaluated. Previously, full-length cDNAs, encoding specific corticotropin-releasing hormone (crh) and proopiomelanocortins (pomca and pomcb), were cloned from whole brain of R. quelen. Crh expression increased after 24h of capture and handling, whereas cortisol and glucose plasmatics enhanced their values in the control group. Transport with EOMS reduced plasma cortisol and lactate levels, while ethanol and EOMS groups increased Na+/K+-ATPase gill activity compared to control. Gene expression of crh, pomcb, prolactin and somatolactin mRNAs were lower after transport with EOMS compared to control. EOMS was able to mitigate the stress pathways activation caused by transport, maintaining a balance in body homeostasis. Thus, EOMS is recommended as sedative in procedures as transport and the pre-transport handling requires greater attention and use of tranquilizers.

Citrate gold nanoparticle exposure in the marine bivalve Ruditapes philippinarum: uptake, elimination and oxidative stress response.

Gold nanoparticles (AuNPs) are considered an important nano-sized component of the twenty-first century. Due to their unique physical and chemical properties, they are being used and developed for a wide range of promising applications in medicine, biology and chemistry. Notwithstanding their useful aspects, in recent years concern has been raised over their ability to enter cells, organelles and nuclei and provoke oxidative stress. In a laboratory-based experiment, the non-target marine bivalve Ruditapes philippinarum was used as a model organism. Uptake, elimination and molecular effects under short-term and sub-chronic exposure conditions to an environmental relevant concentration (0.75 µg L(-1)) of weakly agglomerating citrate AuNPs (∼20 nm) were studied. Our results demonstrate that at the tested concentration, the particles are readily taken up into the digestive gland > gills and can produce significant changes (p < 0.05) in oxidative stress and inflammatory response markers, as measured by phase II antioxidant enzymes and q-PCR gene expression analysis. However, the overall magnitude of responses was low, and oxidative damage was not provoked. Further, a significant elimination of Au from the digestive tract within a 7-day purification period was observed, with excretion being an important pathway. In conclusion, short-term and sub-chronic exposure to an environmental relevant concentration of citrate-stabilized AuNPs cannot be considered toxic to our model organism, while some further consideration should be given to chronic exposure effects.

Cortisol modulates vasotocinergic and isotocinergic pathways in the gilthead sea bream.

In the present study, we assessed the responses of the vasotocinergic and isotocinergic systems to chronic stress induced by cortisol administration in the gilthead sea bream (Sparus aurata). Pituitary and plasma arginine vasotocin (AVT) and isotocin (IT) levels, as well as hypothalamic pro-vasotocin (pro-VT) and pro-isotocin (pro-IT) mRNA expression levels, were analysed. In addition, the mRNA levels of three receptors, AVTR type V1a2, AVTR type V2 and ITR, were analysed in several target organs associated with the following physiological processes: (i) integration and control (hypothalamus), (ii) metabolism and its control (liver and hypothalamus), (iii) osmoregulation (gills) and (iv) stress response (head kidney). Specimens were injected intraperitoneally with slow-release implants (5 µL g(-1) body mass) containing coconut oil alone (control group) or with cortisol (50 µg g(-1) body mass; cortisol group). Both AVT and IT synthesis and release were correlated with plasma cortisol values, suggesting a potential interaction between both hormonal systems and cortisol administration. Our results suggest that the activation of hepatic metabolism as well as the hypothalamic control of metabolic processes provide the energy necessary to overcome stress, which could be partly mediated by AVTRs and ITR. Upregulation of branchial AVT and IT receptor expression following cortisol treatment suggests an involvement of the vasotocinergic and isotocinergic systems in the regulation of ion channels/transporters during stressful situations. Finally, changes in AVT and IT receptor mRNA expression in the head kidney suggest these nonapeptides participate in feedback mechanisms that regulate the synthesis/release of cortisol. Our results indicate a relationship between cortisol and both the vasotocinergic and isotocinergic systems during simulated chronic stress in S. aurata.

A holistic view of dietary carbohydrate utilization in lobster: digestion, postprandial nutrient flux, and metabolism.

Crustaceans exhibit a remarkable variation in their feeding habits and food type, but most knowledge on carbohydrate digestion and utilization in this group has come from research on few species. The aim of this study was to make an integrative analysis of dietary carbohydrate utilization in the spiny lobster Panulirus argus. We used complementary methodologies such as different assessments of digestibility, activity measurements of digestive and metabolic enzymes, and post-feeding flux of nutrients and metabolites. Several carbohydrates were well digested by the lobster, but maize starch was less digestible than all other starches studied, and its inclusion in diet affected protein digestibility. Most intense hydrolysis of carbohydrates in the gastric chamber of lobster occurred between 2-6 h after ingestion and afterwards free glucose increased in hemolymph. The inclusion of wheat in diet produced a slow clearance of glucose from the gastric fluid and a gradual increase in hemolymph glucose. More intense hydrolysis of protein in the gastric chamber occurred 6-12 h after ingestion and then amino acids tended to increase in hemolymph. Triglyceride concentration in hemolymph rose earlier in wheat-fed lobsters than in lobsters fed other carbohydrates, but it decreased the most 24 h later. Analyses of metabolite levels and activities of different metabolic enzymes revealed that intermolt lobsters had a low capacity to store and use glycogen, although it was slightly higher in wheat-fed lobsters. Lobsters fed maize and rice diets increased amino acid catabolism, while wheat-fed lobsters exhibited higher utilization of fatty acids. Multivariate analysis confirmed that the type of carbohydrate ingested had a profound effect on overall metabolism. Although we found no evidence of a protein-sparing effect of dietary carbohydrate, differences in the kinetics of their digestion and absorption impacted lobster metabolism determining the fate of other nutrients.

Different stressors induce differential responses of the CRH-stress system in the gilthead sea bream (Sparus aurata).

The hypothalamus-pituitary-interrenal (HPI) axis, involved in the regulation of the neuroendocrine stress responses, presents important players such as corticotropin-releasing hormone (CRH, generally considered as the initiator of this pathway) and CRH-binding protein (CRH-BP, considered as an antagonist of CRH function). CRH and CRH-BP full-length cDNA sequences were obtained from Sparus aurata by screening a brain cDNA library, and their phylogenetic analysis as well as their roles during acute and chronic stress responses were assessed. mRNA expression levels and plasma cortisol concentrations were measured by RT qPCR and ELISA, respectively, in S. aurata juveniles submitted to: i) different environmental salinities in a short-time course response; and ii) food deprivation during 21 days. In addition, osmoregulatory and metabolic parameters in plasma corroborated a clear reorganization depending on the stress source/period. Salinity transfer induced stress as indicated by enhanced plasma cortisol levels, as well as by up-regulated CRH and down-regulated CRH-BP expression values. On the other hand, food deprivation did not affect both expression levels, although plasma cortisol concentrations were enhanced. These results suggest that different stressors are handled through different stress pathways in S. aurata.