Understanding how high stocking densities and concurrent limited oxygen availability drive social cohesion and adaptive features in regulatory growth, antioxidant defense and lipid metabolism in farmed gilthead sea bream (Sparus aurata).

The study combined the use of biometric, behavioral, physiological and external tissue damage scoring systems to better understand how high stocking densities drive schooling behavior and other adaptive features during the finishing growing phase of farmed gilthead sea bream in the Western Mediterranean. Fish were grown at three different final stocking densities (LD, 8.5 kg/m3; MD, 17 kg/m3; HD, 25 kg/m3). Water oxygen concentration varied between 5 and 6 ppm in LD fish to 3-4 ppm in HD fish with the summer rise of water temperature from 19°C to 26°C (May-July). HD fish showed a reduction of feed intake and growth rates, but they also showed a reinforced social cohesion with a well-defined endogenous swimming activity rhythm with feeding time as a main synchronization factor. The monitored decrease of the breathing/swimming activity ratio by means of the AEFishBIT data-logger also indicated a decreased energy partitioning for growth in the HD environment with a limited oxygen availability. Plasma glucose and cortisol levels increased with the rise of stocking density, and the close association of glycaemia with the expression level of antioxidant enzymes (mn-sod, gpx4, prdx5) in liver and molecular chaperones (grp170, grp75) in skeletal muscle highlighted the involvement of glucose in redox processes via rerouting in the pentose-phosphate-pathway. Other adaptive features included the depletion of oxidative metabolism that favored lipid storage rather than fatty acid oxidation to decrease the oxygen demand as last electron acceptor in the mitochondrial respiratory chain. This was coincident with the metabolic readjustment of the Gh/Igf endocrine-growth cascade that promoted the regulation of muscle growth at the local level rather than a systemic action via the liver Gh/Igf axis. Moreover, correlation analyses within HD fish displayed negative correlations of hepatic transcripts of igf1 and igf2 with the data-logger measurements of activity and respiration, whereas the opposite was found for muscle igf2, ghr1 and ghr2. This was indicative of a growth-regulatory transition that supported a proactive instead of a reactive behavior in HD fish, which was considered adaptive to preserve an active and synchronized feeding behavior with a minimized risk of oxidative stress and epidermal skin damage.

The potential of a combination of pungent spices as a novel supplement in gilthead seabream (Sparus aurata) diets to aid in the strategic use of fish oil in aquafeeds: a holistic perspective.

This work studied the potential of a combination of pungent spices (capsicum, black pepper, ginger, and cinnamaldehyde) to be used as a supplement in diets of gilthead seabream (Sparus aurata; 44.1 ± 4.2 g). During 90 days, fish were fed three experimental diets with low inclusion of fish oil and containing poultry fat as the main source of lipids, supplemented with graded levels of the tested supplement: 0 (control), 0.1 (SPICY0.1%), and 0.15% (SPICY0.15%). As a result, the pungent spices enhanced the growth performance, the activity of the bile-salt-activated lipase in the intestine, and decreased fat deposit levels within enterocytes. The SPICY0.1% diet reduced the feed conversion ratio and the perivisceral fat index and lipid deposits in the liver. Moreover, the ratio of docosahexaenoic acid/eicosapentaenoic acid in fillet increased in fish fed the SPICY0.1% diet, while the hepatic levels of docosahexaenoic acid and total n-3 polyunsaturated fatty acids increased in fish fed the SPICY0.15% diet. Furthermore, there was an effect on the expression of some biomarkers related to lipid metabolism in 2-h postprandial fish (fasn, elovl6, scd1b, cyp7a1, lpl, and pparß), and in 48 h fasted-fish fed with the SPICY0.1% diet, a regulation of the intestinal immune response was indicated. However, no significant differences were found in lipid apparent digestibility and proximate macronutrient composition. The spices did not affect biomarkers of hepatic or oxidative stress. No differences in microbial diversity were found, except for an increase in Simpson's Index in the posterior intestine of fish fed the SPICY0.1% diet, reflected in the increased relative abundance of the phylum Chloroflexi and lower relative abundances of the genera Campylobacter, Corynebacterium, and Peptoniphilus. In conclusion, the supplementation of gilthead seabream diets with pungent spices at an inclusion of 0.1% was beneficial to enhance growth performance and feed utilization; reduce fat accumulation in the visceral cavity, liver, and intestine; and improve the fish health status and condition. Results suggest that the tested supplement can be used as part of a nutritional strategy to promote a more judicious use of fish oil in fish diets due to its decreasing availability and rising costs.

Cysteamine improves growth and the GH/IGF axis in gilthead sea bream (Sparus aurata): in vivo and in vitro approaches.

Aquaculture is the fastest-growing food production sector and nowadays provides more food than extractive fishing. Studies focused on the understanding of how teleost growth is regulated are essential to improve fish production. Cysteamine (CSH) is a novel feed additive that can improve growth through the modulation of the GH/IGF axis; however, the underlying mechanisms and the interaction between tissues are not well understood. This study aimed to investigate the effects of CSH inclusion in diets at 1.65 g/kg of feed for 9 weeks and 1.65 g/kg or 3.3 g/kg for 9 weeks more, on growth performance and the GH/IGF-1 axis in plasma, liver, stomach, and white muscle in gilthead sea bream (Sparus aurata) fingerlings (1.8 ± 0.03 g) and juveniles (14.46 ± 0.68 g). Additionally, the effects of CSH stimulation in primary cultured muscle cells for 4 days on cell viability and GH/IGF axis relative gene expression were evaluated. Results showed that CSH-1.65 improved growth performance by 16% and 26.7% after 9 and 18 weeks, respectively, while CSH-3.3 improved 32.3% after 18 weeks compared to control diet (0 g/kg). However, no significant differences were found between both experimental doses. CSH reduced the plasma levels of GH after 18 weeks and increased the IGF-1 ones after 9 and 18 weeks. Gene expression analysis revealed a significant upregulation of the ghr-1, different igf-1 splice variants, igf-2 and the downregulation of the igf-1ra and b, depending on the tissue and dose. Myocytes stimulated with 200 µM of CSH showed higher cell viability and mRNA levels of ghr1, igf-1b, igf-2 and igf-1rb compared to control (0 µM) in a similar way to white muscle. Overall, CSH improves growth and modulates the GH/IGF-1 axis in vivo and in vitro toward an anabolic status through different synergic ways, revealing CSH as a feasible candidate to be included in fish feed.

Modulation of gut microbiota and intestinal immune response in gilthead seabream (Sparus aurata) by dietary bile salt supplementation.

Given their role in lipid digestion, feed supplementation with bile salts could be an economic and sustainable solution to alterations in adiposity and intestinal inflammation generated by some strategies currently used in aquaculture. An important part of the metabolism of bile salts takes place in the intestine, where the microbiota transforms them into more toxic forms. Consequently, we aimed to evaluate the gut immune response and microbial populations in gilthead seabream (Sparus aurata) fed a diet supplemented with a blend of bile salts with proven background as a regulator of lipid metabolism and fat content. After the 90-day feeding trial, a differential modulation of the microbiota between the anterior and posterior intestine was observed. While in the anterior intestine the relative abundance of Desulfobacterota doubled, in the posterior intestine, the levels of Firmicutes increased and Proteobacteria, Actinobacteriota, and Campylobacterota were reduced when supplementing the diet with bile salts. Even so, only in the anterior intestine, there was a decrease in estimated richness (Chao1 and ACE indices) in presence of dietary bile salts. No significant differences were displayed in alpha (Shannon and Simpson indices) nor beta-diversity, showing that bile sales did not have a great impact on the intestinal microbiota. Regarding the gene expression profile in 2 h postprandial-fish, several changes were observed in the analyzed biomarkers of epithelial integrity, nutrient transport, mucus production, interleukins, cell markers, immunoglobulin production and pathogen recognition receptors. These results may indicate the development of an intestinal immune-protective status to tackle future threats. This work also suggests that this immune response is not only regulated by the presence of the dietary bile salts in the intestine, but also by the microbial populations that are in turn modulated by bile salts. After a fasting period of 2 days, the overall gene expression profile was stabilized with respect to fish fed the unsupplemented diet, indicating that the effect of bile salts was transient after short periods of fasting. On the balance, bile salts can be used as a dietary supplement to enhance S. aurata farming and production without compromising their intestinal health.

Client Applications and Server-Side Docker for Management of RNASeq and/or VariantSeq Workflows and Pipelines of the GPRO Suite.

The GPRO suite is an in-progress bioinformatic project for -omics data analysis. As part of the continued growth of this project, we introduce a client- and server-side solution for comparative transcriptomics and analysis of variants. The client-side consists of two Java applications called "RNASeq" and "VariantSeq" to manage pipelines and workflows based on the most common command line interface tools for RNA-seq and Variant-seq analysis, respectively. As such, "RNASeq" and "VariantSeq" are coupled with a Linux server infrastructure (named GPRO Server-Side) that hosts all dependencies of each application (scripts, databases, and command line interface software). Implementation of the Server-Side requires a Linux operating system, PHP, SQL, Python, bash scripting, and third-party software. The GPRO Server-Side can be installed, via a Docker container, in the user's PC under any operating system or on remote servers, as a cloud solution. "RNASeq" and "VariantSeq" are both available as desktop (RCP compilation) and web (RAP compilation) applications. Each application has two execution modes: a step-by-step mode enables each step of the workflow to be executed independently, and a pipeline mode allows all steps to be run sequentially. "RNASeq" and "VariantSeq" also feature an experimental, online support system called GENIE that consists of a virtual (chatbot) assistant and a pipeline jobs panel coupled with an expert system. The chatbot can troubleshoot issues with the usage of each tool, the pipeline jobs panel provides information about the status of each computational job executed in the GPRO Server-Side, while the expert system provides the user with a potential recommendation to identify or fix failed analyses. Our solution is a ready-to-use topic specific platform that combines the user-friendliness, robustness, and security of desktop software, with the efficiency of cloud/web applications to manage pipelines and workflows based on command line interface software.

Genetics and Nutrition Drive the Gut Microbiota Succession and Host-Transcriptome Interactions through the Gilthead Sea Bream (Sparus aurata) Production Cycle.

Fish genetically selected for growth (GS) and reference (REF) fish were fed with CTRL (15% FM, 5-7% FO) or FUTURE (7.5% FM, 10% poultry meal, 2.2% poultry oil + 2.5% DHA-algae oil) diets during a 12-months production cycle. Samples from initial (t0; November 2019), intermediate (t1; July 2020) and final (t2; November 2020) sampling points were used for Illumina 16S rRNA gene amplicon sequencing of the adherent microbiota of anterior intestine (AI). Samples from the same individuals (t1) were also used for the gene expression profiling of AI by RNA-seq, and subsequent correlation analyses with microbiota abundances. Discriminant analyses indicated the gut bacterial succession along the production cycle with the proliferation of some valuable taxa for facing seasonality and different developmental stages. An effect of genetic background was evidenced along time, decreasing through the progression of the trial, namely the gut microbiota of GS fish was less influenced by changes in diet composition. At the same time, these fish showed wider transcriptomic landmarks in the AI to cope with these changes. Our results highlighted an enhanced intestinal sphingolipid and phospholipid metabolism, epithelial turnover and intestinal motility in GS fish, which would favour their improved performance despite the lack of association with changes in gut microbiota composition. Furthermore, in GS fish, correlation analyses supported the involvement of different taxa with the down-regulated expression of pro-inflammatory markers and the boosting of markers of extracellular remodelling and response to bacterium. Altogether, these findings support the combined action of the gut microbiome and host transcriptionally mediated effects to preserve and improve gut health and function in a scenario of different growth performance and potentiality.

Diet and Host Genetics Drive the Bacterial and Fungal Intestinal Metatranscriptome of Gilthead Sea Bream.

The gut microbiota is now recognised as a key target for improving aquaculture profit and sustainability, but we still lack insights into the activity of microbes in fish mucosal surfaces. In the present study, a metatranscriptomic approach was used to reveal the expression of gut microbial genes in the farmed gilthead sea bream. Archaeal and viral transcripts were a minority but, interestingly and contrary to rRNA amplicon-based studies, fungal transcripts were as abundant as bacterial ones, and increased in fish fed a plant-enriched diet. This dietary intervention also drove a differential metatranscriptome in fish selected for fast and slow growth. Such differential response reinforced the results of previously inferred metabolic pathways, enlarging, at the same time, the catalogue of microbial functions in the intestine. Accordingly, vitamin and amino acid metabolism, and rhythmic and symbiotic processes were mostly shaped by bacteria, whereas fungi were more specifically configuring the host immune, digestive, or endocrine processes.

Use of male-to-female sex reversal as a welfare scoring system in the protandrous farmed gilthead sea bream (Sparus aurata).

Gilthead sea bream is a highly cultured marine fish throughout the Mediterranean area, but new and strict criteria of welfare are needed to assure that the intensification of production has no negative effects on animal farming. Most welfare indicators are specific to a given phase of the production cycle, but others such as the timing of puberty and/or sex reversal are of retrospective value. This is of particular relevance in the protandrous gilthead sea bream, in which the sex ratio is highly regulated at the nutritional level. Social and environmental factors (e.g., contaminant loads) also alter the sex ratio, but the contribution of the genetic component remains unclear. To assess this complex issue, five gilthead sea bream families representative of slow/intermediate/fast growth were grown out with control or a plant-based diet in a common garden system from early life to the completion of their sexual maturity in 3-year-old fish. The plant-based diet highly enhanced the male-to-female sex reversal. This occurred in parallel with the progressive impairment of growth performance, which was indicative of changes in nutrient requirements as the result of the different energy demands for growth and reproduction through development. The effect of a different nutritional and genetic background on the reproductive performance was also assessed by measurements of circulating levels of sex steroids during the two consecutive spawning seasons, varying plasma levels of 17ß-estradiol (E2) and 11-ketotestosterone (11-KT) with age, gender, diet, and genetic background. Principal component analysis (PCA) of 3-year-old fish displayed a gradual increase of the E2/11-KT ratio from males to females with the improvement of nutritional/genetic background. Altogether, these results support the use of a reproductive tract scoring system for leading farmed fish toward their optimum welfare condition, contributing to improving the productivity of the current gilthead sea bream livestock.

Immune Status and Hepatic Antioxidant Capacity of Gilthead Seabream Sparus aurata Juveniles Fed Yeast and Microalga Derived ß-glucans.

This work aimed to evaluate the effects of dietary supplementation with ß-glucans extracted from yeast (Saccharomyces cerevisiae) and microalga (Phaeodactylum tricornutum) on gene expression, oxidative stress biomarkers and plasma immune parameters in gilthead seabream (Sparus aurata) juveniles. A practical commercial diet was used as the control (CTRL), and three others based on CTRL were further supplemented with different ß-glucan extracts. One was derived from S. cerevisiae (diet MG) and two different extracts of 21% and 37% P. tricornutum-derived ß-glucans (defined as Phaeo21 and Phaeo37), to give a final 0.06% ß-glucan dietary concentration. Quadruplicate groups of 95 gilthead seabream (initial body weight: 4.1 ± 0.1 g) were fed to satiation three times a day for 8 weeks in a pulse-feeding regimen, with experimental diets intercalated with the CTRL dietary treatment every 2 weeks. After 8 weeks of feeding, all groups showed equal growth performance and no changes were found in plasma innate immune status. Nonetheless, fish groups fed ß-glucans supplemented diets showed an improved anti-oxidant status compared to those fed CTRL at both sampling points (i.e., 2 and 8 weeks). The intestinal gene expression analysis highlighted the immunomodulatory role of Phaeo37 diet after 8 weeks, inducing an immune tolerance effect in gilthead seabream intestine, and a general down-regulation of immune-related gene expression. In conclusion, the results suggest that the dietary pulse administration of a P. tricornutum 37% enriched-ß-glucans extract might be used as a counter-measure in a context of gut inflammation, due to its immune-tolerant and anti-oxidative effects.

Cross-Talk Between Intestinal Microbiota and Host Gene Expression in Gilthead Sea Bream (Sparus aurata) Juveniles: Insights in Fish Feeds for Increased Circularity and Resource Utilization.

New types of fish feed based on processed animal proteins (PAPs), insect meal, yeast, and microbial biomasses have been used with success in gilthead sea bream. However, some drawback effects on feed conversion and inflammatory systemic markers were reported in different degrees with PAP- and non-PAP-based feed formulations. Here, we focused on the effects of control and two experimental diets on gut mucosal-adherent microbiota, and how it correlated with host transcriptomics at the local (intestine) and systemic (liver and head kidney) levels. The use of tissue-specific PCR-arrays of 93 genes in total rendered 13, 12, and 9 differentially expressed (DE) genes in the intestine, liver, and head kidney, respectively. Illumina sequencing of gut microbiota yielded a mean of 125,350 reads per sample, assigned to 1,281 operational taxonomic unit (OTUs). Bacterial richness and alpha diversity were lower in fish fed with the PAP diet, and discriminant analysis displayed 135 OTUs driving the separation between groups with 43 taxa correlating with 27 DE genes. The highest expression of intestinal pcna and alpi was achieved in PAP fish with intermediate values in non-PAP, being the pro-inflammatory action of alpi associated with the presence of Psychrobacter piscatorii. The intestinal muc13 gene was down-regulated in non-PAP fish, with this gene being negatively correlated with anaerobic (Chloroflexi and Anoxybacillus) and metal-reducing (Pelosinus and Psychrosinus) bacteria. Other inflammatory markers (igm, il8, tnfα) were up-regulated in PAP fish, positively correlating the intestinal igm gene with the inflammasome activator Escherichia/Shigella, whereas the systemic expression of il8 and tnfα was negatively correlated with the Bacilli class in PAP fish and positively correlated with Paracoccus yeei in non-PAP fish. Overall changes in the expression pattern of il10, galectins (lgals1, lgals8), and toll-like receptors (tlr2, tlr5, tlr9) reinforced the anti-inflammatory profile of fish fed with the non-PAP diet, with these gene markers being associated with a wide range of OTUs. A gut microbiota-liver axis was also established, linking the microbial generation of short chain fatty acids with the fueling of scd1- and elovl6-mediated lipogenesis. In summary, by correlating the microbiome with host gene expression, we offer new insights in the evaluation of fish diets promoting gut and metabolism homeostasis, and ultimately, the health of farmed fish.

Diet and Exercise Modulate GH-IGFs Axis, Proteolytic Markers and Myogenic Regulatory Factors in Juveniles of Gilthead Sea Bream (Sparus aurata).

The physiological and endocrine benefits of sustained exercise in fish were largely demonstrated, and this work examines how the swimming activity can modify the effects of two diets (high-protein, HP: 54% proteins, 15% lipids; high-energy, HE: 50% proteins, 20% lipids) on different growth performance markers in gilthead sea bream juveniles. After 6 weeks of experimentation, fish under voluntary swimming and fed with HP showed significantly higher circulating growth hormone (GH) levels and plasma GH/insulin-like growth-1 (IGF-1) ratio than fish fed with HE, but under exercise, differences disappeared. The transcriptional profile of the GH-IGFs axis molecules and myogenic regulatory factors in liver and muscle was barely affected by diet and swimming conditions. Under voluntary swimming, fish fed with HE showed significantly increased mRNA levels of capn1, capn2, capn3, capns1a, n3, and ub, decreased gene and protein expression of Ctsl and Mafbx and lower muscle texture than fish fed with HP. When fish were exposed to sustained exercise, diet-induced differences in proteases' expression and muscle texture almost disappeared. Overall, these results suggest that exercise might be a useful tool to minimize nutrient imbalances and that proteolytic genes could be good markers of the culture conditions and dietary treatments in fish.

A Novel Miniaturized Biosensor for Monitoring Atlantic Salmon Swimming Activity and Respiratory Frequency.

The advanced development of sensor technologies has led to the emergence of fish biosensors that are currently used for research and commercial purposes. AEFishBIT is a miniaturized biosensor attached to fish operculum that measures physical activity and respiration frequencies. In this study, we determined the effect of the tagging method and evaluated the use of this biosensor to monitor post-smolt Atlantic salmon in a tank-based system. The use of piercing fish tag had a negative impact on the gills and operculum, unlike the identical protocols used in gilthead sea bream and European sea bass. In contrast, a surgical thread did not show any apparent tissue damage. Two data recording schedules were considered to monitor immediate early reaction to fish handling and light regime changes (records every 15 min over 2 days) or adaptation to new light conditions (records every 30 min over 4 days). Data showed stabilization of physical activity 8 h post-tagging, with different steady states for the activity/respiratory ratio after changes in light intensity that reflected a different time course adaptation to new light conditions. High correlations were observed between AEFishBIT and video recording data. These findings supported the use of AEFishBIT as a promising tool for smart sensing of Atlantic salmon.

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.

The Use of Defatted Tenebrio molitor Larvae Meal as a Main Protein Source Is Supported in European Sea Bass (Dicentrarchus labrax) by Data on Growth Performance, Lipid Metabolism, and Flesh Quality.

OBJECTIVE: This study aims to determine the maximal inclusion level of defatted (d-) Tenebrio molitor larvae meal (TM) able to replace dietary fishmeal (FM) without compromising growth performance, general metabolism, and flesh quality traits in European sea bass, and to evaluate the major underlying physiological mechanisms. MATERIALS AND METHODS: Fish (55 ± 2 g) were fed with diets containing increasing levels of dTM: 0, 40, 80 and 100% (CTRL, TM40, TM80, and TM100, respectively) to replace FM. After 10 weeks of feeding, the growth performance, nutrient and energy balance, intestinal integrity, plasma metabolites and the expression of genes related to growth and nutrient metabolism, in liver and muscle were determined. The fatty acids (FA) profile, textural properties and color were also evaluated in muscle. RESULTS: Protein and lipids digestibility remained unaltered up to 80% dTM inclusion. Growth performance parameters were similar among dietary treatments. The dTM inclusion increased the hepatosomatic index in fish fed TM100. Muscle eicosapentaenoic acid, docosahexaenoic acid and n-3 long-chain polyunsaturated FA levels were maintained up to 80% dTM inclusion, but total cholesterol and non-esterified FA increased with dietary dTM inclusion. In liver, the expression of elongation of very long-chain FA protein 6 (elovl6) and FA desaturase 2 (fads2) did not change in fish fed TM40 and TM80, but elovl6 decreased whilst fads2 increased in fish fed TM100 when compared to those fed CTRL. The expression of cholesterol 7 alpha-monooxygenase (cyp7a1) decreased with dietary dTM inclusion. In muscle, the expression of myoblast determination protein-2 (myod2) decreased in fish fed TM80 and TM100. CONCLUSION: It is feasible to substitute dietary FM by dTM up to 80% in European sea bass without detrimental effects on nutrient digestibility, growth performance and associated genetic pathways, whilst assuring fillet nutritional value for human consumption.

Dietary Histidine, Threonine, or Taurine Supplementation Affects Gilthead Seabream (Sparus aurata) Immune Status.

AAs have become interesting feed ingredients to be used in functional fish feeds as not only are they protein building blocks, but they also participate in several other key metabolic processes. In the present study, a comprehensive analysis of transcriptomics, hematology, and humoral immune parameters (plasma and skin mucus) were measured twice over the course of the feeding trial (four weeks). Plasma antiprotease activity increased in fish fed Thr compared to those fed the CTRL and Tau treatments, regardless of sampling time. The bactericidal activity in skin mucus decreased in fish fed Tau and His treatments compared to those fed the CTRL diet after two weeks. The membrane IgT (mIgT) was upregulated in fish fed Tau after four weeks, while C-type lectin domain family domain 10 member (clec10a) was downregulated in fish fed Thr after two weeks of feeding. By comparing the molecular signatures of head-kidney by means of a PLS-DA, it is possible to visualize that the main difference is between the two sampling points, regardless of diet. Altogether, these results suggest that dietary supplementation with these AAs at the tested levels causes mild immune-modulation effects in gilthead seabream, which should be further studied under disease challenge conditions.

Effects of genetics and early-life mild hypoxia on size variation in farmed gilthead sea bream (Sparus aurata).

The present study evaluated, in an 18-month gilthead sea bream trial, the time course effects of genetics on individual size variation and growth compensation processes in families selected by heritable growth in the PROGENSA® breeding program. Families categorized as fast, intermediate, and slow growing had different growth trajectories with a more continuous growth in fast growth families. This feature was coincident with a reduced size variation at the beginning of the trial that clustered together the half-sib families sharing the same father. Regression analysis evidenced that the magnitude of compensatory growth was proportional to the initial size variation with no rescaling of families at this stage. By contrast, the finishing growth depensation process can mask, at least partially, the previous size convergence. This reflects the different contribution across the production cycle of genetics in growth. How early-life experiences affect growth compensation at juvenile stages was also evaluated in a separate cohort, and intriguingly, a first mild-hypoxia pulse at 60-81 days post-hatching (dph) increased survival rates by 10%, preventing growth impairment when fish were exposed to a second hypoxia episode (112-127 dph). The early hypoxia experience did not have a negative impact on growth compensatory processes at juvenile stages. By contrast, a diminished capacity for growth compensation was found with repeated or late hypoxia experiences. All this reinforces the use of size variation as a main criterion for improving intensive fish farming and selective breeding.

Local DNA methylation helps to regulate muscle sirtuin 1 gene expression across seasons and advancing age in gilthead sea bream (Sparus aurata).

BACKGROUND: Sirtuins (SIRTs) are master regulators of metabolism, and their expression patterns in gilthead sea bream (GSB) reveal different tissue metabolic capabilities and changes in energy status. Since little is known about their transcriptional regulation, the aim of this work was to study for the first time in fish the effect of age and season on sirt gene expression, correlating expression patterns with local changes in DNA methylation in liver and white skeletal muscle (WSM). METHODS: Gene organization of the seven sirts was analyzed by BLAT searches in the IATS-CSIC genomic database (www.nutrigroup-iats.org/seabreamdb/). The presence of CpG islands (CGIs) was mapped by means of MethPrimer software. DNA methylation analyses were performed by bisulfite pyrosequencing. A PCR array was designed for the simultaneous gene expression profiling of sirts and related markers (cs, cpt1a, pgc1α, ucp1, and ucp3) in the liver and WSM of one- and three-year-old fish during winter and summer. RESULTS: The occurrence of CGIs was evidenced in the sirt1 and sirt3 promoters. This latter CGI remained hypomethylated regardless of tissue, age and season. Conversely, DNA methylation of sirt1 at certain CpG positions within the promoter varied with age and season in the WSM. Among them, changes at several SP1 binding sites were negatively correlated with the decrease in sirt1 expression in summer and in younger fish. Changes in sirt1 regulation match well with variations in feed intake and energy metabolism, as judged by the concurrent changes in the analyzed markers. This was supported by discriminant analyses, which identified sirt1 as a highly responsive element to age- and season-mediated changes in energy metabolism in WSM. CONCLUSIONS: The gene organization of SIRTs is highly conserved in vertebrates. GSB sirt family members have CGI- and non-CGI promoters, and the presence of CGIs at the sirt1 promoter agrees with its ubiquitous expression. Gene expression analyses support that sirts, especially sirt1, are reliable markers of age- and season-dependent changes in energy metabolism. Correlation analyses suggest the involvement of DNA methylation in the regulation of sirt1 expression, but the low methylation levels suggest the contribution of other putative mechanisms in the transcriptional regulation of sirt1.

Sex, Age, and Bacteria: How the Intestinal Microbiota Is Modulated in a Protandrous Hermaphrodite Fish.

Intestinal microbiota is key for many host functions, such as digestion, nutrient metabolism, disease resistance, and immune function. With the growth of the aquaculture industry, there has been a growing interest in the manipulation of fish gut microbiota to improve welfare and nutrition. Intestinal microbiota varies with many factors, including host species, genetics, developmental stage, diet, environment, and sex. The aim of this study was to compare the intestinal microbiota of adult gilthead sea bream (Sparus aurata) from three groups of age and sex (1-year-old males and 2- and 4-year-old females) maintained under the same conditions and fed exactly the same diet. Microbiota diversity and richness did not differ among groups. However, bacterial composition did, highlighting the presence of Photobacterium and Vibrio starting at 2 years of age (females) and a higher presence of Staphylococcus and Corynebacterium in 1-year-old males. The core microbiota was defined by 14 Operational Taxonomic Units (OTUs) and the groups that showed more OTUs in common were 2- and 4-year-old females. Discriminant analyses showed a clear separation by sex and age, with bacteria belonging to the phyla Firmicutes, Proteobacteria and Actinobacteria driving the separation. Pathway analysis performed with the inferred metagenome showed significant differences between 1-year-old males and 4-year-old females, with an increase in infection-related pathways, nitrotoluene degradation and sphingolipid metabolism, and a significant decrease in carbohydrate metabolism pathways with age. These results show, for the first time, how intestinal microbiota is modulated in adult gilthead sea bream and highlight the importance of reporting age and sex variables in these type of studies in fish.

Protective effects of seaweed supplemented diet on antioxidant and immune responses in European seabass (Dicentrarchus labrax) subjected to bacterial infection.

European seabass (Dicentrarchus labrax) production is often hampered by bacterial infections such as photobacteriosis caused by Photobacterium damselae subsp. piscicida (Phdp). Since diet can impact fish immunity, this work investigated the effect of dietary supplementation of 5% Gracilaria sp. aqueous extract (GRA) on seabass antioxidant capacity and resistance against Phdp. After infection, mortality was delayed in fish fed GRA, which also revealed increased lysozyme activity levels, as well as decreased lipid peroxidation, suggesting higher antioxidant capacity than in fish fed a control diet. Dietary GRA induced a down-regulation of hepatic stress-responsive heat shock proteins (grp-78, grp-170, grp-94, grp-75), while bacterial infection caused a down-regulation in antioxidant genes (prdx4 and mn-sod). Diet and infection interaction down-regulated the transcription levels of genes associated with oxidative stress response (prdx5 and gpx4) in liver. In head-kidney, GRA led to an up-regulation of genes associated with inflammation (il34, ccr9, cd33) and a down-regulation of genes related to cytokine signalling (mif, il1b, defb, a2m, myd88). Additionally, bacterial infection up-regulated immunoglobulins production (IgMs) and down-regulated the transcription of the antimicrobial peptide leap2 in head kidney. Overall, we found that GRA supplementation modulated seabass resistance to Phdp infection.

Effects of Dietary Bisphenol A on the Reproductive Function of Gilthead Sea Bream (Sparus aurata) Testes.

Bisphenol A (BPA), a known endocrine disrupting chemical (EDC), was administered by diet to gilthead sea bream (Sparus aurata) in order to study its effects on the endocannabinoid system (ECS) and gonadal steroidogenesis. 2-year-old male gilthead sea bream were fed with two different concentrations of BPA (LOW at 4 and HIGH at 4000 µg/kg body weight for 21 days during the reproductive season. Exposure to 4000 µg BPA/kg bw/day (BPA HIGH) reduced sperm motility and altered the straight-line velocity (VSL) and linearity (LIN). Effects on steroidogenesis were evident, with testosterone (T) being up-regulated by both treatments and 11-ketotestosterone (11-KT) down-regulated by BPA HIGH. Plasma levels of 17ß-estradiol (E2) were not affected. The Gonadosomatic Index (GSI) increased in the BPA HIGH group. Interestingly, the levels of endocannabinoids and endocannabinoid-like compounds were significantly reduced after both treatments. Unpredictably, a few changes were noticed in the expression of genes coding for ECS enzymes, while the receptors were up-regulated depending on the BPA dose. Reproductive markers in testis (leptin receptor (lepr), estrogen receptors (era, erb), progesterone receptors (pr) and the gonadotropin releasing hormone receptor (gnrhr)) were up-regulated. BPA induced the up-regulation of the hepatic genes involved in oogenesis (vitellogenin (vtg) and zona pellucida 1 (zp1)).