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.

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.

Stearoyl-CoA desaturase (scd1a) is epigenetically regulated by broodstock nutrition in gilthead sea bream (Sparus aurata).

The aim of this study was to generate new knowledge on fish epigenetics, assessing the effects of linolenic acid (ALA) conditioning of broodstock in the offspring of the marine fish Sparus aurata. Attention was focused on gene organization, methylation signatures and gene expression patterns of fatty acid desaturase 2 (fads2) and stearoyl-CoA desaturase 1a (scd1a). Blat searches in the genomic IATS-CSIC database (www.nutrigroup-iats.org/seabreamdb) highlighted a conserved exon-intron organization, a conserved PUFA response region, and CG islands at the promoter regions of each gene. The analysed CpG positions in the fads2 promoter were mostly hypomethylated and refractory to broodstock nutrition. The same response was achieved after conditioning of juvenile fish to low water oxygen concentrations, thus methylation susceptibility at individual CpG sites seems to be stringently regulated in fish of different origin and growth trajectories. Conversely, the scd1a promoter was responsive to broodstock nutrition and the offspring of parents fed the ALA-rich diet shared an increased DNA-methylation, mainly in CpG sites neighbouring SP1 and HNF4α binding sites. Cytosine methylation at these sites correlated inversely with the hepatic scd1a expression of the offspring. Co-expression analyses supported that the HNF4α-dependent regulation of scd1a is affected by DNA methylation. The phenotypic output is a regulated liver fat deposition through changes in scd1 expression, which would also allow the preservation of fatty acid unsaturation levels in fish fed reduced levels of n-3 LC-PUFA. Collectively, these findings reveal a reliable mechanism by which parent's nutrition can shape scd1a gene expression in the fish offspring.

Effects of Dietary Lipid Composition and Fatty Acid Desaturase 2 Expression in Broodstock Gilthead Sea Bream on Lipid Metabolism-Related Genes and Methylation of the fads2 Gene Promoter in Their Offspring.

Polyunsaturated fatty acids (PUFA) in parental diets play a key role in regulating n-3 LC-PUFA metabolism of the offspring. However, it is not clear whether this metabolic regulation is driven by the precursors presented in the diet or by the parental ability to synthesize them. To elucidate this, broodstocks of gilthead sea bream with different blood expression levels of fads2, which encodes for the rate-limiting enzyme in the n-3 LC-PUFA synthesis pathway, were fed either a diet supplemented with alpha-linolenic acid (ALA, 18:3n-3) or a control diet. The progenies obtained from these four experimental groups were then challenged with a low LC-PUFA diet at the juvenile stage. Results showed that the offspring from parents with high fads2 expression presented higher growth and improved utilization of low n-3 LC-PUFA diets compared to the offspring from parents with low fads2 expression. Besides, an ALA-rich diet during the gametogenesis caused negative effects on the growth of the offspring. The epigenetic analysis demonstrated that methylation in the promoter of fads2 of the offspring was correlated with the parental fads2 expression levels and type of the broodstock diet.

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.

Tissue-Specific Orchestration of Gilthead Sea Bream Resilience to Hypoxia and High Stocking Density.

Two different O2 levels (normoxia: 75-85% O2 saturation; moderate hypoxia: 42-43% O2 saturation) and stocking densities (LD: 9.5, and HD: 19 kg/m3) were assessed on gilthead sea bream (Sparus aurata) in a 3-week feeding trial. Reduced O2 availability had a negative impact on feed intake and growth rates, which was exacerbated by HD despite of the improvement in feed efficiency. Blood physiological hallmarks disclosed the enhancement in O2-carrying capacity in fish maintained under moderate hypoxia. This feature was related to a hypo-metabolic state to cope with a chronic and widespread environmental O2 reduction, which was accompanied by a differential regulation of circulating cortisol and growth hormone levels. Customized PCR-arrays were used for the simultaneous gene expression profiling of 34-44 selected stress and metabolic markers in liver, white skeletal muscle, heart, and blood cells. The number of differentially expressed genes ranged between 22 and 19 in liver, heart, and white skeletal muscle to 5 in total blood cells. Partial Least-Squares Discriminant Analysis (PLS-DA) explained [R2Y(cum)] and predicted [Q2Y(cum)] up to 95 and 65% of total variance, respectively. The first component (R2Y = 0.2889) gathered fish on the basis of O2 availability, and liver and cardiac genes on the category of energy sensing and oxidative metabolism (cs, hif-1α, pgc1α, pgc1ß, sirts 1-2-4-5-6-7), antioxidant defense and tissue repair (prdx5, sod2, mortalin, gpx4, gr, grp-170, and prdx3) and oxidative phosphorylation (nd2, nd5, and coxi) highly contributed to this separation. The second component (R2Y = 0.2927) differentiated normoxic fish at different stocking densities, and the white muscle clearly promoted this separation by a high over-representation of genes related to GH/IGF system (ghr-i, igfbp6b, igfbp5b, insr, igfbp3, and igf-i). The third component (R2Y = 0.2542) discriminated the effect of stocking density in fish exposed to moderate hypoxia by means of hepatic fatty acid desaturases (fads2, scd1a, and scd1b) and muscle markers of fatty acid oxidation (cpt1a). All these findings disclose the different contribution of analyzed tissues (liver ≥ heart > muscle > blood) and specific genes to the hypoxic- and crowding stress-mediated responses. This study will contribute to better explain and understand the different stress resilience of farmed fish across individuals and species.

Somatotropic Axis Regulation Unravels the Differential Effects of Nutritional and Environmental Factors in Growth Performance of Marine Farmed Fishes.

The Gh/Prl/Sl family has evolved differentially through evolution, resulting in varying relationships between the somatotropic axis and growth rates within and across fish species. This is due to a wide range of endogenous and exogenous factors that make this association variable throughout season and life cycle, and the present minireview aims to better define the nutritional and environmental regulation of the endocrine growth cascade over precisely defined groups of fishes, focusing on Mediterranean farmed fishes. As a result, circulating Gh and Igf-i are revitalized as reliable growth markers, with a close association with growth rates of gilthead sea bream juveniles with deficiency signs in both macro- or micro-nutrients. This, together with other regulated responses, promotes the use of Gh and Igf-i as key performance indicators of growth, aerobic scope, and nutritional condition in gilthead sea bream. Moreover, the sirtuin-energy sensors might modulate the growth-promoting action of somatotropic axis. In this scenario, transcripts of igf-i and gh receptors mirror changes in plasma Gh and Igf-i levels, with the ghr-i/ghr-ii expression ratio mostly unaltered over season. However, this ratio is nutritionally regulated, and enriched plant-based diets or diets with specific nutrient deficiencies downregulate hepatic ghr-i, decreasing the ghr-i/ghr-ii ratio. The same trend, due to a ghr-ii increase, is found in skeletal muscle, whereas impaired growth during overwintering is related to increase in the ghr-i/ghr-ii and igf-ii/igf-i ratios in liver and skeletal muscle, respectively. Overall, expression of insulin receptors and igf receptors is less regulated, though the expression quotient is especially high in the liver and muscle of sea bream. Nutritional and environmental regulation of the full Igf binding protein 1-6 repertoire remains to be understood. However, tissue-specific expression profiling highlights an enhanced and nutritionally regulated expression of the igfbp-1/-2/-4 clade in liver, whereas the igfbp-3/-5/-6 clade is overexpressed and regulated in skeletal muscle. The somatotropic axis is, therefore, highly informative of a wide-range of growth-disturbing and stressful stimuli, and multivariate analysis supports its use as a reliable toolset for the assessment of growth potentiality and nutrient deficiencies and requirements, especially in combination with selected panels of other nutritionally regulated metabolic biomarkers.

Co-expression Analysis of Sirtuins and Related Metabolic Biomarkers in Juveniles of Gilthead Sea Bream (Sparus aurata) With Differences in Growth Performance.

Sirtuins (SIRTs) represent a conserved protein family of deacetylases that act as master regulators of metabolism, but little is known about their roles in fish and livestock animals in general. The present study aimed to assess the value of SIRTs for the metabolic phenotyping of fish by assessing their co-expression with a wide-representation of markers of energy and lipid metabolism and intestinal function and health in two genetically different gilthead sea bream strains with differences in growth performance. Fish from the fast-growing strain exhibited higher feed intake, feed efficiency and plasma IGF-I levels, along with higher hepatosomatic index and lower mesenteric fat (lean phenotype). These observations suggest differences in tissue energy partitioning with an increased flux of fatty acids from adipose tissue toward the liver. The resulting increased risk of hepatic steatosis may be counteracted in the liver by reduced lipogenesis and enhanced triglyceride catabolism, in combination with a higher and more efficient oxidative metabolism in white skeletal muscle. These effects were supported by co-regulated changes in the expression profile of SIRTs (liver, sirt1; skeletal muscle, sirt2; adipose tissue, sirt5-6) and markers of oxidative metabolism (pgc1α, cpt1a, cs, nd2, cox1), mitochondrial respiration uncoupling (ucp3) and fatty acid and triglyceride metabolism (pparα, pparγ, elovl5, scd1a, lpl, atgl) that were specific to each strain and tissue. The anterior intestine of the fast-growing strain was better suited to cope with improved growth by increased expression of markers of nutrient absorption (fabp2), epithelial barrier integrity (cdh1, cdh17) and immunity (il1ß, cd8b, lgals1, lgals8, sIgT, mIgT), which were correlated with low expression levels of sirt4 and markers of fatty acid oxidation (cpt1a). In the posterior intestine, the fast-growing strain showed a consistent up-regulation of sirt2, sirt3, sirt5 and sirt7 concurrently with increased expression levels of markers of cell proliferation (pcna), oxidative metabolism (nd2) and immunity (sIgT, mIgT). Together, these findings indicate that SIRTs may play different roles in the regulation of metabolism, inflammatory tone and growth in farmed fish, arising as powerful biomarkers for a reliable metabolic phenotyping of fish at the tissue-specific level.

Sodium salt medium-chain fatty acids and Bacillus-based probiotic strategies to improve growth and intestinal health of gilthead sea bream (Sparus aurata).

BACKGROUND: The increased demand for fish protein has led to the intensification of aquaculture practices which are hampered by nutritional and health factors affecting growth performance. To solve these problems, antibiotics have been used for many years in the prevention, control and treatment against disease as well as growth promoters to improve animal performance. Nowadays, the use of antibiotics in the European Union and other countries has been completely or partially banned as a result of the existence of antibiotic cross-resistance. Therefore, a number of alternatives, including enzymes, prebiotics, probiotics, phytonutrients and organic acids used alone or in combination have been proposed for the improvement of immunological state, growth performance and production in livestock animals. The aim of the present study was to evaluate two commercially available feed additives, one based on medium-chain fatty acids (MCFAs) from coconut oil and another with a Bacillus-based probiotic, in gilthead sea bream (GSB, Sparus aurata), a marine farmed fish of high value in the Mediterranean aquaculture. METHODS: The potential benefits of adding two commercial feed additives on fish growth performance and intestinal health were assessed in a 100-days feeding trial. The experimental diets (D2 and D3) were prepared by supplementing a basal diet (D1) with MCFAs in the form of a sodium salt of coconut fatty acid distillate (DICOSAN®; Norel, Madrid, Spain), rich on C-12, added at 0.3% (D2) or with the probiotic Bacillus amyloliquefaciens CECT 5940, added at 0.1% (D3). The study integrated data on growth performance, blood biochemistry, histology and intestinal gene expression patterns of selected markers of intestinal function and architecture. RESULTS: MCFAs in the form of a coconut oil increased feed intake, growth rates and the surface of nutrient absorption, promoting the anabolic action of the somatotropic axis. The probiotic (D3) induced anti-inflammatory and anti-oxidant effects with changes in circulating cortisol, immunoglobulin M, leukocyte respiratory burst, and mucosal expression levels of cytokines, lymphocyte markers and immunoglobulin T. DISCUSSION: MCFA supplementation showed positive effects on GSB growth and intestinal architecture acting mainly in the anterior intestine, where absorption takes place. The probiotic B. amyloliquefaciens CECT 5940 exhibited key effects in the regulation of the immune status inducing anti-inflammatory and anti-oxidant effects which can be potentially advantageous upon infection or exposure to other stressors. The potential effects of these feed additives in GSB are very promising to improve health and disease resistance in aquaculture.

Under control: how a dietary additive can restore the gut microbiome and proteomic profile, and improve disease resilience in a marine teleostean fish fed vegetable diets.

BACKGROUND: The constant increase of aquaculture production and wealthy seafood consumption has forced the industry to explore alternative and more sustainable raw aquafeed materials, and plant ingredients have been used to replace marine feedstuffs in many farmed fish. The objective of the present study was to assess whether plant-based diets can induce changes in the intestinal mucus proteome, gut autochthonous microbiota and disease susceptibility of fish, and whether these changes could be reversed by the addition of sodium butyrate to the diets. Three different trials were performed using the teleostean gilthead sea bream (Sparus aurata) as model. In a first preliminary short-term trial, fish were fed with the additive (0.8%) supplementing a basal diet with low vegetable inclusion (D1) and then challenged with a bacteria to detect possible effects on survival. In a second trial, fish were fed with diets with greater vegetable inclusion levels (D2, D3) and the long-term effect of sodium butyrate at a lower dose (0.4%) added to D3 (D4 diet) was tested on the intestinal proteome and microbiome. In a third trial, the long-term effectiveness of sodium butyrate (D4) to prevent disease outcome after an intestinal parasite (Enteromyxum leei) challenge was tested. RESULTS: The results showed that opposed forces were driven by dietary plant ingredients and sodium butyrate supplementation in fish diet. On the one hand, vegetable diets induced high parasite infection levels that provoked drops in growth performance, decreased intestinal microbiota diversity, induced the dominance of the Photobacterium genus, as well as altered the gut mucosal proteome suggesting detrimental effects on intestinal function. On the other hand, butyrate addition slightly decreased cumulative mortality after bacterial challenge, avoided growth retardation in parasitized fish, increased intestinal microbiota diversity with a higher representation of butyrate-producing bacteria and reversed most vegetable diet-induced changes in the gut proteome. CONCLUSIONS: This integrative work gives insights on the pleiotropic effects of a dietary additive on the restoration of intestinal homeostasis and disease resilience, using a multifaceted approach.

Skin Mucus of Gilthead Sea Bream (Sparus aurata L.). Protein Mapping and Regulation in Chronically Stressed Fish.

The skin mucus of gilthead sea bream was mapped by one-dimensional gel electrophoresis followed by liquid chromatography coupled to high resolution mass spectrometry using a quadrupole time-of-flight mass analyzer. More than 2,000 proteins were identified with a protein score filter of 30. The identified proteins were represented in 418 canonical pathways of the Ingenuity Pathway software. After filtering by canonical pathway overlapping, the retained proteins were clustered in three groups. The mitochondrial cluster contained 59 proteins related to oxidative phosphorylation and mitochondrial dysfunction. The second cluster contained 79 proteins related to antigen presentation and protein ubiquitination pathways. The third cluster contained 257 proteins where proteins related to protein synthesis, cellular assembly, and epithelial integrity were over-represented. The latter group also included acute phase response signaling. In parallel, two-dimensional gel electrophoresis methodology identified six proteins spots of different protein abundance when comparing unstressed fish with chronically stressed fish in an experimental model that mimicked daily farming activities. The major changes were associated with a higher abundance of cytokeratin 8 in the skin mucus proteome of stressed fish, which was confirmed by immunoblotting. Thus, the increased abundance of markers of skin epithelial turnover results in a promising indicator of chronic stress in fish.

Tissue-specific gene expression and fasting regulation of sirtuin family in gilthead sea bream (Sparus aurata).

The seven sirtuin (SIRT) counterparts of higher vertebrates were identified and molecularly characterized in a farmed fish of the Sparidae family, order Perciformes. These proteins are NAD+-dependent deacetylases that couple protein deacetylation with the energy status of the cell via the cellular NAD+/NADH ratio with a strict conservation of the characteristic catalytic domain surrounded by divergent N- and C- terminal regions. Phylogenetic analysis showed three major clades corresponding to SIRT1-3, SIRT4-5, and SIRT6-7 that reflected the present hierarchy of vertebrates and the accepted classification of SIRTs. Transcriptional studies revealed a ubiquitous SIRT gene expression that was tissue-specific for each SIRT. This was evidenced by multivariate analyses, which established two main clusters corresponding to SIRTs with relatively high (SIRT1, 2, and 5) and low (SIRT3, 4, 6, and 7) gene expression levels. A nutritional regulation was also evidenced in 10-day fasted fish, and SIRT2-4 exhibited an overall downregulated expression. SIRT1, 5, 6, and 7 were mostly upregulated, although clustering analyses evidenced a highly regulated response that was different for each SIRT according to the different tissue metabolic capabilities. These findings supported the use of SIRTs alone or in combination with other biomarkers for the metabolic phenotyping of farmed fish and gilthead sea bream in particular.