The Expansion of Sirtuin Gene Family in Gilthead Sea Bream (Sparus aurata)-Phylogenetic, Syntenic, and Functional Insights across the Vertebrate/Fish Lineage.

The Sirtuin (SIRT1-7) family comprises seven evolutionary-conserved enzymes that couple cellular NAD availability with health, nutrition and welfare status in vertebrates. This study re-annotated the sirt3/5 branch in the gilthead sea bream, revealing three paralogues of sirt3 (sirt3.1a/sirt3.1b/sirt3.2) and two of sirt5 (sirt5a/sirt5b) in this Perciform fish. The phylogeny and synteny analyses unveiled that the Sirt3.1/Sirt3.2 dichotomy was retained in teleosts and aquatic-living Sarcopterygian after early vertebrate 2R whole genome duplication (WGD). Additionally, only certain percomorphaceae and gilthead sea bream showed a conserved tandem-duplicated synteny block involving the mammalian-clustered sirt3.1 gene (psmd13-sirt3.1a/b-drd4-cdhr5-ctsd). Conversely, the expansion of the Sirt5 branch was shaped by the teleost-specific 3R WGD. As extensively reviewed in the literature, human-orthologues (sirt3.1/sirt5a) showed a high, conserved expression in skeletal muscle that increased as development advanced. However, recent sirt3.2 and sirt5b suffered an overall muscle transcriptional silencing across life, as well as an enhanced expression on immune-relevant tissues and gills. These findings fill gaps in the ontogeny and differentiation of Sirt genes in the environmentally adaptable gilthead sea bream, becoming a good starting point to advance towards a full understanding of its neo-functionalization. The mechanisms originating from these new paralogs also open new perspectives in the study of cellular energy sensing processes in vertebrates.

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.

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.

Gene expression profiling of whole blood cells supports a more efficient mitochondrial respiration in hypoxia-challenged gilthead sea bream (Sparus aurata).

BACKGROUND: Acclimation to abiotic challenges, including decreases in O2 availability, requires physiological and anatomical phenotyping to accommodate the organism to the environmental conditions. The retention of a nucleus and functional mitochondria in mature fish red blood cells makes blood a promising tissue to analyse the transcriptome and metabolic responses of hypoxia-challenged fish in an integrative and non-invasive manner. METHODS: Juvenile gilthead sea bream (Sparus aurata) were reared at 20-21 °C under normoxic conditions (> 85% O2 saturation) followed by exposure to a gradual decrease in water O2 concentration to 3.0 ppm (41-42% O2 saturation) for 24 h or 1.3 ppm (18-19% O2 saturation) for up to 4 h. Blood samples were collected at three different sampling points for haematological, biochemical and transcriptomic analysis. RESULTS: Blood physiological hallmarks remained almost unaltered at 3.0 ppm, but the haematocrit and circulating levels of haemoglobin, glucose and lactate were consistently increased when fish were maintained below the limiting oxygen saturation at 1.3 ppm. These findings were concurrent with an increase in total plasma antioxidant activity and plasma cortisol levels, whereas the opposite trend was observed for growth-promoting factors, such as insulin-like growth factor I. Additionally, gene expression profiling of whole blood cells revealed changes in upstream master regulators of mitochondria (pgcß and nrf1), antioxidant enzymes (gpx1, gst3, and sod2), outer and inner membrane translocases (tom70, tom22, tim44, tim10, and tim9), components of the mitochondrial dynamics system (mfn2, miffb, miro1a, and miro2), apoptotic factors (aifm1), uncoupling proteins (ucp2) and oxidative enzymes of fatty acid ß-oxidation (acca2, ech, and hadh), the tricarboxylic acid cycle (cs) and the oxidative phosphorylation pathway. The overall response is an extensive reduction in gene expression of almost all respiratory chain enzyme subunits of the five complexes, although mitochondrial-encoded catalytic subunits and nuclear-encoded regulatory subunits of Complex IV were primarily increased in hypoxic fish. CONCLUSIONS: Our results demonstrate the re-adjustment of mitochondrial machinery at transcriptional level to cope with a decreased basal metabolic rate, consistent with a low risk of oxidative stress, diminished aerobic ATP production and higher O2-carrying capacity. Taken together, these results suggest that whole blood cells can be used as a highly informative target tissue of metabolic condition.

Effects of dietary NEXT ENHANCE®150 on growth performance and expression of immune and intestinal integrity related genes in gilthead sea bream (Sparus aurata L.).

Gilthead sea bream juveniles were fed different doses (0, 50, 100, 200, 300 ppm) of NEXT ENHANCE®150 (NE) for 9 weeks. Feed gain ratio (FGR) was improved by a 10% with all the doses, but feed intake decreased in a dose dependent manner. The optimum inclusion level to achieve maximum growth was set at 100 ppm. The hepatosomatic index did not vary and only at the highest dose, viscerosomatic and splenosomatic indexes were significantly decreased. No significant changes were found in haematological parameters, plasma biochemistry, total antioxidant capacity and respiratory burst. In a second trial, NE was given at 100 ppm alone (D1) or in combination with the prebiotic PREVIDA® (0.5%) (PRE) (D2) for 17 weeks. There were no differences in the growth rates, and FGR was equally improved for D1 and D2. No significant changes in haematology and plasma antioxidant capacity were detected. The histological examination of the liver and the intestine showed no outstanding differences in the liver, but the number of mucosal foldings appeared to be higher in D1 and D2 vs CTRL diet and the density of enterocytes and goblet cells also appeared higher, particularly in the anterior intestine. A 87-gene PCR-array was constructed based on our transcriptomic database (www.nutrigroup-iats.org/seabreamdb) and applied to samples of anterior (AI) and posterior (PI) intestine. It included 54 new gene sequences and other sequences as markers of cell differentiation and proliferation, intestinal architecture and permeability, enterocyte mass and epithelial damage, interleukins and cytokines, pattern recognition receptors (PRR), and mitochondrial function and biogenesis. More than half of the studied genes had significantly different expression between AI and PI segments. The functional significance of this differential tissue expression is discussed. The experimental diets induced significant changes in the expression of 26 genes. The intensity of these changes and the number of genes that were significantly regulated were higher at PI than at AI. At PI, both diets invoked a clear down-regulation of genes involved in cell differentiation and proliferation, some involved in cell to cell communication, cytokines and several PRR. By contrast, up-regulation was mostly found for genes related to enterocyte mass, cell epithelial damage and mitochondrial activity at AI. The changes were of the same order for D1 and D2, except for fatty acid-binding proteins 2 and 6 and the PRR fucolectin, which were higher in D2 and D1 fed fish, respectively. Thus, NE alone or in combination with PRE seems to induce an anti-inflammatory and anti-proliferative transcriptomic profile with probable improvement in the absorptive capacity of the intestine that would explain the improved FGR.

Interleukin gene expression is strongly modulated at the local level in a fish-parasite model.

The goal of this work was to identify interleukin (IL)-related genes in the gilthead sea bream (GSB) (Sparus aurata L.) and how they are modulated by the parasite Enteromyxum leei, a myxozoan that causes severe enteritis with a strong inflammatory response. A Blast-X search of our transcriptomic GSB database (www.nutrigroup-iats.org/seabreamdb) identified 16 new sequences encompassing seven ILs (IL-7, IL-8, IL-10, IL-12ß, IL-15, IL-18, and IL-34), the interleukin enhancer-binding factor 2 (ILF2), and eight IL receptors (IL-R); IL-R1, IL-6RA, IL-6RB, IL-8RA, IL-10RA, IL-10RB, IL-18R1, and IL-22R. Except for ILF2, their expression, plus that of IL-1ß, IL-1R2, IL-6, and TNF-α (from public repositories), were analysed by 96-well PCR array of samples of blood, spleen, head kidney, and intestine of GSB that were anally intubated with E. leei (recipient group, RCPT). Only the expression profile of the intestine of RCPT fish showed significant difference as compared to samples from PBS-inoculated fish. At 17 days post intubation (dpi), the expression of key pro-inflammatory ILs, such as IL-8, IL-8R, IL-12ß, and TNFα was significantly up-regulated, whereas at 64 dpi, anti-inflammatory IL expression (IL-6, IL-6RB, IL-7, IL-10, IL-10RA, and IL-15) was predominant. These results indicate a modification of the IL expression at late times post infection, probably to protect the fish intestine from the parasite and damage inflicted by an excessive inflammatory response. Furthermore, the response is mainly mediated at the local level as no significant changes were detected in blood, spleen and head kidney.

Tissue-specific gene expression and functional regulation of uncoupling protein 2 (UCP2) by hypoxia and nutrient availability in gilthead sea bream (Sparus aurata): implications on the physiological significance of UCP1-3 variants.

The aim of this study was to assess in an integrative manner the physiological regulation of uncoupling protein 2 (UCP2) in gilthead sea bream. A contig of 1,325 nucleotides in length with an open reading frame of 307 amino acids was recognized as UCP2 after searches in our transcriptome reference database ( http://www.nutrigroup-iats.org/seabreamdb ). Gene expression mapping by quantitative real-time PCR revealed a ubiquitous profile that clearly differs from that of UCP1 and UCP3 variants with the greatest abundance in liver and white skeletal muscle, respectively. The greatest abundance of UCP2 transcripts was found in the heart, with a relatively high expression level in blood cells, where UCP1 and UCP3 transcripts were practically undetectable. Functional studies revealed that UCP2 mRNA expression remains either unaltered or up-regulated upon feed restriction in glycolytic (white skeletal muscle) and highly oxidative muscle tissues (heart and red skeletal muscle), respectively. In contrast, exposure to hypoxic conditions (18-19% oxygen saturation) markedly down-regulated the UCP2 mRNA expression in blood cells in a cellular environment with increased haematocrit, blood haemoglobin content, and circulating levels of glucose and lactate, and total plasma antioxidant activity. These findings demonstrated that UCP2 expression is highly regulated at the transcriptional level, arising this UCP variant as an important piece of the complex trade-off between metabolic and redox sensors. This feature would avoid the activation of futile cycles of energy wastage if changes in tissue oxidative and antioxidant metabolic capabilities are able to maintain the production of reactive oxygen species at a low regulated level.

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.

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.

Molecular characterization and expression analysis of six peroxiredoxin paralogous genes in gilthead sea bream (Sparus aurata): insights from fish exposed to dietary, pathogen and confinement stressors.

The aim of this work was to underline the physiological role of the antioxidant peroxiredoxin (PRDX) family in gilthead sea bream (Sparus aurata L.), a perciform fish extensively cultured in the Mediterranean area. First, extensive BLAST searches were done on the gilthead sea bream cDNA database of the AQUAMAX European Project (www.sigenae.org/iats), and six contigs were unequivocally identified as PRDX1-6 after sequence completion by RT-PCR. The phylogenetic analysis evidenced three major clades corresponding to PRDX1-4 (true 2-Cyst PRDX subclass), PRDX5 (atypical 2-Cys PRDX subclass) and PRDX6 (1-Cys PRDX subclass) that reflected the present hierarchy of vertebrates. However, the PRDX2 branch of modern fish including gilthead sea bream was related to the monophyletic PRDX1 node rather than to PRDX2 cluster of mammals and primitive fish, which probably denotes the acquisition of novel functions through vertebrate evolution. Transcriptional studies by means of quantitative real-time PCR evidenced a ubiquitous PRDX gene expression that was tissue specific for each PRDX isoform. In a second set of transcriptional studies, liver and head kidney were chosen as target tissues in fish challenged with i) the intestinal parasite Enteromyxum leei, ii) a plant oil (VO) diet with deficiencies in essential fatty acids and iii) prolonged exposure to high-rearing densities. These studies showed that PRDX genes were highly and mostly constitutively expressed in the liver and were not affected by dietary intervention or high density. In contrast, head kidney was highly sensitive to the different experimental challenges: significantly lower values were found for PRDX5 in the three trials, for PRDX6 in parasitized and high density fish and for PRDX1 in parasitized and VO fish. PRDX2, 3 and 5 were decreased only in VO, high density and parasitized animals, respectively. These findings would highlight the role of PRDXs as integrative and highly predictive biomarkers of health and welfare in fish and gilthead sea bream in particular.

Feed restriction up-regulates uncoupling protein 3 (UCP3) gene expression in heart and red muscle tissues of gilthead sea bream (Sparus aurata L.) New insights in substrate oxidation and energy expenditure.

The physiological regulation of the mitochondrial uncoupling protein 3 (UCP3) remains practically unexplored in fish and the aim of this study was to examine the effects of ration size on the regulation of UCP3 in heart, red skeletal muscle and white skeletal muscle of gilthead sea bream (Sparus aurata L.). Juvenile fish were fed at three different levels for 11 weeks: i) full ration until visual satiety (R(100) group), ii) 70% of satiation (R(70) group) and iii) 70% of satiation with two finishing weeks at the maintenance ration (20% of the satiation level) (R(70-20) group). The thirty percent feed restriction improved fish performance, increasing feed conversion efficiency and circulating levels of insulin-like growth factor-I (IGF-I). Fish of the R(70-20) group showed reduced growth and low circulating levels of IGF-I in combination with increased circulating concentrations of growth hormone and free fatty acids. Feed restriction did not alter UCP3 transcript levels in white skeletal muscle, but improved this tissue's oxidative capacity as assessed by changes in glycolytic and oxidative mitochondrial enzyme activities. In contrast, in cardiac and red skeletal muscle tissues, this dietary treatment primarily increased UCP3 mRNA expression. The respiratory control ratio of freshly isolated heart mitochondria was slightly lower in R(70-20) fish than in R(100) fish, which suggests that there was an increase in mitochondrial uncoupling concomitant with the enhanced UCP3 mRNA expression. Altogether, these findings highlight the different adaptive mechanism of glycolytic and highly oxidative muscle tissues for their rapid adjustment to varying feed intake.

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.

Dynamics of liver GH/IGF axis and selected stress markers in juvenile gilthead sea bream (Sparus aurata) exposed to acute confinement: differential stress response of growth hormone receptors.

The time courses of liver GH/IGF axis and selected stress markers were analyzed in juvenile gilthead sea bream (Sparus aurata) sampled at zero time and at fixed intervals (1.5, 3, 6, 24, 72 and 120 h) after acute confinement (120 kg/m(3)). Fish remained unfed throughout the course of the confinement study, and the fasting-induced increases in plasma growth hormone (GH) levels were partially masked by the GH-stress inhibitory tone. Hepatic mRNA levels of growth hormone receptor-I (GHR-I) were not significantly altered by confinement, but a persistent 2-fold decrease in GHR-II transcripts was found at 24 and 120 h. A consistent decrease in circulating levels of insulin-like growth factor-I (IGF-I) was also found through most of the experimental period, and the down-regulated expression of GHR-II was positively correlated with changes in hepatic IGF-I and IGF-II transcripts. This stress-specific response was concurrent with plasma increases in cortisol and glucose levels, reflecting the cortisol peak (60-70 ng/mL), the intensity and duration of the stressor when data found in the literature were compared. Adaptive responses against oxidative damage were also found, and a rapid enhanced expression was reported in the liver tissue for mitochondrial heat-shock proteins (glucose regulated protein 75). At the same time, the down-regulated expression of proinflammatory cytokines (tumour necrosis factor-alpha) and detoxifying enzymes (cytochrome P450 1A1) might dictate the hepatic depletion of potential sources of reactive oxygen species. These results provide suitable evidence for a functional partitioning of hepatic GHRs under states of reduced IGF production and changing cellular environment resulting from acute confinement.

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.

Ultra-Low Power Sensor Devices for Monitoring Physical Activity and Respiratory Frequency in Farmed Fish.

Integration of technological solutions aims to improve accuracy, precision and repeatability in farming operations, and biosensor devices are increasingly used for understanding basic biology during livestock production. The aim of this study was to design and validate a miniaturized tri-axial accelerometer for non-invasive monitoring of farmed fish with re-programmable schedule protocols. The current device (AE-FishBIT v.1s) is a small (14 mm × 7 mm × 7 mm), stand-alone system with a total mass of 600 mg, which allows monitoring animals from 30 to 35 g onwards. The device was attached to the operculum of gilthead sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax) juveniles for monitoring their physical activity by measurements of movement accelerations in x- and y-axes, while records of operculum beats (z-axis) served as a measurement of respiratory frequency. Data post-processing of exercised fish in swimming test chambers revealed an exponential increase of fish accelerations with the increase of fish speed from 1 body-length to 4 body-lengths per second, while a close relationship between oxygen consumption (MO2) and opercular frequency was consistently found. Preliminary tests in free-swimming fish kept in rearing tanks also showed that device data recording was able to detect changes in daily fish activity. The usefulness of low computational load for data pre-processing with on-board algorithms was verified from low to submaximal exercise, increasing this procedure the autonomy of the system up to 6 h of data recording with different programmable schedules. Visual observations regarding tissue damage, feeding behavior and circulating levels of stress markers (cortisol, glucose, and lactate) did not reveal at short term a negative impact of device tagging. Reduced plasma levels of triglycerides revealed a transient inhibition of feed intake in small fish (sea bream 50-90 g, sea bass 100-200 g), but this disturbance was not detected in larger fish. All this considered together is the proof of concept that miniaturized devices are suitable for non-invasive and reliable metabolic phenotyping of farmed fish to improve their overall performance and welfare. Further work is underway for improving the attachment procedure and the full device packaging.

Confinement exposure induces glucose regulated protein 75 (GRP75/mortalin/mtHsp70/PBP74/HSPA9B) in the hepatic tissue of gilthead sea bream (Sparus aurata L.).

Glucose regulated protein 75 (GRP75/mortalin/mtHsp70/PBP74/HSPA9B) is a molecular chaperone that was partially cloned and sequenced in gilthead sea bream (Sparus aurata L.) using a RT-PCR and 3'RACE approach. The deduced amino acid sequence supported the early vertebrate divergence of the heat shock protein 70 family into cytoplasmic Hsp70/Hsc70 group, endoplasmic reticulum-resident group and the mitochondrial-type group of GRP75. The tissue-specific regulation of GRP75 was analyzed by real-time PCR and Western blot after acute (24 h, 120 kg/m(3)) and prolonged confinement exposure (3 weeks-trial, 45-50 kg/m(3)). In both experiments, GRP75 gene expression was not significantly altered in brain, head kidney and gills. By contrast, hepatic transcripts of GRP75 were up-regulated and the magnitude of the response was dependent on the intensity of stressor. Furthermore, similar increments in hepatic transcripts and protein levels of GRP75 were found after prolonged confinement exposure. In addition, these stressed animals exhibited a 10% reduction in feed efficiency, significantly increased glycaemia and plasma peroxidases, and their plasma transaminases and respiratory burst of circulating leucocytes were significantly decreased. This stress-mediated response may act in concert with the increased production of hepatic GRP75 to protect metabolically active tissues against oxidative damage.

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 of IGFs and GH receptors (GHRs) in gilthead sea bream (Sparus aurata L.): sequence analysis of the GHR-flanking region.

The tissue-specific expression of IGFs and GH receptors (GHRs) was analyzed in gilthead sea bream (Sparus aurata L.) as an attempt to understand the functional partitioning of duplicated GHRs on the regulation offish growth by season and aging. Gene transcripts were measured in liver, muscle, and adipose tissue by means of quantitative real-time PCR assays. In juvenile fish, concurrent increases in circulating levels of GH and IGF-I and hepatic mRNA levels of IGF-I and GHR-I were evidenced with the summer growth spurt. Conversely, muscle and adipose tissue expression of GHR-I and IGF-II were significantly upregulated by over wintering. The aging decrease of growth rates was accompanied by a reduced activity of the liver GH/IGF axis, and parallel increases in muscle IGF expression would be dictated at the local tissue level by the enhanced expression of GHR-I. Extra-hepatic expression of IGFs and GHR-II did not correlate seasonally in juvenile fish, and nonsignificant effects of aging were found on the summer expression of GHR-II in any analyzed tissue. One transcription start site was identified by RLM-RACE in GHR-I and GHR-II. Sequence analyses indicated that both genes have TATA-less promoters containing consensus initiator sequences and downstream promoter elements surrounding the transcription start site. Conserved CCAAT-boxes and GC-rich regions were retrieved in the GHR-I promoter, whereas stress- and redox-sequence elements (cAMP-responsive element-binding protein, activator proteins; AP-1, and AP-4) were characteristic features of GHR-II. All this supports the functional partitioning of fish GHRs regardless of fish species differences.

Tumour necrosis factor (TNF)alpha as a regulator of fat tissue mass in the Mediterranean gilthead sea bream (Sparus aurata L.).

The study was undertaken to analyze the lipolytic effect and transcriptional regulation of tumour necrosis factor (TNF)alpha in gilthead sea bream (Sparus aurata L.). The study was also focused on the transcriptional regulation and analysis of the 5-flanking region of lipoprotein lipase (LPL) in an attempt to identify cis-regulatory elements that would support the TNFalpha-mediated effects. The lipolytic effect of TNFalpha was evidenced by the increased release of glycerol in the culture medium of freshly isolated adipocytes. This observation, in addition to the summer up-regulation of TNFalpha transcripts in liver and mesenteric adipose tissue, supported a key role of TNFalpha as a fish limiting factor of tissue fat mass. Accordingly, TNFalpha expression in liver and mesenteric adipose tissue was reduced by fasting. Furthermore, the up-regulated expression of TNFalpha in the skeletal muscle of older fish can represent an adaptive response to limit the enhanced lipid influx towards muscle. A close positive association between LPL and TNFalpha transcripts supported the contribution of TNFalpha as a part of a regulatory network that exerts an inhibitory tonus upon the expression of LPL, which in turns limits the tissue uptake of fatty acids and the ultimate increase of tissue lipid reservoirs. The precise mechanism for the inhibition of LPL gene expression by TNFalpha remains to be established in fish, but analysis of the 5'-flanking region evidenced the conservation through vertebrate evolution of a functional OCT-1/NF-Y site that would mediate the TNFalpha effects on LPL expression.

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.