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High-quality sequencing reads from the intestine of European sea bass were assembled, annotated by similarity against protein reference databases and combined with nucleotide sequences from public and private databases. After redundancy filtering, 24,906 non-redundant annotated sequences encoding 15,367 different gene descriptions were obtained. These annotated sequences were used to design a custom, high-density oligo-microarray (8 × 15 K) for the transcriptomic profiling of anterior (AI), middle (MI), and posterior (PI) intestinal segments. Similar molecular signatures were found for AI and MI segments, which were combined in a single group (AI-MI) whereas the PI outstood separately, with more than 1900 differentially expressed genes with a fold-change cutoff of 2. Functional analysis revealed that molecular and cellular functions related to feed digestion and nutrient absorption and transport were over-represented in AI-MI segments. By contrast, the initiation and establishment of immune defense mechanisms became especially relevant in PI, although the microarray expression profiling validated by qPCR indicated that these functional changes are gradual from anterior to posterior intestinal segments. This functional divergence occurred in association with spatial transcriptional changes in nutrient transporters and the mucosal chemosensing system via G protein-coupled receptors. These findings contribute to identify key indicators of gut functions and to compare different fish feeding strategies and immune defense mechanisms acquired along the evolution of teleosts. ; This work was funded by the EU seventh Framework Programme by the ARRAINA (Advanced Research Initiatives for Nutrition and Aquaculture; KBBE-2011-288925) project. It does not necessarily reflect the views of the EU and in no way anticipates the Commission's future policy in this area. The funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. Additional funding was obtained from the Spanish Ministerio de Economía y Competitividad through the MI2-FISH project (Unraveling Metabolic, Intestinal and Immunopathological Fish Status; AGL2013-48560) and Generalitat Valenciana (PROMETEO FASE II-2014/085). ; Peer Reviewed

Mitochondrial oxidative phosphorylation provides over 90% of the energy produced by aerobic organisms, therefore the regulation of mitochondrial activity is a major issue for coping with the changing environment and energy needs. In fish, there is a large body of evidence of adaptive changes in enzymatic activities of the OXPHOS pathway, but less is known at the transcriptional level and the first aim of the present study was to define the molecular identity of the actively transcribed subunits of the mitochondrial respiratory chain of a livestock animal, using gilthead sea bream as a model of farmed fish with a high added value for European aquaculture. Extensive BLAST searches in our transcriptomic database (www.nutrigroup-iats.org/seabreamdb) yielded 97 new sequences with a high coverage of catalytic, regulatory and assembly factors of Complex I to V. This was the basis for the development of a PCR array for the simultaneous profiling of 88 selected genes. This new genomic resource allowed the differential gene expression of liver and muscle tissues in a model of 10 fasting days. A consistent down-regulated response involving 72 genes was made by the liver, whereas an up-regulated response with 29 and 10 differentially expressed genes was found in white skeletal muscle and heart, respectively. This differential regulation was mostly mediated by nuclear-encoded genes (skeletal muscle) or both mitochondrial- and nuclear-encoded genes (liver, heart), which is indicative of a complex and differential regulation of mitochondrial and nuclear genomes, according to the changes in the lipogenic activity of liver and the oxidative capacity of glycolytic and highly oxidative muscle tissues. These insights contribute to the identification of the most responsive elements of OXPHOS in each tissue, which is of relevance for the appropriate gene targeting of nutritional and/or environmental metabolic disturbances in livestock animals. © 2015 Bermejo-Nogales et al. ; This work was funded by the EU AQUAEXCEL (Aquaculture Infrastructures for Excellence in European Fish Research, FP7/2007/2013; grant agreement No. 262336; www.aquaexcel.eu) project. Additional funding was received by Generalitat Valenciana (PROMETEOII/2014/085; www.iats.csic.es/nisaam) and from the Spanish Government through AQUAGENOMICS (Ingenio-2010 Programme; www.aquagenomics.es) project. ; Peer Reviewed

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. © 2013 Springer Science+Business Media Dordrecht. ; This work was funded by the EU ARRAINA project (Advanced Research Initiatives for Nutrition and Aquaculture, FP7/2007–2013; Grant Agreement No. 288925). Additional funding was obtained from Generalitat Valenciana (Research Grant PROMETEO 2010/006) and the Spanish Government through AQUAGENOMICS (Ingenio-2010 Programme) and AQUAFAT (AGL2009-07797) projects. ; Peer Reviewed

35 p., figuras, tablas y bibliografía ; The aim of this work is to underline the biological significance of mitochondrial uncoupling proteins (UCPs) in ectothermic fish using the gilthead sea bream (Sparus aurata L.) as an experimental model. A contig of 1990 bp in length was recognized as a UCP1 ortholog after initial searches in the gilthead sea bream AQUAFIRST database (www.sigenae.org/aquafirst). Additional searches were performed in skeletal muscle by RT-PCR, and the amplified PCR product was recognized as UCP3 after sequence completion by 5´and 3´RACE. UCP1 expression was mostly detected in liver, whereas UCP3 transcripts were only found in skeletal and cardiac muscle fibers (white skeletal muscle > red skeletal muscle > heart). Specific gene regulation of UCP1 (liver) and UCP3 (white skeletal muscle) was addressed in physiological models of age, seasonal growth and energy-metabolic unbalances. Both the increase in energy demand (stress confinement) and the reduction in energy supply during adaptive cold response in winter down-regulated UCP1 expression. Conversely, transcript levels of UCP3 were higher with age, seasonal fattening and dietary deficiencies in essential fatty acids leading to the increase in fatty acid flux towards the muscle. This close association between UCP1 and UCP3 with the oxidative and metabolic tissue status is perhaps directly related to the ancestral protein UCP function, and allows the use of UCPs as lipotoxicity markers in ectothermic fish. ; This work was partially funded by the European Union (FOOD-CT-2006-16249: Sustainable Aquafeeds to Maximise the Health Benefits of Farmed Fish for Consumers, AQUAMAX) and Spanish (Ingenio-2010 Programme; Improvement of Aquaculture Production by the use of biotechnological tools, AQUAGENOMICS) projects. AB-N was recipient of a PhD fellowship from CSIC-BANCAJA. ; Peer reviewed

[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 (pgcβ 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 β-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. ; This work was financially supported by a grant from the European Commission of the European Union under the Horizon 2020 research infrastructure project AQUAEXCEL2020 (652831). Additional funding was provided by Generalitat Valenciana (PROMETEOII/2014/085). JAMS received a Postdoctoral Research Fellowship (Juan de la Cierva-Formación, Reference FJCI-2014-20,161) from MINECO. ; Peer reviewed

[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 (pgcβ 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 β-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. ; This work was financially supported by a grant from the European Commission of the European Union under the Horizon 2020 research infrastructure project AQUAEXCEL2020 (652831). Additional funding was provided by Generalitat Valenciana (PROMETEOII/2014/085). JAMS received a Postdoctoral Research Fellowship (Juan de la Cierva-Formación, Reference FJCI-2014-20,161) from MINECO. ; Peer reviewed

The effect of ration size on muscle fatty acid (FA) composition and mRNA expression levels of key regulatory enzymes of lipid and lipoprotein metabolism have been addressed in juveniles of gilthead sea bream fed a practical diet over the course of an 11-week trial. The experimental setup included three feeding levels: (i) full ration until visual satiety, (ii) 70 % of satiation and (iii) 70 % of satiation with the last 2 weeks at the maintenance ration. Feed restriction reduced lipid content of whole body by 30 % and that of fillet by 50 %. In this scenario, the FA composition of fillet TAG was not altered by ration size, whereas that of phospholipids was largely modified with a higher retention of arachidonic acid and DHA. The mRNA transcript levels of lysophosphatidylcholine acyltransferases, phosphatidylethanolamine N-methyltransferase and FA desaturase 2 were not regulated by ration size in the present experimental model. In contrast, mRNA levels of stearoyl-CoA desaturases were markedly down-regulated by feed restriction. An opposite trend was found for a muscle-specific lipoprotein lipase, which is exclusive of fish lineage. Several upstream regulatory transcriptions were also assessed, although nutritionally mediated changes in mRNA transcripts were almost reduced to PPARα and β, which might act in a counter-regulatory way on lipolysis and lipogenic pathways. This gene expression pattern contributes to the construction of a panel of biomarkers to direct marine fish production towards muscle lean phenotypes with increased retentions of long-chain PUFA. Copyright © The Authors 2012. ; The present study was funded by the Spanish MICINN (AQUAFAT, AGL2009-07797, Predictive modelling of flesh FA composition in cultured fish species with different muscle lipid content; AQUAGENOMICS, CSD2007-00002, Improvement of aquaculture production by the use of biotechnological tools) and the European Union (ARRAINA, FP7-KBBE-2011-5-288925, Advanced research initiatives for nutrition and aquaculture) projects. Additional funding was obtained from the 'GeneralitatValenciana' (research grant PROMETEO 2010/006). ; Peer Reviewed

© 2021 by the authors. ; The advanced development of sensor technologies has led to the emergence of fish biosensors that are currently used for research and commercial purposes. AEFishBI ; This research was funded by European Union's Horizon 2020 Research and Innovation Program under grant agreement no. 652831 (AQUAEXCEL2020, Aquaculture infrastructures for excellence in European fish research towards 2020). This publication only reflects the view of the authors, and the European Commission cannot be held responsible for any use that may be made of the information contained herein. Additional funding was obtained from project "Farmed Animals Welfare Toolbox (FARMWELL)", grant number 194050, The Norwegian Research Council.

The effects of nutrient availability on the transcriptome of cardiac and skeletal muscle tissues were assessed in juvenile gilthead sea bream fed with a standard diet at two feeding levels: (1) full ration size and (2) 70 % satiation followed by a finishing phase at the maintenance ration. Microarray analysis evidenced a characteristic transcriptomic profile for each muscle tissue following changes in oxidative capacity (heart > red skeletal muscle > white skeletal muscle). The transcriptome of heart and secondly that of red skeletal muscle were highly responsive to nutritional changes, whereas that of glycolytic white skeletal muscle showed less ability to respond. The highly expressed and nutritionally regulated genes of heart were mainly related to signal transduction and transcriptional regulation. In contrast, those of white muscle were enriched in gene ontology (GO) terms related to proteolysis and protein ubiquitination. Microarray meta-analysis using the bioinformatic tool Fish and Chips (http://fishandchips.genouest.org/index.php) showed the close association of a representative cluster of white skeletal muscle with some of cardiac and red skeletal muscle, and many GO terms related to mitochondrial function appeared to be common links between them. A second round of cluster comparisons revealed that mitochondria-related GOs also linked differentially expressed genes of heart with those of liver from cortisol-treated gilthead sea bream. These results show that mitochondria are among the first responders to environmental and nutritional stress stimuli in gilthead sea bream, and functional phenotyping of this cellular organelle is highly promising to obtain reliable markers of growth performance and well-being in this fish species. © 2014 Springer Science+Business Media New York. ; This work was funded by the EU seventh Framework Programme by the AQUAEXCEL (Aquaculture Infrastructures for Excellence in European Fish Research, FP7/2007-2012; grant agreement no. 262336) project. Additional funding was obtained from the Generalitat Valenciana (research grant PROMETEO 2010/006) and the Spanish Government through AQUAGENOMICS project (Consolider-Ingenio-2010 Programme). ; Peer Reviewed

Four isoproteic/isolipidic plant protein-based diets were formulated to assess the lasting effects of feed additives and low fish meal (FM) and fish oil (FO) diet formulations on gilthead sea bream growth performance. FM was included at 23% in the control diet (D1) and at 3% in the other three diets (D2, D3, D4). Added oil was either FO (D1) or a blend of vegetable oils replacing 58% (D2) and 84% (D3, D4 diets) of FO. A commercial sodium butyrate preparation (NOREL, 70-BP) was added to the D4 diet at 0.4%. Each diet was allocated to triplicate groups of juvenile fish fed to satiety over an 8-month feeding trial (May-December). All fish grew efficiently from 15. g of initial body weight to 296-320. g with an overall feed efficiency (FE) of 0.95-1.01, although fish fed D3 and D4 diets showed transient growth impairments over the course of the first four weeks of the trial. Data on biometric indexes, whole body composition, haematology and blood biochemistry revealed a strong effect of sampling time in fish sampled at mid-summer (August) and late autumn (December). In contrast, the diet effect was mostly reduced to a few blood parameters. Low inclusion levels of FM reduced plasma haemoglobin levels (D2, D3), but these effects were reversed by butyrate supplementation (D4). The same phenomena occurred for total cholesterol with the highest circulating concentration of choline and IGF-I in fish fed the D4 diet during their summer growth spurt. At the transcriptional level, gene expression profiling of liver and skeletal muscle with a PCR-array of 87 growth markers provided additional evidence for an overall well-growth condition in all of the experimental groups. Up to 73 genes were found at detectable levels in the liver tissue, but only 13 were differentially expressed. Likewise, 84 genes were actively transcribed in the skeletal muscle, but only nine were differentially expressed in at least one experimental group. Butyrate supplementation reversed the up-regulated expression of inflammatory cytokines (TNFα) and muscle markers of cellular morphogenesis and protein breakdown (CDH15, CAPN3, PSMA5, PSMB1, UBE2N) in the muscle of fish fed the extreme D3 diet. These results support the use of low FM/FO diets alone or supplemented with feed additives, which have the potential to improve or reverse metabolic steady-states. Statement of relevance: Butyrate effect on low fish meal/fish oil diets. ; This study was funded by the European Union (ARRAINA, FP7-KBBE-2011-5-288925, Advanced research initiatives for nutrition and aquaculture) projects. Additional funding was obtained from the Spanish MINECO (MI2-Fish, AGL2013-48560) and from Generalitat Valenciana (PROMETEO FASE II-2014/085). ; Peer Reviewed

Gilthead sea bream juveniles were fed different doses (0, 50, 100, 200, 300ppm) 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 100ppm. 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 100ppm 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. © 2015 Elsevier Ltd. ; This work has been carried out with financial support from the Commission of the European Communities, specific RTD programme of Framework Programme 7, (FP7/2007-2013) under grant projects ARRAINA (KBBE-2011-288925) and AQUAEXCEL (262336) under TNA project 0019/02/04/14 to ANDROMEDA. It does not necessarily reflect the EU views and in no way anticipates the Commission's future policy in this area. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Additional funding has been received by Spanish MINECO project no. AGL2013-48560 and Generalitat Valenciana (PROMETEOII/2014/085 and ISIC/2012/003). ; Peer Reviewed

© 2021 by the authors. ; 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. ; This work was financially supported by a grant from the European Commission of the European Union under the Horizon 2020 research infrastructure project AQUAEXCEL2020 (652831) to J.P-S. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH, RTI2018–094128-B-I00). J.A.M.-S. received a Postdoctoral Research Fellowship (Juan de la CiervaFormación, Reference FJCI-2014-20,161).

Reduced oxygen availability generates a number of adaptive features across all the animal kingdom, and the goal of this study was targeting the mild-hypoxia driving force for metabolic and muscle transcriptional reprogramming of gilthead sea bream juveniles. Attention was focused on blood metabolic and muscle transcriptomic landmarks before and after exhaustive exercise. Our results after mild-hypoxia conditioning highlighted an increased contribution of lipid metabolism to whole energy supply to preserve the aerobic energy production, a better swimming performance regardless of changes in feed intake, as well as reduced protein turnover and improved anaerobic fitness with the restoration of normoxia. On-growing juveniles of gilthead sea bream were acclimated for 45 days to mild-hypoxia (M-HYP, 40-60% O-2 saturation), whereas normoxic fish (85-90% O-2 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. ; This work was financially supported by a grant from the European Commission of the European Union under the Horizon 2020 research infrastructure project AQUAEXCEL2020 (652831) to J.P-S. Additional funding was obtained by a Spanish MICINN project (Bream-AquaINTECH, RTI2018-094128-B-I00). J.A.M.-S. received a Postdoctoral Research Fellowship (Juan de la Cierva-Formacion, Reference FJCI-2014-20,161).

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. ; This study was funded by the European Union (AQUAEXCEL, FP7/2007/2013; grant agreement No. 262336, Aquaculture infrastructures for excellence in European fish research) project. The views expressed in this work are the sole responsibility of the authors and do not necessarily reflect the views of the European Commission. Additional funding was obtained from the Spanish Ministerio de Economía y Competitividad (MI2-Fish, AGL2013-48560) and from Generalitat Valenciana (PROMETEO FASE II-2014/085). Proteomics study was done at Proteomics laboratory of University of Valencia, Spain (SCSIE). This laboratory is a member of Proteored, PRB2-ISCIII and is supported by grant PT13/0001, of the PE I+D+i 2013–2016, funded by Instituto de Salud Carlos III and Fondo Europeo de Desarrollo Regional (FEDER). ; Peer reviewed ; Peer Reviewed

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 R ; 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. ; Spanish MINECO: AGL2013-48560-R. . ; Generalitat Valenciana: PROMETEO-FASE II/2014/085. ; MINECO: FPDI-2013-15741. ; CSIC PIE: 201740E013 ; Peer reviewed