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Plastic pollution has become a global problem for marine ecosystems. Microplastics (MPs) are consumed by several marine organisms, including benthic and pelagic fish species that confuse them with food sources, thus contributing to bioaccumulation along the food chain. In addition to structural intestinal damage, ingestion of MPs represents a pathway for fish exposure to potentially hazardous chemicals, too. Most of them are endocrine disrupters, genotoxic or induce immune depression in fish. Accordingly, we assessed the combined toxicological effects of microplastics (MPs) and adsorbed pollutants by adding them to marine fish diet. European sea bass (Dicentrarchus labrax) juveniles were fed for 60 days with feeds containing polypropylene MPs, either virgin or contaminated with chemical pollutants (a blend of dichlorodiphenyldichloroethylene, chlorpyrifos, and benzophenone-3). The data demonstrated a synergic action of MPs and chemical pollutants to induce an inflammatory-like response in distal intestine of sea bass as shown by the up regulation of cytokine il-6 and tnf-α expression. Morphological analysis detected the presence of a focus of lymphocytes in anterior and posterior intestinal segments of fish fed with contaminants in the diet. With regard to microbiota, significant changes in bacterial species richness, beta diversity, and composition of gut microbiota were observed as a consequence of both pollutants and polluted MPs ingestion. These perturbations in gut microbial communities, including the reduction of beneficial lactic acid bacteria and the increase in potential pathogenic microorganism (Proteobacteria and Vibrionales), were undeniable signs of intestinal dysbiosis, which in turn confirmed the signs of inflammation caused by pollutants, especially when combined with MPs. The results obtained in this study provide, therefore, new insights into the potential risks of ingesting MPs as pollutant carriers in marine fish.

The use of low fishmeal/fish oil in marine fish diets affects dietary essential fatty acids (EFAs) composition and concentration and, subsequently, may produce a marginal deficiency of those fatty acids with a direct impact on the fish intestinal physiology. Supplementation of essential fatty acids is necessary to cover the requirements of the different EFAs, including the ones belonging to the n-6 series, such as arachidonic acid (ARA). ARA, besides its structural role in the configuration of the lipid classes of the intestine, plays an important role in the functionality of the gut-associated immune tissue (GALT). The present study aimed to test five levels of dietary ARA (ARA0.5 (0.5%), ARA1 (1%), ARA2 (2%), ARA4 (4%), and ARA6 (6%)) for European sea bass (Dicentrarchus labrax) juveniles in order to determine (a) its effect in selected distal intestine (DI) lipid classes composition and (b) how these changes affected gut bacterial translocation rates and selected GALT-related gene expression pre and post challenge. No differences were found between distal intestines of fish fed with the graded ARA levels in total neutral lipids and total polar lipids. However, DI of fish fed with the ARA6 diet presented a higher (P

Antimicrobial peptides (AMPs) play an important role in the innate immune response of vertebrates by creating a hostile environment for any invading pathogens. Piscidins are potent teleost specific AMPs, which have a broad spectrum activity. We have identified a novel piscidin active peptide, in the greater amberjack, Seriola dumerili, that consists of 25 aa, which forms an amphipathic helix with distinct hydrophobic and positively charged regions. Following homology and phylogenetic analysis the greater amberjack piscidin was deemed to belong to the group 3 family of piscidins. Piscidin was expressed constitutively at immune sites, with transcript level highest in the spleen and gut, at an intermediate level in the gills and lowest in the head kidney. Following in vivo stimulation with PAMPs (poly I:C, LPS and flagellin) piscidin transcript level increased in gills in response to flagellin, in gut and spleen in response to poly I:C, and in head kidney in response to poly I:C, LPS and flagellin. Head kidney and spleen cells were then isolated from greater amberjack and incubated with each of the PAMPs for 4, 12 and 24 h. Piscidin expression was unchanged at 4 and 12 h post PAMP stimulation in head kidney cells but at 24 h transcript level was markedly upregulated compared to control (unstimulated) cells, especially with the bacterial PAMPs. In contrast, spleen cells upregulated piscidin expression by 4 h post stimulation with poly I:C and flagellin, and remained upregulated to 24 h with flagellin exposure, but had returned to baseline levels by 12 h using poly I:C. To determine if piscidin expression could be modulated by diet, greater amberjack were fed diets supplemented with MOS and cMOS for 30 days when transcript level was determined. It was found that MOS supplemented diets increased expression in the spleen, cMOS supplemented diets upregulated transcript levels in the gills and head kidney, whilst a diet containing both MOS and cMOS upregulated transcript in the gut, when compared to fish fed the control diet. Finally, a synthetic greater amberjack piscidin was produced and showed bacteriostatic activity against a number of bacterial strains, including both Gram positive and Gram negative fish pathogens.

Within the food-producing sectors, aquaculture is the one that has developed the greatest growth in recent decades, currently representing almost 50% of the world's edible fish. The diseases can affect the final production in intensive aquaculture; in seabass, aquaculture vibriosis is one of the most important diseases producing huge economical losses in this industry. The usual methodology to solve the problems associated with the bacterial pathology has been the use of antibiotics, with known environmental consequences. This is why probiotic bacteria are proposed as an alternative fight against pathogenic bacteria. The aim of this study was to analyse a strain of Bacillus velezensis D-18 isolated from a wastewater sample collected from a fish farm, for use as probiotics in aquaculture. The strain was evaluated in vitro through various mechanisms of selection, obtaining as results for growth inhibition by co-culture a reduction of 30%; B. velezensis D-18 was able to survive at 1.5-h exposure to 10% seabass bile, and at pH 4, its survival is 5% and reducing by 60% the adhesion capacity of V. anguillarum 507 to the mucus of seabass and in vivo by performing a challenge. Therefore, in conclusion, we consider B. velezensis D-18 isolate from wastewater samples collected from the farms as a good candidate probiotic in the prevention of the infection by Vibrio anguillarum 507 in European seabass after in vitro and biosafety assays.

The present work investigated how the combination of poultry oil with microalgae oils, rich in eicosapentaenoic acid and docosahexaenoic acid (ED diets) or n-6 docosapentaenoic acid and n-3 docosahexaenoic acid (DD diets) modulates hepatic lipid metabolism in gilthead sea bream juveniles. Diets were tested using two different fishmeal contents (15% and 7.5%) and compared against a fish oil-based diet (CTRL) and two negative control diets based on poultry oil as lipid source (PO diets). After 74 days of feeding, sea bream fed 15% FM ED or DD diets showed similar daily growth index to those fed CTRL, while those fed PO diets caused reduced growth. Fish livers reflected the highest contents in n-3 long-chain polyunsaturated fatty acids when fed CTRL, ED or DD diets, which down-regulated fas, scd-1a, fads2, lpl and cpt1, reducing hepatic lipid accumulation and hepatocytes size. In contrast, fish fed PO diets showed the lowest deposition of n-3 long-chain polyunsaturated fatty acids and the highest oleic acid in liver, leading with the highest hepatosomatic index due to increased liver lipids. Therefore, these fish revealed a severe hepatic steatosis associated with an increased expression of lipogenesis-related genes, particularly fas, lpl and sbrep1. Furthermore, PO diets seemed to activate desaturation pathways in fish livers, reflected by the highest accumulation of fatty acids that are products from desaturases and the highest fads2 and scd-1a expressions. The reduction of the dietary fishmeal content to 7.5% lowered fish growth, although hepatic lipid metabolism seemed to be more affected by FO replacement than FM replacement. Combining microalgae with poultry oil could be an alternative lipid and essential fatty acid source to fish oil in marine fish diets.

Author's accepted version (postprint) This is an Accepted Manuscript of an article published by Springer in Probiotics and Antimicrobial Proteins on 03/04/2021 Available online: https://link.springer.com/content/pdf/10.1007/s12602-021-09782-8.pdf Within the food-producing sectors, aquaculture is the one that has developed the greatest growth in recent decades, currently representing almost 50% of the world’s edible fish. The diseases can affect the final production in intensive aquaculture; in seabass, aquaculture vibriosis is one of the most important diseases producing huge economical losses in this industry. The usual methodology to solve the problems associated with the bacterial pathology has been the use of antibiotics, with known environmental consequences. This is why probiotic bacteria are proposed as an alternative fight against pathogenic bacteria. The aim of this study was to analyse a strain of Bacillus velezensis D-18 isolated from a wastewater sample collected from a fish farm, for use as probiotics in aquaculture. The strain was evaluated in vitro through various mechanisms of selection, obtaining as results for growth inhibition by co-culture a reduction of 30%; B. velezensis D-18 was able to survive at 1.5-h exposure to 10% seabass bile, and at pH 4, its survival is 5% and reducing by 60% the adhesion capacity of V. anguillarum 507 to the mucus of seabass and in vivo by performing a challenge. Therefore, in conclusion, we consider B. velezensis D-18 isolate from wastewater samples collected from the farms as a good candidate probiotic in the prevention of the infection by Vibrio anguillarum 507 in European seabass after in vitro and biosafety assays.

Skin mucus is considered the first barrier against diseases in fish. The skin mucus protein profile of the greater amberjack (Seriola dumerili) and its changes due to experimental infection with Neobenedenia girellae were studied by combining 2-DE-MS/MS and gel-free LC-MS/MS proteomic approaches. The 2-DE results led to the identification of 69 and 55 proteins in noninfected and infected fish, respectively, and revealed that keratins were specifically cleaved in parasitized fish. Therefore, the skin mucus of the infected fish showed a higher protease activity due to, at least in part, an increase of metal-dependent protease and serine-type protease activities. Additionally, through a gel-free LC-MS/MS analysis, 1377 and 1251 different proteins were identified in the skin mucus of healthy and parasitized fish, respectively. The functional analysis of these proteins demonstrated a statistical overrepresentation of ribosomal proteins (a well-known source of antimicrobial peptides) in N. girellae-infected fish. In contrast, the components of membranes and protein transport GO categories were underrepresented after infection. Immune system process-related proteins constituted 2.5% of the total skin mucosal proteins. Among these skin mucosal proteins, 14 and 15 proteins exclusive to non-parasitized and parasitized fish were found, respectively, including specific serine-type proteases and metalloproteases in the parasitized fish. Moreover, the finding of tryptic peptides exclusive to some bacterial genera, obtained by gel-free LC-MS/MS, allowed us to construct a preliminary map of the microbiota living in the mucus of S. dumerili, with Pseudomonas and Paracoccus the most represented genera in both noninfected and infected fish.

A sustainable growth of the aquaculture sector implies the use of sustainable novel raw materials as replacers of the traditional fishmeal (FM) and fish oil (FO) ingredients. This fact has led to the development of sustainable and functional diets as part of a management strategy to reduce the effects on fish growth performance and health derived from low FM/FO dietary contents. In this sense, Antarctic krill (Euphausia superba) is considered a potential candidate in dietary inclusions to potentiate fish growth and health status. In this study, European sea bass (Dicentrarchus labrax) were fed a practical diet with either a 15% fishmeal content (KM0; control diet) or the same diet substituted by 30% (KM5; 50 g KM/kg diet) or 50% (KM7.5; 75 g KM/kg diet) Antarctic krill meal (KM) for 12 weeks in triplicates. At the end of the feeding trial, growth performance, liver morphology, liver proximate composition, lipid classes and fatty acid profiles, as well as the expression of hepatic genes related with lipid metabolism were evaluated. Fish fed KM-based diets presented higher (p 0.05) biochemical composition and fatty acid profile. However, smaller hepatocellular area and lower grade of cytoplasm vacuolization as well as a better alignment around sinusoidal spaces were found. The analyses of liver lipid classes revealed a positive correlation between the level of dietary KM and the pigmented material such as astaxanthin and free fatty acid content, as well as a negative correlation with the cholesterol levels. The expression of hepatic genes studied demonstrated a downregulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase (hmgr) and delta-6-desaturase (fads2) expression levels in fish fed KM-based diets. Besides, gene expression levels of fatty acid binding protein 7 (fabp7) and lipoprotein lipase (lpl) were significantly correlated with KM dietary levels. Altogether, these results profile KM as a potential promoter of growth and liver health in European sea bass fed low fish meal and oil diets.

The aim of this study was to evaluate the effects of increasing dietary arachidonic acid (ARA) levels (from 1 up to 6 % of total fatty acids) on European sea bass (Dicentrarchus labrax) juveniles growth performance, tissue fatty acid profile, liver morphology as well as long chain polyunsaturated fatty acids (LC-PUFA) biosynthesis, triglyceride and cholesterol synthesis and lipid transport. A diet with total fish oil (FO) replacement and defatted fish meal (FM) containing a 0.1 g ARA g-1 diet was added to the experimental design as a negative control diet. Dietary ARA inclusion levels below 0.2 g ARA g-1 diet significantly worsened growth even only 30 days after the start of the feeding trial, whereas dietary ARA had no effect on fish survival. Liver, muscle and whole body fatty acid profile mainly reflected dietary contents and ARA content increased accordingly with ARA dietary levels. Tissue eicosapentaenoic acid (EPA), docosapentaenoic acid (DPA) and docosahexaenoic acid (DHA) levels were positively correlated among them. Hepatic lipid vacuolization increased with reduced dietary ARA levels. Expressions of fatty acyl desaturase 2 and 3-hydroxy-3-methylglutaryl-coenzyme genes were up-regulated in fish fed the negative control diet compared to the rest of the dietary treatments denoting the influence of ARA on lipid metabolism. Results obtained highlight the need to include adequate n-6 levels and not only n-3 LC-PUFA levels in European sea bass diets. Keywords: Dicentrarchus labrax, arachidonic acid, growth performance, tissue fatty acid profile, lipid metabolism

11 pages, 6 figures, 1 table, In order to successfully diversify Mediterranean aquaculture, it is necessary to determine optimum culture conditions of potential candidate species such as greater amberjack (Seriola dumerili). Among culture conditions, rearing temperature is a key factor for achieving optimum growth and maintaining fish welfare. However, little is known about the optimum culture conditions of greater amberjack (Seriola dumerili). Thus, the aim of this study was to determine the effect of three different rearing temperatures (17, 22 and 26°C) during 120 days on growth performance, body morphometry, biochemical composition, gut transit and liver morphology of greater amberjack (Seriola dumerili) juveniles. After 120 days of rearing, fish raised at 26°C showed higher (p < .05) body weight and specific growth rate than fish held at lower temperatures, as well as improved feed utilization, protein efficiency and nutrient retention percentages. Fish stomach emptying was faster (p < .05) in fish raised at 26°C than in fish held at 22°C and 17°C. Similar results were obtained for gut transit time, being gut emptying faster (p < .05) in fish reared at 26°C than in fish cultured at lower temperatures. Rearing temperature also induced changes in fish morphology which resulted in a higher (p < .05) caudal propulsion efficiency index for fish reared at 26°C. Based on these results, we conclude that greater amberjack fingerlings perform better at 26°C than at 22°C or 17°C, This work has been funded by the EU seventh Framework Programme by the DIVERSIFY project 7FP‐KBBE‐2013‐GA‐602131

The aim of this study was to evaluate the combination of maximum replacement of fishmeal (FM) and fish oil (FO) by alternative meals (AM) and oils (AO) to develop nutritious, but more eco-friendly diets for European sea bass ( Dicentrarchus labrax ) juveniles. European sea bass of 9.8 ± 1.5 g and 9.1 ± 0.5 cm were fed nine isonitrogenous (45% crude protein) and isolipidic (21%) diets containing gradually reduced levels of FM and FO as follows (%FM/%FO): 58/15, 20/6, 20/3, 10/6, 10/3, 5/6, 5/3 and 0/0. Another diet, 0/0 + , was similar to the 0/0 but supplemented with LC-PUFA from microbial and marine sources. After 90 days of feeding, European sea bass fed the 0/0 diet showed reduced (P + diet). Reduction of FM/FO down to 5/3 did not significantly affect fish survival or condition factor, whereas reduction to 5/6 did neither affect sea bass length or feed conversion and reduction down to 10/3 did not affected body weight, DGI and perivisceral fat or visceral weight. FM reductions down to 5% significantly reduced growth rate. FO substitution by vegetable oils (VO) led to reduced growth and increased liver to body weight ratios, liver lipid deposition and hepatocellular size. In liver and muscle, FO substitution increased n-6 fatty acids (FA) and n-6/n-3 ratio and reduced saturated FA and n-3 LC-PUFA, whereas monounsaturated FA were not increased in proportion to dietary contents. Moreover, FO reduction increased the products from Δ6 desaturase and elongases, Elovl6 and Elovl5. In conclusion, the results of this study show that it is possible to reduce dietary FM and FO levels down to 10% and 3% in mostly plant-based diets without affecting growth performance of European sea bass juveniles.

Early weaning of marine fish larvae with dry diets delays gut maturation and reduces growth rates. In juvenile and adult forms of several marine fish species, inclusion of dietary mannan oligosaccharides (MOS) improves gut integrity and functionality, but the effects of MOS inclusion in gilthead sea bream (Sparus aurata, L.) larval diets have not been addressed yet. Thus, this study assesses the effects of dietary MOS inclusion on survival, growth performance, gut morphology, feed acceptance and quality of gilthead sea bream larvae. For that purpose, 16 days post-hatched gilthead sea bream larvae were fed four graded levels of MOS (Biomos®, Alltech, Nicholasville, KY, USA) in weaning diets as follows: 0 g kg−1 MOS, 0.5 g kg−1 MOS, 1.5 g kg−1 MOS and 2 g kg−1MOS. Dietary MOS did not affect feed acceptance in gilthead sea bream larvae (P > 0.05). MOS supplementation was correlated in a dose-dependent way with higher larval survival (P = 0.026). After 15 days of feeding, dietary MOS increased whole larvae (P

The use of terrestrial raw materials to replace fish meal (FM) and fish oil (FO) in marine fish diets may affect fish growth performance and health. In the last years functional additives have been profiled as good candidates to reduce the effects on health and disease resistance derived from this replacement, via reinforcement of the fish immune system. In the present study, three isoenergetic and isonitrogenous diets with low FM and FO (10% and 6% respectively) were tested based on supplementation either with 0.5% galactomannanoligosaccharides (GMOS diet) or 0.02% of a mixture of essential oils (PHYTO diet), a non-supplemented diet was defined as a control diet. Fish were fed the experimental diets in triplicate for 9 weeks and then they were subjected to a stress by confinement as a single challenge (C treatment) or combined with an experimental intestinal infection with Vibrio anguillarum (CI treatment). Along the challenge test, selected stress and immunological parameters were evaluated at 2, 24 and 168h after C or CI challenges. As stress indicators, circulating plasma cortisol and glucose concentrations were analyzed as well as the relative gene expression of cyp11b hydroxylase, hypoxia inducible factor, steroidogenic acute regulatory protein, heat shock protein 70 and heat shock protein 90 (cyp11b, hif-1α, StAR, hsp70 and hsp90). As immune markers, serum and skin mucus lysozyme, bactericidal and peroxidase activities were measured, as well as gene expression of Caspase-3 (casp-3) and interleukin 1β (il-1ß). The use of functional additives induced a significant (p 0.05) reduction of circulating plasma cortisol concentration when confinement was the unique challenge test applied. Supplementation of PHYTO induced a down-regulation of cyp11b, hif-1α, casp-3 and il-1β gene expression 2h after stress test, whereas StAR expression was significantly (p 0.05) up-regulated. However, when combination of confinement stress and infection was applied (CI treatment), the use of PHYTO significantly (p 0.05) down-regulated StAR and casp-3 gene expression 2h after challenge test, denoting that PHYTO diet reinforced fish capacity of stress response via protection of head kidney leucocytes from stress-related apoptotic processes, with lower caspase-3 gene expression and a higher il-1β gene expression when an infection occurs. Additionally, dietary supplementation with GMOS and PHYTO compounds increased fish serum lysozyme after infection. Both functional additives entailed a better capability of the animals to cope with infection in European sea bass when fed low FM and FO diets.

There is an increasing interest from the aquafeed industry in functional feeds containing selected additives that improve fish growth performance and health status. Functional feed additives include probiotics, prebiotics, organic acids, and phytogenics (substances derived from plants and their extracts). This study evaluated the effects of dietary inclusion of a mucilage extract rich in galactomannan oligosaccharides (GMOS), a mixture of garlic and labiatae-plants oils (PHYTO), and a combination of them (GMOSPHYTO), on gut microbiota composition of European sea bass (Dicentrarchus labrax) fed with a low fishmeal (FM) and fish oil (FO) diet. Three experimental diets and a control diet (plant-based formulation with 10% FM and 6% FO) were tested in a 63-days feeding trial. To analyze the microbiota associated to feeds and the intestinal autochthonous (mucosa-adhered) and allochthonous (transient) microbial communities, the Illumina MiSeq platform for sequencing of 16S rRNA gene and QIIME2 pipeline were used. Metabarcoding analysis of feed-associated bacteria showed that the microbial communities of control (CTRL) feed deeply differed from those of experimental diets. The number of reads was significantly lower in CTRL feed than in other feeds. The OTU (operational taxonomic unit) number was instead similar between the feeds, ranging from 42 to 50 OTUs. The variation of resident gut microbiota induced by diet was lower than the variation of transient intestinal microbiota, because feedstuffs are a major source of allochthonous bacteria, which can temporarily integrate into the gut transient microbiome. However, the composition of transient bacterial communities was not simply a mirror of feed-borne bacteria. Indeed, the microbial profile of feeds was different from both faecal and mucosa profiles. Our findings suggest that the dietary inclusion of GMOS (0.5%) and PHYTO (0.02%) in a low FM and FO diet induces changes in gut microbiota composition of European sea bass. However, if on allochthonous microbiota the combined inclusion of GMOS and PHYTO showed an antagonistic effect on bactericidal activity against Vibrionales, at mucosa level, only GMOSPHYTO diet increased the relative abundance of Bacteroidales, Lactobacillales, and Clostridiales resident bacterial orders. The main beneficial effects of GMOS and PHYTO on gut microbiota are the reduction of coliforms and Vibrionales bacteria, which include several potentially pathogenic species for fish, and the enrichment of gut microbiota composition with butyrate producer taxa. Therefore, these functional ingredients have a great potential to be used as health-promoting agents in the farming of European sea bass and other marine fish.

The main objective of this study was to determine the effect of two feeding additives (cMOS: Actigen®, Alltech Inc., USA) and a phytogenic specifically designed for reducing ectoparasite incidence, combining garlic and labiatae herbal extracts (HERB; Delacon, Austria) on growth performance, stress and immune response of greater amberjack (Seriola dumerili) before and after an experimental parasitization with the skin ectoparasite Neobenedenia girellae, a monogenean with high incidence on Seriola cultured in sea cages. Two parallel trials were conducted in order to determine the effect of 70 days of functional diets supplementation (0.2% cMOS and 200 ppm HERB) on greater amberjack: a) Growth performance and stress resistance and b) disease resistance against N. girellae. Additionally, in order to evaluate the effects of the dietary supplements pre- and post- parasitization challenge against N. girellae, the expression of some immune-related genes was evaluated. The relative expression of Il-1β, piscidin, hepcidin, IgT, tnf-α, muc-2, cathelicidin, complement c3, cd8, and caspase-3 was determined in skin. The activity of protease and lysozyme in serum and skin mucus were also evaluated, as well as parasite load and parasite total size. The use of both functional additives did not affect fish growth performance. However, the dietary supplementation of HERB significantly (p In general, greater amberjack supplementation with cMOS and HERB at the dose used in the present study was effective for reducing stress and for increasing the skin associated lymphoid tissue (SALT) immune response against N. girellae during the infection process.

The main objective of this study was to assess the effects of graded levels of replacement of fishmeal (FM) and fish oil (FO) by terrestrial meals (TM) and vegetable oils (VO) on gut health of European sea bass (Dicentrarchus labrax) based on gut morphology, gut-associated immune system (GALT) and gut microbiota composition. Reducing the dietary FM and FO levels down to 10 and 3%, respectively did not affect the growth of European sea bass. Increase in TM and VO markedly affected anterior and posterior gut morphology and fatty acid composition, which mirrored the dietary fatty acid profile. FO replacement by VO increased lipid deposition in anterior gut lamina propria, denoting an effect in lipid metabolism and lipoprotein synthesis. In posterior gut, FM replacements by TM induced an engrossment of the submucosa, an up-regulation of the proinflammatory cytokines IL-1β and TNFα genes, a relatively high mucus production and changes in mucosal microbiota profiles. Finally, supplementation of 0%FM-0%FO diets with LC-PUFA contributed to maintain gut homeostasis in terms of GALT response and microbiota profiles alterations. Statement of relevance FM and FO appropriate dietary levels for European seabass

European sea bass were fed four low FM/FO (10%/6%) diets containing galactomannan oligosaccharides (GMOS), a mixture of garlic oil and labiatae plants oils (PHYTO), or a combination of both functional products (GMOSPHYTO) for 63 days before exposing the fish to an intestinal Vibrio anguillarum infection combined with crowding stress. In order to evaluate functional diets efficacy in terms of gut health maintenance, structural, cellular, and immune intestinal status were evaluated by optical and electron microscopy and gene expression analyses. A semi-automated software was adapted to determine variations in goblet cell area and mucosal mucus coverage during the challenge test. Feeding with functional diets did not affect growth performance; however, PHYTO and GMOS dietary inclusion reduced European sea bass susceptibility to V. anguillarum after 7 days of challenge testing. Rectum (post-ileorectal valve) showed longer (p = 0.001) folds than posterior gut (pre-ileorectal valve), whereas posterior gut had thicker submucosa (p = 0.001) and higher mucus coverage as a result of an increased cell density than rectum. Functional diets did not affect mucosal fold length or the grade of granulocytes and lymphocytes infiltration in either intestinal segment. However, the posterior gut fold area covered by goblet cells was smaller in fish fed GMOS (F = 14.53; p = 0.001) and PHYTO (F = 5.52; p = 0.019) than for the other diets. PHYTO (F = 3.95; p = 0.049) reduced posterior gut goblet cell size and increased rodlet cell density (F = 3.604; p = 0.068). Dietary GMOS reduced submucosal thickness (F = 51.31; p = 0.001) and increased rodlet cell density (F = 3.604; p = 0.068) in rectum. Structural TEM analyses revealed a normal intestinal morphological pattern, but the use of GMOS increased rectum microvilli length, whereas the use of PHYTO increased (p≤0.10) Ocln, N-Cad and Cad-17 posterior gut gene expression. After bacterial intestinal inoculation, posterior gut of fish fed PHYTO responded in a more controlled and belated way in terms of goblet cell size and mucus coverage in comparison to other treatments. For rectum, the pattern of response was similar for all dietary treatments, however fish fed GMOS maintained goblet cell size along the challenge test.

The main objective of this study was to evaluate the stress response of the greater amberjack, Seriola dumerili, by determining plasma and skin mucus cortisol levels, as physiological indicator of fish stress, and skin mucin-2 gene expression as indicator of skin mucin production, under three different potential stressors related to aquaculture practices: temperature, handling and fasting. For trial 1, 45 greater amberjack juveniles (19.5 ± 4.1 g) were divided in three graded rearing temperatures (17, 22 and 26 °C) and maintained for 120 days, in order to define the basal cortisol level for this species after a long-term thermal acclimation within the range described for this species. Trial 2 was conducted under two different rearing densities and two different handling protocols with 222 fish (425.02 ± 36.9 g) divided in 4 treatments by triplicate for 90 days, in order to study the effect of routine handling protocols on the selected stress markers for this species. Trial 3 used 72 fish (302.27 ± 21.4 g) divided in two experimental groups, one fed to apparent satiety and another one subjected to fasting for 10 days (triplicates for each group). After trials 2 and 3, a shallow water challenge was conducted to further study the adaptive response of greater amberjack juveniles to combined stress situations. Plasma and skin mucus were obtained for cortisol determination at 1, 24 and 72 h (trial 2) and 1, 3 and 24 h (trial 3) post stress challenge. Additionally, skin was dissected out for determining muc-2 gene expression. Long-term acclimation to temperature did not affect plasma or mucus cortisol levels and muc-2 gene expression, being basal levels of circulating plasma cortisol around 7.5 ng/mL. A high stocking density (8 kg m−3) induced an elevation (p

This work has been cofinanced by the “Agencia Canaria de investigacion, Innovacion y Sociedad de la Informacion de la Consejeria de Economia, Industria, Comercio y Conocimiento” and the European social funding, “Programa Operativo Integrado de Canarias 2014-2020”, as funding for the PhD grant of AFM. This project also received funding from the European Union Seventh Framework Programme for research, technological development and demonstration (KBBE-2013-07 single stage, GA 603121, DIVERSIFY).

This study evaluates the effects of dietary mannan oligosaccharides (MOS) on growth, tissue composition, fatty acid profiles and liver morphology of European sea bass (Dicentrarchus labrax) fed diets containing either soybean oil (SBO; SBOMOS) or fish oil (FO; FOMOS) as unique oil source for 8 weeks. Results showed that MOS supplementation enhanced specific growth rate, regardless of the oil source used, and that dietary oil source reduced fish length, regardless of dietary MOS supplementation. Dietary MOS favoured lipid accumulation in muscle and anterior intestine when supplemented in FO-based diets compared to fish fed SBO diet and reduces it in liver in relation to lower hepatocyte area, particularly in fish fed SBOMOS diet. Dietary MOS favoured liver and not muscular ∑n-3 PUFA, DHA, EPA and ARA deposition, when combined with FO but not when included in SBO-based diets. Thus, MOS dietary supplementation favours fish performance and helps to minimize the side effects derived from high dietary SBO supplementation on liver lipid accumulation and hepatocyte vacuolization, which could be of especial interest on long-term feeding trials; however, the effects on favoured deposition ∑n-3 PUFA are limited to FO-based diets.

The decreased availability of fish oil, traditionally used as oil source in marine aquafeeds, has lead to the search for alternatives oils. Vegetable oils (VO) are being extensively used as lipid sources in marine fish diets, inducing an imbalance on certain dietary fatty acids. Alteration on the dietary ratio of w-6/w-3 has been described to have detrimental effects on fish immunity. Senegalese sole has high susceptibility to stress and diseases, and little is known on the effects of dietary VO on its immunity. In this study, Senegalese sole juveniles were fed diets (56% crude protein, 12% crude lipid) containing linseed (100LO), soybean (100SO) or fish (100FO) oils as unique oil source. Growth, cortisol and intestinal fatty acid composition were determined after 90 days. Moreover, at the final of the experiment a stress test (5 min of net chasing) was carried out. To evaluate the effect of diets and stress on intestine immunology, expression profiles of a set of 53 immune-related genes using RT-qPCR was also performed. The use of VO did not induced changes in fish growth, but affected fatty acid profile of intestine and expression of immune-related genes. The use of SO (rich in n-6 fatty acids) induced an over-expression of those genes related to complement pathway, recognizing pathogen associated to molecular patterns, defensive response against bacteria, defensive response against viruses, antigen differentiation, cytokines and their receptors. This general over-expression could indicate an activation of inflammatory processes in fish gut. When a stress was applied, a decrease of mRNA levels of different immune-related genes with respect to the unstressed control could be observed in fish fed 100FO. However, fish fed 100LO, with a higher ALA/LA ratio, seemed to ameliorate the effects of combined effects of FO substitution plus stressful situation whereas fish fed 100SO did not show this type of response.

11 pages, 4 figures, 7 tables, The aim of this study was to assess the effects of dietary mannan oligosaccharides (MOS), Pediococcus acidilactici or their conjunction as a synbiotic in low fish meal (FM) and fish oil (FO) based diets on European sea bass (Dicentrarchus labrax) disease resistance and gut health. For that purpose, sea bass juveniles were fed one of 6 diets containing different combinations of MOS (Biomos® and Actigen©; Alltech, Inc., Kentucky, USA) and Pediococcus acidilactici (BAC, Bactocell®; Lallemand Inc., Cardiff, UK) replacing standard carbohydrates as follows (MOS (%)/BAC (commercial recommendation): high prebiotic level (HP) = 0.6/0, low prebiotic level (LP) = 0.3/0, only probiotic (B) = 0/+, high prebiotic level plus probiotic (HPB) = 0.6/+, low prebiotic level plus probiotic (LPB) = 0.3/+, control (C) = 0/0 for 90 days. After 60 and 90 days of feeding trial, fish were subjected to an experimental infection against Vibrio anguillarum. Additionally, inducible nitric oxide synthase (iNOS) and tumor necrosis factor α (TNFα) gut patterns of immunopositivity and major histocompatibility complex class II (MHCII), transforming growth factor β (TGF-β), regulatory T-cell subset (CD4+T lymphocytes) and effector T cell (CD8α+ T lymphocytes) gene expression patterns in gut by in situ hybridization were evaluated after 90 days of feeding. The effects of both additives on posterior gut through Gut Associated Lymphoid Tissue (GALT) gene expression was also studied. Fish fed the prebiotic and its combination with P. acidilactici presented increased weight regardless of the dose supplemented after 90 days of feeding, however no effect was detected on somatic indexes. For posterior gut, morphometric patterns and goblet cells density was not affected by MOS, P. acidilactici or its combination. Anti-iNOS and anti-TNFα gut immunopositivity patterns were mainly influenced by MOS supplementation and not by its combination with P. acidilactici. MHCII-β, TCR-β, CD4 and CD8-α positive cells distribution and incidence was not affected by diet. Fish fed HP dose presented a clear up-regulation of TNF-α, cyclooxygenase-2 (COX-2), CD4 and IL10, whereas P. acidilactici dietary supplementation increased the number of interleukin-1β (IL1β) and COX-2 gene transcripts. Synbiotic supplementation resulted in a reduction of MOS-induced gut humoral proinflammatory response by increasing the expression of some cellular-immune system related genes. Fish mortality after V. anguillarum infection was reduced in fish fed LPB and LP diets compared to fish fed the non-suppelmented diet after 90 days of feeding. Thus, overall pointing to the combination of a low dose of MOS and P. acidilactici as synbiont (LPB) as a viable tool to potentiate European sea bass juvenile's growth and disease resistance when supplemented in low FM and FO diets., This work was funded by the Spanish Ministry of Economy and Competitiveness (MINECO, “Subprograma de proyectos de investigación fundamental no orientada, call 2012”) by the PROINMUNOIL (AGL2012-39919) project: “Functional additives & alternative oils to fish oils in Aquaculture: an effective tool increase fish disease resistance”. We also acknowledge the complementary funding through a ULPGC predoctoral and postdoctoral fellowships for FR and ST. SBP and RJ were supported by the Spanish Economy and Competitiveness Ministry project AGL2014-52473R.

This study aimed to determine the effects of increasing docosahexaenoic acid (DHA) levels in Sparus aurata diets supplemented with antioxidants vitamins (vitamins C and E). Six isoproteic and isolipidic formulated diets, which varied in the DHA content (38–78 g kg−1 TFA), were fed to 19 dph S. aurata larvae. No significant differences were found in survival rates or growth, which indicates that all the diets covered the minimum essential fatty acid requirements of this species. Stress resistance was not negatively affected by dietary DHA contents. In agreement, two biomarkers of the oxidative stress of the fish and the gene expression of superoxide dismutase and glutathione peroxidase were not affected by the elevation of dietary DHA, denoting that fish were sufficiently protected from oxidative damage even at high dietary DHA contents. The elevation of dietary DHA did affect neither the occurrence of skeletal malformations nor the expression of osteological markers such as run-related transcription factor 2 or alkaline phosphatase. The results of this study have confirmed that increase of the dietary DHA levels in gilthead sea bream diets does not negatively affect larval performance if the larvae are protected from peroxidation risks. Despite not being harmful, increase of DHA over the recommended requirements does not have any significant improvement in larval performance.

Substituting fishmeal (FM) with vegetable meal (VM) can markedly affect the mineral composition of feeds, and may require additional mineral supplementation. Their bioavailability and optimal supplementation levels depend also on the form of delivery of minerals. The aim of the study was to determine the effect of different delivery forms of three major trace elements (Zn, Mn and Se) in a marine teleost. Gilthead sea bream juveniles of 22.5 g were fed a VM-based diet for 12 weeks that was either not supplemented with these minerals or supplemented with inorganic, organic, or encapsulated inorganic forms of minerals in triplicate and compared to a FM-based diet. Our results showed that mineral delivery form significantly affected the biochemical composition and morphology of posterior vertebrae. Supplementation of VM-based diets with inorganic forms of the target minerals significantly promoted growth, increased the vertebral weight and content of ash and Zn, enhanced bone mineralization and affected the vertebral shape. Conversely, encapsulation of inorganic minerals reduced fish growth and vertebral mineral content, whereas supplementation of organic minerals, enhanced bone osteogenesis by upregulating bone morphogenetic protein 2 (bmp2)gene and produced vertebrae with a larger length in relation to height. Furthermore, organic mineral forms of delivery downregulated the expression of oxidative stress related genes, such as Cu/Zn superoxide dismutase (Cu/Znsod) and glutathione peroxidase 1 (gpx-1),suggesting thus that dietary minerals supplemented in the organic form could be reasonably considered more effective than the inorganic and encapsulated forms of supply.

Sex differences between men and women in social anxiety are largely unexplored. This study sought to shed some light on this topic. We administered self-report measures of social anxiety to community samples of 17,672 women and 13,440 men from 16 Latin American countries, Spain and Portugal, as well as to a clinical sample of 601 patients diagnosed with social anxiety disorder. Small but significant differences were found between men and women in the general degree of social anxiety and self-reported fears of interactions with the opposite sex, criticism and embarrassment, and speaking in public-talking to people in authority. These results point to small, but meaningful differences between men and women in social anxiety. Implications of these results for the self-report measurement of social anxiety in men and women are discussed.

The aim of this study was to assess the effects of low levels of dietary fish meal (FM) and fish oil (FO) on disease resistance and gut associated lymphoid tissue (GALT) response after an experimental intestinal infection with V. anguillarum in European sea bass (Dicentrarchus labrax) For that purpose, sea bass juveniles were fed one of four diets containing combined levels of FO and FM as follows: 20%FM/6%FO, 20%FM/3%FO, 5%FM/6%FO and 5%FM/3%FO during 153 days. At the end of the feeding trial, fish were subjected to either an in vivo exposure to a sub-lethal dose of V. anguillarum via anal inoculation or to an ex vivo exposure to V. anguillarum. Additionally, inducible nitric oxide synthase (iNOS) and tumor necrosis factor α (TNFα) gut patterns of immunopositivity were studied. Growth performance was affected by dietary FM level, however ex vivo gut bacterial translocation rates and survival after the in vivo challenge test were affected by dietary FO level. After 5 months of feeding, low dietary FM levels led to a posterior gut up-regulation of interleukin-1β (IL-1β) and TNFα, major histocompatibility complex-II (MHCII) and cyclooxygenase-2 (COX2), which in turn reduced the gut associated lymphoid tissue (GALT) capacity of response after 24 h post infection and conditioned European sea bass capacity to recover gut homeostasis 7 days post infection. Immunoreactivity to anti-iNOS and anti-TNFα presented a gradient of increased immunopositivity towards the anus, regardless of the dietary FM/FO fed. Strong positive anti-TNFα isolated enterocytes were observed in the anterior gut in relation to low levels of dietary FM/FO. Submucosa and lamina propria immunoreactivity grade was related to the amount of leucocyte populations infiltrated and goblet cells presented immunopositivity to anti-iNOS but not to anti-TNFα. Thus, reducing FO content from 6% to a 3% by VO in European sea bass diets increases ex vivo and in vivo gut bacterial translocation rates, whereas reducing FM content from 20% down to 5% up-regulates the expression of several posterior gut inflammation-related genes conditioning fish growth and GALT capacity of response after bacterial infection.

The main objective of this study was to assess the effects of graded levels of dietary arachidonic acid (ARA), supplemented from alternative sources, on fatty acid composition of plasma and head kidney leucocytes of European sea bass (Dicentrarchus labrax). For that purpose, sea bass juveniles were fed four diets containing graded levels of ARA as follows: 0.5% (ARA0.5), 1% (ARA1), 2% (ARA2) and 4% (ARA4) during 60 days. At the end of the feeding trial fatty acid profiles of plasma and head kidney leucocytes were analyzed. Besides, plasma prostaglandins levels, head kidney leucocytes respiratory burst activity; peroxidase activity and phagocytic index were assayed. Reducing dietary ARA levels below 1% markedly reduced European sea bass growth performance. However, fish fed diet ARA0.5 tried to compensate this dietary ARA deficiency by a selective deposition of ARA on plasma and head kidney leucocytes, reaching similar levels to those fish fed diet ARA1 after 60 days of feeding. Nevertheless, head kidney phagocytic capacity was reduced as dietary ARA content in relation not only to variations on membrane composition but also to changes on fish basal prostaglandins levels. Results obtained demonstrated the importance to supply the necessary quantity n-6 LC-PUFA, and not only n-3 LC-PUFA levels, in European sea bass diets, in relation to not only growth performance but also immune system function.

The effect conjugated linoleic acid (CLA) accumulation in fish fillet on sensory and texture attributes has been evaluated. Fish were fed diets with graded levels of CLA, which were administered for different durations prior to harvest. Fish were sampled for fillet proximate composition and fatty acid profile for sensory and texture parameters at the end of the trial. Growth performance, feed conversion ratio and muscle proximate composition were not affected by dietary treatments. Muscle fatty acid profile was not affected by dietary treatments despite the increase in saturated fatty acids in fish fed diet with 10 g kg−1 of CLA for 16 weeks before harvest. Besides, the deposition levels of CLA isomers increased gradually depending on the CLA treatment, reaching its maximum level in fish fed 10 g kg−1 CLA diet for 20 weeks before harvest. Fish fed 10 g kg−1 were juicier (P

Substituting fishmeal (FM) with vegetable meal (VM) can markedly affect the mineral composition of feeds, and may require additional mineral supplementation. Their bioavailability and optimal supplementation levels depend also on the form of delivery of minerals. The aim of the study was to determine the effect of different delivery forms of three major trace elements (Zn, Mn and Se) in a marine teleost. Gilthead sea bream juveniles of 22.5 g were fed a VM-based diet for 12 weeks that was either not supplemented with these minerals or supplemented with inorganic, organic, or encapsulated inorganic forms of minerals in triplicate and compared to a FM-based diet. Our results showed that mineral delivery form significantly affected the biochemical composition and morphology of posterior vertebrae. Supplementation of VM-based diets with inorganic forms of the target minerals significantly promoted growth, increased the vertebral weight and content of ash and Zn, enhanced bone mineralization and affected the vertebral shape. Conversely, encapsulation of inorganic minerals reduced fish growth and vertebral mineral content, whereas supplementation of organic minerals, enhanced bone osteogenesis by upregulating bone morphogenetic protein 2 (bmp2) gene and produced vertebrae with a larger length in relation to height. Furthermore, organic mineral forms of delivery downregulated the expression of oxidative stress related genes, such as Cu/Zn superoxide dismutase (Cu/Zn sod) and glutathione peroxidase 1 (gpx-1), suggesting thus that dietary minerals supplemented in the organic form could be reasonably considered more effective than the inorganic and encapsulated forms of supply.