Proteomic profile and protease activity in the skin mucus of greater amberjack (Seriola dumerili) infected with the ectoparasite Neobenedenia girellae - An immunological approach.

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.

Increased parasite resistance of greater amberjack (Seriola dumerili Risso 1810) juveniles fed a cMOS supplemented diet is associated with upregulation of a discrete set of immune genes in mucosal tissues.

The main objective of this study was to determine the effect of two forms of mannan oligosaccharides (MOS: Bio-Mos® and cMOS: Actigen®, Alltech Inc, USA) and their combination on greater amberjack (Seriola dumerili) growth performance and feed efficiency, immune parameters and resistance against ectoparasite (Neobenedenia girellae) infection. Fish were fed for 90 days with 5 g kg-1 MOS, 2 g kg-1 cMOS or a combination of both prebiotics, in a Seriola commercial base diet (Skretting, Norway). At the end of the feeding period, no differences were found in growth performance or feed efficiency. Inclusion of MOS also had no effect on lysozyme activity in skin mucus and serum, but the supplementation of diets with cMOS induced a significant increase of serum bactericidal activity. Dietary cMOS also reduced significantly greater amberjack skin parasite levels, parasite total length and the number of parasites detected per unit of fish surface following a cohabitation challenge with N. girellae, whereas no effect of MOS was detected on these parameters. Of 17 immune genes studied cMOS dietary inclusion up-regulated hepcidin, defensin, Mx protein, interferon-γ (IFNγ), mucin-2 (MUC-2), interleukin-1ß (IL-1B), IL-10 and immunoglobulin-T (IgT) gene expression in gills and/or skin. MOS supplementation had a larger impact on spleen and head kidney gene expression, where piscidin, defensin, iNOS, Mx protein, interferons, IL-1ß, IL-10, IL-17 and IL-22 were all upregulated. In posterior gut dietary MOS and cMOS both induced IL-10, IgM and IgT, but with MOS also increasing piscidin, MUC-2, and IL-1ß whilst cMOS induced hepcidin, defensin and IFNγ. In general, the combination of MOS and cMOS resulted in fewer or lower increases in all tissues, possibly due to an overstimulation effect. The utilization of cMOS at the dose used here has clear benefits on parasite resistance in greater amberjack, linked to upregulation of a discrete set of immune genes in mucosal tissues.

Effects of thermal stress on the expression of glucocorticoid receptor complex linked genes in Senegalese sole (Solea senegalensis): Acute and adaptive stress responses.

The present study examined the short and mid-term effects of a rise in temperature from 18°C to 24°C on the expression of genes related to the stress response regulation in juveniles of Senegalese sole, Solea senegalensis. The animals were exposed to a temperature increase of 6°C, after 1month of acclimation at 18°C. After this process, samples of different tissues were collected from a total of 96 fish at four sampling points: 1h, 24h, 3days and 1week. The transcript levels of a set of genes involved in the stress response such as glucocorticoid receptors 1 and 2, corticotrophin-releasing factor, corticotrophin-releasing factor binding proteins, proopiomelanocortin A and B, and cellular stress defense (heat shock protein 70, 90AA and 90AB) were quantified at these sampling points. Additionally, blood samples were also taken to measure the circulating plasma cortisol concentration. Thermal stress induced by increasing temperature prompted an elevation of plasma cortisol levels in juvenile Senegalese sole after 1h as a short-term response, and a consecutive increase after one week, as a mid-term response. Senegalese sole seemed to respond positively in terms of adaptive mechanisms, with a rapid over-expression of grs and hsps in liver and brain, significantly higher after one hour post stress, denoting the fast and acute response of those tissues to a rapid change on temperature. The ratio hsp90/gr also increased 24h after thermal shock, ratio proposed to be an adaptive mechanism to prevent proteosomal degradation of GR. As a mid-term response, the elevation of brain crfbp gene expression one week after thermal shock could be an adaptive mechanism of negative feedback on HPI axis. Taken together, these data suggested an initial up-regulation of the glucocorticoid receptor complex linked genes in response to a temperature increase in Senegalese sole, with heat shock protein 90 potentially being a regulatory factor for the glucocorticoid receptor in the presence of cortisol.

Effects of dietary concentrated mannan oligosaccharides supplementation on growth, gut mucosal immune system and liver lipid metabolism of European sea bass (Dicentrarchus labrax) juveniles.

The study assesses the effects of dietary concentrated mannan oligosaccharides (cMOS) on fish performance, biochemical composition, tissue fatty acid profiles, liver and posterior gut morphology and gen expression of selected parameters involved on the intestinal immune response and liver lipid metabolism of European sea bass (Dicentrarchus labrax). For that purpose, specimens of 20 g were fed during 8 weeks at 0 and 1.6 g kg(-1) dietary cMOS of inclusion in a commercial sea bass diet. Dietary cMOS enhanced fish length, specific and relative growth without affecting tissue proximate composition. However, cMOS supplementation altered especially liver and muscle fatty acid profiles by reducing levels of those fatty acids that are preferential substrates for ß-oxidation in spite of a preferential retention of long chain polyunsaturated fatty acids (LC-PUFA), such as 20:4n-6 or 22:5n-6, in relation to the down-regulation of delta 6/5 desaturase gene expression found in liver. Besides, dietary cMOS supplementation reduced posterior gut intestinal folds width and induced changes on the gene expression level of certain immune-related genes mainly by down regulating transforming growth factor ß (TGFß) and up-regulating immunoglobulin (Ig), major histocompatibility complex class II (MHCII), T cell receptor ß (TCRß) and Caspase 3 (Casp-3). Thus, dietary cMOS inclusion at 0.16% promoted European sea bass specific growth rate and length, stimulated selected cellular GALT-associated parameters and affected lipid metabolism in muscle and liver pointing to a higher LC-PUFA accumulation and promoted ß-oxidation.

Dietary vegetable oils: effects on the expression of immune-related genes in Senegalese sole (Solea senegalensis) intestine.

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.

Improved health and growth of fish fed mannan oligosaccharides: potential mode of action.

Nowadays, aquaculture industry still confronts several disease-related problems mainly caused by viruses, bacteria and parasites. In the last decade, the use of mannan oligosaccharides (MOS) in fish production has received increased attention due to its beneficial effects on fish performance and disease resistance. This review shows the MOS use in aquaculture with a specific emphasis on the effectiveness of the several MOS forms available in the market related to disease resistance, fish nutrition and the possible mechanisms involved. Among the main beneficial effects attributed to MOS dietary supplementation, enhanced fish performance, feed efficiency and pathogen protection by potentiation of the systemic and local immune system and the reinforcement of the epithelial barrier structure and functionality are some of the most commonly demonstrated benefits. These combined effects suggest that the reinforcement of the intestinal integrity and functionality, together with the stimulation of the innate immune system, are the primary mode of action of MOS in fish. However, the supplementation strategy related to the structure of the MOS added, the correct dose and duration, as well as fish species, size and culture conditions are determinant factors to achieve improvements in health status and growth performance.

Comparative study of the gut microbial communities collected by scraping and swabbing in a fish model: a comprehensive guide to promote non-lethal procedures for gut microbial studies.

In the present study, we propose the use of swabs in non-lethal sampling procedures to collect the mucosa-adhered gut microbiota from the posterior intestine of fish, and therefore, we compare the bacterial communities collected by conventional scraping and by swabbing methods. For this purpose, samples of the posterior intestine of rainbow trout (Oncorhynchus mykiss) were collected first using the swabbing approach, and after fish euthanasia, by mucosa scraping. Finally, bacterial communities were compared by 16S rRNA gene Illumina sequencing. Results from the current study revealed that similar values of bacterial richness and diversity were found for both sampling procedures. Similarly, there were no differences between procedures when using qualitative metrics (Jaccard and unweighted UniFrac) for estimating inter-individual diversity, but the quantitative metrics (Bray-Curtis and weighted UniFrac) showed a higher dispersion when samples were obtained by swabbing compared to scraping. In terms of bacterial composition, there were differences in abundance for the phyla Firmicutes and Proteobacteria. The cause of these differential abundances may be the inability of the swab to access to certain areas, such as the basal region of the intestinal villi. Moreover, swabbing allowed a higher representation of low abundant taxa, which may also have an important role in host microbiome regardless of their low abundance. Overall, our results demonstrate that the sampling method is a factor to be considered in experimental design when studying gut bacterial communities to avoid potential biases in the interpretation or comparison of results from different studies. In addition, the advantages and disadvantages of each procedure (swabbing vs scraping) are discussed in detail, concluding that swabbing can be implemented as a reliable and non-lethal procedure for posterior gut microbiota studies, which is of particular interest for animal welfare and the 3Rs principle, and may offer a wide range of novel applications.

An In-Depth Study on the Inhibition of Quorum Sensing by Bacillus velezensis D-18: Its Significant Impact on Vibrio Biofilm Formation in Aquaculture.

Amid growing concerns about antibiotic resistance, innovative strategies are imperative in addressing bacterial infections in aquaculture. Quorum quenching (QQ), the enzymatic inhibition of quorum sensing (QS), has emerged as a promising solution. This study delves into the QQ capabilities of the probiotic strain Bacillus velezensis D-18 and its products, particularly in Vibrio anguillarum 507 communication and biofilm formation. Chromobacterium violaceum MK was used as a biomarker in this study, and the results confirmed that B. velezensis D-18 effectively inhibits QS. Further exploration into the QQ mechanism revealed the presence of lactonase activity by B. velezensis D-18 that degraded both long- and short-chain acyl homoserine lactones (AHLs). PCR analysis demonstrated the presence of a homologous lactonase-producing gene, ytnP, in the genome of B. velezensis D-18. The study evaluated the impact of B. velezensis D-18 on V. anguillarum 507 growth and biofilm formation. The probiotic not only controls the biofilm formation of V. anguillarum but also significantly restrains pathogen growth. Therefore, B. velezensis D-18 demonstrates substantial potential for preventing V. anguillarum diseases in aquaculture through its QQ capacity. The ability to disrupt bacterial communication and control biofilm formation positions B. velezensis D-18 as a promising eco-friendly alternative to conventional antibiotics in managing bacterial diseases in aquaculture.

Enhanced intestinal epithelial barrier health status on European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides.

The study assesses the effects of dietary mannan oligosaccharides (MOS) in European sea bass (Dicentrarchus labrax) posterior intestinal lipid class composition and its possible relation to the potential prostaglandins production and Gut Associated Lymphoid Tissue (GALT) stimulation. Fish were fed 4 g kg(-1) MOS (Bio-Mos(®) Aquagrade, Alltech, Inc., USA) for eight weeks. Fish fed MOS presented higher (P ≤ 0.05) weight gain, total length, and specific and relative growth rates than fish fed the control diet. Stimulated posterior gut of fish fed MOS showed higher (P ≤ 0.05) prostaglandins production than fish fed the control diet. Lipid class analyses of posterior gut revealed a reduction (P ≤ 0.05) in the neutral lipid fraction in fish fed MOS compared to fish fed the control diet, particularly due to a reduction (P ≤ 0.05) in triacylglycerols content. The polar lipid fraction increased (P ≤ 0.05) in fish fed MOS compared to fish fed the control diet, mainly due to an increase (P ≤ 0.05) in phosphatidylethanolamine and phosphatidylcoline contents. Light microscopy of posterior gut revealed increased number or goblet cells as well as higher level of infiltrated eosinophilic granulocytes for fish fed MOS. Transmission electron microscopy qualitative observations revealed a better preserved cytoarchitecture of the intestinal epithelial barrier in the posterior gut of fish fed MOS. Posterior gut of fish fed MOS presented more densely packed non-damaged enterocytes, better preserved tight junctions structure, healthier and more organized microvilli, and a higher presence of infiltrated lymphocytes and granulocytes compared fish fed the control diet. The present study indicates that dietary MOS enhances European sea bass posterior gut epithelial defense by increasing membrane polar lipids content in relation to a stimulation of the eicosanoid cascade and GALT, promoting posterior gut health status.

Functional Additives in a Selected European Sea Bass (Dicentrarchus labrax) Genotype: Effects on the Stress Response and Gill Antioxidant Response to Hydrogen Peroxide (H2O2) Treatment.

Functional ingredients have profiled as suitable candidates for reinforcing the fish antioxidant response and stress tolerance. In addition, selective breeding strategies have also demonstrated a correlation between fish growth performance and susceptibility to stressful culture conditions as a key component in species domestication processes. The aim of the present study is to evaluate the ability of a selected high-growth genotype of 300 days post-hatch European sea bass (Dicentrarchus labrax) juveniles to use different functional additives as endogenous antioxidant capacity and stress resistance boosters when supplemented in low fish meal (FM) and fish oil (FO) diets. Three isoenergetic and isonitrogenous diets (10% FM/6% FO) were supplemented with 200 ppm of a blend of garlic and Labiatae plant oils (PHYTO0.02), 1000 ppm of a mixture of citrus flavonoids and Asteraceae and Labiatae plant essential oils (PHYTO0.1) or 5000 ppm of galactomannan-oligosaccharides (GMOS0.5). A reference diet was void of supplementation. The fish were fed the experimental diets for 72 days and subjected to a H2O2 exposure oxidative stress challenge. The fish stress response was evaluated through measuring the circulating plasma cortisol levels and the fish gill antioxidant response by the relative gene expression analysis of nfΚß2, il-1b, hif-1a, nd5, cyb, cox, sod, cat, gpx, tnf-1α and caspase 9. After the oxidative stress challenge, the genotype origin determined the capacity of the recovery of basal cortisol levels after an acute stress response, presenting GS fish with a better pattern of recovery. All functional diets induced a significant upregulation of cat gill gene expression levels compared to fish fed the control diet, regardless of the fish genotype. Altogether, suggesting an increased capacity of the growth selected European sea bass genotype to cope with the potential negative side-effects associated to an H2O2 bath exposure.

More Than an Antioxidant: Role of Dietary Astaxanthin on Lipid and Glucose Metabolism in the Liver of Rainbow Trout (Oncorhynchus mykiss).

This study investigated the influence of dietary astaxanthin (AX) on glucose and lipid metabolism in rainbow trout liver. Two iso-nitrogenous and iso-lipidic diets were tested for 12 weeks in rainbow trout with an initial mean weight of 309 g. The S-ASTA diet was supplemented with 100 mg of synthetic AX per kg of feed, whereas the control diet (CTRL) had no AX. Fish fed the S-ASTA diet displayed lower neutral and higher polar lipids in the liver, associated with smaller hepatocytes and lower cytoplasm vacuolization. Dietary AX upregulated adipose triglyceride lipase (atgl), hormone-sensitive lipase (hsl2) and 1,2-diacylglycerol choline phosphotransferase (chpt), and downregulated diacylglycerol acyltransferase (dgat2), suggesting the AX's role in triacylglycerol (TAG) turnover and phospholipid (PL) synthesis. Dietary AX may also affect beta-oxidation with the upregulation of carnitine palmitoyltransferase 1 (cpt1α2). Although hepatic cholesterol levels were not affected, dietary AX increased gene expression of sterol regulatory element-binding protein 2 (srebp2). Dietary AX upregulated the expression of 6-phosphogluconate dehydrogenase (6pgdh) and downregulated pyruvate kinase (pkl). Overall, results suggest that dietary AX modulates the oxidative phase of the pentose phosphate pathway and the last step of glycolysis, affecting TAG turnover, ß-oxidation, PL and cholesterol synthesis in rainbow trout liver.

Dietary Debaryomyces hansenii promotes skin and skin mucus defensive capacities in a marine fish model.

The present study explores the effects of two supplementation levels of Debaryomyces hansenii (1.1% and 2.2%) as a probiotic in a reference low fish meal-based diet on the skin mucosal tissue in Sparus aurata. This study includes the evaluation of fish performance coupled with a holistic study of the skin mucosa: i) a transcriptomic study of the skin tissue, and ii) the evaluation of its secreted mucus both in terms of skin mucosal-associated biomarkers and its defensive capacity by means of co-culture analysis with two pathogenic bacteria. Results showed that after 70 days of diet administration, fish fed the diet supplemented with D. hansenii at 1.1% presented increased somatic growth and a better feed conversion ratio, compared to fish fed the control diet. In contrast, fish fed the diet including 2.2% of the probiotic presented intermediate values. Regarding gene regulation, the probiotic administration at 1.1% resulted in 712 differentially expressed genes (DEGs), among which 53.4% and 46.6% were up- and down-regulated, respectively. In particular, D. hansenii modulated some skin biological processes related to immunity and metabolism. Specifically, D. hansenii administration induced a strong modulation of some immune biological-related processes (61 DEGs), mainly involved in B- and T-cell regulatory pathways. Furthermore, dietary D. hansenii promoted the skin barrier function by the upregulation of anchoring junction genes (23 DEGs), which reinforces the physical defense against potential skin damage. In contrast, the skin showed modulated genes related to extracellular exosome and membrane organization (50 DEGs). This modulated functioning is of great interest, particularly in relation to the increased skin mucus defensive capacity observed in the bacterial co-culture in vitro trials, which could be related to the increased modulation and exudation of the innate immune components from the skin cells into the mucus. In summary, the modulation of innate immune parameters coupled with increased skin barrier function and cell trafficking potentiates the skin's physical barrier and mucus defensive capacity, while maintaining the skin mucosa's homeostatic immune and metabolic status. These findings confirmed the advantages of D. hansenii supplementation in low fish meal-based diets, demonstrating the probiotic benefits on cultured marine species.

Increased Mauthner cell activity and escaping behaviour in seabream fed long-chain PUFA.

There is limited information on the specific effects of long-chain PUFA (LCPUFA) on neuron development and functioning. Deficiency of those essential fatty acids impairs escape and avoidance behaviour in fish, where Mauthner cells (M-cells) play a particularly important role in initiating this response. Gilthead seabream larvae fed two different LCPUFA profiles were challenged with a sonorous stimulus. Feeding n-3 LCPUFA increased the content of these fatty acids in fish tissues and caused a higher number of larvae to react to the stimulus with a faster burst swimming speed response. This faster startle response in fish fed n-3 LCPUFA was also associated with an increased immune-positive neural response, particularly in M-cells, denoting a higher production of acetylcholine. The present study shows the first evidence of the effect of n-3 LCPUFA on the functioning of particular neurons in fish, the M-cells and the behaviour response that they modulate to escape from a sound stimulus.

Gill Oxidative Stress Protection through the Use of Phytogenics and Galactomannan Oligosaccharides as Functional Additives in Practical Diets for European Sea Bass (Dicentrarchus labrax) Juveniles.

The aim of the present study is to evaluate the potential of two functional additives as gill endogenous antioxidant capacity boosters in European sea-bass juveniles fed low-FM/FO diets when challenged against physical and biological stressors. For that purpose, two isoenergetic and isonitrogenous diets with low FM (10%) and FO (6%) contents were supplemented with 5000 ppm plant-derived galactomannan-oligosaccharides (GMOS) or 200 ppm of a mixture of garlic and labiate plant essential oils (PHYTO). A control diet was void from supplementation. Fish were fed the experimental diet for nine weeks and subjected to a confinement stress challenge (C challenge) or a confinement stress challenge combined with an exposure to the pathogen Vibrio anguillarum (CI challenge). Both GMOS and PHYTO diets attenuated fish stress response, inducing lower circulating plasma cortisol and down-regulating nfκß2 and gr relative gene-expression levels in the gill. This attenuated stress response was associated with a minor energetic metabolism response in relation to the down-regulation of nd5 and coxi gene expression.

Impact of polypropylene microplastics and chemical pollutants on European sea bass (Dicentrarchus labrax) gut microbiota and health.

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.

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

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

Reduced gut bacterial translocation in European sea bass (Dicentrarchus labrax) fed mannan oligosaccharides (MOS).

The objective of this study was to determine the effect of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on mucus production, selected mucus immune parameters activity, gut morphology and in vivo and ex vivo gut bacterial translocation for European sea bass (Dicentrarchus labrax). Specimens were fed 4 g kg⁻¹ dietary MOS level of inclusion in a commercial sea bass diet for eight weeks. At the end of this period, anterior gut mucosal folds height, width and folds surface area were increased by MOS supplementation (P < 0.05, n = 240). Posterior gut presented shorter folds (P < 0.05, n = 240) but wider that those fed control diet (P < 0.05, n = 240) resulting in increased total surface area (P < 0.05, n = 240). For rectum, feeding MOS reduced fold length (P < 0.05, n = 240). Gut morphological analyses showed an enhancement in the number of cells secreting acid mucins by area unit, higher density of eosinophilic granulocytes (ECGs) in the mucosa for fish fed MOS together with an improvement in gut mucus lysozyme activity which could be related to the reduced in vivo and ex vivo gut bacterial translocation found. No differences were found for the skin mucus immune parameters evaluated.

Dietary Phytogenics and Galactomannan Oligosaccharides in Low Fish Meal and Fish Oil-Based Diets for European Sea Bass (Dicentrarchus labrax) Juveniles: Effects on Gill Structure and Health and Implications on Oxidative Stress Status.

An effective replacement for fish meal (FM) and fish oil (FO) based on plant-based raw materials in the feed of marine fish species is necessary for the sustainability of the aquaculture sector. However, the use of plant-based raw materials to replace FM and FO has been associated with several negative health effects, some of which are related to oxidative stress processes that can induce functional and morphological alterations in mucosal tissues. This study aimed to evaluate the effects of dietary oligosaccharides of plant origin (5,000 ppm; galactomannan oligosaccharides, GMOS) and a phytogenic feed additive (200 ppm; garlic oil and labiatae plant extract mixture, PHYTO) on the oxidative stress status and mucosal health of the gills of juvenile European sea bass (Dicentrarchus labrax). The experimental diets, low FM and FO diets (10%FM/6%FO) were supplemented with GMOS from plant origin and PHYTO for 63 days. GMOS and PHYTO did not significantly affect feed utilization, fish growth, and survival. GMOS and PHYTO downregulated the expression of ß-act, sod, gpx, cat, and gr in the gills of the fish compared with that in fish fed the control diet. The expression of hsp70 and ocln was upregulated and downregulated, respectively, in the GMOS group compared with that in the control group, whereas the expression of zo-1 was downregulated in the PHYTO group compared with that in the GMOS group. The morphological, histopathological, immunohistochemical, and biochemical parameters of the fish gills were mostly unaffected by GMOS and PHYTO. However, the PHYTO group had lower incidence of lamellar fusion than did the control group after 63 days. Although the tissular distribution of goblet cells was unaffected by GMOS and PHYTO, goblet cell size showed a decreasing trend (-11%) in the GMOS group. GMOS and PHYTO significantly reduced the concentration of PCNA+ in the epithelium of the gills. The above findings indicated that GMOS and PHYTO in low FM/FO-based diets protected the gill epithelia of D. labrax from oxidative stress by modulating the expression of oxidative enzyme-related genes and reducing the density of PCNA+ cells in the gills of the fish.

Isolation and Characterization of a Bacillus velezensis D-18 Strain, as a Potential Probiotic in European Seabass Aquaculture.

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.

Assessment of dietary supplementation with galactomannan oligosaccharides and phytogenics on gut microbiota of European sea bass (Dicentrarchus Labrax) fed low fishmeal and fish oil based diet.

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.