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Bacillus spp. supplementation as probiotics in cultured fish diets has a long history of safe and effective use. Specifically, B. velezensis show great promise in fine-tuning the European sea bass disease resistance against the pathogenicity caused by several members of the Vibrio family. However, the immunomodulatory mechanisms behind this response remain poorly understood. Here, to examine the inherent immune variations in sea bass, two equal groups were fed for 30 days with a steady diet, with one treatment supplemented with B. velezensis. The serum bactericidal capacity against live cells of Vibrio anguillarum strain 507 and the nitric oxide and lysozyme lytic activities were assayed. At the cellular level, the phagocytic response of peripheral blood leukocytes against inactivated Candida albicans was determined. Moreover, head-kidney (HK) total leukocytes were isolated from previously in vivo treated fish with LPS of V. anguillarum strain 507. Mechanistically, the expression of some essential proinflammatory genes (interleukin-1 (il1b), tumor necrosis factor-alpha (tnfa), and cyclooxygenase 2 (cox2) and the sea bass specific antimicrobial peptide (AMP) dicentracin (dic) expressions were assessed. Surprisingly, the probiotic supplementation significantly increased all humoral lytic and cellular activities assayed in the treated sea bass. In addition, time-dependent differences were observed between the control and probiotic treated groups for all the HK genes markers subjected to the sublethal LPS dose. Although the il1b was the fastest responding gene to a significant level at 48 h post-injection (hpi), all the other genes followed 72 h in the probiotic supplemented group. Finally, an in vivo bacteria challenge against live V. anguillarum was conducted. The probiotic fed fish observed a significantly higher survival. Overall, our results provide clear vertical evidence on the beneficial immune effects of B. velezensis and unveil some fundamental immune mechanisms behind its application as a probiotic agent in intensively cultured European sea bass.

The generation of a variety of new therapeutic agents to control and reduce the effects of pathogen in aquaculture is urgently needed. The antimicrobial peptides (AMPs) are one of the major components of the innate defenses and typically have broad-spectrum antimicrobial activity. However, absorption and distributions of exogenous AMPs for therapeutics application on farmed fish species need to be studied. Previous studies in our laboratory have shown the properties of hepcidin as an effective antimicrobial peptide produced in fish in response to LPS and iron. Therefore, we decided to investigate the antimicrobial activity of four synthetic variants of hepcidin against Vibrio anguillarum in vitro, and using the more effective peptide we demonstrated the pathogen's ability to protect against the infection in European Sea bass. Additionally the uptake of this peptide after ip injection was demonstrated, reaching its distribution organs such as intestine, head kidney, spleen and liver. The synthetic peptide did not show cytotoxic effects and significantly reduced the accumulated mortalities percentage (23.5%) compared to the European Sea bass control (72.5%) at day 21. In conclusion, synthetic hepcidin shows antimicrobial activity against V. anguillarum and the in vivo experiments suggest that synthetic hepcidin was distributed trough the different organs in the fish. Thus, synthetic hepcidin antimicrobial peptide could have high potential for therapeutic application in farmed fish species.

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.

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.

The use of vegetable oils in fish nutrition has been extensively studied; and recent work has focused attention on replacing fish oil with alternative fatty acid sources and their effect on the immune system. However, little is known about the effect of these oils on immune parameters such as the fish interferon system. In this study we evaluate the effect of two vegetable oils (linseed and soybean) on gilthead sea bream Mx expression and other innate immune parameters. Experimental diets were formulated where fish oil was totally replaced by vegetable oils or for a mixture of them (50% linseed and 50% soybean). Another diet prepared with pure fish oil was used as a control. Two experiments were carried out in order to evaluate growth, feed utilization, serum alternative complement pathway activity, serum lysozyme and phagocytic activity of head kidney leucocytes as well as Mx expression in the liver. In the first experiment fish were fed with experimental diets for 6 months and then, growth and feed utilization as well as immune parameters were analyzed. In the second experiment, fish from the previous feeding trial were injected with either a sub-lethal dose of Photobacterium damselae subsp. piscicida (94/99) or a synthetic dsRNA (Poly I:C) in order to stimulate an Mx response. The results show that total substitution of fish oil by vegetable oils decreased the growth of gilthead sea bream juveniles. Furthermore, both phagocytic activity and serum alternative complement pathway activity were significantly reduced by the inclusion of either vegetable oil individually in the sea bream diets, but the diet with mixed vegetable oils had no significant effect. There was no effect on serum lysozyme levels but the basal constitutive levels of Mx transcript expression in the liver were elevated in the fish fed the vegetable oil diets. The time-course of the Mx response to injection of Poly I:C was shorter in the fish fed the fish oil diet and the fish fed the diet based on a mixture of both vegetable oils showed a faster Mx response to bacterial injection. Following stimulation with Poly I:C or PDP the fish fed the vegetable oil based diets still maintained higher basal levels of hepatic Mx expression than the fish fed the fish oil diet which returned to undetectable levels.

The objective of this study was to determine the effect of two levels of inclusion of mannan oligosaccharides derived from the outer cell wall of a select strain of Saccharomyces cerevisiae (Bio-Mos, Alltech Inc, USA) on growth, feed utilization, immune status and disease resistance of European sea bass (Dicentrarchus labrax). Specimens of 35 g at initial density of 3 kg/m3 were fed during 67 days at 0 per thousand, 2 per thousand and 4 per thousand dietary MOS level of inclusion in a commercial sea bass diet. Food conversion rate, specific growth rate, whole body biochemical composition, phagocyctic index of head kidney macrophages, NBT index, lysozyme and alternative complement pathway (ACP) activities as well as gut and liver histological structure were evaluated. Growth significantly increased at both MOS dietary inclusion levels. Histological features of the liver showed lower lipid vacuolization and regular-shaped morphology of hepatocytes around the sinusoidal spaces denoting a better utilization of dietary nutrients. No differences were found on gut histological evaluation. Statistical differences (P

This study investigates the effect of dietary vitamin E on juveniles of gilthead seabream under stressful situations, focusing on the effects on growth, haematology, some immune parameters and plasma cortisol as indicators of stress. Two sardine meal-based experimental diets, one of them supplemented with 150 mg of alpha tocopherol kg(-1) of diet (control) and another one without vitamin E supplementation (diet NE), were assayed under two different stress conditions: overcrowding as a chronic stressor (during 15 weeks) and repetitive chasing as an acute repetitive stressor. Low levels of vitamin E in the diet depleted alternative complement pathway activity [from 167.23 U ml(-1) (control fish) down to 100.99 U ml(-1)] and also nonspecific haemagglutination. Also, fish fed a non-supplemented diet showed an elevation of plasma cortisol basal levels without a stressor influence [from 3.91 ng cortisol ml(-1) plasma (control fish) up to 21.70 ng cortisol ml(-1) plasma]. Low levels of vitamin E in the diet also produced an increase of erythrocyte fragility. Under chronic stress, fish fed the vitamin E-deficient diet showed a reduction in growth and survival, and alterations in haematological parameters, such as an additional haemoconcentration in response to overcrowding when compared with control fish. Under repetitive stress, fish fed the vitamin E deficient diet showed faster elevation of plasma cortisol levels in response to stress and a lower survival rate than control fish. Production of oxygen radicals by blood neutrophils was reduced under repetitive stress in fish fed the non-supplemented diet. These results suggest that fish fed the vitamin E-deficient diet had lower stress resistance.

The greater amberjack Seriola dumerili (Risso 1810) is a marine pelagic species with a high interest for aquaculture due to its rapid growth and high commercial value. However, during grow out, monogenean parasites cause occasional mass mortalities in farmed fish and there is not so much published data on the optimum temperature for growth and immunological parameters of this fish species fingerlings. Two hundred and twenty five juveniles were kept in triplicated in three experimental recirculated aquaculture system (RAS) at 17, 22 and 26 oC, each RAS controlling 3 tanks. Fish were fed a commercial diet to apparent satiety during four months. Growth was monitored each month. At the end of the experimental period samples of intestine, skin, blood and skin mucus were obtained. Growth, feed efficiency and immunological parameters, such as lysozyme and bactericidal activity have been studied to observe the relations between the different growth rates produced by the temperature and the immune system in serum and skin mucus. Besides, samples form skin and intestine were also obtained for histological studies, including the mucus production potential by analysing the goblet cells and the distribution of GALT in posterior intestine. Fish held at 26oC presented the best growth and feed utilization, being the results obtained for immune potential discussed also in terms of temperature influence.

Claudio Alvarez*, Felix Acosta*, B. Vega*, Daniel Montero*, Fanny Guzman *, Elisa Torres* and Luis Mercado* § Grupo de Marcadores Inmunologicos, Laboratorio de Genetica e Inmunologia Molecular. Instituto de Biologia. Facultad de Ciencias, Pontificia Universidad Catolica de Valparaiso. Programa de Doctorado en Biotecnologia, Universidad Tecnica Federico Santa Maria/Pontificia Universidad Catolica de Valparaiso, Chile. Grupo de Investigacion en Acuicultura, Universidad de Las Palmas de Gran Canaria, Espana. Nucleo Biotecnologia Curauma, Pontificia Universidad Catolica De Valparaiso, Chile.

Low dietary levels of raw marine ingredients in marine fish species diets induce variable side-effects on fish growth performance, welfare and health, depending mainly on the terrestrial protein and oil source used, dietary levels and fish species studied. Particularly, high dietary levels of vegetable meals (VM) and oils (VO) blends, may induce intestinal dysfunction by altering tissue morphology, intestinal functionality, microbiota populations or gut associated lymphoid tissue (GALT) homeostasis. On the other hand, alternative feed ingredients such as prebiotics and probiotics have demonstrated to promote fish performance and disease resistance through potentiating GALT efficiency and functionality. Thus, the main objective of this study is to evaluate the effects of dietary mannan oligosaccharides (Biomos® and ActigenTM), Pediococcus acidilactici (BAC; Bactocell ®) and their combination in low FM and FO diets for european sea bass juveniles. Fish of 19.7±0.09g were distributed in 18 tanks and fed for 90 days six experimental diets with a low content of FM and FO, containing graded and combined levels of MOS and BAC as follows: HP; LP; B; HPB; LPB and C (Control). After 60 and 90 days of feeding, fish were sampled for growth, gut morphometry and histopathology, gut mucus production and expression of GALT related genes. Besides, samples were taken for gut iNOS and TNFα immunohistochemistry (IHQ) and for in situ hybridation (ISH) studies of MHCβ, TCRβ, CD4 and CD8. Fish fed diets HP, LP and LPB presented higher final weight. Two-way ANOVA attributed this effect to MOS regardless of dietary BAC inclusion. The use of prebiotic induced an increase on number of goblet cells in posterior gut (Fig.1). IHQ analyses performed against iNOS and TNFα showed the use of prebiotics induce an inflammatory-like pattern in the apical side of gut villi. Results for ISH showed that combined use of pre and probiotic induced a wider distribution of TCR-β marked cells, with special emphasis in the lamina propria. Expression of GALT-related genes, will be also discussed. Fig.1. Posterior gut (Alcian Blue-PAS). (a) LPB and (b) C diets t=90days. Note the higher number of goblet cell for fish fed LPB diet Acknowledgements: This study has been funded by the Spanish Ministry of Economy and Competitiveness, Project AGL2012-39919 (PROINMUNOIL)

International audience; The major economic and environmental consequences of recurring mortalities affecting the Pacific oyster Crassostrea gigas have initiated many research projects aiming at understanding these phenomena. The solutions anticipated to deal with these mortalities are mainly based on mass selection breeding programs but preventive treatments are still lacking. However, over the last decade, studies have been accumulating revealing the adaptive capabilities of innate immunity, the only component of defense mechanisms in invertebrates. Numerous findings have shown that a wide range of invertebrates can develop innate immune memory (also called immune priming) leading to improved survival during a second encounter with a pathogen. In this context, we undertook to study the possibilities of acting against mortalities by stimulating immune capacities of oysters.In the present study, we show that the exposure of oyster juveniles to an immunostimulant (a viral mimic called poly (I: C)) can lead to enhanced survival capacities (up to 100%) following OsHV-1 infection or during a mortality episode in the field. That protection is specific to viral protection as poly(I:C) fails to protect oyster against a pathogenic bacteria. We also show that this priming phenomenon is durable as it can last more than 4 months suggesting for the first time the existence of mechanisms of immune memory in this invertebrate species. Finally, analysis of the molecular pathways underlying that protection using dual RNAseq, revealed that priming was based on the triggering of a strong and sustained antiviral response limiting replication of the virus, thus allowing the protection of oysters on the long term. Altogether these results bring new insights into the oyster capacities to build an innate immune memory, its adaptive capacities and provide a platform to further explore novel strategies to help mitigate disease threats upon marine bivalves.