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We simulated in the laboratory the possible effects on fatty acids and immune status of wild fish arriving for the first time in the vicinity of a sea-cage fish farm, shifting their natural diet to commercial feed consumption, rich in fatty acids of vegetable origin. The flesh fatty acid profile of golden mullet specimens was altered after 2 weeks of commercial feed consumption, showing an increase in fatty acids of vegetable origin. The serum peroxidase and bactericidal activities, and head-kidney leucocyte phagocytic capacity, increased after eight weeks of the new diet, while the respiratory burst activity decreased. The extent of these changes cannot be considered large enough to regard them as compromising the health status of fish. More research is needed in order to elucidate whether the rapid assimilation of the dietary fatty acids could harm the immune status of fish when feeding for longer periods than two months.

Fish skin mucus contains several immune substances that provide the first line of defence against a broad spectrum of pathogens although they are poorly studied to date. Terminal carbohydrate composition and levels of total IgM antibodies, several immune-related enzymes (lysozyme, peroxidase, alkaline phosphatase, esterases, proteases and antiproteases) as well as the bactericidal activity (against fish pathogenic Vibrio harveyi, Vibrio angillarum, Photobacterium damselae and non-pathogenic bacteria Escherichia coli, Bacillus subtilis, Shewanella putrefaciens) were identified and measured in the skin mucus of five marine teleosts: gilthead seabream (Sparus aurata), European sea bass (Dicentrarchus labrax), shi drum (Umbrina cirrosa), common dentex (Dentex dentex) and dusky grouper (Epinephelus marginatus). First, lectin binding results suggests that skin mucus contain, in order of abundance, N-acetylneuraminic acid, glucose, N-acetyl-glucosamine, N-acetyl-galactosamine, galactose and fucose residues. Second, results showed that while some immune activities were very similar in the studied fish (e.g. IgM and lysozyme activity) other such as protease, antiprotease, alkaline phosphatase, esterase and peroxidase activities varied depending on the fish species. High levels of peroxidase and protease activity were found in U. cirrosa respect to the values obtained in the other species while E. marginatus and S. aurata showed the highest levels of alkaline phosphatase and esterase activities, respectively. Moreover, skin mucus of S. aurata revealed higher bactericidal activity against pathogenic bacteria, contrarily, to what happened with non-pathogenic bacteria (E. coli, B. subtilis). Thus, study of the variations in the carbohydrate profile and immune-related components of the fish skin mucus could help to understand the fish resistance as well as the presence and distribution of pathogens and magnitude of infections, aspects that are of major importance for the aquaculture industry., SI

Dehydrated lemon peel (DLP) at two levels (1.5% and 3%) was included for 30days in the diet of gilthead seabream (Sparus aurata L.) and after 15 and 30days the serum and skin mucus of fish were analysed. In serum, both experimental diets led to an initial decrease in glucose and lactate after which the levels became comparable to the control. Dietary DLP modulated the activity of transaminases, alanine aminotransferase and aspartate aminotransferase. The total antioxidant status showed a progressive increase in relation to levels of inclusion of DLP and time of administration (p'.05). In skin mucus, some biomolecular markers related to general stress, oxidative stress and apoptosis exhibited variations that suggest an adaptive response of fish to the new metabolic situation, resulting from the experimental diet. The inclusion of DLP in the diet seems to positively modulate the central metabolism and welfare of gilthead seabream.

The external mucus layer that covers fish skin contains numerous immune substances scarcely studied that act as the first line of defence against a broad spectrum of pathogens. This study aimed to characterize and describe for the first time several humoral immune defence parameters in the skin mucus of the European eel (Anguilla anguilla) after intraperitoneal injection with Vibrio anguillarum or Tenacibaculum soleae. This study evaluated several immune-related enzymes and bactericidal activity against fish pathogenic bacteria in the skin mucus of European eels at 24, 48, and 72 h post-challenge. The results demonstrated that European eel skin mucus showed significant increments in peroxidase and lysozyme activity at 48 and 72 h after V. anguillarum challenge, compared to other experimental groups. In the case of antiprotease activity, an increase was observed at 24 h in the skin mucus of fish challenged with V. anguillarum compared to unchallenged fish, while this activity was undetected at 48 and 72 h. In contrast, protease activity had decreased at 48 and 72 h in the skin mucus of fish challenged with V. anguillarum compared to the unchallenged group. Regarding bactericidal activity, a high growth capacity of T. soleae was observed in the skin mucus of all experimental groups. Interestingly, the skin mucus from fish challenged with V. anguillarum exhibited increased bactericidal activity against this bacterium at 48 h, compared to unchallenged fish. Finally, severe histopathological alterations were observed in the gills and liver at the end of the trial (72 h), whereas the skin showed only an overspread presence of goblet cells in the challenged fish compared to unchallenged fish. The present results may give new insights into the mucosal immune system of this primitive species with potential applications in aquaculture.

The present study was conducted to determine the potential effect of the dietary intake of fenugreek (Trigonella foenum graecum) seeds alone or in combination with Bacillus licheniformis, Lactobacillus plantarum or B. subtilis on gilthead seabream quality and antioxidant response after 2 and 3 weeks of experimental feeding. The results showed that the supplements did not affect the percentage of the fatty acid profiles of muscle, demonstrating that all the additives tested can be administrated without any negative effect on biochemical composition and quality of gilthead seabream. The quantification of thiobarbituric acid reactive substances in muscle demonstrated the significant beneficial effect of the experimental diets compared with the control one. Besides, an increase in superoxide dismutase and catalase in liver was recorded after 3 weeks of administration of experimental diets. Furthermore, real time qPCR revealed that dietary supplementation with FEBS significantly enhances the expression of scavenging enzymes, such as cat and gr genes in the liver after 3 weeks. The findings suggest that the administration of fenugreek supplement alone or combined with probiotic strains could be considered as a good source of natural antioxidants and as a functional aquafeed ingredient for gilthead seabream.

The use of immunostimulants is considered a promising preventive practice that may help to maintain animal welfare and a healthy environment, while increasing production and providing higher profits. The purpose of this study was to evaluate the effects on gilthead seabream (Sparus aurata L.) of the dietary administration of fenugreek (Trigonella foenum graecum) seeds, alone or combined with one of the following probiotic strains: Bacillus licheniformis (TSB27), Lactobacillus plantarum or Bacillus subtilis (B46). Gilthead seabream were fed a control or one of the supplemented diets for 3 weeks. The effects of these supplemented diets on growth performance parameters and the humoral immune response (natural haemolytic complement, peroxidase, total IgM levels, proteases and antiproteases activities) were evaluated after 2 and 3 weeks of feeding. Simultaneously, the expression levels of some immune-relevant genes (igm, tcr-β, csfr1 and bd) were measured in the head-kidney. Interestingly, all probiotic supplemented diets increased seabream growth rates, especially the B. licheniformis supplemented diet. Generally, humoral immune parameters were enhanced by the dietary supplementation at the different time points measured. The results showed a significant increases in the immune parameters, principally in fish fed only fenugreek or fenugreek combined with B. subtilis. Furthermore, real time qPCR revealed that dietary supplementation significantly enhances the expression of immune-associated genes in the head-kidney, particularly igm gene expression. These results suggest that fenugreek alone or combined with one of the probiotic strains mentioned enhances the immune response of gilthead seabream, a species with one of the highest rates of production in marine aquaculture.