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The role of subcutaneous adipose tissue adipocytes and the effects of fatty acids on carrageenan-induced skin inflammation in gilthead seabream (Sparus aurata) were studied. Fish were injected intramuscularly with phosphate-buffered saline (control) or λ-carrageenin (1%), and skin samples collected at the injection site at 3 and 6 h post-injection (p.i.) were processed for histological study. In addition, the presence and levels of lipid classes, fatty acid methyl esters (FAME) and eicosanoids were evaluated in the skin samples obtained from the injected areas. Histological results indicated an increase in adipocyte area in fish sampled at 3 h p.i. with λ-carrageenin compared to fish in the control group. Furthermore, the frequency of adipocytes between 4500 and 5000 μm

Cantharidin is a toxic vesicant terpene used in folk and traditional medicine due to its various therapeutic effects. Since there are no previous data on the effect of cantharidin in fish, this study aimed to investigate the in vitro related-inflammatory effects of cantharidin in gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs). In the first experiment, the HKLs were incubated with 0, 5 and 10 μg mL

To date, the mechanisms of inflammation have been poorly studied in fish of commercial interest, due to the lack of development of appropriate experimental models. The current study evaluated a local inflammation triggered by a polymeric carrageenin mixture (a mucopolysaccharide derived from the red seaweed Chondrus crispus) in the skin of gilthead seabream (Sparus aurata). Fish were injected subcutaneously with phosphate-buffered saline (as control) or λ/κ-carrageenin (1%), and skin samples from the injection sites were collected 1.5, 3 and 6 hr post-injection, processed for inclusion in paraplast and stained with haematoxylin-eosin, Alcian blue or periodic acid-Schiff. Furthermore, immunohistochemistry and expression analyses of several cells' markers and proinflammatory genes were also analysed in samples of the injected sites. Microscopic results indicated an increased number of skin mucus-secreting cells and acidophilic granulocytes in the skin of fish studied at 1.5 hr and 3 hr post-injection with carrageenin, respectively, with respect to the data obtained in control fish. Otherwise, both the gene expression of the non-specific cytotoxic cell marker (granzyme B, grb) and the proinflammatory cytokine (interleukin-1β, il-1β) were up-regulated at 1.5 hr in the skin of fish injected with carrageenin compared with the control fish, whilst the gene expression of acidophilic granulocyte markers (NADPH oxidase subunit Phox22 and Phox40, phox22 and phox40) was up-regulated at 3 and 6 hr in the carrageenin group, compared with the control group. In addition, the gene expression of myeloperoxidase (mpo) was also up-regulated at 6 hr in the skin of fish injected with carrageenin in comparison with control samples. The present results indicate the chronological participation of two important immune cells involved in the resolution of the inflammation in the skin of gilthead seabream.

The yeast Yarrowia lipolytica has been industrially adopted for docosahexaenoic acid and eicosapentaenoic acid production under good manufacturing practices over 2 decades. In recent years, it has claimed attention for novel biotechnological applications, such as a functional feed additive for animals. Studies have demonstrated that this yeast is safe and has probiotic and nutritional properties for mammals, birds, fish, crustaceans, and molluscs. Animals fed Y. lipolytica enhanced productive and immune parameters, as well as modulated microbiome, fatty acid composition, and biochemical profiles. Additionally, some Y. lipolytica-derived compounds have improved productive performance, immune status, and disease resistance in animals. Therefore, the aim of this review is to identify and discuss research advances on the potential use of this yeast for animals of economic interest. Challenges, opportunities, and trends were identified and envisioned in the near future for this industrially produced yeast. KEY POINTS: • Yarrowia lipolytica has probiotic and nutritional effects in animals. • Lipase2, EPA, and β-glucan from Y. lipolytica have health benefits for animals. • Y. lipolytica is envisioned in terrestrial and aquatic animal production systems.

Inflammation is one of the main causes of loss of homeostasis at both the systemic and molecular levels. The aim of this study was to investigate in silico the conservation of inflammation-related proteins in the gilthead seabream (Sparus aurata L.). Open reading frames of the selected genes were used as input in the STRING database for protein–protein interaction network analysis, comparing them with other teleost protein sequences. Proteins of the large yellow croaker (Larimichthys crocea L.) presented the highest percentages of identity with the gilthead seabream protein sequence. The gene expression profile of these proteins was then studied in gilthead seabream specimens subcutaneously injected with carrageenin (1%) or phosphate-buffered saline (control) by analyzing skin samples from the injected zone 12 and 24 h after injection. Gene expression analysis indicated that the mechanisms necessary to terminate the inflammatory response to carrageenin and recover skin homeostasis were activated between 12 and 24 h after injection (at the tested dose). The gene analysis performed in this study could contribute to the identification of the main mechanisms of acute inflammatory response and validate the use of carrageenin as an inflammation model to elucidate these mechanisms in fish.

This study investigated the acute inflammatory response induced by subcutaneous injection of carrageenin (1%) or phosphate-buffered saline (control) in gilthead seabream (Sparus aurata). Skin mucus, serum, head kidney (HK) and liver were sampled at 1.5, 3 and 6 hr post-injection (p.i.) to determine the immune and antioxidant status of this fish species. The skin mucus of the carrageenin group showed increased superoxide dismutase and peroxidase activities, lysozyme abundance, bactericidal activity against Vibrio anguillarum and Photobacterium damselae, and total immunoglobulins compared with those of the control group. However, the carrageenin-injected fish sampled at 6 hr p.i. showed decreased protease activity in the skin mucus and peroxidase activity in the HK leucocytes compared with the control. Moreover, the carrageenin injection had no effects on the systemic immune system, but it reduced the liver catalase activities at both 3 and 6 hr in the carrageenin group relative to those in the control group. The expression levels of several proinflammatory and cell marker genes in the HK and liver were also determined. In the HK, the expression levels of interleukin-1β and prostaglandin D synthase 1 were upregulated at 1.5 and 3 hr, respectively, in the carrageenin group compared with those in the control group. Contrarily, the expression of the NADPH oxidase subunit phox40 (an acidophilic granulocyte marker) in the carrageenin group at 6 hr was downregulated compared with that in the control group. These results suggested that subcutaneous injection of κ/λ-carrageenin in gilthead seabream triggered an acute skin inflammation characterized by the rapid recruitment of acidophilic granulocytes and the release of humoral mediators into the skin mucus.

Marine organisms are affected by the ubiquitous occurrence of microplastics (MPs) in the environment. Several protocols have been described to extract and quantify MPs in seafood, although their complex matrices, with high level of fat, can compromise the efficiency of MPs extraction. To solve this issue, the present study aimed to develop a detailed methodology suitable to process seafood samples with different levels of fat, namely fish and molluscs, from fresh and canned sources, including the immersive liquids from the cans. Sample digestion was tested using different solutions (10% KOH, 30% H2O2), temperatures (40 °C, 65 °C) and incubation times (24, 48, 72 h). For fat removal, three detergents (two laboratory surfactants and a commercial dish detergent) and 96% ethanol were tested, as well as the manual separation of fat. The methodology optimized in this study combined a digestion with 30% H2O2 at 65 °C, during 24 to 48 h, with a manual separation of the fat remaining after the digestion. All steps from the present methodology were tested in six types of polymers (PE-LD, PET, PE, AC, PS, and lycra), to investigate if these procedures altered the integrity of MPs. Results showed that the optimized methodology will allow for the efficient processing of complex seafood samples with different fat levels, without compromising MPs integrity (recoveries rate higher than 89% for all the polymers tested).

The present study sought to investigate the effect of arginine on the involvement of toll-like receptors (TLRs) in skin wound-induced intestinal barrier dysfunction in gilthead seabream (Sparus aurata L.). Two replicates of fish (n = 8) were fed a commercial diet (CON, total 2.75% arginine), CON diet enriched with 1% arginine (ARG1, total 3.65% arginine) and 2% arginine (ARG2, total 4.53% arginine) for 30 days. Half of the fish were sampled, whereas the others were injured and sampled 7 days post-wounding. The intestinal histology results showed that a more intense infiltration of mixed leucocytes was evident in the wounded fish, which was remarkably reduced in fish that were fed the ARG1 diet. Serum IgM levels were significantly higher in the ARG1 group than levels in the CON group at 7 days post-wounding. Compared with the fish in the CON group after wounding, dietary administration of 1% arginine markedly downregulated the gene expression of TLRs (TLR2 and TLR5), MyD88, and proinflammatory cytokines (CSF1R, IL-1β, and TNFα), but significantly enhanced the gene expression of IκBα, the anti-inflammatory cytokine TGF-β1, and tight junction proteins (tricellulin and occludin) in wounded fish. Furthermore, the ARG2 diet demonstrated no additional benefits on intestinal cells, compared to both the ARG1 and the CON diets, and it even appeared to induce negative effects. In summary, dietary administration of 1% arginine significantly inhibited intestinal inflammatory response and tight junction disruption in skin-wounded gilthead seabream by modulating TLR signalling in the intestine.

Due to the natural, innocuous and bioactive compounds that medicinal plants contain, their application in aquafeeds has great potential to ensure more sustainable aquaculture. The aim of the present study was to evaluate the in vitro biological activities of purslane (Portulaca oleracea L.) as well as its in vivo effects after dietary administration to gilthead seabream (Sparus aurata L.) specimens. To carry out this work we first analysed the in vitro effect purslane aqueous and ethanolic extracts on head-kidney (HK) leucocytes from gilthead seabream, as well as their cytotoxic, bactericidal and antioxidant activities. After that, we studied the effect on gilthead seabream growth performance and immune status after the addiction of purslane to its diet. Regarding the in vitro study, purslane aqueous and ethanolic extracts had immunostimulant properties on HK leucocyte phagocytosis, bactericidal activity against marine pathogenic bacteria (Vibrio harveyi, V. anguillarum and Photobacteium damselae) and cytotoxic (mainly against PLHC-1 tumor cells) and antioxidant activities. On the other hand, the dietary inclusion of 2% purslane for 15 or 30 days had no impact on growth performance while improving the fish's immune status. More specifically, skin mucus IgM levels and the phagocytic capacity of HK leucocytes of fish-fed purslane diets for 15 and 30 days, respectively, increased statistically significant with respect to the values recorded for the fish-fed control diet. Overall, the results suggest that purslane extracts, when used in adequate concentrations, have different beneficial properties, and that the dietary incorporation of purslane improves some immune parameters at local and systemic levels while having no negative effect on growth. These results make purslane an interesting candidate for incorporation as an additive in functional diets for farmed fish.

By-products obtained from seaweed processing appear as a promissory source of bioactive compounds that could be included in aquafeed to improve the sustainability of aquaculture. Therefore, the present study aimed to evaluate the effects of dietary supplementation with by-products obtained from the seaweed Gracilaria sp. on the stress response, antioxidant, and immune systems of gilthead seabream (Sparus aurata) exposed to an acute crowding event. For this, fish were fed six experimental diets: (i) a diet containing a commercial antioxidant (CTR), (ii) a diet without antioxidant addition (NO-AOX), (iii) a NO-AOX diet with 0.5% ethanolic extract (0.5% EE), (iv) a NO-AOX diet with 5% ethanolic waste (5% EEW), (v) a NO-AOX diet with 5% agar waste (5% AW), and (vi) a NO-AOX diet with 2.5% agar waste (2.5% AW) for 59 days. After this period, fish were exposed to 1 h of crowding (100 kg m−3) and sampled immediately after 0 and at 24 h post-crowding. Our results showed that the fish-fed AW and EEW diets showed lower plasma cortisol levels than those fed the CTR diet, suggesting an improved stress response to crowding on these groups. No differences were detected on hepatic lipid peroxidation levels between dietary groups after crowding. However, the fish fed 0.5% EE diet showed lower hepatic glutathione reductase (GR) activity than fish fed the NO-AOX diet. Additionally, glutathione peroxidase (GPx) activity was decreased in the liver of fish fed 2.5% and 5% AW diets compared with the other dietary groups. Concerning immune parameters, the plasma peroxidase activity was higher in fish fed the 2.5% AW diet than fish fed NO-AOX or 5% EEW diets, suggesting a modulatory effect of dietary AW at 2.5% in the humoral immune response in crowded fish. Results suggest that dietary supplementation with AW, particularly when supplemented at 2.5%, could be used as a dietary tool to mitigate oxidative stress when improving immune response in gilthead seabream exposed to a stressor as crowding.

The effects of skin wounds on the intestinal barrier function and the beneficial effects of the dietary administration of Shewanella putrefaciens (known as SpPdp11) in gilthead seabream (Sparus aurata L.) were studied. Two replicates of fish were fed a commercial diet (control, CON) or CON diet enriched with 109 cfu g−1 SpPdp11 (SP diet) for 30 days. After this time, half of the fish were sampled, while the others were injured below the lateral line (wounded fish, W) and fed the same diets for an extra week before sampling (CON + W and SP + W groups). The intestinal histology and gene expression of different genes relevant for the intestinal barrier function were studied. The results showed that injured fish had a disordered enterocyte nucleus disposition, a more intense infiltration of mixed leucocytes and a thicker lamina propria in the intestine compared to the control fish. However, the fish in the SP + W group did not present these pathological symptoms in the intestine. No significant variations in the number of goblet cells were detected among the different experimental groups. Pro-inflammatory cytokines (colony-stimulating factor receptor 1, CSF1R, myeloperoxidase, MPO and interleukin-1β, IL-1β), mucins (intestinal mucin, IMUC and mucin 2, MUC2), and immunoglobulin T heavy chain (IGHT) were up-regulated, while tight junction protein occludin was down-regulated in the intestine from fish of the CON + W group. Similarly, the dietary administration of SpPdp11 markedly depressed the gene expression of pro-inflammatory cytokines, MUC2 and IGHT, but increased the gene expression of anti-inflammatory cytokine transforming growth factor-β1 (TGF-β1) and the tight junction proteins tricellulin and occluding after wounding. In brief, the skin wounds provoked an intestinal inflammatory response that included changes in the mucus layer and tight junction disruptions. Besides this, preventive administration of SpPdp11 alleviated the intestinal dysfunctions caused by skin wounds in gilthead seabream.

The current work aimed to carry out an in vivo study of the λ-carrageenin-induced inflammation in the skin of gilthead seabream (Sparus aurata). The fish were injected intramuscularly with phosphate-buffered saline (PBS, as control) or λ-carrageenin (1% in PBS), and the injection zone was evaluated by real-time ultrasonography (Vevo Lab, VisualSonics) at 1.5, 3, 6, 12, and 24 h post-injection (p.i.). Results demonstrated that the skin thickness was increased in fish injected with λ-carrageenin and sampled at 1.5, 3, and 6 h p.i. However, the skin thickness of the injected area decreased to the normal values in those fish sampled at 12 and 24 h p.i. In addition, fish injected with λ-carrageenin and analysed at 1.5, 3, and 6 h p.i. showed, in the underlying muscle at the injection place, several hyperechoic small foci surrounded by an anechoic area which were not observed in control fish. Furthermore, the fish were analysed by X-ray micro-computed tomography (micro-CT). The analysis of the micro-CT acquisitions revealed also a dark area in the place of the injection with λ-carrageenin at 1.5, 3, and 6 h. These areas were smaller in fish analysed at longer times (12 h p.i.) and were almost disappeared in fish sampled at 24 h p.i. These areas had an average density of -850 to -115 HU, which did not correspond with any tissue density of the rest of the body. Furthermore, similar dark areas at the injection zones were never observed in control fish. Present results support the use of both non-invasive techniques to study the inflammatory process in fish of commercial interest such as gilthead seabream.

The λ-carrageenin is a sulphated mucopolysaccharide that has been used for decades to induce experimental inflammation in mammals. However, it has been little considered in fish. We studied the in vitro effects of λ-carrageenin on gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs). For this purpose, HKLs were incubated with serial concentrations (from 0 to 1,000 μg mL-1) of λ-carrageenin for 3, 6, 12 and 24 h to assess its influence on cell viability and morphology, cell activity and modulation of several selected inflammation-related genes. The viability results demonstrated that λ-carrageenin has no negative effects on HKLs. The respiratory burst activity and phagocytic ability of HKLs after being incubated with λ-carrageenin (100 and 1,000 μg mL

The South Lagoon of Tunis (Tunisia) is a Mediterranean lagoon adversely affected by industrial contaminants, harbour activity and untreated urban sewage. In this lagoon, the clam Ruditapes decussatus has been widely used as a biomonitor of seawater pollution through measurements of parameters related to oxidative stress and neurotoxicity. However, few studies have considered parameters of the immune system of this species in the South Lagoon of Tunis. Therefore, the aim of the present work was to evaluate several immune-related parameters in the cell-free haemolymph of carpet shell clams sampled during August and February from three polluted sites in the South Lagoon of Tunis (S1, S2 and S3) and one less polluted site as a reference site (RS) in order to identify suitable biomarkers for environmental quality assessments of this ecosystem. Concerning the immune-related parameters, seasonal factors modulated phenoloxidase, lysozyme, protease and esterase activity, with lower values measured for samples collected in August than for samples collected in February. In fact, bactericidal activity against two of the pathogenic bacteria tested and the activity of most immune-related enzymes were reduced in the cell-free haemolymph of clams collected from the most sampling sites in August compared to February one. In addition, values of abiotic parameters (temperature, salinity and pH) and metal (cadmium, copper, iron, lead and zinc) concentrations in the clams’ soft tissues, previously obtained and published by the authors, as well as the values of immune-related parameters were integrated using principal component analyses. Results indicated that the values of all measured immune-related parameters were negatively correlated with the temperature values and the variations most of these parameters highlighted that the chemical industrial area (S3) was the most impacted location within the South Lagoon of Tunis. The present study illustrates that the immune-related parameters measured in carpet shell clam cell-free haemolymph represent suitable biomarkers for environmental quality assessments because they provide effective seasonal and spatial discrimination.

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.

Fish antimicrobial peptides (AMPs) are small peptides whose actions have great implications in the innate immune response of teleost fish, being considered critical as the first line of defence against a wide spectrum of pathogens. Antibiotics have been widely used in aquaculture, resulting in bacterial resistance. The potential of AMPs as alternative to antibiotics makes them a very interesting tool as they represent lower-risk peptides to develop resistances, or even in the fight against other pathogens such as viruses. Beyond their antimicrobial function, fish AMPs possess several relevant but little-known characteristics and capabilities such as host-defence peptides, antioxidants, active compounds of immunogenic drugs and as adjuvants. Moreover, their antitumour properties make them potential drugs to be used in oncological treatments in human subjects. Novel methodologies as clustered regularly interspaced short palindromic repeats associated proteins system (CRISPR-Cas), or even their use as nutraceutics, seem to be promising instruments to enlarge the functions of fish AMPs. This review discusses these new methodologies, as well as the latest research on fish AMP functions, newly discovered properties of AMPs and their future applications in both the aquaculture industry and human health care.

The increasing replacement of fish oil (FO) by vegetable oils (VO) in aquafeeds for marine carnivorous species may lead to modifications of the dietary arachidonic (ARA), eicosapentaenoic (EPA) and docosahexaenoic (DHA) levels and ratios that may compromise the fish immune status. Therefore, this study aimed to assess how different dietary ratios of ARA, EPA, and DHA modulate the immune response of gilthead sea bream juveniles. For that purpose, four isoproteic and isolipidic plant-based diets were formulated to include different dietary Long-Chain-Polyunsaturated Fatty Acids (LC-PUFA) ratios (%DM): Diet A (2.0% ARA: 0.2% EPA: 0.1% DHA); Diet B (1.0% ARA: 0.4% EPA: 0.4% DHA); Diet C (0% ARA: 0.6% EPA: 0.6% DHA); Diet D (0% ARA: 0.3% EPA: 1.5% DHA). Fish were fed the experimental diets for 56 days and then the haematological profile, humoral innate immune parameters, and expression of immune-related genes in the gut were determined. Our results showed that fish fed diet B exhibited an increase in the monocyte numbers, alternative complement activity, and bactericidal activity against Photobacterium damselae compared to the values found in fish fed other diets. Contrarily, peroxidase, antiproteases, and proteases activity and nitric oxide levels were not affected by the dietary treatments. Immune-related gene expression in the distal intestine did not show differences between dietary treatments. Overall, a diet with a balanced n-6 (ARA) and n-3 (EPA+DHA) LC-PUFA ratio seemed to better modulate the fish innate immune response, thus better preparing the fish innate immune system against potential immunological insults.

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 effect of the probiotic Shewanella putrefaciens Pdp11 (SpPdp11) was studied on the skin healing of experimentally wounded gilthead seabream (Sparus aurata L.). Two replicates (n = 12) of fish were fed CON diet or SP diet for 30 days. Half of the fish were sampled while the others were injured and sampled 7 days post-wounding. Results by image analysis of wound areas showed that SpPdp11 inclusion facilitated wound closure. Compared with the CON group, fish in SP group sampled 7 days post-wounding had a significantly decreased serum AST and increased ALB/GLOB ratio. Furthermore, protease and peroxidase activities were significantly increased in skin mucus from fish in SP group sampled 7 days post-wounding, compared with those fed CON diet. Additionally, SP diet up-regulated the gene expression of antioxidant enzymes, anti-inflammatory cytokines, and re-epithelialization related genes in the fish skin. Furthermore, significant decreases in pro-inflammatory cytokines expression were detected in fish from SP group, respect to control ones. Overall, SpPdp11 inclusion facilitated the wound healing and the re-epithelialization of the damaged skin, alleviated the inflammatory response in the wound area through intensifying the antioxidant system, and enhancing the neo-vascularization and the synthesis of matrix proteins in the skin wound sites of fish.

Most pathogens start the process of infection at the mucosal surfaces and therefore the mucosal immune response plays an essential role in the course of the infection. Due to the Senegalese sole (Solea senegalensis Kaup) condition of flatfish, the present comparative study aimed to analyse several immune-related enzymes as well as the bactericidal activity in the skin mucus from ocular and blind sides. For this purpose, Senegalese sole juveniles were bath challenged with a sub-lethal dose of Tenacibaculum maritimum for 24 h and sampled at 1, 2 and 3 weeks. The haematological profile and immune-related parameters were also measured in plasma in order to evaluate the systemic immune response after T. maritimum challenge. Results from this study showed that most parameters tested increased in skin mucus of bath challenged fish compared to unchallenged ones. In contrast, the sub-lethal dose tested did not influence the haematological profile including peripheral numbers the different leucocyte types. No variations were observed in plasma lysozyme, peroxidase, protease and haemolytic complement activities between unchallenged and bath challenged fish. This study suggests that the studied innate immune-related molecules are constitutively present in both skin mucus sides but at different levels. Interestingly, the levels of most parameters measured were higher on the ocular side than on the blind side, possibly due to the higher exposure to invasion by waterborne microorganisms on this side. Therefore, the present study brings some insights regarding local immune responses after bacterial challenge in skin mucus from the ocular and blind sides in one of the most valuable flatfish species in southern Europe.

Dietary administration of arginine on the wound healing process of gilthead seabream was studied. Two replicates of fish (n = 8) were fed with either a commercial diet [control diet (CON), no arginine added] and the CON diet supplemented with 1% arginine (ARG1) or with 2% arginine (ARG2) for 30 days. Afterward, half of the fish were sampled while the other half were injured and continued to be fed the same diet for an extra week. Results by image analysis showed that the wound closure rate was significantly improved in fish that were fed the ARG1 diet, compared with those in the CON group. After seven days of wound healing, the aminotransferase and creatine kinase levels in the serum and the protease and peroxidase activities in the skin mucus were down-regulated, while the immunoglobulin M level in the skin mucus was up-regulated in the ARG1 group after wounding and in the CON group before wounding. Compared with the CON diet, the ARG1 diet remarkedly depressed the gene expression of mpo, il-8, and tnf-α, and enhanced the gene expression of tgf-β1, igf-1, pcna, krt2, mmp9, fn1α, and colIα and the antioxidant enzyme cat in the skin tissues after wounding. Furthermore, compared with both the ARG1 and the CON groups, negative effects of the ARG2 diet on wound healing were demonstrated. In conclusion, a 1% arginine supplementation facilitates skin wound healing and prevents a systemic inflammation reaction by alleviating the inflammatory response and enhancing the re-epithelialization and ECM biosynthesis in skin wound sites.

Many studies have assessed the effects of incorporation of plant feedstuffs in fish diets on growth performance, whereas few studies have addressed the effects of fish meal replacement by plant protein sources on fish immune parameters. Thus, the aim of this study was to evaluate the effects on immune response of different inclusion levels of carob seed germ meal (CSGM) as partial replacement for fish meal in diets for meagre (Argyrosomus regius) juveniles. Fish were fed four experimental diets with increased CSGM inclusion levels [0% (control), 7.5% (CSGM7.5), 15% (CSGM15) and 22.5% (CSGM22.5)]. After 1, 2, and 8 weeks of feeding fish were sampled to determine haematological profile and several humoral parameters in plasma and intestine. Results showed that dietary inclusion of CSGM did not negatively affect the immune parameters of meagre. In addition, total numbers of red and white blood cells, as well as thrombocytes, lymphocytes, monocytes, and neutrophils counts were not affected by dietary treatments. All parameters evaluated in plasma were unaffected by dietary CSGM inclusion after 1 and 2 weeks of feeding, with only the haemolytic complement activity showing an increase in fish fed diets with CSGM after 1 week and in fish fed CSGM22.5 diet after 2 weeks. Regarding the innate immune parameters analysed in the intestine, it could be highlighted the increase in alkaline phosphatase and antiprotease activities in fish fed the diet with the higher inclusion of CSGM at 8 weeks. Overall, results suggest that high dietary CSGM inclusion do not compromise immune status or induce an inflammatory response in meagre juveniles.

Linking proprieties of adhesion, infectious capacities and antibiotic resistance of pathogen bacteria could help to treat fish diseases. Adhesions of ten fish pathogenic bacteria were tested in microtiter plates vacant, coated with skin or gut mucus, fixed with methanol, stained with 2% crystal-violet and revealed by colorimetric method. Infectious capacity was performed by exposing gilthead seabream fibroblast cell line (SAF-1) to 107-108 CFUmL-1 of pathogen bacteria. Cell viability was measured 3h, 9h and 20 hours post-infection. The sensitivity to antibiotics was executed by disk diffusion. Data showed that all the bacteria tested adhere to polystyrene. For skin mucus, Vibrio harveyi, Vibrio alginolyticus, Halomonas venusta, and Aeromonas bivalvium were moderately adherent. Dietzia maris was strongly adherent. For gut mucus, 60% of tested bacteria were weakly adherent and 40% were non adherent. For infection, D. maris, V. harveyi and A. bivalvium decreased the cells viability to 89% after only 3h. After 20h, the viability percentage ranged between 1% and 32%. All isolates presented resistance to 1000 mg ml-1 of sulphonamide, 60% were resistant to sulfonamide and penicillin G. Present findings could be relevant in fish aquaculture and underscore the importance of the linkage between adhesion, infectious capacity, and antibiotic susceptibility of pathogen bacteria to avoid fish diseases.

β-Glucans are naturally occurring polysaccharides that are produced by bacteria, fungi and yeast. They are considered immunostimulants in fish acting on non-specific defense mechanism. Yeast-derived glucans from cell wall (Sterigmatomyces halophilus, β-Gluc/Sh) have been used for this purpose in this study. Therefore, an in vitro assay using peripheral blood leucocytes (PBLs) from Pacific red snapper was performed to evaluate the stimulant effects of β-Gluc/Sh and zymosan A (positive control) for 12 and 24 h and after bacterial challenge with Aeromonas hydrophila at 24 h. In addition, structural characterization of this marine yeast glucan was performed by proton nuclear magnetic resonance (NMR) revealing structures containing (1-6)-branched (1-3)-β-D-glucan. PBLs responded positively to β-Gluc/Sh where cell viability was higher than 80%. After challenge, β-Gluc/Sh was able to inhibit cytotoxicity caused by A. hydrophila, highlighting that the PBLs incubated with β-Gluc/Sh significantly increased the non-specific immune response, such as phagocytic activity, respiratory burst, nitric oxide and peroxidase activities followed by an increase in superoxide dismutase and catalase activities after 12 and 24 h post-stimulation and after challenge with the pathogen. Regarding induction of antioxidant gene expression, it was more pronounced in stimulated β-Gluc/Sh leucocytes compared to other groups at all experimental times of the trial and after bacterial challenge. Indeed, our results clearly showed the ability of leucocytes to strongly react to β-Gluc/Sh with an increase in cytokine gene expression, particularly the IL-1β, IL-10 and IL-17 genes. These results confirm that S. halophilus yeast-derived β-glucan, isolated from an extreme marine environment, is beneficial for increasing innate immune response and enhancing resistance against A. hydrophila in vitro.

In aquaculture, animals can be continually exposed to environmental stress factors that put their health and even survival at risk. Two experiments were performed to evaluate the impact of different stress conditions (acute crowding and anaesthetic) on the natural haemolytic complement activity in serum and skin mucus of gilthead seabream (Sparus aurata L.). In the first experiment, fish were subjected to 10 kg m−3 (low density, control group) and 50 kg m−3 (high density, crowding group) during 2, 24 and 48 h. In the second experiment, fish were unexposed (control) or exposed to 40 ppm of MS-222 or 5 ppm or 10 ppm of clove oil for 1 h. In fish maintained in acute crowding conditions only an increase of the haemolytic complement activity was observed in the skin mucus after 24 h of exposure. However, a similar statistically significant increase was observed in serum and skin mucus of fish exposed for 1 h to the lowest concentration of clove oil (5 ppm) tested. The results point to a new and alternative way to assess stress in farmed fish by using skin mucus instead of blood serum and confirm that the measurement of natural haemolytic complement activity serves as an indicator of stress in fish.

Clove oil is used as an anaesthetic for many species of fish worldwide; however, relatively few studies have assessed its effects on the innate immune response on these species. The present work aimed to investigate the effects of clove oil-eugenol derived anaesthetic on some humoral and cellular immune response in gilthead seabream (Sparus aurata L.). To compare with an unexposed control group, fish were exposed to 55 ppm clove oil for 5 min, before being sampled at 1, 24 and 48 h post-exposure. Serum glucose level was also measured to obtain information on the fish physiological response after clove oil anaesthesia. One hour after exposure the haemolytic complement activity of fish was lower than in the unexposed group. By contrast, the leucocyte peroxidase activity in head-kidney was significantly stimulated 24 h after exposure to clove oil-eugenol. The rest of innate immune parameters evaluated and the glucose levels not were affected by clove oil exposure at any sampling point. Overall, the use of clove oil at 55 ppm as anaesthetic did not seem to alter the innate immune response and neither did it trigger a stress response. The use of clove oil-eugenol derived had become common practice in aquaculture, and its use with gilthead seabream can be considered safe as it does not cause immunodepression in anesthetized fish.

Improving fish defense through the preventive administration of immunostimulants has an important role in controlling the outbreak of the disease in aquaculture. As a continuity of our previous studies, this paper describes the effects of dietary fenugreek (Trigonella foenum graecum) seeds on the skin mucosal antioxidant status and immune response of gilthead seabream (Sparus aurata L.). Fish were fed with four experimental diets: one a basal diet (control) and three diets with powdered fenugreek seeds incorporated in the fish feed at 1%, 5%, and 10%. After eight weeks of feeding, free radicals scavenging and antioxidant assays were assessed in skin mucus by measuring the peroxidation of phospholipid liposomes, hydroxyl radical and hydrogen peroxide scavenging, measurement of total antioxidant activity and the determination of antioxidant activity in a linoleic acid system. The skin mucosal immune response was evaluated by measuring the IgM levels and some enzymatic activities (peroxidase, antiprotease, protease, esterase, and ceruloplasmin). Our results demonstrated that fenugreek inclusion improved the hydroxyl radical scavenging capacity and conferred very high antioxidant activity. Besides, only the highest supplementation level (10%) was able to augment the peroxidase and protease activities confronted by a general decrement in the antiprotease activity in the experimental fed groups with 1% and 10%. These results suggest that the dietary administration of fenugreek at the higher inclusion dose enhances the skin mucosal immunity response and the antioxidant status of gilthead seabream a species with one of the highest rates of production in marine aquaculture.

Medical plants could be used as a prophylactic method in aquaculture because they are considered safe and so very promising alternatives to the use of chemicals. The aim of the present work was to examine the effects of dietary fenugreek (Trigonella foenum graecum) seeds administered for 8 weeks on the metabolic and immune status of gilthead seabream (Sparus aurata L.). Four experimental groups were designated: one receiving a basal diet (control) and three fed powdered fenugreek seeds incorporated in the fish feed at 1%, 5% and 10%. The results show that significant decreases in aspartate aminotransferase, creatine kinase, potassium and the albumin/globulin ratio were detected in the serum of fish fed 10% fenugreek compared with the values recorded in control fish. As regards the immune status, fish fed the 5% supplemented diet had higher haemolytic complement and peroxidase activities than the control fish whilst antiprotease activity was higher in fish fed the 1% fenugreek level respect to control fish and the fish fed the highest fenugreek supplementation rate. Interestingly, the results also revealed a significant enhancement of most of the cellular immune parameters studied, especially in fish fed the highest level of fenugreek (10%). However, the bacteriostatic activity of serum against fish pathogenic and non-pathogenic bacteria was non-affected to any significant extent in fish fed the supplemented diets. Overall, the results suggest that the high level of dietary fenugreek tested in this work (10%) did not negatively affect any of the metabolic parameters measured in serum but increased some of them. In addition, the inclusion of fenugreek seeds in the gilthead seabream diet at 5% or 10% improved the humoral and cellular immune activities, respectively. Further studies are needed to better understand the effects of this natural product, which may be suitable for use as a feed additive in fish aquaculture.

The trial investigated the effects of dietary supplementation with the commercial prebiotic galactooligosaccharide (1% GOS; GOS diet), the commercial probiotic Pediococcus acidilactici MA18/5 M (6.3 log CFU g− 1 PA; PA diet) and their combination as synbiotic (1% GOS and 6.3 log CFU g− 1 PA; SYN diet) on the growth, innate immunity and disease resistance of juvenile rockfish (Sebastes schlegeli). Triplicate groups of fish (18.2 ± 0.3 g) were fed one of the four diets for 8 weeks and then subjected to a standard challenge with Edwardsiella tarda by intraperitoneal injection. The results showed significant (P

Immune responses relies on an adequate provision of multiple nutrients that sustain the synthesis of key effector molecules. These needs are depicted in the already reported increase of circulating free amino acids in fish under stressful conditions. Since aquaculture and the inherent fish welfare are an emergent call, the immunomodulatory effects of amino acids on gut- and skin-associated lymphoid tissues of the European seabass (Dicentrarchus labrax) were studied under unstressed conditions and after an inflammatory insult. To achieve this goal, fish were distributed in duplicate tanks (fifteen fish per tank) and were fed for 14 days with methionine or tryptophan-supplemented diets at 2× dietary requirement level (MET and TRP, respectively) or a control diet meeting the amino acids requirement levels (CTRL). Afterwards, samples of skin and posterior gut were collected from 6 fish per dietary treatment for the assessment of the immune status while the remaining animals were intraperitoneally-injected with inactivated Photobacterium damselae subsp. piscicida and subsequently sampled either 4 or 24 h post-injection. The immune status of both mucosal surfaces was poorly affected, although a tryptophan effect was denoted after bacterial inoculation, with several immune-related genes up-regulated in the gut at 4 h post-injection, which seems to suggest a neuroendocrine-immune systems interaction. In contrast, skin mucosal immunity was inhibited by tryptophan dietary supplementation. Regarding methionine, results were often statistically non-significant, though increasing trends were denoted in a few parameters. Overall, dietary methionine did not significantly affect neither gut nor skin immunity, whereas tryptophan supplementation seems to induce modulatory mechanisms that might be tissue-specific.

Biomarkers have become crucial tools in modern environmental assessment as they can help to predict magnitude of pollution. The head-kidney (HK) and liver (hematopoietic and xenobiotic metabolism organs, respectively) are the key organs in all fish toxicological studies, although the skin has received less attention in this respect. The impact of two different types of polluted sediment collected from Portman Bay (Spain) on HK, liver and skin gene expression in gilthead seabream (Sparus aurata L.) exposed for two weeks to the sediments was determined by real time-PCR. The expression levels of different antioxidant enzyme genes [superoxide dismutase (sod) glutathione reductase (gr) and catalase (cat)] and immune-related genes [interleukin -1β (il-1b), immunoglobulin M (igm), T-Cell receptor (tcr-b), cyclooxygenase-2 (cox-2), colony-stimulating factor 1-receptor (csf-1r) and hepcidin (hep)] was analysed. Expression varied depending on both the organ and gene studied: tcr-b, csf-1r and hep genes were down-regulated in HK, as were gr, tcr-b and il-1b in liver and gr and il-1b in skin, while cox-2 was up-regulated in skin after exposure to both sediments. Concomitantly, histopathological alterations were also studied in HK, liver and skin. While no significant changes were detected in skin cells of fish reared in aquaria with polluted sediments marked changes in the general morphology of HK and liver were observed, accompanied by a substantial degree of cell death and melano-macrophage centre disorganization. The present study suggests that the biomarkers studied in gilthead seabream could be useful for assessing the impact of pollution in coastal environments.

The effects of vitamin D3 dietary administration on certain innate immune parameters on the expression of immune-related genes in head-kidney (HK) and gut were investigated in European sea bass Dicentrarchus labrax. Vitamin D3 (vD3) was orally administered to fish in a commercial pellet food supplemented with 0 (control); 3750; 18,750; or 37,500Ukg−1. Furthermore, gut histology was considered. This study showed a modulation in the activities examined in fish fed with the addition of vD3. After just 2weeks of administration, diet supplementation with the vitamin resulted in increased phagocytic ability, while serum peroxidase content was increased in fish fed with all experimental diets after 4weeks, no significant differences were observed in protease, anti-protease, natural haemolytic complement activities and total IgM level. At gene level, fbl and rbl transcripts were up-regulated in HK in fish fed with the highest concentration of vD3-supplemented diets after 4weeks, while in the gut, an up-regulation of hep gene was observed in fish fed with the different doses of vD3. These results suggest that vD3 may be of great interest for immunostimulatory purposes in fish farms.

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 2weeks 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.

Recently, interest in mucosal surfaces, more specifically fish skin and its secreted mucus, has greatly increased among immunologists. The abundance of terminal carbohydrates, several enzymes (proteases, lysozyme, peroxidase, alkaline phosphatase, esterases and ceruloplasmin), bactericidal activity against fish pathogenic and non-pathogenic bacteria and several physico-chemical parameters (protein concentration, pH, conductivity, redox potential, osmolarity, density and viscosity) in the skin mucus of Senegalese sole (Solea senegalensis, Kaup) have been evaluated. Present results evidence the abundance of N-acetylneuraminic acid, mannose, glucose and N-acetyl-galactosamine in skin mucus. The levels of lysozyme, proteases, esterases and alkaline phosphatase were very similar (from 20 to 30 Units mg−1 protein). However, 93 Units mg−1 protein were detected of ceruloplasmin and only 4′88 Units mg−1 protein of peroxidase. Skin mucus of S. senegalensis showed high bactericidal activity against the tested pathogen bacteria but weak activity against non-pathogenic bacteria. Finally, a clear relationship between mucus density and temperature was detected, while viscosity showed a direct shear- and temperature-dependent behaviour. These results could be useful for better understanding the role of the skin mucus as a key component of the innate immune system, as well as, for elucidating possible relationships between biological and physico-chemical parameters and disease susceptibility.

The welfare of farmed fish is influenced by environmental and management factors, which may substantially increase stress levels of the animals and even endanger their survival. Three experiments to simulate different stress conditions (acute crowding, anaesthetic agents and air exposure) were developed. Cortisol levels were measured in fish skin mucus and the values obtained were correlated with those obtained in serum from the same fish specimens and also in the water surrounding the fish. Concomitantly, the effect of these stressors on immunoglobulin M (IgM) and several enzymes related to immunity were also determined in the serum and skin mucus of gilthead seabream (Sparus aurata L.) in order to identify potential reliable, non-invasive stress bioindicators. Our results demonstrated that skin mucus and water are good non-invasive matrices for detecting and measuring cortisol in stressed fish. Interestingly, a time lag in cortisol levels between serum, skin mucus and the surrounding water was detected. While IgM levels and protease activity were affected by all the stressors in serum and skin mucus, peroxidase activity increased in both matrices but only in fish exposed to acute crowding. The present findings could be relevant for fish aquaculture and underline the importance of skin mucus not only for assessing fish immune status but also for acting as a potential fish stress biomarker. Some of the studied enzymes could be used as biomarkers not only of fish stress in general, but also for understanding the type of stress suffered by the fish.