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Background Oral immunotherapy is a frequent treatment for the management of food allergies, but adverse events (AE) are common. This study assessed the outcome of cow's milk oral immunotherapy (MOIT) in severe cow`s milk-allergic patients treated with omalizumab in a real-life setting. Methods OmaBASE was a national, multicenter, open, and observational registry that collected clinical, immunologic, and treatment from patients with food allergy receiving omalizumab. Results Data derived from 58 patients aged 10.3 years (IQR 6.3-13.2) and median milk-specific IgE 100 kU(A)/L at the start of omalizumab treatment. Most had experienced anaphylaxis by accidental exposures (70.7%) and had asthma (81.0%). Omalizumab in monotherapy induced tolerance to >= 6000 mg of cow's milk protein (CMP) to 34.8% of patients tested by oral food challenge. Omalizumab combined with MOIT conferred desensitization to >= 6000 mg of CMP to 83.0% of patients. Omalizumab withdrawal triggered more AE (P = .013) and anaphylaxis (P = .001) than no discontinuation. Anaphylaxis was observed in 36.4% of patients who discontinued omalizumab, and more in those with sudden (50.0%) rather than progressive (12.5%) discontinuation. At database closure, 40.5% of patients who had completed follow-up tolerated CMP without omalizumab (7.2% 1500-4500 mg; 33.3% >= 6000 mg). Conclusion Milk oral immunotherapy initiated under omalizumab allows the desensitization of subjects with severe cow's milk allergy even after omalizumab discontinuation. However, discontinuation of omalizumab can lead to severe AE and should be carefully monitored.

Skin mucus is increasingly used as a source for determining immunity-related proteins and enzymes. However, the ability to accurately measure some activities may be modified by inadequate handling and storage of the samples. This study aims to measure the effect of freezing and lyophilization at the time of collection on such activities. Fresh, frozen (immediately after collection at -20 °C and -80 °C) and lyophilized skin mucus samples obtained from the same groups of fish specimens of gilthead seabream (Sparus aurata L.) were analysed in the assays. The amount of total proteins and sugar residues (determined by lectin binding) present in skin mucus samples fell after both freezing and lyophilization of the samples. While no significant differences were exhibited in the levels of some proteins or enzymes (immunoglobulin M, antiprotease, peroxidase, esterase and alkaline phosphatase) determined in fresh or frozen mucus samples, protease and lysozyme activities were lower in frozen mucus samples than in fresh samples. Lyophilization of the mucus samples drastically decreased the total level of proteins obtained, as well as of protease, peroxidase, lysozyme and alkaline phosphatase activities. The results suggest that freezing skin mucus samples is more suitable than lyophilization if samples are stored before determining enzymatic activities.

The in vitro use of fish leucocytes to test the toxicity of aquatic pollutants, and particularly the immutoxicological effects, could be a valuable alternative to fish bioassays but has received little attention. In this study, head-kidney and peripheral blood leucocytes (HKLs and PBLs, respectively) from gilthead seabream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) specimens were exposed to Cd, MeHg (methylmercury), Pb or As for 24 h being evaluated the resulting cytotoxicity. Exposure to metals produced a dose-dependent reduction in the viability, and MeHg showed the highest toxicity followed by Cd, As and Pb. Interestingly, leucocytes from European sea bass are more resistant to metal exposure than those from gilthead seabream. Similarly, HKLs are always more sensitive than those isolated from blood from the same fish species. Moreover, fish leucocytes incubated with metals exhibited alterations in gene expression profiles that were more pronounced in the HKLs in general, being Pb the metal provoking less effects. Concretely, genes related to cellular protection (metallothionein), stress (heat shock protein 70) and oxidative stress (superoxide dismutase, catalase and glutathione reductase) were, in general, down-regulated in seabream HKLs but up-regulated in seabream PBLs and sea bass HKLs and PBLs. In addition, this profile leads to the increase of expression in genes related to apoptosis (Bcl2 associated X protein and caspase 3). Finally, transcription of genes involved in immunity (interleukin-1β and immunoglobulin M) was down-regulated, mainly in seabream leucocytes. This study points to the benefits for evaluating the toxicological mechanisms of marine pollution using fish leucocytes in vitro and insight into the mechanisms at gene level.

Lymphocystis or lymphocystis disease virus (LCDV) is distributed worldwide and affects many fresh and marine water fish species. LCDV is commonly found in aquaria fish species but also in farmed fish species, among them the gilthead seabream (Sparus aurata L.). The immune status of gilthead seabream (S. aurata) specimens under a natural outbreak of LCDV was studied. The replication of the virus was demonstrated in infected fish, but not in control fish. The results showed decreased total serum IgM levels and increased innate cellular immune response (peroxidase and respiratory burst activities) of head kidney leucocytes in LCDV-infected fish, compared to the values obtained in uninfected specimens. In addition, transcription of antiviral genes (ifn and irf3) was down-regulated in the skin of LCDV-positive fish as well as genes involved in cellular immunity (csf1r, mhc2a, tcra and ighm) that were down-regulated in skin and head kidney of infected fish. By contrast, the transcription of nccrp1 was up-regulated in head kidney after LCDV infection. These present results show that head kidney leucocytes are activated to encounter the virus at the sites of replication.

The in vitro use of fish erythrocytes to test the toxicity of aquatic pollutants could be a valuable alternative to fish bioassays but has received little attention. In this study, erythrocytes from marine gilthead sea bream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) specimens were exposed for 24 h to Cd, Hg, Pb and As and the resulting cytotoxicity was evaluated. Exposure to metals produced a dose-dependent reduction in the viability, and mercury showed the highest toxicity followed by MeHg, Cd, As and Pb. Moreover, fish erythrocytes incubated with each one of the metals exhibited alteration in gene expression profile of metallothionein, superoxide dismutase, catalase, peroxiredoxin, glutathione reductase, heat shock proteins 70 and 90, Bcl2-associated X protein and calpain1 indicating cellular protection, stress and apoptosis death as well as oxidative stress. This study points to the benefits for evaluating the toxicological mechanisms of marine pollution using fish erythrocytes in vitro.

As an organism is exposed to pathogens during very early development, specific defense mechanisms must take effect. In this study, we used a germ-free zebrafish embryo model to show that osmotic stress regulates the activation of immunity and host protection in newly hatched embryos. Mechanistically, skin keratinocytes were responsible for both sensing the hyposmolarity of the aquatic environment and mediating immune effector mechanisms. This occurred through a transient potential receptor vanilloid 4/Ca2+/TGF-β–activated kinase 1/NF-κB signaling pathway. Surprisingly, the genes encoding antimicrobial effectors, which do not have the potential to cause tissue damage, are constitutively expressed during development, independently of both commensal microbes and osmotic stress. Our results reveal that osmotic stress is associated with the induction of developmental immunity in the absence of tissue damage and point out to the embryo skin as the first organ with full capacities to mount an innate immune response.

There is increasing concern about the possible effect of pharmaceutical compounds may have on the fish immune system. Bath exposition of 17α-ethynylestradiol (EE2), a synthetic estrogen used in oral contraceptives, altered the immune response of the gilthead seabream (Sparus aurata L.), a marine hermaphrodite teleost. Tamoxifen (Tmx) is a selective estrogen-receptor modulator used in hormone replacement therapy, the effects of which are unknown in fish immunity. This study aims to investigate the effects of dietary administration of EE2 (5 μg/g food) and Tmx (100 μg/g food) on the immune response of gilthead seabream, and the capacity of the immune system to recover its functionality after a recovery period. The results show for the first time the reversibility of the effect of EE2 and Tmx on the fish immune response. Tmx promoted a transient alteration in hepatic vitellogenin gene expression of a different magnitude to that produced by EE2. Both, EE2 and Tmx inhibited the induction of interleukin-1β gene expression while reversed the inhibition of ROI production in leukocytes following vaccination. However, none of these effects were observed after ceasing EE2 and Tmx exposure. EE2 and Tmx stimulated the antibody response of vaccinated fish although Tmx, but not EE2, altered the antibody response and modulated the percentage of IgM(+) B lymphocytes of vaccinated fish during the recovery phase. Taken together, our results suggest that EE2 and Tmx might alter the capacity of fish to appropriately respond to infection and show that Tmx has a long-lasting effect on humoral adaptive immunity.

Heavy metals provoke toxicological effects on aquatic animal species, including fish, though their effects on fish leucocytes and immunotoxicology are still limited. In the present work the effects of heavy metals (Cd, Hg, Pb or As) on viability, oxidative stress and innate immune parameters of isolated head-kidney leucocytes from gilthead seabream (Sparus aurata) are studied. Cytotoxicity results indicated that short exposures (30 min or 2h) to Hg promoted both apoptosis and necrosis cell death of leucocytes whilst Cd, Pb and As did only by apoptosis, in all cases in a concentration- and time-dependent manner. In addition, production of free oxygen radicals was induced by Cd, Hg and As heavy metals. Cd failed to change phagocytosis but Hg and As increased the percentage of phagocytic cells but decreased the number of ingested particles per cell whilst Pb increased both phagocytic parameters. On the other hand, respiratory burst activity was significantly reduced by incubation with Cd, Hg and As but increased with Pb. Furthermore, the gene expression profiles partly support the functional finding of this work. This study provides an in vitro approach for elucidating the heavy metals toxicity, and particularly the immunotoxicity, in fish leucocytes.

Although the skin is one of the main defense barriers of fish to date, very little is known about the immune implications and the properties of the numerous substances present in skin cells. In the present study, terminal carbohydrate composition and some components of the skin immunity (total IgM level, and several enzymatic and bacteriostatic activities) present on aqueous and organic epidermal extracts of European sea bass (Dicentrarchus labrax) were determined. Most of the parameters measured followed a protein concentration dose-response. Curiously, both skin extracts have similar levels of total IgM. However, aqueous extracts showed higher presence of some terminal carbohydrates, alkaline phosphatase and esterase activities and lower proteases and ceruloplasmin activities than epidermal organic extracts. Regarding the bacteriostatic activity, the growth of all the bacterial strains tested was reduced when cultivated in presence of organic extracts, being the observed reduction correlated to the protein concentration present in the extract sample. On the contrary, skin aqueous extracts have no significant effect on bacterial growth or even allow bacteria to overgrow, suggesting that the bacteria could use the extracts as a nutrient source. The results are discussed and compared with the same activities studied on fish skin mucus in order to understand their possible implications on mucosal immunity.

The knowledge about the direct effects of heavy metals on fish leucocytes is still limited. We investigate the in vitro effects of heavy metals (Cd, Hg, Pb or As) on oxidative stress, viability and innate immune parameters of head-kidney leucocytes (HKLs) from European sea bass (Dicentrarchus labrax). Production of free oxygen radicals was induced by Cd, Hg and As, mainly after 30 min of exposure. Cd and Hg promoted both apoptosis and necrosis cell death while Pb and As did only apoptosis, in all cases in a concentration-dependent manner. Moreover, expression of genes related to oxidative stress and apoptosis was significantly induced by Hg and Pb but down-regulated by As. In addition, the expression of the metallothionein A gene was up-regulated by Cd and Pb exposure though this transcript, as well as the heat shock protein 70, was down-regulated by Hg. Cd, methylmercury (MeHg) and As reduced the phagocytic ability, whereas Hg and Pb increased it. Interestingly, all the heavy metals decreased the phagocytic capacity (the number of ingested particles per cell). Leucocyte respiratory burst changed depending on the metal exposure, usually in a time- and dose-manner. Interestingly, the expression of immune-related genes was slightly affected by Cd, MeHg, As or Pb being Hg the form producing the greatest alterations, which included down-regulation of immunoglobulin M and hepcidin, as well as the up-regulation of interleukin-1 beta mRNA levels. This study provides an in vitro approach for elucidating the heavy metals toxicity, and particularly the immunotoxicity, in fish leucocytes.

Although aluminum salts (Alum) have been extensively used in human vaccination for decades, its mechanism of action is controversial. In fish, the use of Alum as a vaccine adjuvant is scarce and there are no studies aimed at identifying its mechanism of action. In the present study we report that Alum is a powerful adjuvant in the gilthead seabream (Sparus aurata L., Sparidae) and the European seabass (Dicentrarchus labrax L. Moronidae). Thus, Alum increased the specific antibody titers to the model antigen keyhole limpet hemocyanin as the commonly used Freund's adjuvant did in both species. In addition, both adjuvants were able to increase the transcript levels of the gene encoding the major pro-inflammatory mediator interleukin-1β (Il1b). Strikingly, however, Alum failed to promote Il1b release by seabream leukocytes and even impaired Il1b induction, processing and release in macrophages. However, it increased NADPH oxidase-dependent reactive oxygen species (ROS) production in gilthead seabream leukocytes and purified granulocytes. In addition, Alum promoted gilthead seabream leukocyte death independently of ROS production and caspases, suggesting that damage-associated molecular patterns release from dying cells mediate Alum adjuvant activity. Our results pave the way for future studies aimed at investigating the relevance of danger signals generated by Alum in vivo on its adjuvant activity in order to increase our understanding of the mechanisms of action of Alum in fish vaccines and to help in the design of new adjuvants for aquaculture.

Viral diseases are responsible for high rates of mortality and subsequent economic losses in modern aquaculture. The nervous necrosis virus (NNV) produces viral encephalopathy and retinopathy (VER), which affects the central nervous system, is considered one of the most serious viral diseases in marine aquaculture. Although some studies have localized NNV in the retina cells, none has dealt with immunity in the retina. Thus, for the first time, we intravitreally infected healthy specimens of European sea bass (Dicentrarchus labrax) with NNV with the aim of characterizing the immune response in the retina. Ultrastructural analysis detected important retinal injuries and structure degradation, including pycnosis, hydropic degeneration and vacuolization in some cell layers as well as myelin sheaths in the optic nerve fibres. Immunohistochemistry demonstrated that NNV re- plicated in the eyes. Regarding retinal immunity, NNV infection elicited the transcription of genes encoding proteins involved in the interferon (IFN) and cell-mediated cytotoxicity (CMC) responses as well as B and T cell markers, demonstrating that viral replication influences innate and adaptive responses. Further studies are needed to understand the retina immunity and whether the main retinal function, vision, is affected by noda- virus., Detección de nodavirus en el mar Mediterráneo. Implicación y riesgo para la acuicultura, NODAMED, SI

Developing viral vaccines through the ultraviolet (UV) inactivation of virus is promising technique since it is straightforward and economically affordable, while the resulting viruses are capable of eliciting an adequate antiviral immune response. Nodavirus (NNV) is a devastating virus that mainly affects European sea bass juveniles and larvae, causing serious economic losses in Mediterranean aquaculture. In this work, a potential vaccine consisting on UV-inactivated NNV (iNNV) was generated and administered to healthy juveniles of European sea bass to elucidate whether it triggers the immune response and improves their survival upon challenge. First, iNNV failed to replicate in cell cultures and its intraperitoneal administration to sea bass juveniles also failed to produce fish mortality and induction of the type I interferon (IFN) pathway, indicating that the NNV was efficiently inactivated. By contrast, iNNV administration induced significant serum non-specific antimicrobial activity as well as a specific antiviral activity and immunoglobulin M (IgM) titres against NNV. Interestingly, few changes were observed at transcriptional level in genes related to either innate or adaptive immunity, suggesting that iNNV could be modulating the immune response at protein or functional level. In addition, the iNNV vaccinated group showed improved survival, reaching a relative survival percentage of 57.9%. Moreover, challenged fish that had been vaccinated presented increased serum antibacterial, antiviral and IgM titres, as well as the higher transcription of mhc1a, ifn, isg15 and cd8a genes in brain, while in the headkidney the transcription of mhc1a, mhc2b and cd8a was down-regulated and mx, isg15 and tcrb was up-regulated. Although the UV-inactivated vaccine against NNV showed promising results, more effort should be addressed to improving this prophylactic method by increasing our understanding of its action mechanisms, thus enabling the mortality rate of NNV to be further reduced, Sí

Inorganic arsenic (As) is one of the most toxic pollutants in the water. We have studied their effects on the marine teleost European sea bass (Dicentrarchus labrax) at 2 and 10 days of 5 μMofAs2O3 (sub-lethal doses) waterborne exposure. Arsenic accumulates in liver and gill tissues. The expression profile of five genes (bax, blc2, casp3, casp8 and casp9) involved in apoptosis cell death confirmed apoptotic effects in liver, slight changes in gill and no effects in skin according with the histopathology findings. Total IgM level and peroxidase activities were increased at 2 and 10 days, respectively. The bactericidal activity was decreased at 2 days after As exposure. A general decrease of cellular immune activities with significant differences in the case of respiratory burst activity was observed after 2 and 10 days of exposure. This work describes for the first time the effects of As exposure on European sea bass., SI

The potential benefits of probiotics when administering to fish could improve aquaculture production. The objective of this study was to examine the modulation of immune status and gut microbiota of gilthead seabream ( Sparus aurata L.) specimens by a probiotic when administered encapsulated. Commercial diet was enriched with Shewanella putrefaciens Pdp11 (SpPdp11, at a concentration of 10 8 cfu g −1 ) before being encapsulated in calcium alginate beads. Fish were fed non-supplemented (control) or supplemented diet for 4 weeks. After 1, 2 and 4 weeks the main humoral and cellular immune parameters were determined. Furthermore, gene expression profile of five immune relevant genes ( il1β , bd , mhcIIα , ighm and tcrβ ) was studied by qPCR in head kidney. On the other hand, intestinal microbiota of fish was analysed at 7 and 30 days by DGGE. Results demonstrated that administration of alginate encapsulated SpPdp11 has immunostimulant properties on humoral parameters (IgM level and serum peroxidase activity). Although no immunostimulant effects were detected on leucocyte activities, significant increases were detected in the level of mRNA of head-kidney leucocytes for mhcIIα and tcrβ after 4 weeks of feeding the encapsulated-probiotic diet. The administration of SpPdp11 encapsulated in alginate beads produced important changes in the DGGE patterns corresponding to the intestinal microbiota. Predominant bands related to lactic acid bacteria, such as Lactococcus and Lactobacillus strains, were sequenced from the DGGE patterns of fish fed the probiotic diet, whereas they were not sequenced from fish receiving the control diet. The convenience or not of probiotic encapsulation is discussed.

Summary Hematopoietic stem cells (HSCs) underlie the production of blood and immune cells for the lifetime of an organism. In vertebrate embryos, HSCs arise from the unique transdifferentiation of hemogenic endothelium comprising the floor of the dorsal aorta during a brief developmental window. To date, this process has not been replicated in vitro from pluripotent precursors, partly because the full complement of required signaling inputs remains to be determined. Here, we show that TNFR2 via TNFα activates the Notch and NF-κB signaling pathways to establish HSC fate, indicating a requirement for inflammatory signaling in HSC generation. We determine that primitive neutrophils are the major source of TNFα, assigning a role for transient innate immune cells in establishing the HSC program. These results demonstrate that proinflammatory signaling, in the absence of infection, is utilized by the developing embryo to generate the lineal precursors of the adult hematopoietic system.

A key goal of a successful vaccine formulation is the strong induction of persistent protective immune responses without producing side-effects. Adjuvants have been proved to be successful in several species at inducing increased immune responses against poorly immunogenic antigens. Fish are not the exception and promising results of adjuvanted vaccine formulations in many species are needed. In this study, over a period of 300 days, we characterized the apparent damage and immune response in gilthead seabream immunized by intraperitoneal injection with the model antigen keyhole limpet hemocyanin (KLH) alone or formulated with Montanide ISA water-in-oil (761 or 763), or Imject™ aluminum hydroxide (aluminium), as adjuvants. Throughout the trial, external tissue damage was examined visually, but no change was observed. Internally, severe adhesions, increased fat tissue, and hepatomegaly were recorded, but, without impairing animal health. At 120 days post priming (dpp), histopathological evaluations of head-kidney, spleen and liver revealed the presence of altered melanomacrophage centers (MMC) in HK and spleen, but not in liver. Surprisingly, in all aluminium treated fish, classical stains unmasked a toxic effect on splenic-MMC, unequivocally characterized by a strong cell depletion. Furthermore, at 170 dpp transmission electron microscopy confirmed this data. Paradoxically, at the same time powerful immune responses were recorded in most vaccinated groups, including the aluminium treatment. Whatever the case, despite the observed adhesions and MMC depletion, fish physiology was not affected, and most side-effects were resolved after 300 dpp. Therefore, our data support adjuvant inclusion, but strongly suggest that use of aluminium must be further explored in detail before it might benefit the rational design of new vaccination strategies in aquaculture.

The increasing use of nanomaterials, e.g. nanosilver, has lead to concerns about environmental contamination and possible toxic effects on aquatic organisms. Here, we present evidence for the impact of silver nanospheres (AgNSs) on fish innate immune cells after in vitro exposure. AgNSs of 20, 50 or 100 nm in diameter were tested with the smallest ones (20 nm) clearly having the most deleterious effects, after an exposure period of 30 min, followed by the medium-sized ones; the NSs of 100 nm had no impact. The effective concentration was determined at 10 μg ml-1 while lower concentrations (1, 2.5 or 5 μg ml-1) were ineffective. Head-kidney mixed leucocyte population showed significant viability reduction which was attributable to diminished viability of macrophages/monocytes and lymphocytes only whereas granulocytes' viability was not affected at the above exposure regime. Furthermore, cellular respiratory burst activity, phagocytic capacity and phagocytic ability were all reduced, with the first two parameters exhibiting the sharper reductions. Finally, transmission electron microscopy revealed that the AgNSs' internalization was brought about via phagocytosis, pinocytosis, receptor-mediated endocytosis and macropinocytosis; also, that cell death could be effected in either an apoptotic or a necrotic manner. It is concluded that AgNSs are potentially very noxious for the teleost fish immune system as they can adversely affect the function and viability of the head-kidney leucocytes.

Nervous necrosis virus (NNV) causes high mortalities in several marine species. We aimed to evaluate the innate cell-mediated cytotoxic (CMC) activity of head-kidney leucocytes (HKLs) isolated from naïve European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata), a very susceptible and resistant fish species to NNV, respectively, against fish cell lines infected with NNV. Seabream HKLs showed significantly increased innate CMC activity against NNV-infected cells, compared to those uninfected, while sea bass HKLs failed to do so. Thus, we performed a RNA-seq study to identify genes related to the CMC activity of sea bass leucocytes. Thus, we found that sea bass HKLs incubated with DLB-1 cells alone (CMC_DLB1) or with NNV-infected DLB-1 cells (CMC_DLB1-NNV) showed very similar transcriptomic profiles and the GO analysis revealed that most of the up-regulated genes were related to immunity. Strikingly, when the CMC samples with and without NNV were compared, GO analysis revealed that most of the up-regulated genes in CMC_DLB1-NNV samples were related to metabolism and very few to immunity. This is also in agreement with the functional data. These data point to the escape of CMC activity by NNV infection as an important factor involved in the high susceptibility to nodavirus infections of European sea bass., SI

Histamine is stored inside hemocytes of the tunicate Styela plicata (Chordata, Tunicata, Ascidiacea), but no evidence on its role in the regulation of the immune response of this species has been reported. We examined whether histamine participated in the regulation of inflammation and host defense in S. plicata. The presence of histamine inside S. plicata hemocytes was confirmed by flow cytometry, and histamine release was detected by ELISA, after in vitro hemocyte stimulation with different PAMPs. In vitro hemocyte treatment with histamine, or specific histamine-receptor agonists, reduced their phagocytic ability. Injection of histamine into the tunic recruited hemocytes to the site of injection. Systemic injection of histamine, or the histamine-releasing agent compound 48/80, decreased the phagocytic ability of hemocytes. Histamine promoted the constriction of tunic hemolymph vessels in vivo, having a direct effect on vasoconstriction in tunic explants. These results provide for the first time clear evidence for the involvement of histamine in the regulation of inflammation and host defense in tunicates.

Inflammation is the first response of animals to infection or tissue damage. Sparus aurata (Perciformes) was the first fish species shown to possess histamine-containing mast cells at mucosal tissues. We report a separation protocol for obtaining highly enriched (over 95% purity) preparations of fish mast cells in high numbers (5-20 million mast cells per fish). The peritoneal exudate of S. aurata is composed of lymphocytes, acidophilic granulocytes, macrophages and mast cells. We separated the lymphocyte fraction through discontinuous density gradient centrifugation. The remaining cells were cultivated overnight in RPMI-1640 culture medium containing 5% fetal calf serum, which allowed macrophages to adhere to the cell culture flasks. Finally, acidophilic granulocytes were separated from the mast cells though a Magnetic-Activated Cell Separation (MACS) protocol, using a monoclonal antibody against these cells. The purity of mast cells-enriched fractions was analyzed by flow cytometry and by transmission electron microscopy. The functionality of purified mast cells was confirmed by the detection of histamine release by ELISA after stimulation with compound 48/80 and the induction of the pro-inflammatory cytokines IL-1β and IL-8 following stimulation with bacterial DNA. This fish mast cells separation protocol is a stepping stone for further studies addressing the evolution of vertebrate inflammatory mechanisms., SI

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.

TLR22 is a fish-specific TLR that recognizes dsRNAs. In the present study, a TLR22 homologue gene from gilthead seabream (sbTLR22) was identified and characterized. The full coding sequence contained a single open-reading frame of 2895 nucleotides encoding a predicted protein of 964 amino acids in length. Its 3′-UTR was relatively long, 1380 nucleotides, and contained three AU-rich sequences frequently associated with mRNA instability. Functional studies showed that the sbTLR22 transcript had a short half-life, although the three AU-rich sequences in its 3′-UTR did not seem to be related with this fact. The sbTLR22 was highly expressed in the spleen, thymus and gills of healthy fish. After Vibrio anguillarum infection, the mRNA levels of sbTLR22 increased greatly in head kidney, blood and peritoneal exudate, but were only moderately induced in spleen and liver, suggesting the involvement of sbTLR22 in the immune response against bacterial infections. In addition, acidophilic granulocytes and macrophages, both considered professional phagocytes in seabream, displayed cell-type-specific sbTLR22 expression profiles when stimulated with different pathogen-associated molecular patterns (PAMPs). Although acidophilic granulocytes expressed sbTLR22, polyinosinic:polycytidylic acid (poly I:C) was unable to up-regulate the expression of this receptor. In contrast, poly I:C induced the expression of sbTLR22 in macrophages, in a process that was partially endosome-dependent. Taken together, our results suggest that sbTLR22 is involved in bacterial infection and might sense bacterial PAMPs.

Deltamethrin, a sintetic pyrethroid, is the insecticide that has been replacing recently to others like organochlorines, organophosphates and carbamates which are less toxic for birds and mammals, although, unfortunately, all of them are highly toxic to various non-targeted aquatic organisms including fish. In the present study, the consequences of the exposition of gilthead seabream (Sparus aurata L.) specimens to sublethal bath dose of deltamethrin (0.1 ppb) on organo-somatic indexes, immunity, seric metabolic parameters, oxidative stress and liver histology were determined after 1, 3, 7 and 14 days of exposure. Deltamethrin alters gilthead seabream immune status, the hepato-somatic index and various seric metabolic parameters since the first exposure day while important progressive deleterious morphological changes in liver were also observed. However, no statistically significant deviation was detected in the expression of oxidative stress-related genes whilst the expression of cytochrome P450 gene was up-regulated in head-kidney and liver of exposed fish. Overall, the present results indicate severe immunotoxicological and metabolic effects of deltamethrin in gilthead seabream, the species with the highest rate of production in Mediterranean aquaculture. In general, the values obtained for the tested parameters during the trial seem to indicate that specimens try to adapt to this adverse situation although the continuous presence of the toxic impede the hypothetic recovery of homoeostasis. The use of deltamethrin in the proximities of seabream farms should be carefully considered.

Redbelly tilapia (Tilapia zillii; Gervais, 1848) is one of the most valuable freshwater species in North Africa representing an important part of the continental production, especially in brackish lakes. In Algeria, T. zillii is distributed in several lakes and tributaries of some rivers in the south. Though some attempts are in progress to culture this species, many investigations covering its biology and farm management are still needed. In this sense, this is the first study attempting to evaluate some of the T. zillii immune parameters and valuable data to assess their health and well-being status. Thus, we have determined the levels of serum peroxidases as well as the alternative complement, antiprotease and bactericidal activities. Furthermore, we have also evaluated the potential impact of two acute stress factors, commonly found in fish farms, in these parameters. Although it was assessed that fish exposed to low temperatures or crowding were stressed, as indicated by their increased serum levels of cortisol and glucose, both acute stressors failed to significantly affect serum peroxidases as well as antiprotease and complement activities. However, the bactericidal activity was reduced in general but only in those exposed to crowding reached statistical significance. Further studies are needed to characterise the immune response in T. zillii as well as the effects that farming stresses may produce.

Neutrophils are major participants in innate host responses. It is well known that estrogens have an immune-modulatory role, and some evidence exists that neutrophil physiology can be altered by these molecules. Traditionally, estrogens act via classical nuclear estrogen receptors, but the identification of a G protein–coupled estrogen receptor (GPER), a membrane estrogen receptor that binds estradiol and other estrogens, has opened up the possibility of exploring additional estrogen-mediated effects. However, information on the importance of GPER for immunity, especially, in neutrophils is scant. In this study, we report that gilthead seabream (Sparus aurata L.) acidophilic granulocytes, which are the functional equivalent of mammalian neutrophils, express GPER at both mRNA and protein levels. By using a GPER selective agonist, G1, it was found that GPER activation in vitro slightly reduced the respiratory burst of acidophilic granulocytes and drastically altered the expression profile of several genes encoding major pro- and anti-inflammatory mediators. In addition, GPER signaling in vivo modulated adaptive immunity. Finally, a cAMP analog mimicked the effects of G1 in the induction of the gene coding for PG-endoperoxide synthase 2 and in the induction of CREB phosphorylation, whereas pharmacological inhibition of protein kinase A superinduced PG-endoperoxide synthase 2. Taken together, our results demonstrate for the first time, to our knowledge, that estrogens are able to modulate vertebrate granulocyte functions through a GPER/cAMP/protein kinase A/CREB signaling pathway and could establish therapeutic targets for several immune disorders in which estrogens play a prominent role. The Journal of Immunology, 2013, 191: 4628–4639., SI

Studies in fish have demonstrated that Cd-exposure produce skeletal deformities and alterations in tissue morphology, enzyme activities, stress response, ion regulation and immune response. In the present work, gilthead seabream (Sparus aurata) specimens were exposed to waterborne Cd (5 μM CdCl2 or 1 mg L(-1)) for 2, 10 or 30 days. Organo-somatic changes, Cd accumulation, liver histology and humoral and cellular immune responses were determined. Results showed that exposure of seabream specimens to Cd induced no alterations on spleen and liver organo-somatic indexes whilst produced progressive deleterious morphological alterations in liver and exocrine pancreas that correlated with the hepatic Cd-accumulation. Regarding the immunotoxicological potential, strikingly, Cd-exposure produced a reduction in the serum complement activity and leucocyte respiratory burst to a significant extent after 10 and 30 days whilst the serum peroxidase activity and leucocyte phagocytosis were increased at different sampling times. On the other hand, serum IgM levels and leucocyte peroxidase activity resulted unaltered. The present results seem to indicate that seabream exposed to Cd in the present conditions suffer toxicity.

Two forms of TLR5, one membrane-anchored and one soluble, have been described in some teleost fish species. However, the exact role of each form has been poorly studied. In the present study, we show that the mRNA levels of soluble gilthead seabream TLR5 (sbTLR5S) are highly induced in head kidney, spleen, liver and blood after Vibrio anguillarum infection, suggesting an important role for sbTLR5S in the innate immune response against bacteria. Comparative genomic and phylogenetic analyses revealed a co-evolution pattern of both genes across fish species and a proximal location in their genomes, further suggesting a functional link between them. To further investigate this issue, the coding sequence of the sbTLR5S was cloned and the corresponding recombinant protein was produced in HEK293 cells. The gene product was secreted to the culture medium as a soluble factor and a physical interaction between flagellin and sbTLR5S was demonstrated. Collectively, these results suggest that sbTLR5S plays an important role in modulating the flagellin-mediated immune response in seabream.

Arsenic (As) has been associated with multitude of animal and human health problems; however, its impact on host immune system has not been extensively investigated. In fish, there are very few works on the potential risks or problems associated to the presence of arsenic. In the present study we have evaluated the effects of exposure (30 days) to sub-lethal concentrations of arsenic (5 μM As₂O₃) in the teleost fish gilthead seabream (Sparus aurata), with special emphasis in the innate immune response. The arsenic concentration was determined using atomic fluorescence spectrometry (AFS) in liver and muscle of exposed fish showing As accumulation in the liver after 30 days of exposure. The hepatosomatic index was increased at significant extent after 10 days but returned to control values after 30 days of exposure. Histological alterations in the liver were observed including hypertrophy, vacuolization and cell-death processes. Focusing on the immunological response, the humoral immune parameters (seric IgM, complement and peroxidase activities) were no affected to a statistically significant extent. Regarding the cellular innate parameters, head-kidney leucocyte peroxidase, respiratory burst and phagocytic activities were significantly increased after 10 days of exposition compared to the control fish. Overall, As-exposure in the seabream affects the immune system. How this might interfere with fish biology, aquaculture management or human consumers warrants further investigations. This paper describes, for the first time, the immunotoxicological effects of arsenic exposure in the gilthead seabream, which is a species with the largest production in Mediterranean aquaculture.

In teleost fish, there are no commercial cell lines for the European sea bass (Dicentrarchus labrax). Thus, we have established the sea bass brain (DLB-1) cell line, using a fish retrovirus for immortalization, which resemble epithelial cells and express glial cells markers. Exposure to metals [Cd, methylmercury (MeHg), Pb or As] produces cytotoxicity and induction of reactive oxygen species (ROS) production. Interestingly, cell cycle analysis of DLB-1 cells shows that exposure to metals alters it significantly. Moreover, all the metals induce apoptosis as indicated by sub-Go/G1 population and annexin V binding. Finally, exposure of DLB-1 cells to metals also produces significant alterations at gene expression level, which confirm the above functional results. This is the first study in which metal cytotoxicity has been evaluated in a fish brain cell line and results seem to support that DLB-1 cells are suitable for toxicological studies., SI

Vaccines for fish need to be improved for the aquaculture sector, with DNA vaccines and the oral administration route providing the most promising improvements. In this study, we have created an oral chitosan-encapsulated DNA vaccine (CP-pNNV) for the nodavirus (NNV) in order to protect the very susceptible European sea bass (Dicentrarchus labrax). Our data show that the oral CP-pNNV vaccine failed to induce serum circulating or neutralizing specific antibodies (immunoglobulin M) or to up-regulate their gene expression in the posterior gut. However, the vaccine up-regulated the expression of genes related to the cell-mediated cytotoxicity (CMC; tcrb and cd8a) and the interferon pathway (IFN; ifn, mx and ifng). In addition, 3 months after vaccination, challenged fish showed a retarded onset of fish death and lower cumulative mortality with a relative survival of 45%. Thus,we created a chitosan-encapsulated DNA vaccine against NNV that is partly protective to European sea bass juveniles and up-regulates the transcription of genes related to CMC and IFN. However, further studies are needed to improve the anti- NNV vaccine and to understand its mechanisms., SI

The effects on histology and microbial ecology in gilthead seabream (Sparus aurata) intestine caused by dietary probiotic and microalgae were studied. Fish were fed non-supplemented (C, control) or supplemented diets with Tetraselmis chuii, Phaeodactylum tricornutum and Bacillus subtilis single or combined (diets T, P, B, BT and BP) for 4 weeks. Curiously, fish fed the experimental diets showed similar morphological alterations when studied by light and electron microscopy and significant signs of intestinal damage were detected. No effect of microalgae or B. subtilis on the intestinal absorptive area was observed, whereas the number of goblet cells and IELs were significantly lower in fish fed the T, P, B and BT diets and T, BT and BP diets, respectively. Interestingly, only the diets containing B. subtilis resulted in a significant reduction of microvilli height. Alterations such as wide intercellular spaces and large vacuoles in enterocytes were observed in fish fed T, B, BT, BT and P in lesser degrees. These observations demonstrate that fish fed experimental diets presented different signs of oedema and inflammation that could compromise their body homeostasis. Moreover, the experimental diets cause important alterations in the intestinal microbiota by a significant decrease in bacterial diversity, as demonstrated by the fall in specific richness, Shannon and range-weighted richness indices. To our knowledge, this is the first in vivo study regarding the implications of the use of probiotics in combination with immunostimulants on fish intestinal morphology and microbiota. More morphofunctional studies are needed in order to correlate the nutritional and immune aspects of fish gut.

Combined or individual effects of two microalgae (Phaeodactylum tricornutum and Tetraselmis chuii) and Bacillus subtilis on immune response, gene expression, and survival to challenge with Photobacterium damselae subsp. piscicida of gilthead sea bream were investigated. To test the capacity of B. subtilis to grow employing the microalgae polysaccharides as energy and carbon source, an in vitro assay was defined, and demonstrated that the digestion product of microalgae, mainly P. tricornutum, supported the growth of B. subtilis much better than glucose. For the in vivo study, fish were distributed in six equal groups (each of two replicates) and received one of the following experimental diets: C) control, non-supplemented diet; T) T. chuii 100 g kg(-1); P) P. tricornutum 100 g kg(-1); B) B. subtilis (10(7) cfu g(-1)); BT) B. subtilis (10(7) cfu g(-1))+T. chuii (100 g kg(-1)); and BP) B. subtilis (10(7) cfu g(-1))+P. tricornutum (100 g kg(-1)). The complement activity, serum IgM level, respiratory burst, phagocytic activity, and expression of seven selected immune-related genes in head-kidney were evaluated following two and four weeks of treatment. At the end of the feeding trial, fish were challenged by intraperitoneal injection of LD(50) concentration of P. damselae subsp. piscicida and mortality was recorded. This is the first study testing the immunomodulatory capacity of the microalgae used in the present work. The dietary applications of B. subtilis, T. chuii, and P. tricornutum, singly or in combination, may exhibit up-regulating effects on gilthead sea bream immune parameters. P. tricornutum demonstrated the highest immunostimulant activity. There were no significant differences between combination feeding and feeding ingredients separately. Our results demonstrated the potential of microalgae as immunostimulants for fish, although further studies regarding the implications and effects of a stimulated immune system against pathogens, especially the protective capacity against specific diseases, are necessary.

The present work evaluates the effects of inulin and Bacillus subtilis, single or combined, on immune parameters, immune-related gene expression and protection against Photobacterium damselae subsp. piscicida in gilthead seabream (Sparus aurata). Three trials were conducted. In the first trial, different concentrations of inulin (10, 15 and 30 g kg(-1)) (as a prebiotic) were administered to determine the optimal concentration for stimulating the seabream's immune system. In the second trial, the optimum concentration of inulin (10 g kg(-1)) was combined with B. subtilis (as a probiotic). Following two and four weeks of the treatment, the main immune parameters, as well as the expression of seven immune-related genes, were measured. In the final trial, fish fed the same diet as in the second trial were challenged intraperitoneally with P. damselae subsp. piscicida (10(9) cfu g(-1)). Treatment groups for the second and third trial were control (non-supplemented diet), inulin (10 g kg(-1)), B. subtilis (10(7) cfu g(-1)) and inulin + B. subtilis (10 g kg(-1) and 10(7) cfu g(-1) respectively). Dietary administration of inulin or B. subtilis for two weeks stimulated the serum complement activity and the IgM level, as well as leucocyte phagocytic activity; furthermore, inulin stimulated leucocyte respiratory burst activity. When inulin and B. subtilis were administered together (as a synbiotic), only the serum complement activity and the IgM level increased in a statistically significant manner. Furthermore, the complement activity showed a significant increase in fish fed the three experimental diets for four weeks. The challenge experiment showed that the fish fed inulin or the synbiotic diet had non-significantly lower or significantly higher cumulative mortality, respectively, compared with the control group (non-supplemented diet). These results suggest that inulin and B. subtilis modulate the immune response of the gilthead seabream, although the combined administration increases susceptibility to infection by P. damselae subsp. piscicida.

In the present study, a feeding trial was conducted to evaluate the effect of inulin and heat-inactivated Bacillus subtilis, single or combined, on several innate immune activities of gilthead seabream (Sparus aurata). Forty-eight specimens were randomly assigned to four dietary treatments: 0 (control), inulin (10 g/kg, prebiotic group), B. subtilis (107 cfu/g, probiotic group), or B. subtilis + inulin (107 cfu/g + 10 g/kg, synbiotic group). After two and four weeks, six fish of each group were sampled, with the main innate immune parameters (natural haemolytic complement activity, serum and leucocyte peroxidase, phagocytosis, respiratory burst, and cytotoxic activities) being determined. Inulin or heat-inactivated B. subtilis failed to significantly stimulate the innate immune parameters assayed, although some activities showed no significant increase through these treatments. A combination of inulin and B. subtilis resulted in an increase of such parameters, with the haemolytic complement activity being the only one significantly stimulated. To conclude, inulin and B. subtilis, when administered as a synbiotic, have a synergistic effect and enhance some innate immune parameters of gilthead seabream.

High stocking density increases the number of emerging diseases triggering economic losses worldwide. Probiotics provide an effective and natural solution for preventing some diseases through an improvement of innate immune system among others. In the present work dietary administration of the probiotic Shewanella putrefaciens (known as Pdp11) was evaluated under stress by high stocking density after 2 and 4 weeks of administration to gilthead seabream (Sparus aurata) specimens. Results showed an increase in cellular peroxidase and respiratory burst activity as well as a modulation of cytokine profile when Pdp11 was administered to fish reared at high stocking density. Overall, our results showed how Pdp11 is not only able to improve to some extent the cellular and humoral immunity but also to increase the gene expression profile of pro-inflammatory cytokines such as il1b or il6 in response to high stocking density in gilthead seabream. These findings may support the potential use of this probiotic as functional feed against stress in fish farms.

Antimicrobial peptides (AMPs) are important mediators of the innate immune response against bacteria and viruses. We have found a β-defensin (BD) gene searching the expressed sequence tags (ESTs) of the teleost fish gilthead seabream ( Sparus aurata ). The clone contains an open reading frame of 201 bp mRNA that encodes a putative seabream β-defensin (saBD) propeptide of 66 amino acids containing the six conserved cysteines as the main signature of this AMP. The phylogenetic tree shows that saBD, and its fish orthologues, are closely related to the human BD-4. Transcripts of the saBD gene were mainly detected by real-time PCR in the skin, peritoneal leucocytes and head–kidney but scarcely expressed in the peripheral blood. Interestingly, head–kidney leucocytes incubation with synthetic unmethylated CpG oligodeoxynucleotides and bacterial DNA up-regulated the saBD gene expression. Recombinant protein (saBD-V5-His) was expressed in the HEK293 cell line and its functional activity determined. First, seabream head–kidney leucocytes showed chemotactic activity towards supernatants containing saBD-V5-His whilst failed to do so to human recombinant BD-1 y BD-4. Moreover, both cell lysates and supernatants containing saBD-V5-His showed strong antimicrobial activity against Vibrio anguillarum (a seabream pathogenic bacterium) and Bacillus subtilis whilst little on other fish pathogens such as Vibrio harvey and Photobacterium damselae . Further studies will elucidate the existence of other BD genes and their implications on the seabream defense against bacteria and virus.

International audience; Specific sites and sequences in collagen to which cells can attach, either directly or through protein intermediaries, were identified using Toolkits of 63-amino acid triple-helical peptides and specific shorter GXX'GEX″ motifs, which have different intrinsic affinity for integrins that mediate cell adhesion and migration. We have previously reported that collagen type I (COL-I) was able to prime in vitro the respiratory burst and induce a specific set of immune- and extracellular matrix-related molecules in phagocytes of the teleost fish gilthead seabream (Sparus aurata L.). It was also suggested that COL-I would provide an intermediate signal during the early inflammatory response in gilthead seabream. Since fibroblasts are highly involved in the initiation of wound repair and regeneration processes, in the present study SAF-1 cells (gilthead seabream fibroblasts) were used to identify the binding motifs in collagen by end-point and real-time cell adhesion assays using the collagen peptides and Toolkits. We identified the collagen motifs involved in the early magnesium-dependent adhesion of these cells. Furthermore, we found that peptides containing the GFOGER and GLOGEN motifs (where O is hydroxyproline) present high affinity for SAF-1 adhesion, expressed as both cell number and surface covering, while in cell suspensions, these motifs were also able to induce the expression of the genes encoding the proinflammatory molecules interleukin-1β and cyclooxygenase-2. These data suggest that specific collagen motifs are involved in the regulation of the inflammatory and healing responses of teleost fish.

Interferons (IFNs) and their receptors exist in all classes of vertebrates, where they represent early elements in innate and adaptive immunity. Both types I and II IFNs have been discovered in fish and type I IFN has recently been classified into two groups based on their primary protein sequences and biological activities. Thus, although groups I and II zebrafish IFN show powerful antiviral activities, only group I (IFNphi1) is able to protect the fish against bacterial infection. In addition, group II IFNs (IFNphi2 and IFNphi3) induce a rapid and transient expression of antiviral genes, while group I IFN exerts a slow but more powerful induction of several antiviral and pro-inflammatory genes. To gain further insight into the IFN system of fish, we have developed a waterborne infection model of zebrafish larvae with the spring viremia of carp virus (SVCV). Larvae were challenged 3 days post-fertilization by immersion, which considerably reduces the manipulation of fish and represents a more natural route of infection. Using this infection model, we unexpectedly found an inability on the part of zebrafish larvae to mount a protecting antiviral response to waterborne SVCV. Nevertheless, zebrafish larvae showed a functional antiviral system since ectopic expression of the cDNA of both groups I and II IFN was able to protect them against SVCV via the induction of IFN-stimulated genes (ISGs). Interestingly, group II IFNs also induced group I IFN, suggesting crosstalk between these two kinds of antiviral IFN. These results further confirm the antiviral activities of type I IFN in the zebrafish and provide the first viral infection model for zebrafish larvae using a natural route of infection. This model, in combination with the powerful gene overexpression and morpholino-mediated knockdown techniques, will help to illuminate the IFN system of teleost fish.

The IFNs and their receptors have existed in early chordates for ∼500 million years and represent the early elements in innate and adaptive immunity. Both types I and II IFNs have been discovered in fish, and type I has recently been classified into two groups based on their primary protein sequences. However, the biological activities of fish IFNs and their roles in infection are largely unknown. Using the zebrafish and manageable bacterial (Streptococcus iniae) and viral (spring viremia of carp virus) infection models, we are reporting in this study that zebrafish IFN (zfIFN) γ failed to induce antiviral and proinflammatory genes when administered in vivo, which correlates with its inability to protect the fish against bacterial and viral infections. We also found that, although both group I (i.e., zfIFN1) and group II zfIFNs (i.e., zfIFN2 and zfIFN3) displayed strong in vivo antiviral activities, only group I zfIFN was able to protect the fish against bacterial infection, which may reflect the different patterns and kinetics of immune-related genes elicited by these two groups of IFNs. Thus, group II zfIFNs induced a rapid and transient expression of antiviral genes, whereas group I zfIFN exerted a slow but more powerful induction of several antiviral and proinflammatory genes. Collectively, our results suggest nonredundant, complementary roles of type I zfIFNs in viral infections and provide evidence for a pivotal role of the recently identified group II IFN of fish in the early stages of viral infections.

It has long been established that lower vertebrates, most notably fish and amphibians, are resistant to the toxic effect of LPS. Furthermore, the lack of a TLR4 ortholog in some fish species and the lack of the essential costimulatory molecules for LPS activation via TLR4 (i.e., myeloid differentiation protein 2 (MD-2) and CD14) in all the fish genomes and expressed sequence tag databases available led us to hypothesize that the mechanism of LPS recognition in fish may be different from that of mammals. To shed light on the role of fish TLRs in LPS recognition, a dual-luciferase reporter assay to study NF-κB activation in whole zebrafish embryos was developed and three different bony fish models were studied: 1) the gilthead seabream (Sparus aurata, Perciformes), an immunological-tractable teleost model in which the presence of a TLR4 ortholog is unknown; 2) the spotted green pufferfish (Tetraodon nigroviridis, Tetraodontiformes), which lacks a TLR4 ortholog; and 3) the zebrafish (Danio rerio, Cypriniformes), which possesses two TLR4 orthologs. Our results show that LPS signaled via a TLR4- and MyD88-independent manner in fish, and, surprisingly, that the zebrafish TLR4 orthologs negatively regulated the MyD88-dependent signaling pathway. We think that the identification of TLR4 as a negative regulator of TLR signaling in the zebrafish, together with the absence of this receptor in most fish species, explains the resistance of fish to endotoxic shock and supports the idea that the TLR4 receptor complex for LPS recognition arose after the divergence of fish and tetrapods.

Microorganisms isolated from fish can be used as prophylactic tools for aquaculture in the form of probiotic preparations. The purpose of this study was to evaluate the effects of dietary administration of the live yeast Debaryomyces hansenii CBS 8339 on the gilthead seabream (Sparus aurata L.) innate immune responses. Seabream were fed control or D. hansenii-supplemented diets (10(6) colony forming units, CFU g(-1)) for 4 weeks. Humoral (seric alternative complement and peroxidase activities), and cellular (peroxidase, phagocytic, respiratory burst and cytotoxic activities) innate immune parameters and antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) were measured from serum, head-kidney leucocytes and liver, respectively, after 2 and 4 weeks of feeding. Expression levels of immune-associated genes, Hep, IgM, TCR-beta, NCCRP-1, MHC-II alpha, CSF-1R, C3, TNF-alpha and IL-1 beta, were also evaluated by real-time PCR in head-kidney, liver and intestine. Humoral immune parameters were not significantly affected by the dietary supplementation of yeast at any time of the experiment. On the other hand, D. hansenii administration significantly enhanced leucocyte peroxidase and respiratory burst activity at week 4. Phagocytic and cytotoxic activities had significantly increased by week 2 of feeding yeast but unchanged by week 4. A significant increase in liver SOD activity was observed at week 2 of feeding with the supplemented diet; however CAT activity was not affected by the dietary yeast supplement at any time of the experiment. Finally, the yeast supplemented diet down-regulated the expression of most seabream genes, except C3, in liver and intestine and up-regulated all of them in the head-kidney. These results strongly support the idea that live yeast Debaryomyces hansenii strain CBS 8339 can stimulate the innate immune parameters in seabream, especially at cellular level.

Unmethylated CpG motifs present in bacterial DNA are recognized by leucocyte receptors triggering an immune response. We have evaluated herein the immunomodulatory actions of a CpG motif in an important commercial fish, the gilthead seabream. Thus, 1, 3 and 7 days after intraperitoneal injection of the CpG motif the seabream immune parameters and gene expression profile were evaluated. Firstly, humoral innate immune responses were unaffected by CpG ODN 1668. On the other hand, ODN injection significantly enhanced the number of peritoneal leucocytes (PELs) 1 day after injection and increased the main innate immune parameters of PELs and HKLs (head-kidney leucocytes). Thus, injection of ODN 1668 significantly increased respiratory burst, peroxidase, cytotoxic and phagocytic activities, with variations in increment and time. The cytotoxic activity of HKLs was the most increased (up to 4.2-fold). Moreover, the expression profile of immune-relevant genes in head-kidney was affected, with substantial up-regulation of TLR9, IL-1β, Mx, TGFβ and Gal8 gene expression. These results demonstrate that unmethylated CpG motifs prime the fish immune response with promising applications for aquaculture.

Melatonin, a molecule produced in the pineal gland and retina of vertebrates, plays a major role in day-night circadian rhythms and other physiological processes, including the immune responses. Because little is known in this respect in fish, we have evaluated the in vivo role of melatonin in the main innate immune activities and the expression of immune-relevant genes in a teleost fish, the gilthead seabream (Sparus aurata L.). An intraperitoneal injection of 1 or 10 mg melatonin/kg of body weight produced a dose-time dependent increase in circulating melatonin serum levels. Several innate immune responses such as peroxidase, phagocytic, reactive oxygen intermediates and cell-mediated cytotoxic activities were significantly enhanced by the administration of melatonin at different sampling times. The expression of immune-relevant genes such as interleukin-1beta, major histocompatibility complex, virus-related response (interferon-regulatory factor-1 and Mx) and lymphocyte markers (immunoglobulin M and T-cell receptor for B and T lymphocytes, respectively), as analysed by semi-quantitative reverse transcription-polymerase chain reaction, was up-regulated in the head-kidney of melatonin-injected fish 1 and 3 days postinjection and decreased after 7 days. These data, together with our previous observations describing how photoperiod and in vitro melatonin treatment may affect seabream and sea bass immunology, confirm melatonin as a regulator of fish immunology. However, further studies are still needed to reveal the mechanisms underlying the direct or indirect interactions of melatonin with the fish immune system.

The potential effects of synthetic unmethylated oligodeoxynucleotides (ODN) containing CpG motifs, mimicking bacterial DNA, has never been evaluated on the immune response in the teleost fish gilthead seabream (Sparus aurata), the most important fish species in Mediterranean aquaculture. First, binding and competition studies have demonstrated that binding is saturated and promiscuous, suggesting the participation of several receptors. Moreover, leucocyte cytotoxic (NCC) activity, production of ROIs (reactive oxygen intermediates), and expression of immune-relevant genes was greatly primed by ODNs. Focusing on the mechanism, the TLR9 gene is widely distributed in seabream tissues and differently regulated in vitro by several stimuli. Moreover, and for the first time in fish, TLR9 mRNA has been detected in lymphocytes as the main cell-source. To conclude, ODNs containing GACGTT, GTCGTT (optimal for mouse and human, respectively) or AACGTT motifs are the most potent inducers of seabream immunity, whilst the involvement of TLR9 is under debate. © 2008 Birkhaueser.

The gilthead seabream is a protandrous hermaphrodite seasonal breeding teleost with a bisexual gonad that offers an interesting model for studying the two first reproductive cycles (RCs), in which the specimens develops as males. During the first RC (RC1), 11-ketotestosterone (11-KT) and testosterone (T), the main androgens in fish, play different and specific roles in the testicular physiology as they peak at different stages of RC1. Moreover, the profiles of T serum levels during the second RC (RC2) demonstrated that this androgen is not essential in the testicular regression process that occurs during this cycle. However, changes in serum levels suggest that 17beta-estradiol (E2) orchestrates this process during both RCs. Moreover, the E2 serum levels recorded during RC2 indicate that there is a threshold level that determines the initiation of ovarian development during this cycle without promoting complete feminization. We analysed triglyceride, protein, glucose and lactate serum levels in order to establish a relationship between the mobilization and transfer of nutrients, the hormonal changes that take place during the RC and body composition, finding that in vivo serum levels of metabolites change significantly throughout the first two RCs, although the physiological relevance of such changes is still unknown. Triglyceride levels seem to be affected by the beginning of ovary development during RC2 but not by testicular recrudescence during RC1. Moreover, glucose and lactate might be important sources of energy during the resting and testicular involution stages.

Summary Two major professional phagocyte populations have been described in fish, namely granulocytes and monocytes/macrophages. Although the distribution and localization of macrophages have been documented in several teleost species using mainly light and/or electron microscopy, the lack of appropriate markers for these cells has hampered our in-depth knowledge of their biology. We report here the generation of a monospecific rabbit polyclonal antibody against the gilthead seabream macrophage colony-stimulating factor receptor (Mcsfr), which is an excellent marker of macrophages in mammals and the zebrafish. The anti-Mcsfr has been found to be very useful in immunohistochemistry (IHC) to specifically immunostain the purified macrophages (adherent cells) obtained from the head-kidney as well as different cell populations in paraffin-embedded organs, including the head-kidney, spleen, thymus, gills and liver. Unexpectedly, however, no Mcsfr immunoreactive (Mcsfr + ) cells were observed in the brain and intestine of the gilthead seabream. We also show that the distribution of Mcsfr + cells in the head-kidney and the spleen is unaltered following infection with the fish pathogenic bacterium Vibrio anguillarum and that the Il1b-producing cells in these two organs after infection are exclusively acidophilic granulocytes. Finally, as the epitope recognized by the anti-Mcsfr is well conserved, we illustrate the potential usefulness of this antibody in other teleost species, such as the European seabass.

Probiotic bacteria are known to exert a wide range of beneficial effects on their animal hosts. Control of intestinal homeostasis, inflammation suppression and a reduction in the incidence of cancer all rely on the antiproliferative potential of probiotics. In this paper, we assess the antiproliferative activity of probiotics in two teleost fish cell lines SAF-1, a fibroblast cell line and EPC, an epithelioma from carp. Cells were grown in the presence of cytoplasmic extracts obtained from two bacterial strains, Lactobacillus delbrueckii subsp. lactis (LDL) and 51M6. Proliferation and apoptosis were measured after 4, 24, 48 or 72 h in culture by the crystal violet or by double staining flow cytometry assays, respectively. Generally, LDL had stronger effects on cell growth than 51M6. Moreover, SAF-1 cells were more susceptible to growth inhibition than EPC cells. Apoptosis took place following growth inhibition, especially when LDL extracts were used. The results are discussed in terms of the biological significance of probiotic bacteria that naturally occur on the fish mucosal surfaces with an emphasis on how dose and species specificity may be determinant factors.

Caspases are a family of cysteine proteases that fulfil critical roles in mammalian apoptosis and in the proteolytic activation of cytokines. In humans, the caspase family includes 13 members whose functions seem to correlate with their phylogenetic relationship. They are classified into two main groups, the cell death (apoptotic) and the inflammatory caspases. Caspase-1 is the best characterized inflammatory caspase and is responsible for the processing of interleukin-1beta (IL-1beta), IL-18 and IL-33. Despite the importance of caspase-1 in inflammation, no information is available on the presence and activity of this enzyme in fish. In this study, we cloned a caspase-1-like gene from the bony fish gilthead seabream (Sparus aurata L.) which shows a conserved N-terminal caspase-recruitment domain (CARD) and a C-terminal caspase catalytic domain. The seabream caspase-1 gene was expressed in 1 day post-hatching larvae and its mRNA levels increased throughout development. In adult fish, caspase-1 was found to be constitutively expressed in all immune tissues analyzed and, unexpectedly, infection of fish and stimulation of professional phagocytes in vitro decreased its mRNA levels. It was also demonstrated that the recombinant seabream caspase-1 ectopically expressed in HEK293 cells was able to cleave a caspase-1 specific substrate, this activity being enhanced upon activation of the rat P2X7 receptor with BzATP. Finally, seabream fibroblast cell line SAF-1 and primary leukocytes showed endogenous caspase-1 activity, which was almost completely inhibited by a caspase-1 specific inhibitor.

Antimicrobial peptides (AMPs) are increasingly recognized as a critical first line of defence against many pathogens. The genes encoding these peptides are expressed in numerous tissue and cell types from a wide variety of different species including mammals, amphibians, fish, and insects. In this study, we report that the AMPs called piscidins were primarily present in the mast cells (MCs) of fish and were only identified in fish belonging to the Order Perciformes. It is striking that histamine was seen to have a similar evolutionary history, since the only piscine MCs endowed with this molecule are in the Perciformes. We also show that both MCs and professional phagocytic granulocytes were armed with different piscidin molecules. In contrast, macrophages were devoid of these AMPs. More importantly, we found by immunoelectron microscopy that piscidins were delivered to the bacteria-containing phagosome of granulocytes upon phagocytosis, suggesting a role for these AMPs in the killing of both extracellular and intracellular pathogenic bacteria.