Synthetic antimicrobial Nkl and Dic peptides are immunomodulatory but only Dic peptide can be therapeutic against nodavirus infection.

Aquaculture is a prosperous economic sector threatened by viral infections. Among the viruses threatening fish culture, Betanodavirus (NNV) is extremely important in the Mediterranean Sea affecting to highly traded species as European sea bass. In this context, application of antimicrobial peptides (AMPs) has arisen as a potential biotechnological tool. The aim of this work was to evaluate the therapeutic application of two European sea bass-derived AMPs, NK-lysin (Nkl) and dicentracin (Dic), against NNV infections. Synthetic Dic peptide was able to significantly reduce NNV-induced mortalities while Nkl failed to do so. Although neither Dic nor Nkl peptides were able to alter the transcriptional levels of NNV and the number of infected cells, Nkl seemed to increase the viral load per cell. Interestingly, both Nkl and Dic peptides showed immunomodulatory roles. For instance, our data revealed an interplay among different AMPs, at both gene and protein levels. Otherwise, Nkl and Dic peptides provoked an anti-inflammatory balance upon NNV infection, as well as the recruitment of macrophages and B cells to the target site of the infection, the brain. In conclusion, Dic can be proposed as a therapeutic candidate to combat NNV.

Regulation and distribution of European sea bass perforins point to their role in the adaptive cytotoxic response against NNV.

Cell-mediated cytotoxicity is a complex immune mechanism that involves the release of several killing molecules, being perforin (PRF) one of the most important effector players. Perforin is synthesized by T lymphocytes and natural killer cells in mammals and responsible for the formation of pores on the target cell membrane during the killing process. Although perforin has been extensively studied in higher vertebrates, this knowledge is very limited in fish. Therefore, in this study we have identified four prf genes in European sea bass (Dicentrarchus labrax) and evaluated their mRNA levels. All sea bass prf genes showed the typical and conserved domains of its human orthologue and were closely clustered by the phylogenetic analysis. In addition, all genes showed constitutive and ubiquitous tissular expression, being prf1.9 gene the most highly expressed in immune tissues. Subsequently, in vitro stimulation of head-kidney (HK) cells with phytohemagglutinin, a T-cell activator, showed an increase of all prf gene levels, except for prf1.3 gene. European sea bass HK cells increased the transcription of prf1.2 and prf1.9 during the innate cell-mediated cytotoxic activity against xenogeneic target cells. In addition, sea bass infected with nodavirus (NNV) showed a similar expression pattern of all prf in HK and brain at 15 days post-infection, except for prf1.3 gene and in the gonad. Finally, the use of a polyclonal antibody against PRF1.9 showed an increase of positive cells in HK, brain and gonad from NNV-infected fish. Taken together, the data seem to indicate that all prf genes, except prf1.3, appear to be involved in the European sea bass immunity, and probably in the cell-mediated cytotoxic response, with PRF1.9 playing the most important role against nodavirus. The involvement of the PRFs and the CMC activity in the vertical transmission success of the virus is also discussed.

Synthetic Antimicrobial Peptides Fail to Induce Leucocyte Innate Immune Functions but Elicit Opposing Transcriptomic Profiles in European Sea Bass and Gilthead Seabream.

Antimicrobial peptides (AMPs) are promising molecules in diverse fields, including aquaculture. AMPs possess lytic effects on a wide range of pathogens, resulting in a potential replacement for traditional antimicrobials in aquaculture. In addition, they also have modulatory effects on host immune responses. Thus, the objective of this work was to evaluate the immunomodulatory capability of three known synthetic AMPs derived from European sea bass, NK-lysin (Nkl), hepcidin (Hamp), and dicentracin (Dic), in head-kidney cell suspensions from European sea bass and gilthead seabream. The tested peptides were neither cytotoxic for European sea bass nor gilthead seabream cells and failed to modulate the respiratory burst and phagocytosis activities. However, they modified the pattern of transcription of immune-related genes differently in both species. Peptides were able to promote the expression of marker genes for anti-inflammatory (il10), antiviral (mx, irf3), cell-mediated cytotoxicity (nccrp1, gzmb), and antibody responses (ighm) in European sea bass, with the Nkl peptide being the most effective. Contrary to this, the effects of those peptides on gilthead seabream mainly resulted in the suppression of immune responses. To conclude, European sea bass-derived peptides can be postulated as potential tools for immunostimulation in European sea bass fish farms, but more efforts are required for their universal use in other species.

Fish HERC7: Phylogeny, Characterization, and Potential Implications for Antiviral Immunity in European Sea Bass.

E3 ubiquitin ligases, key components of the ubiquitin proteasome system, orchestrate protein degradation through ubiquitylation and profoundly impact cellular biology. Small HERC E3 ligases (HERC3-6) have diverse functions in mammals, including roles in spermatogenesis, protein degradation, and immunity. Until now, only mammals' HERC3, HERC5, and HERC6 are known to participate in immune responses, with major involvement in the antiviral response. Interestingly, an exclusive HERC7 has been characterized in fish showing great molecular conservation and antiviral roles. Thus, this study identifies and characterizes the herc7 gene in the European sea bass teleost. The European sea bass herc7 gene and the putative protein show good conservation of the promoter binding sites for interferons and the RCC1 and HECT domains characteristic of HERC proteins, respectively. The phylogenetic analysis shows a unique cluster with the fish-exclusive HERC7 orthologues. During ontogeny, the herc7 gene is expressed from 3 days post-fertilization onwards, being constitutively and widely distributed in adult tissues. In vitro, stimulated leucocytes up-regulate the herc7 gene in response to mitogens and viruses, pointing to a role in the immune response. Furthermore, sea bass herc7 expression is related to the interferon response intensity and viral load in different tissues upon in vivo infection with red-grouper betanodavirus (RGNNV), suggesting the potential involvement of fish HERC7 in ISGylation-based antiviral activity, similarly to mammalian HERC5. This study broadens the understanding of small HERC proteins in fish species and highlights HERC7 as a potential contributor to the immune response in European sea bass, with implications for antiviral defense mechanisms. Future research is needed to unravel the precise actions and functions of HERC7 in teleost fish immunity, providing insights into direct antiviral activity and viral evasion.

Immunity elicited by AMP-encoding plasmids fails to increase the protection of European sea bass against nodavirus.

Antimicrobial peptides (AMPs) are a potent arm of the innate immune system that can directly kill pathogens and induce immunomodulation. In the marine aquaculture, European sea bass (Dicentrarchus labrax L.) is one of the most prosperous species but is highly susceptible to nodavirus (NNV), which produces high rates of mortality in larvae and juvenile stages. Thus, we aimed to evaluate whether AMPs exert immunomodulatory and/or NNV-preventive actions in sea bass. To do this, plasmids encoding the sea bass AMPs dicentracin (pDIC), beta-defensin (pDB1), hepcidin (pHAMP2) or NK-lysin (pNKL) were generated and intramuscularly injected into sea bass juveniles to evaluate their immunomodulatory and anti-NNV roles. Sea bass muscle transcribes the AMPs and produces an increase in their circulating levels, along with an increase of the antibacterial activity. Immune-related gene analysis revealed a great activation of the inflammatory response and the recruitment of neutrophilic granulocytes at the site of injection. However, AMP-encoding plasmids, namely pHAMP2, negatively affected to NNV disease by increasing fish mortality. In conclusion, plasmids encoding AMPs show immunostimulatory effects on European sea bass but do not improve the resistance to NNV.

Antimicrobial Peptides from Photosynthetic Marine Organisms with Potential Application in Aquaculture.

Aquaculture production is at a record level and is estimated to increase in the coming years. However, this production can be negatively affected by infectious diseases produced by viruses, bacteria, and parasites, causing fish mortality and economic losses. Antimicrobial peptides (AMPs) are small peptides that may be promising candidates to replace antibiotics because they are the first line of defense in animals against a wide variety of pathogens and have no negative effects; they also show additional activities such as antioxidant or immunoregulatory functions, which makes them powerful alternatives for use in aquaculture. Moreover, AMPs are highly available in natural sources and have already been used in the livestock farming and food industries. Photosynthetic marine organisms can survive under all kinds of environmental conditions and under extremely competitive environments thanks to their flexible metabolism. For this reason, these organisms represent a powerful source of bioactive molecules as nutraceuticals and pharmaceuticals, including AMPs. Therefore, in this study we reviewed the present knowledge about AMPs from photosynthetic marine organism sources and analyzed whether they could be suitable for use in aquaculture.

Updating Our Knowledge on Fish Immunology.

Fish is the first group of vertebrates to appear during animal evolution [...].

Antigen Presentation and Autophagy in Teleost Adaptive Immunity.

Infectious diseases are a burden for aquaculture. Antigen processing and presentation (APP) to the immune effector cells that fight pathogens is key in the adaptive immune response. At the core of the adaptive immunity that appeared in lower vertebrates during evolution are the variable genes encoding the major histocompatibility complex (MHC). MHC class I molecules mainly present peptides processed in the cytosol by the proteasome and transported to the cell surface of all cells through secretory compartments. Professional antigen-presenting cells (pAPC) also express MHC class II molecules, which normally present peptides processed from exogenous antigens through lysosomal pathways. Autophagy is an intracellular self-degradation process that is conserved in all eukaryotes and is induced by starvation to contribute to cellular homeostasis. Self-digestion during autophagy mainly occurs by the fusion of autophagosomes, which engulf portions of cytosol and fuse with lysosomes (macroautophagy) or assisted by chaperones (chaperone-mediated autophagy, CMA) that deliver proteins to lysosomes. Thus, during self-degradation, antigens can be processed to be presented by the MHC to immune effector cells, thus, linking autophagy to APP. This review is focused on the essential components of the APP that are conserved in teleost fish and the increasing evidence related to the modulation of APP and autophagy during pathogen infection.

Severe Natural Outbreak of Cryptocaryon irritans in Gilthead Seabream Produces Leukocyte Mobilization and Innate Immunity at the Gill Tissue.

The protozoan parasite Cryptocaryon irritans causes marine white spot disease in a wide range of fish hosts, including gilthead seabream, a very sensitive species with great economic importance in the Mediterranean area. Thus, we aimed to evaluate the immunity of gilthead seabream after a severe natural outbreak of C. irritans. Morphological alterations and immune cell appearance in the gills were studied by light microscopy and immunohistochemical staining. The expression of several immune-related genes in the gills and head kidney were studied by qPCR, including inflammatory and immune cell markers, antimicrobial peptides (AMP), and cell-mediated cytotoxicity (CMC) molecules. Serum humoral innate immune activities were also assayed. Fish mortality reached 100% 8 days after the appearance of the C. irritans episode. Gill filaments were engrossed and packed without any space between filaments and included parasites and large numbers of undifferentiated and immune cells, namely acidophilic granulocytes. Our data suggest leukocyte mobilization from the head kidney, while the gills show the up-regulated transcription of inflammatory, AMPs, and CMC-related molecules. Meanwhile, only serum bactericidal activity was increased upon infection. A potent local innate immune response in the gills, probably orchestrated by AMPs and CMC, is triggered by a severe natural outbreak of C. irritans.

Profile of Innate Immunity in Gilthead Seabream Larvae Reflects Mortality upon Betanodavirus Reassortant Infection and Replication.

Historically, gilthead seabream (Sparus aurata) has been considered a fish species resistant to nervous necrosis virus (NNV) disease. Nevertheless, mortality in seabream hatcheries, associated with typical clinical signs of the viral encephalopathy and retinopathy (VER) disease has been confirmed to be caused by RGNNV/SJNNV reassortants. Because of this, seabream larvae at 37 and 86 days post-hatching (dph) were infected by immersion with RGNNV/SJNNV and SJNNV/RGNNV reassortants under laboratory conditions, and mortality, viral replication and immunity were evaluated. Our results show that gilthead seabream larvae, mainly those at 37 dph, are susceptible to infection with both NNV reassortant genotypes, with the highest impact from the RGNNV/SJNNV reassortant. In addition, viral replication occurs at both ages (37 and 86 dph) but the recovery of infective particles was only confirmed in 37 dph larvae,; this value was also highest with the RGNNV/SJNNV reassortant. Larvae immunity, including the expression of antiviral, inflammatory and cell-mediated cytotoxicity genes, was affected by NNV infection. Levels of the natural killer lysin (Nkl) peptide were increased in SJNNV/RGNNV-infected larvae of 37 dph, though hepcidin was not. Our results demonstrate that the seabream larvae are susceptible to both NNV reassortants, though mainly to RGNNV/SJNNV, in an age-dependent manner.

The Effect of 17α-Ethynilestradiol and GPER1 Activation on Body and Muscle Growth, Muscle Composition and Growth-Related Gene Expression of Gilthead Seabream, Sparus aurata L.

Endocrine-disrupting chemicals include natural and synthetic estrogens, such as 17α-ethynilestradiol (EE2), which can affect reproduction, growth and immunity. Estrogen signalling is mediated by nuclear or membrane estrogen receptors, such as the new G-protein-coupled estrogen receptor 1 (GPER1). The present work studies the effect of EE2 and G1 (an agonist of GPER1) on body and muscle parameters and growth-related genes of 54 two-year-old seabreams. The fish were fed a diet containing EE2 (EE2 group) and G1 (G1 group) for 45 days and then a diet without EE2 or G1 for 122 days. An untreated control group was also studied. At 45 days, the shortest body length was observed in the G1 group, while 79 and 122 days after the cessation of treatments, the shortest body growth was observed in the EE2 group. Hypertrophy of white fibers was higher in the EE2 and G1 groups than it was in the control group, whereas the opposite was the case with respect to hyperplasia. Textural hardness showed a negative correlation with the size of white fibers. At the end of the experiment, all fish analyzed in the EE2 group showed a predominance of the gonadal ovarian area. In addition, the highest expression of the mafbx gene (upregulated in catabolic signals) and mstn2 (myogenesis negative regulator) was found in EE2-exposed fish.

NK-lysin is highly conserved in European sea bass and gilthead seabream but differentially modulated during the immune response.

Fish NK-lysin (NKL), an orthologous to human granulysin, exerts a dual role as an antimicrobial peptide (AMP) and as a direct executor of T cytotoxic and natural killer cells during the cell-mediated cytotoxic (CMC) response. Although its best-known function is as AMP against bacteria, recent studies point to a special role of NKL in antiviral responses. Nodavirus (NNV) is a spreading threat in Mediterranean aquaculture. In this study, we have identified and compared the expression pattern of European sea bass and gilthead seabream NKL and evaluated its transcription in different tissues and its regulation in head-kidney leucocyte (HKLs) stimulated in vitro with different immunostimulants, under CMC response and upon an in vivo infection with NNV. Our results showed that nkl transcription is highly expressed in spleen, thymus and skin with species-specific differences. Interestingly, the expression pattern in both species was very different upon treatment. While sea bass nkl transcription was increased in HKLs by the T mitogen phytohemagglutinin all the stimulators inhibited it in seabream HKLs. Similar results occurred in NNV-infected fish where the transcription was increased in sea bass tissues and down-regulated in seabream. Curiously, during CMC assays, nkl transcription was significantly increased in seabream HKLs against NNV-infected fish cell lines but this was not observed in sea bass leucocytes. The potential role of NKL as CMC effector molecule or as AMP in fish will be discussed.

Identification and Regulation of Interleukin-17 (IL-17) Family Ligands in the Teleost Fish European Sea Bass.

Interleukin-17 (IL-17) cytokine comprises a family of six ligands in mammals with proinflammatory functions, having an important role in autoimmune disorders and against bacterial, viral, and fungal pathogens. While IL-17A and IL-17F ligands are mainly produced by Th cells (Th17 cells), the rest of the ligands are expressed by other immune and non-immune cells and have different functions. The identification of IL-17 ligands in fish has revealed the presence of six members, counterparts to mammalian ones, and a teleost-specific form, the fish IL-17N. However, tissue distribution, the regulation of gene expression, and scarce bioactivity assays point to similar functions compared to mammalian ones, though this yet to be investigated and confirmed. Thus, we have identified seven IL-17 ligands in the teleost European sea bass (Dicentrarchus labrax), for the first time, corresponding to IL-17A/F1, IL-17A/F2, IL-17A/F3, IL-17C1, IL-17C2, IL-17D, and IL-17N, according to the predicted protein sequences and phylogenetic analysis. They are constitutively and widely transcribed in sea bass tissues, with some of them being mainly expressed in the thymus, brain or intestine. Upon in vitro stimulation of head-kidney leucocytes, the mRNA levels of all sea bass IL-17 ligands were up-regulated by phytohemagglutinin treatment, a well-known T cell mitogen, suggesting a major expression in T lymphocytes. By contrast, the infection of sea bass juveniles with nodavirus (NNV), a very pathogenic virus for this fish species, resulted in the up-regulation of the transcription of IL-17C1 in the head-kidney and of IL-17C1 and IL-17D in the brain, the target tissue for NNV replication. By contrast, NNV infection led to a down-regulated transcription of IL-17A/F1, IL-17A/F2, IL-17C1, IL-17C2, and IL-17D in the head-kidney and of IL-17A/F1 and IL-17A/F3 in the brain. The data are discussed accordingly with the IL-17 ligand expression and the immune response under the different situations tested.

Recombinant nodavirus vaccine produced in bacteria and administered without purification elicits humoral immunity and protects European sea bass against infection.

Viral necrosis virus (NNV) or nodavirus causes fish viral encephalopathy and retinopathy worldwide. In some cases, mortalities in aquaculture industry can reach up to 100%, some species being especially sensitive as is the case of European sea bass (Dicentrarchus labrax), one of the main cultured species in the Mediterranean, with the consequent economical loses. Development of new vaccines against NNV is in the spotlight though few researches have focused in European sea bass. In this study we have generated a recombinant NNV (rNNV) vaccine produced in Escherichia coli expressing the capsid protein and administered it to European sea bass juveniles by two different routes (intraperitoneal and oral). The last being considered non-stressful and desired for fish farming of small fish, which in fact are the most affected by NNV. Oral vaccine was composed of feed pellets containing the recombinant whole bacteria, and injected vaccine was composed of recombinant bacteria previously lysed. Our results revealed production of specific anti-NNV IgM following the two vaccination procedures, levels that were further increased in orally-vaccinated group after challenge with NNV. Genes related to interferon (IFN), T-cell and immunoglobulin markers were scarcely regulated in head-kidney (HK), gut or brain. Vaccination by either route elicited a relative survival response of 100% after NNV challenge. To our knowledge, this is the first report of a recombinant vaccine followed by no purification steps which resulted in a complete protection in European sea bass when challenged with NNV.

European sea bass brain DLB-1 cell line is susceptible to nodavirus: A transcriptomic study.

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 fish central nervous system. It is considered one of the most serious viral diseases in marine aquaculture, the European sea bass (Dicentrarchus labrax) being amongst the most susceptible. We have evaluated the European sea bass brain derived cell line (DLB-1) susceptibility to NNV genotypes and evaluated its transcriptomic profile. DLB-1 cells supported NNV gene transcription and replication since strains belonging to the four NNV genotypes produce cytopathic effects. Afterwards, DLB-1 cells were infected with an RGNNV strain, the one which showed the highest replication, for 12 and 72 h and an RNA-seq analysis was performed to identify potential genes involved in the host-NNV interactions. Differential expression analysis showed the up-regulation of many genes related to immunity, heat-shock proteins or apoptosis but not to proteasome or autophagy processes. These data suggest that the immune response, mainly the interferon (IFN) pathway, is not powerful enough to abrogate the infection, and cells finally suffer stress and die by apoptosis liberating infective particles. GO enrichment also revealed, for the first time, the down-regulation of terms related to brain/neuron biology indicating molecular mechanisms causing the pathogenic effect of NNV. This study opens the way to understand key elements in sea bass brain and NNV interactions.

Genes related to cell-mediated cytotoxicity and interferon response are induced in the retina of European sea bass upon intravitreal infection with nodavirus.

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 replicated 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 nodavirus.

Transcription of histones H1 and H2B is regulated by several immune stimuli in gilthead seabream and European sea bass.

Histones (H1 to H4) are the primary proteins which mediate the folding of DNA into chromatin; however, and in addition to this function, histones have been also related to antimicrobial peptides (AMPs) activity in vertebrates, in fact, mammalian H1 is mobilized as part as the anti-viral immune response. In fish, histones with AMP activity have been isolated and characterized mainly from skin and gonads. One of most threatening pathogens for wild and cultured fish species nowadays is nodavirus (NNV), which target tissues are the brain and retina, but it is also able to colonize the gonad and display vertical transmission. Taking all this into account we have identified the h1 and h2b coding sequences in European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) fish species and studied their pattern of expression under naïve conditions and NNV in vivo infection. The data obtained prompted us to study their role on the immune response of gonad and head-kidney leucocytes upon viral (NNV), bacteria (Vibrio anguillarum or Photobacterium damselae), pathogen-associated molecular patterns (PAMPs) or mitogens stimulation. The h1 and h2b genes are expressed in a wide range of tissues and their expression is modify by infection or other immune stimuli, but further studies will be needed to determine the significance of these changes. These results suggest that h1 expression is related to the immune response against NNV in the brain, while h2b transcription seems to be more important in the head-kidney. Moreover, the potential role of histones as anti-viral agents is suggested and further characterization is in progress.

Characterization of the IFN pathway in the teleost fish gonad against vertically transmitted viral nervous necrosis virus.

One of the most powerful innate immune responses against viruses is mediated by type I IFN. In teleost fish, it is known that virus infection triggers the expression of ifn and many IFN-stimulated genes, but the viral RNA sensors and mediators leading to IFN production are scarcely known. Thus, we have searched for the presence of these genes in gilt-head sea bream (Sparus aurata) and European sea bass (Dicentrarchus labrax), and evaluated their expression after infection with viral nervous necrosis virus (VNNV) in the brain, the main viral target tissue, and the gonad, used to transmit the virus vertically. In sea bream, a fish species resistant to the VNNV strain used, we found an upregulation of the genes encoding MDA5 (melanoma differentiation-associated gene 5), TBK1 (TANK-binding kinase 1), IRF3 (IFN regulatory factor 3), IFN, Mx [myxovirus (influenza) resistance protein] and PKR (dsRNA-dependent protein kinase receptor) proteins in the brain, which were unaltered in the gonad and could favour the dissemination by gonad fluids or gametes. Strikingly, in European sea bass, a very susceptible species, we also identified, transcripts coding for LGP2 (Laboratory of Genetics and Physiology 2), MAVS (mitochondrial antiviral signalling), TRAF3 (TNF receptor-associated factor 3), TANK (TRAF family member-associated NFκB activator) and IRF7 (IFN regulatory factor 7), and found that all the genes analysed were upregulated in the gonad, but only mda5, lgp2, irf3, mx and pkr were upregulated in the brain. These findings supported the notion that the European sea bass brain innate immune response is unable to clear the virus and pointed to the importance of gonad immunity to control the dissemination of VNNV to the progeny--an aspect that is worth investigating in aquatic animals.

Characterization of the annual regulation of reproductive and immune parameters on the testis of European sea bass.

The European sea bass, Dicentrarchus labrax L., is a seasonal gonochoristic species, the males of which are generally mature during their second year of life. It has been demonstrated that cytokines and immune cells play a key role in the testicular development. This reproductive-immune interaction might be very important in the sea bass since several pathogens are able to colonise the gonad and persist in this tissue, altering further reproductive functions and spreading disease. This study aims to investigate the reproductive cycle of 1-year European sea bass males by analysing cell proliferation and apoptosis and the expression profile of some reproductive and immune-related genes in the testis, as well as the serum sex steroid levels. Our data demonstrate that, in 1-year-old European sea bass males, the testis undergoes the spermatogenesis process and that the reproductive and immune parameters analysed varied during the reproductive cycle. In the testis, the highest proliferative rates were recorded at the spermatogenesis stage, while the highest apoptotic rates were recorded at the spawning stage. We have also analysed, for the first time in European sea bass males, the serum levels of 17ß-estradiol (E2) and dihydrotestosterone and the gene expression profile of the enzymes implied in their production, determining that at least E2 might be involved in the regulation of the reproductive cycle. Some immune relevant genes, including cytokines, lymphocyte receptors, and anti-viral and anti-bacterial molecules were detected in the testis of naïve European sea bass specimens, and their expression profile was related to the stages of the reproductive cycle, suggesting an important role for the defence of the reproductive tissues.

Antimicrobial response is increased in the testis of European sea bass, but not in gilthead seabream, upon nodavirus infection.

Antimicrobial peptides (AMPs) have a crucial role in the fish innate immune response, being considered a fundamental component of the first line of defence against pathogens. Moreover, AMPs have not been studied in the fish gonad since this is used by some pathogens as a vehicle or a reservoir to be transmitted to the progeny, as occurs with nodavirus (VNNV), which shows vertical transmission through the gonad and/or gonadal fluids, but no study has looked into the gonad of infected fish. In this framework, we have characterized the antimicrobial response triggered by VNNV in the testis of European sea bass, a very susceptible species of the virus, and in the gilthead seabream, which acts as a reservoir, both in vivo and in vitro, and compared with that present in the serum and brain (target tissue of VNNV). First, our data show a great antiviral response in the brain of gilthead seabream and in the gonad of European sea bass. In addition, for the first time, our results demonstrate that the antimicrobial activities (complement, lysozyme and bactericidal) and the expression of AMP genes such as complement factor 3 (c3), lysozyme (lyz), hepcidin (hamp), dicentracin (dic), piscidin (pis) or ß-defensin (bdef) in the gonad of both species are very different, but generally activated in the European sea bass, probably related with the differences of susceptibility upon VNNV infection, and even differs to the brain response. Furthermore, the in vitro data suggest that some AMPs are locally regulated playing a local immune response in the gonad, while others are more dependent of the systemic immune system. Data are discussed in the light to ascertain their potential role in viral clearance by the gonad to avoid vertical transmission.