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.

Comparative analysis of blood protein fractions in two mediterranean farmed fish: Dicentrarchus labrax and Sparus aurata.

Total protein levels in fish are widely used in health and welfare studies, providing a simple and accessible measure. However, the multifaceted role of blood proteins makes it sometimes challenging to link total protein content to specific health issues, while specific protein fractions may offer more precise insights into fish biology and health, particularly in farmed fish species where such data is often lacking. Data were gathered from two experiments involving Dicentrarchus labrax and Sparus aurata, key species in European marine aquaculture. The aim was (1) to assess how different globulin fractions contribute to total protein content in blood and (2) how these contributions vary across different sampling times in healthy animals. In D. labrax, the beta1 globulin fraction emerged as the major contributor (34.16%), followed by albumin and alpha2 globulins (18.24% and 16.41%, respectively). In contrast, pre-albumins and alpha1 fractions had the least contribution (5.49% and 7.71%). S. aurata exhibited albumin as the primary contributor (23.39%), followed by beta1 and alpha2 globulins (19.71% and 19.15%, respectively), with gamma and alpha1 fractions contributing the least (5.34% and 8.63%). Notably, the study revealed relatively stable contributions of globulin fractions to total proteins within both species, albeit with minor variations over time, potentially linked to environmental and individual factors. Furthermore, larger fish displayed higher total protein levels. This research underscores the need for further investigation into the diverse factors influencing globulin contributions to total proteins, ultimately enhancing health and welfare monitoring for farmed fish species.

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.

Heterogeneity of otolith chemical composition from two-dimensional mapping: Relationship with biomineralization mechanisms and implications for microchemistry analyses.

Although otoliths are widely used as archives to infer life-history traits and habitat use in fishes, their biomineralization process remains poorly understood. This lack of knowledge is problematic as it can lead to misinterpretation of the different types of signals (e.g., optical or chemical) that provide basic data for research in fish ecology, fisheries management, and species conservation. Otolith calcification relies on a complex system involving a pericrystalline fluid, the endolymph, whose organic and inorganic compositions are spatially heterogeneous for some constituents. This property stems from the particular structure of the calcifying saccular epithelium. In this study, we explored the spatial heterogeneity of elemental incorporation in otoliths for two species of high economic interest, European hake Merluccius merluccius (L. 1758) and European sea bass Dicentrarchus labrax (L. 1758). Two-dimensional mappings of chemical elements were obtained using UV high-repetition-rate femtosecond laser ablation (fs-LA) system coupled to a high-resolution inductively coupled plasma sector field mass spectrometer analyses on transverse sections of sagittae. Results highlighted a clear asymmetry between proximal (sulcus) and distal (antisulcus) concentrations for elements such as magnesium (Mg), phosphorus (P), manganese (Mn), and potassium (K) with concentration gradient directions that varied depending on the element. Strontium (Sr) and barium (Ba) did not show a proximo-distal gradient. These results are discussed in light of current knowledge on the endolymph composition and the mechanisms that lead to its compartmentalization, highlighting the need for further research on otolith biomineralization. Operational implications for studies based on otolith chemical composition are also discussed with emphasis on advice for sampling strategies to avoid analytical biases and the need for in-depth analyses of analytical settings before comparing otolith signatures between species or geographical areas.

Recruitment of European sea bass (Dicentrarchus labrax) in northerly UK estuaries indicates a mismatch between spawning and fisheries closure periods.

European sea bass (Dicentrarchus labrax) is a species of high commercial and recreational value, but it exhibits highly variable recruitment rates and has been subject to recent declines. Emergency management measures put in place to protect spawning stocks include the annual closure of commercial and recreational fisheries over a 2-month, February-March, window. Whether this protection measure is having the desired outcome for this data-poor species remains unclear. Otolith microstructural analyses (counts and widths of daily growth rings and check marks indicative of settlement) were used to estimate (1) spawn timing, (2) pelagic larval duration and settlement timing, (3) growth rate and condition, and (4) the otolith-fish size relationship for juvenile European sea bass caught from two estuaries in Wales (Dwyryd, Y Foryd), located at the northern edge of the species range. We observed a significant mismatch between the timing of fisheries closures and the spawning, with 99.2% of recruits having been spawned after the fishery had reopened (back-calculated median spawn date = May 5 ± 17 days SD), suggesting that the closure may be too early to adequately protect this population. Further, we present the first empirically derived estimates of pelagic larval duration for sea bass recruits settling in UK habitats, which showed a strong negative relationship with spawn date. Finally, we found significant differences in fish condition between the two estuaries, suggesting local variation in habitat quality. The results suggest that the timing of current fisheries closures may not be adequately protecting the spawners supplying these northernmost estuaries, which are likely to become increasingly important as sea bass distributions shift northward in our climate future.

Water temperature explains part of the variation in basal plasma cortisol level within and between fish species.

Within the thermal tolerance range of fish, metabolism is known to escalate with warming. Rapid thermic changes also trigger a series of physiological responses, including activation of the stress axis, producing cortisol. Fish have adapted to their environment by producing a low level of plasmatic cortisol when unstressed (basal), so that thriving in their natural temperature should not impact their basal cortisol levels. Yet, surprisingly, little is known on how temperature affects cortisol within and between fish species. Here, we conducted a phylogenetic meta-analysis to (1) test whether temperature can explain the differences in basal cortisol between species and (2) evaluate the role of temperature on differences in cortisol levels between individuals of a same species. To do this, we retrieved basal plasma cortisol data from 126 studies, investigating 33 marine and freshwater fish species, and correlated it to water temperature. Intra-species variability in basal plasma cortisol levels was further investigated in two species: the European sea bass Dicentrarchus labrax and the Nile tilapia Oreochromis niloticus. Factors such as life stage, sex and weight were also considered in the analyses. Overall, our phylogenetic analysis revealed a clear positive correlation between basal cortisol level and the temperature at which the fish live. The role of temperature has also been confirmed within D. labrax, while it failed to be significant in O. niloticus. In this paper, the influence of habitat, life stage, sex and weight on basal plasma cortisol levels is also discussed. Since some abiotic parameters were not included in the analysis, our study is a call to encourage scientists to systematically report other key factors such as dissolved oxygen or salinity to fully depict the temperature-cortisol relationship in fishes.

Nervous Necrosis Virus Modulation of European Sea Bass (Dicentrarchus labrax, L.) Immune Genes and Transcriptome towards Establishment of Virus Carrier State.

Viral infections of teleost fish have great environmental and economic implications in aquaculture. Nervous necrosis virus (NNV) is a pathogen affecting more than 120 different species, causing high mortality and morbidity. Herein, we studied the course of NNV experimental infection of D. labrax, focusing on survivors which indicated viral carrier state. To determine the carrier state of D. labrax head kidney, we performed a gene expression analysis of selected immune-related genes and we profiled its transcriptome 14 days post infection (dpi). All tested genes showed clear differentiations in expression levels while most of them were up-regulated 14 dpi suggesting that their role is not limited in early antiviral responses, but they are also implicated in disease persistence. To gain a better understanding of the fish that survived the acute infection but still maintained a high viral load, we studied the differential expression of 124 up-regulated and 48 down-regulated genes in D. labrax head kidney, at 14 dpi. Concluding, the NNV virus persistent profile was assessed in D. labrax, where immune-related gene modification was intense (14 dpi) and the head kidney transcriptome profile at this time point offered a glimpse into host attempts to control the infection in asymptomatic carriers.

Non-lethal sampling method for the analysis of white muscle fatty acid profiles in European sea bass (Dicentrarchus labrax).

This study presents a novel non-lethal sampling method for assessing fatty acid (FA) composition in juvenile European sea bass (Dicentrarchus labrax) using subcutaneous white muscle biopsies. This research aimed to evaluate the suitability of the biopsy for FA analysis using two lipid extraction protocols and comparing them to a lethal routine method. The results showed that a mass of fresh tissue as low as 1.4 mg provided good quality FA chromatograms for both reserve and membrane lipids. Although the biopsy method displayed high variability in terms of FA quantity among intra-individual replicates, it showed good FA profile repeatability in both reserve and membrane lipids. The study highlights the potential of this non-lethal approach for studying FA dynamics in fish, with its application being particularly promising for ecological and experimental studies. However, careful biopsy implementation is recommended to account for potential lipid droplet and lipid distribution variability within the tissue.

The extensive transgenerational transcriptomic effects of ocean acidification on the olfactory epithelium of a marine fish are associated with a better viral resistance.

BACKGROUND: Progressive CO2-induced ocean acidification (OA) impacts marine life in ways that are difficult to predict but are likely to become exacerbated over generations. Although marine fishes can balance acid-base homeostasis efficiently, indirect ionic regulation that alter neurosensory systems can result in behavioural abnormalities. In marine invertebrates, OA can also affect immune system function, but whether this is the case in marine fishes is not fully understood. Farmed fish are highly susceptible to disease outbreak, yet strategies for overcoming such threats in the wake of OA are wanting. Here, we exposed two generations of the European sea bass (Dicentrarchus labrax) to end-of-century predicted pH levels (IPCC RCP8.5), with parents (F1) being exposed for four years and their offspring (F2) for 18 months. Our design included a transcriptomic analysis of the olfactory rosette (collected from the F2) and a viral challenge (exposing F2 to betanodavirus) where we assessed survival rates. RESULTS: We discovered transcriptomic trade-offs in both sensory and immune systems after long-term transgenerational exposure to OA. Specifically, RNA-Seq analysis of the olfactory rosette, the peripheral olfactory organ, from 18-months-old F2 revealed extensive regulation in genes involved in ion transport and neuronal signalling, including GABAergic signalling. We also detected OA-induced up-regulation of genes associated with odour transduction, synaptic plasticity, neuron excitability and wiring and down-regulation of genes involved in energy metabolism. Furthermore, OA-exposure induced up-regulation of genes involved in innate antiviral immunity (pathogen recognition receptors and interferon-stimulated genes) in combination with down-regulation of the protein biosynthetic machinery. Consistently, OA-exposed F2 challenged with betanodavirus, which causes damage to the nervous system of marine fish, had acquired improved resistance. CONCLUSION: F2 exposed to long-term transgenerational OA acclimation showed superior viral resistance, though as their metabolic and odour transduction programs were altered, odour-mediated behaviours might be consequently impacted. Although it is difficult to unveil how long-term OA impacts propagated between generations, our results reveal that, across generations, trade-offs in plastic responses is a core feature of the olfactory epithelium transcriptome in OA-exposed F2 offspring, and will have important consequences for how cultured and wild fish interacts with its environment.

Cytokine-like activity of European sea bass ISG15 protein on RGNNV-infected E-11 cells.

IFN-I generates an antiviral state by inducing the expression of numerous genes, called IFN-stimulated genes, ISGs, including ISG15, which is the only ISG with cytokine-like activity. In a previous study, we developed the Dl_ISG15_E11 cell line, which consisted of E11 cells able to express and secrete sea bass ISG15. The current study is a step forward, analysing the effect of secreted sea bass ISG15 on RGNNV replication in E11 cells, and looking into its immunomodulatory activity in order to corroborate its cytokine-like activity. The medium from ISG15-produccing cells compromised RGNNV replication, as it has been demonstrated both, by reduction in the viral genome synthesis and, specially, in the yield of infective viral particles. The implication of sea bass ISG15 in this protection has been demonstrated by ISG15 removal, which decreased the percentage of surviving cells upon viral infection, and by incubation of RGNNV-infected cells with a recombinant sea bass ISG15 protein, which resulted in almost full protection. Furthermore, the immunomodulatory activity of extracellular sea bass ISG15 has been demonstrated, which reaffirms a cytokine-like role for this protein.

Nanoplastics Increase Fish Susceptibility to Nodavirus Infection and Reduce Antiviral Immune Responses.

Nanoplastics (NPs) might cause different negative effects on aquatic organisms at different biological levels, ranging from single cells to whole organisms, including cytotoxicity, reproduction, behavior or oxidative stress. However, the impact of NPs on disease resistance is almost unknown. The objective of this study was to assess whether exposure to 50 nm functionalized polystyrene NPs impacts fish susceptibility to viral diseases both in vitro and in vivo. In particular, we focused on the nervous necrosis virus (NNV), which affects many fish species, producing viral encephalopathy and retinopathy (VER), and causes great economic losses in marine aquaculture. In vitro and in vivo approaches were used. A brain cell line (SaB-1) was exposed to 1 µg mL-1 of functionalized polystyrene NPs (PS-NH2, PS-COOH) and then infected with NNV. Viral titers were increased in NP-exposed cells whilst the transcription of inflammatory and antiviral markers was lowered when compared to those cells only infected with NNV. In addition, European sea bass (Dicentrarchus labrax) juveniles were intraperitoneally injected with the same NPs and then challenged with NNV. Our results indicated that NPs increased the viral replication and clinical signs under which the fish died although the cumulate mortality was unaltered. Again, exposure to NPs produced a lowered inflammatory and antiviral response. Our results highlight that the presence of NPs might impact the infection process of NNV and fish resistance to the disease, posing an additional risk to marine organisms.

Temperature-Biased miRNA Expression Patterns during European Sea Bass (Dicentrarchus labrax) Development.

Environmental effects and, particularly, temperature changes have been demonstrated to influence the activity, function, and well-being of teleosts. Temperature may change seasonally in the wild, and in captivity under aquaculture operations. Moreover, climate change is expected to shift temperature profiles worldwide. MicroRNAs (miRNA) are important temperature-sensitive gene-expression regulators acting at the post-transcriptional level. They are known to be key regulators in development, reproduction, and immune responses. Therefore, early larval development of the European sea bass (Dicentrarchus labrax), one of the most extensively cultured species in Mediterranean aquaculture, was investigated at early rearing temperatures, i.e., 15, 17.5, and 20 °C, in regard to the impact of temperatures on miRNAs through sncRNA high-throughput sequencing but also at the phenotypic level in terms of growth, sex, vision, and skeletal deformities. Expression profiling revealed stage- and temperature-specific miRNA expression targeting genes with roles in reproduction and immune response mainly at the flexion and all-fins stages. Similar stage- and temperature-specific results were also observed concerning the number of rod cells and lower jaw elongation. The present work presents for the first time highly promising results on the influence of early rearing temperature at the post-transcriptional level during European sea bass development, with a putative impact on reproduction and immune response, as well as regarding teleost vision and larval development.

Natural cortisol production is not linked to the sexual fate of European sea bass.

In this study, we aimed to investigate the relationship between cortisol and the determination of sexual fate in the commercially important European sea bass (Dicentrarchus labrax). To test our hypothesis, we designed two temperature-based experiments (19 ℃, 21 ℃ and 23 ℃, experiment 1; 16 ℃ and 21 ℃, experiment 2) to assess the effects of these thermal treatments on European sea bass sex determination and differentiation. In the fish from the first experiment, we evaluated whether blood cortisol levels and expression of stress key regulatory genes were different between differentiating (149 to 183 dph) males and females. In the second experiment, we assessed whether cortisol accumulated in scales over time during the labile period for sex determination as well as the neuroanatomical localisation of brain cells expressing brain aromatase (cyp19a1b) and corticotropin-releasing factor (crf) differed between males and females undergoing molecular sex differentiation (117 to 124 dph). None of the gathered results allowed to detect differences between males and females regarding cortisol production and regulatory mechanisms. Altogether, our data provide strong physiological, molecular and histochemical evidence, indicating that in vivo cortisol regulation has no major effects on the sex of European sea bass.

Gut immunity in European sea bass (Dicentrarchus labrax): a review.

In this review, we summarize and discuss the trends and supporting findings in scientific literature on the gut mucosa immune role in European sea bass (Dicentrarchus labrax L.). Overall, the purpose is to provide an updated overview of the gastrointestinal tract functional regionalization and defence barriers. A description of the available information regarding immune cells found in two immunologically-relevant intestinal compartments, namely epithelium and lamina propria, is provided. Attention has been also paid to mucosal immunoglobulins and to the latest research investigating gut microbiota and dietary manipulation impacts. Finally, we review oral vaccination strategies, as a safe method for sea bass vaccine delivery.

Molecular characterization of the T cell costimulatory receptors CD28 and CTLA4 in the European sea bass.

For the activation of T cells, it is necessary the specific recognition of the peptide by the T cell receptors (TCR) in the surface of antigen-presenting cells (APCs) and additional signals delivered by costimulatory receptors. In fish, knowledge about the presence of these costimulatory signals is limited and functional evidence almost absent. Thus, in this study, we have identified the stimulatory CD28 and the inhibitory cytotoxic T-lymphocyte-associated protein 4 (CTLA4) coreceptors in the European sea bass (Dicentrarchus labrax), and evaluated their transcription. In parallel, the transcription encoding for the T cell markers CD8α and CD4 was also evaluated. Both coreceptors showed the canonical architecture including a signal peptide, an immunoglobulin domain, a transmembrane region and a cytosolic tail. Protein predictions and phylogenetic tree identify them as true mammalian orthologues of CD28 and CTLA4. We found these genes constitutively expressed in all studied organs of European sea bass with high expression in lymphoid organs (thymus, spleen and head-kidney) and liver. The molecular expression pattern of these genes was up-regulated in head-kidney leucocytes stimulated with T mitogens as concanavalin A and phytohemagglutinin (PHA), but not with the B cell mitogen lipopolysaccharide (LPS). Fish challenged with nodavirus (NNV) evidenced a differential and opposing regulation of the cd28 and ctla4 transcription levels in the brain, the target organ for viral replication, and head-kidney. While cd28 transcription tends to decrease over the infection time in both organs the expression of the ctla4 gene tends to increase. Interestingly, the coreceptor expression is highly and significantly correlated to the transcription of the T cell markers. Our results highlight the important role of CD28 and CTLA4 as costimulatory receptors of T cells in European sea bass but further studies are deserved.

First description and diagnostics of disease caused by Piscirickettsia salmonis in farmed European sea bass (Dicentrarchus labrax Linnaeus) from Croatia.

During the winter of 2013 and 2016, several Croatian fish farms experienced mortalities in the fry of European sea bass, Dicentrarchus labrax. Affected fish showed abnormal swimming behaviour and reduced appetite, and death ensued several days after the onset of clinical signs of disease. Necropsy revealed pale liver, empty digestive tract, distended gall bladder, and hyperaemia and congestion of the meninges. Routine bacteriological examination tested negative, and virological examination ruled out nodavirus infection. Histological examination revealed multifocal necrosis and extensive inflammation in the brain with abundant cellular debris in the ventricles. Inflammatory cells displayed intra-cytoplasmic basophilic vacuoles leading to suspicion of Piscirickettsia salmonis infection. Fluorescent in situ hybridization using an oligonucleotide probe targeting Domain Bacterium applied to tissue sections tested positive. The pathogen was identified by 16S rRNA gene sequencing of brain material, and the sequence showed 99% similarity with P. salmonis. This result enabled the design of an oligonucleotide probe specifically targeting P. salmonis. In 2016, P. salmonis was successfully isolated on CHAB from the brain of an affected specimen and identified using 16S rRNA gene sequencing and MALDI-TOF. This study describes the first outbreak of disease caused by P. salmonis in sea bass in Croatia, while new diagnostic tools will enable further research on its epidemiology and pathogenicity.

Effects of dispersant treated oil upon exploratory behaviour in juvenile European sea bass (Dicentrarchus labrax).

Accidental spills are pervasive pollution in aquatic ecosystems. Resorting to chemical dispersant is one of the most implemented strategies in response to oil spills, but it results in an increase in the bio-availability of oil compounds known to disturb fish neurosensory capacities and hence fish habitat use. While it has become well established that acute oil exposure can cause a range of physiological defects, sub-lethal consequences on animal behaviour have only received recent attention. Here we investigated the effect of an exposure to a 62 h- dispersant treated oil on the exploration tendency (exploratory activity, and avoidance of unfamiliar open areas) of juvenile European sea bass. Three different concentrations of chemically dispersed oil were tested, low and medium conditions bracketing the range of likely situations that fish encounter following an oil spill, the high dose representing a more severe condition. Fish recovery capacities were also evaluated during 2 weeks post-exposure. Our results suggest a dose-response relationship; the low dose (0.048 ± 0.007 g L-1 of total petroleum hydrocarbons ([TPH])) had no effect on sea bass behavioural response to a novel environment while medium (0.243 ± 0.012 g L-1 [TPH]) and high (0.902 ± 0.031 g L-1 [TPH]) doses altered fish exploratory activity and their typical avoidance of unfamiliar open areas. Our experiment also suggest signs of recovery capacities in the first 10 days following oil exposure even if fish might need more time to fully recover from observed alterations. We discuss the possibility that observed alterations may result from a neurosensory or physiological known defects of oil exposure, causing anaesthetic-like sedative behaviours. Altogether, this study shows that juvenile sea bass exposed to oil spill exhibit transient behavioural impairments that may have major population-level consequences given the high mortality experienced by juveniles.

Differential immunogene expression profile of European sea bass (Dicentrarchus labrax, L.) in response to highly and low virulent NNV.

European sea bass is highly susceptible to the nervous necrosis virus, RGNNV genotype, whereas natural outbreaks caused by the SJNNV genotype have not been recorded. The onset and severity of an infectious disease depend on pathogen virulence factors and the host immune response. The importance of RGNNV capsid protein amino acids 247 and 270 as virulence factors has been previously demonstrated in European sea bass; however, sea bass immune response against nodaviruses with different levels of virulence has been poorly characterized. Knowing the differences between the immune response against both kinds of isolates may be key to get more insight into the host mechanisms responsible for NNV virulence. For this reason, this study analyses the transcription of immunogenes differentially expressed in European sea bass inoculated with nodaviruses with different virulence: a RGNNV virus obtained by reverse genetics (rDl956), highly virulent to sea bass, and a mutated virus (Mut247+270Dl956, RGNNV virus displaying SJNNV-type amino acids at positions 247 and 270 of the capsid protein), presenting lower virulence. This study has been performed in brain and head kidney, and the main differences between the immunogene responses triggered by both viruses have been observed in brain. The immunogene response in this organ is stronger after inoculation with the most virulent virus, and the main differences involved genes related with IFN I system, inflammatory response, cell-mediated response, and apoptosis. The lower virulence of Mut247+270Dl956 to European sea bass can be associated with a delayed IFN I response, as well as an early and transitory inflammation and cell-mediated responses, suggesting that those can be pivotal elements in controlling the viral infection, and therefore, their functional activity could be analysed in future studies. In addition, this study supports the role of capsid amino acids at positions 247 and 270 as important determinants of RGNNV virulence to European sea bass.

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.

The Diverse Piscidin Repertoire of the European Sea Bass (Dicentrarchus labrax): Molecular Characterization and Antimicrobial Activities.

Fish rely on their innate immune responses to cope with the challenging aquatic environment, with antimicrobial peptides (AMPs) being one of the first line of defenses. Piscidins are a group of fish specific AMPs isolated in several species. However, in the European sea bass (Dicentrarchus labrax), the piscidin family remains poorly understood. We identified six different piscidins in sea bass, performed an in-depth molecular characterization and evaluated their antimicrobial activities against several bacterial and parasitic pathogens. Sea bass piscidins present variable amino acid sequences and antimicrobial activities, and can be divided in different sub groups: group 1, formed by piscidins 1 and 4; group 2, constituted by piscidins 2 and 5, and group 3, formed by piscidins 6 and 7. Additionally, we demonstrate that piscidins 1 to 5 possess a broad effect on multiple microorganisms, including mammalian parasites, while piscidins 6 and 7 have poor antibacterial and antiparasitic activities. These results raise questions on the functions of these peptides, particularly piscidins 6 and 7. Considering their limited antimicrobial activity, these piscidins might have other functional roles, but further studies are necessary to better understand what roles might those be.