Identification of a Novel ß-Defensin Gene in Gilthead Seabream (Sparus aurata).

The excessive use of antibiotics in aquaculture favors the natural selection of multidrug-resistant bacteria, and antimicrobial peptides (AMPs) could be a promising alternative to this problem. The most studied AMPs in teleost fish are piscidins, hepcidins, and ß-defensins. In this work, we have found a new gene (defb2) encoding a type 2 ß-defensin in the genome of gilthead seabream, a species chosen for its economic interest in aquaculture. Its open reading frame (192 bp) encodes a protein (71 amino acids) that undergoes proteolytic cleavage to obtain the functional mature peptide (42 amino acids). The genetic structure in three exons and two introns and the six characteristic cysteines are conserved as the main signature of this protein family. In the evolutionary analysis, synteny shows a preservation of chromosomal localization and the phylogenetic tree constructed exposes the differences between both types of ß-defensin as well as the similarities between seabream and European seabass. In relation to its basal expression, ß-defensin 2 is mostly expressed in the intestine, thymus, skin, and gonads of the gilthead seabream (Sparus aurata). In head kidney leucoytes (HKLs), the expression was very low and did not change significantly when stimulated with various immunocompetent agents. However, the expression was significantly down-regulated in the liver, head-kidney, and blood 4 h post-injection with the fish pathogen Vibrio harveyi. When infected with nodavirus, the expression was downregulated in brain at 7 days post-infection. These results denote a possible complementarity between the expression patterns of ß-defensins and hepcidins. Further studies are needed to analyze gene duplications and expression patterns of ß-defensins and describe their mechanism of action in seabream and other teleost fish.

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.

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.

The susceptibility of shi drum juveniles to betanodavirus increases with rearing densities in a process mediated by neuroactive ligand-receptor interaction.

Nervous necrosis virus (NNV) is one of the greatest threats to Mediterranean aquaculture, infecting more than 170 fish species and causing mortalities up to 100% in larvae and juveniles of susceptible species. Intensive aquaculture implies stressed conditions that affect the welfare of fish and their ability to fight against infections. In fact, a higher susceptibility to NNV has been related to poor welfare conditions. In order to analyze the physiological link between stressed conditions and increased susceptibility to NNV, as well as its possible role in the pathogenesis of this disease, we reared shi drum (Umbrina cirrosa) juveniles (30.7 ± 3.10 g body weight), which are expected to be asymptomatic upon NNV infection, at three stocking densities (2, 15, and 30 kg/m3) for 27 days and subsequently challenged them with NNV. We firstly characterized the stressed conditions of the specimens before and after infection and recorded the mortalities, demonstrating that stressed specimens reared at 30 kg/m3 suffered mortalities. However, the viral loads in different tissues were similar in all experimental groups, allowing horizontal and vertical transmission of the virus from asymptomatic specimens. All of these data suggest that shi drum tolerates wide ranges of culture densities, although high densities might be a setback for controlling NNV outbreaks in this species. In an attempt to understand the molecular pathways orchestrating this susceptibility change in stressed conditions, we performed a transcriptomic analysis of four tissues under mock- and NNV-infected conditions. In addition to the modification of the exceptive pathways such as cell adhesion, leukocyte migration, cytokine interaction, cell proliferation and survival, and autophagy, we also observed a heavy alteration of the neuroactive ligand-receptor pathway in three of the four tissues analyzed. Our data also point to some of the receptors of this pathway as potential candidates for future pharmacological treatment to avoid the exacerbated immune response that could trigger fish mortalities upon NNV infection.

Transcriptional Evaluation of Neuropeptides, Hormones, and Tissue Repair Modulators in the Skin of Gilthead Sea Bream (Sparus aurata L.) Subjected to Mechanical Damage.

The skin of bony fish is the first physical barrier and is responsible for maintaining the integrity of the fish. Lesions make the skin vulnerable to potential infection by pathogens present in the aquatic environment. In this way, wound repair has barely been studied in gilthead sea bream. Thus, this study investigated the modulation of peripheral neuro-endocrine and tissue repair markers at the transcriptional level in the skin of teleost fish subjected to mechanical damage above or below the lateral line (dorsal and ventral lesions, respectively). Samples were evaluated using RT-qPCR at 2-, 4-, and 20-days post-injury. Fish with a ventral lesion presented a trend of progressive increase in the expressions of corticotropin-releasing hormone (crh), pro-opiomelanocortin-A (pomca), proenkephalin-B (penkb), cholecystokinin (cck), oxytocin (oxt), angiotensinogen (agt), and (less pronounced) somatostatin-1B (sst1b). By contrast, fish with a dorsal lesion registered no significant increase or biological trend for the genes evaluated at the different sampling times. Collectively, the results show a rapid and more robust response of neuro-endocrine and tissue repair markers in the injuries below than above the lateral line, which could be attributable to their proximity to vital organs.

Identification and functional characterization of fish IL-17 receptors suggest important roles in the response to nodavirus infection.

Th17 is a lymphocyte T helper (Th) subpopulation relevant in the control and regulation of the immune response characterized by the production of interleukin (IL)-17. This crucial cytokine family acts through their binding to the IL-17 receptors (IL-17R), having up to six members. Although the biology of fish Th17 is well-recognized, the molecular and functional characterization of IL-17 and IL-17R has been limited. Thus, our aim was to identify and characterize the IL-17R repertory and regulation in the two main Mediterranean cultured fish species, the gilthead seabream (Sparus aurata) and the European sea bass (Dicentrarchus labrax). Our in silico results showed the clear identification of six members in each fish species, from IL-17RA to IL-17RE-like, with well-conserved gene structure and protein domains with their human orthologues. All of them showed wide and constitutive transcription in naïve tissues but with highest levels in mucosal tissues, namely skin, gill or intestine. In leucocytes, T mitogens showed the strongest up-regulation in most of the il17 receptors though il17ra resulted in inhibition by most stimulants. Interestingly, in vivo nodavirus infection resulted in alterations on the transcription of il17 receptors. While nodavirus infection led to some increments in the il17ra, il17rb, il17rc and il17rd transcripts in the susceptible European sea bass, many down-regulations were observed in the resistant gilthead seabream. Our data identify the presence and conservation of six coding IL-17R in gilthead seabream and European sea bass as well as their differential regulation in vitro and upon nodavirus infection. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00225-1.

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.

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.

Cytotoxic activity and gene expression during in vitro adaptive cell-mediated cytotoxicity of head-kidney cells from betanodavirus-infected European sea bass.

Cell-mediated cytotoxicity (CMC) is essential in eradicating virus-infected cells, involving CD8+ T lymphocytes (CTLs) and natural killer (NK) cells, through the activation of different pathways. This immune response is well-studied in mammals but scarcely in teleost fish. Our aim was to investigate the adaptive CMC using head-kidney (HK) cells from European sea bass infected at different times with nodavirus (NNV), as effector cells, and the European sea bass brain cell line (DLB-1) infected with different NNV genotypes, as target cells. Results showed low and unaltered innate cytotoxic activity through the infection time. However, adaptive CMC against RGNNV and SJNNV/RGNNV-infected target cells increased from 7 to 30 days post-infection, peaking at 15 days, demonstrating the specificity of the cytotoxic activity and suggesting the involvement of CTLs. At transcriptomic level, we observed up-regulation of genes related to T cell activation, perforin/granzyme and Fas/FasL effector pathways as well as apoptotic cell death. Further studies are necessary to understand the adaptive role of European sea bass CTLs in the elimination of NNV-infected cells.

Evaluation of the Differential Postbiotic Potential of Shewanella putrefaciens Pdp11 Cultured in Several Growing Conditions.

The increased knowledge of functional foods has led to the development of a new generation of health products, including those containing probiotics and products derived from them. Shewanella putrefaciens Pdp11 (SpPdp11) is a strain described as a probiotic that exerts important beneficial effects on several farmed fish. However, the use of live probiotic cells in aquaculture has limitations such as uncertain survival and shelf life, which can limit their efficacy. In addition, its efficacy can vary across species and hosts. When probiotics are administered orally, their activity can be affected by the environment present in the host and by interactions with the intestinal microbiota. Furthermore, live cells can also produce undesired substances that may negatively impact the host as well as the risk of potential virulence reversion acquired such as antibiotic resistance. Therefore, new alternatives emerged such as postbiotics. Currently, there is no knowledge about the postbiotic potential of SpPdp11 in the aquaculture industry. Postbiotic refers to the use of bacterial metabolites, including extracellular products (ECPs), to improve host physiology. However, the production of postbiotic metabolites can be affected by various factors such as cultivation conditions, which can affect bacterial metabolism. Thus, the objective of this study was to evaluate the postbiotic potential of ECPs from SpPdp11 under different cultivation conditions, including culture media, temperature, growth phase, and salinity. We analyzed their hydrolytic, antibacterial, antiviral, and cytotoxic capacity on several fish cell lines. The results obtained have demonstrated how each ECP condition can exert a different hydrolytic profile, reduce the biofilm formation by bacterial pathogens relevant to fish, lower the titer of nervous necrosis virus (NNV), and exert a cytotoxic effect on different fish cell lines. In conclusion, the ECPs obtained from SpPdp11 have different capacities depending on the cultivation conditions used. These conditions must be considered in order to recover the maximum number of beneficial capacities or to choose the appropriate conditions for specific activities.