Elena Chaves-Pozo

Knowledge about fish-pathogen interaction is essential for optimizing technical and biological aspect of culture techniques to guarantee the economic viability of the aquaculture production. Nodavirus (NNV) is one of the most prevalent viruses worldwide causing disease in more than 170 species. In this study, we aimed to develop a non-lethal method to diagnose NNV in the gonad of asymptomatic broodstock specimens of greater amberjack ( Seriola dumerili ) using a RTG cell line expressing the luciferase reporter gene under the control of the Mx promoter (RTG-pmx-luciferase system) and real-time PCR. We also characterize the immune response and reproductive health of both sexes by means of gene expression and functional parameters analysis. Our data showed that NNV is present in both male and female blood and gonads in similar proportions demonstrating that both sexes might develop persistent infection and transfer the virus to their progeny by vertical transmission. Regarding immune response, the levels observed in the testis were higher in infected males. Specifically, we observed an up-regulation of genes related to the interferon pathway, antimicrobial peptides and leucocyte-markers molecules in infected testis, which might lead to a functional alteration. Otherwise, there were no alterations of immune parameters in the infected ovaries suggesting a latent infection. Serum estradiol levels were high in infected females constituting a potential mechanism to fight against the virus. In conclusion, we optimized a non-lethal method for NNV diagnosis involving gonadal biopsies. Moreover, our study revealed sex-biased host-NNV interactions.

Immunovirology in Aquatic Animals 13 Aquatic ecosystems are home to invertebrate and vertebrate animal species and provide important 14 natural resources, including food. Aquatic protein-rich animal foods contribute to global food security 15 and nutrition. In the last decades the production of fish food through aquaculture has overcome fisheries 16 (FAO 2024). Among the present challenges faced by aquaculture are viral infectious diseases that can 17 affect product quality and cause harvest losses.

The influence of algae in a final fattening diet for gilthead seabream, Sparus aurata, was studied in an 87 d feeding trial. Five groups were analysed (three tanks per group). In the first 38 days, one lot was fed with standard algae-free feed (C1) and four batches were fed with an algae-free diet low in fishmeal (FM) and fish oil (FO) and rich in terrestrial vegetables (C2). Subsequently, the C2 diet was changed in three out of the four groups, two of them being fed a diet with a microalgae blend (10%) plus 2% macroalgae (Alaria esculenta), either in raw (C2-R) or hydrolysed (C2-H) form, and a third batch with a diet lacking FM and FO but supplemented with 10% microalgae and 7% algae oil (C2-O) for 49 days. Body length and weight, muscle parameters, and conversion index were analysed after 38 and 87 days. At day 38, no significant differences were observed in any parameter between C1 and C2, but at the end of the trial (day 87), C2 displayed the lowest weight gain and the worst conversion rate, whereas C2-R showed higher body weight, fibrillar hypertrophy, and better conversion rates than the other groups, and C2-H displayed higher hyperplasia values than the other groups.

Nervous necrosis virus (NNV) is the etiological agent of viral encephalopathy and retinopathy in many fish species, including European sea bass (Dicentrachus labrax) and is of great economic losses to fish farmers. To solve this problem in fish production, antimicrobial peptides (AMPs) have been identified as potential candidates for NNV treatment in aquaculture. Hepcidin (Hamp) is one of the most promising AMPs. Thus, we aimed to evaluate the therapeutic application of Hamp synthetic peptide after NNV challenge. Hamp was able to significantly increase survival rates and ameliorate clinical signs of the disease, though the viral levels, determined by viral replication and immunolocalization, were not affected. Synthetic Hamp increased the immunoglobulin M (IgM) and AMP protein levels in serum and some tissues respect to the levels found in NNV-infected fish. However, Hamp peptide decreased the NNV-induced bactericidal activity. At the gene level, Hamp exerted anti-inflammatory properties, reducing the pro-inflammatory response orchestrated by NNV, probably preventing neuronal damage. Apart from this, Hamp up-regulated the expression of adhesion molecules that facilitated the recruitment of immune cells, namely T helper and B cells, probably to orchestrate the adaptive response. To conclude, Hamp immunomodulatory properties and therapeutic application against NNV are very promising for its use in aquaculture.

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.

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.