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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.

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.

Aquaculture is an extremely prosperous market threatened by pathogen outbreaks, including viruses as nodavirus (NNV), which infect fish species with special interest in trading such as European sea bass. Antimicrobial peptides (AMPs) might constitute potential antiviral agents, which had been previously evaluated in fish with positive prospects, based on their properties as immunomodulators or directly killing pathogens. In this line, we aimed to evaluate this dual role by administering two European sea bass synthetic AMPs (Hamp or Dic) prior to NNV infection. Both treatments conferred partial protection against NNV though viral replication and load were not affected. Both AMPs elicited, prior to infection, AMP response and leukocyte mobilization whilst down-regulated pro-inflammatory markers. Upon infection, Hamp and Dic peptides abrogated the inflammatory response provoked by NNV as well as avoid NNV-induced disturbance of the leucocyte distribution in the brain, mainly neutrophils, macrophages and CD8+ T cells. This study points that preventive applications of synthetic Hamp and Dic peptides exert their antiviral actions through the immunomodulatory role and not by a direct action of the antimicrobial on NNV. This work opens the door to the use of AMPs as potential prophylactic tools against NNV as well as immunostimulant in fish farms.

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.

Previous studies have shown that Nannochloropsis gaditana can partially replace fishmeal in the diet of gilthead seabream, Sparus aurata. However, its effect on muscle growth is hardly known. This experiment was carried out with gilthead seabream adults that were fed with N. gaditana at two inclusion levels (2.5 or 5%) either raw (R2.5 and R5 groups) or cellulose-hydrolyzed (H2.5 and H5 groups) for 45 days in the final fattening phase. The body length and body weight were measured in all fish at the beginning and end of the experiment. Also, the white muscle transverse area (WM), size, number and fibrillar density of the white fibers were measured in 9 fish group−1. After 45 days, the body parameters and the WM did not show significant differences among the groups. However, muscle cellularity did show significant differences, such that the hypertrophy values were higher in the H2.5 and H5 than in the R2.5 and R5 groups. On the contrary, R2.5 and R5 showed the highest fibrillar density and hyperplasia values, which are often positively correlated with the fillet firmness and therefore could improve the final quality of the fish. No significant differences attributable to the inclusion levels of N. gaditana were observed

Numerous studies evaluating the effects of the incorporation of microalgae in feeds have reported favourable impacts on different physiological aspects of aquacultured fish. Although productivity is the major goal in terms of profitability in fish farming, qualitative aspects are gaining the attention of producers, given the relevance of quality attributes related to organoleptic parameters, proximal composition, and shelf life on the commercial value of fish. Indeed, microalgae are acknowledged for their richness in substances with potential positive effects on all those quality attributes. In this context, this study assesses the effects of finishing diets enriched with the microalga Nannochloropsis gaditana, either crude or enzymatically hydrolysed, on several quality parameters of gilthead seabream (Sparus aurata) fillets. Two inclusion levels (2.5 and 5%) of raw and enzymatically hydrolysed microalgal biomass were incorporated into diets, plus a microalgae-free control diet, and a 42-day feeding trial was carried out on fish of commercial size (approx. 500 g body weight). The influence of the experimental diets on fish biometry, fillet quality parameters, and shelf life was evaluated. The results indicate, overall, that microalgae-enriched diets yielded favourable, dose-dependent effects on several objective quality parameters of fillets, namely, improved fatty acid profile, reduced microbial counts, enhanced lipid oxidative status, and improved textural and skin colour attributes. Although the enzymatic pre-treatment of the microalgal biomass was expected to impact positively its functional effects on all quality parameters, however, no general trend was observed.

[EN] Currently, microalgae are used in fish diets, but their long-term growth effect is unknown. In this experiment, juvenile seabream specimens were fed with microalgae-enriched diets for three months, and then transferred to a microalgae-free diet for 10 months to assess long-term effects up to commercial size (≈27 cm and ≈300 g). The juvenile diets contained Nannochloropsis gaditana at 2.5 or 5% inclusion levels, either raw (R2.5 and R5 groups) or cellulose-hydrolyzed (H2.5 and H5 groups). The body length and weight were measured in 75 fish group-1 at commercial stage. The size, number, and fibrillar density of white muscle fibers and the white muscle transverse area were measured in nine fish group-1 at commercial stage. The results showed the highest body weight in H5 at commercial stage. The white muscle transverse area and the white fibres hyperplasia and density also showed the highest values in H5, followed by H2.5. In contrast, the highest hypertrophy was observed in C and R2.5, being associated with the lowest muscle growth in both groups. These results showed a microalgae concentration-dependent effect in hydrolyzed diets as well as an advantageous effect of the hydrolyzed versus raw diets on the long-term growth of Sparus aurata.

[EN] Acquiring immunocompetence is essential in the development of fish embryos, as they are exposed to environmental pathogens even before they are fertilized. Despite the importance of the antimicrobial function as the first line of defense against foreign microorganisms, little knowledge is available about its role in larval development. In vertebrates, transgenerational immune priming influences the acquisition of immunocompetence of specimens, regulating the selective allocation of nongenetic resources to their progeny and modulating their development. In this work, we primed teleost European sea bass broodstock females with a viral protein expression vector in order to evaluate the innate immunity development of their offspring. Several antimicrobial functions, the pattern of expression of gene coding for different antimicrobial peptides (AMPs), and their protein levels, were evaluated in eggs and larvae during development. Our data determined the presence of antimicrobial proteins of maternal origin in eggs, and that female vaccination increases antimicrobial activities and the transcription and synthesis of AMPs during larval development.

[EN] 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.

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.