T cell activation is a multifaceted process that depends on the activation of the T cell receptor (TCR). However, other coreceptors are also strictly necessary to provide co-signals and modulate the immune response. However, to date, most of these coreceptors are unknown in fish or their information is very limited. Therefore, in this work, we have identified the cytotoxic and regulatory T cell molecule, CRTAM, and its ligand, the cell adhesion molecule 1, CADM1, in European seabass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata); and evaluated their transcriptional levels. Both putative proteins showed the canonical architecture observed in mammals, where CRTAM exhibited two immunoglobulin domains and CADM1, both the a and b forms, exhibited three of these domains. In addition, phylogeny and synteny analyses showed their conservation throughout vertebrate evolution. We found constitutive expression of all three genes, with crtam and cadm1a being predominant in immune tissues such as spleen, thymus and head-kidney (HK), while cadm1b expression was more limited to the brain. In vitro, only the T cell mitogen phytohemagglutinin (PHA) up-regulated the transcription of crtam and cadm1a in HK leucocytes. Nodavirus (NNV) infection elicited an up-regulation of crtam and cadm1a in brain and HK, appearing earlier in seabream than in seabass, which could explain the resistance of seabream to the development of nodavirus disease. In addition, they are up-regulated during the innate cell-mediated cytotoxic response in seabream but not in seabass. Altogether, our data seem to indicate that CRTAM is more related to the innate cytotoxicity in seabream and more in the specific and T cell-mediated cytotoxicity in seabass. Our results highlight the importance of CRTAM and CADM1 as important molecules in the activation of T lymphocytes in seabass and seabream, but further studies are needed.

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.

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.

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.

The nervous necrosis virus (NNV) leads to viral encephalopathy and retinopathy (VER) disease in more than 170 fish species, mainly from marine habitats. It replicates in the central nervous tissues, reaching up to 100% mortalities after a few days of infection, mainly in the larvae and juvenile stages. This is continuously spreading and affecting more species, both wild and cultured, posing a risk to the development of the aquaculture industry. In the Mediterranean Sea, it mainly affects European sea bass (Dicentrarchus labrax) and some grouper species (Epinephelus spp.). Interestingly, in the gilthead seabream (Sparus aurata), typically resistant to common NNV strains, great mortalities in hatcheries associated with typical clinical signs of VER have been confirmed to be caused by RGNNV/SJNNV reassortants. Thus, we have evaluated the susceptibility of seabream larvae to either RGNNV/SJNNV or SJNNV/RGNNV reassortants, as well as the larval immunity. Based on our results we can conclude that: (i) gilthead seabream larvae are susceptible to infection with both NNV reassortant genotypes, but mainly to RGNNV/SJNNV; (ii) virus replicated and infective particles were isolated; (iii) larval immunity was correlated with larval survival; and (iv) larval resistance and immunity were correlated with age of the larvae. Further investigations should be carried out to ascertain the risks of these new pathogens to Mediterranean larviculture.