Two duplicated piscidin genes from gilthead seabream (Sparus aurata) with different roles in vitro and in vivo.

From the discovery of pleurocidin in skin mucus of winter flounder, many new related sequences have been found, forming a fish-exclusive family of antimicrobial peptides (AMP) called piscidin. Their mature peptides have a broad-spectrum antimicrobial activity and can be involved in the innate immune response. In the present work, two paralogous tripartite piscidin genes are formally described for the first time in gilthead seabream (Sparus aurata), an important marine farmed fish. Gene synteny and protein phylogeny clearly indicated a massive pisc gene expansion in a cluster of the chromosome 22 as well as a special evolution of piscidin in gilthead seabream compared to the rest of piscidins studied in other fish species. Despite being highly similar genes, they show totally different expression patterns in tissues and head-kidney leucocytes under both naïve and Vibrio/nodavirus-stimulated conditions. Moreover, these paralogous genes coded very different proteins according to their physicochemical properties. In this way, these piscidin genes have distinct roles not only related to their microbicide activity but also to their immune modulation. In addition, the present study improves the knowledge of duplication of AMP genes and adaptative diversification of teleost immune system.

Serna-Duque JA ，Cuesta A ，Sánchez-Ferrer Á ，Esteban MÁ ... -  《-》

Hepcidin and piscidin modulation and antibacterial response in gilthead seabream (Sparus aurata) infected with Vibrio harveyi.

Vibriosis is an infectious disease that generates large economic losses in Mediterranean aquaculture. Vibrio harveyi is one of the marine bacteria causing this disease, it is widespread in the Mediterranean Sea and causes ulcers on the skin of the fish it infects. In addition, the skin is a route of entry and colonization of this pathogen. In this study, one group of fish was injected intraperitoneally with phosphate buffered saline (control group) and another with V. harveyi (infected group). At 4 h after injection, samples of skin mucus, blood, skin, head kidney, liver, and spleen were collected to study the immune response generated. Liver histology showed notable alterations in hepatocyte morphology, such as increased vacuolization. Bactericidal activity was measured in skin mucus and serum against V. harveyi and V. anguillarum, different changes in this activity were recorded depending on the bacteria target and sample (skin mucus or serum) used. Gene expression of genes encoding hepcidins and piscidins (antimicrobial peptides) was performed in the mentioned organs. The results indicated a different expression according to the type of AMP and the tissue studied. Hepcidin appeared involved in all tissues studied while piscidins were in the spleen. In this study we have integrated hepcidin-piscidin modulation with the effects of infection on skin mucosa, serum and hepatocyte morphology. Knowing the changes produced in all these parameters improves the understanding of the infection in the first hours in sea bream and could have applications in the diagnosis or treatment of vibriosis in fish farms.

Serna-Duque JA ，Espinosa-Ruiz C ，Esteban MÁ 《-》

Transcription of histones H1 and H2B is regulated by several immune stimuli in gilthead seabream and European sea bass.

Histones (H1 to H4) are the primary proteins which mediate the folding of DNA into chromatin; however, and in addition to this function, histones have been also related to antimicrobial peptides (AMPs) activity in vertebrates, in fact, mammalian H1 is mobilized as part as the anti-viral immune response. In fish, histones with AMP activity have been isolated and characterized mainly from skin and gonads. One of most threatening pathogens for wild and cultured fish species nowadays is nodavirus (NNV), which target tissues are the brain and retina, but it is also able to colonize the gonad and display vertical transmission. Taking all this into account we have identified the h1 and h2b coding sequences in European sea bass (Dicentrarchus labrax) and gilthead seabream (Sparus aurata) fish species and studied their pattern of expression under naïve conditions and NNV in vivo infection. The data obtained prompted us to study their role on the immune response of gonad and head-kidney leucocytes upon viral (NNV), bacteria (Vibrio anguillarum or Photobacterium damselae), pathogen-associated molecular patterns (PAMPs) or mitogens stimulation. The h1 and h2b genes are expressed in a wide range of tissues and their expression is modify by infection or other immune stimuli, but further studies will be needed to determine the significance of these changes. These results suggest that h1 expression is related to the immune response against NNV in the brain, while h2b transcription seems to be more important in the head-kidney. Moreover, the potential role of histones as anti-viral agents is suggested and further characterization is in progress.

Valero Y ，Arizcun M ，Esteban MÁ ，Cuesta A ，Chaves-Pozo E ... -  《-》

Identification of an interferon-stimulated gene, isg15, involved in host immune defense against viral infections in gilthead seabream (Sparus aurata L.).

Interferons (IFNs) play a key role in the innate immunity of vertebrates against viral infections by inducing hundreds of IFN-stimulated genes (ISGs), such as isg15. Isg15 is an ubiquitin-like protein, which can conjugate cellular and viral proteins in a process called ISGylation, although it can also act as a cytokine-like protein. Gilthead seabream (Sparus aurata L.) is an important asymptomatic carrier of viral haemorrhagic septicaemia virus (VHSV) and nodavirus, representing a threat to other co-cultivated susceptible species. In order to better understand virus-host interactions in this fish species, this study addresses the identification and molecular characterization of seabream isg15 (sb-isg15). In addition, the modulation of transcript levels of sb-isg15 was analysed in SAF-1 cells and seabream acidophilic granulocytes (AGs) stimulated in vitro with different pathogen-associated molecular patterns (PAMPs) or inoculated with VHSV and striped jack nervous necrosis virus (SJNNV). The full-length cDNA of sb-isg15 gene, encoding a predicted protein of 155 amino acids, was identified and seen to share the same characteristics as other fish and mammalian isg15 genes. Here we report the clear induction of sb-isg15 transcript levels in SAF-1 cells and AGs stimulated with toll-like receptor (TLR) ligands, such as polyinosinic:polycytidylic acid (poly I:C) or genomic DNA from Vibrio anguillarum (VaDNA), respectively. Furthermore, VHSV and SJNNV inoculation induced a significant degree of sb-isg15 transcription in SAF-1 cells and AGs. However, the relative levels of viral RNA transcription showed that SJNNV replication seems to be more efficient than VHSV in both in vitro systems. Interestingly, sb-isg15 transcript induction elicited by VaDNA was reduced in VHSV- and SJNNV-inoculated AGs, suggesting an interference prompted by the viruses against the type I IFN system. Taken together, these findings support the use of seabream AGs as a valuable experimental system to study virus-host interactions, in which sb-isg15 seems to play an important role.

Álvarez-Torres D ，Gómez-Abellán V ，Arizcun M ，García-Rosado E ，Béjar J ，Sepulcre MP ... -  《-》

In vitro effects of metals on isolated head-kidney and blood leucocytes of the teleost fish Sparus aurata L. and Dicentrarchus labrax L.

The in vitro use of fish leucocytes to test the toxicity of aquatic pollutants, and particularly the immutoxicological effects, could be a valuable alternative to fish bioassays but has received little attention. In this study, head-kidney and peripheral blood leucocytes (HKLs and PBLs, respectively) from gilthead seabream (Sparus aurata L.) and European sea bass (Dicentrarchus labrax L.) specimens were exposed to Cd, MeHg (methylmercury), Pb or As for 24 h being evaluated the resulting cytotoxicity. Exposure to metals produced a dose-dependent reduction in the viability, and MeHg showed the highest toxicity followed by Cd, As and Pb. Interestingly, leucocytes from European sea bass are more resistant to metal exposure than those from gilthead seabream. Similarly, HKLs are always more sensitive than those isolated from blood from the same fish species. Moreover, fish leucocytes incubated with metals exhibited alterations in gene expression profiles that were more pronounced in the HKLs in general, being Pb the metal provoking less effects. Concretely, genes related to cellular protection (metallothionein), stress (heat shock protein 70) and oxidative stress (superoxide dismutase, catalase and glutathione reductase) were, in general, down-regulated in seabream HKLs but up-regulated in seabream PBLs and sea bass HKLs and PBLs. In addition, this profile leads to the increase of expression in genes related to apoptosis (Bcl2 associated X protein and caspase 3). Finally, transcription of genes involved in immunity (interleukin-1β and immunoglobulin M) was down-regulated, mainly in seabream leucocytes. This study points to the benefits for evaluating the toxicological mechanisms of marine pollution using fish leucocytes in vitro and insight into the mechanisms at gene level.

Morcillo P ，Meseguer J ，Esteban MÁ ，Cuesta A ... -  《-》