Insights into Common Octopus (Octopus vulgaris) Ink Proteome and Bioactive Peptides Using Proteomic Approaches.

The common octopus (Octopus vulgaris) is nowadays the most demanded cephalopod species for human consumption. This species was also postulated for aquaculture diversification to supply its increasing demand in the market worldwide, which only relies on continuously declining field captures. In addition, they serve as model species for biomedical and behavioral studies. Body parts of marine species are usually removed before reaching the final consumer as by-products in order to improve preservation, reduce shipping weight, and increase product quality. These by-products have recently attracted increasing attention due to the discovery of several relevant bioactive compounds. Particularly, the common octopus ink has been described as having antimicrobial and antioxidant properties, among others. In this study, the advanced proteomics discipline was applied to generate a common octopus reference proteome to screen potential bioactive peptides from fishing discards and by-products such as ink. A shotgun proteomics approach by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using an Orbitrap Elite instrument was used to create a reference dataset from octopus ink. A total of 1432 different peptides belonging to 361 non-redundant annotated proteins were identified. The final proteome compilation was investigated by integrated in silico studies, including gene ontology (GO) term enrichment, pathways, and network studies. Different immune functioning proteins involved in the innate immune system, such as ferritin, catalase, proteasome, Cu/Zn superoxide dismutase, calreticulin, disulfide isomerase, heat shock protein, etc., were found in ink protein networks. Additionally, the potential of bioactive peptides from octopus ink was addressed. These bioactive peptides can exert beneficial health properties such as antimicrobial, antioxidant, antihypertensive, and antitumoral properties and are therefore considered lead compounds for developing pharmacological, functional foods or nutraceuticals.

Identifying Natural Bioactive Peptides from the Common Octopus (Octopus vulgaris Cuvier, 1797) Skin Mucus By-Products Using Proteogenomic Analysis.

The common octopus is a cephalopod species subject to active fisheries, with great potential in the aquaculture and food industry, and which serves as a model species for biomedical and behavioral studies. The analysis of the skin mucus allows us to study their health in a non-invasive way, by using a hardly exploited discard of octopus in the fishing sector. A shotgun proteomics approach combined with liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using an Orbitrap-Elite instrument was used to create a reference dataset from octopus skin mucus. The final proteome compilation was investigated by integrated in-silico studies, including Gene Ontology (GO), the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway, network studies, and prediction and characterization analysis of potential bioactive peptides. This work presents the first proteomic analysis of the common octopus skin mucus proteome. This library was created by merging 5937 identified spectra of 2038 different peptides. A total of 510 non-redundant proteins were identified. Obtained results show proteins closely related to the defense, which highlight the role of skin mucus as the first barrier of defense and the interaction with the environment. Finally, the potential of the bioactive peptides with antimicrobial properties, and their possible application in biomedicine, pharmaceutical, and nutraceutical industry was addressed.

De novo transcriptome reconstruction in aquacultured early life stages of the cephalopod Octopus vulgaris.

Cephalopods have been considered enigmatic animals that have attracted the attention of scientists from different areas of expertise. However, there are still many questions to elucidate the way of life of these invertebrates. The aim of this study is to construct a reference transcriptome in Octopus vulgaris early life stages to enrich existing databases and provide a new dataset that can be reused by other researchers in the field. For that, samples from different developmental stages were combined including embryos, newly-hatched paralarvae, and paralarvae of 10, 20 and 40 days post-hatching. Additionally, different dietary and rearing conditions and pathogenic infections were tested. At least three biological replicates were analysed per condition and submitted to RNA-seq analysis. All sequencing reads from experimental conditions were combined in a single dataset to generate a reference transcriptome assembly that was functionally annotated. The number of reads aligned to this reference was counted to estimate the transcript abundance in each sample. This dataset compiled a complete reference for future transcriptomic studies in O. vulgaris.

Rag1 immunodeficiency-induced early aging and senescence in zebrafish are dependent on chronic inflammation and oxidative stress.

In mammals, recombination activating gene 1 (RAG1) plays a crucial role in adaptive immunity, generating a vast range of immunoglobulins. Rag1-/- zebrafish (Danio rerio) are viable and reach adulthood without obvious signs of infectious disease in standard nonsterile conditions, suggesting that innate immunity could be enhanced to compensate for the lack of adaptive immunity. By using microarray analysis, we confirmed that the expression of immunity- and apoptosis-related genes was increased in the rag1-/- fish. This tool also allows us to notice alterations of the DNA repair and cell cycle mechanisms in rag1-/- zebrafish. Several senescence and aging markers were analyzed. In addition to the lower lifespan of rag1-/- zebrafish compared to their wild-type (wt) siblings, rag1-/- showed a higher incidence of cell cycle arrest and apoptosis, a greater amount of phosphorylated histone H2AX, oxidative stress and decline of the antioxidant mechanisms, an upregulated expression and activity of senescence-related genes and senescence-associated ß-galactosidase, respectively, diminished telomere length, and abnormal self-renewal and repair capacities in the retina and liver. Metabolomic analysis also demonstrated clear differences between wt and rag1-/- fish, as was the deficiency of the antioxidant metabolite l-acetylcarnitine (ALCAR) in rag1-/- fish. Therefore, Rag1 activity does not seem to be limited to V(D)J recombination but is also involved in senescence and aging. Furthermore, we confirmed the senolytic effect of ABT-263, a known senolytic compound and, for the first time, the potential in vivo senolytic activity of the antioxidant agent ALCAR, suggesting that this metabolite is essential to avoid premature aging.

Interferon-independent antiviral activity of 25-hydroxycholesterol in a teleost fish.

Oxysterols are a family of cholesterol oxygenated derivatives with diverse roles in many biological activities and have recently been linked with the induction of a cellular antiviral state. The antiviral effects of 25-hydroxycholesterol (25HC) extend to several mammalian enveloped and non-enveloped viruses. It has been reported that the expression of the gene encoding cholesterol 25-hydroxylase (CH25H) is induced by interferons (IFNs). In this work, five ch25h genes were identified in the zebrafish (Danio rerio) genome. The ch25h genes showed different tissue expression patterns and differed in their expression after immune stimulation with lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (PolyI:C) and Spring Viremia Carp Virus (SVCV). Only one of the 5 genes, ch25hb, was overexpressed after the administration of the treatments. Synteny and phylogenetic analyses revealed that ch25hb is the putative homolog of mammalian Ch25h in zebrafish, while the remaining zebrafish ch25h genes are products of duplications within the teleost lineage. Interestingly, its modulation was not mediated by type I IFNs, contrasting previous reports on mammalian orthologs. Nevertheless, in vivo overexpression of ch25hb in zebrafish larvae significantly reduced mortality after SVCV challenge. Viral replication was also negatively affected by 25HC administration to the zebrafish cell line ZF4. In conclusion, the interferon-independent antiviral role of 25HC was extended to a non-mammalian species for the first time, and dual activity that both protects the cells and interacts with the virus cannot be discarded.

Antiviral Activity of Myticin C Peptide from Mussel: an Ancient Defense against Herpesviruses.

UNLABELLED: Little is known about the antiviral response in mollusks. As in other invertebrates, the interferon signaling pathways have not been identified, and in fact, there is a debate about whether invertebrates possess antiviral immunity similar to that of vertebrates. In marine bivalves, due to their filtering activity, interaction with putative pathogens, including viruses, is very high, suggesting that they should have mechanisms to address these infections. In this study, we confirmed that constitutively expressed molecules in naive mussels confer resistance in oysters to ostreid herpesvirus 1 (OsHV-1) when oyster hemocytes are incubated with mussel hemolymph. Using a proteomic approach, myticin C peptides were identified in both mussel hemolymph and hemocytes. Myticins, antimicrobial peptides that have been previously characterized, were constitutively expressed in a fraction of mussel hemocytes and showed antiviral activity against OsHV-1, suggesting that these molecules could be responsible for the antiviral activity of mussel hemolymph. For the first time, a molecule from a bivalve has shown antiviral activity against a virus affecting mollusks. Moreover, myticin C peptides showed antiviral activity against human herpes simplex viruses 1 (HSV-1) and 2 (HSV-2). In summary, our work sheds light on the invertebrate antiviral immune response with the identification of a molecule with potential biotechnological applications. IMPORTANCE: Several bioactive molecules that have potential pharmaceutical or industrial applications have been identified and isolated from marine invertebrates. Myticin C, an antimicrobial peptide from the Mediterranean mussel (Mytilus galloprovincialis) that was identified by proteomic techniques in both mussel hemolymph and hemocytes, showed potential as an antiviral agent against ostreid herpesvirus 1 (OsHV-1), which represents a major threat to the oyster-farming sector. Both hemolymph from mussels and a myticin C peptide inhibited OsHV-1 replication in oyster hemocytes. Additionally, a modified peptide derived from myticin C or the nanoencapsulated normal peptide also showed antiviral activity against the human herpesviruses HSV-1 and HSV-2. Therefore, myticin C is an example of the biotechnological and therapeutic potential of mollusks.

Proinflammatory Caspase A Activation and an Antiviral State Are Induced by a Zebrafish Perforin after Possible Cellular and Functional Diversification from a Myeloid Ancestor.

In mammals, perforins play a central role in the granule-dependent cell death induced by natural killer T cells and cytotoxic T lymphocytes, and participate both in the defense against virus-infected and neoplastic cells and in the recognition of nonself molecules by the immune system. Little is known about fish perforin genes. We examined the zebrafish with the aim of increasing our knowledge about the role of perforins. We characterized 6 perforin genes in the zebrafish genome, and we studied them at the evolutionary level in combination with expression patterns in several tissues and cell populations, during both larval development and in the course of a viral infection. Our results suggest the specialization of different cell types in the production of perforins. Moreover, functional diversification during the evolution of these molecules could be inferred from this study. In particular, one of the genes, prf19b, which is mainly produced by myeloid cells, seemed to be involved in antiviral defense, conferring protection after an in vivo infection.

Use of Poly(I:C) Stabilized with Chitosan As a Vaccine-Adjuvant Against Viral Hemorrhagic Septicemia Virus Infection in Zebrafish.

There is an urgent need for more efficient viral vaccines in finfish aquaculture worldwide. Here, we report the use of poly(I:C) stabilized with chitosan as an adjuvant for development of better finfish vaccines. The adjuvant was co-injected with inactivated viral hemorrhagic septicemia virus (VHSV) (CSpIC+iV vaccine) in adult zebrafish and its efficiency in protection against VHSV infection was compared to a live, attenuated VHS virus vaccine (aV). Both free and stabilized poly(I:C) were strong inducers of an antiviral state, measured by transcriptional activation of the genes of viral sensors: toll-like receptors, interferons, and interferon-stimulated genes, such as MXa within 48 h after injection. Both the CSpIC+iV and the aV formulations provided a significant protection against VHSV-induced mortality. However, when plasma from survivors was tested for neutralizing antibodies in an in vitro protection assay, we could not demonstrate any protective effect. On the contrary, plasma from aV vaccinated fish enhanced cytopathic effects, indicating that antibody-dependent entry may play a role in this system. Our results show that poly(I:C) is a promising candidate as an adjuvant for fish vaccination against viral pathogens, and that the zebrafish is a promising model for aquaculture-relevant vaccination studies.

The involvement of cholesterol in sepsis and tolerance to lipopolysaccharide highlighted by the transcriptome analysis of zebrafish (Danio rerio).

Septic shock is the most common cause of death in intensive care units due to an aggressive inflammatory response that leads to multiple organ failure. However, a lipopolysaccharide (LPS) tolerance phenomenon (a nonreaction to LPS), is also often described. Neither the inflammatory response nor the tolerance is completely understood. In this work, both of these responses were analyzed using microarrays in zebrafish. Fish that were 4 or 6 days postfertilization (dpf) and received a lethal dose (LD) of LPS exhibited 100% mortality in a few days. Their transcriptome profile, even at 4 dpf, resembled the profile in humans with severe sepsis. Moreover, we selected 4-dpf fish to set up a tolerance protocol: fish treated with a nonlethal concentration of Escherichia coli LPS exhibited complete protection against the LD of LPS. Most of the main inflammatory molecules described in mammals were represented in the zebrafish microarray experiments. Additionally and focusing on this tolerance response, the use of cyclodextrins may mobilize cholesterol reservoirs to decrease mortality after a LD dose of LPS. Therefore, it is possible that the use of the whole animal could provide some clues to enhance the understanding of the inflammatory/tolerance response and to guide drug discovery.

Transcriptome profiles associated to VHSV infection or DNA vaccination in turbot (Scophthalmus maximus).

DNA vaccines encoding the viral G glycoprotein show the most successful protection capability against fish rhabdoviruses. Nowadays, the molecular mechanisms underlying the protective response remain still poorly understood. With the aim of shedding light on the protection conferred by the DNA vaccines based in the G glycoprotein of viral haemorrhagic septicaemia virus (VHSV) in turbot (Scophthalmus maximus) we have used a specific microarray highly enriched in antiviral sequences to carry out the transcriptomic study associated to VHSV DNA vaccination/infection. The differential gene expression pattern in response to empty plasmid (pMCV1.4) and DNA vaccine (pMCV1.4-G860) intramuscular administration with regard to non-stimulated turbot was analyzed in head kidney at 8, 24 and 72 hours post-vaccination. Moreover, the effect of VHSV infection one month after immunization was also analyzed in vaccinated and non-vaccinated fish at the same time points. Genes implicated in the Toll-like receptor signalling pathway, IFN inducible/regulatory proteins, numerous sequences implicated in apoptosis and cytotoxic pathways, MHC class I antigens, as well as complement and coagulation cascades among others were analyzed in the different experimental groups. Fish receiving the pMCV1.4-G860 vaccine showed transcriptomic patterns very different to the ones observed in pMCV1.4-injected turbot after 72 h. On the other hand, VHSV challenge in vaccinated and non-vaccinated turbot induced a highly different response at the transcriptome level, indicating a very relevant role of the acquired immunity in vaccinated fish able to alter the typical innate immune response profile observed in non-vaccinated individuals. This exhaustive transcriptome study will serve as a complete overview for a better understanding of the crosstalk between the innate and adaptive immune response in fish after viral infection/vaccination. Moreover, it provides interesting clues about molecules with a potential use as vaccine adjuvants, antiviral treatments or markers for vaccine efficiency monitoring.

Cellular visualization of macrophage pyroptosis and interleukin-1ß release in a viral hemorrhagic infection in zebrafish larvae.

Hemorrhagic viral diseases are distributed worldwide with important pathogens, such as dengue virus or hantaviruses. The lack of adequate in vivo infection models has limited the research on viral pathogenesis and the current understanding of the underlying infection mechanisms. Although hemorrhages have been associated with the infection of endothelial cells, other cellular types could be the main targets for hemorrhagic viruses. Our objective was to take advantage of the use of zebrafish larvae in the study of viral hemorrhagic diseases, focusing on the interaction between viruses and host cells. Cellular processes, such as transendothelial migration of leukocytes, virus-induced pyroptosis of macrophages. and interleukin-1ß (Il-1ß) release, could be observed in individual cells, providing a deeper knowledge of the immune mechanisms implicated in the disease. Furthermore, the application of these techniques to other pathogens will improve the current knowledge of host-pathogen interactions and increase the potential for the discovery of new therapeutic targets. Importance: Pathogenic mechanisms of hemorrhagic viruses are diverse, and most of the research regarding interactions between viruses and host cells has been performed in cell lines that might not be major targets during natural infections. Thus, viral pathogenesis research has been limited because of the lack of adequate in vivo infection models. The understanding of the relative pathogenic roles of the viral agent and the host response to the infection is crucial. This will be facilitated by the establishment of in vivo infection models using organisms such as zebrafish, which allows the study of the diseases in the context of a complete individual. The use of this animal model with other pathogens could improve the current knowledge on host-pathogen interactions and increase the potential for the discovery of new therapeutic targets against diverse viral diseases.

The evolution and appearance of C3 duplications in fish originate an exclusive teleost c3 gene form with anti-inflammatory activity.

The complement system acts as a first line of defense and promotes organism homeostasis by modulating the fates of diverse physiological processes. Multiple copies of component genes have been previously identified in fish, suggesting a key role for this system in aquatic organisms. Herein, we confirm the presence of three different previously reported complement c3 genes (c3.1, c3.2, c3.3) and identify five additional c3 genes (c3.4, c3.5, c3.6, c3.7, c3.8) in the zebrafish genome. Additionally, we evaluate the mRNA expression levels of the different c3 genes during ontogeny and in different tissues under steady-state and inflammatory conditions. Furthermore, while reconciling the phylogenetic tree with the fish species tree, we uncovered an event of c3 duplication common to all teleost fishes that gave rise to an exclusive c3 paralog (c3.7 and c3.8). These paralogs showed a distinct ability to regulate neutrophil migration in response to injury compared with the other c3 genes and may play a role in maintaining the balance between inflammatory and homeostatic processes in zebrafish.

Interferon-induced genes of the expanded IFIT family show conserved antiviral activities in non-mammalian species.

Interferon-induced proteins with tetratricopeptide repeats (IFITs) are involved in the protective response to viral infection, although the precise mechanism of IFITs for reducing viral proliferation is currently unknown. The interaction with the translation initiation factor eIF-3 or viral proteins and the sequestering of viral RNA have been proposed as potential antiviral functions for these proteins. In humans, four members of this family have been characterized. Nevertheless, information about these proteins in fish is almost non-existent. Exploiting the conservation of synteny between human and zebrafish genomes, we have identified ten members of the IFIT family located on four different chromosomes. The induction of these genes was examined both in vitro and in vivo after interferon (IFN) administration and rhabdovirus challenge. Whereas an induction of IFIT genes was observed after interferon treatments (IFNΦ1, IFNΦ2 and IFNΦ3), the viral infection did not affect these IFN-induced genes in vitro, and even reduced the IFN-induced expression of these genes. The response was largely different in vivo, with a broad up-regulation of IFIT genes after viral challenge. In addition, three selected IFITs were cloned in an expression vector and microinjected into zebrafish larvae to examine the protective effect of IFITs upon viral infection. Reduction in the mortality rate was observed confirming a conserved antiviral function in non-mammalian species.

Occurrence, seasonality and infectivity of Vibrio strains in natural populations of mussels Mytilus galloprovincialis.

Widespread and large-scale mortalities of bivalve molluscs significantly affect their production. A number of pathogens have been identified as the primary causes of death in oysters or clams, especially bacteria of the genus Vibrio. We evaluated the occurrence, seasonality and infectivity of Vibrio strains associated with natural mussel (Mytilus galloprovincialis) populations. In particular, different isolates of V. splendidus and V. aestuarianus were analysed because they were associated with major oyster mortalities in areas where mussels are cultured without presenting mortalities. The presence of both Vibrio spp. was analysed bimonthly in mussels, water, sediment, plankton and other associated fauna from 2 sites in Galicia (NW Spain), the region with the highest mussel production in Europe. Environmental factors were also considered. The pathogenicity of different Vibrio isolates was analysed by performing experimental infections in mussels with strains isolated from the field. Results showed that Vibrio populations were mainly influenced by changes in water temperature and salinity. V. splendidus was dominant during the warm months and V. aestuarianus was predominant throughout the cold season. The sediment was the most important natural reservoir for bacteria. Experimental infections showed the extreme resistance of mussels to bacterial pathogens. Isolates of V. splendidus and V. aestuarianus were only moderately pathogenic for mussels in intramuscular infections and bath infections, and mortalities only occurred when animals were infected with a high bacterial concentration in adverse environmental conditions (hypoxia and 25°C). Although the pathogenicity of the Vibrio strains isolated from the wild was low for mussels, their potential risk for other bivalves cannot be ignored.

IL-22 is a key player in the regulation of inflammation in fish and involves innate immune cells and PI3K signaling.

IL-22 plays a role in various disorders in mammals, including mucosal-associated infections and inflammatory diseases. No functional IL-22 studies have been conducted on non-mammals to date. In this study, recombinant IL-22 (rIL-22) from turbot was produced to investigate its effects as a bioactive molecule. The expression of several pro-inflammatory cytokines was increased after rIL-22 treatment and reduced by pre-treatment with a JAK/STAT inhibitor. The involvement of the PI3K pathway in IL-22 induction was demonstrated. rIL-22 reduced the mortality in Aeromonas salmonicida-infected turbot, while higher Aeromonas hydrophila- or LPS-induced mortality was observed when IL-22 was blocked in zebrafish embryos. IL-22 knockdown increased pro-inflammatory cytokine expression in bacteria-stimulated fish. In zebrafish, IL-22 expression was detected primarily in the myeloid innate linage. It was found during early developmental stages when the adaptive immune response is not yet functional and in rag1(-)/(-) fish that lack an adaptive immune system. Our results clarify the conserved role of IL-22 in lower vertebrates. We suggest for the first time that IL-22 constitutes a key regulator of inflammatory homeostasis even in distant species such as teleosts, which diverged from mammals more than 350 million years ago.

High-throughput sequence analysis of turbot (Scophthalmus maximus) transcriptome using 454-pyrosequencing for the discovery of antiviral immune genes.

BACKGROUND: Turbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations. METHODOLOGY/PRINCIPAL FINDINGS: Turbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very effective, obtaining an enormous variety of sequences belonging to genes involved in the defense mechanisms.

Mytilus galloprovincialis myticin C: a chemotactic molecule with antiviral activity and immunoregulatory properties.

Previous research has shown that an antimicrobial peptide (AMP) of the myticin class C (Myt C) is the most abundantly expressed gene in cDNA and suppressive subtractive hybridization (SSH) libraries after immune stimulation of mussel Mytilus galloprovincialis. However, to date, the expression pattern, the antimicrobial activities and the immunomodulatory properties of the Myt C peptide have not been determined. In contrast, it is known that Myt C mRNA presents an unusual and high level of polymorphism of unidentified biological significance. Therefore, to provide a better understanding of the features of this interesting molecule, we have investigated its function using four different cloned and expressed variants of Myt C cDNA and polyclonal anti-Myt C sera. The in vivo results suggest that this AMP, mainly present in hemocytes, could be acting as an immune system modulator molecule because its overexpression was able to alter the expression of mussel immune-related genes (as the antimicrobial peptides Myticin B and Mytilin B, the C1q domain-containing protein MgC1q, and lysozyme). Moreover, the in vitro results indicate that Myt C peptides have antimicrobial and chemotactic properties. Their recombinant expression in a fish cell line conferred protection against two different fish viruses (enveloped and non-enveloped). Cell extracts from Myt C expressing fish cells were also able to attract hemocytes. All together, these results suggest that Myt C should be considered not only as an AMP but also as the first chemokine/cytokine-like molecule identified in bivalves and one of the few examples in all of the invertebrates.

New insights into the apoptotic process in mollusks: characterization of caspase genes in Mytilus galloprovincialis.

Apoptosis is an essential biological process in the development and maintenance of immune system homeostasis. Caspase proteins constitute the core of the apoptotic machinery and can be categorized as either initiators or effectors of apoptosis. Although the genes encoding caspase proteins have been described in vertebrates and in almost all invertebrate phyla, there are few reports describing the initiator and executioner caspases or the modulation of their expression by different stimuli in different apoptotic pathways in bivalves. In the present work, we characterized two initiator and four executioner caspases in the mussel Mytilus galloprovincialis. Both initiators and executioners showed structural features that make them different from other caspase proteins already described. Evaluation of the genes' tissue expression patterns revealed extremely high expression levels within the gland and gills, where the apoptotic process is highly active due to the clearance of damaged cells. Hemocytes also showed high expression values, probably due to of the role of apoptosis in the defense against pathogens. To understand the mechanisms of caspase gene regulation, hemocytes were treated with UV-light, environmental pollutants and pathogen-associated molecular patterns (PAMPs) and apoptosis was evaluated by microscopy, flow cytometry and qPCR techniques. Our results suggest that the apoptotic process could be tightly regulated in bivalve mollusks by overexpression/suppression of caspase genes; additionally, there is evidence of caspase-specific responses to pathogens and pollutants. The apoptotic process in mollusks has a similar complexity to that of vertebrates, but presents unique features that may be related to recurrent exposure to environmental changes, pollutants and pathogens imposed by their sedentary nature.

Interaction of the attenuated recombinant rIHNV-Gvhsv GFP virus with macrophages from rainbow trout (Oncorhynchus mykiss).

One of the most important threats to the salmonid aquaculture industry is infection caused by novirhabdoviruses such as infectious haematopoietic necrosis virus (IHNV) or viral haemorrhagic septicaemia virus (VHSV). Using reverse genetics, an avirulent recombinant rIHNV-Gvhsv GFP strain was generated, which was able to replicate as effectively as wild type IHNV in a fish cell line and in macrophages. Although this recombinant virus induced protective responses against IHNV and VHSV, the response did not involve the production of antibodies or modulate the expression of some antiviral genes. To determine the immune mechanisms underlying the protection conferred by the rIHNV-Gvhsv GFP virus, different immune parameters (NO production, respiratory burst activity and the induction of apoptosis) were assessed in the macrophage population. The results obtained in the present work may indicate that the Nv protein could be important in the modulation of NO and ROS production. rIHNV-Gvhsv GFP did not appear to have a clear effect on nitric oxide production or apoptosis. However, an increased respiratory burst activity (with levels induced by the recombinant virus significantly higher than the levels induced by the wild type virus), suggests a stimulation of the macrophage population, which could be related to the protection against virulent viruses.