Sonia Dios

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.

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.

Individual sequence variability and functional activities of fibrinogen-related proteins (FREPs) in the Mediterranean mussel (Mytilus galloprovincialis) suggest ancient and complex immune recognition models in invertebrates.

In this paper, we describe sequences of fibrinogen-related proteins (FREPs) in the Mediterranean mussel Mytilus galloprovincialis (MuFREPs) with the fibrinogen domain probably involved in the antigen recognition, but without the additional collagen-like domain of ficolins, molecules responsible for complement activation by the lectin pathway. Although they do not seem to be true or primive ficolins since the phylogenetic analysis are not conclusive enough, their expression is increased after bacterial infection or PAMPs treatment and they present opsonic activities similar to mammalian ficolins. The most remarkable aspect of these sequences was the existence of a very diverse set of FREP sequences among and within individuals (different mussels do not share any identical sequence) which parallels the extraordinary complexity of the immune system, suggesting the existence of a primitive system with a potential capacity to recognize and eliminate different kind of pathogens.

Evidence of high individual diversity on myticin C in mussel (Mytilus galloprovincialis)

Several antimicrobial peptides (AMP) have been described in Mytilus galloprovincialis. However, only in myticin C a high variability on the nucleotide sequence was detected. To determine the individual variability of this AMP, the myticin C present in more than 100 mussels was analyzed by denaturing gradient gel electrophoresis (DGGE). This technique helped us to describe a very high myticin C diversity as compared with a non-immune related gene such as the beta-actin. Moreover, each mussel showed a specific and exclusive myticin C band pattern. Our results showed that the individual sequences of myticin C are unique for each mussel, independently of their geographic origin, age, sex, gonad maturation stage or aggregate where they group together on the wild. Only the animals belonging to the same family shared myticin C sequences. The comparative analysis of genomic DNA and cDNA sequences from the same individual showed that all detected variants shared a very high homology with the more frequent genomic isoforms, suggesting that all the variations were generated from the more common sequences, through a mechanism not yet determined. The fact that myticin C possesses characteristics of an immune gene, its potential antimicrobial effects, molecular diversity, as well as its early and ubiquitous expression, led us to suggest that myticin C might play an important role in innate immune defense in mussels.

Nodavirus Infection of Sea Bass (Dicentrarchus labrax) Induces Up-Regulation of Galectin-1 Expression with Potential Anti-Inflammatory Activity,

Sea bass nervous necrosis virus is the causative agent of viral nervous necrosis, a disease responsible of high economic losses in larval and juvenile stages of cultured sea bass (Dicentrarchus labrax). To identify genes potentially involved in antiviral immune defense, gene expression profiles in response to nodavirus infection were investigated in sea bass head kidney using the suppression subtractive hybridization (SSH) technique. A total of 8.7% of the expressed sequence tags found in the SSH library showed significant similarities with immune genes, of which a prototype galectin (Sbgalectin-1), two C-type lectins (SbCLA and SbCLB) from groups II and VII, respectively, and a short pentraxin (Sbpentraxin) were selected for further characterization. Results of SSH were validated by in vivo up-regulation of expression of Sbgalectin-1, SbCLA, and SbCLB in response to nodavirus infection. To examine the potential role(s) of Sbgalectin-1 in response to nodavirus infection in further detail, the recombinant protein (rSbgalectin-1) was produced, and selected functional assays were conducted. A dose-dependent decrease of respiratory burst was observed in sea bass head kidney leukocytes after incubation with increasing concentrations of rSbgalectin-1. A decrease in IL-1β, TNF-α, and Mx expression was observed in the brain of sea bass simultaneously injected with nodavirus and rSbgalectin-1 compared with those infected with nodavirus alone. Moreover, the protein was detected in the brain from infected fish, which is the main target of the virus. These results suggest a potential anti-inflammatory, protective role of Sbgalectin-1 during viral infection.

Immunological responses of turbot (Psetta maxima) to nodavirus infection or polyriboinosinic polyribocytidylic acid (pIC) stimulation, using expressed sequence tags (ESTs) analysis and cDNA microarrays.

To investigate the immunological responses of turbot to nodavirus infection or pIC stimulation, we constructed cDNA libraries from liver, kidney and gill tissues of nodavirus-infected fish and examined the differential gene expression within turbot kidney in response to nodavirus infection or pIC stimulation using a turbot cDNA microarray. Turbot were experimentally infected with nodavirus and samples of each tissue were collected at selected time points post-infection. Using equal amount of total RNA at each sampling time, we made three tissue-specific cDNA libraries. After sequencing 3230 clones we obtained 3173 (98.2%) high quality sequences from our liver, kidney and gill libraries. Of these 2568 (80.9%) were identified as known genes and 605 (19.1%) as unknown genes. A total of 768 unique genes were identified. The two largest groups resulting from the classification of ESTs according to function were the cell/organism defense genes (71 uni-genes) and apoptosis-related process (23 uni-genes). Using these clones, a 1920 element cDNA microarray was constructed and used to investigate the differential gene expression within turbot in response to experimental nodavirus infection or pIC stimulation. Kidney tissue was collected at selected times post-infection (HPI) or stimulation (HPS), and total RNA was isolated for microarray analysis. Of the 1920 genes studied on the microarray, we identified a total of 121 differentially expressed genes in the kidney: 94 genes from nodavirus-infected animals and 79 genes from those stimulated with pIC. Within the nodavirus-infected fish we observed the highest number of differentially expressed genes at 24 HPI. Our results indicate that certain genes in turbot have important roles in immune responses to nodavirus infection and dsRNA stimulation.

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.

Gene expression analysis of clams Ruditapes philippinarum and Ruditapes decussatus following bacterial infection yields molecular insights into pathogen resistance and immunity

The carpet shell clam (Ruditapes decussatus) and Manila clam (Ruditapes philippinarum), which are cultured bivalve species with important commercial value, are affected by diseases that result in large economic losses. Because the molecular mechanism of the immune response of bivalves, especially clams, is scarce and fragmentary, we have examined all Expressed Sequence Tags (EST) resources available in public databases for these two species in order to increase our knowledge on genes related with the immune function in these animals. After automatic annotation and classification of the 3784 not-annotated ESTs of R. decussatus and 4607 of R. philippinarum found in GenBank, 424 ESTs of R. decussatus and 464 of R. philippinarum were found to be putatively involved in immune response. These were carefully reviewed and reannotated. As a result, 13 immune-related ESTs were selected and studied to compare the immune response of R. decussatus and R. philippinarum following a Vibrio alginolyticus challenge. Quantitative PCR was performed, and the expression of each EST was determined. The results showed that, in R. philippinarum, the immune response seems to be faster than that in R. decussatus. Additionally, expression of NF-κB activating genes in R. decussatus did not seem to be sufficient to promote an immune response after Vibrio infection. R. philippinarum, however, was able to trigger and efficiently regulate the transcriptional activity of NF-κB, even when low expression values were reported.

Characterisation, expression and ontogeny of interleukin-6 and its receptors in zebrafish (Danio rerio)

Interleukin-6 (IL-6) is one of the most pleiotropic cytokines due to its importance in both innate and adaptive immune responses and other physiological processes. In this study, we identified the zebrafish (Danio rerio) IL-6 homologue by investigating the synteny between the human (Homo sapiens), the fugu (Takifugu rubripes) and the zebrafish genome. Although zebrafish IL-6 showed a low sequence homology with other IL-6 sequences in other species, it presented a high structural similarity to human IL-6. We also analysed IL-6 expression in several different tissues, along with analysis of the expression of the genes that form the IL-6 receptor complex, IL-6R and gp130. After treatment with bacterial or viral stimuli, zebrafish IL-6 expression was modulated in a manner similar to that of other proinflammatory molecules, such as IL-1β and TNF-α. The expression of IL-6, IL-6R and gp130 was also studied during the ontogeny of zebrafish larvae using quantitative PCR and in situ hybridisation. Our results indicated that the transcripts were detected very early, increased during the first week of life and were predominantly expressed in the head, epidermis and neuromasts of the anterior and posterior lateral line system, suggesting their involvement in the normal development of these tissues.

Effect of the temperature during antiviral immune response ontogeny in teleosts

Zebrafish were used to investigate the expression levels of several antiviral and inflammatory genes (IL-1β, iNOS, TNF-α, TLR3, IFN-I, IFNγ, IRF3, MDA-5, Mx) constitutively and after viral stimulation during early development. We also determined how their expression was affected by changes in the temperature. The antiviral genes were almost completely inhibited at 15°C with the exception of TLR3. In contrast, IL-1β, iNOS and TNF-α expression was not obviously different between the two temperatures. At 15°C, most of the genes examined did not differ following stimulation with poly I:C or viral hemorrhagic septicemia virus (VHSV). However, at 28°C, all of the genes showed significant differences in at least some of the sampling points after poly I:C treatment with the largest differences observed for Mx. Mx expression in adult zebrafish was not significantly altered by temperature and poly I:C treatment led to a smaller increase in gene expression when compared to larval Mx levels. Thus, Mx seems to play an important role in viral immunity in larvae, when the adaptive immune response is not fully functional.