Sequence polymorphism and expression variability of Crassostrea gigas immune related genes discriminate two oyster lines contrasted in term of resistance to summer mortalities.

Summer mortalities of Crassostreagigas are a major concern in oyster aquaculture. They are the result of a complex interaction between the host, pathogens and environmental factors. Oyster genetics have been identified as an essential determinant of oyster susceptibility to summer mortalities. As the capability of oysters to circumvent diseases depends in part on their immune defenses, we aimed to analyze the gene expression and sequence polymorphism of 42 immune related genes in two oyster lines selected for their "High" (H) and "Low" (L) survival to summer mortalities. Results showed that the variability of gene expression and the sequence polymorphism acting on particular genes could enable the discrimination between H and L oyster lines. Besides, a higher sequence polymorphism was observed on the L line affecting 11 of the 42 analyzed genes. By analyzing gene expression, sequence polymorphism and gene copy number of two antimicrobial peptide families (Cg-Defs and Cg-Prp), and an antimicrobial protein (Cg-BPI) on individual oysters, we showed that gene expression and/or sequence polymorphism could also discriminate H and L oyster lines. Finally, we observed a positive correlation between the gene expression and the gene copy number of antimicrobials and that sequence polymorphism could be encoded in the genome. Overall, this study gives new insights in the relationship between oyster immunity and divergent phenotypes, and discusses the potential implication of antimicrobial diversity in oyster survival to summer mortalities

Long-lasting antiviral innate immune priming in the Lophotrochozoan Pacific oyster, Crassostrea gigas

Abstract In the last decade, a paradigm shift has emerged in comparative immunology. Invertebrates can no longer be considered to be devoid of specific recognition and immune memory. However, we still lack a comprehensive view of these phenomena and their molecular mechanisms across phyla, especially in terms of duration, specificity, and efficiency in a natural context. In this study, we focused on a Lophotrochozoan/virus interaction, as antiviral priming is mostly overlooked in molluscs. Juvenile Crassostrea gigas oysters experience reoccurring mass mortalities events from Ostreid herpes virus 1 with no existing therapeutic treatment. Our results showed that various nucleic acid injections can prime oysters to trigger an antiviral state ultimately protecting them against a subsequent viral infection. Focusing on poly(I:C) as elicitor, we evidenced that it protected from an environmental infection, by mitigating viral replication. That protection seemed to induce a specific antiviral response as poly(I:C) fails to protect against a pathogenic bacteria. Finally, we showed that this phenomenon was long-lasting, persisting for at least 5 months thus suggesting for the first time the existence of innate immune memory in this invertebrate species. This study strengthens the emerging hypotheses about the broad conservation of innate immune priming and memory mechanisms in Lophotrochozoans

Schematic alignment of transcript sequences of three antimicrobial peptides, <i>Cg</i>-<i>Defh</i>, <i>Cg</i>-<i>Defm</i> and <i>Cg</i>-<i>Prp</i> from H and L oyster lines.

<p>Transcript sequences were obtained by PCR from whole oyster body RNA, from L lines (L) and H line (H) oysters. Numbers between parentheses at the left of sequences indicate the number of identical sequences found. Black bars indicate polymorphic sites compared to the first sequence.</p

Alignment of deduced amino acid sequences of three antimicrobial peptides, <i>Cg</i>-Defh, <i>Cg</i>-Defm and <i>Cg</i>-Prp, from H and L oyster lines.

<p>Amino acid sequences were deduced from transcripts sequences obtained from L lines (L) and H line (H) oysters. Numbers between parentheses at the left of sequences indicate the number of identical sequences found. Black bars indicate substitution sites and dots show identical amino acids compared to the first sequence. Amino acids under positive selection are shown in gray (using the ratio of nonsynonymous to synonymous substitutions per codon).</p

Correlation between basal gene expression and gene copy number for two antimicrobial peptides <i>Cg-Defs</i> and <i>Cg-Prp</i> from two oyster lines.

<p>Relative expression and gene copy number were estimated by qPCR (N=14 or 15) for (a) <i>Cg-Defs</i> and(b) <i>Cg-Prp</i>. Individuals from L oyster line are shown in black, individuals from H oyster line are shown in grey. Significant positive correlations between expression level and gene copy number (Spearman’s rank correlation coefficient) were detected for <i>Cg-Defs</i> (<i>p</i><0.05) and <i>Cg-Prp</i> (<i>p</i><0.1).</p

Melting temperature from transcript amplicons of four antimicrobial peptides and protein and three reference genes in individual oysters from H and L lines.

<p>Graphs represent melting curves of qPCR amplicons of three antimicrobial peptides (<i>Cg-Defs</i>, <i>Cg-Defh</i>, and <i>Cg-Prp</i>), one antimicrobial protein (<i>Cg-BPI</i>), and three constitutively expressed genes (<i>Cg-EF1</i>, <i>Cg-RPL40</i> and <i>Cg-RPS6</i>) from two selected oyster lines (ten oysters per line). H oyster line is represented in grey and L oyster line in black. The three antimicrobial peptides (<i>Cg-Defs</i>, <i>Cg-Defh</i>, and <i>Cg-Prp</i>) display a high variation on their melting temperatures is significantly associated with the L line (Fisher test, <i>p</i><0.05) while other genes present same variations in each oyster lines (Fisher test, <i>p</i>>0.05).</p

Gene expression and sequence polymorphism of 42 immune related genes in two oyster lines.

<p>Selected genes belonging to nine functional categories are listed below the figure and represented as symbols in front of each gene name. <b>A</b>) Hierarchical clustering of the relative expression levels of 42 immune related genes in non-stimulated oysters of H and L line (three groups of ten oysters per line). Each cell in the matrix corresponds to the expression level of one gene in one sample. The intensity of the color from green to red indicates the magnitude of differential expression (see color scale at the bottom of the image). Relative expressions were calculated according the 2^−ΔΔCq method [21]. The dendrogram at the top of the figure indicate relationship among samples; while the dendrogram at the right of the figure indicate relationship among the relative expression levels of selected genes. Hierarchical clustering was constructed with Multiple ArrayViewer software using average linkage clustering with Spearman Rank Correlation as the default distance metric. Significant differences of relative expressions between oyster lines were determined by the Mann-Whitney U test and genes with significant variation are underlined (<i>p</i><0.05). (<b>B</b>) Hierarchical clustering of the melting temperatures of qPCR amplicons of 42 selected genes in non-stimulated oysters of H and L line (three groups of ten oysters per line). Melting temperature of each sample is represented as the deviation from the mean of melting temperatures of all samples for each gene. Each cell in the matrix corresponds to the deviation from the mean of melting temperature of one gene in one sample. The intensity of the color from green to red indicates the magnitude of the deviation of melting temperature from the mean of each gene (see color scale at the bottom of the image). The dendrogram at the top of the figure indicate relationship among samples; while the dendrogram at the right of the figure indicate relationship among variation of melting temperatures of selected genes. Hierarchical clustering was constructed with Multiple Array Viewer software using average linkage clustering with Pearson Correlation as the default distance metric. Genes who present variation equal or superior to 0.5°C between samples and/or a significant differences of melting temperature between oyster lines (Mann-Whitney U test, <i>p</i><0.05) are underlined. Asterisks (*) indicate genes who present a significant differential of expression together with a variation of melting temperature.</p

A Sustained Immune Response Supports Long-Term Antiviral Immune Priming in the Pacific Oyster, Crassostrea gigas

International audienceOver the last decade, innate immune priming has been evidenced in many invertebrate phyla. If mechanistic models have been proposed, molecular studies aiming to substantiate these models have remained scarce. We reveal here the transcriptional signature associated with immune priming in the oyster Cras-sostrea gigas. Oysters were fully protected against Ostreid herpesvirus 1 (OsHV-1), a major oyster pathogen, after priming with poly(I·C), which mimics viral double-stranded RNA. Global analysis through RNA sequencing of oyster and viral genes after immune priming and viral infection revealed that poly(I·C) induces a strong anti-viral response that impairs OsHV-1 replication. Protection is based on a sustained upregulation of immune genes, notably genes involved in the interferon pathway and apoptosis, which control subsequent viral infection. This persistent antiviral alert state remains active over 4 months and supports antiviral protection in the long term. This acquired resistance mechanism reinforces the molecular foundations of the sustained response model of immune priming. It further opens the way to applications (pseudovaccination) to cope with a recurrent disease that causes dramatic economic losses in the shellfish farming industry worldwide. IMPORTANCE In the last decade, important discoveries have shown that resistance to reinfection can be achieved without a functional adaptive immune system, introducing the concept of innate immune memory in invertebrates. However, this field has been constrained by the limited number of molecular mechanisms evidenced to support these phenomena. Taking advantage of an invertebrate species, the Pacific oyster (Crassostrea gigas), in which we evidenced one of the longest and most effective periods of protection against viral infection observed in an invertebrate, we provide the first comprehensive transcriptomic analysis of antiviral innate immune priming. We show that priming with poly(I·C) induced a massive upregulation of immune-related genes, which control subsequent viral infection, and it was maintained for over 4 months after priming. This acquired resistant mechanism reinforces the molecular foundations of the sustained response model of immune priming. It opens the way to pseudovaccination to prevent the recurrent diseases that currently afflict economically or ecologically important invertebrates

Copper homeostasis at the host vibrio interface: lessons from intracellular vibrio transcriptomics

International audienceRecent studies revealed that several vibrio species have evolved the capacity to survive inside host cells. However, it is still often ignored if intracellular stages are required for pathogenicity. Virulence of Vibrio tasmaniensis LGP32, a strain pathogenic for Crassostrea gigas oysters, depends on entry into hemocytes, the oyster immune cells. We investigated here the mechanisms of LGP32 intracellular survival and their consequences on the host-pathogen interaction. Entry and survival inside hemocytes were required for LGP32-driven cytolysis of hemocytes, both in vivo and in vitro. LGP32 intracellular stages showed a profound boost in metabolic activity and a major transcription of antioxidant and copper detoxification genes, as revealed by RNA sequencing. LGP32 isogenic mutants showed that resistance to oxidative stress and copper efflux are two main functions required for vibrio intracellular stages and cytotoxicity to hemocytes. Copper efflux was also essential for host colonization and virulence in vivo. Altogether our results identify copper resistance as a major mechanism to resist killing by phagocytes, induce cytolysis of immune cells and colonize oysters. Selection of such resistance traits could arise from vibrio interactions with copper-rich environmental niches including marine invertebrates, which favor the emergence of pathogenic vibrios resistant to intraphagosomal killing across animal species

Laligation native simplifie l’étude de l’activité etdu mécanismed’action des peptides antimicrobiens à multiples domaines :cas des Big défensines d’huître.

International audienceLesBigdéfensinesd'invertébrésmarinssontdespetitesprotéinesantimicrobiennesdontlastructureparticulièrementoriginaleassocieundomaineN-terminalglobulaireàundomaineC-terminalsemblableauxβ-défensinesdevertébrés.LapremièreBigdéfensineaétédécritechez un chélicérate ancestral, la Limule. Depuis, ces molécules ont été identifiées dansdiverses espèces de mollusques bivalves, notamment les huitres chez lesquelles plusieursformes ont été décrites. Certaines de ces formes, comme la Cg-BigDef1, voient leurexpressiontrèsfortementinduiteenréponseàl'infectionmicrobienne,laissantsupposerunrôle majeur dans la défense antimicrobienne des huitres. Nous avons ici entrepris decaractériserl’activitéantimicrobiennedelaCg-BigDef1etdesesdeuxdomainesstructurauxisolés. Pour cela nous avons synthétisé les deux domaines et la molécule entière parsynthèse en phase solide et ligation native (NCL) en s’appuyant sur une méthodologierécemment développée pour la synthèse du partenaire thioester. Les résultats obtenusmontrent que la Cg-BigDef1 est microbicide sur un large spectre de bactéries et dechampignons filamenteux, incluant des pathogènes des huitres du genre Vibrio. NousobservonségalementdepuissantesactivitésantimicrobiennessurdessouchescliniquesdeStaphylocoques isolées de patients mucoviscidosiques et posant des problèmes demultirésistance aux antibiotiques (MRSA). Ces activités sont stables à salinité élevée.L’activitédesdeuxdomainesséparésestglobalement très faiblepar rapportàlamoléculeentièreetcesdeuxdomainesdéploientdesactivitéssynergiques.Enfin,auxconcentrationsminimales inhibitrices, laCg-BigDef1 induit seulement la perméabilisation desmembranesdesbactériesàGramnégatifalorsquesondomaineglobulaireN-terminalseulperméabiliselesmembranesdesbactériesàGrampositifetdesbactériesà Gramnégatif