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

Gene expression of five antimicrobials in individual oysters from H and L lines.

<p>Relative expression of antimicrobial peptides (<i>Cg-Defs</i>, <i>Cg-Defh</i>, <i>Cg-Defm</i>, and <i>Cg-Prp</i>) and protein (<i>Cg-BPI</i>), were obtained from H and L selected oyster lines (19 oysters per line). Relative expression are expressed as box plot, where central point represents the mean, box represents ± standard error, and plot represents minimum and maximum value. Relative expression levels were obtained according to the 2^−ΔΔCq method [21]. Significant differences of relative expressions between oyster lines are indicated by different lowercase letters (different letters indicate significant difference, a or b) and were determined by the Mann-Whitney U test (<i>p</i><0.05).</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

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

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

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

The Ancestral N-Terminal Domain of Big Defensins Drives Bacterially Triggered Assembly into Antimicrobial Nanonets

β-Defensins are host defense peptides controlling infections in species ranging from humans to invertebrates. However, the antimicrobial activity of most human β-defensins is impaired at physiological salt concentrations. We explored the properties of big defensins, the β-defensin ancestors, which have been conserved in a number of marine organisms, mainly mollusks. By focusing on a big defensin from oyster ( Cg- BigDef1), we showed that the N-terminal domain lost during evolution toward β-defensins confers bactericidal activity to Cg- BigDef1, even at high salt concentrations. Cg- BigDef1 killed multidrug-resistant human clinical isolates of Staphylococcus aureus . Moreover, the ancestral N-terminal domain drove the assembly of the big defensin into nanonets in which bacteria are entrapped and killed. This discovery may explain why the ancestral N-terminal domain has been maintained in diverse marine phyla and creates a new path of discovery to design β-defensin derivatives active at physiological and high salt concentrations. </jats:p