Hyperparathyroïdie primaire et déficit en vitamine D (étude lilloise)

LILLE2-BU Santé-Recherche (593502101) / SudocPARIS-BIUM (751062103) / SudocSudocFranceF

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

Can selection for resistance to OsHV-1 infection modify susceptibility to Vibrio aestuarianus infection in Crassostrea gigas? First insights from experimental challenges using primary and successive exposures

Since 2008, the emergent virus OsHV 1μvar has provoked massive mortality events in Crassostrea gigas spat and juveniles in France. Since 2012, mortality driven by the pathogenic bacteria Vibrio aestuarianus has stricken market sized adults. A hypothesis to explain the sudden increase in mortality observed in France since 2012 is that selective pressure due to recurrent viral infections could have led to a higher susceptibility of adults to Vibrio infection. In our study, two OsHV-1-resistant lines (AS and BS) and their respective controls (AC and BC) were experimentally challenged in the laboratory to determine their level of susceptibility to V. aestuarianus infection. At the juvenile stage, the selected lines exhibited lower mortality (14 and 33%) than the control lines (71 and 80%), suggesting dual resistance to OsHV 1 and V. aestuarianus in C. gigas. Interestingly, this pattern was not observed at the adult stage, where higher mortality was detected for AS (68%) and BC (62%) than AC (39%) and BS (49%). These results were confirmed by the analysis of the expression of 31 immune related genes in unchallenged oysters. Differential gene expression discriminated oysters according to their susceptibility to infection at both the juvenile and adult stages, suggesting that resistance to V. aestuarianus infection resulted in complex interactions between the genotype, stage of development and immunity status. Finally, survivors of the V. aestuarianus challenge at the juvenile stage still exhibited significant mortality at the adult stage during a second and third V. aestuarianus challenge, indicating that these survivors were not genetically resistan

The Pacific Oyster Mortality Syndrome, a polymicrobial and multifactorial disease: state of knowledge and future directions

International audienceThe Pacific oyster (Crassostreae gigas) has been introduced from Asia to numerous countries around the world during the 20th century. C. gigas is the main oyster species farmed worldwide, and represents more than 98% of oyster production. The severity of disease outbreaks that affect C. gigas, which primarily impact juvenile oysters, has increased dramatically since 2008. The most prevalent disease, Pacific oyster mortality syndrome (POMS), has become panzootic and represents a threat to the oyster industry. Recently, major steps towards understanding POMS have been achieved through integrative molecular approaches. These studies demonstrated that infection by Ostreid herpesvirus type 1 µVar (OsHV-1 µvar) is the first critical step in the infectious process, and leads to an immunocompromised state by altering hemocyte physiology. This is followed by dysbiosis of the microbiota, which leads to a secondary colonization by opportunistic bacterial pathogens, which in turn results in oyster death. Host and environmental factors (e.g. oyster genetics and age, temperature, food availability, and microbiota) have been shown to influence POMS permissiveness. However, we still do not understand the mechanisms by which these different factors control disease expression. The present review discusses current knowledge of this polymicrobial and multifactorial disease process, and explores the research avenues that must be investigated to fully elucidate the complexity of POMS. These discoveries will help in decision-making, and will facilitate the development of tools and applied innovations for the sustainable and integrated management of oyster aquaculture

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

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

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

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

Mortalités d'huîtres creuses adultes (Crassostrea gigas) et infection à Vibrio aestuarianus - AESTU

En 2012, le nombre de cas de mortalités d’huîtres creuses adultes rapportés dans le cadre du réseau Repamo, et dans lesquels la bactérie Vibrio aestuarianus a été isolée, a fortement augmenté. Afin d’apporter de premiers éléments de réponse concernant cette augmentation, les objectifs de cette étude étaient de déterminer si l’émergence/la ré-émergence de V. aestuarianus était associée (i) à l’apparition d’un génotype bactérien particulier et/ou (ii) à une sensibilité accrue de certains animaux. L’Ifremer dispose d’une collection de souches bactériennes appartenant majoritairement au genre Vibrio et qui comporte différents isolats de V. aestuarianus, collectés depuis 2001 pendant ou hors épisodes de mortalité. La comparaison de ces isolats en termes de phénotype (virulence estimée en pathologie expérimentale) et de génotype (VNTR et génomes) n'a pas permis de mettre en évidence de génotype plus virulent particulier en 2012, mais plutôt l’existence de 2 clades au sein de l’espèce, dont la signification reste à préciser. Les mécanismes de virulence employés par deux souches « modèle », toutes deux hautement virulentes, ont été comparés in vitro et in vivo. Les résultats obtenus suggèrent des mécanismes d’action divergents, qui doivent être précisés et validés sur un plus grand nombre de souches. Par rapport aux travaux sur l’infection à V. aestuarianus réalisés avant sa ré-émergence (Garnier et al., 2008; Labreuche et al., 2006a) de façon globale, les animaux testés dans le cadre de cette étude se sont révélés tous très sensibles à la bactérie, et en particulier les animaux de plus d’un an. Les animaux de plus d’un an issus de captage naturel ou d’écloserie, diploïdes ou triploïdes, testés au cours de cette étude présentent des sensibilités comparables dans les conditions expérimentales choisies. Au-delà du fond génétique (lot d'animaux), de la ploïdie, de l'origine, le poids des animaux est un critère majeur influençant leur sensibilité. Enfin, la comparaison de lots issus de trois années de sélection massale (opérée sur estran pendant l’été, au moment des épisodes de mortalités associés à la détection de l’herpès virus OsHV-1) suggère une corrélation positive entre les caractères ‘survie sur estran pendant la première année de vie’, ‘survie face à une infection à OsHV-1’ et ‘survie face à une infection à V. aestuarianus’ pour des animaux âgés d’une année. Cependant, le nombre de lots testés dans le cadre de cette étude reste limité et un plus large panel de fonds génétiques doit maintenant être testé pour confirmer ces premières données. Enfin, la comparaison de l'expression de gènes (impliqués dans différents processus dont la réponse immunitaire) dans différents lots d'huîtres a permis de discriminer des huîtres présentant des capacités de survie différentielles avant et à la suite d'une infection à V. aestuarianus. Ces résultats bien qu’ils doivent encore être validés sur de nouveaux lots pour s’assurer de leur robustesse, ouvrent des perspectives pour le développement d’outils de pronostic des capacités de survie d’huîtres et l’analyse de souches d’huîtres d’intérêt en support aux programmes de sélectio