Project description:Ostreid Herpesvirus type 1 (OsHV-1) has become a serious infective agent of the Pacific oyster livestock worldwide. In particular, the OsHV-1 muVar subtype has been associated to severe mortality episodes concerning Crassostrea gigas in France and other regions of the world such as Australia and New Zealand. Factors triggering productive infections and virus interactions with susceptible and resistant bivalve hosts are not completely understood though some studies have been undertaken to explore the genes expressed in oysters after infection. We took advantage of an highly infected oyster sample to perform an in-vivo dual RNA-seq analysis. An extremely high sequencing coverage allowed us to explore in detail the Herpesvirus genome and transcriptome, and to identify viral-activated molecular pathways in Crassostrea gigas, thus expanding the current knowledge on the host-virus interactions.
Project description:Massive mortalities have been observed in France since 2008 on spat and juvenile Pacific oysters, Crassostrea gigas. A herpes virus called OsHV-1, easily detectable by PCR, has been implicated in the mortalities as demonstrated by the results of numerous field studies linking mortality with OsHV-1 prevalence. Moreover, experimental infections using viral particles have documented the pathogenicity of OsHV-1. The physiological responses of host to pathogen are not well known. In this study, a number of genes involved in the response to viral challenge have been identified and can be considered as confirmation of the role of the virus in the observed mortality. The aim of this study was to understand mechanisms brought into play against the virus during infection in the field. A microarray assay has been developed for a major part of the oyster genome and used for studying the host transcriptome. Spat with and without detectable OsHV-1 infection were compared by microarray during mortality episodes. The result allowed establishment of a hypothetic scheme of the host cell’s infection by, and response to, the pathogen. This response seems to be different to “sensu stricto” innate immunity through genic regulation of the virus life cycle. . Some regulatory response against the virus may explain that some oysters are able to survive infection by regulation of the viral genes associated with the OsHV-1 life cycle.
Project description:Massive mortalities have been observed in France since 2008 on spat and juvenile Pacific oysters, Crassostrea gigas. A herpes virus called OsHV-1, easily detectable by PCR, has been implicated in the mortalities as demonstrated by the results of numerous field studies linking mortality with OsHV-1 prevalence. Moreover, experimental infections using viral particles have documented the pathogenicity of OsHV-1. The physiological responses of host to pathogen are not well known. In this study, a number of genes involved in the response to viral challenge have been identified and can be considered as confirmation of the role of the virus in the observed mortality. The aim of this study was to understand mechanisms brought into play against the virus during infection in the field. A microarray assay has been developed for a major part of the oyster genome and used for studying the host transcriptome. Spat with and without detectable OsHV-1 infection were compared by microarray during mortality episodes. The result allowed establishment of a hypothetic scheme of the host cell’s infection by, and response to, the pathogen. This response seems to be different to “sensu stricto” innate immunity through genic regulation of the virus life cycle. . Some regulatory response against the virus may explain that some oysters are able to survive infection by regulation of the viral genes associated with the OsHV-1 life cycle. Gene expression was measured from four individual animals of three sites, an oyster production area where mortalities on spat was observed in Spring (BL: Blainville sur mer) and two sanctuary sites (CRIC, Cricqueville en Bessin without production and CAB, an offshore storage structure)
Project description:To better understand the pathogenesis of OsHV-1 and to determine which cell pathways might be affected during OsHV-1 infection, we used an experimental infection in the Pacific oyster Crassostrea gigas by intra-muscular injection with a low or high load of OsHV-1. Animals were sampled 2 days post-injection (dpi) which corresponds to the incubation period required for OsHV1 to initiate an intense replication phase. Twenty-five abundant proteins linked to infection process were identified using a two-dimensional electrophoresis (2-DE) proteomic approach.
Project description:Hox and ParaHox genes encode transcription factors with conserved similar expression patterns in divergent animals. The Pdx (Xlox) homeobox gene, for example, is expressed in a sharp spatial domain in the endodermal cell layer of the gut in chordates, echinoderms, annelids and molluscs. The significance of comparable gene expression patterns is unclear because it is not known if downstream transcriptional targets are also conserved. We thus conducted experiments to show that a classic transcriptional target of Pdx1 in vertebrates, the insulin gene, is also a direct target of Pdx in the Pacific oyster. We report that oyster has a diversity of insulin-related genes including one co-expressed with Pdx in the endodermal layer of oyster digestive tissue. Transcriptome analysis reveals functional similarity of this tissue to vertebrate pancreas. Using ATAC-seq we identify a Pdx homeodomain binding site upstream of the endodermally-expressed oyster insulin-related gene and using cell culture demonstrate that oyster Pdx acts as a transcriptional activator through this site. These data argue that a classic homeodomain-target gene interaction dates back to the base of Bilateria.
Project description:The N6-methyladenosine (m6A) has recently emerged as an important layer of the gene expression regulatory network with critical implications in vertebrate and insect development. However, despite an m6A-RNA pathway is present in the pacific oyster Crassostrea gigas, the developmental significance of epitranscriptomes in lophotrochozoan organisms remains unknown. We performed RNA sequencing (RNA-seq) and m6A-RNA immunoprecipitation sequencing (MeRIP-seq) on 10 developmental stages, on two distinct developments, to covering the whole oyster developement. The m6A-RNA methylomes show the conservation of m6A consensus motif RRACH, a m6A peak around the stop codon and highlight specific m6A signatures according to RNA species between mRNAs, lncRNAs and transposable elements. The differentially methylated RNAs constitute developmental clusters that correspond to chronological steps of oyster development (cleavage, gastrulation, tissues differentiation and metamorphosis). They are marked with an overall drop of mRNA and lncRNA methylation at the morula stage followed by a global increase up to pre-metamorphosis. Messenger RNA m6A levels are correlated to transcript content and shifts in methyladenine profiles correspond to expression kinetics. The m6A of TE transcripts is also regulated and peaks during the gastrulation. Altogether our results indicate that m6A epitranscriptomes are an important regulator of oyster development. This first epitranscriptome profiling across the development of a lophotrochozoan model brings new insights into the epigenetic control of developmental processes and their evolution.
Project description:Deep sequencing of samples from different development stages, different adult organs and different stress treatments of Pacific oyster Crassostrea gigas Samples of 38 developmental stages from egg to juvenile were analyzed using single-end 49 bp RNA-seq. Two libraries mixed by RNAs from different developmental stages were analyzed using paired-end 90 bp RNA-seq. A total of 11 samples mainly from 8 organs (mantle, gill,adductor muscle, digestive gland, hemocyte, labial palp, female gonad and male gonad) were analyzed using paired-end 90 bp RNA-seq. At the same time, single-end 49 bp RNA-seq was conducted on 61 samples collected from adult oysters subjected to 9 types of environmental stressors (exposure to air, salinity, temperature, and exposure to metals).
Project description:The Pacific oyster (Crassostrea gigas) is a kind of marine bivalve of great economic and ecological importance and is among the animals possessing the highest level of genome DNA variations. Despite large efforts made for the discovery of Pacific oyster SNPs in many research groups, challenge still remains as how to utilize SNPs in a high-throughput, transferable and economical manner. In the study, we constructed an oyster 190K SNP array with Affymetrix Axiom genotyping technology. A total of 190,420 SNPs were designed on the chip, which were selected from 54 M SNPs identified by re-sequencing of more than 400 Pacific oysters. Genotyping results from 96 wild oysters indicated that 133,984 (70.4%) SNPs were polymorphic and successfully converted on the chip. Carrying 133K polymorphic SNPs, the oyster 190K SNP array is the first high density SNP chip with the largest throughput currently in mollusc and is commercially available to the worldwide research community.