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Additional file 6. Oyster sampling data.

Infection with ostreid herpes viruses (OsHv-1) have been causing mass mortalities and reeking havoc on production areas world wide. The resulting production losses have sparked several selective breeding programmes trying to improve resistance to infection. Recent studies showed however that there is genetic variation in OsHv-1 strains between different outbreak areas. Furthermore, it has been suggested that secondary infection by pathogenic bacteria contribute substantially to the observed mortalities. Together this means that the disease differs between regions suggesting that also the genetic determinants of resistance differ them. To test assess the role of environmental variation in disease we employ whole genome sequencing of pools of individuals (poolseq) pairing before and after mortality samples collected during mass mortalities across Europe (France, Germany, Ireland, Norway). We identified 67-109 outlier loci for all pairwise comparisons that showed either signatures of selective sweeps (fixation of major alleles) or allelic pull-ups (increase of minor alle frequency). While we could identify single nucleotide polymorphisms (SNPs) located in close genomic proximity for most pairwise comparisons, that also matched previous QTL-studies, the majority of outlier loci were unique to each location. This supports the polygenic architecture of OsHV-resistance, but also highlights the substantial selective differences between locations. Since the shared outlier loci probably only explained a relatively small variation in resistance, breeding programs should aim at location specific resistance rather than general resistance.

Additional file 3. Review history.

Figure S4. SNV distribution in the OsHV-1-PT genome. (PDF 252 kb)

Computational details of the C. gigas ADAR1 structural modeling. (PDF 457 kb)

Additional file 2. RT-qPCR analysis of selected miRNAs.

Additional file 2. RT-qPCR analysis of selected miRNAs.

Additional file 3. Review history.

Additional file 1. Manuscript data notes. This file includes additional data notes 1–24, along with all the supplementary figures and most supplementary tables referenced in the main text.

Additional file 7 Comparison of two mRNA enrichment methods for dual RNA-seq analysis of C. gigas infected with OsHV-1.

Computational details of the C. gigas ADAR1 structural modeling. (PDF 457 kb)

Figure S4. SNV distribution in the OsHV-1-PT genome. (PDF 252 kb)

Additional file 7 Comparison of two mRNA enrichment methods for dual RNA-seq analysis of C. gigas infected with OsHV-1.

Additional file 1. Manuscript data notes. This file includes additional data notes 1–24, along with all the supplementary figures and most supplementary tables referenced in the main text.

Bivalves are a diverse mollusc group of economic and ecological importance. An evident resilience to pollution, parasites and extreme environments makes some bivalve species important models for studying adaptation and immunity. Despite substantial progress in sequencing projects of bivalves, information on non-coding genes and gene-regulatory aspects is still lacking. Here, we review the current repertoire of bivalve microRNAs (miRNAs), important regulators of gene expression in Metazoa. We exploited available sncRNA data for Pinctada martensii, Crassostrea gigas, Corbicula fluminea, Tegillarca granosa and Ruditapes philippinarum, and we produced new sncRNA data for two additional bivalves, the Mediterranean mussel Mytilus galloprovincialis and the blood clam Scapharca broughtonii. We found substantial heterogeneity and incorrect annotations of miRNAs; hence, we reannotated conserved miRNA families using recently established criteria for bona fide microRNA annotation. We found 106 miRNA families missing in the previously published bivalve datasets and 89 and 87 miRNA complements were identified in the two additional species. The overall results provide a homogeneous and evolutionary consistent picture of miRNAs in bivalves and enable future comparative studies. The identification of two bivalve-specific miRNA families sheds further light on the complexity of transcription and its regulation in bivalve molluscs.This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

Bivalves are a diverse mollusc group of economic and ecological importance. An evident resilience to pollution, parasites and extreme environments makes some bivalve species important models for studying adaptation and immunity. Despite substantial progress in sequencing projects of bivalves, information on non-coding genes and gene-regulatory aspects is still lacking. Here, we review the current repertoire of bivalve microRNAs (miRNAs), important regulators of gene expression in Metazoa. We exploited available sncRNA data for Pinctada martensii, Crassostrea gigas, Corbicula fluminea, Tegillarca granosa and Ruditapes philippinarum, and we produced new sncRNA data for two additional bivalves, the Mediterranean mussel Mytilus galloprovincialis and the blood clam Scapharca broughtonii. We found substantial heterogeneity and incorrect annotations of miRNAs; hence, we reannotated conserved miRNA families using recently established criteria for bona fide microRNA annotation. We found 106 miRNA families missing in the previously published bivalve datasets and 89 and 87 miRNA complements were identified in the two additional species. The overall results provide a homogeneous and evolutionary consistent picture of miRNAs in bivalves and enable future comparative studies. The identification of two bivalve-specific miRNA families sheds further light on the complexity of transcription and its regulation in bivalve molluscs.This article is part of the Theo Murphy meeting issue ‘Molluscan genomics: broad insights and future directions for a neglected phylum’.

Macrophage migration inhibitory factor (MIF) dynamically connects innate and adaptive immune systems in vertebrate animals, allowing highly orchestrated systemic responses to various insults. The occurrence of MIF-like genes in non-vertebrate organisms suggests its origin from an ancestral metazoan gene, whose function is still a matter of debate. In the present work, by analyzing available genomic and transcriptomic data from bivalve mollusks, we identified 137 MIF-like sequences, which were classified into three types, based on phylogeny and conservation of key residues: MIF, D-DT, and the lineage-specific type MDL. Comparative genomics revealed syntenic conservation of homologous genes at the family level, the loss of D-DT in the Ostreidae family as well as the expansion of MIF-like genes in the Mytilidae family, possibly underpinning the neofunctionalization of duplicated gene copies. In M. galloprovincialis, MIF and one D-DT were mostly expressed in haemocytes and mantle rim of untreated animals, while D-DT paralogs often showed very limited expression, suggesting an accessory role or their persistence as relict genes.

Bivalve molluscs comprise more than 9000 extant species. A number of them are traditionally farmed worldwide and are fundamental in the functioning of benthic ecosystems. The peculiarities of marine bivalves have inspired versatile biotechnological tools for coastal pollution monitoring and several new biomimetic materials. Moreover, large amounts of sequence data available for some farmed bivalve species can be used to unveil the organism’s responses to environmental factors (e.g. global climate change, emergence of new infectious agents and other production problems). In bivalves, data from genomics and transcriptomics increases more quickly than data from other omics, and permit new bioinformatics inferences, real comparative genomics and the study of molecules suitable for biotechnological innovations. Bivalves (and their microorganism communities) produce a variety of bioactive peptides, proteins and metabolites. Among them, the numerous families of antimicrobial peptides identified in the Mediterranean mussel likely contribute to its vigour and could assist with the identification of molecular scaffolds for innovative pharmaceuticals, nutraceuticals and constructs suitable for other applications.

Cathelicidins are an important family of antimicrobial peptide effectors of innate immunity in vertebrates. Two members of this group, CATH‐1 and CATH‐2, have been identified and characterized in teleosts (ray‐finned fish). In this study, we investigated the expression of these genes in different tissues of rainbow trout challenged with 4 different inactivated pathogens. By using qPCR, we detected a strong induction of both cath‐1 and cath‐2 genes within 24 hours after intraperitoneal inoculation with Lactococcus garvieae, Yersinia ruckeri, Aeromonas salmonicida, or Flavobacterium psychrophilum cells. Up to 700‐fold induction of cath‐2 was observed in the spleen of animals challenged with Y. ruckeri. Moreover, we found differences in the intensity and timing of gene up‐regulation in the analyzed tissues. The overall results highlight the importance of cathelicidins in the immune response mechanisms of salmonids.

Small non-coding RNAs include powerful regulators of gene expression, transposon mobility and virus activity. Among the various categories, mature microRNAs (miRNAs) guide the translational repression and decay of several targeted mRNAs. The biogenesis of miRNAs depends on few gene products, essentially conserved from basal to higher metazoans, whose protein domains allow specific interactions with dsRNA. Here, we report the identification of key genes responsible of the miRNA biogenesis in 32 bivalves, with particular attention to the aquaculture speciesMytilus galloprovincialisandCrassostrea gigas. In detail, we have identified and phylogenetically compared eight evolutionary conserved proteins: DROSHA, DGCR8, EXP5, RAN, DICER TARBP2, AGO and PIWI. In mussels, we recognized several other proteins participating in the miRNA biogenesis or in the subsequent RNA silencing. According to digital expression analysis, these genes display low and not inducible expression levels in adult mussels and oysters whereas they are considerably expressed during development. As miRNAs play an important role also in the antiviral responses, knowledge on their production and regulative effects can shed light on essential molecular processes and provide new hints for disease prevention in bivalves.