Dynamics of the Pacific oyster pathobiota during mortality episodes in Europe assessed by 16S rRNA gene profiling and a new target enrichment next‐generation sequencing strategy

Abstract

Summary Infectious agents such as the bacteria Vibrio aestuarianus or Ostreid herpesvirus 1 have been repeatedly associated with dramatic disease outbreaks of Crassostrea gigas beds in Europe. Beside roles played by these pathogens, microbial infections in C. gigas may derive from the contribution of a larger number of microorganisms than previously thought, according to an emerging view supporting the polymicrobial nature of bivalve diseases. In this study, the microbial communities associated with a large number of C. gigas samples collected during recurrent mortality episodes at different European sites were investigated by real‐time PCR and 16SrRNA gene‐based microbial profiling. A new target enrichment next‐generation sequencing protocol for selective capturing of 884 phylogenetic and virulence markers of the potential microbial pathogenic community in oyster tissue was developed allowing high taxonomic resolution analysis of the bivalve pathobiota. Comparative analysis of contrasting C. gigas samples conducted using these methods revealed that oyster experiencing mortality outbreaks displayed signs of microbiota disruption associated with the presence of previously undetected potential pathogenic microbial species mostly belonging to genus Vibrio and Arcobacter. The role of these species and their consortia should be targeted by future studies aiming to shed light on mechanisms underlying polymicrobial infections in C. gigas.

Improved microbial community characterization of 16S rRNA via metagenome hybridization capture enrichment

“…Also, metagenomic libraries are more expensive, take longer to prepare, and are much more complex than amplicon libraries, requiring more computational effort (Sekse et al, 2017). In particular, it is difficult to identify low abundance genetic traits and rare taxa using metagenomic libraries, and extensive deep sequencing is often required to do so (Lasa et al, 2019). In summary, shotgun sequencing is less biased and yields data on many genomic regions, but the main tradeoffs are high costs of library preparation, sequencing, and analysis.…”

“…is an enrichment technique that uses a set of biotinylated DNA or RNA baits that are complementary to DNA sequences of interest to increase the proportion of DNA fragments of interest within DNA libraries, subsequently characterizing the DNA by massively parallel sequencing (Lasa et al, 2019).…”

“…Additional hybridization capture bait sets have been designed for a variety of microbial projects, such as sets of defined pathogens or particular genes, including virulence genes for Vibrio spp. that infect oysters (Lasa et al, 2019), bifidobacterial in the gut of mammals (Lugli et al, 2019), and antibiotic resistance genes (Guitor et al, 2019). Importantly, unlike other culture independent techniques, hybridization capture provides greater phylogenetic resolution and increased sensitivity, while requiring fewer sequencing reads (Lasa et al, 2019;Barrett et al, 2020).…”

Vibrio–bivalve interactions in health and disease

“…V. splendidus, V. tasmaniensis, V. cyclitrophicus and V. crassostreae) have been repeatedly isolated from C. gigas juveniles (<12 months old) affected by the Pacific Oyster Mortality Syndrome (POMS) triggered by the Ostreid herpesvirus OsHV-1 μVar (Lemire et al, 2015;Bruto et al, 2017;de Lorgeril et al, 2018). Vibrio aestuarianus (Anguillarum clade) was found to cause mass mortality of adult C. gigas in France since 2001 and since 2011 in Ireland, Scotland and Spain (Garnier et al, 2008;Garcia et al, 2014;Travers et al, 2017) High taxonomic resolution analysis of the bivalve pathobiota applied on healthy and diseased C. gigas samples using a new target enrichment next-generation sequencing approach revealed the dominance of primary pathogens such as V. aestuarianus or OsHV-1 in tissues of diseased C. gigas collected during mass mortality events affecting juveniles and/or adults at different European farming sites (Lasa et al, 2019). Albeit at lower relative abundance, other bacterial species were exclusively identified in infected oysters, particularly Vibrio sp.…”

Spatial epidemiological modelling of infection by Vibrio aestuarianus shows that connectivity and temperature control oyster mortality

“…The Pacific oyster Crassostrea gigas is of primary importance for shellfish farming as it accounts for ap proximately three-quarters of all production in France (~486 million Euros) (FranceAgriMer 2019). Since 2008, the oyster industry has been seriously challenged by mass mortality associated with different pathogens, such as the ostreid herpesvirus OsHV-1 in spat (Segarra et al 2010) and the bac-terium Vibrio aestuarianus in adults in France (Garcia et al 2014, Azéma et al 2017, Ireland, Scotland, and Spain (EFSA Panel on Animal Health and Welfare 2015, Lasa et al 2019). These mortality events have severe economic impacts for the oyster industry.…”