Marine invertebrate microbiomes play important roles in diverse host and ecological processes. However, a mechanistic understanding of host-microbe interactions is currently available for a small number of model organisms. Here, an integrated taxonomic and functional analysis of the microbiome of the eastern oyster, Crassostrea virginica, was performed using 16S rRNA gene-based amplicon profiling, shotgun metagenomics, and genome-scale metabolic reconstruction. Relatively high variability of the microbiome was observed across individual oysters and among different tissue types. Specifically, a significantly higher alpha diversity was observed in the inner shell than in the gut, gill, mantle, and pallial fluid samples, and a distinct microbiome composition was revealed in the gut compared to other tissues examined in this study. Targeted metagenomic sequencing of the gut microbiota led to further characterization of a dominant bacterial taxon, the class Mollicutes, which was captured by the reconstruction of a metagenome-assembled genome (MAG). Genome-scale metabolic reconstruction of the oyster Mollicutes MAG revealed a reduced set of metabolic functions and a high reliance on the uptake of host-derived nutrients. A chitin degradation and an arginine deiminase pathway were unique to the MAG compared to closely related genomes of Mollicutes isolates, indicating distinct mechanisms of carbon and energy acquisition by the oyster-associated Mollicutes. A systematic reanalysis of public eastern oyster-derived microbiome data revealed a high prevalence of the Mollicutes among adult oyster guts and a significantly lower relative abundance of the Mollicutes in oyster larvae and adult oyster biodeposits. IMPORTANCE Despite their biological and ecological significance, a mechanistic characterization of microbiome function is frequently missing from many nonmodel marine invertebrates. As an initial step toward filling this gap for the eastern oyster, Crassostrea virginica, this study provides an integrated taxonomic and functional analysis of the oyster microbiome using samples from a coastal salt pond in August 2017. The study identified high variability of the microbiome across tissue types and among individual oysters, with some dominant taxa showing higher relative abundance in specific tissues. A high prevalence of Mollicutes in the adult oyster gut was revealed by comparative analysis of the gut, biodeposit, and larva microbiomes. Phylogenomic analysis and metabolic reconstruction suggested the oyster-associated Mollicutes is closely related but functionally distinct from Mollicutes isolated from other marine invertebrates. To the best of our knowledge, this study represents the first metagenomics-derived functional inference of Mollicutes in the eastern oyster microbiome.

Marine invertebrate microbiomes play important roles in various host and ecological processes. However, a mechanistic understanding of host-microbe interactions is so far only available for a handful of model organisms. Here, an integrated taxonomic and functional analysis of the microbiome of the eastern oyster, Crassostrea virginica, was performed using 16S rRNA gene amplicon profiling, shotgun metagenomics, and genome-scale metabolic reconstruction. A relatively low number of amplicon sequence variants (ASVs) were observed in oyster tissues compared to water samples, while high variability was observed across individual oysters and among different tissue types. Targeted metagenomic sequencing of the gut microbiota led to further characterization of a dominant bacterial taxon, the class Mollicutes, which was captured by the reconstruction of a metagenome-assembled genome (MAG). Genome-scale metabolic reconstruction of the oyster Mollicutes MAG revealed a reduced set of metabolic functions and a high reliance on the uptake of host-derived nutrients. A chitin degradation and an arginine deiminase pathway were unique to the MAG as compared to other closely related Mycoplasma genomes, indicating a distinct mechanism of carbon and energy acquisition by the oyster- associated Mollicutes. A systematic reanalysis of public eastern oyster-derived microbiome data revealed the Mollicutes as a ubiquitous taxon among adult oysters despite their general absence in larvae and biodeposit samples, suggesting potential horizontal transmission via an unknown mechanism.IMPORTANCEDespite well-documented biological significance of invertebrate microbiomes, a detailed taxonomic and functional characterization is frequently missing from many non-model marine invertebrates. By using 16S rRNA gene-based community profiling, shotgun metagenomics, and genome-scale metabolic reconstruction, this study provides an integrated taxonomic and functional analysis of the microbiome of the eastern oyster, Crassostrea virginica. Community profiling revealed a surprisingly low richness, as compared to surrounding seawater, and high variability among different tissue types and individuals. Reconstruction of a Mollicutes MAG enabled the phylogenomic positioning and functional characterization of the oyster-associated Mollicutes. Comparative analysis of the adult oyster gut, biodeposits, and oyster larvae samples indicated the potentially ubiquitous associations of the Mollicutes taxon with adult oysters. To the best of our knowledge, this study represented the first metagenomics derived functional inference of the eastern oyster microbiome. An integrated analytical procedure was developed for the functional characterization of microbiomes in other non-model host species.