Microbiome Analysis Reveals Diversity and Function of Mollicutes Associated with the Eastern Oyster, Crassostrea virginica

Abstract

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.

Keywords: Chlamydiae; Mollicutes; Spirochaetia; eastern oyster; metagenomics; microbiome.

FIG 1

Diversity of eastern oyster microbiome samples as measured by unique ASV count (A), Shannon index (B), and Simpson index (C). Samples were grouped by tissue type, with abbreviations as follows: Gil (gill), Gut (gut), Hmp (hemolymph), Mtl (mantle), Pfd (pallial fluid), and Ins (inner shell). Pairwise statistical significance was assessed using a pairwise Wilcoxon test with Holm P value adjustment for multiple comparisons: *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001.

FIG 2

Heatmap showing the log fold change (computed with ANCOM-BC) of major oyster microbiome taxa across different tissue samples: Mollicutes (A), Chlamydiae (B), Spirochaetia (C), Fusobacteriia (D), and Gammaproteobacteria (E). Each tissue was assigned an abbreviation as follows: Gil (gill), Gut (gut), Hmp (hemolymph), Mtl (mantle), Pfd (pallial fluid), and Ins (inner shell). Tissue labels representing rows (on the left-hand side of each heatmap) are the target tissue while the labels representing columns (on the bottom of each heatmap) are the reference tissue. For example, a significantly lower relative abundance of the Mollicutes was observed in the hemolymph compared to the gut samples. Pairwise statistical significance was assessed with ANCOM-BC: *, P value < 0.05; **, P value < 0.01; ***, P value < 0.001.

FIG 3

Characterization of a metagenome-assembled genome (MAG) of the Mollicutes from the oyster microbiome. (A) Maximum likelihood phylogenomic reconstruction based on conserved single-copy genes. Members of the Firmicutes were selected as outgroups in the phylogeny (Data Set S2B). Support values were based on 100 iterations of bootstrapping. (B) Visualization of the central metabolic pathways reconstructed from the oyster Mollicutes MAG (created with BioRender). Metabolites are connected with directed edges indicating biochemical conversions or transport and diffusion processes. The edges were coded by circled numbers, with further details represented in Data Set S2D. Additional coding was provided by the edge colors to indicate conservations between the oyster Mollicutes MAG and reference genomes, including the marine host-associated Mycoplasma marinum and Mycoplasma todarodis and the freshwater host-associated Mycoplasma mobile. Green, conserved in all four genomes; blue, conserved between the Mollicutes MAG and the marine Mycoplasma (M. marinum and M. todarodis) but absent in M. mobile; purple, conserved between the Mollicutes MAG and the freshwater M. mobile but absent from the marine Mycoplasma; magenta, unique functions in the Mollicutes MAG. The conversion from pyruvate to acetyl-CoA was marked as black because the function of a pyruvate dehydrogenase was identified outside the MAG from unbinned contigs that had top BLAST hits to members of the Mycoplasma.

FIG 4

Phylogenetic positioning of oyster- and water-associated Mollicutes ASVs. The branches were collapsed to summarize the environments represented by reference 16S rRNA genes in the SILVA database. The four major clades of oyster-associated Mollicutes were labeled with Roman numerals I to IV. The number of SILVA reference sequences (n_REF) and unique ASV sequences (n_ASV) were marked in each collapsed clade. Corresponding counts of unique ASVs in the different sample types were shown as a bubble plot for each clade on the right of the phylogeny, with filled circles sized according to the ASV counts and colored based on sample types. Data from this study and a prior study (39) were included in the analysis. Sample types included water and the following oyster tissues: Gil (gill), Gut (gut), Hmp (hemolymph), Mtl (mantle), Pfd (pallial fluid), and Ins (inner shell). A given ASV may appear in multiple sample types, and thus, the sum of values in each row of the bubble plot is not necessarily the number of ASVs (n_ASV) in the collapsed branch. The orange star indicates the clade where the metagenome-assembled full-length 16S rRNA genes, Mollicutes-1 and Mollicutes-2, and their corresponding ASVs are located (Data Set S2E).