Gut Microbiomes of the Eastern Oyster (Crassostrea virginica) and the Blue Mussel (Mytilus edulis): Temporal Variation and the Influence of Marine Aggregate-Associated Microbial Communities

Abstract

Gut microbial community structure was evaluated for two species of bivalve molluscs, the eastern oyster (Crassostrea virginica) and the blue mussel (Mytilus edulis) collected from Long Island Sound, Connecticut, over the course of a year. These bivalves utilize a shared feeding mechanism, which may result in similar gut microbial communities. Their particle diet, marine aggregates, and surrounding environment, aggregate-free seawater (AFSW), were also collected for comparison. Due to the suspension-feeding activities of bivalves, the potential for aggregate- and AFSW-associated microbiota to influence their microbial communities may be significant. Both taxonomic and functional diversity of the samples were assessed. 16S rRNA gene amplicon sequencing indicated that oysters and mussels maintained similar, but not identical, gut microbiomes, with some temporal variation. Throughout the year, bivalve species had gut microbial community compositions that were more similar to one another than to aggregates. Within a month, bivalves shared on average a quarter of their total operational taxonomic units (OTUs) with each other and a 10th of their total OTUs with aggregates. During months with warm water temperatures, individuals within each of the four sample types had similar alpha diversity, but again, temporal variation was observed. On a functional level, bivalve gut microbial communities exhibited variation attributed to host species and season. Unlike oysters, mussel gut bacterial communities maintained high richness and evenness values throughout the year, even when values for the particle diet and AFSW were reduced. Overall, a core gut bivalve microbiome was present, and it was partially influenced by the marine aggregate microbial community.IMPORTANCE This work investigates the influence that extrinsic factors, diet, and the environment can have on the microbiomes of shellfish. Over the course of a year, the gut microbial communities of two species of bivalves, oysters and mussels, held under identical conditions in coastal marine waters were compared. While the mussels and oysters harbored gut microbial communities with similar composition, on a functional level, they exhibited species and temporal variation. These results indicate that intrinsic factors influence the bivalve microbiome, resulting in species variability, even when environmental conditions, feeding mechanism, and particle diet are constant. Seasonal and multispecies comparisons for bivalve-associated microbial communities are rare, and we believe this research represents an important contribution. The results presented here advance our understanding of the symbiotic interactions between marine invertebrates, the microbial communities they harbor, and the environment.

Keywords: 16S rRNA gene; aggregate; bacteria; bivalve; gut; microbiome; mussel; oyster.

FIG 1

Dissimilarity between samples was measured using the Bray-Curtis distance metric. Each sample type is represented by a unique symbol, and each month (September, November, March, and July) is represented by a unique color. The percent variation explained by each axis is shown in parentheses. Samples cluster according to sample type and month, with some overlap between the two bivalve species. A reduced number of replicates for some samples, in some months, resulted from a failure of samples to amplify.

FIG 2

Community composition of each sample type (mussel gut, oyster gut, aggregate, aggregate-free seawater [AFSW]) by phylum (a) and order (b). The relative abundance of each taxonomic group >1% of the total is listed.

FIG 3

Heat map showing the abundances of different taxa, using the lowest taxonomic level available. Lowercase letters designate taxonomic level (o for order, f for family, g for genus, k for kingdom, etc.). Significant differences in abundance between sample types are indicated by an asterisk next to that taxonomic group (one-way ANOVA, P < 0.05). The number of samples follow: mussels (n = 13), oysters (n = 9), and aggregates (n = 11).

FIG 4

(a and b) Richness of carbon sources utilized by heterotrophic microbial communities (optical density of >0.25 at 168-h reading) (a) and Shannon diversity index (b) by sample type (mussel gut, oyster gut, aggregate, aggregate-free seawater [AFSW]) and month (EcoPlates). Effects of month and sample type were analyzed by means of two-way ANOVA. (c and d) Richness (number of OTUs observed) (c) and Shannon diversity index (d) by sample type and month (16S rDNA). Individual one-way ANOVAs were run due to interaction effects. 16S rDNA data from AFSW samples were not used in the analyses because only July samples amplified. In all panels, different capital letters represent significant differences between months, whereas significant differences between sample types within a month are indicated by lowercase letters (Tukey’s HSD, P < 0.05). Where there are no letters, no significant effects were observed. Data are presented as means plus standard deviations (SD) (error bars) with n = 4 to 6 (EcoPlates) and n = 1 to 6 (16S rDNA) per sample type per month.

FIG 5

(a) Venn diagram of core OTUs for three of the sample types (mussel gut, oyster gut, aggregate) from all months. Core OTUs are defined here as those shared by 95% of samples. AFSW samples were not used in the analyses because only July samples amplified. Sample sizes used are as follows: mussels (n = 13), oysters (n = 9), and aggregates (n = 10). (b) Abundance of the top 10 core OTUs shared by mussels and oysters from all months.

FIG 6

Carbon source utilization ratios of microbial communities, organized by guild groupings, for each of the sampled months. The carbon source guild groups were carbohydrates, polymers, carboxylic and acetic acids, amino acids, and amines/amides. Ratios were calculated by dividing utilization of each guild by total number of wells utilized (richness [S]). Sample types are mussel gut (a), oyster gut (b), aggregate (c), and aggregate-free seawater (d). Different letters denote significant differences in utilization ratios between months (two-way ANOVA, Tukey’s HSD, P < 0.05). Data are presented as means (plus SD) (n = 4 to 6 per carbon source per month).