Abstract

Shellfish, such as the eastern oysters (Crassostrea virginica) are not only valued as seafood but also for the ecosystem services they provide, including improving water quality and reducing eutrophication. Excess N causes eutrophication, harmful algal blooms, fish kills and overall decline of estuarine ecosystems resulting in economic losses. Oyster reefs sequester N and enhance denitrification processes, however, information on the N cycling oyster microbiome is scarce with most studies focusing on random grab samples or on pathogens, such as Vibrio spp. Further, triploid oysters are often used for aquaculture, as they grow faster than diploids, but there is little information on potential microbiome differences with ploidy. To address these knowledge gaps, diploid and triploid farmed oysters were collected at monthly intervals over one year and analyzed using a coupled approach encompassing shotgun metagenomics and quantitative microbial elemental cycling (QMEC) qPCR assays. Overall, the genus Psychrobacter dominated the core microbiome across all samples, regardless of season or ploidy, followed by Synechococcus, Pseudomonas, Pseudoalteromonas and Clostridium. Psychrobacter abundances increased significantly in the colder months; the same trend was also observed in the alpha and beta diversity. However, warmer months had increased bacterial diversity relative to colder months. Gene functional profiles were similar among seasons and ploidy, with respiration and metabolism of carbohydrates, RNA, and proteins as dominant functions. There were strong positive correlations between abundance of the "core" microbiome taxa and gene functions associated with central metabolism, DNA and carbohydrate metabolism, strongly suggesting the functional role of Psychrobacter in the microbiome. Metagenome assembly was performed to characterize dominant species, followed by phylogenetic analysis of select MAGs (metagenome-assembled genomes), further supporting the presence of multiple Psychrobacter spp. Sequence-based identification of denitrification genes in the Pyschrobacter MAGs indicated the presence of norB, narH, narI, nirK, and norB. QMEC analysis indicated C and N cycling genes were most abundant, with no discernable patterns due to seasons or ploidy. Among N cycling genes, the nosZII clade was dominant, which is likely responsible for the eastern oysters potential for bioextraction and enhancing water quality via denitrification.