Symbiotic survey of the bay scallop (Argopecten irradians) from the Gulf coast of Florida, USA.

The bay scallop Argopecten irradians supported a commercial fishery in Florida but their population declined and the fishery closed in 1994. A recreational fishery remains open along the west coast of Florida despite continued threats from overfishing and a changing environment. Disease is among those threats, as it is for bivalve fisheries globally. We examined the relationship between bay scallop population density, its symbiotic microbiome, and geographic location. We focused on three sites within the range of Florida's recreational scallop fishery: St. Joseph Bay (northern extent), offshore of the Steinhatchee River (central), and offshore of Hernando County (southern extent). The study was conducted prior to the seasonal opening of the fishery to minimize the impact of fishing on our results. We also sampled caged scallops that are used for restocking in St. Joseph Bay to assess the effect of artificially high density and confinement on the scallop pathobiome. Using a combination of traditional histological methods, molecular diagnostics, and metagenomics, a suite of 15 symbionts were identified. Among them, RNA-seq data revealed four novel + ssRNA viral genomes: three picorna-like viruses and one hepe-like virus. The DNA-seq library revealed a novel Mycoplasma species. Histological evaluation revealed that protozoan, helminth and crustacean infections were common in A. irradians. These potential pathogens add to those already known for A. irradians and underscores the risk they pose to the fishery.

Development of a multiplex qPCR for the quantification of three protozoan parasites of the eastern oyster Crassostrea virginica.

A multiplex quantitative PCR (qPCR) assay for the simultaneous detection of 3 eastern oyster Crassostrea virginica parasites, Perkinsus marinus, Haplosporidium nelsoni, and H. costale, was developed using 3 different fluorescently labeled hydrolysis probes. The primers and probe from a previously validated singleplex qPCR for P. marinus detection were combined with newly designed primers and probes specific for H. nelsoni and H. costale. The functionality of the multiplex assay was demonstrated on 2 different platforms by the linear relationship of the standard curves and similar cycle threshold (CT) values between parasites. Efficiency of the multiplex qPCR assay on the Roche and BioRad platforms ranged between 93 and 101%. The sensitivity of detection ranged between 10 and 100 copies of plasmid DNA for P. marinus and Haplosporidium spp., respectively. The concordance between the Roche and BioRad platforms in the identification of the parasites P. marinus, H. nelsoni, and H. costale was 91, 97, and 97%, respectively, with a 10-fold increase in the sensitivity of detection of Haplosporidium spp. on the BioRad thermocycler. The concordance between multiplex qPCR and histology for P. marinus, H. nelsoni, and H. costale was 54, 57, and 87%, respectively. Discordances between detection methods were largely related to localized or low levels of infections in oyster tissues, and qPCR was the more sensitive diagnostic. The multiplex qPCR developed here is a sensitive diagnostic tool for the quantification and surveillance of single and mixed infections in the eastern oyster.

The effect of off-bottom versus on-bottom oyster culture on total and pathogenic Vibrio spp. abundances in oyster tissue, water and sediment samples.

Varying culture methods are commonly used for eastern oyster, Crassostrea virginica, aquaculture in the Northeast United States. Vibrio vulnificus and V. parahaemolyticus, two human pathogenic bacteria species, accumulate in this edible, filter feeding shellfish. This study examined the use of two methods in an intertidal area (oysters cultured in trays and in bags on sediment) and two methods in a subtidal area (oysters cultured in trays and loose on the sediment) in Massachusetts over the growing season in 2015. Abundance of total V. vulnificus along with total and pathogenic (tdh+/trh+) V. parahaemolyticus were determined in oysters, sediment and water using real-time PCR. Temperature, salinity, turbidity and chlorophyll were continually measured every 15 min at each location. There were significantly higher abundances of total and pathogenic V. parahaemolyticus in on-bottom cultured oysters, while significantly higher abundances of V. vulnificus were identified in oysters from off-bottom culture in a subtidal location in Duxbury Bay, MA. In an intertidal location, Wellfleet Bay, MA, significantly higher abundances of total and tdh+V. parahaemolyticus were found in off-bottom oysters, but significantly higher abundances of V. vulnificus and trh+V. parahaemolyticus were found in on-bottom oysters. Spearman's correlation indicated that temperature is positively associated with concentrations of Vibrio spp. in oysters, water and sediment, but positive correlations between salinity and Vibrio spp. was also observed. Conversely, turbidity had a negative effect on Vibrio spp. concentrations in all sample types. There was no observed relationship inferred between chlorophyll and Vibrio spp. abundances in oysters, water or sediment.

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

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.