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Synopsis Eastern oysters (Crassostrea virginica) have long been recognized as model organisms of extreme environmental tolerance, showing resilience to variation in temperature, salinity, hypoxia, and microbial pathogens. These phenotypic responses, however, show variability between geographic locations or habitats (e.g., tidal). Physiological, morphological, and genetic differences occur in populations throughout a species’ geographical range, which may have been shaped by regional abiotic and biotic variations. Few studies of C. virginica have explored the combined factors of physiological mechanisms of divergent phenotypes between locations and the genetic relationships of individuals between these locations. To characterize genetic relationships of four locations with aquacultured oysters along the North Carolina and Virginia coast, we sequenced a portion of cytochrome oxidase subunit I (COI) that revealed significant variation in haplotype distribution between locations. We then measured mitochondrial physiology and expression of the innate immunity response of hemocytes to lab acclimation and combined stress conditions to compare basal expression and stress response in oysters between these locations. For stress sensing genes, toll-like receptors had the strongest location-specific response to hypoxia and Vibrio, whereas mannose receptor and a stress-receptor were specific to hypoxia and bacteria, respectively. The expression of stress response genes also showed location-specific and stressor-specific changes in expression, particularly for big defensin and the complement gene Cq3. Our results further suggested that genetic similarity of oysters from different locations was not clearly related to physiological and molecular responses. These results are informative for understanding the range of physiological plasticity for stress responses in this commercially important oyster species. They also have implications in the oyster farming industry as well as conservation efforts to restore endangered native oyster beds.

Estuarine environments are continuously being shaped by both natural and anthropogenic sources which directly/indirectly influence the organisms that inhabit these important niches on both individual and community levels. Human infections caused by pathogenic Vibrio species are continuing to rise, and factors associated with global climate change have been suggested to be impacting their abundance and geographical range. Along with temperature, hypoxia has also increased dramatically in the last 40 years, which has led to persistent dead zones worldwide in areas where these infections are increasing. Thus, utilizing membrane diffusion chambers, we investigated the impact of in situ hypoxia on the gene expression of one such bacterium, Vibrio vulnificus, which is an inhabitant of these vulnerable areas worldwide. By coupling these data with multiple abiotic factors, we were able to demonstrate that genes involved in numerous functions, including those involved in virulence, environmental persistence, and stressosome production, were negatively correlated with dissolved oxygen. Furthermore, comparing 16S ribosomal RNA, we found similar overall community compositions during both hypoxia and normoxia. However, unweighted beta diversity analyses revealed that although certain classes of bacteria dominate in both low‐ and high‐oxygen environments, there is the potential for quantitative shifts in lower abundant species, which may be important for effective risk assessment in areas that are becoming increasingly more hypoxic. This study emphasizes the importance of investigating hypoxia as a trigger for gene expression changes by marine Vibrio species and highlights the need for more in depth community analyses during estuarine hypoxia.

Monitoring of Vibrio vulnificus and V. parahaemolyticus abundance is pertinent due to the ability of these species to cause disease in humans through aquatic vectors. Previously, we performed a multiyear investigation tracking Vibrio spp. levels in five sites along the southeastern North Carolina coast. From February 2013 to October 2015, total V. vulnificus and V. parahaemolyticus abundance was measured in water, oysters and clams. In the current study, pathogenic subpopulations were identified in these isolates using molecular markers, revealing that 5.3% of V. vulnificus isolates possessed the virulence-correlated gene (vcgC), and 1.9% of V. parahaemolyticus isolates harbored one or both of the virulence-associated hemolysin genes (tdh and trh). Total V. parahaemolyticus abundance was not sufficient to predict the abundance of pathogenic subpopulations. Specifically, pathogenic V. parahaemolyticus isolates were more often isolated in cooler waters and were sometimes isolated when no other V. parahaemolyticus strains were detectable. Vibrio vulnificus clinical (C-) genotypes correlated with total V. vulnificus; however, salinity, water depth and total suspended solids influenced C- and E-genotypes differently. Lastly, we documented individual oysters harboring significantly higher V. vulnificus levels for which there was no ecological explanation, a phenomenon that deserves closer attention due to the potentially elevated health hazard associated with these 'hot' shellfish.

Filter feeding shellfish can concentrate pathogenic bacteria, including Vibrio vulnificus and Vibrio parahaemolyticus , as much as 100-fold from the overlying water. These shellfish, especially clams and oysters, are often consumed raw, providing a route of entry for concentrated doses of pathogenic bacteria into the human body. The numbers of foodborne infections with these microbes are increasing, and a better understanding of the conditions that might trigger elevated concentrations of these bacteria in seafood is needed. In addition, if bacterial concentrations in water are correlated with those in shellfish, then sampling regimens could be simplified, as water samples can be more rapidly and easily obtained. After sampling of oysters and clams, either simultaneously or separately, for over 2 years, it was concluded that while Vibrio concentrations in oysters and water were related, this was not the case for levels in clams and water. When clams and oysters were collected simultaneously from the same site, the clams were found to have lower Vibrio levels than the oysters. Furthermore, the environmental parameters that were correlated with levels of Vibrio spp. in oysters and water were found to be quite different from those that were correlated with levels of Vibrio spp. in clams. IMPORTANCE This study shows that clams are a potential source of infection in North Carolina, especially for V. parahaemolyticus . These findings also highlight the need for clam-specific environmental research to develop accurate Vibrio abundance models and to broaden the ecological understanding of clam- Vibrio interactions. This is especially relevant as foodborne Vibrio infections from clams are being reported.

Estuarine environments are continuously being shaped by both natural and anthropogenic sources which directly/indirectly influence the organisms that inhabit these important niches on both individual and community levels. Human infections caused by pathogenic Vibrio species are continuing to rise, and factors associated with global climate change have been suggested to be impacting their abundance and geographical range. Along with temperature, hypoxia has also increased dramatically in the last 40 years, which has led to persistent dead zones worldwide in areas where these infections are increasing. Thus, utilizing membrane diffusion chambers, we investigated the impact of in situ hypoxia on the gene expression of one such bacterium, Vibrio vulnificus, which is an inhabitant of these vulnerable areas worldwide. By coupling these data with multiple abiotic factors, we were able to demonstrate that genes involved in numerous functions, including those involved in virulence, environmental persistence, and stressosome production, were negatively correlated with dissolved oxygen. Furthermore, comparing 16S ribosomal RNA, we found similar overall community compositions during both hypoxia and normoxia. However, unweighted beta diversity analyses revealed that although certain classes of bacteria dominate in both low‐ and high‐oxygen environments, there is the potential for quantitative shifts in lower abundant species, which may be important for effective risk assessment in areas that are becoming increasingly more hypoxic. This study emphasizes the importance of investigating hypoxia as a trigger for gene expression changes by marine Vibrio species and highlights the need for more in depth community analyses during estuarine hypoxia., Key Points Dissolved oxygen was the most variable parameter in a eutrophic North Carolina estuary and occurred cyclicallyNegative correlations were found with respect to dissolved oxygen and in situ gene expression of many gene categories in Vibrio vulnificus Beta analysis of 16S ribosomal RNA sequences suggested increased bacterial diversity during normoxia stressing need for deeper sequencing

Vibrio vulnificus , a pervasive human pathogen, can cause potentially fatal septicemia after consumption of undercooked seafood. Biotype 1 strains of V. vulnificus are most commonly associated with human infection and are separated into two genotypes, clinical (C) and environmental (E), based on the virulence-correlated gene. For ingestion-based vibriosis to occur, this bacterium must be able to withstand multiple conditions as it traverses the gastrointestinal tract and ultimately gains entry into the bloodstream. One such condition, anoxia, has yet to be extensively researched in V. vulnificus . We investigated the effect of oxygen availability on capsular polysaccharide (CPS) production and biofilm formation in this bacterium, both of which are thought to be important for disease progression. We found that lack of oxygen elicits a reduction in both CPS and biofilm formation in both genotypes. This is further supported by the finding that pilA , pilD , and mshA genes, all of which encode type IV pilin proteins that aid in attachment to surfaces, were downregulated during anaerobiosis. Surprisingly, E-genotypes exhibited distinct differences in gene expression levels of capsule and attachment genes compared to C-genotypes, both aerobically and anaerobically. The importance of understanding these disparities may give insight into the observed differences in environmental occurrence and virulence potential between these two genotypes of V. vulnificus .

The ability for bacteria to attach to and detach from various substrata is important for colonization, survival and transitioning to new environments. An opportunistic human pathogen, Vibrio vulnificus, can cause potentially fatal septicemia after ingestion of undercooked seafood. Based on genetic polymorphisms, strains of this species are subtyped into clinical (C) and environmental (E) genotypes. Vibrio vulnificus readily associates with chitin, thus we investigated chitin detachment dynamics in these disparate genotypes. We found that C-genotypes detach significantly more than E-genotypes after 24 hours in aerobic as well as anaerobic conditions. Furthermore, expression of genes involved in type IV pilin production was significantly downregulated in C-genotypes compared to E-genotypes, suggesting an importance in detachment. Interestingly, gbpA, a gene that has been shown to be important in host colonization in V. cholerae, was upregulated in the C-genotypes during detachment. Additionally, we found that C-genotypes detached to a greater extent, and produced more quorum-sensing (QS) autoinducer-2 molecules relative to E-genotypes, which suggests a role for QS in detachment. These findings suggest that for V. vulnificus, QS-mediated detachment may be a potential mechanism for transitioning into a human host for C-genotypes, while facilitating E-genotype maintenance in the estuarine environment.