Elevated temperature and decreased salinity impacts on exogenous Vibrio parahaemolyticus infection of eastern oyster, Crassostrea virginica.

Anthropogenic carbon emissions have resulted in drastic oceanic changes, including increased acidity, increased temperature, and decreased salinity. Anthropogenic carbon emissions have resulted in drastic oceanic changes, including increased acidity, increased temperature, and decreased salinity. Few studies have directly assessed the compounded impact of alterations to oceanic conditions on oyster physiology and the relation to the presence of V. parahaemolyticus. This project investigated the relationship between projected climate scenarios and their influence on both eastern oyster, Crassostrea virginica, and the aquatic bacteria, Vibrio parahaemolyticus. Specifically, we examined whether an increase in water temperature and/or decrease in salinity would impair oyster resistance to V. parahaemolyticus, a human food and waterborne pathogen. Using a culture-dependent approach, our data revealed that the alterations in environmental conditions did not significantly impact the numbers of V. parahaemolyticus numbers within oyster hemolymph or tissues. However, we did observe a dramatic increase in the total amount of bacteria and pathogenic native Vibrio species, Vibrio aestuarianus and Vibrio harveyi. Despite detecting V. parahaemolyticus in most tissues at 7 days post-challenge, oysters were able to reduce bacterial levels below our limit of detection by 28 days of exposure. Furthermore, in our second experimental trial exploring single vs. multiple inoculation of bacteria, we observed that oysters were either able to reduce total bacterial levels to pre-treatment burdens (i.e., below our limit of detection) or die. This study demonstrates that the synergistic effects of elevated temperature and decreased salinity do not inhibit oysters from preventing the long-term colonization of exogenous V. parahaemolyticus. However, our data do show these environmental stressors impact oyster physiology and the native microbiota. This can lead to the proliferation of opportunistic pathogens, which could have impacts on oyster population numbers and ecosystem and human health.

Impacts of elevated temperature, decreased salinity and microfibers on the bioenergetics and oxidative stress in eastern oyster, Crassostrea virginica.

Projected increases in temperature and decreases in salinity associated with global climate change will likely have detrimental impacts on eastern oyster, Crassostrea virginica, as these variables can influence physiological processes in these keystone species. We set out to determine how the interactive effects of temperature (20 °C or 27 °C) and/or salinity (27‰ or 17‰) impacted the energetic reserves, aerobic and anaerobic metabolism, and changes to oxidative stress or total antioxidant potential as a consequence of an altered environment over a 21-day exposure. Gill and adductor muscle were used to quantify changes in total glycogen and lipid content, Electron Transport System and Citrate Synthase activities, Malate Dehydrogenase activity, Protein Carbonyl formation, lipid peroxidation, and total antioxidant potential. A second exposure was performed to determine if these environmental factors influenced the ingestion of microfibers, which are now one of the leading forms of marine debris. Elevated temperature and the combination of elevated temperature and decreased salinity led to an overall decline in oyster mass, which was exacerbated by the presence of microfibers. Changes in metabolism and oxidative stress were largely influenced by time, but exposure to elevated temperature, decreased salinity, the combination of these stressors or exposure to microfibers had small impacts on oyster physiology and survival. Overall these studies demonstrate that oyster are fairly resilient to changes in salinity in short-term exposures, and elevations in temperature or temperature combined with salinity result in changes to the oyster energetic response, which can be further impacted by the presence of microfibers.

Biomagnification of mercury in an estuarine food web.

Methylmercury is a toxin of local, regional, and global concern, with estuarine habitats possessing ecological characteristics that support conversion of inorganic mercury into this methylated form. We monitored Hg concentrations in species within the food web of the lower Cape Fear River (CFR) estuary in 2018-2020. Samples were analyzed for Hg concentrations and nitrogen isotopes (a measure of trophic level), and we found a positive relationship within this food web each year (p < 0.0001), indicating biomagnification is occurring. The highest Hg concentrations were among the upper trophic level species (Royal Terns, 4.300 ppm). While the Hg concentrations we documented are below assumed thresholds for toxic effects, we found spikes in Hg concentrations after Hurricane Florence in 2018 and with other disturbances to the CFR that resuspended bottom sediments. Continued monitoring is needed to understand the cause of annual variations, health implications, and conservation needs.

Haplosporidium nelsoni and Perkinsus marinus occurrence in waters of Great Bay Estuary, New Hampshire.

In Great Bay Estuary, New Hampshire, USA, Haplosporidium nelsoni and Perkinsus marinus are 2 active pathogens of the eastern oyster Crassostrea virginica (Gmelin), that cause MSX (multinucleated sphere with unknown affinity 'X') and dermo mortalities, respectively. Whereas studies have quantified infection intensities in oyster populations and determined whether these parasites exist in certain planktonic organisms, no studies thus far have examined both infectious agents simultaneously in water associated with areas that do and do not have oyster populations. As in other estuaries, both organisms are present in estuarine waters throughout the Bay, especially during June through November, when oysters are most active. Waters associated with oyster habitats had higher, more variable DNA concentrations from these pathogenic organisms than waters at a non-oyster site. This finding allows for enhanced understanding of disease-causing organisms in New England estuaries, where oyster restoration is a priority.

Submarine groundwater discharge interacts with creek geomorphology to affect eastern oyster Crassostrea virginica growth rates in a coastal Georgia creek.

Eastern oysters, Crassostrea virginica, are commercially important coastal species that provide many ecosystem services for coastal communities. Unfortunately, 85% of oyster reefs have been lost globally, prompting investments in restoration efforts to rebuild populations. Managers often consider several well-studied environmental and water quality parameters when making restoration site decisions. However, recent research suggests that submarine groundwater discharge (SGD) may play a role in driving the distribution of oysters in some estuaries. Specifically, SGD may result in localized areas of low dissolved oxygen and low pH that could inhibit oyster recruitment and survival. However, SGD may interact with other potential oyster stressors, including creek geomorphology. On point bars, sediment accumulation could alter growth rates of oysters and physiology, and it is possible that the two factors, SGD and creek geomorphology, could interact to impact oyster growth. We conducted a field experiment to examine the effects of SGD and creek geomorphology on oyster growth rates in a marsh-lined tidal creek in Georgia, USA. High and low SGD sites were paired within point bars and cut banks. Oysters were deployed in cages for 72 days and growth rates were determined. We found a significant interaction between SGD and creek geomorphology on oyster growth rates. Oysters grew at significantly faster rates at locations on accretionary point bars regardless of SGD flux, whereas, on erosional cut banks, high SGD flux significantly reduced oyster growth rate relative to low SGD flux. It appears that SGD may negatively influence oyster growth at specific creek locations, likely due to the presence of other stressors. Therefore, it is important to consider potential interacting and confounding stressors when managing oyster populations. As SGD is still a relatively understudied potential stressor for oysters, it is critical to continue to examine how groundwater might influence oysters in other locations and in combination with other stressors. Regardless, this study provides further evidence that SGD should be considered in future management efforts.

Assessment of Infection Prevalence and Intensity of Disease-Causing Parasitic Protozoans Perkinsus marinus and Haplosporidium nelsoni in Georgia Oysters.

Eastern oysters, Crassostrea virginica, are ecologically and economically important coastal species which provide a commercially valuable food product while also improving water quality through filtration, protecting shorelines, and providing habitat. The protozoan parasites Perkinsus marinus and Haplosporidium nesloni commonly infect oysters along the United States Atlantic and Gulf coasts and have been linked to poor oyster health and mass mortality events. In this study, wild oysters were collected from multiple reefs within four tidal creeks along the coast of Georgia to investigate P. marinus and H. nelsoni prevalence and intensity, their potential impact on oyster health, and identify possible drivers of the parasites. A second study occurred on four sites on Sapelo Island, Georgia, with continuous water quality monitoring data to further elucidate potential drivers. Oyster density and condition index, a proxy for health, were measured, and parasites were quantified using a TaqMan probe based quantitative real-time PCR within gill tissue. Real-time PCR showed that 86% of oysters tested were infected by one or both parasites in the coast-wide survey, and 93% of oysters from Sapelo Island were also infected by one or both parasites. Prevalence and infection intensity for both P. marinus and H. nelsoni varied across sites. Overall impacts on oysters were complex-intensity was not linked to oyster metrics in the coastwide study, but oyster condition was negatively correlated with P. marinus prevalence in the Sapelo Island study. Several relationships between both parasites and water quality parameters were identified, providing valuable information about potential drivers that should be investigated further.

Meta-analysis reveals drivers of restoration success for oysters and reef community.

Restoration aims to reverse the global declines of foundation species, but it is unclear how project attributes, the physical setting, and antecedent conditions affect restoration success. In coastal seas worldwide, oyster reef restoration is increasing to counter historical habitat destruction and associated declines in fisheries production and biodiversity. Yet, restoration outcomes are highly variable and the factors that enhance oyster production and nekton abundance and diversity on restored reefs are unresolved. To quantify the drivers of oyster restoration success, we used meta-analysis to synthesize data from 158 restored reefs paired with unstructured habitats along the United States Gulf and Atlantic coasts. The average recovery of oyster production was 65% greater in subtidal (vs. intertidal) zones, 173% greater in polyhaline (vs. mesohaline) environments and increased with tidal range, demonstrating that physical conditions can strongly influence the restoration success of foundation species. Additionally, restoration increased the relative abundance and richness of nektonic fishes and invertebrates over time as reefs aged (at least 8 years post-construction). Thus, the restoration benefits for provisioning habitat and enhancing biodiversity accrue over time, highlighting that restoration projects need multiple years to maximize ecosystem functions. Furthermore, long-term monitoring of restored and control sites is needed to assess restoration outcomes and associated drivers. Last, our work reveals data constraints for several potential drivers of restoration outcomes, including reef construction material, reef dimensions, harvest pressure and disease prevalence. More experimental and observational studies are needed to target these factors and measure them with consistent methods across studies. Our findings indicate that the assisted recovery of foundation species yields several enhancements to ecosystem services, but such benefits are mediated by time and environmental conditions.

Design and validation of a high-density single nucleotide polymorphism array for the Eastern oyster (Crassostrea virginica).

Dense single nucleotide polymorphism (SNP) arrays are essential tools for rapid high-throughput genotyping for many genetic analyses, including genomic selection and high-resolution population genomic assessments. We present a high-density (200 K) SNP array developed for the Eastern oyster (Crassostrea virginica), which is a species of significant aquaculture production and restoration efforts throughout its native range. SNP discovery was performed using low-coverage whole-genome sequencing of 435 F1 oysters from families from 11 founder populations in New Brunswick, Canada. An Affymetrix Axiom Custom array was created with 219,447 SNPs meeting stringent selection criteria and validated by genotyping more than 4,000 oysters across 2 generations. In total, 144,570 SNPs had a call rate >90%, most of which (96%) were polymorphic and were distributed across the Eastern oyster reference genome, with similar levels of genetic diversity observed in both generations. Linkage disequilibrium was low (maximum r2 ∼0.32) and decayed moderately with increasing distance between SNP pairs. Taking advantage of our intergenerational data set, we quantified Mendelian inheritance errors to validate SNP selection. Although most of SNPs exhibited low Mendelian inheritance error rates overall, with 72% of called SNPs having an error rate of <1%, many loci had elevated Mendelian inheritance error rates, potentially indicating the presence of null alleles. This SNP panel provides a necessary tool to enable routine application of genomic approaches, including genomic selection, in C. virginica selective breeding programs. As demand for production increases, this resource will be essential for accelerating production and sustaining the Canadian oyster aquaculture industry.

Understanding Crassostrea virginica tolerance of Perkinsus marinus through global gene expression analysis.

Disease tolerance, a host's ability to limit damage from a given parasite burden, is quantified by the relationship between pathogen load and host survival or reproduction. Dermo disease, caused by the protozoan parasite P. marinus, negatively impacts survival in both wild and cultured eastern oyster (C. virginica) populations. Resistance to P. marinus has been the focus of previous studies, but tolerance also has important consequences for disease management in cultured and wild populations. In this study we measured dermo tolerance and evaluated global expression patterns of two sensitive and two tolerant eastern oyster families experimentally challenged with distinct doses of P. marinus (0, 106, 107, and 108 parasite spores per gram wet weight, n = 3-5 individuals per family per dose). Weighted Gene Correlation Network Analysis (WGCNA) identified several modules correlated with increasing parasite dose/infection intensity, as well as phenotype. Modules positively correlated with dose included transcripts and enriched GO terms related to hemocyte activation and cell cycle activity. Additionally, these modules included G-protein coupled receptor, toll-like receptor, and tumor necrosis factor pathways, which are important for immune effector molecule and apoptosis activation. Increased metabolic activity was also positively correlated with treatment. The module negatively correlated with infection intensity was enriched with GO terms associated with normal cellular activity and growth, indicating a trade-off with increased immune response. The module positively correlated with the tolerant phenotype was enriched for transcripts associated with "programmed cell death" and contained a large number of tripartite motif-containing proteins. Differential expression analysis was also performed on the 108 dosed group using the most sensitive family as the comparison reference. Results were consistent with the network analysis, but signals for "programmed cell death" and serine protease inhibitors were stronger in one tolerant family than the other, suggesting that there are multiple avenues for disease tolerance. These results provide new insight for defining dermo response traits and have important implications for applying selective breeding for disease management.

Baseline assessment of microplastics in commercially important marine bivalves from New York, U.S.A.

Microplastic (MP) contamination in bivalve mollusks has become a significant concern over the last few years. These ecologically and economically valuable species are popular seafood items for human consumption. As filter feeders, bivalves may ingest MPs in their bodies, possibly impacting their physiology and fitness. Additionally, a considerable amount of the seafood that humans consume comes from coastal areas where MP concentrations tend to be the highest. This research provides the first examination of MPs in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) that were grown locally in coastal areas of New York, contributing to a baseline for the northeast and mid-Atlantic regions of the U.S. A total of 48 eastern oysters (n = 12 per site, at four sites) and hard clams (n = 24 per site, at two sites) were sampled in summer 2021. While MP fibers and fragments (i.e. polyethylene terephthalate, polystyrene, and polypropylene) were found in some oysters, other contaminants (e.g. indigo dye, phthalocyanine, dye 823, etc.) were found in both bivalve species. Particle composition was verified using Raman microspectroscopy. Although mean MP concentrations were low in eastern oysters (i.e. 0.008 MPs g-1 of soft tissue wet weight; 0.125 MPs ind-1) and not found in hard clams, more research is needed to assess the magnitude of contamination in these edible bivalves.

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.

Seasonal gut contents of the eastern oyster, Crassostrea virginica, in the Rhode River, Chesapeake Bay, USA: Growth, phytoplankton and signature pigment data.

A 2-year study was undertaken to understand feeding preferences of the eastern oyster Crassostrea virginica when growing in conditions of eutrophication and variable flow. Oysters were suspended in the Rhode River, a tributary of Chesapeake Bay, Maryland, USA, and a subset of these oysters was collected monthly, measured in height to determine growth, and the phytoplankton in their gut were examined both microscopically and using indicator pigments and compared with phytoplankton abundance and composition in the water column. The data herein summarize the oyster growth and the gut contents with respect to phytoplankton cell numbers and composition and with respect to signature pigments.

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.

Spatial and temporal trends in physiological biomarkers of adult eastern oysters, Crassostrea virginica, within an urban estuary.

Heavy metal contamination and water quality may alter reproductive capacity of oysters in highly urbanized, eutrophic ecosystems. This study assessed physiological biomarkers and heavy metal body burdens in adult oysters, Crassostrea virginica, placed at a highly urban and reference site. Condition index and Vitellogenin-like proteins were significantly different between sites, but protein concentration and activity of the electron transport system were not. Accumulation of Cd and Hg occurred at both sites, and Cd body burden was greater at the urban site. There was a negative relationship between condition index and Cd body burden at the urban site, while no relationship was found between physiological biomarkers and metal burden at the reference site. The results suggest that oyster condition and reproductive potential may be negatively influenced by the biotic and abiotic factors typically found within urban, eutrophic ecosystems.

Variation in global transcriptomic response to Perkinsus marinus infection among eastern oyster families highlights potential mechanisms of disease resistance.

Dermo disease, caused by the protozoan parasite Perkinsus marinus, negatively impacts wild and cultured Eastern oyster populations, yet our knowledge of the mechanistic bases for parasite pathogenicity and the Eastern oyster's response to it is limited. To better understand host responses to the parasite and identify molecular mechanisms underlying disease-resistance phenotypes, we experimentally challenged two families exhibiting divergent Dermo-resistance phenotypes with the parasite, generated global expression profiles using RNAseq and identified differentially expressed transcripts between control and challenged oysters from each family at multiple time points post-parasite injection. The susceptible and resistant families exhibited strikingly different transcriptomic responses to the parasite over a 28-day time period. The resistant family exhibited a strong, focused, early response to P. marinus infection, where many significantly upregulated transcripts were associated with the biological processes "regulation of proteolysis" and "oxidation-reduction process." P. marinus virulence factors are mainly comprised of proteases that facilitate parasite invasion and weaken host humoral defenses, thus host upregulation of transcripts associated with negative regulation of proteolysis is consistent with a Dermo-resistant phenotype. In contrast, the susceptible family mounted a very weak, disorganized, initial response to the parasite. Few transcripts were differentially expressed between control and injected oysters, and no functional enrichment was detected among them. At the final 28 d time point 2450 differentially expressed transcripts were identified and were associated with either "G-protein coupled receptor activity" (upregulated) or "microtubule-based process" (downregulated). A handful of protease inhibitors were differentially expressed between control and injected susceptible oysters, but this function was not enriched in the susceptible data set. The differential expression patterns observed in this study provide valuable insight into the functional basis of Dermo resistance and suggest that the timing of expression is just as important as the transcripts being expressed.

Transcriptomic response of the Crassostrea virginica gonad after exposure to a water-accommodation fraction of hydrocarbons and the potential implications in reproduction.

The Crassostrea virginica oyster has biological and economic importance in the Gulf of Mexico, an area with a high extraction and production of hydrocarbons. Exposure to hydrocarbons affects the reproductive processes in bivalves. In C. virginica, the effect of hydrocarbons on the gonad of the undifferentiated organism has not been evaluated to determine the possible damage during the maturation process. To evaluate this effect, RNA-seq data was generated from C. virginica gonads exposed to a 200 µg/L of hydrocarbons at different exposure times (7, 14 and 21 days) and a control treatment (without hydrocarbons). The analysis of the gonad transcriptome showed the negative effect of hydrocarbons on maturation, with a sub-expression of 22 genes involved in different stages of this process. Additionally, genes in the immune system were down-regulated, which may indicate that exposure to hydrocarbons causes immunosuppression in bivalves. A group of oxidative stress genes was also reduced. These data contribute to a better understanding of the effect of hydrocarbons on the reproductive process in bivalves and, at the same time, allow us to identify possible biomarkers associated with hydrocarbon contamination in the gonad of C. virginica.

Impact of dispersant on early life stages of the water flea Daphnia magna and the eastern oyster Crassostrea virginica.

In response to the 2010 Deepwater Horizon oil spill, over 1 million gallons of dispersant were applied in Gulf of Mexico offshore waters; Corexit 9500 was the most applied dispersant. The impact on organisms in nearshore and freshwaters has received little scrutiny. Acute 48 h toxicity of Corexit 9500 and a new hyperbranched polyethylenimine (HPEI) dispersant-like compound were evaluated for the freshwater indicator organism, Daphnia magna and for larval and early spat stages of the Eastern oyster, Crassostrea virginica. For D. magna, Corexit 9500 demonstrated toxicity (EC50 of 0.14 [0.13, 0.15] ppm) similar to the 10-kDa HPEI (EC50 of 0.16 [0.12, 0.19] ppm). HPEI toxicity increased as a function of molecular weight (1.2 to 750 kDa). The 10 kDa size HPEI was further investigated because it dispersed crude oil with equal effectiveness as Corexit. For Corexit, 100% oyster mortality was detected for the ≤0.2-mm size classes and mortality >50% for the 0.3- and 0.7-mm size classes at the two greatest concentrations (25 and 50 ppm). HPEI (10 kDa) exhibited low mortality rates (<30%) for all concentrations for all oyster size classes except the 0.1-mm class. Although mortality rates for this size class were up to 60%, mortality was still less than the mortality caused by Corexit 9500. The low toxicity of HPEI polymers for C. virginica in comparison with Corexit 9500 suggests that HPEI polymers warrant further study.

Establishment and Validation of RNA-Based Predictive Models for Understanding Survival of Vibrio parahaemolyticus in Oysters Stored at Low Temperatures.

This study developed RNA-based predictive models describing the survival of Vibrio parahaemolyticus in Eastern oysters (Crassostrea virginica) during storage at 0, 4, and 10°C. Postharvested oysters were inoculated with a cocktail of five V. parahaemolyticus strains and were then stored at 0, 4, and 10°C for 21 or 11 days. A real-time reverse transcription-PCR (RT-PCR) assay targeting expression of the tlh gene was used to evaluate the number of surviving V. parahaemolyticus cells, which was then used to establish primary molecular models (MMs). Before construction of the MMs, consistent expression levels of the tlh gene at 0, 4, and 10°C were confirmed, and this gene was used to monitor the survival of the total V. parahaemolyticus cells. In addition, the tdh and trh genes were used for monitoring the survival of virulent V. parahaemolyticus Traditional models (TMs) were built based on data collected using a plate counting method. From the MMs, V. parahaemolyticus populations had decreased 0.493, 0.362, and 0.238 log10 CFU/g by the end of storage at 0, 4, and 10°C, respectively. Rates of reduction of V. parahaemolyticus shown in the TMs were 2.109, 1.579, and 0.894 log10 CFU/g for storage at 0, 4, and 10°C, respectively. Bacterial inactivation rates (IRs) estimated with the TMs (-0.245, -0.152, and -0.121 log10 CFU/day, respectively) were higher than those estimated with the MMs (-0.134, -0.0887, and -0.0732 log10 CFU/day, respectively) for storage at 0, 4, and 10°C. Higher viable V. parahaemolyticus numbers were predicted using the MMs than using the TMs. On the basis of this study, RNA-based predictive MMs are the more accurate and reliable models and can prevent false-negative results compared to TMs.IMPORTANCE One important method for validating postharvest techniques and for monitoring the behavior of V. parahaemolyticus is to establish predictive models. Unfortunately, previous predictive models established based on plate counting methods or on DNA-based PCR can underestimate or overestimate the number of surviving cells. This study developed and validated RNA-based molecular predictive models to describe the survival of V. parahaemolyticus in oysters during low-temperature storage (0, 4, and 10°C). The RNA-based predictive models show the advantage of being able to count all of the culturable, nonculturable, and stressed cells. By using primers targeting the tlh gene and pathogenesis-associated genes (tdh and trh), real-time RT-PCR can evaluate the total surviving V. parahaemolyticus population as well as differentiate the pathogenic ones from the total population. Reliable and accurate predictive models are very important for conducting risk assessment and management of pathogens in food.

Improving management support tools for reintroducing bivalve species (eastern oyster [Crassostrea virginica Gmelin]) in urban estuaries.

Successful reintroduction of "ecologically extinct" bivalve species into anthropogenically impaired urban estuaries is problematic when employing existing management tools used in estuaries where bivalves are present (GIS-based restoration models, expanding existing shellfish beds, placement of shell substrate, physical oceanographic parameters). A significant management challenge is appropriate site selection. We are proposing the inclusion of a biological parameter (evaluation of tissue histopathology) in an inexpensive and rapid site selection model to inform management decision making and identify sites with the greatest potential for reintroduction success. Use of biological biomarkers is not a new concept, but it is important that they be included in a multitiered management approach to bivalve reintroduction. This Case Study tested adult Eastern Oysters (Crassostrea virginica Gmelin) from locations that supported comparable short-term survival rates by evaluating growth and tissue health and/or disease. Biomarkers indicated oyster tissues at one site were normal, the female:male sex ratio was 50:50, and female oysters were in spawning condition. Conversely, oyster tissues at the second site exhibited multiple abnormalities, samples were 100% male, and the incidence of disease was high. Using the biomarker tool, we evaluated 4 additional sites where oysters exhibited short-term (1 year) survival. At 2 locations, we observed chronic health impacts that would preclude reintroduction, including samples from one site where a wild population was surviving. We also analyzed tissue and shell heavy metal contents. Soft tissue metal concentrations in Meadowlands samples were at the high range of scientific literature values, averaging 1.1% of total body weight, whereas tissue metal concentrations at the Keyport site were within acceptable ranges. Although initial survival and growth rates at both locations were comparable, site-specific urban stressors reduced oyster fitness at 1 of the 2 locations. We are proposing an Estuarine Reintroduction Site Selection Model, which includes a biological in situ parameter, to increase the probability of successfully managing a sustainable oyster reintroduction before commencing expensive large-scale restoration activities.

Derelict fishing gear in Chesapeake Bay, Virginia: spatial patterns and implications for marine fauna.

Derelict fishing gear is a source of mortality for target and non-target marine species. A program employing commercial watermen to remove marine debris provided a novel opportunity to collect extensive spatially-explicit information for four consecutive winters (2008-2012) on the type, distribution, and abundance of derelict fishing gear and bycatch in Virginia waters of Chesapeake Bay. The most abundant form of derelict gear recovered was blue crab pots with almost 32,000 recovered. Derelict pots were widely distributed, but with notable hotspot areas, capturing 40 species and over 31,000 marine organisms. The target species, blue crab, experienced the highest mortality from lost pots with an estimated 900,000 animals killed each year, a potential annual economic loss to the fishery of $300,000. Important fishery species were captured and killed in derelict pots including Atlantic croaker and black sea bass. While some causes of gear loss are unavoidable, others can be managed to minimize loss.