Tools and Techniques to Accelerate Crop Breeding.

As climate changes and a growing global population continue to escalate the need for greater production capabilities of food crops, technological advances in agricultural and crop research will remain a necessity. While great advances in crop improvement over the past century have contributed to massive increases in yield, classic breeding schemes lack the rate of genetic gain needed to meet future demands. In the past decade, new breeding techniques and tools have been developed to aid in crop improvement. One such advancement is the use of speed breeding. Speed breeding is known as the application of methods that significantly reduce the time between crop generations, thereby streamlining breeding and research efforts. These rapid-generation advancement tactics help to accelerate the pace of crop improvement efforts to sustain food security and meet the food, feed, and fiber demands of the world's growing population. Speed breeding may be achieved through a variety of techniques, including environmental optimization, genomic selection, CRISPR-Cas9 technology, and epigenomic tools. This review aims to discuss these prominent advances in crop breeding technologies and techniques that have the potential to greatly improve plant breeders' ability to rapidly produce vital cultivars.

Comprehensive Proteomic Analysis of Common Bean (Phaseolus vulgaris L.) Seeds Reveal Shared and Unique Proteins Involved in Terminal Drought Stress Response in Tolerant and Sensitive Genotypes.

This study identified proteomic changes in the seeds of two tolerant (SB-DT3 and SB-DT2) and two sensitive (Merlot and Stampede) common bean genotypes in response to terminal drought stress. Differentially expressed proteins (DEPs) were abundant in the susceptible genotype compared to the tolerant line. DEPs associated with starch biosynthesis, protein-chromophore linkage, and photosynthesis were identified in both genotypes, while a few DEPs and enriched biological pathways exhibited genotype-specific differences. The tolerant genotypes uniquely showed DEPs related to sugar metabolism and plant signaling, while the sensitive genotypes displayed more DEPs involved in plant-pathogen interaction, proteasome function, and carbohydrate metabolism. DEPs linked with chaperone and signal transduction were significantly altered between both genotypes. In summary, our proteomic analysis revealed both conserved and genotype-specific DEPs that could be used as targets in selective breeding and developing drought-tolerant common bean genotypes.

Microplastics and their interactions with microbiota.

As a new pollutant, Microplastics (MPs) are globally known for their negative impacts on different ecosystems and living organisms. MPs are easily taken up by the ecosystem in a variety of organisms due to their small size, and cause immunological, neurological, and respiratory diseases in the impacted organism. Moreover, in the impacted environments, MPs can release toxic additives and act as a vector and scaffold for colonization and transportation of specific microbes and lead to imbalances in microbiota and the biogeochemical and nutrients dynamic. To address the concerns on controlling the MPs pollution on the microbiota and ecosystem, the microbial biodegradation of MPs can be potentially considered as an effective environment friendly approach. The objectives of the presented paper are to provide information on the toxicological effects of MPs on microbiota, to discuss the negative impacts of microbial colonization of MPs, and to introduce the microbes with biodegradation ability of MPs.

Investigating the Relationship between Nitrate, Total Dissolved Nitrogen, and Phosphate with Abundance of Pathogenic Vibrios and Harmful Algal Blooms in Rehoboth Bay, Delaware.

Vibrio spp. and phytoplankton are naturally abundant in marine environments. Recent studies have suggested that the co-occurrence of phytoplankton and the pathogenic bacterium Vibrio parahaemolyticus is due to shared ecological factors, such as nutrient requirements. We compared these communities at two locations in the Delaware Inland Bays, representing a site with high anthropogenic inputs (Torquay Canal) and a less developed area (Sloan Cove). In 2017 to 2018, using light microscopy, we were able to identify the presence of many bloom-forming algal species, such as Karlodinium veneficum, Dinophysis acuminata, Heterosigma akashiwo, and Chattonella subsalsa. Dinoflagellate biomass was higher at Torquay Canal than that at Sloan Cove. D. acuminata and Chloromorum toxicum were found only at Torquay Canal and were not observed in Sloan Cove. Most probable number real-time PCR revealed V. parahaemolyticus and Vibrio vulnificus in environmental samples. The abundance of vibrios and their virulence genes varied between sites, with a significant association between total dissolved nitrogen (TDN), PO4-, total dissolved phosphorus (TDP), and pathogenic markers. A generalized linear model revealed that principal component 1 of environmental factors (temperature, dissolved oxygen, salinity, TDN, PO4-, TDP, NO3:NO2, NO2-, and NH4+) was the best at detecting total (tlh+) V. parahaemolyticus, suggesting that they are the prime drivers for the growth and distribution of pathogenic Vibrio spp. IMPORTANCE Vibrio-associated illnesses have been expanding globally over the past several decades (A. Newton, M. Kendall, D. J. Vugia, O. L. Henao, and B. E. Mahon, Clin Infect Dis 54:S391-S395, 2012, https://doi.org/10.1093/cid/cis243). Many studies have linked this expansion with an increase in global temperature (J. Martinez-Urtaza, B. C. John, J. Trinanes, and A. DePaola, Food Res Int 43:10, 2010, https://doi.org/10.1016/j.foodres.2010.04.001; L. Vezzulli, R. R. Colwell, and C. Pruzzo, Microb Ecol 65:817-825, 2013, https://doi.org/10.1007/s00248-012-0163-2; R. N. Paranjpye, W. B. Nilsson, M. Liermann, and E. D. Hilborn, FEMS Microbiol Ecol 91:fiv121, 2015, https://doi.org/10.1093/femsec/fiv121). Temperature and salinity are the two major factors affecting the distribution of Vibrio spp. (D. Ceccarelli and R. R. Colwell, Front Microbiol 5:256, 2014, https://doi.org/10.3389/fmicb.2014.00256). However, Vibrio sp. abundance can also be affected by nutrient load and marine plankton blooms (V. J. McKenzie and A. R. Townsend, EcoHealth 4:384-396, 2007; L. Vezzulli, C. Pruzzo, A. Huq, and R. R. Colwell, Environ Microbiol Rep 2:27-33, 2010, https://doi.org/10.1111/j.1758-2229.2009.00128.x; S. Liu, Z. Jiang, Y. Deng, Y. Wu, J. Zhang, et al. Microbiologyopen 7:e00600, 2018, https://doi.org/10.1002/mbo3.600). The expansion of Vibrio spp. in marine environments calls for a deeper understanding of the biotic and abiotic factors that play a role in their abundance. We observed that pathogenic Vibrio spp. were most abundant in areas that favor the proliferation of harmful algal bloom (HAB) species. These results can inform managers, researchers, and oyster growers on factors that can influence the growth and distribution of pathogenic Vibrio spp. in the Delaware Inland Bays.

CRISPR-Cas technology a new era in genomic engineering.

The CRISPR-Cas systems have offered a flexible, easy-to-use platform to precisely modify and control the genomes of organisms in various fields, ranging from agricultural biotechnology to therapeutics. This system is extensively used in the study of infectious, progressive, and life-threatening genetic diseases for the improvement of quality and quantity of major crops and in the development of sustainable methods for the generation of biofuels. As CRISPR-Cas technology continues to evolve, it is becoming more controllable and precise with the addition of molecular regulators, which will provide benefits for everyone and save many lives. Studies on the constant growth of CRISPR technology are important due to its rapid development. In this paper, we present the current applications and progress of CRISPR-Cas genome editing systems in several fields of research, we further highlight the applications of anti-CRISPR molecules to regulate CRISPR-Cas gene editing systems, and we discuss ethical considerations in CRISPR-Cas applications.

Uptake and Presence Evaluation of Nanoparticles in Cicer arietinum L. by Infrared Spectroscopy and Machine Learning Techniques.

The aim of this work was to study the applicability of infrared spectroscopy combined with machine learning techniques to evaluate the uptake and distribution of gold nanoparticles (AuNPs) and single-walled carbon nanotubes (CNTs) in Cicer arietinum L. (chickpea). Obtained spectral data revealed that the uptake of AuNPs and CNTs by the C. arietinum seedlings' root resulted in the accumulation of AuNPs and CNTs at stem and leaf parts, which consequently led to the heterogeneous distribution of nanoparticles. principal component analysis and support vector machine classification were applied to assess its usefulness for evaluating the results obtained using the attenuated total reflectance-Fourier transform infrared spectroscopy method of C. arietinum plant grown at different conditions. Specific wavenumbers that could classify the different nanoparticle constituents of C. arietinum plant extracts according to their ATR-FTIR spectra were identified within three specific regions: 450-503 cm-1, 750-870 cm-1, and 1022-1218 cm-1, based on larger PCA loadings of C. arietinum ATR-FTIR spectra with distinct spectral differences between samples of interest. The current work paves a path to the future fabrication strategies for AuNPs and single-walled CNTs via plant-based routes and highlights the diversity of the applications of these materials in bio-nanotechnology. These results indicate the importance of family-plant selection, choice of methods, and pathways for the efficient biomolecule delivery, drug cargo, and optimal conditions in the wide spectrum of bioapplications.

Presence and Plant Uptake of Heavy Metals in Tidal Marsh Wetland Soils.

Marsh grasses have been used as efficient tools for phytoremediation and are known to play key roles in maintaining ecosystem functions by reducing the contamination of coastlines. This study was initiated to understand how human activities in wetlands can impact ion-heavy metal concentrations in relation to native and invasive marsh grasses. The study site, Blackbird Creek (BBC) is a tidal wetland that experiences agricultural, fishing, recreational, residential and other anthropogenic activities throughout the year. Heavy metals cadmium, arsenic, and lead in the soils and marsh grasses were monitored along with the ion compositions of soils. The main objective of this study was to understand if the marsh soils containing monotypic stands of native (Spartina) and non-native (Phragmites) vegetation display similar levels of heavy metals. Differences were observed in the concentrations of heavy metals at study sites with varying marsh vegetation types, and in soils containing vegetation and no vegetation. The soils with dense Spartina and Phragmites stands were anaerobic whereas soil at the boat ramp site was comparatively less anaerobic and also had increased levels of cadmium. Heavy metal concentrations in soil and Phragmites leaves were inversely correlated whereas they were positively correlated in Spartina sites. Electrical conductivity and pH levels in soil also showed increased cadmium and arsenic concentrations. These findings collectively infer that human activities and seasonal changes can increase soil complexities affecting the bioavailability of metals.

Interactive effects of light, CO2 and temperature on growth and resource partitioning by the mixotrophic dinoflagellate, Karlodinium veneficum.

There is little information on the impacts of climate change on resource partitioning for mixotrophic phytoplankton. Here, we investigated the hypothesis that light interacts with temperature and CO2 to affect changes in growth and cellular carbon and nitrogen content of the mixotrophic dinoflagellate, Karlodinium veneficum, with increasing cellular carbon and nitrogen content under low light conditions and increased growth under high light conditions. Using a multifactorial design, the interactive effects of light, temperature and CO2 were investigated on K. veneficum at ambient temperature and CO2 levels (25°C, 375 ppm), high temperature (30°C, 375 ppm CO2), high CO2 (30°C, 750 ppm CO2), or a combination of both high temperature and CO2 (30°C, 750 ppm CO2) at low light intensities (LL: 70 µmol photons m-2 s-2) and light-saturated conditions (HL: 140 µmol photons m-2 s-2). Results revealed significant interactions between light and temperature for all parameters. Growth rates were not significantly different among LL treatments, but increased significantly with temperature or a combination of elevated temperature and CO2 under HL compared to ambient conditions. Particulate carbon and nitrogen content increased in response to temperature or a combination of elevated temperature and CO2 under LL conditions, but significantly decreased in HL cultures exposed to elevated temperature and/or CO2 compared to ambient conditions at HL. Significant increases in C:N ratios were observed only in the combined treatment under LL, suggesting a synergistic effect of temperature and CO2 on carbon assimilation, while increases in C:N under HL were driven only by an increase in CO2. Results indicate light-driven variations in growth and nutrient acquisition strategies for K. veneficum that may benefit this species under anticipated climate change conditions (elevated light, temperature and pCO2) while also affecting trophic transfer efficiency during blooms of this species.

Design and Operation of Effective Landfills with Minimal Effects on the Environment and Human Health.

Totaling at 7.4 billion people, the world's population is rapidly growing, bringing along with it an increase in waste generation. The impact of this exponential increase in waste generation has resulted in the increased formation and utilization of landfills. In the present day, landfills are utilized to dispose of chemical, hazardous, municipal, and electronic wastes. However, despite their convenience, most landfills are improperly managed and face constant changes from the surrounding environment that interfere with their internal landfill processes. The objectives of this mixed review are to highlight the negative impacts landfills have on the environment and public health as well as outline the need for proper management practices to mitigate these effects. Inadequate management of landfills leads to issues concerning leachate collection and landfill gas (LFG) generation, which give rise to groundwater contamination and air pollution. This paper recognizes the disadvantages of utilizing landfills as the main disposal method by focusing on these two primary effects that improper management of landfills has on the environment and human health. Many experts have also reported that communities within close proximity to improperly managed landfills have an increased risk of health issues. Apart from implementing proper landfill management practices, it is important to develop solutions to reduce waste generation altogether. This review discusses some of the innovative methods implemented by other countries to reduce landfill waste and the production of greenhouse gases as well as possible steps individuals can take to minimize their ecological footprints.

Assessment of Vibrio parahaemolyticus levels in oysters (Crassostrea virginica) and seawater in Delaware Bay in relation to environmental conditions and the prevalence of molecular markers to identify pathogenic Vibrio parahaemolyticus strains.

This study identified Vibrio parahaemolyticus in oyster and seawater samples collected from Delaware Bay from June through October of 2016. Environmental parameters including water temperature, salinity, dissolved oxygen, pH, and chlorophyll a were measured per sampling event. Oysters homogenate and seawater samples were 10-fold serially diluted and directly plated on CHROMagarᵀᴹ Vibrio medium. Presumptive V. parahaemolyticus colonies were counted and at least 20% of these colonies were selected for molecular chracterization. V. parahaemolyticus isolates (n = 165) were screened for the presence of the species-specific thermolabile hemolysin (tlh) gene, the pathogenic thermostable direct hemolysin (tdh)/ thermostable related hemolysin (trh) genes, the regulatory transmembrane DNA-binding gene (toxR), and V. parahaemolyticus metalloprotease (vpm) gene using a conventional PCR. The highest mean levels of the presumptive V. parahaemolyticus were 9.63×103 CFU/g and 1.85×103 CFU/mL in the oyster and seawater samples, respectively, during the month of July. V. parahaemolyticus levels in oyster and seawater samples were significantly positively correlated with water temperature. Of the 165 isolates, 137 (83%), 110 (66.7%), and 108 (65%) were tlh+, vpm+, and toxR+, respectively. Among the V. parahaemolyticus (tlh+) isolates, 7 (5.1%) and 15 (10.9%) were tdh+ and trh+, respectively, and 24 (17.5%), only oyster isolates, were positive for both genes. Potential pathogenic strains that possessed tdh and/or trh were notably higher in oyster (39%) than seawater (15.6%) isolates. The occurrence of total V. parahaemolyticus (tlh+) was not necessarily proportional to the potential pathogenic V. parahaemolyticus. Co-occurrence of the five genetic markers were observed only among oyster isolates. The co-occurrence of the gene markers showed a relatedness potential of tdh occurrence with vpm. We believe exploring the role of V. parahaemolyticus metalloprotease and whether it is involved in the toxic activity of the thermostable direct hemolysin (TDH) protein can be of significance. The outcomes of this study will provide some foundation for future studies regarding pathogenic Vibrio dynamics in relation to environmental quality.

Seasonal and Geographical Differences in Total and Pathogenic Vibrio parahaemolyticus and Vibrio vulnificus Levels in Seawater and Oysters from the Delaware and Chesapeake Bays Determined Using Several Methods.

Oyster and seawater samples were collected from five sites in the Chesapeake Bay, MD, and three sites in the Delaware Bay, DE, from May to October 2016 and 2017. Abundances and detection frequencies for total and pathogenic Vibrio parahaemolyticus and Vibrio vulnificus were compared using the standard most-probable-number-PCR (MPN-PCR) assay and a direct-plating (DP) method on CHROMagar Vibrio for total (tlh+ ) and pathogenic (tdh+ and trh+ ) V. parahaemolyticus genes and total (vvhA) and pathogenic (vcgC) V. vulnificus genes. The colony overlay procedure for peptidases (COPP) assay was evaluated for total Vibrionaceae DP had high false-negative rates (14 to 77%) for most PCR targets and was deemed unsatisfactory. Logistic regression models of the COPP assay showed high concordances with MPN-PCR for tdh+ and trh+V. parahaemolyticus and vvhA+V. vulnificus in oysters (85.7 to 90.9%) and seawater (81.1 to 92.7%) when seawater temperature and salinity were factored into the model, suggesting that the COPP assay could potentially serve as a more rapid method to detect vibrios in oysters and seawater. Differences in total Vibrionaceae and pathogenic Vibrio abundances between state sampling sites over different collection years were contrasted for oysters and seawater by MPN-PCR. Abundances of tdh+ and trh+V. parahaemolyticus were ∼8-fold higher in Delaware oysters than in Maryland oysters, whereas abundances of vcgC+V. vulnificus were nearly identical. For Delaware oysters, 93.5% were both tdh+ and trh+, compared to only 19.2% in Maryland. These results indicate that pathogenic V. parahaemolyticus was more prevalent in the Delaware Bay than in the Chesapeake Bay.IMPORTANCE While V. parahaemolyticus and V. vulnificus cause shellfish-associated morbidity and mortality among shellfish consumers, current regulatory assays for vibrios are complex, time-consuming, labor-intensive, and relatively expensive. In this study, the rapid, simple, and inexpensive COPP assay was identified as a possible alternative to MPN-PCR for shellfish monitoring. This paper shows differences in total Vibrionaceae and pathogenic vibrios found in seawater and oysters from the commercially important Delaware and Chesapeake Bays. Vibrio parahaemolyticus isolates from the Delaware Bay were more likely to contain commonly recognized pathogenicity genes than those from the Chesapeake Bay.

Bacteriophages Against Pathogenic Vibrios in Delaware Bay Oysters (Crassostrea virginica) During a Period of High Levels of Pathogenic Vibrio parahaemolyticus.

Eastern oysters (Crassostrea virginica) from three locations along the Delaware Bay were surveyed monthly from May to October 2017 for levels of total Vibrio parahaemolyticus, pathogenic strains of V. parahaemolyticus and Vibrio vulnificus, and for strain-specific bacteriophages against vibrios (vibriophages). The objectives were to determine (a) whether vibriophages against known strains or serotypes of clinical and environmental vibrios were detectable in oysters from the Delaware Bay and (b) whether vibriophage presence or absence corresponded with Vibrio abundances in oysters. Host cells for phage assays included pathogenic V. parahaemolyticus serotypes O3:K6, O1:KUT (untypable) and O1:K1, as well as clinical and environmental strains of V. vulnificus. Vibriophages against some, but not all, pathogenic V. parahaemolyticus serotypes were readily detected in Delaware Bay oysters. In July, abundances of total and pathogenic V. parahaemolyticus at one site spiked to levels exceeding regulatory guidelines. Phages against three V. parahaemolyticus host serotypes were detected in these same oysters, but also in oysters with low V. parahaemolyticus levels. Serotype-specific vibriophage presence or absence did not correspond with abundances of total or pathogenic V. parahaemolyticus. Vibriophages were not detected against three V. vulnificus host strains, even though V. vulnificus were readily detectable in oyster tissues. Selected phage isolates against V. parahaemolyticus showed high host specificity. Transmission electron micrographs revealed that most isolates were ~ 60-nm diameter, non-tailed phages. In conclusion, vibriophages were detected against pandemic V. parahaemolyticus O3:K6 and O1:KUT, suggesting that phage monitoring in specific host cells may be a useful technique to assess public health risks from oyster consumption.

Effects of High Hydrostatic Pressure on the Physical, Microbial, and Chemical Attributes of Oysters (Crassostrea virginica).

The change in the quality attributes (physical, microbial, and chemical) of oysters (Crassostrea virginica) after high hydrostatic pressure (HHP) treatment at 300 MPa at room temperature (RT, 25 °C) 300, 450, and 500 MPa at 0 °C for 2 min and control oysters without treatment were evaluated over 3 wk. The texture and tissue yield percentages of oysters HHP treated at 300 MPa, RT increased significantly (P < 0.05) compared to control. Aerobic and psychrotrophic bacteria in control oysters reached the spoilage point of 7 log CFU/g after 15 d. Coliform counts (log MPN/g) were low during storage with total and fecal coliforms less than 3.5 and 1.0. High pressure treated oysters at 500 MPa at 0 °C were significantly higher (P < 0.05) than oysters HHP treated at 300 MPa at 0 °C in lipid oxidation values. The highest pressure (500 MPa) treatment in this study, significantly (P < 0.05) decreased unsaturated fatty acid percentage compared to control. The glycogen content of control oysters at 3 wk was significantly higher (P < 0.05) when compared to HHP treated oysters [300 MPa, (RT); 450 MPa (0 °C); and 500 MPa (0 °C)]. HHP treatments of oysters were not significantly different in pH, percent salt extractable protein (SEP), and total lipid values compared to control. Based on our results, HHP prolongs the physical, microbial, and chemical quality of oysters.

Effects of High-Hydrostatic Pressure on Inactivation of Human Norovirus and Physical and Sensory Characteristics of Oysters.

The purpose of the study was to determine the effect of high-hydrostatic pressure (HHP) on inactivation of human norovirus (HuNoV) in oysters and to evaluate organoleptic characteristics of oysters treated at pressure levels required for HuNoV inactivation. Genogroup I.1 (GI.1) or Genogroup II.4 (GII.4) HuNoV was inoculated into oysters and treated at 300 to 600 MPa at 25 and 0 °C for 2 min. After HHP, viral particles were extracted by porcine gastric mucin-conjugated magnetic beads (PGM-MBs) and viral RNA was quantified by real-time RT-PCR. Lower initial temperature (0 °C) significantly enhanced HHP inactivation of HuNoV compared to ambient temperature (25 °C; P < 0.05). HHP at 350 and 500 MPa at 0 °C could achieve more than 4 log10 reduction of GII.4 and GI.1 HuNoV in oysters, respectively. HHP treatments did not significantly change color or texture of oyster tissue. A 1- to 5-scale hedonic sensory evaluation on appearance, aroma, color, and overall acceptability showed that pressure-treated oysters received significantly higher quality scores than the untreated control (P < 0.05). Elevated pressure levels at 450 and 500 MPa did not significantly affect scores compared to 300 MPa at 0 °C, indicating increasing pressure level did not affect sensory acceptability of oysters. Oysters treated at 0 °C had slightly lower acceptability than the group treated at room temperature on day 1 (P < 0.05), but after 1 wk storage, no significant difference in sensory attributes and consumer desirability was observed (P > 0.05).

Water-quality parameters and total aerobic bacterial and Vibrionaceae loads in Eastern oysters (Crassostrea virginica) from oyster-gardening sites.

Oyster gardening is a practice designed to restore habitat for marine life and to improve water quality. This study determined physical and chemical water-quality parameters at two oyster gardening sites in the Delaware Inland Bays and compared them with total aerobic bacteria and Vibrionaceae concentrations in Eastern oysters (Crassostrea virginica). One site was located at the end of a man-made canal, whereas the other was located in an open bay. Measured water parameters included temperature, dissolved oxygen (DO), salinity, pH, total nitrogen, nitrate, nitrite, total phosphorus, and total suspended solids. The highest Vibrionaceae levels, as determined by the colony overlay procedure for peptidases, were at the canal site in September (3.5 × 10(5) g(-1)) and at the bay site in August (1.9 × 10(5) g(-1)). Vibrionaceae levels were significantly greater during the duration of the study at the canal site (P = 0.01). This study provides the first baseline levels for total Vibrionaceae in the Delaware Inland Bays. Minimum DO readings at the bay and canal sites were 3.0 and 2.3 mg l(-1), respectively, far less than the state-targeted minimum threshold of 5.0 mg l(-1). Total phosphorus levels exceeded recommendations of ≤0.1 mg l(-1) at the bay and canal sites for all monthly samplings, with mean monthly highs at both sites ≥0.68 mg l(-1) in August. Nitrogen occasionally exceeded the recommended level of 1.0 mg l(-1) at both sites. Overall, waters were highly degraded from high phosphates, nitrogen, and total suspended solids as well as low DO.

Hemocytes are sites of enteric virus persistence within oysters.

The goal of this study was to determine how enteric viruses persist within shellfish tissues. Several lines of novel evidence show that phagocytic blood cells (hemocytes) of Eastern oysters (Crassostrea virginica) play an important role in the retention of virus particles. Our results demonstrated an association of virus contamination with hemocytes but not with hemolymph. Live oysters contaminated overnight with hepatitis A virus (HAV) and murine norovirus (MNV) had 56% and 80% of extractable virus associated with hemocytes, respectively. Transfer of HAV-contaminated hemocytes to naïve (virus-free) oysters resulted in naïve oyster meat testing HAV positive for up to 3 weeks. Acid tolerance of HAV, MNV, poliovirus (PV), and feline calicivirus (FCV) correlated with the ability of each virus to persist within oysters. Using reverse transcription-PCR (RT-PCR) to evaluate persistence of these viruses in oysters, we showed that HAV persisted the longest (>21 days) and was most acid resistant, MNV and PV were less tolerant of acidic pH, persisting for up to 12 days and 1 day, respectively, and FCV did not persist (<1 day) within oysters and was not acid tolerant. This suggests that the ability of a virus to tolerate the acidic conditions typical of phagolysosomal vesicles within hemocytes plays a role in determining virus persistence in shellfish. Evaluating oyster and hemocyte homogenates and live contaminated oysters as a prelude to developing improved viral RNA extraction methods, we found that viruses were extracted more expediently from hemocytes than from whole shellfish tissues and gave similar RT-PCR detection sensitivities.

Seawater and shellfish (Geukensia demissa) quality along the Western Coast of Assateague Island National Seashore, Maryland: an area impacted by feral horses and agricultural runoff.

We evaluated the quality of seawater and ribbed mussels (Gukensia demissa) at six sites along the West Coast of Assateague Island National Seashore (ASIS), a barrier island popular with tourists and fishermen. Parameters evaluated were summertime temperature, pH, salinity, dissolved oxygen, total phosphorus, total ammonia nitrogen, and nitrite levels for seawater and total heterotrophic plate counts and total Vibrionaceae levels for the ribbed mussels. Approximately 150 feral horses (Equus caballus) are located on ASIS and, combined with agricultural runoff from animals and croplands, local wildlife, and anthropogenic inputs, contribute to nutrient loads affecting water and shellfish quality. The average monthly dissolved oxygen for June was 2.65 mg L(-1), below the minimum acceptable threshold of 3.0 mg L(-1). Along Chincoteague Bay, total phosphorus generally exceeded the maximum level of 0.037 mg L(-1), as set by the Maryland Coastal Bays Program management objective for seagrasses, with a high of 1.92 mg L(-1) in June, some 50-fold higher than the recommended threshold. Total ammonia nitrogen approached levels harmful to fish, with a maximum recorded value of 0.093 mg L(-1). Levels of total heterotrophic bacteria spiked to 9.5 x 10(6) cells g(-1) of mussel tissue in August in Sinepuxent Bay, leading to mussels which exceeded acceptable standards for edible bivalves by 19-fold. An average of 76% of the bacterial isolates were in the Vibrionaceae family. Together, these data suggest poor stewardship of our coastal environment and the need for new intervention strategies to reduce chemical and biological contamination of our marine resources.