A study of the eastern oyster, Crassostrea virginica: (1) Dermo tolerance, survival, growth, condition and Hsp70 expression in different geographic stocks; (2) Heat tolerance and effects of sublethal heat shock on survival and Hsp70 expression of infected and uninfected oysters

The diseases Dermo and MSX have devastated Chesapeake Bay populations of the eastern oyster, Crassostrea virginica. The protozoan Perkinsus marinus, which causes Dermo, is particularly problematic since it persists over a wide range of salinities and temperatures. An objective of this dissertation was to determine whether specific wild oyster stocks had developed natural resistance to Dermo and if several parameters (survival, growth, condition and energy reserves) were associated with resistance. Another objective was to characterize heat shock protein (hsp70) expression in the eastern oyster. Heat shock proteins such as hsp70 protect organisms from thermal stress and other stressors, and this function may play an important role in disease resistance in oysters. In field trials a F0 Chesapeake Bay stock from Tangier Sound (CTS) survived similarly to a disease resistant hatchery strain (XB). A Louisiana stock was also resistant to Dermo, but not MSX. Despite high mortality, a disease-susceptible stock (CRB) reached market size the fastest. Growth and condition index varied between stocks, but did not reflect Dermo resistance. Energy reserves were affected strongly by season, but not disease or stock. Results imply that Dermo resistant strains could be developed from these stocks but criteria for optimal strain selection for aquaculture and restoration may be divergent. Mortalities of F1 oysters (CRB, CTS and XB) were similar to F0 parents, demonstrating a genetic basis to Dermo resistance. Total hsp70 did not correlate with seasonal temperatures, while hsp70 isoforms (hsp69 and hsp72) varied inversely across seasons. Hsp70 did not vary significantly between strains, indicating a stronger environmental influence on hsp70 expression. In lab experiments hsp70 in oyster gills was elevated greater than two weeks after a sub-lethal heat shock. Thermal tolerance, but not hsp70, varied between CTS and Louisiana oyster stocks. Heat shock protected oysters experimentally infected with P. marinus and non-infected oysters from lethal heat stress. Infection alone induced expression of hsp70. Observed inherent and induced differences in thermal tolerance suggest that both genotype and phenotype may be manipulated to improve survival in cultured bivalves. The implications of this research for bivalve aquaculture as well as areas for future research are discussed

Seasonal Variation Of Heat Shock Protein 70 In Eastern Oysters (Crassostrea Virginica) Infected With Perkinsus Marinus (Dermo)

Eastern oysters (Crassostrea virginica) inhabit highly variable environments and are exposed to large seasonal shifts in temperature. Prevalence and intensity of oyster diseases, particularly Perkinsus marinas (Dermo), increase during thermally stressful periods, thus posing additional stress on the oyster host. Heat shock proteins (hsps) are important in protecting organisms from thermal and overall environmental stress. Additionally, hsps may play protective roles for both the host and parasite during infection. The interactive effects of temperature and disease on heat shock protein expression in oysters, however, are unknown. In this study, using slot and western blotting assays, seasonal and intraspecific variation in heat shock protein 70 (hsp70) expression was compared among stocks of C. virginica known to be resistant or susceptible to Dermo at two sites in the Chesapeake Bay. Mortalities, shell heights, condition, and P. marinus infections were also compared among stocks to examine relationships between hsp70 and these variables. Hsp70 was analyzed at 4 seasonal samplings (fall, winter, spring, and summer months), while all other variables were measured bimonthly. Patterns and amounts of hsp70 expression varied significantly across different seasons, but did not correspond with seasonal temperature. Total amounts of hsp70 were significantly highest in the fall. Seasonal variation in specific isoforms of hsp70 (69 kDa and 72 kDa) was observed. Highest amounts of each were expressed in the spring and fall, respectively, and they were inversely proportional to each other. Differential expression was observed during the winter and spring, with several individuals expressing only hsp72 in the winter and only hsp69 in the spring. Although hsp72 changed concurrently with seasonal changes in infection, both hsp72 and hsp69 did not vary significantly between stocks or with levels of P. marinus infection. This study reveals that measuring total levels of hsp70 do not sufficiently describe the effect of seasonal temperatures on hsp70 expression. Stock mortalities were consistent with the patterns of disease resistance exhibited by their stock parentage, implying existence of a strong genetic component to resistance to Dermo disease. Differences in shell heights, condition index, and P. marinus infection differences showed significant associations among stock, site, and time. Variation in hsp70 did not reflect differences in infection among oyster stocks, indicating that hsp70 may not be a useful indicator to distinguish the effects of pathogenic stress between resistant and susceptible oyster stocks. Differences in expression between hsp69 and hsp72 suggest that seasonal patterns of specific hsp70 isoforms must be understood to determine the role of hsp70 proteins in stress and disease resistance in oysters

Performance Of Natural Dermo-Resistant Oyster Stocks-Survival, Disease, Growth, Condition And Energy Reserves

To determine if natural populations of the eastern oyster possess resistance to Perkinsus marinus, progeny representing several oyster stocks from the Chesapeake Bay and the Gulf of Mexico were deployed at two sites within the Chesapeake Bay. Mortality, P. marinas infection (prevalence and intensity), shell height, condition index, and energy reserves (glycogen, protein, and lipid) were compared between these stocks. Oyster stocks from the Chesapeake Bay had higher intensities of Dermo infection than Louisiana stocks, with differences among individual stocks. Throughout the 2-y study, a natural Dermo-resistant stock from Tangier Sound (CTS), was identified. Despite infection intensities approaching those of a susceptible Rappahannock River stock (CRB) and higher than a Gulf of Mexico stock (LOB), CTS consistently had lower mortality for the 2-y grow out, and was comparable to a hatchery disease-resistant strain (XB). At a site (Port Kinsale) where the significant parasite was P. marinus, the LOB stock grew to the largest shell heights and bad significantly lower intensities of infection. However, the performance of the LOB stock was comparatively poorer at the other deployment site (Regent Point) where MSX was present. Shell heights were highest overall in the CRB stock at Regent Point, despite high susceptibility to disease. Condition index varied between stocks, although not necessarily along trends of disease resistance since condition was highest in the CRB and XB stocks. Variations in energy reserves were strongly influenced by season, but not disease, or stock origin. The present study shows that differences between stocks contain an underlying genetic component. Differences seen between deployed stocks in mortality, growth, and condition have strong implications for development of selective criteria for an aquaculture-based industry

Heat Shock Protein 70 Expression in Juvenile Eastern Oysters, Crassostrea virginica (Gmelin, 1791), Exposed to Anoxic Conditions

Anoxic water events in conjunction with summer high temperatures are thought to be one of the causes of declines in natural oyster reefs on the eastern shore of Mobile Bay. Work is underway to determine whether tolerance to low oxygen can be selected for in hatchery-produced oysters. As a component of this work, the expression of heat shock protein 70 (HSP 70) was examined in control (normoxia) and anoxia-challenged juvenile oysters. Parental Eastern oysters, Crassostrea virginica were collected from 2 sites, Cedar Point Reef (CP), an area considered to have normoxic conditions, and White House Reef (WH), an area suspected to experience periodic anoxia. F1 generation oysters were produced from CP and WH parents that survived an anoxic exposure of 96 h. Control F1 generation oysters from both parental stocks not exposed to anoxia were also produced. The F1 generation oysters were subsequently exposed to anoxia or control normoxic conditions, and differences in expression of HSP 70 were examined. Nitrogen was used to create the anoxic conditions for both the parental and F1 generations. Three HSP 70 isoforms—2 constitutive forms (HSC 77 and HSC 72) and 1 inducible form (HSP 69)—were expressed in both anoxia- and normoxia-exposed oysters from all groups. Although there were differences among groups of oysters from the 2 sites, there were no differences in the expression of HSC 77 and HSC 72 between the control and anoxia-treated oysters within a group. Interestingly, the expression of HSP 69 was higher in oysters exposed to normoxia than the ones from anoxia treatments. These differences are thought to reflect a combination of responses to nutritional stress in the controls and facultative anaerobiosis and metabolic arrest in the anoxia groups

Cellular and Transcriptional Responses of Crassostrea gigas Hemocytes Exposed in Vitro to Brevetoxin (PbTx-2)

Hemocytes mediate a series of immune reactions essential for bivalve survival in the environment, however, the impact of harmful algal species and their associated phycotoxins upon bivalve immune system is under debate. To better understand the possible toxic effects of these toxins, Crassostrea gigas hemocytes were exposed to brevetoxin (PbTx-2). Hemocyte viability, monitored through the neutral red retention and MTT reduction assays, and apoptosis (Hoechst staining) remained unchanged during 12 h of exposure to PbTx-2 in concentrations up to 1000 µg/L. Despite cell viability and apoptosis remained stable, hemocytes incubated for 4 h with 1000 µg/L of PbTx-2 revealed higher expression levels of Hsp70 (p < 0.01) and CYP356A1 (p < 0.05) transcripts and a tendency to increase FABP expression, as evaluated by Real-Time quantitative PCR. The expression of other studied genes (BPI, IL-17, GSTO, EcSOD, Prx6, SOD and GPx) remained unchanged. The results suggest that the absence of cytotoxic effects of PbTx-2 in Crassostrea gigas hemocytes, even at high concentrations, allow early defense responses to be produced by activating protective mechanisms associated to detoxification (CYP356A1 and possibly FABP) and stress (Hsp70), but not to immune or to antioxidant (BPI, IL-17, EcSOD, Prx6, GPx and SOD) related genes

Heat shock protein (hsp70) expression and thermal tolerance in sublethally heat-shocked eastern oysters Crassostrea virginica infected with the parasite Perkinsus marinus

To investigate whether sublethal heat shock protects Perkinsus marinus (Dermo)infected oysters Crassostrea virginica from lethal heat stress, and the effects of P. marinus infection on sublethal heat shock response, oysters were first experimentally challenged with P. marinus. Then, when infections in oysters progressed to moderate levels (parasite burden = 10(4) to 10(5) cells g(-1) wet tissue weight), oysters were treated with a sublethal heat shock at 40 C for 1 h (heat shock + Dermo challenge). Other treatment groups included heat-shocked, unchallenged (non-P. marinus challenged) oysters and non-heat-shocked, P. marinus-challenged and -unchallenged oysters. Thermal tolerance was compared among these treatments by administering a lethal heat treatment at 44 C for 1 h, 7 d after sublethal heat shock. Sublethal heat shock enhanced survival to lethal heat treatment in both P. marinus-challenged and -unchallenged oysters. Although levels of hsp70 isoforms (hsp69 and hsp72) did not vary significantly by heat shock or infection with P. marinus, responses due to these treatments were apparent when comparing hsp70 levels within infected and uninfected oysters. Infection enhanced expression of hsp69, regardless of whether oysters were heat shocked or not. In uninfected oysters, hsp72 increased due to heat shock 2 and 7 d post heat shock. Overall, this study demonstrates that heat shock can improve survival in oysters, even in oysters infected with P, marinus. Expression of hsp70 varied among isoforms after sublethal and lethal heat shocks and in infected and uninfected oysters. The heat shock response was not negatively affected by P. marinus infection

Evaluating a novel biodegradable lattice structure for subtropical seagrass restoration

While attention in coastal ecosystem restoration has increased over the last two decades, the success rate of efforts remains relatively low. To increase success rates, physical restoration techniques often utilize supporting or protective materials to provide a stable surface for transplantation, and in some cases reduce herbivory and hydrodynamic disturbances. In this study, we evaluated the effectiveness of traditional (staples, burlap) and novel (BESE- elements, a biodegradable potato starch lattice) physical restoration techniques on the growth of transplanted Halodule wrightii seagrass. A first experiment revealed that seagrass planted in both two-stacked BESE structure without planting holes and four-stacked BESE with holes had significantly higher shoot count and blade length than four-stacked BESE without holes, with the latter design losing all seagrass shortly after deployment as shoots could not float through the structure. In a second experiment, the BESE lattice treatment (four-stacked with holes) had three times the shoot count and equal to greater blade length compared to traditional methods of physical restoration (staples and burlap), likely due to BESE providing some protection from hydrodynamic activity. However, disturbances, possibly including herbivory and hydrodynamic activity (culminating with Hurricane Irma), prevented long term study, illustrating the importance of stochastic abiotic factors in seagrass planting success. Overall our study demonstrates the effectiveness of using BESE lattice designs and similar physical techniques in the restoration of seagrass beds

Quantifying Spatial and Temporal Trends of Microplastic Pollution in Surface Water and in the Eastern Oyster Crassostrea virginica for a Dynamic Florida Estuary

Microplastics (MPs) are a ubiquitous pollutant, emphasizing the need to understand their abundance and the factors that influence these patterns around the globe. In a prior study, high numbers of MPs were found in surface waters and tissues of the oyster Crassostrea virginica collected from one location in the Indian River Lagoon (IRL, FL, USA). To better understand spatial and temporal variability of MPs throughout the IRL, for one year, monthly surface water samples were collected from 35 sites, while oysters were collected quarterly from 12 sites. Microscopy and ATR-FTIR were used to quantify MP. In total, 3755 MPs were found in 44% of water samples (mean density &plusmn; CI: 1.47 &plusmn; 0.09 MP/L). South IRL water had the most MPs, likely associated with proximity to urbanization, inlets (MP sinks) and tributaries (MP sources). MP (n = 3181) were found in 70% of examined C. virginica (n = 1402). Abundances of MP in oysters were lower in the spring and in north IRL. The overall mean abundance was 2.26 &plusmn; 0.16 MP/oyster, and the density was 2.43 &plusmn; 0.52 MP/g wet tissue weight. Our results provide a more complete picture of MPs in the IRL, a subtropical, shallow-water estuarine system