NITROGEN LOADING, CLIMATE CHANGE, PATHOGENS: IMPACTS OF MULTIPLE STRESSORS ON RHODE ISLAND SHELLFISH

Filter feeding bivalves, such as the eastern oyster, Crassostrea virginica and the blue mussel, Mytilus edulis are valued for their role in the marine nitrogen cycle; specifically, their ability to facilitate the process of denitrification (reduction of nitrate to an inert N2 gas), and thereby the removal of reactive nitrogen from the system. Historically, these organisms have been victim of overharvesting along much of the east coast, however Rhode Island has undergone a vast expansion of oyster production through the development of a prosperous aquaculture industry within the state, potentially contributing to the restoration of this valuable ecosystem service. However, the success of all of Rhode Island oyster populations (wild, resorted, cultured) are threatened by anthropogenic stressors, such as warming waters and increased nitrogen loads into coastal habitats, which many interrupt and/or alter the rates at which C. virginica and M. edulis are able to perform the process of nitrogen removal. Of particular concern is incomplete processes of denitrification that may lead to an accumulation of nitrous oxide (N2O), a potent greenhouse gas with global warming potential nearly 300 times more powerful than that of carbon dioxide. Warming waters are also known to favor common oyster pathogens such as Haplosporidium nelsoni, H. costale and Perkinsus marinus. The combination of high temperature and nitrogen loads is likely to cause physiological stress to these organisms, leading to increased susceptibility of the organisms to pathogens and, therefore, further impacting the environmental benefits provided by bivalves. The goal of this study was to investigate how the combination of current and projected temperatures and nitrogen loads may impact the health status, rates of nitrogen removal (N2 production), and rates of N2O production of C. virginica and M. edulis. This was accomplished through separate studies conducted on C. virginica and M. edulis. Two types of experiments were performed with C. virginica: Experiment 1, which was a controlled, laboratory-based study, in which organisms were maintained in a gradient of ammonium nitrate levels (20μM, 40μM, 70μM, 100μM), crossed by contrasting temperatures (18°C, 24°C) (i.e. 8 combinations total). Organisms were maintained in these conditions for 3 months, and rates of denitrification and N2O production were measured at 3 time points over the incubation period in order to determine how these gas productions may change with time of exposure to experimental conditions. Upon completion, prevalence of three common oyster pathogens listed above was determined in a subsample of oysters from each treatment. A similar mesocosms experiment was performed with blue mussels with slightly different experimental conditions (5μM, 10μM, 1.5μM, 25μM and 18°C, 21°C), selected based on biological and ecological differences between oysters and mussels. With the realization that C. virginica tolerates much more variation in environmental conditions within their habitat than just temperature and nitrogen, Experiment 2 was performed, and consisted of two (2016 & 2017), 3-month field manipulations in which oysters where maintained in contrasting ends of the estuarine gradient of Point Judith Pond, in Narragansett, RI were performed. At each location, organisms were deployed in experimental setups and left at either ambient or enriched (20μM) conditions. Oyster growth and mortality and water quality measurements were made at selected time points over the 3 month period and, upon completion, experimental organisms were brought back to the laboratory for a single incubation at ambient conditions (18°C, unenriched site water) in Year 1 (2016), and at contrasting temperatures and high nutrient levels (18°C, 24°C, and 100μM) in Year 2 (2017). The goal was to reveal how previous exposure of oysters in the field to different environmental factors (salinity, pH, chlorophyll-a, oxygen, temperature, and nutrient loading) may have impacted the rates of gas production (N2 and N2O) under high temperature and nitrogen loading. In 2017, a random subset of experimental organisms were sacrificed and analyzed for the prevalence of common oyster pathogens. The major hypotheses for this study included: (1) Temperature will initially increase rates of denitrification of both C. virginica and M. edulis. (2) Increased nitrogen loads will increase rates of denitrification and nitrous oxide production of both C. virginica and M. edulis; (3) The combination of warming and high nitrogen levels over long terms will compromise the health of the organism, causing physiological stress for both C. virginica and M. edulis, and higher nitrous oxide accumulation (possibly via incomplete denitrification). Several major conclusions emerged from this study. Based on both mesocosms and field experiments, temperature appeared to be an initial driver of denitrification for C. virginica, however long-term exposure may act as a stressor, possibly inhibiting the process, indicated by the greater level of mortality within warmer laboratory treatments, over time (F48,71=4.80, p=0.001), and general lack of enhanced rates in association with temperature for both experiments. Additionally, increased temperature may lead to increased N2O production, indicated by the field study (F=-2.76, p=0.014). The combination of nitrogen loading and warming appears to promote N2 consumption, as opposed to denitrification (N2 production), (F16,23=5.21, p=0.011; F=-2.92, p=0.010; lab and field study respectively) as well as increased N2O production of C. virginica over time (F16,23= 4.10, p=0.024; lab study). M. edulis generally supported net denitrification (nitrogen removal) in all tested scenarios of nitrogen availability and temperature (average rate across treatments: 28.01 (+/- 23.93) mmol m-2 day-1), which indicates a greater role of N removal than previously reported, as most past studies have focused solely on sedimentary denitrification. Nitrous oxide production was greatest in the cooler treatments (F48,71=5.17, p=0.027) throughout the M. edulis examination. However, with only ¼ of the nitrogen availability, M. edulis produced N2O similar in range of C. virginica. Finally, in most conditions, both species produce N2 at several orders of magnitude greater than N2O, indicating that environmental benefits of filtering feeding bivalves, at this time, greatly outweigh the negative effects caused by the tested anthropogenic stressors