Award: OCE-1505604

Intellectual Merit: A key outcome of this project was novel information regarding fractionation of nitrogen (N) by dinoflagellates when provided micromolar concentrations of NO3-, NH4+, urea, or a combination of the three. The results presented here for dinoflagellates are consistent with the isotopic fractionation published for other taxonomic groups, namely diatoms and coccolithophores, but add new insight into the biochemistry of N assimilation utilization for toxin production by dinoflagellates, and how preferential/multiple source uptake affects d15N. Additionally, our data suggest that understanding the dominant N chemical forms in a system is important for management, i.e., as N chemical form controlled toxicity (but not growth rates), and when utilizing the N isotope ratio of particulate organic matter as a tracer of N source that fueled a dinoflagellate bloom. Toxin content and toxicity of A. fundyense were dependent upon the N chemical form being utilized, as well as the A. fundyense strain, suggesting that eutrophication and dominating strains can affect cellular toxicity of a bloom. High inputs of urea, for example, may lead to large blooms of A. fundyense with relatively low toxicity per cell. Causal analysis of nutrient pollution sources via stable isotopes will advance sustainability for the environment, economy, and human health by helping policy makers make informed decisions about point and nonpoint source pollution control in the United States. Nutrients are only one factor influencing bloom dynamics, but the relative importance of natural or anthropogenic nutrients in the development of a specific toxic bloom is necessary to predict future decadal, annual, and compositional shifts in algal blooms. In summary, this study is the first to show the impact of N chemical form and N preference on N isotope fractionation during uptake by dinoflagellates. Overall, a better understanding of N metabolism within dinoflagellates and the role of N in the synthesis of toxins would enhance prediction and control of harmful algal blooms. Broader Impacts: This project supported the transition of female Lead PI-Smith from postdoc to new faculty member (tenure track) at the Virginia Institute of Marine Science (VIMS), VA. This transition included 1) the transfer of culturing techniques (A. fundyense) to VIMS, 2) the recruitment of the first graduate student, undergraduate, and staff member to the new lab, and 3) the successful completion of a M.S. degree by C. Taylor Armstrong under the new faculty member. C. Taylor Armstrong, a recent graduate (M.S.) of VIMS from the research program of PI-Smith, just defended her final thesis based solely on this NSF project. She begins her next step in her career in February 2017 as a Knauss Fellow with NOAA. Armstrong is currently converting her thesis to a publication. She presented her preliminary results at a national conference, 15-20 November 2015, in Long Beach, CA and expects to present a poster on her final results at ASLO, HI in March 2017. The project has also forwarded the training of two undergraduates. In year one, we provided technical training to an undergraduate student, Jennifer Haskell from Northeastern University, in culturing techniques for Alexandrium, cell enumeration, toxin extraction and purification, preparation of samples and solvent for saxitoxin quantification by HPLC-FLD, and the analysis of 12 saxitoxin congeners. Haskell?s involvement in this project was part of her six-month REU co-op in the Anderson Lab, at Wood Hole Oceanographic Institution. In year two and three, we brought on another undergraduate student, Shannon Jones, also an REU student from Northeastern University. Jones continued the culturing work started by the previous intern, and then took on more independent tasks, such as helping in the development of field sampling protocols, processing of samples post-collection, leading the weekly sampling for this project in Mill Pond and Salt Pond, participating in week-long research cruises in the Gulf of Maine, data management, and sample processing and storage. Through this co-op experience, Jones learned new skills in culturing at VIMS, field sampling on small boats and larger vessels, and laboratory techniques associated with phytoplankton extraction and toxin quantification. Last Modified: 01/29/2017 Submitted by: Juliette L Smith