Award: OCE-1434440

Dynamics of Protistan Grazers: Diversity, Abundance and Prey Relations Intellectual Merit Protists are important as both primary producers and consumers in aquatic microbial ecosystems. Despite decades of studies on protistan grazing, knowledge gaps exist with respect to their abundance, seasonality, and prey selectivity without the bias of cultivation methods. This project built upon an ongoing time series (with hourly resolution since 2006) that utilized a remotely deployed imaging-in-flow cytometer (Imaging FlowCytobot; IFCB) at the Martha?s Vineyard Coastal Observatory (MVCO). An IFCB instrument was modified to permit automated staining and detection of grazing cells, and operated with both standard chlorophyll fluorescence detection mode, as well as staining mode at the Woods Hole Oceanographic Institution (WHOI) dock. Our hypothesis was that the staining capability would allow identification of cells that consumed bacteria or other non-photosynthetic prey, or were not actively grazing, and therefore would be missed by the chlorophyll triggering of the instrument. Results obtained from 2 cruises support the hypothesis of additional cells/cell types detected in the staining mode, but data collected at the WHOI dock did not show a similar increase. It is possible that grazers in the open ocean samples are limited in algal prey and are eating other things, or have not eaten at all. At the dock, there may be enough algal prey that most grazers have consumed something, and are detected in the normal mode of the IFCB. There are also likely differences in the taxa present at the two sites, with small bacterivorous grazers less abundant at the dock site. The maintenance required by the staining instrument made it unsuitable for long-term remote deployment, so the IFCB has now been revised to trigger on side scattering for image collection rather than stain, and new heterotrophic categories have been added to the automated image classifier. Monitoring of both phototrophic and heterotrophic populations have been added to IFCB activity at MVCO and as part spatial monitoring now underway as part of the Northeast U.S. Shelf Long-Term Ecological Research project. The new spatial mapping is supporting hypotheses from the nearshore studies concerning strong seasonal variability in protistan feeding strategies, with early evidence supporting more algal consumption in summer compared to winter. Amplicon sequencing of monthly/bimonthly water samples from MVCO over the study period confirmed a diverse protistan community that exhibits an annual seasonal pattern of recurring taxa, with some inter-annual variation. The patterns of abundance between IFCB images and photosynthetic taxa amplicons were generally similar. As expected, there were a number of taxa present in the amplicon data that were not represented in the regular IFCB data, and the most abundant categories were ciliates and cercozoa. The sequence information was used to identify putative predator/prey and host/parasite interactions by network analysis. A generalist strategy was strongly represented, although specialist taxa were suggested to comprise about a third of the community. Networks based upon syndiniales parasites introduced the potential for novel host taxa, indicating that infectivity testing should include a wider range of organisms. Single cell sequencing further confirmed the diversity of potential prey items for ciliates, suggesting that dinoflagellates and chrysophytes were eaten in addition to diatoms. The single cell analysis also suggested additional novel host/parasite interactions, or the potential for grazing on syndiniales by ciliates. Broader Impacts The results from this project have improved our understanding of grazing communities in situ and their potential prey communities. It has yielded both genetic and morphologic information about the organisms present and the distribution patterns of particular grazer populations. A graduate student obtained a PhD as part of this project, focusing specifically on the ciliate grazers. Several summer undergraduate student researchers participated in analyzing data, and outreach activities were also accomplished to improve community awareness of how oceanographic research is conducted. These included demonstration of the IFCB at a WHOI-sponsored public event, PI participation in a Zephyr cruise with Boston inner city students, lectures and discussion with local Girl Scouts in pursuit of their Oceanography Badge, development of an amplicon sequence project for Dobyns Bennett High School, and an art project where local high school students illustrated door panels that interpreted participating scientist research. Last Modified: 05/23/2020 Submitted by: Rebecca J Gast