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Protists play important roles in grazing and nutrient recycling, but quantifying these roles has been hindered by difficulties in collecting, culturing, and observing these often-delicate cells. During long-term deployments at the Martha’s Vineyard Coastal Observatory (MVCO) (Massachusetts, USA), Imaging FlowCytobot (IFCB) made it possible to study live cells in situ without the need to culture or preserve. IFCB records images of cells with chlorophyll fluorescence above a trigger threshold, so taxonomically resolved analysis of protists is limited to mixotrophs and herbivores, which have eaten recently. To overcome this limitation, I coupled a broad-application ‘live cell’ fluorescent stain with a modified IFCB so that protists which do not contain chlorophyll (such as consumers of unpigmented bacteria and other heterotrophs) can also be recorded. Staining IFCB (IFCB-S) revealed higher abundances of grazers than the original IFCB, as well as some cell types not previously detected. To analyze a 10-year time series of herbivorous ciliates at MVCO and address broad patterns of seasonality of major ciliate classes and their components, I employed a statistical model that estimates a seasonal density pattern and simultaneously accounts for and separates any annual-scale effects. I describe the seasonality of three functional groups: a phototrophic ciliate, a mixotroph, and a group of strict heterotrophs, and comment on potential drivers of these patterns. DNA sequencing has also contributed to the study of protist communities, providing new insight into diversity, predator-prey interactions, and discrepancies between morphologically defined species and genotype. To explore how well IFCB images can be used to detect seasonal community change of the class Spirotrichea, an important and numerous group, I used high-throughput sequencing (HTS), which does not discriminate between chlorophyll-containing cells and the rest of the community. I report on species and genera of ciliates for which morphotype and genotype displayed high congruency. In comparing how well temporal aspects of genotypes and morphotypes correspond, I found that HTS was critical to detect and identify certain ciliates occupying a niche associated with warmer temperatures. I further showed that when these types of analyses are combined with IFCB results, they can provide hypotheses about food preferences. Thesis Supervisor: Dr. Heidi M. Sosik Title: Senior Scientist, Woods Hole Oceanographic Institution

Ciliate micrograzer dynamics of the New England Shelf