(Massachusetts Institute of Technology and Woods Hole Oceanographic Institution, 2017-06)
Brownlee, Emily F.
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 theMartha’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.
