Microbes constitute the bulk of the biodiversity and biomass on Earth and have critical impacts on the function of ecosystems. In order to understand relationships between microbial eukaryotes within the planktonic food web, my goals are to characterize the taxonomic and functional diversity and spatiotemporal variability of the microbial community. This includes, but is not limited to, examining the trophic links between microzooplankton and both phyto- and zooplankton. I also aim to capture species-specific relationships within the planktonic food web through analyses of prey-predator interactions and microcosm experiments. I use a combination of microscopy, molecular tools including single-cell and community genomics and transcriptomics, and bioinformatics to explore these questions. Together, these approaches increase our understanding of the dynamics underlying the microbial portion of marine food webs.
Understanding the planktonic food web is crucial for predicting the impacts of global climate change on ocean biodiversity and biogeochemical cycling. Since Pomeroy (1974) suggested the importance of micro-heterotrophs for carbon fluxes in marine systems, it has become increasingly clear that protists play a key ecological role in the planktonic food web (Fig 1). Heterotrophic protists, the focus of my work, are key members in the food web, as they are the intermediate between small animals and both bacteria and phytoplankton (Azam et al 1983; Sherr & Sherr 1986).
Model of the fluxes estimated and observed during the In situ survey and microcosm experiment in the Eastern English Channel (Grattepanche 2011, PhD defense)
During my graduate training, I examined the community structures and succession of phytoplankton, microzooplankton and mesozooplankton (copepods). This work highlighted: (i) the seasonal and annual variability of heterotrophic protists and its strong relation to phytoplankton succession, bloom magnitude and duration (bottom-up control), and the presence of predators such as copepods (top-down control; Grattepanche et al 2011a); and (ii) the importance of dinoflagellates as major consumers of phytoplankton, particularly with a size > 100µm and ciliates as very efficient grazers of small phytoplankton (<10 µm; Grattepanche et al 2011b). The work indicates the importance of species-specific relationships among microbial eukaryotes and provides greater details on the ‘black boxes’ within the planktonic food web.
A deeper understanding of species-specific links within the planktonic food webs is related to both the spatial scale and ecological drivers of microbial diversity. As a postdoctoral fellow at Smith College, I have been studying the diversity of ciliates and the larger clade SAR (Stramenopiles, Alveolata, and Rhizaria), to assess how biodiversity varies with abiotic and biotic factors. Using DGGE, we observed that changes in ciliate community structure at small scales relate to the tides (Grattepanche et al 2014a), the presence of a large scale assemblage ranging almost everywhere between the coast of Rhode Island and the continental shelf break (160km), and throughout the water column, from the surface to our deepest samples (Fig. 2; up to 850 m; Grattepanche et al 2015).
Fig 2. Ciliate assemblages observed off the coast of the New England using DGGE (Grattepanche et al 2015)
While patterns of abundant community members can be observed by using “classic” tools such as microscopy, clone libraries, DGGE (Grattepanche et al 2014a, 2015), the use of high throughput sequencing (HTS) technologies allow observations of patterns at finer taxonomic scales. One of the more dramatic insights from HTS has been the discovery of numerous rare lineages of both bacteria and archaea in natural environments (i.e. the rare biosphere; Sogin et al 2006) as well as tremendous diversity of eukaryotes in our analyses (e.g. Santoferrara et al 2014, 2016; Grattepanche et al 2014b, 2016a and b). Assessing the diversity of eukaryotic microbes requires accounting for both the technical limits (e.g. bias, accuracy, sample size) and the heterogeneity in units of biodiversity (e.g. species) that vary based on analyses of molecules, morphology and behavior (Grattepanche et al 2014b).
Fig 3. HTS analyses of ciliates throughout the water column reveal unexpected diversity below the photic zone (dashed line). Numbers are OTU richness (Grattepanche et al 2016a).
Analyses of HTS data on ciliates from coastal waters in New England reveals an increase of diversity with the depth, particularly below the photic zone (Fig 3; Grattepanche et al 2015, 2016), which contradicts previous estimates based on microscopy and clone libraries (e.g. Christaki et al 2011, Wickham et al 2011). RNA/DNA comparisons using DGGE show that these diverse communities observed below the photic zone are active (Tucker et al submitted). These HTS analyses also provide evidence for two novel Spirotrichea lineages, named Clusters X and Cluster Y, which lack morphological information (Grattepanche et al 2016a). I am currently using FISH (Fluorescence In Situ Hybridization) to identify these ciliates from marine samples. In my previous studies, we observed that the pattern of communities is generally not well connected to the environmental parameters measured. My last accepted manuscript (Grattepanche et al 2016b) shows that individual OTUs have distinct biogeographies related to the depth (surface, chlorophyll maximum, deep) and to the position on the shore (Fig 4).
Fig 4. Spatial analyses of ciliate diversity reveal patchiness of the communities at inshore stations and distinct biogeographies of individual OTUs (Grattepanche et al 2016b).
While I continue to explore the spatial scale of protist diversity, I have begun to use an experimental approach to elucidate the processes that drive these patterns. I created microcosms, small scale replicates of marine environments that can be subject to varying environmental changes, to assess how bottom-up and top-down controls impact the ciliate community. In a pilot study, I used metatranscriptomic approach to assess the gene involved in the food web under bottom-up and top-down control. Preliminary results show a similar number of gene family expressed in the nanosize and microsize, but nanosize and microsize expressed different genes (e.g. Chlorophyll associated protein are highly expressed in the microsize and Dynein in the nanosize) and nanosize fractions seem to have a greater diversity of gene expressed.
Contrary to conventional wisdom, my diversity studies using DGGE and HTS reveal an incredible diversity of small ciliates in the deep ocean environment (up to 850m of depth; Grattepanche et al 2015, 2016b). I will conduct additional analyses of this extreme environment, which I predict will lead to the discovery of a large number of previously-unknown microbial eukaryotic lineages. I plan to increase my taxonomic focus to understudied microbial lineages such as Foraminifera and Radiolaria (Grattepanche et al in prep). I will sample microbial communities at varying depths below the photic zone to elucidate members and patterns in the deep-water environment. I will also analyze patterns in both DNA and RNA to understand what proportion of the community is most active. Knowledge of these organisms will allow us to have a better picture of the biodiversity on Earth and may fill some gaps in the eukaryotic tree of life. It is even possible that some of these deep-water microbes could represent ‘living fossils’ and thus will help us understand more about the Last Eukaryotic Common Ancestor (LECA).
Another direction that I intend to pursue is the study of the ecological function of plankton communities. Metatranscriptomic approaches allow for investigation of the active genetic content of the planktonic food web in various ecosystems. Such analyses will reveal the ecological functions present in the ecosystem and aid in identifying new genes of ecological and biotechnological importance (e.g. toxin production); SIP-FISH, which combines FISH and Stable Isotope Probing, and in vitro experimentation, can be powerful tools for elucidating ecological function. While SIP-FISH is yet to be developed for microbial eukaryotes, this kind of approach will allow the tracking of gene expression, which can be related back to ecosystem function. Overall, metatranscriptomics will serve as one essential component for assessing ecosystem health.
I will combine my expertise in HTS and experimental (e.g. microcosm) studies to explore how the diversity of the microbial eukaryotic community varies in response to conditions that mimic climate change. Global warming will affect abiotic parameters such as temperature, acidity, and speed of thermohaline circulation, as well as biotic factors such as the magnitude of harmful algal blooms. Understanding how and at which magnitude the microbial diversity will be affected by these changes is of particular interest. These studies will require an interdisciplinary and collaborative approach to: (1) identify species-specific impacts on biogeochemical cycles, (2) capture the abiotic variation within microhabitats and (3) understand what affects microbial diversity at varying spatial and temporal scales.