In the microbial realm, one of the three domains of life -- the Eukarya -- has received little attention in deep-sea research. This stands in contrast to the fact that in all known aquatic environments, and measured by the amount of material and energy transferred, the link between prokaryotic and eukaryotic cells is one of the most significant trophic interactions on Earth. In terms of volume, the deep sea is the largest biome, and despite its tremendous role in long-term biogeochemical cycles, it has largely been neglected. Biological activity in the deep sea is neither negligible nor homogeneous in space and time. Recent data suggest that biological activity in the dark ocean (as evidenced by respiration rates, bacterial secondary production and a variety of other metrics) is much higher than anticipated from all known organic carbon fuel sources combined (i.e., POC flux, DOC convection, in situ production and active transport by zooplankton). Water masses in the deep ocean represent highly-diverse biogeographic regions with distinct communities and particle distributions. Moreover, because of feeding thresholds, cold temperatures, extreme pressures and unique adaptations that deep-sea microbes exhibit, biological activity rules cannot simply be extrapolated from laboratory cultures and from experiments with surface-dwelling microbes. This study focuses on the fundamental role of eukaryotic microbial communities in deep-sea ecology with the overarching hypothesis that protists represent sensitive biological indicators of utilizable organic carbon. There is good reason to believe that microbial eukaryotes and their activities are better indicators of "new" sources of organic carbon than particle inventories, sediment traps, isotope ratios, or models based on surface production and theoretical flux attenuation. For these new biological indicators to work, however, one needs to separate live from the moribund and dead cells, the bacterivores from saprotrophs, the inactive resting stages from those actively feeding on prokaryotes, the gametes and zoospores from vegetative and feeding stages, and those located on particles from the ones freely suspended in the water column. Each of these groups represents different levels of per-cell energy and carbon requirements. Intellectual merit: This study determines the ecological role of eukaryotic microbes in the deep North Atlantic over large geographic regions. The research incorporates two fundamentally different experimental designs that capitalize on different time scales: 1) Short-term incubations (~72 hours) of respiratory activity and bacterivory combined with a high resolution sampling of abundances across large geographic regions performed from a research vessel, and 2) Long-term incubations (=/> 4 weeks) measuring colonization of sinking particles and growth of eukaryotic microbes using free-falling (untethered) vehicles representing the first attempt of physiological rate measurements directly in the deep sea. Methods include new tracers for bacterivory, incubations for single-cell respiration, taxonomic identification using fluorescence in situ hybridization, single-cell genomics, and the first of its kind deep-sea holographic microscope capturing images to a maximum depth of 6000 m at 5 micrometer resolution. Broader Impacts: This project supports undergraduate and graduate research and enhances undergraduate curriculum in biological oceanography courses held by the PI. Workshops for underrepresented groups will be held in Puerto Rico. A permanent exhibit on the role of microbes in the ocean will be installed at a major museum and interpretation center in Hampton Roads. This research contributes significantly to the development of new technology for oceanographic research with emphasis on deep-sea environments and supports international scientific research collaborations.
