Award: OCE-0849578

Award Number: 0849578 Project Outcomes Report Principal Investigator: Virginia P. Edgcomb Organization: Woods Hole Oceanographic Institution Submitted by: Virginia P. Edgcomb Program Officer Name: David L. Garrison Title: Pickled Protists or Community Uniquely Adapted to Hypersalinity Goals: The overall goal of this project was to explore protistan life in the in the seawater-brine interfaces of both the water column and within sediments, as well as the brines, of geochemically contrasting deep hypersaline anoxic basins in the deepest part of the Eastern Mediterranean Sea (~3500-4000m water depth). We sought various lines of evidence for active eukaryotic communities, including visualization of intact cells with intact nuclei using light and epifluorescence microscopy, positive labeling by CellTracker Green which reacts with esterases, positive iTag amplification using extracted RNA as template (cDNA), and detection of expressed metabolic genes within the mRNA fraction of isolated total RNA. Outcomes: We analyzed the first comparative metatranscriptome of a microbial community along a water-column halocline of the deep hypersaline anoxic basin Thetis. This study provided remarkable insights into community activities and biogeochemical cycling in this deep extreme habitat, including the discovery of nitrogen fixation in the anoxic most hypersaline water layer, strategies used for osmotic balance, and insights into carbon fixation pathways utilized under those conditions. Driven by the needs of this project, a new water-column sampler (the Microbial Sampler-Submersible Incubation Device (MS-SID)) was developed for collecting filtered and unfiltered water samples and preserving them in situ in order to minimize biases associated with sample handling and recovery from the deep ocean. This instrument is helping to reshape how microbiologists examine communities in the deep sea and is now available for use by the wider community. Using this instrument we conducted the first grazing study to determine the impacts of protist phagotrophy on microbial communities in deep-sea waters. These studies included DHAB halocline waters and applied in situ approaches to assess impacts on carbon turnover in the deep sea. This showed that protist phagotrophy turns over approximately 25% of the prokaryotic biomass daily, and likely contributes significantly to new sources of labile organic carbon at these sites/depths. Using the MS-SID we conducted the first direct comparison of metatranscriptome results for a deep-water (2200m) sample collected using this technology vs. traditional Niskin-bottle water collection with subsequent on-deck water processing. This revealed the importance of using in situ approaches for precise assessments of in situ activities. Scanning electron microscopy and fluorescent in situ hybridization (FISH) revealed intact protists from water-column haloclines and brines, including numerous ciliates hosting visible epibionts in several halocline and some brine samples. Using the Remotely Operated Vehicle (ROV) Jason, we collected precisely placed cores from where the halocline impinges on the seafloor. These cores were collected from different zones of the halocline, to allow comparisons of their protistan communities. Using microscopy, we visualized living (DAPI-positive, esterase active, and intact) protists in halocline sediments from three different DHABs (LÆAtalante, Urania, Discovery) and we isolated intact messenger and ribosomal RNA from protists in these habitats. Halocline sediments appear to support more abundant communities of protists than the control (normal salinity) and brine sediment samples. A metatranscriptome study of prokaryote and protist communities in DHAB halocline sediments provided additional new lines of evidence for active groups of protists, fungi, bacteria and archaea in these hypersaline habitats, including expression of genes associated with active sulfur, carbon and nitrogen cycl...