Table of Contents

• Dataset Description
  • Methods & Sampling
  • Data Processing Description
• Data Files
• Parameters
• Instruments
• Deployments
• Project Information
• Funding

Pyrotags of bacteria and archaea obtained from Urania, Discovery, and L’Atalante DHAB halocline sediments.

Sequences have been submitted to GenBank under BioProject PRJNA270764. They will be publicly available once the manuscript is accepted (Kormas et al. submitted to Extremophiles)

ROV Jason was used to collect push cores 6.35cm diameter obtained from the Deep Submergence Lab at Woods Hole Oceanographic Institution (www.whoi.edu/groups/DSL/) and configured with a seal to prevent contamination during ascent from the halocline sediments of L’ Atalante, Urania and Discovery basins in the eastern Mediterranean Sea at a depth of about 3500-4000m. Sediments were profiled for oxygen and immediately frozen upon return to surface.

RNA was purified using the MEGAclear kit (Ambion, USA). Reverse transcription of the purified RNA samples was performed using the QuantiTect kit (Qiagen, USA). Tag-pyrosequencing of the 16S rRNA gene was performed using PCR amplification of the V4-V6 region of the 16S rRNA gene and the primer pair S-DBact-0341-b-S-17 (5’-CCTACGGGNGGCWGCAG-3’) and S-D-Bact-0785-a-A-21 (5’-GACTACHVGGGTATCTAATCC-3’) for Bacteria (Klindworth et al. 2012), and archaea349F (5’-GYGCASCAGKCGMGAAW-3’) and archaea806R (5’-GGACTACVSGGGTATCTAAT-3’) for Archaea (Takai & Horikoshi 2000), as described in Dowd et al., (2008). A one-step, 30-cycle PCR reaction was performed using HotStarTaq Plus Master Mix Kit (Qiagen, USA).  Following PCR, all amplicon products (ca. 450 bp) from different samples were mixed in equal concentrations and purified using Agencourt Ampure beads (Agencourt Bioscience Corporation, USA). Samples were sequenced utilizing the Roche 454 FLX Titanium platform and reagents according to the guidelines at the MRDNA Ltd. (Shallowater, TX, USA) sequencing facility. Processing of the resulting sequences, i.e. trimming and quality control, was performed with the MOTHUR software (v 1.30)  including denoising of the flowgrams using PyroNoise and data normalization to the smallest number of sequences in the resulting libraries. Sequences ≥250 bp with no ambiguous base assignments and no homopolymers ≥8 bp were included in downstream analyses. Single singletons, i.e. sequences that appeared only with once in the whole dataset, were excluded from further analysis. These sequences were aligned using the SILVA SSU database (release 108). All sequences were binned into Operational Taxonomic Units (OTUs) and were clustered (average neighbor algorithm) at 97% sequence similarity. Coverage values were calculated with MOTHUR (v 1.30).

BCO-DMO Processing:
- original file: EdgcombDHABprokaryoteTags.xlsx
- replaced positions for Discovery and Urania with those in event log for pushcores
- added html links to GenBank BioProject
- added cruise_id, lat, lon

Protists are an essential component of microbial food webs and play a central role in global biogeochemical cycles, and thus are key players in sustaining the healthy functioning of any ecosystem. Over the past few years a rich diversity of protists has been revealed in a range of extreme environments, indicating that the frontiers of eukaryotic life are still being explored. Only recently, one of the most extreme marine environments known to science was discovered in the eastern Mediterranean Sea at a depth of ~3500m, namely deep hypersaline anoxic basins (DHABs). These basins are characterized by extremely high salt concentrations (up to saturation) that have been considered anathema to life. Instead, highly diverse communities of bacteria exist in the waters of these basins. With the exception of a preliminary study to this proposal that indicated a diverse and active assemblage of protists in the water column along the halocline and below the halocline, these DHABs remain largely unexplored regarding eukaryotic life forms. The sediments of the DHABs have not been explored for protists at all.

The investigators will collect water column and sediment samples on a short cruise to two basins with different brine chemistries. An exciting combination of molecular, cultivation-independent and culture-based approaches will be used to study the microbial communities of two basins. Investigators will use those approaches to determine adaptive strategies of marine protist communities to hypersaline, anoxic environments and the degree of their potential impact on biogeochemical cycling as a result of their predation activities, the degree to which the dominant protists maintain bacterial or archaeal symbionts, and the identity of those symbionts. The original research proposal identified Bannock and Discovery Basins as the field study areas, however the 2009 cruise collected samples at Discovery and Urania Basin. Methods to be employed include RNA-based sequence analysis of diversity based on 18S rDNA genes, statistical analyses of community composition and phylotype richness, geochemical documentation of the water column and sediments using classical and microelectrode approaches, expression profiling using 3'-UTR fragments of mRNAs, sequencing of complete gene transcripts for proteins appearing to confer adaptation to hypersalinity, analysis of the proteome signatures, FISH-SEM to characterize novel extremophiles, CARD-FISH to identify eukaryote prey and putative symbionts, and TEM to assess morphology and endobiont presence in common benthic morphotypes.

Hypersaline environments rank highly in the list of extreme systems that have attracted increasing notice in science as well as by the lay public. For example, considering predictions of increasing temperatures and drought in certain regions of our planet, the number of hypersaline habitats may increase dramatically causing this ecosystem to gain importance on a global scale. Thus, an understanding of the ecosystem in these habitats will help predict future ecosystem functioning due to global change. From a different perspective, revealing the mechanisms of adaptation to high salinity has become a major objective, both for biological science and for potential commercial exploitation of natural products associated with those adaptations.