University of Essex BIODEEP-WP3 Analysis of species diversity, community structures and phylogeny of microorganisms and meiofauna in the Mediterranean deep-sea hypersaline anoxic basins (DHAB) Andrea Sass, Terry McGenity

 Amplification with fluorescent eubacterial primers successful  Water, brine and interface samples were all investigated Not successful:  amplification with Archaeal primers  DNA extraction from most sediment samples  amplification from Discovery brine and interface Summary of achievements up to date: Community structure fingerprinting via t-RFLP of 16S rDNA

Summary of previously presented results:  brines of each basin unique patterns  l’Atalante and Bannock brines more similar  patterns stable over time  same patterns from Urania brines 1 and 2  number of fragments relatively small Brine samples:

new achievements: amplification from l‘Atalante and Bannock basin sediments successful

Bannock sediment sample and brine Sediment top Brine Fragment length RFU Amplification with eubacterial primers, digestion with AluI

Brine samples taken at same location and depth? Brine Sediment L’Atalante sediment and brine Sediment top covered with black ooze Cruise 2002 Cruise 2001 amplification with  -casein Sediment top Sediment bottom amplification with  -caseine extraction with phenol-chloroform RFU Fragment length Amplification with eubacterial primers, digestion with AluI

patterns from sediment samples very similar to brine samples no unique fragments found in sediment samples Community in the sediments widely the same as in brines?  sediments consist mainly of brine  both sediments and brine are anoxic Extraction methods yield DNA only from the brine fraction of the sediment?

Interfaces Patterns from interfaces unique for each basin and different from brines and oxic seawater Certain fragments occur only at certain salinities within the interface Unique community or accumulation of microorganisms from the water column? Bannock interface samples taken in intervals at different salinities Summary of previous presented results:

Salinity 25% = brine Salinity 21.5% Salinities % Salinity 12.7% Salinities % Salinities % Oxic water, 3000 m depth Fragment length Bannock interface Amplification with eubacterial primers, digestion with AluI RFU

Sediment traps 2 weeks 6 months 12 months Amplification with eubacterial primers, digestion with AluI Fragment length RFU

new achievements: investigation of Urania basin interface with greater spatial resolution

Salinity 5.7 ‰ Salinity 15.4 ‰ Salinity 6.3 ‰ Salinity 9.0 ‰ Salinity 13.1 ‰ Salinity 21.1 ‰ Salinity 15.6 ‰ Salinity 18.4 ‰ Brine Urania interface Amplification with eubacterial primers, digestion with AluI Fragment length RFU

 fingerprints change gradually from interface to brine  no succession of microbial communities within interface interface not as stable as Bannock interface?

Bannock and Urania upper interfaces: community fingerprints are different from oxic deep-sea water change in communcity structure starts at seawater salt concentration above the sampled interface?

Amplification with eubacterial primers, digestion with AluI Fragment length Urania interface compared with oxic water samples samples taken 2001 RFU Urania interface oxic water 3500m depth oxic water 3000m depth

Different pattern in water and interface samples due to different amount of water filtered ? Amplification with eubacterial primers, digestion with AluI Patterns from the same DNA extract with different amounts of DNA addes to PCR reaction Fragment length RFU

Summary of newly achieved results: Microbial community in Bannock and l‘Atalante sediments similar to those in corresponding brines Succession of different comminties at different salinities within the interface more pronounced in Bannock interface than in Urania interface Upper interfaces different from sea water