1. 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

2. Genes from different microorganisms Methods:  Cell preservation and lysis  DNA recovery and cleaning  Amplification of 16S rRNA gene with eubacterial and archaebacterial primers, labelled with fluorescent dyes  Digestion with restriction enzymes  Separation of DNA fragments  Detection of label  Community structure profile / fingerprint  Alignment of fragments  Cluster analysis  Find relation between environments University of Essex Total DNA Amplification of 16S rRNA gene Gene fragments Digestion with restriction enzyme

3. Samples included in last report: Interface brine and brine body from l’Atalante, Urania and Bannock basins Oxic water from different locations and depths University of Essex Samples not included: Discovery brine and interface: DNA extracted but no amplification possible Sediments: no DNA could be extracted

4. Clustering of t-RFLP fingerprints, data from restriction digestion with Alu I and Cfo I combined University of Essex Euclidean distance Urania basin brine Bannock basin interface l‘Atalante basin interface Bannock basin brine l‘Atalante basin brine Urania basin interface oxic water near Discovery, 3500 m depth oxic water near Bannock, 3000 m depth oxic water near Discovery, 3300 m depth oxic water near Discovery, 2500 m depth

5. University of Essex  Profiles of brines showed unique peaks not found in oxic water  l‘Atalante and Bannock basin brine similar  Samples from oxic water similar  Similarity of interface samples to each other and oxic or brine water not consistent Observations:

6. University of Essex  The basin brines contain unique microbial communities  The differences in community structure profiles of the brines possibly reflect the difference in their chemical composition  A microbial community unique to the interfaces may exist Conclusions:

7. University of Essex approaches from June 2002: new samples from  All brines and sediments  Different locations within the Urania basin, from different depth within the brine  Differentiated depths within the Bannock and Urania basin interface all sediments  Amplification with archaebacterial primers  Comparison of t-RFLP patterns of sequences from isolates and clones with the patterns from the total community

8. University of Essex Urania brines Urania east brine 1, 2002 Urania east brine 2, 2002 Urania west brine 1, 2002 Urania east brine 1, 2001 t-RFLP fingerprints obtained after digestion with Alu I

9. University of Essex l‘Atalante brine Bannock brine 2001 2002 2001 2002 t-RFLP fingerprints obtained after digestion with Alu I 

10. University of Essex Bannock interface 5 layers salinities 3.8-7.6% 8 layers salinities 8.7-10.8% 1 layer salinity 12.7% 3 layers salinities 13.8-15.9% 1 layer salinity 21.5% 1 layer salinity 25% Niskin layer 1-2 Niskin layer 1-10 Niskin layer 5-5 Niskin layer 5-8 Niskin layer 10-5 Niskin layer 12-5 t-RFLP fingerprints obtained after digestion with Alu I      

11. University of Essex Upper Bannock interface Lower Bannock interface Niskin layer 1-2 salinity 3.7% oxic water near Bannock basin, 3000 m depth t-RFLP fingerprints obtained after digestion with Alu I Niskin layer 12-5 salinity 25% Bannock brine 2001

12. University of Essex Niskin layer 1-2 Niskin layer 1-10 Niskin layer 5-5 Niskin layer 5-8 Niskin layer 10-5 Niskin layer 12-5 Bannock interface 2001 Bannock interface 2002

13. University of Essex Sediment traps from Bannock basin 12 months 6 months 2 weeks t-RFLP fingerprints obtained after digestion with Alu I

14. University of Essex Sediment traps Bannock interface 12 months 2 weeks 6 months Layer 1-2 salinity 3.8 % Layer 1-10 salinity 11.3 % Layer 5-5 salinity 12.7 % Layer 5-8 salinity 15.4 % Layer 10-5 salinity 21.5 % Layer 12-5 salinity 25 %

15. University of Essex Summary of preliminary results:  Bannock and Urania brines from different years very similar  Brines from different locations and depth of the Urania basin very similar  Little changes in l‘Atalante brine  Changes in the profiles of the Bannock interface with rising salinity  Fingerprints from sediment trap material show most similarity to certain samples from within the Bannock interface  No correspondence yet found between profiles from isolates and environmental DNA from Bannock interface (only major peaks considered)

16. Conclusions:  A community inherent to Bannock basin interface exists  Uniform bacterial communities in Urania brines  Relatively stable bacterial communities in brines

17. University of Essex  Closer examination of Urania interface  Profiles from more sediment trap samples  More attempts to obtain profiles from sediments and Discovery brines  Amplification with archaebacterial primers Future work: