Metadata und statistic analysis of archael and bacterial sequences originating from sediments of the Håkon Mosby mud volcano (Z1-Z3, all habitats)

DNA extraction was carried out as described on the MICROBIS project pages (http://icomm.mbl.edu/microbis ) using a commercially available extraction kit. We amplified the hypervariable regions V4-V6 of archaeal and bacterial 16S rRNA genes using PCR and several sets of forward and reverse primers (http://vamps.mbl.edu/resources/primers.php). Massively parallel tag sequencing of the PCR products was carried out on a 454 Life Sciences GS FLX sequencer at Marine Biological Laboratory, Woods Hole, MA, following the same experimental conditions for all samples. Sequence reads were submitted to a rigorous quality control procedure based on mothur v30 (doi:10.1128/AEM.01541-09) including denoising of the flow grams using an algorithm based on PyroNoise (doi:10.1038/nmeth.1361), removal of PCR errors and a chimera check using uchime (doi:10.1093/bioinformatics/btr381). The reads were taxonomically assigned according to the SILVA taxonomy (SSURef v119, 07-2014; doi:10.1093/nar/gks1219) implemented in mothur and clustered at 98% ribosomal RNA gene V4-V6 sequence identity. V4-V6 amplicon sequence abundance tables were standardized to account for unequal sampling effort using 1000 (Archaea) and 2300 (Bacteria) randomly chosen sequences without replacement using mothur and then used to calculate inverse Simpson diversity indices and Chao1 richness (doi:10.2307/4615964). Bray-Curtis dissimilarities (doi:10.2307/1942268) between all samples were calculated and used for 2-dimensional non metric multidimensional scaling (NMDS) ordinations with 20 random starts (doi:10.1007/BF02289694). Stress values below 0.2 indicated that the multidimensional dataset was well represented by the 2D ordination. NMDS ordinations were compared and tested using Procrustes correlation analysis (doi:10.1007/BF02291478). All analyses were carried out with the R statistical environment and the packages vegan (available at: http://cran.r-project.org/package=vegan), labdsv (available at: http://cran.r-project.org/package=labdsv), as well as with custom R scripts. Operational taxonomic units at 98% sequence identity (OTU0.03) that occurred only once in the whole dataset were termed absolute single sequence OTUs (SSOabs; doi:10.1038/ismej.2011.132). OTU0.03 sequences that occurred only once in at least one sample, but may occur more often in other samples were termed relative single sequence OTUs (SSOrel). SSOrel are particularly interesting for community ecology, since they comprise rare organisms that might become abundant when conditions change.16S rRNA amplicons and metagenomic reads have been stored in the sequence read archive under SRA project accession number SRP042162.

Sulphate reduction and methane oxidation rates of Håkon Mosby mud volcano sediments measured at station MSM16/2_855-1 (Z2-S3, aged flow surface)

Sulfate reduction (SR) and anaerobic oxidation of methane (AOM) were measured ex situ by the whole core injection method (doi:10.1080/01490457809377722). We incubated the samples at in situ temperature (1.0°C) for 12 hours with either 14** CH4 (dissolved in water, 2.5 kBq) or carrier-free 35** SO4 (dissolved in water, 50 kBq). Sediment was fixed in 25 ml 2.5% sodium hydroxide (NaOH) solution or 20 ml 20% ZnAc solution for AOM or SR, respectively. Turnover rates were measured as previously described (http://edoc.mpg.de/177065; doi:10.4319/lom.2004.2.171).

Using the critical salinity (Scrit) concept to predict invasion potential of the anemone Diadumene lineata in the Baltic Sea

It is widely assumed that the ability of an introduced species to acclimate to local environmental conditions determines its invasion success. The sea anemone Diadumene lineata is a cosmopolitan invader and shows extreme physiological tolerances. It was recently discovered in Kiel Fjord (Western Baltic Sea), although the brackish conditions in this area are physiologically challenging for most marine organisms. This study investigated salinity tolerance in D. lineata specimens from Kiel Fjord in order to assess potential geographical range expansion of the species in the Baltic Sea. In laboratory growth assays, we quantified biomass change and asexual reproduction rates under various salinity regimes (34: North Sea, 24: Kattegat, 14: Kiel Fjord, 7: Baltic Proper). Furthermore, we used 1H-NMR-based metabolomics to analyse intracellular osmolyte dynamics. Within 4 weeks D. lineata exhibited a 5-fold population growth through asexual reproduction at high salinities (34 and 24). Biomass increase under these conditions was significantly higher (69%) than at a salinity of 14. At a salinity of 7, anemones ceased to reproduce asexually, their biomass decreased and metabolic depression was observed. Five main intracellular osmolytes were identified to be regulated in response to salinity change, with osmolyte depletion at a salinity of 7. We postulate that depletion of intracellular osmolytes defines a critical salinity (Scrit) that determines loss of fitness. Our results indicate that D. lineata has the potential to invade the Kattegat and Skagerrak regions with salinity >10. However, salinities of the Baltic Proper (salinity <8) currently seem to constitute a physiological limit for the species.

Nematode abundance in cold seep sediments of the Darwin mud volcano

During the JC-10 cruise (2007), we sampled the Darwin mud volcano (MV) for meiofaunal community and trophic structure in relation of pore-water geochemistry along a 10 m transect from a seep site on the rim of the crater towards the MV slope. Sediment samples were retrieved by the ROV Isis using push cores. On board and after the pore water extraction, the top 10 cm of the cores were sliced into 1 cm sections and fixed them in 4% formaldehyde for meiofaunal community analysis. In the home laboratory, the formaldehyde-fixed samples were washed over a 32 µm mesh sieve and extracted the meiofauna from the sediment by Ludox centrifugation (Heip et al. 1985). Meiofauna was then sorted, enumerated and identified at coarse taxonomic level. From each slice, ca. 100 nematodes were identified to genus level. Afterwards, abundance of Nematoda were depth integrated over the top 5 cm to gain individual abundances per 10 cm**2. Overall, total nematode biomass in the top 5 cm of the seep sediment core was ~10x higher than that in the core taken 1100 m away. Nematode genus composition varied little among cores and was mainly dominated by Sabatieria.