Bacterial Microflora of the Cold-Water Coral Lophelia pertusa (Scleractinia, Caryophylliidae) Bakterielle Mikroflora der Kaltwasser-Koralle Lophelia pertusa (Scleractinia, Caryophylliidae) Dissertation zur Erlangung des akademischen Grades Doctor rerum naturalium (Dr. rer. nat.) der Mathematisch-Naturwissenschaftlichen Fakultät der Christian-Albrechts-Universität zu Kiel vorgelegt von Sven Christopher Neulinger Kiel, im April 2008 Referentin: Prof. Dr. Karin Lochte Korreferentin: Prof. Dr. Tina Treude Tag der Disputation: 10. Juni 2008 Zum Druck genehmigt: 10. Juni 2008 …………………………………. (Der Dekan) II “I am a firm believer that without speculation there is no good and original observation.” Charles Robert Darwin (1809-1882) in a letter to Alfred Russel Wallace, 22 December 1857 III INDEX Index Summary ............................................................................................................................... 1 Kurzfassung ..........................................................................................................................2 1 Introduction ..................................................................................................................3 1.1 The Cold-Water Coral Lophelia pertusa.........................................................................................3 1.2 Coral-Associated Bacteria..............................................................................................................7 1.3 Aims of this Study ..........................................................................................................................8 1.4 General Strategy............................................................................................................................10 2 Materials and Methods ............................................................................................... 13 2.1 Location and Sampling ................................................................................................................13 2.1.1 Topographic, Hydrological, and Biological Aspects of the Trondheimsfjord ....................13 2.1.2 Sampling and Fixation .................................................................................................................14 2.2 DNA Extraction ...........................................................................................................................17 2.3 T-RFLP ..........................................................................................................................................17 2.4 Cloning and Sequencing ..............................................................................................................19 2.5 Fluorescence In-Situ Hybridisation ...........................................................................................22 3 Results .........................................................................................................................26 3.1 T-RFLP ..........................................................................................................................................26 3.1.1 Comparison of Coral and Environmental Samples.................................................................26 3.1.2 Differences between Coral Samples ..........................................................................................31 3.2 Cloning and Sequencing ..............................................................................................................35 3.2.1 Phylogenetic Analyses..................................................................................................................35 3.2.2 Comparison of T-RFLP Data and 16S rDNA Sequences......................................................46 3.3 Fluorescence In-Situ Hybridisation ...........................................................................................48 3.3.1 Bacteria Associated with Coral Ectoderm ................................................................................48 3.3.2 Bacteria Associated with Coral Endoderm...............................................................................56 4 Discussion ...................................................................................................................58 4.1 Differences in Bacterial Assemblages of L. pertusa..................................................................58 4.1.1 Comparison of Coral and Environmental Samples.................................................................58 IV INDEX 4.1.2 Differences between Coral Samples ..........................................................................................59 4.1.3 Comparison of T-RFLP Data and 16S rDNA Sequences......................................................61 4.2 Dominant Bacterial Groups of L. pertusa and their Ecological Potential.............................62 4.2.1 Phylogenetic and Ecological Inference .....................................................................................62 4.2.2 Sulphur Cycling.............................................................................................................................63 4.2.3 Methylotrophy...............................................................................................................................65 4.2.4 Parallels to Other Symbiotic and Parasitic Associations.........................................................67 4.3 In-Situ Location of L. pertusa-Associated Microbes ................................................................71 4.3.1 Bacteria Associated with Coral Ectoderm ................................................................................71 4.3.2 Bacteria Associated with Coral Endoderm...............................................................................72 4.3.3 Comparison of Sequence Frequencies and Bacterial in-Situ Abundances...........................73 4.4 Relations between L. pertusa and its Associated Bacteria........................................................75 4.4.1 Partitioning and Specificity of the Bacterial Community........................................................75 4.4.2 Implications for Nutrition, Health, and Dispersal of L. pertusa.............................................78 4.4.3 Bacterial Community Composition and Colouring of L. pertusa ...........................................82 4.4.4 The Role of Tissue-Associated Bona Fide TM7 and Mycoplasmataceae..................................83 4.4.5 Proposal of “Candidatus Mycoplasma corallicola” ...................................................................85 4.5 Significance and Outlook ............................................................................................................86 Acknowledgements.............................................................................................................87 References...........................................................................................................................89 Appendix............................................................................................................................111 Declaration of Academic Integrity / Selbstständigkeitserklärung .................................. 128 V SUMMARY Summary The pseudocolonial coral Lophelia pertusa (Scleractinia, Caryophylliidae) is a eurybathic, stenother- mal cosmopolitan cold-water species. It occurs in two colour varieties, white and red. L. pertusa builds vast cold-water coral reefs along the continental margins, which are among the most diverse deep-sea ecosystems. Microbiology of L. pertusa has been in scientific focus for only a few years. The question whether the coral holds a host-specific bacterial community is not finally answered. Possible implications of the two colour varieties for microbial colonisation must be taken into account. Bio imaging can reveal the in-situ location of bacterial groups on and possible interactions with the coral. The present study aimed at investigating these aspects, drawing a more comprehensive picture of community structure, taxonomy, and in-situ location of L. pertusa-hosted microbes. Bacteria on coral samples from the Trondheimsfjord (Norway) were characterised by the culture- independent 16S rDNA- and rRNA-based techniques T-RFLP, cloning and sequencing, and CARD-FISH. L. pertusa revealed a high microbial richness. Clone sequences were dominated by α- and γ-Proteobacteria. Other abundant taxa were Bacteroidetes, Actinobacteria, Verrucomicrobia, Firmi- cutes, and Planctomycetes. The bacterial community of L. pertusa differed conspicuously from that of the environment, but was not ‘specific’ sensu stricto. It was rather divided into a tissue-bound frac- tion that was spatially constant within the sampling area and a “liquid-associated” fraction in the mucus and gastric fluid varying with location and colour variety of its host. Parallels to other coral-bacterial associations suggested existence of certain ‘cold-water coral-specific’ bacterial groups sensu lato. L. pertusa-associated bacteria appeared to play a significant role in the alimen- tation of their host by degradation of sulphur compounds, cellulose, chitin, and end products of the coral’s anaerobic metabolism. Different bacterial groups in red and white L. pertusa could explain the dissimilar dispersal of these two phenotypes by different nutritional strategies. Some microbes were regarded as opportunistic pathogens, others might even be connected to coral colouring. Filamentous bacteria in the gastrocoel were