Molecular identification of microbial ectosymbionts associated with the colonial ciliate Zoothamnium niveum

Andrea D. Nussbaumer^1*, Dávid A. Molnár¹, Katrina Vanura², Monika Bright¹, Joerg Ott¹

¹Marine Biology, Department of Ecology and Conservation Biology, University of Vienna, Althanstr. 14, A-1090 Vienna, Austria

² Department of Internal Medicine I, Division of Hematology, University of Vienna, Währinger Gürtel 18-20, A-1090 Vienna, Austria

Zoothamnium niveum (Ciliophora, Oligohymenophora) lives at mangrove peat walls along banks of tidal channels and ponds in the Caribbean Sea. The feather-shaped sessile colonial ciliate is obligatory associated with chemoautotrophic sulfur oxidizing (thiotrophic) bacteria. The ectosymbionts are arranged in a monolayer covering the whole colony. This monolayer consists of two bacterial morphotypes: stalk, branches, terminal-, and macrozooids are covered with rod-shaped symbionts, whereas microzooids serve as substrate for slightly dumbbell-shaped coccoid symbionts. A series of intermediate shapes between cocci on the oral side and rods on the aboral side of the microzooids provided the first evidence that the two morphotypes belong to the same bacterial species.

In this study the symbionts were phylogenetically analyzed by sequencing the 16S ribosomal RNA gene and were subsequently identified using fluorescence in situ hybridization (FISH) with a symbiont specific probe. FISH conducted on paraffin sections and on whole specimens revealed that both morphological different subpopulations belong to the same phylotype. The symbionts fall within the gamma subclass of the Proteobacteria. They do not cluster with any thiotrophic ecto- or extracellular endosymbionts such as the Laxus / Olavius / Inanidrilus group, or with any thiotrophic endosymbionts like the Riftia / Codakia or the Bathymodiolus / Calyptogena group. Using various group-specific probes we found other bacteria in addition to the symbiont on the lower parts of the colonies, confirming earlier studies that showed the onset of unspecific microbial fouling coupled with senescence of host cells.