NanoSIMS and tissue autoradiography reveal symbiont carbon fixation and organic carbon transfer to giant ciliate host.

The giant colonial ciliate Zoothamnium niveum harbors a monolayer of the gammaproteobacteria Cand. Thiobios zoothamnicoli on its outer surface. Cultivation experiments revealed maximal growth and survival under steady flow of high oxygen and low sulfide concentrations. We aimed at directly demonstrating the sulfur-oxidizing, chemoautotrophic nature of the symbionts and at investigating putative carbon transfer from the symbiont to the ciliate host. We performed pulse-chase incubations with 14C- and 13C-labeled bicarbonate under varying environmental conditions. A combination of tissue autoradiography and nanoscale secondary ion mass spectrometry coupled with transmission electron microscopy was used to follow the fate of the radioactive and stable isotopes of carbon, respectively. We show that symbiont cells fix substantial amounts of inorganic carbon in the presence of sulfide, but also (to a lesser degree) in the absence of sulfide by utilizing internally stored sulfur. Isotope labeling patterns point to translocation of organic carbon to the host through both release of these compounds and digestion of symbiont cells. The latter mechanism is also supported by ultracytochemical detection of acid phosphatase in lysosomes and in food vacuoles of ciliate cells. Fluorescence in situ hybridization of freshly collected ciliates revealed that the vast majority of ingested microbial cells were ectosymbionts.

Thioautotrophic ectosymbiosis in Pseudovorticella sp., a peritrich ciliate species colonizing wood falls in marine mangrove.

Ciliates represent a diversified group of protists known to establish symbioses with prokaryotic micro-organisms. They are mainly phagotrophs and symbiotic relationships with bacteria can give them an important advantage in chemosynthetic environments. The aim of this study is to describe the thiotrophic association that occurs between the peritrich ciliate Pseudovorticella sp. and potential sulfur-oxidizing bacteria. Investigations at microscopic scale (LM, SEM, TEM) showed ectosymbiotic bacteria covering the surface of the body of Pseudovorticella sp. According to 16S rDNA phylogenetic analysis, these ectosymbiotic bacteria belong to γ-proteobacteria and are phylogenetically close to the symbiont of the recently described Zoothamnium ignavum, which inhabits shallow-water wood falls. FISH experiments, using symbiont specific probes, clearly indicate that these ectosymbiotic bacteria are also ingested into food vacuoles. Electron lucent granules observed in TEM in the cytoplasm of the ectosymbiotic bacteria have been identified as sulfur granules by Raman microspectrometry analyses. Raman microspectrometry analyses confirmed the thiotrophic nature of this relationship already suggested by the results obtained by TEM and phylogeny. A complete sulfur map was then performed to investigate the sulfur distribution in the zooid. Results show that the relationship between this protist and its bacterial partner is a thiotrophic ectosymbiosis.

"Candidatus Haloectosymbiotes riaformosensis" (Halobacteriaceae), an archaeal ectosymbiont of the hypersaline ciliate Platynematum salinarum.

The novel ciliate Platynematum salinarum (Scuticociliatia) was isolated only recently from a thalassohaline solar saltern pond (12%) in Portugal. Scanning electron microscopy showed numerous bacterial-shaped cells covering the complete surface of the ciliate. The rod-shaped epibionts were identified and characterized following the "Full-Cycle rRNA Approach". The almost full-length 16S rRNA gene sequence was obtained using archaeal-specific primers and two species-specific probes were designed for fluorescence in situ hybridization. The 16S rRNA gene sequence of the epibiotic cells showed 87% sequence identity with the type strain sequence of the closest characterized species Halolamina pelagica. Phylogenetic reconstructions affiliated the novel organism to the genus Halolamina (Halobacteria, Archaea). Attempts to isolate the epibionts failed and, therefore, growth experiments incorporating the antibiotic anisomycin were conducted in order to investigate the potential symbiotic relationship between P. salinarum and the epibionts. The results suggested an obligate symbiosis between the two organisms and revealed the first symbiotic representative of the Halobacteria. Based on the phylogenetic analyses and growth experiments we propose the classification of this novel organism in a new genus, with the taxon name "Candidatus Haloectosymbiotes riaformosensis".

Sunken wood habitat for thiotrophic symbiosis in mangrove swamps.

Large organic falls to the benthic environment, such as dead wood or whale bones, harbour organisms relying on sulfide-oxidizing symbionts. Nothing is known however, concerning sulfide enrichment at the wood surface and its relation to wood colonization by sulfide-oxidizing symbiotic organisms. In this study we combined in situ hydrogen sulfide and pH measurements on sunken wood, with associated fauna microscopy analyses in a tropical mangrove swamp. This shallow environment is known to harbour thiotrophic symbioses and is also abundantly supplied with sunken wood. A significant sulfide enrichment at the wood surface was revealed. A 72h sequence of measurements emphasized the wide fluctuation of sulfide levels (0.1->100muM) over time with both a tidal influence and rapid fluctuations. Protozoans observed on the wood surface were similar to Zoothamnium niveum and to vorticellids. Our SEM observations revealed their association with ectosymbiotic bacteria, which are likely to be sulfide-oxidizers. These results support the idea that sunken wood surfaces constitute an environment suitable for sulfide-oxidizing symbioses.

High genetic similarity between two geographically distinct strains of the sulfur-oxidizing symbiont 'Candidatus Thiobios zoothamnicoli'.

The giant marine ciliate Zoothamnium niveum (Ciliophora, Oligohymenophora) is obligatorily covered by a monolayer of putative chemoautotrophic sulfur-oxidizing (thiotrophic) bacteria. For Z. niveum specimens from the Caribbean Sea it has been demonstrated that this ectosymbiotic population consists of only a single pleomorphic phylotype described as Candidatus Thiobios zoothamnicoli. The goal of our study was to identify and phylogenetically analyse the ectosymbiont(s) of a recently discovered Z. niveum population from the Mediterranean Sea, and to compare marker genes encoding key enzymes of the carbon and sulfur metabolism between the two symbiont populations. We identified a single bacterial phylotype representing the ectosymbiont of Z. niveum from the Mediterranean population showing 99.7% 16S rRNA gene (99.2% intergenic spacer region) similarity to the Caribbean Z. niveum ectosymbiont. Genes encoding enzymes typical for an inorganic carbon metabolism [ribulose 1,5-bisphosphate carboxylase/oxygenase (RuBisCO)] and for sulfur metabolism (5'-adenylylsulfate reductase, dissimilatory sulfite reductase) were detected in both symbiotic populations. The very high amino acid sequence identity (97-100%) and the high nucleic acid sequence identity (90-98%) of these marker enzymes in two geographically distant symbiont populations suggests that the association of Z. niveum with Cand. Thiobios zoothamnicoli is very specific as well as temporally and spatially stable.

The effects of sulphide on growth and behaviour of the thiotrophic Zoothamnium niveum symbiosis.

Zoothamnium niveum (Ciliophora, Oligohymenophora) is a giant, colonial marine ciliate from sulphide-rich, shallow-water habitats, obligatorily associated with the ectosymbiotic, chemoautotrophic, sulphide-oxidizing bacterium 'Candidatus Thiobios zoothamnicoli'. The aims of this study were to characterize the natural habitat and investigate growth, reproduction, survival and maintenance of the symbiosis from Corsica, France (Mediterranean Sea) using a flow-through respirometer providing stable chemical conditions. We were able to successfully cultivate the Z. niveum symbiosis during its entire lifespan and document reproduction, whereby the optimum conditions were found to range from 3 to 33 micromol l(-1) sigmaH2S in normoxic seawater. Starting with an inoculum of 13 specimens, we found up to 173 new specimens that were asexually produced after only 11 days. Observed mean lifespan of the Z. niveum colonies was approximately 11 days and mean colony size reached 51 branches, from which rapid host division rates of up to every 4.1 hours were calculated. Comparing the ectosymbiotic population from Z. niveum colonies collected from their natural habitat with those cultivated under optimal conditions, we found significant differences in the bacterial morphology and the frequency of dividing cells on distinct host parts, which is most likely caused by behaviour of the host ciliate. Applying different sulphide concentrations we revealed that the symbiosis was not able to survive without sulphide and was harmed by high sulphide conditions. To our knowledge, this study reports the first successful cultivation of a thiotrophic ectosymbiosis.

The microbial community of Ophrydium versatile colonies: endosymbionts, residents, and tenants.

Ophrydium versatile is a sessile peritrichous ciliate (Kingdom Protoctista, class Oligohymenophora, order Peritrichida, suborder Sessilina) that forms green, gelatinous colonies. Chlorophyll a and b impart a green color to Ophrydium masses due to 400-500 Chlorella-like endosymbionts in each peritrich. Ophrydium colonies, collected from two bog wetlands (Hawley and Leverett, Massachusetts) were analyzed for their gel inhabitants. Other protists include ciliates, mastigotes, euglenids, chlorophytes, and heliozoa. Routine constituents include from 50-100,000 Nitzschia per ml of gel and at least four other diatom genera (Navicula, Pinnularia, Gyrosigma, Cymbella) that may participate in synthesis of the gel matrix. Among the prokaryotes are filamentous and coccoid cyanobacteria, large rod-shaped bacteria, at least three types of spirochetes and one unidentified Saprospira-like organism. Endosymbiotic methanogenic bacteria, observed using fluorescence microscopy, were present in unidentified hypotrichous ciliates. Animals found inside the gel include rotifers, nematodes, and occasional copepods. The latter were observed in the water reservoir of larger Ophrydium masses. From 30-46% of incident visible radiation could be attenuated by Ophrydium green jelly masses in laboratory observations. Protargol staining was used to visualize the elongate macronuclei and small micronucleus of O. versatile zooids and symbiotic algal nuclei. Electron microscopic analysis of the wall of the Chlorella-like symbiont suggests that although the Ophrydium zooids from British Columbia harbor Chlorella vulgaris, those from Hawley Bog contain Graesiella sp. The growth habit in the photic zone and loose level of individuation of macroscopic Ophrydium masses are interpretable as extant analogs of certain Ediacaran biota: colonial protists in the Vendian fossil record.