Phylogenetic tree.

<p>Maximum Likelihood tree displaying the phylogenetic relationships between the <i>Cladonema</i> sp. ectosymbiont (<b>in bold</b>) with other endo and ectosymbionts sulphur-oxidizing bacteria based on the analysis of 16S rRNA gene sequences of 926 nucleotides. <i>Methylocystis parvus</i> was used as the outgroup. Only bootstrap values of more than 50% are shown at each node. The white circles with or without black dot indicate whether symbionts are intra- or extracellular respectively. The scale bar corresponds to 0.02 changes per nucleotide.</p

First Description of Sulphur-Oxidizing Bacterial Symbiosis in a Cnidarian (Medusozoa) Living in Sulphidic Shallow-Water Environments

<div><p>Background</p><p>Since the discovery of thioautotrophic bacterial symbiosis in the giant tubeworm <i>Riftia pachyptila</i>, there has been great impetus to investigate such partnerships in other invertebrates. In this study, we present the occurrence of a sulphur-oxidizing symbiosis in a metazoan belonging to the phylum Cnidaria in which this event has never been described previously.</p><p>Methodology/Principal Findings</p><p>Scanning Electron Microscope (SEM), Transmission Electron Microscope (TEM) observations and Energy-dispersive X-ray spectroscopy (EDXs) analysis, were employed to unveil the presence of prokaryotes population bearing elemental sulphur granules, growing on the body surface of the metazoan. Phylogenetic assessments were also undertaken to identify this invertebrate and microorganisms in thiotrophic symbiosis. Our results showed the occurrence of a thiotrophic symbiosis in a cnidarian identified as <i>Cladonema</i> sp.</p><p>Conclusions/Significance</p><p>This is the first report describing the occurrence of a sulphur-oxidizing symbiosis in a cnidarian. Furthermore, of the two adult morphologies, the polyp and medusa, this mutualistic association was found restricted to the polyp form of <i>Cladonema</i> sp.</p></div

Ultrastructural analysis of polyp by TEM.

<p>Thin sections clearly show that the bacteria (arrows) are distributed exclusively on the body surface of polyps. No intracellular bacteria could be observed suggesting that there is no endosymbiosis in <i>Cladonema</i> sp. Higher magnification of the ectosymbionts (B) shows that the cytoplasm of the bacteria located outside the host tissue (b) contained two kinds of inclusions. The non-membrane-bound inclusions correspond to glycogen-like granules (white arrows) distributed throughout the cytoplasm while empty membrane-bound inclusions (curved arrow) correspond to sulphur granules probably located within the periplasm. The ectosymbionts appear to be fixed to the host cytoplasmic membrane (C) though atypical structures (see inset). Fig D displays a higher magnification of an atypical structure which is organized on two levels of “tubes” with a central tuft in contact with the bacteria. The nature of such “tubes” is unknown.</p

Phylogenetic tree.

<p>Maximum Likelihood tree displaying the phylogenetic relationships between <i>Cladonema</i> sp. Guadeloupe F.W.I. (<b>in bold</b>) with other capitata hydrozoans based on the analysis of partial 18S rRNA gene sequences of 895 nucleotides. <i>Bellonella rigida</i> was used as the outgroup. Only bootstrap values of more than 60% are shown at each node. The scale bar corresponds to 0.01 changes per nucleotide.</p

Structural analysis of polyp stage of <i>Cladonema</i> sp.

<p>(A) light micrograph of freshly collected polyps connected by a tube-like hydrocauli. The polyps appear white in colour especially for the tentacles. SEM image (B) shows that the polyp is composed of an entocodon (asterisk), a stalk (S), capitate (star) and filiform tentacles (arrows). A higher magnification of the polyp displays a filiform tentacle and small bacteria that can be clearly distinguished at the surface of the polyp (C). D and E show ectosymbiotic rod-shaped bacteria covering the polyp. Some of these are dividing (arrows) suggesting a high metabolism. The EDX spectrum obtained from the polyp (F) shows a peak of elemental sulphur suggesting that such bacteria are thioautotrophic (Cl: chloride, Na: sodium, Mg: magnesium, C: carbon, O: oxygen, S: elemental sulphur).</p

Ultrastructural analysis of a Medusa.

<p>Light micrograph of the medusoid stage of <i>Cladonema</i> sp. (A) shows that the tentacles extended around the bell while SEM image (D) shows that the tentacles were retracted due to the chemical fixation artefact. No bacterium was observed on the bell surface of the medusa (B, D) or on its tentacles (C-D). The two insets clearly confirmed the absence of ectosymbiotic bacteria on the bell surface (B) and on the tentacles (C) of the medusa.</p

Symbiotic status model in <i>Cladonema</i> sp.

<p>The diagram depicts a model to explain the distinctive symbiotic status existing between polyps and medusae of <i>Cladonema</i> sp. which seems to rely on an inverse gradient of sulphur and oxygen in the environment. The drawing shows that only the polypoid stage of the cnidarian living in a sulphide rich environment (at the oxic/anoxic interface) bears symbiotic sulphur-oxidizing bacteria while the medusoid stage located in the water column depleted in sulphide, is free of such ectosymbiosis. Such medusa were usually observed closed to sea grasses of <i>Caulerpa taxifolia</i> within the mangrove lagoon.</p

Number of samples per species and per sampling site.

Number of samples per species and per sampling site.</p

Phylogenetic tree of <i>Wolbachia</i> detected in Gerroidea and other insects based on their <i>wsp</i> sequences.

Tree was inferred from maximum likelihood method using PhyML program and drawn using iTOL program. Branches are labelled as Supergroup Letter and specie names followed by GenBank accession numbers. Bootstrap values (1,000 replicates) are indicated, except for branches with a value less than 0.5.</p

Geographical localities, substrates, depths and sequences of specimens of <i>I. iwaotakii</i> used in this study.

<p>The number of sequences is indicated in brackets. The superscripts refer to the authors of sequences. The stars indicate the sequences obtained during this study.</p