Spatial Exploration and Characterization of Endozoicomonas spp. Bacteria in Stylophora pistillata Using Fluorescence In Situ Hybridization

Studies of coral-­associated bacterial communities have repeatedly demonstrated that the microbial assemblages of the coral host are highly specific and complex. In particular, bacterial community surveys of scleractinian and soft corals from geographically diverse reefs continually uncover a high abundance of sequences affiliated with the Gammaproteobacteria genus Endozoicomonas. The role of these bacteria within the complex coral holobiont is currently unknown. In order to localize these cells and gain an understanding of their potential interactions within the coral, we developed a fluorescence in situ hybridization(FISH) approach for reef-­building coral tissues. Using a custom small-­subunit ribosomal RNA gene database, we developed two Endozoicomonas-­specific probes that cover almost all known coral-­associated Endozoicomonas sequences. Probe hybridization conditions were quantitatively evaluated against target and non-­target bacterial cultures using fluorescence microscopy. Using these experimentally tested conditions, probes were then hybridized to the branching coral Stylophora pistillata, obtained from the Red Sea, using whole mount and paraffin embedding techniques. This study allowed preliminary spatial exploration and characterization of Endozoicomonas in coral, which has provided insight into their functional role and interactions within the coral holobiont