**5. Conclusions**

Bacterial biodiversity from the past represents a pool of "hidden" bacteria identified in the previous research but poorly characterized by molecular techniques. We provided morphological and molecular description of two endosymbiotic bacteria inhabiting the cytoplasm of *Paramecium nephridiatum*, which resemble those identified 30 years ago. We complemented the description of one of the endosymbionts, recently described as "*Ca*. Megaira venefica" [49], by revealing moderate flagellation, the feature not very common within Rickettsiales. We characterized the second endosymbiont, which had a curved shape and tended to form aggregates with the host mitochondria. Based on morphology and 16S rRNA gene sequence a new taxon "*Ca.* Mystax nordicus" was proposed.

**Supplementary Materials:** The following are available online at http://www.mdpi.com/1424-2818/12/6/251/s1, Figure S1: TEM images of CS-bacteria in the cytoplasm of *P. nephridiatum* strain BMS16-23. A fragment of a putative conglomeration with the host mitochondria. Abbreviations: S—symbiont, M—mitochondria. Scale bar 2 μm. Figure S2: 3D reconstruction of RS-bacterium released from the cytoplasm of *P. nephridiatum* strain BMS16-34, AFM. White arrowheads point to the flagella. Table S1: Primers and FISH probes used in this study. Video S1: Animated FISH Z stack of the endosymbionts in *P. nephridiatum* strain BMS16-23, CLSM. Cells labeled with 16S\_Myst965 probe (red) and counterstained with DAPI (cyan). White arrows point to the bacteria penetrating the cortex.

**Author Contributions:** Conceptualization, E.S.; data curation, A.K. and E.S.; funding acquisition, E.S.; investigation, A.K. and E.S.; supervision, E.S.; visualization, A.K., K.B. and E.S.; writing—original draft, A.K. and E.S.; writing—review and editing, A.K., K.B. and E.S. All authors have read and agreed to the published version of the manuscript.

**Funding:** This study was funded by RFBR, gran<sup>t</sup> number 18-04-00562a to Elena Sabaneyeva.

**Acknowledgments:** The authors are grateful to Aisylu Shaidullina for her help with experimental infections. We would like to thank Natalia Lebedeva for providing the naive strain of ciliates from the RC CCM Culture Collection (Core Facility Centre "Cultivation of Microorganisms"). The study was performed using the equipment of Core Facility Centres of Saint-Petersburg State University "Microscopy and Microanalysis" and "Molecular and Cell Technologies".

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
