Taxonomic classification of the bacterial endosymbiont Wolbachia based on next-generation sequencing: is there molecular evidence for its presence in tardigrades?

We used high-throughput sequencing of 16S rRNA to test whether tardigrade species are infected with Wolbachia parasites. We applied SILVA and Greengenes databases that allowed taxonomic classification of bacterial sequences to OTUs. The results obtained from both databases differed considerably in the number of OTUs, and only the Greengenes database allowed identification of Wolbachia (infection was also supported by comparison of sequences to NCBI database). The putative bacterial endosymbiont Wolbachia was discovered only in adult eutardigrades, while bacteria identified down to the order Rickettsiales were detected in both eutardigrade eggs and adult specimens. Nevertheless, the frequency of Wolbachia in the bacterial communities of the studied eutardigrades was low. Similarly, in our positive control, i.e., a fairy shrimp Streptocephalus cafer, which was found to be infected with Wolbachia in our previous study using Sanger sequencing, only the Rickettsiales were detected. We also carried out phylogenetic reconstruction using Wolbachia sequences from the SILVA and Greengenes databases, Alphaproteobacteria putative endosymbionts and Rickettsiales OTUs obtained in previous studies on the microbial community of tardigrades, and Rickettsiales and Wolbachia OTUs obtained in the current study. Our discovery of Wolbachia in tardigrades can fuel new research to uncover the specifics of this interaction.