Detection of bacterial endosymbionts in freshwater crustaceans: the applicability of non-degenerate primers to amplify the bacterial 16S rRNA gene.

Bacterial endosymbionts of aquatic invertebrates remain poorly studied. This is at least partly due to a lack of suitable techniques and primers for their identification. We designed a pair of non-degenerate primers which enabled us to amplify a fragment of ca. 500 bp of the 16S rRNA gene from various known bacterial endosymbiont species. By using this approach, we identified four bacterial endosymbionts, two endoparasites and one uncultured bacterium in seven, taxonomically diverse, freshwater crustacean hosts from temporary waters across a wide geographical area. The overall efficiency of our new WOLBSL and WOLBSR primers for amplification of the bacterial 16S rRNA gene was 100%. However, if different bacterial species from one sample were amplified simultaneously, sequences were illegible, despite a good quality of PCR products. Therefore, we suggest using our primers at the first stage of bacterial endosymbiont identification. Subsequently, genus specific primers are recommended. Overall, in the era of next-generation sequencing our method can be used as a first simple and low-cost approach to identify potential microbial symbionts associated with freshwater crustaceans using simple Sanger sequencing. The potential to detected bacterial symbionts in various invertebrate hosts in such a way will facilitate studies on host-symbiont interactions and coevolution.