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

Abstract

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.

Wolbachia and Cardinium infection found in threatened unionid species: a new concern for conservation of freshwater mussels?

“…We identified 18 females, out of which 6 were infected by endosymbionts. Nevertheless, given the fact that also for the rest individuals tested, the PCR products were obtained but unspecific (originated from another bacterial species) or weak (most likely due to genome mixing among several bacterial species) sequences were observed, more detailed study is needed (including design species-specific primers; see also Mioduchowska et al 2018). So far, coinfection of Cardinium and Wolbachia was detected in one planthopper species (Nakamura et al 2012), one parasitoid wasp species (e.g.…”

“…Species identification of obtained bacterial sequences were performed on homology searching using the GenBank records-megablast algorithm and nucleotide database were applied. We also used a positive control for DNA extraction using isolates of freshwater crustacean hosts for which we previously discovered Wolbachia and Cardinium infections (see Mioduchowska et al 2018): Branchipus schaefferi isolate, for which Wolbachia infection was detected and Heterocypris incongruens isolate, for which we identified Cardinium. In addition, we used negative control (blank control probes) -PCR amplification of an ultra-pure water sample that allows detection of contaminant DNA and no PCR products were obtained (data not shown).…”

Virulence and transmission biology of the widespread, ecologically important pathogen of zooplankton,Spirobacillus cienkowskii

“…One of the oldest Daphnia pathogens known to science is Spirobacillus cienkowskii, a gram-negative bacterium that belongs to the newly characterized family, Silvanigrellaceae (9). First found in Daphnia magna in Ukraine in the 1880s (10), Spirobacillus cienkowskii (hereafter Spirobacillus) has since been found in a wide range of Cladocera at locations across the globe (11)(12)(13). In Daphnia populations in Michigan, where Spirobacillus' dynamics have been most intensively studied, Spirobacillus causes large epidemics, depleting the numbers of hosts and increasing their vulnerability to predation (4,14,15).…”

Variation in epibiotic bacteria on two squat lobster species of Munidopsidae

“…Others [such as Kiwa puravida ( Thurber et al, 2011 ) and Kiwa hirsute ( Macpherson et al, 2005 )] are concentrated around vents, with distinctive dense setae on their body surfaces. Numerous invertebrate species have formed symbiotic associations with their microbiomes, which can exert a range of effects on their hosts, predominantly beneficial ones ( Mioduchowska et al, 2018 ). Goffredi et al proposed that the bacteria that clustered on the plumose setae of Kiwa hirsute could be used as a nutrient resource to meet the energetic demands of the host ( Goffredi et al, 2008 ).…”