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.

1. The disappearance and deterioration of amphibian breeding habitats is a major cause of their global extinction. In Europe, this problem affects the yellow-bellied toad Bombina variegata, which inhabits small ponds in the early stages of succession, where no predatory invertebrate species normally occur. Nonetheless, as habitats, small temporary ponds are highly endangered, a situation that is intensifying with urban spread. 2. A system of 13 pairs of artificial ponds was constructed in 2012 for the breeding of B. variegata with the aim of reconnecting disjunct populations. During the 2 years of the study, one of the twin ponds was regularly cleared of emergent vegetation and invertebrates. The survival rates of the tadpoles released in the cleared and uncleared ponds were analysed.3. The survival rate of the tadpoles released into the cleared ponds was higher than of those introduced into the uncleared ones. Tadpole survival rates were also higher in new, single ponds, constructed in 2013.4. The lower survival rates of the introduced tadpoles are best explained by the presence of predatory aquatic invertebrates, particularly the large diving beetle Dytiscus marginalis. Experimentally obtained consumption rates indicate that oneDytiscus larva is capable of destroying a typical clutch of B. variegata tadpoles within a few hours, much faster than any of the other insect predators tested.5. The impact of freshwater invertebrates on tadpoles is frequently studied but rarely quantified. For conservation measures to be effective, quantitative standards need to be applied that describe the impact of predation in a predictable manner.6. Construction of artificial small ponds is broadly accepted as an important conservation measure compensating for the disappearance of natural amphibian breeding sites. This function of artificial ponds may be substantially enhanced by the periodic removal of invertebrate predators.

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