Yet, at the same time it was not possible to amplify the PSI-7977 datasheet Rickettsia specific 16S rDNA fragment in the same two species. We thus suppose that the coxA gene sequence is rather conserved among bacteria and may not be adequate for precise Belnacasan datasheet species determination. Supplementary sequence analysis of a range of additional bacterial genes may resolve this issue. Phylogenetic analysis of the Rickettsia endosymbiontic 16S rDNA and coxA gene fragments amplified from Otiorhynchus spp. revealed the relatedness to the rhizobius and/or adalia Rickettsia group as defined by Weinert et al [22]. These subgroups contain Rickettsia bacteria identified in

various beetles, including members of the Curculionidae [22]. Rickettsia endosymbionts act as male-killing agents in leaf mining beetles and ladybirds [23, 24] and play an essential role in the early development of the oocyte and egg production in parthenogenetic book lice [25, 26]. Thus it could be speculated that Rickettsia endosymbionts may also manipulate host reproduction in Otiorhynchus species. Phylogenetic analysis and putative biological function of “Candidatus Nardonella” endosymbionts 454 pyrosequencing detected endosymbionts similar to “Candidatus Blochmannia”

and bacterial endosymbionts of the lice Pedicinus obtusus and P. badii in O. armadillo, O. salicicola and to a lesser extent in O. rugosostriatus. The presence of these putative “Candidatus selleck chemical Blochmannia” like bacteria was verified in these species by using primers specific for the “Candidatus Blochmannia” 16S rDNA [21], which indicated that the obtained sequences are similar to “Candidatus Nardonella”. In addition, a fragment of the same size and sequence was also amplified in O. sulcatus, even though 454 pyrosequencing did not reveal the presence of these bacteria in this weevil species (Table 1). “Candidatus Nardonella” bacteria are often localized in the bacteriome whereas Rickettsia endosymbionts may infect as well different tissues. As we used whole larvae for DNA extraction, the amount of overall isolated DNA might have been lower for “Candidatus Nardonella” than for Rickettsia. Therefore we assume that respective bacterial DNA might have not been

amplified in SSR128129E O. sulcatus with the universal primers used for 454 pyrosequencing due to competition for PCR reagents with taxa such as Rickettsia, having a higher template abundance [27]. However, these results also demonstrate that studies using 454 pyrosequencing can be regarded as a first step towards identifying respective endosymbiotic species in insects, but that for a detailed phylogeny and a more comprehensive insight into endosymbiont-insect-associations, the amplification of specific gene regions is still indispensable. Phylogenetic analysis of the putative “Candidatus Blochmannia” specific 16S rDNA sequence amplified from the four studied Otiorhynchus weevils showed a close relatedness of these bacteria to the genus “Candidatus Nardonella”.