Abstract
BackgroundWeevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide. So far, little is known on the presence of endosymbionts in Otiorhynchus spp.. Investigation of endosymbiosis in this genus may help to understand the evolution of different reproductive strategies in these weevils (parthenogenesis or sexual reproduction), host-symbiont interactions, and may provide a future basis for novel pest management strategy development. Here, we used a multitag 454 pyrosequencing approach to assess the bacterial endosymbiont diversity in larvae of four economically important Otiorhynchus species.ResultsHigh-throughput tag-encoded FLX amplicon pyrosequencing of a bacterial 16S rDNA fragment was used to characterise bacterial communities associated with different Otiorhynchus spp. larvae. By sequencing a total of ~48,000 PCR amplicons, we identified 49 different operational taxonomic units (OTUs) as bacterial endosymbionts in the four studied Otiorhynchus species. More than 90% of all sequence reads belonged either to the genus Rickettsia or showed homology to the phylogenetic group of “Candidatus Blochmannia” and to endosymbionts of the lice Pedicinus obtusus and P. badii. By using specific primers for the genera Rickettsia and “Candidatus Blochmannia”, we identified a new phylogenetic clade of Rickettsia as well as “Candidatus Nardonella” endosymbionts in Otiorhynchus spp. which are closely related to “Candidatus Blochmannia” bacteria.ConclusionsHere, we used multitag 454 pyrosequencing for assessment of insect endosymbiotic communities in weevils. As 454 pyrosequencing generates only quite short sequences, results of such studies can be regarded as a first step towards identifying respective endosymbiotic species in insects. In the second step of our study, we analysed sequences of specific gene regions for a more detailed phylogeny of selected endosymbiont genera. As a result we identified the presence of Rickettsia and “Candidatus Nardonella” endosymbionts in Otiorhynchus spp.. This knowledge is an important step in exploring bacteria-insect associations for potential use in insect pest control.
Highlights
Weevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide
These sequences were summarized into 49 consensus sequences (Table 1), representing the total retrieved endosymbiotic bacterial diversity in the four different Otiorhynchus species
In this study, endosymbiotic bacterial diversity in weevil larvae was assessed via multitag 454 pyrosequencing of a bacterial 16S rRNA fragment
Summary
Weevils of the genus Otiorhynchus are regarded as devastating pests in a wide variety of horticultural crops worldwide. Understanding relationships between insect hosts and their endosymbiotic bacteria is relevant from an evolutionary point of view, but can aid in the identification of new targets for insect pest control [7] as well as for biotechnology and biomedicine [3]. The accessibility of novel genomic techniques, in particular generation sequencing (NGS) technologies represent new, cost-efficient and fast strategies to depict microbial diversity without the need for culturing the respective organisms [8]. With these techniques thousands of sequence reads can be analysed in parallel allowing an extensive assessment of bacterial diversity within insects. As a target for bacterial NGS projects, ribosomal DNA genes (rDNA) like the 16S rDNA, used for the taxonomic classification of bacterial species [9], have frequently been applied for analysing the bacterial microbial community in metagenomic studies of soil [10,11], mines [12], the deep sea [13] or oral human microflora [14]
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