Culture independent analysis of microbiota in the gut of pine weevils

Abstract: In Sweden, the pine weevil causes damages for several hundreds of millions kronor annually. The discouraged use of insecticides has resulted in that other methods to prevent pine weevil feeding needs to be found. Antifeedants found in the pine weevil own feces is one such alternative. The source of the most active antifeedants in the feces is probably from bacterial or fungal lignin degrading symbionts in the pine weevil gut. The aim of the project was to analyze the pine weevil gut microbiota with the help of culture independent methods. DNA (including bacterial DNA) was extracted from both midgut and egg cells. The extracted DNA was amplified with PCR. A clone library was created by cloning the amplified DNA into plasmid vectors and transforming the vector constructs with chemically competent cells. The clones were amplified again with either colony PCR or plasmid extraction followed by PCR, and used for RFLP (Restriction Fragment Length Polymorphism) and sequencing. Species found in the midgut sample included Acinetobacter sp., Ramlibacter sp., Chryseobacterium sp., Flavisolibacter sp. and Wolbachia sp. Species found in the egg sample included Wolbachia sp. and Halomonas sp. Wolbachia sp. and Halomonas sp. were found to be the dominant members of the midgut and egg cells respectively.