Culture independent analysis of microbiota in the gut of pine weevils KTH Biotechnology 2013-January-13 Diploma work by: Tobias B. Ölander Environmental Microbiology, KTH Supervisor: Associate prof. Gunaratna K. Rajarao Examinator: Prof. Stefan Ståhl 1 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. Abbreviations PCR Polymerase Chain Reaction RFLP Restriction Fragment Length Polymorphism T-RFLP Terminal Restriction Fragment Length Polymorphism DGGE Denaturing Gradient Gel Electrophoresis TGGE Temperature Gradient Gel Electrophoresis D-HPLC Denaturing High-Performance Liquid Chromatography RISA Ribosomal Intergenic Spapcer Analysis TAE Tris base, acetic acid and EDTA TBE Tris base, boric acid and EDTA 2 Table of Contents Abstract ...................................................................................................................................... 2 Abbreviations ............................................................................................................................. 2 1 Introduction .......................................................................................................................... 5 1.1 Aim ................................................................................................................................ 5 2 Background .......................................................................................................................... 6 2.1 Pine Weevil .................................................................................................................... 6 2.2 Insecticides .................................................................................................................... 7 2.3 Antifeedants ................................................................................................................... 8 2.3.1 Antifeedant activity of pine weevil feces ................................................................ 8 2.4 Community analysis ...................................................................................................... 9 2.4.1 Genetic fingerprinting ........................................................................................... 10 2.4.2 Sequencing ............................................................................................................ 11 2.4.3 16S rRNA gene ..................................................................................................... 12 2.4.4 Community analysis of insect gut ......................................................................... 12 3 Materials & Methods .......................................................................................................... 13 3.1 Flowchart ..................................................................................................................... 13 3.2 DNA extraction ............................................................................................................ 13 3.3 PCR amplification ....................................................................................................... 14 3.4 Cloning and transformation ......................................................................................... 15 3.5 Plasmid extraction ....................................................................................................... 15 3.6 Colony PCR ................................................................................................................. 16 3.7 RFLP ............................................................................................................................ 17 3.8 Agarose gel electrophoresis ......................................................................................... 17 3.9 Sequencing ................................................................................................................... 17 3.10 Phylogenetic analysis ................................................................................................ 17 4 Results ................................................................................................................................ 18 4.1 DNA extraction ............................................................................................................ 18 4.2 PCR amplification ....................................................................................................... 19 4.3 Cloning and transformation ......................................................................................... 20 4.4 Plasmid DNA extraction .............................................................................................. 21 4.4.1 PCR with plasmid DNA ........................................................................................ 22 4.5 Colony PCR ................................................................................................................. 22 4.6 RFLP ............................................................................................................................ 23 4.7 Sequencing ................................................................................................................... 25 4.8 Phylogenetic analysis .................................................................................................. 28 5 Discussion .......................................................................................................................... 29 3 5.1 PCR amplification ....................................................................................................... 31 5.2 Colony PCR ................................................................................................................. 32 5.3 RFLP ............................................................................................................................ 32 5.4 Phylogenetic analysis .................................................................................................. 33 6 Conclusions ........................................................................................................................ 33 7 Further Studies ................................................................................................................... 34 8 Acknowledgments .............................................................................................................. 34 9 References .......................................................................................................................... 34 10 Appendices ....................................................................................................................... 40 I. PCR Amplification – midgut, hindgut & egg sample .................................................... 40 II. PCR (plasmid template) – midgut sample .................................................................... 43 III. Colony PCR – midgut sample ..................................................................................... 44 IV. Colony PCR – egg sample ........................................................................................... 61 V. Good’s Method ............................................................................................................. 69 VI. Sequence for clones & sample species ........................................................................ 71 4 1 Introduction From an economical perspective, the pine weevil is the most important forest pest in Sweden, as well as for major parts of the rest of Europe [1]. The insect is a serious threat to the regeneration of newly planted conifers (i.e. pines and spruces). Just in Sweden, the pine weevil’s feeding on young conifer plants causes damages for several hundreds of millions kronor annually [1] [2]. Damages caused by the pine weevil is an issue recognized as early as the middle of the 19th century, but the problem with pine weevil feeding increased significantly in Sweden during the 1950s, due to the more and more prevalent forestry practice of clearcutting [2]. Due to the increasing pressure, to abolish the use of traditional insecticides in the forest industry, alternative means for fighting forest pests, like the pine weevil, are required. One alternative would be to search for a more eco-friendly insect repellant or antifeedant to