Sequencing And Phylogenetic Analysis Of The Wsp Gene Of Wolbachia In Spider Mites (Acari:Tetranychidae) In China

The Tetranychidae includes many serious mite pests such as Tetranychus cinnabarinus (Boisduval), T. urticae Koch, T. viennensis Zacher, Panonychus citri (McGregor) and T. kanzawai Kishida. They have a wide range of distribution and host, and high capacity of reproduction, and therefore cause great damages to agriculture and forestry in China as well as many other countries. With the use of more and more miticides, the resistant problem of mites is more serious than those of many other crop pests.In order to effectively control damaging mites, one alternate method is to make use of abnormal reproduction induced by Wolbachia, such as cytoplasmic incompatibility and parthenogenesis. In this paper the infection of Wolbachia in Tetranychidae was examined by PCR, and the wsp sequences of Wolbachia were cloned. In the end the phylogenetic tree of Wolbachia of Tetranychidae in China and other countries was constituted based on the wsp sequences of Wolbachia.1. The collection and rearing of experimental materialField collections have been done and populations of different mites have been keeping at the laboratory separately. These species and populations are 25 geographical populations of T. cinnabarinus (Boisduval), 13 geographical populations of T. urticae Koch, 3 geographical populations of Panonychus citri (McGregor), 2 geographical populations of T. kanzawai Kishida and 4 geographical populations of T. viennensis Zacher.2. The examination to the infection of Wolbachia in Tetranychidae by PCRFor each population, 40 female adult mites were selected to run PCR using a pair of designed primers, 18 geographical populations of T. cinnabarinus (Boisduval) were infected with Wolbachia, 13 geographical populations of T. urticae Koch were infected with Wolbachia. However, T. viennensis Zacher, Panonychus citri (McGregor) and T.kanzawai Kishida were not infected with Wolbachia. The results indicated that the infection of Wolbachia within and between geographical populations were different.3. The clone and comparison to the wsp gene of Wolbachia between geographical populations of TetranychidaeAfter purification, the PCR product was ligated into a PGEM-T Vector (Promega) and transformed into competent Escherichia coli. Three samples infected by Wolbachia were selected to clone and the standard sequences were their identical sequences. The results showed the wsp sequences of Wolbachia between different geographical populations were different, but the difference was not significant, the length of segment was about 596 bp.4. The constitution for the phylogenetic tree of Wolbachia in TetranychidaeThe wsp sequences were aligned with Clustal X program. A distance matrix was calculated with MEGA version 2.0, using a Kimura two- Parameter distance metric. From this matrix, a neighbor-joining tree was produced. The bootstrap analysis was performed with 1000 replications. The results showed most of them were similar to each other or completely identical with the wsp sequences of some strains of Wolbachia of ori group in B supergroup, only 3 geographical populations of T. cinnabarinus (Boisduval) were similar or completely identical with Con group in B supergroup. The findings indicate that they might be the same strain of Wolbachia or at least they were closed-related sister strains. The analysis showed Wolbachia of Tetranychidae in China and other countries might have closer relationship and were homologous.5. The phylogenetic analysis of Wolbachia of T cinnabarinus and T. urticae in China with T. urticae of the red and green form in other countriesThree phylogenetic trees were constituted based on the wsp gene sequences of Wolbachia: (1) The phylogenetic tree of Wolbachia of 13 geographical populations of T. urticae in China and T. urticae of the green form in other countries. (2) The phylogenetic tree of Wolbachia of 25 geographical populations of T. cinnabarinus in China and T. urticae of the red form in other countries. (3) The phylogenetic tree of Wolbachia of 13 geographical populations of T. urticae and 25 geographical populations...