| acts as one of the most important vectors of some human diseases such as filariasis, Japanese encephalitis, West Nile virus, dengue and malaria. Insecticides play a central role in controlling major disease vectors. Some insects are able to survive a wide variety of physical and biological conditions due to their enormous reproductive capacity and genetic flexibility. Resistance to insecticides by mosquitoes is considered to be a recent evolutionary adaptation to environmental changes in response to sequential applications of chemical insecticides ( organochlorines, organophosphates (OPs), carbamates and pyrethroids, etc) and even biological insecticides. The mechanisms involved in mosquito insecticides resistance can be basically classified two groups, metabolic resistance (alterations in the levels or activities of detoxification proteins, such as non-specific carboxylesterases, P450 monooxygenases, Glutathione S-transferases), and target site resistance (mutations in the acetylcholinesterase, GABA receptor and voltage-gated sodium channel genes). Emergence and increase of insecticides resistance problems are not only shortening the life-span of currently available insecticides, but also undermining the efficacy of newly discovered or developed insecticides due to the cross-resistance and multiple resistance mechanisms.Presently, evidences have been shown that the DmαE7a , MdαE7, LcαE7 and in vitro mutantion of carboxylesterases B1, could obtain OP hydrolase actitity. However, there was no report about natural mutation of the carboxylesterases B1 in field mosquito Culex pipiens complex up to now. To make sure whether the mutation of carboxylesterases B1 could cause insecticide resistance in Culex pipiens complex or not, a Drosophila model was used in this study to address how the mutation of carboxylesterases B1 influences the insecticide resistance and its genetic evolutions.Using bioinformatics analysis, we firstly determined that the carboxylesterase B1 of Culex pipines has low homology with the carboxylesterases in Drosophila melanogaster (13%-41%). Then, using GAL4/UAS system, we successfully constructed Culex pipiens carboxylesterase B1W271 and its mutation gene B1L271 transgenic Drosophila lines through microinjection. Twelve and fourteen transgenic lines (both onⅡorⅢchromosome) were obtained after P factor localization respectively. Real-time quantitative PCR showed that both B1W271 and B1L271 were one copy insertion inⅡorⅢchromosome of Drosophila, and the transcriptional level of these transgenic lines was nearly identical. The B1W271 protein level, revealed by western-blot analysis with an anti-C.pipines'carboxylesterase B1 polychonal rabbit antibody, was much higher than of B1L271 in the tested Drosopphila lines. The total carboxylesterase activities of B1W271 transdenic lines were also higher than that of B1W271 much deeper than the B1L271, and were quantified to be around 2-3 times higher than B1L271 by using UV spectrophotometer. These results indicated that the carboxylesterase B1 could lose carboxylesterase activity through mutation. Bioassays showed that B1L271 transgenic flies had higher resistance level to malathion than that of B1W271 transgenic flies, but showed no resistance to parathion and deltamethrin. The result showed that B1L271 had higher malathion carboxylesterase(MCE) activity, which suffested it was possible for C. pipiens to experience the same evolution route as L. cuprina. These results built up a solid foundation for future in deep research of the evolution of carboxylesterases, and it's also helpful to establish reasonable vector-mosquito control measures, and develop new types of insecticides.
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