Basic studies on the insect cytochrome P450 for the development of new insecticides and synergistic agents.

为了寻找杀虫剂或增效剂的新靶点，我对媒介蚊子的抗药性机制进行了研究，特别是对其细胞色素P450单加氧酶的功能进行了分析。首先在实验室用拟除虫菊酯类杀虫剂氯菊酯对成蚊进行筛选。经过8代筛选，SPS8菌株的抗性水平达到敏感菌株的1000倍以上。经胡椒酰丁氧酯预处理后，氯菊酯敏感性明显增强，提示细胞色素P450单加氧酶可能是其抗药性的主要机制。体外代谢研究表明，抗性蚊制备的微粒体蛋白催化氯菊酯对4′ho -氯菊酯的代谢比敏感蚊更快。此外，氯菊酯的羟基化活性依赖于NADPH，并受到胡椒酰丁醇的强烈抑制，这表明细胞色素P450单加氧酶参与了SPS8菌株对氯菊酯的抗性。与氯菊酯抗性相关的P450异构体是杀虫剂或增效剂的新靶点。对2009年在新加坡采集的26只白纹伊蚊进行了钠通道基因分型。令人惊讶的是，超过70%的蚊子拥有抗击倒基因（kdr）。这是白纹伊蚊中存在kdr基因的第一个证据，表明有必要研究该等位基因在这一重要媒介昆虫中的全球分布。

In order to seek novel target sites of insecticides or synergists, I studied on the mechanisms of insecticide resistance in vector mosquitoes, especially on the functional analysis of their cytochrome P450 monooxygenases. First of all, adult mosquitoes were selected in the laboratory with permethrin which is a class of pyrethroid insecticides. After the selections for 8 generations, the resistance level of mosquitoes (SPS8 strain) reached more than 1000-fold compared to an insecticide susceptible strain. Since permethrin susceptibility largely increased with pre-treatment of mosquitoes with piperonyl butoxide, it was suggested that cytochrome P450 monooxygenases have the major role in the resistance. In vitro metabolism study revealed that microsomal protein prepared from resistant mosquitoes catalyzed metabolism of ^<14>C-permethrin more rapidly to 4'HO-permethrin than that of a susceptible strain. Furthremore, permethrin hydroxylation activity was NADPH dependent and was strongly inhibited by piperonyl butoxide strongly suggested that cytochrome P450 monooxygenases are involved in permethrin resistance in SPS8 strain. The P450 isofoms which are associated with permethrin resistance are promising candidates as novel target sites of insecticides or synergists. Sodium channel gene was genotyped for 26 adult Aedes albopictus collected from Singapre in 2009. Surprisingly, more than 70% of mosquitoes possessed the knockdown resistance (kdr) gene. This is the first evidence for the presence of the kdr gene in Aedes albopictus and suggests the need to study the global distribution of this allele in this important vector insect.