Studies On Pesticide Resistance And Population Genetics Of Anopheles Sinensis

Mosquitoes are important health pests distributed worldwide, not only vampire harassment, but also spreading many diseases. Malaria is spread by anopheles mosquitoes carrying malaria parasite to the human body, which cause serious damage to human health. Control of mosquitoes mainly relys on the use of pesticides, however, long-term large-scale use has led to mosquito resistance to pesticides, which has become a major obstacle to mosquito control and malaria prevention.In this study, samples of Anopheles sinensis from southwestern Yunnan province and central Anhui in malaria endemic areas in China were collected, their resistance to four categories of chemical pesticides were tested, binding target resistance gene kdr, ace-1and metabolic detoxification enzyme P450s, GSTs and COEs were analysed in order to investigate the combined effect of these two resistance mechanisms in the mosquito. Further, we collected14national geographic population samples of Anopheles sinensis, and analysed mitochondrial genes co-I, co-II sequence to explore the relationship between target site kdr gene mutation and genetic evolution,The main results are as follows:The results of resistance bioassay of Anhui and Yunnan populations to four kinds of pesticides showed that all are resistant populations. The resistance level of Anhui population to permethrin, DDT and malathion are higher than that of Yunnan population. The resistance level of Anhui and Yunnan populations to deltamethrin and bendiocarb is similar. In Anhui and Yunnan regions, the anopheles mosquitoes exist multiple resistance.The anopheles kdr1014points were detected to have two kinds of non-synonymous mutations:L1014F and L1014C. Kdr gene were found six genotypes:Anhui populations exist five kinds of genotype, while Yunnan populations and laboratory sensitive populations only have one kind of non-mutated wild type. kdr type detection in a large number of samples by the AS-PCR showed that L1014F kdr gene mutation is more and L1014C mutant is less in Anhui samples, especially kdr gene mutation rate is significantly higher at the deltamethrin resistant populations than at the sensitive population, prompting kdr gene mutation in1014and deltamethrin resistance have certain relevance. In Yunnan populations and laboratory sensitive populations, kdr gene mutations are all wild-type L1014.The anopheles ace-1gene119loci was detected to belong to one kind of non-synonymous mutations G119S. Ace-1gene were found in three genotypes: Anhui and Yunnan populations exist two kinds of mutant genotypes, laboratory susceptible population is only one kind of non-mutated wild type. PCR-RFLP assay showed that the both Anhui population and Yunnan population appear moderate frequency of mutations, and the mutations in the two populations are associated with malathion resistance. In laboratory sensitive population, ace-1gene is G119unmutated wild type.The bioassay results of P450s, GSTs and COEs enzyme activity in different populations displayed in Anhui and Yunnan samples, the average P450s activity are significantly higher in deltamethrin, permethrin and malathion resistant populations than in sensitive populations, the average GSTs and COEs activity are also significantly higher in malathion resistant populations than in sensitive populations These results suggest that these three metabolic detoxification enzymes play an important role in Anopheles sinensis resistance to malathion.Multi-factor classification and regression method (CART) analysis showed two target sites and three kinds of metabolic detoxification enzymes play a comprehensive role in Anopheles sinensis resistance to four kinds of pesticides, metabolic role is the most important in resistance mechanism, especially P450s play the biggest role, and target kdr gene and ace-1mutation only play a small role.Using mtDNA co-â… , co-â…¡ as a molecular marker, genetic structure of Anopheles sinensis from14geographical populations in China were analyzed, the results showed that higher genetic diversity exists in all populations. Mantel test showed that there is positive correlation between genetic distance and geographic distance, and there is a certain geographic segregation in Anopheles populations. The analysis of fixed population genetic differentiation coefficient, molecular variation level and the clustering of relationship showed that there is the genetic differentiation between Yunnan populations and the other populations, and there is not obvious genetic differentiation and there may be a high level of gene flow among the other populations. Mismatch analysis showed that the other population of Anopheles sinensis recently experienced expansion, but Yunnan population was stable and not seen a sustained growth.The study results furnish a scientific consultation for resistance mechanism research and malaria control, and provide basic data for the research on mosquito pesticide resistance and genetic evolution.