Polymorphism Of Pyrethroid Knockdown Resistance Gene In Culex Pipiens Pallens Mosquitoes

Current disease vector control strategy is based primarily on the application of insecticides. However, excessive and continuous application of insecticides has caused the development and spread of insecticide resistance, which has become a major obstacle to insect-borne disease control. Accurately monitoring resistance status is essential to guide the rational use of insecticides and resistance management.Target site insensitivity due to point mutations in the para-sodium channel is an important mechanism of resistance to pyrethroid insecticides in mosquitoes and many other insects. It is the current focus to search the molecular markers for pyrethroid resistance detecting in natural mosquito populations from genes associated with knockdown resistance (kdr).In the present study, we examined the nucleotide diversity of kdr gene in 6 Cx. pipiens pallens populations in China, established molecular detection methods for the key mutations associated with resistance, and explored the relationship between kdr allele frequency and resistance level.1.We examined the nucleotide diversity of the para-sodium channel gene which confers knockdown resistance in the Culex mosquitoes in China. The sequence analysis of the para-sodium channel gene identified L1014F and L1014S mutations.We found that the proportion of wild-type(L1014/L1014) genotype was significantly higher in those individuals killed by exposure to 0.05% deltamethrin in Nanjing and Tangkou populations ( p< 0.0001). In contrast, the proportion of mutant genotype(L1014F/L1014F) was significantly higher in the surviving individuals than in the dead dividuals in the two populations (p <0.0001). These data suggest that L1014F allele may be associated in resistance to deltamethrin in Cx. pipens pallens, and L1014F mutations in the kdr gene can be used as a molecular marker for deltamethrin resistance detecting in natural mosquito populations.2. We also found that the proportion of wild-type(L1014/L1014) genotype was significantly higher in those individuals killed by exposure to 0.05% deltamethrin in Wuxi, Weishan, Huaibei and Qingdao populations (p< 0.0001). In contrast, the proportion of mutant genotypes(L014F/L1014F) was significantly higher in the surviving individuals than in the dead individuals in the four populations (p <0.0001). However, the proportion of mutant genotypes(L014S/L1014S) was similar between the dead and surviving individuals (p>0.05). These data confirmed that L1014F allele was associated in resistance to deltamethrin in Cx. pipens pallens mosquitoes, and L1014F mutations in the kdr gene can be used as a molecular marker for deltamethrin resistance detecting and monitoring in natural mosquito populations.3. The frequency of L1014F alleles for the 6 populations varied significantly among the six populations (p< 0.0001) , ranging from 40.3% (Nanjing population) to 71.7% (Tangkou population). A significant positive correlation between the frequency of L1014F allele and survival rate determine by the deltamethrin susceptibly bioassay (y=1.4681x-23.365, r2 = 0.870, p<0.0001) was found with regression analysis, and the correlation between the frequency of L1014S allele and survival rate was not found (r2 = 0.110, p>0.05). This suggests that the frequency of L1014F allele in the kdr gene is an excellent marker for monitoring deltamethrin resistance level in natural mosquito populations.4. Cx. pipiens pallens population collected in Tangkou, Shandong Province, was selected with deltamethrin at larval stage for a total of 12 generations in the laboratory. After six and twelve generations of selection, the 50% lethal concentration (LC50) increased by 2.77 folds and 28.24 folds, respectively.5. In the laboratory selection experiment, it was found that L1014F mutation frequency, but not L1014S mutation frequency, responded to deltamethrin selection, suggesting that the L1014F mutation is a key mutation conferring resistance to deltamethrin, and the L1014S mutation is not likely involved in deltamethrin resistance.6. We developed and validated allele-specific PCR and the real-time TaqMan methods as a mean of molecular resistance diagnosis. The real-time TaqMan assay is more superior to the allele-specific PCR method as evidenced by higher amplification rate and better sensitivity and specificity.