| Mosquitoes transmit many mosquito-borne diseases which threaten the public health, including malaria, dengue fever, etc. The control programs for mosquito-borne diseases are based on control of mosquitoes, and insecticides are the major measure, because of rapid and highly efficient, low cost, and easy to use. However, excessive and continuous usage of insecticides induced the development and spread of mosquito resistance to insecticides, which compromised the effectiveness of control programs, and emergent/re-emergent mosquito-borne diseases continued to occur. Insecticide resistance has become a major obstacle in mosquito-borne diseases control. Hence, research on resistance mechanisms has been the key topics of global prevention and control programs.In this study, we used a field population of Culex. pipiens pollens as experimental object, because they were the most prevalent and important habitat vector in china with high population density. The population was collected from the natural habitats in Shandong province and was selected with the most widely used pyrethroid insecticide deltamethrin for every generation. We dynamically monitored the insecticide susceptibility in mosquito populations under different insecticide selection pressure, and detected the target site resistance(kdr) and three detoxification enzyme activities in the same mosquito sample, by means of a large sample size, in order to reveal the variation of target site resistance and metabolic resistance under different insecticide selection pressure, and explore the heredity and evolution mechanism of insecticide resistance.The main results of this study were as follows:1. The establishment of selected strains with different insecticide selection pressuresA field population of Cx. pipiens pallens had collected and selected with deltamethrin in laboratory. Then, three strains were selected for30generations and28months under different insecticide selection pressure:intense selection strain (IS strain), selected with a LC50concentration of deltamethrin for each generation, reach50-60%mortality; mild selection strain (MS strain), selected with the LC50concentration of deltamethrin for generation6all the way, decreasing mortality; no-selection strain (NS strain), stopped exposing to deltamethrin.2. The dynamically monitoring of the variation of insecticide resistance in the three strains with different insecticide selection pressureIn the IS strain:the level of insecticide resistance was increased generation by generation. At generation30, the LC50of IS strain was increased significantly to0.9713mg/L (p<0.001), and the resistance ratio (RR) was47.15.In the MS strain:the level of insecticide resistance was also increased generation by generation, but the increasing trend was slower than IS strain. At generation30, the LC50of MS strain was increased significantly to0.4367mg/L (p<0.001), and the RR was21.20.In the NS strain:the level of insecticide resistance was reduced slowly generation by generation. The LC50of NS strain was reduced significantly from0.0501mg/L at generation6to0.0241mg/L at generation30(p<0.001), and the RR was also reduced significantly from2.43to1.17, but still higher than the generation1(LC50=0.0206mg/L).The above results suggested that the insecticide resistance of populations was increased significantly after long-term exposure to pyrethroid insecticides. The developing speed of resistance was closely related to the insecticide selection pressure. The higher the selection pressure was, the faster the resistance developed. The pyrethroid resistance was instable, so the resistance level could be decreased without pyrethroid insecticides selection. However, the declining trend of resistance was slow. In addition, long-term exposure to low doses of insecticides would also lead to resistance. For example, the MS strain was selected with the constant low dose of deltamethrin, and the resistance level was also increased with time. Although the increasing trend is slower in MS strain than in IS strain, the RR was increased by21.20folds.3. Simultaneous determination of the variation of target site resistance and metabolic resistance in the same single mosquitoIn our research,3000cases of single mosquito were sequenced and detected for the three metabolic enzymes activities. Some samples had no results due to the failed detection. In the end, we successfully sequenced2427samples, and detected1541samples for P450s,1368samples for ESTs and1665samples for GSTs.In the IS strain, the variation of target site resistance:the frequency of resistant allele L1014F was increased generation by generation; the frequency was increased to100%at generation14, and the strain became kdr resistance gene homozygous population; the frequency of L1014and L1014S was reduced generation by generation. The variation of metabolic resistance:the monooxygenases activities was increased with the development of deltamethrin resistance, and increased by2.30folds at generation30, but the increasing trend was significantly higher after generation14; the carboxylesterase activities was increased with the development of deltamethrin resistance, and increased by1.55folds at generation30, but the increasing trend was relatively stable, and the significant increasing trend was after generation14; the glutathione-S-transferase activities was not involved in deltamethrin resistance.In the MS strain, the variation of target site resistance:the frequency of resistant allele L1014F was increased generation by generation; the frequency was increased to100%at generation26, and the strain became kdr resistance gene homozygous population; the frequency of L1014and L1014S was reduced generation by generation. The variation of metabolic resistance:the monooxygenases activities was increased with the development of deltamethrin resistance, and increased by1.66folds at generation30, but the increasing trend was slower than in IS strain, and the increasing trend was after generation26; the carboxylesterase activities was increased with the development of deltamethrin resistance, and increased by1.18folds at generation30, but the increasing trend was relatively stable and was slower than in IS strain; the glutathioneS-transferase activities was not involved in deltamethrin resistance.In the NS strain, the variation of target site resistance:the frequency of resistant allele L1014F was reduced generation by generation; the frequency was reduced significantly from91.79%at generation6to82.27%at generation30(p<0.001), but still higher than the generation1(72.64%); The frequency of L1014S was increased generation by generation. The variation of metabolic resistance:the monooxygenases activities was reduced generation by generation and reduced by0.81folds at generation30; the carboxylesterase activities was reduced generation by generation and reduced by0.69folds at generation30; the glutathione S-transferase activities was not involved in deltamethrin resistance.The correlation analysis among target resistance kdr gene frequency, the variation of detoxification enzyme activities and the level of insecticide resistance under different insecticide selection pressure, demonstrated as follows:the frequency of resistant allele L1014F was positively correlated resistant phenotype, its frequency increased with the development of deltamethrin resistance, and the increased rate was closely related to selection pressure. Increased monooxygenase and carboxylesterase activities led to metabolic resistance, moreover, the increased monooxygenase activities was the main factors. All the results suggested that kdr, monooxygenase and carboxylesterase could be used as molecular markers for resistance detection. Both of metabolic resistance and target site resistance were involved in insecticide resistance. Early in the development of resistance, target site resistance play a leading role, and when the populations developed to a certain stage, the metabolic resistance began to play the leading role, and the resistance level was increased more significantly. The different intensity of selection caused by insecticides was the major factor that made the two resistance mechanisms plays a role in succession. After removal of insecticide selection pressure, metabolic resistance was reduced more significantly than target site resistance. This result suggested that the fitness cost of metabolic resistance was higher than that of target site resistance in the population, so the insecticide selection pressure preferred to select the mosquito with target site resistance. Furthermore, whether there were other mutations in the sodium ion channel gene, which genes related to metabolic resistance, or the mechanism that enhanced the detoxication of insecticide, still need further research.In this study, we used a field population as object, and dynamically monitored the insecticide susceptibility in mosquitoes populations under different insecticide selection pressure for30generations. Moreover, we detected the target site resistance and three detoxification enzyme activities in the same mosquito sample, by means of a large sample size. The results could guide the use of insecticides, insecticide-resistance detection and management in prevention and control programs. |
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