白纹伊蚊实验室抗溴氰菊酯品系筛选及其形态变化和钠通道基因分析

Screening of deltamethrin resistant strains of Aedes albopictus in laboratory and their morphological changes and sodium channel gene analysis

目的揭示实验室内白纹伊蚊溴氰菊酯抗性汰选中蚊抗性发展趋势、形态表型和蚊击倒抗性相关钠离子通道(VGSC)结构域Ⅲ基因片段(Kdr)的变化。方法实验室严格控制饲养条件,用溴氰菊酯汰选白纹伊蚊至30代,计算幼虫半数致死浓度(LC50)并获得抗性倍数(RR),成蚊接触筒法计算成蚊死亡率。随机选取汰选白纹伊蚊抗性株及敏感株的雌蚊,测量成蚊的体长等8个形态学指标,并对所测形态指标数据进行统计分析。PCR扩增抗性株及敏感株雌蚊钠通道结构域Ⅲ基因片段,TA克隆,测序,进行序列分析。结果经过汰选,抗性株的LC50由0.020 mg/L变成0.048 mg/L,其抗性倍数为2.4倍。幼虫生物测量结果显示抗性株具有低度抗性,成蚊抗药性生物测试表明R-20代白纹伊蚊为抗性群体,且抗性级别为R。18代白纹伊蚊抗溴氰菊酯株(R)与敏感株(S)的体长、前腿长、中腿长、后腿长、翅长、翅宽、触角长和喙长均数差异有统计学意义(P<0.05)。随着代数的增加,白纹伊蚊抗性株在形态学各方面指标的均数呈现降低的趋势。测序显示,Kdr第96、132、204个位点碱基敏感株为C,而抗性株则突变为T;第111位敏感株碱基为A,抗性株则为G;第534位抗性株碱基由T突变为C。然而,敏感株及抗性株均未发现上述位点的氨基酸序列突变,未影响其蛋白质的表达。结论实验室内采用溴氰菊酯抗性汰选抗性白纹伊蚊模型成功。溴氰菊酯汰选抗性白纹伊蚊形态表型发生变化,溴氰菊酯汰选抗性白纹伊蚊的19代、20代蚊Kdr基因一些位点发生碱基突变,但对应的氨基酸序列没有改变。

Objective To reveal the development trend of mosquito resistance and the changes of morphological phenotype and Kdr gene of Aedes albopictus in deltamethrin resistance screening in laboratory. Methods In the laboratory,the living conditions of Aedes albopictus were strictly controlled. Deltamethrin was used to select resistant Aedes albopictus to 30 generations,and the lethal concentration 50(LC50)of larvae and resistance ratio(RR)were obtained,and adult mosquito mortality was calculated by contact tube method. Eight morphological indexes of the resistant and sensitive Aedes albopictus females who were selected by random selection,such as the length of adult mosquitoes,were measured. The morphological indexes data were statistically analyzed. The sodium channel domain Ⅲ gene fragments of resistant and sensitive female mosquitoes were amplified by PCR,cloned by TA cloning and sequenced. Results The LC50 of the resistant strains changed from 0.020 mg/L to 0.048 mg/L;the RR of the resistant strains was 2.4 times of larval bioassay. The results of larval resistance bioassay showed that the resistant strains had low resistance,and the resistance bioassay of adult mosquitoes showed that the R-20 generation of Aedes albopictus was the resistant population,and the resistance level was R.Statistical analysis showed that there were significant differences in body length,foreleg length,middle leg length,hind leg length,wing length,wing width,antenna length and proboscis length between the 18-generation Aedes albopictus resistant(R)and susceptible(S)strains(P<0.05). The morphological changes of Aedes albopictus showed that with the increase of generation,the average level of morphological indexes of the resistant strains demonstrated a decreasing trend. The nucleotide at 96,132,204 sites of Kdr gene of the resistant strain were changed from C to T,while the nucleotide at 111 site was changed from A to G and the 534 site was from T to C. However,these mutations were silence mutations.Conclusion The model of deltamethrin-resistant selection of Aedes albopictus was successfully established in the laboratory.The morphological phenotypes of deltamethrin-resistant population of Aedes albopictus mosquitoes had changed. The Kdr gene of the 19 and 20 generations of deltamethrin-resistant Aedes albopictus mosquitoes had silence mutations at several sites.