云南省三带喙库蚊对DDT和溴氰菊酯抗性群体的击倒抗性基因突变分析

Analysis of knowdown resistance gene mutation in Culex tritaeniorhynchus resistant to DDT and deltamethrin in Yunnan Province, China

目的分析经接触DDT和溴氰菊酯敏感性测定的三带喙库蚊(Culex tritaeniorhynchus)的抗性(存活)与敏感(死亡)表型样本的击倒抗性基因(knockdown resistance,kdr)序列,阐明其抗性表型与kdr基因突变的关系。方法收集云南昭阳、芒市、江城和孟连等地的三带喙库蚊经接触DDT和溴氰菊酯测定后死亡和存活的个体,分别提取单蚊基因组DNA,PCR扩增kdr部分基因片段,测定和分析序列,统计抗性与敏感表型个体中kdr突变的基因型和频率,采用χ~2检验分析kdr基因突变与抗性表型的相互关系。结果共获得411条三带喙库蚊kdr基因序列,检测结果显示,在1014位点存在突变,等位基因有2种,即野生型TTA/L和突变型TTT/F;基因型共3种,分别为野生型纯合子L/L,突变型纯合子F/F,以及野生型/突变型杂合子L/F,频率分别为96.35%(396/411)、0.73%(3/411)和2.92%(12/411)。在接触DDT的抗性与敏感表型个体中,突变基因型频率分别为3.42%(4/117)和3.88%(4/103);在接触溴氰菊酯的抗性与敏感个体中,突变基因型频率为3.96%(4/101)和3.33%(3/90)。接触DDT和溴氰菊酯的三带喙库蚊抗性与敏感表型与kdr基因型频率无相关性(χ~2=0.034,P>0.05;χ~2=0.053,P>0.05)。结论云南省三带喙库蚊中发现kdr新的等位基因L1014F,但频率较低。该蚊对DDT和溴氰菊酯产生抗性与kdr基因突变未见相关性。

Objective To detect the knockdown resistance （kdr） gene mutations in Culex tritaeniorhynchus resistant or susceptible to DDT and deltamethrin, in order to elucidate the relationship between the resistant phenotype and kdr mutations. Methods C. tritaeniorhynchus mosquitoes were captured in Zhaoyang, Mang Shi, Jiang Cheng and Meng Lian in Yunnan Province, and exposed to DDT and deltamethrin, respectively. The resulting alive and dead ones were counted. Genomic DNA was extracted by individual mosquitoes. The kdr gene was amplified by PCR and analyzed. The frequency of kdr genotypes was estimated in resistant and susceptible samples. The relationship between kdr genotypes and resistant phenotypes was analyzed by Chi-square test. Results A total of 411 mosquitoes were genotyped for the kdr gene. Mutation was detected at codon 1014, yielding two alleles, the wild- type TFA/L and the mutant TIT/F, and three genotypes, the wild-type homozygote L/L （frequency, 96.35%; 396/411）, the mutant homozygote F/F （0.73%; 3/411） and the wild/mutant heterozygous L/F （2.92%; 12/411）. The frequencies of mutant genotype in DDT-resistant and -susceptible individuals were 3.42% （4/117） and 3.88% （4/103）, respectively; while those in deltamethrin-resistant and -susceptible individuals were 3.96% （4/101） and 3.33% （3/90）, respeetively. Chi-square testrevealed that there was no correlation between kdr genotype frequency and the resistance phenotypes to either DDT （x2 = 0.034, P〉0.05） or deltamethrin （x2=0.053, P〉0.05）. Conclusion We found a novel L1014F mutant allele for kdr, albeit with a low frequency. There is no correlation between kdr gene mutation and the resistance phenotypes to either DDT or deltamethrin.