Enayati A A, Vatandoost H, Ladonni H, Townson H, Hemingway J
Medical Entomology Group, School of Public Health, Mazandaran University of Medical Sciences, Amir Boulevard, Sari, Iran.
Med Vet Entomol. 2003 Jun;17(2):138-44. doi: 10.1046/j.1365-2915.2003.00418.x.
The mosquito Anopheles stephensi Liston (Diptera: Culicidae) is the urban vector of malaria in several countries of the Middle East and Indian subcontinent. Extensive use of residual insecticide spraying for malaria vector control has selected An. stephensi resistance to DDT, dieldrin, malathion and other organophosphates throughout much of its range and to pyrethroids in the Middle East. Metabolic resistance mechanisms and insensitivity to pyrethroids, so-called knockdown resistance (kdr), have previously been reported in An. stephensi. Here we provide molecular data supporting the hypothesis that a kdr-like pyrethroid-resistance mechanism is present in An. stephensi. We found that larvae of a pyrethroid-selected strain from Dubai (DUB-R) were 182-fold resistant to permethin, compared with a standard susceptible strain of An. stephensi. Activities of some enzymes likely to confer pyrethroid-resistance (i.e. esterases, monooxygenases and glutathione S-transferases) were significantly higher in the permethrin-resistant than in the susceptible strain, but the use of synergists--piperonyl butoxide (PBO) to inhibit monooxygenases and/or tribufos (DEF) to inhibit esterases--did not fully prevent resistance in larvae (permethrin LC50 reduced by only 51-68%), indicating the involvement of another mechanism. From both strains of An. stephensi, we obtained a 237-bp fragment of genomic DNA encoding segment 6 of domain II of the para type voltage-gated sodium channel, i.e. the putative kdr locus. By sequencing this 237 bp fragment, we identified one point mutation difference involving a single A-T base change encoding a leucine to phenylalanine amino acid substitution in the pyrethroid-resistant strain. This mutation appears to be homologous with those detected in An. gambiae and other insects with kdr-like resistance. A diagnostic polymerase chain reaction assay using nested primers was therefore designed to detect this mechanism in An. stephensi.
斯氏按蚊（双翅目：蚊科）是中东和印度次大陆多个国家城市地区的疟疾传播媒介。为控制疟疾传播媒介而广泛使用残留杀虫剂喷洒，已使斯氏按蚊在其大部分分布范围内对滴滴涕、狄氏剂、马拉硫磷和其他有机磷产生了抗性，并在中东地区对拟除虫菊酯产生了抗性。此前已有报道称，斯氏按蚊存在代谢抗性机制以及对拟除虫菊酯的不敏感性，即所谓的击倒抗性（kdr）。在此，我们提供分子数据支持以下假说：斯氏按蚊存在类似kdr的拟除虫菊酯抗性机制。我们发现，与斯氏按蚊的标准敏感品系相比，来自迪拜的拟除虫菊酯选择品系（DUB-R）的幼虫对氯菊酯的抗性高182倍。一些可能赋予拟除虫菊酯抗性的酶（即酯酶、单加氧酶和谷胱甘肽S-转移酶）的活性在氯菊酯抗性品系中显著高于敏感品系，但使用增效剂——胡椒基丁醚（PBO）抑制单加氧酶和/或三丁磷（DEF）抑制酯酶——并不能完全消除幼虫的抗性（氯菊酯LC50仅降低51%-68%），这表明存在另一种机制。从斯氏按蚊的两个品系中，我们获得了一段237 bp的基因组DNA片段，该片段编码para型电压门控钠通道结构域II的第6段，即假定的kdr位点。通过对这237 bp片段进行测序，我们在拟除虫菊酯抗性品系中鉴定出一个点突变差异，涉及单个A-T碱基变化，导致编码的亮氨酸变为苯丙氨酸的氨基酸替换。该突变似乎与在冈比亚按蚊和其他具有类似kdr抗性的昆虫中检测到的突变同源。因此，设计了一种使用巢式引物的诊断聚合酶链反应分析方法来检测斯氏按蚊中的这种机制。
