Chines Journal of Vector Biology and Control ›› 2019, Vol. 30 ›› Issue (2): 158-162.DOI: 10.11853/j.issn.1003.8280.2019.02.010

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An analysis of voltage-gated sodium channel gene mutation in Aedes albopictus resistant populations against pyrethroid insecticides in Ruili, Yunnan province, China

LAN Xue-mei1, XU Jia-bao2, JIANG Jin-yong1   

  1. 1. Yunnan Institute of Parasitic Diseases, Yunnan Provincial Center of Arborvirus Research, Yunnan Provincial Key Laboratory of Vector-borne Diseases Control and Research, Academician Workstation of Professor Jin Ningyi, Training Base of International Scientific Exchange and Education in Tropical Diseases for South and Southeast Asia, Yunnan Innovative Team of Key Techniques for Vector Borne Disease Control and Prevention(Developing), Pu'er 665099, Yunnan Province, China;
    2. Zhejiang Chinese Medical University
  • Received:2018-12-06 Online:2019-04-20 Published:2019-04-20
  • Supported by:
    Supported by the National Key Research and Development Plan (No. 2016YFC1200500), National Natural Science Foundation of China (No. U1602223) and the Yunnan Province Biomedical Major Project (No. 2017ZF007)

云南省瑞丽市白纹伊蚊对拟除虫菊酯类杀虫剂抗性种群的电压门控钠离子通道基因突变分析

兰学梅1, 徐家宝2, 姜进勇1   

  1. 1. 云南省寄生虫病防治所, 云南省虫媒病毒研究中心, 云南省虫媒传染病防控研究重点实验室, 金宁一院士工作站, 面向南亚东南亚热带病国际科技人员交流与教育培训基地, 云南省寄生虫病防治所虫媒传染病防控关键技术省创新团队(培育), 云南 普洱 665099;
    2. 浙江中医药大学, 浙江 杭州 310053
  • 通讯作者: 姜进勇,Email:yipdjiang@126.com
  • 作者简介:兰学梅,女,医师,从事媒介生物学与控制研究,Email:lanxuemei0715@163.com
  • 基金资助:
    国家重点研发计划(2016YFC1200500);国家自然科学基金(U1602223);云南省生物医药重大专项(2017ZF007)

Abstract: Objective To detect the mutations in the knockdown resistance (kdr) gene of the Aedes albopictus populations resistant and susceptible to permethrin, deltamethrin, and α-cypermethrin, and to elucidate the association between the resistance phenotypes and the kdr gene mutations. Methods From June to September, 2016, the Ae. albopictus populations in Ruili, Yunnan province, China, resistant to permethrin, deltamethrin, and α-cypermethrin and the Ae. albopictus populations, and the susceptible lab population of Jiangsu Ae. albopictus strain were collected, respectively, and their resistance phenotypes were detected by a bioassay. Individual genomic DNA was extracted. Three pairs of primers were sgnthetized for PCR amplification of partial gene fragments of voltage-gated sodium channel on the nerve cell membrane to detect the kdr gene mutations. The genotypes and their frequencies of the kdr gene of the resistant and susceptible populations were statistically analyzed. The chi-square test was used to analyze the association between the kdr gene mutations and the resistance phenotypes. Results A total of 500 kdr gene fragments were obtained, including 440 kdr gene fragments of Ae. albopictus resistant populations in Ruili and 60 kdr gene fragments of Ae. albopictus susceptible populations of Jiangsu strain. There was no mutation detected in the gene fragments from the susceptible populations. In Ae. albopictus resistant populations in Ruili, mutations were detected at the 1532, 1534, and 1763 sites. Both F1534S and I1532T mutations were detected in one sample. There were two alleles at the 1532 site, i.e., wild-type ATC/I (isoleucine) and mutant ACC/T (threonine), and their frequencies were 99.32% (292/294) and 0.68% (2/294), respectively; the frequencies of three genotypes-wild-type homozygous I/I, wild/mutant heterozygous I/T, and mutant homozygous T/T were 98.64% (145/147), 1.36% (2/147), and 0 (0/147), respectively. There were five alleles at the 1 534 site, i.e., wild-type TTC/F (phenylalanine), mutant TCC/S (serine), TCG/S (serine), TTG/L (leucine), and TGC/C (cysteine), and their frequencies were 59.53% (175/294), 29.93% (88/294), 0.68% (2/294), 2.72% (8/294), and 7.14% (21/294), respectively; the frequencies of six genotypes-wild-type homozygous F/F, wild-type/mutant heterozygous F/C, F/S, and F/L, mutant homozygous S/S, and mutant heterozygous C/S were 40.14% (59/147), 6.80% (10/147), 26.53% (39/147), 5.44% (8/147), 13.61% (20/147), and 7.48% (11/147), respectively. There were two alleles at the 1 763 site, i.e., wild-type GAC/D (aspartic acid) and mutant TAC/Y (tyrosine), their frequencies were 99.32% (292/294) and 0.68% (2/294), respectively; the frequencies of three genotypes-wild-type homozygous D/D, wild-type/mutant heterozygous D/Y, and mutant homozygous Y/Y were 98.64% (145/147), 1.36% (2/147), and 0 (0/147), respectively. Conclusion In the kdr gene of Ae. albopictus population resistant to pyrethroid insecticides in Ruili, mutations are detected at the 1532, 1534, and 1763 sites and no mutations are detected at the 989 and 1 014 sites. The mutations at the 1534 site are dominant and the mutations at the 1763 site are first discovered. Single mutation is dominant and only one sample contains both the F1534S and I1532T mutations. This study confirms that the kdr mechanism is one of the resistance mechanisms of Ae. albopictus populations against pyrethroid insecticides in Ruili.

Key words: Ruili, Aedes albopictus, Resistance to pyrethroid, Voltage-gated sodium channel, Gene mutation

摘要: 目的 检测云南省瑞丽市白纹伊蚊对氯菊酯、溴氰菊酯和顺式氯氰菊酯杀虫剂抗性种群和敏感种群的击倒抗性(kdr)基因突变,阐明抗性表型与kdr基因突变的关系。方法 2016年6-9月收集瑞丽市白纹伊蚊成蚊对氯菊酯、溴氰菊酯和顺式氯氰菊酯抗药性生物测定的蚊虫样本,江苏白纹伊蚊实验室敏感品系种群,单蚊提取基因组DNA,合成3对引物,PCR扩增神经细胞膜上电压门控钠离子通道(VGSC)部分基因片段,检测kdr基因突变情况。统计抗性和敏感种群kdr突变的基因型和基因频率。采用χ2检验,分析kdr基因突变与抗性表型的相关性。结果 共获得500条kdr基因片段,其中瑞丽市白纹伊蚊抗性种群kdr基因片段440条,江苏敏感品系种群kdr基因片段60条。敏感种群kdr基因片段各位点均未发生突变。瑞丽市白纹伊蚊抗性种群kdr基因在1532、1534和1763位点存在突变。1份样本同时存在F1534S和I1532T突变。1532位点共有2种等位基因,即野生型ATC/I(异亮氨酸)和突变型ACC/T(苏氨酸),频率分别为99.32%(292/294)和0.68%(2/294);3种基因型,即野生型纯合子I/I、野生/突变型杂合子I/T和突变型纯合子T/T,频率分别为98.64%(145/147)、1.36%(2/147)和0(0/147);1534位点共有5种等位基因,即野生型TTC/F(苯丙氨酸),突变型TCC/S(丝氨酸)、TCG/S(丝氨酸)、TTG/L(亮氨酸)和TGC/C(半胱氨酸),频率分别为59.53%(175/294)、29.93%(88/294)、0.68%(2/294)、2.72%(8/294)和7.14%(21/294);6种基因型,即野生型纯合子F/F、野生型/突变型杂合子F/C、F/S、F/L、突变型纯合子S/S和突变型杂合子C/S,频率分别为40.14%(59/147)、6.80%(10/147)、26.53%(39/147)、5.44%(8/147)、13.61%(20/147)和7.48%(11/147);1763位点共有2种等位基因,即野生型GAC/D(天冬氨酸)和突变型TAC/Y(酪氨酸),频率分别为99.32%(292/294)和0.68%(2/294);3种基因型,即野生型纯合子D/D、野生型/突变型杂合子D/Y和突变型纯合子Y/Y,频率分别为98.64%(145/147)、1.36%(2/147)和0(0/147)。结论 瑞丽市对拟除虫菊酯类杀虫剂产生抗性的白纹伊蚊种群中,发现有1532、1534和1763位点存在突变,未发现989和1014等位点的突变,其中以1534位点的突变为主,首次发现了1763位点突变。突变以单一突变为主,仅1份样本同时存在F1534S和I1532T突变。该研究证实了kdr机制是云南省瑞丽市白纹伊蚊对拟除虫菊酯类杀虫剂产生抗性的机制之一。

关键词: 瑞丽市, 白纹伊蚊, 拟除虫菊酯类杀虫剂, 电压门控钠离子通道, 基因突变

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