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
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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。
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 F1534 S and I1532 T 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 were98.64%(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), and7.48%(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 F1534 S and I1532 T mutations. This study confirms that the kdr mechanism is one of the resistance mechanisms of Ae. albopictus populations against pyrethroid insecticides in Ruili.
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 F1534 S and I1532 T 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 were98.64%(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), and7.48%(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 F1534 S and I1532 T mutations. This study confirms that the kdr mechanism is one of the resistance mechanisms of Ae. albopictus populations against pyrethroid insecticides in Ruili.
引文
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[17]Saavedra Rodriguez K,Urdaneta Marquez L,Rajatileka S,et al.A mutation in the voltage gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti[J].Insect Mol Biol,2007,16(6):785-798.DOI:10.1111/j.1365-2583.2007.00774.x.
[18]Kasai S,Ng LC,Lam-Phua SG,et al.First detection of a putative knockdown resistance gene in major mosquito vector,Aedes albopictus[J].Jpn J Infect Dis,2011,64(3):217-221.
[19]Zhu F,Lavine L,O’Neal S,et al.Insecticide resistance and management strategies in urban ecosystems[J].Insects,2016,7(1):2.DOI:10.3390/insects7010002.
[20]唐振华,袁建忠,庄佩君,等.昆虫钠通道的结构和与击倒抗性有关的基因突变[J].昆虫学报,2004,47(6):830-836.DOI:10.3321/j.issn:0454-6296.2004.06.022.
[21]Xu JB,Bonizzoni M,Zhong DB,et al.Multi-country survey revealed prevalent and novel F1534S mutation in voltage-gated sodium channel(VGSC)gene in Aedes albopictus[J].PLo S Negl Trop Dis,2016,10(5):e0004696.DOI:10.1371/journal.pntd.0004696.
[22]师灿南.景洪市登革热媒介伊蚊对常用杀虫剂的抗药性及机制初步研究[D].北京:中国疾病预防控制中心,2017.
[23]Chen HY,Li KL,Wang XH,et al.First identification of kdr allele F1534S in VGSC gene and its association with resistance to pyrethroid insecticides in Aedes albopictus populations from Haikou city,Hainan Island,China[J].Infect Dis Poverty,2016,5:31.DOI:10.1186/s40249-016-0125-x.
[2]Black IV WC,Bennett KE,Gorrochótegui-Escalante N,et al.Flavivirus susceptibility in Aedes aegypti[J].Arch Med Res,2002,33(4):379-388.DOI:10.1016/S0188-4409(02)00373-9.
[3]王永亮,钱成,郭运生,等.全球登革热流行势态及其影响评述[J].口岸卫生控制,2018,23(4):20-26.
[4]林钟宇,潘华峰,王正,等.2014年广东登革热流行趋势与防控对策[J].卫生软科学,2015,29(9):590-592.
[5]刘华兴,王江宁,弥鹏飞,等.西双版纳州2013年登革热流行病学特征分析[J].卫生软科学,2014,28(6):399-402.
[6]潘欢欢,李锋平.2007-2016年福建泉州市登革热流行特征分析[J].公共卫生与预防医学,2017,28(5):30-32.
[7]王传滋,陈文洲.海南省登革热历年流行概况[J].海南医学,1992,3(8):1-4.
[8]郑宇婷,杨明东,周克梅.云南省边境地区2016年登革热媒介监测结果分析[J].中国媒介生物学及控制杂志,2018,29(2):157-160.DOI:10.11853/j.issn.1003.8280.2018.02.010.
[9]Kyaw AK,Ngwe Tun MM,Moi ML,et al.Clinical,virological and epidemiological characterization of dengue outbreak in Myanmar,2015[J].Epidemiol Infect,2017,145(9):1886-1897.DOI:10.1017/S0950268817000735.
[10]刘永华,尹小雄,杨召兰,等.云南省瑞丽市2013年登革热暴发的流行病学分析[J].中国媒介生物学及控制杂志,2014,25(6):524-526.DOI:10.11853/j.issn.1003.4692.2014.06.010.
[11]韩继周,尹正留,刘永华.2014年瑞丽市登革热疫情流行特征分析[J].中国卫生产业,2015(15):186-188.DOI:10.16659/j.cnki.1672-5654.2015.15.070.
[12]王建羽,兰学梅,杨锐,等.云南省耿马县埃及伊蚊对6种常用卫生杀虫剂的抗药性研究[J].中国媒介生物学及控制杂志,2017,28(5):444-446.DOI:10.11853/j.issn.1003.8280.2017.05.008.
[13]高景鹏,陈翰明,马雅军.我国白纹伊蚊对3种拟除虫菊酯类杀虫剂抗性检测诊断剂量的建立[J].昆虫学报,2018,61(1):18-24.DOI:10.16380/j.kcxb.2018.01.003.
[14]兰学梅,郑宇婷,董朝良,等.云南省瑞丽市埃及伊蚊和白纹伊蚊对常用杀虫剂的抗药性调查[J].中国媒介生物学及控制杂志,2017,28(6):572-575.DOI:10.11853/j.issn.1003.8280.2017.06.014.
[15]王晓花,陈辉莹,杨新艳,等.海口市白纹伊蚊对菊酯类杀虫剂的抗药性及击倒抗性基因突变分析[J].第二军医大学学报,2015,36(8):832-838.DOI:10.3724/SP.J.1008.2015.00832.
[16]董学书,周红宁,龚正达.云南蚊类志(下卷)[M].昆明:云南科技出版社,2010:80-82.
[17]Saavedra Rodriguez K,Urdaneta Marquez L,Rajatileka S,et al.A mutation in the voltage gated sodium channel gene associated with pyrethroid resistance in Latin American Aedes aegypti[J].Insect Mol Biol,2007,16(6):785-798.DOI:10.1111/j.1365-2583.2007.00774.x.
[18]Kasai S,Ng LC,Lam-Phua SG,et al.First detection of a putative knockdown resistance gene in major mosquito vector,Aedes albopictus[J].Jpn J Infect Dis,2011,64(3):217-221.
[19]Zhu F,Lavine L,O’Neal S,et al.Insecticide resistance and management strategies in urban ecosystems[J].Insects,2016,7(1):2.DOI:10.3390/insects7010002.
[20]唐振华,袁建忠,庄佩君,等.昆虫钠通道的结构和与击倒抗性有关的基因突变[J].昆虫学报,2004,47(6):830-836.DOI:10.3321/j.issn:0454-6296.2004.06.022.
[21]Xu JB,Bonizzoni M,Zhong DB,et al.Multi-country survey revealed prevalent and novel F1534S mutation in voltage-gated sodium channel(VGSC)gene in Aedes albopictus[J].PLo S Negl Trop Dis,2016,10(5):e0004696.DOI:10.1371/journal.pntd.0004696.
[22]师灿南.景洪市登革热媒介伊蚊对常用杀虫剂的抗药性及机制初步研究[D].北京:中国疾病预防控制中心,2017.
[23]Chen HY,Li KL,Wang XH,et al.First identification of kdr allele F1534S in VGSC gene and its association with resistance to pyrethroid insecticides in Aedes albopictus populations from Haikou city,Hainan Island,China[J].Infect Dis Poverty,2016,5:31.DOI:10.1186/s40249-016-0125-x.