我国白纹伊蚊现场群体击倒抗性基因I1532和F1534突变检测及I1532T突变等位基因报告

doi:10.11853/j.issn.1003.8280.2018.02.002

摘要/Abstract

摘要： 目的检测我国白纹伊蚊现场群体的击倒抗性（kdr）基因突变，初步探讨现场群体kdr基因的适应性进化。方法 2016年10月及2017年6-10月，在上海市杨浦区、江苏省南京市、浙江省杭州市和云南省景洪市外环境或公园白纹伊蚊孳生地舀取蚊幼虫和蛹，带回实验室饲养至成蚊；或直接采集成蚊。鉴定种类后，单只蚊抽提基因组DNA，PCR扩增神经细胞膜上电压门控钠离子通道（VGSC）基因部分片段，测序并进行序列分析，检测kdr基因突变情况，应用GENPOP软件进行连锁不平衡和哈代-温伯格平衡检验。结果共检测4个现场群体299只白纹伊蚊，发现kdr基因I1532和F1534位点突变。1532位点有2种等位基因，分别为野生型ATC/I（91.30%）和突变型ACC/T（8.70%）；1534位点共有5种等位基因，分别为野生型TTC/F，突变型TCC/S、TCG/S、TTG/L和TGC/C，等位基因频率为45.99%、52.01%、0.33%、1.00%和0.67%。2个位点同时突变的个体共有27个（9.03%）。群体符合哈代-温伯格平衡（P > 0.001），2个位点在遗传中为连锁关联关系（P < 0.01）。结论首次记录了我国白纹伊蚊群体在1532位点存在突变I1532T和1534位点新的突变等位基因TCG/S，以及1个个体存在2个位点同时突变的新问题。结果提示，4个白纹伊蚊现场群体对菊酯类杀虫剂已产生抗性，多个位点突变可能与杀虫剂的选择压力有关。

关键词: 白纹伊蚊, 击倒抗性基因, 基因突变, 抗药性

Abstract: Objective To detect the kdr gene mutations in the field populations of Aedes albopictus from China and preliminarily clarify its adaptive evolution. Methods The mosquito larvae and pupae were dip-samples from breeding sites from Yangpu district in Shanghai, Nanjing city in Jiangsu province, Hangzhou city in Zhejiang province, and Jinghong city in Yunnan province, in China, and brought back to insectary to rise till adults. The mosquito adults were also collected directly in the fields using BG-trap or sucking tube. The single individual genomic DNA of Ae. albopictus was extracted after species identification. The VGSC gene fragments were amplified by PCR, which were sequenced and analyzed for detection of the kdr mutations. The genotypic linkage disequilibrium and Hardy-Weinberg equilibrium(HWE) was tested by GENPOP software. Results A total of 299 individuals collected from four field populations of Ae. albopictus were analyzed. There were two positions showed mutations including codon 1532 and 1534 of kdr gene. Two alleles at codon 1532 were wildtype ATC/I and mutant ACC/T, with frequencies 91.30% and 8.70%, respectively. Five alleles at codon 1534 were wildtype TTC/F, mutant allele TCC/S, TCG/S, TTG/L, and TGC/C, with frequencies of 45.99%, 52.01%, 0.33%, 1.00%, and 0.67%, respectively. There were 27 individuals(9.03%) with both mutations in 1532 and 1534 at the same time. The test results showed that the population conformed to HWE(P > 0.001), and the two loci were genetic linkage (P < 0.01). Conclusion This study first reported the novel mutant allele I1532T, and found a mutant allele TCG/S at the codon 1534 of Ae. albopictus in China. More important is a new challenge that an individual harbors two codons of simultaneous mutations. The results suggested that they should be resistant to pyrethroid insecticides. The multiple site mutations may be resulted from the selection pressure of insecticides.

Key words: Aedes albopictus, Knockdown resistance gene, Gene mutation, Resistance

中图分类号:

陈翰明, 高景鹏, 姜进勇, 彭恒, 马雅军. 我国白纹伊蚊现场群体击倒抗性基因I1532和F1534突变检测及I1532T突变等位基因报告[J]. 中国媒介生物学及控制杂志, 2018, 29(2): 120-125.

CHEN Han-ming, GAO Jing-peng, JIANG Jin-yong, PENG Heng, MA Ya-jun. Detection of the I1532 and F1534 kdr mutations and a novel mutant allele I1532T in VGSC gene in the field populations of Aedes albopictus from China[J]. Chines Journal of Vector Biology and Control, 2018, 29(2): 120-125.

参考文献

[1] 杨舒然,刘起勇. 白纹伊蚊的全球分布及扩散趋势[J]. 中国媒介生物学及控制杂志,2013,24(1):1-4.
[2] Kraemer MUG,Sinka ME,Duda KA,et al. The global distribution of the arbovirus vectors Aedes aegypti and Ae. albopictus[J]. eLife,2015,4:e08347.
[3] Xiao JP,He JF,Deng AP,et al. Characterizing a large outbreak of dengue fever in Guangdong province,China[J]. Infect Dis Poverty,2016,5:44.
[4] 上海市卫生和计划生育委员会. 本市报告发现首例本地感染登革热病例[EB/OL]. (2017-09-26)[2018-01-10]. http://www.wsjsw.gov.cn/wsj/n422/n424/u1ai142084.html?from=groupmessage&isappinstalled=0.
[5] 浙江新闻. 杭州市累计报告登革热病例1008例[EB/OL]. (2017-09-29)[2018-01-10]. http://zjnews.zjol.com.cn/zjnews/hznews/201709/t20170929_5237768.shtml.
[6] 嘉祥县人民政府办公室. 嘉祥县人民政府办公室关于终止登革热暴发疫情应急响应的通知[EB/OL].(2017-10-13)[2018-01-10]. http://www.jiaxiang.gov.cn/content.jsp?id=bc5372855f484da0015f57270e0b01cc&classid=2c17a4be401f4817aecce1ab7fb2d1be.
[7] 孟凤霞,王义冠,冯磊,等. 我国登革热疫情防控与媒介伊蚊的综合治理[J]. 中国媒介生物学及控制杂志,2015,26(1):4-10.
[8] Hemingway J,Ranson H. Insecticide resistance in insect vectors of human disease[J]. Annu Rev Entomol,2000,45:371-391.
[9] 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.
[10] 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.
[11] Smith LB,Kasai S,Scott JG. Voltage-sensitive sodium channel mutations S989P + V1016G in Aedes aegypti confer variable resistance to pyrethroids,DDT and oxadiazines[J]. Pest Manag Sci,2017. DOI:10.1002/ps.4771.(in Press)
[12] 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]. PLoS Negl Trop Dis,2016,10(5):e0004696.
[13] 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.
[14] Wang Y,Yu WQ,Shi H,et al. Historical survey of the kdr mutations in the populations of Anopheles sinensis in China in 1996-2014[J]. Malar J,2015,14:120.
[15] Chang XL,Zhong DB,Fang Q,et al. Multiple resistances and complex mechanisms of Anopheles sinensis mosquito:a major obstacle to mosquito-borne diseases control and elimination in China[J]. PLoS Negl Trop Dis,2014,8(5):e2889.
[16] Tan WL,Wang ZM,Li CX,et al. First report on co-occurrence knockdown resistance mutations and susceptibility to beta-cypermethrin in Anopheles sinensis from Jiangsu province,China[J]. PLoS One,2012,7(1):e29242.
[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.
[18] Li YJ,Xu JB,Zhong DB,et al. Evidence for multiple-insecticide resistance in urban Aedes albopictus populations in southern China[J]. Parasit Vectors,2018,11:4.
[19] Pombi M,Guelbeogo WM,Calzetta M,et al. Evaluation of a protocol for remote identification of mosquito vector species reveals BG-Sentinel trap as an efficient tool for Anopheles gambiae outdoor collection in Burkina Faso[J]. Malar J,2015, 14:161.
[20] 陆宝麟,许锦江,俞渊,等. 中国动物志. 昆虫纲. 第9卷. 双翅目:蚊科[M]. 北京:科学出版社,1997:88-106.
[21] 杨明东,姜进勇,郭晓芳,等. 2009-2014年云南省登革热流行病学调查与分析[J]. 中国病原生物学杂志,2015,10(8):738-742.
[22] WHO. Pesticides and their application:for the control of vectors and pests of public health importance[M]. 6th ed. Geneva:World Health Organization,2006.
[23] WHO. Dengue hemorrhagic fever:diagnosis,treatment,prevention, and control[R]. Geneva:World Health Organization,1997.
[24] 段金花,蔡松武,吴军,等. 2014年广东省登革热疫情应急控制后期白纹伊蚊抗药性水平调查[J]. 中国媒介生物学及控制杂志,2017,28(2):141-143.
[25] 李成玲,胡志刚,江毅民,等. 广州地区白纹伊蚊对常用杀虫剂抗药性的初步研究[J]. 热带医学杂志,2010,10(4):429-430,447.
[26] 蔡松武,段金花,卢文成,等. 广州市城区白纹伊蚊抗药性现状与预防对策探讨[J]. 华南预防医学,2004,30(4):40-41.
[27] Brengues C,Hawkes NJ,Chandre F,et al. Pyrethroid and DDT cross-resistance in Aedes aegypti is correlated with novel mutations in the voltage-gated sodium channel gene[J]. Med Vet Entomol,2003,17(1):87-94.
[28] Harris AF,Rajatileka S,Ranson H. Pyrethroid resistance in Aedes aegypti from Grand Cayman[J]. Am J Trop Med Hyg, 2010,83(2):277-284.
[29] Yanola J,Somboon P,Walton C,et al. High-throughput assays for detection of the F1534C mutation in the voltage-gated sodium channel gene in permethrin-resistant Aedes aegypti and the distribution of this mutation throughout Thailand[J]. Trop Med Int Health,2011,16(4):501-509.
[30] Kawada H,Higa Y,Komagata O,et al. Widespread distribution of a newly found point mutation in voltage-gated sodium channel in pyrethroid-resistant Aedes aegypti populations in Vietnam[J]. PLoS Negl Trop Dis,2009,3(10):e527.
[31] Kawada H,Oo SZM,Thaung S,et al. Co-occurrence of point mutations in the voltage-gated sodium channel of pyrethroid-resistant Aedes aegypti populations in Myanmar[J]. PLoS Negl Trop Dis,2014,8(7):e3032.
[32] Marcombe S, Mathieu RB, Pocquet N,et al. Insecticide resistance in the dengue vector Aedes aegypti from Martinique:distribution, mechanisms and relations with environmental factors[J]. PLoS One,2012,7(2):e30989.
[33] Ishak IH,Jaal Z,Ranson H,et al. Contrasting patterns of insecticide resistance and knockdown resistance(kdr) in the dengue vectors Aedes aegypti and Aedes albopictus from Malaysia[J]. Parasit Vectors,2015,8:181.
[34] Kushwah RBS, Dykes CL, Kapoor N, et al. Pyrethroid-resistance and presence of two knockdown resistance (kdr) mutations,F1534C and a novel mutation T1520I,in Indian Aedes aegypti[J]. PLoS Neg Trop Dis,2015,9(1):e3332.
[35] Kushwah RBS,Mallick PK,Ravikumar H,et al. Status of DDT and pyrethroid resistance in Indian Aedes albopictus and absence of knockdown resistance(kdr)mutation[J]. J Vector Borne Dis, 2015,52(1):95-98.
[36] 刘洪霞,朱江,刘曜,等. 上海地区2015-2016年白纹伊蚊幼虫监测及抗药性调查[J]. 中国媒介生物学及控制杂志, 2017,28(4):305-307.
[37] 龚震宇,侯娟,任樟尧,等. 浙江省淡色库蚊和白纹伊蚊对常用化学杀虫剂的抗性调查[J]. 中国媒介生物学及控制杂志, 2012,23(5):458-460.
[38] 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.
[39] Plernsub S,Saingamsook J,Yanola J,et al. Additive effect of knockdown resistance mutations, S989P, V1016G and F1534C, in a heterozygous genotype conferring pyrethroid resistance in Aedes aegypti in Thailand[J]. Parasit Vectors, 2016,9(1):417.
[40] Main BJ,Lee Y,Collier TC,et al. Complex genome evolution in Anopheles coluzzii associated with increased insecticide usage in Mali[J]. Mol Ecol,2015,24(20):5145-5157.