中国寄生虫学与寄生虫病杂志 ›› 2022, Vol. 40 ›› Issue (4): 468-474.doi: 10.12140/j.issn.1000-7423.2022.04.008

• 论著 • 上一篇    下一篇

江苏省白纹伊蚊对溴氰菊酯抗性及击倒抗性突变分析

王冠熙(), 李雅姝, 李月月, 曹园园, 杨蒙蒙, 张梅花, 吴竞瑶, 梁成, 李菊林, 周华云, 唐建霞*(), 朱国鼎   

  1. 江苏省血吸虫病防治研究所、世界卫生组织消除疟疾研究与培训合作中心、国家卫生健康委员会寄生虫病预防与控制技术重点实验室、江苏寄生虫与媒介控制技术重点实验室 无锡 214064
  • 收稿日期:2021-12-27 修回日期:2022-03-15 出版日期:2022-08-30 发布日期:2022-09-07
  • 通讯作者: 唐建霞
  • 作者简介:王冠熙(1995-),男,硕士研究生,从事媒介生物学研究。E-mail: 522534282@qq.com
  • 基金资助:
    国家重点研发计划(2020YFC1200105);江苏省公益类科研院所自主科研项目(BM2018020)

Resistance to deltamethrin and knockdown resistance mutation in Aedes albopictus from Jiangsu Province

WANG Guan-xi(), LI Ya-shu, LI Yue-yue, CAO Yuan-yuan, YANG Meng-meng, ZHANG Mei-hua, WU Jing-yao, LIANG Cheng, LI Ju-lin, ZHOU Hua-yun, TANG Jian-xia*(), ZHU Guo-ding   

  1. Jiangsu Institute of Parasitic Diseases, WHO Collaborating Centre for Research and Training on Malaria Elimination, Key Laboratory of National Health Commission on Parasitic Disease Control and Prevention, Jiangsu Provincial Key Laboratory on Parasite and Vector Control Technology, Wuxi 214064, China
  • Received:2021-12-27 Revised:2022-03-15 Online:2022-08-30 Published:2022-09-07
  • Contact: TANG Jian-xia
  • Supported by:
    National Key R&D Program of China(2020YFC1200105);Jiangsu Provincial Department of Science and Technology(BM2018020)

摘要:

目的 了解江苏省白纹伊蚊对溴氰菊酯的抗性,探究其与击倒抗性(kdr)基因突变的关联,为白纹伊蚊科学防治提供理论依据。 方法 用美国疾病预防与控制中心生物瓶法制备溴氰菊酯含量为1、2、4、6和8 μg/瓶的测试瓶和不含溴氰菊酯的对照瓶,每瓶放10~25只实验室培养的敏感品系白纹伊蚊,确定诊断剂量。2020年8—9月,分别从徐州市睢宁县、淮安市淮阴区和无锡市宜兴市采集白纹伊蚊的幼虫或蛹,培养至子一代(F1代),取10~25只F1代雌蚊置含诊断剂量溴氰菊酯的测试瓶或对照瓶中充分接触30 min,计算白纹伊蚊的死亡率,评价其抗性水平。提取抗性测试后蚊虫的DNA,PCR扩增电压门控钠离子通道(VGSC)跨膜结构域Ⅱ、Ⅲ、Ⅳ的基因编码区并测序,用Bioedit、Seqman等软件分析kdr基因突变情况,统计kdr基因型和突变频率,抗性表型与敏感表型蚊虫kdr突变率的差异使用χ2检验。 结果 用实验室敏感品系白纹伊蚊确定的溴氰菊酯诊断剂量为4 μg/瓶。从睢宁县、淮阴区和宜兴市共采集到白纹伊蚊149只。睢宁县的白纹伊蚊在诊断剂量下的校正平均死亡率为(28.2 ± 1.4)%,淮阴区和宜兴市的平均死亡率分别为(64.4 ± 5.5)%、(56.3 ± 17.7)%,3个县(市、区)的白纹伊蚊对溴氰菊酯均已产生抗药性。共获得DNA序列447条,测序结果显示,3个县(市、区)白纹伊蚊VGSC结构域Ⅳ的基因编码区1532和1534位点均存在kdr基因突变,其中1532位点共有2种等位基因,分别为野生型ATC/I(279/298,93.6%)和突变型ACC/T(19/298,6.4%);组成3种基因型,分别为野生型纯合子I/I(133/149,89.3%)、野生型/突变型杂合子I/T(13/149,8.7%)和突变型纯合子T/T(3/149,2.0%)。1534位点共有4种等位基因,分别为野生型TTC/F(275/298,92.3%)和突变型TCC/S(5/298,1.7%)、TTA/L(14/298,4.7%)和TTG/L(4/298,1.3%);组成4种基因型,分别为野生型纯合子F/F(128/149,85.9%)、突变型/野生型杂合子F/S(5/149,3.4%)和F/L(14/149,9.4%),突变型纯合子L/L(2/149,1.3%)。72只有抗性表型的白纹伊蚊成蚊中,1532、1534位点野生型分别占87.5%(63/72)和86.1%(62/72),kdr基因突变型分别占12.5%(9/72)和13.9%(10/72);77只敏感表型白纹伊蚊成蚊中,1532和1534位点野生型分别占90.9%(70/77)和85.7%(66/77),kdr基因突变型分别占9.2%(7/77)和14.3%(11/77)。抗性表型和敏感表型个体中,1532和1534位点kdr基因突变型间的差异无统计学意义(χ2 = 0.168、4.111,P > 0.05)。 结论 江苏省睢宁县、淮阴区和宜兴市的白纹伊蚊对溴氰菊酯均产生抗药性,但1532和1534位点kdr基因突变频率均较低,未发现抗性表型与1532位点、1534位点kdr基因突变的相关性。

关键词: 白纹伊蚊, 溴氰菊酯, CDC生物瓶法, 抗药性, 击倒抗性突变

Abstract:

Objective To investigate the resistance of deltamethrin in Aedes albopictus in Jiangsu Province, and explore the association with knockdown resistance (kdr) mutation, to provide a theoretical basis for scientific control of Ae. albopictus. Methods The diagnostic doses were determined by preparing test bottles containing deltamethrin of 1, 2, 4, 6 or 8 μg/bottle and control bottles without deltamethrin using the CDC bottle bioassay with 10-25 susceptible laboratory-cultured Ae. albopictus in each bottle. Ae. albopictus larvae and pupae were collected from Suining County in Xuzhou City, Huaiyin District in Huai’an City, and Yixing City in Wuxi City, in Jiangsu Province during August and September 2020 and reared to the first filial generation (F1 generation). The mortality rate of Ae. albopictus was calculated by using 10-25 F1 generation female mosquitoes for exposure to a diagnostic dose of deltamethrin in the test bottle and the blank in the control bottle for 30 min to evaluate the resistance level. The DNA was extracted from the mosquitoes after bioassay, and the gene coding region of voltage-gated sodium channel (VGSC) transmembrane structural domains Ⅱ, Ⅲ and Ⅳwere amplified by PCR and sequenced. Bioedit and Seqman software were used for statistical analysis of the kdr mutations, genotypes and mutation frequency. A Chi-square test was used to compare the kdr mutation frequency in the resistant and susceptible phenotype mosquitoes. Results The diagnostic doses of deltamethrin to Ae. albopictus was 4 μg/bottle. A total of 149 Ae. albopictus were collected from Suining County, Huaiyin District and Yixing City. The corrected mean mortality rate of Suining County was (28.2 ± 1.4)%, and the mean mortality rates in Huaiyin District and Yixing City were (64.4 ± 5.5)% and (56.3 ± 17.7)%, respectively. Ae. albopictus collected from all three counties’ (cities and districts) were resistant to deltamethrin. There were 447 DNA sequences obtained, and the sequencing results showed that the kdr mutations existed in the gene coding region at codons 1532 and 1534 of the VGSC structural domain Ⅳ of Ae. albopictus in three counties (cities and districts). Two alleles at codon 1532 were wild-type ATC/I (279/298, 93.6%) and mutant ACC/T (19/298, 6.4%); three genotypes were the wild-type homozygote I/I (133/149, 89.3%), wild/mutant heterozygote I/T (13/149, 8.7%), and mutant homozygote T/T (3/149, 2.0%). Four alleles at codon 1534 were wildtype TTC/F (275/298, 92.3%), mutant TCC/S (5/298, 1.7%), mutant TTA/L (14/298, 4.7%), and mutant TTG/L (4/298, 1.3%); four genotypes were the wild-type homozygote F/F (128/149, 85.9%), wild/mutant heterozygote F/S (5/149, 3.4%), wild/mutant heterozygote F/L (14/149, 9.4%), and mutant homozygote I/I (2/149, 1.3%). Among the 72 resistant mosquitoes phenotyped, 87.5% (63/72) and 86.1% (62/72) were wild-type at codon 1532 and 1534, respectively, and 12.5% (9/72) and 13.9% (10/72) were kdr mutant, respectively. Among the 77 sensitive adults, 90.9% (70/77) and 85.7% (66/77) were wild type at codon 1532 and 1534, respectively, and 9.2% (7/77) and 14.3% (11/77) were kdr mutant, respectively. Chi-square test revealed no significant difference between kdr mutant genotypes at codon 1532 and 1534 with resistant and susceptible phenotypes Ae. albopictus( χ2 = 0.168, 4.111, P > 0.05). Conclusion In Suining County, Huaiyin District and Yixing City of Jiangsu Province, Ae. albopictus mosquitoes were found resistant to deltamethrin. The frequencies of kdr mutations at codon 1532 and 1534 in Ae. albopictus was low. No significant correlation was found between the resistance phenotype and kdr mutations at codon 1532 and 1534.

Key words: Aedes albopictus, Deltamethrin, CDC bottle bioassay, Insecticide resistance, Knockdown resistance mutation

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