臺灣埃及斑蚊電壓門控鈉離子通道突變對殺蟲劑抗性及病媒競爭力之影響

Abstract

埃及斑蚊因是登革、茲卡和屈公病毒的主要病媒而對熱帶和亞熱帶地區的公共衛生造成威脅。因對這些病原體缺乏有效的疫苗及抗病毒藥，使用除蟲菊酯類殺蟲劑仍是防治斑蚊媒介傳染病的重要策略。然而，重複使用同類型殺蟲劑會導致抗藥性的產生。埃及斑蚊電壓門鈉離子通道（vgsc）突變進而導致與除蟲菊酯殺蟲劑的結合力下降為重要的抗性機制，因此，藉由有效監測vgsc突變及時了解野外蚊蟲的抗藥性狀態對於病媒控制和疾病預防至關重要。另一方面，在選汰壓力下篩選出帶有抗性基因的埃及斑蚊對登革病毒感染的病媒適性對於疾病風險評估提供重要依據。因此，本研究旨在透過分子監測了解vgsc突變在埃及斑蚊抗性的角色並評估野外埃及斑蚊的抗性狀況；此外，亦探討帶有主要抗藥性相關vgsc基因型S989P+V1016G+F1534C異型合子的埃及斑蚊媒介登革病毒的病媒竸爭力。 本研究持續於2016至2023年分別於登革熱流行季節前（3月）和季間（10月）採集臺南市、高雄市和屏東縣埃及斑蚊，並比較2016年3月和10月收集的埃及斑蚊及以賽滅寧（cypermethrin）暴露後活和死的埃及斑蚊vgsc突變情形。接著進行2016至2023年之埃及斑蚊長期監測，以評估野外埃及斑蚊vgsc突變的變化趨勢及抗性現況。我們也自野外埃及斑蚊中分離帶有主要vgsc抗性相關基因型（S989P+V1016G+F1534C異型合子）的品系，藉由評估登革病毒感染該抗性品系的感染率、擴散率、傳染率、傳染效率、潛在傳染效率、病毒量和存活率來評估該品系媒介登革病毒的病媒竸爭力。 根據2016年3月和10月收集之野外監測數據及將經過賽滅寧生物檢定後之死和活的蚊蟲進行分子檢測，我們提出了由S989P、V1016G、F1534C和D1763Y所組成的6種與埃及斑蚊抗性相關的vgsc基因型，並發現埃及斑蚊帶有抗性相關基因型的比例與賽滅寧的抗性程度呈正相關；我們也建議使用vgsc基因型而非等位基因或單倍型作為抗性指標。而在2016至2023年間進行的vgsc基因分子監測中，我們總共自臺南市、高雄市和屏東縣採集並分析了1,761隻埃及斑蚊，除了已於2016年監測之S989P、V1016G、F1534C和D1763Y外，我們首次於台灣偵測到T1520I，並觀察到該些位點突變頻率均增加；此外，除了過去文獻提出的六種單倍型外，我們在臺灣首次監測到另外三種單倍型：PGTFY（分別代表989、1016、1520、1534、1763之胺基酸，底線代表突變）、SVICD和PGTCD，其中，PGTFY為首次在埃及斑蚊中被報導。而SVICD在野外族群中增加了19倍；值得注意的是，未突變的單倍型在野外已有2021年後即未被監測到，這意味著野外埃及斑蚊很有可能都至少含有一種vgsc突變；我們共觀察到了25種vgsc基因型，其中，SVTCD/SVTCD（分別代表二條單倍型，底線代表突變）、SGTFY/PGTFD、SVTCD/SGTFY、PGTFD/PGTFD、SVTCD/PGTFD5種與抗性相關基因型隨著時間增加，共佔野外族群的76%；我們也首次監測到抗性基因型SVICD/PGTFD，該基因型自其首次發現後在野外增加了13倍；另一方面，我們也觀察到vgsc突變在地理分布上有所不同，其中S989P主要分布在高雄；V1016G主要分布在高雄和屏東；T1520I的頻率在高雄明顯較高；而D1763Y主要出現在臺南。此外，我們將所建立帶有主要vgsc抗性相關基因型（SVTCD/PGTFD）的埃及斑蚊感染登革病毒第2型，發現具有該基因型的埃及斑蚊比未突變的埃及斑蚊具有更高的登革病毒感染率、傳播率和潛在傳染率以及類似的存活率，顯示帶有SVTCD/PGTFD的埃及斑蚊比未突變的品系有更好媒介登革病毒的竸爭力。 在本研究中，我們提出了vgsc突變的抗性角色，並發現使用vgsc基因型進行抗性監測比等位基因和單倍型更準確；而八年的抗藥性監測結果也顯示臺灣埃及斑蚊出現新的vgsc突變且這些突變持續擴大，這說明了臺灣乃至世界對埃及斑蚊產生抗藥性所造成的威脅持續增加。本研究闡述了埃及斑蚊vgsc突變的抗性角色和其動態變化，為使用除蟲菊酯殺蟲劑防治埃及斑蚊的地區提供有價值的資訊。此外，我們也證明了帶有SVTCD/PGTFD的埃及斑蚊對媒介登革病毒第2型有較好的病媒竸爭力。因帶有SVTCD/PGTFD的埃及斑蚊廣泛分布於亞洲和非洲，且該抗性基因可能有助於登革病毒的傳播，因此，我們強列建議有關部門持續對蚊蟲的抗藥性情形進行監測；化學防治需進行藥效試驗；並採取整合式病媒管理策略及抗藥性管理，以減緩抗性基因的演化；另一方面，我們也建議開發新病媒防治藥物（包括化學性及生物性）並及早建立並評估新的病媒控制策略，以有效因應未來對病媒的控制及蚊媒傳染病的預防。

Aedes aegypti is the major vector of dengue, Zika, and chikungunya virus that threatens public health in tropical and subtropical regions. Pyrethroid-based control strategies effectively control this vector, but repeated usage of the insecticide leads to resistance and hampered vector control efforts. Understanding the role of the mutant voltage-gated sodium channel (vgsc), the pyrethroid target site, and monitoring the vgsc variation efficiently provides the prompt status of insecticide resistance in local mosquito populations and is critical for vector control and disease prevention. On the other hand, the vector competence of the dengue virus in Ae. aegypti with resistant vgsc genotype selected under selection pressure needs to be clarified for disease risk assessment. Therefore, this study aims to understand the resistance role of vgsc mutations and assess the resistance status of field Ae. aegypti by molecular monitoring. In addition, the vector competence of dengue virus in Ae. aegypti harboring the predominant resistance-associated vgsc genotype, S989P+V1016G+F1534C heterozygous, was assessed in the study. Ae. aegypti from Tainan, Kaohsiung, and Pingtung before (March) and during (October) dengue season between 2016 and 2023 were collected. The resistance role of vgsc mutations in Taiwan Ae. aegypti were evaluated by alive and dead mosquitoes exposed to cypermethrin, and mosquitoes collected in March and October of 2016. Long-term vgsc mutation surveillance was conducted to monitor the vgsc variation and evaluate the resistance status of field Ae. aegypti between 2016 and 2023. On the other hand, we established an Ae. aegypti strain with predominant resistance-associated vgsc genotype, S989P+V1016G+F1534C heterozygous, to assess the vector competence of this strain under dengue virus infection. The infection rate, dissemination rate, transmission rate, transmission efficiency, potential transmission efficiency, viral load, and survival rate were evaluated. According to cypermethrin bioassay and field surveillance data of Ae. aegypti in 2016, we proposed six resistance-associated vgsc genotypes comprising S989P, V1016G, F1534C, and D1763Y and found the proportion of resistance-associated genotypes in the field population positively correlated with the resistance level of cypermethrin. Therefore, we suggest using the vgsc genotype rather than the allele or the haplotype as a resistance indicator. Between 2016 and 2023, molecular surveillance based on the vgsc genotype was conducted. In total, 1,761 field-caught Ae. aegypti from Tainan, Kaohsiung, and Pingtung were genotyped. In addition to S989P, V1016G, F1534C, and D1763Y, T1520I was first detected in Taiwan. All of the mutation frequencies increased since their first detection. In addition to the six previously proposed haplotypes, three further haplotypes, PGTFY (the amino acids in 989, 1016, 1520, 1534, and 1763, the mutant form were underlined), SVICD, and PGTCD, were first detected in Taiwan. Among them, PGTFY was first documented in Ae. aegypti. SVICD increased by 19-fold in the field population. Moreover, the un-mutated haplotype vanished in Taiwan, implying that Ae. aegypti was fixed by at least one vgsc mutation. We observed 25 vgsc genotypes in the field. Among them, five resistance-associated genotypes, including SVTCD/SVTCD, SGTFY/PGTFD, SVTCD/SGTFY, PGTFD/PGTFD, and SVTCD/PGTFD, increased and accounted for 76% of the field population. We also first detected the resistance genotype, SVICD/PGTFD, which increased 13-fold in the field. On the other hand, we also observed that the vgsc mutations were geographically different among the three cities, with S989P mainly found in Kaohsiung and V1016G in Kaohsiung and Pingtung. The proportion of T1520I was noticeably higher in Kaohsiung, and D1763Y occurred mainly in Tainan. We assessed the vector competence of Ae. aegypti with predominant vgsc genotype, SVTCD/PGTFD, under dengue virus 2 infection and found Ae. aegypti with his genotype exhibited higher infection rate, dissemination rate, and potential transmission efficiency and comparable survival rate than the un-mutated counterpart, suggesting Ae. aegypti with SVTCD/PGTFD was a more competent vector than the wild type for dengue virus 2 transmission. In this study, we proposed the resistance role of vgsc mutations and suggested that the vgsc genotype is more accurate for resistance monitoring. The eight-year long-term resistance surveillance also found the emergence and expansion of vgsc mutations, suggesting the elevated threat of insecticide resistance in Taiwan and worldwide. The resistance role and dynamic variation of vgsc mutations in this study provide valuable information for the areas using pyrethroid for Ae. aegypti control. Additionally, we demonstrated the increased vector competence of dengue virus 2 in Ae. aegypti carrying the SVTCD/PGTFD, which is widespread in Asia and Africa. We urge the authority to adopt measures against resistance: 1) Monitor insecticide resistance continuously; 2) Evaluate the efficacy of insecticide for appropriate vector control; 3) Adopt integrated pest management and insecticide resistance management to decelerate the evolution toward resistance; 4) Develop novel chemical- and biological-based vector control agents; 5) Establish and evaluate novel vector control strategies, such the Wolbachia-based technology in advance for future vector control and disease prevention.