Justus Liebig University Giessen, Institute for Food Chemistry and Food Biotechnology, Giessen, Germany.
Justus Liebig University Giessen, Institute for Insect Biotechnology, Giessen, Germany.
PLoS One. 2019 Feb 7;14(2):e0211064. doi: 10.1371/journal.pone.0211064. eCollection 2019.
Different setups and protocols have been developed for investigating insecticide effects on Anopheles (An.) mosquitoes, vectors of malaria. However, chemical uptake resulting from their tarsal contact with insecticide-treated material has seldom been investigated. To address the challenges encountered in the interpretation of bioassay data, a high throughput method for chemical analysis on malaria vectors was developed and validated for five selected insecticides including alpha-cypermethrin (aCYP), deltamethrin (DM), etofenprox (EPX), permethrin (PM), pirimiphos-methyl (PPM).
The method includes a single chemical extraction step via an ultrasound probe on mosquito samples and analysis via liquid chromatography coupled to high-resolution tandem mass spectrometry (UHPLC-MS/MS). The protocol was established for two malaria vector species, Anopheles gambiae senso stricto (s.s.) and An. stephensi, both males and females. Recovery rates ranged from 70 to 100% without any influence of sex or species. The method was efficiently applied to female An. gambiae s.s. of the KISUMU1 reference strain, after susceptibility tests using the World Health Organization's standard protocol.
Susceptibility tests revealed 13.4-18.4 minutes knockdown times for 50% mosquitoes during exposure to EPX and pyrethroids. The mortality rates 24 hours post-exposure to insecticides were mostly 99-100%, except in two PM and three PPM assays suggesting possible or confirmed resistance to these insecticides. The mean insecticide uptake in dead mosquitoes ranged from 23 pg (aCYP) to 1812 pg (EPX) per specimen. However, the mean uptake in survivors to PM and PPM was reduced by at least 25%, suggesting that acute doses were not achieved in these specimens during bioassays.
The developed and validated UHPLC-MS/MS method could be used to address some limitations of bioassays or to assess the penetration of insecticides in mosquito matrix with reference to cuticle thickness and other insecticide resistance mechanisms.
已经开发出不同的设置和方案来研究杀虫剂对疟疾传播媒介按蚊（Anopheles）的影响。然而，很少研究过它们与经杀虫剂处理的材料接触时通过跗节摄入化学物质的情况。为了解决在解释生物测定数据时遇到的挑战，开发并验证了一种针对五种选定杀虫剂的高通量疟疾媒介化学分析方法，包括α-氯氰菊酯（aCYP）、溴氰菊酯（DM）、乙氰菊酯（EPX）、氯菊酯（PM）和吡虫啉（PPM）。
该方法包括通过超声探头对蚊子样本进行单次化学提取，然后通过液相色谱与高分辨率串联质谱（UHPLC-MS/MS）进行分析。该方案针对两种疟疾媒介物种，即冈比亚按蚊敏感型（s.s.）和斯氏按蚊，雄性和雌性均适用。回收率在 70%至 100%之间，不受性别或物种的影响。该方法成功应用于 KISUMU1 参考株雌性冈比亚按蚊 s.s. 的敏感性测试，测试方法采用世界卫生组织的标准方案。
敏感性测试显示，接触 EPX 和拟除虫菊酯后，50%的蚊子在 13.4-18.4 分钟内出现击倒时间。接触杀虫剂 24 小时后的死亡率大多为 99-100%，除了在两个 PM 和三个 PPM 试验中，这表明这些杀虫剂可能存在或已确认的抗性。死亡蚊子中的平均杀虫剂摄入量范围为 23 pg（aCYP）至 1812 pg（EPX）/只。然而，PM 和 PPM 幸存者的平均摄入量减少了至少 25%，这表明在这些标本的生物测定中未达到急性剂量。
开发并验证的 UHPLC-MS/MS 方法可用于解决生物测定的一些限制，或评估杀虫剂在与表皮厚度和其他杀虫剂抗性机制相关的蚊子基质中的渗透情况。
