Department of Biology, College of Natural Sciences, Jimma University, P. O. Box 378, Jimma, Ethiopia.
School of Medical Laboratory Sciences, Faculty of Health Sciences, Jimma University, Jimma, Ethiopia.
Malar J. 2023 Aug 12;22(1):233. doi: 10.1186/s12936-023-04667-3.
Anopheles funestus, which is considered as secondary vector of malaria in Ethiopia, is known to have several morphologically indistinguishable (sibling) species. Accurate identification of sibling species is crucial to understand their biology, behaviour and vector competence. In this study, molecular identification was conducted on the Ethiopian An. funestus populations. Moreover, insecticide resistance mechanism markers were detected, including ace N485I, kdr L1014F, L1014S, and CYP6P9a TaqMan qPCR was used to detect the infective stage of the parasite from field collected adult female An. funestus populations.
Adult female mosquito collection was conducted from Lare, Gambella Regional State of Ethiopia between June 2018 to July 2020 using CDC light traps and HLC. Sub-samples of the morphologically identified An. funestus mosquitoes were molecularly identified using species-specific PCR, and the possible presence of insecticide resistance alleles was investigated using TaqMan qPCR (N485I-Ace-1), PCR-Sanger sequencing (L1014F-kdr), and PCR-RFLP (CYP6P9a resistance allele). Following head/thorax dissection, the TaqMan qPCR assay was used to investigate the presence of the infective stage Plasmodium parasite species.
A total of 1086 adult female An. funestus mosquitoes were collected during the study period. All sub-samples (N = 20) that were morphologically identified as An. funestus sensu lato (s.l.) were identified as An. funestus sensu stricto (s.s.) using species- specific PCR assay. The PCR-RFLP assay that detects the CYP6P9a resistance allele that confers pyrethroid resistance in An. funestus was applied in N = 30 randomly selected An. funestus s.l.
None of the specimens showed a digestion pattern consistent with the presence of the CYP6P9a resistance allele in contrast to what was observed in the positive control. Consequently, all samples were characterized as wild type. The qPCR TaqMan assay that detects the N485I acetylcholinesterase-1 mutation conferring resistance to organophosphates/carbamates in An. funestus was used in (N = 144) samples. All samples were characterized as wild type. The kdr L1014F and L1014S mutations in the VGSC gene that confer resistance to pyrethroids and DDT were analysed with direct Sanger sequencing after PCR and clean-up of the PCR products were also characterized as wild type. None of the samples (N = 169) were found positive for Plasmodium (P. falciparum/ovale/malariae/vivax) detection.
All An. funestus s.l. samples from Lare were molecularly identified as An. funestus s.s. No CYP6P9, N485I acetylcholinesterase 1, kdr L1014F or L1014S mutations were detected in the An. funestus samples. None of the An. funestus samples were positive for Plasmodium. Although the current study did not detect any insecticide resistant mechanism, it provides a reference for future vector monitoring programmes. Regular monitoring of resistance mechanisms covering wider geographical areas of Ethiopia where this vector is distributed is important for improving the efficacy of vector control programs.
冈比亚按蚊被认为是埃塞俄比亚疟疾的次要传播媒介，已知存在几种形态上无法区分的（姊妹）种。准确识别姊妹种对于了解它们的生物学、行为和媒介效能至关重要。在这项研究中，对埃塞俄比亚冈比亚按蚊种群进行了分子鉴定。此外，还检测了杀虫剂抗性机制标记物，包括 ace N485I、kdr L1014F、L1014S 和 CYP6P9a。TaqMan qPCR 用于检测从现场采集的成年雌性冈比亚按蚊种群中的寄生虫感染阶段。
使用 CDC 诱蚊灯和 HLC 于 2018 年 6 月至 2020 年 7 月在埃塞俄比亚 Gambella 地区的 Lare 收集成年雌性蚊子。使用种特异性 PCR 对形态上鉴定的冈比亚按蚊蚊子进行分子鉴定，并使用 TaqMan qPCR（N485I-Ace-1）、PCR-Sanger 测序（L1014F-kdr）和 PCR-RFLP（CYP6P9a 抗性等位基因）调查可能存在的杀虫剂抗性等位基因。进行头部/胸部解剖后，使用 TaqMan qPCR 检测感染阶段疟原虫种的存在。
在研究期间共收集了 1086 只成年雌性冈比亚按蚊。所有（N=20）形态上鉴定为冈比亚按蚊 s.l.的亚样本均使用种特异性 PCR 鉴定为冈比亚按蚊 s.s.。应用 PCR-RFLP 检测 CYP6P9a 抗性等位基因，该等位基因赋予冈比亚按蚊对拟除虫菊酯的抗性，对 N=30 只随机选择的冈比亚按蚊 s.l.进行了检测。
与阳性对照观察到的情况相反，没有一个标本的消化模式与 CYP6P9a 抗性等位基因的存在一致。因此，所有样本均被表征为野生型。用于检测乙酰胆碱酯酶 1 突变（N485I）的 TaqMan qPCR 试剂盒，该突变赋予冈比亚按蚊对有机磷/氨基甲酸酯的抗性，用于（N=144）样本。所有样本均被表征为野生型。在 VGSC 基因中，kdr L1014F 和 L1014S 突变赋予对拟除虫菊酯和滴滴涕的抗性，在用 PCR 进行分析后，直接进行 Sanger 测序，并对 PCR 产物进行了清洁，这些突变也被表征为野生型。在（N=169）样本中均未发现疟原虫（恶性疟原虫/卵形疟原虫/间日疟原虫/疟原虫）检测呈阳性。
Lare 的所有冈比亚按蚊 s.l.样本均经分子鉴定为冈比亚按蚊 s.s.。在冈比亚按蚊样本中未检测到 CYP6P9、N485I 乙酰胆碱酯酶 1、kdr L1014F 或 L1014S 突变。没有冈比亚按蚊样本对疟原虫呈阳性。尽管目前的研究没有检测到任何杀虫剂抗性机制，但它为未来的媒介监测计划提供了参考。定期监测埃塞俄比亚广泛分布这种媒介的地区的抗性机制对于提高媒介控制计划的效果非常重要。
