Species composition, infection rate and detection of resistant alleles in Anopheles funestus (Diptera: Culicidae) from malaria hotspot areas of Ethiopia
Authors: Woyessa Delelegn , Morou Evangelia , Wipf Nadja , Dada Nsa , Mavridis Konstantinos , Vontas John , Yewhalaw Delenasaw 
Published: January 2023

Background: 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, behavior 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. funestuspopulations.
Methods: Adult female mosquito collection was conducted from Lare, Gambella Regional State of Ethiopia between November 2017 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.
Results: 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 s.l were confirmed to belong to An. funestus sensu stricto using species- specific PCR assay. The PCR-RFLP assay that detects the CYP6P9a resistance allele that confers pyrethroid resistance in An. funestus mosquitoes was applied in 30 randomly selected An. funestus s.l specimens. 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 analyzed 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.
Conclusion: Molecular identification of all An. funestus s.l samples from Lare resulted in An. funestus s.s. No CYP6P9, N485I acetylcholinesterase 1, kdr L1014F or L1014S mutations were detected in the An. funestus s.l samples. None of the An. funestus s.l samples were found positive for Plasmodiumdetection. Although the current study did not detect insecticide resistant mechanism possibly due to limited samples and/or L119F-GSTe2 mutation, it provides a reference for future vector monitoring programs. Hence, regular resistance monitoring that involves investigation of L119F-GSTe2 mutation covering wider geographical areas of Ethiopia where this vector is distributed is important for improving the efficacy of vector control programs.