Insecticide resistance in Anopheles mosquitoes is seriously threatening the success of insecticide-based malaria vector control. Surveillance of insecticide resistance in mosquito populations and identifying the underlying mechanisms enables optimisation of vector control strategies. Here, we investigated the molecular mechanisms of insecticide resistance in three Anopheles coluzzii field populations from southern Côte d’Ivoire, including Agboville, Dabou and Tiassalé. All three populations were resistant to bendiocarb, deltamethrin and DDT, but not or only very weakly resistant to malathion. The absence of malathion resistance is an unexpected result because we found the acetylcholinesterase mutation Ace1-G280S at high frequencies, which would typically confer cross-resistance to carbamates and organophosphates, including malathion. Notably, Tiassalé was the most susceptible population to malathion while being the most resistant one to the pyrethroid deltamethrin. The resistance ratio to deltamethrin between Tiassalé and the laboratory reference colony was 1,800 fold. By sequencing the transcriptome of individual mosquitoes, we found numerous cytochrome P450-dependent monooxygenases – including CYP6M2, CYP6P2, CYP6P3, CYP6P4 and CYP6P5 – overexpressed in all three field populations. This could be an indication for negative cross-resistance caused by overexpression of pyrethroid-detoxifying cytochrome P450s that may activate pro-insecticides, thereby increasing malathion susceptibility. In addition to the P450s, we found several overexpressed carboxylesterases, glutathione S-transferases and other candidates putatively involved in insecticide resistance.

The introduction of neonicotinoids for managing insecticide resistance in mosquitoes is of high interest as they interact with a biochemical target not previously used in public health. In this concern, Bayer developed a combination of the neonicotinoid clothianidin and the pyrethroid deltamethrin (brand name Fludora Fusion) as a new vector control tool. Although this combination proved to be efficient against pyrethroid-resistant mosquitoes, its ability to prevent the selection of pyrethroid and neonicotinoid resistance alleles was not investigated. In this context, the objective of this work was to study the dynamics and the molecular mechanisms of resistance of An. gambiae to the separated or combined components of this combination. A field-derived An. gambiae line carrying resistance alleles to multiple insecticides at low frequencies was used as a starting for 33 successive generations of controlled selection. Resistance levels to each insecticide and target site mutation frequencies were monitored throughout the selection process. Cross resistance to other public health insecticides were also investigated. RNA-seq was used to compare gene transcription variations and polymorphisms across all lines. This study confirmed the potential of this insecticide combination to impair the selection of resistance as compared to its two separated components. Deltamethrin selection led to the rapid enrichment of the kdr L1014F target-site mutation. Clothianidin selection led to the over-transcription of multiple cytochrome P450s including some showing high homology with those conferring neonicotinoid resistance in other insects. A strong selection signature associated with clothianidin selection was also observed on a P450 gene cluster previously associated with resistance. Within this cluster, the gene CYP6M1 showed the highest selection signature together with a transcription profile supporting a role in clothianidin resistance. Modelling the impact of point mutations selected by clothianidin on CYP6M1 protein structure showed that selection retained a protein variant with a modified active site potentially enhancing clothianidin metabolism. In the context of the recent deployment of neonicotinoids for mosquito control and their frequent usage in agriculture, the present study highlights the benefit of combining them with other insecticides for preventing the selection of resistance and sustaining vector control activities.

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