Détection des marqueurs moléculaires de la résistance métabolique aux pyréthrinoïdes chez des vecteurs de paludisme à Yaoundé, Cameroun

Abstract

The rapid spay of insecticide resistance among malaria vectors is an obstacle to maintaining the effectiveness of control tools. However, despite a recurrent association between cytochrome P450s and pyrethroid resistance in Africa, the underlying mutations and/or genetic variations capable of modulating the expression of this gene family in Anopheles gambiae s.l. remain poorly documented. To fill this gap, molecular markers that can be used to rapidly identify metabolic resistance have been identified in A. gambiae and A.s coluzzii from Yaoundé, Cameroon, in order to develop a simple diagnostic tool for monitoring and early detection of this type of resistance in the field. To do this, adult mosquitoes of the Anopheles genus were collected from December 2018 to December 2020 in two areas of the city of Yaoundé (urban and peri-urban). After evaluating their level of resistance to insecticides, the different mechanisms of resistance involved were investigated using molecular techniques. The mosquitoes that survived exposure to permethrin were subsequently crossed with a sensitive laboratory strain, and the individuals of the fourth generation obtained were grouped into hyper-resistant (R90) and hyper-sensitive (S20) mosquitoes. Subsequently, two of the most overexpressed genes along with their promoter regions were amplified and then sequenced for polymorphism analysis. The results of the bioassays revealed confirmed resistance to almost all the tested insecticides regardless of the collection season, with a partial restoration of sensitivity after pre-exposure to the synergist piperonyl butoxide (PBO), suggesting the involvement of metabolic enzymes. This hypothesis was confirmed by the results of quantitative PCR (RT-qPCR), which revealed a level of gene overexpression that varied significantly depending on the species, but also from one season to another, with overexpression of the CYP6M2 gene (FC = 4.39 ± 0.49 for A. coluzzii; FC = 9.69 ± 2.58 for A. gambiae) and CYP6P4 (FC = 4.39 ± 0.49 for A. coluzzii; FC = 18.77 ± 7.49 for A. gambiae). Subsequently, the polymorphism results revealed low diversity for the promoter region of the CYP6M2 gene in R90 and S20 mosquitoes (global Hd = 0.49, π = 0.018), while in its gene region, allelic variations were detected but without any sign of haplotype selection. For the CYP6P4 gene, lower diversity was observed in R90 mosquitoes (Hd = 0.48) compared to S20 (Hd = 0.86), with a candidate A/T mutation at position 273 located in nrf2/MAF binding site in A. gambiae, suggesting ongoing selection. This mutation was found at a frequency of 100 % in R90, 44 % in S20. A synonymous mutation in codon 168 (TGC->AGC) was also found in the locus of the CYP6P4 gene with a frequency of 70 % in R90 and 28 % in S20. Moreover, no significant association was observed between any of the alleles identified in this study and the pyrethroid resistance phenotype. This work provides two interesting molecular markers for the CYP6P4 gene in An. gambiae that should be considered in the annual molecular surveillance of resistance and in any study

on markers of metabolic resistance. However, further work is needed to determine their impact on the various vector control tools used in the field.