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Deciphering the molecular drivers of insecticide resistance is paramount to extend the effectiveness of malaria vector control tools. Here, we demonstrated that the E205D amino acid change in a key metabolic resistance P450CYP6P3drives pyrethroid resistance in the major malaria vector,Anopheles gambiae. Spatio-temporal whole genome Poolseq analyses in Cameroon detected a major P450-linked locus on chromosome 2R beside the sodium channel locus.In vitrometabolism assays with recombinantly expressedCYP6P3protein revealed that the catalytic efficiency of 205D was 2.5 times higher than E205 with α-cypermethrin. Similar patterns were observed for permethrin. Overexpression of the 205D allele in transgenic flies confers higher more pyrethroids and carbamates resistance, compared to controls. A DNA-based assay further supported that theCYP6P3-205D variant strongly correlates with pyrethroid resistance in field populations (OR=26.4; P<0.0001) and that it reduces the efficacy of pyrethroid-only LLINs with homozygote RR genotype exhibiting significantly higher survival following PermaNet 3.0 exposure compared to the SS genotype (OR: 6.1, p = 0.0113). Furthermore, theCYP6P3-E205D combines with thekdrtarget-site resistance mechanisms to worsen the loss of bednet efficacy. The 205D mutation is now predominant in West and Central Africa but less abundant or absent in East and South Africa with signs of introgression withAn. coluzziiin Ghana. This study highlights the importance of P450-based resistance and designs field-applicable tools to easily track the spread of metabolic resistance and assess its impact on control interventions.One Sentence Summary:The major obstacle to malaria control and elimination is the spread of parasite resistance to anti-malarial drugs, and mosquito resistance to insecticides. In this study, we identified a key point mutation E205D in the metabolic geneCYP6P3(cytochrome P450) conferring resistance to pyrethroids by enhancing the breakdown of insecticides used for bednets impregnation. DNA-based assays were then designed and used to determine the spread of the resistance across Africa and demonstrate that theCYP6P3-205D allele works together with the knockdown resistance in the voltage-gated Sodium channel to reduce the efficacy of insecticide-treated bednets.
Deciphering the molecular drivers of insecticide resistance is paramount to extend the effectiveness of malaria vector control tools. Here, we demonstrated that the E205D amino acid change in a key metabolic resistance P450CYP6P3drives pyrethroid resistance in the major malaria vector,Anopheles gambiae. Spatio-temporal whole genome Poolseq analyses in Cameroon detected a major P450-linked locus on chromosome 2R beside the sodium channel locus.In vitrometabolism assays with recombinantly expressedCYP6P3protein revealed that the catalytic efficiency of 205D was 2.5 times higher than E205 with α-cypermethrin. Similar patterns were observed for permethrin. Overexpression of the 205D allele in transgenic flies confers higher more pyrethroids and carbamates resistance, compared to controls. A DNA-based assay further supported that theCYP6P3-205D variant strongly correlates with pyrethroid resistance in field populations (OR=26.4; P<0.0001) and that it reduces the efficacy of pyrethroid-only LLINs with homozygote RR genotype exhibiting significantly higher survival following PermaNet 3.0 exposure compared to the SS genotype (OR: 6.1, p = 0.0113). Furthermore, theCYP6P3-E205D combines with thekdrtarget-site resistance mechanisms to worsen the loss of bednet efficacy. The 205D mutation is now predominant in West and Central Africa but less abundant or absent in East and South Africa with signs of introgression withAn. coluzziiin Ghana. This study highlights the importance of P450-based resistance and designs field-applicable tools to easily track the spread of metabolic resistance and assess its impact on control interventions.One Sentence Summary:The major obstacle to malaria control and elimination is the spread of parasite resistance to anti-malarial drugs, and mosquito resistance to insecticides. In this study, we identified a key point mutation E205D in the metabolic geneCYP6P3(cytochrome P450) conferring resistance to pyrethroids by enhancing the breakdown of insecticides used for bednets impregnation. DNA-based assays were then designed and used to determine the spread of the resistance across Africa and demonstrate that theCYP6P3-205D allele works together with the knockdown resistance in the voltage-gated Sodium channel to reduce the efficacy of insecticide-treated bednets.
Background Insecticide resistance is jeopardising malaria control efforts in Africa. Deciphering the evolutionary dynamics of mosquito populations country-wide is essential for designing effective and sustainable national and subnational tailored strategies to accelerate malaria elimination efforts. Here, we employed genome-wide association studies through pooled template sequencing to compare four eco-geographically different populations of the major vector, Anopheles funestus, across a South North transect in Cameroon, aiming to identify genomic signatures of adaptive responses to insecticides. Results Our analysis revealed limited population structure within Northern and Central regions (FST<0.02), suggesting extensive gene flow, while populations from the Littoral/Coastal region exhibited more distinct genetic patterns (FST>0.049). Greater genetic differentiation was observed at known resistance-associated loci, resistance-to-pyrethroids 1 (rp1) (2R chromosome) and CYP9 (X chromosome), with varying signatures of positive selection across populations. Allelic variation between variants underscores the pervasive impact of selection pressures, with rp1 variants more prevalent in Central and Northern populations (FST>0.3), and the CYP9 associated variants more pronounced in the Littoral/Coastal region (FST =0.29). Evidence of selective sweeps was supported by negative Tajima’s D and reduced genetic diversity in all populations, particularly in Central (Elende) and Northern (Tibati) regions. Genomic variant analysis identified novel missense mutations and signatures of complex genomic alterations such as duplications, deletions, transposable element (TE) insertions, and chromosomal inversions, all associated with selective sweeps. A 4.3 kb TE insertion was fixed in all populations with Njombe Littoral/Coastal population, showing higher frequency of CYP9K1 (G454A), a known resistance allele and TE upstream compared to elsewhere. Conclusion Our study uncovered regional variations in insecticide resistance candidate variants, emphasizing the need for a streamlined DNA-based diagnostic assay for genomic surveillance across Africa. These findings will contribute to the development of tailored resistance management strategies crucial for addressing the dynamic challenges of malaria control in Cameroon.
Monitoring the trend of insecticide resistance and understanding associated genetic mechanisms is important for designing efficient malaria vector control strategies. This study was conducted to provide temporal data on insecticide resistance status and mechanisms in the major malaria vector Anopheles gambiae s.l. from Ebolowa, Southern Cameroon. Methods: Larvae of An. gambiae s.l. were collected from typical breeding sites throughout the city and reared to adulthood. Emerging adults were morphologically identified and WHO tube assays were performed to determine their susceptibility to carbamate, organophosphate and pyrethroid insecticides at diagnostic doses. When resistance was observed, its intensity was determined by performing WHO tube tests using 5 and 10 times the concentration of the diagnostic dose. Metabolic resistance mechanisms were investigated using insecticide-synergist assays. Sibling species of the An. gambiae complex were identified using SINE-PCR protocol. TaqMan assay was used to genotype the L1014F and L1014S kdr mutations, and the N1575Y mutation, an amplifier of the resistance conferred by the L1014F mutation. Results: Anopheles coluzzii was by far the dominant (99%) member of the An. gambiae s.l. complex in Ebolowa. The species was fully susceptible to carbamates and organophosphates, but resistant to all pyrethroid insecticides tested. Resistance was of moderate intensity for deltamethrin (mortality: 37%, 70% and 99% for 1×, 5× and 10× insecticide concentration, respectively) but rather of high intensity for permethrin (5% for 1×; 62% for 5× and 75% for 10×) and for alphacypermethrin (4.4% for 1×; 57% for 5× and 80% for 10×). Pre-exposure to the synergist PBO resulted in a full recovery of the susceptibility to delthametrin, but this was not observed for the other two pyrethroids tested. L1014S (kdr-East) and the N1575Y mutations were absent, whereas the L1014F (kdr-West) mutation was present at a high frequency (75%), showing a significant association with resistance to permethrin (OR = 3.8; 95%; CI [1.9–7.4]; p < 0.0001) and alphacypermethrin (OR = 3; 95%; CI [1.6–5.4]; p = 0.0002). Conclusion: The increased resistance of An. gambiae s.l. to pyrethroid insecticides as observed in Ebolowa poses a threat to the efficacy of LLINs used to protect populations from the bites of Anopheles mosquitoes that transmit malaria parasites. The present study further highlights the urgent need to implement resistance management strategies in order to maintain the effectiveness of insecticide-based vector control interventions and prevent a rebound in malaria-related mortality.
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