Cis-regulatory CYP6P9b P450 variants associated with loss of insecticide-treated bed net efficacy against Anopheles funestus

Abstract

Elucidating the genetic basis of metabolic resistance to insecticides in malaria vectors is crucial to prolonging the effectiveness of insecticide-based control tools including long lasting insecticidal nets (LLINs). Here, we show that cis-regulatory variants of the cytochrome P450 gene, CYP6P9b, are associated with pyrethroid resistance in the African malaria vector Anopheles funestus. A DNA-based assay is designed to track this resistance that occurs near fixation in southern Africa but not in West/Central Africa. Applying this assay we demonstrate, using semi-field experimental huts, that CYP6P9b-mediated resistance associates with reduced effectiveness of LLINs. Furthermore, we establish that CYP6P9b combines with another P450, CYP6P9a, to additively exacerbate the reduced efficacy of insecticide-treated nets. Double homozygote resistant mosquitoes (RR/RR) significantly survive exposure to insecticide-treated nets and successfully blood feed more than other genotypes. This study provides tools to track and assess the impact of multi-gene driven metabolic resistance to pyrethroids, helping improve resistance management.

Fig. 1

Comparative transcription profiles of An. funestus between countries. qRT-PCR comparison of the expression profile of major detoxification genes associated with pyrethroid resistance between populations of An. funestus from different African regions supporting a shift in the role of these genes between regions. The data shown are mean + SEM (n = 3). Source data are provided as a Source Data file

Fig. 2

Genetic diversity patterns of the cis-regulatory region of CYP6P9b. a Maximum likelihood phylogenetic tree of CYP6P9a promoter haplotypes across Africa. b Neighbour-joining phylogenetic tree of CYP6P9b-based genetic distance between nine African populations (N_ST estimates). c Africa-wide TCS network for the CYP6P9b haplotypes showing predominant haplotypes in southern Africa. A fixed haplotype is observed in Benin (sky blue) and a nearly fixed haplotype is seen in Ghana (black). Other locations show a greater diversity. Lines connecting haplotypes and each node represent a single mutation event. d A maximum likelihood phylogenetic tree of CYP6P9b showing a cluster of highly diverse haplotypes before scale up of bed nets (pre-intervention) but a nearly fixed haplotype post-intervention. e TCS haplotype network in Mozambique pre- and post-intervention revealing a major resistant haplotype post-intervention but a more diverse set of haplotypes before pre-bed nets

Fig. 3

DNA-based diagnostic assay for CYP6P9b-mediated metabolic resistance to pyrethroids. a Comparative luciferase assay between promoter fragment from the highly resistant FUMOZ and highly susceptible (FANG) lab strains with progressive serial deletions of CYP6P9b 5′ flanking region to detect the causative variants. Bars represent the mean ± S.D. of four independent transfections of three replicates (n = 6). b Agarose gel of PCR-RFLP with NmuCl of CYP6P9b showing the RR, RS, and SS genotypes. c Distribution of the CYP6P9b genotypes between susceptible and resistant mosquitoes showing a very strong correlation between CYP6P9b and resistance phenotype. d Distribution of the combined genotypes of both CYP6P9a and CYP6P9b showing that both genotypes combined to increase the pyrethroid resistance. Source data are provided as a Source Data file

Fig. 4

Geographical distribution of CYP6P9b Africa-wide. a Frequency of the CYP6P9B_R allele across Africa showing that it is highly predominant in southern Africa, moderately present in East Africa but completely absent elsewhere on the continent. b Map of Africa showing the distribution of the CYP6P9b-resistant alleles with a near fixation in southern Africa. The map was generated using a blank map freely available from https://www.rkkerkenschijndel.nl/map-of-africa-drawing.html and pie-charts representing frequency of both alleles in each location were added on the map. c Comparative distribution of the combined genotypes of CYP6P9a and CYP6P9b in Tanzania and RD Congo supporting an independent segregation of genotypes of both genes in the field. Source data are provided as a Source Data file

Fig. 5

Impact of the CYP6P9b-based metabolic resistance on bed nets’ efficacy. a Distribution of CYP6P9b genotypes between dead and alive mosquitoes after exposure to PermaNet 2.0 net in experimental huts showing that CYP6P9b_R significantly allows mosquitoes to survive exposure to this insecticide-treated net. b Association between frequency of CYP6P9b-R and ability to survive exposure to PermaNet 2.0. c Distribution of CYP6P9b genotypes between blood-fed and unfed mosquitoes after exposure to the PBO-based net PermaNet 3.0 showing that CYP6P9b_R allele increases the ability to take a blood meal. Source data are provided as a Source Data file

Fig. 6

CYP6P9b combines with CYP6P9a to further reduce bed nets’ efficacy. a Distribution of the combined genotypes of both CYP6P9a and CYP6P9b showing that genotypes at both genotypes combined to additively increase the ability to survive after exposure to PermaNet 2.0. b Ability to survive exposure to PermaNet 2.0 (Odds ratio) of the double homozygote-resistant (RR/RR) genotypes of CYP6P9a and CYP6P9b compared to other genotypes supporting the additive resistance effect of both genes. Significance is shown by ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, as estimated using Fisher’s exact test. c Ability to survive exposure to PermaNet 2.0 (odds ratio) of the combined homozygote resistant and heterozygote (RR/RS) genotypes of CYP6P9a and CYP6P9b compared to other genotypes. d Distribution of the combined genotypes of both CYP6P9a and CYP6P9b after exposure to PermaNet 3.0 revealing an additive effect of both genes in increasing the ability to blood feed. e Comparison of blood feeding ability of combined genotypes of CYP6P9a and CYP6P9b showing a significantly higher ability (odds ratio) to blood feed for mosquitoes that double homozygote resistant (RR/RR). Source data are provided as a Source Data file