Restriction to gene flow is associated with changes in the molecular basis of pyrethroid resistance in the malaria vector Anopheles funestus

Abstract

Resistance to pyrethroids, the sole insecticide class recommended for treating bed nets, threatens the control of major malaria vectors, including Anopheles funestus Effective management of resistance requires an understanding of the dynamics and mechanisms driving resistance. Here, using genome-wide transcription and genetic diversity analyses, we show that a shift in the molecular basis of pyrethroid resistance in southern African populations of this species is associated with a restricted gene flow. Across the most highly endemic and densely populated regions in Malawi, An. funestus is resistant to pyrethroids, carbamates, and organochlorides. Genome-wide microarray-based transcription analysis identified overexpression of cytochrome P450 genes as the main mechanism driving this resistance. The most up-regulated genes include cytochrome P450s (CYP) CYP6P9a, CYP6P9b and CYP6M7. However, a significant shift in the overexpression profile of these genes was detected across a south/north transect, with CYP6P9a and CYP6P9b more highly overexpressed in the southern resistance front and CYP6M7 predominant in the northern front. A genome-wide genetic structure analysis of southern African populations of An. funestus from Zambia, Malawi, and Mozambique revealed a restriction of gene flow between populations, in line with the geographical variation observed in the transcriptomic analysis. Genetic polymorphism analysis of the three key resistance genes, CYP6P9a, CYP6P9b, and CYP6M7, support barriers to gene flow that are shaping the underlying molecular basis of pyrethroid resistance across southern Africa. This barrier to gene flow is likely to impact the design and implementation of resistance management strategies in the region.

Keywords: Anopheles funestus; cytochrome P450; gene flow; malaria; pyrethroid resistance.

Fig. 1.

Insecticide resistance and gene expression profiling. (A) Map of malaria incidence levels at Malawi collection sites: Chikwawa (CKW), Salima (SLM), and Nkhotakota (NKK). (B) Susceptibility profile of Malawian An. funestus population to the main insecticides and a synergist (PBO). Data are mean ± SEM values of 185–200 mosquitoes (100 for malathion). (C) Fold change determined using microarray and qRT-PCR analyses. For qRT-PCR, error bars represent SD.

Fig. 2.

Population structure of southern Africa. (A) Neighbor-joining tree based on the F_st pairwise scores using 17 microsatellites. (B) Bayesian-based structure patterns of southern Africa. K = 3, 5, 7 shows the separation of CKW, Malawi and Chokwe, Mozambique. (C) Isolation by distance comparing genetic distance versus geographical distance based on the Mantel test and logged scaled. The two outliers are: red dot, Ckw vs. Moz; and blue dot, west vs. north Zambia.

Fig. 3.

Haplotype distribution analysis of CYP6P9a and CYP6P9b. (A) Nucleotide diversity based on Pi × 10⁻³ for CYP6P9a and CYP6P9b. Significance between resistant (red) and susceptible (blue), including SD: *P < 0.05; **P < 0.01. (B and C) The polymorphic amino acid positions for both CYP6P9a and CYP6P9b, respectively, between resistant and susceptible individuals. Three locations in Malawi (CKW, Chikwawa; SLM, Salima; and NKK, Nkhotakota), one location in Mozambique (MOZ), and one location in Zambia (ZB). H denotes haplotypes from more than one location. Blue numbers on the top x-axis are the amino acid position. For resistant amino acids, the susceptible amino acid is listed in red.

Fig. 4.

Comparative analysis of haplotype diversity across Malawi. A TCS network was constructed for the coding region for all samples from Malawi (light blue, CKW; green, NKK; blue, SLM) for CYP6P9a (A), CYP6P9b (B), and CYP6M7 (C). Haplotypes with multiple hits are denoted by “H,” and resistance haplotypes are orange squares. Unique haplotypes are shown as circles with sample information, including a three-digit location code and designation as susceptible (S) or resistant (R).