Discovery of Knock-Down Resistance in the Major African Malaria Vector Anopheles funestus

Abstract

A major insecticide resistance mechanism in insect pests is knock-down resistance (kdr) caused by mutations in the voltage-gated sodium channel (Vgsc) gene. Despite being common in most malaria Anopheles vector species, kdr mutations have never been observed in Anopheles funestus, the principal malaria vector in Eastern and Southern Africa, with resistance mainly being conferred by detoxification enzymes. In a parallel study, we monitored 10 populations of An. funestus in Tanzania for insecticide resistance unexpectedly identified resistance to a banned insecticide, DDT, in the Morogoro region. Through whole-genome sequencing of 333 An. funestus samples from these populations, we found eight novel amino acid substitutions in the Vgsc gene, including the kdr variant, L976F (equivalent to L995F in An. gambiae), in tight linkage disequilibrium with another (P1842S). The mutants were found only at high frequency in one region and were accompanied by weak signatures of a selective sweep, with a significant decline between 2017 and 2023. Notably, kdr L976F was strongly associated with survivorship to exposure to DDT insecticide, while no clear association was noted with a pyrethroid insecticide (deltamethrin). The WHO prequalifies no DDT products for vector control, and the chemical is banned in Tanzania. Widespread DDT contamination and a legacy of extensive countrywide stockpiles may have selected for this mutation. Continued monitoring is necessary to understand the origin of kdr in An. funestus, and the threat posed to insecticide-based vector control in Africa.

Keywords: Anopheles funestus; Tanzania; insecticide resistance; knock‐down resistance; voltage‐gated sodium channel.

Fig. 1:

(A) Map of An. funestus collection locations. Points indicate sample collection locations. The point colour indicates the administrative region from which samples were collected. (B): Phenotypic insecticide resistance profile of An. funestus to DDT using bioassay data adopted from our recent surveillance (17). The colours and the X-axis represent the various regions where the bioassays were conducted, and the error bars are 95% confidence interval. The black and red dotted lines on the Y-axis represent the 98 and 90% mortality thresholds. (C) H12 selection scans of An. funestus chromosome 3RL, coloured and windowed by sample collection region (where n>20 – see Supp Table 2). The X-axis indicates the position (in base-pairs (bp)), and the Y-axis indicates the selection statistic H12. The Grey line indicates the location of the Vgsc gene. Note Mwanza region is absent from panel C as there were too few samples (n<20) to perform a selection scan.

Fig. 2:

(A) Heatmap of Vgsc allele frequencies. Y-axis labels indicate mutation effect, chromosome position, and nucleotide change. X-axis labels indicate the collection date and heatmap intensity indicates frequency where darker = higher, with frequency labelled in each heatmap facet. The heatmap is panelled by the sample collection region. (B) L976F and 1842S frequencies, in the Morogoro region, over time. The y-axis indicates allele frequency, X-axis indicates the date. Line and point colour refer to mutation, specified in the legend. Bars indicate 95% confidence intervals. (C) Denotes the association of L976F and P1842S with resistance to Deltamethrin and DDT. Colour and panelling are by mutation, the x-axis indicates genotype, the y-axis indicates mortality, the point shape indicates the mean for each insecticide and the line indicates the 95% CI based on generalised mixed model prediction. (D) Heatmap of linkage disequilibrium (LD) (Rogers and Huff R) between nonsynonymous variants in the Vgsc gene at frequency > 5%. LD is indicated by fill colour. SNP effects and positions are labelled on the X and Y axes.

Fig. 3:

Clustering of haplotypes at the Vgsc gene (LOC125769886, 3RL:44105643–44156624). The dendrogram branch length corresponds to no. SNPs difference (y-axis). Tips correspond to individual haplotypes (x-axis). The coloured Population bar denotes the administrative region of origin (as described by the legend). Red blocks at the bottom indicate the presence of the labelled non-synonymous SNPs in the Vgsc gene.

Fig. 4:

(A) Tanzanian National Malaria Control Programme (NMCP) indoor residual spraying strategy 2008 – 2012. The colours indicate districts where DDT spraying was planned. (B) DDT stockpile locations with the size of the circle indicating the stockpile quantities. (C) Agro-ecological zones in Tanzania with colours on the map denoting the categories indicated in the figure key.