Substrate promiscuity of key resistance P450s confers clothianidin resistance while increasing chlorfenapyr potency in malaria vectors

Abstract

Novel insecticides were recently introduced to counter pyrethroid resistance threats in African malaria vectors. To prolong their effectiveness, potential cross-resistance from promiscuous pyrethroid metabolic resistance mechanisms must be elucidated. Here, we demonstrate that the duplicated P450s CYP6P9a/-b, proficient pyrethroid metabolizers, reduce neonicotinoid efficacy in Anopheles funestus while enhancing the potency of chlorfenapyr. Transgenic expression of CYP6P9a/-b in Drosophila confirmed that flies expressing both genes were significantly more resistant to neonicotinoids than controls, whereas the contrasting pattern was observed for chlorfenapyr. This result was also confirmed by RNAi knockdown experiments. In vitro expression of recombinant CYP6P9a and metabolism assays established that it significantly depletes both clothianidin and chlorfenapyr, with metabolism of chlorfenapyr producing the insecticidally active intermediate metabolite tralopyril. This study highlights the risk of cross-resistance between pyrethroid and neonicotinoid and reveals that chlorfenapyr-based control interventions such as Interceptor G2 could remain efficient against some P450-based resistant mosquitoes.

Keywords: Anopheles funestus; CP: Microbiology; CYP6P9a and CYP6P9b; RNA interference; chlorfenapyr; cross-resistance; experimental huts; insecticide resistance markers; malaria; metabolism assay; neonicotinoid.

Graphical abstract

Figure 1

Susceptibility profile of the An. funestus from Malawi and the hybrid strain FG/FZ (F₃) to neonicotinoid insecticides and chlorfenapyr (A–F) Mortality rate of F₁ progeny from field-collected An. funestus in Malawi after exposure to chlorfenapyr (A); clothianidin and imidacloprid diluted in acetone only and acetone + MERO (B); time-response mortality after exposure of FG/FZ (F₃) to 100 μg/mL CFP (C); dose-response results of FG/FZ (F₃) after exposure to CFP (D); pyperonyl butoxide (PBO) synergist assay with 20 μg/mL CFP (E); and mortality rate after exposure to PBO/MERO + neonicotinoids (F). In this figure, values represent the mean mortality of 4–5 biological replicates, and error bars represent ±SE of mean. χ² test was used to discern significant differences.

Figure 2

Impact of CYP6P9a/-b on the efficacy of chlorfenapyr using CDC bottle assays (A–F) Distribution of the CYP6P9a genotypes (A) and alleles (B) between alive and dead mosquitoes after exposure to CFP; distribution of the CYP6P9b genotypes (C) and alleles (D) between alive and dead mosquitoes after exposure to CFP; distribution of the combined genotypes at the CYP6P9a_R and CYP6P9b_R alleles between alive and dead mosquitoes after exposure to CFP (E); and odds ratio calculations comparing the ability of double homozygote resistant mosquitoes to survive CFP exposure to other genotype combinations (F). n = total number of mosquitoes from each phenotype that were successfully genotyped. Fisher test was used to discern significant differences.

Figure 3

Impact of the CYP6P9a/-b on the efficacy of CFP-based nets on An. funestus in EHT (A–I) CYP6P9a genotype distribution between alive and dead mosquitoes after exposure to Interceptor (A), IG2 (B), and CFP-100 (C); CYP6P9b genotype distribution between alive and dead mosquitoes after exposure to Interceptor (D), IG2 (E), and CFP-100 (F); and combined CYP6P9a and CYP6P9b genotype distribution between alive and dead mosquitoes after exposure to Interceptor (G), IG2 (H), and CFP-100 (I). For genotype: RR, homozygote resistant; RS, heterozygote; and SS, homozygote susceptible. n = total number from each phenotype that were successfully genotyped. Fisher test was used to discern significant differences.

Figure 4

Impact of the duplicated CYP6P9a/b on the efficacy of clothianidin on An. funestus in CDC bottle assays (A and B) Distribution of the CYP6P9a genotypes and alleles between alive and dead mosquitoes after exposure to CLTD. (C and D) Distribution of the CYP6P9b genotypes and alleles between alive and dead mosquitoes after exposure to CLTD. (E) Distribution of the combined genotypes at the CYP6P9a and CYP6P9b loci between alive and dead mosquitoes after exposure to CLTD. (F) Odds ratio calculations comparing the ability of double homozygote resistant mosquitoes to survive CLTD exposure to other genotype combinations. n = total number from each phenotype that were successfully genotyped. Fisher test was used to discern significant differences.

Figure 5

Impact of CYP6P9a/-b on the efficacy of CFP-based nets on An. funestus in EHT (A–F) CYP6P9a genotype distribution between alive and dead mosquitoes after exposure to deltamethrin (A), Fludora Fusion (B), and clothianidin (C) and CYP6P9b genotype distribution between alive and dead mosquitoes after exposure to deltamethrin (D), Fludora Fusion (E), and clothianidin (F). For genotype: RR, homozygote resistant; RS, heterozygote; and SS, homozygote susceptible. n = total number from each phenotype that were successfully genotyped. Fisher test was used to discern significant differences.

Figure 6

In vivo and in vitro functional validation of the role of CYP6P9a/b in clothianidin and chlorfenapyr resistance (A–F) Mortality pattern of GAL4 x UAS-CYP6Pa and GAL4 x UAS-CYP6P9b transgenic flies exposed to clothianidin (A) and chlorfenapyr (B); mortality of the hybrid FANG/FUMOZ after RNAi knocked down each of the duplicated CYP6P9a genes 7 days post exposure to clothianidin (C) and chlorfenapyr (D); and percentage depletion (mean ± standard deviation [SD]) of clothianidin and chlorfenapyr at 90 min (E) with formation of tralopyril as the primary product of bioactivation of chlorfenapyr (F). Asterisks indicate the difference between each ds-P450 gene in comparison to ds-GFP control and un-injected mosquitoes (^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001). In this figure, values represent the mean of 4–5 biological replicates, and error bars represent ±SD of mean. χ² test was used to discern significant differences.