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

Summary 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.


Figure S1 :
Figure S1: Susceptibility profile of the hybrid strain FG/FZ F3 to the four main classes of insecticides recommended by WHO: A) Mortality rate after exposure to pyrethroids (permethrin, deltamethrin and alpha-cypermethrin), carbamate (bendiocarb) and organophosphate (pirimiphos-methyl) following 60-min exposure; B) PBO synergist assays with pyrethroids; Data are shown as mean ± SEM.

Figure S2 :
Figure S2: Efficacy of chlorfenapyr and clothianidin-based control tools against An.funestus in tunnel assays and experimental hut trials: A) Efficacy of CFP-based nets compared to pyrethroid-only net on the hybrid strain FG/FZ in tunnel tests; B) Efficacy of interceptor G2 and Royal Guard nets on An. funestus from Malawi in tunnel tests; C) Efficacy of CFP-based nets compared to pyrethroid-only net on the hybrid strain FG/FZ in EHT; D) Efficacy of the CLTD-based IRS formulations compared to pyrethroid-only IRS product in EHT.Data are shown as mean ± 95% CI.

Figure S3 :
Figure S3: Impact of CYP6P9a and CYP6P9b on the ability of field An. funestus from Malawi to survive clothianidin and chlorfenapyr exposure.Genotype distribution of CYP6P9a (A) and CYP6P9b (C) between alive and dead after exposure to clothianidin in CDC bottle assays.Genotype distribution of CYP6P9a between dead and alive (B), and blood-fed/unfed (B) after exposure to the CFP-based net IG2 in tunnel test.

Figure S4 :
Figure S4: Impact of the CYP6P9a on the efficacy of CFP-based nets on the hybrid strain FG/FZ after tunnel tests .Genotype distribution between alive and dead after exposure to Interceptor (A), Interceptor G2 (B) and CFP-100 (C); Genotype distribution between blood fed and unfed after exposure to Interceptor (D), Interceptor G2 (E) and CFP-100 (F);

Figure S6 :
Figure S6: Impact of the CYP6P9a on the efficacy of CFP-based nets on the hybrid strain FG/FZ in EHT.Genotype distribution between blood-fed and unfed after exposure to Interceptor (A), Interceptor G2 (B) and CFP-100 (C); Genotype distribution between indoor (Net/Room) and outdoor (veranda) after exposure to Interceptor (D), Interceptor G2 (E) and CFP-100 (F).

Figure S7 :
Figure S7: Impact of the CYP6P9b on the efficacy of CFP-based nets on the hybrid strain FG/FZ in EHT.Genotype distribution between blood-fed and unfed after exposure to Interceptor (A), Interceptor G2 (B) and CFP-100 (C); Genotype distribution between indoor (Net/Room) and outdoor (veranda) after exposure to Interceptor (D), Interceptor G2 (E) and CFP-100 (F).

Figure S8 :
Figure S8: Impact of the CYP6P9a on the efficacy of clothianidin-based IRS formulation on the hybrid strain FG/FZ in EHT.Genotype distribution between blood-fed and unfed after exposure to deltamethrin (A), Fludora Fusion (B) and clothianidin (C); Genotype distribution between indoor (Room) and outdoor (veranda) after exposure to deltamethrin (D), Fludora Fusion (E) and clothianidin (F).

Figure S9 :
Figure S9: Impact of the CYP6P9b on the efficacy of clothianidin-based IRS formulation on the hybrid strain FG/FZ in EHT.Genotype distribution between blood-fed and unfed after exposure to deltamethrin (A), Fludora Fusion (B) and clothianidin (C); Genotype distribution between indoor (Room) and outdoor (veranda) after exposure to deltamethrin (D), Fludora Fusion (E) and clothianidin (F).

Table S1 :
Correlation between genotypes of the CYP6P9a/b and ability of the hybrid strain FG/FZ to survive CFP exposure in CDC bottle assays OR: odd-ratio; CI: confidence interval; RR: homozygote resistant; RS: heterozygote; SS: homozygote susceptible.

Table S2 :
Impact of CYP6P9a and b on the ability of the hybrid strain FG/FZ to survive clothianidin exposure in CDC bottle assay

Table S4 :
Correlation between genotypes at the CYP6P9a/b locus and the ability to survive/blood feed in the presence of various nets in tunnel tests OR: odd-ratio; CI: confidence interval; CFP: chlorfenapyr; RR: homozygote resistant; RS: heterozygote; SS: homozygote susceptible.

Table S5 :
Correlation between genotypes at the CYP6P9a/b locus and the ability to survive exposure/blood feed in the presence of various nets in experimental huts OR: odd-ratio; CI: confidence interval; CFP: chlorfenapyr; RR: homozygote resistant; RS: heterozygote; SS: homozygote susceptible.

Table S6 :
Correlation between genotypes at the CYP6P9a/b locus and the ability to survive exposure/blood feed in the presence of various IRS treatments in experimental huts OR: odds ratio; CI: confidence interval; RR: homozygote resistant; RS: heterozygote; SS: homozygote susceptible.

Table S7 :
Combined impact of CYP6P9a and CYP6P9b on the mortality and blood feeding of the hybrid strain FG/FZ after exposure to various nets in tunnel assays