The highly polymorphic CYP6M7 cytochrome P450 gene partners with the directionally selected CYP6P9a and CYP6P9b genes to expand the pyrethroid resistance front in the malaria vector Anopheles funestus in Africa

Abstract

Background: Pyrethroid resistance in the major malaria vector Anopheles funestus is rapidly expanding across Southern Africa. It remains unknown whether this resistance has a unique origin with the same molecular basis or is multifactorial. Knowledge of the origin, mechanisms and evolution of resistance are crucial to designing successful resistance management strategies.

Results: Here, we established the resistance profile of a Zambian An. funestus population at the northern range of the resistance front. Similar to other Southern African populations, Zambian An. funestus mosquitoes are resistant to pyrethroids and carbamate, but in contrast to populations in Mozambique and Malawi, these insects are also DDT resistant. Genome-wide microarray-based transcriptional profiling and qRT-PCR revealed that the cytochrome P450 gene CYP6M7 is responsible for extending pyrethroid resistance northwards. Indeed, CYP6M7 is more over-expressed in Zambia [fold-change (FC) 37.7; 13.2 for qRT-PCR] than CYP6P9a (FC15.6; 8.9 for qRT-PCR) and CYP6P9b (FC11.9; 6.5 for qRT-PCR), whereas CYP6P9a and CYP6P9b are more highly over-expressed in Malawi and Mozambique. Transgenic expression of CYP6M7 in Drosophila melanogaster coupled with in vitro assays using recombinant enzymes and assessments of kinetic properties demonstrated that CYP6M7 is as efficient as CYP6P9a and CYP6P9b in conferring pyrethroid resistance. Polymorphism patterns demonstrate that these genes are under contrasting selection forces: the exceptionally diverse CYP6M7 likely evolves neutrally, whereas CYP6P9a and CYP6P9b are directionally selected. The higher variability of CYP6P9a and CYP6P9b observed in Zambia supports their lesser role in resistance in this country.

Conclusion: Pyrethroid resistance in Southern Africa probably has multiple origins under different evolutionary forces, which may necessitate the design of different resistance management strategies.

Figure 1

Susceptibility profile of the An . funestus population from the Katete district in Zambia to the main insecticides and when exposed to the synergist piperonyl butoxide (PBO). The data are presented as the mean of at least four replicates and error bars represent standard deviation.

Figure 2

Transcriptional profiling of resistant populations. A) Summary of probes differentially regulated in each of the 3 countries. The Venn diagrams show the number of probes significantly (P < 0.01) up- or down-regulated (FC > 2) in each country as well as the commonly expressed probes. Upward arrows indicate up-regulated probes, and downward arrows represent down-regulated probes. B) Relative expression of the three main detoxification genes (CYP6P9a, CYP6P9b and CYP6M7) by microarray between the three countries (based on probes with highest expression); C) Differential expression of 15 genes up-regulated between permethrin-resistant (R) and -susceptible FANG (S) mosquitoes in the Mozambican (MZ), Malawian (ML) and Zambian field populations (ZB). Fold-change for CYP6M7 was obtained from the average of three independent primer pairs. Error bars represent standard deviation (N = 3). The presence of * on top of the three fold changes for each gene indicates a statistically significant over-expression in all locations compared to the FANG susceptible strain. "ns" is added when the difference was not significant. D) Tissue-specific expression of CYP6P9a, CYP6P9b and CYP6M7 in field permethrin-resistant female An. funestus mosquitoes.

Figure 3

Functional confirmation of the role of CYP6M7 in pyrethroid resistance. A) Transgenic expression of CYP6M7 in Drosophila. Results of a bioassay with 2% permethrin (A) and 0.15% deltamethrin (B) against the transgenic Act5C-CYP6M7 strain (Experimental) and the progeny from the cross between the UAS-CYP6M7 females and w¹¹¹⁸ males (which do not over-express the P450 transgene) (Control). The data shown are the mean ± SEM (n = 6). (C) The proportion of 10 μM insecticide cleared by 0.1 μM P450 with 0.8 μM cyt b5 in the presence of NADPH is indicated by bar height. Error bars represent standard deviation (N = 3).

Figure 4

Turnover and kinetic profiles of CYP6P9a, CYP6P9b and CYP6M7 with type I and type II pyrethroids. The turnover (time course) of the three enzymes with deltamethrin (A) and permethrin (B) is shown; (C) is the Michaelis-Menten plot of CYP6P9a, CYP69b and CYP6M7 with deltamethrin, and (D) is the plot with permethrin. The data are presented as the mean ± S.D. of three replicates.

Figure 5

Comparative analysis of haplotype diversity. The haplotype diversities of CYP6M7 (A)  CYP6P9a (B) and CYP6P9b (C) were compared using a 95% parsimony network based only on coding regions when combining the susceptible (S) and resistant (R) mosquitoes from each country. For CYP6M7, networks are presented by country due to the large size of the combined network. These networks indicate the exceptional diversity of CYP6M7 with high polymorphisms whereas CYP6P9a and CYP6P9b both exhibit reduced diversity, with the presence of a highly predominant haplotype associated with resistance (directional selection). Haplotypes are represented as an oval or a rectangle scaled to reflect their frequencies. The lines connecting haplotypes and each node represent a single mutation event. Gray shapes represent haplotypes unique in susceptible mosquitoes; green shapes represent haplotypes predominantly found in resistant mosquitoes but also in some dead mosquitoes; red shapes represent haplotypes unique to resistant mosquitoes. Some haplotypes with >20 mutation differences from others could not be linked to the major network.