Identification and validation of a gene causing cross-resistance between insecticide classes in Anopheles gambiae from Ghana

Abstract

In the last decade there have been marked reductions in malaria incidence in sub-Saharan Africa. Sustaining these reductions will rely upon insecticides to control the mosquito malaria vectors. We report that in the primary African malaria vector, Anopheles gambiae sensu stricto, a single enzyme, CYP6M2, confers resistance to two classes of insecticide. This is unique evidence in a disease vector of cross-resistance associated with a single metabolic gene that simultaneously reduces the efficacy of two of the four classes of insecticide routinely used for malaria control. The gene-expression profile of a highly DDT-resistant population of A. gambiae s.s. from Ghana was characterized using a unique whole-genome microarray. A number of genes were significantly overexpressed compared with two susceptible West African colonies, including genes from metabolic families previously linked to insecticide resistance. One of the most significantly overexpressed probe groups (false-discovery rate-adjusted P < 0.0001) belonged to the cytochrome P450 gene CYP6M2. This gene is associated with pyrethroid resistance in wild A. gambiae s.s. populations) and can metabolize both type I and type II pyrethroids in recombinant protein assays. Using in vitro assays we show that recombinant CYP6M2 is also capable of metabolizing the organochlorine insecticide DDT in the presence of solubilizing factor sodium cholate.

Fig. 1.

Interwoven microarray experimental loop design for a comparison between DDT-resistant field-collected A. gambiae s.s M-forms from Ghana and two laboratory colonies of M-form A. gambiae originating from West Africa. The Ngoussou (NGOU) colony originates from Cameroon and is fully susceptible to DDT, but the Akron colony was colonized from Benin and displays low level DDT resistance. Each pool, indicated by a circle, represents RNA extracted from 10 female A. gambiae s.s. mosquitoes that were 3–5 d old. Arrows indicate individual microarrays (18 in total), with direction representing microarray Cy dye labeling.

Fig. 2.

Volcano plot showing the reproducibility of differences in gene expression between the three groups (Ghanaian DDT-resistant A. gambiae s.s. from the field and the Akron and Ngoussou control colonies). The figure differs from a conventional volcano plot as there are three treatment groups as opposed to the more usual two. Q values, FDR-adjusted P values (−log₁₀), are shown along with Log₂ fold-changes. A Q value of 4 (q < 0.0001) is shown as the significant test level. A selection of genes from Table 1 found to be significantly overexpressed in the DDT-resistant group are indicated.

Fig. 3.

HPLC trace showing CYP6M2 mediated DDT metabolism. HPLC chromatogram of reaction products of DDT incubated with CYP6M2 membranes in the presence of 1 mM cholate and NADPH. Retention times of suspected metabolites (DDE and dicofol) were confirmed through HPLC. In reactions containing cholate but minus NADPH, no dicofol or DDE was detectable (figure omitted for clarity).