Functional genetic validation of key genes conferring insecticide resistance in the major African malaria vector, Anopheles gambiae

Abstract

Resistance in Anopheles gambiae to members of all 4 major classes (pyrethroids, carbamates, organochlorines, and organophosphates) of public health insecticides limits effective control of malaria transmission in Africa. Increase in expression of detoxifying enzymes has been associated with insecticide resistance, but their direct functional validation in An. gambiae is still lacking. Here, we perform transgenic analysis using the GAL4/UAS system to examine insecticide resistance phenotypes conferred by increased expression of the 3 genes-Cyp6m2, Cyp6p3, and Gste2-most often found up-regulated in resistant An. gambiae We report evidence in An. gambiae that organophosphate and organochlorine resistance is conferred by overexpression of GSTE2 in a broad tissue profile. Pyrethroid and carbamate resistance is bestowed by similar Cyp6p3 overexpression, and Cyp6m2 confers only pyrethroid resistance when overexpressed in the same tissues. Conversely, such Cyp6m2 overexpression increases susceptibility to the organophosphate malathion, presumably due to conversion to the more toxic metabolite, malaoxon. No resistant phenotypes are conferred when either Cyp6 gene overexpression is restricted to the midgut or oenocytes, indicating that neither tissue is involved in insecticide resistance mediated by the candidate P450s examined. Validation of genes conferring resistance provides markers to guide control strategies, and the observed negative cross-resistance due to Cyp6m2 gives credence to proposed dual-insecticide strategies to overcome pyrethroid resistance. These transgenic An. gambiae-resistant lines are being used to test the "resistance-breaking" efficacy of active compounds early in their development.

Keywords: GAL4/UAS; cytochromes P450; functional analysis; glutathione-S-transferase.

Fig. 1.

Multitissue Cyp6 gene up-regulation affects sensitivity to 2 pyrethroids and a carbamate insecticide. (A) Relative transcription levels of Cyp6m2 (m2+) and Cyp6p3 (p3+) in adult females where expression is driven by the Ubi-A10 driver compared with GAL4/+ controls. Bars represent SD (n = 3). Unpaired t test. **P < 0.01; ***P < 0.001. (B) Expression of CYP6M2 and α-tubulin in adult females from Ubi-A10 × UAS-m2 crosses with respective Ubi-A10/+ and +/UAS-m2 controls. Protein extract from the equivalent of 1/10 of a whole female mosquito was loaded in each lane. (C) Sensitivity to insecticides of GAL4/UAS (+) females overexpressing Cyp6m2 or Cyp6p3 ubiquitously under the control of the Ubi-A10 driver compared with GAL4/+ controls (−) measured by WHO tube bioassay. Bars represent SD (n = 4 to 6) (SI Appendix, Table S2). The dotted line marks the WHO 90% mortality threshold for defining resistance. Welch’s t test with P < 0.01 significance cutoff. **P < 0.01; ***P < 0.001.

Fig. 2.

Multitissue Cyp6 gene up-regulation increases sensitivity to the OP insecticide malathion (25-min exposure). Sensitivity to malathion of females overexpressing Cyp6m2 (m2+) or Cyp6p3 (p3+) ubiquitously under the control of the Ubi-A10 driver compared with respective GAL4/+ controls (m2−, p3−) measure by a modified WHO tube bioassay representing mortality rates after 25 min of exposure and 24-h recovery. Bars represent SD (n = 4) (SI Appendix, Table S2). Welch’s t test with P < 0.01 significance cutoff. ***P < 0.001.

Fig. 3.

Multitissue overexpression of GSTE2 affects sensitivity to an organochlorine and an OP insecticide. Sensitivity to insecticides of adult female mosquitoes overexpressing Gste2 (e2+) ubiquitously under the control of the Ubi-A10 driver compared with Ubi-A10 controls (e2−) measured by WHO tube bioassay. Bars represent SD (n = 2 to 6) (SI Appendix, Table S2). The dotted line marks the WHO 90% mortality threshold for defining resistance. Welch’s t test with P < 0.01 significance cutoff. ***P < 0.001.

Fig. 4.

Cyp6 gene up-regulation in the mosquito midgut or oenocytes does not affect sensitivity to insecticides. (A and B) Relative transcription levels of Cyp6m2 (m2+) and Cyp6p3 (p3+) in dissected midguts (A) and abdomens (B) of GAL4/UAS female mosquitoes compared with the equivalent body parts in GAL4/+ controls. Carcass is whole body without the relevant dissected part. Bars represent SD (n = 3). Unpaired t test. *P < 0.05; **P < 0.01; ***P < 0.001. (C and D) Expression of CYP6M2 and α-tubulin in females from the GAL4-mid × UAS-m2 (C) and GAL4-oeno × UAS-m2 (D) crosses. Abd, abdomen cuticle; Car, protein extract from 1/3 of a single female carcass–whole body without the midgut (C) or the abdomen (D); Mid, 2 dissected midguts; Wh, protein extract from 1/3 of a single whole female. +, GAL4/UAS-m2; −, GAL4/+. (E and F) Sensitivity to insecticides of GAL4/UAS females overexpressing (+) Cyp6m2 or Cyp6p3 in the midgut (E) or in the oenocytes (F) compared with GAL4/+ controls (−) measured by WHO tube bioassay. Bars represent SD (n = 2 to 3) (SI Appendix, Table S2). Dotted lines mark the WHO 90% mortality threshold for defining resistance. Welch’s t test with P < 0.01 significance cutoff.