The transcription factor Maf-S regulates metabolic resistance to insecticides in the malaria vector Anopheles gambiae

Abstract

Background: Malaria control in Africa is dependent upon the use insecticides but intensive use of a limited number of chemicals has led to resistance in mosquito populations. Increased production of enzymes that detoxify insecticides is one of the most potent resistance mechanisms. Several metabolic enzymes have been implicated in insecticide resistance but the processes controlling their expression have remained largely elusive.

Results: Here, we show that the transcription factor Maf-S regulates expression of multiple detoxification genes, including the key insecticide metabolisers CYP6M2 and GSTD1 in the African malaria vector Anopheles gambiae. Attenuation of this transcription factor through RNAi induced knockdown reduced transcript levels of these effectors and significantly increased mortality after exposure to the pyrethroid insecticides and DDT (permethrin: 9.2% to 19.2% (p = 0.015), deltamethrin: 3.9% to 21.6% (p = 0.036) and DDT: 1% to 11.7% (p = <0.01), whilst dramatically decreasing mortality induced by the organophosphate malathion (79.6% to 8.0% (p = <0.01)). Additional genes regulated by Maf-S were also identified providing new insight into the role of this transcription factor in insects.

Conclusion: Maf-S is a key regulator of detoxification genes in Anopheles mosquitoes. Disrupting this transcription factor has opposing effects on the mosquito's response to different insecticide classes providing a mechanistic explanation to the negative cross resistance that has been reported between pyrethroids and organophosphates.

Keywords: Anopheles gambiae; Cross resistance; Insecticide resistance; Metabolic resistance; Mosquito; Transcriptional control.

Fig. 1

Maf-S-cnc-Keap1 pathway. The binding of the ubiquitin ligase Keap1 to cnc results in proteasomal degradation of cnc in the absence of oxidative stress. Under oxidative stress conditions, proteolysis is blocked and cnc is released by Keap1, which translocates into the nucleus and binds to Maf-S. The cnc-Maf-S complex then binds to antioxidant response elements (ARE) in the genome, initiating transcription. Adapted from [30]

Fig. 2

Gene expression in Maf-S knockdowns relative to GFP injected controls. qPCR of three transcripts regulated by the cnc-Maf-S pathway in Drosophila (Gnmt, Jheh1, Jheh2) and five selected from the Maf-S co correlated genes in Anopheles (CYP4H17, GSTD3, ABCA3, CYP6M2, GSTD1) was performed on Maf-S knockdown cDNA. Data are shown normalised against expression in GFP-injected controls. Significance (p ≤ 0.05), as determined by a Welch’s t-test, is indicated by an *. Each data point represents the mean of three biological replicates each comprising cDNA from 7 to 10 females, 72 h post injection (actual age of mosquitoes = 7–8 days)

Fig. 3

Insecticide bioassays on Maf-S silenced adults. An. gambiae sl female mosquitoes were injected with Maf-S or GFP dsRNA, and then exposed to insecticides using the WHO tube assay. Exposure times were 60 min to papers coated with 4% DDT, 0.75% permethrin, 0.05% deltamethrin or 0.1% bendiocarb; and 5-min to 5% malathion papers. Significance is represented by: * p ≤ 0.05, ** p ≤ 0.01 and *** p ≤ 0.001 displayed above each bar, as calculated by ANOVA with a Tukey post hoc test. Error bars represent standard error. Number of mosquitoes per test for each insecticide/condition are represented by numbers below the x-axis. Each WHO test had 20–25 female mosquitoes per tube