Abstract
BackgroundMetabolic resistance of the major malaria vector Anopheles gambiae (s.l.) to insecticides is operationally significant, particularly in combination with target site resistance. However, detection of metabolic resistance is not trivial and relies on laborious bioassays, unspecific biochemical methods, or sophisticated and expensive molecular approaches using transcriptomics.MethodsRapid one-step multiplex TaqMan-probe based RT-qPCR assays were developed and optimised to measure the expression levels of genes associated with metabolic insecticide resistance in An. gambiae (s.l.). Primers and probes were designed to target the mRNA of cytochrome P450-dependent monooxygenases CYP6P3, CYP6M2, CYP9K1, CYP6P4 and CYP6Z1, and the glutathione-S-transferase GSTE2. The novel assays were validated versus gold standard methods with a range of phenotyped mosquito specimens. The assays were also tested directly on lysates of RNAlater®-preserved mosquitoes without an RNA extraction step.ResultsThe novel assays are efficient (reaction efficiencies = 95–109%), sensitive (covering a > 10.0 Ct range with R2 values > 0.99), specific (TaqMan chemistry), reproducible (%CV = 4.46–12.07%), as well as readily expandable to capture additional loci as they evolve or to cover additional species. The assays were successfully validated in terms of expression levels against standard two-step singleplex qPCR assays (overall % difference = -17.6%, 95% CI = -38.7–3.43%) and microarrays, using laboratory strains and field-caught samples. The assays can also be applied directly on lysates of mosquito specimens, without RNA extraction or DNase treatment.ConclusionsThe novel multiplex assays for monitoring the levels of major detoxification genes and metabolic resistance in An. gambiae (s.l.) are simple to perform, robust and rapid. They may complement current diagnostic assays to provide evidence-based and operationally relevant information for insecticide resistance management.
Highlights
Metabolic resistance of the major malaria vector Anopheles gambiae (s.l.) to insecticides is operationally significant, in combination with target site resistance
We developed a simple, robust, rapid and cost-effective method for determining the expression levels of major detoxification genes that have been associated with metabolic resistance in the malaria vector An. gambiae
The assay is based on specific TaqMan probes and RT, and it can reliably determine the expression levels of the pyrethroid metabolisers CYP6P3, CYP6M2, CYP9K1, CYP6P4, CYP6Z1 and GSTE2, which have been functionally implicated in metabolic pyrethroid resistance in An. gambiae
Summary
Metabolic resistance of the major malaria vector Anopheles gambiae (s.l.) to insecticides is operationally significant, in combination with target site resistance. In the absence of DNA markers associated with overexpression of detoxification genes for An. gambiae (s.l.), detection methods for metabolic resistance are restricted to either phenotypic bioassays with synergists or biochemical assays. Synergist-insecticide bioassays and biochemical assays are currently recommended follow-up techniques to determine the resistance mechanisms in resistant mosquito populations [17]. These methods have been useful to associate elevated levels of mixed function oxidases with pyrethroid resistance in the field, for example in An. gambiae from Kenya [18] and in An. funestus from southern Africa [19] but they could not specify which P450s were overexpressed. In an attempt to have more specific assays available, chromo- and fluorogenic substrates with a higher preference for insecticide “metabolisers” have been identified or synthesised in some cases [20, 21], with relatively restricted applicability because of the limited availability of substrates and/or complexity of biochemical reactions
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