CYP6 P450 Enzymes and ACE-1 Duplication Produce Extreme and Multiple Insecticide Resistance in the Malaria Mosquito Anopheles gambiae

Abstract

Malaria control relies heavily on pyrethroid insecticides, to which susceptibility is declining in Anopheles mosquitoes. To combat pyrethroid resistance, application of alternative insecticides is advocated for indoor residual spraying (IRS), and carbamates are increasingly important. Emergence of a very strong carbamate resistance phenotype in Anopheles gambiae from Tiassalé, Côte d'Ivoire, West Africa, is therefore a potentially major operational challenge, particularly because these malaria vectors now exhibit resistance to multiple insecticide classes. We investigated the genetic basis of resistance to the most commonly-applied carbamate, bendiocarb, in An. gambiae from Tiassalé. Geographically-replicated whole genome microarray experiments identified elevated P450 enzyme expression as associated with bendiocarb resistance, most notably genes from the CYP6 subfamily. P450s were further implicated in resistance phenotypes by induction of significantly elevated mortality to bendiocarb by the synergist piperonyl butoxide (PBO), which also enhanced the action of pyrethroids and an organophosphate. CYP6P3 and especially CYP6M2 produced bendiocarb resistance via transgenic expression in Drosophila in addition to pyrethroid resistance for both genes, and DDT resistance for CYP6M2 expression. CYP6M2 can thus cause resistance to three distinct classes of insecticide although the biochemical mechanism for carbamates is unclear because, in contrast to CYP6P3, recombinant CYP6M2 did not metabolise bendiocarb in vitro. Strongly bendiocarb resistant mosquitoes also displayed elevated expression of the acetylcholinesterase ACE-1 gene, arising at least in part from gene duplication, which confers a survival advantage to carriers of additional copies of resistant ACE-1 G119S alleles. Our results are alarming for vector-based malaria control. Extreme carbamate resistance in Tiassalé An. gambiae results from coupling of over-expressed target site allelic variants with heightened CYP6 P450 expression, which also provides resistance across contrasting insecticides. Mosquito populations displaying such a diverse basis of extreme and cross-resistance are likely to be unresponsive to standard insecticide resistance management practices.

Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing

“…Afterward, the development of mosquito DNA microarrays allowed screening for resistance genes based on their differential transcription, leading to the identification of detoxification genes conferring resistance (David et al 2005Strode et al 2008;Vontas et al 2010;Edi et al 2014). However, these screenings focused on differential transcription while genomic changes such as CNVs and polymorphisms were neglected.…”

“…A similar role was suggested for the duplication of the acetylcholinesterase gene (carrying the Ace1 mutation) in the resistance of An. gambiae to carbamates (Edi et al 2014). Although such a mechanism is likely contributing to resistance in mosquitoes, no amplification of genes encoding proteins targeted by insecticides were detected in our study.…”

Venom allergen 5is Associated With Deltamethrin Resistance inCulex pipiens pallens(Diptera: Culicidae)

“…many putative genes thought to be involved in pyrethroid resistance . However, by far, the majority of pyrethroid resistance-associated genes belong to the CYP6 family of the CYP3 clan (Edi et al 2014), and the CYP4 family of the CYP4 clan (Reid et al 2014). In contrast, little is currently known about the relationship between the CYP2 clan and insecticide resistance.…”

“…Metabolic resistance occurs via increases in either protein levels or the activity of detoxification enzymes that resist insecticides. For example, esterases (Wu et al 2011), glutathione S-transferases (Lumjuan et al 2011), and cytochrome P450 (CYP or P450; Edi et al 2014) have all been demonstrated to be associated with metabolic resistance. In addition, insecticides induce physiological changes in mosquitoes such as a thickening of the epidermis, which subsequently lowers their permeability to insecticides (Wood et al 2010).…”

RNAi Trigger Delivery into <em>Anopheles gambiae</em> Pupae

“…Although the use of insecticides since the 1960s has proven to be extremely successful, the rise of insecticide resistance has driven a need for development of novel insecticides and alternative vector control strategies [4][5][6][7] . During 2010, a total of 49 of 63 countries reporting to the WHO indicated the occurrence of insecticide resistance in malaria vectors 1 .…”