RNAseq-based gene expression profiling of the Anopheles funestus pyrethroid-resistant strain FUMOZ highlights the predominant role of the duplicated CYP6P9a/b cytochrome P450s.

Charles S Wondji,Gareth Weedall,Jack Hearn,Murielle J Wondji,Helen Irving,J Birchler

doi:10.1093/g3journal/jkab352

Abstract

Insecticide-based interventions, notably long-lasting insecticidal nets, against mosquito vectors of malaria are currently threatened by pyrethroid resistance. Here, we contrasted RNAseq-based gene expression profiling of laboratory-resistant (FUMOZ) and susceptible (FANG) strains of the major malaria vector Anopheles funestus. Cytochrome P450 genes were the predominant over-expressed detoxification genes in FUMOZ, with high expression of the duplicated CYP6P9a (fold-change of 82.23 vs FANG) and CYP6P9b (FC 11.15). Other over-expressed P450s belonged to the same cluster of P450s corresponding to the resistance to pyrethroid 1 (rp1) quantitative trait loci (QTL) on chromosome 2R. Several Epsilon class glutathione S-transferases were also over-expressed in FUMOZ, as was the ATP-binding cassette transporter AFUN019220 (ABCA) which also exhibited between-strain alternative splicing events at exon 7. Significant differences in single-nucleotide polymorphism frequencies between strains occurred in resistance QTLs rp1 (CYP6P9a/b and CYP6AA1), rp2 on chromosome 2L (CYP6Z1, CYP6M7, and CYP6Z3), and rp3 on chromosome 3R (CYP9J5, CYP9J4, and CYP9J3). Differences were also detected in CYP4G17 and CYP4G16 genes on the X chromosome, both of which are associated with cuticular resistance in Anopheles gambiae. A close analysis of nonsynonymous diversity at the CYP6P9a/b loci revealed a drastic loss of diversity in FUMOZ with only a single polymorphism and 2 haplotypes vs 18 substitutions and 8 haplotypes in FANG. By contrast, a lowly expressed cytochrome P450 (CYP4C36) did not exhibit diversity differences between strains. We also detected the known pyrethroid resistance conferring amino acid change N384S in CYP6P9b. This study further elucidates the molecular bases of resistance in An. funestus, informing strategies to better manage widespread resistance across Africa.

Highlights

Malaria prevention still heavily relies on insecticide-based interventions including long-lasting insecticidal nets (LLINs) and indoor residual spraying
This study has revealed a strong association between the over-expression of major resistance genes and the presence of signatures of selective sweep as the duplicated P450 genes CYP6P9a and CYP6P9b are massively over-expressed in FUMOZ while exhibiting a drastically reduced diversity compared to the susceptible FANG strain
The list of upregulated genes is dominated by genes belonging to several gene families associated with insecticide resistance including detoxification genes such as cytochrome P450s, glutathione-S-transferases (GSTs), and carboxylesterases (Table 1; Supplementary Figure S1A)

Summary

Introduction

Malaria prevention still heavily relies on insecticide-based interventions including long-lasting insecticidal nets (LLINs) and indoor residual spraying. These tools have contributed massively to the reduction of malaria burden observed since 2000 (Bhatt et al 2015). Major malaria vectors have developed resistance to pyrethroids as a response to the massive scale up of LLINs (Hemingway et al 2016), and due to ongoing selection from agricultural use of insecticides (WHO 2012). It is vital to implement suitable resistance management strategies to prevent the loss of all the gains recently achieved in reducing malaria burdens (Hemingway et al 2016). Understanding the molecular mechanisms of insecticide resistance in major malaria vectors such as Anopheles funestus, a major malaria vector in sub-Saharan Africa alongside members of the Anopheles gambiae complex is, of great importance for tracking and managing this resistance

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