Genome-Wide Transcriptional Analysis and Functional Validation Linked a Cluster of Epsilon Glutathione S-Transferases with Insecticide Resistance in the Major Malaria Vector Anopheles funestus across Africa.

Mersimine F M Kouamo,Magellan Tchouakui,Jack Hearn,Sulaiman S Ibrahim,Terence Ebai,Thaddée Boudjeko,Jacob M Riveron,Helen Irving,Murielle J Wondji,Michael Kusimo,Fabrice F Boyom,Charles S Wondji,Williams Tchapga

doi:10.3390/genes12040561

Abstract

Resistance is threatening the effectiveness of insecticide-based interventions in use for malaria control. Pinpointing genes associated with resistance is crucial for evidence-based resistance management targeting the major malaria vectors. Here, a combination of RNA-seq based genome-wide transcriptional analysis and RNA-silencing in vivo functional validation were used to identify key insecticide resistance genes associated with DDT and DDT/permethrin cross-resistance across Africa. A cluster of glutathione-S-transferase from epsilon group were found to be overexpressed in resistant populations of Anopheles funestus across Africa including GSTe1 [Cameroon (fold change, FC: 2.54), Ghana (4.20), Malawi (2.51)], GSTe2 [Cameroon (4.47), Ghana (7.52), Malawi (2.13)], GSTe3 [Cameroon (2.49), Uganda (2.60)], GSTe4 in Ghana (3.47), GSTe5 [Ghana (2.94), Malawi (2.26)], GSTe6 [Cameroun (3.0), Ghana (3.11), Malawi (3.07), Uganda (3.78)] and GSTe7 (2.39) in Ghana. Validation of GSTe genes expression profiles by qPCR confirmed that the genes are differentially expressed across Africa with a greater overexpression in DDT-resistant mosquitoes. RNAi-based knock-down analyses supported that five GSTe genes are playing a major role in resistance to pyrethroids (permethrin and deltamethrin) and DDT in An. funestus, with a significant recovery of susceptibility observed when GSTe2, 3, 4, 5 and GSTe6 were silenced. These findings established that GSTe3, 4, 5 and 6 contribute to DDT resistance and should be further characterized to identify their specific genetic variants, to help design DNA-based diagnostic assays, as previously done for the 119F-GSTe2 mutation. This study highlights the role of GSTes in the development of resistance to insecticides in malaria vectors and calls for actions to mitigate this resistance.

Highlights

Malaria is the deadliest vector-borne disease, killing more than 400,000 people every year [1]
In addition to conferring pyrethroid/DDT resistance, it was shown that the L119F-GSTe2-mediated metabolic resistance to pyrethroids/DDT is associated with negative effects on some life-history traits of field populations of An. funestus, supporting that insecticide resistance is associated with a fitness cost [18]
The results have identified genes associated with DDT resistance in the major malaria vector An. funestus Africa-wide

Summary

Introduction

Malaria is the deadliest vector-borne disease, killing more than 400,000 people every year [1]. Vector control interventions through the use of long-lasting insecticide nets and the implementation of indoor residual spray have led to a significant reduction in malaria incidence, between 2000 and 2015 [2] This gain is under threat, as the most recent WHO World Malaria Report revealed there has been increase in annual case numbers since 2016. Several studies, including genome-wide transcriptional analyses using microarray/qPCR and functional validation have linked GST with resistance in the major malaria vectors [8,9,10] Most of these studies have concentrated on GSTe2, neglecting the other GST epsilon genes, even though they have been shown to consistently be overexpressed [11,12,13,14,15]. An experimental hut study, using the same marker in An. funestus population from Mibellon (Cameroon) had confirmed that presence of the L119F-GSTe2 was associated with resistance to DDT and pyrethroids [19] and was reducing the efficacy of bed nets [20]

Methods

Results

Discussion

Conclusion