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

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 <i>Anopheles funestus</i> across Africa including <i>GSTe1</i> [Cameroon (fold change, FC: 2.54), Ghana (4.20), Malawi (2.51)], <i>GSTe2</i> [Cameroon (4.47), Ghana (7.52), Malawi (2.13)], <i>GSTe3</i> [Cameroon (2.49), Uganda (2.60)], <i>GSTe4</i> in Ghana (3.47), <i>GSTe5</i> [Ghana (2.94), Malawi (2.26)], <i>GSTe6</i> [Cameroun (3.0), Ghana (3.11), Malawi (3.07), Uganda (3.78)] and <i>GSTe7</i> (2.39) in Ghana. Validation of <i>GSTe</i> 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 <i>GSTe</i> genes are playing a major role in resistance to pyrethroids (permethrin and deltamethrin) and DDT in <i>An. funestus</i>, with a significant recovery of susceptibility observed when <i>GSTe2</i>, <i>3</i>, <i>4</i>, <i>5</i> and <i>GSTe6</i> were silenced. These findings established that <i>GSTe3</i>, <i>4</i>, <i>5</i> and <i>6</i> 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 <i>119F-GSTe2</i> mutation. This study highlights the role of <i>GSTe</i>s in the development of resistance to insecticides in malaria vectors and calls for actions to mitigate this resistance.