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 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.

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

“…Other members of this epsilon class such as GSTe4 and GSTe5 have been shown to confer pyrethroid resistance either in An. funestus ( Kouamo et al 2021 ) and An. gambiae ( Wilding et al 2015 ).…”

Cytochrome P450 and Glutathione S-Transferase Confer Metabolic Resistance to SYP-14288 and Multi-Drug Resistance in Rhizoctonia solani

“…GSTs are best known for catalyzing the conjugation of reduced glutathione (GS – ) to substrates containing an electrophilic center, hereby increasing the substrate’s water solubility and, thus, facilitating its excretion from the cell ( Oakley, 2011 ). They may participate in chemical pesticide detoxification either by direct metabolism by dehydrochlorination of organochlorines, O-dealkylation/O-dearylation, and glutathione conjugation of pesticides or by passive binding via sequestration ( Gonzalez et al, 2018 ; Georgakis et al, 2020 ; Kouamo et al, 2021 ). Over-expression of the GST gene AG1IA_07383 was observed in resistant mutant 19-7 of R. solani , and heterologous expression of the gene confirmed its contribution in R. solani resistance to SYP-14288.…”

Gene Conversion Explains Elevated Diversity in the Immunity Modulating APL1 Gene of the Malaria Vector Anopheles funestus

“…stephensi, which encodes a single copy of APL1 . In future experiments, the mortality effect of silencing APL1 through RNAi (see [ 71 ] for similar) in response to pathogen challenge would help establish sub-functionalization at this locus. The combinatorial silencing of paralogues would also help define which paralogues act functionally or as wells of genetic diversity alone.…”