287 - Whole transcriptome sequencing reveals distinct insecticide resistance mechanisms across the lifespan of the major malaria vector Anopheles arabiensis

Abstract: Insecticide resistance among Anopheles (An.) malaria vector species is now widespread. To evaluate the spread and intensity of resistance, World Health Organization guidelines mandate that mosquitoes should be phenotypically tested at 3-to-5 days old, and these samples are used in downstream molecular assays to identify underlying insecticide resistance mechanisms. However, the predominant molecular and metabolic mechanisms driving insecticide resistance in young Anopheles vector populations cannot be assumed to be analogous to those in older, malaria transmitting mosquitoes, which are the most epidemiologically important cohort. Differential gene expression in 3-, 6- and 11-day old wild-caught permethrin resistant and unexposed An. arabiensis from Mwagagala, Tanzania was assessed using whole transcriptome sequencing, compared to susceptible colony strain. We identified significant differences in expression of genes encoding detoxification enzymes, cuticular proteins, salivary glands, and gustatory and odorant receptors. In 3-day old mosquitoes, the cytochrome P450 CYP6P3, which metabolises permethrin and deltamethrin in vitro, was expressed over 100 times higher in resistant-, and almost 50 times higher in unexposed-mosquitoes, compared with the susceptible colony strain. Other notable detoxification enzymes included CYP6M2, CYP6Z3, CYP4H19, CYP6Z2 and CYP4H17, the choline-esterase COEJHE2E, and glutathione-S-transferase GSTD7. Expression of these detoxification enzymes was often highest in 3-day old mosquitoes, and declined over time, but in some cases remained significant by 11-days. CYP6Z3 was consistently upregulated across all age groups, while CYP4H17 only displayed increased overexpression in 11-day old An. arabiensis; this gene may represent a putative marker of ageing in An. arabiensis, with the potential to be used for transcriptional age grading. Most cuticular resistance genes were only upregulated in 3-day old mosquitoes, with only CPR86 increasing in expression by 11-days. Dynamic transcriptomic profiles of permethrin resistant An. arabiensis of different ages indicates that insecticide resistance is driven by distinct molecular mechanisms across the mosquito lifespan.