Abstract
BACKGROUND: The swift expansion of the invasive malaria vector Anopheles stephensi throughout Africa presents a major challenge to malaria control initiatives. Unlike the native African vectors, An. stephensi thrives in urban settings and has developed resistance to multiple classes of insecticides, including pyrethroids, organophosphates, and carbamates. METHODS: Insecticide susceptibility tests were performed on field-collected An. stephensi mosquitoes from Awash Sebac Kilo, Ethiopia, to assess insecticide resistance levels. Illumina RNA-seq analysis was then employed to compare the transcriptomes of field-resistant populations and susceptible laboratory strains (STE2). RESULTS: An. stephensi populations exhibited high levels of resistance to both deltamethrin (mortality, 39.4 ± 6.0%) and permethrin (mortality, 59.3 ± 26.3%) in WHO tube bioassays. RNA-seq analysis revealed that both field-resistant and field-unexposed populations exhibited increased expressions of genes associated with pyrethroid resistance, including esterases, P450s, and GSTs, compared to the susceptible STE2 strain. Notably, esterase E4 and venom carboxylesterase-6 were significantly overexpressed, up to 70-fold, compared to the laboratory strain. Functional enrichment analysis revealed a significant overrepresentation of genes associated with catalytic activity under molecular functions and metabolic process under biological process. Using weighted gene co-expression network analysis (WGCNA), we identified two co-expression modules (green and blue) that included 48 genes strongly linked to pyrethroid insecticide resistance. A co-expression network was subsequently built based on the weight values within these modules. CONCLUSIONS: This study highlights the role of esterases in the pyrethroid resistance of an An. stephensi population. The identification of candidate genes associated with insecticide resistance will facilitate the development of rapid diagnostic tools to monitor resistance trends.