Insecticide resistance status of populations Anopheles gambiae s.l. and Anopheles stephensi to Existing and Novel Insecticides in Ethiopia

Background Malaria control in Ethiopia relies mainly on case management with ACT and vector control interventions, notably long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). Anopheles arabiensis is the predominant vector, while the invasive An. stephensi is rapidly spreading across multiple regions, intensifying challenges for malaria elimination. Insecticide resistance further complicates control efforts. Methods This study assessed the susceptibility of An. gambiae s.l. and An. stephensi to commonly used and new public health insecticides at two sites: Awash Sabhat Kilo, where An. stephensi is established, and an area dominated by An. gambiae s.l. WHO tube bioassays tested pirimiphos-methyl (0.25%), propoxur (0.1%), bendiocarb (0.1%), deltamethrin (0.25%), alpha-cypermethrin (0.05%), and clothianidin (2%). CDC bottle bioassays examined susceptibility to pirimiphos-methyl, deltamethrin, chlorfenapyr, clothianidin, and broflanilide. Molecular identification and allele-specific PCR targeted resistant alleles, particularly mutations in the voltage-gated sodium channel (VGSC) gene. Data were analyzed with SPSS v20. Results Results showed An. gambiae s.l. populations were susceptible to propoxur and pirimiphos-methyl but resistant to bendiocarb, deltamethrin, alpha-cypermethrin, and clothianidin. An. stephensi displayed broader resistance, including to pirimiphos-methyl, propoxur, bendiocarb, deltamethrin, alpha-cypermethrin, and clothianidin. Both species remained fully susceptible to chlorfenapyr, clothianidin (at higher doses), and broflanilide across tested concentrations. Resistance to deltamethrin and pirimiphos-methyl was dose-dependent. Molecular identification confirmed An. arabiensis as the major species within the An. gambiae complex. Knockdown resistance ( kdr ) mutations were detected in An. arabiensis with a genotypic frequency of 22.8%. Conclusion These findings highlight the serious threat insecticide resistance poses to Ethiopia’s malaria control efforts. The persistence of resistance in both An. arabiensis and An. stephensi raises concerns about the long-term sustainability of insecticide-based strategies and underscores the need for alternative or integrated approaches.