Uncovering pyrethroid resistance mechanisms in Anopheles punctulatus group mosquitoes: A novel insight from Keerom, Papua, Indonesia

Background and Aim: Malaria control programs in Indonesia heavily rely on insecticide-based interventions such as long-lasting insecticidal nets (LLINs) and indoor residual spraying (IRS). However, the effectiveness of these strategies is increasingly compromised by the emergence of insecticide resistance, particularly in high-transmission regions like Papua Province. The mechanisms underlying pyrethroid resistance in local vector populations remain inadequately characterized. This study aimed to assess the permethrin resistance status and characterize the frequency of L1014F/S knockdown resistance (kdr) alleles in Anopheles punctulatus group mosquitoes in Keerom, Papua, to inform region-specific malaria vector control strategies. Materials and Methods: An entomological and molecular investigation was conducted in five villages across three districts of Keerom Regency. Larval and adult Anopheles mosquitoes were collected through standard World Health Organization techniques. Insecticide susceptibility was evaluated using 0.75% permethrin bioassays. Molecular identification and detection of kdr mutations (L1014F/S) were performed using species-specific polymerase chain reaction and sequencing protocols. Allele frequencies were analyzed using Hardy–Weinberg equilibrium models, and statistical comparisons were made using one-way analysis of variance. Results: Among 163 mosquitoes tested, permethrin resistance was confirmed across all districts, with mortality rates ranging from 50% to 68.66%. Molecular analyses identified Anopheles koliensis, A. punctulatus, and A. peditaeniatus. High frequencies of the L1014S mutation were detected in A. koliensis (0.87) and A. punctulatus (0.66), whereas A. peditaeniatus exhibited only wild-type alleles. No L1014F mutations were observed. The high prevalence of homozygous resistant genotypes indicates intense selection pressure, potentially linked to LLIN and agricultural insecticide use. Conclusion: This study provides the first molecular evidence of widespread permethrin resistance mediated by L1014S mutations in the A. punctulatus group in Papua. These findings underscore the urgent need for enhanced resistance monitoring and the integration of alternative insecticides and non-chemical vector control methods to sustain malaria control efforts in the region. Keywords: Anopheles punctulatus, knockdown resistance, L1014S, malaria vector control, Papua, pyrethroid resistance.