Kaur, T.; Kushwah, R. S.; Pradhan, S.; Das, M. K.; Kona, M. P.; Anushrita, ; Weetman, D.; Dixit, R.; Singh, O. P.

Background Knockdown resistance (kdr) mutations in the voltage-gated sodium channel (VGSC) gene are a key mechanism of insecticide resistance in mosquitoes. In Asian Aedes aegypti populations two main VGSC haplogroups with kdr mutations have been identified: one carrying the F1534C mutation and another with V1016G and/or S989P mutations. Functional studies have demonstrated that these three mutations on a single haplotype confer up to a 1100-fold increase in pyrethroid resistance, underscoring the need to monitor these triple mutations within distinct populations. This study investigates the prevalence of kdr mutations in Indian populations and explores the linkage association between these mutations and two distinct conserved types of introns located between exons 20 and 21. Methods Ae. aegypti specimens collected from eight different locations were genotyped for kdr alleles and intron (between exons 20 and 21) haplotypes using PCR-based assays. Representative samples underwent DNA sequencing of VGSC regions. Results Five kdr mutations namely S989P, V1016G, T1520I, F1534C, and F1534L, were identified, each exhibiting varying distribution and frequencies across different geographical regions. Two distinct and stably-diverged intron haplotypes, designated as intron-A and intron-B, were identified between exons 20 and 21. Seven haplotypes, including two wild-type variants, were observed among Indian populations. The kdr-bearing haplotypes can be classified into three distinct haplogroups: haplogroup G (V1016G with/or without S989P and with intron-A), haplogroup L (F1534L and intron-A), and haplogroup C (F1534C with/or without T1520I and with intron-B). Importantly, no evidence of recombination within Indian populations was detected among these three haplogroups. Conclusions Five kdr mutations were identified in the VGSC of Indian Ae. aegypti populations, each showing a definitive linkage with one of the two types of intron haplotypes. The lack of recombination among haplogroups bearing 1016G with 989P, 1534C and 1534L mutations suggests that the most potent insecticide resistance haplotype, bearing the triple kdr mutation, is currently absent. This finding has significant operational implications, as it may indicate that current vector control measures remain effective against these populations, potentially delaying the emergence of highly resistant phenotypes.