Molecular forms of the Indian Anopheles subpictus complex differ in their susceptibility to insecticides and the presence of knockdown resistance (kdr) mutations in the voltage-gated sodium channel

OBJECTIVES: To investigate the differential insecticide-susceptibility of two molecular forms of Anopheles subpictus complex (A and B) against DDT and pyrethroids, the occurrence of knockdown resistance (kdr) mutations in these forms, and the association of kdr mutations with insecticide resistance. METHODS: Insecticide susceptibility tests of An. subpictus s.l., collected from coastal and inland areas of mainland India, were performed against DDT, permethrin and deltamethrin using the WHO standard insecticide susceptibility test kit. The mosquitoes were characterized for molecular forms using a diagnostic PCR developed in this study. Representative samples of An. subpictus molecular forms A and B were sequenced for a genomic region encompassing the IIS4-5 linker to the IIS6 segments of the voltage-gated sodium channel to identify kdr mutations. A common PIRA-PCR was developed for identifying L1014F-kdr mutation and used for genotyping in both molecular forms of An. subpictus. RESULTS: Molecular form A of An. subpictus was resistant to all three insecticides, i.e., DDT, Permethrin and deltamethrin, whereas Form B was categorized as ‘possibly resistant’ to these insecticides. Significantly higher mortalities in WHO insecticide susceptibility tests were recorded in Form B compared to Form A in sympatric populations. Molecular characterization of the IIS4-5 linker to IIS-6 segments of the voltage-gated sodium channel revealed the presence of two alternative nucleotide transversions at L1014 residue in Form A, both leading to the same amino acid change, i.e., Leu-to-Phe; however, such mutations could not be observed in Form B. PIRA-PCR-based kdr-genotyping of field populations revealed high frequencies of L1014F-kdr mutations in Form A and the absence of this mutation in Form B. The proportion of L1014F mutation was significantly higher in resistant mosquitoes following insecticide-bioassay with DDT (p<0.0001), permethrin (p<0.001) and deltamethrin (p<0.01) as compared to their susceptible counterparts. CONCLUSIONS: Significant differences in insecticide susceptibility were found between two molecular forms of An. subpictus complex in sympatric populations. The L1014F-kdr mutation was observed in Form A only, which was found to be associated with DDT, permethrin and deltamethrin resistance.