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.

Molecular forms of Anopheles subpictus and Anopheles sundaicus in the Indian subcontinent.

BACKGROUND: Anopheles subpictus and Anopheles sundaicus are closely related species, each comprising several sibling species. Ambiguities exist in the classification of these two nominal species and the specific status of members of these species complexes. Identifying fixed molecular forms and mapping their spatial distribution will help in resolving the taxonomic ambiguities and understanding their relative epidemiological significance. METHODS: DNA sequencing of Internal Transcribed Spacer-2 (ITS2), 28S-rDNA (D1-to-D3 domains) and cytochrome oxidase-II (COII) of morphologically identified specimens of two nominal species, An. subpictus sensu lato (s.l.) and An. sundaicus s.l., collected from the Indian subcontinent, was performed and subjected to genetic distance and molecular phylogenetic analyses. RESULTS: Molecular characterization of mosquitoes for rDNA revealed the presence of two molecular forms of An. sundaicus s.l. and three molecular forms of An. subpictus s.l. (provisionally designated as Form A, B and C) in the Indian subcontinent. Phylogenetic analyses revealed two distinct clades: (i) subpictus clade, with a single molecular form of An. subpictus (Form A) prevalent in mainland India and Sri Lanka, and (ii) sundaicus clade, comprising of members of Sundaicus Complex, two molecular forms of An. subpictus s.l. (Form B and C), prevalent in coastal areas or islands in Indian subcontinent, and molecular forms of An. subpictus s.l. reported from Thailand and Indonesia. Based on the number of float-ridges on eggs, all An. subpictus molecular Form B were classified as Species B whereas majority (80%) of the molecular Form A were classified as sibling species C. Fixed intragenomic sequence variation in ITS2 with the presence of two haplotypes was found in molecular Form A throughout its distribution. CONCLUSION: A total of three molecular forms of An. subpictus s.l. and two molecular forms of An. sundaicus s.l. were recorded in the Indian subcontinent. Phylogenetically, two forms of An. subpictus s.l. (Form B and C) prevalent in coastal areas or islands in the Indian subcontinent and molecular forms reported from Southeast Asia are members of Sundaicus Complex. Molecular Form A of An. subpictus is distantly related to all other forms and deserve a distinct specific status.