BackgroundControl of Aedes aegypti, the mosquito vector of dengue, chikungunya and yellow fever, is a challenging task. Pyrethroid insecticides have emerged as a preferred choice for vector control but are threatened by the emergence of resistance. The present study reports a focus of pyrethroid resistance and presence of two kdr mutations—F1534C and a novel mutation T1520I, in Ae. aegypti from Delhi, India.Methodology/Principal FindingsInsecticide susceptibility status of adult-female Ae. aegypti against DDT (4%), deltamethrin (0.05%) and permethrin (0.75%) was determined using WHO's standard insecticide susceptibility kit, which revealed resistance to DDT, deltamethrin and permethrin with corrected mortalities of 35%, 72% and 76% respectively. Mosquitoes were screened for the presence of kdr mutations including those reported earlier (I1011V/M, V1016G/I, F1534C, D1794Y and S989P), which revealed the presence of F1534C and a novel mutation T1520I. Highly specific PCR-RFLP assays were developed for genotyping of these two mutations. Genotyping using allele specific PCR and new PCR-RFLP assays revealed a high frequency of F1534C (0.41–0.79) and low frequency of novel mutation T1520I (0.13). The latter was observed to be tightly linked with F1534C and possibly serve as a compensatory mutation. A positive association of F1534C mutation with DDT and deltamethrin resistance in Ae. aegypti was established. However, F1534C-kdr did not show significant protection against permethrin.Conclusions/SignificanceThe Aedes aegypti population of Delhi is resistant to DDT, deltamethrin and permethrin. Two kdr mutations, F1534C and a novel mutation T1520I, were identified in this population. This is the first report of kdr mutations being present in the Indian Ae. aegypti population. Highly specific PCR-RFLP assays were developed for discrimination of alleles at both kdr loci. A positive association of F1534C mutation with DDT and deltamethrin resistance was confirmed.

BackgroundKnockdown resistance (kdr) in insects, resulting from mutation(s) in the voltage-gated sodium channel (vgsc) gene is one of the mechanisms of resistance against DDT and pyrethroid-group of insecticides. The most common mutation(s) associated with knockdown resistance in insects, including anophelines, has been reported to be present at residue Leu1014 in the IIS6 transmembrane segment of the vgsc gene. This study reports the presence of two alternative kdr-like mutations, L1014S and L1014F, at this residue in a major malaria vector Anopheles stephensi and describes new PCR assays for their detection.MethodsPart of the vgsc (IIS4-S5 linker-to-IIS6 transmembrane segment) of An. stephensi collected from Alwar (Rajasthan, India) was PCR-amplified from genomic DNA, sequenced and analysed for the presence of deduced amino acid substitution(s).ResultsAnalysis of DNA sequences revealed the presence of two alternative non-synonymous point mutations at L1014 residue in the IIS6 transmembrane segment of vgsc, i.e., T>C mutation on the second position and A>T mutation on the third position of the codon, leading to Leu (TTA)-to-Ser (TCA) and -Phe (TTT) amino acid substitutions, respectively. Polymerase chain reaction (PCR) assays were developed for identification of each of these two point mutations. Genotyping of An. stephensi mosquitoes from Alwar by PCR assays revealed the presence of both mutations, with a high frequency of L1014S. The PCR assays developed for detection of the kdr mutations were specific as confirmed by DNA sequencing of PCR-genotyped samples.ConclusionsTwo alternative kdr-like mutations, L1014S and L1014F, were detected in An. stephensi with a high allelic frequency of L1014S. The occurrence of L1014S is being reported for the first time in An. stephensi. Two specific PCR assays were developed for detection of two kdr-like mutations in An. stephensi.

BackgroundKnockdown resistance in insects resulting from mutation(s) in the voltage gated Na+ channel (VGSC) is one of the mechanisms of resistance against DDT and pyrethroids. Recently a point mutation leading to Leu-to-Phe substitution in the VGSC at residue 1014, a most common kdr mutation in insects, was reported in Anopheles culicifacies-a major malaria vector in the Indian subcontinent. This study reports the presence of two additional amino acid substitutions in the VGSC of an An. culicifacies population from Malkangiri district of Orissa, India.MethodsAnopheles culicifacies sensu lato (s.l.) samples, collected from a population of Malkangiri district of Orissa (India), were sequenced for part of the second transmembrane segment of VGSC and analyzed for the presence of non-synonymous mutations. A new primer introduced restriction analysis-PCR (PIRA-PCR) was developed for the detection of the new mutation L1014S. The An. culicifacies population was genotyped for the presence of L1014F substitution by an amplification refractory mutation system (ARMS) and for L1014S substitutions by using a new PIRA-PCR developed in this study. The results were validated through DNA sequencing.ResultsDNA sequencing of An. culicifacies individuals collected from district Malkangiri revealed the presence of three amino acid substitutions in the IIS6 transmembrane segments of VGSC, each one resulting from a single point mutation. Two alternative point mutations, 3042A>T transversion or 3041T>C transition, were found at residue L1014 leading to Leu (TTA)-to-Phe (TTT) or -Ser (TCA) changes, respectively. A third and novel substitution, Val (GTG)-to-Leu (TTG or CTG), was identified at residue V1010 resulting from either of the two transversions–3028G>T or 3028G>C. The L1014S substitution co-existed with V1010L in all the samples analyzed irrespective of the type of point mutation associated with the latter. The PIRA-PCR strategy developed for the identification of the new mutation L1014S was found specific as evident from DNA sequencing results of respective samples. Since L1014S was found tightly linked to V1010L, no separate assay was developed for the latter mutation. Screening of population using PIRA-PCR assays for 1014S and ARMS for 1014F alleles revealed the presence of all the three amino acid substitutions in low frequency.ConclusionsThis is the first report of the presence of L1014S (homologous to the kdr-e in An. gambiae) and a novel mutation V1010L (resulting from G-to-T or -C transversions) in the VGSC of An. culicifacies in addition to the previously described mutation L1014F. The V1010L substitution was tightly linked to L1014S substitution. A new PIRA-PCR strategy was developed for the detection of L1014S mutation and the linked V1010L mutation.

Knockdown resistance (kdr) in insects resulting from mutation(s) in the voltage-gated sodium channel (VGSC) gene is one of the mechanisms of resistance against DDT and the pyrethroid group of insecticides. Earlier, we reported the presence of two classic kdr mutations, i.e., L1014F and L1014S in Anopheles stephensi Liston, a major Indian malaria vector affecting mainly urban areas. This report presents the distribution of these alleles in different An. stephensi populations. Seven populations of An. stephensi from six states of India were screened for the presence of two alternative kdr mutations L1014F and L1014S using allele-specific polymerase chain reaction assays. We recorded the presence of both kdr mutations in northern Indian populations (Alwar and Gurgaon), with the preponderance of L1014S, whereas only L1014F was present in Raipur (central India) and Chennai (southern India). None of the kdr mutations were found in Ranchi in eastern India and in Mangaluru and Mysuru in southern India. This study provides evidence for a focal pattern of distribution of kdr alleles in India.

Background Aedes aegypti is a primary vector of dengue, chikungunya and Zika infections in India. In the absence of specific drugs or safe and effective vaccines for these infections, their control relies mainly on vector control measures. The emergence of insecticide resistance in vectors, especially against pyrethroids, is a serious threat to the insecticide-based vector control programme. This study reports the presence of multiple knockdown resistance (kdr) mutations present in an Ae. aegypti population from Bengaluru (India), including a new mutation F1534L. Methods Aedes aegypti collected from Bengaluru were subjected to insecticide susceptibility tests with DDT, deltamethrin and permethrin. The DNA sequencing of partial domain II, III and IV of the voltage-gated sodium channel (VGSC) was performed to screen kdr mutations present in the population and PCR-based assays were developed for their detection. Genotyping of kdr mutations was done using PCR-based assays, allelic frequencies were determined, and tests of genetic association of kdr mutations with the insecticide resistance phenotype were performed. Results The Ae. aegypti population was resistant to DDT, deltamethrin and permethrin. The DNA sequencing of the VGSC revealed the presence of four kdr mutations, i.e. S989P and V1016G in domain II and two alternative kdr mutations F1534C and F1534L in domain III. Allele-specific PCR assays (ASPCR) were developed for the detection of kdr mutations S989P and V1016G and an existing PCR-RFLP based strategy was modified for the genotyping of all three known kdr mutations in domain III (F1534L, F1534C and T1520I). Genotyping of Ae. aegypti samples revealed a moderate frequency of S989P/V1016G (18.27%) and F1534L (17.48%), a relatively high frequency of F1534C (50.61%) and absence of T1520I in the population. Mutations S989P and V1016G were in complete linkage disequilibrium in this population while they were in linkage equilibrium with kdr mutations F1534C and F1534L. The alleles F1534C and F1534L are genetically associated with permethrin resistance. Conclusions A new kdr mutation, F1534L, was found in an Ae. aegypti population from Bengaluru (India), co-occurring with the other three mutations S989P, V1016G and F1534C. The findings of a new mutation have implications for insecticide resistance management.