Background
Anopheles culicifacies s.l. is the most important malaria vector in the Indian subcontinent, affecting mainly rural areas [
1]. This vector has developed widespread resistance against all the previously used insecticides such as DDT, dieldrin and malathion [
2] and is developing resistance to pyrethroids [
3]--the preferred group of insecticides for indoor residual spraying (IRS) and the only insecticide-class recommended for the impregnation of bed nets due to their relatively low mammalian toxicity and rapid knockdown effect on insects [
4]. DDT and pyrethroids are neurotoxins which act on the voltage gated Na
+ channel (VGSC) leading to paralysis (knockdown) and eventual death of the insect. Knockdown resistance (
kdr) due to reduced target site sensitivity is one of the mechanisms of resistance against these insecticides resulting from mutation(s) in the VGSC. The most common mutation known to be associated with knockdown resistance in insects is found at residue 1014 leading to Leu-to-Phe mutation [
5] commonly referred to as
kdr mutation. In
Anopheles gambiae, two alternative
kdr mutations L1014F [
6] and L1014S [
7] have been reported associated with knockdown resistance, which are referred to as
West African kdr (
kdr-w) and
East African kdr (
kdr-e), respectively. A variant mutation L1014C has been reported in
Anopheles sinensis[
8]. Recently a
kdr-like mutation L1014F has been reported in the Indian malaria vector
An. culicifacies from Surat district of Gujarat, India [
9], which is resistant to both DDT and pyrethroids [
3]. This study reports the presence of two additional amino acid substitutions present in the VGSC of an
An. culicifacies population from Malkangiri district of Orissa, India, one of which is homologous to
kdr-e of
An. gambiae (L1014S) and the other a novel amino acid substitution V1010L resulting from two alternative point mutations.
Results
DNA sequence analysis
A total of 20 samples were sequenced for IIS4-S5 linker-to-IIS5 segments of VGSC using PCR product amplified with PCR-I, where no non-synonymous mutation is recorded.
DNA sequencing of a total of 111 specimens of
An. culicifacies s.l. for IIS6 segment of VGSC (amplified with PCR-II) was successful and revealed the presence of two alternative non-synonymous mutations at residue L1014 leading to amino acid substitutions Leu (TTA)-to-Phe (TTT) or -Ser (TCA) resulting from 3042A>T transversion or 3041T>C transition, respectively, and a novel mutation at residue V1010 leading to Val (GTG)-to-Leu (TTG or CTG) substitution resulting from either 3028G>T or 3028G>C transversions. The numbers of various genotypes recorded after sequencing are shown in Table
1. Sequence analysis also revealed that all the 19 samples with the allele 1014S were also having allele 1010L (17 samples with 3028G>T and two samples with 3028G>C transversion). Few synonymous point mutations, all in heterozygous condition, were recorded which were: 2964A>G (n = 1), 3016C>T (n = 4), 3027A>T (n = 1) and 3045C>A (n = 1).
Table 1
Genotyping results of An. culicifacies samples by PCR-based assays and allelic association between L1014 and V1010 mutations as revealed by DNA sequencing
PCR-based assays
| | | 176 (0.786) | 26 (0.116) | 18 (0.080) | 2 (0.009) | 2 (0.009) | 0 (0.000) | 224 |
| |
V/V
| 64 | 26 | 0 | 0 | 2 | 0 | 92 |
| |
V/L
| 0 | 0 | 17* | 2 | 0 | 0 | 19* |
DNA-sequencing
|
V1010
| | | | | | | | |
| |
L/L
| 0 | 0 | 0 | 0 | 0 | 0 | 0 |
| |
Total
| 64 | 26 | 17 | 2 | 2 | 0 | 111 |
Genotyping results
The results of genotyping of a total of 224 samples for 1014L, 1014F and 1014S alleles as determined by ARMS-F and PIRA-S are shown in Table
1. The allele frequencies of 1014L, 1014F and 1014S, based on PCR-based assays results, were 0.884, 0.071 and 0.045 respectively. The allele frequency of 1010L was not calculated as genotyping of this allele is based on DNA sequencing of selective samples. However, considering the fact that this allele was always found with 1014S in this study, it is presumed that the frequency of 1010L will be same as that of 1014S.
The various genotypes, as revealed by PCR-based assays, were in agreement with HWE (H
O
= 0.20536, H
E
= 0.21205,
p = 0.58537). Results of PCR-based assays were well in agreement with the DNA-sequencing results of 111 samples sequenced successfully (Table
1). One sample with failed DNA sequencing was genotyped as 1014L/S by PCR-based assays.
Allelic association of 1010L and 1014S
It was observed that one of the two alternative mutant alleles at V1010 residue, i.e., 3028G or 3028G>C (both leading to V1010L) and mutant allele 3041T>C (1014S) were found in the same individual. Cloning of seven samples with 1014S (in heterozygous condition) revealed that either of the two alternative mutant alleles 3028G>T or 3028G>C (1010L) and mutant allele 3041T>C (1014S) were located on the same haplotype. The wild allele 3028G (1010V) was found on haplotype with either 1014L (TTA) or 1014F (TTT). Linkage disequilibrium analysis using phased data derived from 79 individuals which were sequenced successfully revealed that the point mutations 3028G>T (V1010L) and 3041T>C (L1014S) are tightly linked (D' = 1.000, chi-square = 158.0, p < 0.001). One sample with 3028G>C mutation was not tested for LD, however, in this sample 3028G>C and 3041T>C were found on same haplotype.
Sibling species composition
Out of a total of 50 samples examined for ovarian polytene chromosomes, 48 were successfully genotyped for the species-specific inversions. Forty-three (90%) samples were identified as species B and five (10%) as species C. Genotyping of these samples revealed that four of the species B and one of the species C were 1014L/1014F heterozygotes. Three of the species B and two of the species C were heterozygous for 1014L/1014S. Remaining samples were homozygous 1014L. Analyses of the data with Fisher's exact test revealed absence of association of any of the two mutations with any sibling species (p > 0.05 for both the mutations).
Discussion
Knockdown resistance resulting from target site insensitivity in insects is one of the mechanisms of resistance against DDT and pyrethroids which is conferred by amino acid substitution(s) in the VGSC. In anophelines, the most commonly reported mutation conferring knockdown resistance is at residue L1014 leading to Leu-to-Phe substitution, often referred to as
kdr mutation and has been reported in several anophelines such as
An. gambiae[
6],
Anopheles arabiensis[
17],
Anopheles stephensi[
18],
Anopheles subpictus[
19],
An. sinensis[
8],
Anopheles sacharovi[
20] and
An. culicifacies[
9]. Variant mutations, Leu-to-Ser and Leu-to-Cys, at this residue have been reported in
An. gambiae[
7] and
An. sinensis[
8], respectively. In
An. culicifacies, only one point mutation (A-to-T transversion) leading to Leu-to-Phe amino-acid substitution at position 1014 was reported from Surat district of Gujarat, west India, which is resistant to both DDT and pyrethroids [
9]. This study revealed the presence of three other non-synonymous point mutations in
An. culicifacies, two at position 1010 (G-to-T or -C), each one leading to Val-to-Leu substitution, and one T-to-C transition at position 1014 leading to Leu-to-Ser substitution in a population from district Malkangiri of Orissa. The two alternative mutations L1014F and L1014S are homologous to mutations found in
An. gambiae, referred to as
kdr-w and
kdr-e, respectively. The V1010L substitution resulting from either of the two alternative point mutations is a novel mutation and was found linked with L1014S irrespective of the types of associated point mutations. Cloning experiment and LD analysis showed that these two amino acid substitutions L1014S and V1010L are found on the same haplotype and not independently.
The role of L1014F and L1014S mutations in conferring DDT/pyrethroid resistance has already been established in many insects including
An. gambiae[
7,
21‐
24]. Their role in
An. culicifacies is uncertain, however, the conserved nature of these mutations particularly that of the classic mutation L1014F in several insects [
25] suggests a similar role in other species. The role of novel mutation V1010L in knockdown resistance is unknown. However, the presence of two alternative point mutations responsible for a same amino acid substitution (V1010L) in a population indicates that this amino acid substitution has been favoured during evolution probably due to their role in protection against knockdown. Further, the fact that V1010L (3028G>T or 3028G>C) always co-existed with L1014S, suggests that the V1010L substitution may probably have complementary role in knockdown resistance in association with L1014S. Their independent role in knockdown resistance is difficult to establish in the population studied because they are found to exist together. Although limited data from one geographic region suggest the absence of recombination, screening of larger population from different geographical localities may reveal recombination and these two alleles may be found independently.
The frequency of
kdr-like mutations L1014F and L1014S (and linked V1010L) is very low in the population studied and these alleles were found mostly in heterozygous conditions with less than 1% homozygotes. The alleles were, however, well in agreement with HWE. This is contrary to a report by Hoti et al [
26] carried out in the same area, where the frequency of homozygous RR (1014F) was too high (71%) as compared to heterozygotes (4%), resulting in significant departure from HWE (
p = 0.00000, Exact-test) due to deficiency of heterozygotes. One possible reason for this departure may be genotyping error due to severe mismatch in flanking primers, which were basically designed for
An. gambiae[
9]. Changes in gene frequency over time due to changes in sibling species composition or insecticide pressure may be other possible reasons that can account for the difference in allele frequency.
The An. culicifacies population in Malkangiri consists mainly species B with low proportion of species C. Preliminary analysis based on limited numbers of samples could not establish any association of any of the kdr-like mutations with sibling species. Since the analysis presented in this study is based on a small sample size, particularly in the case of species C (n = 5), it is emphasized that larger number of samples should be analyzed to establish an association of particular kdr-like mutations with sibling species.
A new PIRA-PCR assay (PIRA-S) was developed for genotyping of L1014S mutation and the results obtained through this assay were in agreement with direct sequencing results. PIRA-PCR was preferred to ARMS for genotyping of L1014S because the ARMS assay tried for this mutation resulted in poor amplification whereas PIRA-PCR provided discrete and better yield of the amplified product. The PIRA-S is very cost effective as the restriction enzyme EcoR I selected for this PIRA-PCR is inexpensive (0.5 cent/unit). No assay was developed for the novel mutation V1010L because it always co-existed with L1014S.
The
kdr factor is reportedly recessive [
27] or incompletely recessive [
22], therefore, the effect of
kdr on phenotypic resistance can best be studied in a population where sufficient number of homozygous individuals for
kdr-like alleles are present. In this study area, the frequency of homozygous mutant alleles is extremely low (<1%) and, therefore, it will be difficult to establish the phenotypic effect of these mutations in this population. Authors are attempting to colonize
An. culicifacies having different
kdr-like mutations to establish their role in phenotypic knockdown resistance.
Acknowledgements
Authors are thankful to Dr SK Sahu of the Vector Control Research Centre, field unit at Malkangiri, for helping out in collecting mosquitoes. Authors thank Mr Uday Kumar, Mr Kanwar Singh and Mr Shri Bhagwan for their excellent technical assistance and field work. The study was partially funded by the Defence Research & Development Establishment (DRDE), Gwalior. SP is supported by University Grant Commission (UGC)-JRF fellowship.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
OPS designed the study, analysed sequences, designed PIRA-PCR strategy, performed statistical analyses and wrote the manuscript; CLD performed genotyping, SP did cloning experiments, MKD and RMB organized field work, OPA and TA contributed to the manuscript. All authors provided critical reviews of the manuscript and approved the final version.