Tropical Medicine and International Health doi:10.1111/j.1365-3156.2009.02323.

volume 14 no 9 pp 10971104 septemper 2009

Molecular analysis of knock down resistance (kdr) mutation

and distribution of kdr genotypes in a wild population of Culex
quinquefasciatus from India
Manas Sarkar1, Aparajita Borkotoki2, Indra Baruah1, Indra Kumar Bhattacharyya3 and Ravi Bihari Srivastava4

1 Medical Entomology Division, Defence Research Laboratory, Tezpur, India

2 Department of Zoology, Gauhati University, Guwahati, India
3 Cotton College, Guwahati, India
4 Directorate of Life Science, Defence Research & Development Organization, New Delhi, India

Summary objective To investigate the presence of knock down resistance (kdr) mutation, its frequency
distribution in the principal vector of bancroftian filariasis, Culex quinquefasciatus from northeastern
India, and to relate kdr genotypes with susceptibility and or resistance to DDT and deltamethrin in this
vectors.
methods Adult female mosquitoes were collected by aspiration from human dwellings in two villages,
Benganajuli and Rikamari, and two military establishments, Field Units I and II. Insecticide susceptibility
tests were performed following WHO methods with 4% DDT and 0.05% deltamethrin. Molecular
identification of kdr mutation and genotyping of kdr locus was performed by allele-specific PCR
(AS-PCR) and direct sequencing in a subset of samples.
results Mosquitoes were resistant to DDT and showed 11.941.2% mortality, whereas the knock
down bioassay for deltamethrin suggests complete susceptibility to this insecticide in all study sites
except Benganajuli. The result of AS-PCR confirmed the presence of three genotypes: susceptible (SS),
resistant (RR) and heterozygous (SR) in the population. Genotype frequencies at kdr locus for DDT-
resistant individuals conformed with the HardyWeinberg proportion, whereas DDT and deltamethrin
susceptible individuals differed significantly (P < 0.05). The efficacy of AS-PCR in detecting the correct
genotype was not encouraging.
conclusion This is the first report from India on kdr genotyping in C. quinquefasciatus, and it
confirms the occurrence of kdr allele in this vector in northeastern India. This finding has serious
implications for the filariasis control programmes in India.

keywords Culex quinquefasciatus, filariasis, kdr genotype, India, knock down resistance, pyrethroid

the agent of LF not only in India, but also throughout

Introduction
Lymphatic or bancroftian filariasis is considered as the
predominant infection in the continental Asia (Gyapong
et al. 2005). In India, the estimates in 2001 indicated that
473 million people are exposed to the risk of bancroftian
infection; 125 million live in urban and 348 million live in
rural areas. Thirty-one million people are estimated to be
harbouring microfilaria (mf) and more than 23 million suffer
from filarial disease manifestations (http://www.whoindia.
org/en/Section3/Section127_370.htm). Lymphatic filariasis
(LF) is a major impediment to socio-economic development
(estimated loss $1 billion per year) and causes immense psy-
chosocial suffering (ICMR 2002). Culex quinquefasciatus is
the main vector of the parasitic worm Wuchereria bancrofti,
continental Asia. In north eastern states of India, especially in
Assam, this species is considered to be an efficient vector of
bancroftian filariasis (Mahanta et al. 2001). The approach to
prevent transmission of filariasis and other mosquito-borne
diseases has extensively relied on the application of insecti-
cides (McCarroll & Hemingway 2002). Hence, the emer-
gence of insecticide resistance is a major concern for effective
management of LF (Ottesen et al. 1997).
In India, DDT is still the insecticide of choice for public
health programme. In army establishments, pyrethroids,
especially deltamethrin, are being used for ITNs (Joshi
et al. 2003). Pyrethroids are also being used in tea agro-
ecosystems in Assam along with malathion. There is
concern that continued and or indiscriminate use of these

2009 Blackwell Publishing Ltd 1097

Tropical Medicine and International Health
M. Sarkar et al. Molecular analysis of kdr mutation
insecticides may result in increased resistance that would
threaten the sustainability of the vector control strategy.
Several mechanisms are involved in insecticide resistance,
including reduced sensitivity of sodium channels (i.e. kdr
or kdr-like mutations) to insecticides and overproduction
of detoxifying enzymes such as esterase, mixed function
oxidases (MFOs) and glutathion-S-transferases (GSTs)
(Georghiou 1986; Roberts & Andre 1994; Nelson et al.
1996, Scott et al. 1998; Feyereisen 1999).
DDT and pyrethroids share a similar target site, the para
type voltage gated sodium channel (vgsc). It alters the normal
functions of the sodium channel, resulting in prolonged
channel opening which causes more nerve impulse transmis-
sions, leading to paralysis and death of the insect (Soderlund
& Bloomquist 1989; Narahashi 1992). A single nucleotide
polymorphism (TTA to TTT) in the S6 hydrophobic trans-
membrane segment of domain-II (IIS6 domain) of vgsc
contributed substitution of leucine to phenylalanine
(L1014F), which reduces the affinity of target site for
insecticides (OReilly et al. 2006). This resistance mechanism
was first identified in the house fly Musca domestica (Milani
1954) and was termed knock down resistance (kdr).
kdr is a well-characterized mechanism of resistance to
pyrethroid and DDT in many insect species (Williamson
et al. 1996; Dong 1997; Martine-Torres et al. 1998, 1999).
In Culex, only few instances of this mutation have so far
been reported from different parts of the world (Chandre
et al. 1998; Martine-Torres et al. 1999; McAbee et al.
2004; Xu et al. 2005; Wondji et al. 2008; Zhou et al.
2009). More than 20 mutations in insect VGSC have been
identified that are involved in reducing channel sensitivity
to insecticides or neurotoxins (Park and Taylor 1997; Liu
& Pridgeon 2002; Pridgeon et al. 2002; Soderlund &
Knipple 2003). However, the kdr mutation in mosquitoes
has attracted a great deal of attention due to the impor-
tance of pyrethroid in the control of mosquito worldwide.
But very little is known about this important insecticide
resistance mechanism in C. quinquefasciatus from India.
We investigated the kdr mutations frequency distribution
and role in insecticide resistance in C. quinquefasciatus
from northeastern India.
Materials and methods
Study site
The study was conducted in army cantonments and
surrounding villages in Assam, India. Assam is the gateway
of northeastern India and strategically the most important
state in northeastern India, having three international
boundaries. The study sites were: Benganajuli village, a
malaria-prone area where DDT is used regularly (three to
1098
volume 14 no 9 pp 10971104 septemper 2009
four times in a year) by the public health workers; two
army animal transport Field Units (I and II) mainly
surrounded by rice fields, deep forest and tea gardens (army
members have been using ITNs regularly for 45 years);
and Rikamari village, which is surrounded by forest. In
Rikamari, too, DDT has been in use for a long time. These
areas are characterized by a long rainy season from April to
August and humid climate. Global Positioning System
(GPS) coordinates of the study site are presented in
Table 1. A Geographical Information System (GIS)-based
map of the study area is presented in Figure 1, which was
created with ArcGIS 9.2 software (ESRIArcMapTM9.2).
Mosquito collection
Adult female mosquitoes were collected pre-monsoon and
post-monsoon by aspiration from human dwellings in
villages and barracks in army cantonments from 0500 to
0700 h and from 1830 to 2030 h. Mosquitoes were
identified as C. quinquefasciatus following standard diag-
nostic keys on morphological characteristic features.
Insecticide susceptibility bioassay
Insecticide susceptibility assays were performed on wild
caught adult female mosquitoes and laboratory reared
(35 days old) known susceptible strain (S-Lab) mosquitoes
to compare the susceptibility levels of the field population.
The age and number of blood feeds were unknown and
variable for wild caught females, which may slightly influence
the bioassay results. Mortality and knock down were
measured using a WHO test kit (WHO 1998). The tests were
performed with 4% DDT for 4 h and 0.05% deltamethrin for
1 h. The number of mosquitoes knocked down was recorded
at 10-min intervals after exposure to deltamethrin for up to
1 h. The mortality was recorded 24 h post-exposure. After
bioassay in the field, dead (susceptible) and alive (resistant)
mosquitoes were collected and stored separately with silica
gel and were carried to the laboratory for molecular study.
DNA isolation
All collected mosquitoes were dried at 95 C for 3 h before
DNA extraction. Genomic DNA from DDT survivors
Table 1 GPS coordinates of field sites where study was conducted
Site GPS coordinates
Benganajuli 265148.4 N 923227.6 E
Field unit-I 265147.4 N 923348.7 E
Field unit-II 265114.1 N 923516.2 E
Rikamari 265045.3 N 923533.2 E
2009 Blackwell Publishing Ltd
Tropical Medicine and International Health volume 14 no 9 pp 10971104 septemper 2009

M. Sarkar et al. Molecular analysis of kdr mutation

Study sites and its eco-environment

923130 E 92330 E 923430 E 92360 E 923730 E

265230 N 265230 N

1 2

3 26510 N
26510 N

264930 N 264930 N

923130 E 92330 E 923430 E 92360 E 923730 E

Legends
N
1. Benganajuli Rivers
Kilometres
2. Field unit I
Teagardens
1 0.5 0 1 2 3. Field unit II
4. Rikamari Settlements
Figure 1 Study sites and its eco-environ-
mental settings. Water bodies

(resistant), DDT dead (susceptible) and deltamethrin dead

(susceptible) population were extracted using the SDS
extraction and ethanol precipitation method as described by
Coen et al. (1982) with modification. The DNA samples was
air dried and suspended in 100 ll of TE buffer (10 mm Tris
Cl, pH 8.0; 1 mm EDTA, pH 8.0) and stored at 20 C.
Primer selection
Four primers (Cq1, Cq2, Cq3 and Cq4) were selected from
region II of the para-type sodium channel gene of C. pipiens,
reported by Martine-Torres et al. (1999). The primers were
renamed as above to maintain the uniformity of our own
primer numbering system. Two primers [Cq1 (Forward),
5-GTGGAACTTCACCGACTTC-3 and Cq2 (Reverse),
5-GCAAGGCTAAGAAAAGGTTAAG-3] were used to
amplify the fragment of sodium channel gene containing the
kdr mutation site. The other two primers [Cq3 (Forward),
5-CCACCGTAGTGATAGGAAATTTA-3 and Cq4 (For-
ward), 5-CCACCGTAGTGATAGGAAATTTT-3] were
allele specific primers used in genotyping of knock down
resistant (kdr) [Cq3] and knock down susceptible (kds)
[Cq4] alleles by allele-specific PCR assay (AS-PCR). These
allele specific primers (Cq3 and Cq4) differed only at the
3-OH end where A in Cq3 is replaced by T in Cq4 and the
rest of the sequences were unchanged and both could amplify
a 380 bp corresponding band.
Allele-specific PCR (AS-PCR) assay to detect knock down
resistance (kdr) mutation
We selected 120 mosquitoes (40 each from DDT resistant,
DDT and deltamethrin susceptible) for AS-PCR assay. The
selection was random after bioassay from each study site,
considering the mosquitoes as a single population, because
the study sites are relatively close to each other and we were
not sure about the distinctness of the mosquito populations.
The PCRs were performed to detect kdr mutation in a single
mosquito following the protocol of Martene-Torres et al.
(1999) with minor modifications. Two PCR reactions were
run in parallel and in each of them, the template was 10 ng of
single mosquito DNA. In one reaction, the primers Cq1, Cq2
and Cq3 were combined (10 pmol each) and in the other one
Cq3 was replaced by Cq4. The reaction mixture contained
1 PCR buffer, 200 lm dNTP mix and 2.5 U Taq Poly-
merase with a final reaction volume of 50 ll. The PCR
conditions were 5 min at 94 C for the first cycle followed by
1 min at 94 C, 2 min at 49 C and 2 min for 72 C for
29 cycles and 10 min at 72 C for the final extension. The
DNA fragments were separated by electrophoresis on 2%
agarose gels and were visualized by ethidium bromide
staining under UV light. All the chemicals used in this study
were purchased from Sigma (Sigma-Aldrich Corporation,
Bangalore, India), Roche (Roche Applied Science,
Mannheim, Germany).

2009 Blackwell Publishing Ltd 1099

Tropical Medicine and International Health volume 14 no 9 pp 10971104 septemper 2009

M. Sarkar et al. Molecular analysis of kdr mutation

Table 2 Results of insecticide resistance bioassay using diagnostic dose of DDT (4%) and deltamethrin (0.05%)

DDT (4%) Deltamethrin (0.05%)

% Mortality % Mortality
Study sites (Sample size) Mean ( SD) (Sample size) KDT50 (95% CI) KDT90 (95% CI) v2 (df)

1. Benganajuli 11.9 (160) 2.38 (1.302) 96.2 (80) 17.8 (12.0523.57) 69.5 (36.26102.83) 2.333 (4)
2. Field Unit I 17.5 (80) 3.50 (1.29) 100 (80) 10.09 (4.3415.82) 44.2 (22.0666.33) 1.235 (3)
3. Field Unit II 30.63 (160) 6.12 (2.031) 100 (80) 10.3 (4.815.89) 41.4 (18.4464.45) 0.116 (2)
4. Rikamari 41.25 (80) 8.25 (1.50) 98.7 (80) 14.5 (9.0720.01) 56.1 (32.2680.13) 1.052 (4)
S-Lab 91.2 (160) 18.25 (1.282) 100 (80) 5.1 (1.2011.54) 27.5 (12.3642.72) 0.0507 (2)

Table shows values of 50% and 90% knock down time, i.e. KDT50 and KDT90 (time in minutes), Chi-square (v2) values with degree of
freedom (df) and percentage mortality after 24 h post-exposure for Culex quinquefasciatus.
Mean value of replicates. Part of the Table has been produced in our previous study (Sarkar et al. 2009).

Amplification and sequencing of kdr flanking region Data analysis

PCR was conducted with Cq1 and Cq2 primers to amplify the
fragment of VGSC-IIS6 domain containing the kdr mutation
site. The PCR reaction contained 10 ng of DNA template and
10 pmol each forward (Cq1) and reverse (Cq2) primer along
with 1 PCR buffer, 200 lm dNTP mix and 2.5 U Taq
Polymerase with a final volume of 50 ll. Amplification was
performed with following conditions: 1 cycle at 94 C for
5 min followed by 29 cycle of PCR at 94 C for 1 min, 48 C
for 2 min and 72 C for 2 min; followed by 1 cycle at 72 C
for 10 min as the final extension.
A total of 18 PCR samples, six numbers each from DDT
resistant, DDT and deltamethrin susceptible phenotypes
were randomly selected for sequencing to cross check the
efficacy of AS-PCR assay in detecting correct kdr genotype.
PCR products were purified using the QIAquick PCR
purification kit (Qiagen) and directly sequenced on both
strands using ABI automated DNA sequencer. Sequences
were analyzed using BLAST search, CLUSTALW and
submitted to the National Center for Biotechnology
Information Open Reading Frame Finder (NCBI ORF
Finder; http://www.ncbi.nlm.nih.gov/projects/gorf/) and
SoftberryBESTORF(http://linux1.softberry.com/
berry.phtml?topic=bestorf&group=programs&sub
group=gfind) to predict the coding regions and introns.
Fully annotated sequences were submitted in GenBank
under accession no. FJ182226 and FJ970025.
Mean mortality was determined across all batches of
mosquitoes tested and WHO (1992) criteria for evaluating
resistance or susceptibility in a mosquito population were
used. Mortality rates of less than 80% indicated resistance,
whereas those greater than 98% indicated susceptibility
and rates between 80% and 98% suggest the possibility of
resistance but this needs further verification. The 50% and
90% knock-down time (KDT50 and KDT90) were esti-
mated by regression analysis between percent knock-down
and exposure time, using the log-probit method (Finney
1971). Analysis of variance (anova) was used to compare
knock-down rates between different study sites.
Genotype frequencies were calculated and deviation from
the HardyWeinberg equilibrium (HWE) was analyzed by
the web-based programme de FINETTI generator version
3.0.5 (2008) (https://finetti.meb.uni-bonn.de/) and plotted
in the de Finetti diagram, which illustrates the genotype
frequencies for a bi-allelic locus within a population. It uses
a triangular plot to represent the distribution of the three
genotype frequencies in relation to each other. The de
Finetti diagram includes a curved line, the HardyWeinberg
parabola, which represents the points where the alleles are
in a state of HWE. The distance between the parabolic curve
and the genotypes indicates the extent of deviation from
the HWE and the significance is calculated by chi-square
test.

Table 3 Confirmation of the knock down resistance (kdr) genotypes determined by AS-PCR followed by DNA sequencing

kdr genotype by PCR kdr genotype by sequencing

Bioassay phenotype TTA (SS) TTA T (SR) TTT (RR) TTA (SS) TTA T (SR) TTT (RR)

DDT resistant (n = 6) 1 2 3 2 2 2
DDT susceptible (n = 6) 3 2 1 2 3 1
Deltamethrin susceptible (n = 6) 1 4 1 2 4 0
Total 5 8 5 6 9 3

1100 2009 Blackwell Publishing Ltd

Tropical Medicine and International Health volume 14 no 9 pp 10971104 septemper 2009

M. Sarkar et al. Molecular analysis of kdr mutation

Phenotype: deltamethrin susceptible

Results

0.40 (0.04)
Susceptibility bioassay

v2 = 8.403, P-value = 0.0037

v2 = 9.183, P-value = 0.0024
Table 4 Distribution of knock down resistance genotypes in relation to HardyWeinberg proportion for DDT survivors (resistance), DDT dead (susceptible) and

Allele frequency (SD)

The percent and mean (SD) mortality against DDT (4%)
R
are shown in Table 2. The percent mortality against DDT
in all the study sites ranged between 11.9 and 41.25,
0.60 (0.04)

P-value = 0.0073
Phenotype: deltamethrin susceptible

whereas in susceptible strain (SLab), mortality was

estimated 91.2%. The data show that C. quinquefasciatus

)0.4583

Table also shows the allele frequencies and statistical significance test for deviations of genotypes from HardyWeinberg Equilibrium (HWE). SD, standard deviation.
S

is resistant to DDT in all the study sites. In Benganajuli a

high rate of resistance to DDT was found.
Chi-square

The percent mortality in knock down bioassay with

1.344
4.033
3.025
(v2)

deltamethrin suggests complete susceptibility to this insec-

ticide in all the study sites, except in Benganajuli, where
10 (14.40)
28 (19.20)
(expected)

df, degree of freedom for test for HardyWeinberg proportions are # genotypes # alleles; Performed according to Elston & Forthofer (1977).
2 (6.40)

further data verification is needed (Table 2). The knock

observed
Number

down rates (KDT50 and KDT90) and percent mortality

against deltamethrin (0.05%) are displayed in Table 2. The
v2 = 3.922, P-value = 0.0477
v2 = 4.014, P-value = 0.0451
Phenotype: DDT susceptible

KDT50 and KDT90 values of all study sites were much higher
0.45 (0.046)

than the SLab of C. quinquefasciatus. In Benganajuli and

Rikamari, higher values of KDT50 and KDT90 possibly
Allele frequency (SD)

P-value = 0.1068

indicate the development of incipient resistance to delta-

R

methrin. The knock down rates against deltamethrin were

0.55 (0.046)

)0.3131

significantly different between the study sites (P < 0.01).

Molecular detection of kdr mutation

Phenotype: DDT susceptible

S
Chi-square

The AS-PCR assay revealed the presence of leucine

phenylalanine kdr mutation in the wild population of
0.794
1.941
1.186
(v2)

C. quinquefasciatus. PCR assay showed three genotypes,

identified by the characteristic 380 bp band corresponding
9 (12.10)
(expected)

26 (19.8)
5 (8.10)
observed
Number

v2 = 0.178, P-value = 0.6733

v2 = 0.178, P-value = 0.6729

to resistant and susceptible specific primers. The 380 bp

Phenotype: DDT resistant

PCR product with both the knock down specific (kds and
kdr) primers in an individual mosquito indicates hetero-
0.56 (0.053)

zygous condition (SR). The appearance of this band only in

P-value = 0.7581

susceptible-specific primer (Cq3) indicates homozygous

susceptible (SS) and in resistant specific primer (Cq4)
Allele frequency (SD)

)0.0667
R

indicates homozygous resistant (RR). The sequencing

results confirmed the presence of one polymorphic site at
0.438 (0.053)

127 (accession no. FJ970025), where A-to-T mutation was

observed, which induces a change in leucine to phenylal-
anine. But the correlation of sequencing results with
deltamethrin dead (susceptible) samples

AS-PCR assay was not satisfactory (Table 3), which is in

Chi-square (v2)

agreement with the findings of Abdalla et al. (2008).

Phenotype: DDT resistant

Log likelihood chi-square (df = 1)

0.056
0.088
0.034

kdr genotyping
Pearsons chi-square (df = 1)
Inbreeding coefficient (F)

Our results demonstrate the presence of different genotypes

(Expected)

21 (19.69)
12 (12.66)

Deviation from HWE

7 (7.66)

for the kdr locus among individuals in the same population

observed
Number

of both resistant and susceptible mosquitoes, including

individuals that were heterozygous for both susceptible (A)
Exact test

and resistant (T) alleles or homozygous for either A or T

Genotype

allele, which agrees with the findings of Xu et al. (2006).

RR
SR
SS

The majority of the individuals were heterozygous

2009 Blackwell Publishing Ltd 1101

Tropical Medicine and International Health volume 14 no 9 pp 10971104 septemper 2009

M. Sarkar et al. Molecular analysis of kdr mutation

(TTA T) (Table 4). Of the three potential genotypes [SS (2008) in the same species. However, we could not find
(A A), SR (A T), RR (T T)], SR were by far the most any A to C mutation as reported by Wondji et al. (2008).
predominant with 75 individuals from a total sample size We also demonstrated the frequency distribution of kdr
of 120, followed by SS (26 individuals) and RR genotype genotype in relation to DDT and deltamehrin resistance
(19 individuals) (Table 4). The genotype frequencies at kdr and or susceptibility in a population of C. quinquefascia-
locus for DDT resistant individuals followed the HWE, but tus from northeastern India, for the first time reporting the
for DDT and deltamethrin susceptible individuals they sequences of vgsc gene in mosquitoes (FJ182226,
differed significantly (P < 0.05) (Table 4). The de Finetti FJ970025).
diagram of genotype frequencies in relation to the HWE in Considering that kdr is a recessive trait (Martine-Torres
Figure 2 adds information not apparent in table of geno- et al. 1998; Ranson et al. 2000) in the DDT-resistant
type data because it reveals the exact distribution pattern C. quinquefasciatus, finding 21 40 (52.5%) genotypic
of kdr genotypes and the degree of distortion from HWE. heterozygous [SR (A T)] individuals and 7 40 (17.5%)
In DDT and deltamethrin susceptible samples the genotype genotypic homozygous susceptible (SS) individuals suggests
frequencies showed significant deviation from HWE, but the involvement of some other mechanisms, especially
not in DDT resistant samples. But the sample size and elevated level of detoxification enzymes, in resistance
design of our study does not allow any strong conclusion development. Therefore, we analyzed the detoxifying
on genetic equilibrium of the population or evolutionary enzyme activities (not addressed in this article) in
pattern of kdr mutation in the population. C. quinquefasciatus population. We found that the levels
of detoxifying enzymes, especially esterases and glutathi-
one-s-transferases, were significantly high in these mos-
Discussion
quito populations which also correlate with the insecticide
In this study, we analyzed the kdr mutation by PCR assay tolerance status (Sarkar et al. 2009). Therefore, we inferred
followed by partial sequencing of sodium channel gene in that mosquitoes from the same study sites may harbour
C. Quinquefasciatus. We observed one polymorphic site at both kdr and metabolic resistance (Sarkar et al. 2009).
position 127 (TTA to TTT) in the IIS6 domain of VGSC, These suppositions are consistent with Djouaka et al.
which induces a change in leucine to phenylalanine as (2008) who demonstrated that the mosquitoes from Benin
previously reported by Xu et al. (2005) and Wondji et al. harboured both kdr and metabolic resistance. We observed

1.0
0.1

0.1

0.2
0.2

0.8
Ge
0.3
0.3

no
S)

3
(S

typ
cy

2
ic
0.4

0.4
en

fre

0.6
qu

qu
fre

en
Genotype frequency (SR)

0.5
0.5

1
cy
ic
typ

Figure 2 De Finetti diagram with Hardy

(R
no

R)

Weinberg equilibrium (HWE) parabola for

0.6
0.6
Ge

0.4 (1) DDT resistant, (2) DDT susceptible and

(3) Deltamethrin susceptible Culex quin-
0.7
0.7

quefasciatus investigated for knock down

resistance (kdr) genotypes. Length of verti-
cal line representing the frequency of
0.8
0.8

0.2 genotype SR, Length of left perpendicular

line representing the frequency of genotype
0.9
0.9

SS, Length of right perpendicular line

representing the frequency of genotype RR,
x-axis showed the frequency of allele S,
HardyWeinberg parabola representing the
0.0 0.2 0.4 0.6 0.8 1.0
point where the alleles are in a state of
Allele frequency (S) HardyWeinberg equilibrium.

1102 2009 Blackwell Publishing Ltd

Tropical Medicine and International Health
M. Sarkar et al. Molecular analysis of kdr mutation
both resistant and susceptible allelic genotypes in suscep-
tible mosquitoes, which confirms the findings of Miyazaki
et al. (1996) who discovered both kdr and susceptible
genotypes in susceptible house fly. The presence of
heterozygous kdr allele in both susceptible and resistant
individuals of the same population makes the conclusion
more difficult. At this point, we found no firm correlation
between kdr genotyping and the level of susceptibility
and or resistance to insecticide in this population, which
conforms with previous studies in mosquitoes and other
insect species where no correlation between the genotype
of the kdr mutation and resistance phenotype was observed
(Miyazaki et al. 1996; Brengues et al. 2003; McAbee et al.
2003; Xu et al. 2006; Matambo et al. 2007; Abdalla et al.
2008).
There is a debate among researchers on whether kdr
mutation can produce a resistance phenotype of signifi-
cance to control interventions. Some authors state that kdr
mutation may not always produce a resistance phenotype
of significance to control interventions (Darriet et al. 2000;
Asidi et al. 2004; Henry et al. 2005); their studies are
contradicted by the finding of NGuessan et al. (2007)
where high-frequency kdr correlates to reduce efficacy of
pyrethroid-based vector control efforts. The data of our
experiment confirm the occurrence of kdr allele in the wild
population of C. quinquefasciatus in northeastern India.
But the exact role of this kdr mutation in developing
insecticide resistance is still to be defined by further studies.
Insecticide resistance is a complex system and a single
mechanism alone is unlikely to contribute solely to the
resistant phenotype. However, kdr is closely associated
with resistance and serves as an effective and apparent
marker. The discovery of kdr mutation in C. quinquefas-
ciatus from India is of great significance at both funda-
mental and applied levels. Our findings have important
implications for understanding the kdr and the genotypic
distribution of kdr allele in the principal vector of
bancroftian filariasis in India.
Acknowledgements
We sincerely acknowledge D. Goswami for his technical
help during molecular work, and thank R. Gopalakrishnan
for his comments and suggestions on the manuscript.
References
Abdalla H, Matambo TS, Koekemoer LL, Hunt RH & Coetzee M
(2008) Insecticide susceptibility and vector status of natural
populations of Anopheles arabiensis from Sudan. Transactions
of the Royal Society of Tropical Medicine and Hygiene 102,
263271.
2009 Blackwell Publishing Ltd
volume 14 no 9 pp 10971104 septemper 2009
Asidi AN, NGuessan R, Hutchinson RA, Traore-Lamizana M,
Carnevale P & Curtis CF (2004) Experimental hut comparisons
of nets treated with carbamate or pyrethroid insecticides,
washed or unwashed, against pyrethroid-resistant mosquitoes.
Medical and Veterinary Entomology 18, 134140.
Brengues C, Hawkes NJ, Chandre F, et al. (2003) Pyrethroid and
DDT cross resistance in Aedes 0aegypti is correlated with novel
mutations in the voltage-gated sodium channel gene. Medical
and Veterinary Entomology 17, 8794.
Chandre F, Darriet F, Darder M, et al. (1998) Pyrethroid resis-
tance in Culex quinquefasciatus from West Africa. Medical and
Veterinary Entomology 12, 359366.
Coen ES, Strachan T & Dover G (1982) Dynamics of concerted
evolution of ribosomal DNA and histone gene families in the
melanogaster species subgroup of Drosophila. Journal of
Molecular Biology 158, 1735.
Darriet F, NGuessan R, Koffi AA, et al. (2000) Impact of pyre-
thrin resistance on the efficacy of impregnated mosquito nets in
the prevention of malaria: results of tests in experimental cases
with deltamethrin SC. Bulletin de la Societe de Pathologie
Exotique 93, 131134 (in French).
Djouaka RF, Bakare AA, Coulibaly ON, et al. (2008) Expression
of the cytochrome P450s, CYP6P3 and CYP6M2 are signifi-
cantly elevated in multiple pyrethroid resistant populations of
Anopheles gambiae s.s. from Southern Benin and Nigeria. BMC
Genomics 9, doi: 10.1186/1471-2164-9-538.
Dong K (1997) A single amino acid change in the para sodium
channel protein is associated with knockdown-resistance (kdr)
to pyrethroid insecticides in German cockroach. Insect Bio-
chemistry and Molecular Biology 27, 93100.
Elston RC & Forthofer R (1977) Testing for HardyWeinberg
equilibrium in small samples. Biometrics 33, 536542.
Feyereisen R (1999) Insect P450 enzymes. Annual Review of
Entomology 44, 507533.
Finney JD (1971) Probit Analysis, 3rd edn. Cambridge University
Press, Cambridge.
Georghiou GP (1986) The Magnitude of Resistance Problem.
Pesticide Resistance: Strategies and Tactics for Management.
National Academy Press, Washington DC, pp. 1443.
Gyapong JO, Kumaraswami V, Biswas G & Ottesen EA (2005)
Treatment strategies underpinning the global programme to
eliminate lymphatic filariasis. Expert Opinion on Pharmaco-
therapy 6, 179200.
Henry MC, Assi SB, Rogier C, et al. (2005) Protective efficacy of
lambda cyhalothrin treated nets in Anopheles gambiae pyre-
throid resistance areas of Cote dlvoire. American Journal of
Tropical Medicine and Hygiene 75, 859864.
ICMR (2002) Prospects of eliminating lymphatic filariasis in India.
Indian Council of Medical Research Bulletin 32, 491503.
Joshi RM, Ghose G, Som TK & Bala S (2003) Study of the impact
of deltamethrin impregnated mosquito nets on malaria incidence
at a military station. Medical Journal Armed Forces India 59,
1214.
Liu N & Pridgeon JW (2002) Metabolic detoxification and the kdr
mutation in pyrethroid resistant house fly, Musca domestica
(L.). Pesticide Biochemistry and Physiology 73, 157163.
1103
Tropical Medicine and International Health
M. Sarkar et al. Molecular analysis of kdr mutation
Mahanta B, Handique R, Narain K, Dutta P & Mahanta J (2001)
Transmission of bancroftian filariasis in tea agro-ecosystem of
Assam, India. Southeast Asian Journal of Tropical Medicine and
Public Health 31, 581584.
Martine-Torres D, Chandre F, Williamson MS, et al. (1998)
Molecular characterization of pyrethroid knockdown resistance
(kdr) in the major malaria vector Anopheles gambiae s.s. Insect
Molecular Biology 7, 179184.
Martine-Torres D, Chevillon C, Brun-Barale A, Berge JB, Pasteur
N & Pauron D (1999) Voltage dependent Na+ channels in
pyrethroid resistant Culex pipiens L mosquitoes. Pesticide
Science 55, 10121020.
Matambo TS, Abdalla H, Brooke BD, et al. (2007) Insecticide
resistance in Anopheles arbiensis and association with the kdr
mutation. Medical and Veterinary Entomology 21, 97102.
McAbee RD, Kang KD, Stanich MA, et al. (2004) Pyrethroid
tolerance in Culex pipiens pipiens var molestus from Marin
Country, California. Pest Management Science 60, 359368.
McCarroll L & Hemingway J (2002) Can insecticide resistance
status affect parasite transmission in mosquitoes? Insect Bio-
chemistry and Molecular Biology 32, 13451351.
Milani R (1954) Comportamento mendeliano della resistenza alla
azione abbatante del DDT: correlazione tran abbatimento e mor-
talia in Musca domestica L. Rivista Parasitologia 15, 513542.
Miyazaki M, Ohyama K, Dunlap DY & Matsumura F (1996)
Cloning and sequencing of the para-type sodium channel gene
from susceptible and kdr-resistant German cockroaches (Blat-
tella germanica) and house fly (Musca domestica). Molecular
and General Genetics 252, 6168.
NGuessan R, Corbel V, Akogbeto M & Rowland M (2007)
Reduced efficacy of insecticide treated nets and indoor residual
spraying for malaria control in pyrethroid resistance area, Benin.
Emerging Infectious Diseases 13, 199206.
Narahashi T (1992) Nerve membrane Na+ channels as targets of
insecticides. Trends in Pharmacological Science 13, 236241.
Nelson DR, Koymans L, Kamataki T, et al. (1996) P450 super-
family: update on new sequences, gene mapping, accession
numbers and nomenclature. Pharmacogenetics 6, 142.
OReilly AO, Khambay BP, Williamson MS, Field LM, Wallace
BA & Davies TGE (2006) Modelling insecticide binding sites in
the voltage gated sodium channel. Biochemistry Journal 396,
255263.
Ottesen EA, Duke BO, Karam M & Behbehani K (1997) Strategies
and tools for the control elimination of lymphatic filariasis.
Bulletin of the World Health Organization 75, 491503.
Park Y & Taylor MFJ (1997) A novel mutation L1029H in
sodium channel gene hscp associated with pyrethroid resistance
for Heliothis virescens (Lepidoptera: Noctuidae), Insect Bio-
chemistry and Molecular Biology 27, 913.
Pridgeon JW, Appel AG, Moar WJ & Liu N (2002) Variability of
resistance mechanisms in three strains of pyrethroid resistant
German cockroach, Blattella germanica (L.). Pesticide Bio-
chemistry and Physiology 73, 149156.
Corresponding Author M. Sarkar, Medical Entomology Division, Defence Research Laboratory, Tezpur 784001, Assam, India.
E-mail: manas_sarkar54491@yahoo.com
1104
volume 14 no 9 pp 10971104 septemper 2009
Ranson H, Jensen B, Vulule JM, Wang X, Hemingway J & Collins
FH (2000) Identification of point mutation in the voltage-gated
sodium channel gene of Kenyan Anopheles gambiae associated
with resistance to DDT and pyrethroids. Insect Molecular
Biology 9, 491497.
Roberts DR & Andre RG (1994) Insecticide resistant issues in
vector-borne disease control. American Journal of Tropical
Medicine and Hygiene 50(6_Suppl), 2134.
Sarkar M, Bhattacharyya IK, Borkotoki A, et al. (2009) Insecticide
resistance and detoxifying enzyme activity in the principal
bancroftian filariasis vector, Culex quinquefasciatus in north-
eastern India. Medical and Veterinary Entomology 23, 122
131.
Scott JG, Liu N & Wen Z (1998) Insect cytochromes P450:
diversity, insecticide resistance and tolerance to plant toxins.
Comparative Biochemistry and Physiology C 121, 147155.
Soderlund DM & Bloomquist JR (1989) Neurotoxic actions of
pyrethroid insecticides. Annual Review of Entomology 34,
7796.
Soderlund DM & Knipple DC (2003) The molecular biology of
knockdown resistance to pyrethroid insecticides. Insect Bio-
chemistry and Molecular Biology 33, 563577.
WHO (1992) Vector Resistance to Insecticides. 15th Report of the
WHO Expert Committee on Vector Biology and Control.
World Health Organization Technical Report Series, Vol. 818,
162, Geneva, Switzerland.
WHO(1998) Test Procedure for Insecticide Resistance Monitoring
in Malaria Vector, Bio-efficacy and Persistence of Insecticides
on Treated Surfaces. Document WHO CDS CPC Mal
98.12.Geneva, Switzerland.
Williamson MS, Martine-Torres D, Hick CA & Devonshire AL
(1996) Identification in mutations in the housefly para-type
sodium channel gene associated with knockdown resistance
(kdr) to pyrethroid insecticides. Molecular and General Genetics
252, 5160.
Wondji CS, De Silva WAPP, Hemingway J, Hilary R &
Karunaratne SHPP (2008) Characterization of knockdown
resistance in DDT and pyrethroid resistant Culex quinquefas-
ciatus population from Sri Lanka. Tropical Medicine and
International Health 13, 548555.
Xu Q, Liu H, Zhang L & Liu N (2005) Resistance in the mos-
quito, Culex quinquefasciatus, and possible mechanisms for
resistance. Pest Management Science 61, 10961102.
Xu Q, Wang H, Zhang L & Liu N (2006) Kdr allelic variation in
pyrethroid resistant mosquitoes, Culex quinquefasciatus (S).
Biochemical Biophysical Research Communication 345, 774
780.
Zhou L, Lawrence GG, Vineis JH, McAllister JC, Wirtz RA &
Brogdon WG (2009) Detection of broadly distributed sodium
channel alleles characteristic of insect pyrethroid resistance in
west nile virus vector Culex pipiens complex mosquitoes in the
United States. Journal of Medical Entomology 46, 321327.
2009 Blackwell Publishing Ltd