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First identification of kdr allele F1534S
in VGSC gene and its association with
resistance to pyrethroid insecticides in
Aedes albopictus populations from Haikou
City, Hainan Island, China
Huiying Chen
1
, Kaili Li
1
, Xiaohua Wang
2,3
, Xinyan Yang
2,3
, Yi Lin
2,3
, Fang Cai
2,3
, Wenbin Zhong
2,3
, Chunyan Lin
2,3
,
Zhongling Lin
2,3
and Yajun Ma
1,3*
Abstract
Background: Aedes albopictus is distributed widely in China, as a primary vector of Dengue fever and Chikungunya
fever in south of China. Chemical insecticide control is one of the integrated programmes to prevent mosquito-borne
diseases. Long-term applications of pyrethroids have resulted in the development of resistance in Ae. albopictus
populations in China. However, the susceptibility of Ae. albopictus to pyrethroids in Hainan Island was unclear. Knockdown
resistance (kdr), caused by point mutations in the VGSC gene, is one of the mechanisms that confer resistance to DDT
and pyrethroids. This study was to investigate the resistance level of Ae. albopictus populationsinHaikouCitytothree
pyrethroid insecticides, and elucidate the relationship between the resistant phenotype and kdr mutations.
Methods: The Aedes albopictus samples were collected in Xinbu Island (XI), Longtang Town (LT), Shishan Town (ST),
Baishamen Park (BP), and Flower Market (FM) from Haikou City, Hainan Island, China. The larval susceptibility to
deltamethrin, permethrin and beta-cypermethrin was tested by larval bioassays, and adult susceptibility to deltamethrin
and DDT was determined by adult bioassays. The degree of resistance was determined by resistance ratio value (RR
50
>3)
for larvae and by mortality for adult. The kdr alleles at codon 1534 of the VGSC gene were genotyped. The relationship
between kdr genotypes and resistant phenotypes was analyzed by Chi-square test.
Results: Out of five populations, assessed by larval bioassays, XI was susceptible to deltamethrin and permethrin; LT was
susceptible to permethrin and beta-cypermethrin; and ST was susceptible to permithrin. FM and BP both were resistant
to all of the three pyrethroids, and FM showed the highest degree of resistance, with RR
50
values from 65.17 to 436.36. A
total of 493 individuals from the larval bioassays were genotyped for kdr alleles. Five alleles were detected, including two
wildtype alleles, TTC(F) (67.04 %) and TTT(F) (0.41 %), and three mutant alleles, TGC(C) (0.30 %), TCC(S) (31.54 %) and TTG(L)
(0.71 %). There was a clear correlation between mutant alleles (or F1534S) and resistant phenotypes (P< 0.01).
Conclusion: Two novel kdr mutant alleles F1534S and F1534L were detected in the pyrethroid resistant populations of
Ae. albopictus in Haikou Hainan, China. For the first time, the mutant F1534S was associated with pyrethroid resistance in
Ae. albopictus.
Keywords: Aedes albopictus, Pyrethroids, Resistance, kdr mutation, China
* Correspondence: yajun_ma@163.com
1
Department of Tropical Infectious Diseases, Faculty of Tropical Medicine and
Public Health, Second Military Medical University, Shanghai 200433, China
3
CDC Key Laboratory of Surveillance and Early-Warning on Infectious Disease,
Haikou 571100, China
Full list of author information is available at the end of the article
© 2016 Chen et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0
International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and
reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to
the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver
(http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
Chen et al. Infectious Diseases of Poverty (2016) 5:31
DOI 10.1186/s40249-016-0125-x
Multilingual abstracts
Please see Additional file 1 for translation of the ab-
stract into the six official working languages of the
United Nations.
Background
Aedes albopictus Skuse is a primary vector of Dengue
fever and Chikungunya fever in China [1, 2]. Mosquito con-
trol is one of the integrated programmes to prevent trans-
mission of mosquito-borne diseases. Chemical insecticides
have been extensively used for vector management since
the 1940s. There were four major categories of insecticides:
organochlorines, organophosphates, carbamates and pyre-
throids [3]. The pyrethroids have been used to indoor/out-
door residual sprays since 1980s for mosquito control in
China. The long-term utilization has resulted in the devel-
opment of resistance in many populations of Ae. albopictus
in China [2, 4–10]. The pyrethroids function as neurotoxins
that target voltage-gated sodium channels (VGSC) and
interfere electronic signaling in the nervous system, which
results in paralysis and death, an effect known as knock-
down [11]. One of the mechanisms that mosquitoes have
developed for the resistance to pyrethroids is the target
insensitivity, which is caused by mutations in the VGSC
gene and generated knockdown resistance (kdr)[12–15]. In
Anopheles mosquitoes, substitution of leucine at residue
position 1014 was correlated to the resistance to pyre-
throids and DDT [14–17]. In Aedes aegypti Linn, mutants
have been detected in several codons of the VGSC gene
from different countries, including three mutants, V1016G/
I and F1534C, all were correlated with kdr [18–27]. In Ae.
albopictus, the relationship between kdr and pyrethroid
resistance was unclear. In a DDT and pyrethroid resistant
population of Ae. albopictus in India, no kdr mutations
were detected [28]. Similarly, no kdr mutations were
found in Ae. albopictus populations in Malaysia where
F1534C and V1016G/I were detected in the populations
of Ae. aegypti [29]. So far, only one study has identified
the F1534C mutant allele in a population of Ae. albopictus
in Singapore with frequency of 73.1 % [13].
Haikou city is a provincial capital of Hainan Island, in
south of China, located at marginal zone of tropic. In
the past, dengue fever outbreaks have occurred twice in
1979–1982 and 1985–1988 in Hainan Island and sur-
rounding areas; the mortality rate was 0.785‰[30–35].
In recent years, dengue fever epidemic situations remain
in Guangdong, Fujian and Yunnan Provinces in China
[30, 36–38]. Especially in 2014, a large-scale outbreak of
dengue fever with more than 45,000 cases occurred in
Guangdong Province [2, 37, 39, 40]. Hainan Island is
near to but separated by a strait from Guangdong Prov-
ince, and there were also reported local cases during the
dengue outbreak in 2014 [2]. Upon the pressure of dengue
epidemics, residual and aerial spraying of pyrethroids have
become a major routine method for the control of Aedes
mosquitoes in the endemic areas in China. The most com-
monly used pyrethroid was deltamethrin [2, 41]. Pyrethroid
resistance has been detected in the populations of Ae.
aegytpi and Ae. albopictus in Hainan [42, 43]. In this study,
we investigated the susceptibility to pyrethroid resistance
and examined the kdr mutations in Ae. albopictus in five
locations in Haikou City, Hainan Island. The bioassays
revealed that resistance to deltamethrin, permethrin and
beta-cypermethrin was developed in certain populations. In
addition to the known kdr mutant, F1534C, two novel mu-
tant alleles, F1534S and F1534L, were detected.
Methods
Ethics statement
No permits were required for the described field studies.
Mosquito collections in breeding sites were consent by
the owners at each location.
Mosquito samples
Mosquito larvae were collected from breeding sites in
Xinbu Island (XI, 110°37′E, 20°06′N), Longtang Town
(LT, 110°42′E, 19°89′N), Shishan Town (ST, 110°22′E,
19°94′N), Baishamen Park (BP, 110°34′E, 20°08′N) and
Flower Marker (FM, 110°29′E, 20°02′N) in Haikou city,
Hainan Province during April and May 2015 (Fig. 1).
The collected larvae were brought back to the insectary
and reared to adults at 26 ± 1 °C and 70 ± 5 % (RH),
under a 14: 10 h (light: dark) photoperiod. The larvae of
F2 generation were used for larval bioassays. The species
of Ae. albopictus was identified by adult morphology [1].
The susceptible laboratory colony of Ae. albopictus was
provided by Department of Tropical Infectious Diseases,
Second Military Medical University, which was established
from a population originally collected from Hangzhou,
China. The colony has been maintained in insectary for
15 years without exposure to any insecticides.
Larval bioassay
The susceptibility of larvae to three pyrethroid insecticides,
deltamethrin (≥98 %, Sigma, USA), permethrin (≥98 %,
Sigma, USA) and beta-cypermethrin (>99 %, Sigma, USA),
was determined using a procedure recommended by WHO
[44]. In the assay, 20–25 late 3rd and early 4th instar larvae
were placed in a glass container that held 199 mL H
2
Oand
1 mL of insecticide solution. Analytical grade insecticides
were diluted five to seven concentrations with acetone. The
solution with no insecticide was used as control. Larval
mortality was recorded 24 h after treatment. The larvae
that were motionless or convulsive upon a sharp stimula-
tion were counted as dead [44]. Larval mortality was deter-
mined by dividing the number of dead larvae by the total
number tested. Dead and survival larvae were collected and
preserved in 95 % alcohol for subsequent DNA analysis. No
Chen et al. Infectious Diseases of Poverty (2016) 5:31 Page 2 of 8
food was provided to larvae during the procedure. If a test
with pupation rate greater than 10 %, or mortality rate in
control greater than 20 %, the test was invalid and was
removed. All bioassays were repeated three times. In the
larval bioassay, the median lethal concentration (LC
50
), the
90 % lethal concentration (LC
90
) and 95 % confidence inter-
val of different pyrethroids were calculated based on the
recorded data using Schoofs and Willhite’s probit analysis
program [45]. The degree of resistance was determined by
theresistanceratio(RR
50
), obtained by the LC
50
value for a
population compared with the LC
50
valuefortheinsecti-
cide for susceptible laboratory colony. The RR
50
≤3was
considered as susceptible, and 3 < RR
50
≤10 as low degree
of resistance, 10 < RR
50
≤20 as median degree of resistance,
and RR
50
> 20 as high degree of resistance [44].
Adult bioassay
Field-collected larvae were reared to adults in the insectary.
Female unfed adults at day 2 or 3 post emergence were
tested for the susceptibility to deltamethrin and DDT, using
the standard WHO tube bioassay [46]. So far, there has
been no sufficient data for a standard diagnostic concentra-
tion for resistance monitoring for Ae. albopictus in China.
The test papers with deltamethrin (0.1 %) and DDT (4 %)
were used for the assay, whichwereprovidedbyNational
Institute for Communicable Disease Control and Preven-
tion, Chinese Center for Disease Control and Prevention.
For each insecticide, approximately 100 female mosquitoes
were tested. Paraffin oil-treated papers without insecticide
were used as control. The knockdown time of individual
mosquitoeswasrecordedat10min,30min,50minand
60 min. Post 1 h exposure, mosquitoes were transferred to
arecoverytubeandmaintainedon6%ofsucrosesolution
for 24 h. Dead and survival mosquitoes were collected and
preserved in 95 % ethanol for subsequent DNA analysis,
respectively. Mosquitoes were considered dead if they were
motionless, when they were mechanically stimulated, fol-
lowing the method of Gonzalez Audino [47].
Detection kdr alleles and correlation with the larval
bioassay
The individual mosquito larvae or adult was used for DNA
extraction with the DNAzol Reagent (Invitrogen, USA). To
identify kdr alleles, a partial sequence of S6 segment of
domain III of the VGSC gene was amplified from 20 to
50 ng genomic DNA using primers aegSCF7 (5’-AGG TAT
CCG AAC GTT GCT GT-3’) and aegSCR8 (5’-TAG CTT
TCA GCG GCT TCT TC-3’) [13]. The PCR kit was from
Aidlab, China. PCR reaction was carried out in Verity 96
well 157 Thermal Cycler (Applied Biosystems, USA). The
cycling parameter included an initial step of denaturation at
94 °C for 2 min, followed by 35 cycles of amplification at
94°Cfor30s,52°Cfor30s,and72°Cfor30s,withafinal
extension step at 72 °C for 8 min. After electrophoresis,
PCR products were purified and directly sequenced in both
directions with the same primers. There were 4 speci-
mens, of which the PCR products were cloned into plas-
mids (pGEMX-T Easy Vector, Aidlab, China), and then
Fig. 1 A map of Hainan province (partial) showing the collecting sites
Chen et al. Infectious Diseases of Poverty (2016) 5:31 Page 3 of 8
sequenced, due to the double peaks at two positions of
the codon 1534.
The codon 1534 was examined by sequence analysis, and
genotypes were determined. In each sample, for a particular
allele, the allele frequency was calculated as: number of
alleles/(sample size × 2). The mutation frequency was de-
fined as frequency of sum of wildtype/mutant heterozygotes
and mutant/mutant homozygotes, which was calculated as:
(sum of wildtype/mutant + mutant/mutant individuals)/
sample size.
Chi-square tests were used to examine the association
between kdr mutation and the resistance phenotype. In
the present study, the dependent variables were the mos-
quito status (alive or dead) at 24 h post larval bioassay.
Results
Insecticide susceptibility bioassays
The larval susceptibility to three pyrethroids was tested for
five populations of Ae. albopictus, which revealed a hetero-
geneous pattern (Table 1). Among five tested populations,
XI (RR
50
= 2.38), LT (RR
50
=1.17) and ST (RR
50
=1.67)
were susceptible to permethrin; BP was resistant with a
median level (RR
50
= 8.83) and FM was resistant with a high
level of resistance (RR
50
= 182.00). Besides, four of the five
populations had developed resistance to deltamethrin and
beta-cypermethrin, only XI was susceptible to deltamethrin
and LT was susceptible to beta-cypermethrin. FM appeared
to be the population having high level of resistance, with
RR
50
= 436.36 to deltamethrin and RR
50
=65.17 to beta-
cypermethrin (Table 1).
The adult bioassay was conducted to determine the sus-
ceptibility to DDT and deltamethrin. The larvae from the 5
locations were pooled and reared to adults in the insectary.
Theadultswereexposedtothe4%DDTtestpaper.The
knockdown percentage was 0.00, 0.02, 0.32 and 0.72 % at
10 min, 30 min, 50 min and 60 min. The mortality was
87.50 %, indicating that the population was resistant to
DDT. There is no standard diagnostic dosage yet for Ae.
albopictus adult bioassay in China. The test paper with
0.1 % of deltamethrin was used for testing, which yielded a
mortality of 98.40 % in the tested sample (Table 2). The
knockdown percentage was 0.32, 0.84, 0.98 and 0.93 % at 4
time nodes,
Detection of mutant kdr gene and correlation with the
bioassay
The VGSC gene was genotyped for kdr alleles. A total of
493 specimens from larval bioassay samples were typed. At
codon 1534, in addition to the wildtype codon TTC encod-
ing phenylalanine (F), four other alleles were detected.
Codon TTT codes also for phenylalanine (F), codon TCC
codes for serine (S), TGC for cysteiine (C) and TTG for
leucine (L). The allele frequency was TTC (F) (67.04 %),
TTT (F) (0.41 %) TGC (C) (0.30 %), TCC (S) (31.54 %), and
TTG(L)(0.71%).Themostfrequentmutantallelewas
TCC (S) (Table 3). A total of eight genotypes were
Table 1 Susceptibility of Aedes albopictus larva to three pyrethroid insecticides in Haikou City, Hainan Island, China
Insecticides Sites LC
50
(mg/L) LC
50
(95%CI) LC
90
(mg/L) LC
90
(95 % CI) RR
50
Deltamethrin XI 0.0001 0.0001–0.0002 0.0003 0.0003–0.0004 1.27
LT 0.0012 0.0011–0.0014 0.0032 0.0027–0.0040 9.09
ST 0.0020 0.0010–0.0020 0.0070 0.0050–0.0100 18.18
BP 0.0080 0.0070–0.0090 0.0210 0.0180–0.0270 72.73
FM 0.0480 0.0420–0.0550 0.1650 0.1300–0.2320 436.36
S 0.0001 0.0001–0.0001 0.0003 0.0003–0.0005
Permethrin XI 0.0143 0.0134–0.0159 0.0259 0.0232–0.0300 2.38
LT 0.0070 0.0060–0.0070 0.0120 0.0110–0.0130 1.17
ST 0.0100 0.0100–0.0110 0.0220 0.0190–0.0270 1.67
BP 0.0530 0.0490–0.0580 0.1130 0.0990–0.1320 8.83
FM 1.0920 0.9540–1.2530 4.6740 3.5090–7.1620 182.00
S 0.0060 0.0050–0.0060 0.0090 0.0080–0.0100
Beta-cypermethrin XI 0.0047 0.0043–0.0052 0.0132 0.0114–0.0158 5.31
LT 0.0020 0.0020–0.0020 0.0040 0.0030–0.0040 2.25
ST 0.0040 0.0030–0.0040 0.0100 0.0080–0.0120 4.49
BP 0.0130 0.0120–0.0140 0.0310 0.0260–0.0400 14.61
FM 0.0580 0.0530–0.0640 0.1740 0.1500–0.2120 65.17
S 0.0009 0.0008–0.0010 0.0020 0.0020–0.0031
The data of deltamethrin and permethrin was from the literature [52]
XI Xinbu Island, LT Longtang Town, ST Shishan Town, BP Baishamen Park, FM Flower Market, S: susceptible colony
Chen et al. Infectious Diseases of Poverty (2016) 5:31 Page 4 of 8
Table 2 kdr alleles in relation to mosquito survival phenotype determined by the deltamethrin and DDT susceptibility adult bioassay
in Aedes albopictus populations in Haikou City, Hainan Island, China
Insecticide Bioassay kdr alleles Mutant
frequency
(%)
Individuals (N) Dead (N) after 24 h
recovery period
Mortality
rate (%)
Bioassay status after
24 h recovery period
Individuals (N) Wildtype Mutant
TTC(F) TCC(S) TGC(C)
Deltamethrin 104 102 98.40 Alive 2 0 4 0 100.00
Dead 17 34 0 0 0.00
DDT 198 173 87.50 Alive 19 15 21 2 60.53
Dead 17 32 2 0 5.89
Table 3 kdr alleles in relation to mosquito survival phenotype determined by three pyrethroids larval bioassay groups in Haikou City,
Hainan Island, China
Insecticides Collecting
sites
Bioassay
status
Individuals
(N)
kdr alleles Mutant
frequency
(%)
Wildtype Mutant
TTC(F) TTT(F) TCC(S) TGC(C) TTG(L)
Deltamethrin XI Alive 17 27 0 7 0 0 20.59
Dead 15 28 0 2 0 0 6.67
LT Alive 21 36 2 2 0 2 9.52
Dead 13 26 0 0 0 0 0.00
ST Alive 20 40 0 0 0 0 0.00
Dead 17 34 0 0 0 0 0.00
BP Alive 17 16 0 16 0 2 52.94
Dead 13 20 0 6 0 0 23.08
FM Alive 19 1 0 36 1 0 97.37
Dead 16 7 0 23 2 0 78.13
Permethrin XI Alive 16 28 0 4 0 0 12.50
Dead 16 29 0 3 0 0 9.38
LT Alive 15 28 2 0 0 0 0.00
Dead 11 22 0 0 0 0 0.00
ST Alive 20 40 0 0 0 0 0.00
Dead 18 36 0 0 0 0 0.00
BP Alive 15 9 0 20 0 1 70.00
Dead 12 14 0 9 0 1 41.67
FM Alive 19 2 0 36 0 0 94.74
Dead 17 9 0 25 0 0 73.53
Beta-cypermethrin XI Alive 13 12 0 14 0 0 53.85
Dead 18 32 0 4 0 0 11.11
LT Alive 19 38 0 0 0 0 0.00
Dead 14 28 0 0 0 0 0.00
ST Alive 14 28 0 0 0 0 0.00
Dead 15 30 0 0 0 0 0.00
BP Alive 20 12 0 27 0 1 70.00
Dead 19 25 0 13 0 0 34.21
FM Alive 20 1 0 39 0 0 97.50
Dead 14 3 0 25 0 0 89.29
XI xinbu Island, LT Longtang Town, ST Shishan Town, BP Baishamen Park, FM Flower Market
Chen et al. Infectious Diseases of Poverty (2016) 5:31 Page 5 of 8
detected, including wildtype genotype TTC/TTC
(57.40 %) and TTC/TTT (0.81 %), wildtype/mutant het-
erozygotes TTC/TCC (17.85 %), TTC/TTG (0.20 %),
TTC/TGC (0.41 %), and mutant genotypes TCC/TCC
(21.91 %), TCC/TTG (1.22 %), TCC/TGC (0.20 %). Over-
all, the frequency of mutant genotypes (S/S, S/L and S/C)
was 23.33 %, and the frequency of wildtype/mutant het-
erozygotes (F/S, F/C and F/L) was 18.46 % (in Additional
file 2: Table S1). The mutant frequency was high in both
BP and FM while low or none in LT and ST populations
of Ae. albopictus (Table 3).
The distributions of wildtype and mutant genotypes in
larval populations were shown in Fig. 2. In Aedes albopictus
resistant population,the frequencies of mutant genotypes
were 41.04 % in deltamethrin group, 56.47 % in permethrin
group and 60.15 % in beta-cypermethrin group. The fre-
quencies of mutant alleles were 35.11 % in alive individuals
and 22.30 % in dead individuals in deltamethrin group,
35.88 % in alive and 25.68 % in dead in permethrin group,
47.09 % in alive and 26.25 % in dead in beta-cypermethrin
group. In each case, the mutant alleles were associated with
resistant alive mosquitoes (P<0.05). There were all
significant differences between the wildtype and mutant al-
leles in every pyrethroid insecticides bioassay groups (P
<0.05). The difference was more significant if the
individuals from all of the pyrethroid bioassays were pooled
together (P<0.01).
In the samples from adult bioassay, three alleles were
detected, namely TTC (F) (73.64 %), TCC (S) (24.55 %)
and TGC (C) (1.82 %), which formed four genotypes:
wildtype homozygote TTC/TTC, and wildtype/mutant
genotypes, TTC/TGC and TTC/TCC and mutant homo-
zygote TCC/TCC (Table 2). The genotypes of the two
resistant mosquitoes that survived the exposure to 0.1 %
deltamethrin were both mutant homozygotes of TCC(S).
The frequency of mutant alleles was 60.53 % in 19 resist-
ant mosquitoes that survived in the 4 % DDT treatment
(Table 2). Significant correlation was detected between
kdr mutations and deltamethrin or DDT resistant phe-
notypes by Chi-test (P< 0.05).
Discussion
In Hainan Island, Aedes mosquitoes are responsible for
the Dengue fever transmissions. The application of ultra
low-volume (ULV) spray of pyrethroids has been a major
measure to control Aedes adults since the 1990s. The
susceptibility to pyrethroids has been monitored, and
pyrethroid resistance has been reported in wild popula-
tions of Ae. albopictus in Hainan in 2005 and 2010,
respectively [42, 43]. In this study, the larval bioassays
showed that the populations in rural areas (XI, ST, LT)
were largely susceptible to the pyrethoids tested; while
BP and FM, two urban populations, were resistant to all
of three pyrethroids. BP represented a population in a
city park, where ULV spraying was applied on a regular
basis. FM was collected from a garden/nursery market,
where containers with aquatic plants, flower pots and
planters with sufficient water constitute a large quantity of
habitats for Aedes larvae. Owners used spray insecticides
frequently to reduce mosquito density in the market. In
those habitats, mosquitoes expose persistently to high
dose of pyrethroids at both larval and adult stages. In rural
Fig. 2 kdr genotype percentage in Aedes albopictus resistant population to deltamethrin, permethrin and beta-cypermethrin in Haikou City, Hainan
Island China
Chen et al. Infectious Diseases of Poverty (2016) 5:31 Page 6 of 8
area, no regular spray was applied, unless dengue patients
were present in a village. This may explain why BP and
FM mosquitoes were resistant to pyrethroids while the
other three rural populations were susceptible.
In the adult assay, adults showed resistance to DDT.
When exposed 0.1 % deltamethrin test paper, 98. 40 % of
adults were dead. Since the concentration was 4 fold higher
than the diagnostic concentration 0.025 % for Ae. aegypti
[48], we rather not to make any conclusion upon the data.
It is an urgent need to develop standard diagnostic concen-
tration for adults of Ae. albopictus in China.
A number of mutations in the VGSC gene have been
reported in pyrethroid resistant strains of Ae. aegypti
[18–25, 49], a few of these mutations (I1011M/V,
V1016G/ I, F1534C) have been clearly associated with
the resistance phenotype [12, 20–23, 25]. However, very
little is known about the molecular or biochemical basis
of resistance in Ae. albopictus.Nokdr mutations were
found in Ae. albopictus resistant populations from India,
Malaysia and Sri Lank [28, 29, 49, 50]. Recently, F1534C
was found in 24 of 26 individuals of Ae. albopictus in
Singapore [13]. In this study, five alleles were identified
in the codon 1534, including two wildtype codons, and
three mutant codons TCC(S), TGC(C) and TTG(L). The
allele TCC(S) was clearly correlated to the resistance to
permethrin and beta-cypermethrin, both belong to Type
I pyrethroids, similar to the situation in Ae. aegypti [51].
This was the first report that kdr mutants, particularly
F1534S, is behind pyrethroid resistance in Ae. albopictus.
Apparently, long term applications of DDT and pyre-
throids have posed selection pressure on VGSC gene in
Ae. albopictus. It is required to examine more loci of
VGSC gene in more populations in different geographic
areas worldwide. In addition, understanding of the re-
sistance mechanisms and development of simple and ac-
curate diagnostic tools to monitor the presence of
resistance gene mutations is critical for effective man-
agement of pyrethroid resistance and sustainable use of
pyrethroid insecticides in the future.
Conclusions
Some Ae. albopictus populations in Haikou City, Hainan
Island of China have developed resistance to deltameth-
rin, permethrin and beta-cypermethrin. The results sug-
gested that Ae. albopictus control should adjust the
usage of insecticides timely based on the resistant status
investigation, and slow down the production and devel-
opment of resistance. Two novel kdr mutant alleles
F1534S and F1534L were detected in the pyrethroid re-
sistant populations of Ae. albopictus in Haikou City,
Hainan Island of China. For the first time, the mutant
F1534S was associated with pyrethroid resistance in Ae.
albopictus.
Additional files
Additional file 1: Multilingual abstract in the six official working
languages of the United Nations. (PDF 370 kb)
Additional file 2: Table S1. kdr genotypes of Aedes albopictus
populations from pyrethroid larval bioassay groups in Haikou City, Hainan
Island, China. Table S2 Frequencies of kdr genotypes in relation to
mosquito survival phenotype determined by the deltamethrin and DDT
susceptibility adult bioassay in Aedes albopictus populations in Haikou
City, Hainan Island, China (ZIP 28 kb)
Competing interests
The authors declared that they have no competing interests.
Authors’contributions
All authors read and approved the final version of the manuscript. YM designed
the study. HC and KL did adult bioassay. XW, XY, YL, FC, WZ, CL and ZL collected
mosquitoes in the field and did larval bioassay. YM, HC, KL and XY did data
analysis. CH and YM wrote the manuscript. The authors would like to thank Prof.
Xu Jiannong to participate in the discussion and to assist in the writing the
manuscript.
Acknowledgements
This work was supported by the YM’s grant 81371848 from the National Natural
Sciences Foundation of China.
Author details
1
Department of Tropical Infectious Diseases, Faculty of Tropical Medicine and
Public Health, Second Military Medical University, Shanghai 200433, China.
2
Haikou Center for Disease Control and Prevention, Haikou 571100, China.
3
CDC Key Laboratory of Surveillance and Early-Warning on Infectious Disease,
Haikou 571100, China.
Received: 30 December 2015 Accepted: 30 March 2016
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