Sodium channel point mutations associated with pyrethroid resistance in Chinese strains of Culex pipiens quinquefasciatus (Diptera: Culicidae)
Parasites & Vectors
Sodium channel point mutations associated with pyrethroid resistance in Chinese strains of Culex pipiens quinquefasciatus (Diptera: Culicidae)
Minghui Zhao 0 1
Yande Dong 0 1
Xin Ran 0
Xiaoxia Guo 0
Dan Xing 0
Yingmei Zhang 0
Ting Yan 0
Xiaojuan Zhu 0
Jianxin Su 0
Hengduan Zhang 0
Gang Wang 0
Wenjun Hou 0
Zhiming Wu 0
Chunxiao Li 0
Tongyan Zhao 0
0 Beijing Institute of Microbiology and Epidemiology, State Key Laboratory of Pathogen and Biosecurity , Beijing , China
1 Anhui Medical University , Hefei , China
Background: Pesticide resistance due to sodium channel point mutations has been well documented in many mosquito species. Methods: We tested the resistance of six, wild, Chinese populations of the mosquito Culex pipiens quinquefasciatus to deltamethrin and cyhalothrin. The full length of the sodium channel gene was cloned and sequenced from a pooled sample of mosquitoes from each population. Results: Seven amino acid alterations were found (V250M, R436K, M943V, I973T, L1035F, L1035S and E1901D). Correlation between the frequencies of these mutations and the level of pesticide resistance (LC50) associated with them indicates that those at position L1035 (corresponding to position L1014F in the house fly, Musca domestica; GenBank Accession No.: X96668) are associated with resistance to deltamethrin and cyhalothrin. The frequency of the L1035F mutation was significantly correlated with resistance to deltamethrin (R2 = 0.536, P = 0.049) and cyhalothrin (R2 = 0.626, P = 0.030), and the combined frequency of the L1035F and L1035S mutations was significantly correlated with resistance to both deltamethrin (R2 = 0.661, P = 0.025), and cyhalothrin (R2 = 0.803, P = 0.008). None of the other mutations were correlated with either deltamethrin or cyhalothrin resistance. Interestingly, an HWE test indicated significant linkage between the M943V and I973T mutations (P < 0.01), but further research is required to determine the biological significance of this linkage. Conclusions: Identifying these mutations may be of practical benefit to the development of pesticide resistance management programs.
Cx. pipiens quinquefasciatus is the predominant
mosquito species in urban environments in southern China.
This species is a major biting nuisance, particularly in
urban areas where it thrives in wet pit-latrines, polluted
puddles, gutters, and blocked open drains. It is also a
major vector of filariasis , West Nile virus (WNV)
, St. Louis encephalitis virus (SLEV) , and Rift Valley
Fever virus (RVFV) [4,5]. Cx. p. quinquefasciatus is
one of the most studied mosquito species with respect
to insecticide resistance.
Pyrethroid is the general term for a group of synthetic
chemicals based on the structure of natural pyrethrins
derived from Chrysanthemum flowers . Pyrethroids are
now becoming the most commonly used insecticides for
mosquito control . Compared to other insecticides, most
pyrethroids are nontoxic to mammals but have a high
knockdown effect on insects. Unfortunately, the extensive
use of pyrethroids has led to the widespread development
of resistance to this group of pesticides in mosquito
populations [8-10]. The increase in resistance can be both large
and rapid; one study reported an approximately 298-fold
increase in resistance in Cx. p. quinquefasciatus after 10
generations of exposure to deltamethrin .
Modification of the active site of the sodium channel
is an important mechanism involved in pyrethroid
resistance [12,13]. Voltage-gated sodium channels are integral
Figure 1 The distribution of collecting sites of Cx. p. quinquefasciatus. - indicated the six field strains of Cx. p. quinquefasciatus in China.
RR was resistance ratio to deltamethrin, and the percentages were mutation frequencies of L1035F.
transmembrane proteins responsible for the rapidly
rising phase of action potentials that is critical for
electrical signaling in most excitable cells. Because of their
crucial role in membrane excitability, sodium channels
are the target of a great variety of neurotoxins,
including insecticidal pyrethrins . Pyrethroid insecticides
bind to insect para-sodium channels locking them in
an open state thereby disrupting neuronal signaling
resulting in paralysis and death.
Point mutations in the para-sodium channel gene
that make sodium channel insensitive to pyrethrins are
an important mechanism of resistance to pyrethroid
insecticides in various insect species [15-22]. The most
common mutation is the substitution of leucine by
phenylalanine at residue L1014, commonly referred to
as the kdr (knockdown resistance) mutation. Resistance
conferred by the L1014F mutation was first described
in the Italian housefly with respect to DDT  and
subsequently named knockdown resistance . Variants
of this mutation such as L1014S, L1014H, L1014W and
L1014C have also been reported [25-28]. Another
mutation, M918T, has also been found in many insects [29-31].
This mutation and the L1014F mutation often occur
together in which case they confer what has been
called super-kdr resistance.
One approach to investigating pesticide resistance in
mosquitoes is to first identify mutations that could
potentially confer resistance and then attempt to correlate
the frequency of these with actual pesticide resistance.
Through a combination of insecticide bioassays and
molecular techniques, we identified mutations associated
with resistance to deltamethrin and cyhalothrin in six
wild Chinese populations of Cx. p. quinquefasciatus and
assessed the similarity between these and previously
documented mutations in the sodium channel gene.
Knowledge of these mutations may have practical benefits
for further research on pesticide resistance in mosquitoes
and for designing resistance management programs.
Cx. p. quinquefasciatus larvae and some female adults
were collected from six different field locations: Haikou
Poxiang (E1101933.79, N195955.07), Haikou Changliu
(E1101150.36, N20050.25), Qionghai Boao (E11034
57.13, N190942.07) and Sanya Fenghuang (E10926
54.38, N18182.91) in 2012; Guangdong Zhanjiang
(E1102353.05, N211130.19) and Hainan Lingshui
(E110215.01, N183021.77) in 2013 (Figure 1). Larvae
were reared to adulthood in captivity but the wild caught
female adults were frozen in liquid nitrogen for subsequent
genetic testing. The control population for insecticide
bioassays was a laboratory strain that had not been exposed to
insecticides for more than 10 years.
Bioassays were conducted by putting thirty late 3rd or
early 4th instar larvae into pans containing 199 ml water
and 1 ml of either deltamethrin or cyhalothrin. The
insecticides were added to each pan using an automatic pipette
according to the methods specified by the WHO . Larval
Figure 2 Schematic diagram of amplification of the sodium
channel gene of Cx. p. quinquefasciatus. The complete gene
sequence was 6450 bp, and the genes six sections are indicated
Table 1 The four pairs of specific primers used to
amplify sodium channel gene mutations detected in
Cx. p. quinquefasciatus
mortality was recorded 24 h after each treatment. No food
was offered to the larvae during bioassays. Larvae were kept
in a laboratory under the following conditions: 14 L:10D
photoperiod, 75% relative humidity and a temperature
of 26 1C during bioassays. Bioassays of each
insecticide were repeated three times. Statistical analyses were
performed using SPSS software version 13.
Extraction of RNA, cDNA synthesis and PCR amplification
Total RNA was extracted from mosquitoes from each
population with Trizol reagent (GBT) following the
manufacturers protocol and cDNA was synthesized
from the extracted total RNA with a cDNA synthesis
kit (Transgen Biotech). The synthesized cDNA was stored
at 20C. Gene-specific primers from Zhao et al. (2014)
used to amplify the sodium channel gene of specimens
from each population . The sodium channel gene is
6450 bp and is divided into six sections (Figure 2).
Cloning and sequencing of PCR products
To identify mutations in the sodium channel gene, the
cDNA of a pooled sample of Cx. p. quinquefasciatus
comprised of specimens from each of the six populations
was cloned and sequenced. PCR products were purified
using a universal DNA purification kit (TIANGEN) and
the purified products were ligated into the pEASY-T1
vector (TRANSGEN). The recombinant plasmids were then
cloned into Trans1-T1 competent cells (TRANSGEN).
The microbials were spread on LB solid medium (including
ampicillin, X-gal, IPTG) and cultured overnight. White
clones were selected, placed in LB liquid medium and
cultured to turbidity. Positive clones were identified by
PCR using M13 forward and reverse primers and
sequenced by Tianyi Biotech . Based on the discovery
of clones, the allele frequency of each mutation was
determined by specific PCR amplification and sequencing
of the mutations found in each population. In this
procedure, a single mosquitos RNA was extracted and
reverse transcribed to cDNA, then amplified by specific
PCR before being sequenced. The gene-specific primers
used to amplify the mutations in the Cx. p. quinquefasciatus
para-sodium channel gene were designed in NCBI Primer
BLAST (Table 1).
Analysis of genetic linkage between mutations
We use the GENEPOP software package to estimate
conformity to the HWE and genetic linkage between
Table 2 Levels of deltamethrin and cyhalothrin resistance measured in Cx. p. quinquefasciatus
LC50 (ppm) (95% CL)1
(LA = Lab strain; CL = Haikou Changliu; PX = Haikou Poxiang; BA = Qionghai Boao; FH = Sanya Fenghuang; ZJ = Guangdong Zhanjiang; LS = Hainan Lingshui.
1CL = confidence limits, 2RR = Resistance Ratio).
0.001 (0.001, 0.001)
0.001 (0.001, 0.001)
0.009 (0.006, 0.011)
0.014 (0.010, 0.017)
0.428 (0.385, 0.476)
0.396 (0.332, 0.468)
0.186 (0.151, 0.231)
0.215 (0.172, 0.264)
1.019 (0.811, 1.268)
1.291 (1.051, 1.560)
0.036 (0.031, 0.041)
0.042 (0.037, 0.048)
0.312 (0.234, 0.413)
0.641 (0.520, 0.800)
Y = 7.412 + 2.371x
Y = 6.819 + 2.356x
Y = 3.028 + 1.475x
Y = 2.898 + 1.551x
Y = 0.842 + 2.284x
Y = 0.610 + 1.517x
Y = 1.314 + 1.799x
Y = 0.761 + 1.139x
Y = 0.012 + 1.439x
Y = 0.165 + 1.484x
Y = 3.073 + 2.127x
Y = 3.048 + 2.215x
Y = 0.630 + 0.541x
Y = 0.221 + 0.497x
Figure 3 Alternative splicing in the sodium channel gene of
Cx. p. quinquefasciatus. CX1 was the sequence of Cx. p.
quinquefasciatus (GenBank Accession No.: AB453977.1). CX2 was
the sequence of Cx. p. quinquefasciatus in China. The dots
indicated the alternative splicings. In the 517570, there were
three kinds of alternative splicings (517550, 542550, and
542570); In the 11061139, there were also three kinds of
alternative splicings (11061118, 11061128, and 11061139).
Correlation of pesticide resistance with the frequencies of
The resistance (LC50) of the six populations to deltamethrin
and cyhalothrin was determined by bioassay and the
allele frequencies of the various mutations determined by
gene-specific amplification and sequencing as described
above. The LC50 of a laboratory strain that had not been
exposed to either pesticide was also determined to serve
as a control. Correlations between resistance and mutation
frequency were analyzed using Graphpad Prism 5.
Resistance to deltamethrin and cyhalothrin
LC50 values of the seven different populations ranged
from 0.009 to 1.019 ppm for deltamethrin and from
0.014 to 1.291 ppm for cyhalothrin (Table 2). The CL
strain was the most susceptible to both insecticides. The
FH strain was the most resistant; 1019 times more resistant
to deltamethrin and 1291 times more resistant to
cyhalothrin than the laboratory strain (LC50 0.001 ppm).
Sodium channel gene mutations
Seven mutations (V250M, R436K, M943V, I973T, L1035F,
L1035S and E1901D) were identified. Because these seven
mutations were identified in a pooled sample they did not
necessarily occur in the same individuals. The seven
mutations were V250M: GTG ATG, R436K: AGG AAG,
M943V: ATG GTG, I973T: ATC ACA, L1035F:
TTA TTT, L1035S: TTA TCA, and E1901D: GAG
GAT. In addition, eleven alternative splices in the sodium
channel gene were detected (Figure 3).
Genetic and genotypic frequencies
Genetic and genotypic frequencies were showed in Table 3.
Analysis of genetic linkage between mutations
The results of HWE were showed in Tables 4. A significant
(P > 0.05) heterozygote deficit was detected in only one
(the LS) population, but there was evidence of heterozygote
excess in some populations. The frequency of four
mutations (R436K, M943V, I973T, and L1035F/S) significantly
deviated from the HWE (P < 0.01) across all populations.
The results of analysis of linkage disequilibrium between
mutations are shown in Table 5. Significant linkage
disequilibrium was found between M943V and I973T (P < 0.01).
Table 3 Gene and genotype frequencies of seven sodium
channel gene mutations calculated in six populations of
Cx. p. quinquefasciatus
Table 3 Gene and genotype frequencies of seven sodium
channel gene mutations calculated in six populations of
Cx. p. quinquefasciatus (Continued)
ZJ 30 83.3 16.7 0.00 8.33
CL 22 40.9 59.1 0.00 29.6
LS 38 34.2 41.1 23.7 44.7
(S = the amino acid sequence of the sodium channel gene listed in GenBank
(Accession No.: AB453977.1). R = mutant variants of S. SS = S
homozygotes, RS = heterozygotes, and RR = R homozygotes).
1SS frequency = SS (SS + RS + RR) 100%; 2RS frequency = RS (SS + RS +
3RR frequency = RR (SS + RS + RR) 100%; 4R frequency = (RR + 1/2RS)
(SS + RS + RR) 100%.
The L1035F/S mutation could also be linked with the
M943V and I973T mutations because the P-values for these
combinations are only slightly above 0.05. And sequencing
data confirms the existence of linkage in some mosquitoes.
Correlation between resistance and mutation frequencies
The degree and significance of correlation between
resistance to deltamethrin and cyhalothrin and the frequency of
each mutation is shown in Table 6. The frequency of the
L1035F mutation was significantly correlated with resistance
to both deltamethrin (R2 = 0.536, P = 0.049) and cyhalothrin
(R2 = 0.626, P = 0.030), and the combined frequency of the
L1035F and L1035S mutations was significantly correlated
with resistance to deltamethrin (R2 = 0.661, P = 0.025),
and even more significantly correlated with resistance
to cyhalothrin (R2 = 0.803, P = 0.008) (Figures 4, 5, 6
and 7). Frequencies of all other mutations were not
correlated with either deltamethrin or cyhalothrin resistance.
Significant cross-resistance between the two insecticides
was also detected (R2 = 0.930, P < 0.01) (Figure 8).
Insecticides have been used to control mosquitoes for
more than half a century. Their indiscriminate use has,
however, resulted in high levels of insecticide resistance
in many mosquito species [8-10]. We tested the resistance
of six Chinese Cx. p. quinquefasciatus populations to
deltamethrin and cyhalothrin. Our results show that,
compared to a susceptible laboratory strain, these six
populations displayed a 9- to 1019-fold resistance to
deltamethrin, and a 14- to 1291-fold resistance to
cyhalothrin. The high resistance of the FH population may
be related to its breeding habitat. This population
frequently breeds in pools of water in peasant courtyards
that are often polluted by insecticides.
The frequent use of insecticides has created an intense
selection pressure for traits that confer resistance to them,
such as changes in behavior, epidermal structure, metabolic
enzymes and target-site mutations. Resistance may be
conferred by the development of one, or more, of these
traits. Osta et al. found that the dramatic reduction in the
Table 4 P-value of HWE test for heterozygote deficiency and excess confirmed at six loci in six Cx. p. quinquefasciatus
frequency of the G119S mutation in Cx pipiens mosquitoes
was probably due to the increased use of pyrethroids over
organosphosphate insecticides . Therefore, alternating
between different kinds of insecticide is one way of
reducing the development of resistance to any one type.
We identified seven point mutations at six loci and
eleven alternative splices in the sodium channel gene of
Chinese Cx. p. quinquefasciatus. The HardyWeinberg
equilibrium (HWE) describes the theoretical frequency
of two alleles at the same locus, in the absence of
mutation and selection, in an indefinitely large population
with discrete generations after one generation of random
mating . The results of HWE tests suggested that
mutations at four of these six loci significantly deviated
from the HWE (P < 0.05). Among these, the R436K
mutation occurred at a frequency of nearly 50% in all
six populations and there was an excess of
heterozygotes with this mutation (P < 0.05). How this mutation
Table 5 Chi-squared test results showed linkage between
pairs of loci across six populations of Cx. p.
quinquefasciatus (Fishers method)
developed and its role, if any, in pesticide resistance
requires further investigation.
Mutations at three other loci (M943, I973 and L1035)
also deviated from the HWE, and some populations had
an excess of heterozygotes with these mutations. L1014
(L1035 according to our sequencing) is a classical
mutation associated with the use of pyrethroid insecticides.
One reason why the previous three mutations deviate
from the HWE may be because a long period of
selection has led to them becoming fixed. Mutations at two
other loci (V250M and E1901D) did not deviate from
We found evidence of significant linkage between the
M943V and I973T mutations (P < 0.01) and P-values
for a Chi-squared test of association between L1014F/S
and M943V and I973T were also only slightly above
0.05. Our sequencing data support linkage between
Table 6 Correlations calculated between frequencies of
sodium channel gene mutations in Cx. p. quinquefasciatus
and resistance to deltamethrin and cyhalothrin
Mutations Insecticide R (95% CL) R2
V250M & R436K
V250M & M943V
R436K & M943V
V250M & I973T
R436K & I973T
M943V & I973T
V250M & L1035F/S
R436K & L1035F/S
M943V & L1035F/S
I973T & L1035F/S
V250M & E1901D
R436K & E1901D
M943V & E1901D
I973T & E1901D
L1035F/S & E1901D
CL = confidence limits.
Figure 4 Linear regression of the relationship between the
frequency of the L1035F mutation and deltamethrin resistance.
these mutations in some mosquitoes but this requires
The V250M mutation is the first to be discovered in the
Cx. p. quinquefasciatus sodium channel genes IS4 domain,
in which mutations rarely occur. The frequency of this
mutation was not correlated with resistance to either
deltamethrin or cyhalothrin so further research is required to
determine its function, if any, in pesticide resistance.
Our results provide the first confirmation of the R436K
mutation, located between the first and second homology
domains of the sodium channel gene near the IS6 domain,
in Cx. p. quinquefasciatus. The E434K mutation was first
identified near the IS6 domain of the sodium channel gene
in the German cockroach. This mutation is associated
with two other mutations, C764R and L993F (L1014F),
that are closely linked to resistance to the pyrethroid
insecticide cypermethrin . We did not find any
correlation between the frequency of this mutation and pyrethroid
Figure 6 Linear regression of the relationship between the
frequency of the L1035 F + S mutation and deltamethrin resistance.
resistance. However, almost all individuals with this
mutation were heterozygotes and its frequency was almost
50% across all populations. The reasons for its prevalence
require further investigation.
The M943V and I973T mutations are located at the
junctions between IIS4 and IIS5, and between IIS5 and
IIS6, in the sodium channel gene and were significantly
linked (P < 0.01). The M943V mutation is only three
amino acids from the M918T site (the amino acid number
in the housefly sodium channel gene, GenBank Accession
No.: X96668). The M918T mutation is usually
associated with the L1014F mutation in which case it confers
super-kdr resistance. However, some studies have
reported super-kdr resistance in the absence of the
L1014F mutation. For example, Benjamin et al. found
that the M918T mutation greatly increased the resistance
of Tetranychus evansi to pyrethroid insecticides in the
absence of the L1014F mutation .
Figure 5 Linear regression of the relationship between the
frequency of the L1035F mutation and cyhalothrin resistance.
Figure 7 Linear regression of the relationship between the
frequency of the L1035 F + S mutation and cyhalothrin resistance.
Figure 8 Linear regression of the relationship between deltamethrin
and cyhalothrin resistance (LC50) in Cx. p. quinquefasciatus.
The I874M mutation (corresponding to the M918
position in the housefly) in mammals can result in a
100-fold desensitization of the sodium channel .
Another study identified a methionine to valine replacement
at this position in the Bemisia tabaci para-sodium channel
gene that was unrelated to resistance . We also detected
a methionine to valine replacement at the fourth amino acid
after the M918 position in all six Cx. p. quinquefasciatus
populations that was not correlated with deltamethrin
or cyhalothrin resistance. How this mutation arose, its
connection with the M918 point mutation, linkage
with the I973T mutation and role, if any, in resistance,
requires further investigation.
L1035 is the site of the classic knockdown resistance
mutation L1014 (amino acid number in the housefly
sodium channel gene, GenBank Accession No.: X96668). It
is located in the IIS6 domain of sodium channel, and
has been relatively well researched. Subsequent studies
have identified mutations associated with knockdown
resistance in a variety of insects, for example, Aphis
gossypii , the German cockroach , Myzus persicae
 Triatoma infestans , Anopheles gambiae, Anopheles
stephensi and Anopheles arabiensis . In addition, recent
studies have that the L1014S mutation is associated
with permethrin, cypermethrin, cyhalothrin and DDT
resistance in Anopheles sinensis, An. vagus and An.
peditaeniatus, ; with deltamethrin resistance in Cx
p. pallens , and with permethrin and DDT resistance
in Anopheles gambiae [13,43].
Another study found a new mutation, V1010L, that is
closely linked to the L1014S mutation in Anopheles
culicifacies but did not mention if it was associated with
resistance . As previously mentioned, the co-occurrence of
the L1014F and M918T mutations can lead to super-kdr
resistance in many insects. For example, these two mutations
are closely related to cyhalothrin and permethrin resistance
in Haematobia irritans ; and resistance to a suite
(permethrin, bifenthrin, tefluthrin, deltamethrin,
cypermethrin, cyhalothrin and fluvalinate) of pyrethoid insecticides
in Myzus persicae . No one has so far, however, detected
super-kdr resistance in a mosquito species.
In Drosophila melanogaster, the L1014F, M918T and
T929I mutations affect the sodium channel currents in the
presence of deltamethrin and permethrin, suggesting that
these mutations confer resistance to those pesticides .
Other authors have found the above three mutations in the
sodium channel gene of pyrethroid-resistant Thrips tabaci
 and identified the L1014H, L1014C and L1014W
Studies have showed mutations in L1014 could lead to
a reduction in susceptibility to a variety of pyrethroids
and a decay of tail current. And then lead to develop
resistance [48,49]. We found that the L1035F and L1035S
mutations were present in all six Cx. p. quinquefasciatus
populations we sampled and that these mutations were
significantly correlated with deltamethrin and cyhalothrin
resistance. This may be related to the frequent use of these
pesticides at our sampling sites. In addition, we found that
the frequency of the leucine to serine replacement in all six
populations was lower than that of the L1035F mutation.
The combined frequency of the L1035F and S mutations
was highly correlated with resistance, especially cyhalothrin
resistance (R2 = 0.803, P < 0.01), which suggests that
mosquitoes in the sampled populations have developed
additional mutations except the L1035F mutation in response
to strong selection pressure.
This study was the first to detect the E1901D
mutation, located on the sodium channel C-terminal tail, in
Cx. p. quinquefasciatus. There has, so far, been
relatively little research on this fragment but it appears to
be unrelated to deltamethrin and cyhalothrin
resistance. Determining the function of such mutations will
require further study.
We confirmed eleven alternative splices in the sodium
channel gene of Cx. p. quinquefasciatus. These reflect
dynamic change and higher level reassembly of genetic
information that can greatly increase the richness of the
coding sequence without changing genomic DNA .
Liu et al. (2012) found thirteen different sodium channel
variants in Cx. p. quinquefasciatus and demonstrated
that this alternative splicing was related to pyrethroid
resistance . The eleven alternative splices we found
differed from those found by Liu et al. They may have
some significance in the rich diversity of the sodium
channel protein, but further research is required to
determine their role, if any, in pyrethroid resistance.
In the study, seven amino acid alterations were
found (V250M, R436K, M943V, I973T, L1035F, L1035S
and E1901D) in the sodium channel gene of Cx. p.
quinquefasciatus from six field populations in China.
Among them, the L1035F and L1035S mutations
frequencies were associated with resistance to
deltamethrin and cyhalothrin. And the M943V and I973T
mutations were significant linkage with each other in the
sodium channel gene. Identifying these mutations may be
of practical benefit to the development of pesticide
resistance management programs.
The authors declare that they have no competing interests.
MHZ carried out the molecular genetic studies, participated in the sequence
alignment and drafted the manuscript. The experiments conceived and
designed by MHZ CXL YDD XXG YMZ DX TYZ, performed the experiments
by MHZ XR ZMW TY XJZ JXS WJH. The data analyzed by MHZ CXL GW HDZ.
All authors read and approved the final manuscript.
This work was funded by grants from the Infective Diseases Prevention and
Cure Project of China (No.2008ZX10004 and No.2012ZX10004219).
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