Insect Molecular Biology (2017) 00(00), 00–00
12309
Point mutations in acetylcholinesterase 1 associated
with chlorpyrifos resistance in the brown planthopper,
Nilaparvata lugens Stål
Y. Zhang*, B. Yang*†, J. Li*, M. Liu* and Z. Liu*
*Key Laboratory of Integrated Management of Crop
Diseases and Pests (Ministry of Education), College of
Plant Protection, Nanjing Agricultural University, Nanjing,
China; and †Rice Technology Research and Development
Center, China National Rice Research Institute,
Hangzhou, China
Abstract
Insecticide resistance frequently results from target-
site insensitivity, such as point mutations in acetyl-
cholinesterases (AChEs) for resistance to organo-
phosphates and carbamates. From a field-originated
population of Nilaparvata lugens, a major rice pest, a
resistant population (R9) was obtained by nine-
generation continuous selection with chlorpyrifos.
From the same field population, a relatively suscepti-
ble population (S9) was also constructed through
rearing without any insecticides. Compared to
the susceptible strain, Sus [medium lethal dose
(LC50) 5 0.012 mg/l], R9 had a resistance ratio (RR) of
253.08-fold, whereas the RR of S9 was only 2.25-fold.
Piperonyl butoxide and triphenyl phosphate syner-
gized chlorpyrifos in R9 less than three-fold, indicat-
ing other important mechanisms for high resistance.
The target-site insensitivity was supported by the key
property differences of crude AChEs between R9 and
S9. Compared to S9, three mutations (G119S, F331C
and I332L) were detected in NlAChE1 from individuals
of the R9 and field populations, but no mutation was
detected in NlAChE2. G119S and F331C could
decreased insecticide sensitivities in recombinant
NlAChE1, whereas I332L took effect through increas-
ing the influence of F331C on target insensitivity.
F331C might be deleterious because of its influence
Correspondence: Zewen Liu, College of Plant Protection, Nanjing Agricul-
tural University, Weigang 1, Nanjing 210095, China. Tel./fax: 1 86 25
84399051; e-mail: liuzewen@njau.edu.cn
C 2017 The Royal Entomological Society
V 1
doi: 10.1111/imb.
on the catalytic efficiency of NlAChE1, whereas I332L
would decrease these adverse effects and maintain
the normal functions of AChEs.
Keywords: Nilaparvata lugens, acetylcholinesterase,
chlorpyrifos, insecticide resistance.
Introduction
The brown planthopper (BPH), Nilaparvata lugens, is a
major insect pest of rice crops throughout Asia, with
direct and indirect deleterious effects such as reducing
plant growth, wilting and leaf chlorosis (Gorman et al.,
2008). In addition to direct sucking, oviposition and virus
disease transmission by N. lugens result in more severe
damage to rice plants (Wang et al., 2008). Chemical
control has thus far been the primary strategy used to
manage N. lugens; however, N. lugens has developed
resistance towards many kinds of insecticides. For
example, high or super-high resistance to imidacloprid
has been detected in both lab-selected strains and field
populations (Liu et al., 2005; Wen et al., 2009; Zhang
et al., 2014; Garrood et al., 2016). Given the high levels
and geographical distribution of imidacloprid resistance
in fields since 2006, imidacloprid application has dramat-
ically reduced and is not recommended for wide use in
controlling N. lugens in China (Wang et al., 2008; Wen
et al., 2009). Pymetrozine, an azomethine pyridine
insecticide, has been commonly used for N. lugens con-
trol in China recently. However, field resistance has also
been reported to pymetrozine (Zhang et al., 2014).
Therefore, insecticides belonging to organophosphate or
carbamate classes have also been used in China in
recent years, such as chlorpyrifos, to provide more
choices of available insecticides for N. lugens control
and decrease the stress due to the only application of
pymetrozine as well as imidacloprid and other neonicoti-
noid insecticides in resistance development (Wang
et al., 2009; Lin et al., 2011).
2 Y. Zhang et al.

Table 1. Chlorpyrifos toxicity against different strains and populations Table 2. Influence of three synergists on chlorpyrifos toxicity against
different populations
strain LC50 (mg/l) Slope RR
strain synergist LC50 (mg/l) Slope RR SR
Sus 0.012 (0.011–0.014) a 3.326 6 0.275 1.00
S9 0.027 (0.024–0.032) b 2.744 6 0.380 2.25 S9 Chlorpyrifos 0.027 (0.024–0.032) a 2.744 6 0.380 2.25 /
R9 3.037 (2.612–3.595) e 2.403 6 0.296 253.08 1PBO 0.022 (0.019–0.027) a 1.965 6 0.224 1.86 1.21
FG 0.202 (0.166–0.247) c 1.873 6 0.282 16.83 1TPP 0.026 (0.022–0.031) a 2.130 6 0.175 2.14 1.05
FN 0.714 (0.573–0.882) d 1.592 6 0.314 59.50 1DEM 0.029 (0.025–0.035) a 2.366 6 2.267 2.39 0.94
FJ 0.881 (0.726–1.060) d 1.618 6 0.321 73.42 R9 Chlorpyrifos 3.037 (2.612–3.595) f 2.403 6 0.296 253.08 /
1PBO 1.133 (0.966–1.301) d 1.536 6 0.228 94.43 2.68
Different letters in medium lethal dose (LC50) column indicate significant 1TPP 1.766 (1.522–2.113) e 1.804 6 0.255 147.14 1.72
differences amongst populations. 1DEM 2.949 (2.630–3.458) f 2.027 6 0.261 245.71 1.03
Data are means of at least three independent experiments. FN Chlorpyrifos 0.714 (0.573–0.882) c 1.592 6 0.314 59.50 /
For LC50 values, 95% confidence intervals are provided in parentheses. 1PBO 0.353 (0.268–0.444) b 1.322 6 0.291 29.46 2.02
For Slope values, SEM values are provided. 1TPP 0.533 (0.411–0.675) bc 1.403 6 0.225 44.40 1.34
FG, FN, FJ, field populations from Guilin, Nanjing and Jiujiang, respectively; 1DEM 0.752 (0.596–0.920) c 1.634 6 0.320 62.63 0.95
RR, resistance ratio; R9, resistant strain; Sus, susceptible strain; S9, rela-
tively susceptible strain. Different letters in medium lethal dose (LC50) column indicate significant
differences amongst populations.
Data are means of at least three independent experiments.
For LC50 values, 95% confidence intervals are provided in parentheses.
Following activation by desulphurases, chlorpyrifos- For Slope values, SEM values are provided.
oxon acts as an inhibitor of insect acetylcholinesterases FN, field population from Nanjing; R9, resistant strain; S9, relatively sus-
(AChEs, EC 3.1.1.7). AChEs hydrolyse the neurotrans- ceptible strain; PBO, piperonyl butoxide; TPP, triphenyl phosphate; DEM,
diethyl maleate; RR, resistance ratio; SR, synergism ratio.
mitter acetylcholine in the cholinergic system and termi-
nate nerve impulses (Toutant et al., 1988). Two mutations in chlorpyrifos resistance were evaluated by
acetylcholinesterase genes (ace) loci are present in comparing NlAChE1 wild-type and mutants in vitro.
many insect species, encoding two distinct AChEs
(AChE1 and AChE2), of which ace1 is orthologous to Results
Drosophila ace (Kim & Lee, 2013). Insect AChE1 has
Susceptibility of N. lugens populations to chlorpyrifos
been proposed as the major catalytic enzyme because
of the high level expression and frequent point mutations To determine the main mechanisms underlying chlorpyri-
associated with insecticide resistance. Kim & Lee (2013) fos resistance in N. lugens, chlorpyrifos toxicity was exam-
compared the catalytic activities between two AChEs ined in a resistant population R9 and a susceptible
amongst 100 insect species. AChE1 showed higher population S9, both derived from the same original popula-
activity than AChE2 in 67 species, but the opposite tion. S9 was relatively susceptible, with a resistance ratio
results were observed in the other 33 species, indicating (RR) of 2.25-fold compared to the susceptible laboratory
that the two AChEs differ in importance amongst insect strain Sus [medium lethal dose (LC50) 5 0.012 mg/l, Table 1].
species and require evaluation in each case. R9 was highly resistant, with an RR of 253.08-fold compared
Several mutations conferring insect target-site insensi- to Sus and 112.48-fold compared to S9. Not surprisingly,
tivity to organophosphate or carbamate insecticides chlorpyrifos resistance was also detected in field populations,
have been reported, and almost all mutations are on with RRs of 16.83- to 73.42-fold (Table 1).
AChE1 (Fournier & Mutero, 1994; Russell et al., 2004;
Fournier, 2005). Hemipteran species are believed to Determination of synergistic effects in different N. lugens
have two AChEs, and AChE1 is the major catalytic populations
enzyme in many species (Kliot & Ghanim, 2012; Kim & The tested synergists did not show any effect on chlorpyri-
Lee, 2013). Point mutations in AChE1 have been found fos toxicity in the susceptible population S9, but both
to be associated with insecticide resistance in Hemiptera piperonyl butoxide (PBO) and triphenyl phosphate (TPP)
such as aphids, rice planthoppers and whiteflies (Nabe- significantly synergized chlorpyrifos against the resistant
shima et al., 2003; Li & Han, 2004; Alon et al., 2008; population R9 with synergistic ratios (SRs) of 2.68- and
Kwon et al., 2012a). In N. lugens, four mutations 1.72-fold, respectively (Table 2). PBO also showed
(G119A, F/Y330S, F331H and I332L) were identified in obvious synergistic effects on chlorpyrifos toxicity in the
NlAChE1 and these mutations were believed to be asso- field population from Nanjing (FN), with an SR of 2.02-fold.
ciated with resistance to carbofuran, a carbamate insec-
ticide (Kwon et al., 2012a,b). In the present study, Characterization of AChE preparations from different
similar mutations (G119S, F331C and I332L) at three populations
loci of NlAChE1 were detected in a laboratory-selected, To examine the potential role of NlAChE insensitivity in
chlorpyrifos-resistant population, and the roles of these chlorpyrifos resistance, the biochemical properties of

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Table 3. Kinetics of substrate hydrolysis (acetylthiocholine iodide) and sensitivity to chlorpyrifos of crude acetylcholinesterases (AChEs)
strain KM (mM)
Sus 21.92 6 2.16 a
S9 23.76 6 2.49 a
R9 39.88 6 3.60 c
FG 24.35 6 3.72 ab
FN 28.48 6 3.24 b
FJ 29.27 6 4.13 b
Different letters in the same column indicate significant differences amongst crude AChEs prepared from different populations.
Data are mean 6 SEM of at least five independent experiments.
KM, Michaelis-Menten constant; Vmax, maximum velocity; IC50, medium inhibitory concentration; FG, FN, FJ, field populations from Guilin, Nanjing and Jiu-
jiang, respectively; R9, resistant strain; Sus, susceptible strain; S9, relatively susceptible strain.
crude NlAChEs prepared from different populations were
determined. As expected, the key parameters in S9 and
Sus were similar (Table 3). The crude NlAChEs in R9
had lower affinity (Michaelis-Menten constant, KM) to the
model substrate acetylthiocholine iodide (ATChI) and a
lower maximum reaction velocity (Vmax) value, which
consequently decreased the catalytic efficiency (Vmax/
KM) (Table 3). Amongst the three field populations (FG:
Guilin, FN: Nanjing, FJ: Jiujiang), the key parameters in
FN and FJ, two populations with relatively high resist-
ance, were different from those of S9 and Sus. These
results indicate that some factors changed the biochemi-
cal properties of endogenous NlAChEs in R9 and these
two field populations. In terms of the inhibition of
chlorpyrifos-oxon, the active metabolite of chlorpyrifos,
on the crude NlAChEs, the medium inhibitory concentra-
tion (IC50) values for AChEs from R9 (14.86 3 1028 M)
were 7.32 times that of S9 (2.03 3 1028 M), displaying
significant insensitivity in endogenous NlAChEs in R9.
AChE insensitivity was also observed in the three field
populations, although the fold-change in IC50 values was
not as high as that in the R9 population (Table 3).
Detection of mutations in NlAChEs in the resistant and
field populations
In order to detect potential mutations in two N. lugens
AChE-encoding genes, specific primers (Table S1) were
designed to amplify the full open reading frames (ORFs)
of Nlace1 (HQ605041) and Nlace2 (JN688930). A com-
parison of the sequences from 30 individuals from R9 and
S9, respectively, revealed potential mutations only in
Nlace1, the gene encoding the protein NlAChE1, such as
the G119S mutation (heterozygote and homozygote, the
same for other mutations) in eight individuals (26.67% in
frequency), F331C in seven individuals (23.33%) and
I332L in five individuals (16.67%), amongst which the dou-
ble mutation F331C/I332L was detected in three individu-
als (10.00%; Fig. 1, Table 4). Interestingly, neither F331C
nor I332L was found in individuals containing the G119S
mutation.
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Chlorpyrifos resistance in Nilaparvata lugens 3
Vmax/KM IC50 (31028 M)
Vmax [nmol
(mg
min)21] [ml
(mg
min)21] for chlorpyrifos-oxon
33.72 6 3.15 a 1538.32 1.59 6 0.22 a
34.24 6 3.10 a 1441.08 2.03 6 0.38 a
12.82 6 2.23 c 321.46 14.86 6 3.27 d
29.51 6 3.76 ab 1211.91 3.19 6 0.53 b
27.32 6 3.45 b 959.27 6.58 6 1.40 c
28.16 6 3.09 b 962.08 7.75 6 1.94 c
Subsequently, specific primers (Table S1) were
designed to amplify the fragments containing sites for
these mutations in NlAChE1. The fragments were
sequenced in 50 individuals from each population. No
mutation mentioned above was detected in S9 com-
pared to the sequences from the Sus strain (Table 4). In
contrast, the frequencies for G119S, F331C and I332L
in R9 were 34, 28 and 22%, respectively. Amongst 50
insects examined from R9, the frequency for the double
mutation F331C/I332L was 16%. These mutations
(G119S, F331C and I332L) were also detected in the
three field populations, although G119S was the only
mutation detected in the FG population. No single indi-
vidual containing the double mutations G119S/F331C
and G119S/I332L or all three mutations was observed in
any population examined.
Kinetics analysis of recombinant NlAChEs
NlAChE1, NlAChE2 and NlAChE1 mutants containing
different mutations (NlAChE1G119S, NlAChE1F331C, NlA-
ChE1I332L and NlAChE1F331C/I332L) were successfully
expressed in insect Spodoptera frugiperda (Sf9) cells.
The peak activities for all recombinant NlAChEs were
detected at 72 h after the infection of Sf9 cells. The key
biochemical properties of the recombinant NlAChEs
were determined by kinetics analysis and subsequently
compared.
Figure 1. Amino acid replacements and nucleotide changes for point
mutations in Nilaparvata lugens acetylcholinesterase 1 (NlAChE1). WT
indicates the sequence of the wild-type of NlAChE1, which is susceptible
to chlorpyrifos. The mutated sequences G119S and F331C/I332L are indi-
cated by underlines, and the bold letters indicated the mutated amino
acids or bases. The asterisks (*) indicate that the nucleotide sequence is
the same as the one above.
4 Y. Zhang et al.

Table 4. The number of individuals containing different mutations in each Table 6. Sensitivity indices (Ki) of recombinant acetylcholinesterases
population (AChEs) to the inhibitor and insecticides

Mutation Sus S9 R9 FG FN FJ Compound Eserine Chlorpyrifos-oxon Carbofuran

G119S 0 0 17 (4) 2 (0) 5 (0) 5 (1) NlAChE1 4.43 6 0.38 a 8.64 6 1.22 a 3.13 6 0.28 a
F331C 0 0 14 (1) 0 4 (0) 5 (0) NlAChE1G119S 2.51 6 0.45 b 2.23 6 0.49 b 1.29 6 0.34 b
I332L 0 0 11 (0) 0 1 (0) 3 (0) NlAChE1F331C 1.37 6 0.24 c 1.39 6 0.33 c 1.02 6 0.25 b
G119S/F331C 0 0 0 0 0 0 NlAChE1I332L 4.06 6 0.53 a 7.01 6 1.61 a 2.82 6 0.40 a
G119S/I332L 0 0 0 0 0 0 NlAChE1F331C/I332L 0.48 6 0.07 e 0.35 6 0.08 d 0.32 6 0.09 c
F331C/I332L 0 0 8 (0) 0 0 1 (0)
G119S/F331C/I332L 0 0 0 0 0 0 Different letters in the same column indicate significant differences
amongst recombinant AChEs.
In each population, 50 individuals were tested. Data are mean 6 SEM of at least five independent experiments.
All digits in the table (except those in parentheses) indicate the heterozy- Nl, Nilaparvata lugens; Ki, inhibition constant.
gote numbers in 50 test individuals from different populations.
The digits in parentheses indicate the mutant homozygote numbers in 50
test individuals from each population. and the two insecticides, but I332L did not show such an
FG, FN, FJ, field populations from Guilin, Nanjing and Jiujiang, respectively;
influence (Table 6). The double mutation F331C/I332L
R9, resistant strain; Sus, susceptible strain; S9, relatively susceptible
strain. could further decreased the sensitivity of NlAChE1 to
both inhibitor and insecticides compared to the two single
mutations F331C and I332L. For chlorpyrifos-oxon,
Of the two wild-type NlAChEs, NlAChE2 showed
F331C increased IC50 (31028 M) from 1.33 6 0.22 for
higher affinity to the model substrate ATChI, but the
NlAChE1 to 9.35 6 1.14 for NlAChE1F331C, whereas the
Vmax of NlAChE1 was 15.9 times higher than that of
double mutation F331C/I332L increased the IC50 value to
NlAChE2, which endowed NlAChE1 with much higher
35.71 6 6.02, although I332L alone did not cause a signif-
catalytic efficiency (4.4 times) than that of NlAChE2
icant change in IC50 (Fig. 2).
(Table 5). Amongst the three point mutations in
NlAChE1, only F331C caused a significant decrease in
the substrate affinity (KM value). Compared to the wild- Discussion
type NlAChE1, all three mutations when present singly
Most insect species possess two different AChEs,
decreased the Vmax value, amongst which F331C
although only one AChE is present in true flies, the
decreased Vmax to less than half (41.54%) of that of
monophyletic Cyclorrhapha in Diptera (Huchard et al.,
NlAChE1. Interestingly, NlAChE1F331C/I332L showed
2006). In insect species with two AChEs, AChE1 has
higher substrate affinity than NlAChE1, with a Vmax
been proposed as a major catalytic enzyme because
value similar to that of NlAChE1, indicating that NlA-
AChE1 has higher catalytic activity than AChE2 and the
ChE1F331C/I332L had a much higher catalytic efficiency
ace1 gene has higher expression level than ace2 (Kim &
than NlAChE1 and other mutants.
Lee, 2013). The importance of insect AChE1 is also indi-
cated by the fact that most of insecticide resistance
Sensitivity of recombinant NlAChEs to inhibitors or associated point mutations are present in AChE1, the
insecticides protein responsible for target-site insensitivity (Lee et al.,
The inhibition constant Ki of serine (a specific inhibitor for 2015). However, the function of insect AChE2 cannot be
AChEs) and two insecticides (chlorpyrifos-oxon and car- overlooked, at least in true flies and Hymenoptera spe-
bofuran) were investigated for NlAChE1 and NlAChE1 cies. In true flies, only AChE2 is present, generating
mutants. The mutations G119S and F331C caused a sig- multiple molecular forms via alternative splicing (Kim &
nificant decrease in the sensitivities of NlAChE1 to serine Lee, 2013). In some social bees, AChE2 possesses the

Table 5. Kinetics of substrate hydrolysis by recombinant acetylcholinesterases (AChEs)

Mutation KM (mM) Vmax [nmol
(mg
min)21] Vmax/KM [ml
(mg
min)21]

NlAChE1 53.16 6 4.85 cd 105.22 6 9.17 a 1979.31

NlAChE1G119S 47.28 6 6.24 c 72.80 6 10.36 b 1539.76
NlAChE1F331C 75.37 6 8.78 e 43.71 6 6.50 c 579.94
NlAChE1I332L 60.25 6 7.43 d 82.50 6 10.36 b 1369.29
NlAChE1F331C/I332L 32.15 6 5.42 b 116.22 6 16.49 a 3614.93
NlAChE2 14.72 6 2.26 a 6.60 6 1.13 d 448.37

Different letters in the same column indicate significant differences amongst recombinant AChEs.
Data are mean 6 SEM of at least five independent experiments.
KM, Michaelis-Menten constant; Vmax, maximum velocity; Nl, Nilaparvata lugens.

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Figure 2. Inhibition curves of chlorpyrifos-oxon against recombinant Nila-
parvata lugens acetylcholinesterase 1 (NlAChE1) and mutants. Data are
mean 6 SEM of at least five independent experiments.
main catalytic activity, and AChE1 may have been speci-
alized to gain non-synaptic functions (Kim et al., 2012;
Kim & Lee, 2013). In many species from Hemiptera,
AChE1 functions as the main catalytic enzyme, although
there are few exceptions, such as Cinara sp. and
Acanthosoma sp. (Kim & Lee, 2013). In N. lugens, a
Hemiptera species with agricultural importance, two
AChEs have been identified. NlAChE1 was found to
have much higher catalytic activity than NlAChE2, and
Nlace1 was expressed at a much higher level than that
of Nlace2 at all developmental stages and in all tissues,
demonstrating that NlAChE1 may be the dominant
AChE form of the two enzymes (Li et al., 2012; Kim &
Lee, 2013). This conclusion was also supported by the
data in the present study derived from the analysis of
the two recombinant NlAChEs. Recombinant NlAChE1
showed a Vmax 15.9 times higher and a catalytic effi-
ciency 4.4 times higher than NlAChE2. In insecticide-
resistant individuals of N. lugens, resistance-associated
mutations have only been observed in NlAChE1,
although the roles of these mutations in insecticide
resistance have not been evaluated in detail (Kwon
et al., 2012a,b). In the present study, three mutations
(G119S, F331C and I332L) in NlAChE1 were detected
in individuals resistant to chlorpyrifos, and these muta-
tions were subsequently confirmed as being associated
with chlorpyrifos resistance based on target-site insensi-
tivity data from recombinant proteins. These results
reveal that NlAChE1 is the main target for insecticides
such as organophosphates and carbamates.
Insecticide resistance often involves several mecha-
nisms including the enhancement of detoxification and
target-site insensitivity through mutations in target pro-
teins (Perry et al., 2011). In terms of resistance to
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Chlorpyrifos resistance in Nilaparvata lugens 5
organophosphate insecticides, point mutations have fre-
quently been identified in target AChEs (Villatte et al.,
2000; Fournier, 2005; Lee et al., 2015). In the present
study, the 1.7-fold synergistic ratio of TPP and 2.68-fold
of PBO in a 253.08-fold resistant population suggest
that mechanisms other than metabolic factors might be
involved in chlorpyrifos resistance in N. lugens, such as
target-site insensitivity. However, the role of metabolism
is a somewhat open question, as the synergist concen-
trations were not optimized for the particular insecti-
cides used in this study. The dual roles of cytochrome
P450 proteins (P450s) on chlorpyrifos toxicities, activat-
ing chlorpyrifos to chlorpyrifos-oxon and afterwards
detoxifying chlorpyrifos-oxon, also made it a little arbi-
trary to confirm the synergistic effects of PBO. Never-
theless, the differences in key properties of the crude
AChEs between chlorpyrifos resistant and susceptible
individuals support the idea that target-site insensitivity
is involved in chlorpyrifos resistance in N. lugens.
Target-site insensitivity has been reported to be respon-
sible for the resistance of N. lugens to carbofuran, a
carbamate insecticide, which involves four mutations
(G119A, F/Y330S, F331H and I332L) only found in
NlAChE1 (Kwon et al., 2012a). Although the roles of the
four putative mutations were not confirmed through
direct evidence, the correlation between the resistance
levels to carbofuran and putative resistance allele fre-
quencies suggested the importance of single point
mutations, such as F331H and I332L (Kwon et al.,
2012b). Here, we identified three mutations (G119S,
F331C and I332L) in NlAChE1 from chlorpyrifos-
resistant individuals at sites identical to the three muta-
tions detected in carbofuran-resistant individuals. How-
ever, G119S was detected instead of G119A, and
F331C was detected instead of F331H. The Y330S
mutation was not detected in chlorpyrifos-resistant indi-
viduals, or in individuals from field populations. This
mutation may be not important in insecticide resistance,
because the correlation coefficients (r2) between insecti-
cide resistance and mutation frequencies were quite
low, with 0.015 for carbofuran and 20.220 for carbosul-
fan (Kwon et al., 2012b).
The precise role of G119A in carbofuran resistance is
ambiguous because the correlation coefficients (r2)
between insecticide resistance and mutation frequencies
are relatively low, with 0.232 for carbofuran and 0.174 for
carbosulfan (Kwon et al., 2012b). Here, we observed that,
although the G119S mutation did not cause a big change in
the catalytic efficiency against the model substrate ATChI,
this mutation significantly decreased the sensitivity of
NlAChE1 to chlorpyrifos-oxon and carbofuran. These
results revealed that the G119S mutation should play impor-
tant roles in chlorpyrifos and carbofuran resistance, and
would also be involved in resistance to other
6 Y. Zhang et al.
organophosphate and carbamate insecticides. Similar to
G119S, F331C also decreased the target-site sensitivity of
NlAChE1 to chlorpyrifos and carbofuran. In contrast, the
I332L mutation did not show obvious effects on the insecti-
cide sensitivity of NlAChE1. Because F331C and I332L
were next to each other, recombinant proteins expressing
these mutations singly or in combination were tested, which
resulted in the finding that the mutant enzyme F331C/I332L
decreased insecticide sensitivity to a much higher extent
than the single mutation F331C. In addition, NlAChE1F331C/
I332L
showed the highest efficiency in catalysing the model
substrate ATChI, whereas NlAChE1F331C showed a much
lower efficiency compared to either NlAChE1 or NlA-
ChE1F331C/I332L. The mutations that altered the activity and
stability of AChEs result in the fitness cost associated with
mutations providing resistance (Shi et al., 2004). F331C
might be deleterious to the normal function of NlAChE1,
whereas I332L would decrease such adverse effects and
maintain the integrated properties. This role of the I332L
mutation, through combination with F331C, might also
reflect the fact that the I332L mutation was primarily
detected in individuals with the F331C mutation. However,
these two mutations did not always co-exist, such as the
presence of F331H and I332L either singly or together in
carbofuran-resistant individuals (Kwon et al., 2012a,b). In
the present study, the three mutations identified (G119S,
F331C and I332L) were not simultaneously detected in any
single individual in any of the tested populations, which
might be because of the fatal effects of mutations that affect
the functioning of NlAChE1. There is a trade-off in
insecticide-resistant insects between the potential advant-
age of mutations conferring insecticide resistance and the
opposing need to conserve normal protein functions. To
resolve this trade-off, insects have adopted special strat-
egies, such as the duplication of a wild-type copy of a target
gene to compensate for the fitness cost caused by muta-
tions in the same target, or by two or more mutations in one
target or different targets (Lenormand et al., 1998; Raymond
et al., 2001; Berticat et al., 2008; Remnant et al., 2013). The
roles of the double mutation F331C/I332L in insecticide
resistance may be also in such compensatory way, although
this idea needs further studies.
Experimental procedures
Insecticides and reagents
Chlorpyrifos (Chemical Abstracts Service (CAS) 80844-07-1),
carbofuran (CAS 1563-66-2), eserine (CAS. 57-64-7), PBO
(CAS 51-03-6), TPP (CAS 115-86-6) and diethyl maleate
(DEM, CAS 141-05-9) were purchased from Sigma-Aldrich (St
Louis, MO, USA). Chlorpyrifos-oxon (CAS 5598-15-2) was pur-
chased from Toronto Research Chemicals (Toronto, Ontario,
Canada).
Brown planthopper (N. lugens) and toxicity bioassay
The susceptible strain (Sus) of N. lugens was a laboratory strain,
obtained from the China National Rice Research Institute in Sep-
tember 2001, and reared as a laboratory strain without any contact
with insecticides for more than 12 years. Resistance selection was
applied on a field population (F) collected from Guilin, China. A
resistant population (R9) was obtained after nine generations of con-
tinuous selection on fourth-instar nymphs using the LC50 dose. In
parallel, the F population was also reared in the laboratory without
contact with any insecticides for nine generations to prepare a rela-
tively susceptible population (S9). In July–August 2015, three field
populations were collected from three areas of China, including Gui-
lin (FG), Nanjing (FN) and Jiujiang (FJ). All insects were reared on
rice seedlings in soilless culture in laboratory cages at 25 6 1 8C, rel-
ative humidity of 70–80% and a 16 h light/8 h dark photoperiod.
The toxicity bioassay was performed with the fourth-instar
N. lugens nymphs using the rice-stem dipping method (Wang
et al., 2008). The rice stems were treated with six concentra-
tions (differently amongst populations) of chlorpyrifos and sub-
sequently used to rear the fourth-instar nymphs from each
population. The mortality of N. lugens was recorded after 48 h,
and the treated insects unable to walk were considered dead
(Wen et al., 2009). The LC50 value was calculated on the basis
of standard probit analysis, using DATA PROCESSING SYSTEM
(DPS) software v. 9.50 (Tang & Zhang, 2013). In the synergism
analysis, 2 mg of the synergist (TPP, PBO or DEM) in 0.08 ml
acetone was topically delivered onto the prothorax notum of the
test insects 1 h before the bioassay. The doses of synergists
were not optimized for this study but were based on our previ-
ous studies (Ding et al., 2013; Zhang et al., 2015, 2016).
Mutation detection of AChEs in N. lugens
Total RNA was isolated from a single female adult using TrizolVR
reagent (Invitrogen, Carlsbad, CA, USA) according to the manu-
facturer’s instructions. First-strand cDNA was synthesized using
M-MLV (Moloney Murine Leukemia Virus) reverse transcriptase
(Promega, Madison, WI, USA) and oligo (dT)18. The primers
(Table S1) were designed to amplify the full ORF of the two
AChE-encoding genes according to the published sequences
for Nlace1 (GenBank: HQ605041) and Nlace2 (GenBank:
JN688930). The potential mutations were determined after com-
paring the ORF sequences of Nlace1 and Nlace2 from S9 and
R9. The PCR reactions were performed with 0.1 mM dNTPs
(deoxy-ribonucleoside triphosphate), 5 lM oligonucleotide pri-
mers, 4 ll cDNA and 1.0 unit of Ex-Taq DNA polymerase
(Promega) in a total volume of 20 ll. Thermal cycling conditions
for the PCR to clone the full ORF were 95 8C for 3 min, fol-
lowed by 35 cycles at 95 8C for 30 s, 56 8C for 40 s and 72 8C
for 3 min.
Genomic DNA (gDNA) was isolated from a single female
R
adult using DNAzolV reagent (Invitrogen) according to the man-
ufacturer’s instructions. The primers (Table S1) were designed
to detect the potential mutations according to a previous report
(Kwon et al., 2012a). The PCR reactions were performed with
0.1 mM dNTPs, 5 lM oligonucleotide primers, 1 ll gDNA and
1.0 unit of Ex-Taq DNA polymerase (Promega) in a total volume
of 20 ll. Thermal cycling conditions for the PCR to clone the
C 2017 The Royal Entomological Society, 00, 00–00
V

full ORF were 95 8C for 3 min followed by 35 cycles at 95 8C
for 30 s, 56 8C for 40 s and 72 8C for 1 min.
Preparation of crude AChE source
Twenty female adults were homogenized in 20 ml ice-cold 0.1 M
phosphate buffer (pH 7.4). After filtering through cheesecloth, the
homogenate was centrifuged at 10 000 g for 20 min. The superna-
tant was directly used as the AChE enzyme source.
Expression of the two NlAChEs in Sf9 cells
Sf9 cells were used for the expression of NlAChEs (with or without
point mutations) and Enhanced Green Fluorescent Protein (eGFP)
(GenBank: AAK15492) using the Bac-to-Bac system (Invitrogen).
The complete coding sequence region of the two NlAChEs was sub-
cloned into the pFastBac-HTa vector (Invitrogen) at the multiple clon-
ing sites BamHI and HindIII using a ClonExpress II One Step
Cloning kit (Vazyme, Nanjing, China) according to the manufac-
turer’s instructions and verified by nucleotide sequencing. Recombi-
nant bacmid DNAs were transfected into Sf9 cells to obtain
recombinant baculovirus for protein expression. Baculovirus culture
supernatants were collected as crude enzymes.
NlAChE activity assay
NlAChE activity was assayed at 25 8C using a Molecular
Devices (Sunnyvale, CA, USA) Thermomax Kinetic Microplate
Reader (Van Asperen, 1960; Devonshire & Moores, 1982; Han
et al., 1998). Enzymes (50 ll of each) were placed in a 96-well
microplate with 100 ll ATChI (1.5 mM) and 100 ll dithiobis-(2-
nitrobenzoic acid) (75 lM) and mixed well. The activity was
measured by monitoring the reactions at 30-s intervals for 20
min at a wavelength of 405 nm. The mean levels of AChE activ-
ity were based on a minimum of three replications.
KM and Vmax were determined by nonlinear regression of the
enzyme activity measurements over 2 min with at least 12 concen-
trations of ATChI, ranging from 10 to 2000 mmol/l. The protein con-
centration was determined using the protein-dye binding method
with standard bovine serum albumin protein (Bradford, 1976).
Measurement of NlAChE sensitivity
NlAChE sensitivity to the specific inhibitor and insecticides was
determined as described previously (Moores et al., 1996). The
enzymes (30 ll of each) were placed in a 96-well microplate with
170 ll phosphate buffer (pH 7.0, 0.02 M) containing 0.1% Triton
X-100 (Sigma-Aldrich, St Louis, MO, USA), followed by adding 50
ll buffer and 100 ll 5, 5-dithio-bis (2-nitrobenzoic acid) (45 lM),
and then incubated with different insecticides for 30 min at 25 8C.
The reaction was triggered after the addition of 100 ll ATChI at
the final concentration of 1.5 mM. The assays were monitored at
405 nm for 20 min, and the results were expressed as the slopes.
Inhibition was summarized and expressed as the mean AChE
activity remaining in the presence of insecticide throughout the
assay as a percentage of that for the same enzyme in the
absence of insecticides. Bimolecular rate constants (Ki) were fit-
ted by a nonlinear regression using ENZFITTER software (Biosoft,
Cambridge, UK).
C 2017 The Royal Entomological Society, 00, 00–00
V
Chlorpyrifos resistance in Nilaparvata lugens 7
Statistical analysis
Data analyses were carried out using DPS software v. 9.50
(Tang & Zhang, 2013). Significant differences were analysed by
one-way analysis of variance with at least three repeats. The
least significant difference (LSD) pairwise comparison of means
was used to analyse the difference between values and the
level of significance of results was set at P < 0.05.
Acknowledgements
We would like to thank Dr Na Yu from Ghent University
(Belgium) for her help in improving the English language.
This work was supported by the National Key Research
and Development Plan (2016YFD0200501-4), National
Natural Science Foundation of China (31601656 and
31322045) and National Key Technology Research and
Development Program (2012BAD19B01).
References
Alon, M., Alon, F., Nauen, R. and Morin, S. (2008) Organophos-
phates’ resistance in the B-biotype of Bemisia tabaci (Hemi-
ptera: Aleyrodidae) is associated with a point mutation in an
ace1-type acetylcholinesterase and overexpression of carbox-
ylesterase. Insect Biochem Mol Biol 38: 940–949.
Berticat, C., Bonnet, J., Duchon, S., Agnew, P., Weill, M. and
Corbel, V. (2008) Costs and benefits of multiple resistance to
insecticides for Culex quinquefasciatus mosquitoes. BMC
Evol Biol 8: 104.
Bradford, M.M. (1976) A rapid and sensitive method for the quan-
titation of microgram quantities of protein utilizing the principle
of protein-dye binding. Anal Biochem 32: 248–254.
Devonshire, A.L. and Moores, G.D. (1982) A carboxylesterase
with broad substrate specificity causes organophosphorus,
carbamate and pyrethroid resistance in peach-potato aphids
(Myzus persicae). Pestic Biochem Physiol 18: 235–246.
Ding, Z., Wen, Y., Yang, B., Zhang, Y., Liu, S., Liu, Z. et al. (2013)
Biochemical mechanisms of imidacloprid resistance in Nila-
parvata lugens: over-expression of cytochrome P450
CYP6AY1. Insect Biochem Mol Biol 43: 1021–1027.
Fournier, D. (2005) Mutations of acetylcholinesterase which con-
fer insecticide resistance in insect populations. Chem-Biol
Interact 157: 257–261.
Fournier, D. and Mutero, A. (1994) Modification of Acetylcholines-
terase as a Mechanism of Resistance to Insecticides, Vol.
108. Elsevier, New York, NY.
Garrood, W.T., Zimmer, C.T., Gorman, K.J., Nauen, R., Bass, C.
and Davies, T.G.E. (2016) Field-evolved resistance to imida-
cloprid and ethiprole in populations of brown planthopper Nila-
parvata lugens collected from across South and East Asia.
Pest Manage Sci 72: 140–149.
Gorman, K., Liu, Z., Denholm, I., Bru €ggen, K.U. and Nauen, R.
(2008) Neonicotinoid resistance in rice brown planthopper,
Nilaparvata lugens. Pest Manage Sci 64: 1122–1125.
Han, Z., Moores, G.D., Denholm, I. and Devonshire, A.L. (1998)
Association between biochemical markers and insecticide
resistance in the cotton aphid, Aphis gossypii Glover. Pestic
Biochem Physiol 62: 164–171.
Huchard, E., Martinez, M., Alout, H., Douzery, E.J.P., Lutfalla, G.,
Berthomieu, A. et al. (2006) Acetylcholinesterase genes
8 Y. Zhang et al.
within the Diptera: takeover and loss in true flies. Proc Biol Sci
273: 2595–2604.
Kim, Y.H. and Lee, S.H. (2013) Which acetylcholinesterase func-
tions as the main catalytic enzyme in the Class Insecta?.
Insect Biochem Mol Biol 43: 47–53.
Kim, Y.H., Cha, D.J., Jung, J.W., Kwon, H.W. and Lee, S.H. (2012)
Molecular and kinetic properties of two acetylcholinesterases
from the western honey bee, Apis mellifera. PLoS One 7:
e48838.
Kliot, A. and Ghanim, M. (2012) Fitness costs associated with
insecticide resistance. Pest Manag Sci 68: 1431–1437.
Kwon, D.H., Cha, D.J., Kim, Y.H., Lee, S.W. and Lee, S.H.
(2012a) Cloning of the acetylcholinesterase 1 gene and identi-
fication of point mutations putatively associated with carbo-
furan resistance in Nilaparvata lugens. Pestic Biochem
Physiol 103: 94–100.
Kwon, D.H., Min, S., Lee, S.W., Park, J.H. and Lee, S.H. (2012b)
Monitoring of carbamate and organophosphate resistance
levels in Nilaparvata lugens based on bioassay and quantita-
tive sequencing. J Asia-Pac Entomol 15: 635–639.
Lee, S.H., Kim, Y.H., Kwon, D.H., Cha, D.J. and Kim, J.H. (2015)
Mutation and duplication of arthropod acetylcholinesterase:
implications for pesticide resistance and tolerance. Pestic Bio-
chem Physiol 120: 118–124.
Lenormand, T., Guillemaud, T., Bourguet, D. and Raymond, M.
(1998) Appearance and sweep of a gene duplication: adaptive
response and potential for new functions in the mosquito
Culex pipiens. Evolution 52: 1705–1712.
Li, B.L., Chen, W., Liu, L., Zhang, X.C., Bao, Y.Y., Cheng, J.A.
et al. (2012) Molecular characterization of two acetylcholin-
esterase genes from the brown planthopper, Nilaparvata
lugens (Hemiptera: Delphacidae). Pestic Biochem Physiol
102: 198–203.
Li, F. and Han, Z. (2004) Mutations in acetylcholinesterase asso-
ciated with insecticide resistance in the cotton aphid, Aphis
gossypii Glover. Insect Biochem Mol Biol 34: 397–405.
Lin, Y.J., Hua, H.X., He, Y.Q., Yang, C.J., Zhai, B.P., Shen, J.L.
et al. (2011) Progress in research on the integrated manage-
ment of the brown planthopper, Nilaparvata lugens (Stål) in
China. Chin J Appl Entomol 48: 1194–1201.
Liu, Z., Williamson, M.S., Lansdell, S.J., Denholm, I., Han, Z. and
Millar, N.S. (2005) A nicotinic acetylcholine receptor mutation con-
ferring target-site resistance to imidacloprid in Nilaparvata lugens
(brown planthopper). Proc Natl Acad Sci USA 102: 8420–8425.
Moores, G.D., Gao, X.W., Denholm, I. and Devonshire, A.L. (1996)
Characterisation of insensitive acetylcholinesterase in
insecticide-resistant cotton aphids, Aphis gossypii Glover
(Homoptera: Aphididae). Pestic Biochem Physiol 56: 102–110.
Nabeshima, T., Kozaki, T., Tomita, T. and Kono, Y. (2003) An
amino acid substitution on the second acetylcholinesterase in
the pirimicarb-resistant strains of the peach potato aphid,
Myzus persicae. Biochem Biophys Res Commun 307: 15–22.
Perry, T., Batterham, P. and Daborn, P.J. (2011) The biology of
insecticidal activity and resistance. Insect Biochem Mol Biol
41: 411–422.
Raymond, M., Berticat, C., Weill, M., Pasteur, N. and Chevillon,
C. (2001) Insecticide resistance in the mosquito Culex
pipiens: what have we learned about adaptation? Genetica
112: 287–296.
Remnant, E.J., Good, R.T., Schmidt, J.M., Lumb, C., Robin, C.,
Daborn, P.J. et al. (2013) Gene duplication in the major
insecticide target site, Rdl, in Drosophila melanogaster. Proc
Natl Acad Sci USA 110: 14705–14710.
Russell, R.J., Claudianos, C., Campbell, P.M., Horne, I.,
Sutherland, T.D. and Oakeshott, J.G. (2004) Two major
classes of target site insensitivity mutations confer resistance
to organophosphate and carbamate insecticides. Pestic Bio-
chem Physiol 79: 84–93.
Shi, M.A., Lougarre, A., Alies, C., Fremaux, I., Tang, Z.H., Stojan, J.
et al. (2004) Acetylcholinesterase alterations reveal the fitness
cost of mutations conferring insecticide resistance. BMC Evol
Biol 4: 5.
Tang, Q.Y. and Zhang, C.X. (2013) Data Processing System
(DPS) software with experimental design, statistical analysis
and data mining developed for use in entomological research.
Insect Sci 20: 254–260.
Toutant, J.P., Arpagaus, M. and Fournier, D. (1988) Native
molecular-forms of head acetylcholinesterase from adult Dro-
sophila melanogaster-quaternary structure and hydrophobic
character. J Neurochem 50: 209–218.
Van Asperen, K. (1960) Toxic action of organophosphorus com-
pounds and esterase inhibition in houseflies. Biochem Phar-
macol 3: 136–146.
Villatte, F., Ziliani, P., Marcel, V., Menozzi, P. and Fournier, D.
(2000) A high number of mutations in insect acetylcholinester-
ase may provide insecticide resistance. Pestic Biochem Phys-
iol 67: 95–102.
Wang, Y., Gao, C., Xu, Z., Zhu, Y.C., Zhang, J., Li, W. et al.
(2008) Buprofezin susceptibility survey, resistance selection
and preliminary determination of the resistance mechanism in
Nilaparvata lugens (Homoptera: Delphacidae). Pest Manage
Sci 64: 1050–1056.
Wang, Y.H., Cang, T., Zhao, X.P., Wu, C.X., Chen, L.P., Yu, R.X.
et al. (2009) Susceptibility to several types of insecticides in
the rice planthoppers Nilaparvata lugens (Stål) and Sogatella
furcifera (Horvath) (Homoptera: Delphacidae). Acta Entomol
Sin 52: 1090–1096.
Wen, Y., Liu, Z., Bao, H. and Han, Z. (2009) Imidacloprid resist-
ance and its mechanisms in field populations of brown plan-
thopper, Nilaparvata lugens Stål in China. Pestic Biochem
Physiol 94: 36–42.
Zhang, X., Liu, X., Zhu, F., Li, J., You, H. and Lu, P. (2014) Field
evolution of insecticide resistance in the brown planthopper
(Nilaparvata lugens Stål) in China. Crop Prot 58: 61–66.
Zhang, Y., Wang, X., Yang, B., Hu, Y., Huang, L., Bass, C. et al.
(2015) Reduction in mRNA and protein expression of a nico-
tinic acetylcholine receptor a8 subunit is associated with
resistance to imidacloprid in the brown planthopper, Nilapar-
vata lugens. J Neurochem 135: 686–694.
Zhang, Y., Meng, X., Yang, Y., Li, H., Wang, X., Yang, B. et al.
(2016) Synergistic and compensatory effects of two point
mutations conferring target-site resistance to fipronil in the
insect GABA receptor RDL. Sci Rep 6: 32335.
Supporting Information
Additional Supporting Information may be found in the
online version of this article at the publisher’s web-site:
Table S1. Specific primers to clone full open reading frame sequences
or fragments containing mutations.
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