Insect Science (2014) 00, 1–8, DOI 10.1111/1744-7917.

12080

ORIGINAL ARTICLE

Abamectin resistance in strains of vegetable leafminer,

Liriomyza sativae (Diptera: Agromyzidae) is linked to
elevated glutathione S-transferase activity

Qing-Bo Wei1,2 , Zhong-Ren Lei1 , Ralf Nauen3 , Du-Cheng Cai2 and Yu-Lin Gao1
1 State
Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural
Sciences, Beijing, 2 Hainan University, Haikou 570228, China, and 3 Bayer CropScience, RD-SMR, Pest Control Biology, 40789, Monheim,
Germany

Abstract Abamectin resistance was selected in the vegetable leafminer, Liriomyza

sativae (Blanchard) (Diptera: Agromyzidae) under laboratory conditions, and cross-
resistance patterns and possible resistance mechanisms in the abamectin-resistant strains
(AL-R, AF-R) were investigated. Compared with the susceptible strain (SS), strain AL-R
displayed 39-fold resistance to abamectin after 20 selection cycles during 25 generations,
and strain AF-R exhibited 59-fold resistance to abamectin after 16 selection cycles during
22 generations. No cross-resistance to cyromazine was found in both abamectin-resistant
strains. However, we failed to select for cyromazine resistance in L. sativae under laboratory
conditions by conducting 17 selection cycles during 22 generations. However, moderate
levels of cross-resistance to abamectin (6–9 fold) were observed in strains which received
cyromazine treatments. Biochemical analysis showed that glutathione S-transferase (GST)
activity in both abamectin-resistant strains (AL-R, AF-R) was significantly higher than
in the susceptible strain (SS), suggesting metabolically driven resistance to abamectinin
L. sativae. Recommendations of mixtures or rotation of cyromazine and abamectin should
be considered carefully, as consecutive cyromazine treatments may select for low-level
cross-resistance to abamectin.
Key words abamectin, cyromazine, GST, insecticide resistance, Liriomyza sativae

Introduction outbreaks of L. sativae (Parrella & Keil, 1984). The suc-

The vegetable leafminer Liriomyza sativae (Burgess)
(Diptera: Agromyzidae) is a damaging pest of vegetables
and ornamentals throughout the world (Parrella, 1987)
with a proven ability to evolve resistance to insecticides
compromising its sustainable control (Parrella, 1987). In-
tense insecticide use is a common strategy to eradicate
Correspondence: Yu-Lin Gao, State Key Laboratory for Bi-
ology of Plant Diseases and Insect Pests, Institute of Plant
Protection, Chinese Academy of Agricultural Sciences, Bei-
jing 100193, China. Tel: 0-106-281-5930; fax: 0-106-281-5981;
email: ylgao@ippcaas.cn
cess of this strategy is dependent on the susceptibility of
L. sativae populations to available insecticide modes of
action.
At present, two of the most effective insecticides for
Liriomyza management are abamectin and cyromazine
(Reitz et al., 2013). Both insecticides target the larval
stage feeding inside the plant foliage. Cyromazine acts
as a growth regulator; whereas abamectin is a neurotoxin
acting as a chloride channel activator. Both insecticides
have translaminar properties, allowing them to reach the
larvae within the plant. Research by Schuster and Everett
(1983) documented the effectiveness of both insecticides
under field conditions. Despite the long history of use of
both abamectin and cyromazine, resistance has not been

1

C 2013 Institute of Zoology, Chinese Academy of Sciences
2 Q. Wei et al.

a major problem yet (Reitz et al., 2013). There are only mazine estimated to result in approximately 50% mortal-
few documented reports of insecticide resistance to cy- ity. The number of nymphs selected per generation ranged
romazine and abamectin in leafminers from commercial between 1 000 and 2 000. Survivors (pupae) were trans-
ornamental greenhouses in the United States (Ferguson, ferred into a glass tube to allow complete development
2004; Ferguson & Pineda, 2010). for next generation selection.
The purpose of the current study was to determine the
response of L. sativae strains to selection by consecutive
Bioassay
abamectin and cyromazine treatments, representing the
two major insecticides used for leafminer control in China.
The bioassay technique used was similar to the lar-
However, knowledge on the development of insecticide re-
val bioassay method described by Cox et al. (1995) and
sistance, cross-resistance and resistance mechanisms are
Ferguson (2004). The larval stage was selected for bioas-
essential for the rational development of resistance man-
say as insecticide applications under field conditions are
agement strategies. In this paper, two abamectin-resistant
normally directed against the larval stage. Ten young (10–
strains of L. sativae were selected in the laboratory and
14 d old after planting) cowpea plants were exposed to sev-
checked for cyromazine cross-resistance and vice versa.
eral hundred 3–4-d-old adult leafminers (3–4 days later
In order to reveal the resistance mechanism, metabolic
after emergence of an adult individual) for an oviposition
enzyme activities were studied to help explain possible
access period (OAP) of 2–4 h. The short OAP allowed
mechanisms involved in resistance to abamectin.
for a synchronous egg hatch and age of the larvae present
at later treatment. After OAP, plants were removed from
cages and held in a leafminer-free greenhouse (26 ◦ C and
Materials and methods
ambient light) for 72 h to allow eggs to hatch and small
mines to develop. The number of small mines with 2nd-
Insect colony
instar larvae (2–3 cm) present were counted under 10×
magnification. Plants were divided into groups contain-
An abamectin/cyromazine-susceptible strain (SS) of
ing equal numbers of mines, 40–100 per dose and every
Liriomyza sativae was collected in 2007 from cowpea
treatment was repeated three times. The leaves and part
in Sanya, and continuously reared on cowpea in the labo-
of the stem were treated by submersion for 5 sec into
ratory at 26 ± 2 ◦ C, 60%–70% RH and L13 : D11 pho-
the appropriate serial dilution of insecticide in distilled
toperiod in the absence of insecticides. The abamectin-
water. Five to seven doses of formulated product were
resistant strain (AL-R) was derived from SS in 2010 by
used in each bioassay, i.e. 1.8% abamectin (Hebei Veyong
conducting 20 selection cycles during 25 generations with
Bio-Chemical Co., Ltd.) and 20% cyromazine (Shandong
abamectin in the laboratory. The cyromazine-selected
Runsheng Bio-Chemical Co., Ltd.). Control leaves were
strain (CL-R) was derived from SS in 2010 by 17 se-
dipped in distilled water only. After treatment, plants were
lection cycles during 22 generations with cyromazine in
held in a greenhouse (26 ◦ C and ambient light) for 2–3
the laboratory, albeit we failed to establish a cyromazine-
d (2–3 later after adult lay egg) to allow larval develop-
resistant strain.
ment. When it became apparent that non-treated larvae
In 2010 another selection experiment with abamectin
were ready to exit the mines, leaves were excised and
was started by using leafminers collected from sponge
placed on plastic frames in small containers with mesh
cucumber in Sanya. However, abamectin and cyromazine
covers to collect pupae. Pupae were counted and held in
have been commonly used at this farm for many years
20 mL glass vials for adult emergence. Larvae were con-
(Gao et al., 2011, 2012), and at the time of collection,
sidered dead if they failed to reach the adult stage.
the resistance ratio to abamectin and cyromazine was
15.1-fold and 1.28-fold, respectively. To maintain and in-
crease abamectin resistance in this strain, it was reared Enzyme preparation and protein quantification
under abamectin selection for 16 selection cycles during
22 generations (AF-R). To increase the cyromazine re- About 30 2nd-instar nymphs were selected and homog-
sistance in this strain, it was also reared separately under enized in 0.5 mL phosphate buffer (0.1 mol/L, pH7.5).
cyromazine selection for 13 selection cycles during 15 After the homogenates were centrifuged at 10 000g for
generations (CF-R), however we again failed to establish 5 min at 4 ◦ C, the supernatants were used as enzyme
a cyromazine-resistant strain. Selection was carried out source for measuring glutathione S-transferase (GST) ac-
by exposing the leaves and part of the stem infested with tivity. For AL-R and AF-R, 25 and 22 generations were
2nd instar larvae to concentrations of abamectin or cyro- used for the enzyme activity study, respectively. Total


C 2013 Institute of Zoology, Chinese Academy of Sciences, 00, 1–8
 GST activity associated with abamectin resistance in L. sativae
protein content in supernatants was determined accord-
ing to Bradford (1976) using bovine serum albumin as a
standard.
GST assay
GST activity was determined using 1-chloro-2,4-
dinitrobenzene (CDNB) as substrate according to the
manufacturer’s instructions (Nanjing Jiancheng, Nanjing,
China). For GST activity measurement 20 μL of enzyme
preparation was mixed with 100 μL of 2 mmol/L CDNB
and 100 μL 12.5 mmol/L reduced glutathione (GSH) in
0.1 mol/L sodium phosphate buffer (pH 6.5). The mi-
croplate was left 2 min to equilibrate and the change
in absorbance was recorded continuously for 5 min at
340 nm using a microplate reader. The activity of GST
was determined as recently described by using an extinc-
tion coefficient of 9.6 mmol/L/cm for the CDNB conju-
gate (Habig et al., 1974). Each sample was repeated three
times.
Data analysis
Bioassay data were analyzed by probit analysis using
the POLO program PC PoloPlus (POLO-PC, 1997). Mor-
tality was corrected using Abbott’s formula for each probit
analysis. A lack of overlap between the 95% CI (confi-
dence intervals) of LC50 values was used as the criterion
for significant differences. Resistance ratios were calcu-
lated by dividing the LC50 value of the resistant strain
by the LC50 value of the susceptible strain. GST activity
measured in all strains was expressed as mean ± stan-
dard error (SE), and subjected to analysis of variance
(ANOVA), with means separated by the Tukey’s mean sep-
aration test at α = 0.05 to determine significance (SAS,
1988).
Results
Abamectin resistance development
The cowpea-collected susceptible strain (SS) of L.
sativae collected in 2007 was used to select for abamectin
resistance under the laboratory conditions. After 20 se-
lection cycles during 25 generations between 2010 and
2012, we obtained a strain of L. sativae showing signif-
icant resistance to abamectin (Table 1). The first nine
consecutive generations selected show a slow increase in
resistance (resistance ratio from 1 to 3.18), but the fol-
lowing thirteen selections revealed a steady increase in

C 2013 Institute of Zoology, Chinese Academy of Sciences, 00, 1–8
3
resistance (resistance ratio from 3.18 to 38.65). We des-
ignated the strain as abamectin resistant strain (AL-R)
which was used in subsequent studies. A second strain
AF-R collected from sponge cucumber in Sanya was sim-
ilarly selected for abamectin resistance in the laboratory.
At the time of collection, the resistance ratio to abamectin
was 15.2-fold. After 16 selection cycles during 22 gener-
ations, we obtained another abamectin-resistant strain of
L. sativae, exhibiting an resistance ratio to abamectin of
59-fold (Table 2).
Cyromazine resistance development
The original SS strain was used to select for cyromazine
resistance in the laboratory as well, and after 17 selection
cycles during 22 generations with cyromazine, no signif-
icant increase in resistance was observed (strain CL-R)
(Table 3). The other strain collected from sponge cucum-
ber in Sanya and showing low abamectin resistant was sus-
ceptible to cyromazine. After 13 selection cycles during
15 generations, we again failed to establish a cyromazine-
resistant strain of L. sativae (CF-R) (Table 3). This result
may indicate a lower risk for cyromazine resistance selec-
tion under field conditions as compared with abamectin
selection.
Cross resistance
Compared to strain SS, both abamectin-resistant strains
AL-R and AF-R exhibited no significant cross-resistance
to cyromazine (Table 4). However, after 13–17 selec-
tion cycles with cyromazine, both strain CF-R and CL-R
were still susceptible to cyromazine, but exhibited 6.41-
to 9.36-fold cross-resistance to abamectin, respectively
(Table 5).
Metabolic enzyme activity
In order to evaluate the possible role of metabolic
detoxification mechanisms glutathione S-transferase ac-
tivity was measured in abamectin-resistant and suscepti-
ble strains (Table 6), since it has been shown in the past
that particularly GST activity is increased in abamectin re-
sistant pest insects. A significant difference in glutathione
S-transferases enzyme activity was found between larvae
of abamectin-resistant strains (AL-R or AF-R) and strain
SS. After 16–20 selection cycles with abamectin, GST
activity was increased by 5.6–6.56 fold in the resistant
strain, possibly indicating a role of GST’s in abamectin
resistance in L. sativae (Table 6).
4 Q. Wei et al.

Table 1 Selection history of the abamectin-resistant strain AL-R of Liriomyza sativae.

Strain LC50 (95% FL)† mg/L Slope (± SE) RR‡ χ2

SS 0.017 (0.011–0.028) 0.539 (± 0.068) 1 0.259

F7 0.046 (0.029–0.065) 1.210 (± 0.179) 2.71 1.468
F9 0.054 (0.035–0.079) 1.118 (± 0.180) 3.18 1.504
F11 0.072 (0.039–0.108) 1.256 (± 0.219) 4.24 2.955
F14 0.147 (0.068–0.237) 1.160 (± 0.214) 8.65 1.784
F17 0.228 (0.164–0.364) 0.954 (± 0.150) 13.4 0.129
F21 0.569 (0.368–0.864) 0.835 (± 0.113) 33.47 0.839
F24 0.537 (0.352–0.792) 0.915 (± 0.103) 31.59 2.007
F25 0.657 (0.385–0.930) 0.922 (± 0.085) 38.65 3.754
†
95% FL = fiducial limits at 95% of probability.
‡
Resistance ratio = LC50 of the AL-R strain/LC50 of the SS strain.

Table 2 Selection history of the abamectin-resistant strain AF-R of Liriomyza sativae.

Strain LC50 (95% FL)† mg/L Slope (± SE) RR‡ χ2

SS 0.017 (0.011–0.028) 0.539 (± 0.068) 1 0.259

F2 0.261 (0.135–0.397) 1.589 (± 0.322) 15.35 1.051
F5 0.336 (0.225–0.588) 0.782 (± 0.125) 19.76 0.974
F7 0.390 (0.252–0.640) 1.040 (± 0.191) 22.94 0.185
F9 0.402 (0.283–0.560) 1.111 (± 0.166) 23.65 1.053
F11 0.425 (0.254–0.747) 0.729 (± 0.142) 25 0.352
F14 0.609 (0.381–1.081) 0.843 (± 0.156) 35.82 0.626
F17 0.721 (0.389–1.141) 0.649 (± 0.087) 42.41 0.149
F22 1.003 (0.667–1.500) 1.078 (± 0.130) 59 1.591
†
95% FL = fiducial limits at 95% of probability.
‡
Resistance ratio = LC50 of the AF-R strain/LC50 of the SS strain.

Table 3 Selection history of the cyromazine-resistant strains of Liriomyza sativae.

Strain LC50 (95% FL)† mg/L Slope (± SE) RR‡ χ2

SS 0.456 (0.177–0.755) 1.037 (± 0.206) 1 2.626

CL-R22 0.477 (0.391–0.563) 2.057 (± 0.189) 1.046 0.195
CF-R15 0.631 (0.431–0.828) 2.051 (± 0.193) 1.384 4.866
†
95% FL = fiducial limits at 95% of probability.
‡
Resistance ratio = LC50 of the CL-R strain/LC50 of the SS strain.

Discussion tion with abamectin, we obtained two abamectin resis-

In vegetables, abamectin and cyromazine are two com-
monly used insecticides for L. sativae control in
China (Gao et al., 2011, 2012). To assess the poten-
tial of resistance development to both compounds in
L. sativae, field-populations collected from cowpea in
2007 and sponge cucumber in 2010 were continuously
selected with abamectin and cyromazine for resistance
under laboratory conditions. After 16–20 cycles of selec-
tant strains with moderate resistance levels between 39 to
59-fold when compared with the susceptible strain. The
results clearly indicates that L. sativae has the capability
of developing resistance to abamectin, as shown for other
pest insects such as L. Trifolii (Freguson, 2004), Plutella
xylostella (Liang et al., 2001; Pu et al., 2010) and
Frankliniella occidentalis (L.) (Bielza, 2008). Our re-
sults suggest that abamectin should be used in rotation
with other modes of action for sustainable resistance


C 2013 Institute of Zoology, Chinese Academy of Sciences, 00, 1–8

C 2013
Table 4 Cross-resistance testing of the abamectin-selected strains of Liriomyza sativae to cyromazine.

SS strain AL-R strain AF-R strain

Insecticides RR† RR‡
§ § §
Slope (± SE) LC50 (95% FL) mg/L Slope (± SE) LC50 (95% FL) mg/L Slope (± SE) LC50 (95% FL) mg/L

Abamectin 0.894 (± 0.152) 0.018 (0.011–0.031) 0.922 (± 0.085) 0.657 (0.385–0.093) 36.5 1.078 (± 0.130) 1.003 (0.667–1.500) 55.7
Cyromazine 1.129 (± 0.194) 0.461 (0.179–0.798) 1.121 (± 0.312) 0.479 (0.185–0.856) 1.04 1.048 (± 0.302) 0.502 (0.356–0.905) 1.09
†
RR (resistance ratio) = LC50 of the AL-R strain/LC50 of the SS strain.
‡
RR (resistance ratio) = LC50 of the AF-R strain/LC50 of the SS strain.
§
95% FL = fiducial limits at 95% of probability

Institute of Zoology, Chinese Academy of Sciences, 00, 1–8

Table 5 Cross-resistance testing of the cyromazine-selected strains of Liriomyza sativae to abamectin.

SS strain CL-R strain CF-R strain

Insecticides RR† RR‡
§ § §
Slope (± SE) LC50 (95% FL) mg/L Slope (± SE) LC50 (95% FL) mg/L Slope (± SE) LC50 (95% FL) mg/L

Cyromazine 1.003 0.402 2.057 0.477 1.186 2.051 0.631 1.569

(± 0.194) (0.125–0.698) (± 0.189) (0.374–0.579) (± 0.193) (0.355–0.901)
Abamectin 0.933 0.022 1.110 0.206 9.36 0.927 0.141 6.41
(± 0.117) (0.009–0.035) (± 0.118) (0.133–0.299) (± 0.093) (0.036–0.321)
†
RR (resistance ratio) = LC50 of the CL-R strain/LC50 of the SS strain.
‡
RR (resistance ratio) = LC50 of the CF-R strain/LC50 of the SS strain.
§
95% FL = fiducial limits at 95% of probability.
GST activity associated with abamectin resistance in L. sativae
5
6 Q. Wei et al.
Table 6 GST activity in different strains of Liriomyza sativae† .
Glutathione-S-transferase (GST)
Strain
Protein (U/mg) Ratio‡
SS 213.4 ± 68.2 a
AL-R 1400.8 ± 175.4 b 6.56
AF-R 1195.3 ± 134.0 b 5.60
†
Mean activity values ± SEM (n = 3) between resistant strain
and SS strain. Values in the same column followed by different
letters are significantly different (P < 0.05).
‡
Ratio = AL-R and AF-R activity/SS strain activity, respectively.
management tactics preventing fast selection for
abamectin resistance in vegetable leafminer. However,
after 13–17 cycles of selection with cyromazine, both
selected strains were still highly susceptible to cyro-
mazine when compared with the susceptible strain. This
result indicates that cyromazine resistance alleles in field-
collected vegetable leafminer populations from both cow-
pea and sponge-cucumber are less frequent compared to
abamectin resistance alleles.
Four general types of mechanism for insecticide resis-
tance have been recently identified: metabolic detoxifi-
cation, reduced penetration of toxicants, target site mu-
tations and behavioral resistance (Brattsten et al., 1986).
Many documented cases of insecticide resistance in in-
sect pest species are conferred by elevated levels of
detoxification enzymes, however often multiple mecha-
nisms were shown to be involved in insecticide resistance
(Bielza et al., 2007; Bielza, 2008; Bielza et al., 2009;
Chen et al., 2011; Gao et al., 2012; Houndete et al., 2010;
Sato et al., 2005; Wang & Wu, 2007). Mechanisms of
abamectin resistance have been reported in several in-
sect as well as spider mite species. In a laboratory se-
lected Bemisia tabaci strain with moderate resistance to
abamectin (RR 14.5) biochemical analysis suggested that
elevated levels of cytochrome P450 and GST activities are
involved in abamectin resistance (Wang & Wu, 2007). In
Tetranychus urticae, detoxification by cytochrome P450
and GST were also described as a key factor confer-
ring abamectin resistance (Sato et al., 2005; Stumpf &
Nauen, 2002). In F. occidentalis it was shown that the
cytochrome P450 monooxygenase activity of the resis-
tant ABA-R strain was 6.66-fold higher than that of the
susceptible ABA-S strain. It appears that enhanced oxida-
tive metabolism mediated by cytochrome P450 monooxy-
genases was a major mechanism for abamectin resis-
tance, at least in western flower thrips (Chen et al.,

C 2013 Institute of
2011). In the present study, biochemical analysis showed
that GST activity in both abamectin-resistant strains
(AL-R, AF-R) was significantly higher than in strain
SS, indicating that increased GST enzyme activity is
likely to be a major factor conferring abamectin re-
sistance in L. sativae. In fact elevated levels of GST
activity were very recently also shown to be associ-
ated with resistance to abamectin in two-spotted spider
mites, T. urticae (Stumpf & Nauen, 2002) and Colorado
potato beetle, Leptinotarsa decemlineata (Argentine
et al., 1992).
No cross-resistance to cyromazine was detected in an
abamectin-resistant strain of L. trifolii, another important
Liriomyza spp. (Freguson, 2004). The current study sup-
ports their findings. Based on data from bioassays in the
current study, there is no cross-resistance to cyromazine
in both abamectin-resistant strains of L. sativae. Due to
the fact that cyromazine and abamectin belong to differ-
ent mode of action classes cross-resistance between both
compounds is quite unlikely (Reitz et al., 2013). In ad-
dition, the AL-R and AF-R strains possessed a moderate
level of resistance to abamectin (36.5–55.7-fold) but al-
most no resistance to cyromazine (1.04–1.09-fold), sug-
gesting that cross-resistance to cyromazine is absent in
both abamectin-resistant strains of L. sativae. However,
it is very interesting that cross-resistance to abamectin
does exist at low-level in cyromazine-selected strains (CL-
R, CF-R) of L. sativae. Both strains CL-R and CF-R
show virtually no resistance to cyromazine (1.186–1.569-
fold) after 15–22 generation selection, but a low level
of resistance to abamectin (6.41–9.36-fold). For strain
CF-R, low cross-resistance to abamectin would be ex-
pected as it was slightly resistant to abamectin at the
time of collection in 2010, i.e. the RR50 to abamectin
was 15.35-fold. However, it is less obvious to explain
low-level cross-resistance to abamectin in strain CL-R,
which was derived from a laboratory susceptible strain
SS with no resistance to abamectin (LC50 0.017 mg/L)
at the beginning of selection with cyromazine. Further
studies are necessary to verify this mechanism as well
as the biochemical mechanisms involved in the observed
resistance.
Recommendations of mixtures or rotation of cyro-
mazine and abamectin should be considered carefully,
as positive cross-resistance may increase the evolution of
metabolic enzyme-mediated resistance. Priority actions
should focus on the regional, rational and concerted use
of insecticides in the whole vegetable-growing area, with
cooperation between professional stakeholders, research
and extension services in communicating with and ex-
plaining strategies to farmers.
Zoology, Chinese Academy of Sciences, 00, 1–8
 GST activity associated with abamectin resistance in L. sativae
Acknowledgments
This research was supported by the national modern agri-
cultural science and technology city industry of Beijing
(Z121100001212006) and China Agriculture Research
System (CARS-25-B-07).
Disclosure
This manuscript and its authors are not involved in any
potential conflicts of interest, including financial interests
and relationships and affiliations.
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Accepted October 22, 2013
Zoology, Chinese Academy of Sciences, 00, 1–8