INSECTICIDE RESISTANCE AND RESISANCE MANAGEMENT
Susceptibility and Detoxifying Enzyme Activity in Two Spider Mite
Species (Acari: Tetranychidae) After Selection with Three Insecticides
XUEMEI YANG, LAWRENT L. BUSCHMAN,1 KUN YAN ZHU,
AND
DAVID C. MARGOLIES
Department of Entomology, Kansas State University, Manhattan, KS 66506 Ð 4004
J. Econ. Entomol. 95(2): 399Ð406 (2002)
ABSTRACT Changes in the susceptibility and detoxifying enzyme activity were measured in laboratory strains of Banks grass mite, Oligonychus pratensis (Banks), and twospotted spider mite,
Tetranychus urticae Koch, that were repeatedly exposed to three insecticides. Three strains of each
mite species were exposed to one of two pyrethroids, bifenthrin, and ␭-cyhalothrin, or an organophosphate, dimethoate, for 10 selection cycles at the LC60 for each insecticide. A reference or
nonselected strain of each mite species was not exposed to insecticides. After 10 cycles of exposure,
susceptibility to the corresponding insecticides, bifenthrin, ␭-cyhalothrin, and dimethoate, decreased
4.5-, 5.9-, and 289.2-fold, respectively, relative to the reference strain in the respective O. pratensis
strains, and 14.8-, 5.7-, and 104.7-fold, respectively, relative to the reference strain in the respective
T. urticae strains. In the bifenthrin-exposed O. pratensis strain, there was a 88.9-fold cross-resistance
to dimethoate. In the dimethoate-exposed T. urticae strain, there was a 15.9-fold cross-resistance to
bifenthrin. These results suggest that there may be cross-resistance between dimethoate and
bifenthrin. The reduced susceptibility to dimethoate remained stable for three months in the absence
of selection pressure in both mites. The decrease in susceptibility in the O. pratensis strains exposed
to bifenthrin, ␭-cyhalothrin, and dimethoate was associated with a 4.7-, 3.0-, and 3.6-fold increase in
general esterase activity, respectively. The decrease in susceptibility in the T. urticae strains exposed
to bifenthrin and ␭-cyhalothrin was associated with a 1.3- and 1.1-fold increase in general esterase
activity, respectively. The mean general esterase activity was signiÞcantly higher in the pyrethroidexposed O. pratensis and T. urticae strains than in the nonselected strain. There was no signiÞcant
increase in esterase activity in the dimethoate-exposed T. urticae strain. The decrease in susceptibility
to insecticides was also associated with reduced glutathione S-transferase 1-chloro-2, 4-dinitrobenzene
conjugation activity, but this did not appear to be related to changes in insecticide susceptibility. These
results suggest that in these mites, the general esterases may play a role in conferring resistance to
pyrethroids. However, some other untested mechanism, such as target site insensitivity, must be
involved in conferring dimethoate resistance.
KEY WORDS Oligonychus pratensis, Tetranychus urticae, insecticide resistance, cross-resistance,
resistance mechanism, general esterase
DEVELOPMENT OF RESISTANCE to miticides in spider mites
(Tetranychidae) is often so rapid that effective spider
mite management is difÞcult in many agricultural systems (Jeppson et al. 1975). In the western high plains
of North America, the Banks grass mite, Oligonychus
pratensis (Banks), is an important pest of maize, sorghum, and other grassy plants, whereas the twospotted spider mite, Tetranychus urticae Koch, is an important pest of maize, sorghum, and many other grassy
and broad-leaf plants (Jeppson et al. 1975). These
spider mites have natural enemies that can suppress
their populations in maize (Dick and Buschman 1996,
Messenger et al. 2000), but pesticides targeting other
1
To whom correspondence should be addressed: SW ResearchExtension Center, Kansas State University, 4500 East Mary Street,
Garden City, KS 67846 Ð913.
pests can suppress populations of natural enemies
(Buschman and DePew 1990). Hot, dry weather conditions may also favor pest mites over their natural
enemies. Under these conditions, spider mites may
become a serious problem and miticides must be used
to manage populations. Many workers have reported
that management of O. pratensis and T. urticae has
become increasingly difÞcult, apparently due to miticide resistance (Perring et al. 1981, Bynum et al.
1990). Field control problems can also be exacerbated
when mite populations become resistant to a miticide
that leads to cross-resistance to chemically related
compounds, or occasionally to a nonrelated compound (Herne et al. 1979). Cross-resistance among
organophosphate (OP), carbamate, and formamidine
insecticides has been reported in T. urticae (Richter
and Schulze 1990).
0022-0493/02/0399Ð0406$02.00/0 䉷 2002 Entomological Society of America
400
JOURNAL OF ECONOMIC ENTOMOLOGY
The mechanism of spider mite resistance to miticides is poorly understood compared with what is
known about insecticide resistance in other arthropods. More information could aid in better understanding the development of resistance and crossresistance, including strategies to avoid resistance and
to manage spider mites when resistance is present. In
this study, we followed the development of resistance
in several strains of two spider mites, O. pratensis and
T. urticae, during repeated exposure to three commonly used miticide/insecticides: the pyrethroids
bifenthrin and ␭cyhalothrin, and the organophosphate, dimethoate. When the experiment started, dimethoate had been in use as a miticide in maize for
about 30 yr, bifenthrin had been in use as a miticide/
insecticide in maize for about 12 yr, and ␭-cyhalothrin
had been in use as an insecticide to control corn
borers, Ostrinia nubilalis (Hübner) and Diatraea grandiosella Dyar (Lepidoptera: Crambidae) in maize for
⬇4 yr. After changes in susceptibility to the insecticides were documented, we examined the patterns of
cross-resistance among the insecticides in each spider
mite strain. The two pyrethroids, bifenthrin and ␭-cyhalothrin, are chemically related, so we hypothesized
that there would be miticidal cross-resistance between the two chemicals. We also evaluated the biochemical mechanisms of resistance associated with
the changes in susceptibility of the different spider
mite strains, and the durability of resistance when no
insecticides were applied for several generations.
Materials and Methods
The initial O. pratensis and T. urticae colonies were
established from mites collected from maize (O. pratensis) or soybean (T. urticae) in September 1997, near
Garden City, KS, (37⬚ 55⬘ N, 100⬚ 50⬘ E, elevation
876 m). They were maintained on maize plants (O.
pratensis) or lima bean plants (T. urticae) in the greenhouse at Kansas State University, Manhattan. As soon
as the two mite populations were established on the
greenhouse hosts (two or three generations later),
they were each divided into four subcolonies for the
selection experiment. The colony strains were maintained in separate mite-proof glass cages with cooling
fans for ventilation.
Technical grade bifenthrin was provided by FMC
(93.5% , A.I., Chicago, IL). CertiÞed grade acetone
was purchased from Fisher (Pittsburgh, PA). Technical grade ␭-cyhalothrin (98%, A.I.) and dimethoate
(98%, A.I.) were purchased from Chem. Service
(West Chester, PA). Analytical quality ␣-naphthol,
␣-naphthyl acetate (␣-NA), tetrazotized O-dianisidine (fast blue B), bicinchoninic acid (BCA) solution,
sodium dodecylsulfate (SDS), reduced glutathione,
1-chloro-2, 4-dinitrobenzene (CDNB), isocitric dehydrogenase, and ␤-nicotinamide adenine dinucleotide
phosphate (␤-NADP⫹) were purchased from Sigma
(St. Louis, MO). 3,4-dichloronitrobenzene (DCNB)
and p-nitroanisole were purchased from Aldrich (Milwaukee, WI). Bovine serum albumin was purchased
from Bio-Rad Laboratories (Hercules, CA).
Vol. 95, no. 2
Susceptibility of spider mites to the insecticides was
evaluated using a vial residue bioassay based on the
method of Bynum et al. (1990). Glass vials (3.7 ml)
were coated with 100 ␮l per vial of an appropriate
concentration of insecticide dissolved in acetone.
Treated vials were set horizontally in a mechanical
roller for 4 min to ensure uniform coverage of the
inside surface as the acetone evaporated. The vials
were removed from the roller and placed upright for
an additional 2 min to permit further evaporation of
acetone before capping. For each bioassay, four replicates with at least six concentrations of insecticide
were tested. Vials treated only with acetone were used
as controls. Twenty adult female mites were transferred into each vial with a Þne brush and vials were
then sealed with paraÞlm. Mortality was assessed under a dissecting microscope after 24 h at 25⬚C with a
16:8 (L:D) h photoperiod. Mites were scored “dead” if
they failed to make active movement after light tapping on the vials. The LC50 values, their 95% CL (95%
CLs), and slopes of regression lines were estimated
using probit analysis (SAS Institute 1996). LC50 values
were considered to be not signiÞcantly different if the
95% CLs of two LC50 values overlapped each other.
Resistance ratios and 95% CL were calculated for each
insecticide and for each strain against the nonselected
strain according to the method of Robertson and Preisler (1992).
In the selection experiment, at least 2,000 adult
female mites were transferred from each strain into
vials (20 mites/vial) treated with an insecticide concentration equal to the LC60 for that strain in that
cycle. More intense selection criteria (e.g., LC90)
resulted in too few survivors to establish an adequate
population in a reasonable period of time. After 24 h,
surviving mites were transferred back to plants and the
populations were allowed to increase. The Þnal mortality in each selection was ⬎90%, because there was
additional postexposure mortality that occurred after
the mites had been transferred back to plants. The
next selection cycle was conducted after the populations had increased, two or three generations later
(⬇1 mo). A bioassay using the selection insecticide
was conducted on the mite strains periodically when
the number of surviving mites changed in the selection
vials. The new LC60 was then applied as the selection
pressure. After 10 cycles of selection, the susceptibility
of each selected strain was evaluated for each of three
insecticides. To evaluate the stability of resistance, the
dimethoate-selected strains from each species, together with their reference (i.e., nonselected) strains,
were maintained without selection pressure for 3 mo,
equivalent to three selection cycles. These strains
were tested because they achieved the highest resistance ratios (ⱖ100 fold). A bioassay using the selection insecticide was conducted on the mite strains
after 3 mo without selection pressure to determine if
dimethoate susceptibility had changed.
Biochemical mechanisms of resistance that potentially may be associated with changes in susceptibility
were evaluated for each spider mite strain by measuring the general esterase, glutathione S-transferase
April 2002
YANG ET AL.: RESISTANCE SELECTION FOR SPIDER MITES
(GST), and cytochrome P450-dependent O-demethylase activities. General esterase activity was determined using the method of van Asperen (1962) as
modiÞed by Zhu and Gao (1998). Forty-eight mites
were individually homogenized in ice-cold 0.1 M
phosphate buffer, pH 7.5, containing 0.3% (vol:vol)
Triton X-100. Because T. urticae was larger than O.
pratensis, the volume of buffer was 180 ␮l for T. urticae
and 140 ␮l for O. pratensis, adjusted to produce similar
protein concentrations in the homogenates for each
mite. Homogenates were centrifuged at 15,000 ⫻ g at
4⬚C for 15 min. The pellets were discarded and the
supernatants were collected as enzyme sources. The
enzyme preparations were kept on ice until needed.
Fifteen ␮l of enzyme were incubated at 37⬚C for 30
min in a Þnal reaction volume of 150 ␮l containing 0.27
mM ␣-NA as substrate. The reaction was stopped by
adding 50 ␮l of freshly prepared fast blue B-SDS solution. The absorbance was determined 15 min later at
600 nm using a Vmax kinetic microplate reader with
SOFTmax software (Molecular Devices, Menlo Park,
CA). QuantiÞcation of enzymatic product was based
on a standard curve prepared with ␣-naphthol.
GST conjugation activities were assayed using
CDNB and DCNB as substrates based on the method
of Habig et al. (1974). A batch of 40 mites from each
mite strain was homogenized in 0.1 M ice-cold phosphate buffer (400 ␮l for T. urticae or 350 ␮l for O.
pratensis), pH 7.5. Each assay was replicated four
times. After centrifugation at 15,000 ⫻ g at 4⬚C for 15
min, supernatants were collected and kept on ice as
the enzyme sources. Fifty microliters of the enzyme
preparations were mixed with 690 ␮l of 10 mM glutathione and 10 ␮l of either 150 mM CDNB or DCNB
in a 1.5-ml glass cuvette. The change in absorbance
was recorded at 340 nm for CDNB-conjugating reaction or at 344 nm for DCNB-conjugating reaction at
20-s intervals for 2 min using the UV/visible spectrophotometer (Ultrospec 3000, Pharmacia). All assays
were corrected for nonenzymatic conjugation that
occurred in a sample that contained substrate and 10
mM glutathione in 0.1 M phosphate buffer, pH 7.5. The
amount of glutathione conjugate formed was calculated using the extinction coefÞcients of 9.6 mM⫺1cm⫺1
for CDNB and 10.0 mM⫺1cm⫺1 for DCNB.
Cytochrome P450-dependent O-demethylase activity was measured in the microplate assay using pnitroanisole as a substrate (Rose and Brindley 1985).
One hundred mites were homogenized in 100 ␮l of 0.1
M ice-cold phosphate buffer, pH 7.5, containing 1 mM
EDTA, and centrifuged at 6,000 ⫻ g for 15 min at 4⬚C.
The supernatant was collected and used as the enzyme
source. Fifty microliters of 10-fold diluted enzyme
aliquots were added to wells in a microplate, each
containing 25 ␮l of p-nitroanisole with 25 ␮l of
NADPH generating system, and incubated at 37⬚C for
30 min. To correct for nonenzymatic catalysis of the
substrate p-nitroanisole, two controls were set up as
follows: (1) 50 ␮l of enzyme was mixed with 25 ␮l of
solvent for dissolving p-nitroanisole (ethyl alcohol:
1,2-propanediol ⫽ 1:4) and 25 ␮l of NADPH generating system; (2) 50 ␮l of enzyme was mixed with 25
401
␮l of p-nitroanisole and 25 ␮l of ddH2O. Reaction
mixture containing 50 ␮l of enzyme, 25 ␮l of p-nitroanisole and 25 ␮l of NADPH regenerating system,
and the two controls were incubated in a microplate
at 37⬚C for 30 min. Absorbance was then determined
at 405 nm using the UV:visible spectrophotometer.
Enzyme activities are presented relative to protein
concentration, which was determined using the
method of Smith et al. (1985), as modiÞed by Zhu and
Clark (1994) with bovine serum albumin as a standard.
Aliquots of 20 ␮l of enzyme sample were decanted into
the wells of a microplate. One hundred eighty ␮l of the
BCA-cupric sulfate mixture was added to each well.
The mixture was incubated at 37⬚C for 30 min. Absorbance was determined at 560 nm using the microplate reader after the sample was allowed to incubate
at room temperature for 5 min. The enzyme activity
data were analyzed by analysis of variance (ANOVA)
and means were separated by least signiÞcant difference (LSD) (SAS Institute 1996).
Results and Discussion
Tetranychus urticae was less susceptible to three
insecticides than was O. pratensis, based on LC50
values in the nonselected strains (Tables 1 and 2). The
susceptibility of T. urticae was 9.5-, 51.8-, and 40.4-fold
lower than that of O. pratensis for bifenthrin, ␭-cyhalothrin, and dimethoate, respectively. This agrees with
Þeld observations of when insecticides are used on
mixed populations; T. urticae will tend to survive and
subsequently dominate in treated Þelds (Sloderbeck
et al. 1988). Bynum et al. (1997) also reported that T.
urticae were 1.4- to 3.3-fold more tolerant to organophosphate insecticides than were O. pratensis.
Resistance to the three insecticides, measured as an
increase in the resistance ratio, appeared to develop
slowly in both spider mites, although resistance to
dimethoate rose rapidly later in the experiment (Figs.
1 and 2). After 10 selection cycles, O. pratensis susceptibility to the insecticide used for selection decreased 4.5-, 5.9, and 289.2-fold for bifenthrin, ␭-cyhalothrin, and dimethoate, respectively (Table 1). T.
urticae susceptibility to the corresponding insecticides
decreased 14.8-, 5.7-, and 104.7-fold, respectively (Table 2). In both species, dimethoate resistance appeared to be quite stable in the absence of selection
pressure. After 3 mo without selection pressure (following 10 cycles of selection) the dimethoate-selected
O. pratensis strain still had a resistance ratio of 266.8fold (Table 1), whereas in the dimethoate-selected T.
urticae strain the resistance ratio was still 125.1-fold
(Table 2).
There appears to be a real risk of cross-resistance
between the two pyrethroids, and also between dimethoate and bifenthrin. Selection with bifenthrin led
to signiÞcant cross-resistance to dimethoate (88.9fold) in O. pratensis (Table 1). At the same time,
selection with dimethoate led to 15.9-fold cross-resistance to bifenthrin in T. urticae (Table 2). Selection
with bifenthrin also led to 8.4-fold cross-resistance to
␭-cyhalothrin in the O. pratensis. However, selection
402
JOURNAL OF ECONOMIC ENTOMOLOGY
Table 1.
Vol. 95, no. 2
Dose-response statistics for selected and non-selected strains of O. peratenais to three insecticides
Mite strain
Nonselected
Bifenthrin-selected
␭-Cyhalothrin-selected
Dimethoate-selected
Nonselected
Dimethoate-selected
Insecticide
n
Bifenthrin
␭-Cyhalothrin
Dimethoate
Bifenthrin
␭-Cyhalothrin
Dimethoate
Bifenthrin
␭-Cyhalothrin
Dimethoate
Bifenthrin
␭-Cyhalothrin
Dimethoate
480
480
480
560
640
720
560
560
560
540
480
640
Slope ⫾ SE
LC50 (95% CL),
␮g/ml
After 10 selection cycles
0.62 ⫾ 0.05
4.8 (4.1Ð5.5)
0.46 ⫾ 0.02
10.2 (7.9Ð12.7)
0.48 ⫾ 0.03
1.4 (1.1Ð1.7)
0.56 ⫾ 0.03
21.8 (18.4Ð25.6)
0.33 ⫾ 0.02
89.8 (67.8Ð116.1)
0.30 ⫾ 0.02
125.7 (95.9Ð165.1)
0.31 ⫾ 0.02
13.2 (10.2Ð16.8)
0.44 ⫾ 0.04
60.7 (47.8Ð75.4)
0.36 ⫾ 0.03
11.0 (8.1Ð14.1)
0.38 ⫾ 0.02
12.5 (10.2Ð15.1)
0.49 ⫾ 0.04
8.7 (5.3Ð12.9)
0.38 ⫾ 0.02
401.5 (318.8Ð503.3)
After selection relaxed for 3 mo following 10 selection cycle
Dimethoate
480
0.42 ⫾ 0.03
1.2 (0.9Ð1.5)
Dimethoate
560
0.36 ⫾ 0.03
322.5 (242.1Ð417.2)
P ⬎ ␹2a
Resistance ratiob
0.11
0.24
0.44
0.76
0.53
0.85
0.80
0.37
0.17
0.37
0.09
0.93
Ñ
Ñ
Ñ
4.5 (3.9Ð5.5)*
8.4 (5.3Ð10.7)*
88.9 (66.5Ð115.9)*
3.1 (2.5Ð3.9)*
5.9 (4.7Ð7.5)*
8.3 (5.8Ð10.1)*
2.7 (2.1Ð3.3)*
0.9 (0.6Ð1.3)ns
289.2 (220.7Ð342.5)*
0.22
0.78
Ñ
266.8 (207.2Ð350.7)*
The value of P ⬎ ␹2 larger than 0.05 indicates a signiÞcant Þt between the observed and expected regression lines.
Resistance ratios and 95% CL were calculated for each insecticide and for each strain against the nonselected strain according to the method
of Robertson and Preisler (1992). *, a signiÞcant difference between this LC50 value and that of the nonselected strain; ns no signiÞcant
difference between this LC50 and that of the nonselected strain, based on the nonoverlapping or overlapping 95% CLs of LC50 values.
a
b
with dimethoate did not lead to measurable crossresistance to ␭-cyhalothrin. Selection with ␭-cyhalothrin led to 3.1- and 2.8-fold cross-resistance to
bifenthrin in O. pratensis and T. urticae, respectively.
Selection with ␭-cyhalothrin led to 8.3-fold cross-resistance to dimethoate in O. pratensis, but not in T.
urticae.
Oligonychus pratensis and T. urticae strains selected
with the two pyrethroids had noticeably more mites
with high general esterase activity compared with the
nonselected strains (Figs. 3 and 4). Mean general
esterase activity was signiÞcantly higher in the pyrethroid-selected strains compared with the nonselected strain (Table 3). The decrease in susceptibility
in the O. pratensis strains exposed to bifenthrin, ␭-cyTable 2.
halothrin and dimethoate was associated with a 4.7-,
3.0-, and 3.6-fold increase in general esterase activity,
respectively. The decrease in susceptibility in the T.
urticae strains exposed to bifenthrin and ␭-cyhalothrin
was associated with a 1.3- and 1.1-fold increase in
general esterase activity, respectively. There was no
signiÞcant increase in esterase activity in the dimethoate-exposed T. urticae strain. The increased general esterase level was more dramatic in O. pratensis
than in T. urticae. Bifenthrin-selected strains of both
species had the highest general esterase activity, followed by ␭-cyhalothrin-selected strains.
General esterase activity may be involved in detoxifying and/or sequestering pyrethroid insecticides in
O. pratensis and T. urticae. For example, plant-induced
Dose-response statistics of selected and non-selected strains of T. articae to three insecticides
Mite strain
Nonselected
Bifenthrin-selected
␭-Cyhalothrin-selected
Dimethoate-selected
Nonselected
Dimethoate-selected
Miticide
n
Bifenthrin
␭-Cyhalothrin
Dimethoate
Bifenthrin
␭-Cyhalothrin
Dimethoate
Bifenthrin
␭-Cyhalothrin
Dimethoate
Bifenthrin
␭-Cyhalothrin
Dimethoate
480
560
480
480
640
720
720
560
560
640
640
560
Slope ⫾ SE
LC50 (95% CL),
␮g/ml
After 10 selection cycles
0.49 ⫾ 0.04
45.7 (36.9Ð56.2)
0.46 ⫾ 0.02
528.4 (429.5Ð644.3)
0.36 ⫾ 0.04
56.5 (39.2Ð74.8)
0.52 ⫾ 0.03
865.6 (711.6Ð1046)
0.42 ⫾ 0.02
872.5 (710.5Ð1065)
0.32 ⫾ 0.03
154.7 (117.9Ð199.9)
0.31 ⫾ 0.02
122.6 (92.8Ð158.7)
0.44 ⫾ 0.03
2856.0 (2288Ð3510)
0.41 ⫾ 0.03
41.5 (32.4Ð51.7)
0.37 ⫾ 0.02
557.5 (443.4Ð708.5)
0.45 ⫾ 0.03
634.1 (518.9Ð777.3)
0.41 ⫾ 0.03
6257.0 (4990Ð7829)
After selection relaxed for 3 mo following 10 selection cycles
Dimethoate
480
0.34 ⫾ 0.03
48.2 (32.1Ð70.0)
Dimethoate
560
0.40 ⫾ 0.03
6075.0 (4890Ð7753)
P ⬎ ␹2a
Resistance ratiob
0.49
0.95
0.29
0.71
0.68
0.35
0.65
0.65
0.37
0.78
0.14
0.10
Ñ
Ñ
Ñ
14.8 (8.1Ð26.9)*
1.9 (1.0Ð3.7)*
2.8 (2.2Ð3.6)*
2.8 (2.0Ð3.5)*
5.7 (4.3Ð6.8)*
0.6 (0.4Ð0.9)ns
15.9 (9.7Ð20.0)*
1.2 (0.9Ð1.4)ns
104.7 (88.7Ð135.6)*
0.30
0.11
Ñ
125.1 (107.2Ð163.9)*
The value of P ⬎ ␹2 larger than 0.05 indicates a signiÞcant Þt between the observed and expected regression lines.
Resistance ratios and 95% CL were calculated for each insecticide and for each strain against the nonselected strain according to the method
of Roberstson and Preisler (1992). *, a signiÞcant difference between this LC50 value and that of the nonselected strain; ns no signiÞcant
difference between this LC50 and that of the nonselected strain, based on the nonoverlapping or overlapping 95% CLs of LC50 values.
a
b
April 2002
YANG ET AL.: RESISTANCE SELECTION FOR SPIDER MITES
Fig. 1. Changes in the resistance ratio, based on LC50
values, in three O. pratensis strains over 10 selection cycles.
changes in the T. urticae general esterase activity were
associated with changes in susceptibility of these mites
to pyrethroids like bifenthrin and ␭-cyhalothrin (Yang
et al. 2001a). Furthermore, there was a signiÞcant
synergism of the pyrethroids bifenthrin and ␭-cyhalothrin in O. pratensis and T. urticae by a synergist that
affects esterase activity (Yang et al. 2001b). This suggests that the esterases may be involved in the detoxiÞcation and/or sequestering of pyrethroid insecticides in these mites. Elevated general esterase activity
has been reported for bifenthrin-resistant silverleaf
whiteßy, Bemisia argentifolii (Bellows and Perring),
and several other pyrethroid-resistant insects (Delorme et al. 1988, Lee and Clark 1996, Zhao et al. 1996,
Riley et al. 2000). These results lead to the conclusion
that cross-resistance among pyrethroids is likely to
occur in spider mites, because there appears to be a
common mechanism of resistance. Thus, use of pyrethroids to control other arthropod pests on maize
could increase the risk of spider mites developing
resistance to pyrethroid insecticides.
The lack of consistency and overall weak esterase
activity for the dimethoate-selected O. pratensis and T.
urticae strains suggests that general esterases may be
less important in the detoxiÞcation of the organophosphate dimethoate. Another mechanism of resistance
must be responsible for the dimethoate resistance
403
Fig. 2. Changes in the resistance ratio, based on LC50
values, in three T. urticae strains over 10 selection cycles.
Resistance ratio was about 1 and is not revealed in the Þgure
at this scale.
(289.2- and 104.7-fold) recorded in these mites. In the
dimethoate-selected O. pratensis strain there were
more mites with high general esterase activity than in
the nonselected strain, but this was not the case in the
dimethoate-selected T. urticae strain (Figs. 3 and 4).
The mean general esterase activity was signiÞcantly
higher in the dimethoate-selected O. pratensis strain
compared with the nonselected strain of O. pratensis,
but this was not the case in the dimethoate-selected T.
urticae strain (Table 3). In previous work, we found
that in these species, the toxicity of dimethoate was
not synergized by a synergist that affected esterase
activity (Yang et al. 2001b).
Involvement of GST in resistance to any of the
pesticides used in this study is equivocal. The decrease
in susceptibility to ␭-cyhalothrin was associated with
reduced glutathione S-transferase CDNB conjugation
activity, but GST did not appear to be related to
changes in susceptibility to bifenthrin or dimethoate.
In O. pratensis, GST activity (with CDNB substrate)
was signiÞcantly lower in the ␭-cyhalothrin- and dimethoate-selected strains than in the nonselected
strain, but it was not signiÞcantly lower in the
bifenthrin-selected strain (Table 3). In T. urticae, GST
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JOURNAL OF ECONOMIC ENTOMOLOGY
Vol. 95, no. 2
Fig. 3. Frequency distribution of general esterase activity of four O. pratensis strains. The general esterase activity
was determined in 48 individual mites for each strain.
Fig. 4. Frequency distribution of general esterase activity of four T. urticae strains. The general esterase activity was
determined in 48 individual mites for each strain.
activity (with CDNB substrate) was signiÞcantly
lower in the bifenthrin- and ␭-cyhalothrin-selected
strains than in the nonselected strain, but it was not
signiÞcantly different in the dimethoate-selected
strain. GST DCNB-conjugation activity and cytochrome P450-dependent O-demethylase activity were
not detectable in O. pratensis or T. urticae (Yang 2000).
These and previous results (Yang 2000) suggest that
metabolic enzymes like glutathione S-transferases and
cytochrome P450-dependent monooxygenases were
less likely to be important in the detoxiÞcation of
insecticides by spider mites. Other mechanisms must
confer resistance to organophosphate insecticides in
these two mites. Decreased target site sensitivity of
acetylcholinesterase (AChE) has been correlated
with resistance to organophosphate pesticides in T.
urticae and southern cattle tick, Boophilus microplus
(Canestrini) (Smissaert et al. 1970; Wharton and Roulston 1970; Zahavi et al. 1970). However, the insensitivity of AChE was not responsible for organophosphate resistance in the bulb mite, Rhizoglyphus robini
Claparede (Kuwahara et al. 1991). Additional studies
with the dimethoate-resistant O. pratensis and T. urticae are needed to determine whether target site
insensitivity (AChE) is the mechanism underlying
dimethoate resistance in spider mites.
April 2002
YANG ET AL.: RESISTANCE SELECTION FOR SPIDER MITES
405
Table 3. Mean (ⴞ SE) general esterase activity (␣-NA hydrolyzing general esterase) and GST activity (CDNB-conjugating glutathione
S-transferase) in O. pratensis and T. articae strains selected with three insecticides
BGM
Mite Strain
Esterase activity,
nmol/min/mg
Nonselected
Bifenthrin-selected
␭-Cyhalothrin-selected
Dimethoate-selected
0.55 ⫾ 0.07a
2.60 ⫾ 0.15d
1.64 ⫾ 0.09b
1.95 ⫾ 0.11c
Nonselected
Dimethoate-selected
TSM
GST activity,
nmol/min/mg
Esterase activity,
nmol/min/mg
GST activity,
nmol/min/mg
0.54 ⫾ 0.02a
0.69 ⫾ 0.03c
0.57 ⫾ 0.01b
0.51 ⫾ 0.02a
1.68 ⫾ 0.07c
1.48 ⫾ 0.01b
1.31 ⫾ 0.06a
1.62 ⫾ 0.04c
After selection relaxed for 3 mo following 10 selection cycles
0.53 ⫾ 0.07a
1.10 ⫾ 0.07b
0.53 ⫾ 0.03a
1.91 ⫾ 0.09c
0.81 ⫾ 0.11a
0.53 ⫾ 0.10a
1.76 ⫾ 0.03c
1.69 ⫾ 0.10c
After 10 selection cycles
1.14 ⫾ 0.07b
0.94 ⫾ 0.08ab
0.85 ⫾ 0.03a
0.87 ⫾ 0.07a
Means followed by the same letter within the column are not signiÞcantly different (P ⬎ 0.05, protected LSD). The esterase activity was
based on the mean for 48 measurements of individual mites. The GST activity (CDNB-conjugating activity) was based on the mean of four
replicates of 40 mites each, with three replicated readings.
The rapid increase in dimethoate resistance in O.
pratensis and T. urticae in this study suggests the presence of a major gene for dimethoate resistance in the
selected strains and in the spider mite samples used to
establish these O. pratensis and T. urticae colonies.
Dimethoate use in the source Þelds was not known to
be particularly different from dimethoate use in other
Þelds in this region. If the resistance allele was present
in this Þeld, we would expect that the same resistance
allele was present in other Þelds as well. If the samples
used to establish these O. pratensis and T. urticae colonies were representative of Þeld populations in the
region, this could mean that this resistance allele could
be widespread in the region, perhaps at a relatively
low frequency. Field experience with dimethoate in
this region is that it can sometimes be used effectively,
particularly in combination with bifenthrin, as the
initial miticide treatment of the season, but that it
becomes less effective later in the season. The presence of a gene for dimethoate resistance would not be
surprising given the long history of dimethoate use on
maize in the western high plains of North America.
There did not appear to be a major gene for
bifenthrin resistance in the O. pratensis and T. urticae
strains selected with bifenthrin. After 10 cycles of
selection with bifenthrin there was only 4.5- and 14.8fold resistance, respectively. If the samples used to
establish these O. pratensis and T. urticae colonies
were representative of Þeld populations in the region,
this could mean that bifenthrin resistance may not be
as common as dimethoate resistance.
There was considerable cross-resistance between
dimethoate and bifenthrin. This suggests that if O.
pratensis populations in the Þeld develop resistance to
one insecticide, they could also be resistant to other
insecticides. There appears to be potential for Þeld
control problems when these insecticides are used in
sequence. There also appears to be cross-resistance
between the two pyrethroids, and this resistance was
associated with higher levels of general esterase activity. This appears to conÞrm the concern that there
may be a risk of selecting for spider mite resistance to
bifenthrin when Þelds are treated with ␭-cyhalothrin
to control corn borers.
Acknowledgments
We thank Shauna Dendy and Fengyou Jia for technical
assistance, and Phil Sloderbeck, Sonny Ramaswamy and Jim
Nechols for insightful reviews of early drafts. Voucher specimens were deposited in the Kansas State University Museum
of Entomological and Prairie Arthropod Research and registered as No. 91. This is Contribution No. 01Ð393-J of the
Kansas Agricultural Experiment Station, Kansas State University, Manhattan, KS 66506.
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Received for publication 16 April 2001; accepted 14 September 2001.