Inheritance and detoxification enzyme levels in Tetranychus urticae Koch (Acari: Tetranychidae) strain selected with chlorpyrifos

Abstract

Changes in insecticide susceptibilities and detoxifying enzyme activities were measured in a strain of Tetranychus urticae Koch following repeated exposure to the organophosphate insecticide, chlorpyrifos. Twelve consecutive selection at the LC60 of the parental strain increased resistance from 8.58 to 91.45 fold. The interaction of some synergists [piperonyl butoxide,
triphenyl phosphate and S-benzyl-O,O-diisopropyl phosphorothioate (IBP)] with chlorpyrifos was analyzed in the selected strain. Solely IBP showed a low synergistic
effect with chlorpyrifos. The selected strain also demonstrated resistance against abamectin, propargite, clofentezine and
fenpyroximate. The mode of resistance inheritance to chlorpyrifos was found to be incompletely dominant, and not sex-linked.
Non-specific esterase enzyme activity was raised from 19.35 to 33.59mOD/min/mg proteins during the selection period and it
was observed that esterase band intensities visualized by polyacrylamide gel electrophoresis increased. This study has investigated
the selection of resistance to chlorpyrifos and documented resistance to abamectin, propargite, clofentezine and fenpyroximate
in Turkish T. urticae. Esterase enzymes may be playing a role in chlorpyrifos resistance while glutathione S-transferase (GST) and P450 enzymes
do not appear to have any significant involvement.

KeywordsChlorpyrifos-Detoxifying enzymes-Inheritance-Resistance-
Tetranychus urticae

Full text

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ORIGINAL PAPER
Inheritance and detoxification enzyme levels in Tetranychus
urticae Koch (Acari: Tetranychidae) strain selected with
chlorpyrifos
Recep Ay Æ Æ Sibel Yorulmaz
Received: 27 January 2009/Accepted: 18 August 2009/Published online: 11 September 2009
? Springer-Verlag 2009
Abstract
detoxifying enzyme activities were measured in a strain of
Tetranychus urticae Koch following repeated exposure to
the organophosphate insecticide, chlorpyrifos. Twelve
consecutive selection at the LC60of the parental strain
increased resistance from 8.58 to 91.45 fold. The interac-
tion of some synergists [piperonyl butoxide, triphenyl
phosphate and S-benzyl-O,O-diisopropyl phosphorothioate
(IBP)] with chlorpyrifos was analyzed in the selected
strain. Solely IBP showed a low synergistic effect with
chlorpyrifos. The selected strain also demonstrated resis-
tance against abamectin, propargite, clofentezine and fen-
pyroximate. The mode of resistance inheritance to
chlorpyrifos was found to be incompletely dominant, and
not sex-linked. Non-specific esterase enzyme activity was
raised from 19.35 to 33.59 mOD/min/mg proteins during
the selection period and it was observed that esterase band
intensities visualized by polyacrylamide gel electrophore-
sis increased. This study has investigated the selection of
resistance to chlorpyrifos and documented resistance to
abamectin, propargite, clofentezine and fenpyroximate in
Turkish T. urticae. Esterase enzymes may be playing a role
in chlorpyrifos resistance while glutathione S-transferase
(GST) and P450 enzymes do not appear to have any sig-
nificant involvement.
Changes in insecticide susceptibilities and
Keywords
Inheritance ? Resistance ? Tetranychus urticae
Chlorpyrifos ? Detoxifying enzymes ?
Introduction
The two-spotted spider mite, Tetranychus urticae Koch, is
a phytophagous mite with a worldwide distribution and a
large number of host plants (Tsagkarakou et al. 2002).
Chemical insecticides are generally utilized against two-
spotted spider mite, as they are easy-to-apply, effective,
and do not generally require identification of the species.
However, heavy and prolonged use of insecticides results
in many problems such as environmental pollution, dis-
ruption of ecological systems, and harm to natural ene-
mies. One of the most significant problems resulting from
heavy use of insecticides is the resistance to these
chemicals developed by the target pests. A major problem
in the control of T. urticae is their ability to rapidly
develop resistance to many important pesticides after only
a few applications (Stumpf et al. 2001; Stumpf and Nauen
2001).
Acquired insecticide resistance presents the greatest
challenge in the control of T. urticae, which is responsible
for economic losses in agricultural fields in many parts of
the world (Van Leeuwen and Tirry 2007). Resistance is
defined as the development of survival ability by the
majority of a normal population when exposed to lethal
doses (Ffrench-Constant and Roush 1990). The resistance
developed to insecticides can be associated with some
specific insect enzymes (Vontas et al. 2001; Yang et al.
2002; Kim et al. 2004; Van Leeuwen et al. 2006). The most
common types of resistance found in insects are increased
enzymatic detoxification and target site insensitivity
(Oppenoorth 1984; Scott 1999).
Communicated by K. J. Gorman.
R. Ay (&) ? S. Yorulmaz
Faculty of Agriculture, Plant Protection Department, Su ¨leyman
Demirel University, 32260 Cunur, Isparta, Turkey
e-mail: recepay@ziraat.sdu.edu.tr
123
J Pest Sci (2010) 83:85–93
DOI 10.1007/s10340-009-0274-9

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Chlorpyrifos
nyl]) is a contact-effect insecticide/acaricide. It is a mem-
ber of the organophosphorus insecticide group, which are
widely used in agriculture to control phytophagous insects
and mites (Picco et al. 2008). Chlorpyrifos acts by inhib-
iting acetylcholinesterase activity, which is necessary for
the proper functioning of the nervous system of insects
(Smegal 2000).
This study examined the resistance characteristics of a
T. urticae strain subjected to chlorpyrifos selection, by
using bioassay and biochemical methods.
(O,O-diethyl-O-[3,5,6-trichloro-2-pyridi-
Materials and method
Materials
The original Turkish strain of T. urticae (termed SAK) was
established from mites collected from a commercial bean
(Phaseolus vulgaris L.) greenhouse in the S ¸arkikaraag ˘ac ¸
District of Isparta Province in June 2002. The SAK strain
was grown continually cultured from June 2002 to Sep-
tember 2006 without being exposed to any pesticides. A
culture line of the SAK strain was exposed to 12 successive
selection with chlorpyrifos from September 2006 to May
2007, and named ‘‘CHLO 12’’. A fully insecticide sus-
ceptible strain (GSS) was obtained from Rothamsted
Research, Harpenden (England), in 2001 and also main-
tained without exposure to pesticides. All of the T. urticae
strains were grown continuously on pinto bean plants,
Phaseolus vulgaris L., under laboratory conditions at
26 ± 2?C, 60 ± 5% RH and a 16-h photoperiod.
The chemical compounds used consisted of chlorpyrifos
(Dursban 4 EC 480 g/l), which is an organophosphate
insecticide/acaricide,andthree
butoxide (PBO); S-benzyl-O,O-diisopropyl phosphoro-
thioate (IBP); and triphenyl phosphate (TPP). IBP and TPP
are inhibitors of esterase enzymes (Kim et al. 2004; Kang
et al. 2006; Wang and Wu 2007) while PBO is an inhibitor
of esterase and P450 (Stumpf and Nauen 2002; Young
et al. 2005; Kim et al. 2006; Van Leeuwen and Tirry 2007).
Chemicals containing amitraz (Kortraz 20 EC 200 g/l),
clofentezine (Apollo SC 500 g/l), fenpyroximate (Meteor
50 g/l), propargite (Komite EC 588 g/l), bifenthrin (Talstar
EC 100 g/l), or abamectin (Agrimec 18 g/l) were used for
the resistance studies conducted only on the resistant strain.
synergists:piperonyl
Bioassays
Tests employed the method described by Ay (2005). The
prepared suspension of chlorpyrifos was sprayed on the
internal surfaces of lids and bases of 60-mm diameter
plastic Petri dishes and allowed to dry for 30 min. For each
application, 1 ml suspension was sprayed on each base and
lid pair by a Potter spray tower (Burckard Manufacturing
Co Ltd, Rickmansworth, Herts, UK) at 100 kPa. Adult
female mites (%30) were transferred to each dish using a
fine brush, and the dishes were closed and sealed with
Parafilm to prevent escape. The mites in the dish were kept
at 26 ± 2?C, 60 ± 5% RH and a 16-h photoperiod for 24 h
after treatment. The survival of individual mites was
determined by touching each mite with a fine brush; mites
that were unable to walk a distance at least equivalent to
their body length were considered as dead. Mortality tests
were performed before each experiment to determine a
range of concentrations that would produce approximately
10–90% mortality. The mortality of the control group never
exceeded 10%. Each experiment was conducted using three
replicates at seven concentrations (plus a distilled water
control). Pooled data were subjected to probit analysis
(POLO PC) (LeOra 1994) and LC50, 60 and 90values with
95% CI were estimated. The LC50values of the selection
strain were compared to those of the susceptible strain
(GSS). The resistance ratio was calculated by dividing the
LC50of the resistant strain by the LC50of the susceptible
strain.
Selection for resistance
Females of the original strain (SAK) were selected for
resistance to chlorpyrifos under laboratory conditions from
September 2006 to May 2007. Selection experiments used
a modified form of the method developed by Yang et al.
(2002). At least 400 adult female mites of the SAK strain
were transferred into Petri dishes (40 mites/Petri dish)
treated with chlorpyrifos concentrations equal to the LC60
for that cycle. After 24 h, surviving mites were transferred
back to untreated plants and the populations were allowed
to regenerate. The next selection cycle was conducted two
or three generations, after the populations had increased
(approximately 15–20 days). A bioassay using the chlor-
pyrifos was conducted periodically on mite populations
when the number of surviving mites changed in the
selection Petri dishes. The new LC60was applied as the
subsequent selection pressure.
Synergism test
The effects of chlorpyrifos ? synergist were tested using
the methods of Kim et al. (2004). PBO, IBP, and TPP were
used to inhibit detoxification mechanisms by non-specific
monooxygenases (P450) and esterases. Synergists were
dissolved in acetone:distilled water (1:1) and 1 ml sus-
pension was sprayed on the base and lid of Petri dishes in
the same manner as the toxicity test, 30 min prior to pes-
ticide application. Distilled water–acetone without a
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synergist was applied to the control group. Synergist
solutions were prepared at the following concentration
(mg/l): PBO (500), IBP (400), and TPP (125). A syner-
gistic ratio (SR) was calculated using the following
formula:
SR ¼LC50of chlorpyrifos without synergist=
LC50of chlorpyrifos with synergist
Toxicity of other pesticides to chlorpyrifos selected
strain
Additional pesticides were evaluated using resistant CHLO
12 and susceptible GSS strains of T. urticae. The pesticides
used were fenpyroximate, amitraz, clofentezine, proparg-
ite, bifenthrin, and abamectin. All pesticides used were
commercially available in Turkey. The bioassay method
used for all pesticides was the same as described previously
for the toxicity test. The mortality data of each pesticide for
the susceptible GSS strain and the selected resistant CHLO
12 strain of T. urticae were subjected to probit analysis
(POLO PC) (LeOra 1994). The resistance ratio was cal-
culated dividing the LC50of the resistant strain by the LC50
of the susceptible strain.
Crossing experiment
To estimate the dominance of resistance, the GSS and
chlorpyrifos resistant CHLO 12 strains were reciprocally
crossed to produce hybrid F1females by placing 15 female
teleiochrysalis of one strain and 30 adult males of the other
strain on the upper side of a primary bean leaf placed on
wet wool in a Petri dish. Directly after molting, the diploid
females were fertilized by the haploid males, and 1 day
later they began to lay eggs. Males and females were
removed after 5 days. If mating was successful, the hap-
loid–diploid mating system resulted in F1 females and
males. The F1females were then transferred to clean bean
plants and bioassay conducted when they had reached
maturity. The bioassay method was the same as previously
described for the toxicity test. The experiment was con-
ducted using three replicates of seven serially diluted
concentrations (plus a distilled water-only control) cover-
ing the range of 10–90% mortality.
The degree of dominance (D) of the resistant trait in the
F1 females from both reciprocal crosses was estimated
using thefollowingformula:
(X1- X3), where: X1is log of the LC50of the resistant
strain, X2is the log of the LC50of the F1females, and X3is
log of the LC50of the susceptible strain (Stone 1968). This
formula gives a value of -1 if resistance is full recessive, a
value of 0 if there is no dominance, and a value of ?1 if
resistance is full dominant.
D = (2X2- X1- X3)/
Biochemical assays
Electrophoresis
Vertical slab polyacrylamide gel electrophoresis was per-
formed following the procedures by Goka and Takafuji
(1992) and Walker (1994). The gels were 1 mm thick and
80 mm 9 80 mm in area. Acrylamide concentrations were
7.5% in separating gels and 3.5% in stacking gels. Adult
female mites were homogenized individually in 10 ll of
32% (w/v) sucrose with 0.1% Triton X-100 in microplates
by a multiple-homogenizer (Moores et al. 1988). Electro-
phoresis was carried out at a constant current of 150 V at
5–8?C for approximately 1.5 h. Esterases were stained by
incubating the gels for 30 min in a 0.02% (w/v) solution of
a-naphthyl acetate in 0.2 M phosphate buffer (pH 6.5),
which contained 1% acetone, and then placing the gels in
0.4 (w/v) fast blue BB salt for 1 h. All staining reactions
were stopped by placing the gel in 7.5% acetic acid. Gels
were prepared simultaneously, and different gels were
simultaneously applied to paternal and resistant strains
under stable conditions.
Photometric esterase assay
Esterase assays were performed according to the method
developed by Stumpf and Nauen (2002). The 10,000g
supernatant of mass homogenates of 100 adult females
prepared in 500 ll ice-cold 0.1 M sodium phosphate buf-
fer, pH 7.5, containing 0.1% (w/v) Triton X-100, was
diluted 10-fold and used as the enzyme source. Twenty five
microliter aliquots (0.5 mite equivalents) were added to the
wells of a 96-well microplate, containing 25 ll of 0.2 M
sodium phosphate buffer, pH 6.0. Wells with buffer-only
served as a control for the nonenzymatic reaction. The
assay was started by adding 200 ll of substrate solution to
each well, giving a final volume of 250 ll. Substrate
solution consisted of 15 mg of fast Blue RR salt dissolved
in 25 ml of sodium phosphate buffer, pH 6.0, and 250 ll of
100 mM a-naphthyl acetate in acetone. The esterase
activity was measured continuously at 450 nm and 25?C in
a Versamax kinetic microplate reader (Molecular Devices)
for 10 min, utilizing Softmax PRO software to fit kinetics
plots by linear regression.
Photometric GST assay using 1-chloro-2, 4-dinitrobenzene
Glutathione S-transferase (GST) activities were measured
according to Stumpf and Nauen (2002). GST activity was
determined using 1-chloro-2,4-dinitrobenzene and reduced
glutathione (GSH) as a substrate. Hundred adult females
were homogenized in 1,000 ll Tris–HCl (0.05 M, pH 7.5).
The total reaction volume per microplate well was 300 ll,
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which consisted of 100 ll of supernatant (10,000g, 5 min),
100 ll of CDNB (dissolved in 0.1% (v/v) ethanol), and
100 ll of GSH in Tris–HCl (0.05 M, pH 7.5), giving final
concentrations of 0.4 mM CDNB and 4 mM GSH.
The change in absorbance was measured continuously
for 5 min at 340 nm and 25?C using the Versamax kinetic
microplate reader (Molecular Devices). The nonenzymatic
reaction of CDNB with GSH measured without homoge-
nate served as a control.
Photometric monooxygenase assay using the O-
demethoxylation of p-nitroanisole
Assays examining the O-demethoxylation of p-nitroanisole
(PNOD) by cytochrome P450 monooxygenases were con-
ducted using the procedures developed by Rose et al.
(1995).
For the PNOD assay, 100 adult females were homoge-
nized on ice in 200 ll of homogenization buffer (0.05 M
Tris–HCl ? 1.15 KCl% ? 1 mM EDTA, pH 7.7). The
homogenate was centrifuged at 4?C, 20,000g for 20 min.
Hundred microliter of 2 mM p-nitroanisole solution, 45 ll
enzyme, and 45 ll homogenization buffer were added to
each well. The microplate was incubated for 5 min at 30?C
and the reaction was initiated by the addition of 10 ll of
9.6 mM NADPH. The absorbance was read in the Versa-
max kinetic microplate reader (Molecular Devices) at
405 nm and 30?C for 15 min.
The activity of enzymes was analyzed by Softmax PRO
software and presented as mOD/min/mg proteins. The data
were analyzed using the General Linear Model (GLM)
procedure of SAS (1999) by using strains in the model and
the PDIFF statement was used to compare strains’ enzyme
activity means for dependent variables. An alpha level of
0.05 was accepted as the significance level.
Results
Selection results
The susceptibility shown by the SAK strain to chlorpyrifos
for each of 12 selections is summarized in Table 1. In the
SAK strain of T. urticae after 12 chlorpyrifos selections the
LC50 value increased from 222.51 to 2,370.44 ll/l.
Chlorpyrifos resistance increased 10.6 fold in CHLO 12
population compared to the main population SAK.
Toxicity of other pesticides to chlorpyrifos selected
strain
LC50values and the rate of resistance of the CHLO 12 strain
tosixdifferentinsecticidesarelistedinTable 2.Itwasfound
that the chlorpyrifos-resistant CHLO 12 strain showed 7.28
fold greater resistance to abamectin and a low-level of
resistance to fenpyroximate, propargite, and clofentezine.
However,thechlorpyrifos-resistant CHLO12 strainwasnot
significantly different in its responses to amitraz and bif-
enthrin when compared to the susceptible strain.
‘‘Synergist ? insecticide’’ results
LC50values and synergist impact rates of chlorpyrifos and
chlorpyrifos ? PBO, IBP, or TPP synergists in CHLO 12
strain are presented in Table 3. PBO, IBP, and TPP were
each applied to the CHLO 12 strain together with chlor-
pyrifos, and solely IBP has shown low synergistic effect
according to the LC50values in 95% confidence interval.
However; PBO, IBP, or TPP synergists applied to the GSS
strain together with chlorpyrifos did not result in any
synergistic effect.
‘‘Resistance-inheritance’’ results
F1LC50values and resistance rates obtained at the end of
the reciprocal crossings between CHLO 12 strain and GSS
(susceptible) strain are listed in Table 4. D values obtained
from both crossings were found to be within the range
0\D[1. Results showed that the ‘‘resistance to chlor-
pyrifos’’ characteristic of CHLO 12 (resistant) 9 GSS
(susceptible) strain was transferred by an incompletely
dominant gene and that it is not dependent on parent.
Biochemical analysis results
Esterase bands of 10 samples, randomly selected from the
SAK and CHLO 12 strains, were analyzed via electro-
phoresis. Differences were detected between SAK and
CHLO 12 strains in terms of the arrangement and intensity
of esterase enzyme bands (Fig. 1). Kinetic activities of
esterase, GST, and P450 enzymes of GSS, SAK and CHLO
12 strains are shown in Table 5. The kinetic activity of
esterase enzymes was found to be significantly higher in
the chlorpyrifos-resistant CHLO 12 strain than in the sus-
ceptible (GSS) strain. In addition, the kinetic activity of
esterase enzymes was also higher than in the parental strain
SAK (P\0.005). Kinetic activity of GST enzymes of the
CHLO 12 strain was found to be the same with that of the
parental strain (SAK) and to be lower than that of the
susceptible strain (GSS) (P\0.005). Compared to that of
the parental strain SAK, the kinetic activity of P450
enzyme increased in CHLO 12 strain, however, the varia-
tion in kinetic activity was not statistically significant
(P[0.05). The P450 activity of the CHLO 12 strain was
found to be significantly higher than that of the susceptible
strain (P\0.005).
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Discussion
The fact that T. urticae has demonstrated a tendency to
develop resistance to insecticides within a short time is a
significant challenge to the effective control of mites via
chemicals. Chlorpyrifos—an organophosphate, wide-spec-
trum insecticide, and acaricide—was used in the study. The
application of chlorpyrifos against spider mites and other
pests increases the selection pressure on spider mites.
Development of resistance by insects and mites to agricul-
tural insecticides is directly related to, among other factors,
the frequency of insecticide application. Spider mites
develop resistance to chlorpyrifos, either directly or indi-
rectly, when exposed to chlorpyrifos or a cross-resisted
product.
The resistance rate increased from 8.58 to 91.45 fold in
the SAK strain of T. urticae after 12 chlorpyrifos selec-
tions. Depending on the number of selections, the SAK
strain developed a significant level of resistance to chlor-
pyrifos. Chlorpyrifos resistance increased 10.6 fold in
CHLO 12 population compared to the main population
SAK. Some researcher determined various levels of
chlorpyrifos resistance in T. urticae populations collected
from field strain (Nauen et al. 2001; Stumpf et al. 2001; Ay
2005).
Chlorpyrifos-resistant CHLO 12 strain showed 7.28,
3.04, 2.68, and 2.60 fold of resistance to abamectin, clof-
entezine, fenpyroximate, and propargite, respectively. Ay
et al. (2005) reported in their previous study that the
parental SAK strain was susceptible to propagite, to
Table 1 Selection for resistance to chlorpyrifos in a strain of T. urticae; estimation of LC50(ll (formulation)/l distilled water) and resistance
ratio
Population
Na
Slope ± SELC50(ll/l)
(95% CIb)
LC60(ll/l)
(95% CIb)
LC90(ll/l)
(95% CIb)
RR LC50c
RR LC90c
SAK 7211.28 ± 0.10222.51
(176.93–276.30)
350.87
(282.57–441.06)
2,227.94
(1,563.17–3,557.37)
8.5816.25
Select 17241.22 ± 0.11307.90
(239.76–391.22)
497.55
(386.52–633.78)
3,489.31
(2,324.93–6,059.41)
11.8725.46
Select 27211.16 ± 0.11 485.63
(371.11–632.15)
802.76
(617.08–1,079.98)
6,172.98
(3,870.18–11,919.51)
18.7345.04
Select 37241.17 ± 0.11513.96
(394.02–663.82)
843.10
(652.93–1,118.11)
6,284.26
(40,301.27–11,671.09)
19.82 45.85
Select 47241.13 ± 0.10 584.67
(453.67–751.74)
968.78
(749.08–1,294.12)
7,796.80
(4,934.77–14,707.48)
22.5556.89
Select 5 7241.11 ± 0.10612.69
(475.93–783.21)
1,034.15
(808.47–1,366.13)
8,654.67
(5,528.10–16,048.77)
23.6363.15
Select 67221.04 ± 0.09739.29
(575.84–947.44)
1,292.49
(1,006.47–1,721.59)
12,475.71
(7,664.35–24,609.97)
28.5291.03
Select 77261.06 ± 0.03 947.61
(728.71–1,224.42)
1,640.72
(1,268.68–2,192.46)
15,226.24
(9,405.39–29,851.82)
36.55 111.10
Select 87271.12 ± 0.10 1,121.49
870.20–1,432.62
1,887.58
(1,476.89–2,475.58)
15,616.10
(10,030.02–28,717.57)
43.26 113.95
Select 9 7271.14 ± 0.101,238.06
(965.13–1,571.71)
2,061.77
(1,623.46–2,677.41)
16,337.74
(10,687.39–29,162.14)
47.76 119.21
Select 10 7281.19 ± 0.101,311.36
(1,030.42–1,646.16)
2,137.73
(1,702.84–2,725.17)
15,534.39
(10,534.98–26,171.05)
50.59111.89
Select 117231.04 ± 0.09 2,067.46
(1,609.62–2,654.72)
3,618.06
(2,811.79–4,837.51)
35,062.57
(21,430.97–69,730.83)
79.76255.85
CHLO 12723 1.19 ± 0.102,370.44
(1,850.78–2,991.09)
3,867.74
(3,065.15–4,956.08)
28,210.23
(19,011.93–48,064.26)
91.45205.85
GSS 720 1.77 ± 0.1325.92
(21.32–30.88)
– 137.04
(109.23–181.83)
––
aTotal number of mites used
bConfidence interval
cResistance ratio = LC50or LC90value of resistance strain/LC50or LC90value of the GSS strain
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