J Pest Sci (2010) 83:85–93 DOI 10.1007/s10340-009-0274-9
ORIGINAL PAPER
Inheritance and detoxification enzyme levels in Tetranychus urticae Koch (Acari: Tetranychidae) strain selected with chlorpyrifos Recep Ay Æ Sibel Yorulmaz
Received: 27 January 2009 / Accepted: 18 August 2009 / Published online: 11 September 2009 Ó Springer-Verlag 2009
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 33.59 mOD/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.
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:
[email protected]
Keywords Chlorpyrifos Detoxifying enzymes Inheritance Resistance Tetranychus urticae
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 twospotted 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, disruption of ecological systems, and harm to natural enemies. 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).
123
86
Chlorpyrifos (O,O-diethyl-O-[3,5,6-trichloro-2-pyridinyl]) is a contact-effect insecticide/acaricide. It is a member of the organophosphorus insecticide group, which are widely used in agriculture to control phytophagous insects and mites (Picco et al. 2008). Chlorpyrifos acts by inhibiting 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.
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 September 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 susceptible strain (GSS) was obtained from Rothamsted Research, Harpenden (England), in 2001 and also maintained 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, and three synergists: piperonyl butoxide (PBO); S-benzyl-O,O-diisopropyl phosphorothioate (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. 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
123
J Pest Sci (2010) 83:85–93
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 90 values with 95% CI were estimated. The LC50 values of the selection strain were compared to those of the susceptible strain (GSS). The resistance ratio was calculated by dividing the LC50 of the resistant strain by the LC50 of 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 chlorpyrifos was conducted periodically on mite populations when the number of surviving mites changed in the selection Petri dishes. The new LC60 was 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 suspension was sprayed on the base and lid of Petri dishes in the same manner as the toxicity test, 30 min prior to pesticide application. Distilled water–acetone without a
J Pest Sci (2010) 83:85–93
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 synergistic ratio (SR) was calculated using the following formula: SR ¼ LC50 of chlorpyrifos without synergist= LC50 of 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, propargite, 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 calculated dividing the LC50 of 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 F1 females 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 haploid–diploid mating system resulted in F1 females and males. The F1 females 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 conducted using three replicates of seven serially diluted concentrations (plus a distilled water-only control) covering 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 the following formula: D = (2X2 - X1 - X3)/ (X1 - X3), where: X1 is log of the LC50 of the resistant strain, X2 is the log of the LC50 of the F1 females, and X3 is log of the LC50 of 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.
87
Biochemical assays Electrophoresis Vertical slab polyacrylamide gel electrophoresis was performed 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). Electrophoresis 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 buffer, 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,
123
88
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 homogenate served as a control. Photometric monooxygenase assay using the Odemethoxylation of p-nitroanisole Assays examining the O-demethoxylation of p-nitroanisole (PNOD) by cytochrome P450 monooxygenases were conducted using the procedures developed by Rose et al. (1995). For the PNOD assay, 100 adult females were homogenized 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 Versamax 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 LC50 values and the rate of resistance of the CHLO 12 strain to six different insecticides are listed in Table 2. It was found
123
J Pest Sci (2010) 83:85–93
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, the chlorpyrifos-resistant CHLO 12 strain was not significantly different in its responses to amitraz and bifenthrin when compared to the susceptible strain. ‘‘Synergist ? insecticide’’ results LC50 values 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 chlorpyrifos, and solely IBP has shown low synergistic effect according to the LC50 values 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 F1 LC50 values 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 chlorpyrifos’’ 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 electrophoresis. 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 susceptible (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 variation 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).
J Pest Sci (2010) 83:85–93
89
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 ± SE
SAK
721
1.28 ± 0.10
Select 1
724
1.22 ± 0.11
LC50 (ll/l) (95% CIb)
LC60 (ll/l) (95% CIb)
LC90 (ll/l) (95% CIb)
222.51
350.87
2,227.94
(176.93–276.30)
(282.57–441.06)
(1,563.17–3,557.37)
307.90
497.55
3,489.31
(239.76–391.22)
(386.52–633.78)
(2,324.93–6,059.41)
RR LC50c
RR LC90c
8.58
16.25
11.87
25.46
Select 2
721
1.16 ± 0.11
485.63
802.76
6,172.98
18.73
45.04
Select 3
724
1.17 ± 0.11
(371.11–632.15) 513.96
(617.08–1,079.98) 843.10
(3,870.18–11,919.51) 6,284.26
19.82
45.85
(394.02–663.82)
(652.93–1,118.11)
(40,301.27–11,671.09) 22.55
56.89
23.63
63.15
28.52
91.03
36.55
111.10
43.26
113.95
47.76
119.21
Select 4 Select 5 Select 6 Select 7 Select 8 Select 9
724 724 722 726 727 727
1.13 ± 0.10 1.11 ± 0.10 1.04 ± 0.09 1.06 ± 0.03 1.12 ± 0.10 1.14 ± 0.10
Select 10
728
1.19 ± 0.10
Select 11
723
1.04 ± 0.09
CHLO 12 GSS
723 720
1.19 ± 0.10 1.77 ± 0.13
584.67
968.78
7,796.80
(453.67–751.74)
(749.08–1,294.12)
(4,934.77–14,707.48)
612.69
1,034.15
8,654.67
(475.93–783.21)
(808.47–1,366.13)
(5,528.10–16,048.77)
739.29
1,292.49
12,475.71
(575.84–947.44)
(1,006.47–1,721.59)
(7,664.35–24,609.97)
947.61
1,640.72
15,226.24
(728.71–1,224.42)
(1,268.68–2,192.46)
(9,405.39–29,851.82)
1,121.49
1,887.58
15,616.10
870.20–1,432.62
(1,476.89–2,475.58)
(10,030.02–28,717.57)
1,238.06
2,061.77
16,337.74
(965.13–1,571.71)
(1,623.46–2,677.41)
(10,687.39–29,162.14)
1,311.36 (1,030.42–1,646.16)
2,137.73 (1,702.84–2,725.17)
15,534.39 (10,534.98–26,171.05)
50.59
111.89
79.76
255.85
91.45
205.85
–
–
2,067.46
3,618.06
35,062.57
(1,609.62–2,654.72)
(2,811.79–4,837.51)
(21,430.97–69,730.83)
2,370.44
3,867.74
28,210.23
(1,850.78–2,991.09)
(3,065.15–4,956.08)
(19,011.93–48,064.26)
25.92
–
137.04
(21.32–30.88) a
(109.23–181.83)
Total number of mites used
b
Confidence interval
c
Resistance ratio = LC50 or LC90 value of resistance strain/LC50 or LC90 value of the GSS strain
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-spectrum 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 agricultural insecticides is directly related to, among other factors, the frequency of insecticide application. Spider mites develop resistance to chlorpyrifos, either directly or indirectly, 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 selections. Depending on the number of selections, the SAK strain developed a significant level of resistance to chlorpyrifos. 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, clofentezine, fenpyroximate, and propargite, respectively. Ay et al. (2005) reported in their previous study that the parental SAK strain was susceptible to propagite, to
123
90 Table 2 Toxicity tests with different acaricides, using the susceptible GSS and chlorpyrifos-resistant CHLO 12 strain of T. urticae
J Pest Sci (2010) 83:85–93
Acaricide
Population
Na
Slope ± SE
Abamectin
GSS
721
1.09 ± 0.09
LC50 (ll/l) (95% CIb)
LC50 RRc
11.03
–
(8.67–14.02) CHLO 12
724
1.06 ± 0.10
80.40
7.28
(61.78–103.87) Amitraz
GSS
724
1.50 ± 0.11
300.35
–
(246.39–362.05) CHLO 12
723
1.26 ± 0.10
244.73
\1
(195.43–301.25) Propargite
GSS
724
1.21 ± 0.10
CHLO 12
724
1.09 ± 0.09
127.55
–
(101.78–158.20) 332.65
2.60
(261.26–422.63) Clofentezine
GSS
716
1.38 ± 0.10
28.64
–
(23.23–34.78) 87.20
3.04
CHLO 12
722
1.18 ± 0.10
GSS
720
1.34 ± 0.10
(68.03–110.18) Fenoxyprimate
66.17
–
(54.23–79.86) CHLO 12 a
Total number of mites used
b
Confidence interval
725
1.21 ± 0.10
Bifenthrin
GSS
527
1.49 ± 0.15
CHLO 12
amitraz, and to abamectin collected from field in 2002. However, CHLO 12 strain was found to be more susceptible to bifenthrin and amitraz than susceptible strain. When the same strain was subjected to chlorpyrifos selection, the resistance was found to be 7.28 fold greater to abamectin and 2.60 fold greater to propargite. Results showed that particularly abamectin resistance improved depending on the chlorpyrifos selection. While IBP has shown a potentially tiny amount of synergistic effect with chlorpyrifos in CHLO12 strain, PBO, and TPP have not presented synergistic effect. However; PBO, IBP, and TPP synergists applied together with chlorpyrifos were found to have no effect on chlorpyrifos effect in the susceptible strain. Kang et al. (2006) stated that PBO and TPP synergists increased chlorpyrifos effect by 2.9 and 2.4 fold, respectively, in Bemisia tabaci (Gennadius) population that is resistant to chlorpyrifos by 21.8 fold. Reciprocal crossings made to determine the inheritance pathway of the resistance between resistant CHLO 12 strain and susceptible GSS strain showed that resistance is transferred by an incompletely dominant gene and is not sex-linked. Devine et al. (2002) suggested that resistance is transferred from father and mother partly dominant in METI-pesticide resistant T. urticae population.
123
2.68
545.31
–
(340.35–855.46)
c
Resistance ratio = LC50 value of the CHLO 12 population/LC50 value of the GSS population
177.45 (139.62–222.87)
722
1.10 ± 0.10
435.98
\1
(336.21–558.92)
Esterase enzyme bands of CHLO 12 strain were found to be different from those of the parental strain (SAK). A considerable increase was observed in the number and the intensity of the esterase bands of the CHLO 12 strain. In addition, kinetic readings of esterase enzyme activities in a microplate reader increased from 19.35 to 33.59 mOD/ min/mg protein. A statistically significant difference was found between kinetic esterase enzyme activities of GSS, SAK, and CHLO 12 strains (P \ 0.005). This supports the theory that as SAK was resistant, esterase enzyme activity was found to be higher than the more susceptible GSS strain. Similarly, esterase enzyme activity of the CHLO 12 strain, the resistance of which increased due to chlorpyrifos selection, was higher than that of both the parental and susceptible strains. Scarf et al. (1998) reported that native PAGE of esterase identified one electromorph (E2) which stained more intensely in Blattella germanica (L.) following chlorpyrifos selection and which inhibited by both chlorpyrifos and chlorpyrifos-oxon. A statistically significant difference was not found between kinetic GST enzyme activities of SAK and CHLO 12 strains (P \ 0.005). In this study, CDNB was used as the substrate to detect GST enzyme activities. However, Tsagkarakou et al. (2002) stated that 3,4-dichloronitro-
J Pest Sci (2010) 83:85–93
91
Table 3 Synergistic effect of PBO, IBP and TPP on chlorpyrifos efficacy in T. urticae strain CHLO 12 and GSS Population
Compound ? synergist
Na
Slope ± SE
LC50 (ll/l) (95% CIb)
LC90 (ll/l) (95% CIb)
SRc
CHLO 12
Chlorpyrifos
723
1.19 ± 0.10
2,370.44
28,210.23
–
(1,850.78–2,991.09)
(19,011.93–48,064.26)
?PBO
723
?IBP
723
?TPP GSS
723
Chlorpyrifos
720
?PBO
720
1.16 ± 0.10 1.22 ± 0.10 1.33 ± 0.11
1,688.56
21,189.35
(1,315.25–2,136.84)
(14,113.15–36,780.08)
1.40
1,337.86
13,222.76
(1,060.44–1,657.48)
(9,423.79–20,633.42)
1.77
1,611.10
14,692.20
(1,270.21–2,006.79)
(10,476.53–22,934.39)
1.72 ± 0.13
25.92
137.04
–
1.44 ± 0.11
(21.32–30.88) 29.03
(109.23–181.83) 224.73
–
(18.89–41.58)
(138.12–487.88)
27.96
214.10
(18.55–39.47)
(134.12–444.48)
28.46
233.35
(18.95–40.43)
(141.61–518.52)
?IBP
723
1.45 ± 0.12
?TPP
722
1.40 ± 0.11
a
Total number of mites used in experiment
b
Confidence interval
c
Synergistic ratio = LC50 for chlorpyrifos alone/LC50 for chlorpyrifos with synergist
1.47
– –
Table 4 Concentration-response data of chlorpyrifos on female adults of the GSS and CHLO 12 strain and their reciprocal crosses Population
Na
Slope ± SE
LC50 (ll/l) (95% CIb)
LC90 (ll/l) (95% CIb)
GSS
720
1.77 ± 0.13
25.92
137.04
1.19 ± 0.10
(21.32–30.88) 2,370.44
(109.23–181.83) 28,210.23
(1,850.78–2,991.09)
(19,011.93–48,064.26)
1,380.31
12,857.77
(1,066.49–1,754.88)
(8,815.12–21,488.26)
1,710.08
23,415.86
(1,293.41–2,248.09)
(14,355.70–47,030.40)
CHLO 12
723
F1 (CHLO 12 $ 9 GSS #)
611
1.32 ± 0.12
F1 (GSS $ 9 CHLO 12 #)
602
1.12 ± 0.11
a
Total number of mites used
b
Confidence interval
c
Resistance ratio = LC50 value of resistance strain/LC50 value of the GSS strain
d
Degree of dominance
LC50 RRc
–
Dd
–
91.45
–
53.25
0.75
65.97
0.85
Fig. 1 The esterase enzyme bands of individual T. urticae SAK and CHLO 12 strains in each well (a SAK, b CHLO 12)
123
92
J Pest Sci (2010) 83:85–93
Table 5 Esterase, GST, and P450 activities in susceptible strain GSS, parental strain SAK and chlorpyrifos resistant strain CHLO 12 of T. urticae (P \ 0.05)a Enzyme
Population
nb
Esterase
GSS
4
10.35 (±0.37) c
1.00
SAK
4
19.35 (±0.37) b
1.86
CHLO 12
4
33.59 (±0.37) a
3.24
GSS
5
13.73 (±0.29) a
1.00
SAK
4
11.91 (±0.32) b
0.86
CHLO 12
4
11.94 (±0.32) b
0.86
GSS SAK
3 3
0.0016 (±0.00071) b 0.0023 (±0.00071) ab
1.00 1.43
CHLO 12
3
0.0042 (±0.00071) a
2.62
GST
P450
Specific activity (±SE) mOD/min/mg proteins
a
Means with different letters in column for each enzyme are significantly different (P \ 0.05)
b
Number of replicates
c
Enzyme activity SAK or CHLO 12/enzyme activity GSS strain
benzene (DCNB) is a weak substrate for the detection of GST activity; that they used CDNB’ in the organophosphates resistant and sensitive T. urticae strain; and that GST activity was not significantly different in the susceptible strain and methyl-parathion or methomyl resistant strains. As in the present study, GST activity, which is detected via CDNB, was not correlated with chlorpyrifos resistance in Malaysian B. germanica (Lee et al. 2000). However, researchers have shown that DCNB-GST activity can be related to organophosphate resistance (Kristensen 2005; Vontas et al. 2001). Therefore, it would be beneficial to use this substrate in future studies of the organophosphate resistance mechanism(s) involved. Wang and Wu (2007) recorded 2.1 fold of monooxygenase and 2 fold of GST enzyme activity in Bemisia tabaci population that was resistant to abamectin by 14.5 fold. In this study, PNOD was used as the substrate in the detection of P450 activity. Generally, O-deethylation of 7-ethoxycoumarin rather than PNOD is used in the detection of P450 activity in most of the insects and mites, 7-ethoxycoumarin (7-EC) is known to produce better results (Stumpf and Nauen 2002; Rauch and Nauen 2003; Sato et al. 2006). Most cases of monooxygenase-mediated resistance results from an increase in detoxification (Scott 1999). Scharf et al. (1998) stated that P450 activity increased in chlorpyrifos-resistant B. germanica. Sato et al. (2006) reported that P450 activity of the medidathion resistant strain of Amblyseius womersleyi Schicha was higher than that of the medidathion susceptible strain. Rauch and Nauen (2003) determined 1.2 fold esterase, 1.2 fold GST, and 2.1 fold P450 enzyme activity in Tetranychus urticae population resistant to spirodiclofen by 13 fold. Decreased sensitivity of AChE is recognized as one of the common mechanisms of resistance to organophosphates in many insects and mites (Stumpf et al. 2001;
123
R/Sc
Huang and Han 2007; Yang et al. 2009; Kang et al. 2006). Stumpf et al. (2001) suggested that an additional resistance mechanism against chlorpyrifos was present in the field collected T. urticae strain. Hemingway and Karunaratne (1998) stated that esterases can also play a role in organophosphate resistance. In conclusion, the present study suggests that there may be relationship between esterase and resistance development in CHLO 12 strain due to chlorpyrifos selection. Interpretation of bioassay and biochemical results together suggests that esterase enzymes may be a minor component of chlorpyrifos resistance in the strains of T. urticae examined, while GST and P450 enzymes have no effect on chlorpyrifos resistance. There are likely to be other more potent mechanism(s) providing the principal levels of protection. Acknowledgment This study was supported by the Scientific and Technological Research Council of Turkey (TUBITAK, TOVAG105O179).
References Ay R (2005) Determination of susceptibility and resistance of some greenhouse populations of Tetranychus urticae Koch to chlorpyrifos (dursban 4) by the Petri dish-potter tower method. J Pest Sci 78:139–143 _ Gu¨rkan MO (2005) Response to some Ay R, So¨keli E, Karaca I, acaricides of the two-spotted spider mite (Tetranychus urticae Koch) from protected vegetables in Isparta. Turk J Agric For 29:165–171 Devine GJ, Barber M, Denholm I (2002) Incidence and inheritance of resistance to METI-acaricides in European strains of the twospotted spider mite (Tetranychus urticae) (Acari: Tetranychidae). Pest Manag Sci 57:443–448 Ffrench-Constant RH, Roush RT (1990) Resistance detection and documentation: the relative roles of pesticidal and biochemical assays. In: Roush RT, Tabashnik BE (eds) Pesticide resistance in arthropods. Chapman and Hall, New York, pp 4–38
J Pest Sci (2010) 83:85–93 Goka K, Takafuji A (1992) Enzyme variations among Japanese populations of the two-spotted spider mites, Tetranychus urticae Koch. Appl Entomol Zool 27:141–150 Hemingway J, Karunaratne SHPP (1998) Mosquito carboxylesterases: a review of the molecular biology and biochemistry of a major insecticide resistance mechanism. Med Vet Entomol 12:1–12 Huang SJ, Han ZJ (2007) Mechanisms for multiple resistances in field populations of common cutworm, Spodoptera litura (Fabricus) in China. Pestic Biochem Physiol 87:14–22 Kang CY, Wu G, Miyata T (2006) Synergism of enzyme inhibitors and mechanisms of insecticide resistance in Bemisia tabaci (Gennadius) (HOM., Aleyrodidae). J Appl Entomol 130:377–385 Kim YJ, Lee SH, Lee SW, Ahn YJ (2004) Fenpyroximate resistance in Tetranychus urticae (Acari: Tetranychidae): cross-resistance and biochemical resistance mechanisms. Pest Manag Sci 60:1001–1006 Kim YJ, Park HM, Cho JR, Ahn YJ (2006) Multiple resistance and biochemical mechanisms of pyridaben resistance in Tetranychus urticae (Acari: Tetranychidae). J Econ Entomol 99:954–958 Kristensen M (2005) Glutathione S-transferase and insecticide resistance in laboratory strains and field populations of Musca domestica. J Econ Entomol 98:1341–1348 Lee CY, Hemingway J, Yap HH, Chong L (2000) Biochemical characterization of insecticide resistance in the German cockroach, Blattella germenica, from Malaysia. Med Vet Entomol 14:11–18 LeOra, Software (1994) POLO—PC: a user’s guide to probit or logit analysis LeOra software. Berkeley, CA, p 28 Moores GD, Devonshire AL, Denholm I (1988) A microtiter plate assay for characterizing insensitive acetylcholinesterase genotypes of insecticide resistant insects. Bull Entomol Res 78:537– 544 Nauen R, Stumpf N, Elbert A, Zebitz CPW, Kraus W (2001) Acaricide toxicity and resistance in larvae of different strains of Tetranychus urticae and Panonychus ulmi (Acari: Tetranychidae). Pest Manag Sci 57:253–261 Oppenoorth FJ (1984) Biochemistry of insecticide resistance. Pestic Biochem Physiol 22:187–193 Picco EJ, Fernandez RH, David DC, Andres MS, Boggio JC, Rodriguez CR (2008) Use of cholinesterase activity in monitoring chlorpyrifos exposure of steer cattle after topical administration. J Environ Sci Health B 43:405–409 Rauch N, Nauen R (2003) Spirodiclofen resistance risk assessment in Tetranychus urticae (Acari: Tetranychidae): a biochemical approach. Pestic Biochem Physiol 74:91–101 Rose R, Barbhaiya L, Roe R, Rock G, Hodgson E (1995) Cytochrome P-450-associated insecticide resistance and the development of biochemical diagnostic assays in Heliothis virescens. Pestic Biochem Physiol 51:178–191 SAS (1999) Statistical analysis systems user’s guide, 8th edn. SAS Institute INC, Raleigh Sato ME, Tanaka T, Miyata T (2006) Monooxygenase activity in methidathion resistant and susceptible populations of Amblyseius wormersleyi (Acari: Phytoseiidae). Exp Appl Acarol 39:13–24
93 Scarf ME, Neal JJ, Bennett GW (1998) Changes of insecticide resistance levels and detoxication enzymes following insecticide selection in the German Cockroach, Blattella germanica (L.). Pestic Biochem Physiol 59:67–79 Scott JG (1999) Cytochrome P450 and insecticide resistance. Insect Biochem Mol Biol 29:757–777 Smegal DC (2000) Human health risk assessments chlorpyrifos, USA, EPA. Available at http://yosemite1.epa.gov/ee/epa/ria.nsf/vwRef/ P.2000.14?OpenDocument Stone BF (1968) A formula for determining degree of dominance in cases of monofactorial inheritance of resistance to chemicals. Bull World Health Organ 38:325–326 Stumpf N, Nauen R (2001) Cross-resistance, inheritance and biochemistry of mitochondrial electron transport inhibitor-acaricide resistance in Tetranychus urticae (Acari: Tetranychidae). J Econ Entomol 94:1577–1583 Stumpf N, Nauen R (2002) Biochemical markers linked to abamectin resistance in Tetranychus urticae (Acari: Tetranychidae). Pestic Biochem Physiol 72:111–121 Stumpf N, Zebitz CPW, Kraus W, Moores GD, Nauen R (2001) Resistance to organophosphates and biochemical genotyping of acetylcholinesterase in Tetranychus urticae (Acari: Tetranychidae). Pestic Biochem Physiol 69:131–142 Tsagkarakou A, Pasteur N, Cuany A, Chevillon C, Navajas M (2002) Mechanisms of resistance to organophosphates in Tetranychus urticae (Acari: Tetranychidae) from Greece. Insect Biochem Mol Biol 32:417–427 Van Leeuwen T, Tirry L (2007) Esterase-mediated bifenthrin resistance in a multi resistant strain of the two-spotted spider mite, Tetranychus urticae. Pest Manag Sci 63:150–156 Van Leeuwen T, Van Pottelberge S, Tirry L (2006) Biochemical analysis of chlorfenapyr—selected resistant strain of Tetranychus urticae Koch. Pest Manag Sci 62:425–433 Vontas JG, Small GJ, Hemingway J (2001) Glutathione S-transferases as antioxidant defence agents confer pyrethroid resistance in Nilaparvata lugens. Biochem J 357:65–72 Walker JM (1994) Nondenaturing polyacrylamide gel electrophoresis of proteins. In: Walker JM (ed) Methods in molecular biology. Humana Press. Inc, Totowa, pp 17–22 Wang L, Wu Y (2007) Cross-resistance and biochemical mechanisms of abamectin resitance in the B-type Bemisia tabaci. J Appl Entomol 131:98–103 Yang X, Buschman LL, Zhu KY, Margolies DC (2002) Susceptibility and detoxifying enzyme activity in two spider mite species (Acari: Tetranychidae) after selection with three insecticides. J Econ Entomol 95:399–406 Yang ML, Zhang JZ, Zhu KY, Xuan T, Liue XJ, Guo YP, Ma EB (2009) Mechanisms of organophosphate resistance in field population of oriental migratory locust, Locusta migratoria manilensis (Meyen). Arch Insect Biochem Physiol 71:3–15 Young SJ, Gunning RV, Moores GD (2005) The effect of piperonyl butoxide on pyrethroid resistance associated esterases in Helicoverpa armigera (Hu¨bner) (Lepidoptera: Noctuidae). Pest Manag Sci 61:397–401
123
