Pesticide Biochemistry and Physiology 98 (2010) 254–262

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Pesticide Biochemistry and Physiology

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Synergism of resistance to phosalone and comparison of kinetic properties of acetylcholinesterase from four ﬁeld populations and a susceptible strain of Colorado potato beetle

M. Malek Mohamadi a,*, M.S. Mossadegh a, M.J. Hejazi b, M.T. Goodarzi c, M. Khanjani d, H. Galehdari e a Department of Plant Protection, Faculty of Agriculture, Chamran University, Ahwaz, Iran b Department of Plant Protection, Faculty of Agriculture, University of Tabriz, Tabriz, Iran c Research Center for Molecular Medicine, Hamedan University of Medical Sciences, Hamedan, Iran d Department of Plant Protection, Faculty of Agriculture, Bu Ali Sina University, Hamedan, Iran e Department of Genetics, Faculty of Science, Chamran University, Ahwaz, Iran article info abstract

Article history: The susceptibility to phosalone and biochemical characteristics of acetylcholinesterase (AChE) were com- Received 16 September 2009 pared between susceptible (SS) strain and four field populations of Colorado potato beetle (CPB) collected Accepted 28 June 2010 from commercial potato fields of Hamedan Province in west of Iran. Bioassays involving topical applica- Available online 19 August 2010 tion of phosalone to fourth instars revealed up to 252 fold resistance in field populations compared with the SS strain. Synergism studies showed that although esterase and/or glutathione S-transferase meta- Keywords: bolic pathways were present and active against phosalone, they were not selected for and did not have Colorado potato beetle a major role in resistance. It is likely that piperonyl butoxide (PBO) reduced phosalone toxicity by inhib- AChE iting bio-activation of phosalone. The affinity (K ) and hydrolyzing efficiency (V ) of AChE to selected Susceptibility m max Inhibition substrates, namely, acetylthiocholine iodide (ATC), propionylthiocholine iodide (PTC), and butyrylthioch- Synergism oline iodide (BTC) were examined. AChE inhibition by higher substrate concentration was evident only in the SS strain. In resistant field populations, Aliabad (Aa), Bahar (B) and Dehpiaz (Dp), substrate inhibition at higher concentrations was not seen. There was no definite optimal concentration found for any of the substrates examined. When ATC, PTC, and BTC were used as substrate, the reaction rates of AChE from Yengijeh (Yg) population increased as the concentration of all three substrates were increased, but were almost constant at concentration of ATC P 3.98, PTC P 2.8, and BTC P 5 mM. The susceptible form of AChE had the most efficient ATC hydrolysis but very low BTC hydrolysis activity. In contrast, AChEs from field populations elicited relatively reduced ATC hydrolysis, but relatively increased BTC hydrolysis. The in vitro inhibition potency of some organophosphates (OPs), on AChEs of the field populations and SS strain was determined. The rank order from the most potent inhibitor to the least as determined by their

bimolecular reaction constants (Ki) was ethyl paraoxon > diazoxon > methyl paraoxon for AChE from Aa, B, Dp, and Yg populations, respectively, whereas the rank order for the susceptible strain was methyl paraoxon > ethyl paraoxon > diazoxon. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction adults and larvae feed on foliage and stems of potato plants resulting in poor yields and/or death of the plants. Adults can also vector Prevalence of resistance to insecticides as a genetic evolution- plants diseases. The need to control these beetles has involved the ary phenomenon is of crucial importance in effectiveness of these use of different insecticides. Presently, the beetle is resistant to chemical agents [1]. Studies on resistance revealed that apart from nearly all classes of insecticides and remains a serious pest in many arthropods, at least 200 species of plant pathogens, 273 species of parts of the world [2]. All resistance mechanisms reported in in- weeds, and several species of nematodes and rodents are also sects have been demonstrated in CPB [3,4]. resistant to one or more pesticides [2]. Colorado potato beetle There are many reports demonstrating elevated efﬁciency or (CPB, Leptinotarsa decemlineata Say), is the major pest of potatoes quantity of mixed function oxidases, esterases, and glutathion-S- in Iran and many other parts of the world. Injury is caused when transferases in insecticide-resistant CPBs [3,5,6]. However, alteration of acetylcholinesterase (AChE; EC 3.1.1.7) to an insensitive form associated with increased AChE activity has been proven as an important mechanism for resistance to organophosphates * Corresponding author. E-mail address: [email protected] (M. Malek Mohamadi). (OPs) and/or carbamates (CBs) in some insect species [7–10].

Measurement of AChE is commonly used as a biomarker of propionylthiocholine iodide (PTC) were purchased from Chem Ser- exposure to different contaminants including pesticides [11]. Ki- vice (West Chester, PA, USA). netic analysis of AChE was used to explain the resistance of some insect strains and the selectivity of some organophosphorous 2.2. Insects insecticides [12–15]. Previous studies have determined OPs and CBs resistance in CPB [16–24]. Resistance to azinphosmethyl has Adult Colorado potato beetles were collected in April 2005 from been reported in CPB [25,26]. The high level of resistance to azin- different commercial potato fields in Hamedan Province before any phosmethyl (136-fold) in a nearly isogenic CPB strain (AZ-R) was insecticides had been applied, and were taken to the greenhouse due to multiple resistance mechanisms, including reduced pene- for rearing. An insecticide-susceptible Colorado potato beetle pop- tration, enhanced xenobiotic metabolism, and target site-insensi- ulation, originally supplied by M. S. Goettel, Agriculture and Agri- tivity [22,27]. Kinetic studies revealed a reduced rate of Food Canada Research Center, Lethbridge was used as the reference hydrolysis of acetylthiocholine, an increased butyrylcholinestrase strain. All insects were reared in the greenhouse at 25 ± 1 °C, 60– activity, and a significantly reduced bimolecular rate constant for 70% RH and a 16:8 h (L:D) photoperiod. After preliminary screen- azinphosmethyl-oxon [22,27]. AChE from the AZ-R strain was less ing, four populations from four different commercial fields (Alia- sensitive to inhibition by azinphosmethyl-oxon and dichlorvos but bad, Bahar, Dehpiaz, and Yengijeh) with least mortalities in was more sensitive to inhibition by paraoxon and diisopropyl flu- response to phosalone treatments were chosen and reared in orophosphate than that from the insecticide-susceptible strain greenhouse and used for further assays. Biochemical characteriza- [22]. Continuous use of organophosphorous (OP) insecticides over tions were performed on larvae using both freshly collected insects a period of many years, as the major measure of control for CPB, and insect materials that had been frozen. however, has led to a growing concern that development of resistance is occurring. In these circumstances, it is important that pop- 2.3. Bioassays ulations under insecticide selection pressure are monitored. To date, very little background information is available concerning Bioassays were done by topical application to determine the either resistance of such populations to OPs, or of the mechanisms resistance level. Twenty-four to 48 h old 4th instars were used in that may be involved. The only study aimed at investigating the all bioassays. Technical phosalone was dissolved in analytical presence and diffusion of insecticide resistance in Iranian popula- grade (Reagent grade) acetone and 1 ll of the solution was applied tions started in 2004. Mohammadi Sharif et al. [28] reported resis- on the dorsum of the metathorax of 4th instars using a Hamilton tance to endosulfan, ranging from 18 to 220 folds, in field microsyringe. Control insects were treated with 1 ll acetone only. populations of CPB in East Azarbaijan Province. Two synergists, Each treatment which consisted of six doses and a control was rep- piperonyl butoxide (PBO) and S,S,S-tributylphosphorotrithioate licated at least four times with 20–25 larvae per replicate. All trea- (DEF), decreased resistance to 2.3- and 3.5-fold in the resistant ted larvae were placed in dishes containing fresh potato foliage and strain, respectively, indicating that metabolic detoxification has a were kept at 25 ± 1 °C, 60–70% relative humidity and photoperiod minor contribution to resistance. Results from the biochemical as- of 16:8 h (L:D). Larval mortality was recorded after 24 h. The larvae says indicated that there is no significant difference in glutathione were recorded as dead, if they did not move when their legs were S-transferase activity between the susceptible and the resistant pinched or if they had shrunken and dark coloured abdomens. strains [28]. Mohammadi Sharif et al. [29], have also identified a Probit option of SPSS was used for analyzing dose–mortality point mutation resulting in the replacement of an alanine by a ser- data and estimating lethal doses, Mortality was corrected based ine in the Rdl gene of Colorado potato beetle that confers resistance on control mortality using Abbott’s formula [30]. If control mortal- to endosulfan. Phosalone (OP) was one of the most common insec- ity was >20%, the results were discarded and the bioassay was re- ticides used for CPB control in Hamedan, Iran. There is anecdotal peated. LD50 values were judged as significantly different, if their evidence from local farmers, of a reduction in the efficacy of con- 95% confidence intervals did not overlap [31]. trol of CPB by phosalone, probably due to reduced susceptibility to the insecticide. 2.4. Synergism tests The aims of this study were: (1) To assess the susceptibility to phosalone of four field populations of CPB (Aliabad, Bahar, Dehpiaz, In synergism tests, PBO, DEF, and DEM were used through top- and Yengijeh, designated Aa, B, Dp, and Yg, respectively) in Hamed- ical application 1 h before phosalone treatment, to determine an Province, in comparison with susceptible strain (designated SS). whether metabolism was involved in phosalone resistance. The (2) To compare the kinetic and inhibitory properties of the AChE doses of PBO, DEF, and DEM were 5, 10 and 150 lg per larva, from each population to selected substrates and inhibitors in order respectively. These were the highest doses that caused no mortal- to determine the level and extent of possible resistance to phos- ity in susceptible strain in preliminary tests. The larvae were trea- alone. (3) To investigate the effects of synergists for testing possi- ted with the synergists only, were used as controls. Other methods ble mechanisms involved in resistance. were the same as above. The synergistic ratio (SR), percentage synergism (%S) and relative percent synergism (R%S) were calculated 2. Materials and methods using methods described by Brindley and Selim [32].

2.1. Chemicals 2.5. AChE preparation and determination of AChE activity

All chemicals used were technical grade products. Phosalone Fourth instars (74–85 mg), were collected from each population (93%, technical grade) was a gift from Shimi Keshavarz Company, and starved for 48 h to remove gut contents. Each larva was cut Ghazvin, Iran. Acetylthiocholin iodide (ATC), S-butyrylthiocholine transversely between the prothorax and the mesothorax. The por- iodide (BTC), Diethyl maleate (DEM), 5,50-dithio-bis-(2-nitroben- tion consisting of the head and prothorax was then homogenized zoic acid) (DTNB), piperonyl butoxide (PBO), Triton X-100 and bo- in 250 ll of 0.1 M sodium phosphate buffer (pH = 8) containing vine serum albumin were purchased from Sigma Chem. (St. Louis, 0.1% (v/v) Triton X-100. The homogenate was centrifuged at MO, USA). S,S,S-tributyl phosphorotrithioate (DEF), diazoxon, ethyl 5000 g and 4 °C for 15 min and the supernatant served as a crude paraoxon (diethyl-p-nitrophenyl phosphate, 99.5% pure), methyl enzyme for determination of AChE kinetic activities by Ellman’s paraoxon (dimethyl-p-nitrophenyl phosphate, 99.6% pure), and colorimetric reaction [33], using three substrates (ATC, PTC, and 256 M. Malek Mohamadi et al. / Pesticide Biochemistry and Physiology 98 (2010) 254–262

BTC). The substrates were dissolved in water to prepare a fresh reader. The median inhibition concentration (I50) for each inhibitor stock solution of 5 106 M concentration. The enzyme reaction was determined based on the log-concentration vs. probit (% inhi- velocity was measured at 25 °C by observing the absorption change bition) regression analysis. Seven replications were conducted for in the first 30 s at 405 nm. Michaelis constant (Km) and the maxi- each treatment. Affinity constant (Kd), phosphorylation constant mum velocity (Vmax), were calculated from the Lineweaver–Burk (kp), and bimolecular rate constant (ki) were then calculated by plot of uninhibited enzyme. using the formula given in Hart and O’Brien [35]. Before all assays, the supernatants were incubated with 100 lM DTNB for at least 15 min at 4 °C to nullify the effects of free thiols 2.7. Assays of protein contents on the AChE activity [34]. All measurements were made at 25 °Cin a kinetic microplate reader .The non-enzymatic reaction measured Protein contents of the enzyme homogenate were determined without homogenate served as control. AChE activities were deter- by the method of Bradford [36], using bovine serum albumin as mined spectrophotometrically at different substrate concentra- standard and Coomassie Brilliant Blue G-250 as a dye. The mea- 5 3 tions (1.56 10 –2.75 10 M) in presence of 0.01 M DTNB by surement was performed at 595 nm wavelength and 25 °C. recording the absorbance (OD) every 6 s over a 2 min interval at 405 nm at 25 °C. Briefly, 10 ll of enzyme source was added to each well of a microplate containing 250 ll of 0.1 M sodium phosphate 2.8. Statistical analysis buffer (pH = 8) and 20 ll of DTNB solution. Activity was expressed as mU/mg protein. All procedures were carried out on ice to min- To estimate parameters of the dose-mortality regression line for imize losses of enzyme activity. each bioassay, the probit analysis was conducted. The values have been presented as means ± standard deviation of mean (SD) of observed data of at least five replicates. The data obtained from en- 2.6. Inhibition kinetic parameters zyme assays were subjected to analysis of variance followed by Duncan’s test or Student t-test. P value of 6 0.05 was considered Because the oxidative analogs of insecticides are effective inhib- significant. All statistical tests were performed by using SPSS com- itors of AChE, three OP oxon analogs (diazoxon, ethyl paraoxon, puter program. and methyl paraoxon), were used to compare the sensitivity levels of AChE between the four field-collected populations and the susceptible strain of CPB. These inhibitors were selected to assess 3. Results in vitro inhibition of AChE based on the method of Hart and O’Brien [35]. ATC–DTNB solution was added to the AChE–OP mixture 3.1. Bioassay resulting in a final assay concentration of 0.075 M ATC and 0.01 M DTNB. Prior to each assay, 10 ll of the enzyme source The results of probit analysis of the mortality data from the field was incubated for 15 min with the appropriate concentration of populations and the susceptible strain exposed to phosalone are the inhibitor, with a reaction without the inhibitor as control. presented in Table 1. The overlap in the 95% CL of the LD50 values The absorbance was recorded every 2 min for a total of 15 min at and the lack of significant differences in the LD50 values indicate 405 nm and at 25 °C, pH:8 by using a UV Max kinetic microplate that there were no large differences in susceptibility between pop-

Table 1 Toxicity of phosalone to larvae of a susceptible strain (SS) and ﬁeld populations of Colorado potato beetle, Aliabad (Aa), Bahar (B), Dehpiaz (Dp), and Yengijeh (Yg) with and without synergists.

a b c 2d e f Population n df Synergist Slope (SE) LD50 (95% CL )(lg/insect) v Resistance ratio Synergist ratio SS 260 4 – 1.3 (0.1) 2.42 (2.1–2.7) 1.2 190 4 DEF 1.60 (0.1) 1.5 (1.3–1.6) 3.7 1.6 270 4 DEM 2.1 (0.2) 1.7 (1.5–1.9) 4.6 1.4 302 4 – 1.7 (0.1) 4.2 (3.6–4.8) 1.8 0.6 Yg 302 4 – 2.8 (0.2) 548.8 (522.1–578.6) 0.2 226.7 270 4 DEF 2.1 (0.2) 184.5 (166.1–200) 1.4 76.2 2.9 230 4 DEM 2.7 (0.2) 206 (183.5–225.3) 1.2 85.1 2.6 230 4 DEM 2.7 (0.2) 206 (183.5–225.3) 1.2 85.1 2.6 270 4 PBO 4.7 (0.4) 785.8 (752.9–818.6) 0.6 324.7 0.7 B 183 4 – 3.1 (0.2) 373.9 (345.9–398.3) 0.8 154.5 270 4 DEF 2.1 (0.2) 330.3 (291.8–361.5) 3.1 136.5 1.1 270 4 DEM 2.7 (0.2) 278.4 (206.4–328.5) 7.6 115 1.3 270 4 PBO 1.6 (0.2) 880.4 (840.9–919.9) 1.2 125.7 0.4 Dp 190 4 – 3.6 (0.3) 610.1 (573.9–642.5) 1.1 252.1 250 4 DEF 1.6 (0.1) 109.7 (93.4–124.2) 1.8 45.3 250 4 DEF 1.6 (0.1) 109.7 (93.4–124.2) 1.8 45.3 5.6 230 4 DEM 3.1 (0.3) 547 (505.7–580.8) 0.9 226 1.1 230 4 PBO 4.3 (0.4) 965.7 (925.9–1008.4) 3.6 237.7 0.6 Yg 270 4 – 3.1 (0.2) 485.9 (463.1–509.3) 0.2 200.8 210 4 DEF 4.5 (0.3) 364.7 (345.5–381.9) 0.5 150.7 1.3 260 4 DEM 2.2 (0.2) 311.8 (272.6–343.1) 2.2 128.8 1.5 258 4 PBO 3.0 (0.3) 926.9 (889.2–966.1) 1.6 383 0.5

a The number of larvae used in each bioassay. b Degree of freedom. c CL, conﬁdence interval limit. d CL, conﬁdence interval limit. e LD50 of resistant population/LD50 of susceptible strain. f LD50 without synergist/LD50 with synergist. M. Malek Mohamadi et al. / Pesticide Biochemistry and Physiology 98 (2010) 254–262 257

ulations Aa, and Dp. However, the difference in the LD50 values for at higher substrate concentrations illustrate the phenomenon of populations B, and Yg was statistically significant based on the cri- substrate activation (Fig. 1E). terion and failure of 95% confidence intervals to overlap. The affinity and hydrolyzing efficiency of AChE from the four A comparison of the susceptibility of these four populations, field populations and the susceptible strain to three substrates showed that, Dp was the most and B the least resistant population, was determined by kinetic analysis (Table 2). The rank order from respectively, compared with the SS strain. Dp population demon- the highest hydrolyzing efficiency to the lowest as expressed by strated 252.1-fold resistance to phosalone when compared at the Vmax value was ATC > PTC > BTC for each population and for LD50 level. Given The long-term, extensive use of phosalone for the susceptible strain. The AChE of the susceptible strain utilized control of CPB in Hamedan, we expected resistance of this ATC and PTC more effectively and BTC less effectively than any of magnitude. the field populations. The AChE of the susceptible strain showed

the lowest Km value with ATC as substrate, indicating that the sensitive form of the AChE had the highest affinity for the substrate 3.2. Synergism studies analog ATC. AChE from population Dp had approximately 1.8- and 1.7-fold lower affinity (i.e. higher K values) to ATC and PTC, DEF, DEM, and PBO were used to inhibit esterase, glutathione-S- m respectively, than that from the susceptible strain. The V value transferase, and microsomal oxidase detoxification mechanisms, max for population Dp was 1.88- and 1.92-fold lower for ATC and PTC, respectively. DEF an inhibitor of estrases, significantly synergized respectively, than that for the susceptible strain. For BTC, however, the activity of phosalone in the three field populations of CPB, that it was 1.4-fold higher. is Aa, Dp, and Yg. With DEF, the highest synergism ratio (5.6) was For the catalytic activity of AChE towards BTC, as expected by observed in population Dp. Synergism with DEF was apparent, also, the V value, there was a similar situation among the field-col- in the susceptible strain. No significant synergism of phosalone max lected populations and the susceptible strain, with the highest va- toxicity was observed in population B when larvae were pretreated lue in B, and least in susceptible strain (i.e. 4.7 vs. 2.3 mU/mg with DEF, as indicated by the overlap in the 95% CL for treatment protein). with phosalone alone or with phosalone + DEF (Table 1). From Table 1, it can be seen that DEM showed significant syn- 3.4. Inhibition of AChEs by OPs ergism on phosalone in populations Aa, Yg and B, synergistic ratios were 2.6, 1.5, and 1.3 respectively. DEM synergism of phosalone Three OP oxon analogs (diazoxon, ethyl paraoxon, and methyl was greater in populations Aa, and Yg as compared with the sus- paraoxon) were used to compare the sensitivity levels of AChE ceptible strain (SR 1.4). Although DEM was synergistic in popula- from the field-collected populations and the susceptible strain of tion Aa, synergism was less pronounced than that caused by DEF CPB. The kinetic parameters of the inhibition process (K , k , and in the other resistant field populations. In the susceptible strain, d p k ) in addition to I were measured for each compound. The results PBO significantly reduced phosalone toxicity. Similar results were i 50 are summarized in Tables 3 and 4, respectively. The rank order as obtained for the dose–mortality responses of the phosalone-resis- determined by their bimolecular reaction constants (k ) from the tant field populations. The highest (0.4) and lowest (0.7) inhibitory i most potent inhibitor to the least was ethyl paraoxon > diazo- effect were observed in populations B and Aa respectively. xon > methyl paraoxon for AChE from populations Aa, B, Dp, and Yg whereas the rank order was methyl paraoxon > ethyl parao- 3.3. Substrate specificity and kinetic properties of AChEs xon > diazoxon for the susceptible strain. AChEs from all four field populations were less sensitive to methyl paraoxon inhibition than The effect of substrate concentration on AChE activity from the the susceptible strain. AChE from Aa was the most insensitive form four field-collected populations and the susceptible strain of CPB and AChE from B was the most sensitive form (most similar to sus- was estimated by measuring AChE activity at different concentra- ceptible strain). The inhibition results according to the I50 values 5 3 tions from 3.1 10 to 5 10 M for ATC, PTC, and BTC are in agreement with the inhibition results according to the ki val- (Fig. 1). AChE inhibition by higher substrate concentrations was ues, with the exception of Yg population. According to I50 value, Yg evident only with the insecticide–susceptible strain. Optimal sub- population was least sensitive to diazoxon, while according to the strate concentrations of the susceptible strain were 0.524 mM for ki value, was least sensitive to methyl paraoxon. According to the ATC and 1.75 mM for PTC. The reaction rate decreased as the con- study of Main [37], ki was generally regarded as the most reliable centrations of ATC and PTC was further increased, indicating sub- criterion to measure the inhibitory power of some organophos- strate inhibition. High concentrations of PTC were less effective phates to esterases. An additional comparison made using the ra- in inhibiting AChE activity from the susceptible strain. When BTC tios of the AChE remaining in the presence of diazoxon, methyl was used as substrate, a linear relationship between the reaction paraoxon, and ethyl paraoxon (Fig. 2). AChE from the SS strain rate and BTC concentration was obtained, thus no definitive opti- was more sensitive to inhibition by methyl paraoxon than the mal concentration for BTC could be identified and high substrate AChEs from field populations (i.e. more than 70% of AChE was inhibition was not observed (Fig. 1A). When ATC, PTC, and BTC inhibited). However, when ethyl paraoxon was examined, the were used as substrate, the reaction rates of AChE from population AChE of the Dp population was 1.75-fold more sensitive to ethyl Yg increased as the concentration of all three substrates were in- paraoxon inhibition than that from the SS strain. The Aa and Yg creased, but were almost constant at concentration of ATC P 3.98, populations elicited respectively the lowest and the most% AChE PTC P 2.8, and BTC P 5Mm(Fig. 1B). AChE from populations Aa activity remaining in the presence of diazoxon. and B were not inhibited by high concentration of ATC, PTC, and The overall inhibitory power determined by ki, would depend BTC, the reaction rates increased as the concentration of substrates on both the affinity of the OP compound for the active site of AChE was further increased (Fig. 1C and D). The specific activities of and the rate of phosphorylation. The phosphorylation constants

AChE from population Dp increased as the concentration of all (kp) of the four field populations by the methyl paraoxon were three substrates was increased. Substrate inhibition at higher con- not significantly different but the affinity constants (kd) of the four centrations was not seen nor was there a definitive optimal con- populations were significantly different from that of the suscepti- centration for any of the substrates examined. The absence of ble strain (Table 3, p > 0.01, t-test). It appeared that the reduction uniform sigmoid curves which would be expected from Michae- in the inhibitory power, as judged by the kis of methyl paraoxon lis–Menten-type kinetics and the drastic increase in AChE activity on the AChEs from field populations was mainly due to their in- 258 M. Malek Mohamadi et al. / Pesticide Biochemistry and Physiology 98 (2010) 254–262

A B

SS Yg

C D

Aa B

Dp Activity (mU/mg protein)

Substrate concentration ( -Log [M] )

Fig. 1. Effect of increasing substrate concentration (right to left in all panels) on AChE from the SS strain (A) and ﬁeld populations of CPB; Yg (B), Aa (C), B (D), and Dp (E). Each points represents the mean of six replicate determinations (n = 6). Vertical bars indicate standard deviations of the mean.

Table 2 Substrate speciﬁcities and kinetic properties of AChEs from the SS and the ﬁeld populations of CPB.

Substrate Kinetic property c SS Aa B Dp Yg

a 1A 2A 3A 4A 3A ATC Km 10.6 ± 0.2 15.6 ± 0.3 12.6 ± 0.3 18.8 ± 0.4 12 ± 0.4 b 1a 2a 3a 2a 4a Vmax 20.2 ± 0.5 11.5 ± 0.43 14.9 ± 0.4 10.7 ± 0.3 18 ± 0.8 1B 2B 1B 2B 2B PTC Km 7.7 ± 0.5 9.2 ± 0.5 8 ± 0.6 13.1 ± 0.6 6.7 ± 0.3 1b 2b 1b 2b 2b Vmax 13.7 ± 0.6 8 ± 0.7 13 ± 0.9 7.1 ± 0.5 7.8 ± 0.6 4C 3C 1C 2B 1C BTC Km 7.1 ± 0.4 11.8 ± 0.6 9.2 ± 0.5 13.5 ± 0.4 8.9 ± .3 2c 1c 1c 1c 1c Vmax 2.3 ± 0.5 3.7 ± 0.5 4.7 ± 0.2 3.3 ± 0.5 4.2 ± 0.3

a Km; Michaelis constant in lM. b Vmax; maximum velocity in mU/mg protein. One unit of AChE hydrolyzes 1 lmole of ATC per min at 25 °C and pH 7.5. c Results are reported as the means ± SD of ﬁve replicates (n = 5). Means in same row and column followed by the same subscript number and letter, respectively, are not statistically different by ANOVA (P > 0.05). M. Malek Mohamadi et al. / Pesticide Biochemistry and Physiology 98 (2010) 254–262 259

Table 3 a Afﬁnity (Kd), phosphorylation (kp), and bimolecular rate (ki) constants of organophosphorus compounds in inhibition reaction with AChEs from the SS strain and the ﬁeld populations of CPB.

b SS (ratio of ki) Aa (ratio of ki) B (ratio of ki) Dp (ratio of ki) Yg (ratio of ki) Methyl paraoxon 6 6* 6* 4* 5* Kd (M) (6.3 ± 0.3) 10 (2.79 ± 1.8) 10 (13.3 ± 0.64) 10 (1.7 ± 0.05) 10 (2.0 ± 1.3) 10

kp 0.2 ± 0.02 0.2 ± 0.05 0.2 ± 0.01 0.2 ± 0.06 0.2 ± 0.01 1 1 4 3* 4* 4* 4* ki (M min ) (3.4 ± 0.09) 10 (1) (8.1 ± 0.57) 10 (4.2) (1.5 ± 0.07) 10 (2.3) (1.4 ± 0.03) 10 (24.2) (1.0 ± 0.1) 10 (3.3) Ethyl paraoxon 5 6* 6* 6* 6* Kd (M) (1.9 ± 1.3) 10 (1.2 ± 0.1) 10 (1.96 ± 0.3) 10 (1.4 ± 0.1) 10 (3.4 ± 0.09) 10 * * * kp 0.2 ± 0.02 0.4 ± 0.03 0.3 ± 0.06 0.7 ± 0.05 0.2 ± 0.02 1 1 4 5* 5* 5* 4* ki (M min ) (1.2 ± 0.09) 10 (1) (3.3 ± 0.3) 10 (0.04) (1.3 ± 0.2) 10 (0.1) (4.9 ± 0.9) 10 (0.02) (6.0 ± 0.6) 10 (0.2) Diazoxon -5 6* 5* 6* 5* Kd (M) (4.5 ± 0.7) 10 (2.1 ± 0.07) 10 (1.0 ± 0.3) 10 (7.0 ± 0.4) 10 (1.1 ± 0.5) 10

kp 0.2 ± 0.01 0.23 ± 0.01 0.2 ± 0.01 0.2 ± 0.02 0.2 ± 0.01 1 1 3 5* 4* 4* 4* ki (M min ) (3.8 ± 0.5) 10 (1) (1.1 ± 0.06) 10 (0.03) (2.3 ± 0.1) 10 (0.2) (3.0 ± 0.2) 10 (0.1) (1.9 ± 0.2) 10 (0.2)

a Results are reported as means ± SD of nine replicates (n = 9). b Ratios of bimolecular rate constants (ki) were ki of SS strain/ki of field populations. Ratios greater than 1 indicate that field populations are less sensitive to the inhibitory action of OP. * Indicates significant difference from the SS strain (P < 0.01, t-test).

Table 4 Inhibition potency of OPs against AChE of the SS strain and the ﬁeld populations of CPB.

a,b I50 (M) Type of inhibition SS Aa B Dp Yg

Methyl paraoxon (1.1 ± 0.1) 103 (9.5 ± 0.4) 103 (2.8 ± 0.2) 103 (1.45 ± 0.1) 104 (4.7 ± 0.2) 103 Mixed Ethyl paraoxon (3.29 ± 0.1) 103 (3.8 ± 0.1) 102 (6.2 ± 0.2) 102 (2.6 ± 0.1) 102 (1.2 ± 0.1) 103 Mixed Diazoxon (3.1 ± 0.1) 104 (2.1 ± 0.1) 103 (8.7 ± 0.1) 103 (6.7 ± 0.1) 103 (1.1 ± 0.04) 104 Mixed

a Concentration that inhibited 50% of the AChE activity. b Each value is the mean ± SD of six replicates. Means in each column and row are signiﬁcantly different at the 0.05 level (Duncan’s multiple-range test).

to others. The inhibition pattern and implied mechanism for all tested compounds, is suggested to be a linear mixed type.

4. Discussion

4.1. Bioassay

Monitoring populations that are under insecticides pressure plays an important role in formulation of effective strategies for CPB control and resistance management. Indeed our bioassay results revealed a 225-fold decrease in susceptibility to phosalone in the field populations studied, and as compared to the susceptible strain (Table 1). Nevertheless, resistance may vary markedly from region to region and from field to field, depending on patterns of use of insecticide and selection pressure. Based on the results obtained in the present study, it is apparent that the continued heavy reliance on OPs, and most likely CBs, will be problematic Fig. 2. Percent AChE activity remaining in the presence of 500 lM diazoxon, ethyl in Hamedan potato fields. paraoxon, and methyl paraoxon, from the SS strain and the field populations of CPB. The results represent the mean ± standard deviation from seven separate replicates. Vertical bars indicate standard deviations of the mean. Different letters on the bars 4.2. Synergism indicate that the means are significantly different (P < 0.05) in Duncan’s multiple- range test. Oxidative, estratic and glutathione-S-transferase metabolism have now been implicated in organophosphate resistance in many creased kds, indicative of a reduction in affinity. AChEs from the insect species [38–40]. four field-collected populations were significantly more sensitive In population Dp, there was no significant change in LD50 value, to ethyl paraoxon and diazoxon than that from the susceptible when DEM was applied to larvae before treatment with phosalone strain. Increases in sensitivity to ethyl paraoxon and diazoxon, (Table 1). This suggested that, under the conditions of this experi- respectively, were 27.5- and 28.9-fold for Aa, 10.8- and 6.05- fold ment, elevated levels of glutathione-S-transferases are not respon- for B, 40.8- and 7.89- fold for Dp and 5-fold for Yg. Thus Yg was sible for the higher tolerance to phosalone in the resistant Dp. the least sensitive field population and most similar in sensitivity However, application of DEF, an inhibitor of esterases to larvae re- to the susceptible strain. Our results are typical of the phenomenon sulted in a LD50 of 109.69 lg/I, approximately one-fifth the phos- of negative cross-insensitivity in which an altered AChE became alone LD50 in the absence of the synergist (Table 1). This less sensitive to some inhibitors while becoming more sensitive observation suggests that esterase based organophosphate meta- 260 M. Malek Mohamadi et al. / Pesticide Biochemistry and Physiology 98 (2010) 254–262 bolic pathways were present and important but could not be solely substrate to the peripheral anionic site to form an enzyme–sub- responsible for resistance in this population. DEF and DEM signifi- strate–substrate complex [46,47]. Zhu and Clark [22] have reported cantly enhanced activity in the population Aa. This suggests that, that AChE from the susceptible strain was significantly inhibited by esterase-, and glutathione-S-transferase-mediated detoxifications higher concentrations of acetylthiocholine and acetyl- (b-methyl) are probably the primary resistance mechanism in the larvae. Since thiocholine, whereas AChE from the azinphosmethyl-resistant the synergists used did not completely eliminate the resistance, strain, AZ-R, was activated by higher concentrations of substrates. there may also be other mechanisms responsible. Although syner- In our study, AChE from the susceptible strain of CPB elicited gism with DEF and DEM was apparent, also, in the susceptible high substrate concentration inhibition (Fig. 1A). The reaction rates strain, it was substantially reduced compared with the resistant of AChE from the population Yg increased as the concentration of populations (Aa and Dp). each substrate was further increased but were almost constant at PBO, as an inhibitor of monooxygenases, has not been generally higher concentrations (Fig. 1B). High substrate concentration inhi- used as an OP synergist because it inhibits the activity of cytochrome bition was not evident with AChE from both populations Aa and B P450s (CYTP450s) [41]. In insect, cytochrome P450s plays an impor- (Fig. 1C and D). For both, AChE, activity increased with increasing tant role in either bio-activation of insecticides from the phosphoro- concentration irrespective of which of the three substrates was dithioate to the more toxic oxon form or more frequently in being tested. Furthermore, there were no definitive optimal sub- insecticide detoxification, the latter process being enhanced in strate concentrations. However, AChE from the DP population elic- many species that have developed metabolic resistance to insecti- ited substrate activation as indicated by the ‘double sigmoidal’ cides [42]. Phosalone belongs to the thiophosphate group of the form of curve obtained for all substrates tested in the activity vs. OPs that requires the CYTP450 based oxidative activation to become substrate concentration plots (Fig. 1E). Therefore, the altered AChE the oxon form of phosalone, with much greater efficiency and po- may occurred some elements of the catalytic properties of butyr- tency in inhibition of AChE activity, the major target of OPs and ylcholinesterase (BuChE, EC 1.1.1.8) [48,49]. AChE from population CBs acute toxicity [41]. Hence, PBO, being an inhibitor of CYTP450s, Dp, hydrolyzed BTC more efficiently than AChE from the suscepti- would inhibit the bio-activation of phosalone, leading to a reduction ble strain. In the susceptible strain, the structure–activity relation- in its toxicity. We demonstrated in this study that PBO did reduce ship based on the kinetic parameters showed that the increase in phosalone toxicity at the dose tested. In other words, the CYTP450s length of the alkyl chain (PTC and BTC) of the substrate could be isomers involved in phosalone activation may be much more sensi- responsible for a decrease in the overall efficiency with which tive to PBO inhibition of phosalone than the ones involved in meta- AChE hydrolyzes the substrates. Thus, compared to the susceptible bolic detoxification of phosalone. form of AChE, which hydrolyzed ATC efficiently, but BTC markedly Studies using PBO on diazinon toxicity to the resistance and less so; AChEs from the field populations elicited reduced ATC susceptible strain of the sheep blowfly Lucilia cuprina and on cou- hydrolysis and increased BTC hydrolysis. maphos toxicity to the southern cattle tick Rhipicephalus (Boophi- lus) microplus have identified PBO differential effects [43,44]. 4.4. Inhibition of AChEs Differential effects of PBO on diazinon or coumaphos occur via the inhibitory effects of PBO on different isomers of CYTP450s in- Inhibition kinetic parameters are mostly used to study the volved in both activation and detoxification of OP insecticides. inhibitory potency of CBs and OPs compounds, e.g., paraoxon However, Li et al. [45] in United States have shown a biphasic effect [34,50], chlorpyrifos-oxon, monocrotophos [40], and methyl parao- of PBO on diazinon toxicity to horn fly, Haematobia irritans (L.). xon [51].

Their study revealed that, PBO synergized diazinon toxicity at low- The rate of the bimolecular inhibition rate constant, ki depends er concentrations by facilitating penetration of diazinon through on both the enzyme affinity towards the inhibitor and rate of en- the cuticle and/or inhibiting the oxidative detoxification of diaz- zyme phosphorylation expressed as Kd and kp, respectively [52]. inon, and reduced diazinon toxicity at high PBO concentration by As far as the biochemical mechanism underlying the reaction be- inhibiting the bio-activation of diazinon. In the present study, we tween OP compounds and AChE noted in our study we conclude showed the inhibitory effect of PBO on phosalone toxicity to CPB that, while the phosphorylation step was slow enough to be a lim- at the dose tested (10 lg per larva). Both the susceptible strain iting factor, it did not appear to be a key factor in determining the and the field populations of CPB studied responded similarly to inhibitory power of the OP compounds. The inhibitory power of the treatments with PBO combined with phosalone. Our aim was to OP compounds was substantially correlated to the affinity of the determine whether PBO could be implicated in phosalone resis- OP compounds to the active site of AChE. tance in CPB but were not able to determine conclusively the Studies on altered AChE, resulting in CBs and OPs insecticide underlying mechanism involved. It is possible that the use of great- resistance due to enzyme insensitivity indicate that insensitive/ er or lesser concentrations than the one we used might have given resistant forms of AChE have increased susceptibility to insecticide different results. More extensive tests should be performed to de- analogs of previous studies. The data from the current study concur velop a more thorough understanding of the dose-dependent bi- with previous findings that the AChEs of the AZ-R and BURTS phasic effects of PBO on phosalone toxicity to CPB. We conclude strains were more sensitive to those inhibitors with larger alkyl that although these metabolic or sequestration pathways were groups (e.g., ethyl paraoxon, anzinphosethyl, DFP, propahos and present for phosalone, they did not have a major role in resistance N-propyl corbofuran vs. azinphosmethyl-oxon and N-methyl car- to phosalone. bofuran) [27,35]. AChEs from the four field populations in our study had relatively greater hydrolysis activities using substrates 4.3. Substrate specificity and kinetic parameters of AChEs with larger alkyl substitutions (e.g., BTC vs. ATC) that were also less sensitive to inhibition by methoxy-substituted insecticides (e.g., Although bioassays are definitely essential to validate the pres- methyl paraoxon) as compared to the susceptible form. The resis- ence of resistance in populations and to quantify the levels of resistant forms of AChEs elicited relatively higher BTC hydrolysis activ- tance associated with particular mechanisms, biochemical and ity and had greater sensitivity to inhibition by insecticides with molecular monitoring techniques enable very accurate assess- larger alkyl groups associated with their acid moieties than the ments of resistance gene frequency to be made. susceptible form. Inhibition at high substrate concentration has been identified as Taken it together, AChEs elicited structure–activity relation- a typical phenomenon for AChE, and is due to the binding of excess ships similar to those previously reported for the native form of M. Malek Mohamadi et al. / Pesticide Biochemistry and Physiology 98 (2010) 254–262 261

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