Pesticide Biochemistry and Physiology 101 (2011) 59–64

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Pesticide Biochemistry and Physiology

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Metabolic resistance mechanisms to phosalone in the common pistachio psyllid,Agonoscena pistaciae (Hem.: Psyllidae)

Ali Alizadeh a, Khalil Talebi a,⇑, Vahid Hosseininaveh a, Mohammad Ghadamyari b

a Department of Plant Protection, Faculty of Agricultural Sciences & Engineering, College of Agriculture and Natural Resources, University of Tehran, Karaj, Iranb Department of Plant Protection, Faculty of Agriculture, University of Guilan, Guilan, Iran

a r t i c l e i n f o a b s t r a c t

Article history:Received 14 March 2011Accepted 31 July 2011Available online 5 August 2011

Keywords:PistachioAgonoscena pistaciaePhosaloneMetabolic resistance mechanismsSynergism

0048-3575/$ - see front matter � 2011 Elsevier Inc. Adoi:10.1016/j.pestbp.2011.07.005

⇑ Corresponding author. Fax: +98 261 2238529.E-mail address: khtalebi@ut.ac.ir (K. Talebi).

The common pistachio psyllid, Agonoscena pistaciae, is the most damaging pest of pistachio in Iran, and isgenerally controlled by insecticides belonging to various classes especially, phosalone. The toxicity ofphosalone in nine populations of the pest was assayed using the residual contact vial and insect-dipmethods. The bioassay results showed significant discrepancy in susceptibility to phosalone among thepopulations. Resistance ratio of the populations to the susceptible population ranged from 3.3 to 11.3.The synergistic effects of TPP, PBO and DEM were evaluated on the susceptible and the most resistantpopulation to determine the involvement of esterases, mixed function oxidases and glutathione S-trans-ferases in resistance mechanisms, respectively. The level of resistance to phosalone in the resistant pop-ulation was suppressed by TPP, PBO and DEM, suggesting that the resistance to phosalone is mainlycaused by esterase detoxification. Biochemical enzyme assays revealed that esterase, glutathione S-trans-ferase and cytochrome P450 monooxygenase activities in the resistant population was higher than that inthe susceptible. Glutathione-S-transferases play a minor role in the resistance of the pest to phosalone.

� 2011 Elsevier Inc. All rights reserved.

1. Introduction

The common pistachio psyllid (CPP), Agonoscena pistaciaeBurckhardt and Lauterer (Hem.: Psyllidae) is a serious insect pestof pistachio trees distributed in all pistachio producing regions ofIran [1,2]. The CPP populations are increasing in many countriesincluding Iran, Turkey, Iraq, Armenia and Turkmenian, in additionto Mediterranean regions such as Syria and Greece [1,3–5]. Bothadults and nymphs suck leaf sap and produce large amounts of awhite powder from dried honeydew. Direct feeding of the pestcauses reduced plant growth, defoliation, stunting, falling of fruitbuds and poor yield [6]. A. pistaciae has a high potential for resis-tance development to insecticides due to its short life cycle andhigh potential reproduction. Due to excessive selection pressurecaused by the intensive use of insecticides on pistachios, somepopulations of CPP have become resistant to synthetic insecticidesin Iran [7]. However, organophosphorus (OPs), neonicotinoids andinsect growth regulators (IGRs) are widely used to control CPP incommercial pistachio orchards and will likely continue to be a pri-mary component of pistachio management programs [8]. CPP hasrecently become a serious problem because of frequent applicationof insecticides resulting in resistance among CPP populations

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through the stimulation of the reproductive potential [2]. Phos-alone (OP) is one of the commonly used insecticides for CPP controlin Kerman province, Iran since 1963. Up to now, there is no avail-able information about the mechanisms of resistance in CPP popu-lations to OPs. The first study on the susceptibility of different CPPpopulations was demonstrated in Iran, in 2001 [7]. Comparisons ofLC50 for both overwintering and aestivating adult’s generationsindicated that populations from Kerman (KR) and Rafsanjan (RF)were the least and that of Jabalbarez the most susceptible. Thelow levels of resistance ratio from 1.3- to 1.8-fold were found,probably due to natural variation of the populations susceptibilityto phosalone [7].

The high level of resistance in some insects is associated with anincrease in general esterase activity or an increased activity ofother detoxification systems, including glutathione S-transferaseand cytochrome P450 monooxygenases [9–11]. Several researchershave reported that resistance to carbamate, organophosphates andpyrethroids is correlated with elevated nonspecific esterase activ-ity [12–14]. Berrada et al. [15] have shown a modified less sensitiveisoform of acetylcholinesterase to the inhibition by the insecticidemonocrotophos accompanied with a slight increase in the oxida-tive metabolism responsible for the high resistance level developedin a selected laboratory strain of Cacopsyllid pyri. Pesticide bioas-says, enzyme assays and application of insecticide synergists arethe common procedures for obtaining the preliminary informationabout potential mechanisms of resistance.

60 A. Alizadeh et al. / Pesticide Biochemistry and Physiology 101 (2011) 59–64

Relying on organophosphate pesticides, especially phosalone, forcontrolling CPP for 15 years has developed the emergence of resis-tant populations in many pistachio growing areas in Iran. The resis-tance level among different CPP populations and mechanism(s)responsible for phosalone resistance has not been fully understood.The aims of the present research were: (1) To evaluate the suscepti-bility of nine field populations of CPP to phosalone in Kerman prov-ince. (2) Determination of detoxification enzymes activitiesincluding esterase, cytochrome P450 monooxygenases and glutathi-one S-transferases among nine CPP populations. (3) To assess theeffects of synergists TPP, BPO and DEM for testing possible mecha-nisms involved in resistance.

2. Materials and methods

2.1. Insects

Nine CPP populations were collected from pistachio orchardslocated in Rafsanjan (RF), Anar (AN), Bam (BA), Kerman (KR), Shah-rbabak (SH), Herat (HE), Sirjan (SI), Pariz (PA) and Paghaleh (PGH)regions of Kerman province, Iran in 2009. The populations, exceptthe population BA, had a history of previous exposure to pesticidesincluding phosalone. The populations were routinely reared inplastic boxes (50 � 60 � 80 cm) under greenhouse conditions at28 ± 2 �C, 45 ± 5 RH and 16:8 (L:D) photoperiod on young, un-treated pistachio plants, Pistacia vera (var. Badami Zarandi).

2.2. Chemicals

Phosalone (99% technical grade) used in all bioassays wasobtained from Research Center of Pesticide and Fertilizer, Iran.a-Naphthyl acetate (a-NA), b-naphthyl acetate (b-NA), q-nitro-phenyl acetate (q-NPA), naphthol, q-nitrophenol (PNP),tris(hydroxymethyl) aminomethane (Tris), 7-ethoxycoumarin (7-EC), glutathione reductase, NADPH, dithiothreitol (DTT), Triton X-100 and bovine serum albumin were purchased from Sigma Chem-ical Co. Fast blue RR salt (o-dianisidine, tetrazolized zinc chloridecomplex) was obtained from Fluka (Buchs, Switzerland) and thesynergist triphenyl phosphate (TPP, 99% purity), diethyl maleate(DEM), ethylenediaminetetraacetic acid (EDTA), and piperonylbutoxide (PBO) were purchased from Merck company (Germany).

2.3. Bioassay

In the current study, two methods were used for determiningmedian lethal concentration (LC50) and synergistic effects.

2.3.1. Insect-dip methodThe toxicity of phosalone to 5th instar nymphs of the nine pop-

ulations of CPP was assayed using the inset-dip method. For bioas-say five or six different concentrations of phosalone, each withthree or four replicates were used. Phosalone was dissolved in amixture of water–acetone (20:80 v/v) and the control receivingthe solvent only. Nymphs were transferred into a piece of net(5 � 2 cm, 40 mesh) and held in the solutions for 2 s while dipping.The treated nymphs were placed on paper tissues using a smallbrush and left for 5 min to absorb extra solution and to be air-dried. Up to 12–15 treated 5th instar nymphs were placed on topistachio leaf discs that was located in clear plastic Petri dishes(5.5 cm diameter). Mortality was assessed after the treatednymphs were maintained in a growth chamber at 28 ± 2 �C,45 ± 5% relative humidity and a photoperiod of 16:8 (L:D) for24 h. The criterion for death was that a nymph was unable to movecorrectly when its leg was probed with soft camel-hair brush.

Resistance ratios (RR) were calculated by dividing the LC50 valuesof the populations by the value of the susceptible population (BA).

2.3.2. Determining LC50 using residual contact vial (RCV) method andcalculation of synergistic effects

The toxicity of phosalone to the susceptible (BA) and most resis-tant (RF) populations was assayed using the residual contact vial(RCV) bioassay. For the determination of LC50, phosalone was dis-solved in acetone at various concentrations. Two hundred microliterof phosalone solution was transferred to glass vials (1 cm in diame-ter and 6 cm high) and the inside wall of the vials was coated withthe phosalone solution by rolling it under a fume hood for 1 h. Aftervials were dried, 15 adults (0–24 h old) were transferred into eachphosalone coated vial and was closed with cotton. Mortality was as-sessed after the treated adults were maintained at 28 ± 2 �C, 45 ± 5RH for 8 h. The criterion for death was that an adult did not moveits appendages when probed with a camel’s hair brush. For determi-nation of synergistic effects, 200 lL of TPP (5 mg L�1), PBO(10 mg L�1) and DEM (5 mg L�1) solutions were transferred intoglass vials to affect esterase, microsomal oxidase, and glutathione-S-transferase detoxifying enzyme activities, respectively. Variousconcentrations of phosalone solution were then added to glass vialsand rolled it under fume cupboard as stated above.

To choose the appropriate dose of synergists we have done apreliminary bioassay using different doses of synergist from highdose to low and for each synergist the highest dose that causedcomparable mortality with the control (acetone treated) was se-lected for final bioassay [16]. Then bioassays were conducted byRCV method, as described above.

2.4. Determination of esterase activity

Two hundred adults of CPP from each population were homog-enized in 300 lL of ice-cold sodium phosphate buffer (10 mM, pH7, containing 0.1% of Triton X-100). The homogenates were centri-fuged at 15,000g at 4 �C for 10 min. The resulting supernatantswere used as the enzyme source in all enzyme assays. Protein con-tent of the enzyme sample was determined following the methodof Lowry et al. [17] using bovine serum albumin (BSA) as thestandard.

Hydrolytic activities against the substrates, a-NA and b-NA weremeasured following the method of Van Asperen [18] with somemodifications. Enzyme assays were started with addition of 50 lLenzyme samples to 100 lL 0.1 M phosphate buffer (pH 7.0) and10 lL of substrates (10 mM in acetone). Fifty microliter fast blueRR (0.5 mg mL�1 in buffer) was then added to the reaction mixtureand the released naphthol was continuously measured at 450 nmfor 25 min using a microplate reader (ELX808 Bio-Tek). A standardcurve of absorbance versus concentrations of naphthol was con-structed to enable calculation of the amount of naphthol producedduring the esterase assay.

Esterase activity was also measured against q-nitrophenyl ace-tate (q-NPA) according to the method of Mackness et al. [19] withsome modification by Mahdavi Moghadam et al. [20]. Briefly, thereaction mixture contained 10 lL of enzyme extract, 172 lL Tris–HCl buffer (20 mM, pH 7.75) and 18 lL of the substrate (50 mMin acetonitrile). Changes in absorbance at 405 nm were monitoredevery 30 s for 6 min and a standard curve was prepared using q-nitrophenol (PNP) as the standard.

2.5. Determination of GSTs activity

Fifty adults were homogenized in 200 lL of ice-cold 10 mMphosphate buffer (pH 7.0). After, the homogenates were centrifugedat 10,000g for 10 min at 4 �C. GST activity was measured using 1-chloro-2,4-dinitrobenzene (CDNB) and reduced GSH as substrates

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with slight modifications of the method used by Habig et al. [21] in96-well microplates. Two hundred microliter reaction mixtures(1 mM CDNB and 5 mM GSH in 0.1 M sodium phosphate buffer,pH 7.0) were placed in a well containing 10 lL of the enzyme sample.Reagents were prepared fresh prior to use. The change in absorbancewas measured continuously for 6 min at 340 nm. At least four repli-cates were performed for each population. Changes in optical den-sity due to GST activity per individual were converted to lmolCDNB conjugated/min/mg protein using the extinction coefficientof 2,4-dinitrophenylglutatione (e340nm = 9.6 mM�1 cm�1).

2.6. Determination of cytochrome P450 monooxygenase activity

Cytochrome P450 monooxygenase activity was measured byO-deethylation of 7-ethoxycoumarin based on the microfluorimet-ric method of De Sousa et al. [22] with minor modifications. Tomeasure the 7-EFC-O-deethylation activity in CPP, 250 adults werehomogenized in 1 mL sodium/potassium phosphate buffer (0.1 M,pH 7.2, containing 1 mM EDTA, 1 mM DTT and 200 mM sucrose)at 4 �C. The homogenates were centrifuged at 5000g for 5 min at4 �C, and the resulting supernatant was centrifuged at 15,000gfor 15 min followed by 100,000g for 60 min. The microsomal pelletwas resuspended in 100 lL of the above buffer and used as the en-zyme source. A quantity of 100 lL of the microsomal fraction and50 lL of sodium/potassium phosphate buffer (0.1 M, pH 7.2) con-

Table 2Effect of PBO, DEM and TPP on phosalone resistant and susceptible populations of Agonos

Populationa Synergist Phosalone

nb v2 (df)d L

RF 317 1.53 (3) 1TPP 254 1.38 (3)PBO 321 0.74 (3) 3DEM 293 0.48 (3) 1

BA 257 0.28 (3)TPP 282 0.67 (3)PBO 266 0.39 (3)DEM 289 0.14 (3)

a RF: Rafsanjan; BA: Bam.b Number of insect tested.c LC50 values are expressed as mg mL�1, LCL: lower confidence limit at 95%; UCL: uppd RR: Resistance ratio = LC50 of the suspected population/LC50 of the susceptible popue SR: Synergist ratio = LC50 of phosalone alone/LC50 of phosalone + synergist.

Table 1Log dose probit-mortality data for phosalone susceptible and resistant populations ofAganoscena pistaciae using insect-dip method.

Populationa nb LC50 (LCL–UCL)c Slope ± SE v2 (df)d RRe

RF 321 622 (501–733) 2.89 ± 0.46 1.99 (4) 11a,*

AN 309 185 (148–230) 2.01 ± 0.25 1.41 (3) 3h

BA 247 55 (31–82) 1.49 ± 0.3 1.6 (3) –i

KR 287 530 (433–637) 2.70 ± 0.48 0.18 (3) 10ab

SH 283 418 (338–503) 2.46 ± 0.4 0.39 (3) 8bcd

HE 262 290 (213–371) 2.108 ± 0.38 0.64 (3) 5efg

SI 250 408 (299–519) 2.242 ± 0.45 0.24 (3) 7bcde

PA 265 471 (372–617) 1.93 ± 0.32 1.76 (3) 9abc

PGH 246 316 (238–402) 2.41 ± 0.36 2.07 (3) 6def

a (RF: Rafsanjan; AN: Anar; BA: Bam; KR: Kerman; SH: Shahrbabak; HE: Herat;SI:Sirjan; PA: Pariz; PGH: Paghaleh).

b Number of insect tested.c The LC50 value are expressed as mg mL�1, LCL: lower confidence limit at 95%;

UCL: upper confidence limit at 95%.d Values of v2, lower than (p 6 0.05) indicate a significant fit between the

observed and expected regression lines.e Resistance ratio: LC50 of resistant population/ LC50 of BA population.

* Means within the same rank followed by different letters are significantly dif-ferent at p < 0.05.

taining 7-ethoxycoumarin and NADPH, resulting in final concen-trations of 1 mM NADPH and 0.4 mM 7-ethoxycoumarin, wereadded in each well. The plate was incubated for 30 min at 30 �Cwhile shaking. The self-fluorescent NADPH was removed by incu-bation with 1.5 mM oxidized glutathione and 0.5 units of glutathi-one reductase at room temperature for 10 min. The reaction wasstopped by adding 150 lL 50% acetonitrile in TRIZMA-base buffer(0.05 M, pH 10). The released amount of 7-hydroxycoumarin wasquantified at 465 nm while exciting at 390 nm. Protein concentra-tion was measured by the method of Bradford [23], using bovineserum albumin as the standard.

2.7. Analysis

Data were analyzed by employing ANOVA and means werecompared by Duncan multiple range test (p < 0.05) using SPSS(2004). LC50 was determined using probit analysis with the soft-ware Polo-PC (LeOra Software) [24].

3. Results

3.1. Phosalone resistance levels

LC50 values for phosalone in nine populations of CPP calculatedfrom probit analysis are given in Table 1. Significant differences(p < 0.05) were observed among the LC50 values of the populations.The lowest (55.18 mg L�1) and highest LC50 values (621.65 mg L�1)were determined in the BA and RF populations, respectively. TheBA population proved to be the most susceptible to phosalone,therefore this population was used as a susceptible reference tocalculate the resistance ratio. Among the field collected popula-tions, the highest and lowest levels of resistance to phosalone weredetected for RF (RR = 11.26-fold) and AN (3.34-fold) populations,respectively. In addition, the resistance levels among KR, SH, PAand SI populations to phosalone were not significantly differentfrom each other.

3.2. Synergists

The synergistic effects of TPP, PBO and DEM were evaluated onphosalone-treated resistant (RF) and susceptible (BA) populationsof A. pistaciae to determine the involvement of esterase, MFO andGST detoxifying enzymes in resistance mechanisms, respectively(Table 2). There was a significant difference between LC50 valuesof TPP-phosalone-treated and phosalone-treated RF populations.Phosalone was approximately three times more toxic in the pres-ence of TPP than in the absence of TPP. The same situation was

cena pistaciae, compared to the susceptible population.

C50 (LCL–UCL)c Slope ± SE RRd SRe

7.84 (11.62–25.5) 1.41 ± 0.26 15.65 –5.68 (3.58–7.98) 1.473 ± 0.27 4.98 3.149.05 (30.06–50.37) 2 ± 0.36 34.25 0.452.65 (9.83–15.78) 2.17 ± 0.37 11.09 1.411.14 (0.62–1.78) 1.22 ± 0.21 – –0.89 (0.49–1.47) 0.99 ± 0.15 – 1.232.2 (1.41–3.28) 1.25 ± 0.22 – 0.971.23 (0.6–2.24) 0.93 ± 0.17 – 1.31

er confidence limit at 95%.lation.

Fig. 1. Esterase activities in nine populations of Agonoscena pistaciae using different substrates. Activity was measured as lmol naphthol/min/mg protein for the substrates a-and b-naphthyl acetate and nmol q-nitrophenol/min/mg protein for the substrate q-nitrophenyl acetate. The means within the same rank followed by different letters aresignificantly different at p < 0.05. RF: Rafsanjan; AN: Anar; BA: Bam; KR: Kerman; SH: Shahrbabak; HE: Herat; SI:Sirjan; PA: Pariz; PGH: Paghaleh.

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observed in the BA population, but the synergistic ratio was lowerthan that of the RF population. The results showed that the addi-tion of TPP to bioassays had a significant effect on phosalonedose–mortality responses in the RF and BA populations.

In the resistant population (RF), PBO significantly reduced phos-alone toxicity (�2-fold) but not in the susceptible populations. Theresistance ratio in the RF population was increased from 15.65 to34.25 suggesting PBO had an antagonistic effect.

3.3. Esterase activity

The activities of general esterases were measured with a-NAand b-NA as substrates. Results showed that there were significantdifferences in esterase activities among the populations (Fig. 1).

Fig. 2. GST activities in nine populations of Agonoscena pistaciae. The means within thRafsanjan; AN: Anar; BA: Bam; KR: Kerman; SH: Shahrbabak; HE: Herat; SI:Sirjan; PA:

Quantitative analysis of general esterase activity revealed highestamounts of esterase in the populations RF and SI 2- to 2.5-foldhigher than those in the susceptible population (BA). The same sit-uation was observed when esterase activity against the substratePNPA was compared. Among the populations, RF and SI showedmore esterase activity (�3-fold) than those in the susceptible pop-ulation (BA).

3.4. GST activity

The results revealed that the highest GST activity that catalyzesthe formation of glutathione-conjugated adducts, is in the SI pop-ulations which is significantly difference than the susceptible pop-ulation (1.49-fold) (Fig. 2). GST activity in the RF, KR and H

e same rank followed by different letters are significantly different at p < 0.05. RF:Pariz; PGH: Paghaleh.

Fig. 3. CYTP450s activities in nine populations of Agonoscena pistaciae. The means within the same rank followed by different letters are significantly different at p < 0.05. RF:Rafsanjan; AN: Anar; BA: Bam; KR: Kerman; SH: Shahrbabak; HE: Herat; SI:Sirjan; PA: Pariz; PGH: Paghaleh.

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populations were also significantly difference than the BA popula-tion. Although, there was no significant difference in the GST activ-ity among AN, SH, PA, PGH and susceptible (BA) populations.

3.5. CYTP450s activity

Significant differences in monooxygenase-mediated O-deethyla-tion of 7-ethoxycoumarin activity were found among the popula-tions. The activity was increased 2.44-, 2.05- and 2.87-fold in RF,SH and SI populations that are resistant to phosalone (Fig. 3). Amongthe tested populations, the BA, KR, HE, PA and PGH populationsshowed lower activities that did not differ significantly from eachother.

4. Discussion

4.1. Bioassay

Among the field collected populations, the highest level of resis-tance to phosalone was detected in the RF population (RR = 11.26-fold) via insect-dip method. The toxicity data resulting from anRCV bioassay on CPP adults indicated that the resistance ratio forRF population was 15.65.

The susceptibility of the CPP to phosalone was first demon-strated by Talebi et al. [7] in Kerman province and the level ofthe resistance ratio was determined 1.8-fold (in RF population).Rafsanjan is the main area of pistachio production in Iran and hugeamounts of pesticide are applied in this area against the CPP. Itseems that repetitive and frequent application of insecticides tocontrol CPP causes higher levels of resistance in this populationcompared with the other populations. The BA population, the mostsusceptible, was collected from the area without any history ofpesticide application. This population indicated the lowest slopevalue among the other populations, suggesting more heterogeneityof the population. This is due to the higher number of heterozygoteindividuals in the BA population.

4.2. Analysis of detoxifying mechanisms

Conventionally, synergism studies and biochemical assays areimportant steps in the reorganization of the detoxifying mecha-

nisms of insecticide resistance. The involvement of esterase, GSHS-transferase and microsomal monooxygenase in insecticide resis-tance has been reported in many insect species [9–12,25,26]. Thedetoxifying mechanisms were investigated in vivo by studying theeffect of three synergists TPP, PBO and DEM on the resistant (RF)and susceptible (BA) CPP populations. TPP and DEM increased thetoxicity of phosalone in the RF population by 3.14- and 1.44-foldrespectively. The results revealed that the general esterase activityin the RF population was approximately 3-fold more than in thesusceptible population. Therefore, the bioassay and biochemical re-sults suggested the involvement of esterase in resistance of CPP tophosalone similar to those revealed in Psylla piricolla [12,15]. In-creased activity of carboxylesterase is a common insecticide resis-tance mechanism against organophosphates, carbamates andpyrethroid insecticides in some insects and mites [10,13,27].

GSTs play a central role in the detoxification of both endoge-nous and xenobiotic compounds. Elevated GST activity has beenassociated with resistance to all the major classes of insecticides[28]. GST activity in the RF and SI populations of CPP were 1.28-and 1.49-fold higher than those in the susceptible one, but a syn-ergistic assay on the RF population showed that the LC50 of phos-alone in the RF and susceptible populations were suppressed byDEM equally. Therefore, GST does not seem to be an important fac-tor in resistance to phosalone.

The mixed function oxidases (MFO) catalyze various types ofreactions in pesticide compounds, playing an important role ininsecticide detoxification and infrequently in activation. Monooxy-genase-mediated resistance is probably the most frequent type ofmetabolism based insecticide resistance [29]. PBO has not com-monly been used as an organophosphate synergist because itinhibits the activity of CYTP450s. CYTP450s plays an important rolein bioactivation of phosalone to the oxon form. Identification of theCYTP450s involved in insecticide resistance has been very difficult.The preferred methods for demonstrating monooxygenase-medi-ated resistance of insect populations to insecticides are bothin vivo and in vitro metabolism studies [29]. In this study PBOhad an antagonistic impact on the phosalone toxicity to the RFpopulation which is consistent with the previous findings by MalekMohamadi et al. [30]. Enzymatic assays with 7-ethoxycoumarinindicated that CYTP450s might also be involved in phosalone resis-tance in the RF population.

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It is already known that multiple isomers of CYTP450s are pres-ent in insects and they all are substrate-specific [31]. In the case oforganophosphorus compounds, a number of isomers of CYTP450sare involved in activation and others are possibly implicated in oxi-dative detoxification of them. Activity of the CYTP450s in SI and RFwas more than that of other CPP populations. Therefore, it seemsmore likely that different isomers of CYTP450s are involved in bothbioactivation and detoxification of phosalone in the CPP. Moreover,it appears that CYTP450s implicated in phosalone activation aremore vulnerable to PBO inhibition than those that are involvedin metabolic detoxification of phosalone. PBO has been regardedan excellent inhibitor of MFO, and it has been shown to inhibitesterase activity in some insect species, however it did not havesignificant effect on the overall esterase activity in the horn flyresistant to diazinon [32]. In addition, the effect of PBO on esteraseactivity is known to be depending upon the applied dose, insecti-cide type, insect population and pre-treatment time [33]. We havedemonstrated the inhibitory effect of PBO on phosalone toxicity toCPP at the dose used (10 mg L�1). It is possible that the use ofgreater or lesser concentrations than the dose we used show differ-ent results. As it was reported for diazinon resistant horn fly thesuggestion of possible involvement of CYTP450s-based metabolicactivation or detoxification of phosalone in the present study isnot conclusive [32].

Detoxifying enzymes activity level in the SI population is com-parable to the RF except for GST that is higher in SI populationwhile the resistance ratio in the SI population is not similar to RFpopulation. Although, previous study showed that the synergisticratio of DEM on phosalone resistant Colorado potato beetle doesnot necessarily correlate with resistant ratio [30], but GST mayhad been induced by various types of pesticides other than phos-alone that applied on SI population during the control period.

The CPP have been survived a long history of different insecti-cides application. No data is available for the susceptibility ofCPP population to insecticides belongs to other classes includingneonicotinoids, insect growth regulators and pyrethroids. Whetheror not resistance to phosalone conferred resistance to other insec-ticides will require further study. This may help to detect the othermechanisms involved in resistance of CPP to phosalone and subse-quently resistance management of this serious pest.

Acknowledgments

The authors are grateful to University of Vali-e-Asr Rafsanjanfor generous support to do bioassay experiments. We thank Prof.John Vontas, Dr. Evangelia Morou and Dr. Vasileia Balabanidoufrom University of Crete for their help to do particular biochemicalexperiments. We are grateful to Thomas Ant from University of Ox-ford and Maryam Jamali for editing and Ebrahim Salari for the col-lection of field CPP populations and assistance in rearing.

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