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Pesticide Biochemistry and Physiology 84 (2006) 135–142 www.elsevier.com/locate/ypest 0048-3575/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.pestbp.2005.06.003 Insensitivity of acetylcholinesterase in a Weld strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith) S.J. Yu ¤ Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, USA Received 29 April 2005; accepted 28 June 2005 Available online 30 September 2005 Abstract Acetylcholinesterase (AChE) was puriWed from adult heads of the fall armyworm (Spodoptera frugiperda) by using a two-step procedure involving gel Wltration on a Sephadex G-200 column and aYnity chromatography on a procain- amide–ECH Sephadex 4B column. Both susceptible and Weld strains possessed two AChE isozymes, namely, AChE-1 and AChE-2, with subunit molecular weights of 63.7 and 66.1 kDa. The puriWed AChE had an apparent K m value of 33.5 M and a V max of 7.07 mol/min/mg protein in the susceptible strain. The apparent K m and the V max were 2.2- and 2.0-fold higher, respectively, in the Weld strain than in the susceptible strain. The puriWed AChE from the Weld strain was 17- to 345-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, eserine, meth- omyl, and bendiocarb) and organophosphates (methyl paraoxon and paraoxon), insensitivity being highest toward car- baryl. The results further support the notion that insensitive AChE played an important role in the insecticide resistance observed in the Weld strain. 2005 Elsevier Inc. All rights reserved. 1. Introduction The fall armyworm, Spodoptera frugiperda (J.E. Smith), is a serious lepidopterous pest of several important crops such as corn, cotton, peanuts, and soybeans. Control of the fall armyworm has depended heavily on insecticides. As a result, this pest has developed resistance to the major classes of insecticides in many areas [1–4]. Recently, we reported that a strain of fall army- worm collected from Citra, Florida, showed high resistance to carbaryl (562-fold) and methyl para- thion (354-fold). Biochemical studies revealed that various detoxiWcation enzyme activities were higher in the Weld strain than in the susceptible strain [4]. In larval fat bodies, activities of microsomal oxidases (epoxidases, hydroxylase, sulfoxidase, N-demethylase, and O-demethylase), Florida Agricultural Experiment Station Journal Series No. R-10853. * Fax: +1 352 392 0190. E-mail address: [email protected]X.edu.

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Page 1: Insensitivity of acetylcholinesterase in a field strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith)

Pesticide Biochemistry and Physiology 84 (2006) 135–142

www.elsevier.com/locate/ypest

Insensitivity of acetylcholinesterase in a Weld strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith) �

S.J. Yu ¤

Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, USA

Received 29 April 2005; accepted 28 June 2005Available online 30 September 2005

Abstract

Acetylcholinesterase (AChE) was puriWed from adult heads of the fall armyworm (Spodoptera frugiperda) by using atwo-step procedure involving gel Wltration on a Sephadex G-200 column and aYnity chromatography on a procain-amide–ECH Sephadex 4B column. Both susceptible and Weld strains possessed two AChE isozymes, namely, AChE-1and AChE-2, with subunit molecular weights of 63.7 and 66.1 kDa. The puriWed AChE had an apparent Km value of33.5 �M and a Vmax of 7.07 �mol/min/mg protein in the susceptible strain. The apparent Km and the Vmax were 2.2- and2.0-fold higher, respectively, in the Weld strain than in the susceptible strain. The puriWed AChE from the Weld strain was17- to 345-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, eserine, meth-omyl, and bendiocarb) and organophosphates (methyl paraoxon and paraoxon), insensitivity being highest toward car-baryl. The results further support the notion that insensitive AChE played an important role in the insecticide resistanceobserved in the Weld strain. 2005 Elsevier Inc. All rights reserved.

1. Introduction

The fall armyworm, Spodoptera frugiperda(J.E. Smith), is a serious lepidopterous pest ofseveral important crops such as corn, cotton,peanuts, and soybeans. Control of the fallarmyworm has depended heavily on insecticides.

� Florida Agricultural Experiment Station Journal Series No.R-10853.

* Fax: +1 352 392 0190.E-mail address: [email protected].

0048-3575/$ - see front matter 2005 Elsevier Inc. All rights reserveddoi:10.1016/j.pestbp.2005.06.003

As a result, this pest has developed resistance tothe major classes of insecticides in many areas[1–4].

Recently, we reported that a strain of fall army-worm collected from Citra, Florida, showed highresistance to carbaryl (562-fold) and methyl para-thion (354-fold). Biochemical studies revealed thatvarious detoxiWcation enzyme activities werehigher in the Weld strain than in the susceptiblestrain [4]. In larval fat bodies, activities ofmicrosomal oxidases (epoxidases, hydroxylase,sulfoxidase, N-demethylase, and O-demethylase),

.

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136 S.J. Yu / Pesticide Biochemistry and Physiology 84 (2006) 135–142

glutathione S-transferases (CDNB,1 DCNB, andp-nitrophenyl acetate conjugation), hydrolases(general esterase, carboxylesterase, �-glucosidase,and carboxylamidase), and reductases (juglonereductase and cytochrome c reductase) were 1.3-to 7.7-fold higher in the Weld strain than in thesusceptible strain. Cytochrome P-450 level was2.5-fold higher in the Weld strain than in the sus-ceptible strain. In adult abdomens, detoxiWcationenzyme activities were generally lower than in lar-val fat bodies. However, activities of microsomaloxidases (S-demethylase), hydrolases (generalesterase and permethrin esterase), and reductases(juglone reductase and cytochrome c reductase)were 1.5- to 3.0-fold higher, respectively, in theWeld strain than in the susceptible strain. In addi-tion, acetylcholinesterase (AChE) from adult headsof the Weld strain was 2- to 85-fold less sensitive thanthat from the susceptible strain to inhibition by vari-ous carbamates and organophosphates; insensitivitywas most pronounced toward carbaryl. Results indi-cated that the insecticide resistance observed in theWeld strain was due to multiple resistance mecha-nisms, including increased detoxiWcation of theseinsecticides by microsomal oxidases, glutathioneS-transferases, hydrolases and reductases, andinsensitive AChE.

In the AChE studies mentioned above, we usedcrude homogenates of adult heads as enzymesource. However, crude head homogenates containnumerous proteins (including enzymes) other thanAChE and these proteins could interfere withAChE inhibition studies. For example, a carboxyl-esterase isolated from green peach aphids wasshown to hydrolyze or sequester AChE inhibitorssuch as organophosphorus and carbamate insecti-cides [5]. Similarly, esterases from green rice leaf-hoppers were capable of playing a dual role: ascatalyst for hydrolysis of malathion and fenvaler-ate, and as binding protein for organophosphorusinsecticides (oxygen analogs) [6]. To avoid suchcomplications in biochemical studies, it is most

1 Abbreviations used: AChE, acetylcholinesterase; CDNB,1-chloro-2,4-dinitrobenzene; DCNB, 1,2-dichloro-4-nitrobenzene;EEDQ, 2-ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electropho-resis; Ki, bimolecular rate constant.

desirable to characterize a puriWed AChE prepara-tion. Furthermore, no information is available onthe biochemical characteristics of puriWed AChEin fall armyworms. Therefore, the purpose of thisresearch was to purify AChE from adult heads ofboth Weld and susceptible strains and characterizethe enzyme biochemically in order to verify theinsensitivity of AChE reported previously [4].

2. Materials and methods

2.1. Insects

The Weld strain was collected from a corn Weldin the Plant Science Research Center, University ofFlorida in Citra, Florida, during the spring of 2002.The susceptible strain, which originated from theUSDA in Tifton, Georgia, has been maintained inthe laboratory without exposure to insecticides.Both strains were maintained on an artiWcial dietin environmental chambers at 25 °C with a 16:8L:D photoperiod as described previously [7]. TheWeld strain was maintained in the laboratory with-out further selection for one generation before theresearch was initiated.

2.2. Chemicals

Acetylthiocholine, paraoxon, eserine, procain-amide hydrochloride, and Sephadex G-200 werepurchased from Sigma (St. Louis, MO). Triton X-100 and 2-ethoxy-1-ethoxycarbonyl-1,2-dihydro-quinoline (EEDQ) were obtained from Aldrich(Milwaukee, WI). Methyl paraoxon was obtainedfrom Chem Service (West Chester, PA). ECHSepharose 4B was purchased from Amersham Bio-sciences (Piscataway, NJ). Other insecticides (tech-nical grade sample) were used as received from themanufacturers. All other chemicals were of analyt-ical quality and purchased from commercialsuppliers.

2.3. Enzyme preparation

Groups of 500 heads were removed from 7- to10-day-old adults and homogenized in 25 ml of ice-cold 0.05 M sodium phosphate buVer, pH 7.5,

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S.J. Yu / Pesticide Biochemistry and Physiology 84 (2006) 135–142 137

containing 0.5% (v/v) Triton X-100, in a motor-driven tissue grinder for 30 s. The crude homoge-nate was Wltered through cheesecloth and theWltered homogenate was centrifuged at 105,000gfor 1 h in a Beckman L5-50E ultracentrifuge. Thesupernatant (soluble fraction) was used as theenzyme source for AChE puriWcation.

For study of subcellular distribution of AChE,the crude homogenate prepared from 80 heads wasWrst centrifuged at 1000g for 15 min to obtain apellet which contained nuclei and cell debris. Thesupernatant was recentrifuged at 10,000g for15 min to obtain mitochondria (pellet). The resul-tant supernatant was again centrifuged at 105,000gfor 1 h to obtain the microsomal pellet and theWnal supernatant representing the soluble fraction.

2.4. PuriWcation of AChE

AChE was puriWed from the soluble fractionwith a two-step procedure involving gel Wltrationand aYnity chromatography. The aYnity columnused in this study was prepared according to themethod of Pasteur et al. [8] with ECH Sepharose 4Bas a matrix and procainamide as a ligand. BrieXy,procainamide solution was prepared by dissolving1.36 g procainamide in 10 ml water. EEDQ solutionwas prepared by dissolving 0.124 g EEDQ in 10 mlethanol. The two solutions were mixed and thenadded to 20 ml ECH Sepharose 4B. The solutionwas mixed gently with a rotator at 4 °C overnight.The procainamide-coupled Sepharose 4B was thenpoured into a 1£20 cm glass column and washedsuccessively with 50% ethanol, 20mM Tris–HClbuVer (pH 7.0) and 1 M NaCl. The column was thenequilibrated with 0.05 M sodium phosphate buVer(pH 7.0) containing 0.1% (v/v) Triton X-100 and0.05 M NaCl (designated as BuVer A).

To purify AChE, the soluble fraction preparedas mentioned above was Wrst applied to a Sepha-dex G-200 column (2.5 £ 40 cm) previously equili-brated with BuVer A. The column was eluted withthe same buVer until no further protein wasdetected. Fractions of 1 ml were collected and ana-lyzed for AChE activity. The active fractions werepooled.

The pooled AChE sample was applied to a pro-cainamide aYnity column previously equilibrated

with BuVer A. The column was washed extensivelywith BuVer A until no further protein was detected(about 220 ml of BuVer A). The bound AChE wasthen eluted with BuVer A containing 0.1 M pro-cainamide. Fractions containing AChE activitywere pooled and dialyzed against 0.05 M sodiumphosphate buVer (pH 7.0) containing 0.1% (v/v)Triton X-100 overnight. PuriWed AChE was thenconcentrated by ultraWltration on an AmiconDiaXo PM-10 membrane. All samples were storedat ¡70 °C for further analysis.

2.5. Protein determination

Protein concentrations were determined by themethod of Peterson [9] using bovine serum albu-min as standard.

2.6. Enzyme assays

Acetylcholinesterase activity was measured withacetylthiocholine as substrate as described by Ell-man et al. [10]. In inhibition studies, inhibitors weredissolved in methyl cellosolve and then dilutedwith 0.1 M sodium phosphate buVer, pH 8.0. Bimo-lecular rate constant (Ki) for inhibition of acetyl-cholinesterase by insecticides was determined bythe method of Aldridge [11].

2.7. Electrophoresis

Sodium dodecyl sulfate–polyacrylamide gelelectrophoresis (SDS–PAGE) was performedaccording to the method of Laemmi [12] in a Bio-Rad Mini-Protein II electrophoresis cell; theseparating gel contained 10% acrylamide and thestacking gel contained 4% acrylamide. Subunitmolecular weights of AChE were determined withthe following standards: lysozyme (14.4 kDa), tryp-sin inhibitor (21.5 kDa), carbonic anhydrase(31 kDa), ovalbumin (45 kDa), serum albumin(66.2 kDa), and phosphorylase b (97.4 kDa). Theprocedure of non-denaturing PAGE was similar tothe SDS–PAGE except that no SDS was used andit was performed at 4 °C. Routinely, the gels werestained for protein with Coomassie brilliant blue Raccording to Fairbanks et al. [13]. In some cases,proteins were detected in the gels with silver stain

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138 S.J. Yu / Pesticide Biochemistry and Physiology 84 (2006) 135–142

(Bio-Rad). The gel was also stained for AChEactivity by the method of Karnovsky and Roots[14].

2.8. Kinetic study

The Michaelis constant (Km) and maximumvelocity (Vmax) for puriWed AChE were determinedby Lineweaver–Burk plot using acetylthiocholineas substrate.

2.9. Statistical analysis

Whenever appropriate, data were analyzed byStudent’s t test.

3. Results

3.1. Subcellular distribution of AChE

AChE activities distributed in diVerent subcellu-lar fractions prepared from head homogenates ofthe susceptible strain in the presence and theabsence of Triton X-100 are shown in Table 1. Inthe absence of detergent, AChE activity wasmainly found in cell debris and mitochondriawhich also contained higher speciWc AChE activ-ity. This suggests that the majority of AChE wasmembrane-bound. However, in the presence ofdetergent (0.05% Triton X-100), the majority ofactivity (92.2%) was detected in the soluble frac-tion, indicating that almost all AChE was solubi-

lized from the membranes. When the detergentincreased to 0.1%, 97.2% of the total enzyme activ-ity was found in the soluble fraction (results notshown).

3.2. PuriWcation of AChE

The results of the puriWcation of AChE fromboth Weld and susceptible strains are summarizedin Table 2. The gel Wltration (Sephadex G-200)yielded 80 and 93% of the initial enzyme activityand puriWcations of 2.1- and 2.0-fold from the sus-ceptible and Weld strain, respectively. The aYnitychromatography step yielded 23 and 31% of theinitial enzyme activity and puriWcations of 57- and56-fold from the susceptible and the Weld strain,respectively. The speciWc activity of AChE was15.38 and 19.18 �mol/min/mg protein in the sus-ceptible and the Weld strain, respectively.

3.3. Characterization of AChE

Analysis of the puriWed AChE by non-denatur-ing PAGE followed by AChE staining revealedtwo isozymes, namely, AChE-1 (Rf D 0.037) andAChE-2 (Rf D 0.15), with AChE-1 being the majorone (Fig. 1). SDS–PAGE of the puriWed AChEpreparation showed two protein bands with molec-ular weights of 63.7 and 66.1 kDa, the former beinga minor subunit (Fig. 2 and Table 3). In allinstances, there was no diVerence in electrophoreticpatterns between the susceptible and the Weldstrain.

Table 1Subcellular distribution of acetylcholinesterase activity in the head of fall armyworm adults

a All fractions were prepared from 80 adults heads of the susceptible strain.b Means § SE of two experiments, each with duplicate determinations.

Subcellular fractiona

Solubilization (0.05% Triton X-100)

SpeciWc activity (nmol/min/mg protein)b

Total activityb

% of total activity

Cell debris, nuclei No 192.3 § 9.2 675.0 § 21.4 31.6Mitochondria No 409.0 § 17.5 1026.4 § 74.6 48.1Microsomes No 26.9 § 1.6 21.82 § 0.96 1.0Soluble fraction No 82.8 § 5.7 409.4 § 16.2 19.2

Cell debris, nuclei Yes — 41.8 § 2.4 1.6Mitochondria Yes — 35.9 § 0.83 1.4Microsomes Yes — 12.0 § 3.56 4.8Soluble fraction Yes — 2327.1 § 113.5 92.2

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Kinetic studies (Table 3) showed that the appar-ent Km for AChE from the Weld strain was 2.2-foldhigher than that from the susceptible strain. TheVmax was 2.0-fold higher in the Weld strain than inthe susceptible strain.

Data in Table 4 show that in the susceptiblestrain eserine was the most potent inhibitor ofAChE, followed by methyl paraoxon, paraoxon,bendiocarb, methomyl, carbaryl, and monocroto-phos based on the Ki value. In the Weld strain, eser-ine was the most potent inhibitor, followed bymethomyl, paraoxon, monocrotophos, bendiocarb,methyl paraoxon, and carbaryl. The results alsoshowed that AChE from Weld strain was less sensi-tive than that from the susceptible strain to inhibi-tion by carbaryl, eserine, methomyl, bendiocarb,methyl paraoxon, and paraoxon ranging from 17-to 345-fold, with insensitivity being most pro-

Fig. 1. Non-denaturing polyacrylamide gel electrophoresis ofpuriWed AChE from heads of fall armyworm adults. The gelwas stained for AChE activity using acetylthiocholine as a sub-strate. Lanes 1–3, Weld strain; lanes 4–6, susceptible strain.

nounced toward carbaryl. There was no diVerencein inhibition by monocrotophos between the twostrains.

4. Discussion

Based on the results of our study with diVeren-tial centrifugation, AChE in adult heads of fallarmyworm was primarily membrane-bound, which

Fig. 2. Sodium dodecyl sulfate–polyacrylamide gel electropho-resis of puriWed AChE from heads of fall armyworm adults.Lane 1, Weld strain; lane 2, susceptible strain.

Table 2PuriWcation of acetylcholinesterase from heads of fall armyworm adults

PuriWcation step Strain Yield (%) SpeciWc activity (�mol/min/mg protein)a

PuriWcation factor

Soluble fraction Susceptible 100 0.27 § 0.03 1.0Field 100 0.34 § 0.01 1.0

Sephadex G-200 Susceptible 80 0.56 § 0.07 2.1Field 93 0.69 § 0.01 2.0

AYnity column Susceptible 23 15.38 § 0.88 57.0Field 31 19.18 § 0.23 56.4

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140 S.J. Yu / Pesticide Biochemistry and Physiology 84 (2006) 135–142

could be solubilized eVectively with Triton X-100.Our results are in agreement with those obtainedfrom other insect species. AChE in the Germancockroach, Blattella germanica, is mainly located inthe 10,000g pellet (mitochondria) [15]. In the hornXy, Haematobia irritans, the activity is primarilyfound in the microsomal fraction [16]. AChE inLygus hesperus is mostly membrane-bound, withonly 23% of the total activity found in the solublefraction [17]. In the greenbug, Schizaphos grami-num, 85% of the AChE activity was found to bemembrane-bound [18]. In contrast, most of theAChE activity in larval Chironomus ripariusappears to be in soluble form, with 76% in the100,000g supernatant [19]. The results of the presentstudy clearly demonstrated that AChE from headsof fall armyworm adults can be puriWed to apparenthomogeneity by a two-step procedure involving gelWltration and aYnity chromatography. AYnity

Table 3Biochemical characteristics of puriWed acetylcholinesterasefrom heads of fall armyworm adults

a Means § SE of three experiments, each with duplicatedeterminations.

b Value signiWcantly diVerent from the susceptible strain(P < 0.05).

Strain Km (�M)a Vmax (�mol/min/mg protein)a

Subunit Mr (kDa)

Susceptible 33.5 § 7.5 7.07 § 1.5 63.766.1

Field 74.5 § 3.5b 14.4 § 2.5b 63.766.1

chromatography appears to be the most eVectivestep, resulting in over 50-fold puriWcation in thesusceptible and the resistant strain. The puriWedAChE consisted of two molecular forms which hadsubunit molecular weights of 63.7 and 66.1 kDa inboth strains.

AChE has been puriWed from various insectspecies. Table 5 shows molecular weights of nativeforms and their subunits of AChE puriWed fromsix species. It is seen that AChE exists in multipleforms (monomers, dimers, and tetramers) withsubunit molecular weights of 60–94 kDa. Thus, thesubunit molecular weights of AChE isolated fromfall armyworms in the present study fall within thepublished range. The subunit composition of these

Table 5Molecular weights of acetylcholinesterase in diVerent species ofinsects

Species Molecular weight (kDa)

References

Native form

Subunit

House Xy 75 75 [20]150 75; 75150 75; 75

Lygus hesperus 199 94; 94 [21]150 79; 7982 86

Colorado potato beetle 130 65; 65 [22]Greater wax moth 240 60; 60; 60; 600 [23]Greenbug 129 72; 72 [24]Oriental fruit Xy 116 61; 61 [25]

Table 4Bimolecular rate constants for the inhibition of acetylcholinesterase by carbamate and organophosphorus insecticides in fall army-worm adults

a PuriWed AChE was prepared from heads of fall armyworm adults.b Values in parentheses denote data from Yu et al. [4] using crude head homogenates as enzyme source.

Insecticide Ki (M¡1 min¡1)a

Susceptible strain (S) Field strain (R) S/R

CarbamateCarbaryl 5.32 £ 104 (2.76 £ 104)b 1.54 £ 102 (3.15 £ 102) 345Eserine 1.28 £ 106 (1.78 £ 105) 6.30 £ 104 (2.24 £ 105) 20Methomyl 2.31 £ 105 (3.65 £ 104) 1.39 £ 104 (3.47 £ 104) 17Bendiocarb 3.65 £ 105 (1.48 £ 105) 2.48 £ 103 (9.37 £ 103) 147

OrganophosphateMethyl paraoxon 6.30 £ 105 (3.47 £ 105) 2.24 £ 103 (3.89 £ 104) 281Paraoxon 4.08 £ 105 (1.54 £ 105) 1.26 £ 104 (6.30 £ 104) 32Monocrotophos 8.89 £ 103 (3.85 £ 103) 7.30 £ 103 (3.01 £ 104) 1.2

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S.J. Yu / Pesticide Biochemistry and Physiology 84 (2006) 135–142 141

two native forms of AChE isolated is not known.Moreover, it is not clear whether these two AChEisozymes represent two gene products since diVer-ent forms can be due to protease or phospholipasedigestion of a main form [26,27]. Inhibition studieswith the puriWed AChE preparation from Weld andsusceptible strains as the enzyme source showedlinear inhibition curves in response to variousinsecticides (Table 4), indicating that these two iso-zymes had similar biochemical characteristics.Insect AChE is believed to exist in globular formsbut not in asymmetric forms [27].

Our results conWrm those reported previously[4] showing insensitive AChE as a resistance factorin the Weld strain. However, some distinct diVer-ences in biochemical characteristics were observedwhen we compared puriWed AChE with crudehomogenates as the enzyme source [4]. Kineticstudies revealed that the apparent Km for the puri-Wed AChE was higher in the Weld strain than in thesusceptible strain, yet the reverse was true withcrude homogenates. It is possible that crudehomogenates contained some factors which alteredthe kinetic activity of AChE in both susceptibleand Weld strains. Table 4 also included the Ki val-ues obtained previously [4] with crude homoge-nates as enzyme source for comparison. It can beseen that in the susceptible strain, Ki values of theseinsecticides for the puriWed AChE were higherthan those for the crude homogenate. However, inthe Weld strain, the reverse was true showing thatKi values of these insecticides for the puriWedAChE were lower than those for the crude homog-enate. In all instances, the insensitivity ratios[Ki (S)/Ki (R)] were higher for puriWed AChE thanfor crude homogenates. In resistant cotton aphids,the puriWed AChE was reported to be more sensi-tive than the crude homogenate to inhibition bymethamidophos and pirimicarb [28]. Thus, crudehomogenates may contain some factors whichinterfere with inhibition studies of AChE resultingin changing AChE sensitivity and may lead to falseobservations. Therefore, using puriWed AChE asenzyme source to verify its insensitivity appears tobe an important procedure in resistance studies.

Insensitive AChE as a resistance mechanism tocarbamates and organophosphates has beenreported in numerous insect species including

S. littoralis, S. exigua, and S. frugiperda [28–30].Insensitive AChE has been shown to be due topoint mutations [31]. For example, in Drosophilamelanogaster, a point mutation of AChE resultingin the replacement of a phenylalanine by a tyrosinein AChE decreased the sensitivity of this enzyme toorganophosphorus insecticides in resistant strains[32]. A novel glycine–serine substitution was foundto be responsible for AChE insensitivity to organo-phosphates in the olive fruit Xy, Bactrocera oleae[33].

Acknowledgments

The author thanks Drs. S.M. Valles (USDA-ARS) and J.L. Nation (University of Florida) forcritical reviews of the manuscript. Technical assis-tance of Sam Nguyen is also appreciated.

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