Archives of Insect Biochemistry and Physiology 45:47–59 (2000)

© 2000 Wiley-Liss, Inc.

Characterization of Esterases Associated With ProfenofosResistance in the Tobacco Budworm, Heliothis virescens (F.)

John A. Harold and James A. Ottea*

Department of Entomology, Louisiana State University Agricultural Center, Baton Rouge

The utility of microplate and electrophoretic assays for detec-tion of biochemical and physiological mechanisms underlyingresistance to profenofos in the tobacco budworm, Heliothisvirescens (F.), was assessed. Esterase (EST) activities were sig-nificantly higher in profenofos-resistant than -susceptible lar-vae, and activities were highly correlated (r2 = 0.87) withresistance to profenofos. Both qualitative and quantitativevariation was observed in electrophoretic gels stained with a-and b-naphthyl acetates. Staining of ESTs was more intensewith resistant larvae than those from a susceptible strain. Inaddition, a band (designated A¢) was expressed in larvae fromprofenofos-resistant strains, but not in larvae from an insecti-cide-susceptible strain. The frequency of expression of A¢ in-creased following selection with profenofos and was detectedin 100% of the individuals from a profenofos-selected strain.The appearance of this band coincided with the decreased ex-pression of a second band (designated A). A similar pattern(overexpression of A¢ and underexpression of A) also was ob-served in larvae from field-collected strains. Finally, reductionin the activity or the sensitivity of acetylcholinesterase to inhibi-tion by chlorpyrifos oxon was observed in laboratory-selectedand field-collected larvae that expressed resistance to profenofos.These results suggest that microplate and electrophoretic assayscan be utilized as complementary tools for detecting and moni-toring profenofos resistance in H. virescens. Arch. Insect Biochem.Physiol. 45:47–59, 2000. © 2000 Wiley-Liss, Inc.

Key words: insecticide; resistance; organophosphate; detection; Heliothisvirescens

Contract grant sponsor: Cotton Incorporated; Contract grantsponsor: Louisiana Agricultural Experiment Station (LAES).

*Correspondence to: Dr. James A. Ottea, Department of Ento-mology, 402 Life Sciences Building, Louisiana State Univer-sity, Baton Rouge, LA 70803. E-mail: jottea@unix1.sncc.lsu.edu

Received 28 July 2000; Accepted 9 August 2000

INTRODUCTION

Over the past 40 years, resistance to all ma-jor classes of insecticides has been a persistentand increasing impediment to effective manage-ment of field populations of the tobacco budworm,Heliothis virescens (F.) (Sparks, 1981; Sparks etal., 1993). All three major mechanisms of insecti-cide resistance (i.e., reduced cuticular penetration,increased metabolic detoxication and altered sites

48 Harold and Ottea

of action) are expressed in organophosphorus(OP)-resistant H. virescens. Reduced cuticularpenetration has been shown as a minor mecha-nisms conferring low levels of resistance tomalathion (Szeicz et al., 1973) and profenofos(Kanga and Plapp, 1994). In addition, decreasedsensitivity of acetylcholinesterase (AChE), the tar-get site for OPs and carbamates, has also been acause of OP resistance in this pest (Brown andBryson, 1992; Harold and Ottea, 1997).

Enhanced metabolic detoxication of insecti-cides by cytochrome P450-dependent monooxygen-ases (P450 MOs), glutathione S-transferases(GSTs) and esterases (ESTs) plays a major rolein insecticide resistance (Oppenoorth, 1985;Abdel-Aal et al., 1993). Enhanced expression ofP450 MO activity is associated with metabolic re-sistance to OP insecticides in H. virescens (Brown,1981; Bull, 1981) and is considered the primarycause of resistance to chlorpyrifos and chlorpyrifosmethyl in some strains of this insect (Whitte andBull, 1974). Similarly, elevated GST activities to-ward non-insecticide (model) substrates weremeasured in field-collected strains of H. virescensand were moderately correlated with frequenciesof resistance to profenofos (Ibrahim and Ottea,1995; Harold and Ottea, 1997). Finally, esterasesare responsible for the hydrolysis of methyl par-athion in laboratory strains of H. virescens (Konnoet al., 1989, 1990). More recent studies suggestthat elevated EST activities (as measured withα-naphthyl acetate [α-NA]) are associated withresistance to carbamate, OP and pyrethroid in-secticides (Goh et al., 1995; Zhao et al., 1996), andare correlated with frequencies of profenofos resis-tance in field-collected and laboratory-selectedstrains of H. virescens (Harold and Ottea, 1997).

Knowledge and biochemical and physiologi-cal mechanisms of resistance is essential for therational development and effective implementa-tion of insecticide resistance management (IRM)strategies (Brown and Brogdon, 1987). Since thesuccess of IRM relies on the ability to diagnosethe nature, frequency, and evolution of insecti-cide resistance in the target population, rapid andsensitive techniques to detect resistance mecha-nisms are needed (ffrench-Constant and Roush,1990). Although results of biological assays pro-vide verification that resistance is present in fieldpopulations, they fail to provide information about

the underlying resistance mechanism(s). Rapid,preliminary diagnosis of metabolic mechanismsof resistance may be obtained using bioassayswith insecticide synergists. Similarly, quantita-tive biochemical assays based on spectrophoto-metric determination of enzyme activities towardmodel substrates have been used as indicators ofmetabolic resistance (Brown and Brogdon, 1987;Rose et al., 1995). However, since detoxifying en-zymes exist in multiple forms (Oppenoorth, 1985;Schoknecht and Otto, 1989) with differing sensi-tivities to synergists, a single synergist may not in-hibit the enzymes responsible for resistance (Brownet al., 1996a). Therefore, absence of synergism can-not be considered as proof that a particular meta-bolic mechanism is absent (ffrench-Constant andRoush, 1990). Similarly, results from studies usingmodel substrates are inconclusive unless enzymeactivities measured are consistently correlated withthe expression of resistance (Brown and Brogdon,1987; Ibrahim and Ottea, 1995). Since EST activi-ties toward α-NA were found previously to be cor-related with frequencies of profenofos resistance(Harold and Ottea, 1997), the objective of this studywas to explore further the utility of this EST assayas a biochemical market to detect and monitorprofenofos resistance in H. virescens.

MATERIALS AND METHODSChemicals

Technical grade profenofos (O-(4-bromo-2-chlorophenyl)-O-ethyl-S-propyl phosphorothioate:89%; Novartis, Greensboro, NC) and chlorpyrifosoxon (O,O-diethyl-O-(3,5,6-trichloro-2-pyridinyl)phosphate; 100%; DowElanco Inc., Indianapolis,IN) were donated by respective manufacturers.Acrylamide, ethylenediaminetetraacetic acid(EDTA) and tris(hydroxymethyl)aminomethane(Tris) were purchased from Gibco BRL (GrandIsland, NY). N,N,N′,N′-tetramethylethylene-di-amine (TEMED), bis acrylamide, ammoniumpresulfate and Coomassie Brilliant Blue R-250were obtained from Amresco (Solon, OH). Bovineserum albumin (fraction 5), acetylthiocholine io-dide (ATChI), 5,5′-dithio-bis(2-nitrobenzoic acid)(DTNB), Fast Blue RR salt, β-naphthyl acetate(β-NA, and α-NA were purchased from SigmaChemical Company (St. Louis, MO). Fast BlueB salt and potassium chloride (KCl) were pur-

Resistance Detection in H. virescens 49

chased from Aldrich Chemical Company (Milwau-kee, WI).

Insects

Results of biological and biochemical assayswith field-collected insects were compared withthose from laboratory strains of OP-susceptibleand -resistant H. virescens. For studies with fieldcollections, eggs and larvae (150–250) were col-lected from a domesticated stand of velvetleaf,Abutilon theophrasti Medicus, at the LouisianaState University Agricultural Center’s NortheastResearch Station/Macon Ridge location (MRS;Winnsboro, LA) on June 12, 1997 (MRS Jun).Other field collections were made on August 22and 25, 1997, from a cotton field near Bayou Ma-con, LA (By Mac) that had been treated with threeapplications of spinosad, two applications each ofcypermethrin and profenofos and one applicationof thiodicarb. Portions of leaves containing eggsand larvae were collected and transported to thelaboratory in styrofoam coolers containing ice andplaced in 1-oz cups containing a pinto bean–basedsemi-synthetic diet (Leonard et al., 1988). Field-collected larvae were identified as H. virescensbased on the presence of spinose cuticle and cha-lazas 1 and 2 on abdominal segments 1, 2, and 8,and a molar area on the oral surface of the man-dible (Oliver and Chapin, 1981). Larvae wereseparated following head capsule slippage at theend of the fourth stadium, and fifth stadium (day1) insects (180 ± 20 mg) were selected for biologi-cal and biochemical assays. Adults were rearedin 3.8-l cardboard cartons covered with cottongauze as a substrate for oviposition and providedwith sucrose (10% in water) as a carbohydratesource. Both larvae and adults were held at 27°C,70% relative humidity, and a 14:10 hr (light:dark)photoperiod.

Larvae from two laboratory strains werestudied. The reference susceptible strain, LSU-S,was established from field collections from cot-ton in 1977 (Leonard et al., 1988) and has beenreared in the laboratory without intentional ex-posure to insecticides. A resistant laboratorystrain (OPR) was originally established by select-ing larvae from a field-collection made at the RedRiver (RR) Research Station (Bossier City, LA)on August 23, 1995 (Harold and Ottea, 1997). Lar-vae from the OPR strain were reared for three

generations without selection (OPR F3) and wereeither selected with profenofos (15.3 µg/larva;OPR F4S) or reared without exposure to insecti-cide (OPR F4). Fifth stadium larvae (day 1) fromeach of the strains were used for biochemical (n= 30) and biological (n ≥ 30) assays.

Biological Assays

Susceptibility of H. virescens to profenofoswas measured in fifth stadium (day 1) larvae fol-lowing application of 1 µl of acetone containingvarying doses of profenofos onto the thoracic dor-sum. The dose-mortality response of larvae wasmeasured with at least 5 doses of profenofos (≥10larvae/dose) and replicated thrice. Treated larvaewere held in 1-oz cups with diet and maintainedat 27°C, 70 ± 5% relative humidity, and a photo-period of 14:10 (light:dark) hr. Mortality was re-corded 72 hr following topical application usingabsence of coordinated movement within 30 secafter being prodded with a pencil as the crite-rion. Data were analyzed by probit analysis(Finney, 1971) using a microcomputer-based pro-gram (SAS, 1985). Frequencies of resistance toprofenofos in larvae (n ≥ 30) from laboratory andfield-collected strains were measured 72 hr fol-lowing topical application of a single dose ofprofenofos (15.3 µg/larva; approximately 20× theLD50 for LSU insects). This dose was high enoughto kill all susceptible larvae but low enough toallow differences in frequencies among the resis-tant strains to be resolved.

Tissue Preparation

Tissue homogenates from individual larvaewere used as enzyme sources for all biochemicalassays. Individual larvae were weighed, decapi-tated, dissected, and the digestive system was re-moved. The opened hemocoel was rinsed withice-cold buffer (0.1 M sodium phosphate, pH 7.0)and fat bodies were obtained by gentle scrapingand aspiration using a Pasteur pipette. Fat bod-ies were homogenized in an all-glass homogenizercontaining 200 µl of ice-cold 1.15% KCl (contain-ing a few crystals of phenylthiourea). Individualheads were homogenized in 200 µl of 0.1 M so-dium phosphate buffer (pH 8.0) containing 0.1%Triton X-100. Homogenates were centrifuged at12,000g for 15 min. The resulting supernatantswere filtered through glass wool then held in ice

50 Harold and Ottea

and used in enzyme assays within 30 min ofpreparation.

Biochemical Assays

Activity of esterases towards α-NA was mea-sured using the assay of Gomori (1953) with modi-fications (van Asperen, 1961; Grant et al., 1989;Ibrahim and Ottea, 1995). Reaction mixtures inindividual wells of a microtiter plate contained240 µl of substrate solution (2.13 mM and 0.56%,final concentrations of α-NA and Fast Blue B, re-spectively), and 10 µl of fat body homogenate (con-taining 0.05 tissue equivalent; 0.016 ± 0.007 mgprotein). The substrate solution was prepared byadding 600 µl of α-NA (0.113 M dissolved in 50%acetone in water) to a solution of Fast Blue Bsalt (18 mg in 30 ml of 0.1 M phosphate buffer,pH 7.0), and then filtered using Whatman no. 3filter paper. Reaction mixtures were incubated at30°C, and the rate of change in absorbance dur-ing the initial 10 min was measured at 595 nmusing a Thermomax microplate reader (Molecu-lar Devices, Palo Alto, CA). Data were correctedfor non-enzymatic activity using incubations with-out protein as the control. Changes in OD wereconverted to nmol/min using an experimentallyderived “extinction coefficient” (3.825 mM–1 250µl–1) for the α-naphthol-Fast Blue B conjugate at595 nm.

Native PAGE was used to visualize ESTfrom individual larvae using a vertical electro-phoresis unit (Hoefer Scientific Instruments, SanFrancisco, CA) and 5% acrylamide (Sambrook etal., 1989; Gunning et al., 1996). The protein con-centrations of homogenates from individual fatbodies were adjusted to contain 30 µg protein in40 µl, which was loaded onto a gel with 5 µl of 6×tracking dye (0.25% Bromophenol Blue and 40%sucrose w/v in water). Electrophoresis occurredin Tris-borate/EDTA buffer (100 mM Tris, 2.4 mMEDTA, and 100 mM boric acid, pH 8.0) at a con-stant voltage (150 V) until the dye marker waswithin 1 cm of the gel base. After electrophore-sis, gels were stained in darkness at 25°C with100 ml of 0.1 M sodium phosphate buffer (pH 7.0)containing 0.5 mM α- and β-NA and 0.2% FastBlue RR salt for 30 min. Gels were destained indistilled water and fixed in 5% acetic acid. Rela-tive mobility (Rm) was calculated by dividing themigration distance of the specific band from the

origin to the center of the band by the migrationdistance of the bromophenol blue tracking dyefrom the origin.

A “squash assay” was performed on filterpaper based on earlier methods (Pasteur andGeorghiou, 1989; Abdel-Aal et al., 1990) withmodifications. Individual second stadium larvae(approximately 10–20 mg) were homogenized ina 1.5 ml microcentrifuge tube (cut open at thedistal end) using a pencil tip capped with a 0.5ml microcentrifuge tube onto a Whatman no. 3filter paper moistened with phosphate buffer (0.1mM; pH 7.0). The filter paper was incubated atopa second filter paper containing α-NA/Fast Bluesubstrate solution (prepared as described above)for 60 sec in darkness. The reaction was stoppedby rinsing the filter paper with water followedby the addition of 5% acetic acid. Esterase activi-ties were visualized as violet strains on theundersurface of the filter paper, which were com-pared with a color scale prepared with α-naph-thol standards (0–1.8 µmol).

Sensitivity of AChE to chlorpyrifos oxon wasmeasured using head homogenates from indi-vidual larvae following the method of Ellman etal. (1961) with modifications (Moores et al., 1988;Byrne and Devonshire, 1991, 1993; Ibrahim andOttea, 1995). As profenofos in an indirect inhibi-tor of AChE (Wing et al., 1998; Byrne and Devon-shire, 1993), it could not be used for these studies.Homogenates of individual heads (30 µl contain-ing 0.15 tissue equivalent; 0.070 ± 0.083 mg pro-tein) were incubated for 10 min at 30°C in wellsof a microplate containing 69 µl of sodium phos-phate buffer (0.1 M, pH 8.0) and 1 µl of chlor-pyrifos oxon (148.8 nM final concentration). Theconcentration of chlorpyrifos oxon used in thesestudies was the I90 for the LSU strain as deter-mined in preliminary experiments (data notshown). Following preincubation, reactions wereinitiated by adding 200 µl of ATChI/DTNB (0.5mM/0.05 mM, final concentration), and the rateof change in OD at 405 nm at 30°C was recorded.Data were corrected for nonenzymatic activity us-ing incubations without protein as the control andexpressed as miliOD min–1 mg protein–1. Activi-ties of AChE measured in the absence of chlorpy-rifos oxon served as the control. Percentageinhibition was calculated as 1-(activity with in-hibitor/activity without inhibitor) × 100.

Resistance Detection in H. virescens 51

Protein concentrations were measured by themethod of Bradford (1976) with bovine serum al-bumin (fraction V; concentrations corrected forimpurities) as the standard. Data were subjectedto analysis of variance followed by Tukey’s mul-tiple comparison test (P = 0.05) using a micro-computer-based program (SAS, 1985). Linearregressions between enzyme activities and sus-ceptibility to profenofos were estimated using themethod of least squares.

RESULTSProfenofos Resistance

Profenofos resistance in laboratory-resistantstrains was statistically higher than in the sus-ceptible LSU strain (Table 1). The highest levelof resistance to profenofos (31-fold relative toLSU) was expressed in OPR larvae (LD50 = 22.8µg/larva). In the absence of selection with profen-ofos, levels of resistance decreased significantlyto 11.8-fold (LD50 = 8.6 µg/larva) after 3 genera-tions (OPR F3) and to 9.7-fold (LD50 = 7.1 µg/larva) after 4 generations (OPR F4). Resistanceincreased significantly to 22-fold (LD50 / 16.0 µg/larva) following selection of OPR F3 larvae withprofenofos (OPR F4S).

Frequencies of resistance to profenofos (asdetermined in topical bioassays with 15.3 µgprofenofos/larva) were higher in larvae from thefield-collected and the laboratory-selected strainsthan the susceptible LSU strain (Table 1). A highfrequency of resistance was measured for OPRlarvae (68%) but decreased to 27% in OPR F3 lar-vae. Resistance frequency decreased further be-

tween OPR F3 to OPR F4, but this difference wasnot statistically significant. However, followingselection of OPR F3 larvae with profenofos, re-sistance frequency increased to 53% in OPR F4S.High frequencies of profenofos resistance alsowere recorded in field-collected insects (60 and97% in MRS Jun and By Mac, respectively).

Esterase Activities

Activities of ESTs were higher in larvae fromboth laboratory-selected and field-collected insectsthan in the susceptible LSU strain (Table 2). Thehighest mean EST activity (294 nmol α-naphtholformed min–1 mg protein–1) was expressed in lar-vae from the OPR strain. Mean EST activity de-

TABLE 1. Profenofos Susceptibility in Larvae From Laboratory and Field-Collected Strains of H. virescens†

Percentage survivalStrain N LD50 (95% CL) Slope ± SE Resistance ratio χ2 @ diagnostic dose

LSU 392 0.73 (0.65–0.81)c 3.72 ± 0.32 — 3.34 0.00OPR 213 22.8 (18.8–27.5)a 2.46 ± 0.30 31.2 3.42 67.7OPR F3 180 8.60 (6.78–10.6)b 2.44 ± 0.30 11.7 1.57 26.8OPR F4 184 7.08 (5.50–8.86)b 2.23 ± 0.28 9.70 1.97 23.3OPR F4S 241 16.0 (13.1–19.1)a 2.44 ± 0.28 22.0 0.85 53.3MRS Jun 30 ND ND ND ND 60.0By Mac 30 ND ND ND ND 96.7†Susceptibility was measured 72 hr following topical application of 1 µl of acetone containing profenofos onto fifth stadium(day 1) larvae. Resistance ratio = LD50 of resistant strain/LD50 of LSU strain. Percentage survival @ diagnostic dose =survival following topical application of 15.3 µg of profenofos/larva (n = 30) in fifth stadium (day 1) larvae. ND, notdetermined.*Expressed as µg/larva; values followed by the same letter are not statistically different (Tukey’s; P = 0.05).

TABLE 2. Enzyme Activities and Sensitivity of AChEto Inhibition by Chlorpyrifos Oxon in Field-Collectedand Laboratory Strains of H. virescens*

EST AChE AcHEStrain (SD) activity (SD) inhibition (SD)LSU 71.6 (38.1)c 0.25 (0.05)a,b 89.8 (2.72)a

OPR 294 (127)a 0.33 (0.16)a 69.6 (14.8)d

OPF F3 88.8 (52.3)c 0.20 (0.18)b,c 78.0 (12.0)b,c

OPR F4 106 (44.5)b,c 0.16 (0.06)c,d,e 88.3 (4.79)a

OPR F4S 163 (49.5)b 0.12 (0.05)d,e 87.4 (5.83)a

MRS Jun 167 (104)b 0.20 (0.09)b,c,d 74.9 (11.4)c,d

By Mac 237 (105)a 0.10 (0.04)e 83.2 (10.7)a,b

*Activities were measured in assays with individual larvae (n≥ 30) from laboratory-susceptible (LSU), -resistant (OPR), andfield-collected (MRS: Macon Ridge, By Mac: Bayou Macon)strains of H. virescens. Values followed by the same letter arenot significantly different (Tukey’s; P = 0.05). Mean EST ac-tivities (±SD) expressed as nmols of α-naphthol formed min–1

mg protein–1. Mean AChE activities (±SD) toward ATChI ex-pressed as miliOD min–1 mg protein–1. Mean of percent inhibi-tion of AChE (±SD) measured following preincubation of headhomogenates with chlorpyrifos oxon for 10 min.

52 Harold and Ottea

creased in the absence of selection to 88.8 nmolin OPR F3 larvae, but increased significantly (1.8-fold) in OPR F4S larvae following selection withprofenofos. In assays with field strains, larvaefrom By Mac expressed EST activity that wascomparable with that of OPR larvae. The ESTactivity in MRS Jun larvae was intermediate rela-tive to LSU and OPR larvae, and did not differsignificantly from that of OPR F4S.

Whereas mean levels of EST activity did notdiffer among the OPR F3, F4, and LSU strains,differences in frequency profiles for this activityin these strains were apparent (Fig. 1). The fre-quency distribution of LSU larvae was narrowwith no individuals expressing activity >90 nmolmin–1 mg protein–1. In contrast, the distributionof EST activity was broad in larvae from OPR,and 97% of the individuals expressed activitiesgreater than that of LSU larvae. In the absenceof selection (OPR F3), the frequency profile nar-rowed, and only 20% of the larvae expressed ac-tivity >90 nmol. In profenofos-selected OPR F4Slarvae, increased frequencies of individuals ex-pressing high EST activity were detected: 83% ofindividuals expressed activities greater than 90nmol. Finally, broad frequency profiles were ob-

served in the field strains with activities greaterthan the LSU strain measured in 62 and 87% ofthe individuals from MRS Jun and By Mac, re-spectively (Fig. 2).

Electrophoretic and Filter Paper Analysis

Qualitative and quantitative variation inbanding patterns of ESTs in electrophoretic gelswas observed between OP-susceptible and -re-sistant larvae. Staining of ESTs appeared moreintense with resistant larvae than those fromthe susceptible strain (Figs. 3–5). In addition,a band (designated A′; Rm = 0.65) was expressedin some OPR larvae, but not in larvae from theLSU strain (Fig. 3). In most cases, the appear-ance of this band coincided with the decreasedexpression of a second band (designated A) withRm = 0.68. In addition, after selection of OPRlarvae with a high dose of profenofos (OPRF4S), expression of band A′ (and underexpres-sion of band A) was measured in 100% of theindividuals tested (Fig. 4). Finally, this pattern(i.e., overexpression of A′ and underexpressionof A) was observed in gels with the majority offield-collected larvae from MRS Jun (67%) andBy Mac (87%) (Fig. 5).

Fig. 1. Frequency distribution of EST activity (expressed as α-naphthol formed min–1 mg protein–1) from fat bodyhomogenates of individual, fifth stadium larvae (day 1; n = 30) in laboratory-susceptible and -resistant strains of H.virescens.

Resistance Detection in H. virescens 53

Fig. 2. Frequency distribution of EST activity (expressed as α-naphthol formed min–1 mg protein–1) from fat body homogenatesof individual, fifth stadium larvae (day 1; n = 30) in laboratory-susceptible and field-collected strains of H. virescens.

Fig. 3. Polyacrylamide gel stained for EST activity in individual larvae from profenofos-susceptible (LSU) and -resistant(OPR) strains of H. virescens. Each lane contains 30 µg of protein from fat body homogenates of individual, fifth stadium(day 1) larvae.

Quantitative differences in EST activity be-tween LSU and OPR F4S larvae were also appar-ent in a filter paper assay using individual, secondstadium larvae (Fig. 6). The color developed usingsusceptible larvae ranged from no color to moder-ate violet color, and staining was more intense withhomogenates from resistant than susceptible lar-vae. In individual homogenates from 15 LSU lar-vae, intensity of staining corresponded to < 0.8 µmol

of α-naphthol. In contrast, intense staining was ob-served with homogenates from OPR F4S larvae andamounts of α-naphthol produced were > 0.8 µmolfor 14 of the 15 homogenates.

Activity of AChE and Sensitivity to Inhibition byChlorpyrifos Oxon

Activities of AChE from individual larvaewere variable in both laboratory and field-col-

54 Harold and Ottea

lected strains (Table 2). The highest activity (0.33miliOD min–1 mg protein–1) were measured inOPR larvae. Activities decreased significantly inthe absence of selection to 0.20 and 0.16 miliODmin–1 mg protein–1 in the OPR F3 and F4 strains,respectively. Although activity was significantlylower in OPR F4S than LSU larvae, activities did

not differ between OPR F4 (unselected) and OPRF4S (selected). The lowest activity was measuredin the field-collected By Mac strain.

Sensitivity of AChE (expressed as percentinhibition following incubation with chlorpyrifosoxon) was significantly lower in larvae from theOPR strain than from the LSU strain (Table 2).Sensitivity increased significantly in the absenceof selection for both the OPR F3 (78%) and OPRF4 (88%) strains. However, as with AChE activity,there were no differences in AChE sensitivity be-tween OPR F4 and OPR F4S larvae. Reduced sen-sitivity of AChE also was measured in field-collectedMRS Jun insects (75%), but there was no signifi-cant difference in AChE sensitivity between By Mac(83%) and LSU (90%) larvae.

Frequency profiles for AChE inhibition alsoreflected differences among strains (Fig. 7). Thesensitivity of AChE in LSU larvae was normallydistributed with more than 88% inhibition mea-sured in 97% of individuals. For OPR and OPRF3 strains, 80 and 30% of individuals, respec-tively, expressed AChE with decreased sensi-tivity (i.e., <88% inhibition). Broad frequencyprofiles of AChE sensitivity were measured withlarvae from the field-collected strains, and de-creased sensitivity of AChE was expressed in67 and 27% of individuals from MRS Jun andBy Mac, respectively.

Fig. 4. Polyacrylamide gel stained for EST activity in indi-vidual larvae from profenofos-susceptible (LSU) and -selected(OPR F4S) strains of H. virescens. Each lane contains 30µg of protein from fat body homogenates of individual, fifthstadium (day 1) larvae.

Fig. 5. Polyacrylamide gel stained for EST activity in individual larvae from field-collected strains of H. virescens. Eachlane contains 30 µg of protein from fat body homogenates of individual, fifth stadium (day 1) larvae.

Resistance Detection in H. virescens 55

Fig. 6. Filter paper assay measuring the intensity of color developed using crude homogenates of second stadium larvaefrom susceptible (LSU) and resistant (OPR F4S) strains of H. virescens. Bottom: Color scale developed with known con-centration of α-naphthol.

Fig. 7. Frequency distribution of AChE sensitivity in profenofos-susceptible (LSU) and -resistant (OPR, OPR F3, F4, and F4S)strains of H. virescens. Sensitivity is expressed as percentage inhibition of AChE activity measured in head homogenates ofindividual, fifth stadium larvae (day 1; n = 30) following 10 min preincubation with 149 nM chlorpyrifos oxon.

56 Harold and Ottea

DISCUSSION

Biochemical assays, including those withmodel substrates, can be utilized successfully fordetecting and monitoring insecticide resistance ininsect populations (Brown and Brogdon, 1987;Devonshire, 1987). Such knowledge of the natureof resistance mechanisms may serve as a foun-dation for the development of field kits to diag-nose metabolic resistance in field populations ofthis pest. This would enable growers to ascertainthe resistance status of H. virescens prior to in-secticide application, and to choose effective in-secticides for control of this pest. The basis forthe present study is a correlation reported be-tween EST activities and frequencies of profenofosresistance in an array of resistant field popula-tions and laboratory strains of H. virescens(Harold and Ottea, 1997).

Elevated EST activities toward model sub-strates have been associated with OP resistancein a number of insect pests (Abdel-Aal et al.,1993). In previous studies, elevated activities ofESTs toward permethrin, α-NA (Dowd et al.,1987) and methyl parathion (Konno et al., 1989,1990) have been documented in larvae from labo-ratory strains of OP-resistant H. virescens. Morerecently, EST activities toward α-NA from masshomogenates of thiodicarb-selected H. virescenswere suggested to be responsible for cross-resis-tance among carbamate, OP, and pyrethroid in-secticides (Goh et al., 1995; Zhao et al., 1996). Inthe present study, a high level of resistance tocypermethrin (17-fold at the LD50) was measuredin OPR larvae (data not shown), suggesting thatmetabolic mechanisms expressed in this strainmay confer cross-resistance between cypermethrinand profenofos.

Results presented in this study provide in-direct evidence for the role of ESTs in OP resis-tance in H. virescens and illustrate the utility ofEST activity toward α-NA as a biochemicalmarker for profenofos resistance in this insect.Frequencies of profenofos resistance in 21 field-collected and laboratory-selected strains of H.virescens were highly correlated (r2 = 0.90) withmean levels of EST activities toward α-NA (thisstudy and Harold and Ottea, 1997). In addition,EST activity toward α-NA decreased in the ab-sence of selection (i.e., from OPR to OPR F4) but

increased following one generation of selection(i.e., from OPR F3 to OPR F4S). Finally, frequen-cies of profenofos resistance and EST activitieswere significantly higher in two field populationsof H. virescens than in the LSU strain.

In addition to elevated EST activities, aunique EST was expressed in larvae from pro-fenofos-resistant laboratory colonies, and the fre-quency of expression increased following selectionwith profenofos. Further, this EST was detectedin field-collected strains and was expressed in 67and 87% of the individuals from MRS Jun andBy Mac, respectively. Corresponding frequenciesof resistance measured in these strains were 60and 97% for MRS Jun and By Mac, respectively.Since detoxifying enzymes exist in multiple forms(Schoknecht and Otto, 1989), the mean interstaindifference in total activity with α-NA might over-lap between susceptible and resistant strains(Devonshire, 1989), masking the expression of aresistance-associated esterase. However, resultsfrom the present study suggest that such es-terases can be accurately and rapidly detected inindividual larvae using electrophoretic separation.In a similar study, discrimination between sus-ceptible and moderately resistant aphids (whosetotal activity overlapped with that of susceptibleaphids) was made possible by electrophoreticseparation of esterase-4 (Sawicki et al., 1978).Since estimations based on intensity of electro-phoretic bands are subjective, electrophoresis maybest be viewed as a complementary tool with spec-trophotometric determinations of total EST ac-tivities (Devonshire and Field, 1991).

Although no ESTs from the profenofos-resis-tant larvae used for this study have been shownto metabolize insecticides directly, the consistentexpression of a unique enzyme in resistant in-sects suggests a potential role in profenofos re-sistance in H. virescens and as a biochemicalmarker. In a similar study, Konno et al. (1990)reported a unique EST (esterase III) that hydro-lyzed methyl parathion in OP-selected H. vires-cens. In the current study, elevated EST activityand electrophoretic banding patterns in resistantH. virescens formed the basis for a preliminaryversion of a “squash assay,” which has potentialutility in the field to assess the contribution ofESTs to resistance in this pest. In the prelimi-

Resistance Detection in H. virescens 57

nary assays reported here, staining was more in-tense with homogenates from profenofos-resistantthan -susceptible strains.

Altered expression and sensitivity of AChEwere measured in the OP-resistant strains. Highfrequencies (>50%) of the individuals tested fromfield-collected and resistant laboratory strainsexpressed both increased EST activity and alteredAChE (data not shown). Similar enhancement ofesterase-based insecticide resistance by insensi-tive AChE has been observed in the aphids,Myzus persicae and M. nicotianae (Moores et al.,1994). In the present study, both decreased ac-tivity of AChE and reduced sensitivity to inhibi-tion by chlorpyrifos oxon were measured at highfrequencies in larvae from field-collected strains:38 and 87% of individuals from MRS Jun and ByMac, respectively, expressed lower AChE activ-ity than LSU larvae. In addition, as in the OPRstrain, AChE with decreased sensitivity to inhi-bition by chlorpyrifos oxon was expressed in 67and 27% of MRS Jun and By Mac larvae, respec-tively. Whereas reduced sensitivity of AChE to OPinhibitors in H. virescens has been documented(Brown, 1991; Brown and Bryson, 1992; Kangaand Plapp, 1994; Wolfenbarger, 1996; Brown etal., 1996b), decreased activity of AChE was not.Lower hydrolyzing efficiency, in conjunction withreduced sensitivity of AChE, has also been ob-served in azinphosmethyl-resistant Colorado po-tato beetles, Leptinotarsa decemlineata (Zhu andClark, 1995), and several other insect species (re-viewed in Oppenoorth, 1985).

In conclusion, the results of this studyshowed that both enhanced esterase activity andaltered expression of AChE are associated withprofenofos resistance in H. virescens. ElevatedEST activities toward α-NA in OP-resistant lar-vae were measured using a microplate assay andwere associated with the appearance of a uniqueelectrophoretic band. These data suggest thatmicroplate and electrophoretic assays can be uti-lized as complementary tools for detecting andmonitoring mechanisms of profenofos resistancein H. virescens.

ACKNOWLEDGMENTS

We thank Dr. Roger Leonard for assistancewith field collections of insects. This manuscript

has been approved for publication by the Direc-tor of the LAES as MS 99-17-0265.

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