Characterization of Esterases Associated with Profenofos Resistance in the Tobacco Budworm, Heliothis Virescens (F.) John A

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

Characterization of Esterases Associated With Profenofos Resistance 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 detection of biochemical and physiological mechanisms underlying resistance to profenofos in the tobacco budworm, Heliothis virescens (F.), was assessed. Esterase (EST) activities were significantly higher in profenofos-resistant than -susceptible larvae, and activities were highly correlated (r2 = 0.87) with resistance to profenofos. Both qualitative and quantitative variation was observed in electrophoretic gels stained with a- and b-naphthyl acetates. Staining of ESTs was more intense with resistant larvae than those from a susceptible strain. In addition, a band (designated A¢) was expressed in larvae from profenofos-resistant strains, but not in larvae from an insecticide-susceptible strain. The frequency of expression of A¢ increased following selection with profenofos and was detected in 100% of the individuals from a profenofos-selected strain. The appearance of this band coincided with the decreased expression of a second band (designated A). A similar pattern (overexpression of A¢ and underexpression of A) also was observed in larvae from field-collected strains. Finally, reduction in the activity or the sensitivity of acetylcholinesterase to inhibition by chlorpyrifos oxon was observed in laboratory-selected and field-collected larvae that expressed resistance to profenofos. These results suggest that microplate and electrophoretic assays can be utilized as complementary tools for detecting and monitoring profenofos resistance in H. virescens. Arch. Insect Biochem. Physiol. 45:47–59, 2000. © 2000 Wiley-Liss, Inc.

Key words: insecticide; resistance; organophosphate; detection; Heliothis virescens

INTRODUCTION Over the past 40 years, resistance to all major classes of insecticides has been a persistent and increasing impediment to effective manage- Contract grant sponsor: Cotton Incorporated; Contract grant ment of field populations of the tobacco budworm, sponsor: Louisiana Agricultural Experiment Station (LAES). Heliothis virescens (F.) (Sparks, 1981; Sparks et *Correspondence to: Dr. James A. Ottea, Department of Ento- al., 1993). All three major mechanisms of insecti- mology, 402 Life Sciences Building, Louisiana State Univer- cide resistance (i.e., reduced cuticular penetration, sity, Baton Rouge, LA 70803. E-mail: [email protected] increased metabolic detoxication and altered sites Received 28 July 2000; Accepted 9 August 2000

© 2000 Wiley-Liss, Inc. 48 Harold and Ottea of action) are expressed in organophosphorus the underlying resistance mechanism(s). Rapid, (OP)-resistant H. virescens. Reduced cuticular preliminary diagnosis of metabolic mechanisms penetration has been shown as a minor mecha- of resistance may be obtained using bioassays nisms conferring low levels of resistance to with insecticide synergists. Similarly, quantita- malathion (Szeicz et al., 1973) and profenofos tive biochemical assays based on spectrophoto- (Kanga and Plapp, 1994). In addition, decreased metric determination of enzyme activities toward sensitivity of acetylcholinesterase (AChE), the tar- model substrates have been used as indicators of get site for OPs and carbamates, has also been a metabolic resistance (Brown and Brogdon, 1987; cause of OP resistance in this pest (Brown and Rose et al., 1995). However, since detoxifying en- Bryson, 1992; Harold and Ottea, 1997). zymes exist in multiple forms (Oppenoorth, 1985; Enhanced metabolic detoxication of insecti- Schoknecht and Otto, 1989) with differing sensi- cides by cytochrome P450-dependent monooxygen- tivities to synergists, a single synergist may not in- ases (P450 MOs), glutathione S-transferases hibit the enzymes responsible for resistance (Brown (GSTs) and esterases (ESTs) plays a major role et al., 1996a). Therefore, absence of synergism can- in insecticide resistance (Oppenoorth, 1985; not be considered as proof that a particular meta- Abdel-Aal et al., 1993). Enhanced expression of bolic mechanism is absent (ffrench-Constant and P450 MO activity is associated with metabolic re- Roush, 1990). Similarly, results from studies using sistance to OP insecticides in H. virescens (Brown, model substrates are inconclusive unless enzyme 1981; Bull, 1981) and is considered the primary activities measured are consistently correlated with cause of resistance to chlorpyrifos and chlorpyrifos the expression of resistance (Brown and Brogdon, methyl in some strains of this insect (Whitte and 1987; Ibrahim and Ottea, 1995). Since EST activi- Bull, 1974). Similarly, elevated GST activities to- ties toward α-NA were found previously to be cor- ward non-insecticide (model) substrates were related with frequencies of profenofos resistance measured in field-collected strains of H. virescens (Harold and Ottea, 1997), the objective of this study and were moderately correlated with frequencies was to explore further the utility of this EST assay of resistance to profenofos (Ibrahim and Ottea, as a biochemical market to detect and monitor 1995; Harold and Ottea, 1997). Finally, esterases profenofos resistance in H. virescens. are responsible for the hydrolysis of methyl parathion in laboratory strains of H. virescens (Konno MATERIALS AND METHODS et al., 1989, 1990). More recent studies suggest Chemicals that elevated EST activities (as measured with α-naphthyl acetate [α-NA]) are associated with Technical grade profenofos (O-(4-bromo-2- resistance to carbamate, OP and pyrethroid in- chlorophenyl)-O-ethyl-S-propyl phosphorothioate: secticides (Goh et al., 1995; Zhao et al., 1996), and 89%; Novartis, Greensboro, NC) and chlorpyrifos are correlated with frequencies of profenofos resis- oxon (O,O-diethyl-O-(3,5,6-trichloro-2-pyridinyl) tance in field-collected and laboratory-selected phosphate; 100%; DowElanco Inc., Indianapolis, strains of H. virescens (Harold and Ottea, 1997). IN) were donated by respective manufacturers. Knowledge and biochemical and physiologi- Acrylamide, ethylenediaminetetraacetic acid cal mechanisms of resistance is essential for the (EDTA) and tris(hydroxymethyl)aminomethane rational development and effective implementa- (Tris) were purchased from Gibco BRL (Grand tion of insecticide resistance management (IRM) Island, NY). N,N,N′,N′-tetramethylethylene-di- strategies (Brown and Brogdon, 1987). Since the amine (TEMED), bis acrylamide, ammonium success of IRM relies on the ability to diagnose presulfate and Coomassie Brilliant Blue R-250 the nature, frequency, and evolution of insecti- were obtained from Amresco (Solon, OH). Bovine cide resistance in the target population, rapid and serum albumin (fraction 5), acetylthiocholine io- sensitive techniques to detect resistance mecha- dide (ATChI), 5,5′-dithio-bis(2-nitrobenzoic acid) nisms are needed (ffrench-Constant and Roush, (DTNB), Fast Blue RR salt, β-naphthyl acetate 1990). Although results of biological assays pro- (β-NA, and α-NA were purchased from Sigma vide verification that resistance is present in field Chemical Company (St. Louis, MO). Fast Blue populations, they fail to provide information about B salt and potassium chloride (KCl) were pur- Resistance Detection in H. virescens 49 chased from Aldrich Chemical Company (Milwau- generations without selection (OPR F3) and were kee, WI). either selected with profenofos (15.3 µg/larva; OPR F4S) or reared without exposure to insecti- Insects cide (OPR F4). Fifth stadium larvae (day 1) from Results of biological and biochemical assays each of the strains were used for biochemical (n with field-collected insects were compared with = 30) and biological (n ≥ 30) assays. those from laboratory strains of OP-susceptible and -resistant H. virescens. For studies with field Biological Assays collections, eggs and larvae (150–250) were col- Susceptibility of H. virescens to profenofos lected from a domesticated stand of velvetleaf, was measured in fifth stadium (day 1) larvae fol- Abutilon theophrasti Medicus, at the Louisiana lowing application of 1 µl of acetone containing State University Agricultural Center’s Northeast varying doses of profenofos onto the thoracic dor- Research Station/Macon Ridge location (MRS; sum. The dose-mortality response of larvae was Winnsboro, LA) on June 12, 1997 (MRS Jun). measured with at least 5 doses of profenofos (≥10 Other field collections were made on August 22 larvae/dose) and replicated thrice. Treated larvae and 25, 1997, from a cotton field near Bayou Ma- were held in 1-oz cups with diet and maintained con, LA (By Mac) that had been treated with three at 27°C, 70 ± 5% relative humidity, and a photo- applications of spinosad, two applications each of period of 14:10 (light:dark) hr. Mortality was re- cypermethrin and profenofos and one application corded 72 hr following topical application using of thiodicarb. Portions of leaves containing eggs absence of coordinated movement within 30 sec and larvae were collected and transported to the after being prodded with a pencil as the crite- laboratory in styrofoam coolers containing ice and rion. Data were analyzed by probit analysis placed in 1-oz cups containing a pinto bean–based (Finney, 1971) using a microcomputer-based pro- semi-synthetic diet (Leonard et al., 1988). Field- gram (SAS, 1985). Frequencies of resistance to collected larvae were identified as H. virescens profenofos in larvae (n ≥ 30) from laboratory and based on the presence of spinose cuticle and cha- field-collected strains were measured 72 hr fol- lazas 1 and 2 on abdominal segments 1, 2, and 8, lowing topical application of a single dose of and a molar area on the oral surface of the man- profenofos (15.3 µg/larva; approximately 20× the dible (Oliver and Chapin, 1981). Larvae were LD50 for LSU insects). This dose was high enough separated following head capsule slippage at the to kill all susceptible larvae but low enough to end of the fourth stadium, and fifth stadium (day allow differences in frequencies among the resis- 1) insects (180 ± 20 mg) were selected for biologi- tant strains to be resolved. cal and biochemical assays. Adults were reared in 3.8-l cardboard cartons covered with cotton Tissue Preparation gauze as a substrate for oviposition and provided Tissue homogenates from individual larvae with sucrose (10% in water) as a carbohydrate were used as enzyme sources for all biochemical source. Both larvae and adults were held at 27°C, assays. Individual larvae were weighed, decapi- 70% relative humidity, and a 14:10 hr (light:dark) tated, dissected, and the digestive system was re- photoperiod. moved. The opened hemocoel was rinsed with Larvae from two laboratory strains were ice-cold buffer (0.1 M sodium phosphate, pH 7.0) studied. The reference susceptible strain, LSU-S, and fat bodies were obtained by gentle scraping was established from field collections from cot- and aspiration using a Pasteur pipette. Fat bod- ton in 1977 (Leonard et al., 1988) and has been ies were homogenized in an all-glass homogenizer reared in the laboratory without intentional ex- containing 200 µl of ice-cold 1.15% KCl (contain- posure to insecticides. A resistant laboratory ing a few crystals of phenylthiourea). Individual strain (OPR) was originally established by select- heads were homogenized in 200 µl of 0.1 M so- ing larvae from a field-collection made at the Red dium phosphate buffer (pH 8.0) containing 0.1% River (RR) Research Station (Bossier City, LA) Triton X-100. Homogenates were centrifuged at on August 23, 1995 (Harold and Ottea, 1997). Lar- 12,000g for 15 min. The resulting supernatants vae from the OPR strain were reared for three were filtered through glass wool then held in ice 50 Harold and Ottea and used in enzyme assays within 30 min of origin to the center of the band by the migration preparation. distance of the bromophenol blue tracking dye from the origin. Biochemical Assays A “squash assay” was performed on filter Activity of esterases towards α-NA was mea- paper based on earlier methods (Pasteur and sured using the assay of Gomori (1953) with modi- Georghiou, 1989; Abdel-Aal et al., 1990) with fications (van Asperen, 1961; Grant et al., 1989; modifications. Individual second stadium larvae Ibrahim and Ottea, 1995). Reaction mixtures in (approximately 10–20 mg) were homogenized in individual wells of a microtiter plate contained a 1.5 ml microcentrifuge tube (cut open at the 240 µl of substrate solution (2.13 mM and 0.56%, distal end) using a pencil tip capped with a 0.5 final concentrations of α-NA and Fast Blue B, re- ml microcentrifuge tube onto a Whatman no. 3 spectively), and 10 µl of fat body homogenate (con- filter paper moistened with phosphate buffer (0.1 taining 0.05 tissue equivalent; 0.016 ± 0.007 mg mM; pH 7.0). The filter paper was incubated atop protein). The substrate solution was prepared by a second filter paper containing α-NA/Fast Blue adding 600 µl of α-NA (0.113 M dissolved in 50% substrate solution (prepared as described above) acetone in water) to a solution of Fast Blue B for 60 sec in darkness. The reaction was stopped salt (18 mg in 30 ml of 0.1 M phosphate buffer, by rinsing the filter paper with water followed pH 7.0), and then filtered using Whatman no. 3 by the addition of 5% acetic acid. Esterase activi- filter paper. Reaction mixtures were incubated at ties were visualized as violet strains on the 30°C, and the rate of change in absorbance dur- undersurface of the filter paper, which were com- ing the initial 10 min was measured at 595 nm pared with a color scale prepared with α-naph- using a Thermomax microplate reader (Molecu- thol standards (0–1.8 µmol). lar Devices, Palo Alto, CA). Data were corrected Sensitivity of AChE to chlorpyrifos oxon was for non-enzymatic activity using incubations with- measured using head homogenates from indi- out protein as the control. Changes in OD were vidual larvae following the method of Ellman et converted to nmol/min using an experimentally al. (1961) with modifications (Moores et al., 1988; –1 derived “extinction coefficient” (3.825 mM 250 Byrne and Devonshire, 1991, 1993; Ibrahim and –1 µl ) for the α-naphthol-Fast Blue B conjugate at Ottea, 1995). As profenofos in an indirect inhibi- 595 nm. tor of AChE (Wing et al., 1998; Byrne and Devon- Native PAGE was used to visualize EST shire, 1993), it could not be used for these studies. from individual larvae using a vertical electro- Homogenates of individual heads (30 µl contain- phoresis unit (Hoefer Scientific Instruments, San ing 0.15 tissue equivalent; 0.070 ± 0.083 mg pro- Francisco, CA) and 5% acrylamide (Sambrook et tein) were incubated for 10 min at 30°C in wells al., 1989; Gunning et al., 1996). The protein con- of a microplate containing 69 µl of sodium phos- centrations of homogenates from individual fat phate buffer (0.1 M, pH 8.0) and 1 µl of chlor- bodies were adjusted to contain 30 µg protein in pyrifos oxon (148.8 nM final concentration). The 40 µl, which was loaded onto a gel with 5 µl of 6× concentration of chlorpyrifos oxon used in these tracking dye (0.25% Bromophenol Blue and 40% studies was the I90 for the LSU strain as deter- sucrose w/v in water). Electrophoresis occurred mined in preliminary experiments (data not in Tris-borate/EDTA buffer (100 mM Tris, 2.4 mM shown). Following preincubation, reactions were EDTA, and 100 mM boric acid, pH 8.0) at a con- initiated by adding 200 µl of ATChI/DTNB (0.5 stant voltage (150 V) until the dye marker was mM/0.05 mM, final concentration), and the rate within 1 cm of the gel base. After electrophore- of change in OD at 405 nm at 30°C was recorded. sis, gels were stained in darkness at 25°C with Data were corrected for nonenzymatic activity us- 100 ml of 0.1 M sodium phosphate buffer (pH 7.0) ing incubations without protein as the control and containing 0.5 mM α- and β-NA and 0.2% Fast expressed as miliOD min–1 mg protein–1. Activi- Blue RR salt for 30 min. Gels were destained in ties of AChE measured in the absence of chlorpy- distilled water and fixed in 5% acetic acid. Rela- rifos oxon served as the control. Percentage tive mobility (Rm) was calculated by dividing the inhibition was calculated as 1-(activity with in- migration distance of the specific band from the hibitor/activity without inhibitor) × 100. Resistance Detection in H. virescens 51

TABLE 1. Profenofos Susceptibility in Larvae From Laboratory and Field-Collected Strains of H. virescens† Percentage survival 2 Strain N LD50 (95% CL) Slope ± SE Resistance ratio χ @ diagnostic dose LSU 392 0.73 (0.65–0.81)c 3.72 ± 0.32 — 3.34 0.00 OPR 213 22.8 (18.8–27.5)a 2.46 ± 0.30 31.2 3.42 67.7 OPR F3 180 8.60 (6.78–10.6)b 2.44 ± 0.30 11.7 1.57 26.8 OPR F4 184 7.08 (5.50–8.86)b 2.23 ± 0.28 9.70 1.97 23.3 OPR F4S 241 16.0 (13.1–19.1)a 2.44 ± 0.28 22.0 0.85 53.3 MRS Jun 30 ND ND ND ND 60.0 By 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, not determined. *Expressed as µg/larva; values followed by the same letter are not statistically different (Tukey’s; P = 0.05).

Protein concentrations were measured by the tween OPR F3 to OPR F4, but this difference was method of Bradford (1976) with bovine serum al- not statistically significant. However, following bumin (fraction V; concentrations corrected for selection of OPR F3 larvae with profenofos, re- impurities) as the standard. Data were subjected sistance frequency increased to 53% in OPR F4S. to analysis of variance followed by Tukey’s mul- High frequencies of profenofos resistance also tiple comparison test (P = 0.05) using a micro- were recorded in field-collected insects (60 and computer-based program (SAS, 1985). Linear 97% in MRS Jun and By Mac, respectively). regressions between enzyme activities and sus- Esterase Activities ceptibility to profenofos were estimated using the method of least squares. Activities of ESTs were higher in larvae from both laboratory-selected and field-collected insects than in the susceptible LSU strain (Table 2). The RESULTS highest mean EST activity (294 nmol α-naphthol Profenofos Resistance formed min–1 mg protein–1) was expressed in lar- Profenofos resistance in laboratory-resistant vae from the OPR strain. Mean EST activity de- strains was statistically higher than in the susceptible LSU strain (Table 1). The highest level of resistance to profenofos (31-fold relative to TABLE 2. Enzyme Activities and Sensitivity of AChE to Inhibition by Chlorpyrifos Oxon in Field-Collected LSU) was expressed in OPR larvae (LD50 = 22.8 and Laboratory Strains of H. virescens* µg/larva). In the absence of selection with profen- EST AChE AcHE ofos, levels of resistance decreased significantly Strain (SD) activity (SD) inhibition (SD) µ to 11.8-fold (LD50 = 8.6 g/larva) after 3 genera- LSU 71.6 (38.1)c 0.25 (0.05)a,b 89.8 (2.72)a a a d tions (OPR F3) and to 9.7-fold (LD50 = 7.1 µg/ OPR 294 (127) 0.33 (0.16) 69.6 (14.8) OPF F3 88.8 (52.3)c 0.20 (0.18)b,c 78.0 (12.0)b,c larva) after 4 generations (OPR F4). Resistance b,c c,d,e a µ OPR F4 106 (44.5) 0.16 (0.06) 88.3 (4.79) increased significantly to 22-fold (LD50 / 16.0 g/ OPR F4S 163 (49.5)b 0.12 (0.05)d,e 87.4 (5.83)a larva) following selection of OPR F3 larvae with MRS Jun 167 (104)b 0.20 (0.09)b,c,d 74.9 (11.4)c,d profenofos (OPR F4S). By Mac 237 (105)a 0.10 (0.04)e 83.2 (10.7)a,b Frequencies of resistance to profenofos (as *Activities were measured in assays with individual larvae (n determined in topical bioassays with 15.3 µg ≥ 30) from laboratory-susceptible (LSU), -resistant (OPR), and profenofos/larva) were higher in larvae from the field-collected (MRS: Macon Ridge, By Mac: Bayou Macon) strains of H. virescens. Values followed by the same letter are field-collected and the laboratory-selected strains not significantly different (Tukey’s; P = 0.05). Mean EST ac- than the susceptible LSU strain (Table 1). A high tivities (±SD) expressed as nmols of α-naphthol formed min–1 –1 frequency of resistance was measured for OPR mg protein . Mean AChE activities (±SD) toward ATChI expressed as miliOD min–1 mg protein–1. Mean of percent inhibi- larvae (68%) but decreased to 27% in OPR F3 lar- tion of AChE (±SD) measured following preincubation of head vae. Resistance frequency decreased further be- homogenates with chlorpyrifos oxon for 10 min. 52 Harold and Ottea creased in the absence of selection to 88.8 nmol served in the field strains with activities greater in OPR F3 larvae, but increased significantly (1.8- than the LSU strain measured in 62 and 87% of fold) in OPR F4S larvae following selection with the individuals from MRS Jun and By Mac, re- profenofos. In assays with field strains, larvae spectively (Fig. 2). from By Mac expressed EST activity that was comparable with that of OPR larvae. The EST Electrophoretic and Filter Paper Analysis activity in MRS Jun larvae was intermediate rela- Qualitative and quantitative variation in tive to LSU and OPR larvae, and did not differ banding patterns of ESTs in electrophoretic gels significantly from that of OPR F4S. was observed between OP-susceptible and -re- Whereas mean levels of EST activity did not sistant larvae. Staining of ESTs appeared more differ among the OPR F3, F4, and LSU strains, intense with resistant larvae than those from differences in frequency profiles for this activity the susceptible strain (Figs. 3–5). In addition, in these strains were apparent (Fig. 1). The fre- a band (designated A′; Rm = 0.65) was expressed quency distribution of LSU larvae was narrow in some OPR larvae, but not in larvae from the with no individuals expressing activity >90 nmol LSU strain (Fig. 3). In most cases, the appear- min–1 mg protein–1. In contrast, the distribution ance of this band coincided with the decreased of EST activity was broad in larvae from OPR, expression of a second band (designated A) with and 97% of the individuals expressed activities Rm = 0.68. In addition, after selection of OPR greater than that of LSU larvae. In the absence larvae with a high dose of profenofos (OPR of selection (OPR F3), the frequency profile nar- F4S), expression of band A′ (and underexpres- rowed, and only 20% of the larvae expressed ac- sion of band A) was measured in 100% of the tivity >90 nmol. In profenofos-selected OPR F4S individuals tested (Fig. 4). Finally, this pattern larvae, increased frequencies of individuals ex- (i.e., overexpression of A′ and underexpression pressing high EST activity were detected: 83% of of A) was observed in gels with the majority of individuals expressed activities greater than 90 field-collected larvae from MRS Jun (67%) and nmol. Finally, broad frequency profiles were ob- By Mac (87%) (Fig. 5).

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

Quantitative differences in EST activity be- of α-naphthol. In contrast, intense staining was ob- tween LSU and OPR F4S larvae were also appar- served with homogenates from OPR F4S larvae and ent in a filter paper assay using individual, second amounts of α-naphthol produced were > 0.8 µmol stadium larvae (Fig. 6). The color developed using for 14 of the 15 homogenates. susceptible larvae ranged from no color to moder- ate violet color, and staining was more intense with Activity of AChE and Sensitivity to Inhibition by homogenates from resistant than susceptible lar- Chlorpyrifos Oxon vae. In individual homogenates from 15 LSU lar- Activities of AChE from individual larvae vae, intensity of staining corresponded to < 0.8 µmol were variable in both laboratory and field-col-

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. 54 Harold and Ottea

not differ between OPR F4 (unselected) and OPR F4S (selected). The lowest activity was measured in the field-collected By Mac strain. Sensitivity of AChE (expressed as percent inhibition following incubation with chlorpyrifos oxon) was significantly lower in larvae from the OPR strain than from the LSU strain (Table 2). Sensitivity increased significantly in the absence of selection for both the OPR F3 (78%) and OPR F4 (88%) strains. However, as with AChE activity, there were no differences in AChE sensitivity between OPR F4 and OPR F4S larvae. Reduced sensitivity of AChE also was measured in field-collected MRS Jun insects (75%), but there was no significant difference in AChE sensitivity between By Mac (83%) and LSU (90%) larvae. Frequency profiles for AChE inhibition also Fig. 4. Polyacrylamide gel stained for EST activity in individual larvae from profenofos-susceptible (LSU) and -selected reflected differences among strains (Fig. 7). The (OPR F4S) strains of H. virescens. Each lane contains 30 sensitivity of AChE in LSU larvae was normally µg of protein from fat body homogenates of individual, fifth distributed with more than 88% inhibition mea- stadium (day 1) larvae. sured in 97% of individuals. For OPR and OPR F3 strains, 80 and 30% of individuals, respec- lected strains (Table 2). The highest activity (0.33 tively, expressed AChE with decreased sensi- miliOD min–1 mg protein–1) were measured in tivity (i.e., <88% inhibition). Broad frequency OPR larvae. Activities decreased significantly in profiles of AChE sensitivity were measured with the absence of selection to 0.20 and 0.16 miliOD larvae from the field-collected strains, and de- min–1 mg protein–1 in the OPR F3 and F4 strains, creased sensitivity of AChE was expressed in respectively. Although activity was significantly 67 and 27% of individuals from MRS Jun and lower in OPR F4S than LSU larvae, activities did By Mac, respectively.

Fig. 5. Polyacrylamide gel stained for EST activity in individual larvae from field-collected strains of H. virescens. Each lane 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 larvae from susceptible (LSU) and resistant (OPR F4S) strains of H. virescens. Bottom: Color scale developed with known concentration 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 of individual, fifth stadium larvae (day 1; n = 30) following 10 min preincubation with 149 nM chlorpyrifos oxon. 56 Harold and Ottea

DISCUSSION increased following one generation of selection Biochemical assays, including those with (i.e., from OPR F3 to OPR F4S). Finally, frequen- model substrates, can be utilized successfully for cies of profenofos resistance and EST activities detecting and monitoring insecticide resistance in were significantly higher in two field populations insect populations (Brown and Brogdon, 1987; of H. virescens than in the LSU strain. Devonshire, 1987). Such knowledge of the nature In addition to elevated EST activities, a of resistance mechanisms may serve as a foun- unique EST was expressed in larvae from pro- dation for the development of field kits to diag- fenofos-resistant laboratory colonies, and the fre- nose metabolic resistance in field populations of quency of expression increased following selection this pest. This would enable growers to ascertain with profenofos. Further, this EST was detected the resistance status of H. virescens prior to in- in field-collected strains and was expressed in 67 secticide application, and to choose effective in- and 87% of the individuals from MRS Jun and secticides for control of this pest. The basis for By Mac, respectively. Corresponding frequencies the present study is a correlation reported be- of resistance measured in these strains were 60 tween EST activities and frequencies of profenofos and 97% for MRS Jun and By Mac, respectively. resistance in an array of resistant field popula- Since detoxifying enzymes exist in multiple forms tions and laboratory strains of H. virescens (Schoknecht and Otto, 1989), the mean interstain α (Harold and Ottea, 1997). difference in total activity with -NA might over- Elevated EST activities toward model sub- lap between susceptible and resistant strains strates have been associated with OP resistance (Devonshire, 1989), masking the expression of a in a number of insect pests (Abdel-Aal et al., resistance-associated esterase. However, results 1993). In previous studies, elevated activities of from the present study suggest that such es- ESTs toward permethrin, α-NA (Dowd et al., terases can be accurately and rapidly detected in 1987) and methyl parathion (Konno et al., 1989, individual larvae using electrophoretic separation. 1990) have been documented in larvae from labo- In a similar study, discrimination between sus- ratory strains of OP-resistant H. virescens. More ceptible and moderately resistant aphids (whose recently, EST activities toward α-NA from mass total activity overlapped with that of susceptible homogenates of thiodicarb-selected H. virescens aphids) was made possible by electrophoretic were suggested to be responsible for cross-resis- separation of esterase-4 (Sawicki et al., 1978). tance among carbamate, OP, and pyrethroid in- Since estimations based on intensity of electro- secticides (Goh et al., 1995; Zhao et al., 1996). In phoretic bands are subjective, electrophoresis may the present study, a high level of resistance to best be viewed as a complementary tool with spec- cypermethrin (17-fold at the LD50) was measured trophotometric determinations of total EST ac- in OPR larvae (data not shown), suggesting that tivities (Devonshire and Field, 1991). metabolic mechanisms expressed in this strain Although no ESTs from the profenofos-resis- may confer cross-resistance between cypermethrin tant larvae used for this study have been shown and profenofos. to metabolize insecticides directly, the consistent Results presented in this study provide in- expression of a unique enzyme in resistant indirect evidence for the role of ESTs in OP resis- sects suggests a potential role in profenofos re- tance in H. virescens and illustrate the utility of sistance in H. virescens and as a biochemical EST activity toward α-NA as a biochemical marker. In a similar study, Konno et al. (1990) marker for profenofos resistance in this insect. reported a unique EST (esterase III) that hydro- Frequencies of profenofos resistance in 21 field- lyzed methyl parathion in OP-selected H. vires- collected and laboratory-selected strains of H. cens. In the current study, elevated EST activity virescens were highly correlated (r2 = 0.90) with and electrophoretic banding patterns in resistant mean levels of EST activities toward α-NA (this H. virescens formed the basis for a preliminary study and Harold and Ottea, 1997). In addition, version of a “squash assay,” which has potential EST activity toward α-NA decreased in the ab- utility in the field to assess the contribution of sence of selection (i.e., from OPR to OPR F4) but ESTs to resistance in this pest. In the prelimi- Resistance Detection in H. virescens 57 nary assays reported here, staining was more in- has been approved for publication by the Direc- tense with homogenates from profenofos-resistant tor of the LAES as MS 99-17-0265. than -susceptible strains. Altered expression and sensitivity of AChE REFERENCES were measured in the OP-resistant strains. High Abdel-Aal YAI, Wolff MA, Roe RM, Lampert EP. 1990. Aphid frequencies (>50%) of the individuals tested from carboxylesterases: biochemical aspects and importance field-collected and resistant laboratory strains in the diagnosis of insecticide resistance. Pestic expressed both increased EST activity and altered Biochem Physiol 38:255–266. AChE (data not shown). Similar enhancement of Abdel-Aal YAI, Ibrahim SAE, Lampert EP, Rock GC. 1993. esterase-based insecticide resistance by insensi- Detection methodology of esterase-mediated insecticide tive AChE has been observed in the aphids, resistance from bioassay to biotechnology. 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