Examination of esterases from insecticide resistant and susceptible strains of the German cockroach, Blattella germanica (L.)

Pergamon

PIh S0965-1748(97)00023-4

Insect Biochem. Molec. BioL Vol. 27, No. 6, pp. 489-497, 1997 © 1997 Elsevier Science Ltd All rights reserved. Printed in Great Britain 0965-1748/97 $17.00 + 0.00

Examination of Esterases from Insecticide Resistant and Susceptible Strains of the German Cockroach, Blattella germanica (L.) MICHAEL E. SCHARF,*t JANET HEMINGWAY,~: GRAHAM J. SMALL,:~ GARY W. BENNETTt Received 1 November 1996; revised and accepted 6 March 1997

Esterases from insecticide resistant and susceptible Blattella germanica (L.) were examined biochemically. Two strains were utilized: Johnson Wax (JWax; susceptible), and Munsyana (MA; chlorpyrifos LDso and LD9s resistance ratio 5.2 and 10.0). On native polyacrylamide gel electrophoresis (PAGE), MA had four visible esterase electromorphs (El, E2, E3 and E4), whereas JWax had three (El, E2 and E4). Esterases E1 and E4 were more intense in the MA strain, and none of these esterase electromorphs were acetylcholinesterases. Insecticide inhibition of native esterases within polyacrylamide gels showed an interaction of all electromorphs with the carbamate insecticide propoxur and complete inhibition of all electromorphs by the carbamate bendiocarb and the organophosphate insecticides chlorpyrifos oxon, malaoxon and paraoxon. The pyrethroid insecticides permethrin and cypermethrin had no inhibitory effects. Sequential Q-Sepbarose and hydroxyapatite column chromatography was used to fractionate esterases from each strain into two groups (I and H). Following hydroxyapatite fractionation of these esterase groups, inhibition kinetic constants (k~ and k3), and molecular weights were estimated. Results for ka (the rate of enzyme inhibition) indicated a greater affinity for organophosphate insecticides by MA esterases. Results for k3 (the rate of enzyme recovery) indicated lengthened times of MA esterase-inhibition by organophosphate insecticides. Therefore the role, if any, in organophosphate resistance played by MA esterases must be by sequestration. Molecular weight estimates were within the range (55--65 kDa) previously observed for esterases from both B. germanica and Culex quinquefasciatus. © 1997 Elsevier Science Ltd Blatella germanica

Esterases

INTRODUCTION Esterases have diverse functions in insects, including proteolysis, nervous system function, hormone metabolism, and xenobiotic metabolism/sequestration (Aldridge, 1993). Studies of insecticide resistance have indicated the specific relevance of esterases with regard to xenobiotic metabolism in several insect species, including Blattella germanica. Recent studies of insecticide resistance in B. germanica have used colorimetric assays (i.e. hydrolysis of p-nitrophenyl or naphthyl esters) to identify elevated esterase activity (Siegfried *Author for correspondence. Tel.: + 1 317 494 8646; Fax: + 1 317 494 0535; E-mail: [email protected] tCenter for Urban and Industrial Pest Management, Department of Entomology, Purdue University, West Lafayette,IN 47907-1158, U.S.A. :~Departmentof Pure and Applied Biology,Universityof Wales Cardiff, P.O. Box 915, Cardiff, CF1 3TL U.K.

and Scott, 1992; Hemingway et al., 1993; Prabhakaran and Kamble, 1993, 1994; Anspaugh et al., 1994; Lee et al., 1994; Scharf et al., 1996). Native polyacrylamide gel electrophoresis (PAGE; Siegfried and Scott, 1992; Prabhakaran and Kamble, 1993, 1994; Scharf et al., 1996) and thin-agarose gel electrophoresis (Lee et al., 1994) have been used to identify esterase electromorphs with novel properties in resistant B. germanica. Prabhakaran and Kamble (1994) used native PAGE to separate, concentrate, characterize, and estimate molecular weights for esterases from resistant and susceptible strains of B. germanica. Whereas this method is effective for identifying the activity of single esterase electromorphs and estimating molecular weight, it is not useful for isolating the stable quantities of esterase necessary for kinetic characterizations. Devonshire (1977) purified the E4 esterase from a resistant strain of Myzus persicae by column chromatography. Column chromatography has also been used to isolate resistance-conferring ester-

489

490

MICHAELE. SCHARFet al.

ases from Culex quinquefasciatus (Ketterman et al., 1992; Karunaratne et al., 1993); Myzus nicotinae (Wolff et al., 1994); Schizaphis graminum (Siegfried and Zera, 1994); and Nilaparvata lugens (Chen and Sun, 1994). Additionally, Prabhakaran and Kamble (1995) purified three esterase electromorphs from B. germanica and found one (E6) to be potentially responsible for resistance via sequestration rather than hydrolysis. The present study was undertaken to determine the nature of organophosphate resistance in a field-collected, multi-resistant strain of B. germanica from the U.S.A. In this study, we have used electrophoretic, chromatographic, and kinetic techniques to characterize nymphal esterases from susceptible and resistant B. germanica strains. MATERIALS AND METHODS

Chemicals

fuged at 15,000g for 15 min and 4°C. The pellet was discarded and a volume of supernatant containing 270/~g protein (approximately 3/~1) was mixed with 3/xl of marker (bromophenol blue + xylene cyanol in sucrose) and loaded onto a 7.5% polyacrylamide gel within a BioRad Protean Mini Gel System (Richmond, CA, U.S.A.). Electrophoresis occurred in electrode buffer (containing 100 mM Tris, 2.4 mM ethylene diamine tetraacetic acid (EDTA), and 100 mM boric acid; pH 8.0) and initially lasted for 5 min at 150 V. The wells of the gel were flushed and electrophoresis continued under identical conditions until the marker ran to within 1 cm of the gel base. After electrophoresis, the gels were incubated in 100 ml of 200 mM phosphate buffer (pH 6.8) containing 0.45 mM 1-naphthyl acetate (NA), and 0.45 mM 2-NA. After 5 min, 25 mg Fast Blue BN salt (in 1.0 ml water) was added to visualize esterase bands. Approximately 5 min after adding Fast Blue BN, the gels were removed and fixed in 10% acetic acid.

All chemicals were purchased from Sigma unless stated otherwise. Malaoxon, paraoxon, propoxur, and permethrin were purchased from British Greyhound (Birkenhead, U.K.). Chlorpyrifos oxon was provided by DowElanco Inc. (Indianapolis, IN, U.S.A.), cypermethrin was provided by Zeneca Inc. (Richmond, CA, U.S.A.), and bendiocarb was provided by Nor-Am Inc. (Wilmington, DE, U.S.A.).

For inhibition studies, 100 mM of each insecticide was prepared in 1.0 ml acetone. After electrophoresis, native gels were removed from electrophoresis units and placed in 100 ml sodium phosphate buffer (200 mM, pH 6.8) containing 1.0 ml (1 mM final concentration) of a single inhibitor solution. After 5 min, NA solutions were added, and 5 min later the gels were stained as described above.

Insects

Native PAGE of acetylcholinesterase

Two strains of B. germanica (second-fourth instar) were used in this study. The Johnson Wax (JWax) strain of B. germanica is considered susceptible to insecticides (Koehler and Patterson, 1986). JWax has previously been shown to possess elevated levels of esterase activity over another susceptible strain (Prabhakaran and Kamble, 1993), however, that was a reproductively isolated population from that which we have employed here. The Munsyana (MA) strain of B. germanica was collected in 1994, is resistant to organophosphate and pyrethroid insecticides, and possesses elevated levels of esterase activity and increased cytochrome P450 monooxygenase levels (Scharf et al., 1997). Shortly after collection from the field and 3 months before the initiation of this study, topical application resistance ratios of chlorpyrifos for the MA strain (over JWax) were 5.2 at LDso and 10.0 at LD95 (Scharf et al., 1997). Two years subsequent to this study, levels of chlorpyrifos tolerance had not changed significantly in the MA strain (Scharf and Bennett, unpublished results). Such levels of tolerance (> 10-fold) are considered to be associated with control failures under field conditions (Ballard et al., 1984; Rust and Reierson, 1991).

To view acetycholinesterase banding patterns, the method of Karnovsky and Roots (1964) was used. After preparing and running a native gel as previously described, the gel was placed in 100 ml of a potassiumphosphate buffer solution (6.0 mM, pH 6.0) containing 0.3 mM acetylthiocholine iodide, 1.2 mM sodium citrate, 0.7 mM copper sulfate, and 0.1 mM potassium ferricyanide. Gels were removed from the staining solution after sufficient visualization had occurred (approximately 60 min) and fixed in a solution of 10% acetic acid.

Inhibition of esterase electromorphs

Protein assay All protein concentrations were determined using the method of Bradford (1976) in a UVmax kinetic microplate reader (Molecular Devices, Sunnyvale, CA). Bovine serum albumin (made from 1 mg/ml stock in water) was used as a standard. Protein samples (10/xl) and Bio-Rad protein reagent (300/zl; prepared as specified by the manufacturer) were mixed in microplate wells. After 5 min the absorbance was measured at 570 nm and converted to protein concentration by comparison to a standard curve. Esterase activity assays

Native PAGE of esterases For native PAGE, 25 frozen fourth instar cockroaches of each strain were homogenized in 9.0 ml of ice cold 200 mM sodium phosphate buffer (pH 7.6) and centri-

Esterase activity for all protein fractions and crude homogenates was measured using the substrate p-nitrophenyl acetate (PNPA). Stock solutions of PNPA were prepared in acetonitrile at concentrations of either

B. GERMANICA

100 mM (for purification assays) or 200 mM (for inhibition assays). Protein sample (10/zl) and 200/xl of 1 mM PNPA solution (prepared by diluting PNPA stock solution in 500 mM sodium phosphate buffer; pH 7.4) were mixed in microplate wells. The rate of hydrolysis of PNPA was monitored for 2 min at 405 nm in a UVmax kinetic microplate reader. Absorbance was converted to /xmol product/min/mg protein using a molar extinction coefficient of 6.53 mM -~, which was corrected for a path length of 0.6 cm (measurement provided by manufacturer).

Partial purification of esterases Esterases were purified using sequential column chromatography at 16°C. Separate preparations were used from those described previously for native PAGE. German cockroach nymphs (fourth instar; JWax wet weight 1.68 g; MA wet weight 2.40 g) were homogenized in 10 ml of 20 mM bis-tris propane buffer (BTP; pH 7.0 and conductivity 2.0 mS/cm) containing 25 mM dithiothreitol (DTT). Homogenates were centrifuged at 10,000g for 5 min, supernatants were filtered through a Whatman no. 1 filter paper (Maidstone, U.K.), and adjusted to a pH and conductivity identical to that of the homogenization buffer. Q-Sepharose chromatography. Supematant was applied to a Q-Sepharose Fast Flow column (2.2 × 7.0 cm, Pharmacia, U.K.) equilibrated with homogenization buffer. The column was washed with six bed volumes of homogenization buffer, followed by a 10-bed volume gradient of increasing sodium chloride concentration (0-500 mM in BTP buffer) in which the esterase activity eluted. Protein concentration and esterase activity were determined for fractions eluting from the column as described above. Esterase activity eluted in two main peaks (identified as groups I and II). Hydroxyapatite chromatography. Hydroxyapatite columns (Bio-Rad, U.K.) were used to fractionate esterases further from Q-Sepharose groups I and II. Hydroxyapatite columns (1.5 x 5 cm) were equilibrated with 20 mM BTP buffer containing 500 mM sodium chloride (pH 7.0) and 10 mM dithiothreitol (DTI'). Before loading, the QSepharose fractions were raised to a sodium chloride concentration of 500 mM and pH 7.0. After sample loading, the columns were washed with five-bed volumes of equilibration buffer. Esterase activity was eluted with a five-bed volume gradient of 10-200 mM sodium phosphate buffer (pH 7.0) containing 10 mM DTT. The group I and group II esterases were run individually, and both eluted as single peaks of esterase activity which were well separated from the general protein peaks. Fractions with high activity were pooled and concentrated in Amicon Centriprep 10 concentrators (Amicon Inc., Beverly, MA, U.S.A.). The group I and group II concentrated fractions were stored in 50% (v/v) glycerol containing 25 mM DTT. Identity of the partly purified esterases within each group was confirmed by native PAGE

ESTERASES

491

as previously described, with 150 ~g (group I) and 90/xg (group II) of protein being loaded per lane.

Determination of kinetic constants (ka and k3) Kinetic constants were determined for each of four partly purified samples: JWax groups I and II; and MA groups I and II. Kinetic constants were determined on the basis of the model describing inhibition and re-activation of esterases after exposure to organophosphate and carbamate insecticides (Aldridge and Reiner, 1972). The bi-molecular rate constant (k,) gives a measure of the rate of binding of the insecticide to the esterase active site, whereas the rate of enzyme reactivation is measured by k~. On the basis of the lack of interaction of these esterases with pyrethroids in native PAGE (permethrin and cypermethrin), only malaoxon, paraoxon, and propoxur were utilized in kinetic assays. ka was determined by pre-incubating esterases with inhibitors at 0.5, 1.5, and 2.5 min before determining activity in the presence of the substrate PNPA. Inhibitors were dissolved in acetonitrile and assay concentrations varied (malaoxon and propoxur 0.20/xM, and paraoxon 1 x 10 4p, M). Percentage remaining activities were determined in comparison to the uninhibited enzyme at each time point, as described by Aldridge and Reiner (1972). For each of four replicates at each pre-incubation time, ko was determined and all results were then pooled to give mean k, ( + SE). Paraoxon inhibited the cockroach esterases too rapidly to determine k~ accurately as previously described. As a result, ka values for paraoxon were determined in the presence of the substrate (PNPA) using the method of Main and Dauterman (1963). To determine the reactivation time for these esterases, k3 data was obtained by using the rate of PNPA hydrolysis at 0.5, 2.5, and 4.5 h after a 15 min pre-incubation with inhibitor. Before measurement, unbound insecticide and the enzyme-insecticide complex were separated using Nick-Spin columns (Pharmacia, U.K.) after the manufacturers' instructions. The slope of the line which plots time vs log percentage remaining activity in comparison to uninhibited enzyme at each time point gave the reactivation constant k3 (in molecules recovered per hour). For k3 determinations, differing concentrations of malaoxon and propoxur (0.20/xM), vs paraoxon (0.0125 p,M) were used. Blanks contained acetonitrile alone and four replicates were used per esterase groupinhibitor combination.

Estimation of molecular weight Estimates of native molecular weight for cockroach esterases in groups I and II were obtained by running samples of the partly purified esterases and Sigma standard proteins (14.2-54.5 kDa) on pre-cast Bio-Rad MiniProtean II gradient acrylamide gels (4-20%). Gels were run and cut so that esterases could be visualized as previously described, and standard proteins visualized by staining with Coomassie Blue R250. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-

492

MICHAEL E. SCHARF et al.

PAGE) was performed using pre-stained Bio-Rad standard proteins (18.5-10.6kDa) on pre-cast Bio-Rad gradient acrylamide gels (4-20%) by the method of Laemmli (1970). In SDS-PAGE, Coomassie Blue R250 stain was used to visualize proteins. For each native PAGE and SDS-PAGE gel, log molecular weight vs the relative mobility of each standard was plotted to obtain a regression line. The molecular weights of esterases were determined using their relative mobility in comparison to the standards. RESULTS AND DISCUSSION

Native PAGE

Results of native PAGE of esterases from susceptible and resistant, fourth instar B. germanica are shown in Fig. 1. Three esterase electromorphs from both B. germanica strains were visible (i.e. El, E2 and E4). Two electromorphs stained much more intensely in the MA strain (El and E4) whereas one electromorph (E2) appeared only in the MA strain. These results indicate increased catalytic activity, increased quantity of enzyme, or a combination of each for these electromorphs in the MA strain (as concluded for several electromorphs in the Muncie '86 strain; Scharf et al., 1996). These electromorph patterns are similar to patterns reported previously which were obtained using adult females (Lee et al., 1994) and first instar nymphs (Scharf et al., 1996), but different from those observed using adult males (Siegfried and Scott, 1992; Prabhakaran and Kamble, 1993). Polymorphism was reported previously from mass homogenates of adult male cockroaches (Siegfried and Scott, 1992; Prabhakaran and Kamble, 1993, 1994) but not with mass homogenates of first instar

A JW

B MA

JW

C MA

JW

MA

E1 E2 E3 E4

FIGURE 1. Native PAGE results of: (A) uninhibited; (B) propoxur inhibited; (C) chlorpyrifos oxon, malaoxon, paraoxon or bendiocarb inhibited esterases from B. germanica. Identities for figure captions are: JW, Johnson Wax strain (susceptible); MA, Munsyana strain (resistant); and El, E2, E3 and EA, esterase electromorphs. See text for methods.

nymphs (Scharf et al., 1996). Native acetylcholinesterase gels (not shown) indicate that none of the esterases viewed in Fig. 1 are acetylcholinesterase. On native gels, acetylcholinesterases are less mobile than the uppermost electromorphs shown in Fig. 1. Inhibition of esterase electromorphs

Results of native esterase gel inhibition are also shown in Fig. 1. The inhibitors examined included two pyrethroid insecticides (permethrin and cypermethrin), two carbamate insecticides (bendiocarb and propoxur), and three organophosphate insecticides (chlorpyrifos oxon, malaoxon and paraoxon). Neither permethrin nor cypermethrin had inhibitory effects on esterases from either cockroach strain (gels not shown). Propoxur partly inhibited all electromorphs whereas bendiocarb and all three organophosphate insecticides completely inhibited all electromorphs. As all esterase electromorphs interacted with the organophosphate analogues examined, sequestration by the three electromorphs (El, E2 and E4) showing elevated activity in the MA strain may be a factor in chlorpyrifos resistance. Partial purification of esterases

Sequential column chromatography was used partly to purify esterases from the susceptible (JWax) and resistant (MA) strains of B. germanica. Q-Sepharose (ionexchange) columns were used to separate esterases into two groups (referred to as I and II). Hydroxyapatite columns were then used to further fractionate esterases from groups I and II (separately). Q-Sepharose elution profiles for the JWax and MA strains are shown in Fig. 2(A) and (B), respectively. QSepharose elution profiles were different for each strain, with JWax having two major peaks and MA having three major peaks of eluting activity. However, for each strain, esterases eluted from the Q-Sepharose column in opposite order of their native PAGE mobility. For JWax, each of the two main peaks were pooled separately for further fractionation by hydroxyapatite chromatography. For MA, the two peaks eluting in fractions 30-55 were pooled together as one group, separately from the second (later eluting) group. These two initial MA peaks were later determined to contain separate electromorphs (El and E2). Hydroxyapatite elution profiles for each B. germanica strain are shown in Fig. 3(A) and (B) (group I esterasesl and Fig. 4(A) and (B) (group II esterases). Esterases eluted from hydroxyapatite columns as a single peak which was separate from the majority of other proteins. Identities of esterases from each group, fractionated by Q-Sepharose and hydroxyapatite chromatography, are shown in comparison to crude homogenate in Fig. 5. Equivalent amounts of protein were loaded per lane for each of the esterase groups (group I 150/xg; group I1 90/xg). Table i shows the purification table for the partial purifications executed in this study. Crude homogenates

493

B. GERMANICA ESTERASES Esterase Activity .......

Protein'Content

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FIGURE 2. Q-Sepharose elution profiles for esterases from (A) the Johnson Wax (susceptible) and (B) the Munsyana (resistant) strains of B. germanica. See text for methods.

from the MA strain had a specific activity (in /xM pnitrophenol produced/min/mg protein) of 7.63 whereas the value for JWax was 2.02, equating to a 3.8-fold elevation in the MA strain. The rank (and specific activity) of esterase groups after hydroxyapatite purification is: MA group II (67.1) > MA group I (14.5) > JWax group I (12.42) > and JWax group II (4.06). In Fig. 5, the E4 electromorph which appears in MA group II is the probable cause of the elevated specific activity of this group. Increased catalytic activity resulting from a change in

amino acid sequence, increased expression, gene amplification, or a combination of these are possible causes for the elevation of activity in the MA strain.

Kinetic constants Values of kinetic constants (i.e. ka and k3) for group I and II esterases from the JWax and MA strains are shown in Table 2. Values of the bi-molecular rate constant ka (Aldridge and Reiner, 1972) represent the rate of esterase inhibition (moles of inhibitor sequestered per min) and

494

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et al.

E. SCHARF

Esterase Activity .......

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3. H y d r o x y a p a t i t e e l u t i o n p r o f i l e s tor g r o u p 1 e s t e r a s e s f r o m ( A ) the J o h n s o n W a x ( s u s c e p t i b l e } a n d ( B ) the M u n s y a n a

(resistant) strains o f

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values of the reactivation constant k3 represent the rate of re-activation after 90% inhibition (molecules inhibitor recovered per hour). Summarized k, results (by inhibitor) are as follows. Results for malaoxon and paraoxon indicated that MA esterases of each group were more rapidly inhibited. However, MA group I (for malaoxon) and MA group II esterases (for paraoxon) were the only groups which were significantly different from the other, less rapidly inhibited esterases of each strain (a = 0.05, n = 4). Results for propoxur indicated that all groups were significantly different (a = 0.05, n = 4), with MA esterases being more slowly inhibited in each case (i.e. JW group I > MA group I and JW group II > MA group II). These results suggest that, generally, MA esterases have a greater affinity for malaoxon and paraoxon but less affinity for propoxur. Values of k3 for malaoxon showed no significant differences in recovery time between esterase groups from either strain (a = 0.05, n = 4). Paraoxon k3 values were not significantly different with the exception of JWax

S e e text for m e t h o d s .

group II esterases (a = 0.05, n = 4), which recovered more quickly, indicating a potential for organophosphate resistance via sequestration by esterases of MA group II. For propoxur, esterases of the JWax group II recovered most rapidly, and esterases of MA group II had significantly similar k~ values to both JWax group II and MA group 1. However, JWax group Il activity had not yet recovered at 4.5 h after propoxur inhibition. Inhibition results have shown both greater affinity for, and lengthened times of, inhibition by oxon analogues of organophosphate insecticides. Also, earlier gel electrophoresis and examinations of hydrolytic activity towards p-nitrophenyl acetate showed an additional electromorph, and elevation of specific activity for MA esterases (respectively). In conjunction, these results suggest that there may be both qualitative and quantitative differences in esterase composition between resistant and susceptible strains. These differences may be sufficient to confer organophosphate insecticide resistance to the MA strain.

8. GERMANICA ESTERASES

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FIGURE 4. Hydroxyapatite elution profiles for group II esterases from (A) the Johnson Wax (susceptible) and (B) the Munsyana (resistant) strains of B. germanica. See text for methods.

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FIGURE 5. Native PAGE results for crude and partly purified esterases from insecticide-resistant and susceptible B. germanica. C, crude homogenate; I, group I esterases; II, group II esterases; MA, Munsyana strain (resistant); JW, Johnson Wax strain (susceptible); and El, E2, E3 and E4, esterase ele~tromorphs.

MICHAEL E. SCHARF et al.

496

T A B L E 1. Partial purification table for esterases from susceptible and resistant B. germanica strains Strain"

Fraction b

Total protein (mg)

Specific activity'

JWax

S 10 QI QII HI HII SI0 QI QII HI HII

1.70 1.10 I. 10 1/.03 0.01 3.63 0.36 0.57 0.02 0.01

2,152 2.23 2.45 12.42 4.06 7.63 10.26 8.74 14.5(/ 67. 117

MA

Purification J

% Recovery'"

1.11) 1.21 5.57 1.66

38.0 5(/.17 100.0 100.0

1.34 1.15 1,41 7.67

38.0 25.0 100.0 100.0

"Strain identities: JW, Johnson Wax (susceptible); MA, Munsyana (resistant). hFractions are defined as: S10, supernatant from crude homogenate spun at 10,000g; Q, Q-Sepharose derived; H, hydroxyapatite derived" 1 and II, the original activity peaks which eluted from Q-Sepharose columns. '/~mol p-nitrophenol produced per min per mg protein. aQ-sepharose results are in relation to S10 fractions. Hydroxyapatite results are in relation to Q-Sepharose fractions. See text tbr mcthods.

T A B L E 2. Average k, and k3 values ( _+ SE) for inhibitors in the presence of partly puritied esterasesfrom susceptible and resistant B. germanica Strain"

Esterase group ~'

Constant'

lnhibitors Malaoxon

JWax MA JWax MA

I I1 I II I II I I1

k,

k,, k, k,, k~ k~ k~ k~

0.60 0.25 1.82 1.22 9.00 7.00 9.00 9.00

(0.52) (0.23) 11.101 ((I.611 /0.305 (0.20) (0.265 (0.40)

ab b a ab a a a a

Paraoxon

Propoxur

259.00 1157.91 b 216.20 1176.11 b 338.10 (60.9) ab 541.20 (58.6) a 8.00 10.191 b 31.00 (3.40) a 15.00 (3.90) b 8.00 (0.00) b

33.40 16.481 b 41.60 10.331 a 1.10 ((/.905 d 20.40 (2.77) ¢ 38.00 115.001 a 0.00 (0.001 b 11.00 11.9151 ab 7.00 12.01/7 b

Values shown for ka are x 10° (M ' min '). Values shown for k3 are molecules recovered ( x 102)/h. For each kinetic constant, means within the same column followed by the same letter are not significantly different by the Ryan-Q test (SAS Institute, 1990; a = 0.05; n = 4). "Strain identities: JW, Johnson Wax (susceptible); MA, Munsyana (resistant). bEsterase groups are the original activity peaks which eluted using Q-sepharose chromatography. Each group was further purified (separately~ using hydroxyapatite chromatography before determination of kinetic constants. CKinetic constants (k,, and k0 have been determined separately. See text for details.

Molecular weight estimation

Using 4-20% native and SDS gradient gels with both partly purified esterase samples and molecular weight protein standards, molecular weight estimates were obtained. Under native conditions, peak I esterases had estimated molecular weights of 63.6 + 0.3 kDa, whereas peak II esterases had native estimates of 60.1 + 1.0 kDa. Using SDS gels, denatured peak I esterases had estimated molecular weights of 60.1 + 1.4 kDa, whereas peak II molecular weight estimates were 63.0 + 2.1 kDa. The similarities in the molecular weight estimates between each peak for each of the native and denaturing methodologies show that all esterases examined here are monomeric in their active forms. These molecular weight values were similar to those obtained for the purified esterases PelRR/32 (62.8 + 2.4; Karunaratne et al., 1993) and c~2 (60.0 + 9.0; Ketterman et al., 1992) respectively, which are elevated in Culex mosquitoes. Using a similar native methodology, Prab-

hakaran and Kamble reported molecular weight estimates for 10 B. germanica esterase electromorphs (E~-Elo) which ranged from 48.0 to 81.0 kDa (Prabhakaran and Kamble, 1994), and in another report (Prabhakaran and Kamble, 1995) for three electromorphs (E5-E7; 5357 kDa). Two of the 10 esterase electromorph molecular weights reported by Prabhakaran and Kamble were similar to those reported in this study ( E 6 = 60.0 and E7 = 63.0). In summary, our findings of enhanced esterase expression are consistent with results and conclusions of Prabhakaran and Kamble (1995). On the basis of the differing Q-Sepharose elution profiles and differing kinetic constants, active site and/or structural polymorphisms also appear to exist between the JWax and MA strains. Previously, Prabhakaran and Kamble (1993, 1994) have shown 10 esterase electromorphs, one activity peak eluting from Q-Sepharose columns (Prabhakaran and Kamble, 1995), and no differences in paraoxon inhibition pro-

B. GERMANICA ESTERASES

files (Prabhakaran and Kamble, 1995). In this report, we have shown fewer esterase electromorphs (four in total), two eluting peaks of esterase activity in Q-Sepharose chromatography, and differences in paraoxon inhibition profiles. It is unclear if these differences in reports are related to experimental techniques, age class/sexual differences, natural variation, or resistance. Because we have found carbamate insecticides to interact with esterases that are also probably involved in organophosphate resistance, one proposal for resistance management appears most logical in this case. By using mixtures of carbamates (i.e. propoxur or bendiocarb) and organophosphates (i.e. chlorpyrifos, diazinon, or acephate) at rates which are within the limits of legality, the efficacy of each active ingredient may be synergized. Specifically, as each component of the mixture is sequestered over time by different resistance-associated esterases, larger quantities of non-sequestered insecticide would remain available to inhibit acetylcholinesterase. Further research is in progress to determine the practicality of such a strategy. REFERENCES Aldridge W. N. and Reiner E. (1972) Enzyme inhibitors as substrates. In Frontiers of Biology (Edited by Neuberger A. and Tatum E. L.), North Holland, Amsterdam. Aldridge W. N. (1993) The esterases: perspectives and problems. Chem. Biol. Interact. 87, 5-13. Anspaugh D. D., Rose R. L., Koehler P. G., Hodgson E. and Roe R. M. (1994) Multiple mechanisms of pyrethroid resistance in the German cockroach. Pestic. Biochem. Physiol. 50, 138-148. Ballard J. B., Gold R. E. and Rauscher J. D. (1984) Effectiveness of six insecticide treatment strategies in reduction of German cockroach populations in infested apartments. J. Econ. Entomol. 77, 10921094. Bradford M. M. (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analyt. Biochem. 72, 248-254. Chen W. L. and Sun C. N. (1994) Purification and characterization of carboxylesterases of a rice brown planthopper, Nilaparvata lugens. Insect Biochem. Molec. Biol. 24, 347-355. Devonshire A. L. (1977) The properties of a carboxylesterase from the peach-potato aphid, Myzus persicae, and its role in conferring insecticide resistance. Biochem. J. 167, 675-683. Hemingway J., Small G. J. and Monro A. G. (1993) Possible mechanisms of organophosphorous and carbamate insecticide resistance in German cockroaches from different geographical areas. J. Econ. Entomol. 86, 1623-1630. Karnovsky M. J. and Roots L. (1964) A direct coloring thiocholine method for cholinesterases. J. Histochem. Cytochem. 12, 219-226. Karunaratne S. H. P. P., Jayawardena K. G. I., Hemingway J. and Ketterman A. J. (1993) Characterization of a B-type esterase involved in insecticide resistance from the mosquito Culex quinquefasciatus. Biochem. J. 294, 575-579.

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Acknowledgements--This research was partially supported by Zeneca Professional Products (U.S.A.) and Zeneca Public Health (U.K.). Appreciation is extended to Jonathan Neal and Peter Dunn for critical review of manuscript drafts. This is journal paper no. 14,822 of the Agricultural Research Program of Purdue University, West Lafayette, IN, U.S.A.