Genetics and biochemical mechanisms of abamectin resistance in two isogenic strains of Colorado potato beetle

PESTICIDE

BIOCHEMISTRY

AND

PHYSIOLOGY

44, 191-207 (1992)

Genetics and Biochemical Mechanisms of Abamectin Resistance Two lsogenic Strains of Colorado Potato Beetle’

in

JOSEPHA. ARGENTINE,' J. MARSHALL CLARK,~ AND HAO LIN Department

of Entomology,

University

of Massachusetts

Amherst,

Massachusetts

01003

Received April 28, 1992; accepted September 9, 1992 Abamectin resistance in two isogenic strains of Colorado potato beetle was determined to be autosomal, incompletely recessive, and polygenic. Both resistant strains exhibited high levels of synergism to piperonyl butoxide and moderate levels to S,S,S,-tributyl phosphorotrithioate compared to that of a susceptible strain as judged by synergistic ratio and relative percentage synergism values. Both resistant strains had significantly elevated levels of cytochrome P,,, and oxidative metabolites of [3H]abamectin B,, under both in vivo and in vitro conditions compared to those of the susceptible strain. High levels of oxidative synergism, elevated cytochrome Pd5a levels, and increased amounts of oxidative metabolites of [3H]avermectin B,, substantiate a monooxygenasebased resistance mechanism. Additionally, the polygenetic form of resistance, esteratic synergism, and elevated hydrolytic activity indicate the possibility of a carboxylesterase-based resistance mechanism. The lack of hydrolytic metabolites of [3H]avermectin B,, and low inhibitory action of abamectin on carboxylesterase activity, however, suggest principally a sequestration role for the resistant carboxylesterase. Penetration and excretion factors play no significant role in resistance nor does there appear to be a significant glutathione-S-transferase component in the abamectinresistant strains. 0 1992 Academx Press. Inc.

INTRODUCTION

The avermectins are novel insecticides and drugs that have had tremendous impact in veterinary medicine and whose potential in human medicine and insect control is enormous (1, 2). It is therefore vital that resistance management strategies be devised prior to the appearance of resistance in field populations when it is too late to preserve a species’ susceptibility. The availability of abamectin-resistant insect strains would greatly assist in the development of such programs. Resistance management strategies (e.g., proper application schemes, use of effective synergists and compounds eliciting negative crossresistance, etc.) and accurate, sensitive, biochemical monitoring schemes (3, 4) could be developed before commercial use of abamectin. In a previous study, selection for abamectin resistance in Tetranychus urticae was unsuccessful (5), although selection of ’ This work was supported by research Grant USDA(NAPIAP)-TPSU-UM-3361-529, and MAES(Hatch Grant 617).

Metaseiulus occidentalis resulted in low to modest levels of resistance (6). No crossresistance to abamectin could be detected in resistant strains of Musca domestica (7), cockroach (8), or Colorado potato beetle (CPB;4 Leptinotarsa decemlineata (Say)) (9). Cross-resistance to abamectin in pyre’ Current address: Department of Molecular Biology and Biochemistry, University of California-Irvine, Irvine, CA 92717. 3 To whom correspondence and reprint request should be addressed. 4 Abbreviations used: AB-Fd, abamectin-resistant isogenic strain from field selections; AB-L, abamectinresistant isogenic strain from EMS mutation in conjunction with abamectin selection of a laboratory susceptible strain; CDNB, chlorodinitrobenzene; CPB, Colorado potato beetle; DCNB, dichloronitrobenzene; 3’-desmethyl, 3’-desmethyl-avermectin B,,; DEF, S,S,S-tributyl phosphorotrithioate; DEM, diethyl malate; DFP, diisopropyl fluorophosphate; DTT, dithiothreitol; E64, epoxysuccinyl-L-leucylamido-(4guanidino)-butane; EMS, ethyl methanesulfonate; GST, glutathione-S-transferase; 24-OH, 24-hydroxymethyl-avermectin B,,; PAGE, polyacrylamide gel electrophoresis; PBO, piperonyl butoxide; PHMB, parahydroxymercuriobenzoate; PMSF, phenylmethylsulfonyl fluoride; R%S, relative percentage synergism; SR, synergistic ratio; SS, susceptible strain of CPB. 191 0048-3575192 $5.00 Copyright All rights

D 1992 by Academic Press. Inc. of in any form reserved.

reproductmn

192

ARGENTINE,

CLARK,

throid-resistant house flies (LPR and Dairy strains), however, appears to be due to oxidative and penetration factors (10) and a field strain of Plutella xylostella, resistant to a number of insecticides, showed a low level of cross-resistance to abamectin (11). Most recently, laboratory selections have resulted in extremely high levels of abamectin resistance (26- to 60,000-fold) in field-collected house flies (12). This particular resistance was not synergised by piperonyl butoxide (PB0),4 S,S,S,-tributyl phosphorotrithioate (DEF),4 or diethyl malate (DEM).4 Also, no increases in crossresistance levels to a number of organophosphosphates, permethrin, or cyclodienes were evident after laboratory selections of the highly abamectin-resistant strains. Subsequent biochemical and genetic studies on the highly abamectinresistant AVER strain of house fly has determined this resistance to be autosomal, recessive, and polyfactorial (13). Two biochemical mechanisms have been associated with this resistance; decreased cuticular penetration and altered abamectin binding due to a reduction in the number of specific binding sites. Lastly, hydrolysis appears to be a resistance mechanism in T. urticae to milbemycin, a close analog of the avermectins (14, 15). Resistant mites had a high level of synergism to esterase inhibitors, an altered esterase zymogram, and inactivated milbemycin more effectively in vitro than susceptible mites. The CPB is one of the most harmful pests of potato, particularly in the northeast United States. It is also a paradigm of insecticide resistance, invariably being one of the first agricultural pests to develop resistance to any new insecticide class. Argentine and Clark (9) have developed two strains of abamectin-resistant CPB using field selection (AB-Fd strain)4 and the mutagen, ethyl methanesulfonate (EMS),4 in conjunction with laboratory selection (AB-L strain).4 Resistance appears to be autosomal and incompletely recessive. Re-

AND

LIN

sistance levels for AB-Fd and AB-L were 23- and 15fold at the LD,, and 38- and 21fold at the LD,,, respectively. Both resistant strains had little mortality at 10 ng/ larva, while this dose caused approximately 99% mortality in a susceptible strain. The F, crosses for both abamectin-resistant strains were backcrossed to the SS strain for four generations to create isogenic strains (i.e., approximately 94% of the genomic background of these abamectinresistant strains is derived from the susceptible strain). The aim of this study is to determine the biochemical mechanisms of resistance to abamectin and to complete the population genetics of abamectin resistance in CPB. The use of isogenic strains greatly assists this endeavor, since any biochemical differences will be due to resistance mechanisms rather than unrelated strain differences. Such information is necessary in designing effective resistance management strategies and in directing future work on the molecular genetics of resistance in CPB. MATERIALS

AND

METHODS

Chemicals. Abamectin (L676, MK-936), 93% purity, 13H]avermectin B,, labeled at carbon 5 (sp act, 11.31 mCi/pmol), 14C-24hydroxymethyl-avermectin Bi, (24-OH),4 and [‘4C]-3’-desmethyl-avermectin B,, (3’desmethyl)4 labeled at carbon 5, 3, 7, 11, 13, and 23 (sp act of each, 1.4 mCi/mol), were donated by Merck, Sharp, and Dohme Research Laboratories (Three Bridges, NJ). All other chemicals were purchased commercially and were of the highest grade available. Dieldrin (99%) was purchased from Chem Services (West Chester, PA). Insect strains, rearing conditions, bioassay, and statistical procedures. A susceptible strain (SS)4 of CPB was supplied by G. G. Kennedy (North Carolina State University, Raleigh). Rearing conditions and selection schemes for the two abamectinresistant strains (AB-Fd and AB-L) of CPB are given in a previous publication (9). Ab-

ABAMECTIN

RESISTANCE

IN COLORADO

amectin bioassays were performed and analyzed using the methods described previously (16). For dieldrin bioassays, larvae were placed on cut potato slices and mortality assessed 48 hr after treatment. Data were subjected to logit analysis and a likelihood ratio test was used to test the hypotheses of parallelism and equality (P = 0.05) (17, 18). The number of genetic factors involved in abamectin resistance was established using the backcross method of Georghiou (19). Metabolic synergists were applied as in Argentine et al. (16). The effects of metabolic synergists were measured using synergist ratio (SR)4 and relative percentage synergism (R%S)4 (20). Esterase enzyme preparation. The abdomens of fourth-instar larvae were homogenized in Tris-base/HCl (0.2 M, pH 7.8, l/l abdomen/v) containing 1% Triton X- 100 for solubilization of membrane-bound esterases (21). The 13,000g supernatant was used for all esterase assays. Glutathione-S-transferase (GSlJ4 enzyme preparation. The abdomens of fourth-

instar terase GSH zyme

larvae were homogenized as for espreparation above except 4 mM was added for protection of the en(21).

Microsome of cytochrome

preparation and estimation levels. The preparation of

microsomes was essentially based on that of Leonova et al. (22) except fourth-instar larvae were fed potato tubers for 16 hr to purge potato leaf pigments from the midgut. The potassium phosphate homogenizing buffer contained 0.4 rniV phenylmethylsulfonyl fluoride (PMSF),4 0.1 mM dithiothreito1 (DTT),4 and 0.028 mM epoxysuccinylL-leucylamido-(4-guanidino)-butane (E-64,4 a cysteine specific-protease inhibitor (23, 24). The 105,OOOg pellet was resuspended in 0.05 M Tris-base/HCl (pH 7.9) containing 20% glycerol. The resulting protein concentration was approximately 24 mg/ml (25). Cytochrome P,,, and P,,, levels were assayed following the methods of Omura and

Sato (26) (27). The centration of Omura

POTATO

BEETLE

193

as modified by Jesudason et al. microsomal cytochrome bS conwas measured using the methods and Takesue (28).

NADPH-dependant cytochrome c reductase assay. Microsomal cytochrome c re-

ductase activity was assayed by using the method of Omura and Takesue (28). 0-demethylase assays. Two O-demethylase assays were used. The first reaction assay (29) used the substrate, p-nitroanisole. 0-demethylase activity using methoxyresorufin and pentoxyresorufin as substrates was also measured by the method of Meyer et al. (30). Microsomal and oxidative ester cleavage assays. The overall ester cleavage activity

of the microsomal preparation was measured by the method of Kao et al. (31) using p-nitrophenylacetate as a substrate. To assess oxidative ester cleavage activity in microsomes, 0.1 mA4 DEF was added to inhibit membrane-associated esterases. Biphenyl hydroxylation assay. Aromatic hydroxylation was measured using biphenyl as a substrate in a method adapted from Yu and Ing (32). Aliesterase, L‘nonspecific” terase, and carboxylesterase

general esassays. Ali-

esterase activity was measured using methylthiobutyrate as the substrate (31). The procedures for general (nonspecific) esterase activity were based on those of van Asperen (34). a-Naphthyl butyrate was also used to examine if any strain elicited increased activity in comparison to a-naphthy1 acetate which is indicative of a lipophilic catalytic center of the esterase (3). To measure carboxylesterase activity, the above reaction mixture was incubated with eserine (10e4 Ikl) and p-hydroxymercuriobenzoate (PHMB)4 (10e4 M) for 10 min at 25°C prior to substrate addition (35). A potent carboxylesterase inhibitor, diisopropyl fluorophosphate (DFP)4, was used to determine any differences in the specific carboxylesterase activities (a-naphthyl butyrate hydrolysis) of the SS strain compared to the

194

ARGENTINE,

CLARK,

abamectin-resistant strains in a DFPleveling experiment (36). To determine the effect of abamectin on carboxylesterase activity from the SS and abamectin-resistant strains, five substrate concentrations of c*-naphthyl butyrate were examined (0.049, 0.073, 0.097, 0.194, 0.388 mM). Abamectin (0.1 mM) was added as a lo-p.1 acetone aliquot to eserine- and PHMB-preincubated esterase enzyme preparation. Control tubes received only acetone. This was further incubated at 32°C for 15 min prior to the start of the reaction by the addition of o-naphthy1 butyrate as above. The data was analyzed as a Lineweaver-Burk double reciprocal plot to determine the apparent kinetics of inhibition. Analysis of carboxylesterase activities by native polyacrylamide gel electrophoresis (PAGE).4 Proteins in the esterase enzyme

preparations from the SS and AB-L strains were separated by nondenaturing (native) PAGE (mini-protean II (2-D), Bio-Rad, Richmond, CA). A 4% stacking and a 7.5% seperating gel composition was used. Samples were electrophoresed for 45 min at 200 V constant voltage. Total hydrolytic (i.e., general esterase) and carboxylesterase activity (incubated with eserine and PHMB as above) were determined by ol-naphthyl butyrate hydrolysis in conjunction with dianisidine (fast blue) staining as detailed by Sparks and Hammock (37). Relative hydrolytic activity in the gel was assessed using a scanning densitometer (Soft Laser, Model SLR-2D/lD, Zenith Corp.). Glutathione-S-transferase

(GST) assays.

The procedures to determine GST activity using dichloronitrobenzene (DCNB)4 were based on those of Habig et al. (38) and Yu et al., (35) for chlorodinitrobenzene (CDNB)4. Pharmacokinetics and in vivo metabolism of [3H]avermectin B,,. Fourth-instar

larvae fed on potato slices for 16 hr were dosed with [3H]avermectin B,, (6 nCi/ larvae, 0.46 &larvae). Individual larvae were kept at 22°C for 0, 1, 2, 6 hr postapplication in 20-ml carbowax-treated vials.

AND

LIN

The 22°C temperature regime was selected in part to reduce the mobility of the larvae and prevent loss of topically-applied [3H]avermectin B,, due to surface abrasion with the vial. At each interval, groups of five larvae were washed twice in 4 ml acetone to remove surface [3H]avermectin B,,. The acetone washings were evaporated just to dryness under N2 and the amount of unpenetrated (i.e., external rinse) [3H]avermectin B ,a was determined in an emulsifier scintillator 299 fluid (Packard, Downers Grove IL) using a LKB-Wallac 1209 liquid scintillation spectrometer. The solventrinsed larvae were then homogenized using a glass-glass homogenizer in 5 ml of 0.1 M acetate buffer (pH 5.0) and ethyl acetate (2:3). The carcasses of the larvae were further extracted with 4 ml of ethyl acetate which was subsequently used to extract the acetate buffer. The ethyl acetate fractions were combined and evaporated just to dryness under N, at 50°C. The dried residues were resuspended into 1 ml acetonitrile: H,O (9:l). The acetonitrile:H,O was washed twice with 2 ml isooctane to remove most of the carotinoid pigments extracted from CPB. After washing, 1.5 ml of a 0.1 M phosphate buffer (pH 7.0) was added to the acetonitrile:H,O fraction. This fraction was then extracted twice with 2 ml ethyl acetate. The ethyl acetate was evaporated just to dryness at 40°C under N, in a scintillation vial and the amount of radioactivity (i.e., internal extract) determined as described above. Extraction efficiencies (total cpm recovered of topically applied dose) for the above procedures were all 80% or more. Radioactivity associated with excrement was extracted by two 2.5-ml aliquots of ethyl acetate and one 2.5-ml aliquot of methanol collected sequentially from the five holding vials. The combined extracts were centrifuged at 500g and the supernatant concentrated under N, in a scintillation vial. The radioactivity (i.e., excrement extract) was determined as described previously. For identification of metabolites, 10 larvae were held in vials for

ABAMECTIN

RESISTANCE

IN

6 hr and the excrement extracted and centrifuged as above. The extracts were washed with isooctane and evaporated just to dryness as with the larval carcasses. Avermectin B ,a and its metabolites were determined using the methods of Crouch et al. (39) by reversed-phase C18HPLC. In vitro metabolism of avermectin B,,. Oxidative and hydrolytic metabolism of avermectin Bra were examined using microsomes which were prepared as described previously except no glycerol was added to the homogenation buffer. The reaction mixture for oxidative metabolism of 13H]avermectin Br, consisted of 1.0 ml microsomes, the NADPH regenerating system previously described, and DFP (10e4 It4) in carbowax-treated tubes (40). The hydrolytic component of [3H]avermectin B,, metabolism in microsomes was measured using 1.O ml microsomes and PBO ( lop4 M) at a final volume of 1.5 ml in carbowaxtreated tubes. Both assay mixtures were preincubated at 32°C for 5 min prior to the addition of 46 nCi [3H]avermectin B,, (2.7 n&I, 0.33% acetone final cont.) to the reaction mixture and incubated at 32°C for an additional 60 min. Methylene chloride (2.0 ml) was added to terminate the reaction and extract avermectin and its metabolites. This step was repeated once, and then 2.0 ml ethyl acetate was used for a final extraction. The pooled solvent fractions were evaporated just to dryness under N, and prepared for HPLC analysis as in the in vivo studies described above. RESULTS

Inheritance of abamectin resistance. Classic backcrossing techniques were used to determine the number of factors involved in abamectin resistance. However, determination of the exact number of factors with this method is difficult when the SS and F, mortality lines overlap (19) or in the case of moderate resistance levels (41). The second backcross of the AB-Fd strain (AB-Fd Bc,) showed no significant difference from the predicted monogenic curve

COLORADO

POTATO

195

BEETLE

(x2 = 0.91; df = 2; P = 0.633) (Fig. 1, top). The following backcross generation (ABFd Bc,) was significantly different from the predicted monogenic curve, although the probability value was only slightly less than 0.05 (x2 = 6.30; df = 2; P = 0.043). Nevertheless, the AB-Fd Bc, cross was also significantly different from the AB-Fd Bc, (x2 = 14.08; df = 2; P = 0.001). These two differences indicate that abamectin resistance is probably polyfactorial in this strain. The AB-L Bc, backcross showed no significant difference from the predicted monogenic curve (x2 = 3.45; df = 2; P = 0.178) (Fig. 1, bottom). The AB-L Bc, backcross also was not significantly different from the predicted monogenic curve (x2 = 5.18; df = 2; P = 0.075), although the slopes of these two curves were signifi-

,‘A&Fd 24 0 A&Fd EC, 0 AB-Fd BcJ - Predicted Bc 0.001

0.010

0 100

1

z 97 .. x 91 -76 -50 -24 -~

-----a0.001

0.010

ILog Dose Abamectln

i 0.100

1.ooo

(pg/lawa)

FIG. I. Log dose versus logit mortality regressions offourth-instar CPBs of the susceptible (SS) and abamectin-resisianf strains (AB-Fd and AB-L), pooled reciprocal F1 crosses (F,), backcross generations 2 (Ec2) and 3 (Bc,), and the predicted backcross response for monofactorial inheritance (dashed line). Top, Findings for the AB-Fd strain. Bottom, Findings for the AB-L strain.

196

ARGENTINE,

CLARK,

cantly different (x2 = 4.29; & = 1; P = 0.038). As indicated, the two P values generated in this comparison were both very close to 0.05, making it difficult to distinguish inheritance as mono- or polyfactorial. However, comparison of the AB-L Bc, and the AB-L Bc3 demonstrated a significantly high level of difference between the two crosses (x2 = 22.94; df = 2; P < O.OOl), indicating a possible polyfactorial inheritance of abamectin resistance in the AB-L strain.

AND

LIN

No significant cross-resistance to dieldrin was evident in the MA-R- or abamectin-resistant strains as measured by LD,, or LD,, resistance ratios (Table 1). In vivo synergism of abamectin toxicity.

PBO produced very high levels of synergism as judged by their synergistic ratios (SRs) to abamectin in both abamectinresistant strains (Table 2). The AB-Fd strain has a slightly elevated SR value compared to that of the AB-L strain (19 and 15, respectively). PBO also synergized abamectin toxicity in the SS strain but at a much more reduced level relative to the abamectin-resistant strains. This is shown in the high relative percentage synergism (R%S) values of the abamectin-resistant strains compared to those of the SS strain (68 and 70, AB-Fd and AB-L, vs 21, SS) (Table 2). The log dose versus logit mortality curves of the PBO-treated AB-Fd (ABFd + PBO) and AB-L (AB-L + PBO) strains were not significantly different (P = 0.05) from the SS strain treated with abamectin alone (SS) by the likelihood ratio test of parallelism and equality (x2 = 8.80; df = 2; P = 0.12; x2 = 3.11; df = 2; P = 0.21, respectively). The esteratic synergist, DEF, produced a

Abamectin resistance levels and dieldrin cross-resistance. Abamectin was deter-

mined to be a very effective insecticide against both field (MA-R) and susceptible laboratory (SS) strains of CPB (Table 1, LD,, = 1.95-1.98 @beetle, respectively). The MA-R strain, which is highly resistant to organophosphate and pyrethroid insecticides (16)) was not significantly different in its mortality response to abamectin compared to the SS strain (x2 = 1.57; df = 2; P = 0.46). This indicates an absence of crossresistance to abamectin in the MA-R field strain. The AB-Fd strain is more resistant (23- vs 15-fold, Table 1) and significantly different from the AB-L strain (x2 = 25.83; df = 2; P < 0.001, Table 1). TABLE

1

Comparative Topical Toxicities of Fourth-lnstar Larvae of the Susceptible (SS). Multiply Resistant Massachusetts (MA-R) and Abamectin-Resistant (AB-Fd and AB-L) Strains of CPB to Abamectin and Dieldrin L&o

n

ss MA-R AB-Fd AB-Fd AB-L AB-L

F, F,

ss MA-R AB-Fd AB-L a Resistance

(95%

CI)

540 251 936 482 1006 586

1.95 1.98 12.7 45.3 11.1 29.4

(1.47, (1.69, (11.5, (32.6, (10.1, (23.9,

2.31) 2.39) 14.0) 56.2) 12.2) 34.4)

219 267 256 340

0.72 1.32 0.52 0.70

(0.53, (0.89, (0.14, (0.53,

0.94) 1.85) 15.33) 0.89)

ratio

(RR)

= LD,

RR MW’ Abamectin 7 23 6 15 Dieldrin -

of each strain/LD,

RR

LD9,

(95%

CI)

(&larva) 6.62 4.95 39.2 249.8 28.1 142.2

(4.92, (3.61, (33.7, (145.6, (24.7, (112.4,

12.3) 10.4) 47.4) 1291) 32.9) 200.9)

(LD97Y

6 38 4 21

Slope

2 SE

6.56 8.76 4.51 4.68 5.47 5.08

2 f + -+ 2 t

0.97 1.89 0.32 0.65 0.39 0.56

2.66 2.53 1.73 2.57

t f f *

0.43 0.36 0.25 0.28

(&larva) 14.5 31.4 53.2 15.9

2 1 1 SS strain.

(7.1, 55.5) (15.8, 100.1) (7.7, 7186.2) (9.2, 35.9)

ABAMECTIN

Effect of Metabolic

RESISTANCE

IN

COLORADO

POTATO

197

BEETLE

TABLE 2 Synergists on the Toxici@ of Abamectin in Fourth-lnstar Larvae on the Susceptible (SS) and Abamectin-Resistant (AB-Fd and AB-L) Strains of CPB LD,,(95% CI) (&beetle) 1.95 (1.47-2.31) 0.96 (0.80-1.15) 1.62 (1.08-2.39) 0.61 (0.34-1.01)

Slope GE) 6.56 (0.97) 3.73 (0.45) 3.26 (0.37) 5.37 (0.67)

Strain ss DEF DEM PBO

n 540 287 287 288

AB-Fd DEF DEM PBO

482 305 311 245

45.30 8.87 23.72 2.40

(32.60-56.20) (5.84-11.51) (19.67-28.68) (1.92-2.88)

4.68 5.89 3.42 4.85

(0.65) (1.06) (0.34) (0.70)

-

AB-L DEF DEM PBO

568 268 302 237

29.40 5.66 10.40 1.94

(23.90-34.42) (2.84-7.90) (8.74-12.34) (1.40-2.62)

5.08 4.66 3.90 4.84

(0.56) (0.91) (0.43) (0.64)

-

a SR = LD,dsLDsO, where S = synergized. b R%S (S) = lOO[log LD,,,(S)-log sLD,,(S)]i[log

SR” 2 1 3

R%Sb 21 18 21

5 2 19

42 19 68

-

5 2 1.5

48 35 70

LD,,(R)-log sLD,,(S)]; R%S(R) = lOOtlog LD,,(R)-log

sLD,,(R)l~tlog L&,(R)-log sLD,,(S)I. moderate level of synergism in both abamectin-resistant strains (SR = 5) and little in the SS strain (SR =: 2) (Table 2). Although DEF synergism is not of the same magnitude as PBO synergism, the increase in the SR values of the abamectin-resistant strains is indicative of a possible esterase involvement in abamectin resistance. The glutathione-S-transferase inhibitor, DEM, had a small but similar effect on abamectin toxicity in both the SS and the abamectin-resistant strains (Table 2). Pharmacokinetics of [3H]avermectin B,,. Cuticular penetration of [3H]avermectin B,, was similar for all strains and resulted in only 15-25% of the topically applied insecticide remaining on the surface after 6 hr (Fig. 2, external rinse, top). Although there was a statistical difference between the AB-L and SS strain at 6 hr in the amount of compound left on the cuticle, a similar situation was not present in the AB-Fd strain and the actual level of difference in the AB-L strain was slight. However, at 6 hr, both the AB-Fd and the AB-L strains had significantly lower levels of radioactivity internally and signif-

icantly higher levels in the excrement (Fig. 2, internal extract, middle, and excrement extract, bottom). This could be caused by a higher rate of excretion of the parent compound, an increase in the level of watersoluble metabolites which are then more rapidly excreted, or a combination of both. Cytochrome levels and general metabolic substrate assays. Cytochrome P,,, levels were significantly elevated (P < 0.01, t test) in both the AB-Fd and the AB-L strains (457 k 140 and 389 + 113 pmol/mg protein, mean 5 SD, respectively) compared to those of the SS strain (240 k 79 pmol/mg protein). Figure 3 illustrates a typical cytochrome Pa5e and PdzOpatterns from microsomes prepared from the AB-L and SS strains. There was no significant difference (P > 0.05, t test) in the cytochrome P,,, levels of the AB-Fd or AB-L strains (109 2 32 and 116 + 29 pmol/mg protein, mean + SD, respectively) compared to those of the SS strain (139 rt 80 pmoYmg protein, mean k SD) (Fig. 3), nor were there any significant differences (P > 0.05, t test) in the levels of cytochrome b5 levels of the AB-Fd, AB-L, and SS strains (221 k

198

ARGENTINE,

CLARK,

AND

LIN

(Table 3). This was most apparent in the carboxylesterase assay where there was over a twofold increase in hydrolytic activity to a-naphthyl butyrate in both abamectin-resistant strains compared to that in the SS strain. There were no significant differences (P > 0.05, t test) in any of the strains in aliesterase activity as determined by methylthiobutyrate hydrolysis (Table 3). There were no significant differences in GST activities between the SS and the abamectin-resistant strains in their ability to metabolize CDNB or DCNB (Table 3), nor was there any increase in oxidative activities (i.e., oxidases) for any of the general oxidative substrates tested and there was no concurrent increase in cytochrome c reductase activity levels (Table 3). In vivo metabolism of [3H]avermectin B,,. Unaltered 13H]avermectin Br, was the major radioactive component extracted from excrement at 6 hr post-topical application of the AB-Fd, AB-L, and SS strains

CPB

STRAINS

2. Pharmacokinetics of [3Hjavermectin B,, (0.46 &larva, $ve larva/treatment) in fourth-instar larvae of the susceptible (SS) and abamectin-resistant (AB-Fd and AB-L) strains of CPB. *Significant difference from the SS strain, t test, P < 0.05 (N = 4). FIG.

32, 205 -+ 63, and 264 +- 35 pmol/mg protein, mean + SD, respectively) (data not shown). General e&erase (i.e., nonspecific) and carboxylesterase activities in the abamectin-resistant strains were significantly enhanced compared to those in the SS strain

425

450

475

500

WAVELENGTH (mb) FIG. 3. Typical carbon monoxide-reduced cytochrome P450 differences spectrum for CPB microsomes prepared from fourth&star larvae of the susceptible (SS) and abamectin-resistant strain

(M-L).

ABAMECTIN

RESISTANCE

IN COLORADO TABLE

In Vitro

Metabolic

Activities

of Fourth-Insrar Larvae (AB-Fd and AB-L)

POTATO

3

of the Strains

Susceptible of CPB

ss

CDNB DCNB

AB-L

(nmohminlmg protein f SD) 129.4 2 12.9 8.0 k 0.9

Glutathione-S-transferase 143.9 ? 10.5 9.1 k 0.9 General

a-Naphthyl acetate a-Naphthyl butyrate

(SS) and Abamectin-Resistant

AB-Fd

Assays

356.5 -+ 39.9 450.0 k 12.3

esferases

77.7 ?

6.1

73.3 k 17.2

O-demethylation p-nitroanisole Methoxyresorufin Microsomal ester cleavage Oxidative ester cleavage’ Biphenyl hydroxylation NADPH-reductase

1.7 t

138.2 f ND’ 124.5 2 25.6 2 ND 36.9 +

0.7

45.0 51.6 6.7 9.2

140.2 2 6.5 8.6 f 0.3 (4)

505.7 5 37.46 711.0 5 0.36 (4)

97.6 2 12.6d 150.7 2 43.8’ Aliesrerase

Methylthiobutyrate

(5)”

516.6 k llSb 689.4 2 63.5’ Carboxylesterases

a-Napthyl acetate a-Napthyl butyrate

199

BEETLE

117.2 -r- 8.1b 193.7 Z!I20.3’

(4)

7.2 It

1.7

Oxidases

(4)

158.8 k 45.0 ND 123.4 t 26.9 28.3 k 9.2 ND 39.2 2 13.6

8.6 2

1.9

118.9 + 42.5 ND 118.0 ? 17.5 20.9 k 5.7 ND 43.3 2 6.5

a Number of replicates in parentheses (n). b Significantly different from SS strain, t test, P < 0.01. ’ Carboxylesterase activity measured by inhibiting acetylcholinesterase with eserine (0.1 mM) and arylesterases with PHMB (0.1 mkfl. d Significantly different from SS strain, t test, P < 0.05. e Not detected. r Oxidative ester cleavage was measured by inhibiting membrane-associated esterase activity to p-nitrophenyl acetate with DEF (0.1 mkfl.

(35.8 + 5.5, 33.4 k 4.5, and 32.7 + 3.6% of

topically-applied dose, mean k SD, respectively). However, significantly higher levels of 3’desmethyl avermectin B,, (3’desmethyl), 24-hydroxymethyl avermectin B,, (24-OH),4 and an unidentified metabolite which eluted off the reverse-phase column at 14-15 min (i.e., fraction 14) were associated with the abamectin-resistant strains compared to the SS strains (Fig. 4). The AB-Fd strain had slightly elevated levels of all metabolites which is consistent with the slightly higher level of resistance to abamectin compared to that of the AB-L strain (Table 1). The AB-L strain had significantly higher 24-OH and fraction 14 me-

tabolite levels but did not have a significantly higher level of the 3’-desmethyl metabolite compared to that of the SS strain. Fraction 14 was detected at the same or greater levels as 24-OH regardless of the strain but was particularly evident in the abamectin-resistant strains (Fig. 4). Apparently, fraction 14 has a water solubility intermediary between 24-OH and 3’desmethyl, since these two metabolites eluted off the reverse-phase HPLC column at 6 and 19 min, respectively. In vitro metabolism of [3H]avermectin

B,,. The results of the above in vivo metabolism studies of [3H]avermectin B,, have been corroborated in vitro. The 3’-

200

ARGENTINE,

0” n

-1

,-gz a’” [1 fl

4

D

ss

17 IIXI

AB-Fd AB-L

24-Hydroxy Avermectin

CLARK,

Fraction

AND

14

B, (l

Elution

Time

LIN

3”Des-methyl Avermectin

01 (I

(min.)

4. In vivo metabolism of [‘Hlavermectin B,, in fourth-instar larvae of the susceptible (SS) and abamectin-resistant (AB-Fd and AB-L) strains of CPB. Groups offive larvae were topically treated with [3H]avermectin B,, (0.46 ngllarva) and metabolites and parent compound recovered from excrement after 6 hr. Solid bar, SS strain; open bar, AB-Fd strain; cross hatch bar, AB-L strain. *Significant difference from the SS strain, t test (*. P < 0.01; **, P < 0.05), n = 3. FIG.

desmethyl metabolite formation was elevated 2.3- and 2.0-fold in the AB-Fd and AB-L strains, respectively (Fig. 5). The fraction 14 and 24-OH metabolites were not

z +I I

0

AB-Fd

24-Hydroxy Avermectin

B,,

detectable in the SS strain, amectin-resistant strains levels of these metabolites. lites are apparently formed

Fraction

Elution

Time

14

3”Des-methyl Avermectin

while both abhad detectable These metaboby monooxyge-

BID

(min.)

FIG. 5. In vitro metabolism of [‘Hlavermectin B,, (2.7 nM) by microsomes and abamectin-resistant (AB-Fd and AB-L) strains of CPB. ND, Compound Solid bar, SS strain; open bar, AB-Fd strain: cross hatch bar, AB-L strain. from the SS strain, t test, P < 0.01 (n = 3).

from susceptible (SS) below detection limits. *Signijicant difference

ABAMECTIN

RESISTANCE

IN COLORADO

POTATO

201

BEETLE

TABLE 4 Kinetic Analysis of Carboxylesterase Activity to a-Naphthyl Butyrate for Fourth-lnstar Larvae of the Susceptible (SS) and Abamectin-Resistant (AB-Fd and AB-L) Strains of CPB Apparent kinetic constants

ss

AB-Fd

Km (iA0 V,,, (nmol/min/mg protein)

150.2 * 22.2” 118.2 2 26.9

144.3 -c 32.0 281.7 2 18.8’

AB-L 132.8 2 33.2 296.7 L 35.8b

LIMean values ? standard deviation of four separate experiments. * Significantly different from the SS strain, f test, P < 0.01, n = 3.

nases, since PBO-treated microsomes produced no metabolites in any strain, including fraction 14 (unpublished data, J. A. Argentine). Role of carboxylesterase(s) in abamectin resistance. To substantiate a possible role

in abamectin resistance for carboxylesterase as indicated previously by DEF synergism (Table 2) and enhanced ol-naphthy1 butyrate hydrolysis (Table 3), carboxylesterase activities from SS and abamectin-resistant strains were examined kinetically. Analysis of Lineweaver-Burk double reciprocal plots indicated that there was no apparent change in substrate affinities between the SS and the abamectin-resistant strains (i.e., apparent K, values are approximately equal, Table 4). However, the apparent V,,, values were approximately 2.5fold higher in the abamectin-resistant strains compared to those in the SS strain (Table 4). Similar results were obtained from a DFP inhibition experiment. DFP is a potent inhibitor of CPB carboxylesterase activity as judged by its effect on relative activity in both abamectin-resistant and SS strains. This finding indicates the presence of a common serine hydroxyl moiety in the catalytic center of each of these esterases (Fig. 6, bottom). However, approximately lOOtimes more DFP is necessary to inhibit the specific enzyme activities of the resistant strains to a similar level obtained in the SS strain (i.e., compare activities between 10e9 to 10d7 M DFP, Fig. 6, top). Avermectin B,, (0.1 nuI4) increased the apparent K,,, values of carboxylesterase activities in the abamectin-resistant strains

but had little effect on the apparent V,,, values (Fig. 7, top and middle). The extent of change of the apparent K, value in the presence of abamectin was greatly reduced in the SS strain (Fig. 7, bottom). These results indicate that avermectin B,, under these experimental conditions acts as a competitive inhibitor of carboxylesterase activity in the AB-Fd and AB-L strains, but

OL 100 -

w

ss

V-

AB-F AB-L

!

!

0 0 v 6080 -

\ \

40 -

\

20 -

\

_j

\ \

0

7

-;og

DF8P Concentzatio:

(M)

FIG. 6. Effect of DFP on relative and specific carboxylesterase activities of the susceptible (SS) and abamectin-resistant (AB-Fd and AB-L) strains of CPB.

ARGENTINE,

B-F

-32

-24

-16

-6

0

8

16

24

CLARK,

+ Abamectln

32 Abamectm

AND

LIN

dicates only a low affinity interaction under these experimental conditions. Native (nondenaturing) PAGE was used to separate the proteins from the 105,OOOg supernatant of the esterase enzyme preparation. This allowed us to assess strain differences in their ability to hydrolyze a-naphthyl butyrate by dianisidine staining (Fig. 8). Total hydrolytic activity in the major staining band of the gel (i.e., largest peak identified by scanning desitometry) increased 1.3-fold in the AB-L strain compared to that in the SS strain. The difference in carboxylesterase activity was even more pronounced (2.6-fold higher) in the AB-L strain compared to the SS strain. Indeed, carboxylesterase activity accounted for 0.86 of the total hydrolytic activity of the AB-L strain, while it only made up 0.58 of the total hydrolytic activity of the SS strain. TOTAL ACTIVITY

12

SS

+

I

CARBOXYLESTERASE ACTIVITY

Abamectin

/

FIG. 7. Effect of avermectin B,, (0.1 mMJ on carboxylesterase activity of the susceptible (SS) and abamectin-resistant (AB-Fd and AB-L) strains of CPB. The jagged lines represent the actual data set from which the Lineweaver-Burk plots were drawn. Inflection points indicate actual data points

has less inhibitory effect on the SS strain. Also, the high concentration of avermectin B,, necessary for inhibition (i.e., inhibition was not evident at lower concentrations) in-

FIG. 8. Scanning densitometry patterns of native PAGE of total esterase and carboxylesterase activities in the susceptible (SS) and abamectin-resistant strains (AB-Fd and AB-L) of CPB. The y-axis is a relative scale set to approximated full-scale deflection for the largest peak (total activity, AB-L strain). The x-axis is a I:1 representation (65 mm prior to reduction) of a gel lane running top (left side) to bottom (right side) of the scan.

ABAMECTIN

RESISTANCE

IN COLORADO

DISCUSSION

Abamectin selection schemes and abamectin selection in conjunction with EMSpretreatment resulted in two abamectinresistant strains of CPB, AB-Fd, and ABL, respectively. Resistance to abamectin in both resistant strains appears to be autosomal, incompletely recessive, and polygenie, and to involve both monooxygenases and possibly carboxylesterases. The high levels of PBO synergism, elevated levels of cytochrome P,,,, and increased levels of oxidative metabolites substantiate that monooxygenase-based oxidative metabolism is partially responsible for abamectin resistance in these two strains. In all strains, the major metabolite detected was 3’-desmethyl. The level of this oxidative metabolite was significantly elevated in both resistant strains compared to the SS strain under both in vivo and in vitro conditions. This is similar to the metabolism of abamectin by rats which is principally oxidative and results in the production of the 3’-desmethyl metabolite as the major oxidative product (42,43). Interestingly, a new but structurally unidentified oxidative metabolite (i.e., fraction 14) was found and its formation was enhanced principally in the abamectin-resistant strains. The lack of increased activity to general oxidative substrates indicates that the particular cytochrome P,,,(s) produced in the abamectin-resistant strains may have an increased specificity or higher activity for abamectin with no similar increase in activity toward the substrates used in the chromogenic assays. Also, the overall levels of abamectin metabolites were low in all strains, particularly under in vitro conditions. This may be due to a number of factors. First, the levels of resistance achieved by the abamectin-resistant strains (23- to 15fold) were only modest in both cases. Second, the low levels of radiolabeled abamectin administered in both experimental protocols (i.e., 0.46 pg in vivo and 2.7 nM in vitro) and the short time span of these experi-

POTATO

BEETLE

203

ments would not have resulted in maximal levels of metabolism. Third, the amphipathic nature of abamectin is likely to have resulted in a large proportion of the abamectin being nonspecifically bound, especially in the microsomal fraction used in the in vitro assays. However, even a modest increase in oxidative metabolism coupled with other resistance mechanisms (e.g., sequestration) could easily account for the 23to 15fold resistance levels to abamectin. Nonetheless, the differences between the SS and the abamectin-resistant strains in the amount of oxidative abamectin metabolites produced make it clear that oxidative metabolism is a mechanism of abamectin resistance in these strains. The effectiveness of PBO as a synergist in the abamectin-resistant strains indicates that PBO and other oxidative synergists could be effective in preventing or delaying enhanced oxidative mechanisms from evolving in pest populations (44). Additionally, new avermectins could be developed that hinder oxidative metabolism as sites prone to attack, such as the 3’-methoxy site (45). Since abamectin resistance is most likely polyfactorial for both strains (see Fig. l), monooxygenases may not be the only mechanism involved in resistance in these strains. Evidence suggests carboxylesterases may be also involved in abamectin resistance. Synergism to DEF was not as high as with PBO, but it was substantially higher in the abamectin-resistant strains compared to the SS strain, particularly when the R%S values are examined (see Table 2). Carboxylesterase activity was significantly higher in the abamectinresistant strains than in the SS strain, particularly to the more lipophilic substrate, a-naphthyl butyrate (see Table 3). This difference was due to an elevated apparent Vmax value rather than to a change in affinity (apparent K,) of the enzyme (see Table 4). These results indicate that abamectin resistance may be attributed, in part, to increased levels of carboxylesterase(s).

204

ARGENTINE,

CLARK,

The above findings are corroborated by both the DFP inhibition studies and the nondenaturing, native PAGE experiments. The lack of difference in the ability of DFP to inhibit the relative activity of any of the strains demonstrates that the carboxylesterases involved all have a common serine hydroxyl moiety in their catalytic centers and that they all have a similar affinity for this inhibitor. However, approximately loo-times more DFP is needed to inhibit the specific activity of the abamectin-resistant strains to a level of inhibition comparable to that achieved in the SS strain. This indicates that the resistant beetles may be producing a greater amount of carboxylesterase(s) rather than an altered form of the carboxylesterase(s). In the native gel experiments, the AB-L strain demonstrated not only a greater level of carboxylesterase activity (0.86 of the total hydrolytic activity) but this activity was associated with a single major band in the gel indicative of an increase in a single carboxylesterase or of a few very similar carboxylesterases. Abamectin also appears to interact with resistant form of the carboxylesterase(s) in more or less a competitive fashion but does so only at high concentrations (0.1 mM). Although this suggests a low affinity interaction, the hydrophobic nature of the catalytic center associated with the resistant forms of the carboxylesterase(s) [as judged by the more rapid hydrolysis of c;u-naphthyl butyrate compared to the corresponding acetate ester, Table 3 and Ref. (45)] and the lipophilicity associated with abamectin make a proper assessment of this interaction difficult if not impossible using standard Michaelis and Menten kinetics. As discussed by Devonshire and Moores (46), such an interaction is most efficiently studied by determining K, values and inhibition constants (ki) at low substrate concentrations. Nevertheless, these results do indicate an interaction of the carboxylesterase(s) with abamectin and the interaction is more pronounced in the resistant forms. Whether such an inter-

AND

LIN

action is involved in any aspect of abamectin resistance (e.g., sequestration, hydrolysis) in these strains is, however, only speculative at this time. Enzyme purification, characterization, and tissue distribution experiments will help clarify such a function. It also still remains to be seen if siteinsensitivity is a mechanism in either of the abamectin-resistant strains. Clearly, GABA-stimulated chloride flux studies need to be done to determine if siteinsensitivity is a mechanism in abamectin resistance. So far, no cross-resistance to abamectin has been observed in dieldrinresistant strains where site insensitivity has been established as a mechanism of resistance (7, 47). Recently, an abamectinresistant strain of house fly (AVER) with altered abamectin binding was shown not to be cross-resistant to dieldrin or lindane (12, 13). Nevertheless, differential penetration does not contribute significantly to overall abamectin resistance (see Fig. 2) nor does there appear to be a substantial glutathioneS-transferase component (see Table 3). Finally, comparison of the similarities of the two resistant strains demonstrates the utility of using EMS in conjunction with appropriate selection techniques (i.e., abamectin selection at an LD, dose level) as a means to generate resistance prior to the commercial use of an insecticide. In addition to the selection of polygenic resistant insect strains, drug resistant cell lines that contain both a point mutation and gene amplification mechanisms have also been developed by using EMS in conjunction with specific selection procedures (48). This demonstrates the importance of utilizing proper selection techniques in conjunction with EMS-induced mutations. The selection scheme used to produce the AB-L strain (9) cannot discount the possibility that resistance was due solely to abamectin selection pressure (i.e., an abamectin-only selection scheme was not included in the laboratory selection process). However, it is highly unlikely that EMS pretreatment played no role in the production of the

ABAMECTIN

RESISTANCE

IN

AB-L strain given that the initial resistant gene frequency for the process is calculated at 1 in 400. This number is at least one to two orders of magnitude larger than the highest estimate of spontaneous resistant gene frequencies currently reported (19, 49). Clearly, many resistance mechanism can be produced through the use of EMS in conjunction with proper selection techniques, making this a potentially powerful tool for resistance management studies and strategies. ACKNOWLEDGMENTS

The authors thank Nicole Boudreau and Evan Murray for their assistance in the bioassays. The authors also thank Drs. Richard Dybas and Rabi Babu of Merck, Sharp, and Dohme for their support of this project. Special appreciation is given to Dr. L. S. Crouch, Dept. of Animal and Exploratory Drug Metabolism, Merck, Sharp, and Dohme Res. Lab., for sharing his HPLC method and supplying standard abamectin metabolites. This work was supported by research Grant USDA-TPSU-UM-2977-403. REFERENCES

1. J. Putter, J. G. MacConnell, F. A. Preiser. A. A. Haidri, S. S. Ristich, and R. A. Dybas, Avermectins: Novel insecticides, acaricides and nematicides from a soil microorganism, Experientia 37, 963 (1981).

2. W. C. Campbell, M. H. Fisher, E. 0. Stapley, G. Albers-Schonberg, and T. A. Jacob, Ivermectin: A potent new antiparasitic agent, Science 221, 823 (1983). 3. R. H. ffrench-Constant and A. L. Devonshire, Monitoring frequencies of insecticide resistance in Myzus persicae (Sulzer) (Hemiptera: Aphididae) in England during 1985-86 by immunoassay, Bull. Entomol Res. 78, 163 (1988). 4. L. M. Field, A. L. Devonshire, R. H. ffrenchConstant, and B. G. Forde, The combined use of immunoassay and a DNA diagnostic technique to identify insecticide-resistant genotypes in the peach-potato aphid, Myzus persicae (Sulz.), Pestic. Biochem. Physiol. 34, 174 (1989). 5. M. A. Hoy and J. Conley, Selection for abamectin resistance in Teranychus urticae and T. pacificus, J. Econ. Entomol. 80, 221 (1987). 6. M. A. Hoy and Y. L. Ouyang, Selection of the western predatory mite, Metaseiulus occidentalis (Atari: Phytoseiidea), for resistance to abamectin, J. Econ. Entomol. 82, 35 (1989). 7. R. T. Roush and J. E. Wright, Abamectin: Toxic-

COLORADO

POTATO

BEETLE

205

ity to house flies (Diptera: Muscidae) resistant to synthetic organic insecticides, J. Econ. Entomol.

79, 562 (1986).

8. D. Cl. Cochran, Efficacy of abamectin fed to German cockroaches (Dictyoptera: Blattellidae) resistant to pyrethroids, J. Econ. Entomol. 83, 1243 (1990). 9. I. A. Argentine and J. M. Clark, Selection for abamectin resistance in Colorado potato beetle (Coleoptera: Chrysomelidae). Pestic. Sci. 28, 17 (1990). 10. J. G. Scott. Cross-resistance to the biological insecticide abamectin in pyrethroid-resistant house flies. Pestic. Biochem. Physiol. 34, 27 (1989). 11. G. H. Abro, R. A. Dybas, A. St. J. Green, and D. J. Wright, Toxicity of avermectin B, against a susceptible laboratory strain and an insecticide-resistant strain of P/ate//a xylostella (Lepidoptera: Plutellidae), J. Econ. Entomol. 81, 1575 (1988). 12. J. G. Scott. R. T. Roush, and N. Liu, Selection of high-level of abamectin resistance from fieldcollected house flies, Musca domestica, Experientia 47, 288 (1991). 13. Y. Konno and J. Cl. Scott, Biochemistry and genetics of abamectin resistance in the house fly, Pestic. Biochem. Physiol. 41, 21 (1991). 14. S. Yamamoto and H. Nishida, Acaricidal studies on milbemycins, a new family of macrolide antibiotics. I. Studies on a milbemycin-resistant strain of the two-spotted spider mite, Tetranythus urticae (Koch). J. Jpn. Sot. Appl. Anim. Entomol. 25, 286 (1981). 15. s Yamamoto, K. Ishikawa, and H. Nishida, Acaricidal studies on milbemycins, a new family of macrolide antibiotics. II. Joint effects of milbemycins and organophosphorus insecticides on a milbemycins-organophosphate-resistant strains of the two-spotted spider mite, Tetrunychus articae (Koch), J. Jpn. Sot. Appl. Entomol. Zool. 26, 29 (1982). 16. J A. Argentine, J. M. Clark, and D. N. Ferro, Genetics and synergism of azinphosmethyl and permethrin resistance in the Colorado potato beetle (Coleoptera: Chrysomelidae), J. Econ. Entomol. 82, 698 (1989). 17. N. E. Russell, J. L. Robertson, and R. M. Russell, POLO: A new computer program for computer analysis, Bull. Entomol. Sot. Am. 23, 257 (1977). 18. N. E. Savin, J. L. Robertson, and R. M. Russell, A critical evaluation of bioassay in insecticide research: Likelihood ratio test of dosemortality regression, Bull. Entomol. Sot. Am. 23, 257 (1977). 19. G. P. Georgiou, Genetics of resistance to insecti-

206

20.

21.

22.

23.

24.

25.

26.

27.

28.

29.

30.

31.

ARGENTINE,

CLARK,

tides in houseflies and mosquitoes, Exp. Parasitol. 26, 224 (1969). W. A. Brindley and A. A. Selim. Synergism and antagonism in the analysis of insecticide resistance, Environ. Enfomol. 13, 348 (1984). K. F. Armstrong and D. M. Suckling, Investigations into the biochemical basis of azinphosmethyl resistance in the light brown apple moth Epiphyus postvittuna (Lepidoptera: Tortricidae), Pestic. Biochem. Physiol. 32, 62 (1988). I. N. Leonova, S. V. Nedel’kina, and R. I. Salganik. Investigation of the enzyme systems of detoxification of insecticides in the Colorado beetle, Biokhimiya 51, 426 (1986). L. L. Murdock, G. Brookhart, P. E. Dunn, D. E. Foard, S. Kelly, L. Kitch, R. E. Shade, R. H. Shukle. and J. L. Wolfson, Cysteine digestive proteinases in Coleoptera, Camp. Biochem. Physiol. B 87, 783 (1987). N. M. Thie and J. G. Houseman, Identification of cathepsin B, D and H in the larval midgut of Colorado potato beetle, Leptinoiarsa decemlineuru Say (Coleoptera: Chrysomelidae), Insect Biochem. 20, 313 (1990). P. K. Smith. R. I. Krohn, G. T. Hermanson, A. K. Mallia, F. H. Gartner. M. D. Provenzano, E. K. Fujimoto, N. M. Goeke, B. J. Olson, and D. C. Klenk, Measurement of protein using bicinchoninic acid, Anal. Biochem. 150, 76 (1985). T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes. II. Solubilization, purification, and properties, J. Biol. Chem. 239, 2379 (1964). P. Jesudason, P. E. Levi, M. Weiden, and R. M. Roe, Developmental changes in the microsomal monooxygenase system and the metabolism of aldrin in larvae of the Mexican bean beetle (Coleoptera: Coccinellidae), J. Econ. Entomol. 81, 1598 (1988). T. Omura and S. Takesue, A new method for simultaneous purification of cytochrome b, and NADPH-cytochrome c reductase from rat liver microsomes, J. Biochem. 67, 249 (1970). B. V. Madhukar and F. Matsumura, Comparison of induction patterns of rat hepatic microsomal mixed-function oxidases by pesticides and related chemicals, Pesfic. Biochem. Physiol. 11, 301 (1979). R. T. Mayer, J. W. Jermyn, M. D. Burke, and R. A. Prough, Methoxyresorufin as a substrate for the fluorometric assay of insect microsomal 0-dealkylases. Pestic. Biochem. Physiol. 7, 349 (1977). L. R. Kao, N. Motoyama, and W. C. Dauterman, Multiple forms of esterases in mouse, rat and rabbit liver, and their role in hydrolysis of or-

32.

33.

34. 35.

36.

37.

38.

39.

40.

41. 42.

43.

44.

45.

AND LIN ganophosphorus and pyrethroid insecticides. Pestic. Biochem. Physiol. 23, 66 (1985). S. J. Yu and R. T. Ing, Microsomal biphenyl hydroxylase of fall armyworm larvae and its induction by allelochemicals and host plants, Camp. Biochem. Physiol. C 78, 145 (1984). G. L. Ellman, K. D. Courtney, V. Andres, and R. M. Featherstone. A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Phurmucol. 7, 88 (1961). K. van Asperen, A study of house fly esterase by means of a sensitive calorimetric method, J. Znsect Physiol. 8, 401 (1962). S. J. Yu. F. A. Robinson, and J. L. Nation, Detoxication capacity in the honey bee, Apis melliferu L., Pestic. Biochem. Physiol. 22, 360 (1984). A. L. Devonshire and G. D. Moores, A carboxylesterase with broad substrate specificity causes organophosphorus, carbamate and pyrethroid resistance in peach-potato aphids (Myzus persicue). Pesric. Biochem. Physiol. 18, 235 (1982). T. C. Sparks and B. D. Hammock, A comparison of induced and naturally occurring juvenile hormone esterases from last instar larvae of Trichoplosiu Ni, Insect Biochem. 9, 411 (1979). W. H. Habig, M. J. Pabst. and W. B. Jakoby, Glutathione S-transferases: The first enzymatic step in mercapturic acid formation, J. Biol. Chem. 249, 7130 (1974). L. S. Crouch, L. R. Gordon. W. F. Feely, M. S. Mayrand, L. D. Wise, and P. G. Wislocki, Nontoxicity of polar abamectin degradates from citrus fruit and then film photolysis, J. Agric. Food Chem. 40, 88 (1992). D. W. Helmuth, S. M. Ghiasuddin, and D. M. Soderlund, Polyethylene glycol pretreatment reduces pyrethroid adsorption to glass surfaces, J. Agric. Food Chem. 31, 1127 (1983). B. E. Tabashnik, Determining the mode of inheritance of pesticide resistance with backcross experiments, J. Econ. Enfomol. 83, 703 (1991). M. S. Maynard, B. A. Halley, M. Green-Erwin, R. Alvaro, V. F. Gruber, S. C. Hwang, B. W. Bennett, and P. G. Wislocki. Fate of avermectin B,, in rats. J. Agric:Food Chem. 38, 864 (1990). S. L. Chiu, E. Sestokas, R. Taub, J. L. Smith, B. Arison, and A. Y. Lu, The metabolism of avermectin-H,b,, and -H,B,s by pig liver microsomes. Drug Metub. Dispos. 12, 464 (1984). T. E. Anderson, J. R. Babu, R. A. Dybas, and H. Mehta, Avermectin B,: Ingestion and contact toxicity against Spodopferu eriduniu and Hefiothis virescens (Lepidoptera: Noctuidea) and potentiation by oil and piperonyl butoxide, J. Econ. Entomol. 79, 197 (1986). H. Mrozik, P. Eskola, B. 0. Linn, A. Lusi, T. L.

ABAMECTIN

RESISTANCE

IN

Shih, F. S. Waksmunski, M. J. Wyvratt, N. J. Hilton, T. E. Anderson, J. R. Babu, R. A. Dybas, F. A. Preiser, and M. H. Fisher, Discovery of novel avermectins with unprecedented insecticidal activity. Experientia 45, 315 (1989). 46. A. L. Devonshire and G. D. Moores, Detoxication of insecticides by esterase from Myzus persicae-Is hydrolysis important? In “Enzymes Hydrolysing Organophosphorus Compounds” (E. Reiner, W. N. Aldridge, and F. C. Hoskin, Eds.), pp. 180-192, Wiley, Chichester, England, 1989. 47. R. W. Beeman and J. J. Stuart, A gene for lindane

COLORADO

POTATO

BEETLE

207

+ cyclodiene resistance in the red flour beetle (Coleoptera: Tenebrionidae). J. Econ. Entomol. 83, 1745 (1990). 48. D. W. Shen, A. Fojo, J. E. Chin, I. B. Roninson, N. Richert, I. Pastan, and M. M. Gottesman, Human multidrug-resistant cell lines: Increased m&l expression can precede gene amplification. Science 232, 643 (1986). 49. M. J. Whitten and J. A. McKenzie, The genetic basis for pesticide resistance, in “Proceedings, 3rd Australian Conference on Grassland Invertebrate Ecology” (K. E. Lee, Eds.), pp. l-16 Government Printer, Adelaide, Australia, 1982.