Pesticide Biochemistry and Physiology 71, 124132 (2001) doi:10.1006/pest.2001.2568, available online at http://www.idealibrary.

com on

Altered Acetylcholinesterase Confers Organophosphate Resistance in the Olive Fruit Fly Bactrocera oleae
John G. Vontas,*,1 Nikos Cosmidis, Michael Loukas, Spyridon Tsakas, Mir Jalil Hejazi, Anna Ayoutanti, and Janet Hemingway*
*Cardiff School of Biosciences, Cardiff University, Cardiff CF10 3TL, Wales United Kingdom; Department of Genetics, Agricultural University of Athens, Athens, Greece; and Department of Plant Protection, College of Agriculture, University of Tabriz, Tabriz, Iran Received March 23, 2001; accepted July 2, 2001 An organophosphate-resistant strain of the olive fruit fly Bactrocera oleae, the most important pest for olive orchards worldwide, was obtained by laboratory selection with dimethoate. Resistance mechanisms were investigated in comparison with the colonized parental strain and a field population collected from the same area after 12 years of continuous dimethoate-based insecticide pressure. Combined biochemical and bioassay data suggested that, although esterase and/or glutathione S-transferase metabolic pathways were present and active against dimethoate, they were not selected for and did not have a major role in resistance. There was no evidence of increased oxidase activity in the resistant strains or significant synergism of dimethoate toxicity by piperonyl butoxide; thus, oxidative metabolism was not a major component of resistance. An altered acetylcholinesterase (AChE) with poorer catalytic efficiency for the substrate acetylthiocholine iodide and 5- to 16-fold lower sensitivity to inhibition by omethoate was the major resistance mechanism. Dimethoate selected the insensitive AChE allele(s) in the resistant insects, which were also insensitive to paraoxon, but the altered AChE mechanism conferred negative cross-resistance to the carbamate propoxur. 2001 Academic Press Key Words: Bactrocera oleae; dimethoate; organophosphate resistance; altered acetylcholinesterase.

INTRODUCTION

The olive fruit fly, Bactrocera oleae (formerly Dacus oleae), is the most important olive orchard pest in southern Europe, Asia, and Africa. It causes severe economic damage to olives, due to quantitative losses and reduced quality of both olives and olive oil. Although sterile insect release, mass trapping, and sex pheromone mating disruption technologies have been employed to manage B. oleae, chemicals remain the principal control tool. In the Mediterranean basin for more than three decades, control has been based on the intense use of the organophosphorus insecticide dimethoate, because of its low residual persistence in olive oil (1). In Greece, economic losses brought about by B. oleae are annually estimated at 30% of total production and the cost of B. oleae chemical
To whom correspondence should be addressed. E-mail: vontasj@cardiff.ac.uk.
1

control in 1994 was estimated at U.S. $20 million, involving almost 75% of the olive orchards in the country (2). The appearance of B. oleae insecticide-resistant populations in olive groves has seriously threatened olive production; however, no investigation of the spectrum of resistance mechanisms developed has been undertaken. In the early 1970s the selective action of dimethoate on an acetylcholinesterase (AChE; EC 3.1.1.7) polymorphism of B. oleae was reported (3, 4). It was concluded that dimethoate selected for quantitatively but not qualitatively altered AChE isoenzymes and killed preferentially flies which had less AChE (5). A duplication of the AChE gene was hypothesized to be involved in resistance. AChE is a key enzyme in the insect nervous system. It terminates nerve impulses by catalyzing the hydrolysis of the neurotransmitter acetylcholine (6). It is the major molecular target for

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organophosphate and carbamate insecticides, which inhibit enzyme activity by covalently phosphorylating or carbamylating the serine residue within the active site gorge. AChE is restricted to the insect central nervous system and is composed of a single molecular form which is a glycosylated dimer attached to the membrane via a glycolipid anchor (7). Quantitative and qualitative changes in AChE confer resistance to insecticides. Fournier et al. (8) showed that insecticide resistance is correlated with overexpression of AChE, in a manner similar to that described for scavenger or metabolizing proteins. However, point mutations accompanied by a modification of kinetic parameters of acetylcholine hydrolysis have more often been identified as being responsible for insecticide resistance (6, 911). In the present investigation we show from both in vivo and in vitro results that modification of AChE is the dominant factor in organophosphate resistance in B. oleae.
MATERIALS AND METHODS

the abdominal sternum of 1-day-old adult insects with a 10-l Hamilton microsyringe. Controls were exposed to acetone alone. Treated insects were maintained at 25C for 24 h until mortality was scored. For selection experiments the LD80 value for dimethoate was determined for each generation. Mass selection was then undertaken at this dosage and survivors were used to establish the next generation. Two synergists, PB and DEF, were used to determine whether metabolism was involved in dimethoate resistance. PB inhibits monooxygenases (MFO), and DEF inhibits both esterase (Est) and glutathione S-transferase (GST) metabolic pathways. Insects were pretreated with the maximum sublethal doses of synergist, determined after a 24-h recovery period, i.e. 0.5 g/insect of PB and 0.2 g/insect of DEF. Insecticide treatment was administered 1 h after synergist treatment. Data from bioassays were subjected to log-time probit mortality data regression analysis with a program written by Dr. C. Schofield (WHO, Geneva). Biochemical Assays One-day-old adults were frozen and stored at 70C until used for biochemical assays. Soluble protein extracts were prepared by homogenization of 10 insects in 1.4 ml of 50 mM sodium phosphate buffer, pH 7.2. A 10,000g supernatant was obtained by centrifugation for 10 min at 4C and used as the source of enzymes for determination of Est, GST, and Se-independent glutathione peroxidase (GPOX) activities. In preliminary experiments, AChE activities of various fractions resulting from differential centrifugation of B. oleae crude homogenates, prepared in the presence and absence of Triton X-100, were assayed (13). In the absence of detergent, more than 60% of AChE activity precipitated after centrifuging at 100,000g, indicating that the majority of the enzyme was membrane bound. Thus, extracts of total proteins for the AChE study were prepared as described for the soluble protein, except that decapitated insects (heads) were homogenized, in the presence of 1% Triton X-100. Microsomal proteins were obtained from

Chemicals Insecticides and their oxon analogues used in this study were of analytical grade and purchased from British Greyhound (Birkenhead, Merseyside, UK). Piperonyl butoxide (PB), S,S,S-tributyl phosphorothioate (DEF), and all other reagents were purchased from Sigma (Poole, Dorset, UK) except as stated. Insects The LS strain was established from wild B. oleae collected from Attiki, Greece in 1987 and maintained under artificial conditions (12). Adult dimethoate selection at the LD80 (log dose that kills 80% of the insects) for seven generations resulted in the LR dimethoate-resistant strain. A field population was collected from Attiki in September 1999, after 12 years of continuous dimethoate-based insecticide pressure (two to three sprays per year). Bioassays Bioassays were done by topical application. Insecticide was delivered in 0.4 l acetone to

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adult abdomens, to avoid eye pigments and high quantities of mitochondria rich in cytochromes present in the thoracic muscles. Briefly, 10 abdomens were homogenized in 1 ml of sodium phosphate buffer, pH 7.2, containing 1 mM EDTA, 0.1 mM dithiothreitol, and 0.4 mM phenylmethylsulfonyl fluoride. Total cytochrome content was measured in two replicates of 20 l of homogenate. The remaining homogenates were centrifuged at 10,000g for 10 min at 4C and the supernatant was centrifuged at 100,000g for 1 h at 4C to obtain the microsomal pellet, which was resuspended in the same buffer before being used to determine monooxygenase activity. Est and GST activities were determined in a UVmax microtiter plate reader (Molecular Devices, USA), as detailed by Penilla et al. (14). Briefly, for the Est activity assay, 5 l of the soluble protein extract was incubated for 30 min at 25C, in 200 l of - or -naphthyl acetate solution (0.3 mM - or -naphthyl acetate, 20 mM sodium phosphate buffer, pH 7.2). At the end of the reaction, 50 l of 20 mM fast blue garnet was added to the incubation mixture and the optical density was recorded at 570 nm. Units are given in nmol - or -naphthol/min/mg protein. For the GST activity assay, 5 l of the soluble protein extract was mixed with 200 l of 1-chloro-2,4-dinitrobenzene (CDNB) or 1,2dichloro-4-dinitrobenzene (DCNB) substrate solution (10 mM reduced glutathione, 3 mM CDNB or 1 mM DCNB, 20 mM sodium phosphate buffer, pH 6.5 or 8) and the activity was measured at 340 nm for 5 min. Units are given in mmol CDNB or DCNB conjugated/min/mg protein. Se-independent GPOX activity was measured by a method adapted from Lawrence and Burk (15), as detailed before (16). Briefly, 60 l of the soluble extract was added to a reaction mixture which consisted of 1 mM EDTA, 0.2 mM NADPH, 1 E.U./ml glutathione reductase, and 1 mM GSH, in a total volume of 300 l. The mixture was incubated for 5 min at 25C, before the initiation of the reaction by the addition of cumene hydroperoxide (1.5 mM). Oxidation of NADPH at 25C was measured by the decrease in absorbance at 340 nm for 4 min. Activity values were substracted from blanks for

the nonenzymatic oxidation of NADPH by the peroxide substrate and nonspecific oxidation of NADPH by the crude homogenate. Units are given as mol NADPH oxidized/min/mg protein. MFO activities were studied by measurement of the amounts of cytochromes P450 and oxidase activity with a fluorogenic substrate. An estimate of total cytochrome P450 content was obtained by the hemeperoxidase assay, as modified for use in microtiter plates (17), and units are give in cytochrome P450 equivalents (103). Oxidase activity was determined in microsomal preparations, according to Ulrich and Weber (18), as modified by Stumpf and Nauen (19) with 7-ethoxycoumarin as a substrate. The liberation of fluorescent 7-hydroxycoumarin was detected at 380 nm (excitation) and 480 nm (emission) on a F2 fluorescence absorbence reader (Labtech, UK). Units are given in nmol 7-hydroxycoumarin/min/mg protein. Protein was assayed with the Bio-Rad protein assay kit (Bio-Rad, UK), with bovine serum albumin (BSA) as the standard protein (20). AChE activity was assayed by the spectrophotometric method of Ellman et al. (13) at 25C, with acetylthiocholine iodide (ATChI) as substrate. Apparent Km values were determined at 25C by nonlinear regression (Micromath Scientific Software Inc.) of enzyme activity measurements over 2 min with ATChI concentrations from 0.05 to 2 mM. Results comprise the mean values of three separate preparations with two determinations for a minimum of 10 concentrations. The bimolecular rate constant (ki) was estimated in the absence of substrate by a preincubation method adapted from Devonshire (21) and Moores et al. (22). In brief, 2530 l of the total protein extract was incubated in 50 mM phosphate buffer, pH 7.2, 0.5% Triton X-100, with various concentrations of the inhibitor. At short time intervals, residual activity was measured by placement of the inhibition mixture into an excess of ATChI and dithiobis 2-nitrobenzoic acid (DTNB). The plot of the ln of residual activity (Vi/Vo) against time was linear for a

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given inhibitor concentration (up to 3 min). Slopes and standard errors of inhibition lines were calculated by linear regression and converted to bimolecular rate constants as described by Main and Iverson (23). This constant was estimated at five different concentrations for each inhibitor, with three determinations for each time point, in five independent experiments and 10 different extracts. For the interaction of Est with the insecticides tested, stopped-time inhibition assays were performed with -nitrophenyl acetate (-NPA) as the substrate. Briefly, soluble protein extracts were preincubated with a series of concentrations of the inhibitor, and at 0.5- to 5-min intervals aliquots were withdrawn and diluted with the substrate immediately to stop further inhibition. Residual activity was determined by measurement of the rate of substrate hydrolysis, divided by those measured in the absence of insecticide, and the bimolecular rate constants ka for the formation of the acylated enzyme were estimated according to Aldridge and Reiner (24). All biochemical data were subjected to analysis of variance (ANOVA). Sceffes or Dunnets method were used as the post hoc test. The differences described in the results and shown in the figures were statistically significant at P 0.01. Nondenaturating gel electrophoresis was performed according to methods adapted from Siegfried and Scott (25) and Zhu and Brindley (26), in a Bio-Rad Mini-Protean II vertical electrophoresis (7.5% polyacrylamide gel, 2.5% stacking gel, 5 mM tris/38 mM glycine buffer, pH 8.3). Individual heads were homogenized in 0.1 M sodium phosphate buffer (pH 7.6) containing 1% Triton X-100 and 10% sucrose and mixed

with gel running buffer 1:1 prior to electrophoresis. Approximately equal amount of protein (equivalent to one head) were loaded onto the gel. The electrophoresis was performed in the presence or absence of 0.5% Triton X-100 (both in running buffer and in the gel). At the end of the migration (10 V/cm, 4 h, 4C) the gels were rinsed in sodium acetic acid (0.1 M, pH 6.0) and stained for AChE activity according to the method of Karnovsky and Roots (27).
RESULTS

A five-fold resistant strain (LR) of B. oleae was obtained by laboratory selection with dimethoate, after seven generations of continuous selection pressure at the 80% mortality level. The Attiki field population, collected from the same place after 12 years of insecticide pressure by dimethoate, was 9-fold resistant (Table 1). No significant synergism of dimethoate toxicity was observed in any strain, when adults were pretreated with PB (Table 1). Similarly, there was no evidence of an increase in cytochrome P450 content or oxidase (O-dealkylate) activity in either resistant strains (Table 2). Hence oxidative metabolism is not a major component of dimethoate resistance in these strains. The GST activities with both CDNB and DCNB were similar in the parental (LS) and selected (LR) strains (Table 2) and significantly lower in the field population. There was no significant difference in GPOX activities in strains tested. Est activities were slightly lowered by insecticide selection (LR). DEF, an inhibitor of both Est and GST activity, significantly synergized dimethoate toxicity in bioassays to an almost equal extent in all the

TABLE 1 The Response of Bactrocera oleae to the Organophosphate Dimethoate Alone or after Synergist Exposure Strain and LD50 (fiducial limits, ng/insect) Treatment Parental (LS) Slope SRa Selected (LR) Slope SR RRb 1.8 1.9 1.7 1.1 1.7 5 5 5 Field (Attiki) 0.94 (0.890.98) 0.81 (0.760.85) 0.60 (0.570.62) Slope SR RR 2.6 2.3 2.6 9 1.1 9 1.6 10

Dimethoate 0.11 (0.070.14) 2.3 Dimethoate PB 0.09 (0.070.014) 2.2 Dimethoate DEF 0.06 (0.020.08) 2.2
a b

0.55 (0.520.57) 1.2 0.50 (0.440.55) 1.8 0.32 (0.280.35)

Synergistic ratio LD50 of insecticide alone/LD50 of insecticide with synergistic. Resistance ratio LD50 of insecticide of resistant strain/LD50 of insecticide of susceptible strain (LS).

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VONTAS ET AL. TABLE 2 The Effect of Dimethoate Selection on Resistance Ratios and Enzyme Activities on Bactrocera olea
Mean esterase specific activity Dimethoate Mean GST specific activities CDNB
a a

MFO Cytochrome content

a

Strain LS LR Attiki

LD50 0.11 0.55 0.94

RR 1 5 9

-Naphthylacetate
a

-Naphthylacetate

DCNB
a

Cumene hydroperoxide 0.117 0.009 0.120 0.010a 0.106 0.012a

a

ECOD

0.130 0.008 0.136 0.008 0.17 0.006 0.012 0.001 0.112 0.007b 0.119 0.009a 0.19 0.008a 0.011 0.001a 0.123 0.007a 0.134 0.009a 0.13 0.009b 0.008 0.001b

0.053 0.01 0.21 0.04a 0.052 0.01a 0.22 0.04a 0.049 0.02a 0.20 0.05a

Note. Values are the mean SE; n 4060 samples. All specific activities are given in units/mg protein. In the same column, different superscript letters indicate a significant difference.

strains (Table 1). These results are consistent with those for in vitro Est and GST activities. Hence, although these metabolic or sequestration pathways were present for dimethoate, they did not have a major role in resistance. AChE activity toward the substrate ATChI in the LS strain was 40% higher than either enzyme preparation from the resistant strains, which did not differ significantly from each other (Table 3). Comparison of the apparent Km of AChE from the resistant strains with that of the LS strain shows a reduced affinity for the substrate acetylthiocholine (Table 3). This reduced catalytic efficiency was associated with a marked insensitivity of the resistant AChE alleles to the dimethoate oxon analogue, omethoate (Table 4). Comparison of the ki values for the LR and Attiki strains to that for the LS strain shows that the former were 5- and 16-fold less sensitive to inhibition by omethoate. The omethoate-resistant AChE allele(s) were insensitive to paraoxon, but conferred a significant level of negative crossresistance to the carbamate propoxur (Table 4). Interaction studies of Est with the insecticides tested, based on the comparison of bimolecular

rate constants, ka, showed homogeneity among the strains tested (data not shown). This suggests that the differences in AChE sensitivity observed were genuine and not a result of differential binding of the inhibitor by other Est. The linearity of the inhibition curves (Fig 1), determined by use of much lower concentrations of insecticides and longer preincubation times than those used for the determination of ki values, suggests that the resistant strains are homozygous. Even after 15 generations of selection, the ki of the AChE of the selected strain and the slope of this curve were the same. Thus, the presence of a different insensitive AChE allele in the Attiki population that might not be present in the LR strain was possibly indicated. A plateau at 5% remaining activity, in assays based on coincubation with substrate and omethoate (22), suggested the presence of a rare insensitive enzyme component in the LS strain that was selected after seven generations of insecticide pressure (data not shown). The linearity of the inhibition curves presented in Fig. 1 may also suggest the presence of only one gene coding for AChE in B. oleae, although

TABLE 3 Km and Specific Activity of AChE from Resistant and Susceptible Bactrocera oleae with Acetylthiocholine Iodide Specific activity Strain LS LR Attiki Km (mM) 0.32 0.03a 0.42 0.02b 0.45 0.07b nmol/min/mg protein 97.4 8.4a 60.6 4.9b 56.0 8.3b nmol/min/head 30.3 2.7a 18.5 1.5b 17.5 2.4b

Note. Values are the mean SE of three determinations. In the same column, different superscript letters indicate a significant difference.

ORGANOPHOSPHATE RESISTANCE IN Bactrocera oleae TABLE 4 Bimolecular Rate Constants (103 ki, M1 min1 SE) for Inhibition of AChE from Resistant and Susceptible Bactrocera olea Insects Strain LS LR Attiki Omethoate 4.50 0.41 0.89 0.09b 0.29 0.06c
a

129

Paraoxon 68.2 5.2 18.7 2.0b 12.3 2.6c

a

Propoxur 93.8 12.3a 135.5 15.9b 146.2 14.1b

Note. Kinetic constants derived from three to five separate experiments. In the same column, different superscript letters indicate a significant difference.

FIG. 2. Nondenaturating PAGE of AChE from heads of adult female Bactrocera olea (lane 1, LS, lane 2, LR, lane 3, Attiki) in the presence and absence of Triton X-100 (both in the buffer and in the gel). The running conditions and the staining are described in the text.

based on these data we cannot exclude the possible presence of a second gene with similar kinetic and insecticide-affinity properties. AChE enzyme preparations from resistant and susceptible B. oleae adults (heads) behaved as a single moiety (Fig. 2) and could not be distinguished electrophoretically, whether or not Triton X-100 was incorporated into the gels and the running buffer. In gels containing no Triton X-100, AChE electrophoretic mobility was slightly lower than that containing 0.5% Triton X-100.
DISCUSSION

Although chemical control remains the principal tool for B. oleae control in olive orchards worldwide, no investigations of the spectrum of the actual or potential resistance mechanisms have been undertaken.

FIG. 1. Inhibition of acetylcholinesterase from Bactrocera oleae heads by 0.03 mM omethoate concentration in preincubation assays.

We investigated the physiological mechanisms responsible for organophosphate resistance in B. oleae, present after laboratory and natural field selection. From both in vivo and in vitro results, modification of AChE appears to be the dominant factor in resistance. Previous studies in B. oleae reported that dimethoate tolerance was correlated with overexpression of AChE, via a possible duplication of the AChE gene (35). In our study, dimethoate pressure, both in the laboratory and in nature, resulted in the selection for mutant AChE allele(s) with reduced catalytic activity for the substrate acetylthiocholine iodide and great insensitivity to the dimethoate oxon analogue, omethoate. Hence we showed a qualitative rather than a quantitative change associated with organophosphate resistance, although our data do not allow us to exclude the latter. However, a decrease in AChE activity can originate from point mutations in the active site, but not from an increased amount of enzyme. Although insensitive AChE is an important organophosphate resistance mechanism (28, 29), it has been suggested that it is not common in insect species (6). Genetic parameters that affect intracistronic recombination explain this insect specificity (6). However, altered AChE has recently been identified in species such as the olive fruit fly B. oleae, the greenbug Schizaphis graminum (30), and the green rice leafhopper Nephotettix cincticeps (31), showing that this mechanism is not restricted to a few species. Resistance-associated mutations in insecticide-insensitive AChE in various species

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involve seven or more conserved key amino acid replacements which reduces the enzyme phosphorylation rate by the organophosphate (6, 10, 11). Generally the levels of organophosphate insensitivity conferred by each individual replacement are low, but combinations of these replacements produce increasingly resistant enzymes (32). This alteration of AChE is usually, but not always, associated with a decrease in the efficacy of acetylthiocholine iodide hydrolysis (30, 33), as demonstrated in Musca domestica (21), Cacopsylla pyri (29), and Myzus persicae (22). Our results correspond with this observation. The dimethoate-selected AChE-insensitive allele(s) in resistant B. oleae insects were insensitive to paraoxon, the oxon analogue of parathion, which has also been employed in the control of B. oleae. However, they conferred negative cross-resistance to the carbamate propoxur. Similar negative cross-resistance to carbamates occurs in the lesser grain borrer, Rhyzopertha dominica (34). Our in vitro analysis of AChE inhibition suggests that a single gene codes for AChE in B. oleae, as in most but not all insects (35), and that more than one form of insensitive AChE is present in the resistant strains, as in other species (10, 36). However, genetic crosses and molecular data are required to confirm this hypothesis. Synergist and MFO activity analysis showed that oxidative metabolism is not a major component of resistance. However, the oxidative activation of dimethoate by monooxygenase to produce an efficient inhibitor of AChE complicates this analysis. Est- and/or GST-based organophosphate metabolic pathways were present, but they were not selected for and did not have a major role in resistance. Interestingly, GSTs were significantly increased in the laboratory strain. Our combined biochemical and bioassay data indicate that this difference is probably attributable to the artificial rearing, as has been shown before in B. oleae for other enzymes (37), rather than being resistance related. Recent studies suggest that mechanisms other than changes in the protein structure by amino

acid substitution, such as alternate transcriptional variation, posttranscriptional modifications, or the presence of multiple AChE loci, might be involved in insensitive AChE-mediated insecticide resistance (38, 39). Comparison of cDNA sequences of individual resistant and susceptible insects in B. oleae, a new species with altered AChE based organophosphate resistance, would be of great interest.
ACKNOWLEDGMENTS
We thank Professor Alan Devonshire for his expert advice and two anonymous reviewers for their constructive suggestions. A European Union Marie Curie TMR Grant (MCFI1999-00259) supported J.V.

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