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Nov 26, 2010 - Insecticide-resistant strains of the brown planthopper,. Nilaparvata lugens, from four locations all had a single diffuse elevated esterase band ...
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Characterization of the elevated esteraseassociated insecticide resistance mechanism in Nilaparvata lugens (Stal) and other planthopper species S. H. P. P. Karunaratne , G. J. Small & J. Hemingway Published online: 26 Nov 2010.

To cite this article: S. H. P. P. Karunaratne , G. J. Small & J. Hemingway (1999) Characterization of the elevated esterase-associated insecticide resistance mechanism in Nilaparvata lugens (Stal) and other planthopper species, International Journal of Pest Management, 45:3, 225-230, DOI: 10.1080/096708799227833 To link to this article: http://dx.doi.org/10.1080/096708799227833

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INTERNATIONAL JOURNAL OF P EST MANAGEME NT, 1999, 45(3) 225± 230

Characterization of the elevated esterase-associated insecticide resistance mechanism in Nilaparvata lugens (StaÊ l) and other planthopper species (Keywords: Brown planthopper, esterase, organophosphate resistance)

S. H. P. P. KARUNARATNE ² , G. J. SMALL³ and J. HEMINGWAY*³

² Department of Zoology, University of P eradeniya, P eradeniya, Sri Lanka

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³ Cardiff School of Biosciences, University of Wales Cardiff, P O Box 915,C ardiff, C F 1 3TL, UK

Abs tract. Insecticide-resistant strains of the brown planthopper, Nilaparvata lugens, from four locations all had a single diffuse elevated esterase band on native polyacrylamide gel electrophoresis. The white backed planthopper Sogatella furcifera had two elevated esterases with lower relative mobilities than the N. lugens esterase, while the insecticide-susceptible N. bakeri and the grass-associated sibling species of N. lugens sensu lato all had a single low intensity staining esterase with a lower relative mobility than the resistanceassociated N. lugens esterase. All the esterases were inhibited by pre-incubation with 0.1 mM paraoxon, but were not affected by permethrin up to its solubility limit, indicating their possible role in organophosphorus, but not pyrethroid insecticide, resistance. Partial purification of the elevated esterase from insecticide resistant Sri Lankan N. lugens showed that despite its diffuse nature on gels it purified as a single isoform, with a specific activity of 5.85 l mol 1 1 min mg and an estimated molecular weight of approximately 60 kDa. Insecticide resistance was conferred by this elevated esterase through rapid binding and slow turnover of the carbamate or the insecticidally active oxon analogues of the phosphorothioates, i.e. sequestration rather than metabolism is the primary resistance mechanism. In contrast to earlier studies on the elevated esterases of N. lugens from Japan and the Philippines, we were unable to detect any malathion metabolism by this esterase, nor did malathion inhibit the esterase, although it was sensitive to inhibition by malaoxon. The elevated N. lugens esterase did not cross-react with antisera raised to the elevated mosquito or aphid esterases. The efficacy of the partially purified N. lugens esterase in insecticide sequestration and turnover is compared with other well characterized amplified esterases from mosquitoes and aphids. Ð

1.

Ð

Introduction

The brown planthopper Nilaparvata lugens (StaÊl) is a major rice pest in many parts of Asia. Extensive use of insecticides has selected for resistance in populations of this pest collected from rice in Japan, the Philippines, the Solomon islands, Sri Lanka and Taiwan (Tranter, 1983; Hasui and Ozaki, 1984). Until the late 1970s, N. lugens was thought to be a single species attacking only wild species of rice and the cultivars of Oryza. However, populations of the insects have now been found widely living and feeding on a common semi-aquatic grass, Leersia hexandra Sw. These insect populations were at first regarded as a biotype of the N. lugens, but have since been shown to represent a distinctly sibling species which does not interbreed in the field with riceassociated N. lugens (Claridge et al., 1985). Insecticide resis-

tance has not, as yet, been detected in the Leersia-associated sibling species (Hemingway et al., 1999). The major insecticide resistance mechanism reported to date in N. lugens collected from rice is elevation of one or more esterases (Chen and Sun, 1994). We recently determined the resistance spectrum and probable underlying resistance mechanism of two strains of N. lugens collected from rice in Sri Lanka and Indonesia (Hemingway et al. 1999). These resistant strains had low level (2 ± 5-fold) resistance to the insecticides malathion, permethrin and propoxur associated with an elevation of esterase activity and, on native 7.5% polyacrylamide gels (PAGE), only a single diffuse elevated esterase band was apparent compared with the insecticide-susceptible sibling species collected from Leersia (Hemingway et al. 1999). Early studies on N. lugens using starch gel electrophoresis suggested a single monofactorially inherited elevated esterase was involved in resistance (Chung and Sun, 1983). Later work demonstrated more than ten forms of esterase on isoelectric focusing, at least three of which were elevated in resistant insects from Japan and the Philippines, although one esterase, E1, was predominant (Chen and Sun, 1994). It has been proposed that the elevated esterases in N. lugens confer both malathion and permethrin resistance by increased rates of hydrolysis of these insecticides, and more broad spectrum organophosphate resistance through sequestration of the oxon analogues of the organophosphorus insecticides (OPs) (Chen and Sun, 1994). The aphid Myzus persicae contains an amplified esterase E4 which hydrolyses permethrin and sequesters the oxon analogues of the OPs, although this esterase is unable to metabolize malathion (Devonshire and Moores, 1982). Many insecticide-resistant Culex quinquefasciatus mosquito strains contain two amplified esterase genes Esta 2 and Estb 2, or various forms of amplified Estb 1 (Vaughan and Hemingway, 1995; Vaughan et al., 1995). All these genes give broad spectrum resistance through enzymes which sequester the carbamates or the oxon analogues of the OPs (Karunaratne et al., 1993, 1995; Ketterman et al., 1993). None of the Culex esterases are able to metabolize either permethrin or any of the organophosphorus insecticides. The apparent low levels of resistance present in the Sri Lankan and Indonesian strains of N. lugens from rice, coupled

*To whom correspondence should be addressed. e-mail: [email protected] International Journal of P est Management ISSN 0967-0874 print/ISSN 1366-5863 online http://www.tandf.co.uk/JNLS/tpm.htm http://www.taylorandfrancis.com/JNLS/tpm.htm

Ó

1999 Taylor & Francis Ltd

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with high frequencies of individuals with elevated esterase activity in these strains suggests that resistance is due to sequestration rather than metabolism in this species. To test this hypothesis, further purification and characterization of the elevated esterase from N. lugens was undertaken. A number of other field collections of planthoppers were also analysed for their esterases banding patterns on native polyacrylamide gel electrophoresis to determine how common the elevation of this specific esterase is throughout Asia.

2.

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2.1.

Materials and methods Insects

Nine strains of planthopper were used. These were N. lugens from Batalagoda, Sri Lanka; Garvi and Sulawesi, Indonesia; Puri, India; and Ingham, Australia collected from rice, the sibling species of N. lugens from Bogor, Indonesia and Goa, India collected from the wild grass Leersia hexandra, N. bakeri from Puri, India collected from Leersia, and Sogatella furcifera, the white-backed planthopper from Suramandi, W. Java, Indonesia collected from rice. The Culex quinquefasciatus Say mosquito strains Pel RR, in which all individuals have the 1 1 amplified esterases Esta 2 and Estb 2 , and Pel SS, in which all 3 mosquitoes have the non-amplified Esta 3 and Estb 1 alleles, were used as standard reference strains for electrophoretic and immunoblotting work.

Size estimations of the native elevated esterase was undertaken on pre-cast 4 ± 20% gradient gels. For each gradient native PAGE log molecular weight was plotted against log relative mobility to determine the esterase molecular weight against a standard curve of known molecular weight proteins.

2.3.

Partial purification of elevated esterases

Q-Sepharose Fast flow and nick spin columns were purchased from Pharmacia, UK. Hydroxyapatite and the protein assay kits were purchased from Bio-Rad, UK. Chemicals were purchased from Sigma, UK except where stated otherwise. Permethrin [60 : 40 trans : cis ratio] (3-phenoxybenzyl(1RS,3RS;1RS,3SR )-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropane carboxylate), (97.2% pure), malaoxon (92% pure) and paraoxon (98% pure), the oxon analogues of the insecticides malathion (diethyl (dimethoxythiophosphoroylthio) succinate) and parathion (diethyl-4-nitrophenyl phosphate) were purchased from ChemServices, Birkenhead, UK. Propoxur (2-isopropoxyphenyl methylcarbamate) was a gift from Bayer, Monheim, Germany. During the purification, esterase activity was followed using the substrate p-nitrophenol acetate. Ten l l of the crude homogenate was mixed with 200 l l of 1 mM substrate solution in 50 mM phosphate buffer (pH 7.4) in a microtitre plate well and the increase in absorbance at 405 nM was continuously monitored for 1 ± 2 min in a UVma x microtitre plate reader (Molecular Devices, USA) at 228 C. An extinction co-efficient of 1 6.53 mM (corrected for a path length of 0.6 cm) was used to convert the absorbance to moles. Protein concentrations of the fractions were determined by the method of Bradford (1976) using bovine serum albumin as the standard protein. In a microtitre plate well, 10 l l of protein sample was mixed with 300 l l of working solution (prepared according to the instructions of the manufacturer) and the absorbance was read at 570 nm after a 5 min incubation at 228 C. The planthoppers (1 ± 2 g) were homogenized in 25 ml of bistris propane buffer (pH 6.5) containing 15 mM DTT. The homogenate was centrifuged at 14 000 g for 10 min and the supernatant was applied to a Q-Sepharose fast flow column (4.4 cm ´ 4 cm) equilibrated with 25 mM bistris propane buffer (pH 7.0). The esterase was eluted with a five-bed-volume salt gradient (0 ± 0.5 M NaCl in homogenization buffer) and the elution profiles were determined for esterase activity, conductivity and protein. The esterase activity eluted in a single peak. The active fractions were combined and dialysed against dry sucrose. Buffer exchange into the hydroxyapatite buffer was performed on PD10 columns according to manufacturers’ instructions. The sample was applied to a 2.2 ´ 5.4 cm hydroxyapatite column equilibrated with 10 mM phosphate buffer, pH 6.8 containing 50 mM NaCl and 10 mM DTT. The esterase activity was eluted with a five-bed-volume gradient of 10 ± 200 mM phosphate buffer pH 6.8. A single peak of esterase activity eluted. The active fractions were collected and concentrated in an Amicon centricon 10 concentrator unit. Ð

2.2.

Electrophoresis and immunoblotting

Electrophoresis of native protein samples was performed in 7.5% polyacrylamide gel electrophoresis (PAGE) in tris/ borate buffer, pH 8.0 by the method of Davis (1964). Five adult planthoppers or three adult mosquitoes were homogenized in 100 ± 150 l l of 50 mM phosphate buffer pH 7.4 containing 10 mM dithiothreitol (DTT). Homogenate volumes containing 13 l g of protein were loaded into each well. The gels were stained for esterase activity with 0.4% (w/v) a - and b -naphthyl acetate and 0.1% (w/v) Fast blue B in 20 mM phosphate buffer pH 7.4. For inhibition studies, gels were incubated after electrophoresis in 0.1 mM paraoxon or permethrin in 50 mM phosphate buffer pH 7.4 for 15 min, and then stained for esterase activity as above in the presence of the insecticide. For immunoblotting native PAGE was performed as above. The gels were then equilibrated in transfer buffer (25 mM Tris, 0.2 M glycine, pH 8.3) for 15 min, and blotted onto Hybondenhanced chemoluminescent (ECL) membranes using a transblot SD semi-dry electrophoretic transfer cell (Bio-Rad, UK). Non-specific sites on the membrane were blocked with 5% (w/v) ECL blocking powder in T-PBS [0.1% (v/v) Tween-20 in phosphate buffered saline] for 1 h. The membrane was washed with T-PBS and incubated with primary antibody (1 : 2000 1 dilution of antibody raised to mosquito Esta 2 or aphid E4 esterase in T-PBS) for 1 h. The membrane was then washed with T-PBS and incubated with secondary antibody (Rabbit IgG, horseradish peroxidase-linked whole antibody at 1 : 1000 dilution in T-PBS). The membrane was again washed and incubated with ECL detection reagent for 1 min before exposure to hyperfilm ECL for 15 ± 30 s.

2.4.

Influence of effectors

Solutions of EDTA (1 mM) and various metal ions (1 mM) were prepared in either 50 mM phosphate buffer pH 7.4 or

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Insecticid e resistanc e mechanism in plant hoppers

25 mM bistris propane buffer pH 7.4 depending on their solubility. Each effector was pre-incubated with the partially purified esterase for 30 min at 228 C. Esterase activity was then measured with 1 mM p-nitrophenyl acetate in the presence of each effector.

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2.5.

activity to the N. lugens from rice, but on electrophoresis the esterases were resolved into two distinct bands, one b -naphthyl acetate and the other a -naphthyl acetate specific. Both Sogatella esterase bands migrated more slowly than the N. lugens electromorphs (figure 1).

Kinetic constants

The partially purified esterase was incubated with a series of concentrations of malaoxon (0.5 ± 2 l M), paraoxon (0.025 ± 0.1 l M) or propoxur (200 ± 800 l M). Stock solutions of insecticide (100 mM) were prepared in acetonitrile and diluted in phosphate buffer pH 7.4 immediately prior to each experiment. The concentration of acetonitrile in the mixture never exceeded 1% (v/v). At various time intervals aliquots of the incubation mixture were withdrawn and residual activity was determined by measuring the rate of substrate hydrolysis. The activities were divided by those measured in the absence of inhibitor (control). The bimolecular rate constants for the formation of the acylated enzyme (ka ) were derived from these data by the method of Aldridge and Reiner (1972). Reactivation experiments were performed by incubating the partially purified enzyme with insecticide for 10 ± 15 min, so that the enzyme was > 90% inhibited. The unbound insecticide and enzyme ± insecticide complex were separated on Nick spin columns. Aliquots of the enzyme were then removed over a 5 h time course to measure the residual activity in the inhibited and control (uninhibited) preparations. The slope of the curve, obtained by plotting the percentage remaining activity against time, gave the reactivation constant k3 .

F igure 1.

Esterase banding patterns with a - and b - naphthyl acetate from

equal loading of crude homogenate proteins of several planthopper strains and species compared with the C ulex quinquefasciatus Pel R R resistant and P elSS

2.6.

susceptible strains. SL= Sri Lankan strain, R = rice, G= grass.

Malathion metabolism

14

C -labelled malathion was obtained from Amersham, UK 1 with a specific activity of 4.6 mCi mM . The insecticide was labelled in both carbon atoms of the succinyl moiety. Radiolabelled insecticide was diluted 1 : 5 with unlabelled insecticide. Aliquots of the partially purified esterase, or replicates of pooled crude homogenates from five N. lugens adults with either high or 14 low esterase activity were incubated with 1 nM C-labelled malathion in 50 mM phosphate buffer pH 7.4 for 3 h at 288 C. Malathion and its metabolites were extracted and analysed as described previously (Hemingway, 1982). Ð

3.

Results

Native 7.5% PAGE revealed that all five N. lugens strains collected from rice had a heavily staining diffuse b -naphthyl acetate specific band, which had a relative mobility between that 1 1 of the Esta 2 and Estb 2 elevated esterases of the Pel RR strain of C. quinquefasciatus. In contrast, the two strains of the N. lugens sibling species, collected from L. hexandra, and the strain of N. bakeri collected from grass, all had a much fainter slower migrating b -naphthyl acetate specific band (see figure 1). The level of esterase activity in these strains, although significantly lower than that of N. lugens from rice, was greater than that of the insecticide-susceptible Pel SS mosquito strain for the same loading of protein (13 l g of the crude homogenate per well), where no esterase bands were visible. S. furcifera collected from rice had a comparable level of general esterase

F igure 2.

Western blot of an identical gel to that in figure 1 screened with 1 rabbit antisera raised to elevated Esta 2 from Culex quinquefasciatus. AChE = acetylcholinesterase.

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S. H. P. P. Karunaratne et al.

Immunoblots of the esterases from the different strains and species of planthopper showed that, under the conditions used, 1 none of the planthopper esterases interacted with the Esta 2 1 1 antisera, while both the Culex elevated Esta 2 and Estb 2 from Pel RR and the non-elevated co-migrating esterases from Pel SS give clear bands on the immunoblot (see figure 2). The only planthopper proteins to interact with the antisera were slow migrating bands with a mobility corresponding to that of 1 acetylcholinesterase. The cross-reactivity of the Esta 2 antisera to acetylcholinesterase has already been noted previously (Karunaratne et al., 1995). Similarly an E4 esterase antiserum did not cross-react with the elevated planthopper esterases. On the basis of their esterase banding patterns five planthopper strains; N. lugens collected from rice in Sri Lanka and Indonesia, and from Leersia in Indonesia, and the N. bakeri and S. furcifera strains, were selected for further study. On native PAGE 0.1 mM paraoxon completely inhibited the activity of the esterase bands of all five strains, while the pyrethroid permethrin had no observable effect on the intensity of any of the esterase bands up to the solubility limit of the insecticide. The elevated esterases from all the rice strains of N. lugens were all estimated to have a native molecular weight of 60 ± 65 kDa, which is in the same range as those of the resistanceassociated Culex and aphid esterases. The Sri Lankan N. lugens strain associated with rice was used for further purification of the elevated esterase(s). Pilot studies with different column chromatography methods failed to resolve the esterase activity in this strain into more than one enzyme band. Larger scale preparations were then undertaken using QSepharose and hydroxylapatite column chromatography, which gave a 10-fold purification with 78% recovery of esterase activity (see table 1). After chromatography the partially purified esterase preparation was stable at room temperature without glycerol or DTT for more than 24 h without any decrease in specific activity. From 1 ± 2 g of planthoppers (wet weight) we obtained approximately 2.8 mg of partially purified esterase with a specific activity 1 1 of 5.85 l mol min mg . This esterase preparation was used for further characterization. Table 2 shows the interaction of the 1 esterase with different effectors. The data for the purified Esta 2 1 and Estb 2 esterases with the same effectors are given for comparison. Activity of the planthopper esterase was completely inhibited by HgCl2 . The only other effectors to show any significant interactions with the planthopper esterase were the 2+ 2+ metal ions Cu and Zn and the metal chelator EDTA. The bimolecular rate constants (ka ) for the interaction of the partially purified esterase were determined with paraoxon, malaoxon, permethrin and propoxur (see table 3). Paraoxon was the most effective inhibitor of the esterase, followed by malaoxon and propoxur. The pyrethroid permethrin was unable

to inhibit the esterase, confirming our observations on native PAGE. The deacylation rates (k3 ) for malaoxon, paraoxon and propoxur are given in table 3. The turn-over rates of the insecticides were very slow, hence all three can be considered as inhibitors of the esterase rather than as substrates. Neither the crude N. lugens homogenates with high or low esterase activity or the partially purified esterase preparation were able to metabolize malathion to any significant extent. Malathion was also unable to inhibit the partially purified esterase at concentrations of up to 1 mM. Hence the elevated esterase from Sri Lankan N. lugens appears to be unable to either metabolize or sequester malathion, although it is clearly sensitive to inhibition by its insecticidally active oxon analogue.

Table 1.

Table 3.

Ð

Purification table for the partial purification of the major

Crude homogenate Q-Sepharose Hydroxyapatite

Discussion

Nine strains of brown planthopper were analysed for esterase activity levels. The six strains collected from rice cultivars all had a significant frequency of individuals with elevated levels of esterase activity compared with three strains collected from wild grass. The five N. lugens strains from rice all had a single elevated diffuse b -naphthyl acetate specific band on native PAGE, while the S. furcifera from rice had two distinct electromorphs. The two planthopper collections from wild grass had lower levels of esterase activity, which on PAGE resolved as a single electromorph which migrated more slowly than those from the rice strains of N. lugens. The lack of any strong immunological cross-reactivity between the planthopper esterases in their native form and the Culex Esta 21 antisera suggest that the two enzymes do not share any common surface epitopes, despite being functionally

Table 2.

The influence of various effectors (1.0 mM) on partially purified

elevated Nilaparvata lugens esterase [data for purified resistance associated esterases Est a 21 and Est b 21 (Karunaratne et al. 1993)

Ð

elevated esterase from Sri Lankan Nilaparvata lugens

Step

4.

Specific activity 1 (l mol mg 1 min ) Ð

0.56 3.85 5.85

Ð

Ð 6.9 10.5

% remaining activity Effector CaCl2 CuCl2 FeCl3 HgCl2 MgCl2 MnCl2 NiCl2 ZnCl2 EDTA

N. lugens esterase

Est a 21

Est b 21

99% 75% 96% 3% 107% 94% 89% 80% 84%

103% 37% 101% 0.5% 104% 98% 101% 63% 100%

106% 5% 4% 12% 113% 97% 98% 102% 104%

The bimolecular rate constants (k a s) and deacylation rates

(k3 s) for insecticide interactions with partially purified elevated esterase

Protein Purification % (mg) factor Recovery

2.15 0.25 0.16

are given for comparison]

Ð 79.9 77.7

from Nilaparvata lugens Insecticide Propoxur Paraoxon Malaoxon Permethrin

K a ´ 10 Ð

5

(M Ð

1

min

0.0076 6 0.0027 82.06 23.0 3.26 1.7 No interaction

Ð

1

)

k 3 ´ 10 (min 4

15.06 0.6 4.06 0.2 8.96 0.8

Ð

1

)

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Insecticid e resistanc e mechanism in plant hoppers

similar. This confirms earlier studies, which showed that an antisera raised to the elevated E 1 esterase of N. lugens did not cross-react with the Culex esterases, but possibly showed weak cross-reactivity with the aphid E4 esterase (Chen and Sun, 1994). However, an antiserum raised to the E4 esterase did not cross-react with the elevated esterases in any of our strains of planthopper. The Culex and aphid esterases have ~ 22% homology at the amino acid level (Vaughan and Hemingway, 1995), but antisera to the esterases from either insect shows no cross-reactivity with enzymes from the other species. The partially purified esterase from N. lugens was progressively inhibited by organophosphates, hence it can be classified as a B-type serine hydrolase (EC 3.1.1.1.) (Aldridge, 1953; Manco et al., 1994). The rapid binding and slow turnover of the three insecticides used indicate that the role of this esterase in resistance is sequestration. The bimolecular rate constants (ka ) for the planthopper esterase show that its specificity is in the order paraoxon > malaoxon > propoxur. This is the same order as seen in Culex mosquitoes and aphids, but when compared with the Culex esterases it is slightly less efficient at binding paraoxon, but slightly more efficient at binding malaoxon (see table 3). The E4 esterase from the aphid Myzus persicae is an order of magnitude better at binding paraoxon than either the Nilaparvata or Culex esterases (Devonshire, 1977; Karunaratne et al., 1993). Reactivation rates (k3 ) were readily measured for the N. lugens esterase, unlike earlier reports where no reactivation was observed over a 3-h time course (Chen and Sun, 1994). The k3 values for N. lugens with propoxur are similar to those for the E4 and Estb 2, while the rate for malaoxon is slower (Devonshire and Moores, 1982; Ketterman et al., 1992; Karunaratne et al., 1993). However the rates are still slow for all three esterases with both the oxon analogues and carbamates. Hence the efficacy of the resistance mechanism to these insecticides in all these insects is a function of the amount of esterase available for sequestration. In contrast to the earlier study, where metabolism of malathion and trans-permethrin were observed for three N. lugens elevated esterases (Chen and Sun, 1994), we were unable to detect any significant metabolism of malathion, nor was this insecticide able to inhibit the partially purified esterase. This is in agreement with the low level (2 ± 5-fold) of malathion resistance observed in the rice N. lugens strain from Sri Lanka and suggests that resistance is conferred solely by sequestration of the active oxon analogue. 1 The Culex Esta 2 and N. lugens elevated esterase had a very similar pattern of response to a range of effectors. The inhibition of the partially purified N. lugens esterase by HgCl2 suggests the involvement of a thiol group in catalysis. A similar 2+ pattern of complete inhibition with Hg and partial inhibition with 2+ Cu was shown by a phosphotriester hydrolase from Heliothis virescens (Konno et al., 1990) and the resistance-associated 1 esterase Estb 2 from C. quinquefasciatus (Karunaratne et al., 1993). The slight inhibition of the N. lugens esterase by EDTA suggests that it may require a metal ion for catalysis. Further studies to characterize this enzyme at a molecular level are now in progress. Bioassays and biochemical assays of the brown planthopper reported on here and in the companion paper by Hemingway et al. (1999) have shown that widespread and heavy usage of organophosphorus and carbamate insecticides for the control of rice pests in Asia has selected one major

229

resistance mechanism based on insecticide sequestration by elevated esterases. Whilst this mechanism gives only low levels of resistance (2 ± 5-fold for malathion and propoxur), its spread in field populations of the insect has compromised control programmes based on insecticidal control and changed the status of N. lugens from that of a minor pest of rice to a major one over the last 15 years. The introduction and large-scale use of rice varieties with resistance to the brown planthopper has had little effect, resistance rapidly breaking down as the insect overcomes the resistance mechanism of the variety employed. All this is testament to the enormous genetic flexibility and reproductive capacity of the brown planthopper and of insects in general. The resistance to permethrin detected in this study (see companion paper by Hemingway et al., 1999) was of a low order and showed that use of pyrethroids is a viable alternative to organophosphorus and carbamate insecticides. Imidacloprid, a chloronicotinyl insecticide, has also been shown to be effective against the brown planthopper (Ishii et al., 1994) whilst having a lesser impact on at least one possible biological control agent, the mermithid parasite Agamermis unka (Nematoda: Mermithidae), than insecticides currently used in pest control (Choo et al., 1998). These insecticides should, however, be used wisely and with measures in place to monitor for the appearance of, and change in frequency of, resistance mechanisms. The future introduction of resistant rice varieties having more than one resistance gene and with different modes of action should overcome the `boom and bust’ associated with the use of resistant varieties. The use of these new varieties combined with the use of insecticidal control measures against the brown planthopper should, if integrated into a rational control programme, reduce the selection pressures on individual components and would hence represent a sustainable approach to brown planthopper control.

Acknowledgements We thank Professor Alan Devonshire for providing the E4 antiserum, Professor M. F. Claridge for constructive criticism of this manuscript and J. Morgan for technical assistance. J. H. has project grant support for this work from the BBSRC , S. H. P. P. K. was supported by funding from the Sir Halley Stewart Trust. Insects were imported and maintained in culture under licence from the Welsh Office Department of Agriculture.

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