Effects of Toxic and Deterrent Terpenoids on Digestive Protease and Detoxication Enzyme Activities of Colorado Potato Beetle Larvae

Pesticide Biochemistry and Physiology 63, 76–84 (1999) Article ID pest.1998.2386, available online at http://www.idealibrary.com on

Effects of Toxic and Deterrent Terpenoids on Digestive Protease and Detoxication Enzyme Activities of Colorado Potato Beetle Larvae ´ ´ ´ ´ ˜ Felix Ortego, Jesus Lopez-Olguın, Marisa Ruız, and Pedro Castanera1 ´ ´ CSIC, C.I.B., Departamento de Biologıa de Plantas, Velazquez 144, 28006 Madrid, Spain Received July 6, 1998; accepted October 20, 1998 Larvae of Colorado potato beetle, Leptinotarsa decemlineata, were fed on potato leaf disks treated with the toxic limonoids azadirone and a mixture of 1,7-di-O-acetylhavanensin and 3,7-di-O-acetylhavanensin (F18) from Trichilia havanensis (Meliaceae) and a deterrent neo-clerodane (scutalpin-B) from Scutellaria alpina (Labiatae). Azadirone, a toxicant with low mortality action, decreased esterase activity and increased glutathione S-transferase activity during the treatment period, but these detoxication enzymes reached normal levels of activity when the larvae were shifted to untreated leaf disks during the post-treatment period. The mixture of limonoids F18, a strong toxicant that produces high levels of larval mortality, significantly reduced digestive protease and esterase activities and increased glutathione S-transferase and polysubstrate monooxygenases during the treatment period. All enzymatic activities but glutathione S-transferase remained altered during the post-treatment period. Scutalpin-B, with a deterrent mode of action, did not have any significant effect on any of the enzymatic processes. We concluded that the effects of these compounds on digestive proteases and detoxication enzymes in the larval midgut of L. decemlineata larvae reflect their postulated mode of action. 䉷1999 Academic Press

INTRODUCTION

The developmental effects of azadirachtin are attributed to a disruption of ecdysteroid and juvenile hormone titters through a blockage of morphogenetic peptide hormone release (2). The antifeedant activity of limonoids and neo-clerodanes appears to be due to their effects on chemosensory mouthparts (deterrent activity), and through their postingestive toxic effects on other tissues and organs resulting in an overall loss of fitness of the insect and a reduction in food intake (2,9–11). The mechanisms of action of these terpenoids at the enzymatic level are mostly unknown. Smith and Mitchell (12) reported in vitro inhibition by azadirachtin of ecdysone 20-monooxygenase, the cytochrome P-450-dependent hydroxylase responsible for the conversion of ecdysone to 20-hydroxyecdysone, in Drosophila melanogaster, Aedes aegypti, and Manduca sexta. Furthermore, azadirachtin appears to disturb the digestive process in insects, inhibiting the activity of digestive proteases in larvae of Spodoptera litura (11) and M. sexta (13). Smirle et al. (14) reported that ingestion of neem oil significantly reduced esterase activities in larvae and adults of Choristoneura rosaceana.

The plant family Meliaceae has been the subject of study as one of the most promising sources of compounds with insect control properties (1). Azadirachtin, a limonoid (tetranortriterpene) obtained from the seed kernels of the neem tree Azadirachta indica (Meliaceae), possesses a wide range of biological activities, including insect antifeedant and growth regulator properties (2). Information on the antifeedant activity of other limonoids has also been reviewed (1,3). Clerodane diterpenes have attracted interest recently on account of their biological activities against some economically important pests (4). An abundant source of this kind of compound is the plants belonging to the genus Scutellaria (family Labiatae), from which several neo-clerodanes with insect antifeedant activity have been isolated (5–8). 1

To whom correspondence should be addressed at CSIC, ´ ´ C.I.B., Departamento de Biologıa de Plantas, Velazquez 144, 28006 Madrid, Spain. Fax: (34-91) 562-7518. E-mail: [email protected]. 76 50048-3575/99 $30.00 Copyright 䉷 1999 by Academic Press All rights of reproduction in any form reserved.

TERPENOIDS ON DIGESTIVE ENZYMES

´ ´ Recently, Lopez-Olguın (15) has shown that the limonoids azadirone and a mixture of 1,7di-O-acetylhavanensin and 3,7-di-O-acetylhavanensin (F18), isolated from the seed kernels of Trichilia havanesis (Meliaceae), and the neoclerodane scutalpin-B, from Scutellaria alpina (Labiatae), exhibit strong antifeedant activity against the Colorado potato beetle (CPB), Leptinotarsa decemlineata (Say). Nutritional tests, antifeedant simulation assays, and post-treatment studies indicated that scutalpin-B acts as an insect feeding deterrent, whereas the antifeedant activity of azadirone and F18 is likely associated with a toxic mode of action. It is expected that toxicants should display postingestive effects on some biochemical process, whereas no such effects should appear with deterrents. We report here the effects of these compounds with different modes of action on digestive proteases and detoxication enzymes in the larval midgut of the Colorado potato beetle. MATERIALS AND METHODS

Chemicals and equipment. The limonoids azadirone and a mixture (4:1) of 1,7-di-O-acetylhavanensin and 3,7-di-O-acetylhavanensin (F18) (Fig. 1) were isolated from the seed kernels of Trichilia havanesis Jacq. (Meliaceae) (15). The neo-clerodane diterpenoid scutalpinB (Fig. 1) was obtained from S. alpina subsp. javalambrensis (Pau) (Labiatae) in a previous study (16). Azadirone and scutalpin-B were purified to homogeneity, whereas the purity of F18 was 95%. The chemical structure of the natural products was determined by comparison of their 1 H NMR spectra. All substrates were supplied by Sigma Chemical Co. (St. Louis, Mo). Spectrophotometric measurements were made using a Hitachi U-2000 spectrophotometer. Insects. A colony of Colorado potato beetle was established by collecting over 200 adults from a potato field located in Toledo (Spain) during the fall of 1996. The laboratory population was reared on potato plants, Solanum tuberosum cv. Kennebec, at 24 ⫾ 1⬚C, 90 ⫾ 10% RH, and 16:8 (L:D) h photoperiod in an environmental chamber.

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Feeding assays. Twenty newly emerged fourth-instar larvae were fed over 20 h on 8 potato leaf disks treated with 0, 3, 10, 30, 100, 300, or 1000 ppm of F18, azadirone, or scutalpin-B. At the end of this period,10 larvae per treatment were allowed to continue feeding on 8 untreated potato leaf disks for another 20 h. Larval ingestion was calculated for the 20 h that the larvae were feeding on treated leaf disks (treatment period), and for the following 20 h that the larvae were feeding on untreated leaf disks (post-treatment period). In each assay, 20 additional larvae were starved during the 20 h of the treatment period and allowed to feed on 8 untreated potato leaf disks during the 20 h of the post-treatment period. The arenas for the assays consisted of plastic petri dishes (15 ⫻ 90 mm), coated on their bottom half with a 2.5% agar solution, as previously described (10). Potato leaf disks (1.77 cm2) were treated on the upper surface with 12 ␮l of an acetone solution containing the test compound or the solvent carrier alone. After complete evaporation of the solvent, newly emerged fourthinstar larvae (less than 24 h old and starved for 6 h) were individually placed in each dish in a growth chamber at 26 ⫾ 0.5⬚C and 85 ⫾ 10% RH, where they were allowed to feed. Enzyme assays. At the end of the treatment and post-treatment periods, the 10 larvae of each treatment were dissected in 0.15 M NaCl and the midgut with contents were removed and stored frozen (⫺20⬚C) until needed. Each midgut (with contents) was homogenized in 500 ␮l of 0.15 M NaCl and centrifuged at 10, 000g for 5 min at room temperature. The supernatants were frozen individually to provide 10 samples of each treatment for enzymatic activity assays. Protease and detoxication enzyme activities were conducted at their optimum pH of activity. The optimum pHs for the detoxication enzymes of this species were determined as a plateau in the range 7.0–8.5 for CDNB (1-chloro-2,4dinitrobenzene) conjugation (glutathione Stransferase activity); 6.0 for 1-NA (1-naphthyl acetate) hydrolysis (general esterase activity); and 7.5 for cytochrome c reduction by NADPHcytochrome P-450 reductases (polysubstrate

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FIG. 1. Chemical structure of compounds tested against Colorado potato beetle larvae.

monooxygenases). Blanks were used to account for spontaneous breakdown of substrates. Protease activity was determined by adding 1 ml of 0.1% sulfanilamide-azocasein solution (0.1 M citrate buffer, 1 mM L-cysteine, 0.15 M NaCl, 5 mM MgCl2, pH 5.5) to 50 ␮l of midgut extract, as described by Novillo et al. (17). The reaction mixture was incubated at 30⬚C for 4 h and stopped by the addition of 500 ␮l of 10% ice-cold trichloroacetic acid. The solution was centrifuged at 10,000g for 5 min and the

absorbance of the supernatant measured at 420 nm. Glutathione S-transferase activity was measured in 1 ml of reaction mixture containing 50 ␮l of midgut extract, 1 mM CDNB added in 10 ␮l of ethylene glycol monomethyl ether, 5 mM reduced glutathione, and 0.1 M phosphate buffer, 0.15 M NaCl, 5 mM MgCl2, pH 7.0. The reaction buffer containing the midgut extract and the reduced glutathione was incubated at 30⬚C for 15 min, and the reaction started by the addition

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of the substrate solution. The increment in absorbance at 340 nm was recorded during 2 min to determine the nanomole substrate conjugated per minute per milligram protein, using a molar extinction coefficient of 9.6 mM⫺1 cm⫺1 (18). General esterase activity was determined using 1-NA as substrate based on the procedure described by Gomori (19). The reaction was started by the addition of 20 ␮l of 1/10 diluted midgut extract to a total 1 ml of reaction mixture containing 0.25 mM 1-NA (added in 1 ␮l ethanol) in 0.1 M phosphate buffer, 0.15 M NaCl, 5 mM MgCl2, pH 6.0. The mixture was incubated at 30⬚C for 30 min, and the reaction was terminated by the addition of 500 ␮l of an aqueous solution containing 0.4 mg Fast blue B salt and 15 mg sodium dodecyl sulfate/ml. The solution was incubated for 1 h at room temperature, centrifuged at 10,000g for 5 min and the absorbance of the supernatant was determined at 600 nm. The activity was expressed as nanomole substrate hydrolyzed per minute per milligram protein, using 1-naphthol as standard. Polysubstrate monooxygenases were assayed by the reduction of cytochrome c by NADPHcytochrome P-450 reductases according to Masters et al. (20). The 1 ml of incubation mixture contained 50 ␮l of midgut extract, 50 ␮M cytochrome c, NADPH-generating system (0.5 mM NADP, 2.5 mM glucose-6-phosphate, and 0.25 units of glucose-6-phosphate dehydrogenase), and 0.1 M Tris-HCl, 0.15 M NaCl, and 5 mM MgCl2, pH 7.5. The reaction mixture was incubated at 30⬚C for 1 h and stopped by the addition of 500 ␮l of methanol. The solution was centrifuged at 10,000g for 5 min and the absorbance of the supernatant measured at 550 nm to determine the nanomole substrate reduced per minute per milligram protein, using a molar extinction coefficient of 27.6 mM⫺1 cm⫺1 for the reduced form of cytochrome c (21). Total protein in the midgut extracts was determined according to the method of Bradford (22) using bovine serum albumin as the standard. Statistical analysis. We found that protease, glutathione S-transferase, and monooxygenases

activities were dependent on the larval ingestion rate and/or protein content. A regression of each enzymatic activity on protein content was run with 60 fourth-instar larvae, representing the range of larval weights obtained in the assays. A regression of each enzymatic activity on larval ingestion rate was obtained by feeding newly molted fourth-instar larvae with different amounts of food, ranging from total starvation to surplus of food. Ten larvae were individually presented for 20 h with one of five different arrangements: 0, 2, 4, 6, and 8 potato leaf disks. Analysis of covariance was used to exclude the effects of ingestion and protein content of the larvae after the treatment and post-treatment periods. Means were compared between each treatment and the control by Dunnett two-tailed tests, using as covariates their protein content and larval ingestion for protease activity, larval ingestion for glutathione S-transferase activity, and protein content for polysubstrate monooxygenase activity. RESULTS

A regression of each enzymatic activity on larval protein content showed that protease (r 2 ⫽ 0.745, P ⱕ 0.0001) and polysubstrate monooxygenases specific activities (r 2 ⫽ 0.605, P ⱕ 0.0001) were dependent on protein content, whereas glutathione S-transferase (r 2 ⫽ 0.077, P ⫽ 0.171) and esterase specific activities (r 2 ⫽ 0.002, P ⫽ 0.976) were not. In addition, a regression of each enzymatic specific activity on larval ingestion was done. In the case of protease and polysubstrate monooxygenases, which were previously found to be dependent on protein content, the data were compared to the expected enzymatic activities according to their protein content and the difference (residuals) used for the regression on larval ingestion. We found that protease (r 2 ⫽ 0.360, P ⱕ 0.0001) and glutathione S-transferase specific activities (r 2 ⫽ 0.378, P ⱕ 0.0001) were dependent on larval ingestion, but not polysubstrate monooxygenases (r 2 ⫽ 0.042, P ⫽ 0.301) and esterase specific activities (r 2 ⫽ 0.073, P ⫽ 0.108). According to these results, we exclude the effects of protein content and larval ingestion

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on the enzymatic activities using protein content and larval ingestion as covariates for protease activity, larval ingestion as covariate for glutathione S-transferase activity, and protein content as covariate for polysubstrate monooxygenase activity. Ingestion of azadirone-treated potato disks for 20 h (treatment period) significantly reduced esterase activity and increased glutathione Stransferase activity at 1000 and 300 ppm, whereas protease and polysubstrate monooxygenases activities were not affected (Table 1). At 100 ppm, only esterase activity was significantly reduced. When the larvae were placed on untreated leaf disks for another 20 h (posttreatment period), all enzymatic activities were not significantly different from controls, except for a reduction in esterase activity at 1000 ppm, a concentration at which 30% mortality was observed (Table 1). Dietary F18 caused 100% mortality during the treatment period at 1000 ppm. At 300 ppm, 30% mortality occurred during the treatment

period and 50% at the post-treatment period. No mortality occurred during the treatment period with 100 ppm of F18, but 30% mortality was recorded during the post-treatment period. Digestive protease and esterase activities were reduced and glutathione S-transferase and polysubstrate monooxygenases significantly increased in larvae feeding during the treatment period on potato disks treated with F18 at 30, 100, and 300 ppm (Table 2). Protease, esterase, and polysubstrate monooxygenases activities remained altered during the post-treatment period, whereas no significant differences from control were observed for glutathione S-transferase activity (Table 2). Larvae feeding on scutalpin-B-treated disks consumed 18, 20, and 40% less than controls at 100, 300, and 1000 ppm, respectively. All enzymatic activities remained unaltered during the treatment and post-treatment periods for larvae feeding on potato disks treated with scutalpin-B (Table 3). No differences between starved and control

TABLE 1 Digestive Protease and Detoxication Enzyme Activities in Fourth-Instar Larvae of L. decemlineata Fed for 20 h on Potato Leaf Disks Treated with Azadirone (Treatment Period) and Subsequently on Untreated Potato Leaf Disks for Another 20 h (Posttreatment Period) Specific activitya Dose (ppm) Treatment 1000 300 100 Starved Control Posttreatment 1000 300 100 Starved Control a

Mortality (%)

b

Protease (azocasein)

c

GST (CDNB)

Esterased (1-NA)

PSMOse (cytochrome c)

0 0 0 0 0

25.7 19.9 20.8 28.9 29.4

⫾ ⫾ ⫾ ⫾ ⫾

3.6 3.1 1.6 3.2 2.1

367 377 267 249 171

⫾ ⫾ ⫾ ⫾ ⫾

58* 21* 24 25 19

94 81 132 163 201

⫾ ⫾ ⫾ ⫾ ⫾

16* 18* 13* 22 18

2.59 2.22 1.76 2.96 1.85

⫾ ⫾ ⫾ ⫾ ⫾

0.20 0.16 0.26 0.18 0.17

30 0 0 0 0

15.9 16.8 14.9 23.4 14.9

⫾ ⫾ ⫾ ⫾ ⫾

1.9 1.1 0.7 2.0 0.8

216 242 162 184 157

⫾ ⫾ ⫾ ⫾ ⫾

27 22 11 23 10

165 213 299 296 290

⫾ ⫾ ⫾ ⫾ ⫾

35* 26 39 37 21

1.77 1.92 1.48 2.33 1.63

⫾ ⫾ ⫾ ⫾ ⫾

0.31 0.26 0.17 0.13 0.10

Specific activities as nmoles of substrate hydrolyzed (esterase), conjugated (glutathione S-transferase, GST), or reduced (polysubstrate monooxygenases, PSMOs)/min/mg protein, except for general proteolytic activity against azocasein as mU ⌬ Abs 420 nm/min/mg protein. Values are the means ⫾ SE (n ⫽ 10, except for 1000 ppm in post-treatment with n ⫽ 7). b Means were compared by analysis of covariance using as covariates protein content and ingestion. c Means were compared by analysis of covariance using as covariate ingestion. d Means were compared by analysis of variance. e Means were compared by analysis of covariance using as covariate protein content. * Significantly different from control (Dunnett two-tailed test, P ⱕ 0.05).

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TABLE 2 Digestive Protease and Detoxication Enzyme Activities in Fourth-Instar Larvae of L. decemlineata Fed for 20 h on Potato Leaf Disks Treated with a Mixture of 1,7-di-O-acetylhavanensin and 3,7-di-O-acetylhavanensin (F18) (Treatment Period) and Subsequently on Untreated Potato Leaf Disks for Another 20 h (Posttreatment Period) Specific activitya Dose (ppm) Treatment 300 100 30 10 3 Starved Control Posttreatment 300 100 30 10 3 Starved Control

Mortality (%)

Proteaseb (azocasein)

GSTc (CDNB)

Esterased (1-NA)

PSMOse (cytochrome c)

30 0 0 0 0 0 0

12.6 14.1 13.8 24.3 21.1 26.1 23.1

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

3.4* 2.5* 2.0* 2.1 1.0 2.1 2.1

314 297 313 198 157 234 147

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

26* 25* 25* 19 17 18 14

126 178 188 389 339 260 372

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

30* 57* 29* 50 48 34 27

4.72 4.19 2.79 2.98 2.56 3.36 1.99

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.25* 0.26* 0.24 0.31 0.28 0.15 0.20

50 30 0 0 0 0 0

5.8 14.5 14.6 19.1 16.6 20.2 18.9

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

2.0* 2.3* 1.4* 2.0 0.5 1.3 0.8

238 228 178 130 97 109 105

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

33 41 11 8 11 10 9

151 261 311 357 486 486 409

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

46* 46* 20 54 17 24 21

4.13 3.86 3.56 2.04 1.52 2.14 1.30

⫾ ⫾ ⫾ ⫾ ⫾ ⫾ ⫾

0.39* 0.48* 0.52* 0.25 0.04 0.18 0.06

a Specific activities as in Table 1. Values are the means ⫾ SE (n ⫽ 10, except for those doses in which dead larvae were not analyzed). b–e Means were compared as in Table 1. * Significantly different from control (Dunnet two-tailed test, P ⱕ 0.05).

larvae were found for protease and detoxication enzyme activities during the treatment and posttreatment periods (Tables 1, 2, and 3). DISCUSSION

Toxic (antibiosis) and deterrent (antixenosis) modes of action have been suggested as responsible for the antifeedant activity of limonoids and neo-clerodanes on CPB (9,10). Thus, the limonoid limonin seems to possess postingestive toxic effects (9,23), whereas epilimonol and limonin diosphenol appear to act as deterrents (24,25). Likewise, Ortego et al. (10) found that the neo-clerodane teuscorolide acts as a feeding deterrent against CPB larvae, whereas the antifeedant activity of teucrin-A, teucvin, and eriocephalin is likely associated with a toxic mode of action. According to their mode of action, it is expected that the toxic azadirone and the mixture of limonoids F18 should display postingestive effects on some biochemical process, whereas

no such effects should appear with the deterrent scutalpin-B (15). We have shown that the effects of these compounds on digestive proteases and detoxication enzymes in the larval midgut of CPB larvae matched their postulated mode of action. F18, a strong toxicant that produces high mortality, affected the activity of both protease and detoxification enzymes during the treatment and post-treatment periods. Azadirone, a toxicant with low mortality action, affected esterase and glutathione S-transferase activities during the treatment period, but these detoxication enzymes reach normal levels of activity when the larvae were shifted to untreated leaf disks during the post-treatment period. In contrast, scutalpin-B, with a deterrent mode of action, did not have any significant effect on these enzymatic processes. The effects of the limonoid azadirachtin on gut physiology have been mostly related to efficiency of diet conversion and inhibition of digestive enzymes (11,13). Timmins and Reynolds

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TABLE 3 Digestive Protease and Detoxication Enzyme Activities in Fourth-Instar Larvae of L. decemlineata Fed for 20 h on Potato Leaf Disks Treated with Scutalpin-B (Treatment Period) and Subsequently on Untreated Potato Leaf Disks for Another 20 h (Posttreatment Period) Specific activitya Dose (ppm) Treatment 1000 300 100 Starved Control Posttreatment 1000 300 100 Starved Control

Mortality (%)

Proteaseb (azocasein)

GSTc (CDNB)

Esterased (1-NA)

PSMOse (cytochrome c)

0 0 0 0 0

23.6 26.2 20.9 23.5 22.1

⫾ ⫾ ⫾ ⫾ ⫾

2.3 1.9 1.8 1.4 1.4

173 179 187 229 140

⫾ ⫾ ⫾ ⫾ ⫾

18 22 29 23 12

340 324 270 186 256

⫾ ⫾ ⫾ ⫾ ⫾

24 39 39 33 18

1.98 2.11 1.94 3.14 2.21

⫾ ⫾ ⫾ ⫾ ⫾

0.11 0.16 0.07 0.11 0.22

0 0 0 0 0

18.1 20.5 19.0 20.8 16.3

⫾ ⫾ ⫾ ⫾ ⫾

0.8 2.2 2.2 1.3 0.6

149 149 117 114 124

⫾ ⫾ ⫾ ⫾ ⫾

20 23 17 13 15

286 316 243 344 237

⫾ ⫾ ⫾ ⫾ ⫾

26 44 21 30 32

1.51 1.69 1.87 2.18 1.43

⫾ ⫾ ⫾ ⫾ ⫾

0.05 0.10 0.17 0.13 0.07

Specific activities as in Table 1. Values are the means ⫾ SE (n ⫽ 10). Means were compared as in Table 1. * Significantly different from control (Dunnet two-tailed test P ⱕ 0.05).

a

b–e

(13) showed that azadirachtin reduced growth of M. sexta larvae due to impaired protein digestion by inhibition of trypsin synthesis and/or secretion by midgut cells. Furthermore, Koul et al. (11) showed a drastic reduction of gut trypsin activity in S. litura larvae treated with azadirachtin, whereas the limonoids salannin and nimbinene did not interfere with the trypsin activity of the gut. The most toxic limonoid we tested, the mixture F18, decreased protease activity in CPB larvae during both treatment and post-treatment periods, indicating that inhibition of digestive proteases may play a role in its toxicity. However, protease activity was not affected by azadirone, indicating that other postingestive effects may account for its toxic mode of action. Dysfunction of midgut due to necrosis of midgut epithelial cells following azadirachtin treatment in locusts has also been reported (26). The walls of the digestive tract in insects have a high content of detoxication enzymes, as a barrier to allelochemicals that they may consume with the diet (27). Our data showed increased levels of glutathione S-transferase and polysubstrate monooxygenases activities after consumption of azadirone and F18, suggesting that both

enzymatic systems might be involved in the detoxification of these limonoids. Information regarding the detoxification enzymes involved in the inactivation of limonoids is scarce. Interestingly, the toxicity of neem seed kernel extracts to CPB and Plutella xylostella larvae increased after the synergist piperonyl butoxide (PBO) was added (28,29). Since PBO is a well-known polysubstrate monooxygenase inhibitor, the blockage of the metabolic inactivation of limonoids via polysubstrate monooxygenases, as suggested by our results, may account for the synergistic effects. It has been pointed out that compounds, such as limonoids, that reduce esterase activity may be useful in the management of insect populations where insecticide resistance has developed as a result of elevated esterase activity (14). The Colorado potato beetle is a good example of an insect that has developed resistance to most classes of synthetic insecticides (30). Biochemical mechanisms of insecticide resistance in CPB have developed as a result of elevated polysubstrate monooxygenase (31–33), glutathione S-transferase (32,34), and esterase activities (35–37). Our finding that ingestion of azadirone

TERPENOIDS ON DIGESTIVE ENZYMES

and F18 significantly reduced esterase activities in CPB larvae is an indication that these limonoids may be useful control agents and thereby play a relevant role in the management of this economically important pest. ACKNOWLEDGMENTS ´ ´ We thank Drs. Benjamın Rodrıguez and Mari Carmen de la Torre for providing the terpenoids. This work was sup´ ´ ported by “Comunidad de Madrid, Consejerıa de Educacion y Cultura” (Grant CCAM 06G/001/96).

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