Insecticide resistance in the fall armyworm, Spodoptera frugiperda (J. E. Smith)

PESTICIDE

BIOCHEMISTRY

Insecticide

AND

PHYSIOLOGY

39, 84-91 (1991)

Resistance in the Fall Armyworm, (J. E. Smith)’

Spodoptera

frugiperda

S. J. Yu Department of Entomology and Nematology,

University of Florida, Gainesville, Florida 32611

Received June 8, 1990; accepted September 18, 1990 A strain of the fall armyworm, Spodoptera frugiperda (J. E. Smith), collected from corn in North Florida showed resistance to commonly used insecticides. Resistance to pyrethroids (permethrin, cypermethrin, cyhalothrin, fenvalerate, tralomethrin, bifenthrin, tetramethrin, and fluvalinate) ranged from 2- to 216fold; the highest resistance level observed was to fluvalinate. Resistance to organophosphorus insecticides (chlorpyrifos, methyl parathion, diazinon, sulprofos, dichlorvos, and malathion) ranged from 12- to 271-fold; the highest resistance level observed was to methyl parathion. Resistance to carbamates (methomyl, carbaryl, and thiodicarb) ranged from 14 to >192-fold with the highest resistance level being observed with carbaryl. Detoxication enzyme assays revealed that activities of microsomal oxidases (aldrin epoxidase, heptachlor epoxidase, biphenyl hydroxylase, p-nitroanisole 0-demethylase, and phorate sulfoxidase) and hydrolases (helicin g-glucosidase and acetylcholinesterase) were 1.4- to 6.6fold higher in the field strain than in the susceptible strain. Levels of cytochrome P450 and cytochrome b, were 1.7- and l.ffold higher, respectively, in the field strain than in the susceptible strain. In addition, the bimolecular rate constants for inhibition of acetylcholinesterase by dichlorvos and carbaryl were 2.8- and 35fold higher, respectively, in the susceptible strain than in the field strain. There was no diierence in the rate of cuticular penetration of carbaryl in both strains. The results indicated that the broad spectrum of insecticide resistance observed in the field strain was due to multiple resistance mechanisms, including increased detoxication of these insecticides by microsomal oxidases and target site insensitivity such as insensitive acetylcholinesterase. 8 1%~ACXICIICC PMS, IN. INTRODUCTION

also reported by Wood er al. (3). According to these authors, larvae collected from corn and signalgrass previously treated with carbaryl near Hamond in 1979 showed greater resistance to carbaryl (41x), methyl parathion (113x), permethrin (17x), and trichlorfon (31x) than the laboratory (susceptible) strain. Resistance of fall armyworms to insecticides has never been studied in Florida even though control of these pests has depended exclusively on these chemicals. The present studies were carried out to learn the current status of resistance in this insect in the north Florida area. The detoxication capacity, acetylcholinesterase sensitivity, and rate of cuticular penetration of a selected insecticide in field-collected and susceptible strains were also investigated.

Insecticide resistance in fall armyworms was first reported by Young and McMillian (1) who found that a population of this insect collected from a corn field in Tifton, Georgia, was resistant to carbaryl. Resistant larvae were able to detect and avoid feeding upon carbaryl-treated leaf surfaces resulting in low mortalities compared with the susceptible larvae. On the basis of their observations, these authors suggested that behavioral, and physiological, factors were involved in resistance. The biochemical basis of resistance was later found to be mainly due to enhanced detoxication of carbaryl by microsomal oxidases (via hydroxylation and epoxidation) in this insect (2). Fall armyworm resistance to various classes of insecticides in Louisiana was r Florida Agricultural No. RXIO706.

Experiment

MATERIALS

Station Series

Insects. 84

0048-3575/91 $3.00 Copyri&t Au rigbts

0 1991 by Academic Press, Inc. of repmduction in any form nserved.

AND METHODS

The field strain was collected

INSECTICIDE

RESISTANCE

from a corn field in Gainesville, Florida, during the spring of 1989. The susceptible strain was obtained from the USDA in Gainesville, Florida. This strain was originally obtained from the USDA in Tifton, Georgia, where the strain was collected from a field in 1975 and had been maintained in the laboratory without exposure to insecticides. Both strains were maintained on an artificial diet in environmental chambers at 25°C with a 16:8 L:D photoperiod as described previously (4). Chemicals. [naphthyl-l-14C]Carbaryl with a specific activity of 21 mCi/mmol was purchased from California Bionuclear Corp. (Sun Valley, CA). Pure transpermethrin was a gift from ICI Americas (Wilmington, DE). 3-Phenoxybenzyl alcohol (98% purity) was obtained from Aldrich Chemical Co. (Milwaukee, WI). Methoxyresorufin and ethoxyresorufin were purchased from Molecular Probes (Eugene, OR). All insecticides (technical grade samples) were used as received from the manufacturers. All other chemicals were of analytical quality and purchased from commercial suppliers. Enzyme assays. When microsomal oxidases were assayed, groups of 25 midguts were dissected from 2-day-old six instar larvae and their gut contents were removed. They were then washed in 1.15% KC1 and homogenized in 25 ml of ice-cold 0.1 M sodium phosphate buffer, pH 7.5, in a motordriven tissue grinder for 30 sec. The crude homogenate was filtered through cheesecloth and the filtered homogenate was centrifuged at 1O,OOOg, for 15 min in a Beckman L5-50E ultracentrifuge. The pellet which contained cell debris, nuclei, and mitochondria was discarded. The supematant was recentrifuged at 105,OOOg,, for 1 hr to obtain the microsomal pellet which was suspended in ice-cold 0.1 M sodium phosphate buffer, pH 7.5 (unless otherwise stated), to make a final concentration of 1 mg protein/ml and used as the enzyme source. In the case of the O-dealkylase assays, microsomes were suspended in 0.1 M

IN FALL

ARMYWORMS

85

sodium phosphate buffer, pH 7.8 (for methoxyresorufin or ethoxyresorufin O-dealkylase) or 0.1 M Tris-HCl buffer, pH 7.8 (for p-nitroanisole 0-demethylase). For cytochrome P450 and cytochrome bS measurements, microsomes were suspended in 0.07 M sodium phosphate buffer, pH 7.5, containing 30% glycerol. The above procedures were conducted at 0 to 4°C. Activities of microsomal epoxidase, Ndemethylase, and S-demethylase were measured with aldrin (or heptachlor), pchloro-N-methylaniline (PCMA), and 6methylthiopurine (6-MTP) as substrates, respectively (4, 5). Microsomal p-nitroanisole (PNA) 0-demethylase activity was measured as described previously (4). Activities of microsomal methoxyresorufin (MR) 0-demethylase and ethoxyresorufin (ER) 0-deethylase were assayed using a method slightly modified from that of Mayer et al. (6). The 2-ml reaction mixture which contained 0.1 ml of microsomal suspension and 1.9 ml of 0.1 M sodium phosphate buffer, pH 7.8, was first incubated for 3 min at 30°C. After incubation, 5 ~1 of 0.5 mM methoxyresorutin or ethoxyresorufin (in Methyl Cellosolve) and 10 pl of 50 mM NADPH was added to the mixture and mixed well. The rate of reaction was followed immediately in a Shimazu RF-5000 spectrofluorophotometer. Excitation and emission wavelengths were set at 560 and 580 nm, respectively. Microsomal sulfoxidase activity was determined with phorate as substrate as described previously (7). Microsomal hydroxylase activity was determined with biphenyl as substrate as described by Yu and Ing (8). Cytochrome P450 and cytochrome b, contents were measured by the method of Omura and Sato (9) using a Beckman Model 5260 uv/ vis spectrophotometer equipped with a scattered transmission accessory. In some experiments, individual larvae were used to assay for aldrin epoxidase activity. To this end, cleaned midguts from 2-day-old larvae were prepared individually as described above and each midgut was

86

S. J. YU

then homogenized in 4.6 ml of 0.1 M sodium phosphate buffer, pH 7.5, for 30 sec. The crude homogenate was poured directly in an incubation tube which contained 250 nmol of aldrin in 0.1 ml Methyl Cellosolve. The reaction was initiated by addition of an NADPH-generating system consisting of 1.8 u.mol of NADP, 18 pmol of glucose 6phosphate, and 1 unit of glucose-6-phosphate dehydrogenase (in 0.3 ml of 0.1 M sodium phosphate buffer, pH 7.5). The incubations were carried out with shaking at 30°C in an atmosphere of air for 15 min and aldrin epoxidase activity was measured as described previously (4). When glutathione transferases were assayed, the midgut soluble fraction (i.e., 105,OOOg supernatant) prepared as described above in different buffers was used as enzyme source: for 1,2-dichloro4-nitrobenzene (DCNB) conjugation, 0.1 M Tris-HCl buffer, pH 9.0; for I-chloro-2, 4-dinitrobenzene (CDNB) conjugation, 0.1 M sodium phosphate buffer, pH 6.5; and for p-nitrophenyl acetate (PNPA) conjugation, 0.1 M sodium phosphate buffer, pH 7.0. Glutathione transferase activities toward DCNB and CDNB were determined as described previously (10). Glutathione transferase activity toward PNPA was measured by the method of Keen and Jakoby (10 a-Naphthyl acetate (a-NA) esterase and carboxylesterase activities were measured by using midgut crude homogenate as the enzyme source as described previously (12). Permethrin esterase was determined as described by Yu (13). Helicin B-glucosidase activity was assayed as described previously (14). Styrene oxide epoxide hydrolase activity was determined by the method described by Yu ef al. (12). When acetylcholinesterase activity was measured, groups of 20 heads were removed from 3- to 5-day-old adults and homogenized in 10 ml of ice-cold 0.1 M sodium phosphate buffer, pH 8.0, in a motordriven tissue grinder for 30 sec. The homogenate was filtered through cheese-

cloth. The filtered homogenate was then centrifuged at lOOOg,, for 15 min and the supernatant was used as enzyme source. Acetylcholinesterase activity was measured with acetylthiocholine as the substrate as described by Ellman et al. (15). Bimolecular rate constant (Ki) for inhibition of acetylcholinesterase by insecticides was determined by the method of Aldridge (16). Cytochrome c reductase activity was measured according to Masters et al. (17) as modified by Yu (7). Juglone reductase activity was measured using the juglonedependent NADPH oxidation method (18). Briefly, the 4.6-ml reaction mixture which contained 0.1 mg microsomal protein, 0.1 M sodium phosphate buffer, pH 6.0, was first incubated for 3 min at 30°C and then 0.5 pmol NADPH in 0.4 ml of the same buffer was added. After the addition of 0.25 p,mol juglone in 5 ~JJof Methyl Cellosolve to 2.5 ml of the mixture, the rate of NADPH oxidation was recorded at 340 nm against the same reaction mixture in the absence of substrate in a Beckman Model 5260 uv/vis spectrophotometer. The 30-min preincubation described in the original paper was a typographical error. Protein concentration was determined by the method of Bradford (19) using bovine serum albumin as standard. Cuticular penetration. Groups of 2day-old sixth instar larvae were topically treated with 1 ~1 acetone containing 0.191 kg (44,000 dpm) [‘4C]carbaryl and placed individually in scintillation vials. At various time intervals after treatment, duplicate groups of two larvae each were rinsed three times with 5 ml of acetone each. The combined extracts were concentrated and unpenetrated carbaryl was determined by liquid scintillation counting. Bioassuys. Groups of l-day-old fourth instar larvae were collected from the culture and treated on the dorsal prothorax with various insecticides in 1 pJ acetone. All larvae were held in separate scintillation vials at 25°C and provided with approximately 1 g of artificial diet. Each insecticide was

INSECTICIDE

RESISTANCE

tested at a minimum of five doses. All tests were replicated twice with 10 larvae per replicate. Mortality counts were made after 48 hr. Statistical analysis. Probit analysis was performed by a computer program (20). Whenever appropriate, data were analyzed by Student’s t test. RESULTS

Bioassays. The results of the bioassays of various classes of insecticides with tieldcollected and susceptible strains of fall armyworm are shown in Table 1. It can be seen that field-collected fall armyworms were resistant to different classes of insecticides. Resistance to the pyrethroids ranged from 2- to 216-fold depending on the insecticide; the highest resistance level observed was to fluvalinate. Resistance to the organophosphates ranged from 12- to 271fold; the highest resistance level observed was to methyl parathion. Resistance to the

IN FALL ARMYWORMS

87

carbamates ranged from 14- to >192-fold with the highest resistance level being observed with carbaryl. In fact, this field strain was highly resistant to carbaryl; no mortality was observed at doses a80 pg of carbaryl per larva. Therefore, the LDSo value of the field strain was calculated from the sublethal dose of carbaryl. The generally low slope values of the log dose-probit lines in the field strain compared with the susceptible strain indicated the heterogeneity of the field population. Enzyme assays. Detoxication capacity was studied in both field and susceptible strains of fall armyworm. From Table 2, it is seen that among the microsomal oxidases studied, activities of the epoxidases, hydroxylase, 0-demethylase (PNA), and sulfoxidase were higher in the field strain, ranging from 1.4- to 6.6-fold, than in the susceptible strain. However, activities of two other 0-dealkylases (MR and ER) were significantly lower in the field strain than in

TABLE 1 Toxicity of Insecticides Applied Topically to Fall Armyworm Larvae” Field strain (R) Insecticide Pyrethroid Permethrin Cypermethtin Cyhalothrin Fenvalerate Tralomethrin Bifenthrin Tetramethrin Fluvalinate Organophosphate Chlorpyrifos Methyl parathion Diazinon Sulprofos Dichlorvos Malathion Carbamate Methomyl Carbaryl Thiodicarb

Slope 1.9 2.4 1.8 2.1 2.2 2.9 1.4 2.9

(95ZL) 1.53 (O.W.35)b 0.48 (0.31-0.82) 0.76 (0.38-l 52) 6.53 (3.71-10.6) 0.70 (0.037-l .37) 1.59 (0.94-2.89) 97.7 (45.3-625.6) 7.13 (4.43-12.6)

6.5 2.8 7.2 4.0 5.1 3.5

36.3 48.7 141.6 55.1 375.9 1201.6

(3O.ti5.8) (32.6-l 16.8) (97.6-209.6) (33.4-207.5) (29.5-958.9) (8318-2672.2)

2.9 5.3

8.23 (4.86-l 1.6) 1600’ 58.8 (39.3-91.6)

LI48-hr mortality; n, 20 for a minimum of each of five doses. b ug/g insect. c No mortality was observed at this dose.

Susceptible strain (S) Slope

(95ZL)

L&,(R)/ LD,,W

3.3 3.2 4.1 2.8 5.4 11.0 3.5 6.5

0.11 0.086 0.061 3.74 0.017 0.054 21.1 0.033

(0.060-0.14) (0.053Jl.12) (0.04-0.08) (1.37-5.64) (0.010-0.020) (0.042-0.069) (9.76-300.1) (0.026-0.041)

13.9 5.6 12.5 1.7 41.2 29.4 4.6 216.1

8.1 6.5 3.8 3.6 4.0 3.8

1.45 0.18 6.78 4.68 19.7 17.4

(1.11-1.66) (0.114.28) (3.22-9.39) (2.79-27.00) (9.81-26.9) (12.9-23.6)

25.0 270.6 20.9 11.8 19.1 68.9

4.9 3.8 1.6

0.57 (0.45-0.78) 8.33 (5.79-11.4) 2.25 (0.30-3.89)

14.4 >192 26.1

S. J. YU

88

TABLE Detoxication

Enzyme

Levels

in Field

2

and Susceptible

Strains

of Fall Annyworm

Specific activity (nmol/min/mg protein)” Detoxication enzyme Microsomal oxidases Aldrin epoxidase Heptachlor epoxidase Biphenyl hydroxylase Phorate sulfoxidase PCMA N-demethylase PNA O-demethylase MR 0-demethylase ER 0-deethylase 6-MTP S-demethylase Cytochrome P-450 (nmol/mg protein) Cytochrome b, (nmol/mg protein) Glutathione transferases DCNB conjugation CDNB conjugation PNPA conjugation Hydrolases a-NA esterase a-NA carboxylesterase Permethrin esterase Helicin B-glucosidase Styrene oxide epoxide hydrolase Acetylcholinesterase’ Reductases Juglone reductase Cytochrome c reductase

Field strain (RI 1.47 0.35 6.69 3.40 1.41 0.42 0.030 0.0035 0.41

f f 2 2 f 2 f 2 ”

0.44 0.40

o.O6**b 0.01*** 0.75* 0.26* 0.07 0.02*** 0.003* o.ooo5* 0.04

Susceptible strain (9 0.03 0.005 0.02 0.06 0.14 0.01 0.007 O.oool 0.03

R/S

0.55 0.094 1.01 2.46 1.61 0.17 0.076 0.0054 0.42

2 ” 2 2 * k ” + +

2.7 3.7 6.6 1.4 0.9 2.5 0.4 0.6 1.0

+ 0.02***

0.26

+ 0.01

1.7

+ 0.01*

0.31

2 0.01

1.3

33.6 369.2 1341.0

k 4.1 t 15.1* 2 40.9

31.2 503.0 1105.8

f 3.6 f 22.9 f 99.1

1.1 0.7 1.2

1185.2 286.6 9.15 28.8 35.6 354.5

f ? 2 2 2 2

1090.0 245.2 10.2 11.6 33.0 244.6

f 40.4 ” 7.0 f 1.5 f 0.62 + 2.3 x!z 1.7

1.1 1.2 0.9 2.5 1.1 1.4

50.9 86.0

2 0.93 +- 5.8

1.1 1.2

56.4 99.7

24.4 9.7 0.55 2.7* 0.88 1.1*

+ 1.3 iz 1.0

a Mean 2 SE of two experiments, each with duplicate determinations. b Value significantly different from the susceptible strain. *P < 0.05; **P < 0.01; ***P c Enzyme was prepared from heads of 3- to 5-day-old adults.

the susceptible strain and activities of the N-demethylase and S-demethylase remained the same in both strains. Levels of cytochrome P450 and cytochrome b, were 1.7- and 1.3-fold higher, respectively, in the field strain than in the susceptible strain. Table 2 also shows that glutathione transferase activities toward DCNB and PNPA were similar in the field strain and the susceptible strain. In contrast, the transferase activity toward CDNB in the field strain was only 73% of that of the susceptible strain. The hydrolase activities of helicin @glucosidase and acetylcholinesterase in the field strain were 1.4- to 25fold higher than in the susceptible strain. However, ac-

<

0.001.

tivities of CX-NA esterase, carboxylesterase, permethrin esterase, and epoxide hydrolase were similar in both strains. Finally, there was no marked difference between the two strains in the activities of two reductases, juglone reductase and cytochrome c reductase. Figure 1 shows the histogram for aldrin epoxidase activity in 50 individual larvae of the field and susceptible strains of fall armyworm. It is seen that aldrin epoxidase activity was quite homogeneous in the susceptible population; activity was mainly in the range 0.2-0.4 nmol/min/larva. However, in the resistant population it was very heterogeneous, ranging from 0.2 to 3.3

INSECTICIDE

RESISTANCE

IN

FALL

89

ARMYWORMS

I6 S strain

r: 4 0 0

0.1

Aldrm

0.8

1.2

epoxldase

1.6

2.0

(nmol

2.4

/mm

2.8

/larva

3.2

1

FIG. 1. Histogram for aldrin epoxidase activity in 50 individual larvae of the susceptible (S) andfield (R) strains of fall armyworm.

nmol/min/larva. The average activity in the field strain (1.26 nmol/min/larva) was four times higher than that in the susceptible strain (0.31 nmoYmin/larva). Data in Table 3 show that the bimolecular rate constants for inhibition of acetylcholinesterase by dichlorvos and carbaryl were 2.8-and 35fold higher, respectively, in the susceptible strain than in the field strain. Cuticular penetration. Figure 2 shows that there was no difference in the rate of penetration of carbaryl into the cuticle of the field and susceptible larvae up to 24 hr after treatment.

I 0

4

12 II) 0 HOURS AFTER TREATUEnT

20

2.

FIG. 2. Rate of penetration of carbaryl in larvae of the susceptible (S) and field (R) strains of fall armyworm.

secticides, including pyrethroids, organophosphates, and carbamates. Among the three classes, the highest levels of resistance observed were to fluvalinate (216x), methyl parathion (271x), and carbaryl (~192~). Detoxication enzyme assays revealed that activities of microsomal oxidases including epoxidases, 0-demethylase, hydroxylase, and sulfoxidase were higher in the field strain than in the susceptible strain. Since microsomal oxidases are known to be actively involved in the metabolism of insecticides and in resistance (21DISCUSSION 23), it is highly likely that increased activiThe results of the present study clearly ties of these detoxication enzymes in the demonstrated that the fall armyworm col- field strain play important roles in the oblected from the north Florida area has de- served resistance. The widely individual veloped resistance to various classes of in- variation of aldrin epoxidase activity in the field population (Fig. 1) would explain the generally low slope values of log doseTABLE 3 Bimolecular Rate Constants for the Znhibition of probit lines for the insecticides observed in Acetylcholinesterase by Dichlorvos and Carbaryl in this strain (Table 1). The results suggest Fall Armyworm Adults that this strain can achieve even higher levels of resistance to different classes of inKi (M-’ min-I)” secticides if continuing selection pressure is Strain Dichlorvos Carbaryl applied. Increase in microsomal oxidase ac2.4 x 16 2.1 x 104 tivity in insects, which can result from inField 7.3 x 104 secticide selection, is one of the most comSusceptible 6.8 x 16 LIAcetylcholinesterase was prepared from heads of mon mechanisms of resistance to different 3- to S-day-old adults. Mean of two experiments. classes of insecticides (21).

90

S. J. YU

In agreement with the previous study (2), I found that only certain microsomal oxidase activities were higher in the field strain than in the susceptible strain. Furthermore, among the 0-dealkylases, PNA Odemethylase activity was higher but MR Odemethylase and ER 0-deethylase activities were lower in the field strain than in the susceptible strain. The results strongly support the notion that cytochrome P450 exists in multiple forms in fall armyworms and that some forms are more sensitive to selection pressure than others. It is interesting to note that helicin B-glucosidase was 2.5-fold higher in the field strain than in the susceptible strain. l3-Glucosidase, which is an activation enzyme, is capable of hydrolyzing plant glucosides to release toxic allelochemicals (14). Therefore, a vulnerable point in the field strain may be resistant plant varieties if the resistance mechanism involves antibiosis. The low bimolecular rate constants for the inhibition of acetylcholinesterase by dichlorvos and carbaryl in the field strain as compared with those in the susceptible strain also indicated that the observed resistance to the organophosphates and carbamates in this strain was partially due to an insensitive acetylcholinesterase. The high acetylcholinesterase activity observed in the field strain may reflect the difference in the biochemical characteristics of the enzyme between the two strains. Insensitive acetylcholinesterase as a resistance mechanism to organophosphates and carbamates has been reported in numerous insect species including Spodoptera littoralis (24).

My results were also in agreement with those of McCord and Yu (2) showing that cuticular penetration did not play any significant role in carbaryl resistant in fall armyworms. The high resistance levels to carbaryl and methyl parathion observed in the field strain of fall armyworm in this study could be due to the extensive applications of

these insecticides to control agricultural insects previously in Florida. Being a highly polyphagous and mobile species, this insect must have been exposed heavily to these insecticides in the field and thus under high selection pressure for developing resistance. Why this insect has become highly resistant to fluvalinate, a relatively new pyrethroid insecticide which has been registered for cotton insect control, is not known. Since resistance to pyrethroids caused by a kdr (knockdown resistance) factor is rather nonspecific (23, the observed resistance is unlikely due to the kdr mechanism. It is possible that crossresistance caused by increased levels of specific microsomal oxidases from exposure to other insecticides is a major mechanism for the resistance. Additional research will be required to determine the biochemical mechanisms responsible for the multiple resistance in the field strain of fall armyworm. ACKNOWLEDGMENTS

This work was supported by USDA (Southern Regional Pesticide Impact Assessment Program) and ICI Americas, Inc. I thank Drs. S. M. Ferkovich and J. L. Nation for reviewing the manuscript. The technical assistance of Sam Nguyen is also appreciated. REFERENCES

1. J. R. Young and W. W. McMillian, Differential feeding by two strains of fall armyworm larvae on carbaryl treated surfaces, .I. &on. Entomol. 72, 202 (1979). 2. E. McCord, Jr., and S. J. Yu, The mechanisms of carbaryl resistance in the fall armyworm, Spodopteru frugiperdu (J. E. Smith), Pestic. Biochem. Physiol. 27, 114 (1987). 3. K. A. Wood, B. H. Wilson, and J. B. Graves, Intluence of host plant on the susceptibility of the fall armyworm to insecticides, J. Econ. Entomol. 74, 96 (1981). 4. S. J. Yu, Induction of rnicrosomal oxidases by host plants in the fall armyworm, Spodoptera frugiperda (J. E. Smith), Pestic. Eiochem. Physiol. 17, 59 (1982). 5. S. J. Yu, Microsomal S-demethylase activity in four lepidopterous insects, Pestic. Biochem. Physiol. 31, 182 (1988). 6. R. T. Mayer, J. W. Jermyn, M. D. Burke, and R. A. Prough, Methoxyresorutin as a substrate

INSECTICIDE

RESISTANCE

for the fluorometric assay of insect microsomal 0-dealkylases, Pestic. Biochem. Physiol. 7,349 (1977). 7. S. J. Yu, Microsomal sulfoxidation of phorate in the fall armyworm, Spodoptera frugiperdu (J. E. Smith), Pestic. Biochem. Physiol. 23,273 (1985). 8. S. J. Yu and R. T. Ing, Microsomal hydroxylase of fall armyworm larvae and its induction by allelochemicals and host plants. Comp. Biochem. Physiol. C: Comp. Pharmacol. Toxicol. 78, 145 (1984). 9. T. Omura and R. Sato, The carbon monoxidebinding pigment of liver microsomes, .I. Biol. Chem. 239, 2370 (1964). 10. S. J. Yu, Interactions of allelochemicals with detoxication enzymes of insecticide-susceptible and resistant fall armyworms, Pestic. Biochem. Physiol. 22, 60 (1984). 11. J. H. Keen and W. B. Jakoby, Glutathione transferases: Catalysis of nucleophilic reactions of glutathione, J. Biol. Chem. 253, 5654 (1978). 12. S. J. Yu, F. A. Robinson, and J. L. Nation, Detoxication capacity in the honey bee, Aphis mellifera L., Pestic. Biochem. Physiol. 22, 360 (1984). 13. S. J. Yu, Liquid chromatographic determination of permethrin esterase activity in six phytophagous and entomophagous insects, Pestic. Biothem. Physiol. 36, 237 (1990). 14. S. J. Yu, g-Glucosidase in four phytophagous Lepidoptera, Insect Eiochem. 19, 103 (1989). 15. G. L. Ellman, K. D. Courtney, V. Anders, Jr., and R. M. Featherstone, A new and rapid colorimetric determination of acetylcholinesterase activity, Biochem. Pharmacol. 7, 88 (1961). 16. W. N. Aldridge, Some properties of specific cholinesterase with particular reference to the mechanism of inhibition by diethyl p-nitro-

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