induction of detoxification enzymes by triazine herbicides in the fall armyworm, spodoptera...

Pesticide Biochemistry and Physiology 80 (2004) 113–122

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PESTICIDEBiochemistry & Physiology

Induction of detoxification enzymes by triazine herbicidesin the fall armyworm, Spodoptera frugiperda (J.E. Smith)q

S.J. Yu*

Department of Entomology and Nematology, University of Florida, Gainesville, FL 32611, USA

Received 13 April 2004; accepted 21 June 2004

Available online 3 August 2004

Abstract

The inductive effect of six triazine herbicides on a variety of detoxification enzymes was investigated in fall army-

worm (Spodoptera frugiperda) larvae maintained on an artificial diet. Dietary atrazine induced nine microsomal oxidase

activities ranging from 1.3- to 21.6-fold, 12 glutathione S-transferase activities ranging from 1.3- to 4.2-fold, four hy-

drolase activities ranging from 1.3- to 2.9-fold, and two reductase activities ranging from 1.5- to 5.1-fold, depending on

the enzyme assayed and tissue source (midgut vs. fat body) used. Simazine, cyanazine, ametryn, tebutryn, and terbu-

thylazine also induced these detoxification enzymes. The induction of microsomal oxidase (aldrin epoxidase) ranged

from 1.2- to 11-fold, glutathione S-transferase (CDNB) ranged from 1.3- to 4-fold, and general esterase ranged from

1.4- to 4.1-fold, depending on the tissue source examined. In general, fat bodies were more inducible than midguts with

respect to these detoxification enzymes, especially the microsomal oxidases. The induction by atrazine was associated

with decreased toxicity of carbaryl, permethrin and indoxacarb, but increased toxicity of methyl parathion, phorate,

and trichlorfon.

� 2004 Elsevier Inc. All rights reserved.

1. Introduction

Detoxification enzymes such as microsomal ox-

idases, glutathione S-transferases and hydrolases

play important roles in the metabolism of and resis-

tance to insecticides in insects [1–4]. These enzymes

0048-3575/$ - see front matter � 2004 Elsevier Inc. All rights reserve

doi:10.1016/j.pestbp.2004.06.005

q Florida Agricultural Experiment Station Journal Series

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E-mail address: [email protected].

possess the capacity to rapidly increase their activ-

ity in response to chemical stress, the phenomenon

of enzyme induction. It is now well established that

the induction of microsomal oxidases involves syn-

thesis of new enzyme, i.e., de novo protein synthe-

sis, rather than activation of preexisting enzyme or

a block in the rate of degradation [1]. The purposeof the induction of detoxification enzymes is to en-

hance the metabolism of toxicants.

In insects, microsomal oxidases can be in-

duced by a variety of organic chemicals including

d.

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114 S.J. Yu / Pesticide Biochemistry and Physiology 80 (2004) 113–122

insecticides such as DDT and cyclodienes; insect

hormones and growth regulators such as 20-hy-

droxyecdysone and juvenile hormone; drugs such

as phenobarbital, 3-methylcholanthrene, butylat-

ed hydroxytoluene and triphenyl phosphate; andallelochemicals such as terpenoids, indoles, flavo-

noids, and furanocoumarins. On the other hand,

glutathione S-transferases are induced by various

xenobiotics including insecticides such as DDT

and dieldrin; methylenedioxyphenyl compounds

such as piperonyl butoxide; barbiturates such as

phenobarbital; and allelochemicals such as xan-

thotoxin and indole 3-acetonitrile. Hydrolases al-so are induced by xenobiotics, but to a lesser

extent, by juvenile hormone, juvenoids and allelo-

chemicals such as terpenoids, indoles, and flavo-

noids [5,6].

Very little is known about the induction of de-

toxification enzymes by herbicides. Recently, atra-

zine was shown to induce microsomal oxidases in

insects [7–9]. Therefore, the purpose of this re-search was to study the induction of various detox-

ification enzymes by six triazine herbicides in fall

armyworm larvae. The effect of dietary atrazine

on the toxicity of several insecticides also was ex-

amined in this insect.

2. Materials and methods

2.1. Insects

Larvae of the fall armyworm, Spodoptera fru-

giperda (J.E. Smith), were reared on an artificial

diet and maintained in environmental chambers

at 25 �C with a 16:8 L:D photoperiod as described

previously [10].

2.2. Chemicals

The chemicals (analytical grade) used in this

study and their sources were reduced glutathi-

one (GSH), glutathione reductase, 1-chloro-2,4-

dinitrobenzene, cumene hydroperoxide, juglone,

p-nitroacetanilide, ethacrynic acid, a-naphthylacetate, p-nitrophenyl acetate, helicin, and eser-

ine (Sigma, St. Louis, MO); trans-4-phenyl-3-bu-

ten-2-one, 2,4-hexadienal, trans,trans-nonadienal,

trans,trans-decadienal, trans-2-octenal, trans-2-

nonenal (Aldrich, Milwauke, WI), p-nitroanisole

and 1,2-dichloro-4-nitrobenzene (Eastman Kodak,

Rochester, NY), methoxyresorufin (Molecular

Probes, Eugene, OR); dichlorvos, atrazine, sima-zine, cyanazine, tebutryn, ametryn, and terbuthyl-

azine (Chem Service, West Chester, PA). Other

pesticides (technical grade sample) were used as re-

ceived from the manufacturers. All other chemi-

cals were of analytical quality and purchased

from commercial suppliers.

2.3. Treatment of insects

In induction experiments, groups of newly

molted sixth instars were fed artificial diets

containing 0.1% of the triazine herbicides. This

concentration was used because in preliminary ex-

periments dietary atrazine at the 0.1% level was

found to be the optimum concentration for induc-

ing microsomal oxidase activity in the insects; theminimum effective concentration was 0.01%.

Controls were fed the artificial diet only. After

feeding for 48h, the larvae were removed from

their respective diet and used for enzyme assays.

No mortality was observed due to the treatment.

2.4. Enzyme preparation

Groups of midguts and fat bodies were dissect-

ed from 2-day-old sixth instars. In the case of

midguts, gut contents were removed. Larval mid-

guts and fat bodies were then washed in 1.15%

KCl and homogenized in 25ml of ice-cold 0.1M

sodium phosphate buffer, pH 7.5, in a motor-driv-

en tissue grinder for 30s. The crude homogenate

was filtered through cheese cloth and the filteredhomogenate was centrifuged at 10,000gmax for

15min in a Beckman L5-50E ultracentrifuge.

The pellet (cell debris, nuclei, and mitochondria)

was discarded and the supernatant was recentri-

fuged at 105,000gmax for 1h to obtain the soluble

fraction (supernantant) and microsomes (pellet).

The microsomal pellet was suspended in 0.1M so-

dium phosphate-buffer, pH 7.5, and used immedi-ately. The above procedures were conducted at

0 �C.

S.J. Yu / Pesticide Biochemistry and Physiology 80 (2004) 113–122 115

2.5. Enzyme assays

Unless otherwise stated, microsomal oxidase ac-

tivities were determined with the crude homogenate

of midguts or fat bodies as enzyme source. Micro-somal epoxidase activity was measured with aldrin

or heptachlor as substrate as described previously

[10]. Microsomal hydroxylase activity was assayed

with biphenyl as subtrate as described by Yu

and Ing [11]. Microsomal sulfoxidase activity was

determined with phorate as substrate as described

previously [12]. Microsomal N-demethylase activi-

ty was measured with p-chloro-N-methylaniline(PCMA)1 as substrate as described by Yu [10]. Mi-

crosomal O-demethylase activity was determined

with p-nitroanisole (PNA) or methoxyresorufin

(MR) as substrate as described previously [10,13].

Microsomal S-demethylase activity was assayed

with 6-methylthiopurine as substrate as described

previously [14]. Microsomal desulfurase activity

was measured using the modified method of Burattiet al. [15]. Briefly, the 1.35-ml reaction mixture con-

tained 0.5ml of microsomal suspension; 0.5ml

0.1M sodium phosphate buffer, pH 7.5, 0.3ml

of an NADPH-generating system consisting of

1.8lmol NADP, 18lmol glucose-6-phosphate,

and 1U glucose-6-phosphate dehydrogenase; and

50ll parathion (1mg/ml in methyl cellosolve). A

complete reaction mixture containing 0.5ml 0.1Msodium phosphate, pH 7.5, instead of an NADPH-

generating systemwas used as a blank.Duplicate in-

cubations were conducted in a water bath with

shaking at 30 �C for 15min. The reaction was

stopped by adding 0.64ml of 12.5% Triton X-100.

The inhibitory activity of paraoxon produced to-

ward acetylcholinesterase (prepared from head ho-

mogenate of fall armyworm adults) was determinedas described by Yu et al. [16]. The amount of parao-

xon formed was then determined by referring to a

standard curve obtained with known amounts of

paraoxon, plotting the percentage inhibition vs.

the logarithm of paraoxon concentration. Cyto-

1 Abbreviations used: PCMA, p-chloro-N-methylaniline;

PNA, p-nitroanisole; MR, methoxyresorufin; GST, glutathione

S-transferase; CDNB, 1-chloro-2,4-dinitrobenzene; DCNB,

1,2-dichloro-4-nitrobenzene; PNPA, p-nitrophenyl acetate; a-NA, a-naphthyl acetate; 6-MTP, 6-methylthiopurine; TPBO,

trans-4-phenyl-3-buten-2-one.

chrome P450 and cytochrome b5 contents were

measured by the method of Omura and Sato [17]

with a Beckman Model 5260 UV/Vis spectropho-

tometer equipped with a scattered transmission ac-

cessory.Glutathione S-transferase activities were deter-

mined with the soluble fraction as enzyme source.

GST activities toward 1-chloro-2,4-dinitrobenzene

(CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB)

were measured as reported previously [18]. GST

activity toward p-nitrophenyl acetate was deter-

mined by the method of Keen and Jakoby [19].

GST activities toward ethacrynic acid and trans-4-phenyl-3-buten-2-one (TPBO) were measured

as described by Habig et al. [20]. GST activities to-

ward trans,trans-2,4-alkadienals and trans-2-alke-

nals were measured by the method of Brophy

et al. [21]. Glutathione peroxidase activity was de-

termined with cumene hydroperoxide as substrate

as described previously [22].

a-Naphthyl acetate (a-NA) esterase and a-NAcarboxylesterase activities were determined with

the crude homogenate as enzyme source [23]. Per-

methrin esterase activity was assayed with the crude

homogenate as enzyme source as described by Yu

[24]. Carboxylamidase activity was measured with

p-nitroacetanilide as substrate as described previ-

ously [25]. Helicin b-glucosidase activity was deter-mined as described previously [26].

Juglone reductase activity was measured with

the juglone-dependent NADPH oxidation method

[27]. Cytochrome c reductase was determined ac-

cording toMasters et al. [28] as modified byYu [12].

Protein concentration was determined by the

method of Bradford [29] with bovine serum albu-

min as a standard.

2.6. Bioassays

Newly molted sixth instars were divided into two

groups. One reared on an artificial diet containing

0.1% atrazine and the other on the artificial diet as

control. After 48 h, larvae were placed individually

in a scintillation vial and starved for 2 h. They werethen fed individually a leaf disk of crisphead lettuce

(Lactuca sativa L.), 0.7cm in diameter, which had

been topically treated with 1ll of acetone contain-ing various insecticides. Preliminary experiments


established a dosage for each insecticide which

showed a maximum toxicity difference between

control and atrazine-fed larvae. All tests were rep-

licated twice with 10 larvae per replicate. Mortality

counts were made after 48 h.

2.7. Statistical analysis

Whenever appropriate, data were analyzed by

Student�s t test.

Table 1

Effect of atrazine on detoxification enzyme activities in fat bodies of

Detoxification enzyme Specific activit

Control

Microsomal oxidases

Aldrin epoxidase 0.047 ± 0.005

Heptachlor epoxidase 0.009 ± 0.0005

Biphenyl hydroxylase 0.023 ± 0.004

Phorate sulfoxidase 0.033 ± 0.003

Parathion desulfurase 0.01 ± 0.001

p-Chloro-N-methylaniline N-demethylase 0.33 ± 0.01

p-Nitroanisole O-demethylase 0.031 ± 0.003

Methoxyresorufin O-demethylase NDb

6-Methylthiopurine S-demethylase 0.032 ± 0.009

Cytochrome P450 (nmol/mg protein) 0.036 ± 0.002

Cytochrome b5 (nmol/mg protein) 0.073 ± 0.0003

Glutathione S-transferases (substrate)

CDNB 512.6 ± 20.9

DCNB 5.96 ± 0.30

p-Nitrophenyl acetate 43.3 ± 6.3

Ethacrynic acid 16.7 ± 2.3

trans-4-Phenyl-3-buten-2-one 2.91 ± 2.3

trans-2-Octenal ND

trans-2-Nonenal ND

2,4-Hexadienal 1.79 ± 0.08

trans,trans-2,4-Heptadienal 2.66 ± 0.13

trans,trans-2,4-Nonadienal 2.00 ± 0.09

trans,trans-2,4-Decadienal 3.75 ± 0.07

Cumene hydroperoxide 47.5 ± 2.8

Hydrolases

a-NA esterase 48.7 ± 4.6

a-NA carboxylesterase 22.3 ± 2.7

Permethrin esterase ND

Helicin b-glucosidase ND

p-Nitroacetanilide carboxylamidase 0.74 ± 0.04

Reductases

Juglone reductase 3.20 ± 0.01

Cytochrome c reductase 6.02 ± 0.31

a Means ± SE of 2–3 experiments, each with duplicate determinatb Not detected.

3. Results

Data presented in Table 1 show that various de-

toxification enzymes were induced by dietary atra-

zine (0.1%) in fat bodies of fall armyworm larvae.The induction of microsomal oxidases including

epoxidases, hydroxylase, sulfoxidase, desulfurase,

N-demethylase, O-demethylases and S-demethyl-

ase ranged from 2.5- to 21.6-fold with the highest

induction level being observed with p-nitroanisole

fall armyworm larvae

y (nmol/min/mg protein)a

Atrazine (0.1%) Percentage of control

0.57 ± 0.02 1212

0.11 ± 0.01 1222

0.31 ± 0.007 1348

0.69 ± 0.03 2091

0.060 ± 0.002 600

0.81 ± 0.09 245

0.67 ± 0.07 2161

0.023 ± 0.002 —

0.15 ± 0.01 469

0.18 ± 0.003 500

0.16 ± 0.001 219

859.3 ± 59.6 168

24.0 ± 1.51 403

116.5 ± 16.3 269

31.3 ± 5.11 187

8.27 ± 0.35 284

ND —

10.3 ± 0.95 —

5.03 ± 0.37 281

6.87 ± 0.35 258

8.48 ± 0.95 424

15.9 ± 0.71 424

90.8 ± 6.0 191

141.1 ± 4.6 290

52.9 ± 4.5 237

ND —

ND —

0.75 ± 0.08 101

16.4 ± 1.5 513

28.1 ± 1.3 467

ions.


O-demethylase. Cytochrome P450 and cytochrome

b5 were induced by 5.0- and 2.2-fold, respectively.

The induction of glutathione S-transferases ranged

from 1.7- to 4.2-fold with the highest induction le-

vel being observed with trans,trans-2,4-decadienaland trans,trans-2,4-nonadienal conjugation. Ester-

ases were induced by atrazine causing 2.9- and

2.4-fold increases in general esterase and a-NA

carboxylesterase activities, respectively. However,

this treatment had no effect on carboxylamidase

Table 2

Effect of atrazine on detoxification enzyme activities in midguts of fa

Detoxification enzyme Specific activi

Control

Microsomal oxidases

Aldrin epoxidase 0.16 ± 0.01

Heptachlor epoxidase 0.017 ± 0.008

Biphenyl hydroxylase 0.33 ± 0.009

Phorate sulfoxidase 0.17 ± 0.02

Parathion desulfurase 0.019 ± 0.002

p-Chloro-N-methylaniline N-demethylase 0.42 ± 0.07

p-Nitroanisole O-demethylase 0.10 ± 0.007

Methoxyresorufin O-demethylase 0.12 ± 0.02

6-Methylthiopurine S-demethylase 0.055 ± 0.01

Cytochrome P450 (nmol/mg protein) 0.17 ± 0.02

Cytochrome b5 (nmol/mg protein) 0.19 ± 0.02

Glutathione S-transferases (substrate)

CDNB 192.2 ± 11.2

DCNB 16.0 ± 3.5

p-Nitrophenyl acetate 323.4 ± 40.8

Ethacrynic acid 17.0 ± 1.1

trans-4-Phenyl-3-buten-2-one 3.00 ± 0.25

trans-2-Octenal 2.27 ± 0.19

trans-2-Nonenal 5.54 ± 0.41

2,4-Hexadienal 3.56 ± 0.10

trans,trans-2,4-Heptadienal 5.09 ± 0.15

trans,trans-2,4-Nonadienal 6.57 ± 0.09

trans,trans-2,4-Decadienal 8.11 ± 0.55

Cumene hydroperoxide 51.1 ± 3.7

Hydrolases

a-NA esterase 389.8 ± 1.4

a-NA carboxylesterase 103.4 ± 4.6

Permethrin esterase 0.91 ± 0.11

Helicin b-glucosidase 3.70 ± 0.01

p-Nitroacetanilide carboxylamidase 2.57 ± 0.11

Reductases

Juglone reductase 21.5 ± 1.5

Cytochrome c reductase 51.6 ± 2.0

a Means ± SE of 2–3 experiments, each with duplicate determinat

activity in this insect. Reductases also were in-

duced by atrazine causing 5.1- and 4.7-fold in-

creases in juglone reductase and cytochrome c

reductase activities, respectively.

Similarly, the induction of these detoxificationenzymes by dietary atrazine (0.1%) was ob-

served in larval midguts, although they were less

inducible as compared with larval fat bodies

(Table 2). The induction of microsomal oxidases

ranged from 1.3- to 2.6-fold with the highest

ll armyworm larvae

ty (nmol/min/mg protein)a

Atrazine (0.1%) Percentage of control

0.24 ± 0.01 150

0.040 ± 0.004 235

0.46 ± 0.01 139

0.31 ± 0.02 182

0.049 ± 0.003 258

0.72 ± 0.04 171

0.26 ± 0.001 260

0.052 ± 0.009 43

0.070 ± 0.01 127

0.19 ± 0.017 112

0.24 ± 0.02 126

385.8 ± 19.4 201

26.8 ± 4.0 168

546.2 ± 54.5 169

20.3 ± 2.3 119

6.61 ± 1.7 220

4.44 ± 0.04 196

12.1 ± 1.5 218

5.02 ± 0.35 141

8.83 ± 0.22 173

9.79 ± 0.71 149

12.9 ± 0.65 160

64.8 ± 4.5 127

1092.1 ± 10.8 280

271.4 ± 12.2 262

2.31 ± 0.25 254

4.82 ± 0.20 130

2.04 ± 0.11 79

35.3 ± 3.5 164

77.8 ± 7.9 151

ions.


induction level being observed with p-nitroanisole

O-demethylase and parathion desulfurase. How-

ever, this treatment caused a 57% decrease in

methoxyresorufin O-demethylase activity. Cyto-

chrome P450 content was not altered by atrazine,but cytochrome b5 level was increased by 26% as

compared with the control. The induction of

glutathione S-transferases ranged from 1.3- to

2.2-fold with the highest induction level being

observed with TPBO and trans-2-nonenal conju-

gation. Interestingly, ethacrynic conjugation was

not affected by the treatment. The induction of

hydrolases ranged from 1.3- to 2.8-fold with thehighest induction level being observed with a-NA esterase. However, this treatment caused a

21% reduction in p-nitroacetanilide carboxylami-

dase activity. Reductases also were moderately in-

duced by atrazine causing 1.6- and 1.5-fold

increases in juglone reductase and cytochrome c

reductase activity, respectively.

Table 3

Effect of triazine herbicides on detoxification enzymes in fall armywo

Tissue Detoxification enzyme % of control activitya

Simazine Cyana

Fat body Aldrin epoxidase 517 ± 40 351 ± 1

GST (CDNB) 144 ± 6 147 ± 6

a-NA esterase 206 ± 16 200 ± 7

Midgut Aldrin epoxidase 130 ± 7 82 ± 2

GST (CDNB) 128 ± 2 212 ± 1

a-NA esterase 166 ± 13 114 ± 5

a Means ± SE of 2–3 experiments, each with duplicate determinat

Table 4

Effect of atrazine on the toxicity of insecticides to fall armyworm lar

Insecticidea Dose (lg/larva)

Carbaryl 2

Permethrin 1

Indoxacarb 0.05

Methyl parathion 2.5

Phorate 20

Trichlorfon 3

Dichlorvos 3

a Newly molted sixth instars were fed an artificial diet containingb Means ± SE of three experiments.* Value significantly different from the respective control (P < 0.05

Table 3 shows the induction of three detoxifica-

tion enzymes (aldrin epoxidase, glutathione S-

transferase [CDNB], and general esterase) by five

other triazine herbicides in fall armyworm larvae.

It is seen that simazine, cyanazine, ametryn, tebut-ryn, and terbuthylazine all induced these detoxifi-

cation enzymes with the exceptions that aldrin

epoxidase was not induced by cyanazine and te-

butryn, and general esterase was not induced by

cyanazine in midguts. Among these herbicidies,

the induction of aldrin epoxidase ranged from

2.5- to 11-fold in fat bodies and 1.2- to 1.6-fold

in midguts. The induction of glutathione S-trans-ferase (CDNB) ranged from 1.4- to 4.0-fold in

fat bodies and 1.3- to 2.4-fold in midguts. The in-

duction of a-NA esterase ranged from 2.0- to 4.1-

fold in fat bodies and 1.4- to 1.7-fold in midguts.

The results also showed that fat bodies were more

inducible than midguts with respect to the detoxi-

fication enzymes.

rm larvae

zine Ametryn Tebutryn Terbuthylazine

3 1097 ± 204 254 ± 11 520 ± 12

400 ± 83 142 ± 11 147 ± 9

410 ± 30 217 ± 28 243 ± 20

155 ± 12 92 ± 6 124 ± 8

8 232 ± 3 241 ± 13 182 ± 10

151 ± 11 135 ± 7 172 ± 5

ions.

vae

Percent mortalityb

Control Atrazine (0.1%)

88 ± 12 18 ± 12*

75 ± 5 33 ± 3*

80 ± 2 11 ± 1*

0 60 ± 10*

4 ± 4 88 ± 8*

14 ± 3 55 ± 5*

73 ± 8 73 ± 7

atrazine (0.1%) for 2 days prior to insecticide treatments.

).


The results obtained from the bioassays of var-

ious insecticides including carbamate, pyrethroid,

oxadiazine and organophosphate with fall army-

worm fed atrazine are summarized in Table 4. It

can be seen that dietary atrazine at the 0.1% leveldecreased the toxicity of carbaryl, permethrin and

indoxacarb, but increased the toxicity of methyl

parathion, phorate and trichlorfon. However, the

toxicity of dichlorvos was not affected by the treat-

ment.

4. Discussion

The results of this study clearly demonstrated

that triazine herbicides such as atrazine, simazine,

cyanazine, ametryn, tebutryn, and terbuthylazine

(Fig. 1) induced detoxification enzymes including

microsomal oxidases, glutathione S-transferases,

and hydrolases in fall armyworm larvae. Atrazine

also induced reductases in this insect. In general,atrazine and ametryn were better inducers of these

enzymes among those herbicides tested, although

there is no clear indication of structure/activity re-

lationship. Atrazine was shown to induce micro-

somal oxidase (aldrin epoxidase) activity in the

cabbage moth, Mamestra brassica L. [7], the

southern armyworm, Spodoptera eridania Cram.

[8], and the aquatic midge, Chironomus tentans

(Fabricius) [9]. Atrazine also induced glutathione

S-transferase activity toward CDNB and DCNB

Fig. 1. Structures of atrazine, simazine, cyanazi

in cabbage moths [7]. Moreover, this herbicide

was found to alter the subunit composition of

GSTs in Orthosia gothica [30].

Our results showed that various detoxification

enzymes were more inducible in fat bodies thanin midguts. This trend also was observed in our

previous report showing that the induction of

GST in this species by xanthotoxin and indole 3-

acetonitrile was higher in fat bodies than in mid-

guts [22]. Apparently, fat bodies are more sensitive

to induction than midguts in this insect. In cotton

bollworms, total cytochrome P450 and microsom-

al O-demethylase activity also were more inducibleby naphthalene and pentamethylbenzene in the fat

body than in the midgut [31]. However, several mi-

crosomal oxidase activities in the southern army-

worm were more inducible by xenobiotics in the

midgut than in the fat body [32].

In the previous report [16], we showed that the

orders of eight microsomal oxidase activities (from

the highest activity to the lowest activity) in larvalmidguts were different from those of fat bodies

supporting the notion that cytochrome P450 en-

zymes are different in midguts and fat bodies of fall

armyworm larvae. This notion is further support-

ed by the results of the present study showing that

the orders of nine microsomal oxidase activities in

midguts were different from those of fat bodies af-

ter induction by atrazine in fall armyworm larvae.We reported previously that p-nitroacetanilide

carboxylamidase was different from carboxylester-

ne, ametryn, tebutryn, and terbuthylazine.


ases in the fall armyworm based on the purifica-

tion factor, substrate specificity and sensitivity to

hydrolase inhibitors [33]. This finding is further

confirmed by the present study showing that

dietary atrazine induced a-NA esterase and carb-oxylesterase but not p-nitroacetanilide carboxy-

lamidase in this insect.

Interestingly, the GST activity toward cumene

hydroperoxide was also inducible by atrazine in

fall armyworms. This GST peroxidase which is

an antioxidant enzyme detoxifies lipid peroxida-

tion products formed by the free-radical-mediated

attack on membrane lipids. Therefore, the induc-tion of GST peroxidase activity by atrazine would

reduce levels of lipid peroxidation products and

protect insects from membrane destruction and

DNA damage [34,35] caused by lipid peroxidation.

The decreased toxicity of carbaryl, permethrin

and indoxacarb was apparently due to enhanced

detoxification by microsomal oxidases, esterases

and glutathione S-transferases since these enzymesare involved in the metabolism of these insecticides

[36]. On the other hand, the increased toxicity of

methyl parathion and phorate was likely due to

enhanced microsomal desulfuration and sulfoxi-

dation, respectively, caused by atrazine. These

proinsecticides are known to be activated by mi-

crosomal oxidation to become more potent acetyl-

cholinesterase inhibitors [37,38]. The results are inagreement with our previous finding showing that

host plant induction of microsomal desulfurase

and sulfoxidase resulted in enhancing the toxicity

of numerous phosphorothioate and thioether-con-

taining insecticides in fall armyworm larvae [39]. It

is unclear as to how induction of these detoxifica-

tion enzymes by atrazine increased the toxicity of

trichlorfon in fall armyworm larvae. Trichlorofonwas found to be dehydrochlorinated to become

dichlorvos in mammals, which increased anticho-

linesterase activity [36]. Since glutathione S-trans-

ferases catalyze a dehydrochlorination reaction

[40], it is possible that the enhanced toxicity of tri-

chlorfon caused by atrazine was attributed to in-

creased GST activity in the fall armyworm. The

induction of detoxification enzymes by atrazinedid not decrease the toxicity of dichlorvos in this

insect even though this insecticide is degraded to

dimethyl phosphate possibly by phosphatase and

to desmethyl dichlorvos by glutathione S-transfer-

ase [41,42].

Finally, it should be mentioned that these six

triazine herbicides are commonly used in corn

fields to control weeds. Since corn is the preferredhost plant for fall armyworm larvae, it is likely

that through exposure, these herbicides will induce

detoxification enzymes in this insect and hence

change the susceptibility of this insect to various

insecticides in the field. Moreover, atrazine also

induced GST activities toward many toxic a,b-unsaturated carbonyl allelochemicals including

trans,trans-2,4-alkadienals and trans-2-alkenals infall armyworms. These allelochemicals are com-

monly present in corn, wheat and oats [43–45],

so induction of these GST activities may play an

important role in the feeding strategies of the lep-

idopterous insects.

Acknowledgments

The author thanks Drs. S.M. Valles (USDA-

ARS) and S.M. Ferkovich (USDA-ARS) for criti-

cal reviews of the manuscript. Technical assistance

of Sam Nguyen is also appreciated.

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