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In vivo penetration and metabolism of methyl parathion in larvae of the tobacco budworm, Heliothis virescens (F.), fed different host plants
PESTICIDE
BIOCHEMISTRY
AND
PHYSIOLOGY
33, 49-56 (1989)
In Vivo Penetration
and Metabolism of Methyl Parathion in Larvae of the Tobacco Budworm, Heliothis virescens (F.), Fed Different Host Plants S. Toxicology
F.
ABD-ELGHAFAR,’
Program,
Box 7633, North
W. C. Carolina
DAUTERMAN, State
AND
University,
E.
Raleigh,
HODGSON North
Carolina
27695
Received July 7, 1988; accepted September 9, 1988 The effect of wild tomato, Lycopersicon hirslrtum f. glabratum (accession PI 134417), and peppermint, Mentha piperita L., on the susceptibility, penetration, and metabolism of methyl parathion, O,f%dimethyl O-p-nitrophenyl phosphorothioate, was studied in larvae of the tobacco budworm, Heliothis virescens F. Third-instar larvae fed wild tomato or peppermint leaves for 1 day were 3.3- and 2.7-fold more tolerant to methyl parathion, respectively, as compared to insects fed an artificial diet. These tolerance levels were only 2.2- and 1.7-fold, respectively, in fifth-instar larvae when fed leaves of the same plant species for 1 day. Penetration studies did not indicate any differences in the rate of penetration of methyl parathion in larvae fed different diets. Methyl parathion injected into fifth-instar larvae was converted into three chloroform-soluble and five water-soluble metabolites. Five hours after injection, the extent of methyl parathion metabolism was greater in larvae fed wild tomato leaves (77.4%) or peppermint leaves (72.8%), than in those fed an artificial diet (64.55%). The major metabolite was p-nitrophenol and its formation was higher in larvae fed wild tomato leaves (49.4%) than in larvae fed peppermint leaves (44.23%), which in turn were higher than those fed an artificial diet (38.19%). These data suggested that the two plants induced enzymes responsible for detoxifying methyl parathion. o 1989 Academic press, hc. INTRODUCTION
as well as in mammals. There is evidence that induction by secondary plant substances found in host plants results in the reduction of insecticide susceptibility (6-9, 12, 13, 15) due to stimulation of the metabolism of the insecticide in question (18, 19). Many reports indicate the presence of certain monoterpenes in the peppermint plant, Mentha piperita L. (7, 12-14) and 2tridecanone in the glandular trichomes of a wild species of the tomato plant, Lycopersicon hirsutum f. glabratum (accession PI 134417) (20-23). The present study investigated the effects of both peppermint and wild tomato plants on the tobacco budworm larvae Heliothis virescens (F.) in relation to the toxicity, absorption, and metabolism of methyl parathion (O,Odimethyl 0-p-nitrophenyl phosphorothioate).
A wide variety of chemicals representing insecticides (l-3), insect hormones (4, 5), and allelochemicals present in host plants (6-9) have been shown to induce detoxication enzyme systems in insects. A number of insect species have been shown to respond to inducers by producing high levels of these enzymes (10). The Japanese beetle (1 l), the alfalfa and cabbage loopers (12), the southern armyworm (6, 8), the variegated cutworm (7, 9), the fall armyworm (13-16), the two spotted spider mite (17), and the tobacco budworm (18, 19) are representative of these species. The cytochrome P450-dependent monooxygenase system, carboxylesterases, and the glutathione S-transferases play an important role in the metabolism of various xenobiotics including insecticides in insects
MATERIALS
’ Present address: Auburn University, Entomology Department, 301 Funchess Hall, Auburn, AL 36849. ’ Abbreviation used: TOCP, tri-o-tolyl phosphate.
AND
METHODS
Insects. Larvae of the tobacco budworm, H. virescens (F.), were obtained from a col49 0048-3575189 $3.00 Copyright B 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
50
ABD-ELGHAFAR,
DAUTERMAN,
ony established at North Carolina State University and reared for approximately 3 years on a corn-soybean diet (24). This colony was maintained in the laboratory at 26.7”C with a 16:8 hr (1ight:dark) photoperiod. Chemicals. Methyl parathion was obtained from Chem Service Inc. (West Chester, PA) and methyl paraoxon was supplied by American Cyanamid Co. (Princeton, NJ). p-Nitrophenyl B-D-glucopyranoside was purchased from Sigma Chemical Co. (St. Louis, MO), both p-nitrophenol and tri-o-tolyl phosphate (TOCP) were obtained from Eastman Kodak Co. (Rochester, NY), and piperonyl butoxide was purchased from Fluka Chemical Corp. (New York, NY). p-[2,6-‘4C]Nitrophenol (Pathfinder Laboratories Inc., St. Louis, MO) was obtained with a specific activity of 8.5 mCi/mmol and was used to synthesize both i4C-labeled methyl parathion and methyl paraoxon. Radioactive methyl parathion was prepared by heating and stirring p-[2,614C]nitrophenol with an equimolar amount of anhydrous powdered K&O, in acetone for 1 hr at 400°C. An equimolar amount of 0,0-dimethyl phosphorochloridothionate was added dropwise to the reaction and heated and stirred at 700°C for 4 hr. The product was purified by silica gel 60 column chromatography using benzene:methanol (9:1, v/v) as the eluting solvent. “C-Ringlabeled methyl parathion was obtained with a specific activity of 6.07 pCi/mg and a yield of 85.7%. 14C-Ring-labeled methyl paraoxon was prepared and purified in a similar manner except that 0,0-dimethyl phosphorochloridate was substituted for O,O-dimethyl phosphorochloridothionate. The purification procedure was similar to that used with methyl parathion and the final yield was 90.85% with a specific activity of 6.43 &i/mg. Both desmethyl methyl parathion and desmethyl methyl paraoxon were prepared by reacting trimethylamine with methyl parathion and methyl paraoxon, respec-
AND
HODGSON
tively, according to the method of Crayford and Huston (25). The purities of both radiolabeled and nonradiolabeled compounds were greater than 98% as determined by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Treatment of insects. Third-instar larvae (25-30 mg) maintained on an artificial diet were randomly divided into three groups. One group was fed on fresh mature wild tomato leaves, Lycopersicon hirsutum F. glubrutum (accession PI 134417), the second group was fed on peppermint leaves, Mentha piperita L., and the third was fed on an artificial diet as a control (18). All plants were grown under greenhouse conditions. Plant leaves were placed on moist filter paper in lo-cm petri dishes, and a group of 20-30 larvae were placed in each dish. After 24 hr, larvae were removed from their respective diets and used for either toxicity (18) or penetration tests. In the metabolism studies, fifth-instar larvae (250300 mg) were confined in pairs with plant leaves in petri dishes to minimize cannibalism. Absorption studies. Third-instar larvae that had fed on the three different diets were treated topically with a sublethal dose (0.2 mg/larva) of [i4C]methyl parathion. The treated larvae were then held without food in covered glass containers for specific times. The unabsorbed radioactivity was removed from the larvae by rinsing them with 2 ml acetone (6 per sample). The larvae were then immediately homogenized in 4 ml distilled water. The homogenate was then partitioned against 10 ml of chloroform (1:2.5, v/v). Excreted radioactive material was recovered by rinsing the holding containers with 2 ml water followed by 2 ml acetone. Radioassays were made of the external rinse, the partitioned fractions of the homogenate, and the excreta wash (26). The larvae were analyzed at 1, 2, and 4 hr after treatment with methyl parathion as described by Whitten and Bull (27). Toxicity tests. Various doses of methyl parathion dissolved in 1.0 ml acetone were
METHYL
PARATHION
PENETRATION
applied to the dorsum of the thorax of thirdinstar larvae by means of an Arnold hand microapplicator (Burkard Manufacturing Co. Ltd., Rickmansworth, England). Five to seven concentrations were applied to three to five replicates of 10 larvae each. When the synergist was used, it was applied to the dorsum of the abdomen at a dose of 25 &larva 4 hr prior to the insecticide application. Mortality was recorded 24 hr after treatment and correction was made according to the method of Abbott (28). The LD,, values (mg/g body weight) were computed with probit analysis using SAS (29). In vivo metabolism. Fifth-instar larvae were anesthetized with CO, and two larvae/ replicate were injected with 2 mg [14C]methyl parathion through the first abdominal proleg (18). The radioactive insecticide was dissolved in 1 ml acetone (ca. 30,000 dpm/ml); preliminary studies showed that this quantity of acetone had no toxic effect on the larvae. Treated larvae were held in a glass container for 1, 3, or 5 hr and then homogenized in 5 ml of distilled water with a motor-driven all-glass tissue homogenizer. The homogenate was partitioned immediately against 12.5 ml chloroform and the resulting two phases were separated by centrifugation at 1OOOgfor 5 min. The aqueous phase was reextracted with chloroform three times. The amount of radioactivity in the organic and aqueous fractions was determined using a Packard Model 3330 liquid scintillation counter (Packard Instrument Co., Downers Grove, IL) using Scinti Verse II cocktail (Fisher Scientific Company, Fair Lawn, NJ). The counting efficiency was corrected by internal standardization with 14C-labeled IIhexadecane. Extraction efficiency from larval homogenates was ca. 97% of the injected radioactivity. Separation and identzjkation of metabolites. Chloroform extracts were separated, dried over anhydrous sodium sulfate, and the residues were extracted five to six times with 5-ml portions of methanol. The combined methanol extracts were concentrated
AND
METABOLISM
IN H. virescens
51
and the unextracted residual material was combusted in a Biological Oxidizer (Radiomatic Instruments and Chemical Co. Inc., Tampa, FL) to determine the amount of nonextractable radioactivity. More than 93% of the radioactivity in the water phase was extracted into methanol. The radioactivity in the organic extract was separated by TLC on 5 x 20-cm silica gel G/uv,,, (0.25 mm thick) precoated plates (Brinkman Instruments, Inc., New York). The water-soluble metabolites were separated using precoated thin-layer 20 x 20-cm silica gel channeled plates (0.25 mm thick) (Analtech, Newark, DE). Radioactive metabolites were detected and quantified by scanning the plates with an Automatic TLC linear analyzer (LB2832 Berthold). Identification of metabolites was based on cochromatography with standards which were visualized at 254 nm, by exposure to iodine vapor, or by spraying with 0.5% solution of 2,6-dibromo-Nchloro-p-quinonimine in cyclohexane (30) or with 0.25% palladium chloride in dilute HCI (0.1-0.2 N). The solvent systems used for the separation of various metabolites are given in Table 1. A number of solvent systems were used for the verification of each of the radioactive metabolites. RESULTS
AND
DISCUSSION
Toxicity Studies The toxicity of methyl parathion to H. virescens larvae fed on wild tomato or peppermint plants is presented in Table 2. Both third- and fifth-instar larvae of H. virescens became less susceptible to methyl parathion after 1 day of feeding on the host plants compared to larvae fed on the artificial diet. The tolerance levels were slight in the case of the fifth-instar larvae (2.2- and 1.7-fold, respectively) but were more obvious in the third-instar larvae (3.3- and 2.7-fold, respectively). These data agree with those obtained in similar studies with carbaryltreated variegated cutworm larvae (7) and diazinon-treated tobacco budworm larvae (18). Previous reports have shown that in-
52
ABD-ELGHAFAR,
DAUTERMAN, TABLE
R, Values
Solvent
of Methyl
Methyl parathion
system
1 Acetonitrile:water:ammonium hydroxide (40:9: I) 2 Benzene:ethyl acetate (8:2) 3 Benzene:ethyl acetate (3:2) 4 Benzene:methanol (9: 1) 5 Ethyl acetate:ethanol:ammonia (80:15:5) 6 Hexane:ethyl acetate:benzene (24: 1) 7 Petroleum ether:ethyl etheracetic acid (8O:lS:S)
1
Parathion
Methyl paraoxon
Diet” Artificial
of Methyl
and Its Metabolites
p-Nitrophenol
Desmethyl methyl parathion
Desmethyl methyl paraoxon
p-Nitrophenyl P-glucoside
0.92 0.88 0.67 0.90
0.89 0.47 0.26 0.71
0.70 0.60 0.52 0.64
0.58 0.21 0.00 0.03
0.64 0.08 0.00 0.01
0.00
0.93
0.84
0.24
0.00
0.00
0.04
0.91
0.46
0.65
0.00
0.00
0.00
0.68
0.26
0.47
0.34
0.58
0.70
sects fed peppermint leaves are more tolerant to insecticides due to the presence of high concentrations of certain monoterpenes, such as a-pinene, P-pinene, menthol, and peppermint oil (7, 13, 18). These monoterpenes increased the cytochrome P450 levels in larvae of the variegated cutworm (7), alfalfa looper and cabbage looper (12), and the tobacco budworm (18). The data obtained in this study with wild tomato plants agree with data obtained with diazinon-treated H. virescens (18) and was due to the presence of 2-tridecanone in the glandular trichomes (20-23). Our data also showed that fifth-instar larvae fed wild tomato leaves were more tolerant to methyl parathion than those fed peppermint leaves, whereas, overall, third-instar larvae
Toxicity
AND HODGSON
Parathion
Web
to Third-
0.00
were less tolerant than fifth-instar larvae. Synergists with different modes of action are commonly used to identify the induced enzymes. The effects of both piperonyl butoxide (an oxidase inhibitor) and tri-o-tolyl phosphate (TOCP;* an esterase inhibitor) on the third- and fifth-instar larvae of H. virescens are presented in Table 3. In all cases, slight or no synergism was observed with larvae fed the artificial diet as compared to larvae fed plant leaves. Higher degrees of synergism were observed with piperonyl butoxide compared with that of TOCP. Data in Table 3 shows that piperonyl butoxide caused synergistic factors of 2.5 and 1.9 in third-instar larvae fed tomato and peppermint leaves, respectively, and 2.5 and 2.3 in fifth-instar larvae.
TABLE and Fifth-Instar
Tolerance’ level
2 Larvae
of H. virescens
Fed Different
LDmb
Diets
Tolerance’ level
23.2 (9.1-41.0)
1.0
916.6 (706.4-1132.2)
1.0
Wild tomato
76.7 (55.8-104.1)
3.3
2031.3 (1604.13372.4)
2.2
Peppermint
62.7 (44.4-84.6)
2.7
1552.9 (1249.8-1994.2)
1.7
n Third-instar (25-30 mg) and fifth-instar larvae (250-300 mg) were fed on the diet indicated 24 hr prior to bioassay. ’ Micrograms per gram body weight (95% fiducial limits). c LD,, for larvae fed plant leave&D,, for larvae fed on artificial diet.
METHYL
PARATHION
PENETRATION
AND
METABOLISM
IN
H. virescens
53
TABLE 3 The Effect of Piperonyl Butoxide and TOCP on Methyl Parathion Toxicity to Third- and Fifth-Instar Larvae of H. virescens Fed Different Diets Third Instar
Fifth Instar MP alone
MP + PB
MP + TOCP
MP alone
S.F.’
LDmb
S.F.’
LD,ab
S.F.’
22.1 (8.5-36.9)
1.1
20.3 (7.2-34.0)
1.1
916.6 (706.4-1132.2)
857.7 (584.3-1075.9)
1.1
942.1 (658.0-1184.5)
1.0
76.1 (55.8-104.1)
30.4 (17.3-43.0)
2.5
41.7 (25.7-W. 1)
1.8
2031.1 (1604.8-3372.4)
813.1 (679.1-1235.3)
2.5
1022.9 (947.9-1575.9)
2.0
62.7 144.4-84.6)
32.7 (16.Wi2.5)
1.9
59.2 (20.492.6)
1.1
1522.9 (1249.8-1994.2)
662.4 (457.7-1038.3)
2.3
1041.4 (937.1-1637.9)
1.5
LDsob 23.2 (9.141.0)
Tomato Peppermint
LD,,,b
MP + TOCP
LDmb
Data” Artificial
S.F.”
MP + PB LD,ob
a Third-instar larvae (25-30 mg) and f&h-instar larvae. (25&300 mg) were fed on the diet indicated for 24 hr prior to bioassay. b Microgram per gram body weight (95% fiducial limits). c Synergistic factor: LDSo of methyl parathion alonelLDsO of methyl parathion + synergist. Synergist applied to the dorsum of the abdomen at a dose of 25 llgflarva 4 hr prior to application of the insecticides.
The synergistic factor obtained with piperonyl butoxide was higher than that obtained with TOCP for both instars tested. These data suggest that methyl parathion is metabolized in H. virescens larvae mainly by oxidative enzymes which are induced in larvae feeding on tomato and peppermint plants and that these enzymes are induced to a greater degree by wild tomato plants than by peppermint plants.
ference in the rates at which the insecticide was absorbed from the surface of the cuticle (Table 4). However, the proportion of dose that was recovered from all the larvae decreased with time. Four hours after the application of the insecticide, only about 35% of the original dose was recovered from all the larvae fed the various diets. These findings are similar to those obtained by Whitten and Bull (26, 27), who indicated that there was no significant difference in the rate of absorption of methyl parathion between resistant and susceptible H. virescens larvae. From our data it can be concluded that the secondary plant sub-
Absorption Studies
The results of experiments on the penetration of methyl parathion into larvae fed different diets indicated no significant dif-
TABLE 4 Penetration of Methyl Parathion into Third-Instar Larvae of Tobacco Budworm Fed Different Diets Percentage Hr after treatment 1 hr
Artificial
Ext. 63.13 (213.67)
recovery
2 hr
55.34 (23.62)
recovery
4 hr recovery
34.45 (23.11)
of applied
diet
dose as indicated
Tomato
fractionO’.b
leaves
Peppermint
Int.
Excreta
Ext.
Int.
Excreta
Ext.
22.12 (k2.15) 96.80%
14.75 (21.61)
64.55 (24.88)
22.99 (22.01) 94.41%
12.46 (k1.72)
65.83 (23.73)
26.80 (k2.67) 95.20%
17.86 (k2.10)
35.13 (23.09) 96.20%
30.42 (23.53)
36.98 (23.18)
mean
5 standard
L?Average of three to five replicates; b Dose applied was 0.2 &larva.
50.76 (k2.18)
29.54 (k3.11) 92.36% 37.81 (23.53) 91.70% deviation
leaves
Int. 20.50 (k1.85)
Excreta 13.67 (+l.ol)
91.98% 19.70
(22.37)
56.86 (k3.93)
25.88
17.26
90.80% 25.21 (22.14)
37.37 (53.29)
37.58 (23.91) 92.17%
25.05 (22.93)
54
ABD-ELGHAFAR,
DAUTERMAN,
stances have little or no effect on the penetration rate of methyl parathion in H. Studies
In vivo metabolism studies of methyl parathion in the fifth-instar larvae of H. virescens indicate that the insecticide was converted into three chloroform-soluble and five water-soluble metabolites (Table 5). The chloroform-soluble compounds were identified as methyl parathion, its oxygen analog, and p-nitrophenol. The five water-soluble metabolites were identified as p-nitrophenol, desmethyl methyl parathion, desmethyl methyl paraoxon, pnitrophenyl p-glucoside, and a minor unknown product. These data showed that the amount of metabolites increased with time in all tested larvae. After 5 hr, the percentTABLE
Vivo Metabolism
of Injected
Methyl
Parathion
Percentage Chloroform Hr after treatment
1 hr
3 hr
5 hr
Methyl parathion
Methyl
A.D.
69.% (f3.62)
11.31 (T1.21)
T.P.
59.05 (23.25)
8.85 (e2.23)
P.P.
63.56 (53.56)
9.79 (k-2.79)
A.D.
52.32 (22.75)
7.41 (23.41)
T.P.
43.55 (k3.82)
6.41 (21.56)
P.P.
48.45 (+2.-B)
6.17 (kl.87)
A.D.
35.45 (e-2.46)
5.74 (t 1.26)
T.P.
22.64 (21.41)
2.04 (20.49)
P.P.
27.18 (k2.38)
3.25 (?l.Ol)
Dietb
of dose
5
by Fifth-Instar recovered
soluble
p-Nitrophenol
* Average of three replicated tests; mean b Fifth-instar larvae were fed the indicated P.P., peppermint plant.
9.94 (f2.25) (12.69 14.36 (k2.39) (21.31 13.15 (21.89) (17.90 13.11 (23.35) (23.96 14.28 (~2.63) (29.98 13.44 (kl.98) (25.45 16.98 (kl.83) (38.19 19.60 (-t1.60) (49.42 16.89 (kl.78) (44.23
p-Nitrophenol
+
k
2
f.
c
f
2
t
t
HODGSON
age of remaining unmetabolized methyl parathion was 35.5,22.6, and 27.2 in larvae fed artificial diet, wild tomato leaves, and peppermint leaves, respectively. Also, the rate of metabolism was not that much higher in larvae fed wild tomato leaves than in larvae fed peppermint or artificial diet. The principal metabolite recovered from both treatments was p-nitrophenol, which was distributed between the chloroform and water phases. The total amount of pnitrophenol recovered after 5 hr was 38.2, 49.4, and 44.2% of initially injected radiolabeled methyl parathion for larvae fed artificial diet, wild tomato leaves, and peppermint leaves, respectively. These results indicate that cleavage of the aryl phosphate linkage is the primary detoxification route for methyl parathion in H. virescens larvae and the enzymes involved are induced
virescens. Metabolism
AND
2.75 (kO.48) 2.73) 6.95 (LO.70) 3.09) 4.75 (kO.53) 2.42) 10.85 (?1.64) 4.99) 15.70 (kl.09) 3.72) 13.01 (kl.15) 3.13) 21.21 (kl.35) 3.18) 29.82 (22.61) 3.87) 27.34 (k2.06) 3.84)
+ standard deviation. diet 24 hr prior to insecticide
H. virescens
after
treatment0
Water
soluble
Larvae
Desmethyl methyl parathion
Desmethyl methyl paraoxon
p-Nitrophenyl P-glucoside
Unknown
3.56 (?0.57)
0.92 (AO.34)
1.08 (20.19)
0.49 (kO.43)
6.71 (~0.81)
1.73 (k0.31)
1.91 (50.36)
0.37 (20.17)
5.09 (10.50)
1.22 (+0.26)
1.30 (20.27)
1.14 (AO.34)
9.53 (f0.82)
1.60 (?0.12)
3.35 (kO.45)
1.83 (kO.37)
11.24 (5 1.33)
2.39 (kO.85)
5.45 (+0.90)
0.99 (kO.30)
10.81 (Zl.13)
1.91 (kO.41)
4.36 (20.82)
1.55 (-cO.Sl)
12.92 (21.46)
0.79 (-cO.12)
5.39 (20.51)
1.49 (kO.53)
17.57 (kl.97)
0.91 (r0.32)
6.86 (+0.16)
0.55 (?0.05)
15.87 (~1.67)
1.20 (kO.54)
5.87 (k1.45)
2.40 (20.73)
treatment.
A.D.,
artificial
diet;
T.P.,
tomato
plant;
METHYL
PARATHION
PENETRATION
when the larvae are fed on tomato or peppermint leaves. These findings are in agreement with Whitten and Bull (26, 27), who indicated that the rate of metabolism of methyl parathion and chlorpyrifos was greater in resistant larvae of H. virescens than susceptible larvae. Also, they indicated that the cleavage of the aryl phosphate linkage was the primary metabolic route for both methyl parathion and chlorpyrifos. MethyI paraoxon was detected in all cases. However, the amount found in larvae fed artificial diets was always higher than that found in the larvae which were fed plant leaves. Also an increase in the amount of desmethyl methyl parathion and desmethyl methyl paraoxon was found in larvae fed wild tomato or peppermint leaves when compared to larvae fed artificial diets. The observed differences in the rates of methyl parathion degradation suggest that a difference in susceptibility between larvae fed plant leaves and larvae fed the artificial diet is due to increased metabolism. The importance of our findings indicate that the selection of plants with high levels of allelochemicals for resistance to insects should be done with caution. Our data also suggest that plants with high levels of allelochemicals that are resistant to some insects could also induce tolerance to certain insecticidal chemicals. This interaction should be investigated when developing plants resistant to insect pests: this favorable resistance in the plant species to a primary pest may be detrimental to insecticide treatments applied to control other pests. ACKNOWLEDGMENTS Paper No. 11672 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. S. F. Abd-Elghafar is grateful to Amideast for financial support during this study.
AND METABOLISM
3.
4.
5.
6.
7.
8. 9.
10. 11.
12.
13.
14.
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IN H. virescens
55
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17. 18.
19.
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23.
ABD-ELGHAFAR,
DAUTERMAN,
detoxication enzymes of insecticide-susceptible and resistant fall armyworms, Pestic. Biochem. Physiol. 22, 60 (1984). C. A. Mullin and B. A. Croft, Host-related alterations of detoxification enzymes in Terrunychus urticae, Environ. Entomol. 12, 1278 (1983). M. R. Riskallah, W. C. Dauterman, and E. Hodgson, Host plant induction of microsomal monooxygenase activity in relation to diazinon metabolism and toxicity in larvae of the tobacco budworm Heiiothis virescens (F.), Pestic. Biothem. Physiol. 25, 233 (1986). M. R. Riskallah, W. C. Dauterman, and E. Hodgson, Nutritional effects on the induction of cytochrome P-450 and glutathione transferase in larvae of the tobacco budworm, Heliothis virescens (F.), Insect Biochem. 16, 491 (1986). G. G. Kennedy and W. R. Henderson, A laboratory assay for resistance to tobacco homworm in Lycopersicon and Solanum spp., J. Amer. Sot. Hortic. Sci. 26, 121 (1979). G. G. Kennedy and R. T. Yamamoto, A toxic factor causing resistance in a wild tomato to the tobacco homworm and some other insects, Entomof. Exp. Appt. 26, 121 (1979). W. G. Williams, G. G. Kennedy, R. T. Yamamoto, J. D. Thacker, and J. Bordner, 2Tridecanone: A naturally occurring insecticide from the wild tomato Lycopersicon hirsutum F. glabratum, Science 207, 888 (1980). G. G. Kennedy, R. T. Yamamoto, M. B. Dimock,
AND
24. 25.
26.
27.
28. 29. 30.
HODGSON
W. G. Williams, and J. Bordener, Effect of the day length and light intensity on 2-tridecanone levels and resistance in Lycopersicon hirsutum F. glabratum to Manduca sexta, .I. Chem. Ecol. 7, 707 (1981). R. L. Burton, A low cost diet for the corn earworm, J. Econ. Entomol. 63, 1969 (1970). J. V. Crayford and D. H. Huston, The metabolism of the herbicide 2-chloro-4-(ethylamine)6-( I-cyano-7-methyl(ethylamino)-S-triazine in the rat, Pestic. Biochem. Physiot. 2,295 (1972). C. J. Whitten and D. L. Bull, Comparative toxicity, absorption, and metabolism of chlorpyrifos and its dimethyl homologs in methyl parathionresistant and -susceptible tobacco budworm, Pestic. Biochem. Physiol. 4, 266 (1974). C. J. Whitten and D. L. Bull, Metabolism and absorption of methyl parathion by tobacco budworms resistant or susceptible to organophosphorus insecticides, Pestic. Biochem. Physiol. 9, 196 (1978). W. S. Abbott, A method of computing the effectiveness of an insecticide, J. Econ. Entomol. 18, 265 (1925). “SAS User’s Guide: Statistics,” SAS Institute, Cat-y, NC, 1985. J. J. Menn, W. R. Erwin, and H. T. Gordon, Color reaction of 2.6-dibromo-N-chlorop-quinoneimine with thiophosphate insecticides on paper chromatogram, J. Agric. Food Chem. 5, 601 (1957).
BIOCHEMISTRY
AND
PHYSIOLOGY
33, 49-56 (1989)
In Vivo Penetration
and Metabolism of Methyl Parathion in Larvae of the Tobacco Budworm, Heliothis virescens (F.), Fed Different Host Plants S. Toxicology
F.
ABD-ELGHAFAR,’
Program,
Box 7633, North
W. C. Carolina
DAUTERMAN, State
AND
University,
E.
Raleigh,
HODGSON North
Carolina
27695
Received July 7, 1988; accepted September 9, 1988 The effect of wild tomato, Lycopersicon hirslrtum f. glabratum (accession PI 134417), and peppermint, Mentha piperita L., on the susceptibility, penetration, and metabolism of methyl parathion, O,f%dimethyl O-p-nitrophenyl phosphorothioate, was studied in larvae of the tobacco budworm, Heliothis virescens F. Third-instar larvae fed wild tomato or peppermint leaves for 1 day were 3.3- and 2.7-fold more tolerant to methyl parathion, respectively, as compared to insects fed an artificial diet. These tolerance levels were only 2.2- and 1.7-fold, respectively, in fifth-instar larvae when fed leaves of the same plant species for 1 day. Penetration studies did not indicate any differences in the rate of penetration of methyl parathion in larvae fed different diets. Methyl parathion injected into fifth-instar larvae was converted into three chloroform-soluble and five water-soluble metabolites. Five hours after injection, the extent of methyl parathion metabolism was greater in larvae fed wild tomato leaves (77.4%) or peppermint leaves (72.8%), than in those fed an artificial diet (64.55%). The major metabolite was p-nitrophenol and its formation was higher in larvae fed wild tomato leaves (49.4%) than in larvae fed peppermint leaves (44.23%), which in turn were higher than those fed an artificial diet (38.19%). These data suggested that the two plants induced enzymes responsible for detoxifying methyl parathion. o 1989 Academic press, hc. INTRODUCTION
as well as in mammals. There is evidence that induction by secondary plant substances found in host plants results in the reduction of insecticide susceptibility (6-9, 12, 13, 15) due to stimulation of the metabolism of the insecticide in question (18, 19). Many reports indicate the presence of certain monoterpenes in the peppermint plant, Mentha piperita L. (7, 12-14) and 2tridecanone in the glandular trichomes of a wild species of the tomato plant, Lycopersicon hirsutum f. glabratum (accession PI 134417) (20-23). The present study investigated the effects of both peppermint and wild tomato plants on the tobacco budworm larvae Heliothis virescens (F.) in relation to the toxicity, absorption, and metabolism of methyl parathion (O,Odimethyl 0-p-nitrophenyl phosphorothioate).
A wide variety of chemicals representing insecticides (l-3), insect hormones (4, 5), and allelochemicals present in host plants (6-9) have been shown to induce detoxication enzyme systems in insects. A number of insect species have been shown to respond to inducers by producing high levels of these enzymes (10). The Japanese beetle (1 l), the alfalfa and cabbage loopers (12), the southern armyworm (6, 8), the variegated cutworm (7, 9), the fall armyworm (13-16), the two spotted spider mite (17), and the tobacco budworm (18, 19) are representative of these species. The cytochrome P450-dependent monooxygenase system, carboxylesterases, and the glutathione S-transferases play an important role in the metabolism of various xenobiotics including insecticides in insects
MATERIALS
’ Present address: Auburn University, Entomology Department, 301 Funchess Hall, Auburn, AL 36849. ’ Abbreviation used: TOCP, tri-o-tolyl phosphate.
AND
METHODS
Insects. Larvae of the tobacco budworm, H. virescens (F.), were obtained from a col49 0048-3575189 $3.00 Copyright B 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.
50
ABD-ELGHAFAR,
DAUTERMAN,
ony established at North Carolina State University and reared for approximately 3 years on a corn-soybean diet (24). This colony was maintained in the laboratory at 26.7”C with a 16:8 hr (1ight:dark) photoperiod. Chemicals. Methyl parathion was obtained from Chem Service Inc. (West Chester, PA) and methyl paraoxon was supplied by American Cyanamid Co. (Princeton, NJ). p-Nitrophenyl B-D-glucopyranoside was purchased from Sigma Chemical Co. (St. Louis, MO), both p-nitrophenol and tri-o-tolyl phosphate (TOCP) were obtained from Eastman Kodak Co. (Rochester, NY), and piperonyl butoxide was purchased from Fluka Chemical Corp. (New York, NY). p-[2,6-‘4C]Nitrophenol (Pathfinder Laboratories Inc., St. Louis, MO) was obtained with a specific activity of 8.5 mCi/mmol and was used to synthesize both i4C-labeled methyl parathion and methyl paraoxon. Radioactive methyl parathion was prepared by heating and stirring p-[2,614C]nitrophenol with an equimolar amount of anhydrous powdered K&O, in acetone for 1 hr at 400°C. An equimolar amount of 0,0-dimethyl phosphorochloridothionate was added dropwise to the reaction and heated and stirred at 700°C for 4 hr. The product was purified by silica gel 60 column chromatography using benzene:methanol (9:1, v/v) as the eluting solvent. “C-Ringlabeled methyl parathion was obtained with a specific activity of 6.07 pCi/mg and a yield of 85.7%. 14C-Ring-labeled methyl paraoxon was prepared and purified in a similar manner except that 0,0-dimethyl phosphorochloridate was substituted for O,O-dimethyl phosphorochloridothionate. The purification procedure was similar to that used with methyl parathion and the final yield was 90.85% with a specific activity of 6.43 &i/mg. Both desmethyl methyl parathion and desmethyl methyl paraoxon were prepared by reacting trimethylamine with methyl parathion and methyl paraoxon, respec-
AND
HODGSON
tively, according to the method of Crayford and Huston (25). The purities of both radiolabeled and nonradiolabeled compounds were greater than 98% as determined by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC). Treatment of insects. Third-instar larvae (25-30 mg) maintained on an artificial diet were randomly divided into three groups. One group was fed on fresh mature wild tomato leaves, Lycopersicon hirsutum F. glubrutum (accession PI 134417), the second group was fed on peppermint leaves, Mentha piperita L., and the third was fed on an artificial diet as a control (18). All plants were grown under greenhouse conditions. Plant leaves were placed on moist filter paper in lo-cm petri dishes, and a group of 20-30 larvae were placed in each dish. After 24 hr, larvae were removed from their respective diets and used for either toxicity (18) or penetration tests. In the metabolism studies, fifth-instar larvae (250300 mg) were confined in pairs with plant leaves in petri dishes to minimize cannibalism. Absorption studies. Third-instar larvae that had fed on the three different diets were treated topically with a sublethal dose (0.2 mg/larva) of [i4C]methyl parathion. The treated larvae were then held without food in covered glass containers for specific times. The unabsorbed radioactivity was removed from the larvae by rinsing them with 2 ml acetone (6 per sample). The larvae were then immediately homogenized in 4 ml distilled water. The homogenate was then partitioned against 10 ml of chloroform (1:2.5, v/v). Excreted radioactive material was recovered by rinsing the holding containers with 2 ml water followed by 2 ml acetone. Radioassays were made of the external rinse, the partitioned fractions of the homogenate, and the excreta wash (26). The larvae were analyzed at 1, 2, and 4 hr after treatment with methyl parathion as described by Whitten and Bull (27). Toxicity tests. Various doses of methyl parathion dissolved in 1.0 ml acetone were
METHYL
PARATHION
PENETRATION
applied to the dorsum of the thorax of thirdinstar larvae by means of an Arnold hand microapplicator (Burkard Manufacturing Co. Ltd., Rickmansworth, England). Five to seven concentrations were applied to three to five replicates of 10 larvae each. When the synergist was used, it was applied to the dorsum of the abdomen at a dose of 25 &larva 4 hr prior to the insecticide application. Mortality was recorded 24 hr after treatment and correction was made according to the method of Abbott (28). The LD,, values (mg/g body weight) were computed with probit analysis using SAS (29). In vivo metabolism. Fifth-instar larvae were anesthetized with CO, and two larvae/ replicate were injected with 2 mg [14C]methyl parathion through the first abdominal proleg (18). The radioactive insecticide was dissolved in 1 ml acetone (ca. 30,000 dpm/ml); preliminary studies showed that this quantity of acetone had no toxic effect on the larvae. Treated larvae were held in a glass container for 1, 3, or 5 hr and then homogenized in 5 ml of distilled water with a motor-driven all-glass tissue homogenizer. The homogenate was partitioned immediately against 12.5 ml chloroform and the resulting two phases were separated by centrifugation at 1OOOgfor 5 min. The aqueous phase was reextracted with chloroform three times. The amount of radioactivity in the organic and aqueous fractions was determined using a Packard Model 3330 liquid scintillation counter (Packard Instrument Co., Downers Grove, IL) using Scinti Verse II cocktail (Fisher Scientific Company, Fair Lawn, NJ). The counting efficiency was corrected by internal standardization with 14C-labeled IIhexadecane. Extraction efficiency from larval homogenates was ca. 97% of the injected radioactivity. Separation and identzjkation of metabolites. Chloroform extracts were separated, dried over anhydrous sodium sulfate, and the residues were extracted five to six times with 5-ml portions of methanol. The combined methanol extracts were concentrated
AND
METABOLISM
IN H. virescens
51
and the unextracted residual material was combusted in a Biological Oxidizer (Radiomatic Instruments and Chemical Co. Inc., Tampa, FL) to determine the amount of nonextractable radioactivity. More than 93% of the radioactivity in the water phase was extracted into methanol. The radioactivity in the organic extract was separated by TLC on 5 x 20-cm silica gel G/uv,,, (0.25 mm thick) precoated plates (Brinkman Instruments, Inc., New York). The water-soluble metabolites were separated using precoated thin-layer 20 x 20-cm silica gel channeled plates (0.25 mm thick) (Analtech, Newark, DE). Radioactive metabolites were detected and quantified by scanning the plates with an Automatic TLC linear analyzer (LB2832 Berthold). Identification of metabolites was based on cochromatography with standards which were visualized at 254 nm, by exposure to iodine vapor, or by spraying with 0.5% solution of 2,6-dibromo-Nchloro-p-quinonimine in cyclohexane (30) or with 0.25% palladium chloride in dilute HCI (0.1-0.2 N). The solvent systems used for the separation of various metabolites are given in Table 1. A number of solvent systems were used for the verification of each of the radioactive metabolites. RESULTS
AND
DISCUSSION
Toxicity Studies The toxicity of methyl parathion to H. virescens larvae fed on wild tomato or peppermint plants is presented in Table 2. Both third- and fifth-instar larvae of H. virescens became less susceptible to methyl parathion after 1 day of feeding on the host plants compared to larvae fed on the artificial diet. The tolerance levels were slight in the case of the fifth-instar larvae (2.2- and 1.7-fold, respectively) but were more obvious in the third-instar larvae (3.3- and 2.7-fold, respectively). These data agree with those obtained in similar studies with carbaryltreated variegated cutworm larvae (7) and diazinon-treated tobacco budworm larvae (18). Previous reports have shown that in-
52
ABD-ELGHAFAR,
DAUTERMAN, TABLE
R, Values
Solvent
of Methyl
Methyl parathion
system
1 Acetonitrile:water:ammonium hydroxide (40:9: I) 2 Benzene:ethyl acetate (8:2) 3 Benzene:ethyl acetate (3:2) 4 Benzene:methanol (9: 1) 5 Ethyl acetate:ethanol:ammonia (80:15:5) 6 Hexane:ethyl acetate:benzene (24: 1) 7 Petroleum ether:ethyl etheracetic acid (8O:lS:S)
1
Parathion
Methyl paraoxon
Diet” Artificial
of Methyl
and Its Metabolites
p-Nitrophenol
Desmethyl methyl parathion
Desmethyl methyl paraoxon
p-Nitrophenyl P-glucoside
0.92 0.88 0.67 0.90
0.89 0.47 0.26 0.71
0.70 0.60 0.52 0.64
0.58 0.21 0.00 0.03
0.64 0.08 0.00 0.01
0.00
0.93
0.84
0.24
0.00
0.00
0.04
0.91
0.46
0.65
0.00
0.00
0.00
0.68
0.26
0.47
0.34
0.58
0.70
sects fed peppermint leaves are more tolerant to insecticides due to the presence of high concentrations of certain monoterpenes, such as a-pinene, P-pinene, menthol, and peppermint oil (7, 13, 18). These monoterpenes increased the cytochrome P450 levels in larvae of the variegated cutworm (7), alfalfa looper and cabbage looper (12), and the tobacco budworm (18). The data obtained in this study with wild tomato plants agree with data obtained with diazinon-treated H. virescens (18) and was due to the presence of 2-tridecanone in the glandular trichomes (20-23). Our data also showed that fifth-instar larvae fed wild tomato leaves were more tolerant to methyl parathion than those fed peppermint leaves, whereas, overall, third-instar larvae
Toxicity
AND HODGSON
Parathion
Web
to Third-
0.00
were less tolerant than fifth-instar larvae. Synergists with different modes of action are commonly used to identify the induced enzymes. The effects of both piperonyl butoxide (an oxidase inhibitor) and tri-o-tolyl phosphate (TOCP;* an esterase inhibitor) on the third- and fifth-instar larvae of H. virescens are presented in Table 3. In all cases, slight or no synergism was observed with larvae fed the artificial diet as compared to larvae fed plant leaves. Higher degrees of synergism were observed with piperonyl butoxide compared with that of TOCP. Data in Table 3 shows that piperonyl butoxide caused synergistic factors of 2.5 and 1.9 in third-instar larvae fed tomato and peppermint leaves, respectively, and 2.5 and 2.3 in fifth-instar larvae.
TABLE and Fifth-Instar
Tolerance’ level
2 Larvae
of H. virescens
Fed Different
LDmb
Diets
Tolerance’ level
23.2 (9.1-41.0)
1.0
916.6 (706.4-1132.2)
1.0
Wild tomato
76.7 (55.8-104.1)
3.3
2031.3 (1604.13372.4)
2.2
Peppermint
62.7 (44.4-84.6)
2.7
1552.9 (1249.8-1994.2)
1.7
n Third-instar (25-30 mg) and fifth-instar larvae (250-300 mg) were fed on the diet indicated 24 hr prior to bioassay. ’ Micrograms per gram body weight (95% fiducial limits). c LD,, for larvae fed plant leave&D,, for larvae fed on artificial diet.
METHYL
PARATHION
PENETRATION
AND
METABOLISM
IN
H. virescens
53
TABLE 3 The Effect of Piperonyl Butoxide and TOCP on Methyl Parathion Toxicity to Third- and Fifth-Instar Larvae of H. virescens Fed Different Diets Third Instar
Fifth Instar MP alone
MP + PB
MP + TOCP
MP alone
S.F.’
LDmb
S.F.’
LD,ab
S.F.’
22.1 (8.5-36.9)
1.1
20.3 (7.2-34.0)
1.1
916.6 (706.4-1132.2)
857.7 (584.3-1075.9)
1.1
942.1 (658.0-1184.5)
1.0
76.1 (55.8-104.1)
30.4 (17.3-43.0)
2.5
41.7 (25.7-W. 1)
1.8
2031.1 (1604.8-3372.4)
813.1 (679.1-1235.3)
2.5
1022.9 (947.9-1575.9)
2.0
62.7 144.4-84.6)
32.7 (16.Wi2.5)
1.9
59.2 (20.492.6)
1.1
1522.9 (1249.8-1994.2)
662.4 (457.7-1038.3)
2.3
1041.4 (937.1-1637.9)
1.5
LDsob 23.2 (9.141.0)
Tomato Peppermint
LD,,,b
MP + TOCP
LDmb
Data” Artificial
S.F.”
MP + PB LD,ob
a Third-instar larvae (25-30 mg) and f&h-instar larvae. (25&300 mg) were fed on the diet indicated for 24 hr prior to bioassay. b Microgram per gram body weight (95% fiducial limits). c Synergistic factor: LDSo of methyl parathion alonelLDsO of methyl parathion + synergist. Synergist applied to the dorsum of the abdomen at a dose of 25 llgflarva 4 hr prior to application of the insecticides.
The synergistic factor obtained with piperonyl butoxide was higher than that obtained with TOCP for both instars tested. These data suggest that methyl parathion is metabolized in H. virescens larvae mainly by oxidative enzymes which are induced in larvae feeding on tomato and peppermint plants and that these enzymes are induced to a greater degree by wild tomato plants than by peppermint plants.
ference in the rates at which the insecticide was absorbed from the surface of the cuticle (Table 4). However, the proportion of dose that was recovered from all the larvae decreased with time. Four hours after the application of the insecticide, only about 35% of the original dose was recovered from all the larvae fed the various diets. These findings are similar to those obtained by Whitten and Bull (26, 27), who indicated that there was no significant difference in the rate of absorption of methyl parathion between resistant and susceptible H. virescens larvae. From our data it can be concluded that the secondary plant sub-
Absorption Studies
The results of experiments on the penetration of methyl parathion into larvae fed different diets indicated no significant dif-
TABLE 4 Penetration of Methyl Parathion into Third-Instar Larvae of Tobacco Budworm Fed Different Diets Percentage Hr after treatment 1 hr
Artificial
Ext. 63.13 (213.67)
recovery
2 hr
55.34 (23.62)
recovery
4 hr recovery
34.45 (23.11)
of applied
diet
dose as indicated
Tomato
fractionO’.b
leaves
Peppermint
Int.
Excreta
Ext.
Int.
Excreta
Ext.
22.12 (k2.15) 96.80%
14.75 (21.61)
64.55 (24.88)
22.99 (22.01) 94.41%
12.46 (k1.72)
65.83 (23.73)
26.80 (k2.67) 95.20%
17.86 (k2.10)
35.13 (23.09) 96.20%
30.42 (23.53)
36.98 (23.18)
mean
5 standard
L?Average of three to five replicates; b Dose applied was 0.2 &larva.
50.76 (k2.18)
29.54 (k3.11) 92.36% 37.81 (23.53) 91.70% deviation
leaves
Int. 20.50 (k1.85)
Excreta 13.67 (+l.ol)
91.98% 19.70
(22.37)
56.86 (k3.93)
25.88
17.26
90.80% 25.21 (22.14)
37.37 (53.29)
37.58 (23.91) 92.17%
25.05 (22.93)
54
ABD-ELGHAFAR,
DAUTERMAN,
stances have little or no effect on the penetration rate of methyl parathion in H. Studies
In vivo metabolism studies of methyl parathion in the fifth-instar larvae of H. virescens indicate that the insecticide was converted into three chloroform-soluble and five water-soluble metabolites (Table 5). The chloroform-soluble compounds were identified as methyl parathion, its oxygen analog, and p-nitrophenol. The five water-soluble metabolites were identified as p-nitrophenol, desmethyl methyl parathion, desmethyl methyl paraoxon, pnitrophenyl p-glucoside, and a minor unknown product. These data showed that the amount of metabolites increased with time in all tested larvae. After 5 hr, the percentTABLE
Vivo Metabolism
of Injected
Methyl
Parathion
Percentage Chloroform Hr after treatment
1 hr
3 hr
5 hr
Methyl parathion
Methyl
A.D.
69.% (f3.62)
11.31 (T1.21)
T.P.
59.05 (23.25)
8.85 (e2.23)
P.P.
63.56 (53.56)
9.79 (k-2.79)
A.D.
52.32 (22.75)
7.41 (23.41)
T.P.
43.55 (k3.82)
6.41 (21.56)
P.P.
48.45 (+2.-B)
6.17 (kl.87)
A.D.
35.45 (e-2.46)
5.74 (t 1.26)
T.P.
22.64 (21.41)
2.04 (20.49)
P.P.
27.18 (k2.38)
3.25 (?l.Ol)
Dietb
of dose
5
by Fifth-Instar recovered
soluble
p-Nitrophenol
* Average of three replicated tests; mean b Fifth-instar larvae were fed the indicated P.P., peppermint plant.
9.94 (f2.25) (12.69 14.36 (k2.39) (21.31 13.15 (21.89) (17.90 13.11 (23.35) (23.96 14.28 (~2.63) (29.98 13.44 (kl.98) (25.45 16.98 (kl.83) (38.19 19.60 (-t1.60) (49.42 16.89 (kl.78) (44.23
p-Nitrophenol
+
k
2
f.
c
f
2
t
t
HODGSON
age of remaining unmetabolized methyl parathion was 35.5,22.6, and 27.2 in larvae fed artificial diet, wild tomato leaves, and peppermint leaves, respectively. Also, the rate of metabolism was not that much higher in larvae fed wild tomato leaves than in larvae fed peppermint or artificial diet. The principal metabolite recovered from both treatments was p-nitrophenol, which was distributed between the chloroform and water phases. The total amount of pnitrophenol recovered after 5 hr was 38.2, 49.4, and 44.2% of initially injected radiolabeled methyl parathion for larvae fed artificial diet, wild tomato leaves, and peppermint leaves, respectively. These results indicate that cleavage of the aryl phosphate linkage is the primary detoxification route for methyl parathion in H. virescens larvae and the enzymes involved are induced
virescens. Metabolism
AND
2.75 (kO.48) 2.73) 6.95 (LO.70) 3.09) 4.75 (kO.53) 2.42) 10.85 (?1.64) 4.99) 15.70 (kl.09) 3.72) 13.01 (kl.15) 3.13) 21.21 (kl.35) 3.18) 29.82 (22.61) 3.87) 27.34 (k2.06) 3.84)
+ standard deviation. diet 24 hr prior to insecticide
H. virescens
after
treatment0
Water
soluble
Larvae
Desmethyl methyl parathion
Desmethyl methyl paraoxon
p-Nitrophenyl P-glucoside
Unknown
3.56 (?0.57)
0.92 (AO.34)
1.08 (20.19)
0.49 (kO.43)
6.71 (~0.81)
1.73 (k0.31)
1.91 (50.36)
0.37 (20.17)
5.09 (10.50)
1.22 (+0.26)
1.30 (20.27)
1.14 (AO.34)
9.53 (f0.82)
1.60 (?0.12)
3.35 (kO.45)
1.83 (kO.37)
11.24 (5 1.33)
2.39 (kO.85)
5.45 (+0.90)
0.99 (kO.30)
10.81 (Zl.13)
1.91 (kO.41)
4.36 (20.82)
1.55 (-cO.Sl)
12.92 (21.46)
0.79 (-cO.12)
5.39 (20.51)
1.49 (kO.53)
17.57 (kl.97)
0.91 (r0.32)
6.86 (+0.16)
0.55 (?0.05)
15.87 (~1.67)
1.20 (kO.54)
5.87 (k1.45)
2.40 (20.73)
treatment.
A.D.,
artificial
diet;
T.P.,
tomato
plant;
METHYL
PARATHION
PENETRATION
when the larvae are fed on tomato or peppermint leaves. These findings are in agreement with Whitten and Bull (26, 27), who indicated that the rate of metabolism of methyl parathion and chlorpyrifos was greater in resistant larvae of H. virescens than susceptible larvae. Also, they indicated that the cleavage of the aryl phosphate linkage was the primary metabolic route for both methyl parathion and chlorpyrifos. MethyI paraoxon was detected in all cases. However, the amount found in larvae fed artificial diets was always higher than that found in the larvae which were fed plant leaves. Also an increase in the amount of desmethyl methyl parathion and desmethyl methyl paraoxon was found in larvae fed wild tomato or peppermint leaves when compared to larvae fed artificial diets. The observed differences in the rates of methyl parathion degradation suggest that a difference in susceptibility between larvae fed plant leaves and larvae fed the artificial diet is due to increased metabolism. The importance of our findings indicate that the selection of plants with high levels of allelochemicals for resistance to insects should be done with caution. Our data also suggest that plants with high levels of allelochemicals that are resistant to some insects could also induce tolerance to certain insecticidal chemicals. This interaction should be investigated when developing plants resistant to insect pests: this favorable resistance in the plant species to a primary pest may be detrimental to insecticide treatments applied to control other pests. ACKNOWLEDGMENTS Paper No. 11672 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, NC 27695-7643. S. F. Abd-Elghafar is grateful to Amideast for financial support during this study.
AND METABOLISM
3.
4.
5.
6.
7.
8. 9.
10. 11.
12.
13.
14.
REFERENCES 1. C. F. Wilkinson and L. B. Brattsten, Microsomal drug metabolizing enzymes in insects, Drug Metab. Rev. 1, 153 (1972). 2. E. Hodgson, Microsomal monooxygenases, in
15. 16.
IN H. virescens
55
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