Induction of detoxifying enzymes by allelochemicals and host plants in the fall armyworm

The effect of various plant substances and host plants on the microsomal oxidases and glutathione S-transferase was investigated in the fall armyworm (Spodoptera frugiperda (J. E. Smith)) maintained on a meridic diet. The glucosinolate, sinigrin, and the hydrolytic products of glucosinolates, β-phenylethylisothiocyanate, indole 3-acetonitrile, and indole 3-carbinol, and flavone were found to be potent inducers of the glutathione S-transferase in the armyworm. An 18-fold increase in the transferase activity was observed when larvae were fed a diet containing 0.2% indole 3-acetonitrile for 2 days. These compounds, with the exception of β-phenylethylisothiocyanate which appeared to be inhibitory, also stimulated the microsomal aldrin epoxidase significantly. In all instances, no induction of the microsomal oxidase or glutathione S-transferase was observed by the plant hormones, indole 3-acetic acid and gibberellic acid; the terpenoids, stigmasterol, sitosterol, and β-carotene; the polyphenolic gossypol; and the flavonol, quercetin; some of them were found to be inhibitory. Using corn, potato, and sweet potato as inducers of various microsomal oxidases, it was found that the inducing pattern of the N-demethylase was different from the two epoxidases and O-demethylase. Corn leaves were the most active compared with other aerial parts of corn (silks, developing corn, and husk) in inducing the microsomal oxidase. The microsomal oxidase in the younger larvae appeared to be less inducible by host plants than in the older larvae.     

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

The inductive effect of six triazine herbicides on a variety of detoxification enzymes was investigated in fall armyworm (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 hydrolase 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 terbuthylazine 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.     

Induction of microsomal oxidases by host plants in the fall armyworm,Spodoptera frugiperda (J. E. Smith)

Larvae of the fall armyworm (Spodoptera frugiperda (J. E. Smith)) were maintained on a semidefined artificial diet until the end of the fifth instar and the newly molted sixth-instar larvae were then fed fresh leaves of various host plants for 2 days prior to microsomal oxidase assays. The order of the midgut aldrin epoxidase activity of larvae after feeding on these plants was: corn > Bermuda grass > cotton > cowpeas > cabbage > peanuts > sorghum > alfalfa > millet > soybean. The ratios of aldrin epoxidase, p-nitroanisole O-demethylase, and p-chloro-N-methylaniline N-demethylase activities for soybean- and corn-fed larvae were 4.5, 6.3, and 2.7-fold, respectively. The induction was affected by the age and developmental stage of the corn plant with mature leaves from old plants being more active. Higher epoxidase activities were also observed in mature larvae reared on corn plants since egg stage as compared with those on soybean plants. Monoterpenes such as α-pinene, β-pinene, limonene, menthol, and peppermint oil were found to induce the midgut epoxidase activity. Corn-fed larvae were more tolerant of the insecticides methomyl, acephate, methamidophos, diazinon, trichlorfon, monocrotophos, permethrin, and cypermethrin than soybean-fed larvae.     

Detoxication capacity in the honey bee,Apis mellifera L

Various detoxifying enzymes, including microsomal oxidases, glutathione S-transferases, esterases, epoxide hydrolase, and DDT-dehydrochlorinase, were assayed in adult worker bees (Apis mellifera L.) using midguts as the enzyme source. A cell-free system was used for all enzyme assays, except that microsomal oxidases required intact midgut because of the inhibitor encountered. Midgut microsomal preparations contained mainly cytochrome P-420, the inactive form of cytochrome P-450, which may explain the low microsomal oxidase activity in microsomes. All enzymes studied were active, suggesting that the high susceptibility of honey bees to insecticides is not due to low detoxication capacity. Sublethal exposure of honey bees to various insecticides had no effect on these enzyme activities, with the exception of permethrin which significantly stimulated the glutathione S-transferase, and malathion, which significantly inhibited the α-naphthylacetate esterase and carboxylesterase.     

Host plant induction of glutathione S-transferase in the fall armyworm

The influence of various host plants on glutathione S-transferase activity was studied in the fall armyworm, Spodoptera frugiperda (J. E. Smith). Fall armyworm larvae were maintained on a semidefined artificial diet until the end of the fifth instar. The newly molted sixth instar larvae were then fed fresh leaves of various host plants for 2 days prior to glutathione S-transferase assays using 3,4-dichloronitrobenzene as substrate. The order of the midgut glutathione S-transferase activity of larvae after the worms fed on these plants was: mustard > turnip > cowpeas > peanuts > cotton > corn > cucumber > potato > Bermudagrass > millet > sorghum > soybeans. The difference in the transferase activity between soybean- and mustard-fed larvae was 10-fold. Kinetic study revealed a quantitative, but no qualitative difference in the glutathione S-transferase between soybean- and cowpea-fed larvae. Monoterpenes, such as α-pinene, β-pinene, menthol, and peppermint oil, had no effect on the enzyme. Cowpea-fed larvae were more tolerant of the insecticides diazinon, methamidophos, and methyl parathion than soybean-fed larvae were. These new observations help explain what has been happening in the field and might be of use in the development of pest management programs.     

Resistance selection and biochemical mechanism of resistance to two Acaricides in Tetranychus cinnabarinus (Boiduval)

A Tetranychus cinnabarinus strain was collected from Chongqing, China. After 42 generations of selection with abamectin and 20 generations of selection with fenpropathrin in the laboratory, this T. cinnabarinus strain developed 8.7- and 28.7-fold resistance, respectively. Resistance to abamectin in AbR (abamectin resistant strain) and to fenpropathrin in FeR (fenpropathrin resistant strain) was partially suppressed by piperonyl butoxide (PBO), diethyl maleate (DEM) and triphenyl phosphate (TPP), inhibitors of mixed function oxidase (MFO), glutathione S-transferases (GST), and hydrolases, respectively, suggesting that these three enzyme families are important in conferring abamectin and fenpropathrin resistance in T. cinnabarinus. The major resistant mechanism to abamectin was the increasing activities of carboxylesterases (CarE), glutathione-S-transferase (GST) and mixed function oxidase (MFO), and the activity in resistant strain developed 2.7-, 3.4- and 1.4-fold contrasted to that in susceptible strain, respectively. The activity of glutathione-S-transferase (GST) in the FeR strain developed 2.8-fold when compared with the susceptible strain, which meant the resistance to fenpropathrin was related with the activity increase of glutathione-S-transferase (GST) in T. cinnabarinus. The result of the kinetic mensuration of carboxylesterases (CarE) showed that the structure of CarE in the AbR has been changed.     

In vitro metabolism of EPN and EPNO in susceptible and resistant strains of house flies

EPN is twice as toxic as EPNO to house flies from both the Diazinon-resistant strain and the susceptible strain. EPN and EPNO are also eight times more toxic to the susceptible than the resistant strain. This is due to the ability of the resistant strain to metabolize these compounds to a greater extent. Metabolism by the glutathione S-transferases present in the 100,000g supernatant is more extensive than that by the NADPH-dependent microsomal mixed-function oxidases. The glutathione S-transferases are the major route of metabolism for EPN and appear to be the principal mechanism conferring resistance. EPN was metabolized by the microsomal fraction via oxidative desulfuration to the oxygen analog, EPNO, and by oxidative dearylation to p-nitrophenol. EPNO was metabolized by the same system to p-nitrophenol and desethyl EPNO as well as to an unknown metabolite. The soluble fraction metabolized EPN to p-nitrophenol, S-(p-nitrophenyl)glutathione, O-ethyl phenylphosphonothioic acid, and S-(O-ethyl phenylphosphonothionyl)glutathione. The identification of the latter conjugate demonstrates a new type of metabolite of organophosphorus compounds. EPNO was metabolized by the soluble fraction to p-nitrophenol and S-(p-nitrophenyl)glutathione.     

Microsomal oxidase activity of three blowfly species and its induction by phenobarbital and β-naphtoflavone

Induction of the microsomal oxidase system by dietary phenobarbital and β-naphthoflavone was examined in three blowflies, Phormia regina (Mg.), Lucilia illustris (Mg.), and Eucalliphora lilica (Walk.). Responses were similar in adults and larvae of all species. Phenobarbital increased cytochrome P-450 levels up to 9-fold and aldrin epoxidase up to 138-fold. Increases in cytochrome P-450 and aldrin epoxidase caused by β-naphthoflavone were minor relative to those produced by phenobarbital. In toxicity experiments with carbaryl and propoxur tolerance was associated with the amount of microsomal oxidase activity. Using piperonyl butoxide to synergize carbaryl and propoxur there was no clear indication for the use of either the synergist ratio or synergist difference as an indicator of microsomal oxidase activity.     

Metabolism of fenitrothion by organophosphorus-resistant and -susceptible house flies, Musca domestica L

The metabolism of fenitrothion was investigated in highly resistant (Akita-f) and susceptible (SRS) strains of the house fly, Musca domestica L. The Akita-f strain was 3500 times more resistant to fenitrothion than the SRS strain. Fenitrothion, topically applied to the flies, was metabolized in vivo far faster in the Akita-f strain than in the SRS strain. In vitro studies revealed that fenitrothion was metabolized by a cytochrome P-450-dependent monooxygenase system and glutathione S-transferases. The former oxidase system metabolized fenitrothion in vitro into fenitrooxon and 3-methyl-4-nitrophenol as major metabolites, and into 3-hydroxymethyl-fenitrothion and 3-hydroxymethyl-fenitrooxon as minor metabolites. Glutathione S-transferases metabolized fenitrothion into desmethylfenitrothion. The cytochrome P-450-dependent monooxygenase system and glutathione S-transferases of the resistant Akita-f strain had 1.4 to 2.2 times and 9.7 times, respectively, as great activities as those of the susceptible SRS strain. These results suggest the importance of glutathione S-transferases in fenitrothion resistance in the Akita-f strain.     

Induction of glutathione S-transferase by phenobarbital and pesticides in various house fly strains and its effect on toxicity

The effect of phenobarbital and certain pesticides on glutathione S-transferase activity was investigated. The maximum amount of enzyme induction occurred 96 hr after phenobarbital treatment. Chlorinated hydrocarbons were more effective inducers than the other pesticides evaluated. Phenobarbital treatment did not alter the apparent K_m value but altered the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ value of glutathione S-transferase to 3,4-dichloronitrobenzene. The amount of reduced glutathione was not increased by phenobarbital treatment. Pretreatment of house flies with phenobarbital provides some protection against methyl parathion, methyl paraoxon, azinphosmethyl, and methidathion toxicity.     

Metabolism of substituted diphenylether herbicides in plants. I. Enzymatic cleavage of fluorodifen in peas (Pisum sativum L.)

A “soluble” glutathione S-transferase that catalyzes the cleavage of the herbicide, 2,4′-dinitro-4-trifluoromethyl diphenylether (fluorodifen), was isolated and partially characterized from epicotyl tissues of pea seedlings. A 32-fold purification of the enzyme was achieved by differential centrifugation, ammonium sulfate precipitation, Sephadex gel filtration, and DEAE-cellulose ion exchange chromatography. The enzyme had a pH optimum of 9.3–9.5 and was specific for reduced glutathione, with an estimated apparent K_m value of 7.4 × 10^?4M. Limited specificity studies with four substituted ¹⁴C-labeled diphenylether compounds indicated that fluorodifen was the only effective substrate, with an estimated apparent K_m value of 1.2 × 10^?5M. Differences and similarities between the pea epicotyl enzyme and other plant and animal glutathione S-transferases were discussed from the standpoint of substrate specificity, pH optima, distribution, stability, and inhibitor studies.     

Host plant stimulation of detoxifying enzymes in a phytophagous insect

The midgut microsomal aldrin epoxidase of variegated cutworm larvae (Peridroma saucia, Hübner) fed bean or peppermint leaves was up to 10 and 45 times more active, respectively, than that of larvae fed a basic control diet. Large increases in oxidase activity and cytochrome P-450 levels also occurred in larvae fed mint plant constituents such as menthol menthone, α-pinene, and β-pinene. Mint-fed larvae were more tolerant of the insecticide, carbaryl, than bean-fed larvae.     

The metabolism of 6,7-dihydroisodrin by microsomes and southern armyworm larvae

Dihydroisodrin is hydroxylated to 6-exo-hydroxy-6,7-dihydroisodrin in southern armyworm gut and vertebrate liver microsomal preparations. The sedimentation characteristics of the hydroxylase, its requirement for NADPH and oxygen, and its inhibition by carbon monoxide and 4,5,6,7-tetrachloro-1,3-benzodioxole indicate that the enzyme system may be classed as a typical microsomal oxidase. The enzyme system is susceptible to substrate inhibition, but in other respects it is similar to microsomal aldrin epoxidase and p-chloro-N-methylaniline demethylase.     

Induction of glutathione S-transterase by fumigants in larvae of the Khapra beetle,Trogoderma granarium (E.)

The effect of fumigants on glutathione and glutathione S-transferase in the Khapra beetle larvae (Trogoderma granarium) was studied by fumigating for 1, 3, and 5 hr with a dose causing 100% mortality at 24 hr of exposure. Glutathione and glutathione S-transferase were assayed in the cytosol at 1, 3, and 5 hr of exposure. Time-dependent depletion of glutathione was seen for all fumigants except carbon tetrachloride and phosphine. The depletion was maximum (60–70%) in the cases of methyl bromide, methyl iodide, and acrylonitrile, and least (20–30%) in the cases of ethylene dibromide and ethylene oxide. The order of glutathione depletion by various fumigants at 5 hr exposure was methyl iodide > methyl bromide = acrylonitrile > ethylene dichloride > ethylene oxide > ethylene dibromide. Glutathione S-transferase was induced by all fumigants except ethylene dibromide, methyl bromide being more potent than methyl iodide. The enzyme induction ranged from 186% by acrylonitrile to 40% by carbon tetrachloride. Mortality above 10% correlated well with the degree of GSH depletion (r = 0.729) whereas the latter did not correlate with the transferase induction.     

Biochemical characteristics of insecticide resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith)

A strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from corn in Citra, Florida, showed high resistance to carbaryl (562-fold) and methyl parathion (354-fold). Biochemical studies revealed that various detoxification enzyme activities were higher in the field strain than in the susceptible strain. In larval midguts, activities of microsomal oxidases (epoxidases, hydroxylase, sulfoxidase, N-demethylase, and O-demethylase) and hydrolases (general esterase, carboxylesterase, β-glucosidase) were 1.2- to 1.9-fold higher in the field strain than in the susceptible strain. In larval fat bodies, various activities of microsomal oxidases (epoxidases, hydroxylase, N-demethylase, O-demethylases, and S-demethylase), glutathione S-transferases (CDNB, DCNB, and p-nitrophenyl acetate conjugation), hydrolases (general esterase, carboxylesterase, β-glucosidase, and carboxylamidase) and reductases (juglone reductase and cytochrome c reductase) were 1.3- to 7.7-fold higher in the field strain than in the susceptible strain. Cytochrome P450 level was 2.5-fold higher in the field strain than in the susceptible strain. In adult abdomens, their detoxification enzyme activities were generally lower than those in larval midguts or fat bodies; this is especially true when microsomal oxidases are considered. However, activities of microsomal oxidases (S-demethylase), hydrolases (general esterase and permethrin esterase) and reductases (juglone reductase and cytochrome c reductase) were 1.5- to 3.0-fold higher in the field strain than in the susceptible strain. Levels of cytochrome P450 and cytochrome b₅ were 2.1 and 1.9-fold higher, respectively, in the field strain than in the susceptible strain. In addition, acetylcholinesterase from the field strain was 2- to 85-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, propoxur, carbofuran, bendiocarb, thiodicarb) and organophosphates (methyl paraoxon, paraoxon, dichlorvos), insensitivity being highest toward carbaryl. Kinetics studies showed that the apparent K_m value for acetylcholinesterase from the field strain was 56% of that from the susceptible strain. The results indicated that the insecticide resistance observed in the field strain was due to multiple resistance mechanisms, including increased detoxification of these insecticides by microsomal oxidases, glutathione S-transferases, hydrolases and reductases, and target site insensitivity such as insensitive acetylcholinesterase. Resistance appeared to be correlated better with detoxification enzyme activities in larval fat bodies than in larval midguts, suggesting that the larval fat body is an ideal tissue source for comparing detoxification capability between insecticide-susceptible and -resistant insects.     

Endogenous inhibitors of glutathione S-transferases in house flies

The inhibition of glutathione S-transferase by endogenous compounds present in the soluble fraction of house fly homogenates was investigated. The highest inhibition was found with the female abdomen and increased with incubation time and with an increase in the tissue concentration. The correlation of increased inhibition with a parallel increase in the darkening of the soluble fraction indicated a possible association with melanization, thereby suggesting quinones as the possible endogenous inhibitiors of glutathione transferase. In vitro experiments demonstrated that quinones produced by mushroom tyrosinase did indeed inhibit glutathione S-transferase. Inhibition by quinones can be prevented by including glutathione or bovine serum albumin in the homogenization buffer. The inhibitory activity of a variety of quinones and related compounds on purified glutathione S-transferase was investigated. Oxygenated aromatics with hydroxy groups in the 1,2- or 1,4-position or ketonic carbonyls in the 1,4-position are good inhibitors of glutathione S-transferase.     

Microsomal oxidases in the mole crickets,Scapteriscus acletus Rehn and Hebard and Scapteriscus vicinus Scudder

An active microsomal oxidase system was found in various tissues of the mole cricket adults, Scapteriscus acletus Rehn and Hebard and Scapteriscus vicinus Scudder. Aldrin epoxidase activity was mainly located in the midgut and Malpighian tubules. p-Nitroanisole O-demethylase and p-chloro-N-methylaniline N-demethylase activities were also detected in these two tissues. The overall oxidase activities in the two species were generally similar. Seasonal variation in epoxidase activity was evident in the two species with the spring populations having more activity than the fall populations. Spectral characterization of the cytochrome P-450 revealed that the enzyme was not of “resistant type” in both species. S. vicinus was four- to fivefold more susceptible to the insecticide propoxur than S. acletus.     

The effect of phenobarbital induction on glutathione conjugation of diazinon in susceptible and resistant house flies

The relationship between glutathione S-transferase activity toward 3,4-dichloronitrobenzene and O-alkyl or O-aryl conjugation of diazinon was investigated in eight strains of house flies. No significant difference was found in the amount of O-aryl conjugation. In contrast, house flies which had higher glutathione S-transferase activity toward 3,4-dichloronitrobenzene also had higher O-alkyl conjugating activity toward diazinon. The glutathione S-transferase(s) in phenobarbital-pretreated flies degraded diazinon faster than those in the nontreated ones. The present results showed that the formation of the O-alkyl conjugate was enhanced by phenobarbital pretreatment, while the formation of the O-aryl conjugate was not affected by induction. Based on these findings, it would appear that one of the multiple forms of glutathione S-transferase is specifically induced and responsible for the increase in O-alkyl conjugation.     

Status of Insecticide Resistance in Spodoptera litura in Andhra Pradesh,India

Twenty-two strains of the tobacco caterpillar, Spodoptera litura (F.) (Lepidoptera: Noctuidae), collected from groundnut crops of eight locations in Andhra Pradesh, India, between 1991 and 1996 were assayed in the F1 generation for resistance to commonly used insecticides. Resistance levels ranged as follows: cypermethrin, 0·2- to 197-fold; fenvalerate, 8- to 121-fold; endosulfan, 1-to 13-fold; quinalphos, 1- to 29-fold; monocrotophos, 2- to 362-fold and methomyl, 0·7- to 19-fold. In nearly all strains pre-treatment with the metabolic inhibitor, piperonyl butoxide, resulted in complete suppression of cypermethrin resistance (2- to 121-fold synergism), indicating that enhanced detoxification by microsomal P450-dependent monooxygenases was probably the major mechanism of pyrethroid resistance. Pre-treatment with the synergist DEF, an inhibitor of esterases and the glutathione S-transferase system, resulted in a 2- to 3-fold synergism with monocrotophos indicating that esterases and possibly glutathione S-transferases were at least to some extent contributing to organophosphate resistance. © 1997 SCI.     

Metabolic resistance mechanisms to phosalone in the common pistachio psyllid, Agonoscena pistaciae (Hem.: Psyllidae)

The common pistachio psyllid, Agonoscena pistaciae, is the most damaging pest of pistachio in Iran, and is generally controlled by insecticides belonging to various classes especially, phosalone. The toxicity of phosalone in nine populations of the pest was assayed using the residual contact vial and insect-dip methods. The bioassay results showed significant discrepancy in susceptibility to phosalone among the populations. Resistance ratio of the populations to the susceptible population ranged from 3.3 to 11.3. The synergistic effects of TPP, PBO and DEM were evaluated on the susceptible and the most resistant population to determine the involvement of esterases, mixed function oxidases and glutathione S-transferases in resistance mechanisms, respectively. The level of resistance to phosalone in the resistant population was suppressed by TPP, PBO and DEM, suggesting that the resistance to phosalone is mainly caused by esterase detoxification. Biochemical enzyme assays revealed that esterase, glutathione S-transferase and cytochrome P450 monooxygenase activities in the resistant population was higher than that in the susceptible. Glutathione-S-transferases play a minor role in the resistance of the pest to phosalone.