S.J. Yu

Host plant stimulation of detoxifying enzymes in a phytophagous insect

Abstract The midgut microsomal aldrin epoxidase of variegated cutworm larvae (Peridroma saucia, Hubner) 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.

Insect juvenile hormones: Induction of detoxifying enzymes in the housefly and detoxication by housefly enzymes☆

Abstract The cecropia juvenile hormone and three of its analogs were compared as inducers of microsomal epoxidase, O -demethylase, and DDT dehydrochlorinase in the housefly, Musca domestica L. The compounds were the cecropia juvenile hormone, methoprene, hydroprene, 6,7-epoxy-3,7-diethyl-1-[3,4-(methylenedioxy)phenoxy]-2-octene, and piperonyl butoxide, a well known insecticide synergist. The compounds were administered by feeding at levels up to 1% in the diet for 3 days to 1-day-old female adults. Enzymes were then prepared and assayed for their activity using heptachlor, p -nitroanisole, and DDT as substrates. There was approximately a twofold increase in the microsomal oxidases and a 50% increase in DDT dehydrochlorinase after the treatment with the cecropia juvenile hormone, while methoprene had some activity as an inducer of the epoxidase (30% increase) but no activity in the case of the O -demethylase or the dehydrochlorinase. Hydroprene had no effect on any of the enzyme systems, while 6,7-epoxy-3,7-diethyl-1-[3,4-(methylenedioxy)phenoxy]-2-octene was an inhibitor of the two microsomal oxidases. The latter compound and piperonyl butoxide were strong inducers of DDT dehydrochlorinase, causing approximately twofold increases in the activity of this enzyme. There was evidence that the microsomal preparations were able to metabolize and inactivate methoprene and hydroprene, the action being oxidative in the case of methoprene and both oxidative and hydrolytic in the case of hydroprene. The oxidative metabolism of the two juvenile hormone analogs by the microsomal preparations was inducible by the cecropia juvenile hormone and by phenobarbital and dieldrin.

Metabolism of juvenile hormone I by microsomal oxidase, esterase, and epoxide hydrase of Musca domestica and some comparisons with Phormia regina and Sarcophaga bullata

Abstract House fly ( Musca domestica L.) microsomes prepared from larvae, pupae, or adults contain three enzyme system which can metabolize juvenile hormone I: an esterase, an oxidase, and epoxide hydrase. The presence of the oxidase is indicated by the increased metabolism when microsomes are supplemented with NADPH and by the occurrence of additional metabolites tentatively identified as products arising from oxidation of the 6, 7 double bond. Additional evidence of the activity of the oxidase system is the increased metabolism of juvenile hormone I by the NADPH-dependent system from phenobarbital-induced insects, by inhibition of the oxidation by piperonyl butoxide and carbon monoxide, and by the greater metabolism of the hormone by microsomes from insecticide-resistant (high oxidase) strains. In vivo studies of house fly adults treated with 3 H-labeled juvenile hormone I reveal a pattern of metabolism similar to that seen during NADPH-supplemented in vitro metabolism. The three enzymes have somewhat different patterns of activity during the larval stage of the house fly, juvenile hormone esterase and epoxide hydrase beginning at a high level of activity in the young larvae while the juvenile hormone oxidase is low at this stage. In the late larval stage all three enzymes show increased activity followed by declines during the pupal stage and further increases in the adult stage. Comparison of in vitro enzyme levels of the house fly, flesh fly ( Sarcophaga bullata Parker), and blow fly [ Phormia regina (Meigen)] showed that, although the enzymes were present in the latter two species, their activity on a per insect basis was considerably less than that of the house fly.

Ecdysone metabolism by soluble enzymes from three species of diptera and its inhibition by the insect growth regulator TH-6040☆

Abstract Homogenates of larvae, pupae, and adults of house flies ( Musca domestica L.), flesh flies ( Sarcophaga bullata Parker), and blow flies ( Phormia regina (Meigen)) have been examined for enzymes which convert α- and β-ecdysone to apolar products. Most of the activity was found in the soluble fraction from house flies and flesh flies but none of the blow fly fractions was active. Two enzymes seem to be involved in the ecdysone metabolism, one requiring NADPH and the other functioning without this cofactor. The product of the latter enzyme is thought to be the 3-dehydro-ecdysone. This product is further converted to the 3α-hydroxy isomer of ecdysone by the NADPH-requiring enzyme. On feeding the insect growth regulator TH-6040 (1-(4-chlorophenyl)-3-(2,6-difluorobenzoyl)-urea) to larvae at dietary levels ranging from 0.3 to 10 ppm, the activity of the enzyme producing the 3-dehydro product is reduced by 20 to 82%. It is suggested that the growth regulator exerts its effect on pupal-adult ecdysis through its inhibition of ecdysone metabolism.

Cytochrome P-450 in insects: 1. Differences in the forms present in insecticide resistant and susceptible house flies

Abstract Soluble cytochrome P -450 prepared from the microsomal fraction of abdomen homogenates of an insecticide resistant strain (Rutgers) and a susceptible strain (NAIDM) of the house fly, Musca domestica L., was characterized by spectral and electrophoretic methods. Six chromatographically distinct fractions were obtained after chromatography on DEAE-cellulose and hydroxylapatite. Examination of the six fractions by difference spectrophotometry indicated that the wave lengths for maximum absorption of the cytochrome P -450-carbon monoxide complexes were at 450, 451, and 452 nm for the NAIDM fractions and at 449, 450, and 451 nm for the Rutgers fractions. The type II binding spectra of the cytochrome P -450 in each fraction were measured with n -octylamine. Several of these resembled spectra which, in studies of hepatic cytochrome P -450, have been shown to be due to the presence of the high spin form of this hemoprotein. Four of the fractions from the resistant strain were of this type compared to one from the susceptible strain. Electrophoresis experiments indicated that there were at least three hemoproteins in the 40,000–60,000 molecular weight range in the fractions from the resistant strain while four could be detected in those from the susceptible strain. The specific aldrin epoxidase activity of the most active Rutgers fractions was considerably higher than that of similar fractions from the NAIDM microsomes in reconstitution experiments.

Phenobarbital induction of detoxifying enzymes in resistant and susceptible houseflies

Abstract Houseflies, Musca domestica , L., were treated with the drugs phenobarbital and 3-methylcholanthrene to study the effects of these compounds as inducing agents of the microsomal oxidases, heptachlor epoxidase, and p -nitroanisole O -demethylase, and of DDT-dehydrochlorinase. Phenobarbital was active when applied by injection or as part of the diet but inactive when topically applied. The resulting increases in heptachlor epoxidase activity were as much as 25-fold that of the untreated controls. The net increase in enzyme activity after phenobarbital treatment was greater in an insecticide-susceptible strain, WHO-SRS strain, than in a carbamate-resistant strain. However, the phenobarbital induced increases in DDT-dehydrochlorinase were greater, about 2-fold, in the resistant strains than in the susceptible strain. The optimum dose for phenobarbital was 1% in the diet for a period of 3 days. None of the treatments with 3-MC, feeding, injection, exposure to residues, or topical, were effective in induction.

Aldrin epoxidase activity and cytochrome P-450 content of microsomes prepared from alfalfa and cabbage looper larvae fed various plant diets

Abstract The alfalfa looper ( Californica autographica ) and the cabbage looper ( Trichoplusia ni ) were reared through the fourth instar on a semidefined artificial diet and for the first 60 hr of the last instar on fresh leaves of one of their host plants. Microsomes prepared from midguts and a carcass segment which included the fat body were then assayed for aldrin epoxidase activity and cytochrome P -450 content. Epoxidation by midgut microsomes of alfalfa loopers fed peppermint leaves was at a rate up to eight times that of larvae fed alfalfa, snap beans, or broccoli. The epoxidase activity of the carcass microsomes was induced about fourfold by the peppermint diet. Midgut microsomes prepared from cabbage looper larvae reared on peppermint leaves were also more active (about four times) than those reared on alfalfa, broccoli, or cabbage. Although the epoxidase activity of the carcass microsomes of the cabbage loopers was low, about one-eighth that of the midgut microsomes, this enzyme was greatly induced by peppermint leaves. In neither species did the peppermint leaf diet cause a corresponding increase in the cytochrome P -450 content of the microsomes, the maximum difference between peppermint and the other plants being about twofold. Bioassays of cabbage looper larvae reared on broccoli or peppermint and of alfalfa loopers reared on alfalfa or peppermint indicated that the stimulation of microsomal oxidase activity by the peppermint constituents provided increased tolerance for carbaryl and methomyl but not acephate.

Microsomal oxidases in the flesh fly (Sarcophaga bullata Parker) and the black blow fly [Phormia regina (Meigen)]

Abstract The microsomal oxidase system in the flesh fly ( Sarcophaga bullata Parker) and the black blow fly [ Phormia regina (Meigen)] was examined using aldrin as substrate. In both species the oxidase requires NADPH and is inhibited by CO and by piperonyl butoxide. The enzyme activity changes significantly during larval development, reaching a maximum shortly before puparium formation then declining during the pupal stage and increasing again in adults. Dietary sodium phenobarbital at 0. 1– 0.5% increases the oxidase activity up to 60- and 56-fold in the larvae of blow flies and flesh flies, respectively.

Enzyme induction in the housefly: The specificity of the cyclodiene insecticides☆

Abstract Eleven cyclodiene compounds, including the insecticides aldrin, dieldrin, heptachlor, and endrin, were compared as inducers of microsomal oxidases in a cyclodieneresistant strain of the housefly. All of the compounds except dihydroisodrin caused increases in enzyme activity. The epoxide members of the group appeared to be more effective inducers than their olefin precursors. Isodrin and endrin were effective at approximately 1 300 th the dose of the other compounds tested although neither compound enhanced enzyme activity as much as the others in the group. The maximum increase in enzyme activity was approximately 100% for aldrin and about 30% for isodrin and endrin. There was no additive effect when each of the compounds was combined with dieldrin in the induction treatments. In addition to microsomal epoxidation and hydroxylation, O-demethylation was enhanced by the cyclodiene treatments. Furthermore, DDT dehydrochlorinase, a nonmicrosomal enzyme, was increased as much as 137% by the treatments. The results indicate that within this group of compounds there are some structural requirements for activity but that the overall effect on enzyme activity is rather nonspecific.

Microsomal metabolism of juvenile hormone analogs in the house fly, Musca domestica L.☆

Abstract Of six juvenile hormone analogs of the alkyl 3,7,11-trimethyl-2,4-dodecadienate type, only the isopropyl ester was strongly morphogenic in the house fly, Musca domestica L. In vitro assays revealed that house fly microsomes contain B-esterases as well as oxidases which metabolize such analogs. However, these esterases did not hydrolyze the isopropyl ester, ZR-515. Enzymes prepared from larvae, pupae, and adults were all active and there was evidence that in the late larval stage the esterase activity was cyclic, showing a minimum in the early third instar and a maximum a few hours later. When microsomes from two susceptible and two resistant house fly strains were compared for metabolic activity against the juvenile hormone analogs, those from the resistant strains were 1.3 to 20 × higher in oxidase activity but there was no difference in esterase activity. The oxidative metabolism of two analogs ZR-515 and 512 was greatly enhanced when the flies were induced with phenobarbital but there was no enhancement in metabolism of three of the remaining analogs and only a slight enhancement of a fourth. It is concluded that the insecticidal action of ZR-515 is largely due to its stability in the presence of the house fly esterases.