L.C. Terriere

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.

Hormonal modification of microsomal oxidase activity in the housefly

Abstract The effect of treatments with synthetic juvenile hormone and ecdysterone on microsomal heptachlor epoxidase activity was investigated in female adults of the Isolan-B strain of the housefly. Flies were treated topically or by injection and the microsomes prepared and assayed at intervals up to 36 hours later. Both hormones caused increased epoxidase activity, up to 65% above controls, with the maximum for ecdysterone occuring at 6 hours and for the juvenile hormone at 18 to 24 hours after treatment. Enhancement in the case of the juvenile hormone was preceded by an inhibitory phase at 12 to 18 hours. The increased activity of these enzymes could be prevented by simultaneous treatment with actinomycin D. The enhancement caused by the juvenile hormone could also be blocked by treatment with cycloheximide but this inhibitor had no effect on the activity of ecdysterone, presumably because of the shorter time involved. Microsomal epoxidase assay of untreated larvae revealed a burst of activity in the late third instar about 11 hours before pupation. Attempts to induce this activity by treatment with ecdysterone were unsuccessful.

A possible rôle for microsomal oxidases in metamorphosis and reproduction in the housefly.

Abstract Housefly larvae and adults of the Isolan-B strain, insecticide resistant because of high microsomal oxidase activity, and the susceptible WHO Standard Reference (SR) strain, were reared on diets containing phenobarbital or piperonyl butoxide. Periodically throughout the treatments groups of insects were assayed for microsomal aldrin epoxidase activity. This activity was compared with such growth and development parameters as pupation, adult emergence, and reproduction in similar groups of insects. At diet levels of 0·01 to 0·50 per cent both chemicals caused large increases, as much as elevenfold in microsomal oxidase activity in third instar larvae and 15 to 100 per cent inhibition of pupation and emergence. In the adult diet at 1 per cent, both compounds caused at least 50 per cent decrease in egg production. Phenobarbital enhanced the enzyme system in both larval and adult stages of both strains but piperonyl butoxide, while enhancing enzyme activity in larvae of the Isolan-B strain, was an inhibitor in the adult stage. The results are interpreted as an indication of a direct connexion between microsomal oxidase activity and the action of hormones in the housefly, probably through regulation of hormone titre by these enzymes.

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.

Enzyme Induction, Gene Amplification and Insect Resistance to Insecticides

Perhaps the most fully understood mechanism of insecticide resistance in insects is that due to increased metabolism of the toxicant. We will refer to this as biochemical resistance. It is obvious that any metabolism that inactivites an insecticide will be beneficial to the insect and that such traits will be transmitted genetically. What is not so obvious, however, is how the organism achieves the observed increase in enzyme activity. We will consider some possibilities in this chapter.

Activities of hormone metabolizing enzymes in house flies treated with some substituted urea growth regulators

Abstract The insect growth regulators (IGR) TH 6038 and TH 6040 affect larvae of various species by interfering with cuticle development. In a biochemical study of their effects, larvae of the house fly, Musca domestica L. were reared for 2 days on diets containing 1.7 to 166.7 ppm of these compounds, then assayed for activities of the microsomal oxidases and the enzyme(s) which metabolize β-ecdysone. The activities of these enzymes were compared with the percentage of treated larvae completing pupal-adult ecdysis. The two compounds reduced the activity of the β-ecdysone metabolizing enzyme(s) by as much as 57%, reduced pupal-adult ecdysis by 43% to 100%, and stimulated microsomal oxidase activity 4- to 12-fold. Supplementation of the diet of the treated insects with the Cecropia juvenile hormone, JH I, partially restored pupal-adult ecdysis but supplementation with β-ecdysone had no effect. The mode of action indicated by these results is that the IGRs cause an accumulation of β-ecdysone in the treated larvae. This stimulates the enzyme, chitinase, which degrades chitin in preparation for formation of the new cuticle. The hormone may also cause a JH deficiency and the stimulation of DOPA decarboxylase and phenol oxidase which would further disrupt the normal molting process.