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

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.     

Cross resistance to juvenile hormone analogues in insecticide-resistant strains of Musca domestica L

Five juvenile hormone analogues (JHAs) were tested by topical application to prepupae of a susceptible (S) and 8 insecticide-resistant (R) strains of the housefly. Activity was measured by the inability to completely emerge from the puparium. Aitosid (isopropyl 11-methoxy-3,7,1l-trimethyldodeca-2,4-dienoate) was the most active compound against the S strain (ED₅₀ 0.0033 μg/prepupa) followed by Ro 7-9767 [6,7-epoxy-3,7-diethyl-(3,4-(methylenedioxy)phenoxy)-2-cis/trans-octene], R-20458 [trans l-(4-ethylphenoxy)-6,7-epoxy-3,7-dimethyl-2-octene], sesamex, and NIA 23509 (10,11 -epoxy-N-ethyl-3,7,11-trimethyI-2,6-dodecadienamide). The R strains, designated by the name of the selecting insecticide, have been under pressure for over 10 years and are considered maximally resistant. The dimethoate-R and OMS-15-R (carbamate-resistant) strains exhibited high levels of cross resistance to all JHAs often exceeding 100x at the ED₉₅ The fenthion-R strain showed high cross resistance toward all JHAs except Altosid, toward which it manifested an intermediate level (17.5x). The DDT/lindane-R demonstrated only negligible tolerance to Aitosid but an intermediate response to all the other JHAs. The OMS-12-R strain (phosphoramidothioate-R) exhibited intermediate to high levels of cross resistance toward all JHAs, whereas the parathion-R, Chlorthion-R and a multi-resistant field-collected strain showed only low to intermediate levels of cross resistance. On the basis of known degradative mechanisms of the OMS-15-R strain, mixed function oxidases apparently play an important role in deactivating JHAs.     

The metabolism of R-20458 [(E)-6,7-epoxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene] in rat hepatocyte suspensions

The metabolism of R-20458 [(E)-6,7-epoxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene] by rat hepatocytes has been analyzed and compared with that of juvenile hormone I [methyl-(E,E)-cis-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate] under identical conditions. The metabolism of R-20458 is characterized by the predominance of NADPH-dependent cytochrome P-450 and epoxide hydrolase reactions; whereas, JH I is metabolized mainly by carboxylesterase, epoxide hydrolase, and glutathione S-transferases. The metabolites of R-20458 have been shown to correspond to (E)-6,7-epoxy-1-(4-hydroxyethylphenoxy)-3,7-dimethyl-2-octene; (E)-6,7-epoxy-1-(4-acetylphenoxy)-3,7-dimethyl-2-octene; (E)-6,7-dihydroxy-1-(4-ethylphenoxy)-3,7-dimethyl-2-octene; and, (E)-6,7-dihydroxy-1-(4-acetylphenoxy)-3,7-dimethyl-2-octene. The production of the α-hydroxyethyl, p-acetylphenoxy, and acetylphenoxy-6,7-diol metabolites is markedly inhibited by SKF 525-A. No dramatic effects are produced by diethylmaleate and 1,2-epoxy-3,3,3-trichloropropane.     

Phenobarbital induction of detoxifying enzymes in resistant and susceptible houseflies

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.     

An Example of Cross-resistance to Pyrethroids in DDT-resistant Aedes aegypti

Comparisons of the susceptibility of several strains of adult Aedes aegypti were made. Mosquitoes from Bangkok and Jakarta were found to be highly resistant to DDT and resistant to pyrethroids relative to a laboratory strain. A strain from Singapore, where less DDT has been used, was susceptible to DDT and pyrethroids. Two strains from the Caribbean had LC₅₀ values to DDT 3 times that of the reference strain while the LC₅₀ values against bioresmethrin synergised with piperonyl butoxide were 1 1/2 times raised. Another two strains from central Africa were 2 times tolerant of DDT and 1 1/2 times tolerant of bioresmethrin plus piperonyl butoxide. Agents which block DDT-dehydrochlorinase, esterases and oxidases each caused small increases in the mortality of the Bangkok strain due to DDT and bioresmethrin as well as augmenting toxicity to the susceptible reference strain. It is tentatively suggested that resistance in the Bangkok strain is due to a combination of the actions of these and perhaps other resistance mechanisms.     

Albumin and cytochrome P450 binding characteristics of juvenile hormone and its analogs

Binding data were gathered for the cecropia juvenile hormone (methyl(E, E cis)-10,11-epoxy-7-ethyl-3,11-dimethyl-2,6-tridecadienoate) and two of its analogs {isopropyl(2E, 4E)-11-methoxy-3,7,11-trimethyl-2,4-dodecadienoate; (E)-4-[(6,7-epoxy-3,7-dimethyl-2-nonenyl)-oxyl]-1,2-(methylenedioxy)benzene} with bovine serum albumin and rat hepatic microsomal cytochrome P₄₅₀. The proteins were found to bind the juvenile hormone and juvenile hormone analogs with affinity constants ranging from 10⁵ to 10⁶M^?1. Thermodynamic calculations suggest that the binding of all three compounds is electrostatic in nature and that the size of the ether and ester substituents can greatly influence the binding to proteins. The juvenile hormone and its analogs all formed spectrally apparent Type I complexes with oxidized cytochrome P₄₅₀; one of the juvenile hormone analogs formed a spectrally observable product adduct with reduced cytochrome P₄₅₀. The product complex may contribute many of the hormonal effects observed for this compound.     

Changes in cross-resistance patterns of houseflies selected with natural pyrethrins or resmethrin (5-benzyl-3-furylmethyl (±)-cis-trans-chrysanthemate)

A pyrethrins-resistant strain of houseflies, 213ab, previously selected with a 1:10 (by wt.) mixture of natural pyrethrins and piperonyl butoxide, was further selected either with natural pyrethrins alone (strain NPR) or with resmethrin (strain 104). After 50 generations the two populations differed in their resistance to the natural and synthetic esters. Both were resistant to all pyrethroids. Part of strain NPR was immune and very much more resistant than strain 104 to the natural pyrethrins and allethrin, but it was only 2–3 times more resistant than strain 104 against the new synthetic esters resmethrin (5-benzyl-3-furylmethyl (±)-cis-trans-chrysanthemate), bio-resmethrin (5-benzyl-3-furylmethyl (+)-trans-chrysanthemate), pyresmethrin (5-benzyl-3-furylmethyl pyrethrate) and 5B2Me3FC (5-benzyl-2-methyl-3-furylmethyl (±)-cis-trans-chrysanthemate). Pretreatment of both strains with sesamex diminished but did not eliminate resistance. Synergism was greater in strain NPR, especially with natural pyrethrins and allethrin. Both strains had great resistance to DDT indicating that resistance to DDT and pyrethroids is linked. Differences in resistance to different compounds suggest that at least three factors can confer resistance, one of which, pen, delays penetration and two others involve detoxication, one py a on the acid side of the ester linkage and the other, py b, on the alcohol side. Natural pyrethrins and resmethrin select for different groupings of these factors. Treatment with resmethrin does not select for py b presumably because this mechanism cannot attack the resmethrin molecule. Similarly when piperonyl butoxide is added to the natural pyrethrins py b is inhibited and so removed from selection pressure. Under these conditions, the strain produced contains the same factors as one selected by resmethrin and so shows the same small resistance to natural pyrethrins alone.     

Resistance mechanisms to DDT and transpermethrin in Aedes aegypti

Penetration, metabolism and excretion of [¹⁴C]DDT and [³H]transpermethrin were studied in three strains of Aedes aegypti L. after topical application of 10 nl of a solution of the insecticide in 2-ethoxyethanol. The standard susceptible strain was compared with a DDT-selected strain (BKS) and a permethrin-selected strain (BKPM). No significant penetration resistance was observed in either resistant strain, but both showed high DDT-dehydrochlorinase activity which contributed to the DDT resistance. A non-metabolic factor was also involved. Rates of transpermethrin metabolism were very similar in all three strains and substantially higher internal concentrations of transpermethrin were required to produce toxic effects in both BKS and BKPM mosquitoes. By analogy with other insect species, it is concluded that transpermethrin resistance in these strains is of the kdr type, while the DDT resistance is a mixture of kdr and DDT-dehydrochlorinase.     

Insecticidal and repellent properties of nine volatile constituents of essential oils against the American cockroach,Periplaneta americana (L.)

The toxic and repellent properties of nine major constituents of essential oils, comprising benzene derivatives and terpenes, were evaluated against Periplaneta americana (L.). Contact and fumigant toxicities to adult females and repellency to nymphs were determined. The decreasing order of knockdown activity via contact was methyl-eugenol>isosafrole=eugenol>safrole. The killing effect via contact was in the order eugenol=methyl-eugenol=isosafrole>safrole. Fumigant toxicity was only observed for safrole and isosafrole, with safrole being the more potent. Isoeugenol and the tested terpenes had neither contact nor fumigant toxic effect. The decreasing order of repellency to nymphs was safrole>isosafrole>methyl-eugenol=α-pinene> eugenol>isoeugenol. The benzene derivatives were generally more toxic and repellent to P. americana than the terpenes. The distance of the side chain double bond from the aromatic ring and the substitution of a methoxy group to these compounds appeared to be important determinants of their toxicity and repellency. © 1998 Society of Chemical Industry     

Neurophysiological effects of insecticides on the labellar taste receptors of Lucilia cuprina Wied

The effect of insecticide concentration on the nerve impulses at the labellar receptors of the blowfly Lucilia cuprina Wied was tested and found to be significant for the DDT type of insecticide, but not for the DDT-pyrethroid type. The experiments were performed at three different times of the day, but time of day had no effect on the multiplicity indices calculated from nerve spikes induced by the compounds. No effects on the nerve response were elicited by the synergistic action of sesamex.     

Cross-resistance spectrum in pyrethroid-resistant Culex quinquefasciatus

Strains of Culex quinquefasciatus Say, selected with biopermethrin [(1R)-trans-permethrin] or with (1R)-cis-permethrin, were examined in the larval stage for crossresistance to 30 pyrethroids, DDT, dieldrin, temephos, propoxur, and two organotin compounds. The (1R)-trans-Permethrin-R strain [resistance factor (RF) = 4100-fold] and the (1R)-cis-Permethrin-R strain (RF= 450-fold) of C. quinquefasciutus were cross-resistant to all pyrethroids tested [RF= 12-fold for an allethrin isomer to about 6000-fold for (RS,RS)-fenvalerate] as well as to DDT (RF= about 2000-fold). However, they were not significantly Cross-resistant to dieldrin, temephos, propoxur, and the two organotin compounds. Changes in the alcohol moiety, structural isomerism, and susceptibility of the cyclopropane C-3 side chain to oxidative attack are important factors in determining the level of cross-resistance to various pyrethroids. Limited synergism of the pyrethroids by S,S,S-tributyl phosphorotrithioate and piperonyl butoxide (PB), and of DDT by chlorfenethol and PB, suggested that some non-metabolic mechanism, such as kdr, may be an important component of resistance to pyrethroids as well as to DDT in this mosquito.     

Oxidative cleavage of a carboxyester bond as a mechanism of resistance to malaoxon in houseflies

The synergistic effect of triphenyl phosphate (a carboxyesterase inhibitor), sesamex (inhibitor of microsomal oxidation) and O,O-diethyl O-phenyl phosphorothioate on the toxicity of malathion and malaoxon for one susceptible and two resistant strains of housefly was studies. It was found that in the resistant strain G (characterized by high carboxyesterase activity) both malathion and malaoxon were synergized by triphenyl phosphate, but only malaoxon (and not malathion) by sesamex. The other resistant strain E 1, moderately tolerant for malathion but highly resistant to malaoxon, differed from strain G in that triphenyl phosphate had no effect; its response to sesamex was similar to that of strain G. The third synergist, O,O-diethyl O-phenyl phosphorothioate, combined the properties of triphenyl phosphate and sesamex. It was found to be the best of the three compounds used.Biochemical in vitro studies showed that both resistant strains could degrade malaoxon oxidatively at a rate at least 10 × higher than that of the susceptible strain. This oxidation could be inhibited by very low concentrations of the thiono analogue; a malaoxon to malathion ratio of 10:1 gave an inhibition of about 70% at a malaoxon concentration of 5 × 10^?6M. The product of this oxidation is malaoxon β-monocarboxylic acid. This metabolite was also found 1 hr after application of malaoxon in vivo.The results mentioned in this paper indicate that houseflies may become resistant to malaoxon by an increased rate of oxidative carboxyester bond cleavage.     

Present Status of Research on Insect Growth Regulators for the Protection of Stored Grain1

Two insect growth regulators of the juvenile hormone type, namely 6,7-epoxy-3-ethyl-(p-ethylphenoxy)-7-methylnonane, cis/trans mixture (compound A) and 6,7-epoxy-3,7-dimethyl-1-[3,4-(methylendioxy)-phenoxy]-2-nonene (compound B) have been tested for their persistence when treated on stored grain. The interim results of this trial showed inhibition of adult emergence of Ephestia kühniella Zell., Plodia interpunctella Hübn., Tribolium castaneum Herbst and Rhyzopertha dominica F. even 8 months after treatment. The gas-chromatography residue determination of compound A showed a reduction of 27 % of the original concentration within 8 months. No effect of compound A on the endosperm of the grain or taste of flour and bread could be found in the Bakery School in Lucerne.     

The epoxidation of aldrin by microsomes from the Fc strain of housefly: A genetic survey

The DDT-resistant housefly strain, Fe, known to resist DDT by biochemical oxidation, is also resistant to carbamate insecticides and has a high in vitro microsomal epoxidase activity. The purpose of this investigation was to determine whether the DDT resistance, associated with chromosome V, is also responsible for the resistance to carbamates and for the high epoxidase levels. Genetic procedures for segregating the R factors were employed using a multimarker insecticide susceptible strain designated acbco. The technique involved backcrossing the F₁ hybrid of the resistant and susceptible parents to the susceptible parent. The genotypes with a single R chromosome from the Fc parent were retained for further development as substrains and for toxicological and biochemical studies.These studies revealed that both resistance to the carbamate insecticide, propoxur, and the high in vitro microsomal epoxidation of aldrin were lost during the genetic isolation of the R factors. However, the resistance to DDT, associated with chromosome V, was present in the substrain carrying this chromosome from the Fc parent. All of the substrains were induced five- to seven-fold, by feeding phenobarbital at 1% in the diet for 3 days.Additional substrains synthesized from the substrains carrying chromosomes II and V or III and V from the Fc parent did not possess sufficient propoxur resistance or aldrin epoxidase activity to account for that present in the R parent.The interpretation of these rseults is that neither the carbamate resistance nor the microsomal epoxidase of the Fc strain is due to the factor which oxidizes DDT. Furthermore, the factor responsible for the high microsomal epoxidase activity is not due to a single chromosome such as chromosome II which is the case in other housefly strains with high oxidase activities.     

Antagonism of iprodione toxicity to Botrytis cinerea by mixed function oxidase inhibitors

The fungitoxicity of iprodione to a sensitive strain of Botrytis cinerea was antagonised by a variety of cytochrome P-450 mixed function oxidase inhibitors. Piperonyl butoxide, metyrapone and N-(2-ethylhexyl)-8,9,10-trinorborn-5-ene-2,3-dicarboximide (MGK-264) at non-fungitoxic concentrations were strongly antagonistic, whereas sesamex, nuarimol, fenarimol, etaconazole and 6-nitro or 6-methoxy 1,2,3-benzothiadiazole were moderately antagonistic. Phenobarbital and 2-diethylaminoethyl 2,2-diphenylvalerate (SKF 525-A) were slightly antagonistic. The results suggest that fungitoxicity of iprodione may be dependent on an activation catalysed by a cytochrome P-450 mixed function oxidase.     

Selection of Anopheles stephensi with DDT and dieldrin and cross-resistance spectrum to pyrethroids and fipronil

The field strain of Anopheles stephensi, the main malaria vector in south of Iran, was colonized in laboratory and selected with DDT and dieldrin in two separate lines for 3 generations to a level of 19.5- and 14-fold for DDT and dieldrin resistance, respectively. Synergist tests with chlorofenethol (DMC) and piperonyl butoxide (PBO) on the selected strains indicated that dehydrochlorination and oxidative detoxification might be the underlying mechanisms involved in the resistance to dieldrin and DDT in selected strains. DDT selection decreased susceptibility to DDT and pyrethroids including lambdacyhalothrin, permethrin deltamethrin and cyfluthrin. The result also showed that selection with dieldrin caused negative and positive cross-resistance to pyrethroid and fipronil, respectively. Based on these results, it can be concluded that besides metabolic resistance mechanisms, other factors such as mutation in γ aminobutyric acid (GABA) and voltage-gated sodium channels (Kdr) might be involved.     

The resistance to eighteen toxicants of a strain of Musca domestica L. collected from a farm in England

A sample of houseflies initially collected from a pig farm and found to be resistant to bendiocarb, DDT, gamma-HCH, pyrethrins + piperonyl butoxide (PB), tetrachlorvinphos and trichlorfon, was tested for resistance to knockdown by other toxicants. At the KD₅₀ response level, resistance factors were obtained for: permethrin (× 141), deltamethrin (×96), bioresmethrin + PB (×37), resmethrin + PB (×33), fenitrothion (×94), bromophos (×58), iodofenphos (×42), pirimiphos-methyl (×30), dichlorvos (×22), dimethoate (×9), diazinon (×8), methomyl (×4) and methomyl + PB (×4). The slopes of the dose–response lines were lower for the farm strain than for a susceptible strain. This resulted in an increase of resistance factors at the KD₉₅ level by an average of × 1.6. The houseflies on the farm could not be controlled using space sprays of pyrethrins + PB, although resistance to this toxicant was only ×12. However, control was achieved with a methomyl bait.     

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.     

Metabolic O-dealkylation of 1-(4′-ethylphenoxy)-3,7-dimethyl-7-methoxy or ethoxy-trans-2-octene,potent juvenoids

1-(4′-Ethylphenoxy)-3,7-dimethyl-7-methoxy or ethoxy-trans-2-octene (the ethyl-methoxide and ethyl-ethoxide) are more stable in some biological systems and less stable in others than 1-(4′-ethylphenoxy)-3,7-dimethyl-6,7-epoxy-trans-2-octene (the ethyl-epoxide). In housefly adults and mealworm pupae the persistence increases in the order of the ethyl-epoxide, -methoxide and -ethoxide but with adult stable flies the epoxide is of intermediate stability. The alkoxides are metabolized in living insects and microsomal oxidase systems of houseflies and mouse liver mainly by O-dealkylation, at a higher rate for the methoxide than the ethoxide, but benzylic oxidation of the ethyl group also occurs and is more important in the degradation of the ethyl-epoxide than the ethyl-alkoxides. The photostability on silica gel is slightly better for the ethyl-ethoxide than the -methoxide or -epoxide.     

Synergism of teflubenzuron and chlorfluazuron in an acylurea-resistant field strain of Plutella xylostella L. (lepidoptera: Yponomeutidae)

Topical application of the synergists piperonyl butoxide (PB) and S,S,S-tributyl phosphorotrithioate (DEF) to second-instar larvae of a standard laboratory strain (FS) and an unselected Malaysian field strain (CH) of the diamondback moth Plutella xylostella had no significant effect on the toxicity of the acylurea insecticides, chlorfluazuron and teflubenzuron, in a subsequent leafdip bioassay. In contrast, pre-treatment with PB or DEF in acylurea-selected subpopulations of the CH strain with varying levels of cross-resistance to chlorfluazuron and teflubenzuron significantly increased (up to 34-fold and 28-fold, respectively) the toxicity of both compounds, suggesting that microsomal monooxygenases and esterases may be involved in resistance. The addition of a mineral oil, ‘Sunspray 6E’, to topically-applied chlorfluazuron consistently reduced its LD₅₀ value, and the effect of the oil appeared to be greatest on the most resistant population of P. xylostella. However, the effects of the oil were not significant (P > 0·05) and further studies are necessary to determine whether a penetration factor is present in the CH strain.