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.     

Resynthesis of multiple resistance to organophosphorus insecticides from strains with factors of resistance isolated from the SKA strain of house flies

Individual factors of resistance to insecticides attributable to chromosomes II, III and ^V of the SKA strain of houseflies (Musca domestica L) were combined in pairs to determine how their presence affects resistance. The re-synthesised strains with resistance factors on chromosomes II and V, and on chromosomes III and V, were tested with several organophosphorus insecticides and DDT. The penetration delaying mechanism Pen on chromosome III, which alone gives little or no resistance, slightly increased the resistance of flies with the microsomal detoxifying factor Ses on chromosome V to diazinon and malaoxon-ethyl (c. × 1.5), but was more effective in increasing resistance to DDT (× 6). There was no effect on the response to other insecticides tested. The combined effect of the mechanisms of resistance on chromosome II (glutathione S-ethyl transferase and phosphatase) and on chromosome V (microsomal detoxication) approximated to the product of the resistance conferred by each of these mechanisms singly, suggesting that the mechanisms of resistance on the two chromosomes act independently. Therefore, most of the strong resistance to organophosphorus insecticides in the SKA strain results from the interaction between delayed penetration (chromosome III) and the factors of resistance on chromosome II, and the independent action of the resistance factors on chromosomes II and V.     

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.     

Biochemical genetics of oxidative resistance to diazinon in the house fly

The genetics and biochemistry of oxidative resistance to diazinon were investigated in a diazinon-resistant strain of the house fly, Musca domestica L. The resistant strain was crossed with a multimarker susceptible strain and substrains containing portions of the resistant strain genome were prepared. Resistance, microsomal oxidase, and cytochrome P-450 spectral characteristics were then compared in the different strains. The major gene for resistance to diazinon is semidominant and is located on chromosome II, 13 crossing over units from the recessive mutant stubby wing. Additional resistance genes occur on chromosome II and on other chromosomes as well. Resistance to diazinon was introduced into a susceptible mutant-marked strain via genetic crossing over. Increases in parathion oxidase, total and P-450-specific N- and O-demethylase activity, and resistant strain type I binding spectrum were introduced along with resistance, indicating genes controlling these parameters and resistance are either identical or closely linked. No increase in activity of cytochrome P-450 itself was introduced into the mutant strain. Additional genes controlling the amount of cytochrome P-450 and several spectral changes characteristic of the resistant strains are apparently controlled by genes located at different loci on chromosome II. Resistance factors on other chromosomes are also present, but were not characterized.     

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.     

Cytochrome P-450 in insects: 2. Multiple forms in the flesh fly (Sarcophaga bullata,Parker), and the blow fly (Phormia regina (Meigen))

Microsomes prepared from the abdomens of the flesh fly (Sarcophaga bullata, Parker) and the blow fly (Phormia regina (Meigen)) contain approximately one-fifth and one-eighth as much cytochrome P-450, respectively, as those prepared from house fly (Musca domestica, L.) abdomens. These values correlate well with the microsomal aldrin epoxidase activity of the three species and with their respective susceptibilities to the insecticide, propoxur. When the microsomes of the flesh fly and the blow fly are solubilized by treatment with deoxycholate and resolved by ion-exchange chromatography on DEAE-cellulose and hydroxylapatite, four chromatographically distinct fractions containing cytochrome P-450 are obtained. Spectrophotometric assays of the cytochrome P-450 in these fractions indicate purifications of two-to sixfold for the flesh fly hemoprotein and two-to eightfold for that of the blow fly. SDS-Polyacrylamide gel electrophoresis of the four column fractions from the flesh fly microsomes indicates that six hemoproteins in the 40,000–60,000 molecular weight range are present. In similar experiments with blow fly fractions containing approximately the same amount of cytochrome P-450 no high molecular weight hemoproteins could be detected. This result is interpreted, with other evidence, as an indication of the greater instability of the blow fly hemoprotein. The results indicate that multiple forms of cytochrome P-450 are present in both species but there is insufficient data on which to estimate the number of such forms.     

Juvenile hormone analogs: Toxicity and cross-resistance in the housefly

The toxicity of several juvenile hormone analogs (JHAs) to susceptible and insecticide-resistant housefly (Musca domestica L.) strains was determined by an assay procedure in which larvae were exposed to residues of JHAs in glass vials. All JHAs tested were toxic and the most active compound, isopropyl 11-methoxy-3, 7, 11-trimethylododeca-2, 4-dienoate, was 100 times as toxic to the susceptible Orlando Regular strain as methyl parathion and 600 times as toxic as DDT.A 5- to 30-fold tolerance to the different JHAs was present in an insecticide resistant strain in which resistance is associated with a high level of NADPH-dependent microsomal oxidase activity controlled by a gene(s) on chromosome II. Cross-resistance was less marked in a strain with a chromosome V high oxidase gene and absent in strains with other resistance mechanisms.The data indicate that cross-resistance to JHAs in insects may occur in certain strains with high levels of oxidative detoxifying activity. Even so, the most active JHA was far more toxic to both susceptible and resistant strains than methyl parathion or DDT.     

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

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.     

Bimodal effect of methylenedioxyphenyl compounds on detoxifying enzymes in the housefly

Thirteen methylenedioxyphenyl (MDP) compounds, including commercial insecticide synergists and juvenile hormone analogs, were compared in their effect on detoxifying enzymes in the housefly (Musca domestica). Flies were fed a diet containing 1% of the compounds for 3 days. Enzymes were then assayed in vitro for their activity using aldrin and DDT as substrates. Piperonyl butoxide (PB), sesamex, propyl isome, sulfoxide, safrole, isosafrole, 6,7-epoxy-3,7-diethyl-1-[3-4(methylenedioxy) phenoxy]-2-octene (MDP-JH I) and 6,7-epoxy-3-methyl-7-ethyl-1-[3,4-(methylenedioxy) phenoxy]-2-octene (MDP-JH II) all caused a bimodal effect, inhibiting microsomal epoxidase and inducing DDT-dehydrochlorinase in the resistant Isolan-B strain. Two of these, PB and MDP-JH I, gave similar results with the susceptible strain, stw;w⁵ and two resistant strains, Fc-B and Orlando-DDT. However, o-safrole, piperonylic acid, piperonal, 3,4-methylenedioxybenzyl acetate and methyl-(3,4-methylenedioxy) benzoate had little or no effect on the enzyme systems studied. The standard susceptible strain (WHO-SRS) responded to these compounds very differently. Among those tested, piperonyl butoxide, sesamex, safrole, and isosafrole were inducers of microsomal epoxidase, a 4-fold increase occurring after treatment with sesamex. Only MDP-JH II showed a marked inhibition of the epoxidase. These treatments did not effect DDT-dehydrochlorinase activity in this strain.The enhancement of DDT-dehydrochlorinase activity by the MDP compounds is associated with an increased rate of DDT dehydrochlorination in vivo. The stimulatory effect could be blocked by treatment with actinomycin D or cycloheximide.     

Genetic studies on glutathione-dependent reactions in resistant strains of the house fly,Musca domestica L

Genetic studies of glutathione-dependent reactions were conducted with a diazinon-resistant house fly strain in which resistance is controlled primarily by genes on chromsome II. The resistant strain was crossed with a susceptible strain which had mutant markers on chromosomes II, III, and V, and the F₁ was backcrossed to the susceptible strain. Glutathione transferase activities of the resultant eight phenotypes were measured using 3,4-dichloronitrobenzene, methyl iodide, and γ-benzene hexachloride as substrates. High levels of all these activities are controlled by gene(s) on chromosome II. Further analysis was made by introducing diazinon resistance into a susceptible strain via genetic crossing-over. Intermediate activity levels for 3,4-dichloronitrobenzene and methyl iodide conjugations were introduced along with intermediate levels of resistance. Assays of individual flies of the synthesized strain revealed they were heterogeneous for glutathione-dependent activities, consisting of individuals with low, intermediate, and high transferase activity. Based on these results, high levels of the glutathione-dependent enzymes are not a major biochemical mechanism responsible for diazinon resistance. It was also demonstrated that glutathione S-aryltransferase and S-alkyltransferase in the house fly, as measured with 3,4-dichloronitrobenzene and methyl iodide, are inseparable genetically and may, therefore, be the same enzyme.     

The mechanism of resistance to lindane and hexadeuterated lindane in the Third Yumenoshima strain of house fly

The factors which cause lindane resistance in the Third Yumenoshima strain, a strain of house flies highly resistant to insecticides, were studied using hexadeuterated lindane. Hexadeuterated lindane has the same physicochemical properties as lindane, but the former is much less biodegradable than the latter. The LD₅₀ ratio of lindane to hexadeuterated lindane in this strain, deuterium isotope effect on LD₅₀ values, was larger than that in S_NAIDM, a susceptible (nonresistant) strain. The penetration rates of labeled and nonlabeled lindane through the insect cuticle were about the same for both strains. Thus, penetration rate does not cause resistance. The metabolic degradation of lindane in the resistant strain in vivo occurred much faster than in the susceptible strain. This was also the case for lindane degradation processes in vitro such as microsomal oxidation and glutathione conjugation. In both strains, significant isotope effects were observed in the degradation rates in vitro of labeled and nonlabeled lindane. Therefore, principal biodegradation and detoxication pathways should include reactions which cleave the CH bonds. When the much less biodegradable d₆ counterpart of lindane was applied to both strains, the susceptible strain became much more highly intoxicated than the other within 20 to 30 min. This indicates that a combination of both greater degradability and probably lower sensitivity at the action site are the main factors underlying resistance in the Third Yumenoshima strain.     

German cockroach resistance: Propoxur selection induces the same resistance spectrum as diazinon selection

A resistant laboratory strain of the German cockroach, Blattella germanica, was developed from a normal laboratory strain by selection with propoxur. Resistance to all insecticides except chlordane began increasing after 15 generations of selection and reached a plateau for most insecticides by generation 27. The resistant colony, designated B-strain, developed significant resistance to carbamates, organophosphorus compounds, pyrethrins and DDT, developed low resistance to gamma-BHC and no resistance to chlordane. The resistance spectrum, effect of synergists and inheritance of resistance of this propoxur resistant strain are similar to a diazinon resistant strain. Therefore, diazinon and propoxur may select for the same resistance mechanism(s) in this species. The practical implications of this research are discussed.     

Metabolism and absorption of methyl parathion by tobacco budworms resistant or susceptible to organophosphorus insecticides

Factors involved in insecticide resistance were evaluated by using ¹⁴C-labeled methyl parathion and aldrin to compare rates of absorption and metabolism by Heliothis virescens (F.) larvae that were resistant (R) and susceptible (S) to methyl parathion. Tests with third-stage R and S larvae suggested that the rate of insecticide absorption from the cuticular surface was not a major resistance factor. Further evidence for this assumption was demonstrated by the resistance of R larvae to injected and orally administered doses of methyl parathion. Smaller amounts of unmetabolized methyl parathion and aldrin were recovered from S larvae, an indication that differences in metabolism were probably related to the resistance.     

A comparative study of insecticide resistance assays with the German cockroach

Females of a diazinon-resistant strain of German cockroach exhibiting cross resistance to propoxur were evaluated for resistance to diazinon and propoxur by six assays: LD₅₀s for topical application and injection; LC₅₀, KT₅₀ and LT₅₀ for deposits on wood and KT₅₀ for deposits on glass. Also investigated were certain variables in the topical application technique and the experimental conditions for topical synergism studies. Among all assays, diazinon resistance ratios (resistant:susceptible) ranged from 3- to 145-fold; propoxur varied from 1- to 14-fold. Resistance ratios based on KT₅₀ data were consistently low (<4x) for both insecticides. Resistance to diazinon was 13x when applied topically and 6x when injected. Resistance to propoxur was 14x by injection compared to 8x for topical application. The LC₅₀ assay exhibited the greatest difference in resistance to the two insecticides: namely, 145x to diazinon and 1x(no resistance) to propoxur. By LT₅₀ analysis, resistance to propoxur was 10x compared with 100x resistance to diazinon. Thus, the method used had a significant effect on the final value of the resistance ratio. Even though resistance ratios may vary widely among different techniques, the final choice of a method depends upon many factors. The LT₅₀ method may provide a more realistic appraisal of resistance in wild populations of German cockroaches especially when experimental residues are similar to those used in control programmes.     

Relation of acetylcholinesterase sensitivity to cross-resistance of a resistant house fly strain to organophosphates and carbamates

Acetylcholinesterase (AChE, E.C. 3.1.1.7) from an organophosphate-resistant strain of house fly, Musca domestica (L.) exhibited a decrease in sensitivity towards four organophosphates and two carbamates in comparison with enzyme from the parent susceptible strain. Sensitivity was less, as measured by the bimolecular reaction constant (k_i), by a factor of 117 for dichlorvos, 94 for paraoxon, 11 for diazoxon, 7 for Tetram, 62 for propoxur, and 50 for dimetilan. These differences in bimolecular reaction constants were attributed entirely to differences in their affinity for the enzyme, as measured by the dissociation constant, K_d. It is suggested that the cross resistance to these inhibitors is due at least in part to insensitive acetylcholinesterase.     

The biochemistry of insecticide resistance in Anopheles sacharovi: Comparative studies with a range of insecticide susceptible and resistant Anopheles and Culex species

Fourth instar larvae, the progeny from wild-caught Anopheles sacharovi females, were subjected to a number of biochemical tests and the results were compared to those from similar tests on laboratory insecticide resistant and susceptible strains of anopheline and culicine mosquitoes. DDT resistance in An. sacharovi is associated with the ability to rapidly metabolise DDT to DDE. The organophosphorus and carbamate resistance was not associated with quantitative changes in esterases, multifunction oxidases, or glutathione S-transferase. The acetylcholinesterase was less sensitive to malaoxon and propoxur than laboratory susceptible An. albimanus, and plots of inhibition suggest that the population was polymorphic for more than one form of acetylcholinesterase. Metabolism studies on malathion and pirimiphos methyl did not indicate resistance due to increased metabolism. There was no evidence of penetration barriers contributing to resistance to either DDT or malathion, and there was no indication of any resistance to pirimiphos methyl in our tests.     

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.     

Interactions of allelochemicals with detoxication enzymes of insecticide-susceptible and resistant fall armyworms

The induction of glutathione S-transferases and microsomal oxidases by host plants and allelochemicals was examined in sixth-instar larvae of insecticide-susceptible and resistant strains of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Among 11 host plants studied, parsnip and parsley were the best inducers of glutathione S-transferase, resulting in increases of 39- and 19-fold, respectively, compared with the artificial diet. The inducer in parsnip leaves was identified by mass spectrometry, high-pressure liquid chromatography, gas chromatography, and thin-layer chromatography as xanthotoxin, a furanocoumarin. Xanthotoxin also showed a bimodal effect on the microsomal oxidase systems, increasing cytochrome P-450 content and heptachlor epoxidase activity but inhibiting aldrin epoxidase, biphenyl 4-hydroxylase, and p-chloro-N-methylaniline N-demethylase. Using indole 3-acetonitrile, indole 3-carbinol, and flavone as inducers, the inducing pattern of glutathione S-transferases was the same toward 3,4-dichloronitrobenzene, 1-chloro-2,4-dinitrobenzene, and methyl iodide. Microsomal oxidase and glutathione S-transferase were also inducible by host plants and allelochemicals in larvae of a carbaryl-resistant strain.