Arguments against a fifth chromosomal factor in control of aldrin epoxidation and propoxur resistance in the Fc strain of the house fly

The DDT-resistant Fc strain of house flies, Musca domestica L., was analyzed genetically by means of crosses with a susceptible strain carrying a recessive mutant marker for each of the five autosomes. Progeny (substrains) retaining combinations of two, three, or four chromosomes of the resistant parent were selected for measurement of their microsomal aldrin expoidase activity and its correlation with chromosomal makeup and level of resistance to DDT and propoxur. There was no evidence that microsomal epoxidation of aldrin or resistance to propoxur, is associated with chromosome V in the Fc strain as has been reported. Instead, the well-known oxidase regulating factor on chromosome II was of major importance in the strain's microsomal oxidation of aldrin. There was also evidence, though not conclusive, that a factor on chromosome I has an influence on the oxidative metabolism of insecticides in this strain, possibly through an interaction with the factor on chromosome II. The reasons for the conflicting reports on the genetic control of microsomal oxidation in the Fc strain are discussed.     

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.     

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.     

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.     

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.     

Insecticide resistance in the tropical bedbug Cimex hemipterus

Insecticide resistance in the bedbug Cimex hemipterus was investigated using 4211 bedbugs collected from three districts of Sri Lanka. Insecticide bioassays were carried out with discriminating dosages of deltamethrin, permethrin, DDT, malathion, and propoxur. Activity levels of insecticide metabolizing enzymes and the insecticide target site acetylcholinesterase were monitored using biochemical assays. Percentage survivals after DDT, malathion, and propoxur exposure were 41-88%, 18-64%, and 11-41%, respectively. For deltamethrin and permethrin, KT₅₀/KT₉₀ (time to knock-down 50%/90% of the population) values were 0.5-24/1.0-58 and 1.3-10/2.5-47 h, respectively. Both elevated esterase and malathion carboxylesterase mechanisms were present in bedbug populations. Monooxygenase levels were heterogeneous. Organophosphate and carbamate target site acetylcholinesterase, was insensitive in 29-44% of the populations. High DDT resistance was probably due to glutathione S-transferases. Malathion carboxylesterases are mainly responsible for high malathion resistance. High tolerance to both DDT and pyrethroids suggests the presence of ‘kdr’ type resistance mechanism in one population.     

Why does DDT toxicity change after a blood meal in adult female Culex pipiens?

The toxicity of topically applied DDT to adult female anautogenous mosquitoes (Culex pipiens L.) showed dramatic variations in blood-fed insects. It decreased very rapidly about twofold to a minimum at 24 hr after a blood meal, then increased within 72 hr back to values typical of non-blood fed insects. A comparison of the metabolism of [¹⁴C]DDT in vivo revealed an increase in DDT dehydrochlorination to DDE at 72 hr after a blood meal, but this increase was not responsible for the variations in DDT toxicity at 24 hr. Changes in penetration rates were not observed and changes in the distribution of DDT could likewise not be related to the short period of decreased toxicity of DDT. Fenvalerate and trans-permethrin, two pyrethroid insecticides which are believed to have a mode of action similar to that of DDT, were also significantly less toxic 24 hr after a blood meal. By contrast, the cyclodiene insecticide aldrin and the carbamate insecticide propoxur were not less toxic 24 hr after a blood meal. The results suggest that after a blood meal an unidentified and transient change in C. pipiens specifically decreases DDT/pyrethroid toxicity. A hypothesis concerning this transient change is advanced. The results illustrate the difficulties in explaining physiological changes in insecticide toxicity.     

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.     

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.     

Cross-resistance to pyrethroids and other insecticides in Aedes aegypti

Two substrains of Aedes aegypti, already resistant to DDT and pyrethroids, were further selected using either DDT or permethrin by mass exposure of the females only. DDT selection over 14 generations raised the resistance to DDT so far that no accurate LC₅₀ values could be determined. Selection with permethrin raised the tolerance to an irregular plateau 7–10 times the original. DDT selection in the adults raised the DDT resistance of the larvae, but this could be partly overcome using a dehydrochlorinase inhibitor. The resistance to pyrethroids was increased but tolerance of dieldrin, malathion and propoxur compounds was little changed. Permethrin selection of the adults raised resistance to pyrethroids more than DDT selection but also increased DDT resistance. Similar patterns were found for the larval insects. A strain from Demerara in Guyana showed both DDT and pyrethroid resistance, including strong resistance to pyrethrins together with dieldrin and propoxur. It was concluded that two major independent resistance mechanisms existed in the selected strains, a dehydrochlorinase affecting DDT alone, and an unknown mechanism, probably nerve insensitivity (kdr) affecting both DDT and pyrethroids.     

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.     

Biochemical study of resistance to imidacloprid in B biotype Bemisia tabaci from Guatemala

Systemic uptake bioassays using excised cotton leaves confirmed resistance to imidacloprid in a Guatemalan population of the tobacco whitefly Bemisia tabaci Gennadius. Polyacrylamide gel electrophoresis of naphthyl esterases identified the insects as B-types. Upon collection from the field, resistance was determined to be 58-fold relative to a susceptible strain originating in the Imperial Valley of California. Resistance levels increased to 126-fold in this population during its continuous exposure to systemically treated cotton. In biochemical investigations, there was no detectable NADPH-dependent mixed function oxidase metabolism of 14C-imidacloprid at any time during the selection process. In contrast, microsomal preparations from housefly abdomens readily produced significant amounts of the mono-hydroxy and olefin derivatives of the parent compound. Detoxification of imidacloprid by housefly MFOs may account for reports of lower toxicity of the insecticide towards this insect compared with whiteflies, despite similar binding properties between imidacloprid and the nicotinic acetylcholine receptors in both species.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.