GABA receptors of insects susceptible and resistant to cyclodiene insecticides

Toxicity tests revealed up to 40-fold resistance to a number of cyclodiene insecticides in a laboratory-reared, cyclodiene-resistant (CYW) housefly strain (Musca domestica L.). Using [³⁵S] TBPS as a probe for convulsant sites in insects, saturable specific binding was detected in thorax and abdomen membranes prepared from housefly strains susceptible (CSMA) and resistant (CYW) to cyclodienes. Scatchard analysis of[³⁵S] TBPS binding data to CSMA and CYW membranes failed to provide evidence for significant differences between the two strains in either the affinity (K_d) or density (B_max) of saturable binding sites. For several polychlorocycloalkane insecticides, the ligand displacement profile of [³⁵S] TBPS binding was almost identical for the CSMA and CYW houseflies. Therefore, using [³⁵S] TBPS as a probe for convulsant sites, a 40-fold resistance to cyclodienes in the CYW housefly strain cannot be accounted for only in terms of alterations in TBPS binding sites.     

Mixed-function oxidase activity toward cyclodiene insecticides in bass and bluegill sunfish

Microsomal mixed-function oxidase (MFO) was characterized in liver of freshwater fishes, bass, Micropterus dolomeiux, and bluegill, Lepomis macrochirus, using the chlorinated cyclodiene insecticide aldrin as a substrate.The MFO activity of bluegill adults, bluegill fry, and bass fry using cyclodienes as substrates was compared with that of mouse. Aldrin epoxidase activity was found to be high in bluegill fry (73% of the activity in mouse) compared to bluegill adults (13% of the activity in mouse).Chlordene was found to be metabolized by both fish and mouse MFO to chlordene 2,3-epoxide and 1-hydroxychlordene.Competitive inhibition between various cyclodiene substrates studied in mouse and fish showed that endrin was a potent inhibitor of aldrin epoxidation in vitro.     

The biochemical mechanisms of resistance to insecticides with especial reference to the housefly,Musca domestica and aphid,Myzus persicae

The mechanisms by which insects chemically modify and thereby resist insecticides, and the techniques used to study this metabolism arc described. The enzymes associated with the endoplasmic reticulum of the cell (the microsomal enzymes) play a major and ubiquitous role in this metabolism which can produce molecules either more or less toxic than the insecticide applied. “Soluble” enzymes in the cytoplasm of the cell also metabolise insecticides, and it is the balance between these types of metabolism and such factors as the rate of penetration of the insecticide and the susceptibility of the target which determines the tolerance of an insect to a particular insecticide. A very small change in one of these factors can cause a large change in the dose of insecticide required to kill an insect.     

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.     

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.     

The photoreactions of carbamate insecticides

The photochemical reactions of the O-phenyl-N-methyl carbamates are reviewed. Most of the members of this series absorb radiation available in the solar region on the surface of the earth. The bulk of the work in this area has been carried out in the laboratory although some field experiments have also been reported. The products of the photolysis of carbamates in solution suggest that the reaction proceeds via a photo-Fries mechanism. Methods, based on emission properties for distinguishing between different carbamates in a mixture, are discussed and extensions are proposed.     

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.     

Inhibition of microsomal oxidoses from the housefly

The hydroxylation of naphthalene, O-demethylation of p-nitroanisole, and N-demethylation of N,N-dimethyl-p-nitrophenylcarbamate by housefly microsomes were inhibited by CO, sulfhydryl reagents, Cu (II), methylene dioxyphenyl compounds and β-diethylaminoethyldiphenylvaleric acid (SKF 525-A). Kinetic analysis indicates that the inhibitory properties of SKF 525-A and piperonyl butoxide are changed during the incubation period. Bovine serum albumen changes the kinetic constants in a way which suggests binding of an endogenous inhibitor(s). Patterns of inhibition indicate p-nitroanisole oxidation is effected by a system with at least one component distinct from the system which oxidizes naphthalene and N,N-dimethyl-p-nitrophenylcarbamate.     

Inhibitors of cyclodiene epoxide ring hydrating enzymes of the blowfly,Calliphova erythvocephala

A cyclodiene epoxide notable for its susceptibility to enzymic epoxide ring hydration in living organisms has been used to investigate potential inhibitors of the enzyme involved (epoxide hydrase), which is mainly microsomal, but distinct from the microsomal oxidases. Compounds with varying degrees of inhibitory efficiency include insect hormone analogues containing epoxide rings, certain other epoxides, methylenedioxybenzene-derived insecticide synergists, tri-o-cresyl phosphate, triphenyl phosphate and the microsomal oxidase inhibitor SKF 525A. The simple epoxides 1,l,l-trichloro-2,3-epoxy-propane and l,2-epoxy-l,2,3,4-tetrahydronaphthalene are the best inhibitors of this enzyme in blowfly pupal homogenates.     

A PCR diagnostic for cyclodiene insecticide resistance in the red flour beetle Tribolium castaneum

A molecular diagnostic was used to examine the conservation of cyclodiene resistance associated mutations between different strains of Tribolium castaneum (Herbst.). An improved insecticide bioassay for discrimination between resistant genotypes was developed and seven resistant strains were established from five different continents. In order to develop a molecular diagnostic a partial cDNA of the cyclodiene insecticide resistance gene Rdl, a γ-aminobutyric-acid-gated chloride-ion channel, was cloned and sequenced. This cDNA spans exon 7, the region containing the resistance-associated mutation, and part of exon 8. An ‘allele-specific’ oligonucleotide primer, carrying the resistance-associated mutation at its 3′ end, was used in combination with a flanking ‘allele-independent’ primer in the polymerase chain reaction to selectively amplify a single resistance-associated mutation from all seven strains collected worldwide. The implications of these findings for the population genetics of insecticide resistance and its management in pest insects via quarantine are discussed.     

Toxicological significance of acetylcholinesterase of the housefly thorax

Polyacrylamide gel electrophoresis of the 100,000g supernatant fraction of thoracic homogenates from the DDT/S housefly strain separated five bands having acetylcholinesterase activity. Ferguson plots indicated that the three bands of slowest mobility constituted members of the heterologous-size isomer family described for the head enzyme. The two faster migrating bands appeared as electrophoretic artifacts of juxtaposition with a 3.6 S nonacetylcholinesterase component; in the absence of juxtaposition, a single form was characterized with identical molecular properties to the 5.3 S fundamental unit of head acetylcholinesterase. The kinetic properties of isolated forms were consonant with the molecular data and no major differences in K_m values for acetylthiocholine were discovered between the thoracic and head enzymes. We suggest the preferential in vivo inhibition of thoracic acetylcholinesterase by organophosphate pesticides is unlikely the consequence of biochemical differences between the enzymes from the two regions.     

Characterisation of spinosad resistance in the housefly Musca domestica (Diptera: Muscidae)

BACKGROUND: Spinosad, a relatively new, effective and safe pesticide, has been widely used in pest control over the last 10 years. However, different levels of resistance to this insecticide have developed in some insects worldwide. RESULTS: After continuous selection for 27 generations, a strain (SpRR) of the housefly developed 247‐fold resistance to spinosad compared with the laboratory susceptible strain (CSS). The estimated realised heritability (h²) of spinosad resistance was 0.14. There was no significant difference in the LD₅₀ values and slopes between reciprocal progenies F₁ and F₁′, and values of 0.33 (F₁) and 0.30 (F₁′) were obtained for the degree of dominance. Chi‐square analysis from responses of self‐bred (F₂) and backcrosses (BC₁ and BC₂) were highly significant, suggesting that the resistance was probably controlled by more than one gene. Synergists piperonyl butoxide (PBO), diethyl maleate (DEM) and S,S,S‐tributyl phosphorotrithioate (DEF) affected the toxicity of spinosad at a low level, and demonstrated that metabolic‐mediated detoxification was not an important factor in conferring resistance to spinosad in the SpRR strain. CONCLUSION: It was concluded that spinosad resistance in the housefly was autosomal and incompletely dominant, and the resistance was probably controlled by more than one gene. These results provide the basic information for designing successful management programmes for the control of houseflies. Copyright © 2011 Society of Chemical Industry     

Inheritance of beta-cypermethrin resistance in the housefly Musca domestica (Diptera: Muscidae)

BACKGROUND: Beta-cypermethrin, a synthetic pyrethroid insecticide, was applied frequently in the control of health pests including houseflies, Musca domestica L., in China. However, different levels of resistance to beta-cypermethrin were monitored in field strains of houseflies. A strain of M. domestica, 4420-fold resistant to beta-cypermethrin after continuous 25 generations of selection, was used in this paper to determine the mode of inheritance of pyrethroid resistance. RESULTS: The estimated realized heritability (h(2)) of beta-cypermethrin resistance was 0.30 in this resistant strain. Results of bioassays showed no significant difference in values of LD(50) and slope of log dose-probit lines between reciprocal progenies F(1) and F'(1), and yielded values of - 0.10 (F(1)) and - 0.11 (F'(1)) for the degree of dominance (D). Chi-square analysis from responses of self-bred and backcross progenies (F(2), BC(1) and BC(2) respectively) indicated that the null hypothesis, a single gene responsible for resistance, was accepted. The minimum number of independent segregation genes was 0.93 for F(1) by Lande's method. CONCLUSION: It was concluded that beta-cypermethrin resistance in the housefly was inherited as a single, major, autosomal and incompletely recessive factor. These results would provide the basic information for pest management programmes.     

Binding activity of substituted benzyl derivatives of chloronicotinyl insecticides to housefly‐head membranes,and its relationship to insecticidal activity against the housefly Musca domestica

Variously substituted benzyl derivatives of chloronicotinyl insecticides were synthesized with a wide range of substituents including halogens, NO₂, CN, CF₃ and small alkyl and alkoxy groups at the ortho, meta and para positions, as well as multiple‐substituted benzyl analogues. Their binding activity to the α‐bungarotoxin binding site in housefly (Musca domestica) head membrane preparations was measured. Among the compounds tested, the activity of the meta‐CN derivative was the highest, being 20–100 times higher than those of imidacloprid, acetamiprid and nitenpyram. The synergized insecticidal activity against houseflies was also measured for selected compounds with the metabolic inhibitor, NIA16388 (propargyl propyl phenylphosphonate). For the nitromethylene analogues, including both benzyl and pyridylmethyl analogues, higher binding activity usually resulted in higher insecticidal activity. © 2000 Society of Chemical Industry     

The mode of entry of contact insecticides

Experiments have been carried out to test the generally accepted view that contact insecticides enter into the central nervous system via the haemolymph. So far, they have only produced evidence to the contrary As an alternative route, it is suggested that these insecticides reach the site of action via the integumental tissues of the body wall and the tracheal system. The evidence for this is discussed.     

The cuticle,growth and moulting in insects: The essential background to the action of acylurea insecticides

The functions, structure and biochemistry of the insect cuticle in relation to the moulting cycle are briefly reviewed as an introduction to the actions of insecticides that act on the cuticle, particularly acylureas. The symptoms of poisoning with diflubenzuron (DFB) and other acylureas are consistent with ultra-structural and biochemical evidence that these insecticides inhibit the formation of chitin microfibrils in newly synthesised cuticle. It is probable that DFB acts at a late stage in chitin biosynthesis, perhaps inhibiting chitin synthase (CS) itself. However, the results of studies using cell-free preparations of CS have not, on the whole, supported this hypothesis. A number of alternative suggestions as to the mode of action of DFB are reviewed. Among the most attractive of these is the possibility that DFB may inhibit the transmembrane transport of chitin synthesis precursors from their site of production within the epidermal cells to the site of the final poly condensation reaction, presumably at the apical membrane of the epidermal microvilli.     

The status and development of insecticide resistance in Danish populations of the housefly Musca domestica L

Samples of housefly (Musca domestica) field populations were collected from Danish livestock farms in 1997. The tolerance of the first‐generation offspring was determined for a number of insecticides. Dose‐response values were obtained by topical application for the pyrethroids bioresmethrin and pyrethrum, both synergised with piperonyl butoxide, and the organophosphate dimethoate. The organophosphates azamethiphos and propetamphos and the carbamate methomyl were tested in discriminating dose feeding bioassays. Resistance was low to moderate in most of the populations for most of the compounds tested, but this study also revealed the existence of high resistance to pyrethroid, organophosphate and carbamate insecticides in some populations. The resistance factors at LD₅₀ for bioresmethrin/piperonyl butoxide ranged between 2 and 98, and for pyrethrum/piperonyl butoxide between 2 and 29. Our results indicate that pyrethroid resistance in Denmark is increasing, since four of the 21 farms showed more than 100‐fold resistance at LD₉₅, a level of resistance only observed once before. Resistance factors at LD₅₀ for dimethoate ranged from 9 to 100, and showed two distinct trends: populations with either decreasing or increasing resistance. Resistance to azamethiphos was found to be widespread and high. Although two strains with high methomyl and propetamphos resistance were observed, methomyl and propetamphos resistance is moderate and appears not to be increasing. © 2001 Society of Chemical Industry     

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.     

Characterization of imidacloprid resistance in the housefly Musca domestica (Diptera: Muscidae)

In recent years, imidacloprid was introduced to control the housefly in China and it was documented that the housefly indeed showed signs of resistance to imidacloprid somewhere but not in China. Therefore, a housefly population collected from filed (IFS) was selected continuously with imidacloprid to establish the resistant strain (IRS) and the basic characteristics were investigated in this study. After continuous selection over 21 generations, the resistance ratio increased from 9.01 to 140, and different levels of cross-resistance were developed to beta-cypermethrin, chlorpyrifos, chlorfenapyr, acetamiprid and azamethiphos in the IRS strain. The realized heritability of resistance was 0.10. The synergistic ratios for IRS pretreated with DEF, DEM and PBO were 1.68, 1.52 and 2.53, and the corresponding ones for IFS were 3.17, 1.87 and 2.67, respectively. Synergistic and biochemical assays suggested that the cytochrome P450 may play an important role in the imidacloprid resistance comparing with GSTs- and carboxylesterases-mediated detoxification in the IRS strain, and there might be additional mechanisms (e.g. reduced target-site sensitivity) contributed to imidacloprid resistance in the IRS strain.