Cytochrome P-450 in insects. 6. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase,and monooxygenase activities in susceptible and resistant strains of Musca domestica

Development and phenobarbital (PB) induction of microsomal cytochrome P-450, cytochrome P-450 reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of insecticide-susceptible (NAIDM) and -resistant (Rutgers) house flies. Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Untreated insects of both strains had comparable reductase levels, whereas cytochrome P-450 and associated monooxygenase activities were 1.5-fold or more higher in Rutgers. Maximum induction, as well as toxicity, occurred at a lower PB concentration in NAIDM than Rutgers. The drug caused consistently higher increases in enzymes and activities within 12 hr of starting treatment in both strains. When PB was withdrawn from treated flies (both strains) 48 hr after treatment began, specific activities (product min^?1 mg protein^?1) in all enzymes returned to control values in 24 hr while metabolic capacity (product min^?1 insect^?1) achieved control values within 48 hr. The changes in turnover numbers (pmol product min^?1 pmol P-450^?1), in conjunction with the differences in the monooxygenation of the four substrates, suggest that PB treatment induced both a quantitative and qualitative change in NAIDM monooxygenation but only a quantitative change in Rutgers monooxygenation.     

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.     

Absence of enhanced detoxication of permethrin in pyrethroid-resistant house flies

The mechanisms of resistance to pyrethroids were studied in a permethrin-selected (147-R) strain of the house fly, Musca domestica L. Approximately 12-fold synergism was obtained with a mixture of (1R)-trans-permethrin:piperonyl butoxide (1:5) so that the resistance decreased from 97-fold to 22-fold. Tests with the esterase inhibitor S,S,S-tributyl phosphorotrithioate produced very little synergism in either the resistant (R) strain (1.6-fold) or the susceptible (S) strain (1.9-fold). An investigation of the microsomal components revealed that compared to the S strain, the R strain demonstrated twice as much cytochrome P-450 and cytochrome b₅ and double the rate of NADPH-cytochrome c reductase activity. In addition, the rate of p-nitroanisole O-demethylation was found to be six times greater in the R strain. An in vivo accumulation study showed that the R strain displayed a decreased rate of penetration of trans-[¹⁴C]permethrin. When treated at equitoxic doses the R strain was found to tolerate 50-fold more internal permethrin than the S strain. An in vitro metabolism study indicated that there was no difference between strains in the overall rate of metabolism of trans-[¹⁴C]permethrin. The evidence obtained supports the conclusion that several resistance factors are involved but that decreased sensitivity of the nervous system to the action of pyrethroids is the principal mechanism of resistance in the 147-R strain.     

Laboratory studies on the mechanisms of resistance to permethrin in a field-selected strain of house flies

Enhanced oxidative metabolism appeared to be a major factor involved in resistance to permethrin in a field strain of house flies, selected with permethrin over 4 years. This was shown in the 7.8-fold synergism by piperonyl butoxide which reduced the resistance ratio from 97 to 15. The rate of permethrin detoxication was significantly higher (P=0.05) in the resistant flies compared with a susceptible strain or resistant flies pretreated with piperonyl butoxide. The esterase inhibitor S,S,S-tributyl phosphorotrithioate did not reduce the level of resistance to permethrin in the resistant strain, although some hydrolytic metabolism was apparent. Rates of penetration were similar in susceptible and resistant flies and in resistant flies pre-treated with piperonyl butoxide. A minor unidentified resistance factor, possibly reduced sensitivity of the nervous system, may also have been present in the resistant strain.     

The mechanisms of carbaryl resistance in the fall armyworm,Spodoptera frugiperda (J. E. Smith)

The physiological mechanisms of resistance to carbaryl were investigated in a carbaryl-resistant strain of the fall armyworm, Spodoptera frugiperda (J. E. Smith). Piperonyl butoxide greatly reduced the resistance level from 90- to 6-fold, indicating that microsomal cytochrome P-450-dependent monooxygenases may play a major role in resistance. This finding is consistent with metabolic data in which the oxidative metabolism of carbaryl by midgut homogenates was five times more active in the resistant strain than in the susceptible strain. In addition, the resistant strain showed increased activities of microsomal hydroxylation and epoxidation compared to the susceptible strain. Cuticular penetration studies using [¹⁴C]carbaryl revealed that 55% of the applied radioactivity remained on the cuticle of resistant larvae while 32% remained on susceptible larvae 24 hr after topical treatment. The resistance appeared to be unrelated to target site insensitivity. It is concluded that the high level of resistance to carbaryl in this insect was mainly due to enhanced oxidative metabolism of the insecticide (via hydroxylation and epoxidation) with reduced cuticular penetration playing a very minor role, if any.     

The effect of host plant on colorado potato beetle (coleoptera: Chrysomelidae) susceptibility to pyrethroid insecticides

In laboratory studies, Colorado potato beetle (CPB) (Leptinotarsa decemlineata Say) reared on eggplant were significantly more susceptible to permethrin than CPB reared on tomato. Field studies conducted in 1984 and 1985 showed that the percentage reduction of CPB larvae reared on eggplant due to foliar applications of fenvalerate was significantly greater than the percentage reduction of CPB larvae reared on either potato or tomato. Plant defoliation was greater on eggplant and potato than on tomato. Eggplant and potato yields increased as the dose of fenvalerate increased and the potato beetle population decreased, but tomato yields were unaffected by fenvalerate treatments. The effects and biological activity of individual plant chemical compounds (i.e., glvcoalkaloids) in various Solanum species need to be examined in detail before the full significance of the role of these compounds in host-plant resistance and their interaction with CPB insecticide resistance are understood.     

Polysubstrate monooxygenases (cytochrome P-450) in larvae of susceptible and resistant strains of house flies

The polysubstrate monooxygenases (PSMO or cytochrome P-450) of house fly larvae were studied at the mature larval or “clear gut” stage. Fat body and gut tissues were most efficient in the conversion of aldrin to dieldrin. Microsomal fractions of larval homogenates had the highest PSMO activities, with lower PSMO activities also found associated with mitochondrial fractions. Microsomes from Rutgers (resistant) larvae had higher levels of NADPH:cytochrome c reductase (2×), cytochrome P-450 (2×), aldrin (4×), and heptachlor (9×) epoxidases than microsomes from CSMA (susceptible) larvae. Cytochrome P-450 of Rutgers larvae had an absorption maximum at 449 nm, 2 nm lower than the cytochrome P-450 of CSMA larvae. n-Octylamine spectra showed that the level of high-spin cytochrome P-450 was higher in Rutgers larvae. NADPH:cytochrome c reductase, cytochrome P-450, and aldrin epoxidase were induced by phenobarbital, and Rutgers larvae were shown to be more sensitive to this inducer than CSMA larvae. Induction of larval PSMO by phenobarbital did not affect the expression or the inducibility of PSMO in adults.     

Behavioural and electrophysiological investigation of 1-(7-ethoxygeranyl)-2-methylbenzimidazole on the house fly Musca domestica

The insecticidal properties of 1-(7-ethoxygeranyl)-2-methylbenzimidazole (EGMB) were investigated on larval and adult house flies. Unsynergised EGMB gave topical LD₅₀ values of 0.53 μg per female fly on NAIDM strain house flies. When flies were pretreated with 5.2 μg piperonyl butoxide, susceptibility was increased (LD₅₀ 0.12 μg per female fly). House fly larvae were less susceptible to EGMB (LD₅₀ 2.2 μg). Poisoning with EGMB resulted in a rapid reduction in locomotor activity of both larval and adult house flies. This reduction in locomotion was progressive and led to complete paralysis. Various parameters of larval nervous system function were investigated in larvae during these early phases of poisoning. As early as 15 min after dosing larvae with LD₉₅ doses of EGMB, sensory nerves were less responsive. Over a somewhat longer time (2–4 h), neurally evoked contractures were adversely affected by EGMB. In some cases, this effect appeared to be due to reduced postsynaptic potential amplitude; in other instances, it appeared to be due to an effect independent of neuromuscular transmission. The close temporal correlation between behavioural and electrophysiological observations suggests that the nervous and muscular systems are important sites of action of EGMB.     

Mechanisms of resistance to the juvenoid methoprene in the house fly Musca domestica L

The mechanisms of resistance and cross resistance to the juvenoids methoprene and R-20458 in the house fly, Musca domestica, were examined. Radiolabeled methoprene was found to be metabolized faster in resistant and cross-resistant house fly larvae than in susceptible larvae, and methoprene and R-20458 penetrated more slowly into larvae of the resistant strain. In vivo and in vitro metabolism of methoprene was largely by oxidative pathways followed by conjugation in all strains examined, and little or no ester change of methoprene was noted in vitro. In vitro oxidative metabolism of methoprene, R-20458, juvenile hormone I, and several model substrates was higher in resistant and cross-resistant larvae than in susceptible larvae. Juvenoid functionalities susceptible to metabolic attack by resistant strains are indicated.     

苏云金芽胞杆菌在马铃薯甲虫防治上的研究进展

马铃薯甲虫是重要的入侵害虫,严重威胁着我国粮食作物马铃薯的生产。苏云金芽胞杆菌是重要的农业害虫生防细菌,对马铃薯甲虫有良好的防治效果。本文围绕苏云金芽胞杆菌在马铃薯甲虫防治上的研究进展与应用进行综述。主要从马铃薯甲虫的入侵与防治手段、苏云金芽胞杆菌的晶体蛋白结构与杀虫机制、对马铃薯甲虫有活性的Bt毒蛋白研究进展、Bt毒蛋白对马铃薯甲虫的作用机制以及马铃薯甲虫对Bt毒蛋白的抗性机制等方面进行了综述。最后,从Bt新基因的挖掘和杀虫机理方面对苏云金芽胞杆菌在马铃薯甲虫防治上的研究进行了展望。     

Target site insensitivity and mutational analysis of acetylcholinesterase from a carbofuran-resistant population of Colorado potato beetle, Leptinotarsa decemlineata (Say)

The BERTS strain of Colorado potato beetle (CPB) was found to be highly resistant to N-methyl carbofuran but relatively susceptible to azinphosmethyl. N-Methyl carbofuran resistance was found to correlate well with acetylcholinesterase (AChE) insensitivity. In becoming resistant to N-methyl carbofuran, the AChE of the BERTS strain became more sensitive to N-propyl carbofuran inhibition. This negative cross-insensitivity correlated well to the increased relative toxicity of the BERTS strain to N-propyl carbofuran compared to the susceptible SS strain. BERTS beetles were sorted into BERTS-R and BERTS-S substrains using their AChE activity profiles. Sequence comparisons of AChE cDNAs from the two substrains revealed the presence of the point mutation that results in the S291G substitution previously found in the AChE of the azinphosmethyl-resistant AZ-R strain of CPB. A novel mutation present only in BERTS-S CPB, however, resulted in an additional I392T substitution in the AChE and apparently reverses the resistance conferring properties of the S291G substitution.     

Antibody Capture Immunoassay for the Detection of Permethrin Carboxylesterase in Colorado Potato Beetle,Leptinotarsa decemlineata Say

Polyclonal antibodies were generated against the three denatured forms (30-, 48-, and 59-kDa proteins) of permethrin carboxylesterase from Colorado potato beetle. Each antiserum showed cross-reactivities to all the denatured forms of permethrin carboxylesterase as judged by antibody capture immunoassay. An antibody capture immunoassay was developed using the immunoglobulin purified from the 30-kDa antiserum in conjunction with denatured Colorado potato beetle hemolymph as the antigen source. In this antibody capture immunoassay, the hemolymph from the permethrin-resistant strain produced 1.1–5.3 times higher signal levels than that from the susceptible strain depending on assay conditions, suggesting that the hemolymph from the resistant strain contains more permethrin carboxylesterase. These results corroborate our original findings that permethrin carboxylesterase is overexpressed in the hemolymph of the permethrin-resistant strain of Colorado potato beetle and contributes to overall permethrin resistance. The relative quantities of permethrin carboxylesterase in four different strains of Colorado potato beetle with different levels of permethrin resistance were assessed with this immunoassay and with a carboxylesterase activity assay using α-naphthyl caproate as substrate. The average levels of permethrin carboxylesterase determined by the antibody capture immunoassay were better correlated with the levels of permethrin resistance than those determined by the carboxylesterase activity. Thus, the antibody capture immunoassay can be utilized as a sensitive means to detect the levels of permethrin carboxylesterase as a resistance marker in field populations of Colorado potato beetle.     

Cytochrome P-450 in insects. 7. Age dependency and phenobarbital induction of cytochrome P-450, P-450 reductase,and monooxygenase activities in the black blow fly (Phormia regina,Meigen)

Development and phenobarbital (PB) induction of microsomal cytochrome P-450, NADPH-cytochrome c (P-450) reductase, two epoxidation, and two O-demethylation activities were examined in chronologically timed populations of female black blow flies (Phormia regina, Meigen). Measurements of these enzymes started with the pharate adult stage and ended 5 days following eclosion. Induction occurred in all enzymes, even at 0.005% PB, and was maximum at 0.15%. Dramatic induction of the O-demethylation of 7-methoxy-4-methylcoumarin was observed in flies dosed with the maximum concentration of the drug. This monooxygenase activity increased to nearly 1400 times the level in control flies, whereas the other O-demethylation (methoxyresorufin) and the two epoxidation reactions exhibited considerably less change. Induction of the structural enzymes of this enzyme system were 10-fold for cytochrome P-450 and 5-fold for NADPH-cytochrome c (P-450) reductase. These data suggest that PB induces several P-450's in the blow fly, particularly one bearing a high degree of specificity for 7-methoxy-4-methycoumarin.     

Microsomal oxidase activities in relation to age and chlorcyclizine induction in American cockroach,Periplaneta americana,fat body,midgut and hindgut

Female adult American cockroaches, Periplaneta americana L., showed definite age-dependent changes in levels of activity of the microsomal mixed-function oxidases. Cytochrome P-450 levels, EPN-detoxication, and p-nitroanisole O-demethylation activities were very low in young adult insects but increased steadily reaching a natural peak at about 100 days in fat body and at about 90 days in midgut and hindgut. The activities then declined rapidly reaching levels of young insects at about 130 to 140 days of age. NADPH-neotetrazolium-reductase activity was high in young insects, declined later in adult life, and returned to a peak at about 100 days.Injections of chlorcyclizine, a known microsomal enzyme inducer, significantly increased levels of cytochrome P-450, EPN-detoxication, p-nitroanisole O-demethylation, and NADPH-NT-reductase activities in young cockroaches. The drug injections were effective, however, only before the natural activity peak was reached. Beyond this point the injections had no inductive effect indicating that the microsomal oxidases in this insect are uninducible when normal enzyme levels are falling.NADPH-NT-reductase activity in male cockroaches, while being somewhat higher than in females, showed a similar age-dependent curve with the peak occurring at about 120 days.Age-dependent carbaryl resistance in male and female insects tended to follow levels of the microsomal oxidase activities. Fifty to 60-day-old insects, however, tended to be more resistant to the insecticide than microsomal enzyme levels would indicate.RNA levels of normal female insects showed age-dependent curves similar to those of the microsomal enzyme activities, being low in young adults and reaching a peak at about 100 days. Chlorcyclizine injections had little or no effect on total microsomal RNA levels.     

In vitro metabolism of etrimfos by house flies

The metabolism of etrimfos, O,O-dimethyl-O-(6-ethoxy-2-ethyl-4-pyrimidinyl) phosphorothioate was studied in vitro in a diazinon-resistant (Rutgers) and a susceptible (CSMA) strain of house flies. Practically no metabolism of etrimfos occurred without the addition of cofactors. However, the addition of the cofactor, reduced glutathione, resulted in a substantial amount of metabolism in both strains, the metabolism being higher in the resistant strain. The major route of metabolism was via the glutathione transferase system and the predominant metabolite was desmethyl etrimfos. Although the oxygen analog could not be isolated, microsomal oxidation of etrimfos resulted in the inhibition of acetylcholinesterase, suggesting the formation of the oxygen analog. Bovine serum albumin also degraded etrimfos yielding desmethyl etrimfos and 6-ethoxy-2-ethyl-4-hydroxypyrimidine.     

Toxicological and biochemical characterization of coumaphos resistance in the San Roman strain of Boophilus microplus (Acari: Ixodidae)

The San Roman strain of the southern cattle tick, Boophilus microplus, collected from Mexico was previously reported to have a high level of resistance to the organophosphate acaricide coumaphos. An oxidative detoxification mechanism was suspected to contribute to coumaphos resistance in this tick strain, as coumaphos bioassay with piperonyl butoxide (PBO) on larvae of this resistant strain resulted in enhanced coumaphos toxicity, while coumaphos assays with PBO resulted in reduced toxicity of coumaphos in a susceptible reference strain. In this study, we further analyzed the mechanism of oxidative metabolic detoxification with synergist bioassays of coroxon, the toxic metabolite of coumaphos, and the mechanism of target-site insensitivity with acetylcholinesterase (AChE) inhibition kinetics assays. Bioassays of coroxon with PBO resulted in synergism of coroxon toxicity in both the San Roman and the susceptible reference strains. The synergism ratio of PBO on coroxon in the resistant strain was 4.5 times that of the susceptible strain. The results suggested that the cytP450-based metabolic detoxification existed in both resistant and susceptible strains, but its activity was significantly enhanced in the resistant strain. Comparisons of AChE activity and inhibition kinetics by coroxon in both susceptible and resistant strains revealed that the resistant San Roman strain had an insensitive AChE, with a reduced phosphorylation rate, resulting in a reduced bimolecular reaction constant. These data indicate a mechanism of coumaphos resistance in the San Roman strain that involves both insensitive AChE and enhanced cytP450-based metabolic detoxification.     

Mechanisms of resistance to diflubenzuron in the house fly, Musca domestica (L.)

The mechanisms of resistance to the chitin synthesis inhibitor diflubenzuron were investigated in a diflubenzuron-selected strain of the house fly (Musca domestica L.) with > 1000 × resistance, and in an OMS-12-selected strain [O-ethyl O-(2,4-dichlorophenyl)phosphoramidothioate] with 380 × resistance to diflubenzuron. In agreement with the accepted mode of action of diflubenzuron, chitin synthesis was reduced less in larvae of the resistant (R) than of a susceptible (S) strain. Cuticular penetration of diflubenzuron into larvae of the R strains was about half that of the S. Both piperonyl butoxide and sesamex synergized diflubenzuron markedly in the R strains, indicating that mixed-function oxidase enzymes play a major role in resistance. Limited synergism by DEF (S,S,S-tributyl phosphorotrithioate) and diethylmaleate indicated that esterases and glutathione-dependent transferases play a relatively small role in resistance. Larvae of the S and R strains exhibited a similar pattern of in vivo cleavage of ³H- and ¹⁴C-labeled diflubenzuron at N₁C₂ and N₁C₁ bonds. However, there were marked differences in the amounts of major metabolites produced: R larvae metabolized diflubenzuron at considerably higher rates, resulting in 18-fold lower accumulation of unmetabolized diflubenzuron by comparison with S larvae. Polar metabolites were excreted at a 2-fold higher rate by R larvae. The high levels of resistance to diflubenzuron in R-Diflubenzuron and R-OMS-12 larvae are due to the combined effect of reduced cuticular penetration, increased metabolism, and rapid excretion of the chemical.     

CNS insensitivity to pyrethroids in the resistant kdr strain of house flies

Solutions of tetramethrin, RU 11679, or cismethrin caused uncoupled convulsions in 30–40 min in exposed thoracic ganglia from S_NAIDM house flies at concentrations down to 10^?10M: whereas these same compounds at 10^?6M concentrations failed to produce poisoning symptoms when perfused onto the exposed ganglia of the kdr strain of house fly. The pyrethroid analogs examined had a negative temperature coefficient of action on the exposed thoracic ganglia from S_NAIDM flies. DDT and GH-74 possessed positive temperature coefficients of action on the exposed thoracic ganglion of susceptible house flies. It is concluded that the central nervous system of the kdr strain of house fly is resistant to pyrethroid action; furthermore, the resistance appears to be widespread throughout the house fly nervous system, involving sensory, motor, and central neural elements.     

辛硫磷对氰戊菊酯在抗性家蝇体内的增效机制

作者就辛硫磷对氰戊菊酯在抗拟除虫菊酯类杀虫剂的家蝇体内的增效作用进行了初步的研究。结果表明：在辛硫磷与氰戊菊酯同时存在时（质量比为２：１），家蝇体内未被降解的氰戊菊酯原化合物含量显著高于只使用等剂量氰戊菊酯时的对照处理（２．６－４．６倍）。同时，家蝇对氰戊菊酯的氧化代射速率降低（１７－４８％）。这些结果可能是辛硫磷对氰戊菊酯增效的主要作用机制，并对延缓害虫抗药性发展有积极的作用。     

Resistance to neonicotinoid insecticides in field populations of the Colorado potato beetle (Coleoptera: Chrysomelidae)

BACKGROUND: Neonicotinoid insecticides were first used commercially for Colorado potato beetle [Leptinotarsa decemlineata (Say), Coleoptera: Chrysomelidae] control in the United States in 1995, and since then have been critical for management of this pest. Field populations from the northeastern and midwestern United States were tested from 1998 to 2010 for susceptibility to imidacloprid and thiamethoxam using standard topical dose assays with adults. RESULTS: From 1998 to 2001, imidacloprid resistance was present in only a few locations in the eastern United States. By 2003, imidacloprid resistance was common in the northeastern Unites States. In 2004, imidacloprid resistance in Colorado potato beetle was detected for the first time in the midwestern United States. In 2003, the first case of resistance to thiamethoxam was found in a population from Massachusetts. Neonicotinoid resistance in summer‐generation adults was higher than in overwintered adults from the same locations. By 2009, 95% of the populations tested from the northeastern and midwestern United States had significantly higher LD₅₀ values for imidacloprid than the susceptible population. CONCLUSIONS: The increasing resistance to neonicotinoid insecticides raises concerns for the continued effective management of Colorado potato beetles in potatoes and highlights the need for more rigorous practice of integrated pest management methods. Copyright © 2011 Society of Chemical Industry