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 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.     

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.     

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.     

Selective inhibition of the metabolism of diazinon and diazoxon in vitro by piperonyl butoxide,NIA 16824, and 1-(2-isopropylphenyl)imidazole

Several pesticide synergists known to be mixed-function oxidase inhibitors were found to inhibit the in vitro metabolism of diazinon by mouse liver microsomes. Piperonyl butoxide and NIA 16824 (O-isobutyl-O-propargyl phenylphosphonate) inhibit all oxidative reactions of diazinon to the same extent. In contrast, 1-(2-isopropylphenyl)imidazole selectively inhibits oxidative dearylation and thiophosphate to phosphate conversion without significant effect on ring side chain hydroxylation. This selectivity suggests that two different mechanisms of oxidative detoxification may be operating, mechanisms which may involve either two cytochrome P-450s or two different binding sites on the same cytochrome.     

Metabolism of fenitrothion by organophosphorus-resistant and -susceptible house flies, Musca domestica L

The metabolism of fenitrothion was investigated in highly resistant (Akita-f) and susceptible (SRS) strains of the house fly, Musca domestica L. The Akita-f strain was 3500 times more resistant to fenitrothion than the SRS strain. Fenitrothion, topically applied to the flies, was metabolized in vivo far faster in the Akita-f strain than in the SRS strain. In vitro studies revealed that fenitrothion was metabolized by a cytochrome P-450-dependent monooxygenase system and glutathione S-transferases. The former oxidase system metabolized fenitrothion in vitro into fenitrooxon and 3-methyl-4-nitrophenol as major metabolites, and into 3-hydroxymethyl-fenitrothion and 3-hydroxymethyl-fenitrooxon as minor metabolites. Glutathione S-transferases metabolized fenitrothion into desmethylfenitrothion. The cytochrome P-450-dependent monooxygenase system and glutathione S-transferases of the resistant Akita-f strain had 1.4 to 2.2 times and 9.7 times, respectively, as great activities as those of the susceptible SRS strain. These results suggest the importance of glutathione S-transferases in fenitrothion resistance in the Akita-f strain.     

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.     

多功能氧化酶系与甜菜夜蛾对氯氟氰菊酯抗药性的关系

比较了甜菜夜蛾Spodoptera exigua (Hübner)抗氯氟氰菊酯品系和敏感品系5龄幼虫中肠、脂肪体及体壁微粒体细胞色素P450的含量,结果表明,抗性和敏感品系不同组织细胞色素P450的含量均为中肠＞脂肪体＞体壁,抗性品系中肠、脂肪体及体壁细胞色素P450的含量分别是敏感品系的1.78、1.54及1.37倍。中肠微粒体甲氧试卤灵-O-脱甲基酶、乙氧试卤灵-O-脱乙基酶、乙氧香豆素-O-脱乙基酶、芳香基羟基化酶及艾氏剂环氧化酶的活性测定结果表明,抗性品系中肠5种酶的活性分别比敏感品系的酶活性提高1.33、1.73、1.40、1.51及1.30倍,说明甜菜夜蛾对氯氟氰菊酯的抗药性与微粒体多功能氧化酶活性的提高密切相关。     

Resistance to insecticides in winter- and summer-forms of pear psylla,Psylla pyricola

Summer-form pear psylla, Psylla pyricola Foerster, from sprayed pear were resistant to endosulfan (2·4-fold), methiocarb (2·5-fold), ethylan (5·8-fold), azinphos-methyl (7·7-fold), and fenvalerate (40·1-fold). Esterase (3·8-fold), glutathione transferase (1·8-fold), and cytochrome P-450 monooxygenase (1·6-fold) detoxification enzyme activity was higher in resistant than in susceptible summer forms. Synergism by piperonyl butoxide and S,S,S-tributylphosphorotrithioate (DEF) was added evidence for cytochrome P-450 monooxygenases and esterases as resistance mechanisms. Reduced penetration may also have contributed to resistance, as indicated by a 1·6-fold slower penetration of azinphos-methyl in resistant than susceptible summer-forms. Similar differences in insecticide toxicity and esterase and glutathione transferase activities were observed between winter-forms of resistant and susceptible pear psylla. Winter-forms of P. pyricola were up to three times more tolerant to insecticides than summer-forms. Higher cytochrome P-450 monooxygenase activity (1·7-fold) and slower azinphosmethyl penetration (2·1-fold) in winter-forms may have contributed to their greater insecticide tolerance; however, sequestration may also have been involved.     

Spectral interactions of insecticide synergists with microsomal cytochrome P-450 from insecticide-resistant and susceptible house flies

The spectral interactions of 45 insecticide synergists and related compounds with oxidized and reduced cytochrome P-450 from microsomes of insecticide-resistant and -susceptible house flies were investigated. The type III interaction typical of piperonyl butoxide was the most common spectral interaction for the compounds studied. In addition to this, several other varients of the type III interaction were noted. In general these responses with house fly microsomes were similar to those reported for mammals, although some minor species and strain differences were observed. The cytochrome P-450 from susceptible house flies, although reported previously not to exhibit type I difference spectra with many xenobiotics, was found to elicit this spectral response with several methylenedioxyphenyl compounds.     

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.     

Identification of cytochrome P450 differentiated expression related to developmental stages in bromadiolone resistance in rats (Rattus norvegicus)

Adult, 20-week-old, rats from a Danish bromadiolone-resistant strain of rats (Rattus norvegicus) over-express the cytochrome P450 genes Cyp2e1, Cyp3a2 and Cyp3a3 upon bromadiolone exposure. Furthermore, adult female rats of this strain over-express the Cyp2c13 gene and suppress Cyp2c12, while males over-express the Cyp2a1 gene. The altered gene expression has been suggested to be involved in the bromadiolone resistance by facilitating enhanced anticoagulant metabolism. To investigate the gene expression of these cytochrome P450 genes in rats of different developmental stages we compared expression profiles from 8-, 12- and 20-week-old resistant rats of the Danish strain to profiles of anticoagulant-susceptible rats of same ages. The three age-groups were selected to represent a group of pre-pubertal, pubertal and adult rats. We found expression profiles of the pre-pubertal and pubertal resistant rats to concur with profiles of the adults suggesting that cytochrome P450 enzymes are involved in the Danish bromadiolone resistance regardless of developmental stage. We also investigated the relative importance of the six cytochrome P450s in the different development stages of the resistant rats. The P450-3a2 and -3a3 isoforms were proposed to be of higher importance in adult male resistance than in pre-pubertal resistance. In contrast, the P450-2c13 and -3a2 isoforms were proposed to be more important in sexual immature female resistance, while the P450-2e1 and -3a3 isoforms were suggested to play a more significant role in adult female resistance.     

Differential expression of cytochrome P450 genes between bromadiolone-resistant and anticoagulant-susceptible Norway rats: a possible role for pharmacokinetics in bromadiolone resistance

BACKGROUND: Anticoagulant resistance in Norway rats, Rattus norvegicus (Berk.), has been suggested to be conferred by mutations in the VKORC1 gene, encoding the target protein of anticoagulant rodenticides. Other factors, e.g. pharmacokinetics, may also contribute to resistance, however. To examine the involvement of pharmacokinetics in bromadiolone resistance in male and female rats, liver expression profiles of seven cytochrome P450 genes from a Danish bromadiolone-resistant rat strain (with an Y139C-VKORC1 mutation) were compared with profiles from an anticoagulant-susceptible strain. RESULTS: In the presence of bromadiolone, the Cyp2e1, Cyp2c13, Cyp3a2 and Cyp3a3 genes were significantly overexpressed, while Cyp2c12 expression was suppressed in resistant female rats compared with susceptible females. Relative to susceptible males, resistant males showed significant overexpression of the Cyp2a1, Cyp2e1, Cyp3a2 and Cyp3a3 genes. On exposure to bromadiolone, females had higher Cyp2e1 expression than males, which possibly explains why female rats are generally more tolerant to anticoagulants than male rats. CONCLUSION: Results suggest that bromadiolone resistance in a Danish strain of Norway rats involves enhanced anticoagulant metabolism catalysed by cytochrome P450-2e1, -3a2 and -3a3. This pharmacokinetically based bromadiolone resistance is to some extent sex differentiated, as female resistance furthermore seems to involve overexpression of cytochrome P450-2c13 and suppression of P450-2c12, whereas male resistance appears to involve P450-2a1 overexpression.     

Evolutionary plasticity of monooxygenase-mediated resistance

The cytochrome P450 monooxygenases are an important metabolic system involved in the detoxification of xenobiotics, and are thus one of the major mechanisms by which insects evolve insecticide resistance. However, comparatively little is known about the evolutionary constraints of this insecticide resistance mechanism. We investigated the genetic basis of resistance in a strain of house fly (NG98) from Georgia, USA that had evolved 3700-fold resistance to the pyrethroid insecticide permethrin, and compared this to other permethrin resistant strains of house flies from the US and Japan. Resistance in NG98 was due to kdr on autosome 3 and monooxygenase-mediated resistance on autosomes 1, 2, and 5. These results indicate that the genes which evolve to produce monooxygenase-mediated resistance to permethrin are different between different populations, and that the P450 monooxygenases have some degree of plasticity in response to selection. Monooxygenase-mediated resistance appears to evolve using different P450s, and possibly different regulatory signals controlling P450 expression, even in strains selected with the same insecticide.     

Synergism and stability of acetamiprid resistance in a laboratory colony of Plutella xylostella

The involvement of metabolic enzymes in the resistance of a laboratory colony of diamondback moth, Plutella xylostella (L), to the neonicotinoid insecticide acetamiprid was determined with the synergists piperonyl butoxide (PBO), which suppresses the activity of cytochrome P-450 monooxygenases, and S,S,S-tributyl phosphorotrithioate (DEF), an inhibitor of esterases, using the leaf-dipping method. Both PBO and DEF enhanced the insecticidal activity of acetamiprid significantly in the resistant P. xylostella strain but not in a reference strain, suggesting that cytochrome P-450 monooxygenases and esterases play an important role in the resistance of P. xylostella to acetamiprid. The resistant P. xylostella strain was also reared without further exposure to acetamiprid to determine the stability of resistance. Maintaining the resistant strain for seven generations in the absence of selection pressure resulted in a drop in resistance ratio from 110 to 2.42, indicating that acetamiprid resistance in P. xylostella is not stable.     

Spinosad resistance in female Musca domestica L. from a field-derived population

BACKGROUND: Bait-formulated spinosad is currently being introduced for housefly (Musca domestica L.) control around the world. Spinosad resistance was evaluated in a multiresistant field population and strains derived from this by selection with insecticides. Constitutive and spinosad-induced expression levels of three cytochrome P450 genes, CYP6A1, CYP6D1 and CYP6D3, previously reported to be involved in insecticide resistance, were examined. RESULTS: In 2004 a baseline for spinosad toxicity of Danish houseflies where all field populations were considered to be susceptible was established. In the present study, females of a multiresistant field population 791a were, however, 27-fold spinosad resistant at LC₅₀, whereas 791a male houseflies were susceptible. Strain 791a was selected with spinosad, thiamethoxam, fipronil and imidacloprid, resulting in four strains with individual characteristics. Selection of 791a with spinosad did not alter spinosad resistance in either males or females, but counterselected against resistance to the insecticides thiamethoxam and imidacloprid targeting nicotinic acetylcholine receptors. A synergist study with piperonyl butoxide, as well as gene expression studies of CYP6A1, CYP6D1 and CYP6D3, indicated a partial involvement of cytochrome P450 genes in spinosad resistance. CONCLUSION: This study reports female-linked spinosad resistance in Danish houseflies. Negative cross-resistance was observed between spinosad and neonicotinoids in one multiresistant housefly strain. Spinosad resistance involved alterations of cytochrome P450 gene expression. Copyright © 2011 Society of Chemical Industry     

Resistance selection and biochemical mechanism of resistance to two Acaricides in Tetranychus cinnabarinus (Boiduval)

A Tetranychus cinnabarinus strain was collected from Chongqing, China. After 42 generations of selection with abamectin and 20 generations of selection with fenpropathrin in the laboratory, this T. cinnabarinus strain developed 8.7- and 28.7-fold resistance, respectively. Resistance to abamectin in AbR (abamectin resistant strain) and to fenpropathrin in FeR (fenpropathrin resistant strain) was partially suppressed by piperonyl butoxide (PBO), diethyl maleate (DEM) and triphenyl phosphate (TPP), inhibitors of mixed function oxidase (MFO), glutathione S-transferases (GST), and hydrolases, respectively, suggesting that these three enzyme families are important in conferring abamectin and fenpropathrin resistance in T. cinnabarinus. The major resistant mechanism to abamectin was the increasing activities of carboxylesterases (CarE), glutathione-S-transferase (GST) and mixed function oxidase (MFO), and the activity in resistant strain developed 2.7-, 3.4- and 1.4-fold contrasted to that in susceptible strain, respectively. The activity of glutathione-S-transferase (GST) in the FeR strain developed 2.8-fold when compared with the susceptible strain, which meant the resistance to fenpropathrin was related with the activity increase of glutathione-S-transferase (GST) in T. cinnabarinus. The result of the kinetic mensuration of carboxylesterases (CarE) showed that the structure of CarE in the AbR has been changed.     

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.     

抗阿维菌素小菜蛾的细胞色素P450酶系研究

通过对不同发育时期敏感和抗阿维菌素小菜蛾品系细胞色素P450含量的测定,以及使用不同模式底物对P450单加氧酶活性的比较研究发现:除成虫期外,不同发育时期抗性品系小菜蛾中P450和细胞色素b5的含量都高于敏感品系;抗性品系还原型辅酶Ⅱ(NADPH)-细胞色素P450还原酶活性是敏感品系的1.97倍;同时发现抗性品系中甲氧试卤灵-O-脱甲基酶(MROD)、乙氧试卤灵-O-脱乙基酶(EROD)、乙氧基香豆素-O-脱乙基酶(ECOD)以及对硝基苯甲醚-O-脱甲基酶(PNOD)的活性均明显高于敏感品系,分别为敏感品系的9.41、4.15、1.67和2.94倍。研究结果表明,细胞色素P450含量和单加氧酶活性的增高是小菜蛾对阿维菌素产生抗性的一个重要机制。     

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.