Insecticide toxicity, synergism and resistance in the German cockroach (Dictyoptera: Blattellidae)

The toxicity of synergism of and resistance to insecticides in four strains of German cockroach, Blattella germanica (L.), were investigated. Toxicity of nine insecticides by topical application to the susceptible strain varied greater than 2,000-fold, with deltamethrin (LD50 = 0.004 micrograms per cockroach) and malathion (LD50 = 8.4 micrograms per cockroach) being the most and least toxic, respectively. Resistance to pyrethrins (9.5-fold) in the Kenly strain was unaffected by the synergists piperonyl butoxide (PBO) or S,S,S-tributylphosphorotrithioate (DEF), suggesting that the metabolism is not involved in this case. Malathion resistance in the Rutgers strain was suppressible with PBO, implicating oxidative metabolism as a resistance mechanism. The Ectiban-R strain was resistant to all the pyrethroids tested, and cypermethrin resistance was not suppressible with PBO or DEF. These findings support results of previous studies that indicated this train has a kdr-like mechanism. Bendiocarb resistance in both the Kenly and Rutgers strains was partially suppressed by either PBO or DEF, suggesting that oxidative and hydrolytic metabolism are involved in the resistance. Trends between the effects of the synergists on the susceptible versus resistant strains are discussed.     

Susceptibility of spinosad in Musca domestica (Diptera: Muscidae) field populations

The toxicity of spinosad was determined in one susceptible and five insecticide-resistant laboratory strains of house fly, Musca domestica L. Spinosad was relatively slow-acting, but highly toxic to house flies. In a feeding bioassay, spinosad LC50 at 72 h was 0.51 microg of spinosad per gram of sugar, making it 6.3- and 3.5-fold more toxic to house flies compared with azamethiphos and methomyl, respectively. In topical application bioassay, the LD50 at 48 h of spinosad in susceptible house flies was 40 ng per 20 mg of house fly, making spinosad less toxic than the pyrethroid bioresmethrin synergized by piperonyl butoxide and the organophosphate dimethoate. The insecticide-resistant laboratory strains had resistance factors to spinosad at LC50 in feeding bioassay from 1.5 to 5.5 and at LD50 in topical application bioassay from 2.5 to 4.7, indicating that in house fly cross-resistance to the major insecticide classes will not initially be of major concern for the use of spinosad for house fly control. The toxicity of spinosad was also evaluated against 31 field populations of house flies collected from livestock farms across Denmark. The field populations were 2.2- to 7.5-fold resistant to spinosad at 72 h in feeding bioassay, but based on steep slopes in the bioassay and the limited variation of spinosad toxicity against the various field populations, we consider the field populations to be spinosad-susceptible. We propose a diagnostic dose of 12 microg of spinosad per gram of sugar in feeding bioassay with impregnated sugar for determination of resistant house flies, which is 10x the LC95 of the susceptible strain WHO and approximately = 2x the LD95 of the field populations. Spinosad showed no substantial cross-resistance to the pyrethroid bioresmethrin synergized by piperonyl butoxide, the anticholinesterases dimethoate, azamethiphos, methomyl, and spinosad in house fly field populations.     

Studies on some enzymes involved in insecticide resistance in fenitrothion-resistant and -susceptible strains of Musca domestica L. (Dipt, Muscidae)

Abstract: The toxicity of fenitrothion and fenitrothion plus synergists was determined by topical application to adults of fenitrothion-resistant (571ab) and -susceptible (Cooper) strains of Musca domestica L. The strain 571ab was 232-fold resistant to fenitrothion when compared with the Cooper strain. Co-administration of fenitrothion with three synergists, namely piperonyl butoxide (PBO), tributylphosphorotrithioate (DEF) and diethyl maleate (DEM) was investigated, respectively, at 1 : 5, 1 : 5 and 1 : 10 ratio. This co-administration of fenitrothion with PBO, DEF and DEM caused a decrease in the doses which produced 50% lethality (LD₅₀s) in 571ab but had no synergistic effect on fenitrothion toxicity was observed in the Cooper strain. The effect of topical application of fenitrothion alone and in combination with PBO, DEF and DEM at the LD₅₀ level on some enzyme activities in 571ab and Cooper strains was examined. The application of fenitrothion alone and in combination with DEF and DEM at LD₅₀ level caused a significant decrease in activities of total esterases, acetylcholinesterase (AChE) and glutathione S-transferase (GST) in the 571ab strain. The decrease in GST activity was not significant in treated flies of the Cooper strain when compared with GST activity of control flies. A non-significant effect on total cytochrome P450 level was observed with fenitrothion alone and the fenitrothion + PBO treatment. No increase in activity level of total esterases, AChE and GST was found, which might suggest that changes in activity level of these enzymes are not related to fenitrothion resistance in the 571ab strain.     

Interactions of carbaryl with susceptible and multiresistant house flies (Diptera: Muscidae).

The in vivo and in vitro fate of [14C]carbaryl was compared in adult male and female house flies from an insecticide-susceptible (S) strain and a resistant (R) strain with multiple resistance to different classes of insecticides. Cuticular penetration of topically applied carbaryl (0.01 microgram/insect) was very rapid and rates were essentially the same among males and females of both strains. Rates of penetration were dramatically reduced as the concentration of applied carbaryl was increased over a range of 0.01-5.0 micrograms/insect. In vivo and in vitro tests demonstrated that the R strain had an enhanced capability for the metabolic degradation of carbaryl. In evaluations of topical toxicity and in vitro metabolic degradation, coadministration of the metabolic synergists piperonyl butoxide (a microsomal oxidase inhibitor) and S,S,S-tributyl phosphorothioate (DEF, an esterase inhibitor) with carbaryl provided conclusive evidence that microsomal oxidases were the major factor in enhanced metabolism and that hydrolytic enzymes had only a minor effect. Studies of the in vitro inhibition of acetylcholinesterase (AChE) activity by carbaryl demonstrated that there was no difference between males and females of a given strain and that the R strain AChE was considerably less sensitive to inhibition. These tests also indicated that homogenates of brains from the R strain contained more than one form of AChE with different sensitivities to the inhibitor. This information and results of toxicity tests with other insecticides suggest that the R strain is not homozygous in its resistance to carbaryl.     

Insecticide resistance and cross-resistance in the house fly (Diptera: Muscidae)

A house fly strain, ALHF, was collected from a poultry farm in Alabama after a control failure with permethrin, and further selected in the laboratory with permethrin for five generations. The level of resistance to permethrin in ALHF was increased rapidly from an initial 260-fold to 1,800-fold after selection. Incomplete suppression of permethrin resistance by piperonyl butoxide (PBO) and S,S,S,-tributylphosphorotrithioate (DEF) reveals that P450 monooxygenase- and hydrolase-mediated detoxication, and one or more additional mechanisms are involved in resistance to permethrin. The ALHF strain showed a great ability to develop resistance or cross-resistance to different insecticides within and outside the pyrethroid group including some relatively new insecticides. Resistance to beta-cypermethrin, cypermethrin, deltamethrin, and propoxur (2,400-4,200-, 10,000-, and > 290-fold, respectively, compared with a susceptible strain, aabys) in ALHF house flies was partially or mostly suppressed by PBO and DEF, indicating that P450 monooxygenases and hydrolases are involved in resistance to these insecticides. Partial reduction in resistance with PBO and DEF implies that multiresistance mechanisms are responsible for resistance. Fifteen- and more than fourfold resistance and cross-resistance to chlorpyrifos and imidacloprid, respectively, were not effected by PBO or DEF, indicating that P450 monooxygenases and hydrolases are not involved in resistance to these two insecticides. Forty-nine-fold cross-resistance to fipronil was mostly suppressed by PBO and DEF, revealing that monooxygenases are a major mechanism of cross-resistance to fipronil. Multiresistance mechanisms in the ALHF house fly strain, however, do not confer cross-resistance to spinosad, a novel insecticide derived from the bacterium Saccharopolyspora spinosa. Thus, we propose that spinosad be used as a potential insecticide against house fly pests, especially resistant flies.     

甜菜夜蛾对氯氟氰菊酯抗性的表皮穿透机理

用三种方法测定了采自南京江浦的甜菜夜蛾Spodoptera exigua对氯氟氰菊酯的抗性。结果表明,抗性水平的次序为3龄幼虫点滴法（5 499.5倍）和5龄幼虫点滴法（3 973.2倍）＞3龄幼虫浸叶法（1 041.6倍）＞5龄幼虫叶片夹毒法（24.7倍）,因此该品系触杀毒力的抗性水平至少为胃毒毒力LD50的160倍。用¹⁴C标记氯氟氰菊酯测定甜菜夜蛾抗性和敏感品系5龄幼虫表皮穿透率结果表明,处理后8h,抗性品系5龄幼虫的表皮穿透率仅为敏感品系幼虫表皮穿透率的55.5%。证实表皮穿透率的降低是产生抗性的一个重要机理。     

Effects of the synergists piperonyl butoxide and S,S,S-tributyl phosphorotrithioate on propoxur pharmacokinetics in Blattella germanica (Blattodea: Blattellidae).

Effects of the synergists piperonyl butoxide (PBO) and S,S,S-tributyl phosphorotrithioate (DEF) on propoxur pharmacokinetics were examined in the German cockroach, Blattella germanica (L.). Treatment of adult male German cockroaches with the cytochrome P450 monooxygenase inhibitor, PBO, or the esterase inhibitor, DEF, increased propoxur toxicity by 2- and 6.8-fold, respectively, implicating hydrolysis as a major detoxification route of propoxur in the German cockroach. However, significant hydrolytic metabolism could not be demonstrated conclusively in vitro resulting in a conflict between in situ bioassay data and in vitro metabolic studies. In vitro propoxur metabolism with NADPH-fortified microsomes produced at least nine metabolites. Formation of metabolites was NADPH-dependent; no quantifiable metabolism was detected with cytosolic fractions. However, microsomal fractions lacking an NADPH source did produce a low, but detectable, quantity of metabolites (1.6 pmol). PBO inhibited NADPH-dependent propoxur metabolism in a dose-dependent fashion, implicating cytochrome P450 monooxygenases as the enzyme system responsible for the metabolism. Interestingly, DEF also inhibited the NADPH-dependent metabolism of propoxur, albeit to a lower extent. Treatment with PBO or DEF also caused a significant reduction in the cuticular penetration rate of propoxur. The data demonstrate that unanticipated effects are possible with synergists and that caution must be exercised when interpreting synergist results.     

Glutathione S-transferase and insecticide resistance in laboratory strains and field populations of Musca domestica

Glutathione S-transferase (GST) activity of 10 house fly laboratory strains and 21 field populations are described with two substrates, 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB), commonly associated with resistance. Three laboratory strains selected by tetrachlorvinphos, lindane (gamma-HCH) and dimethoate, respectively, had significantly elevated CDNB- and DCNB-GST activities. The multiresistant field population 791a had significantly elevated CDNB- and DCNB-GST activities. Many strains recorded several individuals with high CDNB- and/or DCNB-GST activity and to evaluate these differences, phenotypes were defined by cluster analysis. A phenotype, GST-R, indicating high CDNB- and DCNB-GST activities, was found in 23 of 31 laboratory strains or field populations. GST-R was likely to be involved in gamma-HCH resistance in strain 17e, tetrachlorvinphos resistance in strain 39m2b, and dimethoate resistance in strain 49r2b. The frequency of GST-R in selected laboratory strains and field population correlated with the frequency of house flies surviving the organophosphate azamethiphos either topically applied or by ingestion. There was no significant correlation between GST activity and the toxicity of the organophosphate dimethoate or the pyrethroids bioresmethrin and pyrethrin.     

Decapitation impacting effect of topically applied chlorpyrifos on acetylcholinesterase and general esterases in susceptible and resistant German cockroaches (Dictyoptera: Blattellidae)

The effect of topically applied chlorpyrifos on acetylcholinesterase and other esterases in heads and decapitated bodies of CSMA and Crawford German cockroaches was examined with spectrophotometric enzyme assay and native polyacrylamide gel electrophoresis. The toxicity of chlorpyrifos was greatly reduced in decapitated CSMA male cockroaches with LD50 value 17.1-fold higher than that of normal CSMA cockroaches. Acetylcholinesterase activity from heads was significantly higher in the Crawford compared with the CSMA strain and did not change until 24 h after chlorpyrifos in vivo treatment in both strains. The p-nitrophenyl butyrate (NPB) esterase activities from both heads and decapitated bodies of the resistant Crawford strain were significantly greater than the susceptible CSMA strain. The p-NPB esterase activity was significantly inhibited by chlorpyrifos in vivo treatment, and total p-NPB esterase activity was significantly reduced in decapitated bodies compared with heads of both strains. Native polyacrylamide gel electrophoresis (PAGE) analysis of extracts solubilized with Triton X-100 from heads and decapitated bodies revealed five major esterase bands and an acetylcholinesterase (AChE) band with a high capability of hydrolyzing alpha-naphthyl butyrate and acetylthiocholine, respectively. In the heads of susceptible CSMA male cockroaches, the activity of mobile isozymes d1 and d2 was completely inhibited at 24 h after chlorpyrifos application, and isozyme e was partially inhibited. In contrast, isozymes c1 and c2 from the decapitated bodies of CSMA cockroaches were mostly affected at 24 h after the topical application of chlorpyrifos. The activities of acetylcholinesterase and esterase isozymes a and b from the decapitated body remained uninhibited in both strains. Inhibition of isozymes d1 and d2 seems to be more important in chlorpyrifos intoxication than acetylcholinesterase.     

拟除虫菊酯抗性家蝇的交互抗药性研究

拟除虫菊酯的广泛使用使家蝇普遍产生了抗药性,为有效的控制家蝇的危害,需要了解家蝇对轮换或新杀虫剂的交互抗性状况。作者用点滴法测定了两个实验室汰选的拟除虫菊酯抗性家蝇品系对几种杀虫剂的交互抗性,结果表明:二氯苯醚菊酯和溴氰菊酯之间存在较高程度的交互抗药性;拟除虫菊酯抗性较高的家蝇对作用机制不同的新农药(多杀菌素、氟虫腈)表现较低程度的交互抗性。     

Studies on the resistance to various insecticides of a house fly strain (Diptera: Muscidae) selected with azamethiphos.

A colony of azamethiphos-resistant house flies, Musca domestica (L.), was obtained from Denmark and further selected in the laboratory with azamethiphos for four generations. LD50s for various insecticides were determined and compared with those of a susceptible house fly strain. The selected flies showed cross-resistance to all insecticides evaluated. The flies were highly resistant to most organophosphorus, carbamate, and chlorinated hydrocarbon insecticides except prothiophos, p,p'-DDT, and the pyrethroids. We conclude that the main mechanisms responsible for resistance are presumed to be factors other than acetylcholinesterase sensitivity and nerve sensitivity due to knockdown resistance.     

Enhanced esterase gene expression and activity in a malathion-resistant strain of the tarnished plant bug, Lygus lineolaris

Extensive use of insecticides on cotton in the mid-South has prompted resistance development in the tarnished plant bug, Lygus lineolaris (Palisot de Beauvois). A field population of tarnished plant bugs in Mississippi with 11-fold higher resistance to malathion was used to examine how gene regulation conferred resistance to this organophosphate insecticide. In laboratory bioassays, synergism by the esterase inhibitors S,S,S,-tributylphosphorotrithioate (DEF) and triphenylphosphate (TPP) effectively abolished resistance and increased malathion toxicity by more than 80%. Esterase activities were compared in vitro between malathion susceptible and resistant (selected) strains. More than 6-, 3- and 10-fold higher activities were obtained with the resistant strain using alpha-naphthyl acetate, beta-naphthyl acetate, and p-nitrophenyl acetate, respectively. Up to 95% and 89% of the esterase activity in the susceptible and resistant strains, respectively, was inhibited by 1 mM DEF. Inhibition of esterase activity up to 75% and 85% in the susceptible and resistant strains, respectively, was obtained with 0.03 mM TPP. Esterase activities in field populations increased by up to 5.4-fold during the fall season. The increase was synchronized with movement of the insect into cotton where exposure to pesticides occurred. Esterase cDNA was cloned and sequenced from both malathion susceptible and resistant strains. The 1818-nucleotide cDNA contained a 1710-bp open reading frame coding a 570 amino acid protein which was similar to many insect esterases conferring organophosphate resistance. No amino acid substitution was observed between susceptible and resistant strains, indicating that esterase gene mutation was not involved in resistance development in the resistant strain in Mississippi. Further examination of esterase gene expression levels using quantitative RT-PCR revealed that the resistant strain had a 5.1-fold higher level of esterase mRNA than the susceptible strain. The results of this study indicated that up-regulation of the esterase gene appeared to be related to the development of resistance in the tarnished plant bug.     

Selection and reversion of azamethipos-resistance in a field population of the housefly Musca domestica (Diptera: Muscidae), and the underlying biochemical mechanisms

The organophosphorus insecticide, azamethiphos, is widely used throughout the world to control the housefly, Musca domestica (L.). Since its commercial introduction to Denmark in 1983 for this purpose, we have monitored the toxicity of azamethiphos to housefly populations at livestock farms throughout the country and carried out regular field studies. The findings of our field studies, which have revealed a strong potential for resistance development, have been born out by regular surveys showing that resistance has increased in recent years. Through the analysis of a field derived laboratory strain, we have implicated oxidative and hydrolytic mechanisms together with altered acetylcholinesterase in this resistance. Our field and laboratory studies have also indicated that resistance is relatively unstable, and can revert in the absence of selection. The implications of our findings for the continued efficacy of azamethiphos are discussed.     

Synergism of chlorpyrifos against the German cockroach, Blattella germanica

Of fifteen compounds tested as synergists for chlorpyrifos against susceptible and resistant strains of Blattella germanica, the German cockroach, eleven were active against the resistant strain but only seven were synergistic against the susceptible strain. Overall, the most effective synergist was S,S,S-tributyl phosphorotrithioate (DEF) followed by phenyl saligenin cyclic phosphonate (PSCP) and two substituted N,N-dimethylcarbamates: SK-102 and SK-37. The most effective synergist for overcoming chlorpyrifos resistance was SK-37 which reduced the resistance ratio by 3.2-fold. Four other synergists which reduced chlorpyrifos resistance, in order of their effectiveness, were: SK-102 > PSCP > DEF > tridiphane. The potential usefulness of these synergists for German cockroach control is discussed.     

Thiamethoxam Resistance in the House Fly,Musca domestica L.: Current Status,Resistance Selection,Cross-Resistance Potential and Possible Biochemical Mechanisms

The house fly, Musca domestica L., is an important ectoparasite with the ability to develop resistance to insecticides used for their control. Thiamethoxam, a neonicotinoid, is a relatively new insecticide and effectively used against house flies with a few reports of resistance around the globe. To understand the status of resistance to thiamethoxam, eight adult house fly strains were evaluated under laboratory conditions. In addition, to assess the risks of resistance development, cross-resistance potential and possible biochemical mechanisms, a field strain of house flies was selected with thiamethoxam in the laboratory. The results revealed that the field strains showed varying level of resistance to thiamethoxam with resistance ratios (RR) at LC₅₀ ranged from 7.66-20.13 folds. Continuous selection of the field strain (Thia-SEL) for five generations increased the RR from initial 7.66 fold to 33.59 fold. However, resistance declined significantly when the Thia-SEL strain reared for the next five generations without exposure to thiamethoxam. Compared to the laboratory susceptible reference strain (Lab-susceptible), the Thia-SEL strain showed cross-resistance to imidacloprid. Synergism tests revealed that S,S,S-tributylphosphorotrithioate (DEF) and piperonyl butoxide (PBO) produced synergism of thiamethoxam effects in the Thia-SEL strain (2.94 and 5.00 fold, respectively). In addition, biochemical analyses revealed that the activities of carboxylesterase (CarE) and mixed function oxidase (MFO) in the Thia-SEL strain were significantly higher than the Lab-susceptible strain. It seems that metabolic detoxification by CarE and MFO was a major mechanism for thiamethoxam resistance in the Thia-SEL strain of house flies. The results could be helpful in the future to develop an improved control strategy against house flies.     

斜纹夜蛾抗性种群中酶抑制剂对杀虫剂的增效作用（英文）

采用浸液生测法研究了斜纹夜蛾Spodoptera litura巴基斯坦抗性种群中酶抑制剂［胡椒基丁醚（PBO）和脱叶膦（DEF)］对丙溴磷、灭多威、硫双灭多威、氯氰菊酯、氯氟氰菊酯、联苯菊酯、茚虫威和多杀菌素等杀虫剂的增效作用。结果表明：PPO和DEF对氨基甲酸酯杀虫剂灭多威和硫双灭多威均具有增效作用,但对有机磷杀虫剂丙溴磷不具有增效作用。两种抑制剂对氯氰菊酯均产生增效作用,但对联苯菊酯没有增效作用。PPO 和DEF增加了氯氟氰菊酯对Multan种群的毒性,但没有增加其对Mailsi种群的毒性。DEF对多杀菌素具有增效作用,但PBO对其没有增效作用。PBO和DEF对氨基甲酸酯杀虫剂、拟除虫菊酯杀虫剂、茚虫威和多杀菌素具有明显的增效作用,这说明细胞色素P450单加氧酶和酯酶的解毒作用至少部分参与了斜纹夜蛾对这些杀虫剂的抗性过程。不过,两种增效剂对杀虫剂增效作用范围有限,暗示对于斜纹夜蛾巴基斯坦种群而言,其他的机制（如靶位点不敏感、表皮穿透作用降低）可能是更重要的抗性机制。     

Factors affecting the toxicity of diazinon to Musca domestica L

Processes affecting the toxicity of diazinon to a susceptible and a resistant strain of houseflies were examined. More evidence was obtained to show that slower penetration of diazinon through the integument of resistant flies is a cause of resistance. Small amounts of two decomposition products were found in both strains. The decomposition mechanisms, in these strains were differently distributed and, although detoxication of diazinon in the two strains is quantitatively similar and small, it may contribute to resistance. Traces of diazoxon were detected when diazinon was incubated with tissue extracts of either strain. Tissue extracts of resistant, but not of susceptible, flies decomposed significant amounts of diazinon in 1 hr. and the ability to decompose diazoxon seems to be an important cause of resistance. Tissues of both strains sorbed diazinon from aqueous solution similarly; the quantities sorbed were large and suggest that sorption may increase the amount of poison needed inside the insects to kill, by between five and forty times.     

Strain-differences and inducibility of microsomal oxidative enzymes in Drosophila melanogaster flies

Some basic characteristics of the enzyme system involved in the oxidative metabolism of xenobiotic compounds were investigated in Drosophila melanogaster flies. Attention was focussed on (1) the normal levels of these enzymes and their activities in whole flies, in different parts of the fly's body and in different sexes, (2) the changes in levels and activities of the enzymes elicited by pretreatment of the flies with known enzyme inducers and (3) differences between strains.Four commonly used wild-type (WT) strains, three insecticide resistant strains (IR) and one white-eyed mutant strain were employed. Except in those experiments on sex differences and in spatial distribution in the fly's body of the enzymatic activities, microsomes were isolated from whole-body homogenates of mixtures of female and male flies. Microsomal cytochrome P-450, benzo[a]pyrene (BP) hydroxylation, p-nitroanisole (pNA) demethylation and aminopyrine (AP) demethylation were measured in control flies and in flies pretreated with Aroclor 1254 (AC), phenobarbital (PB) or butylated hydroxytoluene (BHT).In flies of the WT strain Berlin-K, there were no significant differences in BP hydroxylation activity and its inducibility between the two sexes. In males, inducibility of BP hydroxylation activity was similar in the head, thorax and abdomen, but significantly lower in testis. Considerable differences in some enzyme activities were found between the strains. pNA demethylation and AP demethylation were substantially higher in all IR strains, while no correlation could be found between their increased insecticide resistance and BP hydroxylating capacity or cytochrome P-450 content of the microsomes.Response to enzyme inducing compounds was found to be strain-dependent. PB proved to be a more efficient inducer of BP hydroxylation than AC, which does induce pNA demethylation. BHT has inducing properties that are intermediate between PB and AC. IR strain Hikone-R turned out to be an exception, possessing very low BP hydroxylating capacity and a low degree of inducibility of mixed-function oxidase activities. Differential temperature dependence was found for BP hydroxylation as compared with pNA demethylation. While BP hydroxylation was doubled when raising the temperature from 25°C to 35°C, pNA demethylation was reduced by 50%.     

Separation of multiple forms of acidic glutathione S-transferase isozymes in a susceptible and a resistant strain of house fly,Musca domestica (L.)

The acidic glutathione S-transferases from a CSMA (susceptible) strain and a Cornell-R (resistant) strain of houseflies were purified and separated utilizing affinity chromatography followed by chromatofocusing. Nine fractions were isolated from each house fly strain. Fraction 1 had the highest 1-chloro-2,4-dinitrobenzene vs. 1,2-dichloro-4-nitrobenzene ratio (CDNB/DCNB ratio) in both strains and the ratio of all the other fractions tended to decrease as the isoelectrical points decreased except for fractions 4 and 9. Most fractions from the CSMA strain had higher CDNB conjugation activities than the fractions from the Cornell-R strain, but all the fractions from the CSMA strain had lower DCNB conjugation activities than fractions from the Cornell-R strain. Steady-state kinetics of all the fractions were examined. The Km values obtained from both strains ranged from 0.36 to 1.12 mM, while the Vmax value ranged from 3.0 to 32.6 μmol/min/mg. In the 100,000 g supernatant, the CDNB specific activities in the CSMA strain was about 1/3 of the activity in the Cornell-R strain but it was about 1.5-fold following affinity chromatography. The specific activity for DCNB measured in the CSMA strain was only 1/5 of the activities of the Cornell-R strain in the 100,000 g supernatant, but was about the same after affinity chromatography. The difference was due to the selectivity of the affinity column used in the current study. © 1995 Wiley-Liss, Inc.     

Adult specific expression and induction of cytochrome P450tpr in house flies

Cytochrome P450_tpr is a xenobiotic metabolizing P450 that is found in house flies (Musca domestica). To better understand the regulation of cytochrome P450_tpr, the effects of 21 potential monooxygenase inducers were examined for their ability to induce total cytochromes P450 and cytochrome P450_tpr levels in adult flies. Six compounds caused induction of total cytochromes P450 per mg protein in adult susceptible (CS) house flies: ethanol (1.6-fold), phenobarbital in food (1.5-fold) or water (1.5-fold), naphthalene (1.3-fold), DDT (1.3-fold), xanthotoxin (1.4-fold), and α-pinene (1.2-fold). Six compounds were found to be inducers of cytochrome P450_tpr: piperonyl butoxide in food (1.9-fold), phenobarbital in food (1.4-fold) and water (3.4-fold), clofibrate (1.3-fold), xanthotoxin (1.3-fold), methohexital (1.3-fold), and isosafrole (1.3-fold). Comparison of our results with house fly P450 6A1 indicates that there are specific inducers for each of these individual P450s as well as compounds that induce both P450s. Total P450s were inducible by PB in CS house fly larvae, but not in LPR larvae. Immunoblotting revealed no detectable P450_tpr in control or PB-treated larvae in either strain. Thus, although total P450s are inducible in the susceptible strain larvae, P450_tpr does not appear to be normally present or inducible with PB in larvae of either strain. Northern blots of phenobarbital (in water) treated CS flies indicated that there was a 4.2-fold increase in the P450_tpr (i.e., CYP6D1) mRNA levels over the untreated flies. In the multiresistant LPR strain there was no apparent induction of CYP6D1 mRNA by phenobarbital. Following phenobarbital induction, the level of CYP6D1 mRNA in the CS strain was about half of the level in the LPR strain. © 1996 Wiley-Liss, Inc.