Metabolic insecticide defenses in the boll weevil compared to those in a resistance-prone species

Activities of cytochrome P-450-dependent microsomal monooxygenases (PMSO) are up to 46-fold higher in insecticide-susceptible fifth instar tobacco budworm larvae than in adult boll weevils. Glutathione transferase and esterase activities are likewise higher in susceptible tobacco budworm larvae than in adult boll weevils, the life stages of the two species that are exposed to selection pressure from insecticide treatments. Good metabolic defenses may facilitate insecticide resistance development in the tobacco budworm. Differences in ecological, biological, and other biochemical factors, not quantified in this study, are also likely to contribute to the observed differences in resistance development in the two species. PSMO activities as well as glutathione transferase and esterase activities are 3- to 11-fold higher in boll weevil larvae than in adults. Exposure of the life stage with the weakest metabolic defenses to insecticide selection pressure in the boll weevil may contribute to its relative lack of resistance development.     

Biochemical characteristics of insecticide resistance in the fall armyworm, Spodoptera frugiperda (J.E. Smith)

A strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith), collected from corn in Citra, Florida, showed high resistance to carbaryl (562-fold) and methyl parathion (354-fold). Biochemical studies revealed that various detoxification enzyme activities were higher in the field strain than in the susceptible strain. In larval midguts, activities of microsomal oxidases (epoxidases, hydroxylase, sulfoxidase, N-demethylase, and O-demethylase) and hydrolases (general esterase, carboxylesterase, β-glucosidase) were 1.2- to 1.9-fold higher in the field strain than in the susceptible strain. In larval fat bodies, various activities of microsomal oxidases (epoxidases, hydroxylase, N-demethylase, O-demethylases, and S-demethylase), glutathione S-transferases (CDNB, DCNB, and p-nitrophenyl acetate conjugation), hydrolases (general esterase, carboxylesterase, β-glucosidase, and carboxylamidase) and reductases (juglone reductase and cytochrome c reductase) were 1.3- to 7.7-fold higher in the field strain than in the susceptible strain. Cytochrome P450 level was 2.5-fold higher in the field strain than in the susceptible strain. In adult abdomens, their detoxification enzyme activities were generally lower than those in larval midguts or fat bodies; this is especially true when microsomal oxidases are considered. However, activities of microsomal oxidases (S-demethylase), hydrolases (general esterase and permethrin esterase) and reductases (juglone reductase and cytochrome c reductase) were 1.5- to 3.0-fold higher in the field strain than in the susceptible strain. Levels of cytochrome P450 and cytochrome b₅ were 2.1 and 1.9-fold higher, respectively, in the field strain than in the susceptible strain. In addition, acetylcholinesterase from the field strain was 2- to 85-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, propoxur, carbofuran, bendiocarb, thiodicarb) and organophosphates (methyl paraoxon, paraoxon, dichlorvos), insensitivity being highest toward carbaryl. Kinetics studies showed that the apparent K_m value for acetylcholinesterase from the field strain was 56% of that from the susceptible strain. The results indicated that the insecticide resistance observed in the field strain was due to multiple resistance mechanisms, including increased detoxification of these insecticides by microsomal oxidases, glutathione S-transferases, hydrolases and reductases, and target site insensitivity such as insensitive acetylcholinesterase. Resistance appeared to be correlated better with detoxification enzyme activities in larval fat bodies than in larval midguts, suggesting that the larval fat body is an ideal tissue source for comparing detoxification capability between insecticide-susceptible and -resistant insects.     

Detoxication capacity in the honey bee,Apis mellifera L

Various detoxifying enzymes, including microsomal oxidases, glutathione S-transferases, esterases, epoxide hydrolase, and DDT-dehydrochlorinase, were assayed in adult worker bees (Apis mellifera L.) using midguts as the enzyme source. A cell-free system was used for all enzyme assays, except that microsomal oxidases required intact midgut because of the inhibitor encountered. Midgut microsomal preparations contained mainly cytochrome P-420, the inactive form of cytochrome P-450, which may explain the low microsomal oxidase activity in microsomes. All enzymes studied were active, suggesting that the high susceptibility of honey bees to insecticides is not due to low detoxication capacity. Sublethal exposure of honey bees to various insecticides had no effect on these enzyme activities, with the exception of permethrin which significantly stimulated the glutathione S-transferase, and malathion, which significantly inhibited the α-naphthylacetate esterase and carboxylesterase.     

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.     

Effect of fraxinellone on growth and digestive physiology of Asian corn borer, Ostrinia furnacalis Guenee

A limonoid, fraxinellone significantly reduced the relative growth rate, food consumption rate as well as the efficiency of conversion of ingested food of the Asian corn borer, Ostrinia furnacalis when incorporated into artificial diets at concentration of 10 ppm and above. After being fed with diets that contained fraxinellone for 24 h, the larval midguts of O. furnacalis possess significantly lower activities of α-amylase and non-specific proteases and higher activities of cytochrome P450s. In vitro, the compound did not inhibit the activities of α-amylase and non-specific proteases extracted from the larval midguts. The modes of action of fraxinellone against the insect were discussed.     

Host plant induction of microsomal monooxygenase activity in relation to diazinon metabolism and toxicity in larvae of the tobacco budworm Heliothis virescens (F.)

The effect of the wild tomato, Lycopersican hirsutam F. glabratam (accession PI 134417), on susceptibility to and metabolism of diazinon, 0,0-diethyl-0-(2-isopropyl-4-methyl-6-pyrimidinyl) phosphorothioate, in larvae of the tobacco budworm, Heliothis virescans F., was studied. The larvae were over 4-fold more tolerant to diazinon toxicity when fed on leaves of wild tomato than when fed on an artificial diet. Diazinon injected into fifth instar larvae is converted into two chloroform-soluble and five water-soluble metabolites. Degradation of diazinon was faster in larvae fed tomato leaves (88.1%) than in larvae fed on the artificial diet (68.4%). Some oxon (13.0%) was detected in the latter case but none in larvae fed tomato leaves. The major metabolite was hydroxypyrimidine and its formation was higher (73.2%) in larvae fed tomato leaves than in larvae fed the artificial diet (49.2%). In vitro studies revealed that both diazinon and its oxon were metabolized primarily by the microsomal cytochrome P-450-dependent monooxygenase system which was induced 2.5- to 3.7-fold by feeding on tomato leaves. It was concluded that diazinon was degraded in H. virescens larvae through desulfuration, hydroxylation of the ring-alkyl side chain, and oxidative dearylation reactions, all of which were increased by varying amounts after feeding on tomato leaves. Treatment of the larvae with 2-tridecanone, a naturally occurring toxin in tomato leaves, resulted in increased tolerance to diazinon and increased in vitro degradation of diazinon and its oxon, the induction being dependent on the magnitude of 2-tridecanone treatment. The microsomes of tomato fed larvae had a 1.5- to 2.1-fold higher concentration of cytochrome P-450, accompanied by a 1–2 nm shift in the λmax of the cytochrome P-450 carbon monoxide complex.     

Blood meal and cytochrome P-450 monooxygenases in the northern house mosquito,Culex pipiens

Conditions for the measurement of aldrin epoxidation by microsomes prepared from abdominal tissues (fat body + integument) of adult female Culex pipiens were characterized. The enzyme activity had a pH optimum of 7.2 and an apparent K_m of 3.4 μM. Aldrin epoxidation and NADPH-cytochrome c reductase had similar patterns of inhibition by a rabbit antiserum to house fly NADPH-cytochrome P-450 reductase, thus implicating cytochrome P-450 monooxygenase(s) in the epoxidation of aldrin. Low (71 pmol/mg protein) levels of cytochrome P-450 were detected in abdominal tissue microsomes. In non-blood-fed insects, aldrin epoxidation and NADPH-cytochrome c reductase activities did not change between Day 1 and Day 12 after adult emergence, except for a small peak on Day 2. In insects fed a blood meal on Day 6 after emergence both activities increased (two- to threefold) to a plateau maintained between 2 and 4 days after the blood meal. Aldrin epoxidation and NADPH-cytochrome c reductase activities decreased to normal values between 4 and 6 days after the blood meal.     

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。     

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.     

Temperature and diet modulate cytochrome P-450 activities in southern armyworm,Spodoptera eridania (Cramer), caterpillars

Larvae of the southern armyworm, Spodoptera eridania (Cramer), grew well in the 15–30°C temperature range. Pupae survived poorly at 15°C but moths emerged from 85% of the pupae at 30°C. The time for development was prolonged at 15°C and larvae grew significantly bigger than at 30°C. Cytochrome P-450 content, cytochrome P-450 reductase, p-chloro N-methylaniline N-demethylation, methoxyresorufin 0-demethylation, and aldrin epoxidation activities were higher at 15°C than at 30°C. All cytochrome P-450 activities were more inducible by dietary pentamethylbenzene at 30°C than at 15°C. High cytochrome P-450-catalyzed activities were associated with increases in microsomal protein rather than with changes in membrane lipid or phospholipid content. Phosphatidylcholine was the major midgut membrane phospholipid. There was only a tendency towards increased unsaturation of the phospholipid fatty acyl moieties and lowered membrane phase transition temperature in cold-adapted larvae. Acute oral carbaryl toxicity was generally inversely correlated with cytochrome P-450 catalyzed activities. Carbaryl toxicity was decreased about 10-fold by pentamethylbenzene induction and about 3-fold by the lower acclimatization temperature.     

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.     

Prochloraz,a potent inducer of the microsomal cytochrome P-450 system

Prochloraz (N-propyl-N-[2-(2,4,6-trichlorophenoxy)ethyl]-imidazole-1-carboxamide), a recently developed agricultural fungicide, is a potent inducer of microsomal enzymes. Rats fed 7 days with a prochloraz-contaminated diet (2500 ppm) showed an increase in hepatic cytochrome P-450, cytochrome b₅, and microsomal protein level; aniline hydroxylase, 7-ethoxycoumarin dealkylase, 7-ethoxyresorufin dealkylase, NADPH-cytochrome c reductase, and epoxide hydrolase were significantly induced. At a lower dose (100 ppm), only an increase in cytochrome P-450 and 7-ethoxyresorufin dealkylase was noticed. As shown with aniline hydroxylase and 7-ethoxycoumarin dealkylase, prochloraz is also a potent inhibitor of drug-metabolizing enzymes. The interaction of prochloraz with hepatic microsomal fraction from rat liver was also studied. Prochloraz binds to oxidized cytochrome P-450 to produce a type II spectral change; the compound also binds to reduced cytochrome P-450. The binding of some ligands (7-ethoxycoumarin, n-octylamine, aniline, and imidazole) to oxidized cytochrome P-450 was determined after induction by prochloraz. Japanese quails (Coturnix coturnix) fed 7 days with a prochloraz-contaminated diet (2000 ppm) showed a dramatic increase in liver weight (2.5-fold) and both hepatic and duodenal cytochrome P-450 (9- and 12-fold, respectively).     

Induction of the mixed-function oxidase system in the liver of the barn owl Tyto alba by PCBs

Injection of 30 mg/kg body wt of polychlorinated biphenyl (Aroclor 1254) into liver parenchymal tissue of nestling and adult barn owls Tyto alba resulted in increases in the level of cytochrome P-450. Concomitantly, there were increases in catalytic activity of the microsomal enzyme system as measured by aldrin epoxidation and aminopyrine N-demethylation. However, the ratio $\text{455}\text{430}\text{nm}$ in the ethylisocyanide-difference spectrum remained unchanged. Of particular interest is the sudden drop in the level and catalytic activity of cytochrome P-450 in nestling owls at age 40 days. Treatment with Aroclor 1254 produced small hemorrhages in the liver of nestling owls and the liver appeared much enlarged (hepatomegaly), indicating a toxic effect and resulting in little induction of microsomal enzymes. In adult owls the inductive effect was much greater. Aroclor 1254 produced a spectral shift in the cytochrome P-450-difference spectrum from 450 to 448 nm and in the ethylisocyanide-difference spectrum from 455 to 453 nm and from 430 to 427 nm.     

Hepatic microsomal oxidative metabolism of pesticides and other xenobiotics in pregnant CD1 mice

Pregnancy-related changes in oxidative metabolism of several xenobiotics including pesticides were examined in the hepatic microsomes of CD₁ mice. The effect of pregnancy on hepatic microsomal cytochrome P-450-catalyzed substrate oxidation was found to be dependent upon the type of reaction examined. Not all substrates undergoing the same reaction showed identical changes during pregnancy. Those enzyme activities which exhibited a decline in specific activity during pregnancy generally exhibited no change in total hepatic capacity. Enzymes posting no change in specific activity throughout gestation generally showed large increases in total hepatic activity. Phorate S-oxidation was catalyzed by both microsomal flavin-containing monooxygenase (MFMO) and cytochrome P-450. Moreover, there was no pregnancy-related change in either MFMO or total enzymatic (MFMO plus cytochrome P-450) phorate S-oxidation.     

In-vivo effects of 2,4-D and atrazine on cytochrome P-450 and insecticide toxicity in southern armyworm (Spodoptera eridania) larvae

After feeding 2,4-D or atrazine in a diet to southern armyworm (Spodoptera eridania Cram.) larvae for three days, the effect on total content and activities of cytochrome P450 and on insecticide toxicity were determined. Both 2,4-D and atrazine induced cytochrome P450-catalyzed aldrin epoxidation (AE) and methoxyresorufin O-demethylatin (MROD). The 2,4-D was a more potent inducer for total cytochrome P450 content, whereas atrazine disproportionately increased AE. Both compounds increased MROD significantly. The apparent kinetic characteristics of AE indicates that 2,4-D and atrazine induced similar P450 isozymes (K_m 8.78 and 7.80 μM, respectively), which may differ from the constitutive isozyme (K_m 3.14 μM). The 2,4-D-induced cytochrome P450 contributed to decreased carbaryl and permethrin toxicity, whereas the atrazine-induced cytochrome P450 caused decreased parathion and permethrin toxicity. The carbaryl toxicity correlated directly with 2,4-D-induced total P450 content and activities but not with atrazine-induced changes. The 2,4-D and atrazine also induced nonspecific esterase activity which may contribute to permethrin detoxification.     

Morphological basis of resistance to spotted bollworm, Earias vittella (Fabricius) in Asiatic cotton

Ten genotypes of Asiatic cotton, Gossypium arboreum, viz. C-353, G 17, G 33, AC-3545, LD 491, LD 575, B 11 A, SAG-BLY (Red), Sargosi and LD 694, were screened under epiphytotic conditions against spotted bollworm, Earias vittella, at the cotton farm of the Department of Plant Breeding and Genetics, Punjab Agricultural University, Ludhiana, India, and their morphological basis of resistance to spotted bollworm was studied. The plant characters examined were plant color, leaf shape, size and shape of bract, density of gossypol glands, nectaries, hair density and hair length. Hair density was positive (r?=?0.81, P?=?0.005) and significantly correlated with the number of eggs laid by E. vittella, whereas a significantly negative (r?=?–0.66, P?=?0.038) correlation was obtained with gossypol glands. Similarly, correlation of larval population with hair density was significantly positive (r?=?0.68, P?=?0.030) and gossypol glands were negative (r?=?–0.63, P?=?0.050) and significantly correlated with larval population. The R² values, computed together for multiple regression analysis, indicated that the morphological plant traits, viz., gossypol glands and hair density, cumulatively contributed 67% and 50% variability in the egg and larval population, respectively. Genotype B 11 A, with the lowest egg and larval population, had a high number of gossypol glands, low hair density and smaller bracts and proved to be the best, followed by LD 491. Genotypes with low hair density, smaller bracts and a greater number of gossypol glands are desirable for low oviposition and larval population of E. vittella. These characteristics should be taken into consideration when planning a resistance breeding program against spotted bollworm in arboreum cotton.     

The effects of dicofol on induction of hepatic microsomal metabolism in rats

In a comparative study, the induction effects of dicofol, technical Kelthane, and DDT on hepatic microsomal and cytosolic enzyme activities in rats were compared with those effects produced by phenobarbital (PhB) and β-naphthoflavone (BNF). Male rats (ca. 250 g) were injected (ip) for 4 consecutive days with 1.0 ml of vehicle containing either dicofol (1.5, 15.0, 29.5, or 59.0 mM, Kelthane (dicofol content equal to 29.5 or 59.0 mM), DDT (59.0 mM), or BNF (36.7 mM). Liver weights, microsomal protein, and cytochrome P-450 concentrations and microsomal and cytosolic enzyme specific activities were measured. Dicofol produced dose-related increases in all of the parameters measured except liver weight and cytosolic epoxide hydrolase activity. At a concentration of 59.0 mM, dicofol increased the concentrations of microsomal protein (1.7-fold) and cytochrome P-450 (2.9-fold), and the specific activities of cytochrome c reductase (1.6-fold), ethoxycoumarin O-deethylase (2.3-fold), aminopyrine N-demethylase (3.0-fold), microsomal epoxide hydrolase (2.6-fold), and glutathione S-transferase (2.9-fold). The induction potency of dicofol was equivalent to Kelthane, DDT, and PhB at equimolar (59.0 mM) concentrations of chemical.     

Inhibition of neurotoxic esterase and acetylcholinesterase by organophosphorus compounds in selected ectothermic vertebrates

The sensitivity of brain acetylcholinesterase and neurotoxic esterase to inhibition by several organophosphorus compounds was studied in selected ectothermic vertebrates. These enzymes are associated with organophosphorus compound acute and delayed toxicity, respectively. In addition, the susceptibility of several of these species to delayed neurotoxicity induced by organophosphorus compounds was studied. Larvae of the gray treefrog, Southern leopard frog, and narrow-mouthed toad were exposed dermally to tri-o-tolyl phosphate or phenyl saliginen cyclic phosphate (PSCP); no symptoms of delayed neurotoxicity were observed in any of these animals up to 2 weeks after metamorphosis. No symptoms of delayed neurotoxicity were seen in juvenile bullfrogs exposed to multiple ip doses of PSCP. The specific activity of neurotoxic esterase was highest in the larval bullfrog, with juvenile channel catfish and adult mosquitofish demonstrating intermediate levels. The larval Southern leopard frog, adult Northern leopard frog, juvenile green treefrog, and adult marine toad exhibited extremely low activities. The specific activity of acetylcholinesterase was highest in the juvenile channel catfish. Neurotoxic esterase in the larval bullfrog was more sensitive to organophosphate inhibition than that in either fish. PSCP was a more potent neurotoxic esterase inhibitor than leptophos-oxon. The juvenile channel catfish had the acetylcholinesterase most sensitive to organophosphate inhibition. Under the conditions tested, no evidence of in vivo sensitivity to the organophosphate-induced delayed neurotoxicity phenomenon was observed.     

Cytochrome P-450 in insects: 4. Reconstitution of cytochrome P-450-dependent monooxygenase activity in the house fly

Two cytochrome P-450-containing fractions were isolated from detergent-solubilized house fly microsomes by hydrophobic chromatography on a tryptamine-Sepharose gel. These fractions (designated P-450-1 and P-450-2) were distinctive in their spectral characteristics and in their profiles following electrophoresis in the presence of sodium dodecyl sulfate. Both fractions exhibited NADPH-dependent epoxidase activity when reconstituted with purified house fly cytochrome P-450 reductase and phospholipid. The aldrin epoxidase activity of fraction P-450-1 was twice that of P-450-2 even though heptachlor epoxidase activity of the fractions was equivalent. O-Demethylase activity with 7-methoxy-4-methylcoumarin was detectable only in the P-450-2 fraction.