Fipronil metabolism,oxidative sulfone formation and toxicity among organophosphate‐ and carbamate‐resistant and susceptible western corn rootworm populations

Fipronil toxicity and metabolism were studied in two insecticide‐resistant, and one susceptible western corn rootworm (Diabrotica virgifera virgifera, LeConte) populations. Toxicity was evaluated by exposure to surface residues and by topical application. Surface residue bioassays indicated no differences in fipronil susceptibility among the three populations. Topical bioassays were used to study the relative toxicity of fipronil, fipronil + the mono‐oxygenase inhibitor piperonyl butoxide, and fipronil's oxidative sulfone metabolite in two populations (one resistant with elevated mono‐oxygenase activity). Fipronil and fipronil‐sulfone exhibited similar toxicity and application of piperonyl butoxide prior to fipronil resulted in marginal effects on toxicity. Metabolism of [¹⁴C]fipronil was evaluated in vivo and in vitro in the three rootworm populations. In vivo studies indicated the dominant pathway in all populations to be formation of the oxidative sulfone metabolite. Much lower quantities of polar metabolites were also identified. In vitro studies were performed using sub‐cellular protein fractions (microsomal and cytosolic), and glutathione‐agarose purified glutathione‐S‐transferase. Oxidative sulfone formation occurred almost exclusively in in vitro microsomal reactions and was increased in the resistant populations. Highly polar metabolites were formed exclusively in in vitro cytosolic reactions. In vitro reactions performed with purified, cytosolic glutathione‐S‐transferase (MW = 27 kDa) did not result in sulfone formation, although three additional polar metabolites not initially detectable in crude cytosolic reactions were detected. Metabolism results indicate both cytochromes P450 and glutathione‐S‐transferases are important to fipronil metabolism in the western corn rootworm and that toxic sulfone formation by P450 does not affect net toxicity. © 2000 Society of Chemical Industry     

Effects of the synergist S,S,S-tributyl phosphorotrithioate on indoxacarb toxicity and metabolism in the European corn borer, Ostrinia nubilalis (Hübner)

The toxicity of the oxadiazine insecticide indoxacarb to the European corn borer, Ostrinia nubilalis (Hübner) (Lepidoptera: Crambidae), was evaluated in the presence and absence of S,S,S-tributyl phosphorotrithioate (DEF), an inhibitor of hydrolytic metabolism. Bioassays involving topical application of different concentrations of indoxacarb to third instars of a susceptible O. nubilalis laboratory strain were performed, and in vitro metabolism experiments involving [¹⁴C] indoxacarb were examined to determine the role of hydrolytic metabolism in indoxacarb activation. Indoxacarb toxicity decreased in the presence of DEF indicating antagonism of toxicity. Results of in vivo and in vitro inhibition experiments indicated a reduction of indoxacarb activation and formation of the hydrolytic metabolite. These results are consistent with the proposed mechanism of hydrolytic activation for this compound.     

Disposition and metabolism of [14C]chlorpyrifos in the black imported fire ant, Solenopsis richteri Forel

The short-term disposition and metabolism of topically administered [¹⁴C]chlorpyrifos was assessed in the black imported fire ant (Solenopsis richteri Forel) in the presence and absence of the mixed-function oxidase inhibitor piperonyl butoxide. Chlorpyrifos is readily absorbed into an internal organosoluble fraction which was quickly converted into a water-soluble fraction. The radioactivity was slowly excreted over a 24-hr period. Piperonyl butoxide slowed the conversion of the internal organosoluble radioactivity to the water-soluble fraction. Thin-layer chromatography indicated that piperonyl butoxide slowed the conversion of chlorpyrifos to material remaining at the origin, presumably water-soluble metabolites. The results of acid hydrolysis studies indicated that the water-soluble radioactivity was comprised mainly of conjugates. Although very little chlorpyrifos oxon was recovered in the metabolism experiments, in vitro studies on fire and head homogenates showed the compound to be an extremely potent anticholinesterase, with an I₅₀ of 4.6 × 10^?10M, while a major metabolite, 3,5,6-trichloropyridinol, was an ineffective acetylcholinesterase inhibitor.     

In vivo metabolic fate of [14C]-acetamiprid in six biological compartments of the honeybee, Apis mellifera L

The in vivo metabolism of acetamiprid was studied in the honeybee, Apis mellifera L. The distribution of acetamiprid and its metabolites was monitored over a 72-h period in six biological compartments: head, thorax, abdomen, haemolymph, midgut and rectum. Honeybees were treated orally with 100 microg [14C]-acetamiprid kg(-1) bee, a dose which is about 1500 times lower than the median lethal dose. After 72 h, only 40% of the total radioactivity was eliminated, suggesting that acetamiprid and its metabolites tended to persist in the honeybee. Acetamiprid was rapidly distributed in all compartments and metabolized. Just after administration, radioactivity was mainly localized in the abdomen and subsequently in the rectum. After 72 h, the maximum amount of radioactivity (about 20% of the ingested dose) was detected again in the abdomen, whereas the lowest level of total radioactivity was detected in the haemolymph. Radioactivity in the head did not exceed 7.6% of total ingested radioactivity. More than 50% of acetamiprid was metabolised in less than 30 min, indicating a very short half-life for the compound. During the first hours, acetamiprid was mainly detected in nicotinic acetylcholine receptor-rich tissues: abdomen, thorax and head. Of the seven metabolites detected, the major ones were 6-choronicotinic acid and an unknown metabolite called U1, which was present mainly in the rectum, the thorax and the head. Our results indicate that the low toxicity of acetamiprid may reflect its rapid metabolism.     

Utilization of common ditch vegetation in the reduction of fipronil and its sulfone metabolite

BACKGROUND: Fipronil, a phenylpyrazole insecticide, and its oxidative sulfone metabolite are two potential pollutants from treated rice and cotton production. A consequence of these pollutants occurring in surface runoff is degradation of downstream aquatic ecosystems. Utilization of primary intercept drainage ditches as management practices to reduce fipronil concentrations and loads has not been examined. This study used ditch mesocosms planted with monospecific stands of common emergent wetland vegetation to determine if certain plant species were more proficient in fipronil mitigation. RESULTS: Three replicates of four plant species were compared against a non-vegetated control to determine differences in water column outflow concentrations (microg L(-1)) and loads (microg). There were no significant differences between vegetated and control treatments in outflow concentrations (F = 0.35, P = 0.836) and loads (F = 0.35, P = 0.836). The range of fipronil reduction was 28-45% for both concentration and load. Unlike fipronil, fipronil sulfone concentrations and load increased by 96-328%. CONCLUSION: The increase in fipronil sulfone was hypothesized as a direct consequence of oxidation of fipronil within each mesocosm. The type of ditch vegetation had no effect on fipronil reduction. Future research needs to examine initial concentrations and hydraulic retention times to examine potential changes in reduction capacities.     

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.     

亚洲玉米螟幼虫膜结合海藻糖酶基因RNAi效应

海藻糖是昆虫的血糖,为昆虫提供能量;海藻糖酶催化海藻糖分解为葡萄糖,是几丁质生物合成的原料。围食膜（peritrophic membranes,PMs）是昆虫消化道特有结构,对昆虫消化食物、保护肠道表皮细胞具有重要作用;几丁质是PMs的重要组分。海藻糖酶（trehalase,Tre）和几丁质合成酶（chitin synthase,CHS）是几丁质合成途径的第1个和最后1个酶。本研究通过饲喂亚洲玉米螟（Orstrinia furnacalis,Asian corn borer,ACB）膜结合海藻糖酶（OfMT）基因特异的干扰dsRNA,研究RNAi对ACB幼虫中肠CHSB（OfCHSB）基因表达及幼虫发育的影响。发现处理48 h后,OfMT基因和OfCHSB基因表达量分别下降了52%和53%,幼虫发育迟缓。通过对处理及对照ACB幼虫中肠石蜡切片进行苏木精-伊红染色（hematoxylin-ethanol,HE染色）和几丁质标记,发现血腔内脂肪体组织减小、中肠围食膜组织中几丁质含量减少。推测OfMT基因有可能成为ACB的生物防治的靶标基因。     

Examination of bound (non-extractable) residues of MCPA and flamprop in wheat straw

The long term metabolism of [¹⁴C]MCPA and [¹⁴C]flamprop in wheat (Triticum aestivum) straw was found to involve incorporation of radioactivity as residues that were insoluble in acetone+water (1+1 by volume). A chemical and an enzymic solubilisation procedure were critically evaluated in attempts to release these residues for further examination. The chemical procedure resulted in complete solubilisation of all the radioactivity of both compounds in association with more than one cell wall fraction. However, routine quantitative analysis was found to be difficult for some fractions. Furthermore, the extracts did not appear to be suitable for investigation of the nature of the binding with the plant constituents. None of the enzymes employed in the enzymic procedures released significant amounts of the residues insoluble in the aqueous acetone. Despite these problems, the residues of MCPA that were insoluble in aqueous acetone were found to contain both the parent MCPA and its major metabolite 4-chloro-α-hydroxy-o-tolyloxyacetic acid.     

氟虫腈对二化螟生长发育的影响及对解毒酶的诱导效应

研究了氟虫腈对不同虫态二化螟Chilo suppressalis的作用特点、持续毒性及对解毒酶的诱导效应。结果表明:氟虫腈对二化螟幼虫具有很强的毒杀作用,且毒杀作用随幼虫龄期的增长而逐渐减弱;对二化螟蛹的羽化具有较低的抑制作用;对卵孵化无抑制作用。亚致死剂量氟虫腈能引起幼虫持续死亡,降低存活试虫体重增长率和蛹重。用亚致死剂量氟虫腈处理4龄幼虫后,对其体内羧酸酯酶活性具有明显的诱导效应和时间效应,对谷胱甘肽S--转移酶活性的诱导效应仅在药剂处理后3h内较为显著,对多功能氧化酶活性表现出较强的诱导效应,比活力约为对照的1.37倍。     

甜玉米田玉米螟发生危害及防治措施

甜玉米在我国大面积种植历史较短,对甜玉米田亚洲玉米螟发生危害和防治技术研究较少。本文对国内外甜玉米上玉米螟成虫产卵习性、幼虫危害和防治技术研究和应用现状进行了综述,为我国甜玉米田亚洲玉米螟的研究和防治提供参考。     

亚洲玉米螟和黏虫幼虫取食Cry1F蛋白后的中肠组织病理变化

为了明确Cry1F杀虫蛋白对亚洲玉米螟和黏虫两种重要玉米害虫的作用机制,本文通过透射电镜观察了亚洲玉米螟和黏虫4龄幼虫取食含Cry1F蛋白人工饲料后的中肠组织病理变化,并与其取食含Cry1Ab蛋白人工饲料后的组织病理变化进行了比较。亚洲玉米螟和黏虫4龄幼虫取食Cry1F蛋白48 h后中肠杯状细胞发生了明显的病变,主要表现为：细胞微绒毛肿胀、脱落;细胞核变形,质膜和核膜界限不清晰;染色质发生固缩、贴近核膜;线粒体拉伸变形数量减少,严重的发生空泡化;内质网肿胀与断裂、数量减少,与取食含Cry1Ab蛋白人工饲料相比,玉米螟和黏虫中肠组织发生的病变程度相似。本研究可为Cry1F作为转基因玉米重要的杀虫蛋白在未来亚洲玉米螟和黏虫防治中更好地发挥作用提供理论依据。     

The biochemical basis of resistance to organophosphorus insecticides in the sheep blowfly, Lucilia cuprina

The metabolism in vivo and in vitro of [¹⁴C]parathion and [¹⁴C]paraoxon was studied in a susceptible (LS) and an organophosphorus-resistant (Q) strain of the sheep blowfly, Lucilia cuprina. Both strains detoxified the insecticides in vivo via a number of pathways, but the resistant strain produced more of the metabolites diethyl phosphate and diethyl phosphorothionate. No difference was found between strains in the rate of penetration of the compounds used. Also, in vitro studies showed no difference between strains in the sensitivity of head acetylcholinesterase to inhibition by paraoxon. Both the microsomal and the 100,000g supernatant fractions degraded paraoxon, but resistance in Q could be explained by the eightfold greater rate of diethyl phosphate production with or without added NADPH. Parathion was also degraded to diethyl phosphorothionate by an NADPH-requiring enzyme in microsomal preparations from both strains. However, Q produced significantly more diethyl phosphorothionate in vivo than LS. It was concluded that organophosphorus resistance in Q was due mainly to a microsomal phosphatase hydrolyzing phosphate but not phosphorothionate esters, probably enhanced by a microsomal oxidase detoxifying the latter.     

In-vitro Metabolism of a Novel Monocrotophos Derivative by Rat and Japanese Quail

NADPH-dependent inhibition of hepatic microsomal carboxylesterase by a derivative of monocrotophos (coded as RPR-5) was studied in rat and Japanese quail as a measure of monooxygenase-catalysed activation of RPR-5. There was NADPH-dependent inhibition of hepatic microsomal α-naphthyl acetate esterase (carboxylesterase) both in rat and quail, indicating monooxygenase-catalysed formation of an oxon that subsequently phosphorylated α-NaE. The pattern of in-vitro metabolism of ¹⁴C-labelled RPR-5 by 11000g supernatant (11-S), microsomes and 105000g supernatant (105-S) fractions of rat and quail livers suggested the involvement of microsomal monooxygenases and carboxylesterases. A radiolabelled metabolite (M2) was tentatively identified as an acid produced by carboxyl esterase attack. In rat, metabolism by microsomal and cytosolic (105-S) carboxylesterases appeared to predominate with relatively little oxidative metabolism. In quail, putative microsomal carboxylesterase hydrolysis of RPR-5 was much lower than in the rat with almost neglible hydrolysis by cytosolic fractions. Also, production of M₂ by quail microsomes was substantially reduced after addition of NADPH, suggesting inhibition of a carboxyl esterase by the oxon of RPR-5. Differences in this detoxification of RPR-5 between rat and quail may be an important factor in determining selective toxicity and the results underline the importance of relating metabolism to toxicity when selecting animal models for toxicity testing.     

Studies on the metabolism of vamidothion and its thioanalog in insecticide-resistant and susceptible house fly strains

The in vivo and in vitro metabolism of vamidothion [O,O-dimethyl S-[2-(1-methylcarbamoyl)-ethylthio] ethylphosphorothiolate] as well as the in vitro metabolism of thiovamidothion [O,O-dimethyl S-[2-(1-methylcarbamoyl)ethylthio] ethylphosphorodithioate] was investigated in insecticide-resistant and susceptible house fly strains. Vamidothion was converted in vivo to the sulfoxide, the principle metabolite, and subsequently to the sulfone at a slower rate. Vamidothion and vamidothion sulfoxide were hydrolyzed at the PS and SC bond. The resulting primary alcohol metabolite was further oxidized to a carboxylic acid followed by decarboxylation. No metabolism of vamidothion or thiovamidothion occurred in vitro without the addition of NADPH. The addition of NADPH resulted in rapid conversion of vamidothion to the sulfoxide, and thiovamidothion was oxidatively metabolized to six metabolic products. No qualitative differences were found between resistant and susceptible strains, but there were signficant quantitative differences. The metabolism was highest in the Rutgers strain followed by Cornell-R, Hirokawa, and then CSMA strain. The route of vamidothion and thiovamidothion metabolism was via the cytochrome P-450-dependent monooxygenase system, and none of the resistant strains showed glutathione S-transferase activity toward vamidothion or thiovamidothion. No further oxidation of vamidothion sulfoxide to the sulfone was observed and also no hydrolysis products were formed, in vitro.     

Metabolism of cisanilide (Cis-2,5-dimethyl-1-pyrrolidinecarboxanilide) by rat liver microsomes

Metabolism of [phenyl-¹⁴C] and [(2,5) pyrrolidine-¹⁴C] cisanilide was investigated in vitro with microsomal preparations from rat liver. Microsomal activity was associated with a mixed-function oxidase system that required O₂ and NADPH and was inhibited by CO. Two major ether-soluble metabolites were isolated. They were identified as primary oxidation products: 2-hydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide (A) and 4′-hydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide (B). Minor ether-soluble metabolites were also isolated. Precursor product studies and qualitative thin layer chromatography analysis of [pyrrolidine-¹⁴C] and methylated [phenyl-¹⁴C] hydrolysis products suggested that these metabolites were secondary oxidation products formed from metabolites A or B. One of these metabolites appeared to be the dihydroxy product 2,4′-dihydroxy-2,5-dimethyl-1-pyrrolidinecarboxanilide. Crude microsomal preparations (postmitochondrial supernatant fractions) also formed small quantities (<10%) of polar metabolites. Enzyme hydrolysis with β-glucuronidase (Escherichia coli) indicated that approximately 50% of these metabolites were glucuronides. Similarities and differences in cisanilide oxidation in vivo in plants and in vitro with rat liver microsomal preparations were discussed.     

Nitroreductases in the Madagascar cockroach,Gromphadorhina portentosa

Nitrobenzene reduction by tissue preparations of the Madagascar cockroach, Grompphadorhina portentosa, was studied in vitro. Active enzyme preparations were obtained from midgut, hindgut, fat body, and Malpighian tubules. Anaerobic conditions were essential for activity which was found in all subcellular fractions tested. The microsomal enzymes were strongly NADPH-dependent whereas the soluble enzymes were strongly NADH-dependent. Flavin addition stimulated activity greatly and it appeared that the true substrate for the nitroreductases was FMN and that nitrobenzene was nonenzymatically reduced by FMH₂.     

Perspektiven der Kontrolle des Maiszünslers (Ostrinia nubilalis Hbn.) mit Insektiziden – Versuchsergebnisse aus dem Oderbruch

With rise of the European corn borer (Ostrinia nubilalis Hbn.) in the Oderbruch area in the 1990s the necessity of an effective control of this particular pest in the agricultural field became even more important. Since 1997 experiments on controlling the increase of the European corn borer by using insecticides took place in the Oderbruch area. Between 2000–2002 different control methods (Insecticide use, Trichogramma use, Bt-corn use) were tested referring to the local conditions in the Oderbruch area. By cultivating Bt-corn types highest efficiency rates against European corn borer could be detected. Employed insecticides reached out to efficiency rates ranging from 60% to 90%. The application of Trichogramma under local conditions in the Oderbruch area was not satisfying at all. Since 2003 insecticides of different groups of active agents were examined concerning their biological effect. That is because of insufficient permission status (indication for European corn borer) and development of anti-resistant strategies. This knowledge is a necessary requirement for permission of insecticides to come.     

Toxicity and metabolism of carbaryl in the European corn borer

Larvae from two strains of the European corn borer, Ostrinia nubilalis (Hübner), were compared for differences in their tolerance and metabolism of carbaryl (1-naphthyl N-methylcarbamate). The Geneva strain was about twice as susceptible to carbaryl, but both Valley and Geneva borers converted carbaryl to oxidative metabolites at similar rates in vivo and in vitro. Maximum carbaryl-metabolizing activity was present in last-instar larvae, particularly in the fat body and gut tissues. However, the specific activity of gut homogenates was highest in the Geneva strain and the specific activity of fat body was highest in the Valley strain. Other differences in the mixed-function oxidase systems of gut and fat body were also found. The major metabolite in vivo and in vitro was hydroxymethyl carbaryl.     

亚洲玉米螟幼虫对不同寄主植物的取食选择性

为深入了解亚洲玉米螟Ostrinia furnacalis(Guenée)不同龄期幼虫的取食特性,采用叶碟法对6个科的24种植物进行了测定.结果显示,亚洲玉米螟初孵幼虫选择性试验中,酸模叶蓼上幼虫百分率最高,为29.81％;菜豆上次之,为27.36％.3龄幼虫选择性试验中,菜豆上幼虫百分率最高,为28.66％;酸模叶蓼上次之,为26.21％;该虫较喜食的其它几种植物上幼虫百分率由多到少依次是葎草、向日葵和苘麻,而玉米叶片上的幼虫取食百分率最低,初孵幼虫为0,3龄幼虫为4.28％.5龄幼虫在上述6种植物茎秆或果实上取食停留的时间没有显著差异.试验说明,亚洲玉米螟幼虫保持了对原始寄主的取食特性.     

Isolation and characterization of efficient isoxaben-transforming Microbacterium sp strains from four European soils

Nutrient-agar plates containing isoxaben (500 mg litre(-1)) were used to isolate isoxaben-metabolising bacteria from four European soils incubated with the herbicide under laboratory conditions. In flask experiments, inoculation of a basal salts medium containing nitrogen and [phenyl-U-14C]isoxaben with an isolate (B2b) resulted in 33% recovery of the initial radioactivity as [14C]carbon dioxide after 2 weeks. A major metabolite identified by GC-MS and NMR analysis as 3-(1-ethyl-1-methylpropyl)isoxazol-5-ylamine accumulated both in basal salts and nutrient broth media. 2,6-Dimethoxybenzoic acid, a suspected metabolite of isoxaben, was not detected in either liquid media. However, the capability of the B2b isolate to use 2,6-dimethoxybenzoic acid as a source of carbon was demonstrated. Soil inoculation with the B2b strain resulted in an increase in the recovery of [14C] carbon dioxide from both [phenyl-U-14C] and [isoxazole-5-14C]isoxaben. The metabolite identified as 3-(1-ethyl-1-methylpropyl)isoxazole-5-ylamine only accumulated if the soil was autoclaved before inoculation. This metabolite was rapidly mineralized by the microflora of a natural soil without history of isoxaben treatment. Homology patterns of sequenced 16S rDNA between isoxaben-transforming isolates and reference strains showed that the four isolates identified belonged to the genus Microbacterium.