Insect epoxide hydrolase: Properties of a purified enzyme from the southern armyworm (Spodoptera eridania)

An epoxide hydrolase purified from midgut microsomes of southern armyworm (Spodoptera eridania) larvae exhibited high activity toward monosubstituted epoxides (1,2-epoxyoctane, 1,2-epoxypropane, and styrene oxide) and lower activity toward cis-1,2-disubstituted epoxides (cyclohexene oxide, and the cyclodienes HEOM, HCE, and chlordene epoxide). Trisubstituted epoxides (2-methyl-2,3-epoxyheptane and JH-1) as well as several cyclodiene insecticides (dieldrin, endrin, endo-epoxyaldrin, and anti-heptachlor epoxide) were refractory to enzymatic attack. It is concluded that both lipophilic and steric factors dictate the substrate specificity of the enzyme. With cyclohexene oxide the enzyme yields the 1R, 2R enantiomer of the trans-diol. The purified enzyme is inhibited by several epoxides and mixed-function oxidase inhibitors and the potency of 3,3,3-trichloro-1,2-epoxypropane and sodium picrylsulfonate suggest the importance of electronic factors in the inhibitory mechanism. Studies with specific amino acid modifiers suggest the presence of an essential lysine or histidine residue at the active site and indicate that the enzyme lacks a metal ion requirement and an essential cysteine residue. The purified enzyme has a molecular weight of 46,000 daltons and amino acid analysis and immunochemical studies show it to be very similar to, but not identical with, the epoxide hydrolase from mammalian liver microsomes.     

Induction of microsomal enzymes in the southern armyworm (Prodenia eridania)

Levels of microsomal epoxidation, N-demethylation, and cytochrome P-450 in the gut tissues of sixth instar southern armyworm larvae were considerably enhanced following oral in vivo treatment with a series of methylbenzenes. Induction increased with increasing methyl substitution and was maximal with pentamethylbenzene. The increase in microsomal activity occurred rapidly after initiation of treatment and the final levels of induction achieved were dependent on the concentration of the inducer in the diet and the time of exposure. Microsomal enzyme activity returned to control levels following termination of exposure and induction was blocked by puromycin and cycloheximide but not by actinomycin D. The in vivo tolerance of induced worms to orally administered carbaryl was increased in a manner reflecting the enhanced microsomal enzyme activity.     

甜菜夜蛾的抗寒力研究

抗寒力的强弱决定了甜菜夜蛾的分布范围及越冬存活情况,并直接影响下一代的种群数量.通过测定甜菜夜蛾三个地理种群(河北隆尧、湖南衡阳和江苏南京)的过冷却点和结冰点及田间埋蛹越冬试验,以明确甜菜夜蛾的抗寒能力.研究结果表明,在各虫态中甜菜夜蛾蛹的过冷却点最低(-16.61～-15.18 ℃),其次为成虫、卵和幼虫.蛹的过冷却现象也最明显,因而蛹是其最适宜越冬的虫态.三个地理种群中,以湖南衡阳的抗寒能力最强,其次为河北隆尧和江苏南京.在2001和2002连续两年的田间埋蛹越冬试验表明,甜菜夜蛾的越冬死亡率均为100%.分析南京多年的冬季气温,表明某些年份甜菜夜蛾不大可能在南京越冬.因此甜菜夜蛾的常年越冬区范围应在南京以南的区域.     

Monitoring of beet armyworm Spodoptera exigua (Lepidoptera: Noctuidae) resistance to chlorantraniliprole in China

Beet armyworm, Spodoptera exigua is a major insect pest of vegetables in China, and has been reported to develop resistance to many broad-spectrum insecticides. Recently registered chlorantraniliprole provides a novel option for control of this pest resistant to other conventional insecticides. The susceptibilities of field collected populations were measured by diet incorporation assay with neonate, obvious variation of susceptibility was observed among the 18 field populations with LC₅₀ values varying from 0.039 to 0.240 mg/liter. Moderate resistant level was discovered in 8 of 18 field populations, other 8 populations had become low level tolerance to chlorantraniliprole, and only one population in all the field colonies remained susceptible. Biochemical assays were performed to determine the potential mechanisms involved in tolerance variation. Field populations displayed varied detoxification enzyme activities, but the regression analysis between chlorantraniliprole toxicities and enzyme activities demonstrated each field population might have specific biochemical mechanisms for tolerance. Artificial selection in laboratory with chlorantraniliprole was carried out, 23 generations of continuous selections resulted in 11.8-fold increase in resistance to chlorantraniliprole, and 3.0-fold and 3.7-fold increases in mixed function oxidase and esterase, respectively. Compared with the susceptible strain kept in laboratory the selection strain had developed 128.6-fold resistance to this insecticide. Synergism assays showed the detoxification enzymes might not involved in the resistance observed in field collected populations and the selected strain.     

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.     

The effects of azadirachtin and nucleopolyhedrovirus on midgut enzymatic profile of Spodoptera litura Fab. (Lepidoptera: Noctuidae)

The effects of azadirachtin (AZA) and nucleopolyhedrovirus (NPV) on midgut enzyme activity in Spodoptera litura Fabricius (Lepidoptera: Noctuidae) (Tobacco cutworm) were evaluated. Gut enzyme activities were decreased by AZA and NPV individually and in combination. When S. litura larvae were fed a diet of castor leaves treated with AZA and NPV in bioassays, gut enzyme—acid phosphatases, alkaline phosphatases, adenosine triphosphatases, and lactate dehydrogenase—activities were decreased. There were statistically significant differences (P ? 0.05) in enzyme activities between combined and individual treatment. A synergistic effect of botanical insecticides and virus was found when combined in low doses. These effects are most pronounced in early instars. Maximum weight loss (59-72%) occurred, when AZA and NPV were combined.     

Induction of detoxification enzymes by triazine herbicides in the fall armyworm, Spodoptera frugiperda (J.E. Smith)

The inductive effect of six triazine herbicides on a variety of detoxification enzymes was investigated in fall armyworm (Spodoptera frugiperda) larvae maintained on an artificial diet. Dietary atrazine induced nine microsomal oxidase activities ranging from 1.3- to 21.6-fold, 12 glutathione S-transferase activities ranging from 1.3- to 4.2-fold, four hydrolase activities ranging from 1.3- to 2.9-fold, and two reductase activities ranging from 1.5- to 5.1-fold, depending on the enzyme assayed and tissue source (midgut vs. fat body) used. Simazine, cyanazine, ametryn, tebutryn, and terbuthylazine also induced these detoxification enzymes. The induction of microsomal oxidase (aldrin epoxidase) ranged from 1.2- to 11-fold, glutathione S-transferase (CDNB) ranged from 1.3- to 4-fold, and general esterase ranged from 1.4- to 4.1-fold, depending on the tissue source examined. In general, fat bodies were more inducible than midguts with respect to these detoxification enzymes, especially the microsomal oxidases. The induction by atrazine was associated with decreased toxicity of carbaryl, permethrin and indoxacarb, but increased toxicity of methyl parathion, phorate, and trichlorfon.     

虫螨腈胁迫对甜菜夜蛾保护酶系和解毒酶系的诱导效应

为探明虫螨腈对甜菜夜蛾幼虫体内生化代谢的影响及酶抑制剂对虫螨腈的增效潜力,采用点滴法测定了虫螨腈对甜菜夜蛾3、4、5龄幼虫的毒力和顺丁烯二酸二乙酯(DEM)、增效醚(PBO)、磷酸三苯酯(TPP)对虫螨腈的增效作用,并采用生化分析法测定了LD₂₅和LD₅₀剂量虫螨腈对甜菜夜蛾体内保护酶系和解毒酶系的影响。结果表明,虫螨腈对甜菜夜蛾不同龄期幼虫的毒力差异不大,处理后24h和48h的LD₅₀基本相同。PBO对虫螨腈有拮抗作用,增效比为0.21,而DEM和TPP对虫螨腈无增效作用。虫螨腈对甜菜夜蛾幼虫体内超氧化物歧化酶具有激活作用,对过氧化氢酶、过氧化物歧化酶及羧酸酯酶具有先抑制后诱导的作用;虫螨腈处理后6~24h,谷胱甘肽-S-转移酶活性显著提高,最高为对照的1.85倍;处理6h后,甜菜夜蛾体内细胞色素P450含量明显下降,而12~24h后,又明显上升,最高时为对照的1.86倍。     

The metabolism of 6,7-dihydroisodrin by microsomes and southern armyworm larvae

Dihydroisodrin is hydroxylated to 6-exo-hydroxy-6,7-dihydroisodrin in southern armyworm gut and vertebrate liver microsomal preparations. The sedimentation characteristics of the hydroxylase, its requirement for NADPH and oxygen, and its inhibition by carbon monoxide and 4,5,6,7-tetrachloro-1,3-benzodioxole indicate that the enzyme system may be classed as a typical microsomal oxidase. The enzyme system is susceptible to substrate inhibition, but in other respects it is similar to microsomal aldrin epoxidase and p-chloro-N-methylaniline demethylase.     

Insensitivity of acetylcholinesterase in a field strain of the fall armyworm, Spodoptera frugiperda (J.E. Smith)

Acetylcholinesterase (AChE) was purified from adult heads of the fall armyworm (Spodoptera frugiperda) by using a two-step procedure involving gel filtration on a Sephadex G-200 column and affinity chromatography on a procainamide-ECH Sephadex 4B column. Both susceptible and field strains possessed two AChE isozymes, namely, AChE-1 and AChE-2, with subunit molecular weights of 63.7 and 66.1 kDa. The purified AChE had an apparent K_m value of 33.5 μM and a V_max of 7.07 μmol/min/mg protein in the susceptible strain. The apparent K_m and the V_max were 2.2- and 2.0-fold higher, respectively, in the field strain than in the susceptible strain. The purified AChE from the field strain was 17- to 345-fold less sensitive than that from the susceptible strain to inhibition by carbamates (carbaryl, eserine, methomyl, and bendiocarb) and organophosphates (methyl paraoxon and paraoxon), insensitivity being highest toward carbaryl. The results further support the notion that insensitive AChE played an important role in the insecticide resistance observed in the field strain.     

Phenobarbital induction of permethrin detoxification and phenobarbital metabolism in susceptible and resistant strains of the beet armyworm Spodoptera exigua (Hübner)

The permethrin resistant strain (TR-strain) of the beet armyworm, Spodoptera exigua (Hübner), has 92.5-fold resistance to permethrin (at LD₅₀ level) compared to the permethrin susceptible strain (TS-strain). Bioassay involving permethrin mixed with piperonyl butoxide, an inhibitor of microsomal cytochrome P450s, significantly reduced the resistance ratio from 92.5- to 7.9-fold. However, S,S,S-tributylphosphorotrithioate and diethylmaleate which are inhibitors of esterases and glutathione S-transferase, respectively, did not affect the resistance level. These results indicate that the detoxification of permethrin in the TR-strain was primarily due to the cytochrome P450 monooxygenases. LD₅₀ for permethrin was increased to 4.5-fold by the pre-treatment of phenobarbital in the TS-strain. The effect of induction by phenobarbital was almost completely overcome by the piperonyl butoxide treatment. However, it was observed that phenobarbital treatment did not cause any change in the toxicity of permethrin to TR strain. Since this result deviated from the expectation that the metabolism of phenobarbital in the TR-strain should be greater than that in the TS-strain, it was deemed necessary to compare the metabolism of phenobarbital between the TS- and TR-strains. Comparison was made based on the concentration of phenobarbital in the hemolymph and whole body. The results showed no significant difference in phenobarbital treatment between the two strains used in this study suggesting the possibility that the induction system in TS-strain is different from the TR-strain.     

寄主植物对甜菜夜蛾生长发育和消化酶活性的影响

通过室内饲养和生化测定,研究了甜菜、玉米和黄瓜3种寄主植物的营养物质及其对甜菜夜蛾幼虫发育、成虫产卵及5龄幼虫体内蛋白质和总氨基酸含量、淀粉酶和脂肪酶活力的影响.结果表明:与取食玉米和黄瓜的个体相比,取食甜菜的甜菜夜蛾,其幼虫期和蛹期较短,分别为11.4天和5.6天;蛹期存活率最高,达100%;成虫寿命较长,为26.3天;单雌产卵量最高,为301.8粒.取食3种寄主植物,甜菜夜蛾5龄幼虫体内的蛋白质含量无差异,但总氨基酸含量差异显著,此外,幼虫体内淀粉酶和脂肪酶活力也存在显著差异,取食甜菜的个体>取食玉米的个体>取食黄瓜的个体.取食不同寄主植物,甜菜夜蛾幼虫的消化酶和脂肪酶活力与其发育适合度呈正相关.     

Selection and characterization of spinosad resistance in Spodoptera exigua (Hübner) (Lepidoptera: Noctuidae)

The cross-resistance and biochemical mechanism of the beet armyworm, Spodoptera exigua (Hübner), to spinosad was studied in the laboratory. S. exigua population were collected from Shanghai suburb. After five generations of selection, the resistance of S. exigua to spinosad increased 345.4 times compared with the susceptible strain. There was no cross-resistance between spinosad and fenvalerate, phoxim, methomyl, abamectin, and cyfluthrin. When the inhibitors, PBO, TPP, DEF, and DEM were used as synergist in the susceptible strain and resistant strain, the synergistic ratio was 0.7-, 0.5-, 1.0-, and 0.6- fold for the susceptible strain, and 9.8-, 1.5-, 2.6-, and 1.5-fold for the resistant strain, respectively. The results revealed that PBO had significant synergistic effect on the resistant strain. The activity in vitro of microsomal-O-demethylase and glutathione S-transferase in the resistant strain was 5.2- and 1.0-fold of the susceptible strain, respectively. The results implied that microsomal-O-demethylase might be important in conferring spinosad resistance in the S. exigua population.     

虫酰肼对苜蓿银纹夜蛾核型多角体病毒的增效作用

研究了以甜菜夜蛾为靶标害虫时,昆虫生长调节剂虫酰肼对苜蓿银纹夜蛾核型多角体病毒(AcNPV)的增效作用.结果表明:虫酰肼在作用于甜菜夜蛾时对AcNPV有显著增效作用,不仅可提高杀虫毒力,而且也能提高杀虫速度.作用于3龄和4龄幼虫时,6.67 mg/L虫酰肼对AcNPV的增效比分别是1.31和3.62,使害虫因表现感染病毒症状而死亡的LT50(致死中时)分别缩短10.8%和6.5%,使害虫因病毒和虫酰肼作用而死亡的LT50分别缩短25%和20.4%.虫酰肼和病毒协同作用还显著抑制了幼虫的生长发育,在协同作用1天后,幼虫体重即显著低于单用病毒或对照,并且随着作用时间的延长,协同作用组幼虫体重与病毒单剂组及对照组的差异越来越大.但6.67mg/L虫酰肼和2.7×105PIB/mL AcNPV协同作用于幼虫期时,其对存活幼虫发育为成虫后的繁殖力无影响,成虫产卵量、交配率、卵孵化率以及卵孵化幼虫数等均与病毒单剂及虫酰肼单剂无显著差异,也与对照无显著差异.     

几种因素对斜纹夜蛾性信息素诱捕效果的影响

采用田间诱集效果测定,就不同来源诱芯、诱捕器不同置放高度、不同类型诱捕器、诱芯不同储藏期等因素对信息素诱集斜纹夜蛾数量以及时序动态的影响进行了研究,以探讨蔬菜害虫斜纹夜蛾性信息素应用的关键技术。结果表明:3种不同来源的斜纹夜蛾性诱剂诱捕效果无显著差异;斜纹夜蛾性信息素置放高度依据不同寄主田间生长高度,以1～1.5m效果较好;3种诱捕器诱集效果相当,诱集代次清晰、峰谷明显,均可作为斜纹夜蛾性信息素配套的诱捕器;斜纹夜蛾橡皮塞诱芯置放冰箱贮藏1年或2年,诱蛾活性显著下降,诱蛾量下降60%以上。     

Inhibition of the fucosterol-24(28)-epoxide cleavage enzyme of sitosterol dealkylation in Spodoptera littoralis larvae

The conversion of sitosterol into cholesterol and the effect of inhibitors on the dealkylation reaction has been studied in Spodoptera littoralis Boisd. Using a microsomal preparation from the midgut of the larvae [³H]fucosterol and [³H]fucosterol-24,28-epoxide were converted into desmosterol and cholesterol in vitro, and a series of inhibitors were tested using the latter substrate. Triadimefon [1-(4-chlorophenox)-3,3-dimethyl-1-(1H-1,2,4-triazol-1-yl)butan-2-one] strongly inhibited the cleavage enzyme at 2 μM. In a functional group study we observed that (i) the phenoxy substituent was more strongly inhibitory than the corresponding benzyl, and (ii) when the ketone is reduced to the secondary alcohol in either phenoxy or benzyl series, inhibition decreased markedly. In the benzyl series of compounds we found that (iii) a 2,4- or 2,6-dichlorophenyl gave greater inhibition than mono-, tri-, or pentachlorophenyl derivatives, and (iv) changing the heterocyclic ring gave activity in the order 2-pyridyl=3-pyridyl=imidazole>pyrazole>triazole. In the triazole series, substitution of the hetero ring did not abolish inhibition. When either triadimefon or 22,25-diazacholesterol was incorporated into a diet (1000 ppm) containing sitosterol plus campesterol as the only sterol supplements and fed to final larval instar insects, the weight of the insects throughout the instar was reduced and the duration of the instar increased. Insects reared from the early third-instar stage on such a diet containing amounts of triadimefon had reduced percentages of cholesterol and desmosterol in the final larval instar with a proportional increase in the sitosterol level. Thus, inhibition of side chain cleavage has been obtained in vitro and in vivo, but the high concentrations of inhibitor required preclude the enzyme as a target for a novel class of insecticide.     

杀菌剂丙环唑对斜纹夜蛾的生物活性

为从现有农药中寻找杀虫剂先导化合物，研究了11种农药对斜纹夜蛾细胞的毒杀活性。结果表明，杀菌剂丙环唑的活性最高，处理后24h和48h，丙环唑对斜纹夜蛾细胞的LC50值分别为23．89μg／mL和13．62μg／mL。在活体试验中，以0．5μg／头剂量的丙环唑注射斜纹夜蛾4龄幼虫，处理后24h和48h，试虫血淋巴中血细胞数分别下降了19．67％、21．38％，血淋巴含量分别下降了20．42％、23．87％。以0．5μg／头和1．0μg／头剂量的丙环唑注射处理后，斜纹夜蛾幼虫体重显著降低。在注射处理后48h和72h，丙环唑对斜纹夜蛾4龄幼虫的LD50值分别为0．91μg/头和0．63μg/头。丙环唑对斜纹夜蛾细胞和斜纹夜蛾幼虫均具有较好的毒杀活性，显示出杀菌剂丙环唑衍生物控制害虫的可能性。     

Immunocytochemical localization of the Bacillus sphaericus binary toxin components in Culex quinquefasciatus (Diptera: Culicidae) larvae midgut

This study describes the immunocytochemical localization of the Bacillus sphaericus 2362 binary toxin components, BinA and BinB, in Culex quinquefasciatus larvae that had been intoxicated with this entomopathogen. Ultrathin sections of C. quinquefasciatus larvae midgut embedded in the hydrophilic resin L.R. White were incubated with the antibodies anti-BinA or anti-BinB and then revealed with goat anti-rabbit IgG coupled to gold particles. Immunocytochemical detection demonstrated the presence of specific labeling in ultra-thin sections that had been incubated with the BinA antiserum. Gold particles were detected on the apical areas of cell membranes and inside the epithelial cell cytoplasm, particularly the mitochondria, of cells from the gastric caeca and posterior stomach in larvae exposed during 2 or 24 h to the entomopathogen. A similar labeling pattern was observed in ultrathin sections from both midgut regions when incubated with BinB antiserum.     

Mechanisms for multiple resistances in field populations of common cutworm, Spodoptera litura (Fabricius) in China

The mechanisms for multiple resistances had been studied with two field resistant strains and the selected susceptible and resistant strains of Spodoptera litura (Fabricius). Bioassay revealed that the two field strains were both with high resistance to pyrethroids (RR: 63-530), low to medium resistance to organophosphates and carbamates, AChE targeted insecticides (RR: 5.7-26), and no resistance to fipronil (RR: 2.0-2.2). Selection with deltamethrin in laboratory could obviously enhance the resistance of this pest to both pyrethroids and AChE targeted insecticides. Synergism test, enzyme analysis and target comparison proved that the pyrethroid resistance in this pest associated only with the enhanced activity of cytochrome P450 monooxygenase (MFO) and esterase. However the resistance to the AChE targeted insecticides depended on the target insensitivity and also the enhanced activity of MFO and esterase. Thus, the cross-resistance between pyrethroids and the AChE targeted insecticides was thought to be resulted from the enhanced activity of MFO and esterase.     

三种不同波长绿-黄光对甜菜夜蛾产卵生物学的影响

甜菜夜蛾Spodoptera exigua(Hübner)属鳞翅目夜蛾科,是一种世界性分布、间歇性大发生的杂食性农业害虫,近年来,在全国部分省区发生逐渐加重[1]。目前,国内外已有应用黄色光对农业害虫进行有效防治的报道,如八濑顺也[2]报道利用黄色灯防治蔬菜上的棉铃虫、甘蓝夜蛾和斜纹夜蛾等