Effects of chlordimeform on mitochondria from eggs of Spodoptera littoralis

Mitochondria were isolated from eggs of Spodoptera littoralis. With succinate (+pyruvate) as substrate, respiratory control ratios between 1.70 and 2.54 were obtained. Uncouplers and the energy transfer inhibitor oligomycin influenced these mitochondria in the well-known manner. The uncoupling activity of chlordimeform in vitro was very weak and decreased with increasing age of the eggs. Electron transport in mitochondria from chlordimeform-treated eggs was not uncoupled. Therefore, it is concluded that the ovicidal effect of this pesticide is not due to its uncoupling effect.     

Pyrethroid resistance in a strain of Spodoptera littoralis is correlated with decreased sensitivity of the CNS in vitro

The effects of permethrin and cypermethrin on the isolated abdominal nerve cord of insecticide-resistant [R] and -susceptible [S] strains of Spodoptera littoralis larvae have been studied. Above ca. 19°C, permethrin at 10^?7M caused a prolonged spike train to follow electrical stimulation of the nerve cord. The time of onset of this repetitive firing was significantly greater for the [R] strain. Moreover, cypermethrin, to which this strain shows negligible resistance, did not cause such repetitive discharges. Thus, resistance to permethrin but not to cypermethrin appears to be based on a qualitative difference between the pyrethroids. Nerve blockage by the two pyrethroids was also investigated, with particular reference to temperature. Once again, differences were apparent: when considered relative to untreated controls, permethrin caused quicker nerve blockage as temperature was reduced whereas the blocking action of cypermethrin was not affected by temperature. However, the times taken to cause nerve blockage by permethrin in [R] and [S] larvae were not significantly different, making it unlikely that nerve blockage plays a major role in this resistance. Two methods were employed to reduce the resistance factor in vitro. The synergist dodecyl imidazole failed to significantly reduce the time taken for permethrin to cause either repetitive firing or nerve blockage. However, reducing the calcium concentration in the saline did significantly reduce the latency of repetitive firing caused by permethrin in [R] larvae, thus increasing the nerve sensitivity to approximately the same level as normal calcium, [S] insects.     

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。     

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.     

Toxicity and biochemical study of two insect growth regulators, buprofezin and pyriproxyfen, on cotton leafworm Spodoptera littoralis

Lethal and sublethal effects of two insect growth regulators (IGRs) buprofezin and pyriproxyfen were evaluated on larvae of cotton leafworm Spodoptera littoralis. Activity of chitinase and polyphenol oxidase (PPO) in surviving larvae after treatment was carried out in order to investigate the biochemical influences of these compounds. The compounds were low toxic against the larvae at 0.05, 0.1, 0.25, 0.5, and 1.0-fold of the field application rate. However, the overall mortalities within 6 days of feeding at 2.0-fold were 46.67% and 100% for buprofezin and pyriproxyfen, respectively. Larval weight gain was considerably decreased as concentration increased. Pyriproxyfen showed high antifeedant activity in a concentration-dependent manner, and larvae stopped to eat from the third day with high dose. Conversely, buprofezin did not significantly show antifeedant except with high concentration (3000 mg (a.i.)/kg diet) that gave 80.68%. The high doses of both compounds showed adverse effects on pupae, and emergence of adults. Buprofezin at the recommended dose (1500 mg (a.i.)/kg diet) caused 93.33% pupation and 53.33% emergence of adults. Otherwise, pyriproxyfen caused 21.33% pupation and zere emergence of adults at the recommended dose (75 mg (a.i.)/kg diet) compared to 100% pupation, and 96.30% emergence of adults in the control. Both compounds varied in their influences on chitinase and PPO activity, and these enzymes could have relation with toxicity of buprofezin and pyriproxyfen against S. littoralis larvae.     

Inhibition of permethrin-hydrolyzing esterases from Wiseana cervinata larvae

Inhibition of permethrin-hydrolyzing enzymes from larvae of the porina moth Wiseana cervinata has been examined in vivo and in vitro. Significant inhibition was shown by carbaryl and pirimiphos-methyl. 1-Dodecylimidazole substantially inhibited permethrin hydrolysis only in liver insects. The triphenylmethane dye tetrabromophenolphthalein was a moderate inhibitor only in vitro. TMDM (bis(N-dimethyl-4-aminophenyl)methane) had little effect on hydrolysis. These observations extend the range of species and substrates for which the triphenylmethane dyes and 1-dodecylimidazole are useful inhibitors of insecticide metabolism.     

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.     

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.     

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.     

Spectral and inhibitory interactions of methylenedioxyphenyl compounds with southern armyworm (Spodoptera eridania) midgut microsomes

Characteristics of the Type III optical difference spectra of 13 methylenedioxyphenyl compounds in NADPH-fortified armyworm midgut microsomes varied with the nature of the substituents in the aromatic ring. Compounds with electron-donating substituents yielded spectra with large $\text{427}\text{458}\text{nm}$ peak ratios, whereas those with electron-withdrawing groups exhibited low $\text{427}\text{458}\text{nm}$ peak ratios. Small amounts of carbon monoxide were generated during incubation of the 4,5-dihalo derivatives with midgut microsomes, and cis- and trans-methylenedioxycyclohexanes exhibited spectra with a major Soret peak at about 430 nm and a very weak absorbance maximum at about 480 nm. Formation of the Type III spectral complex occurred very rapidly and was associated with a marked decrease (up to 72%) in cytochrome P-450 levels as measured by carbon monoxide binding. Although a 24% reduction of cytochrome P-450 was observed in the absence of any measureable 458-nm spectral complex a linear relationship existed between further decreases in the cytochrome and the increase in Type III complex formation (458 nm). Inhibitory potencies of the compounds towards aldrin epoxidase and benzopyrene hydroxylase activities were not clearly correlated with either spectral complex formation or decrease in cytochrome P-450 and it is apparent that different factors are involved in the inhibition of different monooxygenase reactions.     

Protein metabolism in Spodoptera litura (F.) is influenced by the botanical insecticide azadirachtin

Azadirachtin, as a botanical insecticide, affects a wide variety of biological processes, including reduction of feeding, suspension of molting, death of larvae and pupae, and sterility of emerged adults in a dose-dependent manner. However, the mode of action of this toxin remains obscure. By using proteomic techniques, we analyzed changes in protein metabolism of Spodoptera litura (F.) induced by azadirachtin. Following feeding 4th instar larvae of Spodoptera litura (F.) with an artificial diet containing 1 ppm azadirachtin until pupation, 48 h old pupae were collected and protein samples prepared. Total soluble protein content was measured and the results showed that azadirachtin significantly influenced protein level. Moreover, the proteins were separated by 2-DE (two-dimensional polyacrylamide gel electrophoresis) and 10 proteins were significantly affected by azadirachtin treatment when compared to an untreated control. Six of these proteins were identified with peptide mass fingerprinting using MALDI-TOF-MS after in-gel trypsin digestion. These proteins are involved in various cellular functions. One identified protein may function as an ecdysone receptor, which regulates insect development, and reproduction. It is suggested that the botanical insecticide azadirachtin affects protein expression and the azadirachtin-related proteins would be essential for a better understanding of the mechanisms by which neem toxins exert their effects on insects.     

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.     

溴虫腈对甜菜夜蛾的毒力及应用研究简报

室内毒力测定结果表明,溴虫腈EC对甜菜夜蛾卵的孵化有一定的抑制作用,但作用较小,20 mg/L药液浸卵后,卵孵化抑制率为43.11%。溴虫腈EC对甜菜夜蛾1~2龄幼虫的毒力很高,3龄后随着虫龄的增大,甜菜夜蛾对溴虫腈的敏感性显著降低。室内钵栽试验表明,20 mg/L溴虫腈EC对甜菜夜蛾2龄幼虫的减退率在91.43%~100%之间,持效期15 d以上。田间试验表明,溴虫腈对甜菜夜蛾有优异的防治效果,每公顷施用5%溴虫腈EC 1 200 mL,防治效果在95％以上,持效期为20 d。     

Effect of dihydrodillapiole on pyrethroid resistance associated esterase inhibition in an Indian population of Spodoptera litura (Fabricius)

Resistance in Spodoptera litura (Fabricius) has been attributed to enhanced detoxification of insecticides by increased levels of esterases, oxidases and/or glutathione S-transferases. Enzyme inhibiting insecticide synergists can be employed to counter increased levels of such enzymes in S. litura. Dihydrodillapiole induced synergism of pyrethroid toxicity was examined in the laboratory-reared third instar larval population of S. litura collected in Delhi (susceptible), and Guntur (resistant) region of Andhra Pradesh, India. The Guntur population was found to be 7.04 and 10.19 times resistant to cypermethrin and lambdacyhalothrin, respectively. The activity of cypermethrin, lambdacyhalothrin and profenophos against susceptible and resistance populations of S. litura, was gradually increased when used along with a plant-derived insecticide synergist dihydrodillapiole. The α-naphthyl acetate hydrolysable esterase activity in Delhi population was less as compared to the Guntur population. Resistance associated esterases in Delhi population were inhibited by pre-treatment with dihydrodillapiole. The esterase level in insect was instantly reduced initially, sustained for about 3 h and equilibrated at 4 h post treatment. The esterase activity of Guntur population was increased to 1.28 μmoles/mg/min at 2 h post treatment and subsequently reduced to lower than 0.70 μmoles at 4-12 h post treatment. The variation in esterase activity is suggestive of its homeostatic regulation in test populations. Dihydrodillapiole thus caused significant reduction of resistance in S. litura to cypermethrin, lambda cyhalothrin and profenophos.     

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.     

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.     

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。     

Physiological effect of chitinase purified from Bacillus subtilis against the tobacco cutworm Spodoptera litura Fab.

An extracellular chitinase was purified from Bacillus subtilis. The lethal concentration (LC₅₀) was determined by using chitinase in first, second, and third instars of Spodoptera litura Fab. Chitinase showed the highest insecticidal activity at 6 μM concentration within 48 h. The nutritional indices were also significantly affected by the 6 μM concentration (P < 0.05). Food consumption, efficiency of conversion of ingested and digested food, relative growth rate, and consumption values declined significantly while approximate digestibility was increased. Our study indicates that treatment of host plant leaves with the chitinase can regulate (reduce) larval growth and weight, and enhance the mortality. This may serve as an effective biocide and alternative to Bt toxin.