Mechanism of resistance to spinosyn in the tobacco budworm, Heliothis virescens

Topical laboratory selection of tobacco budworm larvae, Heliothis virescens, with technical spinosad for multiple generations resulted in larvae 1068-fold resistant to topical applications of the insecticide and 316.6-fold resistant to insecticide treated diet as compared to the parental strain. The penetration of 2′-O-methyl[¹⁴C]spinosyn A across the cuticle of the susceptible (parental) and selected (resistant) tobacco budworms increased with time 3-12 h after application. A trend of reduced penetration in the resistant strain was found but the differences were not statistically significant. 2′-O-methyl[¹⁴C]spinosyn A when injected into the hemocoel was not metabolized 96 h after treatment in both the susceptible and resistant strain, suggesting that a change in metabolism was not the mechanism of resistance. Electrophysiological studies indicated that dose-dependent spinosyn A-induced currents occurred in neurons from spinosyn resistant and susceptible (adult) tobacco budworms. At both 10 and 100 nM spinosyn A, however, the amplitude of these currents in the resistant insects was significantly smaller than the amplitude of currents observed from neurons from susceptible tobacco budworm adults. This suggests that neurons from resistant insects have decreased sensitivity to spinosyn A. However, the reduced inward currents in the resistant strain may or may not be related to the mode of action of the spinosyns. No statistically significant cross-resistance was noted for the spinosad resistant tobacco budworms for topical applications of permethrin (Pounce^®), profenofos (Curacron^®), emamectin benzoate (Denim^®), or indoxacarb (Steward^®). A statistically significant reduction in susceptibility to acetamiprid (Mospilan^®) in artificial diet as determined from a resistance ratio of 0.482 was found.     

Acephate toxicity,metabolism, and anticholinesterase activity in Heliothis virescens (F.) and Anthonomus grandis grandis (Boheman)

The toxicity (72 hr) of acephate and methamidophos to fourth-instar larvae of the tobacco budworm, Heliothis virescens (F.), was nearly equivalent. In contrast, toxicity (72 hr) of methamidophos to adult boll weevils, Anthonomus grandis grandis (Boheman), was substantially greater than that of acephate. The internal accumulation of acephate was greater for A. grandis grandis than for H. virescens at 24 and 48 hr post-treatment, as was excretion. Acephate was metabolized to methamidophos both in vivo and in vitro by H. virescens but not by A. grandis grandis. In vitro acetylcholinesterase inhibition by methamidophos was greater than that of acephate, but less than that of paraoxon for H. virescens, A. grandis grandis, and the electric eel. Treatment of H. virescens larvae with acephate resulted in increased in vivo acetylcholinesterase inhibition between 24 and 72 hr post-treatment, which was associated with a large increase in mortality. H. virescens treated with methamidophos showed greater mortality and greater acetylcholinesterase inhibition at earlier time periods than those treated with acephate. However, by 72 hr post-treatment, in vivo acetylcholinesterase inhibition by LD₅₀ doses of acephate and methamidophos were approximately equivalent. These results indicate that, for H. virescens, toxicity of acephate is directly related to its metabolism to methamidophos and subsequent acetylcholinesterase inhibition. Likewise, the differential toxicity of acephate and methamidophos to A. grandis grandis adults appears to be due to their inability to metabolize acephate to methamidophos.     

Mixed function oxidase and glutathione transferase activity in last instar Heliothis virescens larvae

Methyl parathion-resistant and -susceptible strains of Heliothis virescens were assayed for mixed function oxidase and glutathione transferase activity at four time periods within the last instar. No significant differences were found between the mixed function oxidase or glutathione transferase activities of the resistant and susceptible strains. However, significant differences did appear among time periods within the last instar. Larvae assayed during the postfeeding time period (60–80 hr) had the highest mixed function oxidase activity, which was 28 times that of last instar larvae assayed during the first time period (0–19 hr). Glutathione transferase activity appeared to be highest during the 20- to 39-hr and 40- to 59-hr time periods, but the differences among time periods were small. The importance of these findings to studies of insecticide resistance and plant herbivore interactions is discussed.     

Resistance selection and mechanisms of oriental tobacco budworm (Helicoverpa assulta Guenee) to indoxacarb

The oriental tobacco budworm, Helicoverpa assulta, is one of the most destructive pests for numerous commercial crops, and these organisms are responsible for enormous economic losses in Chinese agriculture. Insect larvae often feed within host plant fruits, providing protection from many currently used insecticides and making field control of H. assulta very difficult. Owing to its novel mode of action, high insecticidal activity, and low mammalian toxicity, the nonsystemic insecticide indoxacarb has been considered a promising alternative for the control of lepidopterous pests of agricultural significance. Indoxacarb evidences an elevated insecticidal activity against H. assulta. After 13 generations of selection with indoxacarb and bifenthrin insecticides under laboratory conditions, the LC₅₀ of these compounds for H. assulta increased by 4.19-fold and 10.67-fold, respectively. The synergists diethyl maleate (DEM) and triphenyl phosphate (TPP) increased indoxacarb toxicity by 2.76-fold and 4.10-fold in resistant strains and, comparatively, 1.58-fold and 1.75-fold in susceptible strains, suggesting that carboxylesterase (CarE) and glutathione-S-transferases (GSTs) may be involved in the development of indoxacarb resistance in H. assulta. Activity and kinetic parameters observed in detoxification enzymes further demonstrated that the enhanced activity of CarE and GSTs may be critical in development of indoxacarb resistance in H. assulta. The data provides a foundation for further study of the indoxacarb resistance mechanism observed in H. assulta and the rational use of indoxacarb as a rotation insecticide with other insecticide classes for the control of H. assulta.     

Toxicity of chlordimeform and amitraz to the egyptian cotton leafworm (Spodoptera littoralis) and the tobacco budworm (Heliothis virescens)

The relative toxicity (μg a.i. g^?1 body wt) of the formamidine insecticide chlordimeform (CDM) and the triazapentadiene insecticide amitraz was examined in two species of noctuid moth Spodoptera littoralis and Heliothis virescens. When applied topically, there was an unexpected and marked difference in the toxicity of CDM base and its hydrochloride to adults of both species, the salt being appreciably more toxic. For H. virescens at least, this difference in toxicity could not be explained by differences in penetration. This trend was reversed for larval instars of S. littoralis; while there was relatively little difference in the toxicity of the base to adult and larval stages, the salt was at least 1000-fold more toxic to adults than to larvae. N¹-Demethylchlordimeform (DCDM) was the only metabolite of CDM to show biological activity against either species, but was much less toxic than the parent compound. Amitraz was far less toxic than either CDM or DCDM; like the CDM salt, it appeared to be more toxic to adult than larval stages of S. littoralis. Application of piperonyl butoxide significantly increased the toxicity of the CDM salt, DCDM and amitraz to adult H. virescens, the synergist being particularly effective with DCDM and amitraz. In contrast, piperonyl butoxide had no significant effect on the toxicity of DCDM, and slightly antagonised the toxicity of DCDM to fourth-instar larvae of S. littoralis.     

Resistance in the highly DDT-resistant 91-R strain of Drosophila melanogaster involves decreased penetration,increased metabolism,and direct excretion

Resistance to 4,4′-dichlorodiphenyltrichloroethane (DDT) in the 91-R strain of Drosophila melanogaster is extremely high compared to the susceptible Canton-S strain (>1500 times). In addition to enhanced oxidative detoxification, the 91-R strain also has a reduced rate of DDT penetration, increased levels of reductive and conjugative metabolism, and substantially more excretion than the Canton-S strain. Contact penetration of DDT was ∼30% less with 91-R flies, which also had significantly more cuticular hydrocarbons and a thicker, more laminated cuticle compared to Canton-S flies, possibly resulting in penetration differences. DDT was metabolized ∼1.6-fold more extensively by 91-R than Canton-S flies, resulting in dichlorodiphenyldichloroethane (DDD), two unidentified metabolites and polar conjugates being formed in significantly greater amounts. 91-R flies also excreted ∼4-fold more DDT and metabolites than Canton-S flies. Verapamil pretreatment reduced the LD₅₀ value for 91-R flies topically dosed with DDT by a factor of 10-fold, indicating that the increased excretion may involve, in part, ATP-binding cassette (ABC) transporters. In summary, DDT resistance in 91-R is polyfactorial and includes reduced penetration, increased detoxification and direct excretion.     

The metabolism of DDT in vivo by the Japanese quail (Coturnix coturnix japonica)

Male and female Japanese quail (Coturnix coturnix japonica) were given intraperitoneal injections of [¹⁴C]DDT in ethanol at a rate of 13.4 mg/kg body wt. Fifty-six days later the tissues and droppings were analysed for total ¹⁴C and metabolites. The rate of loss of ¹⁴C in droppings was very similar in males and females. The maximal rate was reached on the third day, and 65–66% of the injected dose was voided by the fifty-sixth day. Ninety-three to ninety-four percent of the ¹⁴C in droppings and 83–90% of the ¹⁴C in tissues were extracted by solvents. Combined extracts from males and females were used for determination of DDT and its metabolites. Expressing all results as percentages of injected dose, the following were isolated from droppings: DDA (24%), DDT (3%), DDD (5.1%), DDE (11%), and uncharacterised polar metabolites (17%). Twenty-five percent of the dose was retained in the tissues and this was largely accounted for as DDT (10.4%) and DDE (10.5%). Of the total metabolites found 31% was DDE (almost equally divided between tissues and droppings) and 35% was DDA (almost entirely in droppings). Since DDD was not found in significant quantities in tissues, the substantial quantities in droppings were probably produced from DDT by the action of microorganisms.     

Toxicity and tolerance of sesquiterpene lactones in the migratory grasshopper, Melanoplus sanguinipes (Acrididae)

Sesquiterpene lactones, natural constitutents of the Asteraceae, are toxic to the grasshopper, Melanoplus sanguinipes, when injected into the hemocoel at doses greater than 0.25 μmol/300 mg insect. Dose-dependent sublethal symptoms range from a slight reduction in normal locomotory ability to severe locomotory impairment or paralysis, leading to death. The symptoms appear to be irreversible. Of the four compounds subjected to dose-response testing, parthenin was the most toxic with and LD₅₀ in adult male grasshoppers of 0.55 μmol/insect. Structure-activity relationships for four of the six lactones tested indicate that compounds containing a cyclopentenone ring are equitoxic to males and females, whereas those lacking this functional group are approximately 4 × more toxic to males than females. In contrast to their sensitivity to injected lactones, adult male grasshoppers can tolerate ingestion or topical administration of up to 4 μmol of these compounds without any adverse effects. These results suggest that the integument and alimentary canal in this species provide effective barriers limiting the bioavailability of sesquiterpene lactones to the hemolymph in nature. Possible target sites and mechanisms-of-action for sesquiterpene lactones (once in the bloodstream) are discussed.     

阿维菌素对黄胸散白蚁Reticulitermes speratus的毒效

在室内采用毒土法、滤纸法和毒土柱法测定了0.2%阿维菌素乳油对黄胸散白蚁的毒杀效果。 结果表明,阿维菌素对黄胸散白蚁的触杀作用速度较慢,在处理的96 h内,随时间推移毒性逐渐增强;黄胸散白蚁接触或取食药剂96 h后,其LC50值均低于20 mg/L;在供试条件下,用2.5 mg/L以上的浓度处理土壤,即能阻止黄胸散白蚁在土柱中的穿透,显示阿维菌素是一种值得进一步研究的白蚁预防药剂。     

Activity and metabolism of cyano-oxime derivatives in various strains of Botrytis cinerea

The fungitoxicity of cymoxanil, 4 cyano-oxime analogs (in which the acetylurea group was replaced with various groups: amide, ester, propargyl, and cyanomethyl-amide), and 2 cymoxanil-metabolites was studied against various strains of Botrytis cinerea. The fungicidal effect was measured on germ-tube elongation and mycelial growth. Cymoxanil and the analogs bearing the amide and ester groups showed the best anti-botrytis activity. The strains studied can be classified into 3 groups according to the germ-tube sensitivity to cyano-oximes. These groups fitted well with the 3 phenotypes of sensitivity to cymoxanil previously characterized in our laboratory: CyaS₁ (highly sensitive phenotype); CyaS₂ (moderately sensitive phenotype); and CyaR (tolerant phenotype). The bio-transformations of all the cyano-oximes were monitored in the culture-medium of the different strains using HPLC- and IP-HPTLC-methods. HPLC-studies showed that cymoxanil and the analogs bearing the groups amide and ester were quickly metabolized in the culture-medium of the CyaS₁ strain. Moreover, these studies allowed us to correlate disappearance of these cyano-oximes, to their fungicidal activity towards the CyaS₁ strain. This suggests that cyano-oximes and particularly cymoxanil are probably activated in a fungitoxic compound. IP-HPTLC-studies strengthened the precedent results and allowed us to correlate disappearance of cyano-oximes studied with appearance of two acids metabolites of hydrolysis. These metabolites could be the actual active-principles.     

Biological effects and metabolism of fenoxycarb after treatment of the fourth and the fifth instars of the tobacco budworm,Heliothis virescens F.

Treatment of 4th and 5th instar larvae of Heliothis virescens with different amounts of fenoxycarb induced an increase in weight of the 5th larval instar, prolongation of the 5th larval instar period, or the formation of dauer larvae. No effects were observed during the 4th instar and the moult to the 5th larval instar. To check whether these effects were due to persistence of fenoxycarb in treated larvae, metabolism of this chemical was analysed. Larvae were fed with radio-labelled fenoxycarb at the first day of the 4th or at the first day of the 5th larval instar. Behaviour of the radio-labelled compounds was followed during each instar. At the time of gut purge, the whole of the radio-labelled material was excreted. The effects observed during the 5th instar of 4th-instar-treated larvae do not appear to be due to the persistence of fenoxycarb. The main metabolites of fenoxycarb were identified using coupled gas chromatography and mass spectrometry.     

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.     

Stereoselective metabolism of fipronil in water hyacinth (Eichhornia crassipes)

The enantioselective metabolism of racemic fipronil in water hyacinth (Eichhornia crassipes) had been investigated. In this study, the degradation data and the enantiomer fraction (EF) were determined by chiral high-performance liquid chromatography (HPLC) with a column cellulose-tri-(3, 5-dimethylphe-nylcarbamate)-based chiral stationary phase (CDMPC-CSP). During the uptake phase, the EF value of plant sample increased from 0.50 at 1st day to 0.72 at 63rd day, while it was almost unchanged in water. For the depuration phase, the S- and R-enantiomer of fipronil in water hyacinth plants were degraded 92.22% and 82.07% after 17 days, respectively. The process of the degradation of the two enantiomers was followed first-order kinetics (R² ? 0.94). Stereoselective behavior was observed in both accumulation and degradation process. In this study, fipronil-sulfone and fipronil-sulfide, the metabolites of fipronil, were detected by GC-MS to show the main metabolic pathway of fipronil in water hyacinth.     

新烟碱类等杀虫剂对葱蝇的毒力及其对生长发育和繁殖的影响

采用定量滴加法和浸渍法测定了8种药剂对葱蝇1龄和3龄幼虫的毒力,用噻虫胺和噻虫嗪等新烟碱类杀虫剂的亚致死浓度分别处理葱蝇1龄和3龄幼虫,观察其对葱蝇生长发育和繁殖的影响,并进行了田间药效试验。结果表明,噻虫胺和噻虫嗪对3龄幼虫48 h的LC₅₀分别为14.7839 mg/L和13.3055 mg/L,毒力为毒死蜱的0.41倍和0.50倍。但LC_30-40剂量处理葱蝇3龄幼虫后,第6天存活幼虫体重分别较对照降低35.47%和36.34%,幼虫发育历期延长3.68天和3.98天,化蛹率降低71.91%和78.65%,雌蛹重降低13.36%和14.72%,雄蛹重降低15.97%和18.32%,羽化率降低12.50%和17.86%,单雌产卵量降低47.71%和51.68%。噻虫胺和噻虫嗪处理1龄幼虫,对其生长发育也有显著不利影响。田间药效试验结果显示,1 200 g/hm²噻虫胺和噻虫嗪处理后7天防效分别达98.16％和92.46％,表明这两种药剂对葱蝇防治效果高、推广应用价值大。     

Toxicity and neurophysiological effects of selected insecticides on the mole cricket, Scapteriscus vicinus (Orthoptera: Gryllotalpidae)

Mole crickets (Scapteriscus spp.) are severe subterranean pests of turfgrasses, commonly targeted with neurotoxic insecticides. Ideally insecticides used against mole crickets should induce quick knockdown or mortality to minimize damage caused by their tunneling. However, neurophysiological effects of insecticides on mole crickets are mostly unknown. The aims of this study were to investigate neurophysiological and toxic effects of several insecticides on tawny mole cricket (Scapteriscus vicinus Scudder) adults and nymphs, and potential synergy between pyrethroid and neonicotinoid insecticides. Bifenthrin, fipronil, and the combination of bifenthrin + imidacloprid provided the fastest median mortality when injected. The combination of bifenthrin + imidacloprid elicited faster toxicity than either active ingredient alone. Imidacloprid, bifenthrin, and bifenthrin + imidacloprid caused immediate knockdown, whereas fipronil immobilized mole crickets within 1-2 h. Acephate, bifenthrin, fipronil, imidacloprid, and bifenthrin + imidacloprid caused significant neuroexcitation. Bifenthrin + imidacloprid resulted in greater increases of spontaneous neural activity than the additive effects of imidacloprid and bifenthrin alone. Excitatory compounds acting at sodium and chloride channels (bifenthrin and fipronil) were the most toxic against S. vicinus. Combining a sodium channel toxin (bifenthrin) and a synaptic toxin (imidacloprid) led to greater than additive neurophysiological and toxic effects, which to our knowledge provides the first documented evidence of synergistic neurological “potentiation”.     

Influence of oil droplet size on the toxicity of bifenthrin on cotton to tobacco budworm (Heliothis virescens)

Sprays of bifenthrin and paraffinic oil mixtures were applied to cotton leaves (Gossypium hirsutum L.) to investigate droplet size effects on the mortality of Heliothis virescens (F.). An air-assisted nozzle applied mixtures at 9.4 litre ha ?1 spray rate with volume median diameters (VMD) of 96 and 337 μm, as verified with a Malvern laser diffraction analyzer. An hour after spraying, susceptible 3rd-instars walked for 2-min through wet droplets of spray mixtures applied at bifenthrin rates of 22, 45, 67, 90. and 112 g a.i. ha ?1. Mortality was assessed at 21, 26, 45, 50, 74, and 117h after exposure. Final mean mortality was affected (P =0.01) by bifenthrin rate and droplet size. Bifenthrin applied in 337 μm VMD droplets caused 75.8% mortality, whereas 96-μm VMD droplets resulted in a mortality of 67.9%. Also, the 337 μm VMD droplets killed larvae in 13% less time than the 96μm VMD droplets, based on analysis (P =0.01) using the mean elapsed time till death as the dependent factor.     

嘧菌酯与吡唑醚菌酯对烟草白粉病菌的抑制作用及在烤烟漂浮育苗阶段的防治效果

采用小苗法测定了嘧菌酯和吡唑醚菌酯对烟草白粉病菌Erysiphe cichoracearum的毒力,以及对苗期烟草白粉病的预防和治疗作用及持效期。结果表明:嘧菌酯和吡唑醚菌酯对烟草白粉病菌抑制作用的EC50值分别为7.62和9.90 μg/mL,对该病害的预防和治疗作用的EC50值分别为8.76、12.34 μg/mL和17.32、23.40 μg/mL;2种药剂对烟草白粉病菌均具有较长的持效期,在有效成分56.25 g/hm2 剂量下、2次喷雾处理烟苗,30 d后的防效仍分别达到94.45%和88.53%;2种药剂对新生烟叶均具有良好的保护作用,且在实验剂量范围内对烟苗安全。     

Toxicity of novel aromatic and aliphatic organic acid and ester analogs of trypsin modulating oostatic factor to larvae of the northern house mosquito, Culex pipiens complex, and the tobacco hornworm, Manduca sexta

Eight non-peptidic chemical analogs of trypsin modulating oostatic factor (TMOF, NH₂-YDPAP₆), an insect hormone inhibiting trypsin biosynthesis in mosquitoes, were synthesized based on the structure of the native peptide. The median lethal concentration (LC₅₀) for the chemical analogs, TMOF and FDPAP (a peptidic analog of TMOF) was estimated for larvae of the northern house mosquito, the Culex pipiens complex, using a static 5-day bioassay. Four of these compounds demonstrated the same larvicidal activity as TMOF, while three of these compounds were 1.2-2.5-fold more active than TMOF. The compounds introduced by injection were toxic to fourth instars of the tobacco hornworm, Manduca sexta, except for TMOF, FDPAP, and PPHEN. Injection of TMOF and FDPAP into fourth stadium and TMOF into second stadium M. sexta had no effect on trypsin activity, growth, or mortality. Apparently the mosquito hormone is inactive in the tobacco hornworm at the developmental stages examined. Three TMOF analogs (CHEA, PHEA, and PHA) demonstrating the highest activity by injection in M. sexta were also found to be toxic by injection in fourth instars of the tobacco budworm, Heliothis virescens, and the cotton bollworm, Helicoverpa zea, as well as adult male German cockroaches, Blattela germanica. A two-choice feeding bioassay with H. virescens indicated that at least one of the TMOF analogs, PHEA, has anti-feeding properties.     

刺探电位图谱(EPG)技术的原理与发展

刺探电位图谱(EPG)技术是用来记录刺吸式口器昆虫口针在寄主组织中刺探行为的电信号变化特征的技术,主要用于昆虫刺探(取食)、传毒行为,植物抗虫机制等的研究,本文对其发展历史、基本原理、波形特征和生物学意义进行了综述,并讨论了该技术目前存在的问题及应用前景。     

Herbicide and inhibitor effects on cell leakage in suspension cultures of tobacco (Nicotiana tabacum)

Effects of selected herbicides and respiratory inhibitors on leakage from tobacco (Nicotiana tabacum) cell suspension cultures were studied. Leakage was monitored by quantitation of fluorescein dye released from preloaded cells and by measuring conductivity changes in the suspension medium. The herbicides ioxynil, Barban, 2,4,5-T, MCPB, and PCP (10^?6 to 10^?4M) caused leakage of fluorescein dye and electrolytes within 2 hr of exposure to the herbicides. Potassium cyanide and 2,4-DNP caused appreciable leakage at the same concentrations. Similar responses were induced by anaerobiosis. Atrazine, metolachlor, paraquat, and nitrofen did not induce leakage when tested at concentrations of 10^?6 to 10^?4M.