菜蛾绒茧蜂和小菜蛾对杀虫剂的敏感性及酶学特性的比较研究

分别采用药膜法和浸叶法测定了菜蛾绒茧蜂Apanteles plutellae和小菜蛾Plutella xylostella对杀虫剂的敏感度。结果显示： 有机磷、氨基甲酸酯、拟除虫菊酯类杀虫剂、阿维菌素和锐劲特对菜蛾绒茧蜂高毒,而抑太保和Bt为低毒,然而,短时间(1 h)接触常规防治剂量的锐劲特、氰戊菊酯、氯氰菊酯和乙酰甲胺磷对菜蛾绒茧蜂低毒。增效剂胡椒基丁醚（PB）、磷酸三苯酯（TPP）和马来酸二乙酯（DEM）对菜蛾绒茧蜂的甲胺磷、克百威、氰戊菊酯、氯氰菊酯、阿维菌素和锐劲特敏感性增效显著,但对抑太保无增效作用。PB的增效作用显著高于TPP 和DEM。PB和TPP对菜蛾绒茧蜂羧酸酯酶(CarE),以及DEM对谷胱甘肽S转移酶(GST)具显著的活体抑制作用,但PB,TPP和 DEM对菜蛾绒茧蜂乙酰胆碱酯酶（AChE）无抑制作用。菜蛾绒茧蜂AChE的米氏常数(K_m)、最大反应速度(V_max)、CarE和GST活性分别为小菜蛾的0.22、2.08、4.60和0.45倍,甲胺磷、敌敌畏和克百威对菜蛾绒茧蜂AChE的双分子速度常数(Ki)分别为对小菜蛾的14.7、10.5 和26.0倍。酶与抑制剂反应温度增高将导致酶抑制率增高,尤其对菜蛾绒茧蜂AChE的抑制作用更为显著。上述结果表明,菜蛾绒茧蜂对有机磷和氨基甲酸酯类杀虫剂的高敏感性与其显著高的AChE敏感性有关,氧化代谢的解毒作用对菜蛾绒茧蜂耐药性的影响大于水解作用。此外,对小菜蛾和菜蛾绒茧蜂杀虫剂敏感性差异的毒理学原因进行了讨论。     

小菜蛾及菜蛾绒茧蜂乙酰胆碱酯酶敏感性的相关变化

用生物测定和生化检测的方法,对福州地区小菜蛾Plutella xylostella和菜蛾绒茧蜂Apanteles plutellae的抗药性及两种昆虫乙酰胆碱酯酶对杀虫剂的敏感性进行了田间监测。结果显示,从1998年9月至1999年4月,小菜蛾乙酰胆碱酯酶对6种有机磷和氨基甲酸酯杀虫剂敏感性逐渐恢复,寄生于同一虫源的菜蛾绒茧蜂乙酰胆碱酯酶敏感性的变化也呈明显的相关性,但菜蛾绒茧蜂乙酰胆碱酯酶的敏感性高于其寄主小菜蛾。脱离选择压力后,两种昆虫对杀虫剂的敏感性迅速恢复,乙酰胆碱酯酶的K_i值显著增高。对乙酰胆碱酯酶的K_m、V_max和K_i值测定结果表明,两种昆虫对有机磷和氨基甲酸酯杀虫剂的抗性与乙酰胆碱酯酶对杀虫剂的不敏感性有关。此外还研究了不同发育期小菜蛾乙酰胆碱酯酶活性及其K_i值的变化。探讨了在杀虫剂选择压力下,两种昆虫乙酰胆碱酯酶敏感性的环境适应性变化机制。     

Effects of synergists on toxicity of six insecticides in parasitoid Diaeretiella rapae (Hymenoptera: Aphidiidae)

The resistance to and the effects of synergists on the toxicity of six insecticides in Diaeretiella rapae (M'Intosh) (Hymenoptera: Aphidiidae), a parasitoid of vegetable aphid collected in Jianxin at Fuzhou-City, Fujian, China, were studied. In comparison with susceptible F21 progeny, the resistance ratios in resistant F0 parents were 27.6 for methamidophos, 20.8 for fipronil, 47.5 for avermectin, 3.3 for fenvalerate, 4.5 for cypermethrin, and 74.7 for imidacloprid. Piperonyl butoxide (PB), triphenyl phosphate (TPP), and diethyl maleate (DEM) were chosen to be applied in susceptible F21 progeny, as well as in resistant F11 progeny and F0 parents. Significant synergistic effects on the toxicity of the six insecticides were found by using PB, TPP, and DEM in F0 parents; on methamidophos, avermectin, and imidacloprid by PB, TPP, and DEM in F11 progeny; on fipronil by PB and DEM in F11 progeny; and on fenvalerate and cypermethrin by PB in F11 progeny. PB also showed significant synergism on the six insecticides in susceptible F21 progeny, although the synergism was far less in F21 progeny than those in resistant F0 parents. TPP and DEM showed little or no synergistic effects on the toxicity of the six insecticides in F21 progeny. Compared with TPP and DEM, the highest synergistic ratios of PB for methamidophos, fipronil, avermectin, fenvalerate, cypermethrin, and imidacloprid were observed in F0 parents, and F11 and F21 progeny. The resistance levels to methamidophos, fipronil, avermectin, fenvalerate, and cypermethrin could be inhibited strongly by applying PB in F0 parents. From the results, oxidative degradation is believed to play a critical role in resistance to methamidophos, fipronil, avermectin, fenvalerate, and cypermethrin in D. rapae. To a lesser extent, hydrolytic reactions also were partially involved in the resistance to these five insecticides by using the synergists PB, TPP, and DEM. However, although high synergism of PB, TPP, and DEM on imidacloprid was found, the resistance levels to imidacloprid remained high in the presence of PB, TPP, and DEM. The mediated detoxification of oxidative degradation and hydrolytic reactions was thought to be involved in the resistance to imidacloprid in F0 parents.     

小菜蛾有机磷抗性季节性变化及毒理机制研究

1998～ 2 0 0 3年期间的田间监测结果显示 ,福州地区小菜蛾 Plutella xylostella对甲胺磷和敌敌畏的抗性水平呈季节性变化 ,每年春秋季的抗性水平显著高于夏季 7月期间的抗性水平。与 2 5℃ 2 d的饲养条件相比 ,35℃ 2 d和 5℃ 30 d可使小菜蛾羧酸酯酶 (Car E)和谷胱甘肽 - S-转移酶 (GST)活力显著抑制 ,35℃ 2 d还可使小菜蛾乙酰胆碱酯酶 (ACh E)活力显著抑制 ,但 5℃4 d对小菜蛾 ACh E、Car E和 GST活力影响差异不显著。当反应温度均为 2 5℃时 ,在 35℃ 2 d、2 5℃ 2 d和 5℃ 30 d下饲养的小菜蛾 ACh E对甲胺磷和克百威的敏感性 (K i值 )没有明显变化 ,但酶与杀虫剂在 37℃的反应温度下 ,甲胺磷、敌敌畏和克百威对在 2 5℃下饲养的小菜蛾 ACh E的抑制作用显著增高。上述结果表明环境温度会影响小菜蛾的解毒代谢及杀虫剂对 ACh E的抑制作用。探讨了环境温度的季节性变化对田间小菜蛾有机磷抗性水平影响的毒理学机制。     

Effects of Bt plants on the development and survival of the parasitoid Cotesia plutellae (Hymenoptera: Braconidae) in susceptible and Bt-resistant larvae of the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae)

A range of crops have been transformed with delta-endotoxin genes from Bacillus thuringiensis (Bt) to produce transgenic plants with high levels of resistance to lepidopteran pests. Parasitoids are important natural enemies of lepidopteran larvae and the effects of Bt plants on these non-target insects have to be investigated to avoid unnecessary disruption of biological control. This study investigated the effects of Cry1Ac-expressing transgenic oilseed rape (Brassica napus) on the solitary braconid endoparasitoid Cotesia plutellae in small-scale laboratory experiments. C. plutellae is an important natural enemy of the diamondback moth (Plutella xylostella), the most important pest of brassica crops world-wide. Bt oilseed rape caused 100% mortality of a Bt-susceptible P. xylostella strain but no mortality of the Bt-resistant P. xylostella strain NO-QA. C. plutellae eggs laid in Bt-susceptible hosts feeding on Bt leaves hatched but premature host mortality did not allow C. plutellae larvae to complete their development. In contrast, C. plutellae developed to maturity in Bt-resistant hosts fed on Bt oilseed rape leaves and there was no effect of Bt plants on percentage parasitism, time to emergence from hosts, time to adult emergence and percentage adult emergence from cocoons. Weights of female progeny after development in Bt-resistant hosts did not differ between plant types but male progeny was significantly heavier on wildtype plants in one of two experiments. The proportion of female progeny was significantly higher on Bt plants in the first experiment with Bt-resistant hosts but this effect was not observed again when the experiment was repeated.     

Detection of insecticide resistance in Plutella xylostella (L.) (Lepidoptera: Plutellidae) populations in South Australian crucifer crops

In response to reports of failures of sprays to control Plutella xylostella infestations in vegetable-growing areas around Adelaide in 1991, the extent of resistance to six insecticides in several South Australian populations of P. xylostella was determined. Populations sampled from vegetable crops in the Adelaide region were five- to 200-fold resistant to organophosphates (chlorpyrifos, methamidophos and mevinphos), eight- to 400-fold resistant to pyrethroids (esfenvalerate and permethrin) and six-fold resistant to the carbamate methomyl compared with a susceptible strain. Mevinphos and esfenvalerate were the most effective of the tested insecticides. These, together with Bacillus thuringiensis products, have continued until 1998 to be used almost exclusively for the control of P. xylostella in South Australian vegetable crops in the absence of any resistance management strategy.     

Comparison of Diadegma insulare (Hymenoptera: Ichneumonidae) and Microplitis plutellae (Hymenoptera: Braconidae) as biological control agents of Plutella xylostella (Lepidoptera: Plutellidae): field parasitism, insecticide susceptibility, and host-searching

Parasitism of Plutella xylostella (L.) third and fourth instars was evaluated in a cabbage field in Geneva, NY, in 1999. Over the entire season, average parasitism was 33.6% for third instars and 53.6% for fourth instars, and the main parasitoids were Diadegma insulare (Cresson) and Microplitis plutellae Muesbeck. In the early season, total parasitism was low, and mainly caused by D. insulare. However, later in the season, parasitism reached >80% for the fouth instars and 50% for the third instars. Our survey indicated that M. plutellae heavily parasitized P. xylostella, and provided higher parasitism rates than D. insulare in the late season. Comparison of these two species in laboratory bioassays indicated there were no significant differences in susceptibility to four insecticides commonly used in crucifer fields. For both parasitoids, an experience with P. xylostella on a damaged leaf increased their host-searching efficacy. Compared with M. plutellae, D. insulare was a better host-searcher both for the naive and the experienced adults. Although both parasitoids can cause high mortality rates of P. xylostella, D. insulare may be more suitable to be released in fields to enhance natural control against P. xylostella.     

不同地理种群小菜蛾对甲胺磷的抗药性

采用浸叶法研究了福州建新镇、福建农林大学校园内、闽侯县上街镇和闽侯县甘蔗镇等地田间小菜蛾种群对甲胺磷的抗性水平及抗性机制。结果表明,与敏感小菜蛾相比,上述4个田间小菜蛾种群已对甲胺磷产生了34-82倍的抗性水平。敏感小菜蛾乙酰胆碱酯酶(AChE)的Ki值是田间抗性小菜蛾AChE的Ki值的17.3-39.1倍。不同小菜蛾种群对甲胺磷的抗性水平也存在着差异,小菜蛾种群AChE对甲胺磷的敏感性大小与其对甲胺磷的抗性水平高低显著相关。     

抑制剂存在下不同种群烟粉虱乙酰胆碱酯酶残余活性频率分布及其与抗药性的关系

为了探讨烟粉虱Bemisia tabaci不同种群个体乙酰胆碱酯酶敏感性差异及其与抗药性的关系, 我们选用室内饲养的烟粉虱SUD S敏感品系和6个田间抗性种群, 采用酶标板酶动力学法测定了各品系 (种群）乙酰胆碱酯酶对抑制剂的敏感性反应以及抑制剂存在时各抗性种群个体乙酰胆碱酯酶残余活性频率分布。结果表明: 在抑制剂浓度为300 μmol/L时, 敏感品系乙酰胆碱酯酶的活性基本上被完全抑制, 可以明显地区分敏感品系与田间抗性种群。在抑制剂浓度为2 000 μmol/L时, 各抗性种群个体乙酰胆碱酯酶残余活性频率分布差异明显, 其中ZZ-R种群和FZ-R种群的乙酰胆碱酯酶残余活性频率分布相似, 大部分个体的乙酰胆碱酯酶残余活性分布在1.00~1.80 mOD/min之间; SM-R种群和ND-R种群的乙酰胆碱酯酶残余活性频率分布也相似, 大部分个体的乙酰胆碱酯酶残余活性分布在0.40~1.00 mOD/min之间; LY-R和NP-R种群大部分个体的乙酰胆碱酯酶残余活性分别分布在1.00~1.60 mOD/min和0.80~1.20 mOD/min之间。各抗性种群乙酰胆碱酯酶高残余活性 (大于1.00 mOD/min）个体频率与对敌敌畏的抗性水平之间具有明显相关性, 相关系数为0.86 (P<0.05）。考虑到乙酰胆碱酯酶对抑制剂作用不敏感是一些昆虫对有机磷和氨基甲酸酯类杀虫剂抗性的重要机制之一, 建议可以将乙酰胆碱酯酶对敌敌畏的敏感性作为烟粉虱抗药性生化检测的一个参考指标。     

B型烟粉虱田间种群对毒死蜱和敌敌畏抗性的生化机制

通过增效剂生物测定和生化分析,探讨了采自福建省的B型烟粉虱Bemisia tabaci 6个田间种群对毒死蜱和敌敌畏抗性的生化机制。结果表明：与敏感品系SUD-S相比,6个田间种群对毒死蜱和敌敌畏分别具有54.53～78.43倍和6.23～11.25倍的抗性。TPP、PBO和DEM对毒死蜱的增效比分别为3.61～24.94倍、1.14～1.76倍和1.04倍,对敌敌畏的增效比分别为1.67～2.64倍、1.33～1.65倍和1.09倍,表明羧酸酯酶的解毒代谢在烟粉虱对毒死蜱的抗性中起着重要作用。烟粉虱抗性种群乙酰胆碱酯酶的K_m值是敏感品系的1.83～4.0倍,V _max值是敏感品系的0.34～0.62倍; 敏感品系乙酰胆碱酯酶的活性在底物浓度大于1.0 mmol/L时受抑制,抗性种群乙酰胆碱酯酶的活性在底物浓度大于16 mmol/L时受抑制;抗性种群乙酰胆碱酯酶对敌敌畏和毒死蜱的敏感度分别比敏感品系低119.92～161.33倍和10.11～14.24倍,表明烟粉虱田间抗性种群乙酰胆碱酯酶可能已发生了变构,由变构引起的乙酰胆碱酯酶不敏感是烟粉虱田间种群对毒死蜱和敌敌畏产生抗性的重要原因。结果提示,乙酰胆碱酯酶不敏感性和羧酸酯酶的解毒代谢在烟粉虱对毒死蜱的抗性中均起着重要作用,而乙酰胆碱酯酶不敏感性在对敌敌畏的抗性中起重要的作用,多功能氧化酶和谷胱甘肽S转移酶在烟粉虱对毒死蜱和敌敌畏抗性中所起的作用不大。     

Characteristics Of Parasitism Of Plutella Xylostella (Lep., Plutellidae) By Oomyzus Sokolowskii (Hym., Eulophidae)

Laboratory and greenhouse studies were conducted on Oomyzus sokolowskii Kurdjumov, a parasite of the crucifer pest Plutella xylostella ( L.). O. sokolowskii preferred the 3^rd and 4^th instar P. xylostella larvae over fresh pupae for parasitization. It is thus a larval parasite. Within the range of 10?C to 35?C, parasitism was positively correlated with temperature. High parasitism at temperatures of 30?C and 35?C indicates that this insect is suitable for the control of P. xylostella in the tropical lowlands. In a no- choice test where only fresh pupae of Cotesia plutellae Kurdjumov, another potentially competing larval parasite of P. xylostella, were offered, O. sokolowskii failed to parasitize pupae of C. plutellae. In a choice test where the 4^th instar P. xylostella larvae and fresh C. plutellae pupae were offered, O. sokolowskii parasitized only P. xylostella larvae. This parasite, therefore, is not a hyperparasite of P. xylostella. When C. plutellae-ovvposited P. xylostella larvae were offered at intervals, O. sokolowskii, parasitized only freshly oviposited host larvae. The longer the period that elapsed after C. plutellae oviposition of P. xylostella larvae, the lesser was the parasitism of these larvae by O. sokolowski.     

Monitoring insecticide resistance in Australian Frankliniella occidentalis Pergande (Thysanoptera: Thripidae) detects fipronil and spinosad resistance

Abstract  Insecticide resistance monitoring using a Potter precision spray tower with discriminating concentration and log dose probability techniques underpins the Australian insecticide management strategy for Frankliniella occidentalis Pergande. Abamectin, acephate, chlorpyrifos, dichlorvos, dimethoate, endosulfan, fipronil, malathion, methamidophos methidathion, methiocarb, methomyl, pyrazophos and spinosad are recommended for use against F. occidentalis but abamectin, methiocarb and pyrazophos are the only chemicals where insecticide resistance has not been detected. Although not registered, chlorfenapyr was effective against F. occidentalis and should be pursued for that purpose. In contrast, chlorpyrifos, dichlorvos and malathion resistance were detected at low to moderate levels throughout the study period putting their sustainable use for F. occidentalis control in doubt . Although it appears that acephate, dimethoate, endosulfan, fipronil, methamidophos, methidathion and spinosad remain effective, some populations contained a small percentage of thrips that survived exposure to a concentration that killed 100% of the susceptible strain. Subsequent laboratory selection of one such population separately with fipronil and spinosad caused an increase in resistance to these insecticides. These products must now be considered at risk. This is the first report of fipronil or spinosad resistance in populations of F. occidentalis.     

Lack of intraspecific biological variation between two geographical populations of Oomyzus sokolowskii (Hymenoptera: Eulophidae), a gregarious larval-pupal parasitioid of Plutella xylostella (Lepidoptera: Plutellidae)

The chalcid, Oomyzus sokolowskii Kurdjumov has been recorded in many parts of the world as a major larval-pupal, gregarious endoparasitoid of the diamondback moth, Plutella xylostella (Linnaeus), a serious pest of brassica vegetable crops worldwide. This study investigated intraspecific variation between two populations of O. sokiolowskii, one from Cape Verde Islands, West Africa and the other from Hangzhou, China. In all crosses and backcrosses between the two geographical populations, the numbers of progeny and sex ratio of progeny were similar to those obtained within each of the populations, demonstrating complete reproductive compatibility between the two populations. The two populations showed similar responses to temperature with respect to development time and survival of immature stages. Observations on the interactions between the two O. sokolowskii populations and Cotesia plutellae (Kurdjumov), another major parasitoid of P. xylostella, showed that neither population could achieve successful parasitism of P. xylostella larvae already parasitized by C. plutellae. However, both O. sokolowskii populations could achieve hyperparasitism by ovipositing into a mid-late stage larva of C. plutellae developing inside the primary host. Contrary to earlier reports, no evidence of intraspecific variations in ability to hyperparasitize between these two populations of O. sokolowskii was found.     

小菜蛾抗性个体不敏感乙酰胆碱酯酶的鉴定

乙酰胆碱酯酶(AChE)敏感性降低是小菜蛾对有机磷和氨基甲酸酯产生抗性的重要机制之一,已得到广泛的承认和报道。一种用硝酸纤维素膜的斑点法鉴定个体小菜蛾的抗性AChE不敏感性的应用技术,提供了早期侦测和随后监测田间种群抗性的可能性。此法操作简便灵敏。小菜蛾抗性品系(GBR)和田间种群成虫头部AChE活力,在残杀威抑制时,抑制率分别为50.97%和43.96%,有对氧磷存在时,分别为63.78%和35.87%,较敏感品系的AChE为不敏感。     

寄主抗药性对菜蛾绒茧蜂抗药性发展的影响

室内筛选小菜蛾中抗品系（SRP）、高抗品系（RP）、以敏感小菜蛾幼虫（SP）为寄主的菜蛾绒茧蜂SRC品系和以SRP幼虫为寄主的菜蛾绒茧蜂RSC品系对氰戊菊酯的抗性。分别筛选了13、14、14和13代。小菜蛾SRP和RP品系分别获得了68.9和605.8倍的抗性,菜蛾绒茧蜂SRC和RSC品系分别获得了4.3和11.0倍的抗性。上述结果表明通过施药于体内有寄生蜂的小菜蛾幼虫筛选寄生蜂抗性,可以获得具有抗性的寄生蜂。以SRP为寄主的RSC品系的抗性水平高于以SP为寄主的SRC品系的抗性水平,表明和抗性较高的寄主同步筛选,寄生蜂的抗性发展更快。小菜蛾SP、SRP和RP三个品系幼虫的多功能氧化酶（MFO）活性比为1∶1.15∶1.50;菜蛾绒茧蜂SC、SRC和RSC三个品系幼虫的MFO活性比为1∶1.10∶1.49,成蜂的MFO活性比为1∶1.18∶1.54;而每种昆虫不同品系的羧酸酯酶（CarE）、总酯酶（Es）活性水平与其抗性水平变化不一致,表明抗性与MFO活性升高有关,而与CarE和Es的活性无关。     

Seasonal abundance of the parasitoid complex associated with the diamondback moth, Plutella xylostella (Lepidoptera: Plutellidae) in Hangzhou, China

An investigation of insect parasitoids of the diamondback moth, Plutella xylostella (Linnaeus), in brassica vegetable crops in the suburbs of Hangzhou was conducted during five periods from 1989 to 1997. Eight species of primary parasitoids were recorded: Trichogramma chilonis Ishii, Cotesia plutellae Kurdjumov, Microplitis sp., Oomyzus sokolowskii Kurdjumov, Diadromus collaris (Gravenhorst), Itoplectis naranyae (Ashmead), Exochus sp. and Brachymeria excarinata Gahan. Seven species of hyperparasitoids were also collected. Rates of parasitism of eggs of P. xylostella were usually very low. However, rates of parasitism of larvae and pupae were substantial and showed two peaks each year, around June-July and September-November respectively. Rates of parasitism during peaks were usually 10-60% and reached over 80% on a few occasions. Cotesia plutellae, O. sokolowskii and D. collaris were the major larval, larval-pupal and pupal parasitoids respectively. In the field, C. plutellae was active throughout the year. Oomyzus sokolowskii was active from May to October, entered a quiescent pupal stage in October-November to overwinter and did not emerge until next April-May. Diadromus collaris was recorded from April to July and October. Rates of parasitism of P. xylostella in radish and mustard fields were usually higher than those in cabbage and Chinese cabbage fields in the same locality. Negative correlations of parasitism rates between C. plutellae and O. sokolowskii indicate a competitive relationship for host larvae between these two larval parasitoids.     

菜蚜茧蜂对甲胺磷和灭多威的抗性机制

采用试管药膜法测定了菜蚜茧蜂 Diaeretiella rapae 对甲胺磷和灭多威的抗性及增效剂的增效作用,并测定了菜蚜茧蜂和菜缢管蚜 Lipaphis erysimi 乙酰胆碱酯酶动力学参数、解毒酶活性及增效剂对菜蚜茧蜂酶活性的体内抑制作用。福州地区菜蚜茧蜂已对甲胺磷和灭多威产生5.6和9.1倍的抗性,胡椒基丁醚、磷酸三苯酯和马来酸二乙酯对两种杀虫剂的抗性品系均有显著增效作用,胡椒基丁醚的作用最为显著。胡椒基丁醚对抗性菜蚜茧蜂杀虫剂敏感性的增效作用远高于对敏感菜蚜茧蜂的影响。3种增效剂对菜蚜茧蜂乙酰胆碱酯酶均无抑制作用;磷酸三苯酯和胡椒基丁醚对羧酸酯酶,马来酸二乙酯对谷胱甘肽S转移酶有显著抑制作用。抗性和敏感菜蚜茧蜂乙酰胆碱酯酶的米氏常数K_m）、最大反应速度（V_max）、羧酸酯酶及谷胱甘肽S转移酶活性值相近,但敏感菜蚜茧蜂乙酰胆碱酯酶的双分子速度常数（K_I）值远高于抗性的。此外,对菜蚜茧蜂和菜缢管蚜乙酰胆碱酯酶、羧酸酯酶和谷胱甘肽S转移酶进行了比较研究。结果表明菜蚜茧蜂对甲胺磷和灭多威的低水平抗性与乙酰胆碱酯酶的不敏感性及解毒酶的解毒代谢有关。     

Inhibitory effects of four carbamate insecticides on acetylcholinesterase of male and female Carassius auratus in vitro

The inhibitory effects of four carbamate insecticides, methomyl, thiodicarb, carbofuran and carbosulfan, on acetylcholinesterase of male and female Carassius auratus were investigated in the laboratory. Kinetic constants, biomolecular rate constant (k(i)), carbamylation rate constant (k2) and decarbamylation rate constant (k3) were determined in vitro. The ratios of bimolecular rate constant (female/male) for AChE reacting with methomyl, thiodicarb, carbofuran and carbosulfan were 1.03, 2.44, 1.03 and 1.106, respectively. The k(i) of AChE for thiodicarb was significantly higher in female fish than in male fish (P<0.05). The ratios of carbamylation rate constant (female/male) for methomyl, thiodicarb, carbofuran and carbosulfan were 1.18, 4.29, 3.53, and 2.07, respectively. The k2 values of AChE for the above four carbamates were significantly higher in females than in males. The ratios of the decarbamylation rate constant (female/male) for methomyl, thiodicarb, carbofuran and carbosulfan were 1.02, 1.39, 1.06, and 1.98, respectively. Only for carbosulfan, the decarbamylation rate of AChE was significantly higher in the female than in the male, indicating that AChE of females inhibited by carbosulfan recovered more quickly than that of males. The above results suggest that the female in the sensitivity of AChE to carbamates was different from the male fish.     

Comparative investigation between acetylcholinesterase obtained from commercial sources and genetically modified Drosophila melanogaster: application in amperometric biosensors for methamidophos pesticide detection

Genetically modified acetylcholinesterase (AChE) from Drosophila melanogaster (dm) and from commercial sources, Electric eel (ee), Bovine erythrocites (be) and Human erythrocites (he), were investigated as biological receptors for the detection of methamidophos pesticide based on inhibition studies. Most engineered variant of AChE from dm showed enhanced sensitivity toward methamidophos pesticide. Among 24 dmAChE variants tested, 12 presented a sensitivity comparable to the commercially available eeAChE, but higher than AChEs from be and he. Four were found more sensitive and six others were insensitive to methamidophos insecticide. The D375G,Y370F,Y374A,F376L mutant was the most sensitive, with a ki value of 2.2 X 10(6) mol(-1) L min(-1), three orders of magnitude higher than eeAChE (1.1 X 10(3) mol(-1) L min(-1)). The sensor constructed with genetically modified enzyme showed better characteristics with respect to detection limit and sensitivity compared with those using commercial eeAChE. Differential pulse polarography and chronoamperometry were used as electrochemical techniques to characterize the AChE biosensors. The lower detection limit of 1 ppb was obtained with D375G,Y370F,Y374A,F376L mutant of dmAChE, compared to 90 ppb for the commercial eeAChE. This study may stimulate scientists to develop more sensitive and selective procedures for organophosphorus insecticides detection by using engineered variant of dmAChE.     

Selective spectrophotometric detection of insecticides using cholinesterases,phosphotriesterase and chemometric analysis

Enzyme spectrophotometric assays based on acetylcholinesterase (AChE) inhibition were used in combination with Artificial Neural Network (ANN) chemometric analysis for the resolution of pesticides mixtures of chlorpyriphos, dichlorvos and carbofuran. Electric eel (EE) AChE and the recombinant B394-AChE from Drosophila melanogaster were selected due to their different sensitivities to insecticides. These enzymes were used in association with phosphotriesterase (PTE), an enzyme allowing to discriminate between organophosphate and carbamate insecticides. The combined response of three enzymes systems composed of EE-AChE, EE-AChE + PTE, and B394-AChE + PTE was modelled by means of ANN. Specifically, an ANN was constructed where the structure providing the best modelling was a single hidden layer containing four neurons. To prove the concept, a study to resolve pesticide mixtures was done with spectrophotometric measurements. Finally the developed system was successfully applied to the determination of carbofuran, CPO and dichlorvos pesticides in real water samples.