植物次生物质和杀虫剂对分月扇舟蛾谷胱甘肽S-转移酶的抑制作用

本论文采用分光光度计法研究了植物次生物质和杀虫剂对分月扇舟蛾Closteraanastomosis(L.)谷胱甘肽S-转移酶(GSTs)的体外抑制作用。结果表明各植物次生物质和杀虫剂对分月扇舟蛾GSTs活性的体外抑制作用存在差异。当植物次生物质和杀虫剂终浓度为8.34×10-5mol/L时,槲皮素和单宁酸对分月扇舟蛾GSTs活性抑制作用最强,分别为64.41%和58.61%;三唑磷、毒死蜱、辛硫磷、氟铃脲、氟虫腈和哒螨灵都有较强的抑制作用,对GSTs活性抑制率超过20%;氧化乐果、水胺硫磷、丙溴磷、马拉硫磷、灭多威、联苯菊酯、高效氯氰菊酯、甲氰菊酯、高效氯氟氰菊酯、吡虫啉和啶虫脒对分月扇舟蛾GSTs有中等抑制作用;其他杀虫剂对分月扇舟蛾GSTs有较弱(或没有)抑制作用。此外,在一定的浓度范围内,槲皮素、单宁酸和辛硫磷对分月扇舟蛾GSTs活性的抑制作用存在明显的剂量效应关系。槲皮素、单宁酸和辛硫磷对分月扇舟蛾GSTs的抑制中浓度分别为2.19×10-5,2.62×10-5,1.49×10-4mol/L。因此,此研究明确了槲皮素和单宁酸作为防治分月扇舟蛾的新型防治剂或增效剂具有很好的潜力。     

6种植物次生物质对斜纹夜蛾解毒酶活性的影响

草食性昆虫取食植物时遇到宿主植物中大量次生物质的化学防御,研究昆虫适应植物毒素的反防御策略具有重要的科学意义。分别添加0.01%肉桂酸、0.01%水杨酸、0.01%花椒毒素、0.02%槲皮素、0.05%黄酮和0.1%香豆素等6种植物次生物质的人工饲料饲养斜纹夜蛾(Spodoptera litura)五龄幼虫48 h后,测定斜纹夜蛾幼虫中肠和脂肪体中谷胱甘肽S-转移酶(GSTs)、羧酸酯酶(CarE)、P450的酶含量及头部乙酰胆碱酯酶(AChE)的活性,利用半定量RT-PCR检测中肠和脂肪体中CYP4M14和CYP4S9的基因表达水平。结果表明:取食肉桂酸和香豆素后,斜纹夜蛾中肠中CarE的酶活性分别提高了1.67和1.37倍,取食6种次生物质均能显著提高斜纹夜蛾脂肪体中GSTs酶活性。取食肉桂酸和香豆素48 h后,脂肪体中P450酶含量比对照增加2.93和14.50倍。取食肉桂酸、花椒毒素、槲皮素和香豆素后,斜纹夜蛾头部AchE酶活性与对照相比提高了1.53、1.80、2.36和1.56倍。6种次生物质均可诱导脂肪体中CYP4M14基因表达,槲皮素、肉桂酸和香豆素强烈诱导CYP4S9在脂肪体中表达。表明,斜纹夜蛾具有利用植物次生物质诱导其解毒酶的能力,进而提高其对毒素的抗性。     

单宁酸对棉铃虫谷胱甘肽S转移酶的影响

通过培养基混药法,研究了植物次生物质单宁酸对棉铃虫Helicoverpa armigera（Hübner）谷胱甘肽S-转移酶活性的影响。谷胱甘肽S-转移酶活性随棉铃虫发育期的进程而变化,在卵期最低,5龄、6龄幼虫和成虫期最高。用含0.005%单宁酸的饲料饲喂棉铃虫后,5龄和6龄幼虫的谷胱甘肽S-转移酶活性明显降低,分别为对照的59%和67%。单宁酸低剂量、短时间处理棉铃虫幼虫,可诱导中肠和脂肪体中的谷胱甘肽S-转移酶的活性增加,高剂量或低剂量长时间处理没有诱导增加作用,甚至还有抑制作用。单宁酸连续处理4代,对棉铃虫6龄幼虫中肠谷胱甘肽S 转移酶均有抑制作用,对脂肪体谷胱甘肽S-转移酶活性无明显影响或有抑制作用。单宁酸处理的第4代幼虫对溴氰菊酯的敏感度有增加的趋势,对甲基对硫磷的敏感度没有明显改变。     

七种抑制剂对两种白蚁谷胱甘肽S-转移酶活性抑制作用的比较

利用分光光度酶动力学方法,确定了白蚁谷胱甘肽S-转移酶（GSTs）的最适反应条件,并进一步研究了7种抑制剂对黑翅土白蚁Odontotermes formosanus (Shiraki)和黑胸散白蚁Reticulitermes chinensis Snyder GSTs活性的体外影响。结果表明：白蚁GSTs测定的最适反应条件为pH 6.5,温度25℃,最适反应时间2 min。黑翅土白蚁GSTs的米氏常数(K_mCDNB和K_mGSH)分别为0.11±0.02 mmol/L和0.81±0.16 mmol/L,最大反应速度(V_maxCDNB和V_maxGSH)分别为425.92±19.67 nmol/(min·mg)和534.86±39.05 nmol/(min·mg)。黑胸散白蚁GSTs的米氏常数(K_mCDNB和K_mGSH)分别为0.12±0.03 mmol/L和1.03±0.31 mmol/L,最大反应速度(V_maxCDNB和V_maxGSH)分别为544.39±37.19 nmol/(min·mg)和715.45±83.68 nmol/(min·mg)。浓度为2×10^-5 mol/L时,槲皮素和辛硫磷对黑胸散白蚁GSTs活性的抑制作用要强于黑翅土白蚁,对黑胸散白蚁GSTs活性的抑制作用分别为62.28％和44.89％,对黑翅土白蚁GSTs活性的抑制作用分别为54.96％和28.36％。高效氯氰菊酯、甲氰菊酯、啶虫脒和单宁酸对黑翅土白蚁GSTs活性的抑制作用要强于黑胸散白蚁,对黑翅土白蚁GSTs活性的抑制作用分别为39.43％,72.07％,52.24％和82.19％;对黑胸散白蚁GSTs活性的抑制作用分别为14.96％,40.23％,39.96％和57.80％。阿维菌素对黑翅土白蚁和黑胸散白蚁GSTs活性的抑制作用没有显著差异,对黑翅土白蚁和黑胸散白蚁GSTs活性的抑制作用分别为76.21％和76.88％。这表明两种白蚁对药剂的敏感性完全不同。实验结果还表明,在3.2×10^-8～2×10^-5 mol/L内,上述植物次生物质和杀虫剂对两种白蚁GSTs活性的抑制率存在明显的剂量-效应关系。     

棉铃虫的谷胱甘肽S—转移酶(GSTs)：杀虫药剂和植物次生性物质的诱导与GSTs对杀虫药剂的代谢

棉铃虫Helicoverpa armigera(Hubner)中肠谷胱甘肽S-转移酶(GSTs)对甲基对硫磷和灭多威的代谢能力明显高于对马来酸二乙酯(DEM)和两个混剂。LD₅剂量的对硫磷和灭多威对棉铃虫3龄幼虫GSTs的活性均没有诱导增加的影响,用LD₅₀的选择剂量仅对硫磷组GSTs活性增加15%。用含0.01%的芸香苷、2-十三烷酮和槲皮素的人工饲料饲养棉铃虫经1～4代后,GSTs活性提高4～18倍。3种植物次生性物质诱导组对灭多威和溴氰菊酯的敏感度均没有明显的变化,而槲皮素组对甲基对硫磷的敏感度则降低近一半,芸香苷和2-十三烷酮组对甲基对硫磷的敏感度略有降低。这种对甲基对硫磷敏感度的变化可能与上述GSTs活性的变化有关。     

棉铃虫的谷胱甘肽S-转移酶(GSTs):杀虫药剂和植物次生性物质的诱导与GSTs对杀虫药剂的代谢

棉铃虫Helicoverpaarmigera（Hubner）中肠谷胱甘肽S-转移酶（GSTs）对甲基对硫磷和灭多威的代谢能力明显高于对马来酸二乙酯（DEM）和两个混剂。LD5剂量的对硫磷和灭多威对棉铃虫3龄幼虫GSTs的活性均没有诱导增加的影响,用LD50的选择剂量仅对硫磷组GSTs活性增加15％。用含0．01％的芸香苷、2-十三烷酮和槲皮素的人工饲料饲养棉铃虫经1～4代后,GSTs活性提高4～18倍。3种植物次生性物质诱导组对灭多威和溴氰菊酯的敏感度均没有明显的变化,而槲皮素组对甲基对硫磷的敏感度则降低近一半,芸香苷和2-十三烷酮组对甲基对硫磷的敏感度略有降低。这种对甲基对硫磷敏感度的变化可能与上述GSTs活性的变化有关。     

单宁酸对杨小舟蛾谷胱甘肽S-转移酶活性的诱导

利用分光光度酶动力学的方法,确定杨小舟蛾Micromelalopha troglodyte(Graeser)谷胱甘肽S-转移酶(GSTs)的最适反应条件,并进一步研究单宁酸对杨小舟蛾GSTs活性的诱导。结果表明:杨小舟蛾GSTs测定的最适反应pH为6.5,最适反应温度为25℃。杨小舟蛾GSTs的米氏常数(KmCDNB和KmGSH)为2.63±2.32和0.61±0.10mmol/L,最大反应速度(VmaxCDNB和Vmax GSH)分别为556.26±380.02和234.12±12.84nmol/(min.mg)。单宁酸对杨小舟蛾GSTs诱导具有明显的剂量效应和时间效应关系。有效成分为0.01,0.05,0.10,0.50和1.00mg/mL的单宁酸作用48h后,杨小舟蛾体内GSTs活性分别增加了1.13,0.89,0.94,0.86和0.85倍;同样有效成分的单宁酸作用72h后,杨小舟蛾GSTs活性分别增加了1.49,1.06,1.55,1.63和0.93倍;而作用96h后,GSTs活性则分别增加了2.04,1.61,1.12,1.56和2.03倍。     

植物次生代谢物对甜菜夜蛾生长发育及解毒酶的影响

【目的】明确植物次生代谢物对甜菜夜蛾Spodoptera exigua生长发育及解毒酶的影响,探索利用植物次生物质防控甜菜夜蛾的潜在途径。【方法】本研究选用3种含量（0.01%、0.1%和1.0%）的槲皮素、山奈酚和香豆素,分别与人工饲料混合均匀后饲养甜菜夜蛾3龄初幼虫,观察植物次生代谢物对幼虫生长发育的影响;并测定幼虫取食添加0.1%的槲皮素、山奈酚和香豆素的人工饲料24、48和72 h后,幼虫羧酸酯酶（Caboxylesterase,CarE）、谷胱甘肽-S-转移酶（Glutathione-S-transferase,GSTs）和P450解毒酶活性。【结果】添加不同次生物质的人工饲料显著影响甜菜夜蛾幼虫生长和解毒酶活性。与对照组相比,3种次生代谢物均显著提高了幼虫死亡率。幼虫取食添加1%槲皮素的人工饲料后,蛹重显著降低,发育历期明显延长。而取食添加0.1%山奈酚的人工饲料后,可诱导幼虫CarE活性显著增强,0.1%槲皮素和0.1%香豆素对幼虫CarE活性均有显著抑制作用。添加槲皮素对幼虫GSTs活性无显著性影响,添加0.1%山奈酚和0.1%香豆素可诱导幼虫GSTs活性显著升高。0.1%槲皮素和0.1%香豆素可促进幼虫P450活性增强但未达到显著水平,但0.1%山奈酚处理48h后,幼虫P450活性显著降低。【结论】植物次生代谢物种类与含量对甜菜夜蛾生长发育及解毒酶活性存在不同程度的影响。     

杨扇舟蛾性引诱行为的观察

杨扇舟蛾羽化高峰在19～21时,羽化后4小时左右性成熟,雌蛾分泌性外激素,对雄蛾产生性引诱;以羽化当日性引诱力最强;这种性行为直至交配或死亡为止。活雌蛾性外激素粗提液对雄蛾也有引诱力,但诱雄蛾效果不如活雌蛾。     

亚洲小车蝗痘病毒与化学杀虫剂混用的杀虫效果及对寄主主要解毒酶活性的影响

亚洲小车蝗痘病毒（Oedaleus asiaticus entomopoxvirus, OaEPV）作为一种增效剂,分别与马拉硫磷、毒死蜱、高效氯氰菊酯、氟氯氰菊酯 、溴氰菊酯化学杀虫剂混合饲喂亚洲小车蝗若虫,统计致死中浓度 LC₅₀ 和其混合使用后的增效比;测定虫体内与抗性有关的两种重要酶——羧酸酯酶（CarE）和谷胱甘肽 S-转移酶（GSTs）的比活力。结果表明：OaEPV 与化学杀虫剂混合饲喂亚洲小车蝗,OaEPV 与毒死蜱 、高效氯氰菊酯、氟氯氰菊酯、溴氰菊酯混用对亚洲小车蝗无明显的增效作用,OaEPV 与马拉硫磷混用,具有一定的增效作用,增效比为 1.42 倍。混剂感染亚洲小车蝗,除与溴氰菊酯混用外,虫体的中肠部位 CarE 的比活力都受到了明显的抑制作用,其中 OaEPV 与马拉硫磷混用下降了 4.21 倍,抑制作用最大。当 OaEPV 与氟氯氰菊酯、溴氰菊酯化学杀虫剂混用后,中肠部位 GSTs 受到了明显的抑制作用,而其脂肪体部位 CarE 和 GSTs 的变化无一定的规律性。结果提示痘病毒与农药混合处理时,病毒主要通过抑制中肠部位 CarE 比活力而增加了农药的杀虫效果。     

Purification of fat body glutathione S‐transferase from the desert locust Schistocerca gregaria: investigation of flavonoid inhibitory effects on enzyme activity

Focus on the development of botanical insecticides such as polyphenols may represent an alternative method to chemical control. In the present study, total glutathione concentration and its related antioxidant enzymes in foregut, midgut, hindgut and fat body homogenates of the desert locust Schistocerca gregaria are examined. Glutathione S‐transferase (GST) activity exhibits a significantly higher value in fat bodies compared with other tissues. A simple and reproducible procedure for the purification of S. gregaria fat body GST is established and the purified enzyme is shown to be homogenous. The purified GST displays a typical Michaelis behaviour with respect to its substrates. Characterization of the GST, including optimum pH, substrate specificity and inhibitor effects, is carried out. The ability of some flavonoids to inhibit S. gregaria fat body GST activity is examined. High‐performance liquid chromatography analysis indicates that the major components in Glycyrrhiza glabra roots are 18α‐glycyrrhetinic acid, quercetin and rutin, and the major components in Hibiscus sabdariffa calyx are cyanidin 3‐O‐glucoside chloride and delphinidin. Quercetin and delphinidin chloride exhibit strong GST inhibition and the inhibition type is determined for both. Rutin shows a smaller inhibitory effect, whereas 18α‐glycyrrhetinic acid and cyanidin have no effect. Inhibition of S. gregaria fat body GST activity would be expected to prevent, or at least delay, the development of resistance to chemical pesticides. Among the examined levels of the antioxidant enzymes, total glutathione concentration and its related enzymes in foregut, midgut, hindgut and fat body crude homogenates of S. gregaria GST activity exhibit a significantly higher value in fat bodies compared with other tissues. Some flavonoids that are detected in H. sabdariffa calyx and G. glabra root extracts are the most effective inhibitors of the purified S. gregaria fat body GST activity. Inhibition of S. gregaria fat body GST activity by quercetin and delphinidin (major compounds detected by HPLC) would be expected to prevent, or at least delay, the development of resistance to chemical pesticides.     

Inhibition of <Emphasis Type="Italic">Naja naja</Emphasis> Venom Hyaluronidase by Plant-Derived Bioactive Components and Polysaccharides

The inhibitory effect of several bioactive compounds on the activity of hyaluronidase enzyme purified from Naja naja venom was investigated in vitro. Compounds were found to inhibit the hyaluronidase activity dose dependently. Among glycosaminoglycans, heparin, heparan sulfate, and dermatan sulfate showed maximum inhibition compared to chondroitin sulfates. Different molecular forms of chitosan inhibit the enzyme, and inhibition appears to depend on the chain length. In addition, plant-derived bioactive compounds also inhibited the activity of hyaluronidase dose dependently. Among those tested, aristolochic acid, indomethacin, quercetin, curcumin, tannic acid, and flavone exhibited inhibition, with aristolochic acid and quercetin completely inhibiting the enzyme activity. It is concluded that the inhibitors of hyaluronidase could be used as potent first aid agents in snakebite therapy. Furthermore, these inhibitors not only reduce the local tissue damage but also retard the easy diffusion of systemic toxins and hence increase survival time.     

Characterization and molecular cloning of a glutathione S-transferase from the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)

Glutathione S-transferases (GST) catalyzing the conjugation of reduced glutathione to a vast range of xenobiotics including insecticides were characterized in the whitefly Bemisia tabaci. GST activities were determined in susceptible and resistant strains of B. tabaci towards artificial substrates, i.e. 1-chloro-2,4-dinitrobenzene (CDNB) in a photometric microplate assay and monochlorobimane (MCB) in a fluoroemtric microplate assay and characterized by their Michaelis-Menten kinetics. The inhibitory potential of ethacrynic acid was very effective with IC50-values between 0.9 and 5.8 microM depending on substrate and strain. The inhibitory effect of dicumarol was 10 times lower. Glutathione-affinity chromatography purified GST enzymes of two different B. tabaci strains appeared as a single band on SDS-PAGE and had a molecular mass of 23.5 kDa determined by MALDI mass spectrometry. The N-terminus of the purified enzyme was sequenced by Edman degradation. The nearly full-length cDNA of the enzyme was isolated by RT-PCR using a degenerate primer derived from the N-terminal amino acid sequence and contained an open reading frame encoding a 194-amino-acid protein. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to insect class sigma GSTs.     

Purification and partial characterization of glutathione S-transferase from insecticide-resistant field populations of Liposcelis paeta Pearman (Psocoptera: Liposcelididae)

Enzymes that possess glutathione S-transferase (GST) activity were purified to homogeneity by glutathione-agarose affinity chromatography from three field populations of Liposcelis paeta (Pearman). These populations were collected from Nanyang city of Henan Province (NY), Wuzhou (WZ) and Hezhou (HZ) cities of Guangxi Province, China, and had different susceptibilities to dichlorvos [LC₅₀s of the NY (281.48 mg/m²), the WZ (285.07 mg/m²), and the HZ (243.52 mg/m²), respectively]. The specific activities of purified enzymes from these three populations increased 32.24-, 99.81-, and 42.52-fold, respectively. Kinetic analyses showed that the catalytic activity of purified GST from NY population towards GSH was much higher than the others, while WZ population reached the highest in V. SDS–polyacrylamide electrophoresis revealed that the purified GST had two subunits with a molecular mass of 23.31 and 20.43 kDa for NY, 53.14 and 20.13 kDa for WZ, and 50.79 and 19.42 kDa for HZ, respectively. The in vitro inhibition studies of GSTs indicated that three kinds of insecticides (chlorpyrifos, carbosulfan, and cypermethrin) and five metallic ions (Zn²⁺, Ba²⁺, Ca²⁺, Hg²⁺, Mn²⁺, and Mg²⁺) all possessed inhibitory effects on purified GST, and ethacrynic acid (EA, a specific inhibitor of GST) expressed inhibitory effects. In the bioassay, three populations of L. paeta had different susceptibilities to different insecticides, even after they were reared on diets consisting of 25% EA. The GST activities of L. paeta from different areas also showed different temperature and pH stabilities. The differences in GST among the three populations may be attributed partially to the differences in control practices for psocids between Henan and Guangxi Provinces. © 2009 Wiley Periodicals, Inc.     

Biochemical Warfare on the Reef: The Role of Glutathione Transferases in Consumer Tolerance of Dietary Prostaglandins

Background

Despite the profound variation among marine consumers in tolerance for allelochemically-rich foods, few studies have examined the biochemical adaptations underlying diet choice. Here we examine the role of glutathione S-transferases (GSTs) in the detoxification of dietary allelochemicals in the digestive gland of the predatory gastropod Cyphoma gibbosum, a generalist consumer of gorgonian corals. Controlled laboratory feeding experiments were used to investigate the influence of gorgonian diet on Cyphoma GST activity and isoform expression. Gorgonian extracts and semi-purified fractions were also screened to identify inhibitors and possible substrates of Cyphoma GSTs. In addition, we investigated the inhibitory properties of prostaglandins (PGs) structurally similar to antipredatory PGs found in high concentrations in the Caribbean gorgonian Plexaura homomalla.

Principal Findings

Cyphoma GST subunit composition was invariant and activity was constitutively high regardless of gorgonian diet. Bioassay-guided fractionation of gorgonian extracts revealed that moderately hydrophobic fractions from all eight gorgonian species examined contained putative GST substrates/inhibitors. LC-MS and NMR spectral analysis of the most inhibitory fraction from P. homomalla subsequently identified prostaglandin A₂ (PGA₂) as the dominant component. A similar screening of commercially available prostaglandins in series A, E, and F revealed that those prostaglandins most abundant in gorgonian tissues (e.g., PGA₂) were also the most potent inhibitors. In vivo estimates of PGA₂ concentration in digestive gland tissues calculated from snail grazing rates revealed that Cyphoma GSTs would be saturated with respect to PGA₂ and operating at or near physiological capacity.

Significance

The high, constitutive activity of Cyphoma GSTs is likely necessitated by the ubiquitous presence of GST substrates and/or inhibitors in this consumer''s gorgonian diet. This generalist''s GSTs may operate as ‘all-purpose’ detoxification enzymes, capable of conjugating or sequestering a broad range of lipophilic gorgonian compounds, thereby allowing this predator to exploit a range of chemically-defended prey, resulting in a competitive dietary advantage for this species.     

Glutathione S-transferases in the adaptation to plant secondary metabolites in the Myzus persicae aphid

Glutathione S-transferases (GST) in insects play an important role in the detoxification of many substances including allelochemicals from plants. Induction of GST activity in Myzus persicae in response to secondary metabolites from Brassica plants was determined using different host plant species and confirmed using artificial diet with pure allelochemicals added. The 2,4-dinitro-1-iodobenzene (DNIB) was found to be a useful substrate for identifying particular GSTs in insects. GSTs from M. persicae were purified using different affinity chromatography columns and related kinetic parameters were calculated. GST isoenzymes were characterised using electrophoretic methods. Although SDS-PAGE results indicated similarity among the purified enzymes from each affinity column, biochemical studies indicated significant differences in kinetic parameters. Finally, the GST pattern of M. persicae was discussed in terms of insect adaptation to the presence of plant secondary substances such as the glucosinolates and the isothiocyanates, from Brassicaceae host plants.