Thyroid hormones regulate lipid peroxidation and antioxidant enzyme activities in Anabas testudineus (Bloch)

Glutathione S-transferases (GST) play an important role in the detoxification of many substances including allelochemicals from plants. Brassicaceae plants contain glucosinolates and emit volatile isothiocyanates which affect the GST system. A comparison of the GST of two aphid species, the generalist Aulacorthum solani found on Brassicaceae and the Fabaceae specialist Acyrthosiphon pisum, was made to try to explain their respective feeding behaviour. Differences of GST were determined among the two aphid species based on purification by affinity chromatography, SDS-PAGE and on kinetic studies. Purification yields using an epoxy-activated Sepharose 6B column were highly different for the two aphid species (18% and 34% for A. solani and A. pisum, respectively). These variations were confirmed by SDS-PAGE. While only a 27-kDa band was observed for A. pisum, two bands of approximately 25-kDa were visualized for the generalist aphid, A. solani. Considering the kinetic results, differences of Km and Vmax were observed following the aphid species when a range of substrates (CDNB and DCNB) and GSH concentrations were tested. Studies on the detoxification enzymes of generalist and specialist herbivores would be undertaken to determine accurately the effect of the host plant on the organisms eating them, particularly in terms of biochemical and ecological advantages.     

Plant secondary substances and insects behaviour

Allelochemicals are storing in different location in plant tissues as inactive form. Number of identified compounds may now exceed 100,000. Environmental factors have an effect on allelochemicals concentration in plants. Many allelochemicals classified as toxins or deterrents for herbivorous insects. Allelochemicals play a major role in feeding or ovipositing stimulants for some specialist insects. Consumption and assimilation of herbivorous insects had affected by the type of allelochemicals in host plants. Allelochemicals have an acute or chronic toxicity on herbivorous insects. Most specialist herbivorous insects rely heavily of ingested plant allelochemicals. Plant allelochemicals may influence an insect's susceptibility to pathogens such as bacteria, fungi and nematode. Specialists herbivorous insect can be using the allelochemicals in their host plants as protection against natural enemies. Some herbivorous insects are synthesising the aggregation, attracting, alarm or mating pheromone from the allelochemicals in their host plants.     

Proteomics in Myzus persicae: effect of aphid host plant switch

Chemical ecology is the study of how particular chemicals are involved in interactions of organisms with each other and with their surroundings. In order to reduce insect attack, plants have evolved a variety of defence mechanisms, both constitutive and inducible, while insects have evolved strategies to overcome these plant defences (such as detoxification enzymes). A major determinant of the influence of evolutionary arms races is the strategy of the insect: generalist insect herbivores, such as Myzus persicae aphid, need more complex adaptive mechanisms since they need to respond to a large array of different plant defensive chemicals. Here we studied the chemical ecology of M. persicae associated with different plant species, from Brassicaceae and Solanaceae families. To identify the involved adaptation systems to cope with the plant secondary substances and to assess the differential expression of these systems, a proteomic approach was developed. A non-restrictive approach was developed to identify all the potential adaptation systems toward the secondary metabolites from host plants. The complex protein mixtures were separated by two-dimensional electrophoresis methods and the related spots of proteins significantly varying were selected and identified by mass spectrometry (ESI MS/MS) coupled with data bank investigations. Fourteen aphid proteins were found to vary according to host plant switch; ten of them were down regulated (proteins involved in glycolysis, TCA cycle, protein and lipid synthesis) while four others were overexpressed (mainly related to the cytoskeleton). These techniques are very reliable to describe the proteome from organisms such as insects in response to particular environmental change such as host plant species of herbivores.     

昆虫对植物次生物质的代谢适应机制及其对昆虫抗药性的意义

植物次生物质(plant secondary metabolites)对昆虫的取食行为、生长发育及繁殖可以产生不利影响,甚至对昆虫可以产生毒杀作用。为了应对植物次生物质的不利影响,昆虫通过对植物次生物质忌避取食、解毒代谢等多种机制,而对寄主植物产生适应性。其中,昆虫的解毒代谢酶包括昆虫细胞色素P450酶系（P450s）及谷胱甘肽硫转移酶（GSTs）等,在昆虫对植物次生物质的解毒代谢及对寄主植物的适应性中发挥了重要作用。昆虫的解毒酶系统不仅可以代谢植物次生物质,还可能代谢化学杀虫剂,因而昆虫对寄主植物的适应性与其对杀虫剂的耐药性甚至抗药性密切相关。昆虫细胞色素P450s和GSTs等代谢解毒酶活性及相关基因的表达可以被植物次生物质影响,这不仅使昆虫对寄主植物的防御产生了适应性,还影响了昆虫对杀虫剂的解毒代谢,因而改变昆虫的耐药性或抗药性。掌握昆虫对植物次生物质的代谢适应机制及其在昆虫抗药性中的作用,对于明确昆虫的抗药性机制具有重要的参考意义。本文综述了植物次生物质对昆虫的影响、昆虫对寄主植物次生物质的代谢机制、昆虫对植物次生物质的代谢适应性对昆虫耐药性及抗药性的影响等方面的研究进展。     

21种杀虫剂和3种植物次生物质对杨扇舟蛾各组织谷胱甘肽S-转移酶的抑制作用

为了比较杨扇舟蛾Clostera anachoreta (Fabricius)各组织谷胱甘肽S 转移酶（GSTs）的差异,利用分光光度酶动力学的方法,研究了21种杀虫剂和3种植物次生物质对杨扇舟蛾4个组织（中肠、脂肪体、头部和体壁）GSTs活性的体外影响。结果表明:21种杀虫剂和3种植物次生物质对杨扇舟蛾4个组织GSTs活性的抑制作用不同。毒死蜱、氟虫腈、槲皮素和单宁酸对于杨扇舟蛾头GSTs活性抑制作用最强;槲皮素和单宁酸对中肠GSTs活性的抑制作用最强;单宁酸对脂肪体GSTs活性的抑制作用最强;辛硫磷、高效氯氟氰菊酯、溴氰菊酯和硫丹对皮GSTs活性的抑制作用最强。杨扇舟蛾4个组织GSTs对杀虫剂和植物次生物质敏感性存在的这种差异,可能是由于其在同工酶组成上的差异造成的。     

植物抗虫性次生物质的研究概况

综述了国内外与植物抗虫性有关的次生物质的主要类型和植物次生物质对昆虫的寄主选择、取食和产卵等作用的研究进展,对次生物质在生态系统中的作用也作了介绍,并展望了植物次生物质的应用前景。     

植物抗虫性次生物质的研究概况

综述了国内外与植物抗虫性有关的次生物质的主要类型和植物次生物质对昆虫的寄主选择、取食和产卵等作用的研究进展,对次生物质在生态系统中的作用也作了介绍,并展望了植物次生物质的应用前景。     

Comparison of glutathione S-transferase and general esterase in two bugs, Eurygaster integriceps Puton (Heteroptera: Scutelleridae) and Brachynema germari Kolenati (Heteroptera: Pentatomidae)

Glutathione S -transferases (GSTs) and general esterases play important roles in the detoxification of many substances including allelochemicals from plants and chemical pesticides. In this study, to determine the feeding status and effects of agronomical practices on GST and general esterase, two bug species, Eurygaster integriceps and Brachynema germari , were selected with differences in these cases. GST and general esterase responses varied when switching from 1-chloro-2,4-dinitrobenzene (CDNB) and α -naphtyl to 1,2-dichloro-4-nitro-benzene (DCNB) and β -naphtyl in E. integriceps and B. germari , respectively. The activities of GST and esterase by using CDNB and α -naphtyl were higher than that of DCNB and β -naphtyl for both insects. It was shown that the optimal pH for GST and general esterase activity varied between pH 5.5 and 6. While the Michaelis constant (K_m) value related to E. integriceps toward CDNB and α -naphtyl was lower than K_m for B. germari , K_m values calculated for E. integriceps toward DCNB and β -naphtyl corresponding to those related to A. hejeri were similar to CDNB and α -naphtyl values. The maximal reaction velocity (V_max) values related to E. integriceps for both substrates (CDNB and α -naphtyl) were always higher than those from B. germari and showed significant differences. At the basement of the native-PAGE electrophoresis, five bands were observed in for GST and three were visualized for B. germari , with a large, darker band for E. integriceps in the case of esterases. Studies on the detoxification enzymes of herbivores should be undertaken to determine accurately the effect of the host plants on the organisms eating them, particularly in terms of biochemical and ecological advantages.     

Proteomic identification, cDNA cloning and enzymatic activity of glutathione S-transferases from the generalist marine gastropod, Cyphoma gibbosum

Glutathione S-transferases (GST) were characterized from the digestive gland of Cyphoma gibbosum (Mollusca; Gastropoda), to investigate the possible role of these detoxification enzymes in conferring resistance to allelochemicals present in its gorgonian coral diet. We identified the collection of expressed cytosolic Cyphoma GST classes using a proteomic approach involving affinity chromatography, HPLC and nano-spray liquid chromatography-tandem mass spectrometry (LC-MS/MS). Two major GST subunits were identified as putative mu-class GSTs; while one minor GST subunit was identified as a putative theta-class GST, apparently the first theta-class GST identified from a mollusc. Two Cyphoma GST cDNAs (CgGSTM1 and CgGSTM2) were isolated by RT-PCR using primers derived from peptide sequences. Phylogenetic analyses established both cDNAs as mu-class GSTs and revealed a mollusc-specific subclass of the GST-mu clade. These results provide new insights into metazoan GST diversity and the biochemical mechanisms used by marine organisms to cope with their chemically defended prey.     

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。     

植物次生物质对烟青虫和棉铃虫食物利用及中肠解毒酶活性的影响

用分别添加0-5%(干重比)棉酚、烟碱、番茄苷和辣椒素4种植物次生物质的人工饲料饲养烟青虫Helicoverpa assulta和棉铃虫H. armigera 5龄幼虫48 h,测定这些次生物质对烟青虫和棉铃虫的营养效应和中肠谷胱甘肽S-转移酶（GST）及羧酸酯酶（CarE）活性的影响。结果表明：在实验浓度下,棉酚可显著降低烟青虫的相对消化率,但对棉铃虫却有助食作用;番茄苷抑制烟青虫的取食和生长,对其近似消化率和食物利用率也有显著的抑制作用,但食物转化率有明显升高,对棉铃虫的各营养指标无显著影响;烟碱对烟青虫和棉铃虫的相对生长率均无影响;辣椒素使烟青虫的取食量有大幅度的提高,对棉铃虫的取食量无影响,但引起其相对消化率的提高。由此可见,棉铃虫对4种次生物质有普遍的适应性,而烟青虫只对寄主植物所含的烟碱和辣椒素有较好的适应性。烟青虫和棉铃虫幼虫中肠CarE活性不受4种次生物质的影响,烟碱和辣椒素对烟青虫GST有显著的诱导作用,番茄苷对烟青虫GST活性则有抑制作用,4种次生物质对棉铃虫GST均无显著影响。     

Purification and Characterisation of Hepatic Glutathione Transferases from a Herbivorous Marsupial, the Brushtail Possum (Trichosurus vulpecula)

A single glutathione transferase isoenzyme was purified from hepatic cytosol of the brushtail possum and shown to represent 3.6 ± 0.3% of the total cytosolic protein. Characterisation of the enzyme, termed Possum GST 1–1, indicated that it possessed similar catalytic activity and structural homology with isoenzymes belonging to the alpha class of glutathione transferases. This homodimeric GST exhibited a single band with an apparent molecular mass of 25.4 kDa on sodium dodecyl sulphate-polyacrylamide gels and an apparent pI of 9.8. Inhibition studies demonstrated that Possum GST 1–1 displays binding affinity for a range of inhibitors similar to that shown by alpha class GSTs purified from other mammals. Immunoblot analysis demonstrated immuno-cross reactivity between Possum GST 1–1 and antisera raised against human alpha GST, while this GST did not cross-react with antisera raised against human mu and pi GST. N-terminal sequencing of purified Possum GST 1–1 revealed that the N-terminus of the protein is chemically blocked. Sequence analysis of three internal peptide sequences demonstrated homology with mammalian alpha GSTs. Of particular interest is the significant substrate specificity that Possum GST 1–1 displays with both organic and inorganic hydroperoxides. It is proposed that this substrate specificity is an evolutionary adaptation to a diet high in potentially toxic plant allelochemicals.     

寄主植物影响害虫药剂敏感性的研究进展

害虫取食不同寄主植物后,对杀虫剂的反应可归为3类：敏感性降低、增高和无明显变化。害虫对药剂的敏感性变化与不同植物中次生物质诱导激活/抑制昆虫体内相关解毒酶活性有关。这种诱导作用可受到植物营养、次生物质种类及其含量分布、害虫种类与发育阶段、以及环境温度等多种因素影响。经诱导的昆虫解毒酶对不同类型杀虫剂的代谢能力并不相同,进而导致对不同药剂的敏感性变化有明显差异。解毒酶系的诱导激活在害虫抗药性形成早期被认为有利于提高隐性抗性基因频率,从而可促进害虫抗药性的发展。最后,就寄主植物影响害虫对药剂敏感性在害虫治理中的应用作了探讨。     

Characterization of Cytosolic Glutathione S-Transferase in Spruce Needles

Active glutathione S-transferase (GST) has been purified from needles of Norway spruce (Picea abies L. Karst.). Two isoforms of the enzyme which exhibit different physico-chemical and catalytic properties were separated by (NH₄)₂SO₄ fractionation, affinity chromatography on epoxy-activated 4% cross-linked beaded agarose, using glutathione as the ligand, ion-exchange chromatography, and isoelectric focusing. The isozymes have pI values of 5.5 (GST I) and 4.3 (GST II). Both GST isozymes are homodimeric proteins with subunit sizes of 26 kD (GST I), and 23 kD (GST II). The kinetic properties of the enzymes are described and compared with other plants GSTs. Only GST II is able to conjugate the pesticides fluorodifen and alachlor.     

Purification and characterization of glutathione S-transferases from two syrphid flies (Syrphus ribesii and Myathropa florae)

Glutathione S-transferases (GST) play an important role in the detoxification of many substances including organic pollutants and plant secondary metabolites. We compared the GST of two syrphid species, the aphidophagous Syrphus ribesii and the saprophagous Myathropa florea to assess the relation between feeding type and GST patterns. Differences between the GST of the hoverfly species were observed after purification by affinity chromatography, SDS-PAGE and kinetic studies. While the specific activities of the purified enzymes were different, the purification yields were similar. The variation in specific activities was related to the presence of different isoenzymes in both syrphid species by SDS-PAGE. While two bands of 24 and 32 kDa were observed for M. florea, one more band of 26 kDa was present in S. ribesii. When a range of substrate and glutathione concentrations was tested, differences in Km and Vmax between the glutathione S-transferases from both hoverfly species were also observed. These results are discussed in terms of adaptations to the feeding habit and the habitat of the two syrphid species.     

灰飞虱唾液腺三大解毒酶家族的转录组分析

灰飞虱Laodelphax striatellus （Fallén）是危害多种禾本科经济作物的重要刺吸式害虫。唾液腺对刺吸式口器昆虫取食植物尤其重要, 其分泌的唾液可以帮助刺吸式口器昆虫刺穿植物、 消化食物、 解毒植物的次生物质。细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）、 谷胱甘肽S 转移酶（glutathione S transferase, GST）和羧酸酯酶（carboxylesterase, CarE）是昆虫主要的解毒酶系。为了分析解毒酶基因在灰飞虱唾液腺中的表达谱, 本研究对灰飞虱成虫唾液腺进行转录组测序、 重头组装和注释, 并与豌豆蚜Acyrthosiphon pisum和西方蜜蜂Apis mellifera的同源蛋白进行系统发育分析。发现有9个谷胱甘肽S 转移酶（glutathione S transferase, GST）基因、 22个羧酸酯酶（carboxylesterase, CarE）基因和39个细胞色素P450单加氧酶（cytochrome P450 monooxygenase, P450）基因在灰飞虱唾液腺中表达。通过对同源蛋白进行系统发育分析, 发现灰飞虱唾液腺大部分的CarE是参与消化/解毒和激素/信息素的加工, 而参与神经/发育的CarE很少; 灰飞虱唾液腺表达的P450基因远远少于豌豆蚜和西方蜜蜂基因组的P450基因数, 且只有CYP6和CYP4家族的成员; GST家族在3种昆虫的保守性最高。研究结果为灰飞虱对寄主植物和杀虫剂的适应性研究奠定了基础。     

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。     

Is the (E)‐β‐farnesene only volatile terpenoid in aphids?

Abstract: Herbivore insects use a broad range of chemical cues to locate their host to feed or to oviposit. Whether several plant volatiles are effective allelochemicals for insects, the latter also emit molecules which have infochemical role. The (E)‐β‐farnesene (EBF) is a well‐known aphid alarm pheromone commonly found in all previously tested species. Analysis of the released molecules from 23 aphid species, mainly collected on their natural host plant from May to July, was performed by gas chromatography–mass spectrometry. While EBF was identified as the main volatile substance in 16 species, alone or associated with other molecules, the alarm pheromone was only a minor component of the volatile molecule pattern of five other species. Moreover, two species, Euceraphis punctipennis and Drepanosiphum platanoides, did not release EBF at all but other terpenes were identified. This original observation raised the question on the utility and the source of the non‐EBF volatiles. Are these potential infochemical substances produced by the aphid or only absorbed from the host plant? Here we determined that terpenes released by insects were not only provided by the host plants. Indeed, Megoura viciae emitted additional molecules than the ones from several aphid species reared on the same host plant. Moreover, no systematic relation between the feeding behaviour of the aphid species and the volatile releases was observed. Aphid terpene composition and proportion would provide reliable cues to identify the emitting organism, plant or insect. The next step of this work will be to determine the infochemical role of terpenes found in the range of tested aphid samples to better understand the relations between the different tritrophic levels.     

蚜虫雄蚜与雌性母对性信息素及植物挥发物的田间反应

田间观察了桃蚜Myzus persicae (Sulzer)、绣线菊蚜Aphis spiraecola Patch 、山楂圆疣蚜Ovatus crataegarius (Walker)等3种蚜虫对性信息素[(4aS,7S,7aR)-荆芥内酯和 (1R,4aS,7S,7aR)-荆芥醇]的反应,并且调查了性信息素与植物挥发物对桃蚜的田间引诱活性的相互作用.在有冬寄主或夏寄主植物的田中,性信息素诱捕器诱捕到桃蚜雄蚜与雌性母的数量显著多于对照诱捕器的诱捕数;但在非寄主植物的田中,却引诱不到桃蚜.苯甲醛(冬寄主植物桃树Prunus persica的主要挥发物组分之一)能够增强桃蚜雄蚜的引诱作用.绣线菊蚜雄蚜和雌性母对植物中提取的荆芥内酯有反应,而山楂圆疣蚜雄蚜和雌性母对植物中提取的和人工合成的荆芥内酯都没有反应,但对荆芥醇有反应.并且在荆芥醇中添加荆芥内酯之后对山楂圆疣蚜雄蚜引诱活性显著提高.还讨论了雌性蚜产生化合物被雄蚜作为性信息素、被雌性母作为聚集信息素以及性信息素与寄主植物挥发物的相互作用.