我国刺吸电位技术三十年应用及创新

刺吸电位技术(Electricalpenetrationgraph,EPG)是研究刺吸式昆虫取食行为的电生理仪器及其配套技术体系。我国从20世纪90年代开始将刺吸电位技术引入生物学和农业科学领域,主要应用于昆虫与植物的关系、植物抗性机理、昆虫传毒机理、转基因安全性评价、全球气候变化等方面,研究所涉及的昆虫种类包括半翅目的蚜虫、粉虱、叶蝉、飞虱、介虫、木虱和缨翅目的蓟马等。自2007年以来举办了5次全国性的EPG技术培训和研讨会,并对EPG仪器进行了创新设计,实现了EPG仪器的国产化,大部分消耗品(如金丝、银胶等)可以实现国内供应。人工饲料上的EPG记录、指标体系的优化等与EPG相配套的技术体系不断完善。目前我国主要是应用EPG技术进行各类农林相关刺吸式昆虫的研究,而在欧洲和美国,除农林害虫外,EPG近年来也开始应用于医学昆虫和畜牧昆虫方面的研究。欧美在波形库构建、仪器原理等方面比我们具有优势,我国今后应该加强在EPG仪器自动化和可视化、波形自动识别、指标体系完善等方面的工作,同时培养能够应用EPG的年轻一代,尝试将EPG应用到医学昆虫领域。     

刺吸电位技术实验中固定昆虫的三种方法比较研究

刺吸电位技术(Electrical penetration graph,EPG)是用来记录刺吸式口器昆虫的口针在其寄主植物组织中刺探所引起的电信号变化特征的技术,其核心在于建立昆虫取食行为与EPG波形的对应关系。近年来,EPG技术在昆虫取食行为及其传毒行为,以及植物抗虫机制等研究中得到越来越广泛的应用。其中,昆虫固定是影响EPG电极连接成功与否的关键技术,进而影响EPG实验结果。本文以灰飞虱Laodelphax striatellus(Fallén)为例,就EPG实验中电极连接时常用的3种昆虫固定技术(麻醉法、冷冻法和负压法)进行了详细描述与比较。研究结果表明,固定灰飞虱最优的方法为负压法,其次为麻醉法和冷冻法。     

EPG即时显示软件Realdisplay的开发和利用

研究和开发在刺吸式植食性昆虫取食行为研究中,刺吸电位技术（EPG）的一种新的应用软件Realdisplay。该软件可任意选择EPG信号采集频率和显示的通道数,也可以在同一屏幕上同时以不同的信号采焦频率即时清晰地显示1个通道的EPG信号,使研究者能准确地了解和控制昆虫的即时行为细节。此外,该软件存储的EPG信息为实验过程中每一时刻的电热值,因而后续分析和统计极为方便。因此,Realdisplay强化了EPG技术的功能,拓宽了EPG技术的应用领域。为研究刺吸式植食性昆虫的取食行为细节,昆虫与病毒、宿主植物的关系提供了有力的工具。     

褐飞虱取食人工饲料的刺吸电位波形鉴别

昆虫刺吸电位技术(Electrical penetration graph,EPG)与人工饲料体系相结合,能为昆虫与植物相互关系中的行为、生理/毒理和分子机制等多个领域提供定量的研究手段。由于人工饲料体系与植物组织在结构和成分方面有较大差异,以现有的植物EPG波形作为参照标准的研究方法存在局限性。本研究应用EPG技术记录褐飞虱Nilaparvata lugens(Stl)在人工饲料上的取食行为,结合刺孔检测和饲料染色等方法,鉴定各波形的生物学意义。研究发现,人工饲料EPG波形与褐飞虱取食水稻产生的7种EPG波形相比存在差异。人工饲料EPG波形包括4种基本波形:np波,非刺探波;P波,口针刺入饲囊膜;S波,口针分泌唾液,并在人工饲料中移动;I波,吸食人工饲料。研究结果可为建立准确的人工饲料-刺吸电位技术体系提供基础。     

EPG技术在昆虫学研究中的应用及进展

刺吸行为电图(electrical penetration graph, EPG) 技术在植食性刺吸式昆虫,如蚜虫、粉虱、叶蝉和蓟马等研究中的应用日益深入,尤其是对蚜虫和粉虱的研究,已涉及寄主专化性、植物的抗虫机制以及病毒传播等领域。该文从蚜虫和粉虱等典型的刺吸式昆虫入手,介绍EPG技术的研究进展和应用前景。     

灰飞虱取食行为刺吸电位波形的初步研究

利用刺吸电位技术(EPG)研究了灰飞虱Laodelphax striatellus(Fallén)EPG波形与其取食行为之间的对应关系,并对不同虫龄、性别灰飞虱的取食行为进行EPG特征参数的比较。结果表明,灰飞虱取食行为的EPG波形可分为NP、N1、N2-a、N2-b、N3、N4和N5 7种类型,波谱的形状和频率与已报道的褐飞虱Nilaparvata lugens(Stl)的EPG取食波形基本相同。首次发现灰飞虱在口针刺探水稻维管束进行唾液分泌时可明显分为两个阶段即N2-a和N2-b。N2-a波表示口针移动并伴随唾液的分泌;N2-b波与灰飞虱持续分泌唾液的行为有关。经对比,不同虫龄、性别灰飞虱的EPG取食波形基本相同,但EPG特征参数存在差异。雌虫刺探次数显著多于雄虫和低龄若虫;成虫口针在临近韧皮部细胞外移动的时间显著长于若虫。     

麦蚜在不同抗级小麦上刺吸行为的比较

应用EPG(E1ectrical Penetration Graph)技术记录了麦长管蚜Sitobion avenae F.及禾谷缢管蚜Rhopalosiphum padi L.在不同抗性级别的小麦品种(红芒红、小白冬麦、kok1679和L₁)上的取食行为。结果表明,麦蚜在参试品种上刺吸的各种特征波型基本相同,但其出现时间、重复出现的次数及历期长短存在差异,其中自开始记录至第一次出现E₁：波的时间及E₂。波历期比率可做为参试品种是否抗蚜的主要生理性指标。依据主要波型统计参数用聚类分析方法对参试品种抗蚜性进行分类,其结果与室内生命表参数研究及田间鉴定结果基本一致。本文还对EPG在抗性因子定位、抗蚜鉴定中作用及EPG记录的影响因素略作讨论。     

扩增片段长度多态性技术及其在植物研究中的应用

详述了扩增片长度多态性技术（ＡＦＬＰ）的原理及在植物研究中的应用,并探讨了此项技术在植物检疫中应用的可通用性和在实验操作中应注意的一些问题。     

可可西里藏羚羊、野牦牛、藏野驴和藏原羚冬季蠕虫卵粪检研究初报

2004年12月25日~2005年1月5日,在可可西里地区收集到藏羚羊、野牦牛、藏野驴和藏原羚的新鲜粪便各36、16、13和20份带回实验室,利用漂浮法、沉淀法对其在冬季的寄生蠕虫卵进行检查。结果显示:藏羚羊、藏原羚粪便中均存在有细颈属线虫、马歇尔属线虫和莫尼茨属绦虫虫卵,藏羚羊的感染率在19.4%~94.4%之间,感染强度EPG分别为5.58、5.11和2.86;藏原羚感染率为15%~65%,感染强度EPG各自为11.1、2.45和12.65。野牦牛粪样有古柏属和毛圆属线虫卵,感染率在75%~87.5%之间,感染强度EPG为9.06和5.93;藏野驴粪样有裸头属绦虫卵和园形科线虫卵,感染率在31%~100%之间,感染强度EPG分别为3.77和104。粪检表明:冬季藏羚羊、野牦牛、藏野驴和藏原羚的蠕虫感染率高、感染种类少和感染强度低,动物处于带虫免疫状态。     

刺吸电波图实验中金丝与烟粉虱的粘连技术

刺吸电波图(EPG)是用于研究刺吸式口器昆虫取食的电生理技术。实验中,昆虫粘连技术是一项非常关键的技术,直接影响实验结果。该文以烟粉虱Bemisiatabaci(Gennadius) ,为例,介绍了粉虱成虫和若虫粘连的具体步骤和注意细节,比较了不同粘连方法的优缺点。结果表明,冷冻法是粘连粉虱最好的一种方法。     

Choice of Tethering Material Influences the Magnitude and Significance of Treatment Effects in Whitefly Electrical Penetration Graph Recordings

The electrical penetration graphing or electropenetrography (EPG) technique is essential for understanding interactions of hemipteran insects with their host plants. Typically, 10–12.5 μm diameter gold wire is used as the tethering material in EPG studies. This wire was originally chosen based on suitability for aphids, but application of the EPG technique to other insects necessitates testing of alternative tethering materials that permit natural foraging and probing behavior. Whiteflies are one group for which EPG studies are increasing, with most researchers using 10 or 12.5 μm diameter gold wire even though these insects are smaller than aphids and very different in mobility. However, 2.5 μm diameter Wollaston process platinum wire has been used for a subset of EPG studies and seems to permit more natural movement and feeding behaviors. Here, we compared EPG variables derived from recordings of the sweet potato whitefly (Bemisia tabaci) tethered with 12.5 μm diameter gold wire or 2.5 μm diameter platinum wire. On a suitable host, gold-tethered whiteflies had reduced phloem phases, which are indicative of host plant acceptance, compared to platinum-tethered whiteflies. When we included a treatment known to reduce plant quality (methyl jasmonate application), platinum-tethered whiteflies exhibited expected reductions in EPG variables related to host acceptance, while gold-tethered whiteflies either had no response, or the opposite response. Our results indicate that tethering material strongly influences the outcome of EPG experiments, with important consequences for evaluations of host plant resistance, putative instances of plant virus manipulation, and feeding variables associated with virus transmission.     

Effect of Manipulations on the Host Selection Behavior of Sitobion avenae (Homoptera: Aphididae)

The study of aphid host selection and feeding behavior is difficult because aphids have to penetrate the plant to reach their feeding site, phloem tissue. The activity of the stylets, salivation or food intake, can not be observed externally and requires an indirect visualization technique such as the Electric Penetration Graph (EPG). The plant selection behavior of Sitobion avenae on potato varied depending on whether an ethological or EPG method was used to study it. A similar variation did not occur with Myzus persicae or Rhopalosiphum padi. The application of water-based silver conductive paint onto the thorax, as normally used for EPG, or onto the abdomen of Sitobion avenae alates resulted in increased duration and frequency of probing compared to results from ethological observations. Our results indicated that EPG manipulations might have different effects on different species of aphids and that a comparison of EPG and ethological data is required to confirm that the EPG method does not bias aphid feeding behavior.     

EPG-Calc: a PHP-based script to calculate electrical penetration graph (EPG) parameters

Electrical penetration graph (EPG) technique is a powerful tool to investigate the hidden feeding behavior of piercing–sucking insects allowing to link recorded EPG waveforms to stylet penetration and complex behaviors related to feeding activities occurring within plant tissue. Calculating the numerous EPG parameters necessary to unravel the complex insect–plant interactions is very time consuming, and few tools have been developed to automate it. EPG-Calc is a rich internet application intended to fill this gap, providing a fast and user-friendly web-based interface that uses analysis files from dedicated software (STYLET⁺) or database-compatible CSV text files containing waveform codes and cumulative time as input, and produces output files in database-compatible CSV text or Microsoft Excel^® XLS format that are directly usable by different statistical analysis softwares. EPG-Calc greatly reduces the time needed for EPG parameters calculation and allows to calculate more than 100 different parameters based on standardized definitions and calculus methods in such a way that avoid confusion between all kinds of definitions and calculations by individual authors.     

New structure in cell puncture activities by aphid stylets: a dual‐mode EPG study

Intracellular punctures by aphid stylets appear as potential drop (pd) waveforms in DC electrical penetration graph (EPG) recordings. We used a dual‐EPG device that recorded in one channel the ‘full EPG’ with R‐plus emf‐components (i.e., the usual DC EPG) and concurrently in a second channel the ‘R‐EPG’ with R‐components only. The circuit of the latter channel was an optimised amplitude modulation (AM) version derived from early (before 1990) AC systems. We also made some ‘emf‐EPG’ recordings using a separate high input resistance ‘emf‐amplifier’ sensitive to emf‐components only. The intracellular pd waveforms have previously been divided into three subphases, and we aimed to distinguish and separate these subphases more accurately by the dual‐EPG recordings than with the normal full EPG only. In this study, we temporarily distinguished five subphases (α–ε), but unequivocal distinction of only a few of these appeared possible, in spite of the information coming from the two signals. The lack of clearly separable features in R‐EPG signals often provided serious difficulties in pd recognition without the concurrent full EPG, but once located, only subphase II‐2 features were clear and supported the II‐2 data from the full EPG. Consequently, we could not distinguish subphases of complete pd waveforms better with additional R‐EPG information during cell punctures by Aphis gossypii Glover (Hemiptera: Aphididae). In Brevicoryne brassicae (L.) (Hemiptera: Aphididae), however, distinguishing II‐2 subphases in the full EPG was sometimes a problem. Our detailed dual‐EPG observations showed some waveform continuity from halfway into the II‐1 subphase (start of the newly recognised subphase β) until the end of the pd, with a strong but variable emf origin. This waveform tended to overrule other subphase waveforms in B. brassicae more than in A. gossypii and Myzus persicae (Sulzer) (Hemiptera: Aphididae). Subphase waveforms in full EPGs were especially difficult to recognise when pd periods had been interrupted in a virus inoculation experiment and additional R‐EPG information could then be useful. This inoculation experiment showed again that only the first subphase (II‐1) contributes to virus (Cucumber mosaic virus) inoculation by A. gossypii. In B. brassicae, the benefit of concurrent R‐EPG information in such virus experiments is presently under further investigation. Apart from this special application to virus experiments, we do not recommend the routine use of the dual‐EPG device. Furthermore, we do not advocate the distinction of more than the previously recognised three intracellular pd subphases as a feasible option in future studies. Analysis of EPGs with concurrent R‐EPGs requires substantially more analysis work without yielding consistently useful additional insights. This confirms earlier dual‐EPG results from thrips.     

Effects of treatments for electrical penetration graph recordings on behaviour and biology of Aphis craccivora (Aphididae)

Abstract. Two laboratory studies were conducted to investigate effects of treatments for direct "current electrical penetration graph (DC-EPG) recordings or 'tether effect', on behaviour and reproductive performance of cowpea aphid Aphis craccivora Koch. The experiments constituted a control study in application of DC-EPGs to analyse cowpea aphid feeding behaviour and host plant resistance mechanisms. Resistant (ICV-12) and susceptible (ICV-1) cultivars of cowpea Vigna unguiculata (L.) Walp were used. EPG treatments included two groups of aphids: tethered aphids that were exposed to DC electricity via an attachment of a thin, flexible gold wire on their dorsum using a droplet of adhesive silver paint, and 'free' (untethered) aphids with a dorsal spot of silver paint only. EPGs of the tethered aphids were recorded continuously for c. ! h, whereas from the 'free' aphids recordings were done only for brief periods of 2–5 min, by temporarily contacting a gold wire to the spot of silver paint. Waveform signals generated from resistance fluctuations and electromotive forces, and representing aphid stylet penetration behaviour were recorded. A separate experiment was conducted to investigate effects of EPG treatments on aphid survivorship and population growth. Overall, EPG treatments did not significantly affect aphid stylet penetration behaviour or life-table parameters. However, effects of crop cultivar on those characteristics were significant. Waveform E2, which denotes aphid ingestion in phloem sieve elements, and non-penetration behaviour were important indicators of aphid resistance in ICV-12. Also, apart from the number of aphid generations, other life-table parameters were useful indicators of ICV-12 resistance. Thus, DC-EPGs provided a reliable technique for studying aphid stylet behaviour, and investigation of aphid resistance in cowpeas.     

Correlation of stylet activities by the glassy-winged sharpshooter, Homalodisca coagulata (Say), with electrical penetration graph (EPG) waveforms

Glassy-winged sharpshooter, Homalodisca coagulata (Say), is an efficient vector of Xylella fastidiosa (Xf), the causal bacterium of Pierce's disease, and leaf scorch in almond and oleander. Acquisition and inoculation of Xf occur sometime during the process of stylet penetration into the plant. That process is most rigorously studied via electrical penetration graph (EPG) monitoring of insect feeding. This study provides part of the crucial biological meanings that define the waveforms of each new insect species recorded by EPG. By synchronizing AC EPG waveforms with high-magnification video of H. coagulata stylet penetration in artifical diet, we correlated stylet activities with three previously described EPG pathway waveforms, A1, B1 and B2, as well as one ingestion waveform, C. Waveform A1 occured at the beginning of stylet penetration. This waveform was correlated with salivary sheath trunk formation, repetitive stylet movements involving retraction of both maxillary stylets and one mandibular stylet, extension of the stylet fascicle, and the fluttering-like movements of the maxillary stylet tips. Waveform B1 was ubitquious, interspersed throughout the other waveforms. B1 sub-type B1w was correlated with salivation followed by maxillary tip fluttering. This tip fluttering also occurred before and during B1 sub-type B1s, but was not directly correlated with either the occurrence or frequency of this waveform. Waveform B2 was correlated with sawing-like maxillary stylet movements, which usually occurred during salivary sheath branching. Waveform C was correlated with ingestion. Fluid outflow was also observed as a mechanism to clear the maxillary tips from debris during waveform C. This detailed understanding of stylet penetration behaviors of H. coagulata is an important step toward identifying the instant of bacterial inoculation which, in turn, will be applied to studies of disease epidemiology and development of host plant resistance.