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。     

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。     

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。     

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。     

刺吸式口器昆虫吸食量测定

<正> 就咀嚼式口器昆虫取食研究,由于其取食行为较为直观,经常采用称重法,直接测定昆虫某一龄期或整个一生的取食量。但刺吸式口器的昆虫,其取食对象是寄主的汁液,而且取食行为较为复杂,加上对昆虫人工饲料筛选的难度,尽管许多学者采用了许多方法,但还不能精确测定其吸食量。本文就刺吸式口器昆虫吸食量的研究方法作一概述。     

基于EPG的三种刺吸式害虫在苹果苗上的取食行为比较

【目的】苹果绵蚜Eriosoma lanigerum、绣线菊蚜Aphis citricola和梨网蝽Stephanitis nashi是苹果园的一类重要害虫,它们以刺吸式口器对苹果树造成不同程度的危害。本研究旨在明确这3种刺吸式昆虫在苹果树上取食行为差异。【方法】利用刺吸电位(electrical penetration graph, EPG)技术对苹果绵蚜和绣线菊蚜成虫在苹果苗韧皮部和非韧皮部上的EPG指标,以及苹果绵蚜、绣线菊蚜和梨网蝽成虫在苹果苗上的取食行为进行了比较分析,分析了这3种害虫的成虫在苹果苗上取食8 h各种波形平均持续时间的占比。【结果】结果表明,苹果绵蚜和绣线菊蚜成蚜在苹果苗上均产生6种取食波形,即非刺探波(np)、路径波(C)、意外穿刺细胞非主动取食细胞波(pd)、木质部取食波(G)、韧皮部唾液分泌波(E1)和韧皮部取食波(E2);而梨网蝽成虫取食过程中只产生非刺探波(np)、表皮刺穿波(A)、叶肉细胞取食波(Gc)和木质部取食波(E)4种波形。从蚜虫在苹果苗非韧皮部上取食的EPG指标看,苹果绵蚜成蚜pd波平均时间显著高于绣线菊蚜的,而刺探次数、np波总时间和pd...     

韧皮部取食昆虫诱导的植物防御反应

刺吸式昆虫与寄主植物之间具有特殊的生物互作关系。本文对刺吸式昆虫取食韧皮部诱导的植物防御反应类型、 防御物质变化、 信号途径以及植物反应转录组学研究等方面进行综述。韧皮部取食昆虫取食诱导的植物防御反应机制主要包括： (1)改变自身的营养状况; (2)产生有毒的次生化合物; (3)产生防御蛋白。防御反应与植物水杨酸、 茉莉酸、 乙烯等信号分子密切相关。研究表明, 刺吸式昆虫取食诱导的植物防御反应主要引发以水杨酸为主的信号途径, 但相关分子互作机制还有待明确。日益丰富的基因组资源和不断发展的分子生物学技术为揭示植物防御反应中信号分子的作用机制、 找出植物内生抗性的特异因子以及阐明诱导防御机制奠定了基础。了解刺吸式昆虫取食诱导的植物防御反应, 为深入理解植物-昆虫间协同进化关系提供了依据, 为害虫治理和抗虫植物的培育提供了新的思路。     

臭氧浓度升高对昆虫影响的研究进展

臭氧(O3)是最具危害性的空气污染物之一。目前流层中的臭氧水平从100多年前的10ppb到今天的40ppb,预计到2050年将达到68ppb左右。臭氧通过改变植物"质量"而影响植食性昆虫的取食偏嗜性、行为、生长和发育,进而影响天敌昆虫的适合度。臭氧还通过改变化学信息物质而影响昆虫的行为。本文根据国内外研究进展,结合作者的研究,论述了大气臭氧浓度升高对刺吸式昆虫、咀嚼式昆虫和天敌昆虫的影响,展望了未来研究的前景。     

唾液成分在刺吸式昆虫与植物关系中的作用

近年来,人们对刺吸式昆虫唾液成分的研究,揭示出其在刺吸式昆虫与植物关系中的重要作用。对多数刺吸式昆虫而言,他们取食时会分泌胶状和水状两种唾液,其中胶状唾液会在取食早期分泌形成唾液鞘来围绕并保护口针,通过直接和间接的作用来帮助取食;而水状唾液中则包含了果胶酶、纤维素酶、多酚氧化酶、过氧化物酶、碱性磷酸酯酶、蔗糖酶等组分,来帮助刺吸式昆虫对植物穿刺、消化食物、解毒次生物质并破坏植物的防御反应。有趣的是,唾液成分同时还可以诱导植物的防御反应,包括诱导植物的伤信号引起直接防御反应和诱导植物产生挥发物吸引植食者的天敌引起间接防御反应。并且,许多刺吸式昆虫取 食能够特异性地引发植物的病理反应,有研究推测刺吸式昆虫唾液中多聚半乳糖醛酸酶、碱性磷酸酯酶、蔗糖酶、多酚氧化酶等成分可能是某些植物特定病理反应的激发子,但是目前还没有定论,同时许多刺吸式昆虫唾液中的氨基酸和蛋白酶还是引起植物虫瘿的原因之一。 迄今的研究表明,刺吸式昆虫会根据不同的寄主植物和不同的生理需要,通过唾液组分的改变,来达到取食和发育的目的。对刺吸式昆虫唾液成分和作用机理的研究,可以为揭示刺吸式昆虫致害机理特别是传毒机理、指导害虫有效治理、阐明其与植物的协同进化等提供一定的思路。     

基于EPG的三种刺吸式害虫在苹果苗上的取食行为比较

【目的】苹果绵蚜Eriosoma lanigerum、绣线菊蚜Aphis citricola和梨网蝽Stephanitis nashi是苹果园的一类重要害虫,它们以刺吸式口器对苹果树造成不同程度的危害。本研究旨在明确这3种刺吸式昆虫在苹果树上取食行为差异。【方法】利用刺吸电位(electrical penetration graph, EPG)技术对苹果绵蚜和绣线菊蚜成虫在苹果苗韧皮部和非韧皮部上的EPG指标,以及苹果绵蚜、绣线菊蚜和梨网蝽成虫苹果苗上的取食行为进行了比较分析,分析了这3种害虫的成虫在苹果苗上取食8 h各种波形平均持续时间的占比。【结果】结果表明,苹果绵蚜和绣线菊蚜成蚜在苹果苗上均产生6种取食波形,即非刺探波(np)、路径波(C)、意外穿刺细胞非主动取食细胞波(pd)、木质部取食波(G)、韧皮部唾液分泌波(E1)和韧皮部取食波(E2);而梨网蝽成虫取食过程中只产生非刺探波(np)、表皮刺穿波(A)、叶肉细胞取食波(Gc)和木质部取食波(E)4种波形。从蚜虫在苹果苗非韧皮部上取食的EPG指标看,苹果绵蚜成蚜pd波平均时间显著高于绣线菊蚜的,而刺探次数、np波总时间和pd波次数均显著低于绣线菊蚜的。从蚜虫在苹果苗韧皮部上取食的EPG指标来看,除第1次出现E2波的时间外,各指标没有显著差异。从3种害虫在苹果苗上取食8 h各波形的占比来看,梨网蝽成虫的np波总时间所占比例最高(53%),其次是绣线菊蚜成虫的(24%),苹果绵蚜成虫的最低(为1%)。同时,苹果绵蚜和绣线菊蚜成虫取食波为E2波,所占总时间比例分别为35%和25%,而梨网蝽的取食波为Gc波(占总时间比例为36%)和E波(占总时间比例为11%)。【结论】本研究阐释了苹果园刺吸式口器害虫的生态位分离和取食行为学机制,为果园刺吸式口器害虫的综合治理提供了理论依据。     

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.     

Electrical Penetration Graphs from Peregrinus maidis on a susceptible maize hybrid

The feeding behavior of Peregrinus maidis (Ashmead) (Homoptera: Delphacidae), vector of maize mosaic virus (MMV) and maize stripe virus (MStpV) in maize (Zea mays L.), has been studied by Electrical Penetration Graph (EPG). The different recordings collected have allowed the temporal distinction of three EPG signal classes. These class 1, class 2 and class 3 signals are correlated through histological sections to the feeding activities of probing, xylem ingestion and phloem ingestion, respectively. Although these signals are described by various statistical parameters, only the median allows significant differentiation between class 2 and class 3 signals, the others varying from one insect to the next. On the other hand, spectral analysis is used to describe the signal classes by associating a characteristic frequency spectrum to each. This study treats the importance of such analysis in characterizing and comparing the signals of various piercing and sucking insects.     

Perception of insect feeding by plants

The recognition of phytophagous insects by plants induces a set of very specific responses aimed at deterring tissue consumption and reprogramming metabolism and development of the plant to tolerate the herbivore. The recognition of insects by plants requires the plant’s ability to perceive chemical cues generated by the insects and to distinguish a particular pattern of tissue disruption. Relatively little is known about the molecular basis of insect perception by plants and the signalling mechanisms directly associated with this perception. Importantly, the insect feeding behaviour (piercing‐sucking versus chewing) is a decisive determinant of the plant’s defence response, and the mechanisms used to perceive insects from different feeding guilds may be distinct. During insect feeding, components of the saliva of chewing or piercing‐sucking insects come into contact with plant cells, and elicitors or effectors present in this insect‐derived fluid are perceived by plant cells to initiate the activation of specific signalling cascades. Although receptor–ligand interactions controlling insect perception have yet not been molecularly described, a significant number of regulatory components acting downstream of receptors and involved in the activation of defence responses against insects has been reported. Some of these regulators mediate changes in the phytohormone network, while others directly control gene expression or the redox state of the cell. These processes are central in the orchestration of plant defence responses against insects.     

EPG technique as a tool to reveal host plant acceptance by xylem sap‐feeding insects

The discovery of a new exotic insect herbivore triggers responses from biosecurity agencies, one of which is the decision of whether or not to attempt eradication. Rapid determination of the host range of the new invader is necessary, but when sap‐sucking insects are first collected from plants, the lack of visible signs of feeding damage makes it difficult to determine their host status. We investigated the Electrical Penetration Graph (EPG‐DC) technique as tool to assess host range of a xylem sap‐feeding invader, using Carystoterpa fingens (Hemiptera: Aphrophoridae), a New Zealand endemic xylem feeder, as a model insect. Real‐time probing and feeding events over a 12‐h recording period were compared for adult C. fingens on 18 plant species. Hebe azure, a known host, was designated the ‘reference plant species’ against which events on all other plants were statistically compared. EPG waveforms were categorized on their amplitude, frequency, voltage and electrical origin, and six parameters (time taken to first probe, time to first xylem ingestion from first probe, total probing time, number of xylem‐ingesting events, duration of the longest xylem‐ingesting event and total xylem ingestion time) were measured. The total xylem ingestion period (i.e. the actual feeding period) on each plant species expressed as a percentage of total probing time was considered the best parameter for comparing the host status of plants with H. azure. Although the EPG data overestimated the actual host range of C. fingens, we consider that they provided a reasonable first guide to the potential host status of the unknown plants, and so might usefully be used to rapidly assess whether a plant from which a new invader was collected was a host, or whether the association was merely incidental.     

Effect of elevation on the insect herbivory of Mongolian oaks in the high mountains of southern South Korea

Elevation is strongly associated with the diversity and ecology of plants and animals. We explored how elevation affects herbivores on one of the major cool-temperate trees, Quercus mongolica, in southern South Korea. The feeding activities of insect herbivores were measured through insect-feeding damage on leaves. Of 78 types of insect-feeding damage that were observed from Jirisan Mountain (Mount.) and Hallasan Mount. in Korea, 61.6% was associated with externally feeding insects. The sum of feeding damages per leaf was significantly higher on Jirisan Mount. than on Hallasan Mount., and higher at higher elevation in both mountains. Two feeding guilds, externally feeding, and piercing and sucking were strongly related with elevation, but the relationship was opposite depending on feeding guild. Leaf damage by the internal feeding guild showed no effect of elevation at the two mountains. These study results showed that insect herbivory responds to elevational differences in temperate forests, and that such response can vary among feeding guilds.     

Effects of jasmonate-induced defenses in tomato on the potato aphid, Macrosiphum euphorbiae

Jasmonates such as jasmonic acid (JA) are plant‐signaling compounds that trigger induced resistance (IR) to a broad range of arthropod herbivores. JA‐dependent defenses are known to reduce the growth and survivorship of many chewing insects, but their impact on piercing–sucking insects such as aphids has not been extensively investigated. In this study, induced resistance was activated in tomato (Lycopersicon esculentum Mill) (Solanaceae) using a foliar application of synthetic JA, and control plants were treated with carrier solution. The life parameters of individual potato aphids and their progeny (Macrosiphum euphorbiae Thomas) (Hemiptera: Aphididae) were evaluated on the unsprayed leaves of plants in order to access the systemic effects of the foliar treatments. IR significantly reduced the longevity and net reproduction of adult aphids, as well as the percentage of juveniles to survive to maturity. These results indicate that JA application induces systemic defenses in tomato that have a direct negative impact on aphid survivorship. This study also examined aphid honeydew excretion, in order to evaluate the potential influence of induced resistance on aphid feeding behavior. The average honeydew production per aphid was comparable on plants with or without JA treatment, indicating that JA‐dependent defenses did not deter feeding. This suggests that the observed effects of JA on aphid survivorship were due to antibiotic rather than antixenotic factors. In addition to studying the effects of JA treatment on a tomato cultivar that is susceptible to aphids, this study also examined the effects of exogenous application of JA on tomato plants that carry the aphid resistance gene, Mi‐1.2. JA application did not significantly enhance or inhibit aphid control on resistant tomato. These findings expand our understanding of the effects of JA‐dependent defenses on piercing–sucking insects, and of the potential interactions between induced resistance and R‐gene mediated aphid resistance in tomato.     

Drought stress and pathogen infection alter feeding behavior of a phytopathogen vector

The impact of drought stress on tripartite plant-pathogen-vector interactions constitutes a complex and largely understudied field of plant-insect interaction. A number of studies explored these topics using aphid vectors of plant pathogens, but few have considered the interactions between drought-stressed plants and pathogen-transmitting psyllids. The potato psyllid, Bactericera cockerelli (Šulc) (Hemiptera: Triozidae), is one of the key pests of solanaceous crops in the USA that causes direct injury as well as indirect injury through transmission of a bacterial pathogen, Candidatus Liberibacter solanacearum (Lso), the causal agent of zebra chip. Previous studies explored the impact of Lso infection and drought stress on B. cockerelli development and reproductive rate separately, but no research to date has evaluated whether drought stress and Lso infection alter feeding behavior of the insects. We explored this using the electrical penetration graph (EPG) technique and monitored feeding behavior of Lso-infected and uninfected potato psyllids on well-watered and drought-stressed tomato (Solanum lycopersicum L., Solanaceae). We found that drought stress had a significant effect on feeding behavior associated with salivation into the phloem and phloem ingestion, both linked to Lso transmission. Furthermore, infected potato psyllids in particular produced a higher number of events associated with these feeding behaviors and remained in these phases longer in well-watered plants than in plants that were under drought stress. We also reported a new and previously undescribed waveform H of unknown biological function that was produced by the psyllids. This is the first study that considered the impact of bacterial infection and concomitant drought stress on feeding behavior of an insect quantified using EPG.     

Mouthpart morphology and feeding behavior of the invasive kudzu bug,Megacopta cribraria (Hemiptera: Plataspidae)

The invasive kudzu bug, Megacopta cribraria, was first reported in North America in 2009 and has subsequently spread through most of the southeastern United States, causing yield loss in soybean. Since detection in the USA, research has focused mainly on managing this newly established pest, but many important characteristics of the pest's mouthpart morphology and feeding behavior are unknown. Qualitative and quantitative comparisons of nymph and adult mouthparts and sensilla were made through scanning electron microscopy and light microscopy, and feeding behavior was examined using electropenetrography (EPG) and paraffin histology. Morphologies observed were similar to what has previously been reported for other piercing–sucking hemipterans. The relationship between rostrum length and body size (pronotum width and dorsal length) exhibited negative allometry. Rostrum length exhibited an isometric relationship with interocular width. Adult females (n=9) probed soybean stems 1.3±0.8 times in 9 h, with an average probe time of 2.3±1.3 h. EPG waveforms were characterized and correlated with behavior. Salivary sheaths were shown to terminate in the vascular tissue; four of five sheaths terminated in the phloem. This is the first time that the feeding behavior of a member of the Plataspidae has been recorded using EPG. Results add to our current limited knowledge of plataspid mouthpart morphology and provide a baseline for further research on the feeding behaviors of M. cribraria and other soybean‐feeding hemipterans.