Intracellular punctures by the adult whitefly Bemisia argentifolii on DC and AC electronic feeding monitors

One of the most biologically important electrical penetration graph (EPG) waveforms recorded from aphids on DC EPG systems is the potential drop (pd), which is correlated with intracellular punctures by the stylet tips. In this study, pds of the adult female Bemisia argentifolii Bellows & Perring (Homoptera: Aleyrodidae), recorded on a DC EPG, are characterized and compared to pds of aphids. Whitefly pds consisted of 3 phases similar to those recorded from probing aphids. The major difference between aphid pds and whitefly pds was that whitefly pds lacked any observable subphases within the second phase of the pd. In addition, whitefly pds differed from aphid pds in that they: (1) did not occur frequently during stylet penetration, (2) did not occur early within probes, (3) did not occur during brief probes (<1 min). Pds produced by probing whiteflies always were preceded by a variant of waveform C which we named the pre-pd. The differences between pds of aphids and whiteflies are discussed in terms of their implications for virus transmission and host selection. Using a technique where EPG recordings can be switched back and forth between DC and AC systems, we demonstrated that the AC EPG pseudotransition waveform (Pt) was equivalent to the DC pd, and thus was correlated with intracellular punctures. Previously, intracellular punctures by whiteflies had not been detectable on AC EPG systems. The AC Pt consisted of three distinct phases (Pt1, Pt2, and Pt3) and our observations suggest that AC Pt1 correlates with the pre-pd waveform in DC EPGs and that AC Pt 2 and 3 correlate with the intracellular phase of the DC pd. AC Pts (n=47) and DC pds (n=43) were recorded on three separate plant species and were similar on all plant species.     

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.     

EPG waveform characteristics of solenopsis mealybug stylet penetration on cotton

The solenopsis mealybug, Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae), is a polyphagous insect known to cause severe damage to cotton (especially transgenic varieties) in South Asia, and currently poses a serious threat in Asia and potentially elsewhere. Stylet penetration behavior of P. solenopsis on cotton was monitored using the electrical penetration graph (EPG) technique (DC system) and the EPG characteristics were compared with those previously published from Phenacoccus manihoti Matile‐Ferrero and Planococcus citri (Risso). We identified and further characterized typical waveforms of A, B, C, and pd (together pathway), E1 and E2 (phloem), F (derailed stylet mechanics), and G (xylem). Five novel EPG aspects were distinguished in the EPG waveforms from P. solenopsis: (1) obvious B waveforms occurred following waveform A, (2) during waveform C, some aphid‐like E1e waveforms were observed, (3) prolonged potential drops (pd) up to >1 h occurred with two continuously alternating sub‐phases pd1 and pd2, (4) the pd1 waveform always occurred as the first waveform related to phloem sieve elements, preceding the other phloem waveforms (E), the labeling of which we changed to achieve a better comparison to the aphid E waveforms, and (5) waveform F, related to derailed stylet mechanics occurred but was not reported from other mealybugs so far. This is mainly a waveform morphology study to extend existing knowledge on mealybug EPGs to investigate mealybug‐host plant interactions. Further experimental verification of waveform correlations with plant tissue positions of stylet tips and insect activities is still needed.     

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.     

Effect of saponins and apigenin mixtures on feeding behavior of the pea aphid,Acyrthosiphon pisum Harris

Electrical penetration graphs (DC EPG) were used to monitor the feeding behavior of the pea aphid Acyrthosiphon pisum Harris (Hemiptera: Aphididae) exposed to mixtures of saponins: 3GlcA, 28AraRhaXyl medicagenic acid glycoside (MAG) and zanhic acid tridesmoside (ZAD) with flavonoid apigenin, on agarose-sucrose gels. In general, mixtures of saponins with apigenin incorporated into agarose-sucrose gels resulted in a reduction in both the number of aphid probes and delayed their duration. Aphid feeding on gels impregnated with mixtures of the tested chemicals also showed a reduction in salivation into the gels and elongation of passive ingestion from the gels (EPG patterns g-E1 and g-E2, respectively). There were significant differences among the feeding behaviors of the pea aphids on gels containing mixtures of either MAG or ZAD with apigenin. These differences were also depended on the concentration and proportions of these compounds.     

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。     

Feeding behavior of two exotic aphid species on their original hosts in a new invaded area

Greenidea ficicola Takahashi and Greenidea psidii van der Goot (Aphididae: Greenideinae) are Asian aphid species newly introduced in Brazil associated with Moraceae and Myrtaceae. The feeding behavior of G. ficicola and G. psidii was investigated on their respective host plants, Ficus benjamina (Moraceae) and Psidium guajava (Myrtaceae), using the Electrical Penetration Graph (EPG). Fifteen females of each aphid species were monitored during 24h using a DC-EPG GIGA-4 monitor. The time spent in phloem phase (waveforms E1 and E2) was 13.6% of the total recording time for G. ficicola and 0.8% for G. psidii. The average time in the pathway phase (waveforms C and pd) represented 50% of the total time for both species. Aphids spent more time in non-penetration and stylet pathway activities than in the phloem phase or actual feeding. The principal component analysis (PCA) showed that the two species formed different groups in relation to EPG parameters, despite some overlapping. The probing patterns with multiple penetrations of short duration in the sieve elements for both species may indicate apparent unsuitability for sustained feeding on their respective host plants. These results suggest that these two exotic species are in the process of adaptation to their host plants in their new environment and/or the plants may present either chemical or physical barriers against these insects.     

Xylem ingestion by winged aphids

When aphids and their host plant are incorporated in a DC electrical circuit, phloem and xylem ingestion register as separate waveforms of the electrical penetration graph (EPG) signal. Aphids are primarily phloem feeders; xylem ingestion is seldom reported but can be induced experimentally by fasting the insects in desiccating conditions. In experiments with the black bean aphid, Aphis fabae Scop., young winged (alate) and unwinged (apterous) virginoparous adults were collected from their natal host plants (broad bean, Vicia faba L.) and allowed 3-h continuous EPG-recorded access to V. faba seedlings. Several aphids (47% of both morphs) showed ingestion from phloem sieve elements. Alate aphids also showed frequent xylem ingestion (60% of individuals), but no apterous aphids exhibited this activity. The EPG technique involves attachment of a fine gold wire electrode to each insect, a process that may affect normal behaviour at the plant surface. However, when the technique was modified to monitor the stylet activities of freely-settled aphids, high levels of xylem ingestion by alates were also recorded. The results suggest that the developmental physiology of winged aphids somehow predisposes them to xylem ingestion, possibly as a result of dehydration during the teneral period. Alate aphids may reduce their weight by fasting before take-off, giving aerodynamic benefits, but making rehydration, via xylem uptake, a priority following plant contact.     

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 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.     

Mi‐mediated aphid resistance in tomato: tissue localization and impact on the feeding behavior of two potato aphid clones with differing levels of virulence

The Mi‐1.2 gene in tomato, Solanum lycopersicum L. (Solanaceae), confers resistance against several herbivores, including the potato aphid, Macrosiphum euphorbiae (Thomas) (Hemiptera: Sternorrhyncha: Aphididae) and the sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Sternorrhyncha: Aleyrodidae). Previous studies on the tissue localization of resistance have given varying results; whitefly resistance was attributed to factors localized in the mesophyll or epidermis, whereas aphid resistance was attributed to factors localized in the phloem. Our study utilizes the direct current electrical penetration graph (DC‐EPG) technique to compare aphid feeding behavior on resistant (Mi‐1.2+) and susceptible (Mi‐1.2?) tomato plants. This study also compares the impact of resistance on the feeding behavior of two aphid clones that vary in their virulence, or their ability to survive and reproduce on resistant plants. Previous work had shown that the avirulent WU11 clone is almost completely inhibited by resistance, whereas the semi‐virulent WU12 clone can colonize resistant hosts. Here, DC‐EPG analysis shows that both aphid clones take longer to initiate cell sampling and to establish a confirmed sieve element phase on resistant plants than on susceptible hosts, and have shorter ingestion periods on resistant plants. However, the magnitude of these deterrent effects is far less for the semi‐virulent clone than for the avirulent aphids. In particular, the WU12 clone is less sensitive to factors that limit sieve element ingestion, showing shorter non‐probe duration and rapidly establishing sustained phloem ingestion on resistant plants when compared to the WU11 clone. We conclude that, in addition to previously described factors in the phloem that inhibit ingestion, Mi‐mediated aphid resistance also involves factors (possibly in the mesophyll and/or epidermis) that delay initiation of phloem salivation, and that act in the intercellular spaces to deter the first cell sampling. Furthermore, the relative effectiveness of these components of resistance differs among insect populations.     

Electrical penetration graphs of thrips revised: combining DC- and AC-EPG signals

Within thrips feeding behaviour, sequences of four waveforms have been distinguished earlier in the DC-EPG, i.e. P, Q, R and S, representing mandibular stylet insertion, maxillary stylet insertion, ingestion, and repetitive mandibular insertion, respectively. During signal analysis it appeared that transitions from one waveform to the next were difficult to establish, making results ambiguous. In order to improve the quantitative reliability of the thrips' EPG data, the DC-EPGs were recorded concurrently with AC-EPG signals, thus providing two signals from the same activities containing different information. The additional AC information did not solve most quantification problems, however. We now propose to merge waveforms P, Q, and S, into 'puncture phase' (indicated by PQ) and waveforms R, T, and U, into 'feeding phase' (indicated by R), rather than trying to analyse all separate waveforms. This will provide a more reliable and much less laborious analysis of thrips probing behaviour. Waveforms T and U are two novel waveforms identified here by combining DC- and AC-EPG recordings with concurrent video recordings. Waveform T represents a single mandibular thrust embedded in waveform R and waveform U represents the end of a probe, presumably the retraction of the maxillary stylets.     

Pathway phase waveform characteristics correlated with length and rate of stylet advancement and partial stylet withdrawal in AC electrical penetration graphs of adult whiteflies

A technique was developed for measuring the length of stylet insertion during adult whitefly probing. The distance that the labium shortens during a probe was shown to be equal to the length of stylets that were inserted into the plant tissue. The length of labial shortening then was measured in high-magnification video recordings of adult female silverleaf whitefly, Bemisia argentifolii, in conjunction with recording electrical penetration graphs (EPGs – AC method). Using a split-screen device, video images of the whitefly's labium during a probe and the EPG waveforms produced during the probe were recorded simultaneously on the same video tape. On playback, changes in labial length could be measured during specific EPG waveforms to determine the length of stylet insertion that occurred during the waveforms. The focus of the study was on two characteristics of the pathway phase sawtooth waveform: the frequency of voltage peaks and the increase in voltage level that occurs over time during sawtooth waveforms. The rate of stylet penetration was significantly and positively correlated with frequency of sawtooth waveform voltage peaks (r ²=0.33) and the length of stylet penetration was significantly and positively correlated (second-order polynomial) with the relative difference in voltage level between the beginning and end of the sawtooth waveform (r ²=0.43). Stylet advancement did not appear to occur during the few low-flat waveforms (unknown behavioral correlation) and high-flat waveforms (phloem phase) that were observed. Voltage drops occur sporadically during sawtooth waveforms, and these were associated with partial stylet withdrawal (indicated when the labium increased in length, but the probe was not terminated) with an accuracy of 99%.     

基于模糊识别和刺探电位技术鉴定小麦种质资源抗蚜性

利用田间抗蚜性鉴定模糊识别技术,结合室内刺探电位(EPG)植物抗性鉴定技术,比较分析了不同遗传背景的小麦种质资源的抗蚜性,为筛选新型小麦抗蚜种质材料提供依据。2年田间抗蚜性鉴定结果表明: 小偃麦多表现为中抗或低抗,而小黑麦多为中感或低感。选取抗性性状稳定且抗性级别不同的小偃麦21(中抗)、小偃麦22(低抗)、小黑麦31(中感)和小黑麦32(低感)进行麦长管蚜取食行为分析。对非刺探波(Np)、刺探波(P)、电势落差(Pd)、水溶性唾液分泌波(E1)、韧皮部取食波(E2)、细胞机械阻碍波(F)和木质部取食波(G)等基本波形的分析显示,麦长管蚜在小偃麦上首次开始刺探的时间显著长于小黑麦,且在小偃麦上的E1波的持续时间显著大于小黑麦;麦长管蚜在小偃麦21上的F波和小偃麦22上Np波的持续时间最长,在小黑麦31上的P波和小黑麦32上的G波的持续时间最长。以E1、F和Np波的持续时间为指标,基于刺探电位的小麦种质资源抗性水平鉴定结果与田间鉴定结果基本一致。因此,使用EPG技术筛选抗蚜小麦材料时,建议采用E1、F和Np波作为评价小麦抗性水平的指标。小偃麦21、22对麦长管蚜的抗性水平较高,可作为小麦抗蚜育种的种质材料。     

Modulation of a gated ion channel admittance in lipid bilayer membranes.

A future class of amperometric biosensors may utilize gated ion channels such as acetylcholine and glutamate receptors as chemical detection components. In this study, bilayer lipid membranes containing voltage-dependent anion channels (VDAC) were used to model an ion-channel-based biosensor which could continuously monitor AC amperometric changes resulting from induced changes in channel conductance. The in-phase and quadrature components of the induced alternating membrane current were monitored as a function of the applied DC offset voltage which was superimposed on the sinusoidal test voltage. The accuracy and sensitivity of the AC-measured VDAC response was dependent on the magnitude of the AC test voltage relative to the DC offset necessary for channel closure. The VDAC channel appears to be a suitable model protein for AC impedance-based biosensor fabrication.     

Characterization of the feeding behavior of three Erythroneura species on grapevine by histological and DC‐electrical penetration graph techniques

Feeding behavior of three leafhopper species – Erythroneura vitis (Harris), Erythroneura ziczac (Walsh), and Erythroneura elegantula (Say) (Hemiptera: Cicadellidae) – reared on grapevine, Vitis vinifera L. cv. ‘Seyval blanc’ (Vitaceae), was investigated using histological techniques and DC‐electrical penetration graphs (DC‐EPG). Histological studies revealed that the Erythroneura species induced white stipples on the leaves and that these leafhoppers produced thin salivary sheaths in grapevine leaf tissues. The DC‐EPG system allowed the characterization of five waveforms associated with stylet penetration and feeding in leaf tissues. These waveforms were characteristic of feeding phases corresponding to epidermis penetration pathway, salivation, and ingestion. We calculated 28 parameters (e.g., number of probes, duration of phases, and time spent in the various tissues) to describe and compare the feeding behavior of the Erythroneura species. We conclude that the three Erythroneura species are mainly mesophyll feeders but may probably also feed in other tissues such as xylem.     

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.     

Resistance of potato cultivars to Myzus persicae (Sulz.) (Hemiptera: Aphididae)

Aphids are the most important vectors of viruses infecting potato (Solanum tuberosum). We focused on the response of the aphid vector Myzus persicae (Sulzer) to five commercial potatocultivars: ágata, Jaette Bintje, Mondial, Monalisa and Santè, by traditional antibiosis and antixenosis tests and by the EPG (Electrical Penetration Graph) technique, as a step forward to the design of effective management practices. Our aim was to identify plant factors involved in resistance of these cultivars against M. persicae, both at the surface and in deeper plant tissues. Results from the antixenosis test confirmed a strong preference of M. persicae for the Mondial cultivar. The antibiosis study indicated a lower population development of the aphid in 'Monalisa' when compared to 'ágata' and 'Jaette Bintje'. EPG assays indicated that 'Santè' inhibited the initial feeding process of M. persicae, whereas 'Monalisa' showed a physical-type of resistance as demonstrated by a very high number of short probes. The cultivar Mondial showed average values for all EPG variables analyzed. The behavior in 'Jaette Bintje' indicated this cultivar was an ideal host for aphid feeding and reproduction. Together, the EPG data revealed the existence of pre and post-phloematics factors in the cultivars under study, which have important implications on the efficiency of transmission and spread of virus in potato by M. persicae.