Analysis of tea plant resistance to tea green leafhopper,Empoasca onukii,by detecting stylet‐probing behavior with DC electropenetrography

The tea green leafhopper, Empoasca onukiiMatsuda (Hemiptera: Cicadellidae: Typhlocybinae), is a serious pest of tea plants in East Asia. Previous work has shown that two tea germplasms, Cd19 and Cd289, sustain less hopperburn damage by E. onukii than does Camellia sinensis (L.) O. Kuntze cv. ‘Yabukita’ (Theaceae), and E. onukii excretes less honeydew on these germplasms than on the susceptible Yabukita. This study investigated the feeding behavior of E. onukii with a direct current electropenetrograph (DC EPG) to compare feeding behaviors, including ingestion, on resistant tea germplasms and Yabukita. Feeding behaviors on the resistant germplasms were significantly restricted, with few bouts of active ingestion of short duration and long periods of non‐probing, whereas E. onukii engaged in active ingestion of long duration many times on the susceptible cv. Yabukita. The tea germplasms, Cd19 and Cd289, therefore showed strong resistance to E. onukii. Furthermore, the shape of puncture holes left after probing was compared between the susceptible Yabukita and the resistant germplasms. The puncture holes on Cd19 and Cd289 were indistinct in shape and closed compared with those on Yabukita.     

假眼小绿叶蝉（Empoasca vitis Gothe）在不同品种茶树上的取食行为

应用可视DC-EPG方法研究了假眼小绿叶蝉(Empoasca vitis Gothe)在9个品种茶苗上的口针刺探行为,共发现并初步确定了7种主要波型,即A波、S波、C波、E波、F波和R波、以及非刺探波NP波。A波为刺探波,S波为口针向韧皮部刺探和进入韧皮部中的分泌唾液波,C波为口针到达韧皮部之前的主动取食波,E波和F波为口针在韧皮部中吸收波,R波为取食间歇波。以该叶蝉在不同品种茶树上的平均刺探次数和各波形平均持续时间为指标,或者以其在不同品种茶树上含有各波形的单次刺探平均持续时间为指标,分别进行聚类分析,均将9个品种分为3个不同的组。S、E和F波对应着假眼小绿叶蝉在茶树上的主要取食活动,可能与茶树抗假眼小绿叶蝉的取食密切相关。以S、E和F各波的平均持续时间、以及含有各波形的单次刺探的平均持续时间为指标,对品种的抗性强弱排序,评判9个茶树品种抗叶蝉取食能力由强至弱的顺序为:龙井长叶、黄旦、政和大白茶、黔湄601、红芽佛手、中茶102、中茶302、龙井43和安吉白茶。该顺序与田间查得的9个品种茶树上假眼小绿叶蝉种群密度由小到大的顺序一致,表明DC-EPG方法简捷、可信度高,可作为检测茶树品种对叶蝉抗性的有效手段之一。     

An analysis of the feeding behavior of three stages of Toxoptera citricida by DC electrical penetration graph waveforms

With the purpose of studying the feeding behavior of the brown citrus aphid pest, Toxoptera citricida (Kirkaldy) (Hemiptera: Aphididae), we compared stylet probing behaviors of third and fourth instars and adults on Citrus unshiu Marc (Rutaceae) seedlings using the electrical penetration graph (EPG) technique. EPG waveforms exhibited the full suite of stylet behaviors – stylet pathway, intracellular stylet puncture, phloem salivation (E1), sieve ingestion (E2), and xylem sap ingestion activities, plus the non‐penetration (Np) waveform. Before the phloem phase, the number of probes was significantly higher for third‐instar nymphs than for adults. Overall duration of Np events by adults was significantly lower than the duration of third and fourth instars. The number of short probes of the fourth instars was significantly higher than that of the adults. In the phloem phase, adults made more frequent and longer E1 events than the third and fourth instars. Third instars made more frequent but shorter E2 events, whereas adults made fewer but longer events. These results showed adults gained nutrients by increasing feeding time during phloem ingestion. Thus, the probability of phloem‐associated virus acquisition and transmission of T. citricida was higher in adults than in nymphs.     

Characterization of electrical penetration graphs of the Asian citrus psyllid, Diaphorina citri, in sweet orange seedlings

Detailed information on probing behavior of the Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is critical for understanding the transmission process of phloem‐limited bacteria (Candidatus Liberibacter spp.) associated with citrus ‘huanglongbing’ by this vector. In this study, we investigated stylet penetration activities of D. citri on seedlings of Citrus sinensis (L.) Osbeck cv. Pêra (Rutaceae) by using the electrical penetration graph (EPG‐DC system) technique. EPG waveforms were described based on amplitude, frequency, voltage level, and electrical origin of the observed traces during stylet penetration into plant tissues. The main waveforms were correlated with histological observations of salivary sheath termini in plant tissues, to determine the putative location of stylet tips. The behavioral activities were also inferred based on waveform similarities in relation to other Sternorrhyncha, particularly aphids and whiteflies. In addition, we correlated the occurrence of specific waveforms with the acquisition of the phloem‐limited bacterium Ca. Liberibacter asiaticus by D. citri. The occurrence of a G‐like xylem sap ingestion waveform in starved and unstarved psyllids was also compared. By analyzing 8‐h EPGs of adult females, five waveforms were described: (C) salivary sheath secretion and other stylet pathway activities; (D) first contact with phloem (distinct from other waveforms reported for Sternorrhyncha); (E1) putative salivation in phloem sieve tubes; (E2) phloem sap ingestion; and (G) probably xylem sap ingestion. Diaphorina citri initiates a probe with stylet pathway through epidermis and parenchyma (C). Interestingly, no potential drops were observed during the stylet pathway phase, as are usually recorded in aphids and other Sternorrhyncha. Once in C, D. citri shows a higher propensity to return to non‐probing than to start a phloem or xylem phase. Several probes are usually observed before the phloem phase; waveform D is observed upon phloem contact, always immediately followed by E1. After E1, D. citri either returns to pathway activity (C) or starts phloem sap ingestion, which was the longest activity observed.     

Leafhoppers on leaves: An analysis of feeding behavior using conditional probabilities

Feeding behavior of four deltocephaline leafhoppers,Graminella nigrifrons, G. oquaka, Amblysellus grex, andDalbulus maidis on maize and johnsongrass was analyzed using an electronic monitoring device. Five distinct waveform patterns were identified: secretion of sheath saliva (salivation), nonvascular probing, nonsieve element ingestion, x-waveform, and phloem ingestion. Waveforms were associated with feeding activities by correlation with light microscopic examination of salivary sheath termination points in leaf tissue and analysis of honeydew excreted by monitored leafhoppers. In previous studies x-waveforms have been reported to occur only when the stylets of homopterans are in contact with the phloem; the function of x-waveforms is poorly understood. There were no differences in time spent salivating or ingesting from nonsieve elements amongG. nigrifrons, G. oquaka andA. grex on either plant.D. maidis differed from other species in phloem probing and feeding behavior; only a small proportion produced x-waveforms, although those that did spent significantly more time in this behavior than other species. Also,D. maidis spent more time than other leafhoppers ingesting from tissues other than sieve elements. Kinetic diagrams of transition probabilities show that probing activities of all species were not random regarding the sequence of behaviors culminating in phloem ingestion. Thirty-five percent ofG. nigrifrons x-waveforms were followed by nonsieve element ingestion. This was consistent with observations showing that salivary sheaths of leafhoppers producing x-waveforms sometimes do not terminate in the phloem, but rather in nearby cells. Phloem ingestion was always preceeded by x-waveforms. The quantitative differences in probing behavior are discussed in relation to ability of these leafhoppers to transmit the phloem-associated maize chlorotic dwarf waikavirus.     

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.     

The role of sinigrin in host plant recognition by aphids during initial plant penetration

Plant penetration behaviour (probing) of the cabbage aphid, Brevicoryne brassicae, and the pea aphid, Acyrthosiphon pisum, was studied on excised leaves of broad beans, Vicia faba, kept in water or in a 1% aqueous solution of sinigrin. Using the DC EPG (Electrical Penetration Graph) technique it was shown that the cabbage aphid on sinigrin-untreated bean leaves showed numerous short probes into epidermis and mesophyll. None of these aphids showed either phloem salivation or ingestion waveforms on untreated leaves. In contrast, on sinigrin-treated bean leaves, 35% of the probing time was spent on phloem sap ingestion (E2) and almost all aphids reached phloem vessels and started feeding. The duration of phloem salivation before phloem ingestion and the mean duration of phloem ingestion periods were similar on a host and a sinigrin-treated non-host plant. However, the total probing time by B. brassicae was 10% longer, the total phloem sap ingestion time was twice as long, and the time to the first phloem phase within a probe was three times shorter on the host plant compared to sinigrin-treated broad beans. Acyrthosiphon pisum also responded to the addition of sinigrin to broad beans, but in this case sinigrin acted as a deterrent. On sinigrin-treated leaves, A. pisum terminated probes before ingestion from phloem vessels, and none of these aphids showed phloem salivation and ingestion on treated leaves. Glucosinolates were detected in the mesophyll cells of the brassicaceous plant, Sinapis alba. Based on this finding and in addition to the foregoing EPG analysis of aphid probing on these plants and broad beans, our hypothesis is that aphids may recognise their host plants as soon as they probe the mesophyll tissue and before they start ingestion from phloem vessels.     

Feeding disruption of potato psyllid,Bactericera cockerelli,by imidacloprid as measured by electrical penetration graphs

The potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), is a serious pest of potatoes that can cause yield loss by direct feeding and by transmitting a bacterial pathogen, Candidatus Liberibacter psyllaurous (also known as Candidatus L. solanacearum), which is associated with zebra chip disease of this crop. Current pest management practices rely on the use of insecticides for control of potato psyllid to lower disease incidences and increase yields. Imidacloprid is typically applied at potato planting, and it remains unknown if imidacloprid has any effect on potato psyllid feeding behavior. Thus, our specific objectives of this study were to determine and characterize the effects of imidacloprid treatment (0.11 ml l^?1) to potato plants on adult potato psyllid feeding behavior 1, 2, and 4 weeks post‐application. Electrical penetration graph (EPG) recordings of potato psyllid feeding revealed six EPG waveforms, which include non‐probing (NP), intercellular stylet penetration (C), initial contact with phloem tissue (D), salivation into phloem sieve elements (E1), phloem sap ingestion (E2), and ingestion of xylem sap (G). The number of NP events and the duration of individual NP events significantly increased on plants treated with imidacloprid compared with untreated controls. Potato psyllids exhibited significant decreases in the number of phloem salivation events on plants treated with imidacloprid. Waveform durations and waveform durations per event for E2 and G were significantly decreased for psyllids on plants treated with imidacloprid compared with untreated controls. These data suggest that the effective use of imidacloprid to reduce transmission of Ca. Liberibacter psyllaurous is related to the negative effects of imidacloprid on psyllid feeding.     

Electrical penetration graphs indicate that tricin is a key secondary metabolite of rice,inhibiting phloem feeding of brown planthopper,Nilaparvata lugens

Tricin (5,7,4′‐trihydroxy‐3′,5′‐dimethoxyflavone) is a valuable secondary metabolite which is widely present in gramineous plants, including cultivated rice (Oryza sativa L.) (Poaceae). It can defend the rice plant against damage by the brown planthopper (BPH), Nilaparvata lugens (Stål) (Hemiptera: Delphacidae), one of the most important pests of rice. This study was conducted to elucidate the mechanisms of action of tricin on BPH feeding behavior. BPH feeding behavior in resistant (Rathu Heenati, RHT) and susceptible (Taichuang native 1, TN1) rice varieties and artificial diets was monitored using the electrical penetration graph (EPG) technique. Tricin concentrations in leaves of varieties RHT and TN1 were quantitatively analyzed by liquid chromatography, coupled to tandem mass spectrometric techniques. Six (NP and N1‐5) and four (NP, N1, N2, and N4) types of waveforms occurred during feeding on rice plants and artificial diets, respectively. The tricin concentration of rice varieties was correlated with total and average durations of N4. Moreover, EPG data indicated that tricin significantly increased the duration of non‐probing and pathway periods and strongly inhibited phloem ingestion (N4). The inhibition was strongly dose dependent, resulting in complete suppression of activity in the phloem region when the tricin concentration was increased to 1 g l^?1. This study revealed that tricin disturbed the feeding behavior of BPH mainly by increasing the non‐probe period and inhibiting phloem ingestion. We confirmed the hypothesis that tricin is a ‘stylet probing stimulant’ of rice planthoppers as proposed in previous studies. The information on the ecological effect of tricin from this study may be useful to clarify the resistance mechanism against BPH of RHT and other tricin‐containing rice varieties.     

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.     

Probing behavior of the tea green leafhopper on different tea plant cultivars

The probing behaviors of the tea green leafhopper, Empoasca vitis (Gothe), on 9 tea cultivars were studied using video-text Direct Current-Electrical Penetration Graph, i.e., DC-EPG. The following 7 types of waveforms produced by the leafhopper stylet probing were determined: A, stylet pathway formation; S, salivation when stylets pierce into and stay in phloems; C, active ingestion before stylets reach phloems; E and F, passive ingestion in phloems; R, the insect resting with its stylet inserted into the leaf tissue and NP without probing. The 9 tested tea cultivars were categorized into 3 groups by the cluster analysis according to the number of probes per insect, waveform durations, or duration per probe of various waveforms on different tea cultivars. Waveforms S, E and F correlated to the main feeding activity of the leafhopper and may provide valuable information on predicting the resis-tance level of the tea plants to the leafhopper. The resistance level of the 9 tea cultivars to the leafhopper was ranked based on the durations of waveforms S, E and F, as well as the duration per probe including various waveforms. The ranking order of the resis-tance was: Longjingchangye > Hangdan > Zhenghedabaicha > Qianmei 601 > Hongyafoshuo > Zhongcha 102 > Zhongcha 302 > Longjing 43 > Anjibaicha, which corresponded to the resistance level determined by the population density (infestation) of the leaf-hopper on the 9 tea cultivars in the tea fields. Our study suggests that this simple and convenient DC-EPG technique might have great potential as a reliable tool to predict the resistance of tea cultivars to the tea leafhopper.     

Probing behavior of the tea green leafhopper on different tea plant cultivars

The probing behaviors of the tea green leafhopper, Empoasca vitis (Gothe), on 9 tea cultivars were studied using video-text Direct Current-Electrical Penetration Graph, i.e., DC-EPG. The following 7 types of waveforms produced by the leafhopper stylet probing were determined: A, stylet pathway formation; S, salivation when stylets pierce into and stay in phloems; C, active ingestion before stylets reach phloems; E and F, passive ingestion in phloems; R, the insect resting with its stylet inserted into the leaf tissue and NP without probing. The 9 tested tea cultivars were categorized into 3 groups by the cluster analysis according to the number of probes per insect, waveform durations, or duration per probe of various waveforms on different tea cultivars. Waveforms S, E and F correlated to the main feeding activity of the leafhopper and may provide valuable information on predicting the resis-tance level of the tea plants to the leafhopper. The resistance level of the 9 tea cultivars to the leafhopper was ranked based on the durations of waveforms S, E and F, as well as the duration per probe including various waveforms. The ranking order of the resis-tance was: Longjingchangye > Hangdan > Zhenghedabaicha > Qianmei 601 > Hongyafoshuo > Zhongcha 102 > Zhongcha 302 > Longjing 43 > Anjibaicha, which corresponded to the resistance level determined by the population density (infestation) of the leaf-hopper on the 9 tea cultivars in the tea fields. Our study suggests that this simple and convenient DC-EPG technique might have great potential as a reliable tool to predict the resistance of tea cultivars to the tea leafhopper.     

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.     

Comparison of Acyrthosiphon pisum probing behaviors on different alfalfa cultivars

Acyrthosiphon pisum (Harris) is a major pest of alfalfa worldwide. Here, we evaluated the resistance of eight alfalfa cultivars to A. pisum to identify resistant cultivars. The Giga-8 DC-EPG technique was used to record differences in electrical waveform patterns of probing behavior to identify cultivars with higher resistance. The frequency and average duration of EPG waveforms significantly differed among the cultivars, with eight documented waveforms (np, C, pd, E1, E2, sE2, F, and G). The longer duration of np, C, F and E1 waveforms reflected stronger resistance, whereas longer sE2 waveforms reflected weaker resistance. The resistance-related waveforms, np and C, occurred most frequently with Sardi 7, the time of the first E waveforms were the latest, duration of G waveforms and F waveforms were the longest, the feeding waveforms E2 and sE2 were the shortest, the time of reaching phloem for feeding was the latest, with notable resistance in leaf epidermis, mesophyll and phloem. The number of C waveforms occurring with Gibraltar were the least, duration of np waveforms was short, feeding waveforms were long, time to reach the phloem for feeding was early, and the resistance was not pronounced in various tissues. After comprehensive evaluation, the resistance levels of 8 alfalfa cultivars were as follows: High resistance, Sardi 7; Resistance, Zhongcao No. 3, Derby, WL319HQ; Low resistance, Golden Empress, Algonquin, Zhaodong; Susceptible, Gibraltar. In conclusion, evaluation of cultivar resistance to aphids can provide baseline data for selecting and planting cultivars with high local resistance.     

烟粉虱和温室粉虱在甘蓝上的刺探取食行为比较

利用刺吸电波图技术研究B型、ZHJ_1型烟粉虱Bemisia tabaci(Gennadius)和温室粉虱Trialeurodes vaporariorum(Westwood)在甘蓝上的取食行为,将3种粉虱的电波图进行比较,其中,B型和ZHJ_1型烟粉虱记录到np,C,pd,E1,E2,F和G波7种波形,温室粉虱只记录到刺探波形,少有取食波形。B型的20个记录中只有1个没有持续吸食波形;ZHJ_1型的25个记录中有10个记录没有持续吸食波形;温室粉虱没有持续吸食记录。甘蓝叶片韧皮部的结构或汁液的化学成分与温室粉虱的抗性密切相关。     

Plant penetration activities by the flatid planthopper Metcalfa pruinosa (Hemiptera: Fulgoroidea): an electrical penetration graph‐histology analysis

The citrus flatid planthopper, Metcalfa pruinosa Say (Hemiptera: Flatidae), is a very polyphagous native insect in North America and currently a serious pest in Europe and South Korea. To understand the feeding behaviour of M. pruinosa, stylet penetration behaviour of M. pruinosa was investigated with an electrical penetration graph (EPG) system. This study reports seven EPG waveforms related to M. pruinosa feeding behaviour: np (no stylet penetration), Mp1 (initiation of stylet penetration), Mp2 (stylet movement and salivation), Mp4 (phloem feeding), Mp4‐H (honeydew excretion), Mp5 (xylem feeding) and Mp6 (unknown). To determine respective feeding behaviour related to the Mp4 and Mp5 waveforms, stylets were cut with a laser beam, and the location of the stylet tip within plant tissue was examined. We found plant sap was exuded from the severed stylets only when the Mp4 waveform was observed, suggesting phloem sap ingestion. The stylet tip was located in the xylem region, indicating xylem‐feeding activity, when the Mp5 waveform was observed. The analysis of 24 different EPG parameters suggests that M. pruinosa stylets reached the vascular bundle of a plant within ca. 5 min and spend ca. 70% of the time feeding on xylem and phloem feeding. This is the first study that reports seven distinctive EPG waveforms with respect to the feeding behaviour of M. pruinosa which could help determine host specificity and host plant susceptibility.     

Characterization of EPG waveforms for the tea green leafhopper, Empoasca vitis Göthe (Hemiptera: Cicadellidae), on tea plants and their correlation with stylet activities

The stylet probing activities of the tea green leafhopper Empoasca vitis Gothe (Hemiptera: Cicadellidae) were studied using the DC electrical penetration graph (EPG) technique. Seven different EPG waveforms (i.e., Np, E1, E2, E3, E4, E5 and E6) were distinguished and characterized on susceptible tea leaves. In addition, four of them (i.e., Np, E1, E2, E3), together accounting for 97.08% of the total recording time, were behaviorally correlated with probing and non-probing activities using artificial diet observation with high-magnification video recording. At the start of stylet probing, waveform E1 always occurred at a variable voltage. E1, with all three of its waveform sub-types (E1-A to E1-C), was correlated with production of the salivary sheath trunk, stylet laceration, and channel cutting in viscous artificial diet. Afterwards, two types of high-amplitude waveforms, E2 and E3, followed. E2 had a highly regular, quasi-square wave, repetitive appearance, and lasted the longest duration of all E. vitis probing waveforms. E3 usually appeared after E2, and also exhibited a quasi-square wave feature similar to E2, but had much higher amplitude. Both waveforms E2 and E3 were correlated with active ingestion in liquid artificial diet. In addition, secretion of watery, enzymatic saliva was likely during E2. The active stylet movements and channel-cutting observed during the probing process indicate that E. vitis is a cell rupture feeder, not a salivary sheath feeder, as aphids and other leafhoppers. Thus, hopperburn damage to the tea plant is probably due to the cell rupture feeding strategy, similar to other hopperburning Empoasca species.     

Characterization of the electrical penetration graphs of the psyllid Bactericera trigonica on carrots

The psyllid Bactericera trigonica Hodkinson (Hemiptera: Triozidae) is a carrot and celery pest recently described as a vector of the plant pathogenic bacterium Candidatus Liberibacter solanacearum (Lso) on Apiaceae. Detailed information on vector stylet penetration activities is essential in the study of Lso transmission. In this study we used the electrical penetration graph (EPG) technique, characterized waveforms produced during the various stylet penetration activities in carrot leaves, and correlated them with stylet tracks and salivary sheath termini on plant tissues as well as with Lso inoculation. In addition, the effect of Lso in B. trigonica on the stylet penetration activities was tested. The EPG waveforms identified were: waveforms C1 and C2 detected in the mesophyll, waveforms D, E1, and E2 near or in the phloem sieve elements, and waveform G in the xylem vessels. A waveform pattern not previously reported for psyllids was the ‘pseudo‐potential drop’ (pseudo‐pd), characterized by sudden voltage dips similar to potential drops. However, the lowered voltage appeared to be inverted when the plant voltage is negative, indicating that it is caused by an increased resistance period and not due to a cell puncture. A direct correlation is shown between the waveform E1 and salivation into phloem sieve elements by B. trigonica as the inoculation of Lso occurred in a period as short as 30 s of E1; Lso transmission occurred in 17 of 35 plants (48%). Stylet activities during waveforms C or D had no consequences on the inoculation of Lso. In conclusion, Lso infection directly affects the probing behaviour of B. trigonica by increasing the total duration of C and D waveforms, but not variables related to phloem salivation (Lso inoculation) or ingestion (Lso acquisition). The reported information here is fundamental for identifying the psyllid vector traits of behaviour associated with transmission of Lso to Apiaceae.     

Characterization and correlation of DC electrical penetration graph waveforms with feeding behavior of beet leafhopper, Circulifer tenellus

Feeding behavior of beet leafhopper, Circulifer tenellus (Baker) (Homoptera: Cicadellidae), was studied with a DC electrical penetration graph. Nine different electrical penetration graph waveforms associated with feeding were identified and characterized. Waveforms were correlated with specific feeding behaviors using a number of techniques, including high magnification video recording, honeydew analysis, stylectomy, and histological processing. Waveforms were grouped into three phases based on feeding behavior: pathway phase (waveforms A, B1, B2, and C), non-phloem ingestion phase (waveform G), and phloem phase (waveforms D1, D2, D3, and D4). No ingestion was found to occur during waveforms A, B1, B2, and C. Waveform G was associated primarily with ingestion of xylem sap and occasionally with ingestion of mesophyll sap. Stylet tips were located in phloem during waveforms D1, D2, and D3, and waveforms D2 and D3 were correlated with ingestion of phloem sap. Waveform D4 probably also plays a role in phloem ingestion, because D4 is very brief and always occurs embedded in either waveform D2 or D3. In contrast to most other homopteran insects, rate of honeydew production (and hence rate of ingestion) was much lower on phloem than on xylem. More rapid rates of ingestion are expected on phloem, because its high turgor pressure drives sap into the feeding insect whereas the negative pressure of xylem sap is expected to cause a slow rate of ingestion. The very slow ingestion rate of beet leafhopper feeding on phloem suggests that it is not able to exploit the high turgor pressure of phloem to achieve the high rate of ingestion that is typical of phloem ingestion by other insects.