Monitoring and Characterization of DC Electrical Penetration Graph Waveforms of Tea Green Leafhopper, Empoasca Onukii,Onteaplants

Entomological Science (2016) 19,401–409 doi: 10.1111/ens.12224

ORIGINAL ARTICLE

Monitoring and characterization of DC electrical penetration graph waveforms of tea green leafhopper, Empoasca onukii,onteaplants

Hiroshi YOROZUYA Institute of Fruit Tree and Tea Science, National Agriculture and Food Research Organization, Makurazaki, Kagoshima, Japan

Abstract The tea green leafhopper, Empoasca onukii (Homoptera: Cicadellidae), is a serious pest of “Yabukita”, the most popular tea cultivar in Japan. This study investigated its stylet-probing behavior with a direct current (DC) electrical penetration graph (EPG) system. The EPG signals were classified into four distinct waveforms according to amplitude, frequency, voltage level and electrical origin. The waveforms were then characterized by fast Fourier transformation. The waveforms were correlated with distinct feeding behaviors: Np, non- probing, when stylets were not inserted; Eo1, putative pathway and channel-cutting phase, when stylets were inserted but not ingesting sap; Eo2, putative phloem phase, when the leafhoppers were probably ingesting from phloem; and Eo3, putative non-phloem phase, when the leafhoppers were probably ingesting from mesophyll. Mean durations of waveforms showed that E. onukii likely ingested plant fluid mainly from phloem and partly from mesophyll. The description of DC EPG waveforms associated with feeding behaviors of this serious pest constitutes a fundamental step toward the understanding of the resistance mechanisms of the host plants against herbivorous insects. Key words: Camellia sinensis, feeding behavior, resistance mechanism, sap-sucking insects, Typhlocybinae.

INTRODUCTION pest management tactics, including use of host-plant resistance (Hazarika et al. 2009), have been developed. The tea green leafhopper, Empoasca onukii Matsuda However, development of resistance to leafhoppers in tea (Homoptera: Cicadellidae: Typhlocybinae), is one of the has lagged behind that in other crops because of the lack most serious insect pests of tea (Camellia sinensis (L.) O. of understanding of stylet-probing behaviors (Banerjee Kuntze) in Japan. The nymphs and adults pierce young 1992). Therefore, understanding the probing behavior of tea shoots and ingest plant fluids, causing classical E. onukii is the first step toward understanding host-plant symptoms of hopperburn (Backus et al. 2005) such as vein resistance (Jin et al. 2012). reddening, leaf margin yellowing, leaf curling, stunted An effective method of investigating hemipteran shoot growth and leaf drop (Fig. 1). The leafhopper can feeding is the electrical penetration graph (EPG) system, significantly reduce tea production and quality and can which measures voltage in a circuit that contains a cause economic losses of up to 33% (Xu et al. 2005). piercing–sucking insect and a plant. When a gold- Empoasca onukii is controlled mainly by insecticides, but wire-tethered insect inserts its stylet into an electrified multiple sprays are needed and these are costly. Broad-scale plant, an electrical circuit is completed. Voltage use of pesticides on tea has resulted in resurgence of primary fluctuations from resistance or biopotentials of the pests, outbreaks of secondary pests, development of insect–plant interface in the circuit are amplified and resistance and presence of pesticide residues in the product displayed on a computer monitor. These fluctuations (Hazarika et al. 2009). To reduce these problems, integrated produce stereotypical waveforms that can be correlated with stylet activities within plant tissues (Walker 2000). Correspondence: Hiroshi Yorozuya, Institute of Fruit Tree and Tea EPG studies have often been supported by data obtained Science, National Agriculture and Food Research Organization, 87 Seto-cho, Makurazaki, Kagoshima 898-0087, Japan. through observation of the salivary sheath, honeydew Email: [email protected] production and stylet position (Seo et al. 2009). Received 27 May 2015; accepted 14 November 2015; first McLean and Kinsey (1964) first developed the published 16 August 2016. alternating current (AC) EPG system to monitor the stylet

2012). Among Empoasca species, E. fabae (Harris) and E. kraemeri Ross and Moore have been studied by AC EPG (Backus et al. 2005), and E. vitis Göthe on tea plants has been studied by DC EPG (Miao & Han 2007; Jin et al. 2012; Miao et al. 2014). Recently, Jin et al. (2012) associated EPG waveforms with three E. vitis behaviors: (i) the salivary sheath channel-cutting phase, when the stylets are inserted but are not ingesting sap; (ii) the combined ingestion/salivation phase; and (iii) the active ingestion phase. Therefore, the objectives of this study were: (i) to monitor DC EPG waveforms produced by E. onukii on the most popular Japanese tea cultivar, “Yabukita”; (ii) to characterize and distinguish the waveforms by electrical characteristics and spectral analysis; and (iii) to hypothesize possible associations Figure 1 Young tea shoots damaged from probing by Empoasca onukii. between waveforms and the behaviors proposed by Jin et al. (2012) based on past studies and their histological evidence (e.g. Lett et al. 2001; Miao & Han 2007; Stafford penetration behavior of aphids. Tjallingii (1978) later & Walker 2009; Jin et al. 2012). developed the direct current (DC) EPG system, which can show both the electrical resistance and electromotive force (emf) components of the waveforms and give MATERIALS AND METHODS excellent waveform details that allow measurement of subfrequencies within a waveform (Tjallingii 2000; Insect and plant materials Walker 2000). By identifying more waveforms than the Young adult females (5–7 days after eclosion) were AC EPG system can (Reese et al. 2000), the DC EPG obtained from stock colonies reared according to the system allows more detailed conclusions about feeding method of Yorozuya and Tanaka (2012) and were starved behavior (van Helden & Tjallingii 2000). It has been used for 30 min before the experiments. All experiments used to compare resistant and susceptible cultivars (Annan five- to six-leaf shoots of “Yabukita” collected from the et al. 2000; Diaz-Montano et al. 2007; Crompton & Ode same field. The shoots, cut to about 10 cm long, were 2010) and locate the resistance factors in plant tissues inserted in flasks filled with water. As leafhoppers favor (Alvarez et al. 2006; Marchetti et al. 2009). A third the first leaf from the apical bud (Yorozuya & Tanaka – generation of monitor (AC DC EPG) has been developed 2012), all leaves except for the first were removed. One – (Backus & Bennett 2009), and the new AC DC monitor leafhopper was placed per shoot. allows the user to choose settings to replicate either a classical AC EPG monitor (input resistor Ri = 106 Ω)ora classical DC EPG monitor (Ri = 109 Ω), or to blend the EPG system capabilities of them (intermediate Ri). However, it has A Giga-8 DC EPG system (EPG Systems, Wageningen, the not yet been applied for Empoasca leafhoppers. Netherlands) with a 1 GΩ input resistance was used to After initial studies of aphids (van Helden & Tjallingii record EPGs in a Faraday cage, which can exclude 2000), the DC EPG system has since been applied to other electromagnetic noise from outside of the study system, sap-sucking insects, including leafhoppers (Lett et al. in a climate-controlled room (25 ± 2°C). Output from the 2001; Miranda et al. 2009; Stafford & Walker 2009; EPG at 100× gain was digitized at a rate of 100 samples Stafford et al. 2009; Trębicki et al. 2012), planthoppers from each of the four channels using a DI-710 analog-to- (Seo et al. 2009; Ghaffar et al. 2011), whiteflies (Walker digital board (Dataq Instruments, Akron, OH, USA). & Janssen 2000; Fereres & Moreno 2009), psyllids EPG signals were acquired and analyzed in the Stylet+ (Civolani et al. 2011) and thrips (Joost & Riley 2005; software (EPG Systems). The substrate voltage electrode Kindt et al. 2006). These studies associated EPG was inserted into the flask, and the substrate voltage was waveforms with stylet-probing behaviors in salivary adjusted to fit the EPG signals into the +5 to À5Vwindow sheath-feeding hemipterans. However, previous studies allowed by Stylet+. indicated that several Empoasca leafhoppers are cell- Leafhoppers were anesthetized with CO2 for 5 s. They rupture feeders that make a partial salivary sheath only were then held by their wingtips in tweezers and tethered or do not make it at all (Backus et al. 2005; Jin et al. to a gold wire (18 μm diameter, 3 cm length) with a

402 Entomological Science (2016) 19,401–409 © 2016 The Entomological Society of Japan EPG waveforms of tea green leafhopper droplet of water-based silver glue (EPG Systems) placed on the pronotum by a fine entomological pin. After about 30 s, a second droplet of glue was added and allowed to dry. The other tip of the gold wire was attached to a copper electrode (1 mm diameter, 5 cm length) connected to the EPG head stage amplifier. The tethered leafhopper was then placed on the leaf. Each leafhopper was recorded for 5 h (10:00–15:00 h) and 15 individuals were analyzed.

Characterization of EPG waveforms EPG waveforms were described on the basis of amplitude (maximum peak voltage per waveform, mV), frequency (Hz), voltage level (extracellular, positive; or intracellular, negative) and electrical origin (resistance (R) or emf). To determine the electrical origin, the applied signal polarity was switched from positive to negative; if the waveform polarity and amplitude and/or shape changed, it indicated the R component; if not, it indicated the emf component (Walker 2000; Jin et al. 2012). Amplitude, frequency and voltage level were identified from the appearance of output signals (Jin et al. 2012). The means and SE of amplitude and frequency were calculated from 40 waveform events from all experiments. The fast Fourier transform (FFT) tool of Stylet+ was used to identify the fundamental frequency in every waveform, providing an auto power spectrum graph (i.e. a frequency analysis of the first 10 s of the waveform). After the waveform categories were determined, the mean duration of each waveform (per insect), mean proportion of recording time per waveform, mean number of waveform events per recording and mean duration per waveform event (per recording) were calculated. These variables were compared among waveform categories by aKruskal–Wallis test with Steel–Dwass multiple- comparison test at α < 0.05 in R v3.1.0 software (R Development Core Team 2014).

RESULTS

Description of waveforms The leafhoppers produced three distinct DC EPG waveforms, labeled Eo1, Eo2 and Eo3, and a non-probing (Np) phase, in accordance with their general order of appearance during recordings (Fig. 2).

Np waveform The recordings showed a flat baseline; this is the Np phase Figure 2 Typical DC EPG waveforms produced by Empoasca onukii feeding on a tea shoot. (A) Feeding behavior over (Fig. 2B). During the Np waveform, the voltage remained 60 min; (B) non-probing (Np) and Eo1 waveform over 10 s. around 0 V; the small irregularities were caused by Waveform Eo1 is composed of the subtypes Eo1-A and Eo1-B; walking or labial dabbing. (C) Eo2 waveform over 10 s; (D) Eo3 waveform over 10 s.

Eo1 waveform Eo1 was the first waveform produced in all stylet probes, and was characterized by a sharp increase in the voltage from the baseline and a jagged and irregular shape (Fig. 2B). Eo1 usually occurred in every feeding probe, not only at the beginning but also at the end of a probe. The Eo1 waveform was composed of two waveform sub-types, Eo1-A and Eo1-B (Fig. 2B). Eo1-A always appeared at the beginning of a probe, accompanied by a rapid increase of voltage. Eo1-B usually occurred following Eo1-A and continued to the end of the Eo1 event. The amplitude of Eo1 was higher than that of Eo2 and lower than that of Eo3 (Table 1). The frequency of Eo1 was mixed (Table 1), as indicated by the irregular shape of the waveform. The FFT spectrum of Eo1 generally showed the predominance of a low frequency under 1 Hz (Fig. 3A). The voltage level of all Eo1 waveform sub-types was always extracellular and the electrical origin was R (Table 1).

Eo2 waveform Eo2 was a highly regular waveform with squarish peaks (Fig. 2C). The amplitude of Eo2 was lower than those of Eo1 and Eo3 (Table 1). The voltage level was always extracellular and the electrical origin was emf (Table 1). The FFT spectrum of Eo2 generally showed a fundamental peak between 3 and 4 Hz and a second peak around 7 Hz (Fig. 3B). Eo2 was usually preceded by Eo1.

Eo3 waveform Eo3 was also a highly regular waveform characterized by rapid sequences of sharp peaks with high amplitude (Fig. 2D). The frequency of Eo3 was higher than that of Eo2 (Table 1). The voltage level was always extracellular and the electrical origin was emf (Table 1). The FFT spectrum revealed a major frequency around 5 Hz and a Figure 3 Frequency spectra of EPG waveforms. (A) Eo1; (B) Eo2; (C) Eo3. Table 1 Summary of electrical characteristics of each waveform from DC EPG recordings of Empoasca onukii on tea plants second peak between 9 and 10 Hz (Fig. 3C). Eo3 was usually preceded by Eo1. Amplitude Frequency Vo lt ag e Electrical † † Waveform (mV) (Hz) level origin Np 0 0 0 – Duration and number of events of waveforms Eo1 There were significant differences between waveforms in sub-type Eo1-A 1668.4 ± 60.6 Mixed e R α < sub-type Eo1-B 676.8 ± 27.0 Mixed e R mean duration (Table 2, 0.05). Np and Eo2 were Eo2 557.8 ± 25.5 3.8 ± 0.1 e emf dominant, at 63.8% and 28.6%, respectively, of recording Eo3 2717.4 ± 70.7 4.8 ± 0.1 e emf time. Eo3 occurred least frequently, at 3.5% (Table 2, † α < Data indicate the mean values ± SE. e, extracellular (positive); emf, 0.05). Np and Eo2 had the longest duration and Eo1 electromotive force; R, resistance. the shortest (Table 2). Empoasca onukii probed many

Table 2 Mean waveform duration per insect (±SE), mean proportion of recording time of every waveform, mean number of each † waveform in one recording (5 h) and mean duration of one event

Mean duration Proportion of Mean number Mean duration Waveform per insect (min) recording time (%) of events in 5 h of an event (s) Np 191.4 ± 14.6a 63.8 ± 4.9a 46.5 ± 5.3a 287.6 ± 36.7a Eo1 18.2 ± 2.4c 6.1±0.8c 50.9 ± 5.5a 28.9 ± 6.9c Eo2 85.8 ± 13.1b 28.6 ± 4.4b 41.5 ± 3.8a 126.5 ± 15.1b Eo3 10.5 ± 2.2d 3.5±0.7d 17.5 ± 2.9b 67.6 ± 32.5d † Significance differences within columns are marked with different letters by Steel–Dwass multiple-comparison test (α < 0.05). times; the mean numbers of events in 5 h of Eo1 and Eo2 fabae and E. kraemeri produce no salivary sheath, or were more than 50 times and 40 times, respectively rarely only a sparse pseudosheath after performing one (Table 2). The mean durations per event of Eo1 and Eo2 of three cell rupturing tactics (Backus et al. 2005). In were very short, about 30 s and 120 s, respectively contrast, E. vitis produces incomplete sheaths, the trunk (Table 2). and sometimes the base of branches as the stylets fan out in multiple channels (Jin et al. 2012). Vigorous protraction and retraction of stylets during channel-cutting cause DISCUSSION mechanical laceration and wounding of plant tissues, contributing to hopperburn symptoms (Backus et al. Association between EPG waveforms and feeding 2005). It is presently unknown whether E. onukii behaviors produces any sheath saliva, i.e. an incomplete sheath like Four distinctive waveforms from DC EPG recordings of E. vitis (Jin et al. 2012) or absent like E. fabae (Backus E. onukii on tea were identified, and some of their et al. 2005). Only histological analysis of E. onukii characteristics showed similarities to those of other probing can resolve this question. leafhoppers, planthoppers, aphids (Tjallingii 1978; Lett Waveform Eo2 was characterized by a highly regular et al. 2001; Seo et al. 2009; Stafford & Walker 2009) shape, a relatively low amplitude, an extracellular and especially the congener, E. vitis (Miao & Han 2007; voltage level and a low frequency. These features Jin et al. 2012). showed that the major component of this waveform Generally, the stylet access time started with Np. The was emf, which is likely produced by the activity of the flat waveform indicates that no probing is occurring or cibarial pump (Lett et al. 2001). Eo2 resembles phloem that the stylet has not penetrated the leaf. ingestion waveforms reported in salivary sheath-feeding Waveform Eo1 always occurred after an Np phase, leafhopper species such as C. mbila (waveform 5, Lett starting with a sudden increase in voltage and showing et al. 2001) and O. orientalis (O5, Trębicki et al. 2012). variations in frequency and amplitude and an irregular These characteristics show similarities to those of other waveform shape. Similar waveforms have been observed sucking insects such as aphids (E2, van Helden & in work with aphids (Tjallingii 1978), Nilaparvata lugens Tjallingii 2000) and brown planthopper (N4, Seo et al. Stål (Seo et al. 2009; Ghaffar et al. 2011), Cicadulina 2009). However, cell rupture feeders, like typhlocibine mbila Naude (waveform 1, Lett et al. 2001), Orosius leafhoppers, may or may not ingest from phloem. Jin orientalis (Matsumura) (O1, Trębicki et al. 2012) and E. et al. (2012) found that their E2 waveform, which vitis (A, Miao & Han 2007; E1, Jin et al. 2012). resembles Eo2 in the present study, represents active The Eo1 waveforms recorded in this study are very ingestion combined with watery salivation. Studies of similar to the waveform of the pathway phase of Circulifer E. fabae and E. kraemeri identified the waveforms tenellus (Baker) (Stafford & Walker 2009) and channel- representing active ingestion: Ic and spiky Ic (Backus cutting and salivary sheath phase of E. vitis (Jin et al. et al. 2005). Ic could occur in mesophyll or general 2012). Unlike C. tenellus, E. vitis produces gelling saliva phloem cells, not restricted to sieve elements, whereas only at the beginning of the probe, creating an incomplete spiky Ic was thought to represent ingestion from leaking salivary sheath trunk to surround the base of stylets as sieve elements. Neither the present study nor that of Jin they enter the tissues (Jin et al. 2012). Thereafter, the et al. (2012) can identify whether phloem ingestion stylets perform active laceration movements and occurs in E. onukii or E. vitis, respectively. channel-cutting, also during the phase analogous to Eo1. The amplitude of Eo2 was lower than that of Eo3, In the studies of E. fabae and E. kraemeri with AC EPG, similar to E2 and E3, respectively, of E. vitis in Jin et al. the active stylet movements and channel-cutting were (2012). This could be caused by R-component revealed in waveform Ia, which resembles Eo1. Empoasca salivation waveforms interacting with emf-component

Entomological Science (2016) 19,401–409 405 © 2016 The Entomological Society of Japan H. Yorozuya ingestion waveforms. Watery salivation plus active emf component of waveforms, and has been shown to be ingestion could occur either in mesophyll or phloem, especially sensitive to streaming potentials from active but likely not in xylem. ingestion (Walker 2000; Backus & Bennett 2009). Waveform Eo3 resembles active ingestion waveforms of Consequently, the Eo2 (active ingestion with salivation) other leafhopper species: C. mbila (waveform 2, Lett et al. and Eo3 (active ingestion without salivation) waveforms 2001), O. orientalis (O2, Trębicki et al. 2012) and E. vitis can be distinguished in this study with the DC system, (E3, Jin et al. 2012). The highly regular shape, high although they were not clearly distinguished in AC amplitude and high frequency all show that the major recordings. However, similar waveforms have been seen component of this waveform was emf, probably produced recently, in unpublished recordings (EA Backus, pers. by the activity of the cibarial pump (Lett et al. 2001; comm., 2015) of an Empoasca species at Ri 109 Ω using Dugravot et al. 2008). Thus, it is likely that Eo3 the new AC–DC monitor (Backus & Bennett 2009). Given corresponds with active ingestion without salivation. This its duration, sequence in a probe and similarity, the Eo2 waveform occurred in E. vitis on tea plants following the waveform may be the same as the older Ic waveform channel-cutting phase to ingest the contents of artificial (ingestion, Backus et al. 2005) and the Eo3 waveform diets (Jin et al. 2012). A chromatographic study showed may be the same as the “spiky” sub-type of Ic waveform. that E. fabae consumes chlorophyll, indicating ingestion If more studies using the new AC–DC monitor are of whole mesophyll cell contents as a result of channel- conducted, we can compare waveforms at different Ri cutting (Backus et al. 2005). In addition, E. onukii levels and correlate probing behaviors and waveforms produced green, not transparent, honeydew (Fig. 4); this recorded by both AC and DC systems. It is likely that lower supports that E. onukii is a cell-rupture feeder in mesophyll, Ri levels (emphasizing R components) will be necessary to at least part of the time of feeding. There is no other feeding correlate E. onukii and E. vitis waveforms with specific behavior that results in green honeydew drops (EA Backus, tissues, in addition to (or instead of) fluid flow. pers. comm., 2015). Thus, it is possible that E. onukii may ingest from mesophyll, wholly or in part, during Eo2 and/or Eo3. Thus, the waveforms observed in this study Quantitative analysis of E.onukii probing corresponded to E. vitis, almost exactly the three phases behaviors of probing behavior proposed by Jin et al. (2012). Empoasca onukii ingested actively, both with salivation The key difference between most of the older AC (Eo2, 28.6% of recording time) and without salivation systems and the DC system was the proportion of emf (Eo3, 3.5%) (Tables 2,3). The feeding behavior of E. vitis vs. R present at the measurement point, caused by Ri on tea cultivar “Anjibaicha” was similar: waveform E values (Backus & Bennett 2009). Ri of the older AC (similar to Eo2, 20.0%) and waveform C (similar to monitor was 106 Ω, whereas that of the DC monitor was Eo3, 2.7%) (Miao & Han 2007). Empoasca fabae and 109 Ω. The AC monitor’sRiof106 Ω emphasized the R E. kraemeri ingested sap from both phloem (spiky I )and component of waveforms, so that the I (multiple cell c a mesophyll (I ) on common bean, but only from phloem laceration, Backus et al. 2005) shows taller peaks and b on alfalfa (Serrano & Backus 1998). It is possible that greater variability in voltage level than Eo1 from the DC Empoasca spp. leafhoppers switch their probing systems. However I and Eo1 occur at the same time in a behaviors on different host plants and cultivars. the probing behavior sequence, so an I waveform of older a Empoasca onukii may also change its probing behavior AC systems likely corresponded to the Eo1 waveform. such as the frequency of channel-cutting (Eo1) and The higher Ri level of the DC monitor emphasizes the Table 3 Summary of putative stylet-probing behaviors of Empoasca onukii in EPG waveforms

† Waveform Phase Activity Np Non-probing Eo1 Channel-cutting Cuticle penetration, some sheath salivation, stylet laceration Eo2 Ingestion and Active ingestion combined salivation with watery salivation Figure 4 Honeydew drops excreted by Empoasca onukii.The color of honeydew drops is green, not transparent. One adult Eo3 Ingestion Active ingestion without female was left to ingest and produce honeydew drops on the salivation terminal bud of a young shoot for 48 h and collected in 2 mL †Based on comparison with published studies on other Empoasca microtube. leafhoppers.

406 Entomological Science (2016) 19,401–409 © 2016 The Entomological Society of Japan EPG waveforms of tea green leafhopper the duration time of ingestion (Eo2 and/or Eo3) among putatively resistant genotypes and contribute to the tea cultivars. breeding of new resistant cultivars. Certain typhlocybine leafhopper species, such as E. abrupta DeLong, cause an unusual and characteristic symptom on leaves after probing: round, silvery- ACKNOWLEDGMENTS white marks called stipples. Thus, these species are called stipplers. Several other species such as E. fabae I thank Dr Elaine A Backus (USDA Agricultural Research and E. kraemeri are recognized as not causing stipples Service) for her valuable comments about the EPG system and to be associated with hopperburn symptoms; and cell-rupture feeding leafhoppers, and critical reading thus, they are called burners (Backus et al. 2005). of this paper. I also acknowledge Dr Tamotsu Murai Because E. onukii leaves only hopperburn symptoms, (Japan Agri-Clinic Institute Corporation) for his guidance not stipple marks, on tea leaves after probing and advice on this study. I am grateful to Mr Yasushi Sato (Fig. 1), it is likely that E. onukii is a burner, similar (Institute of Fruit Tree and Tea Science, NARO) for his to E. fabae and E. kraemeri. Moreover, using AC valuable comments. EPG monitoring, it is now known that Empoasca spp. leafhoppers are plastic in their probing behavior REFERENCES and perform a repertoire of three different feeding sub-strategies (called tactics); lacerate-and-sip, Alvarez AE, Tjallingii WF, Garzo E, Vleeshouwers V, Dicke M, lacerate-and-flush and lance-and-ingest. Furthermore, Vosman B (2006) Location of resistance factors in the leaves there are two variants of laceration-and-sip, called of potato and wild tuberbearing Solanum species to the pulsing laceration and sawing laceration (Backus aphid Myzus persicae. Entomologia Experimentalis et – et al. 2005). Pulsing laceration is composed of bouts Applicata 121,145 157. Annan IB, Tingey WM, Schaeferers GA, Tjallingii WF, Backus of many repeated, short-duration probes; for EA, Saxena KN (2000) Stylet penetration activities by Aphis example, each probe lasts about 1–2minforE. craccivora (Homoptera: Aphididae) on plants and excised – kraemeri or 2 6minforE. fabae (Backus et al. plant parts of resistant and susceptible cultivars of cowpea 2005). This study with the DC EPG system also (Leguminosae). Annals of the Entomological Society of revealed that E. onukii repeated short durations of America 93,133–140. laceration (Eo1) and active ingestion (Eo2 and Eo3) Backus EA, Bennett WH (2009) The AC–DC correlation many times (Table 2). It is reported that burners monitor: new EPG design with flexible input resistors to perform pulsing laceration during short-duration, detect both R and emf components for any piercing- primarily vascular probes, while stipplers perform sucking hemipteran. Journal of Insect Physiology 55, – sawing laceration during long-duration, usually 869 884. interval probes into mesophyll (Backus et al. 2005). Backus EA, Serrano MS, Ranger CM (2005) Mechanisms of hopperburn: an overview of insect taxonomy, behavior Therefore, I hypothesize that E. onukii performs and physiology. Annual Review of Entomology 50, primarily pulsing laceration of the lacerate-and-sip 125–151. tactic, similar to E. fabae and E. kraemeri.This Banerjee B (1992) Botanical Classification of Tea: Cultivation to hypothesis will be more rigorously tested in future Consumption. Chapman and Hall, London. research of resistant genotypes. Three tea genotypes Civolani S, Leis M, Grandi G et al. (2011) Stylet penetration of sustained less feeding damage by E. onukii than Cacopsylla pyri; an electrical penetration graph (EPG) study. “Yabukita” (Yorozuya & Tanaka 2012), and E. Journal of Insect Physiology 57,1407–1419. onukii excreted less honeydew on them than on Crompton DS, Ode PJ (2010) Feeding behavior analysis of “Yabukita” (Yorozuya & Ogino 2014). the soybean aphid (Hemiptera: Aphididae) on resistant soybean ‘Dowling’. Journal of Economic Entomology 103, 648–653. Diaz-Montano J, Reese JC, Louis J, Campbell LR, Schapaugh CONCLUSIONS WT (2007) Feeding behavior by the soybean aphid This study provided detailed information about the (Hemiptera: Aphididae) on resistant and susceptible soybean genotypes. Journal of Economic Entomology 100, probing behavior of E. onukii on tea plants. The 984–989. description of DC EPG waveforms associated with the Dugravot S, Backus EA, Reardon BJ, Miller TA (2008) feeding behaviors of this serious pest constitutes a Correlations of cibarial muscle activities of Homalodisca fundamental step toward the use of EPG in studies of spp. sharpshooters (Hemiptera: Cicadellidae) with EPG host-plant resistance. Application of the DC EPG ingestion waveform and excretion. Journal of Insect technique will help to reveal resistance mechanisms of Physiology 54,1467–1478.

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