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 © 2016 The Entomological Society of Japan H. Yorozuya 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