ARTICLE IN PRESS Journal of Insect Physiology 52 (2006) 327–337 www.elsevier.com/locate/jinsphys Correlation of stylet activities by the glassy-winged sharpshooter, Homalodisca coagulata (Say), with electrical penetration graph (EPG) waveforms P. Houston Joosta, Elaine A. Backusb,Ã, David Morganc, Fengming Yand aDepartment of Entomology, University of Riverside, Riverside, CA 92521, USA bUSDA-ARS Crop Diseases, Pests and Genetics Research Unit, San Joaquin Valley Agricultural Sciences Center, 9611 South Riverbend Ave, Parlier, CA 93648, USA cCalifornia Department of Food and Agriculture, Mt. Rubidoux Field Station, 4500 Glenwood Dr., Bldg. E, Riverside, CA 92501, USA dCollege of Life Sciences, Peking Univerisity, Beijing, China Received 5 May 2005; received in revised form 29 November 2005; accepted 29 November 2005 Abstract 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. r 2005 Elsevier Ltd. All rights reserved. Keywords: Insecta; Hemiptera; Electronic monitoring of insect feeding; Probing; Stylet penetration 1. Introduction among applied entomologists and plant pathologists because it is a recently invasive, exotic insect in California. The glassy-winged sharpshooter, Homalodisca coagulata H. coagulata is an efficient vector of Xylella fastidiosa (Xf), (Say) (Hemiptera, Cicadellidae, Cicadellinae), is a xylo- the causal bacterium of Pierce’s disease in grape, leaf phagous leafhopper reported to feed on 100 species of scorch in almond and oleander, as well as citrus variegated plants in 37 families (Turner and Pollard, 1959; Adlerz, chlorosis (Hopkins, 1989). Xf naturally lives in the xylem 1980; Hoddle et al., 2003). This species has gained attention vessels of plants and in the foregut of cicadelline leafhoppers and other xylophagous auchenorrhynchans ÃCorresponding author. Tel.: +1 559 596 2943; fax: +1 559 596 2921. (Redak, 2004). H. coagulata acquires and inoculates Xf E-mail address: [email protected] (E.A. Backus). during stylet penetration (probing). Presumably, 0022-1910/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.jinsphys.2005.11.012 ARTICLE IN PRESS 328 P. Houston Joost et al. / Journal of Insect Physiology 52 (2006) 327–337 sharpshooters acquire Xf during ingestion of xylem sap and with EPG correlations with salivary sheath location in the inoculate plants when they release Xf into the xylem vessels plant (Backus et al., 2005), define the major H. coagulata of plants. waveforms, and provide a dynamic and detailed picture of There is copious data in the literature about the stylet stylet penetration behavior of this species while it is being penetration behaviors of hemipterans (Forbes, 1977; electronically monitored. Backus, 1985; Miles, 1987; Nickel, 2003). However, little has been published about xylophagous auchenorrhynchans 2. Materials and methods except a recent study of the blue-green sharpshooter, Graphocephala atropunctata (Say) (Almeida and Backus, 2.1. Insect maintenance 2004). Leopold et al. (2003) described the ultrastructure of H. coagulata mouthparts and the salivary sheaths they H. coagulata were greenhouse-reared to adulthood on leave behind in plants during stylet penetration. These healthy cowpea, Vigna unguiculata (L.) and sorghum, findings give us a picture of where the insect probes in the Sorghum bicolor (L.) at the California Department of plant and the function of its mouthparts based on Food and Agriculture’s Mt. Rubidoux Field Station, morphology. However, because of the static nature of Riverside, CA. Once insects reached adulthood, they were ultrastructure and plant histology studies, they provide transported to the USDA-Agricultural Research Service/ only limited insight to the precise types, movements and California State University-Fresno H. coagulata quaran- durations of such behaviors as ingestion and bacterial tine research laboratory in Fresno, CA. Sharpshooters expulsion during stylet penetration. A detailed under- were kept in 61 Â 61 Â 61 cm cages with cowpea and standing of stylet penetration behaviors of H. coagulata is sorghum under artificial and natural light at approximately an important step toward identifying the instant of 27 1C, L:D 16:8 h. All insects used in this study were bacterial inoculation which, in turn, will be applied to relatively young females of unknown age, and presumed to studies of disease epidemiology and development of host be non-inoculative for Xf. plant resistance (see Backus, 1994, for overview). A technique called electrical penetration graph (EPG) 2.2. EPG recordings monitoring of insect feeding can provide a real-time, dynamic picture of stylet penetration within a plant EPG waveforms were recorded with an AC/DC correla- (McLean and Kinsey, 1964; Tjallingii, 1978). In general, tion monitor (Backus ms., in preparation) that provided EPG is a measurement of voltage in a circuit that contains two simultaneous views of the insect’s waveform trace a piercing-sucking insect and substrate (i.e. plant or using AC and DC signal processing, respectively. The AC artificial diet). When a gold wire-tethered insect inserts its channel, whose design was based on the Missouri AC stylets into an electrified substrate, an electrical circuit is monitor v. 2.2 (Backus and Bennett, 1992), was used for all completed. Voltage fluctuations from resistance and/or analysis. Preliminary research (E.A.B., unpublished) has biopotentials of the insect–plant interface in the circuit are shown that H. coagulata waveforms recorded on plants are amplified and displayed on a computer monitor. These the same for the AC channel of the correlation monitor as voltage fluctuations produce stereotypical waveforms that those recorded on plants using the older Missouri monitor can be correlated with biological events at the plant–insect used for Backus et al. (2005). interface (Walker, 2000). Waveforms were acquired using a WinDaq DI-720 Backus et al. (2005) identify and categorize stereotypical analog-to-digital converter (DATAQ instruments, Akron, waveforms produced by H. coagulata during stylet OH) and displayed with WinDaq Pro+ software (DATAQ penetration on grape, and correlate these waveforms with instruments, Akron, OH) on a PC computer. All EPG salivary sheath branch terminations in plant cells. As a traces were recorded at 100 samples per second, with a result, they are able to follow step-by-step the progress of substrate voltage ranging from 50 to 80 mV, and an input stylet penetration into grape solely by viewing EPG impedance of 106 O. These are the same measurement waveforms. Though their study gives excellent spatial and parameters used for EPG recordings in Backus et al. temporal information on the stylet locations during (2005). To reduce handling of insects, we removed probing in grape, it does not correlate any stylet activities sharpshooters from holding cages in paper cups and with the EPG waveforms. immobilized them with CO2 for approximately 10 s. Silver Traditionally, insect stylet activity has been correlated conducting paint with N-butyl acetate solvent (Ladd with EPG waveforms by viewing stylet activities in clear Research Industries, Burlington, VT) was used to glue a artificial diets (McLean and Kinsey, 1964; Tjallingii, 1978; 50 mm diameter, 1.5 cm gold wire (Sigmund Cohn, Mount Hunter and Ullman, 1989; Kindt et al., 2003). The Vernon, NY) to the sharpshooter’s mesonotum. Insects objective of this work was to correlate the stylet activities were allowed to recover and acclimate to the tether on a of H. coagulata with EPG waveforms by viewing videos of cowpea plant for an hour after tethering. stylet movement in clear artificial diets