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ARTICLE IN PRESS

Journal of Physiology 52 (2006) 327–337 www.elsevier.com/locate/jinsphys

Correlation of stylet activities by the glassy-winged , 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 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 , 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 (Xf), the causal bacterium of Pierce’s disease, and 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 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 plant resistance. r 2005 Elsevier Ltd. All rights reserved.

Keywords: Insecta; ; 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, ), is a xylo- the causal bacterium of Pierce’s disease in grape, leaf phagous reported to feed on 100 species of scorch in almond and oleander, as well as variegated plants in 37 families (Turner and Pollard, 1959; Adlerz, chlorosis (Hopkins, 1989). Xf naturally lives in the 1980; Hoddle et al., 2003). This species has gained attention vessels of plants and in the foregut of cicadelline 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 52 (2006) 327–337 sharpshooters acquire Xf during ingestion of xylem 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, 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 -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 of Riverside, CA. Once 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 61 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 simultaneously After an hour, the individual wired insect was placed synchronized with EPG waveforms. Correlation of stylet into a diet chamber (see description below). The insect was activities with EPG waveforms from this study, combined attached to the EPG amplifier by connecting the gold wire ARTICLE IN PRESS P. Houston Joost et al. / Journal of Insect Physiology 52 (2006) 327–337 329 to the EPG input. The diet well was filled with 5% rectangle and an electrode connector was glued with epoxy solution and a wire conveying the substrate voltage was into the wall. A 301 angle cut was made on the 6 cm long placed in the solution. Sharpshooters were recorded walls, 2 cm from wall with the electrode connector. The throughout the day under artificial light at approximately piece of the rectangular frame with the electrode connector 25 1C. Individual recordings ranged from approximately was glued to an 8 8 cm Plexiglas base. The other piece of 15 min to 2.5 h. Duration of observation of an individual the rectangular frame, the diet well, bottom edges, and insect was based on the amount of probing and duration of edges on the angled walls were coated with dental wax. We the probes. The number of probes that we recorded per attached an unstretched piece of Parafilm (these sharp- sharpshooter ranged from 1 to 12 probes with an average shooters preferred unstretched, not stretched, Parafilm) on of 3.3. these waxed edges. The diet well then was placed on the base, flush with the angle of the other part. A wired sharpshooter was attached to the electrode and placed on 2.3. Diet chambers the Parafilm on the angle edges. The diet well was filled with a 5% sucrose solution and the chamber was placed Two different designs of artificial diet chambers enabled under the stereomicroscope to observe stylet activities from us to video record sharpshooter stylets in artificial diet a partial side-view. from two angles. The first artificial diet chamber was designed for observing the stylets at a 01 angle (i.e. viewed 2.4. High magnification video straight-on) (Fig. 1A). A 4 cm diameter hole was drilled into the center of a 6 6 0.6 cm square of Plexiglas. The High magnification video was made by placing either hole served as the sidewalls of the diet well. To make the diet chamber onto the stage of a Leica MZ16 stereomicro- bottom of the diet well, we sealed Parafilm to the bottom scope (Leica Microsystems Ltd., Switzerland) with PlanA- face of the Plexiglas with dental wax (Longs Drugstore PO 1.0 or 2.0 objectives. The stereoscope image of the Corporation, Walnut Creek, CA). The Parafilm was diet chamber was recorded with a CCD camera (JK- trimmed to the edges of the Plexiglas face. A wired insect TU52H Toshiba Corporation, Japan) attached to the was placed on the bottom (Parafilm-covered) face. The stereoscope. A fiber optic ‘cold light’’ (Schott 150 H chamber with the insect then was placed on 0.7 cm Universal, Schott Fiberoptik, Germany) was used as the Plexiglas risers underneath the objective. We filled the well light source. We viewed stylet movements and salivation in with 5% sucrose solution and observed the stylet activity the diet by moving the diet chamber into the plane of view by directly viewing the stylets through the diet. by hand and focusing the stereoscope. To observe fluid The second diet chamber was designed for observing the movement, we pipetted a highly concentrated solution of stylets at a 301 angle (i.e. viewed from the side) during ground blue Chinese stick ink particles into the sucrose probing (Fig. 1B). A 4 6 2 cm rectangular frame was solution during probes. These particles became suspended constructed from 0.6 cm thick Plexiglas. The pieces of the in the solution and their movement was used as a marker rectangle were glued together with IPS Weld-On 16 cement for fluid movement. (IPS Corporation, Gardena, CA). A 2 mm diameter hole was drilled into the center one of the short 4 cm sides of the 2.5. Synchronization of video and EPG waveforms

The sharpshooter’s EPG waveforms were synchronized in real time to video of its stylets by cabling the signal from the Windaq Pro+waveform display (DATAQ instruments, Akron, OH) on the computer monitor through a scan converter (Corioscan Pro S, TV One, Erlanger, KY), then into a video mixing board (Panasonic Digital AV Mixer WJ-MX20, Panasonic Corporation, Japan) (Fig. 2). The video signal from the CCD camera was also fed into the mixing board, where both signals were combined to produce a single, split screen view of a time-synchronized, real time video of both stylets and EPG signals. This mixed image was then fed into a Dell Optiplex computer (Dell Corporation, Austin, TX) through a video card ( MVR1000, Canopus Corporation, San Jose, CA) and recorded at 30 frames per second with MediaCruise software (Canopus Corporation, San Jose, CA) in an Fig. 1. Diagram of artificial diet chambers. (A) Side and stereoscope view Mpeg-2 format. We confirmed that the high magnification of the 01 diet chamber. (B) Side and stereoscope view of the 301 diet video and EPG signal were synchronized by touching a chamber. finger to an electrode that was connected to the EPG ARTICLE IN PRESS 330 P. Houston Joost et al. / Journal of Insect Physiology 52 (2006) 327–337

wave-like flat areas interspersed among B1s ‘‘spikelet bursts,’’ which are chains of highly repetitive, very low- amplitude but high frequency (15–25 Hz) spikes. Waveform B2: A highly uniform waveform that is composed of smooth, medium-sized and frequency, peaks (3–4 Hz) that collectively form a chevron, or sometimes half-chevron, pattern. Waveform C: A very regular pattern consisting of low- amplitude plateaus and squared valleys.

3. Results

Sixty-one probes were recorded from 19 individual sharpshooters. We observed three stylet pathway phase waveform types, A1, B1 and B2, and one ingestion phase Fig. 2. Equipment configuration for split screen video of synchronized H. waveform , C, all known to occur when H. coagulata coagulata stylet activities and EPG waveforms. Arrows indicate direction probes on grape plants (Fig. 3). Highly irregular wave- of the signal. Arrows with a barbed back on a solid line represent the forms unique to artificial diets (labeled ‘?’ in Fig. 3) were signal from the EPG recording. Small arrows with a flat-back on a dashed occasionally also observed but not correlated, because they line indicate signal from the video. Large, flat-back arrows on a dashed represented 10% of the total duration of probing, were line exiting the top of the video mixer represent the video and EPG signals o combined. Image in monitor is an actual split screen video with H. not consistently associated with observable stylet activities, coagulata stylets in the top frame and EPG waveform in the bottom frame. and did not resemble plant-derived waveforms seen in studies to date (Backus et al., 2005). Generally, waveforms in this study resembled plant-recorded waveforms, with the exception of a reduced percentage of probes with waveform monitor while recording the test under high magnification C. Other, minor differences between diet and plant video. waveforms are considered in Section 4.

2.6. Waveform terminology and analysis of data 3.1. Pathway phase: waveform A1 Stylet movement, salivary sheath formation, and fluid We observed stylet activities and salivary sheath forma- movement into and out of stylets were correlated with EPG tion during waveform A1 in 28 probes from 15 sharp- waveforms by reviewing split screen video using Canopus shooters. The A1 waveform typically occurred at the onset Media Cruise software. We also used Windaq Browser of the probe in clusters of 2–5 peaks at a frequency of software (DATAQ Instruments, Akron, OH) to view 0.66 Hz. Occasionally, a single A1 peak was observed in the detailed waveform patterns that corresponded to video. middle of the probe. A salivary sheath bubble was formed Only waveforms defined by Backus et al. (2005) from H. coagulata probing on plants were correlated with the video. We used the hierarchical naming convention for waveforms introduced by Almeida and Backus (2004) and Backus et al. (2005), consisting of (1) phases: interpretable with coarse-structure amplification, representing overarching behaviors such as stylet pathway and ingestion, (2) families: interpretable with medium-structure amplification, desig- nated by alphabetic letters, and (3) types: interpretable with fine-structure amplification designated by numerals ap- pended to the family letter. In brief, the waveform families and types we observed are as follows. Images of the waveforms and more detailed descriptions are found in Backus et al. (2005). Waveform A1: A moderately regular, high-amplitude waveform that is composed of slow peaks with low valleys Fig. 3. Waveform trace of one H. coagulata probe in 5% sucrose diet. The that ride on a large waveform hump (i.e. positive voltage trace in the top panel is 333 s long and compressed 40 times. Scale bar, 1 s. offset). A1 is the waveform that occurs at the beginning of Waveforms A1, B1, B2 and C are indicated by divisions and labels. Waveforms labeled ‘‘?’’ are undefined waveforms. The bottoms panels are the probe. 3.8 s long, 2 compressed traces from the top panel. For all traces in the Waveform B1: A very low-amplitude waveform that bottom panel, scale bar, 1 s. The ‘‘*’’ in the top panel indicates the point contains two subparts, B1w and B1s. B1w is composed of which each bottom panel trace was enlarged. ARTICLE IN PRESS P. Houston Joost et al. / Journal of Insect Physiology 52 (2006) 327–337 331 during each A1 peak, however the precise moment of tional to large changes in voltage level, i.e. voltage valleys salivation during A1 could not be identified. often indicated deeper penetration, while voltage peaks Each A1 peak represented a repetitive series of stylet represented partial stylet withdrawal. activities (Fig. 4). First, both maxillary stylets (maxillaries) Consequently, the single peak of A1 waveform repre- simultaneously retracted at the beginning of the A1 spike’s sented a cycle of stylet activities: (1) retraction of both incline (Fig. 4b and g). Approximately 0.4 s after the maxillary styles and one mandibular stylet, (2) the maxillaries retracted, a single mandibular stylet retracted extension of the entire stylet fascicle, up to and past its while the other mandibular stylet extended slightly (Fig. 4c previous furthest extension, and (3) the fluttering-like and h). The mandibular stylet that retracted was alternated movements of the maxillary stylet tips. Each cycle caused for each peak (compare Fig. 4c and h). Extension of the the stylets to advance deeper into the substrate, with previously retracted stylets followed, beginning at the apex accompanying sheath salivation. A1 always occurred at the of the A1 peak (just after Fig. 4c and h), progressing start of a probe; thus the sheath salivation formed the through the voltage decline (Fig. 4d, e, and i), and ending sheath trunk (described more fully in Backus et al., 2005). at the start of the waveform valley (Fig. 4f and j). A short Because this early stage of penetration often occurs bout of B1 s, or a spikelet burst, plus maxillary stylet through bark or other tough tissues, and the alternating fluttering (i.e. short, alternating extensions and retractions retraction/extension of the mandibular stylets resembles within the salivary sheath, see further description of B1 sawing, we termed this action ‘mandibular stylet sawing’ events, below), always occurred between A1 peaks (see further interpretation in Section 4). After mandibular (Fig. 4d–f and k). The exact voltage level of B1 s varied, stylet sawing and sheath trunk formation were completed, sometimes occurring during the voltage decline (Fig. 4d only the maxillary stylets enacted the remaining activities and e), or in the valley (Fig. 4k and beyond), or during in the probe; the mandibulars remained in a fixed position, both waveform sub-types. When the stylets were fully shallowly braced just beneath the Parafilm. extended, the maxillary stylet tips were slightly extended beyond the mandibulars (Fig. 4a), and the once-retracted 3.2. Pathway phase: waveform B1 mandibular stylet was slightly extended further than the other mandibular stylet (not shown in Fig. 4). This position Forty-nine probes with B1 waveforms were observed of the stylet was correlated with the valley of the A1 from 15 H. coagulata. As on plants (Backus et al., 2005), waveform. Thus, depth of stylets was inversely propor- B1 was divided into two alternating sub-types: (1) B1w (also termed B1 wave), a smooth waveform of increasing voltage level (Fig. 5a–d), and (2) B1s (also termed B1 spikelet burst), with a frequency of 15–25 Hz (Fig. 5e). Secretion of a bubble of sheath saliva was correlated with B1w, especially during the rising portion of the wave, in all cases ðn ¼ 9Þ when resolution allowed a clear view of the transparent saliva (Fig. 5a). Sheath salivation stopped approximately 1–2 s before a spikelet burst, B1s (Fig. 5c). At this point, the salivary bubble diminished in size (i.e. possibly contracted) (Fig. 5d). During B1, the mandibular stylets were motionless. The maxillaries, extended at varying lengths beyond them and spread apart longitudinally (i.e. staggered or offset), made very short, alternating extensions and retractions within the salivary sheath (Fig. 5a–c and e). This maxillary stylet tip fluttering occurred in this manner during both sub-types of B1, i.e. during B1w and B1s. Neither the frequency nor precise occurrence of maxillary flutterings were correlated with the frequency or occurrence of B1s spikelets. For example, a compressed spikelet burst is shown between Fig. 4. A1 waveform correlated with H. coagulata stylet activities. Top Fig. 6a and b, yet there was no tip fluttering at this time; in panel is an uncompressed waveform trace of two A1 peaks. Arrows with letters are 1 s. apart. The middle panel shows the actual images of the contrast, tip fluttering occurred later, in Fig. 6c–e,in stylets at the point on the waveform marked by the corresponding letters absence of a B1 s multi-spikelet burst. The maxillaries in the top panel. Actual video views on the computer monitor were in fluttered inside the salivary sheath during B1, while color and were of higher resolution. The bottom panel shows interpretive gradually both maxillaries and the salivary sheath were drawings of the stylets from the middle panel, without surrounding sheath extended forward, making a very narrow path within a saliva, which was present in all cases. The larger arrows in the bottom panel indicate movement of the individual stylets; the small double-ended narrow salivary sheath. arrows indicate maxillary tip fluttering. Salivary sheath not shown. Scale B1s spikelet bursts were embedded within or interspersed bar, 1 s. among most other waveform types. For instance, B1s was ARTICLE IN PRESS 332 P. Houston Joost et al. / Journal of Insect Physiology 52 (2006) 327–337

Fig. 5. B1 waveform correlated with H. coagulata stylet activities, movements and saliva secretion. Top panel shows a spread-out (i.e. uncompressed) waveform trace of a B1 waveform. Arrows with letters are Fig. 6. B2 waveform correlated with H. coagulata stylet activities. Top 1 s apart, except ‘‘d’’ and ‘‘e’’ which are 2 s apart. The middle panel shows panel shows a 2 compressed trace of waveforms prior to and including the actual images of the stylets, and the bubbles of sheath saliva at their the first part of B2. The boxed panel at the beginning of the traces tips, at the point on the waveform marked by the corresponding letters in indicates waveform B1. The B2 waveform begins at the dotted line, with the top panel. Note expansion of the salivary sheath from points ‘‘a’’ to the waveform in between being undefined. Points ‘‘a’’ and ‘‘b’’ are 2 s ‘‘c’’ and its slight diminishing in size from point ‘‘c’’ to ‘‘d’’. Actual video apart and points ‘‘b’’ and ‘‘c’’ are 3 s apart. The remaining points are 1 s views on the computer monitor were in color and were of higher apart. The middle panel shows the actual images of the stylets and salivary resolution. Sheath saliva surrounding the length of the stylets is not shown sheath at the point on the waveform marked by the corresponding letters in the middle, video images, although it is depicted in the bottom panel of in the top panel. Note the retraction of the maxillary stylet from point ‘‘a’’ drawings, showing interpretations of stylets from the middle panel. Dotted to ‘‘c’’ and the branch in the salivary sheath from points ‘‘e’’ to ‘‘k’’. Also lines indicates extant salivary sheath; solid-lined saliva indicates the note the change in angle in the maxillary stylet from point ‘‘a’’ to point portion of the sheath that is forming during B1w. The larger arrows in the ‘‘d’’ and the in-and-out sawing of the maxillary stylet during the B2 bottom panel indicate movement of the individual stylets; the small waveform. The left mandibular stylet (on the right side) appears broken, double-ended arrows indicate maxillary tip fluttering. Scale bar, 1 s. but is merely refracted by the edge of the nearly invisible salivary sheath bubble. Actual video views on the computer monitor were in color and were of higher resolution. The bottom panel shows interpretive drawings observed within: (1) the A1 waveform (Fig. 4d and e), (2) of stylets from the middle panel. The larger arrows in the bottom panel non-descript waveforms (not shown), (3) waveform C (not indicate movement of the individual stylets; the small double-ended shown), and (4) before and after B2 events (Fig. 6a–e). arrows indicate maxillary fluttering. Scale bar, 1 s.

3.3. Pathway phase: B2 waveform

Twenty-one probes with B2 waveforms were observed Together both would then retract strongly and abruptly from 11 H. coagulata. In all observations, the B2 waveform (Fig. 6g). This coordinated, very extensive, in-and-out was associated with a highly stereotypical and repetitive action of both maxillaries was termed maxillary stylet series of rapid, large retractions and extensions of sawing. This action was different from maxillary fluttering the maxillary stylets, which (in all cases in this artificial because (in the latter case) the maxillary stylets alternated substrate) caused branching of the salivary sheath (Fig. 6). very minor extensions and retractions at a small amplitude, Prior to B2, the maxillaries (which previously had been as though only the tips rather than the full length of the extended beyond the mandibulars within the salivary stylets were moving. Although the stylet activities pictured sheath; Fig. 6a), retracted to the tip or below the tip of in Fig. 6, lower panel, are not very abrupt, nor are the mandibular stylets (Fig. 6b and c). This partial stylet extensions and retractions made very deeply, most typical withdrawal occurred between Fig. 6a and c. It was not maxillary sawing was much more so. During both B2 possible to discern whether the stylets withdrew only at the extension and B2 retraction, maxillary tip-fluttering also instant of the small, abrupt voltage increases (Fig. 6, before occurred. The maxillary stylets would eventually saw (or and after b). During stylet withdrawal, B1s and B1w were chisel) through the side of the existing (presumably both recorded. hardened) salivary sheath and form a new branch in the Fig. 6c–f and h–k show maxillary tip fluttering simulta- sheath (Fig. 6f–k). B2 sawing ended when the insect neous with the following stylet activities. At the beginning stopped extending and retracting its stylets, usually when of the B2 waveform (Fig. 6, dotted line) one maxillary the maxillary fascicle punctured through the tip of the stylet, followed by the other maxillary stylet, would extend newly formed sheath branch. Typically, a B1 waveform in a direction that was skewed from their original position. and new salivation followed B2 sawing. ARTICLE IN PRESS P. Houston Joost et al. / Journal of Insect Physiology 52 (2006) 327–337 333

3.4. Ingestion phase: waveform C 4. Discussion

A highly repetitive waveform, C, was observed in three Artificial diets frequently have been used to correlate probes from three different H. coagulata probing into insect stylet activity with EPG waveforms (McLean, 1964; artificial diet. No stylet movement was observed during C. Kawabe and McLean, 1978, 1980; Tjallingii, 1978; Raman The maxillary stylets were extended beyond the mandib- et al., 1979; Triplehorn et al., 1984; Hunter and Backus, ulars and their tips were offset (Fig. 7). The precise moment 1989; Kindt et al., 2003). Observation of stylets in diets is of fluid uptake was indicated when Chinese stick ink established as one requirement to complete the ‘‘triangle of particles moved toward and into the maxillary stylet tips. correlations’’ to define new waveforms (i.e. stylet activities, This action was correlated with the waveform’s valley (Fig. waveforms, and plant locations, all correlated with one 7a, c and d). Particles ceased to move, or moved away from another; Backus, 1994). Although artificial diet correla- the maxillary stylet tips during the plateau phase of the tions provide excellent observational data of stylet waveform, indicating intermittent fluid uptake (suction) activities during probing, extending these findings to actual during continuous waveform C (Fig. 7b and e). Sometimes probing on plants must be done cautiously (Walker, 2000). particles collected around the maxillary tips. These Researchers have reported that waveform durations, particles became compressed together over the tip opening appearances and waveform progression differ when insects during the valley phase and uncompressed during the probe into artificial diets, compared with plants (Van plateau portion (Fig. 7a and b). In one observation, Helden and Tjallingii, 2000). In part to avoid this problem, particles that collected around the stylet tips were blown we describe only the stylet activities and salivary sheath away from the stylets, indicating a fluid outflow event formation for three clearly recognizable pathway wave- during the plateau portion of the waveform (not shown). forms, A1, B1 and B2, and an ingestion waveform, C This event could not be clearly correlated with a specific known to occur when H. coagulata stylets penetrate plants, waveform sub-type. In another observation, the insect specifically grape (Backus et al., 2005). In addition, the bypassed the particles at its stylet tips by making a new correlated waveforms together represented most probing salivary sheath around it. The insect initiated B2 sawing (90%) on artificial diets. and, once a new branch was made, re-commenced with The waveform trace of an adult H. coagulata probe in waveform C. artificial diet (Fig. 3) bears a strong resemblance to one made on grape (Backus et al., 2005), but with a few differences. Interestingly, the trace is flatter, with less positional information (i.e. fewer increases and decreases in voltage level) when recorded on diet compared with grape. Flatter diet waveforms may reflect penetration through an electrically uniform substrate. In contrast, background electrical signals may differ slightly among different layers of a structurally more complex, electrified plant. This is supported by the finding that voltage level changes in AC recordings on plants have been correlated with depth of stylet position (Backus et al., 2005). Other differences in waveform appearance between diet and grape involve size of the waveform types. B2 and C are taller on diet, while A1 is shorter and B1 is about the same. The electrical meaning of these differences is unknown.

4.1. Pathway phase: waveform A1 Fig. 7. C waveform correlated with H. coagulata stylet activities and fluid movement. Top panel is an uncompressed waveform trace of a C In this study, probes always began with the A1 wave- waveform. Arrows with letters are 1 s apart. The *’s in the top panel form, similar to H. coagulata’s probes on plants (Backus et indicate the beginning and end of the movement of a Chinese stick ink al., 2005). Salivary sheaths in plants begin with a thick, particle from further out in the diet solution to the maxillary stylet tips. short trunk (Leopold et al., 2003). A1 waveform represents This particle is shown in the middle panel by * and a dashed line around it. The middle panel shows the actual images of the stylets at the point on the the behaviors when the stylets enter into the plant and form waveform marked by the correspond letters in the top panel. Note the the trunk of the salivary sheath. Chinese stick ink particles around the maxillary tips. The bottom panel We were able to correlate the precise extensions and shows interpretive drawings of the middle panel. Actual video views on the retractions of mandibular and maxillary stylets with the computer monitor were in color and were of higher resolution. In the AC EPG waveforms during A1, and overall with sheath bottom panel the larger arrows indicate movement of fluid, the stars represent Chinese stick ink particles, while the oval represents the particle salivation. Unfortunately, individual spurts of sheath outlined in the middle panel. Note the fluid movement and compression of saliva could not be precisely correlated with any recogniz- particles around the maxillary tips to the waveform plateaus. Scale bar 1 s. able stages of a repetitive waveform. The large voltage ARTICLE IN PRESS 334 P. Houston Joost et al. / Journal of Insect Physiology 52 (2006) 327–337 incline of the A1 spike was visually correlated with partial the mandibular stylets (in the maxillaries-ahead approach stylet withdrawal, specifically the insect retracting one of Backus, 1985) to penetrate through the interior tissues. mandiblular stylet and both maxillary stylets. This In that latter case, it is likely that the mandibular stylets act repetitive stylet movement has also been observed for G. as a both a brace and a fulcrum for the further movement atropunctata (Crane, 1970)andHomalodisca liturata of the maxillary stylets, as has been observed in other (Joost, unpublished), and may be typical of all cicadelline hemipterans (Cohen, 2000; Wheeler, 2000). probing. There are several functions to the stylet movements 4.2. Pathway phase: waveform B1 during the A1 waveform. An important requirement for directional movement of the stylets is that they are Waveform B1, especially sub-type B1s, was ubiquitous incurved (Pollard, 1969). Like those of fasciatus throughout H. coagulata’s stylet penetration in artificial (Dallas), H. coagulata’s mandibular stylets are also diet, as is typical during plant probing. B1 was associated incurved at their tips (Leopold et al., 2003). Miles (1959) with both salivation and independent maxillary tip flutter- reported that O. fasciatus used one mandibular stylet ahead ing. For example, we observed B1 s spikelet bursts at the during probing also. However, instead of retracting a end of each A1 peak’s decline, when the fascicle was fully mandibular stylet and both maxillary stylets followed by or nearly fully extended. The maxillaries would indepen- extension of the whole fascicle, O. fasciatus works in dently flutter prior to and during these spikelet bursts, reverse, by extending a single mandible beyond the rest of while sometimes extending beyond the mandibular tips. the fascicle followed by extension of the remaining three Thus, the function of maxillary tip fluttering is to create a stylets. Miles (1959) proposed that by extending one narrow path through the salivary sheath. Sometimes the incurved mandibular stylet beyond the rest of the fascicle maxillary stylet tips would exit the sheath. O. fasciatus could guide the trailing maxillary stylets in a B1 occurred regardless of the position of the maxillary determined direction. This is called the mandibular stylets- stylets. Sometimes maxillary stylets would extend, retract ahead approach (Backus, 1985), commonly used by insects or remain in the same position during B1. We correlated in the infraorder . It seems likely to the B1 subpart B1w with sheath salivation. Also, most rises work best in soft plant tissues. in voltage level were correlated with partial stylet with- In contrast, H. coagulata stylet movements during the drawal, although resolution did not allow us to precisely earliest stages of penetration are different from those of O. correlate very tiny stylet movements with tiny, abrupt fasciatus in that the entire stylet fascicle is used to break the voltages, as in Fig. 6b. Backus et al. (2005) reported that path, then three stylets are retracted. Retraction plus salivation occurs during B1 in grape. At the end of B1w the fixation of a single mandibular stylet in place serve several salivary sheath shrank. This could indicate uptake of additional function (s) besides directional movement. First, unhardened sheath saliva, or shrinkage due to hardening of as described above, H. coagulata, unlike most leafhoppers, the extant sheath. However, we observed no ink particle is known to penetrate tough bark and wood with its stylets movement during B1. (Blua et al., 1999). Leopold et al. (2003) observed scallop- Neither the B1 spikelet burst frequency nor occurrence like flanges on the tips of H. coagulata mandibular (not was directly correlated with frequency or occurrence of maxillary) stylets that resemble the triangular teeth of a maxillary tip fluttering. However, tip fluttering always saw blade, not backward-pointing barbs as on the stylets occurred near and sometimes during B1s. Because no most leafhoppers (Backus, 1985). We propose that rapid external correlation was observed with B1s, the B1 spikelet alternation of the mandibular stylets during A1, causes a burst could be correlated with actions that occur inside the sawing-like action that cuts through the first layers of the insect. The exact nature of such actions is unknown at this toughest bark and plant material. A sharpshooter’s time. Hypothetically, streaming potential interruptions triangular stylet teeth would allow cutting action in either caused by the rapid opening and closing of either the direction, for maximum efficiency. Furthermore, these precibarial valve or cibarial dilators could cause the insects can achieve a stronger power stroke for penetration spikelet burst. These phenomena would likely have an by withdrawing the entire stylet fascicle first, then followed emf electrical origin, and therefore would not be detectable by extension to a deeper level. by most AC monitors. However, the Missouri monitor Second, the protracted mandibular stylet probably design used for this research does not have high-pass filters supports the excavated pathway after the maxillaries and (Backus and Bennett, 1992; Backus et al., 2000). Therefore, single mandibular stylet are retracted. By doing so, there in theory, it allows detection of both resistance and emf would be less physical resistance during subsequent components of the electrical signal (Backus et al., 2000). protraction of the retracted fascicle, and greater momen- B1 salivation (during B1w) and subsequent maxillary tip tum to move forward. fluttering (during both B1w and B1s) made a narrow Third, once the tough bark has been cut through, the salivary sheath branch path in a stepwise fashion. These mandibular stylets then are braced in the peripheral tissues, narrow paths extended various lengths beyond a thicker probably again using the mandibular stylet flanges seen by sheath trunk where the mandibles were enclosed in the Leopold et al. (2003). The maxillary stylets then push past salivary sheath. Only the maxillary stylet tips exited the ARTICLE IN PRESS P. Houston Joost et al. / Journal of Insect Physiology 52 (2006) 327–337 335 sheath. Leakage protection during ingestion is thought to sheaths became very stiff, presumably to accommodate be an important function of the salivary sheath (Miles, ingestion from xylem. 1972). Leopold et al. (2003) describes histological evidence that leakage protection is important in H. coagulata 4.4. Ingestion phase: waveform C ingestion from xylem vessels. Piercing only the maxillary stylet tips through the salivary sheath probably keeps the The regular, squared valley-and-plateau C waveform has length of the fascicle sealed better than piercing the whole been correlated with ingestion in G. atropunctata (Almeida stylet fascicle through the salivary sheath. In this way, for and Backus, 2004)andH. liturata (Joost, unpublished example, only the tip of the maxillary stylet fascicle data). We used ink particles to correlate fluid movement penetrates into the terminal, ingestion cell. Evidence in into the maxillary stylet tips with the C waveform. Walker Backus et al. (2005) supports that initial penetration into a (2000) cautioned researchers about using particles in diet xylem cell occurs during B1 in most cases. Sealing only the because the Brownian movement of the particles can be maxillary stylet tips into the cell via sheath saliva and misinterpreted as movement caused by the insect. How- very narrow puncturing aids in ingestion of fluid under ever, because we were able to consistently correlate the negative pressure in xylem vessels, and to prevent possible successive movement of particles to and into the tips of the cavitation. stylets only during the valley subpart of C, we are confident that we observed fluid uptake during C. 4.3. Pathway phase: waveform B2 This discontinuous movement of particles towards the stylets during the valley of the C waveform suggests The B2 waveform was correlated with maxillary stylet rhythmic muscular contraction of the cibarial dilator sawing within the salivary sheath. Backus et al. (2005) has muscles (cibarial pumping) during continuous ingestion. correlated the number of chevron-shaped B2 episodes in a Crane (1970) observed a similar, regular pattern of H. coagulata probe with large salivary deposits within the rhythmic suction in G. atropunctata. Such suction is the salivary sheath. Also, B2 and B1 events within a stereo- ingestion strategy for many , because typical pattern (B1–B2–B1 in a voltage-drop-then-rise active ingestion waveforms often show highly regular, trench; termed a B2 trench) were correlated with sheath rhythmic series of peaks or plateaus (Lett et al., 2001). We branching. Interestingly, although the B2 trench is highly could not determine whether fluid outflow occurred during stereotypical on plants, it was poorly or not seen on the plateau phase of the C waveform, although such artificial diets. Perhaps this was due to the flatness of outflow was observed in G. atropunctata (Crane, 1970). artificial diet waveforms compared with those on plants, As H. coagulata ingested, ink particles accumulated in discussed previously. Therefore, the B2 trench is not shown the salivary sheath around its stylet tips. This accumulation herein (see Backus et al., 2005). Yet our work contributes appeared to impede the intake of fluid into the stylets. to understanding the B2 trench on plants by showing that Sharpshooters remedied this problem by blowing the H. coagulata stylet activity during B2 consists of maxillary particles away from its stylet tips via outflow of a clear, stylet sawing through the hardened salivary sheath prior to non-gelling fluid. In this study, we could not determine if creating a new sheath path. Branched salivary sheaths are the fluid outflow was watery salvia or fluid previously commonly found in plants where H. coagulata probed taken up into the food canal. However, expulsion of fluid (Leopold et al., 2003). from the anterior alimentary canal (especially from the The B2 waveform appears to be unique to cicadellines. area of the precibarium) is particularly interesting to vector This distinctive waveform has been observed in EPG biologists, because it demonstrates a mechanism that could recordings of G. atropunctata (Almeida and Backus, 2004) inoculate a plant with a pathogen such as Xf. Xf are found and H. liturata (Joost, unpublished data). Crane (1970) in the precibarium of H. coagulata, and it is possible that described a similar stylet behavior to what we observed during this clearing behavior Xf could be released into the during sheath branching by G. atropunctata. Crane’s (1970) xylem. Obviously, further investigation is needed to clearly EPG traces show highly compressed waveforms that might demonstrate this hypothesis. have been B2 (discussed further in Almeida and Backus, 2004). Other leafhoppers make branched salivary sheaths 5. Conclusions without maxillary stylet sawing. H. coagulata produces very solid salivary sheaths that Detailed understanding of stylet activities via EPG has can remain intact in grapes for over 90 days before been used to answer research questions about hemipterans dissolving (J. Habibi and E.A.B., unpublished). In con- other than H. coagulata, such as the mechanisms of trast, salivary sheaths of , Circulifer tenellus pathogen transmission by (Powell, 1991; Prado (Baker), dissolve in less than 30 days post-deposition (Day and Tjallingii, 1994; Powell et al., 1995; Martin et al., and Irzykiewicz, 1954). Perhaps H. coagulata must saw 1997), whiteflies (Johnson et al., 2002), and deltocephaline through its own, highly solidified sheath in create a leafhoppers (Wayadande and Nault, 1993). Such studies new sheath branch. This suggests that the cicadelline also have been used to evaluate plant resistance to leafhoppers evolved this behavior when their salivary leafhoppers (Serrano et al., 2000). We have shown that ARTICLE IN PRESS 336 P. Houston Joost et al. / Journal of Insect Physiology 52 (2006) 327–337 the precise movements of individual stylets and fluid flow Blua, M.J., Phillips, P.A., Redak, R.A., 1999. A new sharpshooter into maxillary stylets by H. coagulata can be correlated threatens both and ornamentals. California Agriculture 53, with AC EPG waveforms. This baseline information will 22–25. be built upon in future studies of more applied questions. Cohen, A.C., 2000. How carnivorous bugs feed. In: Schaefer, C.W., Panizzi, A.R. (Eds.), of Economic Importance. CRC In particular, we seek to identify the moments when Press, Boca Raton, FL, pp. 563–570. sharpshooters acquire and inoculate plants with Xf during Crane, P.S., 1970. The feeding of the blue-green sharpshooter Hordnia stylet penetration. Ultimately, our goal is to develop a circellata (Baker) (: Cicadellidae). Ph.D. Dissertation, Stylet Penetration Index (Serrano et al., 2000), a rapid, University of California, Davis, CA. non-destructive, high-throughput system for screening Day, M.F., Irzykiewicz, H., 1954. On the mechanism of transmission of non-persistent phytopathogenic by aphids. Australian Journal plant genotypes for resistance to transmission of Xf. Such of Biological Sciences 7, 251–273. an index that will help breeders determine whether or to Forbes, A.R., 1977. The mouthparts and feeding mechanism of aphids. In: what degree a certain plant genotype will stimulate H. Harris, K.F., Maramorosch, K. (Eds.), Aphids as Vectors. coagulata to probe in a manner that enhances acquisition Academic Press, Inc., New York, pp. 83–103. or inoculation Xf. Hoddle, M.S., Triapitsyn, S.V., Morgan, D.J.W., 2003. Distribution and host plant records fpr Homalodisca coagulata (Say) (Hemiptera: Cicadellidae). Florida Entomologist 86, 89–91. Hopkins, D.L., 1989. Xylella fastidosa: xylem-limited bacterial pathogen Acknowledgements of plants. Annual Review of Phytopathology 27, 271–290. Hunter, W.B., Backus, E.A., 1989. Mesophyll-feeding by the We thank Holly Shugart and Jose Gutierrez for plant leafhopper, fabae (Homoptera: Cicadellidae): results from electronic monitoring and thin-layer chromatography. Environmental rearing and sharpshooter maintenance. We also thank Entomology 18, 465–472. Greg Walker and Holly Shugart for advice and insights in Hunter, W.B., Ullman, D.E., 1989. Analysis of mouthpart movements preparation of this manuscript, as well as two anonymous during feeding of Frankliniella occidentalis (Pergande) and F. schultzei reviewers whose comments significantly improved the Trybom (Thysanoptera: Thripidae). International Journal of Insect manuscript. 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