ARTHROPODS IN RELATION TO PLANT DISEASES Sharpshooter X Wave: Correlation of an Electrical Penetration Graph Waveform With Xylem Penetration Supports a Hypothesized Mechanism for Xylella fastidiosa Inoculation ELAINE A. BACKUS,1 WENDY J. HOLMES,2 FRED SCHREIBER,2 BRENDON J. REARDON,3,4 3 AND GREGORY P. WALKER Ann. Entomol. Soc. Am. 102(5): 847Ð867 (2009) ABSTRACT Electrical penetration graph (EPG) monitoring is the most rigorous means of obser- vation and quantiÞcation of feeding by piercingÐsucking arthropods. Previous EPG studies with aphids and leafhoppers have demonstrated that the X wave identiÞes when the stylets of these phloem ßuid-ingesting insects make contact with their preferred plant vascular cell, phloem sieve elements. This article presents the Þrst direct evidence of an X wave identifying ingestion from a xylem tracheary element by a xylem ßuid-ingesting type of leafhopper Homalodisca liturata Ball (Hemiptera: Cicadel- lidae: Cicadellinae), whose waveforms are nearly identical to those of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar). We document consistent association of the sharpshooter X wave with salivary sheath termini in xylem, especially ligniÞed secondary xylem cells, and absence of the X wave in the rare instance of ingestion from a nonxylem cell. The sharpshooter X wave is a complex, multicomponent waveform, composed of X wave-speciÞc variants of waveform subtypes B1w (rep- resenting salivation), B1s (representing precibarial valve movement for tasting), types C1 (a new waveform type that may represent egestion) and C2 (a new designation for the waveform type representing ingestion/cibarial pumping). It is proposed that the sharpshooter X wave represents a blended suite of behaviors that function to 1) physically seal stylet tips into the cell via sheath salivation, 2) repeatedly taste then eject (egest) chemical constituents of the cell to determine acceptability, and 3) mechanically test the strength of the stylet seal via trial cibarial pumping (ingestion). It is further hypothesized that the X wave represents vector behaviors that control inoculation of the PierceÕs disease bacterium, Xylella fastidiosa. The ingestion-(salivation and eges- tion) hypothesis is stated for the mechanism of transmission of X. fastidiosa. KEY WORDS electrical penetration graph, Homalodisca spp., PierceÕs disease, probing, stylet pen- etration Sharpshooters (Hemiptera: Cicadellidae: Cicadelli- tor of X. fastidiosa in California on host plants such as nae) are large, xylem ßuid-ingesting leafhoppers that oleander, almond, citrus, and nursery trees (Redak et are capable of transmitting (i.e., acquiring and inoc- al. 2004). These crops and others in California are at ulating) the bacterium Xylella fastidiosa, the causative risk of epidemic PD due to introduction of H. vitrip- agent of PierceÕs disease (PD) of grape (Vitis spp.) ennis. Efforts are underway to develop host plant re- and other leaf scorches into host plants. The glassy- sistance to the vectorÕs ability to transmit of X. fastid- winged sharpshooter, Homalodisca vitripennis (Ger- iosa; however, lack of fundamental understanding of mar), formerly Homalodisca coagulata (Say) (Takiya the transmission mechanism and its impact on PD et al. 2006), is an exotic, economically important vec- epidemiology currently impedes progress. For the past 6 yr, Backus and colleagues have been researching the basic question of how sharpshooter Mention of trade names or commercial products in this article is solely for the purpose of providing speciÞc information and does not feeding controls transmission (especially inoculation) imply recommendation or endorsement by the U.S. Department of of X. fastidiosa, with the goals of deÞning the speciÞc Agriculture. inoculation behaviors and identifying the instant of 1 Corresponding author: USDAÐAgricultural Research Service, San inoculation during the feeding process, in real-time. Joaquin Valley Agricultural Sciences Center, 9611 S. Riverbend Ave., Parlier, CA 93648 (e-mail: [email protected]). Our primary tool is electrical penetration graph 2 Department of Biology, California State University, Fresno, CA (EPG) monitoring, the most rigorous and detailed 93710. method of observing and quantifying the feeding of 3 Department of Entomology, University of California, Riverside, piercingÐsucking arthropods (Walker 2000). This CA 92521. 4 Current address: USDAÐAnimal and Plant Health Inspection Ser- study is the fourth in a series of articles that have 1) vice, 350 Corporate Blvd., Robbinsville, NJ 08691. characterized the main sharpshooter waveforms (rep- 848 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 102, no. 5 resenting stylet penetration [probing]) of the blue- The uniqueness of the stylet activities during the green sharpshooter, Graphocephala atropunctata (Si- aphid X wave was supported by the discovery that the gnoret) (Almeida and Backus 2004), and the glassy- pds immediately preceding sieve element phase (later winged sharpshooter (Backus et al. 2005); 2) termed R-pds; Tjallingii and Gabrys 1999) were longer performed histological correlations to identify the in duration in some aphid species than pds elsewhere host plant tissues in which each waveform occurs in the probe. R-pds are thought to represent intracel- (Backus et al. 2005); and 3) performed correlations of lular punctures into phloem cells, as opposed to non- waveforms with speciÞc stylet activities by observing phloem cells along the pathway. Reese et al. (2000) stylet movements in transparent, artiÞcial diet (Joost also rediscovered the Þnding of McLean and Kinsey et al. 2006). (1967) that their “X waves” (actually pathway pds) are The present work builds on the previous studies by recognizable during pathway, in addition to immedi- characterizing more waveform types, completing our ately preceding sieve element phase. However, be- histological correlations, and using them to hypothe- cause these partial X waves were correlated with in- size the mechanism of X. fastidiosa inoculation. The tracellular punctures of mesophyll/parenchyma cells Backus et al. (2005) study left some unanswered ques- along the stylet pathway (McLean and Kinsey 1967), tions such as: 1) During which ingestion events in a rather than with sieve elements, Reese et al. (2000) probe are the stylets deÞnitely in and ingesting from felt their existence confused the deÞnition of X waves xylem? 2) Is there any point during stylet penetration, and did not advocate use of the term. before ingestion ensues, when the stylets penetrate In contrast, we believe that the concept of the X xylem cells? and 3) Can stylet penetration and inges- wave has great practical and heuristic value for EPG tion speciÞcally from xylem cells be reliably deter- researchers, if it is deÞned carefully and used as part mined via EPG waveforms alone, without histology? of a broader, behavioral/ecological concept for The speciÞc objectives of this study were to answer hemipteran probing behavior. McLean and Kinsey, the above-mentioned questions, by histologically cor- due primarily to the limitations of their instruments relating individual pathway and ingestion events in (Backus et al. 2000, Tjallingii 2000), used a “top-down” identiÞed probes with cell type penetrated. research approach of identifying broad concepts and The ultimate goal of this study was to search for a phases of stylet penetration, of necessity leaving the putative “sharpshooter X wave.” In EPG parlance, the details to later researchers. Tjallingii and colleagues X wave is a complex, stereotypically repeating pattern used the bottom-up approach of elegantly and pains- of waveforms that represents stylet contact and sub- takingly characterizing all waveform types and sub- sequent activities (such as ingestion or salivation) in types Þrst, then assembling their blended meanings a hemipteran speciesÕ preferred ingestion cell type; up into phases and concepts. Over time, these comple- to now, strictly phloem sieve elements. X waves are mentary styles have provided greater richness of in- important because they, or their components, have formation about aphid stylet activities than could ei- been shown to control inoculation of noncirculative ther method alone. In light of this ultimate success, we plant pathogens. Our study uses deductive, “bottom- have applied both top-down and bottom-up ap- up” reasoning, by Þrst identifying when xylem ele- proaches to our studies of sharpshooter waveforms. ments were penetrated, the stylet activities that oc- The current study employs the bottom-up approach. curred at that time, then searching for a stereotypical Based on the evidence here, we advocate restored use waveform that could be called the X wave, and Þnally, of the X wave concept but more precisely deÞned than hypothesizing its role in inoculation of X. fastidiosa. in previous literature. The concept of the X wave as a landmark waveform The X wave concept has proven invaluable for EPG was one of the earliest and most signiÞcant contribu- studies of leafhoppers and planthoppers. In recordings tions of the original inventors of EPG (McLean and of 10 species of phloem ßuid-ingesting Deltocephaline Kinsey 1965, 1967). McLean and Kinsey (1967) cor- leafhoppers (Triplehorn et al. 1984, Rapusas and Hei- related both the aphid X wave and the regular, long- nrichs 1990, Wayadande and Nault 1993) and one duration waveform following it with stylet tips in a species of delphacid planthopper, Nilaparvata lugens phloem sieve element, the preferred