Do Sharpshooters From Around the World Produce the Same EPG Waveforms? Comparison of Waveform Libraries From Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae) Vectors Kolla paulula (Hemiptera: Cicadellidae) From Taiwan and Graphocephala atropunctata From California.

When an exotic invasive species is a vector-borne plant pathogen, vector feeding behavior must be studied to identify potential host plant range and performance of specialized pathogen transmission behaviors. The most rigorous tool for assessing vector feeding behavior is electropenetrography (EPG). Xylella fastidiosa Wells et al. is a gram-negative bacterium native to the Americas, where it is the causal agent of lethal scorch-type diseases such as Pierce's disease (PD) of grapevines. In 2002, a PD strain of X. fastidiosa invaded Asia for the first time, as confirmed from grape vineyards in Taiwan. Kolla paulula (Wallker), a native Asian species of sharpshooter leafhopper, was found to be the primary vector in Taiwanese vineyards. This study used an AC-DC electropenetrograph to record stylet probing behaviors of K. paulula on healthy grapevines. The main objective was to create an EPG waveform library for K. paulula. Waveform description, characterization of R versus emf components (electrical origins), and proposed biological meanings of K. paulula waveforms are reported. In addition, comparison of K. paulula waveforms with those from the most efficient, native vector of X. fastidiosa in California vineyards, Graphocephala atropunctata, is also reported. Overall, both species of sharpshooters had similar-appearing waveforms. Five new findings were identified, especially that the previously described but rare waveform subtype, B1p, was extensively produced in K. paulula recordings. Sharpshooter waveforms from species worldwide share a high degree of similarity. Thus, EPG methods can be rapidly applied to potential vectors where X. fastidiosa is newly introduced.

Review of the EPG Waveforms of Sharpshooters and Spittlebugs Including Their Biological Meanings in Relation to Transmission of Xylella fastidiosa (Xanthomonadales: Xanthomonadaceae).

Electropenetrography (EPG) is one of the most rigorous methods to study stylet probing behaviors of piercing-sucking insects whose mouthparts move invisibly inside hosts. EPG is particularly useful for identifying vector behaviors that control transmission (acquisition, retention, and inoculation) of plant pathogens, comparing those behaviors among vector species, and aiding in development of novel vector and disease management tactics. Xylella fastidiosa (Wells et al.) is a gram-negative, invasive bacterium native to the Americas, where it is the causal agent of lethal scorch-type diseases such as Pierce's disease of grapevines. Xylella fastidiosa is transmitted by sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) and spittlebugs (Hemiptera: Aphrophoridae). Despite over 75 yr of study, details of the inoculation mechanism of X. fastidiosa were unknown until the advent of EPG research with sharpshooters. Herein, the following topics are presented: 1) review of key EPG principles and waveforms published to date, emphasizing sharpshooters and spittlebugs; 2) summary of present understanding of biological meanings of sharpshooter waveforms; 3) review of mechanisms of transmission for X. fastidiosa illuminated by EPG; and 4) recommendations of the most useful waveform categories for EPG use in future, quantitative comparisons of sharpshooter stylet probing on various treatments such as infected versus uninfected plants, resistant varieties, or insecticide treatments. In addition, new work on the functional anatomy of the precibarial valve is discussed in the context of X. fastidiosa transmission and EPG waveforms. Also, the first block diagram of secondary, signal-processing circuits for the AC-DC EPG is published, and is discussed in relation to EPG signals appearances and meanings.

Feeding Behavior of Soybean Aphid (Hemiptera: Aphididae) Biotype 2 on Resistant and Susceptible Soybean.

Host plant resistance to the soybean aphid, Aphis glycines Matsumura, is an effective means of controlling populations of this introduced pest species in the United States. Rag (Resistance to Aphis glycines) genes identified in soybean germplasm have been incorporated into commercial cultivars, but differential responses by soybean aphid biotypes to the Rag genes have made understanding mechanisms underlying resistance associated with Rag genes increasingly important. We compared the behavior of biotype 2 aphids on the resistant soybean line PI243540, which is a source of Rag2, and the susceptible cultivar Wyandot. Scanning electron microscopy revealed that the abaxial surface of leaves from resistant plants had a higher density of both long and glandulartrichomes, which might repel aphids, on veins. Time-lapse animation also suggested a repellent effect of resistant plants on aphids. However, electropenatography (EPG) indicated that the time to first probe did not differ between aphids feeding on the resistant and susceptible lines. EPG also indicated that fewer aphids feeding on resistant plants reached the phloem, and the time before reaching the phloem was much longer relative to susceptible soybean. For aphids that reached the phloem, there was no difference in either number of feedings or their duration in phloem. However, aphids feeding on resistant soybean had fewer prolonged phases of active salivation (E1) and many more pathway activities and non-probing intervals. Together, the feeding behavior of aphids suggested that Rag2 resistance has strong antixenosis effects, in addition to previously reported antibiosis, and was associated with epidermal and mesophyll tissues.

O antigen modulates insect vector acquisition of the bacterial plant pathogen Xylella fastidiosa.

Hemipteran insect vectors transmit the majority of plant pathogens. Acquisition of pathogenic bacteria by these piercing/sucking insects requires intimate associations between the bacterial cells and insect surfaces. Lipopolysaccharide (LPS) is the predominant macromolecule displayed on the cell surface of Gram-negative bacteria and thus mediates bacterial interactions with the environment and potential hosts. We hypothesized that bacterial cell surface properties mediated by LPS would be important in modulating vector-pathogen interactions required for acquisition of the bacterial plant pathogen Xylella fastidiosa, the causative agent of Pierce's disease of grapevines. Utilizing a mutant that produces truncated O antigen (the terminal portion of the LPS molecule), we present results that link this LPS structural alteration to a significant decrease in the attachment of X. fastidiosa to blue-green sharpshooter foreguts. Scanning electron microscopy confirmed that this defect in initial attachment compromised subsequent biofilm formation within vector foreguts, thus impairing pathogen acquisition. We also establish a relationship between O antigen truncation and significant changes in the physiochemical properties of the cell, which in turn affect the dynamics of X. fastidiosa adhesion to the vector foregut. Lastly, we couple measurements of the physiochemical properties of the cell with hydrodynamic fluid shear rates to produce a Comsol model that predicts primary areas of bacterial colonization within blue-green sharpshooter foreguts, and we present experimental data that support the model. These results demonstrate that, in addition to reported protein adhesin-ligand interactions, O antigen is crucial for vector-pathogen interactions, specifically in the acquisition of this destructive agricultural pathogen.

Direct Evidence of Egestion and Salivation of Xylella fastidiosa Suggests Sharpshooters Can Be "Flying Syringes".

Xylella fastidiosa is unique among insect-transmitted plant pathogens because it is propagative but noncirculative, adhering to and multiplying on the cuticular lining of the anterior foregut. Any inoculation mechanism for X. fastidiosa must explain how bacterial cells exit the vector's stylets via the food canal and directly enter the plant. A combined egestion-salivation mechanism has been proposed to explain these unique features. Egestion is the putative outward flow of fluid from the foregut via hypothesized bidirectional pumping of the cibarium. The present study traced green fluorescent protein-expressing X. fastidiosa or fluorescent nanoparticles acquired from artificial diets by glassy-winged sharpshooters, Homalodisca vitripennis, as they were egested into simultaneously secreted saliva. X. fastidiosa or nanoparticles were shown to mix with gelling saliva to form fluorescent deposits and salivary sheaths on artificial diets, providing the first direct, conclusive evidence of egestion by any hemipteran insect. Therefore, the present results strongly support an egestion-salivation mechanism of X. fastidiosa inoculation. Results also support that a column of fluid is transiently held in the foregut without being swallowed. Evidence also supports (but does not definitively prove) that bacteria were suspended in the column of fluid during the vector's transit from diet to diet, and were egested with the held fluid. Thus, we hypothesize that sharpshooters could be true "flying syringes," especially when inoculation occurs very soon after uptake of bacteria, suggesting the new paradigm of a nonpersistent X. fastidiosa transmission mechanism.

Differential Probing Behavior of Blissus insularis (Hemiptera: Blissidae) on Resistant and Susceptible St. Augustinegrasses.

Southern chinch bug, Blissus insularis Barber, is a severe pest of St. Augustinegrass throughout the southern United States. Host plant resistance is an environmentally friendly method to manage chinch bug infestations and is increasingly important, as the southern chinch bug develops resistance to insecticides. In this study, in an effort to understand resistance mechanisms in two varieties of St. Augustinegrass ('FX-10' and 'NUF-76'), we used the electrical penetration graph method to quantify stylet probing behaviors in two resistant and two susceptible St. Augustinegrass varieties. Overall, chinch bugs spent less time probing on resistant FX-10 and NUF-76 than on susceptible 'Floratam' and 'Palmetto', and individual probes were shorter in average duration but more numerous in resistant varieties than in susceptible varieties. During probing, chinch bugs spent more time in pathway-associated stylet activities (i.e., penetration through epidermal and mesophyll tissue) in the resistant varieties than in the susceptible varieties, likely indicating difficulty in finding and accessing an ingestion site. As a consequence, chinch bugs spent proportionately much less time engaged in xylem ingestion in both resistant varieties than in susceptible varieties but only in FX-10 were phloem-associated activities significantly reduced compared with those in susceptible varieties. We conclude that there is evidence for non-phloem-associated chinch-bug resistance factors in both NUF-76 and FX-10, and phloem-associated factors in FX-10.

Plant water stress effects on stylet probing behaviors of Homalodisca vitripennis (Hemiptera: Cicadellidae) associated with acquisition and inoculation of the bacterium Xylella fastidiosa.

ABSTRACT The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae), is a xylem fluid-ingesting leafhopper that transmits Xylella fastidiosa Wells et al., a plant-infecting bacterium that causes several plant diseases in the Americas. Although the role of plant water stress on the population density and dispersal ofH. vitripennis has been studied, nothing is known about the effects of plant water stress on the transmission of X. fastidiosa by H. vitripennis. A laboratory study was conducted to determine the influence of plant water stress on the sharpshooter stylet probing behaviors associated with the acquisition and inoculation of X. fastidiosa. Electrical penetration graph was used to monitor H. vitripennis feeding behaviors for 20-h periods on citrus [Citrus sinensis (L.) Osbeck] and almond [Prunus dulcis (Miller) D.A. Webb] plants subjected to levels of water stress. Adult H. vitripennis successfully located xylem vessels, then performed behaviors related to the evaluation of the xylem cell and fluid, and finally ingested xylem fluid from citrus and almond plants under the tested fluid tensions ranging from -5.5 to -33.0 bars and -6.0 to -24.5 bars, respectively. In general, long and frequent feeding events associated with the acquisition and inoculation of X. fastidiosa were observed only in fully irrigated plants (i.e., >-10 bars), which suggests that even low levels of plant water stress may reduce the spread of X. fastidiosa. Results provided insights to disease epidemiology and support the hypothesis that application of regulated deficit irrigation has the potential to reduce the incidence of diseases caused by X.fastidiosa by reducing the number of vectors and by decreasing pathogen transmission efficiency.

Probing behavior of the leafhopper analyzed through DC electropenetrography and microscopy.

Yamatotettix flavovittatus Matsumara is a new leafhopper species vector of sugarcane white leaf (SCWL) phytoplasma that causes sugarcane chlorosis symptoms. The effects of probing behavior of Y. flavovittatus on sugarcane and its implication for SCWL phytoplasma transmission are yet to be studied. In this research, we used DC electropenetrography (EPG) to define waveforms produced by adult and fifth-instar nymphal Y. flavovittatus on sugarcane and correlated them with salivary sheath termini (likely stylet tip locations) via light and scanning electron microscopy. The following six waveforms and associated activities are described: (NP) non-probing, (Yf1) stylet probing into epidermal cells, (Yf2) stylet probing through mesophyll/parenchyma, (Yf3) stylet contact with phloem and likely watery salivation, (Yf4) active ingestion of sap from phloem, probably sieve elements, and (Yf5) unknown stylet activity in multiple cell types. Study findings reveal that the Y. flavovittatus vector ingests sieve tube element more frequently and for longer durations than any other cell type, supporting that Y. flavovittatus is primarily a phloem feeder. Adult Y. flavovittatus show a longer total probing duration and produces a high density of puncture holes on sugarcane leaves. Moreover, probing behaviors revealed that adults typically ingest phloem sap more frequently and for longer durations than fifth-instar nymphs, enhancing sap ingestion. Furthermore, we propose that adults are more likely to acquire (during Yf4) and inoculate (during Yf3) higher amounts of phytoplasma than fifth-instar nymphs. This information on the penetration behavior of leafhopper Y. flavovittatus serves as a basis for advanced studies on the transmission mechanism of SCWL phytoplasma.

Xylella fastidiosa inoculation behaviors (EPG X wave) are performed differently by blue-green sharpshooters based on infection status of prior probing host.

Does Xylella fastidiosa, a bacterial plant pathogen with noncirculative foregut-borne transmission, manipulate behavior of its sharpshooter vector to facilitate its own inoculation? To answer this question, blue-green sharpshooters, Graphocephala atropunctata (Signoret), were reared on basil to clean their foreguts, then removed from the colony and given one of four pre-electropenetrography (EPG) treatments: i) old colony adults on basil, ii) young colony adults on basil, iii) young colony adults held on healthy grapevine for 4 days, and iv) young colony adults held on Xf-infected (symptomatic) grapevine for 4 days. After treatments, stylet probing behaviors were recorded on healthy grapevine via AC-DC electropenetrography. Waveforms representing putative Xf inoculation (XB1 [salivation and rinsing egestion] and XC1 [discharging egestion]) and other behaviors were statistically compared among treatments. Mean number of events per insect and 'total' duration per insect of XB1 and XC1 were highest for insects from healthy grape, lowest for basil (regardless of insect age), and intermediate for Xf-infected grape. The surprising results showed that prior exposure to healthy grapevines had a stronger effect on subsequent performance of inoculation behaviors on healthy grapevine than did prior exposure to Xf-infected grapevine. It is hypothesized that non-Xf microbes were acquired from healthy grapevine, causing greater clogging of the precibarium, leading to more performance of inoculation behaviors. This study shows for the first time that presence of noncirculative, foregut-borne microbes can directly manipulate a vector's behavior to increase inoculation. Also, EPG can uniquely visualize the dynamic interactions between vectors and the microbes they carry.

An electropenetrography waveform library for the probing and ingestion behaviors of Culex tarsalis on human hands.

Culex tarsalis Coquillett (Diptera: Culicidae) mosquitoes are capable of vectoring numerous pathogens affecting public and animal health. Unfortunately, the probing behaviors of mosquitoes are poorly understood because they occur in opaque tissues. Electropenetrography (EPG) has the potential to elucidate these behaviors by recording the electrical signals generated during probing. We used an AC-DC EPG with variable input resistors (Ri levels) to construct a waveform library for Cx. tarsalis feeding on human hands. Biological events associated with mosquito probing were used to characterize waveforms at four Ri levels and with two electrical current types. The optimal settings for EPG recordings of Cx. tarsalis probing on human hands was an Ri level of 107 Ohms using an applied signal of 150 millivolts alternating current. Waveforms for Cx. tarsalis included those previously observed and associated with probing behaviors in Aedes aegypti L. (Diptera: Culicidae): waveform families J (surface salivation), K (stylet penetration through the skin), L (types 1 and 2, search for a blood vessel/ingestion site), M (types 1 and 2, ingestion), N (type 1, an unknown behavior which may be a resting and digestion phase), and W (withdrawal). However, we also observed variations in the waveforms not described in Ae. aegypti, which we named types L3, M3, M4, and N2. This investigation enhances our understanding of mosquito probing behaviors. It also provides a new tool for the automated calculation of peak frequency. This work will facilitate future pathogen acquisition and transmission studies and help identify new pest and disease management targets.

Fluid dynamic simulations at the interface of the blue-green sharpshooter functional foregut and grapevine xylem sap with implications for transmission of Xylella fastidiosa.

Xylella fastidiosa is a multi-continental, lethal, plant pathogenic bacterium that is transmitted by sharpshooter leafhoppers (Insecta: Hemiptera: Cicadellidae: Cicadellinae) and adult spittlebugs (Hemiptera: Aphrophoridae). The bacterium forms biofilms in plant xylem and the functional foregut of the insect. These biofilms serve as sources of inoculum for insect acquisition and subsequent inoculation to a healthy plant. In this study, 3D fluid dynamic simulations were performed for bidirectional cibarial propulsion of xylem sap through tube-like grapevine xylem and an anatomically accurate model of the functional foregut of the blue-green sharpshooter, Graphocephala atropunctata. The analysis supports a model of how fluid dynamics influence X. fastidiosa transmission. The model supports the hypothesis that X. fastidiosa inoculation is mostly driven by detachment of bacteria from the foregut due to high-velocity flow during egestion (outward fluid flow from the stylets). Acquisition occurs by fluid dynamics during both egestion and ingestion (fluid uptake through the stylets and swallowing). These simulation results are supported by previously reported X. fastidiosa colonization patterns in the functional foregut and sharpshooter stylet probing behaviors. The model indicates that xylem vessel diameter influences drag forces imposed on xylem wall-adherent bacteria; thus, vessel diameter may be an important component of the complex transmission process. Results from this study are directly applicable to development of novel grapevine resistance traits via electropenetrographic monitoring of vector acquisition and inoculation behaviors.

Spatiotemporal colonization of Xylella fastidiosa in its vector supports the role of egestion in the inoculation mechanism of foregut-borne plant pathogens.

The pathogen that causes Pierce's disease of grapevine, Xylella fastidiosa, is the only known bacterial, arthropod-transmitted plant pathogen that does not circulate in the vector's hemolymph. Instead, bacteria are foregut-borne, persistent in adult vectors but semipersistent in immatures (i.e., bacteria colonize cuticular surfaces of the anterior foregut, are retained for hours to days, but are lost during molting). Yet, exactly how a sharpshooter vector inoculates bacteria from foregut acquisition sites is unknown. The present study used confocal laser-scanning microscopy to identify locations in undissected, anterior foreguts of the glassy-winged sharpshooter colonized by green fluorescent protein-expressing X. fastidiosa. Spatial and temporal distributions of colonizing X. fastidiosa were examined daily over acquisition access periods of 1 to 6 days for both contaminated field-collected and clean laboratory-reared Homalodisca vitripennis. Results provide the first direct, empirical evidence that established populations of X. fastidiosa can disappear from vector foreguts over time. When combined with existing knowledge on behavior, physiology, and functional anatomy of sharpshooter feeding, present results support the idea that the disappearance is caused by outward fluid flow (egestion) not inward flow (ingestion) (i.e., swallowing). Thus, results support the hypothesis that egestion is a critical part of the X. fastidiosa inoculation mechanism. Furthermore, results suggest a cyclical, spatiotemporal pattern of microbial colonization, disappearance, and recolonization in the precibarium. Colonization patterns also support two types of egestion, termed rinsing and discharging egestion herein. Finally, comparison of acquisition results for field-collected versus laboratory-reared sharpshooters suggest that there may be competitive binding for optimum acquisition sites in the foregut. Therefore, successful inoculation of X. fastidiosa may depend, in large part, on vector load in the precibarium.

AC-DC electropenetrography unmasks fine temporal details of feeding behaviors for two tick species on unsedated hosts.

Ticks are significant nuisance pests and vectors of pathogens for humans, companion animals, and livestock. Limited information on tick feeding behaviors hampers development and rigorous evaluation of tick and tick-borne pathogen control measures. To address this obstacle, the present study examined the utility of AC-DC electropenetrography (EPG) to monitor feeding behaviors of adult Dermacentor variabilis and Amblyomma americanum in real-time. EPG recording was performed during early stages of slow-phase tick feeding using an awake calf host. Both tick species exhibited discernable and stereotypical waveforms of low-, medium-, and high-frequencies. Similar waveform families and types were observed for both tick species; however, species-specific waveform structural differences were also observed. Tick waveforms were hierarchically categorized into three families containing seven types. Some waveform types were conserved by both species (e.g., Types 1b, 1c, 2b, 2c) while others were variably performed among species and individually recorded ticks (e.g., Types 1a, 2a, 2d). This study provides a proof-of-principle demonstration of the feasibility for using EPG to monitor, evaluate, and compare tick feeding behaviors, providing a foundation for future studies aimed at correlating specific feeding behaviors with waveforms, and ultimately the influence of control measures and pathogens on tick feeding behaviors.

AC-DC electropenetrography: fundamentals, controversies, and perspectives for arthropod pest management.

Studying the intimate association of arthropods with their physical substrate is both important and challenging. It is important because substrate is a key determinant for organism fitness; challenging because the intricacies of this association are dynamic, and difficult to record and resolve. The advent of electropenetrography (EPG) and subsequent developments allowed researchers to overcome this challenge. Nonetheless, EPG research has been historically restricted to piercing-sucking hemipteran plant pests. Recently, its potential use has been greatly broadened for additional pests with instrument advances. Thus, blood-feeding arthropods and chewing feeders, as well as non-feeding behaviors like oviposition by both pests and parasitoids, are novel new targets for EPG research, with critical consequences for integrated pest management. EPG can explain mechanisms of crop damage, plant or animal pathogen transmission, and the effects of insecticides, antifeedants, repellents, or transgenic plants and animals, on specific behaviors of damage or transmission. This review broadly covers the principles and development of EPG technology, emphasizing controversies and challenges remaining with suggested research to overcome them. In addition, it summarizes 60+ years of basic and applied EPG research, and previews future directions for pest management. The goal is to stimulate new applications for this unique enabling technology. Published 2020. This article is a U.S. Government work and is in the public domain in the USA.

Alteration in the Stylet Probing Behavior and Host Preference of the Vector Matsumuratettix hiroglyphicus (Hemiptera: Cicadellidae) After Infection with Sugarcane White Leaf Phytoplasma.

The leafhopper (Matsumuratettix hiroglyphicus (Matsumura) (Hemiptera: Cicadellidae)) is a crucial insect vector of the phytoplasma associated with sugarcane white leaf (SCWL) disease. The aim of this study was to compare the stylet probing behaviors of M. hiroglyphicus on healthy sugarcane plants, asymptomatic, and symptomatic SCWL-infected sugarcane plants, using DC electropenetrography. We also used host-selection preference (free-choice) assays to identify the preferred types of host plants, and scanning electron microscopy to observe stylet puncture holes and salivary flanges after leafhopper probing. According to a quantitative analysis of M. hiroglyphicus stylet probing, mean durations per insect of both phloem ingestion (waveform D; the phytoplasma-acquisition behavior) and phloem salivation (waveform C; the phytoplasma-inoculation behavior) were significantly longer on both types of infected sugarcane than on healthy plants. These longer overall durations were mainly because the same number of significantly longer-duration C and D events was performed on infected sugarcane compared with healthy plants. On free-choice tested plants, M. hiroglyphicus displayed a significantly greater preference to settle on the infected plants (both types) than the healthy sugarcane. These results provide the first empirical evidence that acquiring the SCWL phytoplasma alters the host selection and stylet probing behaviors of its main vector (M. hiroglyphicus). Our study thus contributes to a better understanding of the interactions between the insect vector and SCWL phytoplasma-infected plants, and will aid in developing novel disease management tactics for sugarcane.

Field-Collected Glassy-Winged Sharpshooters (Hemiptera: Cicadellidae) Perform More Xylella fastidiosa-Inoculating Behaviors on Susceptible Vitis vinifera cv. 'Chardonnay' Than on Resistant Vitis champinii Grapevines.

The glassy-winged sharpshooter, Homalodisca vitripennis (Germar) (Hemiptera: Cicadellidae: Cicadellinae), is an introduced vector of the xylem-dwelling bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae) in California. Once acquired, X. fastidiosa colonizes the functional foregut of the vector. Bacteria can be inoculated directly into grapevine xylem during the xylem cell acceptance process in sharpshooter stylet probing, represented by the X wave using electropenetrography (EPG). Since 2001, an effort has been underway to develop PD-resistant grapevines, Vitis vinifera L., through classical breeding of various species of resistant wild grapevines with more susceptible V. vinifera. The present study used EPG to compare H. vitripennis stylet probing behaviors in a factorial experiment between V. champinii (a V. candicans/V. rupestris natural hybrid with moderate trichomes) and V. vinifera cv. 'Chardonnay' (which lacks trichomes) that had been gently scraped to remove trichomes or was not scraped. Results showed that sharpshooters performed significantly more X waves/X. fastidiosa inoculation behaviors of overall longer duration on Chardonnay than on V. champinii, regardless of shaving or not-shaving to remove trichomes. In addition, trichomes caused more frequent standing/walking/test-probing behaviors on V. champinii, whose xylem was rapidly accepted for sharpshooter ingestion once probing began. Thus, EPG can detect a novel type of grapevine resistance to X. fastidiosa-to the vector's probing process and inoculation of bacteria-in addition to the bacterial infection and symptom development processes that are the basis for most resistance breeding today. Future research could use EPG to screen grapevines for this novel type of resistance.

Functional foregut anatomy of the blue-green sharpshooter illustrated using a 3D model.

Sharpshooter leafhoppers (Hemiptera: Cicadellidae: Cicadellinae) are important vectors of the plant pathogenic bacterium Xylella fastidiosa Wells et al. (Xanthomonadales: Xanthomonadaceae). This pathogen causes economically significant diseases in olive, citrus, and grapes on multiple continents. Bacterial acquisition and inoculation mechanisms are linked to X. fastidiosa biofilm formation and fluid dynamics in the functional foregut of sharpshooters, which together result in egestion (expulsion) of fluids likely carrying bacteria. One key X. fastidiosa vector is the blue-green sharpshooter, Graphocephala atropunctata (Signoret, 1854). Herein, a 3D model of the blue-green sharpshooter functional foregut is derived from a meta-analysis of published microscopy images. The model is used to illustrate preexisting and newly defined anatomical terminology that is relevant for investigating fluid dynamics in the functional foregut of sharpshooters. The vivid 3D illustrations herein and supplementary interactive 3D figures are suitable resources for multidisciplinary researchers who may be unfamiliar with insect anatomy. The 3D model can also be used in future fluid dynamic simulations to better understand acquisition, retention, and inoculation of X. fastidiosa. Improved understanding of these processes could lead to new targets for preventing diseases caused by X. fastidiosa.

Waveforms From Stylet Probing of the Mosquito Aedes aegypti (Diptera: Culicidae) Measured by AC-DC Electropenetrography.

Electropenetrography (EPG) has been used for many years to visualize unseen stylet probing behaviors of plant-feeding piercing-sucking insects, primarily hemipterans. Yet, EPG has not been extensively used with blood-feeding insects. In this study, an AC-DC electropenetrograph with variable input resistors (Ri), i.e., amplifier sensitivities, was used to construct a waveform library for the mosquito arbovirus vector, Aedes aegypti (Linneaus), while feeding on human hands. EPG waveforms representing feeding activities were: 1) electrically characterized, 2) defined by visual observation of biological activities, 3) analyzed for differences in appearance by Ri level and type of applied signal (AC or DC), and 4) quantified. Electrical origins of waveforms were identified from five different Ri levels and AC versus DC. Mosquitoes produced short stylet probes ('bites') that typically contained five waveform families. Behaviors occurred in the following order: surface salivation (waveform family J), stylet penetration through the outer skin (K), penetration of deeper tissues and location of blood vessels/pathway activities (L), active ingestion with engorgement (M), and an unknown behavior that terminated the probe (N). Only K, L, and M were performed by every insect. A kinetogram of conditional probabilities for waveform performance demonstrated plasticity among individuals in L and M, which were alternated. Now that EPG waveforms for mosquito feeding have been defined, EPG can be used as a tool for improved biological understanding of mosquito-borne diseases.

Targeted Mutations in Xylella fastidiosa Affect Acquisition and Retention by the Glassy-Winged Sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae).

Xylella fastidiosa (Wells) is a xylem-limited bacterium that causes Pierce's disease of grapevines. The bacterium is transmitted by insect vectors such as the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis (Germar). Experiments were conducted to compare the role of selected X. fastidiosa genes on 1) bacterial acquisition and retention in GWSS foreguts, and 2) transmission to grapevines by GWSS. Bacterial genotypes used were: mutants Xf-ΔpilG, Xf-ΔpilH, Xf-ΔgacA, and Xf-ΔpopP; plus wild type (WT) as control. Results showed that Xf-ΔpilG had enhanced colonization rate and larger numbers in GWSS compared with WT. Yet, Xf-ΔpilG exhibited the same transmission efficiency as WT. The Xf-ΔpilH exhibited poor acquisition and retention. Although initial adhesion, multiplication, and retention of Xf-ΔpilH in GWSS were almost eliminated compared with WT, the mutation did not reduce transmission success in grapevines. Overall, Xf-ΔgacA showed colonization rates and numbers in foreguts similar to WT. The Xf-ΔgacA mutation did not affect initial adhesion, multiplication, and long-term retention compared with WT, and was not significantly diminished in transmission efficiency. In contrast, numbers of Xf-ΔpopP were variable over time, displaying greatest fluctuation from highest to lowest levels. Thus, Xf-ΔpopP had a strong, negative effect on initial adhesion, but adhered and slowly multiplied in the foregut. Again, transmission was not diminished compared to WT. Despite reductions in acquisition and retention by GWSS, transmission efficiency of genotypes to grapevines was not affected. Therefore, in order to stop the spread of X. fastidiosa by GWSS using gene-level targets, complete disruption of bacterial colonization mechanisms is required.

Quantitative Differences in Feeding Behavior of Lygus lineolaris (Hemiptera: Miridae) on Transgenic and Nontransgenic Cotton.

Lygus lineolaris (Palisot de Beauvois) is one of the most important pests on cotton in the United States. Previous research showed that transgenic cotton plants expressing the Bacillus thurigiensis (Bt) crystalline protein Cry51Aa2.834_16 (designated MON 88702) have insecticidal effects on nymphal L. lineolaris. The present study is the first to examine effects of a Bt-expressing cotton on feeding by a heteropteran like L. lineolaris. We compared stylet probing behaviors of third-instar nymphs on pin-head squares (i.e., buds <3 mm wide) of MON 88702 cotton versus nontransgenic (control) DP393 plants using AC-DC electropenetrography. Waveforms were quantified based on appearances previously characterized and correlated with adult L. lineolaris feeding behaviors; nymphal and adult waveforms had the same appearance. Generalized third-instar feeding included maceration of tissues during cell rupturing (waveform CR), tasting/testing during a waveform called transition (T), and ingestion (I); all were similar between MON 88702 and DP393 plants. However, the number of events and duration of each waveform were different between treatments. Relative to nymphs on DP393, those on MON 88702 spent more time overall in stylet probing, due to increased number of maceration events per probe and longer durations of tasting/testing, per waveform event, per probe, and per insect; yet, ingestion events were shorter and more frequent. These findings support that MON 88702 cotton plants were less palatable and/or preorally digestible to L. lineolaris nymphs than DP393, suggesting antixenosis for MON 88702. Transgenic cotton antixenosis could positively affect cotton pest management by reducing feeding of L. lineolaris nymphs and protecting crop yield.