Elaine A. Backus

Stylet Penetration by Adult Homalodisca coagulata on Grape: Electrical Penetration Graph Waveform Characterization, Tissue Correlation, and Possible Implications for Transmission of Xylella fastidiosa

Abstract The sharpshooter Homalodisca coagulata (Say) (Hemiptera: Cicadellidae: Cicadellinae) is an exotic vector of the Pierce’s disease (PD) bacterium, Xylella fastidiosa (Wells et al.), that was first observed in California in 1989. H. coagulata has since greatly increased the threat of PD to the grape industry as well as stone fruit, nursery, and ornamental industries in California. This is the first in a series of articles that together describe how sharpshooter stylet penetration behaviors (especially intricate stylet activities, salivation, and ingestion) control transmission (i.e., acquisition and inoculation) of X. fastidiosa. Herein, we categorized and characterized alternating current electrical penetration graph (EPG) waveforms from glass-winged sharpshooter stylet penetration on petiole of susceptible grape (‘Cabernet Sauvignon’), paying special attention to waveform fine structures that are likely to be the key to detecting the instant of inoculation. We also correlated waveforms with salivary sheath termini in grape tissues. For the first time in any EPG study of leafhopper or planthopper feeding, we demonstrate through case studies of individual probes how to follow the process of stylet penetration step by step as it is occurring, including salivary sheath branching and when the stylets first puncture a xylem cell. Finally, we discuss the implications of our findings for understanding the transmission mechanism of X. fastidiosa, in comparison with hypothesized mechanisms in the literature.

The AC–DC Correlation Monitor: New EPG design with flexible input resistors to detect both R and emf components for any piercing–sucking hemipteran

Much of what is known today about hemipteran feeding biology, as well as mechanisms of their host plant interactions and transmission of phytopathogens, has been learned via use of electrical penetration graph (EPG) technology, originally called electronic monitoring of insect feeding. Key to all of this information has been the electronic designs of EPG monitors. It has been 45 years since the publication of the original EPG, the AC monitor, and 30 years since introduction of the DC monitor, an important improvement for EPG science. Herein we describe our new AC-DC Correlation Monitor, the first major improvement in design since the DC monitor. We provide the monitors block diagram and circuit description, and discuss (as a first example) its application to aphid feeding waveforms. Our instrument combines design features from the existing AC Missouri monitor and the DC Tjallingii monitor, plus several new innovations. It can produce three simultaneous, time-synchronized, output signals from a single insect, via AC and DC signal processing circuitry, as well as using either AC, DC, AC-plus-DC, or 0V substrate voltage. Our research conclusively demonstrates that AC signal processing can be designed to duplicate the level of detail and fidelity of aphid waveforms previously provided solely by the DC monitor, including all R- and emf-component waveforms. Availability of either AC or DC applied voltages will allow similar high-resolution recording of insects that appear to be sensitive to DC applied voltages. We also begin to determine the subtle reasons why published waveforms from older AC and DC monitors appear to differ so greatly. Our instrument is a single, flexible, universal monitor that can provide maximum, R-plus-emf waveform information from any piercing-sucking species, especially non-aphid species with sensitivity to DC applied voltage.

Correlations of cibarial muscle activities of Homalodisca spp. sharpshooters (Hemiptera: Cicadellidae) with EPG ingestion waveform and excretion

Fluid flow into and out of the stylets of xylem-ingesting sharpshooters (Hemiptera: Cicadellidae: Cicadellinae) is powered by muscles of the cibarial pump. Such fluid flow is crucial for transmission of Xylella fastidiosa, the Pierces Disease bacterium, yet has not been rigorously studied via electrical penetration graph (EPG) technology. We correlated EPG waveforms with electromyographically (EMG) recorded muscle potentials from the cibarial dilator muscles, which power the piston-like cibarial diaphragm. There was a 1:1 correspondence of each cycle of cibarial muscle contraction/relaxation with each plateau of EPG waveform C. Results definitively showed that the C waveform represents active ingestion, i.e. fluid flow is propelled by cibarial muscle contraction. Moreover, each C waveform episode represents muscular diaphragm uplift, probably combined with a bounce from cuticular elasticity, to provide the suction that pulls fluid into the stylets. Fine structure of the EPG ingestion waveform represents directionality of fluid flow, supporting the primary role of streaming potentials as the electrical origin of the C waveform. Rhythmic bouts of cibarial pumping were generally correlated with sustained production of excretory droplets. However, neither the onset nor cessation of ingestion was correlated with onset or cessation of excretion, respectively. Volume of excreta is an inexact measure of ingestion. Implications for using EPG to understand the mechanism of X. fastidiosa transmission are discussed.

Characterization of EPG waveforms for the tea green leafhopper, Empoasca vitis Göthe (Hemiptera: Cicadellidae), on tea plants and their correlation with stylet activities

The stylet probing activities of the tea green leafhopper Empoasca vitis Gothe (Hemiptera: Cicadellidae) were studied using the DC electrical penetration graph (EPG) technique. Seven different EPG waveforms (i.e., Np, E1, E2, E3, E4, E5 and E6) were distinguished and characterized on susceptible tea leaves. In addition, four of them (i.e., Np, E1, E2, E3), together accounting for 97.08% of the total recording time, were behaviorally correlated with probing and non-probing activities using artificial diet observation with high-magnification video recording. At the start of stylet probing, waveform E1 always occurred at a variable voltage. E1, with all three of its waveform sub-types (E1-A to E1-C), was correlated with production of the salivary sheath trunk, stylet laceration, and channel cutting in viscous artificial diet. Afterwards, two types of high-amplitude waveforms, E2 and E3, followed. E2 had a highly regular, quasi-square wave, repetitive appearance, and lasted the longest duration of all E. vitis probing waveforms. E3 usually appeared after E2, and also exhibited a quasi-square wave feature similar to E2, but had much higher amplitude. Both waveforms E2 and E3 were correlated with active ingestion in liquid artificial diet. In addition, secretion of watery, enzymatic saliva was likely during E2. The active stylet movements and channel-cutting observed during the probing process indicate that E. vitis is a cell rupture feeder, not a salivary sheath feeder, as aphids and other leafhoppers. Thus, hopperburn damage to the tea plant is probably due to the cell rupture feeding strategy, similar to other hopperburning Empoasca species.

Salivary enzymes are injected into xylem by the glassy-winged sharpshooter, a vector of Xylella fastidiosa

A few phytophagous hemipteran species such as the glassy-winged sharpshooter, Homalodisca vitripennis, (Germar), subsist entirely on xylem fluid. Although poorly understood, aspects of the insects salivary physiology may facilitate both xylem-feeding and transmission of plant pathogens. Xylella fastidiosa is a xylem-limited bacterium that causes Pierces disease of grape and other scorch diseases in many important crops. X. fastidiosa colonizes the anterior foregut (precibarium and cibarium) of H. vitripennis and other xylem-feeding vectors. Bacteria form a dense biofilm anchored in part by an exopolysaccharide (EPS) matrix that is reported to have a β-1,4-glucan backbone. Recently published evidence supports the following, salivation-egestion hypothesis for the inoculation of X. fastidiosa during vector feeding. The insect secretes saliva into the plant and then rapidly takes up a mixture of saliva and plant constituents. During turbulent fluid movements in the precibarium, the bacteria may become mechanically and enzymatically dislodged; the mixture is then egested back out through the stylets into plant cells, possibly including xylem vessels. The present study found that proteins extracted from dissected H. vitripennis salivary glands contain several enzyme activities capable of hydrolyzing glycosidic linkages in polysaccharides such as those found in EPS and plant cell walls, based on current information about the structures of those polysaccharides. One of these enzymes, a β-1,4-endoglucanase (EGase) was enriched in the salivary gland protein extract by subjecting the extract to a few, simple purification steps. The EGase-enriched extract was then used to generate a polyclonal antiserum that was used for immunohistochemical imaging of enzymes in sharpshooter salivary sheaths in grape. Results showed that enzyme-containing gelling saliva is injected into xylem vessels during sharpshooter feeding, in one case being carried by the transpiration stream away from the injection site. Thus, the present study provides support for the salivation-egestion hypothesis.

Effects of Soil-Applied Imidacloprid on Asian Citrus Psyllid (Hemiptera: Psyllidae) Feeding Behavior

ABSTRACT The Asian citrus psyllid, Diaphorina citri Kuwayama (Hemiptera: Psyllidae), is one of the most important pests of citrus (Citrus spp.) because of its status as a vector of Candidatus Liberibacter asiaticus (Las), the bacterium associated with citrus greening disease. The use of insecticides for vector control is the primary method of managing the spread of this pathogen. Imidacloprid is an insecticide commonly applied to the root zone of young citrus trees to provide systemic protection from pests. The effects of imidacloprid on feeding behavior of D. citri have not been studied in much detail. The purpose of this study was to examine the effects of imidacloprid application on feeding behavior of D. citri and to determine whether use of this systemic insecticide could have any effect on pathogen transmission by D. citri. A direct current electrical penetration graph monitor was used to record D. citri feeding behaviors for 12-h periods on mature and young leaves of imidacloprid-treated and -untreated citrus seedlings. Overall, compared with untreated plants, the feeding behavior of D. citri was disrupted on imidacloprid-treated plants via reduction in the number of probes, as well as durations of average probes, initial stylet contact with phloem, phloem salivation, and phloem ingestion. The results of this study demonstrate that soil applications of imidacloprid can reduce the probability of citrus plants becoming inoculated with Las through a reduction in the number and duration of phloem salivation events by D. citri. Furthermore, Las acquisition from infected citrus is greatly reduced as a result of decreased phloem ingestion by D. citri on imidacloprid-treated plants.

Characterization and Correlation of EPG Waveforms of Bactericera Cockerelli (Hemiptera: Triozidae): Variability in Waveform Appearance in Relation to Applied Signal

ABSTRACT n The tomato-potato psyllid, Bactericera cockerelli (Sulc) (Hemiptera: Triozidae), was recently shown to be a vector of “Candidatus Liberibacter solanacearum” (Lso), a phloem-limited bacterium that is the putative causal agent of “zebra chip” in potato and unnamed diseases in other solanaceous species. Despite its importance, very little is known about B. cockerelli stylet probing behaviors that control transmission of Lso to its host plants. Herein, we report the first study characterizing the electrical penetration graph waveforms representing stylet penetration behaviors of the B. cockerelli feeding on potato. Waveforms produced by adult B. cockerelli on potato were also correlated using light microscopy of salivary sheath termini in plant tissue after probes were artificially terminated during the identified waveforms. In addition, behavioral activities were inferred based on electrical origins of waveforms as well as similarities in waveform appearances with those of other psyllids, aphids, and whiteflies. Adult B. cockerelli produced six waveform families and four types, which represent the following proposed biological meanings: family A, initial penetration and sheath salivation; family B, penetration of epidermal cells; family C, secretion of most of the salivary sheath and stylet pathway in mesophyll and parenchyma, with two types, C1 and C2, of unknown meaning; family D, initial contact with phloem cells; family E, activities in phloem cells, with two types, El, putative phloem salivation, and E2, phloem sap ingestion; and family G, xylem ingestion. In addition, a previously unreported variant of waveform G was characterized and correlated with applied signal type. Variation in D and G waveform appearances was correlated with polarity, type, and magnitude of applied signal. Results suggest that active phloem sap ingestion during E2 may play a critical role in acquisition of Lso bacterial cells. E1 may be important in inoculation of the bacterium into phloem sieve elements because it may represent salivation into phloem sieve elements. Analysis of B. cockerelli waveforms could lead to faster development of resistant host plant varieties, strengthen integrated pest management strategies by incorporating alternative plant hosts, and maximize the efficiency of pesticides.

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 Pierces disease of grapevine, Xylella fastidiosa, is the only known bacterial, arthropod-transmitted plant pathogen that does not circulate in the vectors 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.

Characterization of an EPG Waveform Library for Redbanded Stink Bug, Piezodorus guildinii (Hemiptera: Pentatomidae), on Soybean Plants

Abstract Feeding behaviors of the redbanded stink bug, Piezodorus guildinii (Westwood), on vegetative (stem and leaflet) and reproductive (pod) tissues of soybean, Glycine max (L.), were recorded using an AC-DC electropenetrograph (EPG) apparatus. Eight different probing waveforms were characterized and defined: Pg1a, Pg1b, Pg1c, Pg1d, Pg2, Pg3a, Pg3b, and Pg4 grouped into three different families, P, I, and N. Histological studies of intact stylets within salivary sheaths observed during Pg1b, Pg1c, Pg2, and Pg3 waveforms were correlated with the specific penetration sites. Waveforms Pg1a, Pg1b, Pg1c, and Pg1d (pathway—family P) occurred at the start of probing activities and represent stylet penetration deep into plant tissue. Waveforms Pg2, Pg3a, and Pg3b (family I) represent the food ingestion phase. Pg2 waveform represents xylem sap ingestion primarily on leaves and stems. During Pg3a, stylets were moving, lacerating deeply into pod tissue and partially retracting; during Pg3b, stylets were motionless inside the pod tissue. Pg3b occurred interspersed with waveform Pg3a. Waveform Pg4 (family N) represented short interruptions that occurred within waveform Pg2. The study demonstrated that P. guildinii uses the cell rupture strategy to ingest from endosperm in soybean pod, and the same insect could switch to salivary sheath feeding to ingest from xylem in soybean leaves and stems. This unusual behavior explains symptoms of stink bug damage to soybean. The P. guildinii waveforms defined herein will allow future EPG studies to aid in development of soybean varieties that resist the feeding and damage caused by this and other stink bug pests.

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 vectors 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 vectors 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.