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The precibarial valve is a tiny structure in the functional foregut (cibarium and precibarium) of hemipteran insects. Piercing-sucking insects like hemipterans use straw-like mouth parts (stylets) to ingest fluid from plant vascular cells like phloem or xylem. Suction is provided by the cibarium (or cibarial pump), which draws fluid through the stylets into a short canal called the precibarium, then into the cibarium from which the fluid is swallowed into the pharynx then esophagus. The precibarium houses two sets of chemosensilla separated by the tiny precibarial valve, which is operated by its own muscle independent of the muscles powering the cibarium. Despite intensive study, the operational mechanism of the precibarial valve in sharpshooter leafhoppers is not known because the muscle attachment to the valve has not been clearly identified. Such an operational mechanism is important because it controls inoculation of the lethal bacterial pathogen Xylella fastidiosa into healthy crop plants, causing economically devastating diseases such as Pierce’s disease of grape, citrus variegated chlorosis, olive quick decline, and numerous leaf scorches. Together, these diseases cause many $billions of damage and control costs worldwide, annually. The present study provides definitive knowledge of how the precibarial valve muscle attaches to the valve in sharpshooter vectors of Xylella fastidiosa . It also proposes a new operation model to control fluid flows responsible for inoculating the pathogen into healthy plants. Such information will aid in development of novel disease management methods such plant resistance to vector performance of inoculation behaviors.

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.

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.

The glassy-winged sharpshooter (GWSS) is an invasive insect species that transmits Xylella fastidiosa, the bacterium causing Pierce's disease of grapevine and other leaf scorch diseases. X. fastidiosa has been shown to colonize the anterior foregut (cibarium and precibarium) of sharpshooters, where it may interact with other naturally-occurring bacterial species. To evaluate such interactions, a comprehensive list of bacterial species associated with the sharpshooter cibarium and precibarium is needed. Here, a survey of microbiota associated with the GWSS anterior foregut was conducted. Ninety-six individual GWSS, 24 from each of 4 locations (Bakersfield, CA; Ojai, CA; Quincy, FL; and a laboratory colony), were characterized for bacteria in dissected sharpshooter cibaria and precibaria by amplification and sequencing of a portion of the 16S rRNA gene using Illumina MiSeq technology. An average of approximately 150,000 sequence reads were obtained per insect. The most common genus detected was Wolbachia; sequencing of the Wolbachia ftsZ gene placed this strain in supergroup B, one of two Wolbachia supergroups most commonly associated with arthropods. X. fastidiosa was detected in all 96 individuals examined. By multilocus sequence typing, both X. fastidiosa subspecies fastidiosa and subspecies sandyi were present in GWSS from California and the colony; only subspecies fastidiosa was detected in GWSS from Florida. In addition to Wolbachia and X. fastidiosa, 23 other bacterial genera were detected at or above an average incidence of 0.1%; these included plant-associated microbes (Methylobacterium, Sphingomonas, Agrobacterium, and Ralstonia) and soil- or water-associated microbes (Anoxybacillus, Novosphingobium, Caulobacter, and Luteimonas). Sequences belonging to species of the family Enterobacteriaceae also were detected but it was not possible to assign these to individual genera. Many of these species likely interact with X. fastidiosa in the cibarium and precibarium.

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 .

Hopperburn is a noncontagious disease of plants caused by the direct feeding damage of certain leafhoppers and planthoppers. Although long studied, especially with Empoasca spp. leafhoppers (Cicadellidae: Typhlocybinae), the mechanisms underlying hopperburn have only recently been elucidated. Hopperburn is caused by a dynamic interaction between complex insect feeding stimuli (termed hopperburn initiation) and complex plant responses (termed the hopperburn cascade). Herein we review the nature of the feeding stimuli in hopperburn initiation, especially for Empoasca spp., which we also compare with the planthopper Nilaparvata lugens. Contrary to previous reports, Empoasca hopperburn is not caused solely by toxic saliva. Instead, it is caused by a plant wound response triggered by a unique type of stylet movement, which is then exacerbated by saliva. Electrical penetration graph monitoring has revealed that all Empoasca spp. are cell rupture feeders, not sheath feeders, and that certain tactics of that feeding strategy are more damaging than others. Measuring the proportions of the most damaging feeding led to development of a resistance index, the Stylet Penetration Index, which can predict hopperburn severity in different plants or under different environmental conditions and can supplement or replace traditional, field-based resistance indices.

Lygus lineolaris is an important native pest of cotton in the mid-southern USA and a potential invasive species in other parts of the world. L. lineolaris feeds on more than 300 plant species, especially preferring reproductive plant organs but also feeding on young terminal leaves. There is little known about feeding behaviors performed on cotton leaves nor of associated injury symptoms triggered. Herein, we used the most accurate way to study feeding of piercing–sucking insects, i.e., electropenetrography (EPG). EPG was used to quantify L. lineolaris feeding behaviors on young terminal leaves of cotton, for third, fourth, and fifth instars plus male adults. Both non-probing (combined walking, standing and antennation) as well as probing behaviors (cell rupturing, transition, and ingestion) were compared with a time course of digitally measured leaf damage. Overall, L. lineolaris , especially adults, spent most time in non-probing behaviors. For probing behaviors, the longest duration was cell rupturing, especially for fourth and fifth instars followed by the third instars. The greatest damage to cotton leaves occurred when high numbers of wound-inducing cell-rupturing probes, which inject macerating saliva, were combined with minimal subsequent ingestion of saliva. On cotton leaves, this style of cell rupturing matches that of older nymphs. Thus, even small amounts of cell rupture feeding and ingestion by older L. lineolaris nymphs are damaging to cotton leaf terminals. These results help to understand the cause of damage to L. lineolaris hosts and consequently aid in developing strategies to reduce crop loss.

Abstract 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.

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.

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.