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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

EPG waveform library for Graphocephala atropunctata (Hemiptera: Cicadellidae): Effect of adhesive, input resistor, and voltage levels on waveform appearance and stylet probing behaviors.

Blue-green sharpshooter, Graphocephala atropunctata, is a native California vector of Xylella fastidiosa (Xf), a foregut-borne bacterium that is the causal agent of Pierce's disease in grapevines. A 3rd-generation, AC-DC electropenetrograph (EPG monitor) was used to record stylet probing and ingestion behaviors of adult G. atropunctata on healthy grapevines. This study presents for the first time a complete, updated waveform library for this species, as well as effects of different electropenetrograph settings and adhesives on waveform appearances. Both AC and DC applied signals were used with input resistor (Ri) levels (amplifier sensitivities) of 106, 107, 108 and 109 Ohms, as well as two type of adhesives, conducting silver paint and handmade silver glue. Waveform description, characterization of electrical origins (R versus emf components), and proposed biological meanings of waveforms are reported, as well as qualitative differences in waveform appearances observed with different electropenetrograph settings and adhesives. In addition, a quantitative study with AC signal, using two applied voltage levels (50 and 200 mV) and two Ri levels (107 and 109 Ohms) was performed. Intermediate Ri levels 107 and 108 Ohms provided EPG waveforms with the greatest amount of information, because both levels captured similar proportions of R and emf components, as supported by appearance, clarity, and definition of waveforms. Similarly, use of a gold wire loop plus handmade silver glue provided more definition of waveforms than a gold wire loop plus commercial conducting silver paint. Qualitative/observational evidence suggested that AC applied signal caused fewer aberrant behaviors/waveforms than DC applied signal. In the quantitative study, behavioral components of the sharpshooter X wave were the most affected by changes in Ri and voltage level. Because the X wave probably represents X. fastidiosa inoculation behavior, future studies of X. fastidiosa inoculation via EPG will require carefully determined instrument settings. An intermediate Ri level such as 108 Ohms with low voltage, AC applied signal, and gold wire loop plus silver glue is recommended as the best electropenetrograph methods to conduct future EPG studies of sharpshooter inoculation behaviors on Xf-resistant and -susceptible grapevine.

Handling Artificially Terminated Events in Electropenetrography Data.

Electropenetrography a.k.a. electrical penetration graph or EPG is a rigorous technique for studying arthropod behavior. Essentially, the arthropod and host are part of an electrical circuit that generates patterns of voltage (waveforms), whose biological meanings are defined by correlation with histology and behavior. EPG is used for studying stylet probing behavior of hemipterans, and blood-feeding arthropods. These results are applied to understanding pesticide action, host plant resistance, and vector-pathogen-host interactions. At the end of all recordings, the arthropod begins a behavior that ends because the scientist stopped recording. An argument for keeping this event in the data has been made based on the assumption that the insect is adapting to laboratory conditions. In this adaptation process, the expected durations of ingestion behaviors will increase as the insect adapts. We show that this assumption can cause problems in data analysis and interpretation of the data. If the assumption is false, then there are more options for analyzing the data. Deleting artificially terminated events can be advantageous, but the best approach needs to consider the biology of the arthropod and align with research objectives.

Behavioral Plasticity in Probing by Diaphorina citri (Hemiptera, Liviidae): Ingestion from Phloem Versus Xylem is Influenced by Leaf Age and Surface.

Diaphorina citri is a major pest of citrus because it transmits Candidatus Liberibacter asiaticus, a phloem-limited bacterium that putatively causes Huanglongbing (HLB). The disease moves slowly through a tree, and the vector facilitates further within-tree movement via transmission of the pathogen. However, this only happens when D. citri stylets contact the phloem, to inoculate bacteria during phloem salivation and acquire bacteria during phloem sap ingestion. Behavioral changes in D. citri associated with different plant parts would affect how long it takes to reach phloem and how long the psyllids stays in phloem to ingest, thereby influencing the risk of disease spread. D. citri feeding was recorded on the abaxial and adaxial surfaces of mature and immature citrus leaves. Adults in the field can be found on these surfaces at all times of year. On abaxial surface of immature leaves, phloem salivation would occur after 11 h on average, but rarely as soon as 0.56 h. The corresponding values on mature leaves were 16 and 2.7. In general, psyllids spent more time ingesting phloem sap on immature leaves than on mature leaves. Psyllids on abaxial surfaces spent more time ingesting from phloem, though the strength of this effect was less than for immature versus mature leaves. In contrast, xylem ingestion increased on mature leaves compared with young. The biological differences that could produce this outcome are discussed. The results discussed herein are of relevance to further studies on the efficacy of an insecticide to act quickly enough to prevent pathogen transmission.

Certain applied electrical signals during EPG cause negative effects on stylet probing behaviors by adult Lygus lineolaris (Hemiptera: Miridae).

This study is the first to fully evaluate whether electrical signals applied to large insects during electropenetrography (EPG; also called electrical penetration graph) negatively affect insect behavior. During EPG, electrical signals are applied to plants, and thus to the gold-wire-tethered insects feeding on them. The insect completes an electrical circuit whose changes in voltage reflect the insect's stylet probing/penetration behaviors, recorded as waveform output. For nearly 50 years of EPG science, evidence has supported that there are no or negligible effects on tiny insects from applied electricity during EPG. Recently however, EPG studies of large-bodied hemipterans such as heteropterans and sharpshooter leafhoppers have been published. The wider stylet diameters of such large insects cause them to have lower inherent resistances to applied signals compared with smaller insects, conveying more electrical current. The present study asked whether such increased currents would affect insect stylet probing, by comparing Lygus lineolaris behaviors on pin-head cotton squares using an AC-DC electropenetrograph. Effects of AC or DC applied signals were separately examined in two factorial studies, each comparing four input resistor (Ri) levels (106, 107, 108 and 109 Ω) and four applied voltage levels (2, 60, 150 and 250 mV). Results showed that changes in both probing and non-probing behaviors were indeed caused by changing signal type, Ri level, or applied voltage. Negative effects on feeding were numerically greater overall for DC than AC applied signals, perhaps due to muscular tetany from DC; however, AC versus DC could not be statistically tested. Results strongly support the need for flexible Ri and applied voltage levels and types, to tailor instrument settings to the size and special needs of each insect subject. Our findings will facilitate further EPG studies of Lygus spp., such as host plant resistance or insecticidal assays/bioassays to assess mode of action and appropriate dosage. It is hoped that this study will also inform EPG studies of similar, large heteropterans in the future.

Correlation of Electropenetrography Waveforms From Lygus lineolaris (Hemiptera: Miridae) Feeding on Cotton Squares With Chemical Evidence of Inducible Tannins.

Probing behavior of Lygus lineolaris (Palisot de Beauvois) has previously been characterized with electropenetrography (EPG). Cell rupturing (CR) and ingestion (I) EPG waveforms were identified as the two main stylet-probing behaviors by adult L. lineolaris. However, characterization and identification of EPG waveforms are not complete until specific events of a particular waveform are correlated to insect probing. With the use of EPG, histology, microscopy, and chemical analysis, probing behavior of L. lineolaris on pin-head cotton squares was studied. Occurrences of waveforms CR and I were artificially terminated during the EPG recording. Histological samples of probed cotton squares were prepared and analyzed to correlate specific types and occurrences of feeding damage location and plant responses to insect feeding. Both CR and I occurred in the staminal column of the cotton square. Cell rupturing events elicited the production of dark-red deposits seen in histological staining that were demonstrated via chemical analysis to contain condensed tannins. We hypothesize that wounding and saliva secreted during CR triggered release of tannins, because tannin production was positively correlated with the number of probes with single CR events performed by L. lineolaris. Degraded plant tissue and tannins were removed from the staminal column during occurrence of waveform I. These results conclude the process of defining CR and I as probing waveforms performed by L. lineolaris on pin-head cotton squares. These biological definitions will now allow EPG to be used to quantitatively compare L. lineolaris feeding among different plant treatments, with the goal of improving pest management tactics against this pest.

First finding of a dual-meaning X wave for phloem and xylem fluid ingestion: Characterization of Scaphoideus titanus (Hemiptera: Cicadellidae) EPG waveforms.

The leafhopper Scaphoideus titanus (Hemiptera: Cicadomorpha: Cicadellidae), an invasive deltocephaline species introduced into Europe from North America, is the vector of the most important phytoplasma disease in European viticulture, flavescence dorée. In this first electropenetrography (EPG) study of S. titanus, we characterized its feeding waveforms and defined their biological meanings. Four typical waveform phases (pathway, X wave, sustained ingestion, and interruption) and four families within those phases (A, B, C, and N) were characterized using DC EPG technology. We proposed biological meanings for these waveforms based on excreta pH-ingestion correlations, presence of X waves, and comparison with previous AC, DC, and AC-DC EPG waveforms conducted on Cicadomorpha. We observed that sustained (i.e., >10min) ingestion by a deltocephaline leafhopper can occur from both xylem and phloem vascular cells. Waveform C2x represented ingestion of xylem fluid, and two waveforms represented behaviors when stylets were inserted into phloem sieve elements: C2p variant 1 (C2p-1), which may represent salivation (perhaps simultaneous with ingestion), and C2p variant 2 (C2p-2), which represented active ingestion. Furthermore, we found that the EPG-recorded X wave has a dual meaning by occurring prior to sustained ingestion from either phloem or xylem. This X wave was very similar in appearance to the model X wave of sharpshooters, an entirely different leafhopper subfamily, Cicadellinae. All cicadellines are obligate xylem-ingesters. Such a "dual-meaning X wave" will provide insights into how the feeding tactics of S. titanus relate to other sheath-feeding hemipterans, and will provide support for future research to clarify the role of this leafhopper as a vector of plant pathogens.