New Tool Opens a Bigger Window to Insect-Plant Warfare

When an aphid, leafhopper, or psyllid lands on a plant to feed, it begins a process of chamical welfare. As piercing-sucking insects, they use needlelike stylets to insert saliva into plant tissues and open a pathway to ingest fluids critical to the plant’s survival. When punctured, the plant senses the attack and secretes proteins and other chemical defenses to prevent fluids from being pulled out, thus creating a stress on the plant. This warfare costs growers billions of dollars each year in lost ornamentals, vegetables, citrus, and other important agricultural crops. Because much of the action takes place in the plant’s interior, a scientific tool called an “electrical penetration graph” (EPG) is critical for peering into the process. To use it, researchers connect the insect and plant to an electronic monitor that, like an electrocardiogram, reads electrical charges produced by tiny changes in voltage that occur as the insect feeds. A new type of EPG, developed by Elaine Backus, an ARS entomologist at the San Joaquin Valley Agricultural Sciences Center, in Parlier, California, and the late William Bennett, formerly from the University of Missouri, is giving scientists the clearest window yet into the wars waged between piercing sucking insects and the plants they infest. Because these insects are often carriers of plant pathogens that are transmitted through feeding, EPG can also illuminate how pathogens are injected into the plant to start the infection process

New Tool Opens a Bigger Window to Insect-Plant Warfare

When an aphid, leafhopper, or psyllid lands on a plant to feed, it begins a process of chamical welfare. As piercing-sucking insects, they use needlelike stylets to insert saliva into plant tissues and open a pathway to ingest fluids critical to the plant’s survival. When punctured, the plant senses the attack and secretes proteins and other chemical defenses to prevent fluids from being pulled out, thus creating a stress on the plant. This warfare costs growers billions of dollars each year in lost ornamentals, vegetables, citrus, and other important agricultural crops. Because much of the action takes place in the plant’s interior, a scientific tool called an “electrical penetration graph” (EPG) is critical for peering into the process. To use it, researchers connect the insect and plant to an electronic monitor that, like an electrocardiogram, reads electrical charges produced by tiny changes in voltage that occur as the insect feeds. A new type of EPG, developed by Elaine Backus, an ARS entomologist at the San Joaquin Valley Agricultural Sciences Center, in Parlier, California, and the late William Bennett, formerly from the University of Missouri, is giving scientists the clearest window yet into the wars waged between piercing sucking insects and the plants they infest. Because these insects are often carriers of plant pathogens that are transmitted through feeding, EPG can also illuminate how pathogens are injected into the plant to start the infection process

Waveform Library for Chinch Bugs (Hemiptera: Heteroptera: Blissidae): Characterization of Electrical Penetration Graph Waveforms at Multiple Input Impedances

Electrical penetration graph (EPG) monitoring has been used extensively to elucidate mechanisms of resistance in plants to insect herbivores with piercing-sucking mouthparts. Characterization of waveforms produced by insects during stylet probing is essential to the application of this technology. In the studies described herein, a four-channel Backus and Bennett AC-DC monitor was used to characterize EPG waveforms produced by adults of two economically important chinch bug species: southern chinch bug, Blissus insularis Barber, feeding on St. Augustinegrass, and western chinch bug, Blissus occiduus Barber, feeding on buffalograss. This is only the third time a heteropterans species has been recorded by using EPG; it is also the first recording of adult heteropterans, and the first of Blissidae. Probing of chinch bugs was recorded with either AC or DC applied voltage, no applied voltage, or voltage switched between AC and DC mid-recording, at input impedances ranging from 106 to 1010Ω, plus 1013 Ω, to develop a waveform library. Waveforms exhibited by western and southern chinch bugs were similar, and both showed long periods of putative pathway and ingestion phases (typical of salivary sheath feeders) interspersed with shorter phases, termed transitional J wave and interruption. The J wave is suspected to be an X wave, that is, in EPG parlance, a stereotypical transition waveform that marks contact with a preferred ingestion tissue. The flexibility of using multiple input impedances with the AC-DC monitor was valuable for determining the electrical origin (resistance vs. electromotive force components) of the chinch bug waveforms. It was concluded that an input impedance of 107Ω, with either DC or AC applied voltage, is optimal to detect all resistance- and electromotive force–component waveforms produced during chinch bug probing. Knowledge of electrical origins suggested hypothesized biological meanings of the waveforms, before time-intensive future correlation experiments by using histology, microscopy, and other techniques

Stylet Penetration Activities by Aphis craccivora (Homoptera: Aphididae) on Plants and Excised Plant Parts of Resistant and Susceptible Cultivars of Cowpea (Leguminosae)

Direct current electrical penetration graphs (DC-EPGs) were used to analyze the stylet penetration activities of cowpea aphid, Aphis craccivora Koch, on plants of aphid-resistant (ICV-12) and aphid-susceptible (ICV-1) cultivars of cowpea, Vigna unguiculata (L.) Walpers. Aphid stylet penetration on whole plants at seedling, flowering, and podding stages were studied in one experiment, and in another experiment excised leaves from seedling plants, excised flowers, and excised pods were tested. Electrical signals depicting the aphid stylet penetration activities on their host plants were amplified, recorded onto a paper chart recorder, and scored for specific waveform patterns. Compared with similar tissues of ICV-1, intact leaves and excised seedling foliage of ICV-12 plants caused severe disruption of aphid stylet penetration activities. This was manifested in frequent penetration attempts that were abruptly terminated or unsustained, and in shorter penetration times, signifying antixenosis resistance in ICV-12. There was reduced occurrence of E waveforms, which represent stylet activity in plant vascular tissues. Also, prior exposure of test aphids to plants of one cultivar did not significantly influence the expected stylet penetration activities on plants of the other cultivar. Overall, ICV-12 exhibited high levels of resistance against A. craccivor

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.Peer reviewedEntomology and Plant Patholog

The New, Third-generation, AC-DC Electrical Penetration Graph (EPG) Monitor and Its Usefulness for IPM Research on Vectors of Plant Pathogens

The most rigorous method to identify feeding behaviors of hemipteran vectors of plant pathogens is electrical penetration graph (EPG) monitoring. The purpose of this talk was to review: 1) principals of EPG as a tool for developing novel integrated pest management tools against vectors, and 2) application of EPG to identify feeding behaviors leading to inoculation of Xylella fastidiosa. X. fastidiosa is a xylem-limited bacterium that causes several scorch diseases in important crops, such as Pierce’s disease of grape. Bacteria form a dense biofilm on the foregut cuticle of the glassy-winged sharpshooter, Homalodisca vitripennis (Germar) and other xylem-feeding vectors. Bacteria are inoculated directly from sites in the foregut into a host plant during sharpshooter feeding (i.e. probing of the mouthparts, stylets, into the plant). However, despite nearly 70 years of research, no one had associated specific sharpshooter stylet probing behaviors with inoculation until EPG was employed for such research. Development of the third generation (AC-DC) EPG monitor from the first two generations of monitors (AC and DC) helped define the mechanism of X. fastidiosa inoculation. EPG and other evidence for the salivation-egestion hypothesis for X. fastidiosa inoculation, in which salivation combined with egestion [outward fluid flow] carries bacteria into the xylem, was reviewed. Understanding the inoculation mechanism will aid development of grape varieties resistant to inoculation of X. fastidiosa by sharpshooter vectors

Waveform Library for Chinch Bugs (Hemiptera: Heteroptera: Blissidae): Characterization of Electrical Penetration Graph Waveforms at Multiple Input Impedances

Electrical penetration graph (EPG) monitoring has been used extensively to elucidate mechanisms of resistance in plants to insect herbivores with piercing-sucking mouthparts. Characterization of waveforms produced by insects during stylet probing is essential to the application of this technology. In the studies described herein, a four-channel Backus and Bennett AC-DC monitor was used to characterize EPG waveforms produced by adults of two economically important chinch bug species: southern chinch bug, Blissus insularis Barber, feeding on St. Augustinegrass, and western chinch bug, Blissus occiduus Barber, feeding on buffalograss. This is only the third time a heteropterans species has been recorded by using EPG; it is also the first recording of adult heteropterans, and the first of Blissidae. Probing of chinch bugs was recorded with either AC or DC applied voltage, no applied voltage, or voltage switched between AC and DC mid-recording, at input impedances ranging from 106 to 1010Ω, plus 1013 Ω, to develop a waveform library. Waveforms exhibited by western and southern chinch bugs were similar, and both showed long periods of putative pathway and ingestion phases (typical of salivary sheath feeders) interspersed with shorter phases, termed transitional J wave and interruption. The J wave is suspected to be an X wave, that is, in EPG parlance, a stereotypical transition waveform that marks contact with a preferred ingestion tissue. The flexibility of using multiple input impedances with the AC-DC monitor was valuable for determining the electrical origin (resistance vs. electromotive force components) of the chinch bug waveforms. It was concluded that an input impedance of 107Ω, with either DC or AC applied voltage, is optimal to detect all resistance- and electromotive force–component waveforms produced during chinch bug probing. Knowledge of electrical origins suggested hypothesized biological meanings of the waveforms, before time-intensive future correlation experiments by using histology, microscopy, and other techniques

Anterior Foregut Microbiota of the Glassy-Winged Sharpshooter Explored Using Deep 16S rRNA Gene Sequencing from Individual Insects

<div><p>The glassy-winged sharpshooter (GWSS) is an invasive insect species that transmits <i>Xylella fastidiosa</i>, the bacterium causing Pierce's disease of grapevine and other leaf scorch diseases. <i>X. fastidiosa</i> 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 <i>Wolbachia</i>; sequencing of the <i>Wolbachia ftsZ</i> gene placed this strain in supergroup B, one of two <i>Wolbachia</i> supergroups most commonly associated with arthropods. <i>X. fastidiosa</i> was detected in all 96 individuals examined. By multilocus sequence typing, both <i>X. fastidiosa</i> subspecies <i>fastidiosa</i> and subspecies <i>sandyi</i> were present in GWSS from California and the colony; only subspecies <i>fastidiosa</i> was detected in GWSS from Florida. In addition to <i>Wolbachia</i> and <i>X. fastidiosa</i>, 23 other bacterial genera were detected at or above an average incidence of 0.1%; these included plant-associated microbes (<i>Methylobacterium</i>, <i>Sphingomonas</i>, <i>Agrobacterium</i>, and <i>Ralstonia</i>) and soil- or water-associated microbes (<i>Anoxybacillus</i>, <i>Novosphingobium</i>, <i>Caulobacter</i>, and <i>Luteimonas</i>). 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 <i>X. fastidiosa</i> in the cibarium and precibarium.</p></div

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

International audienceFluid 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 Pierce’s 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 discusse