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

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

Characterization of electropenetrography waveforms for the invasive heteropteran pest, Halyomorpha halys, on Vicia faba leaves

peer reviewedThe brown marmorated stink bug (BMSB), Halyomorpha halys Stål, has dispersed throughout the northern hemisphere, causing disturbances in agroecosystems and urban areas. Studies are, therefore, needed on the biology, physiology, and ecology of BMSB. This project characterized and partially correlated (via histology of plant tissues) the DC electropenetrography (EPG) waveforms related to non-probing (NP and Z) and both salivary sheath and cell rupture feeding. BMSB feeding behaviors were analyzed in comparison with the five other EPG waveform characterization papers published to date for heteropteran insects. Waveforms consisted of epidermal scanning (Hh1), pathway of stylets through leaf tissue (Hh2), X waves (Hh3), and sustained vascular ingestion (Hh4). In addition, a cell-rupturing phase in mesophyll (Hh2) and putative ingestion of resulting macerate (Hh5) were characterized. The most striking results were (1) the similarities of waveforms with other heteropterans, (2) performance of both sheath and cell rupture feeding strategies from all three plant cell types (xylem, likely phloem, and parenchyma macerate) on the same plant part, and (3) two different-appearing, dual-meaning X waves according to ingestion from xylem (Hh3a) or likely from phloem (Hh3b). This study provides a crucial framework for future, quantitative research on feeding behavior of this important invasive pest, to aid in the development of novel pest management tools

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

BGPI : équipe 6The 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., &gt;10 min) 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

Genera most commonly associated with <i>H. vitripennis</i> cibarium and precibarium.

<p>A) Average abundance of non-<i>Wolbachia</i> genera over all 96 insects; B–E) Abundance of all classifications from B) Bakersfield individuals; C) Ojai individuals; D) Florida individuals; E) colony individuals. Color legend in panel A applies to all panels (A–E); order of classifications in the legend is the same as the order around the pie graph in A (starting with the dark red <i>Anoxybacillus</i> in the upper right) and in the bar graphs B–E (starting with the dark blue <i>Wolbachia</i> on the left).</p

<i>X. fastidiosa</i> genomes per GWSS head.

<p>Quantitative RT-PCR was used to measure the number of <i>X. fastidiosa</i> genomes associated with 24 different dissected GWSS heads from the colony.</p