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

Primer modification improves rapid and sensitive in vitro and field-deployable assays for detection of High plains virus variants

A high consequence pathogen, High plains virus (HPV) causes considerable damage to wheat if the crop is infected during early stages of development. Methods for the early, accurate, and sensitive detection of HPV in plant tissues are needed for the management of disease outbreaks and reservoir hosts. In this study, the effectiveness of five methods—real-time SYBR green and TaqMan reverse transcription-quantitative PCR (RT-qPCR), endpoint RT-PCR, RT-helicase dependent amplification (RT-HDA) and the Razor Ex BioDetection System (Razor Ex)—for the broad-range detection of HPV variants was evaluated. Specific PCR primer sets and probes were designed to target the HPV nucleoprotein gene. Primer set HPV6F and HPV4R, which amplifies a product of 96 bp, was validated in silico against published sequences and in vitro against an inclusivity panel of infected plant samples and an exclusivity panel of near-neighbor viruses. The primers were modified by adding a customized 22 nucleotide long tail at the 5′ terminus, raising the primers' melting temperature (Tm; ca. 10°C) to make them compatible with RT-HDA (required optimal Tm = 68°C), in which the use of primers lacking such tails gave no amplification. All of the methods allowed the detection of as little as 1 fg of either plasmid DNA carrying the target gene sequence or of infected plant samples. The described in vitro and in-field assays are accurate, rapid, sensitive, and useful for pathogen detection and disease diagnosis, microbial quantification, and certification and breeding programs, as well as for biosecurity and microbial forensics applications.Peer reviewedEntomology and Plant Patholog