A waveform library for the study of probing and ingestion behaviors from Culicoides sonorensis using AC-DC electropenetrography

Authors: COOPER, ANASTASIA - Kansas State University; OSBORNE, CAMERON - Kansas State University; PICKENS, VICTORIA - Kansas State University; PIFFIER, KAITLIN - Kansas State University; JAMESON, SAMUAL - Kansas State University; RODRIGO DA SILVA, ANDERSON - Instituto Federal Goiano; REIF, KATHRYN - Auburn University; Mitzel, Dana; SILVER, KRISTOPHER - Kansas State University

Submitted to: Parasites & Vectors
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/14/2025
Publication Date: 7/7/2025
Citation: Cooper, A.M., Osborne, C., Pickens, V., Piffier, K., Jameson, S., Rodrigo Da Silva, A., Reif, K.E., Mitzel, D.N., Silver, K. 2025. A waveform library for the study of probing and ingestion behaviors from Culicoides sonorensis using AC-DC electropenetrography. Parasites & Vectors. 18. Article 265. https://doi.org/10.1186/s13071-025-06899-5.
DOI: https://doi.org/10.1186/s13071-025-06899-5

Interpretive Summary: Biting midges transmit pathogens that negatively affect animal welfare and production in livestock operations. However, little is known about the biting and feeding behaviors that occur inside host tissues, even though these behaviors may directly affect pathogen acquisition and transmission. Electropenetrography (EPG), is a technology that allows for indirect visualization and quantification of these behaviors by measuring the changes in electrical signals that arise during feeding. This technology was used to create a library of the waveforms caused by the changes in electrical signals. The library was created utilizing different electrical settings, allowing for the identification of the different waveforms. Videos and interruption of feeding studies were used to correlate behaviors with the different waveforms. Five major waveforms were identified and the optimal EPG setting for recording the feeding behavior of biting midges was identified. The creation of this waveform library establishes the foundation for future studies examining the effects of various factions including pathogen infection, pesticides, and genetic modifications.

Technical Abstract: Background: Culicoides sonorensis biting midges transmit arboviruses that negatively affect animal welfare and production in ruminant livestock operations. However, little is known about the probing and ingestion (i.e., biting) behaviors that occur inside host tissues, even though these behaviors may directly affect pathogen acquisition and transmission. Electropenetrography (EPG) allows for indirect visualization and quantification of these behaviors by measuring the changes in electrical signals that arise during probing. Methods: A waveform library for C. sonorensis was constructed using data from 70 adult females feeding to repletion on human hands recorded using an AC-DC electropentrograph with variable input resistors (Ri levels). The waveforms were characterized using each combination of four Ri levels (107, 108, 109, 1010 Ohms) and two electrical current types (AC, DC). Five response variables related to the count and duration of the waveforms were analyzed for each waveform family using one-way ANOVA models and likelihood ratio tests to detect significant effects between the eight treatment groups. The probability of transitioning between the waveform families was assessed based on a frequency table of transition events and multiple exact binomial tests used to identify non-random transition events. Videography and interruption experiments were used to correlate behaviors with waveforms. Results: Waveforms generated by C. sonorensis included waveform families J/K (stylet penetration through the skin), L (types 1, 2, 3, 4, and 5; preparation of an ingestion site), M (types 1, 2, 3, 4, and 5; ingestion), N (types 1, and 2; an unknown behavior which may be a resting and digestion phase), and W (withdrawal). The waveforms generally occurred in that order, sometimes with multiple transitions between L, M, and N. Significant differences in NWEi for J, K, and L were between some DC treatment groups. The optimal settings for EPG recordings of C. sonorensis probing on human hands was a Ri level of 108 Ohms using an applied signal of 75 millivolts direct current. Conclusions: EGP uniquely enhances our understanding of C. sonorensis probing and ingestion behaviors, which will facilitate further exploration and guide development of EPG procedures for other biting midges and telmophagous insects.