We mist-netted eight big brown bats (E. fuscus) in southwest Idaho under Idaho Department of Fish and Game permit #110615. All bats were housed and cared for at Boise State University following the Institutional Animal Care and Use Committee protocol #006-AC14-014 and care protocols established by Lollar and Schmidt-French (52). The light regime was 10-hour dark/14-hour light. Before behavioral trials, we trained bats to hunt greater wax moths (Galleria mellonella) suspended from a monofilament tether. Wild, male fireflies (P. pyralis) were collected in Lehigh County, PA and Douglas County, KS, while control scarab beetles (Callistethus marginatus and Dyscinetus morator) were collected in Alachua County, FL in June to July 2015. Big brown bats occur throughout North America and are considered “nonmigratory” (53), while fireflies that flash as adults are generally only found east of the Rocky Mountains. We thus assumed that the bats used in these experiments were naïve to flashing, noxious fireflies.

We conducted bat-firefly behavioral experiments in a sound-attenuated flight room (6.8 × 5.6 × 3.9 m) illuminated by Wildlife Engineering infrared light-emitting diode arrays and red-filtered lighting. To record bat-prey interactions, we captured streaming video from three high-speed, infrared-sensitive cameras [Basler scout, 100 frames per second (fps)] using a National Instruments PCIe-8235 GigE Vision frame grabber and a customized LabVIEW program. Simultaneously, we recorded echolocation behavior using four ultrasonic Avisoft microphones [three CM16, ±3 dB(Z), 20 to 140 kHz; one USG Electret Ultrasound Microphone, ±9 dB(Z), 20 to 120 kHz] connected to a four-channel Avisoft UltraSoundGate 416H (sampling at 300 kHz) via XLR cables and recording to a desktop computer running Avisoft-RECORDER software. We mounted microphones on the ceiling in the center of the room, placing each CM16 microphone 1.5 m apart from and encircling the central USG Electret mic. For all interactions, we synchronized audio and video recordings by triggering both with a National Instruments 9402 digital I/O module.

We presented P. pyralis fireflies to bats in one of three treatments: (i) intact, free-flying, and flashing (n = 3 bats); (ii) intact, free-flying, and nonflashing (n = 4 bats); and (iii) intact, tethered, and flashing (n = 1 bat). To occlude bioluminescence for treatment #2, we painted all bioluminescent abdominal terga and adjacent terga, black or red (Testors Enamel Paints “GI Black” and “Red Cherry,” respectively). Both colored paints effectively blocked bioluminescent transmittance. We also presented painted scarabs (n = 2) to bats to control for the effect of paint. Once it was apparent that painted scarabs were caught and ingested with vigor, we then presented unpainted scarabs to minimize bat exposure to the enamel paints. Flight controls were wax moths, scarab beetles, or a combination of the two. Free-flying prey were hand-released, while tethered insects were suspended from an ~75-cm-long monofilament line attached to the ceiling. To avoid contaminating control prey, experimenters’ hands and the tether were washed following contact with each firefly. A trial began once we released the prey and we allowed bats to hunt for 1 min after release. We assessed capture behavior during experiments and confirmed captures by reviewing recordings of all interactions from all three cameras.

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