2.3. Video-Tracking Recordings

Changes in honey bee feeding behavior were observed using a video-tracking protocol as previously described [26]. Behavior was recorded using EthoVision XT video recording software and a Basler acA-1300-60gm camera (Noldus, Leesburg, VA, USA). The recording arena was draped in a black plastic tarp to block ambient light, and illuminated solely with light in the red spectrum, to avoid inducing light bias, using Cryon LED lights (Model HTP904E, Chatsworth, CA, USA). HCA compounds were evaluated, relative to untreated controls and DEET, for their ability to deter feeding when suspended in 1.5 × 1.5 × 0.3 cm sugar-agarose cube. Sugar-agarose cubes were prepared by combining 8 g of sucrose, 20 mL deionized water, and 170 mg agarose, then adding DEET and HCAs to yield a final concentration of 10 µL/mL within the solution (controls were left untreated), before finally transferring aliquots of the solution to individual molds and allowing each to set.

For behavioral video-tracking experiments, standard, transparent plastic Petri dishes were arranged beneath the camera in a 4 × 4 grid pattern. A sugar-agarose cube was placed in the center of each dish, along with a single bee, and then covered with a lid that had holes drilled it for ventilation. Eight treated and untreated sugar-agarose cubes were tested each time, and bee movement was recorded and tracked for 10 min. The amount of time spent by each bee in the area of the sugar-agarose cube, designated the feeding zone, was calculated using EthoVision video-tracking software (Noldus Information Technology, Wageningen, the Netherlands). The differences between each treatment group were statistically analyzed using one-way ANOVA with Tukey’s post-hoc test (GraphPad Prism 8.3.0, La Jolla, CA, USA).