Motion detection software

All experiments were performed using an automated, computerized fear conditioning chamber as previously described (Uhernik et al. 2018 (link)). In brief, the apparatus consisted of two plexiglass chambers, each with a sound-attenuating isolation cubicle equipped with a ventilation fan, a top-mounted USB camera, and a house light mounted on the side wall. Foot shocks and auditory cues were delivered through a removable floor grid by the Actimetrics FreezeFrame software. Freezing responses were captured on digital video and analyzed using motion detection software (Actimetrics).

Larval growth (i.e., length and weight) and mortality were monitored at 2-day intervals after being transferred to individual container until pupation. To examine if CBD affects larval behaviors, diet consumption and mobility were also monitored. The diet consumption was measured by weighing the diet loss of the container between 1^st instar larvae and pupation. All mobility experiments were performed using an automated, computerized system (Coulbourn Instruments, Holliston, MA). The system consisted of two Plexiglas chambers (203 mm × 228 mm × 254 mm), each equipped with a top-mounted 60 frame per second USB camera, and a LED light mounted on the side wall. Each chamber was placed inside a sound-attenuating isolation cubicle, which was equipped with a ventilation fan that produced 60 dB white noise. The mobility response of 5^th-instar M. sexta larvae was captured at 60 frames per second for 10 mins., and the video was analyzed using motion detection software (Actimetrics) to generate a motion index. For multiple group statistical comparisons, the differences of larval growth (i.e., size and weight) and motion index were analyzed by one-way ANOVA with Tukey’s post-test. The log-rank (Mantel-Cox) test was used for survival curve comparisons. All the statistical analyses were performed using the GraphPad Prism software package (GraphPad Software, Inc., La Jolla, CA).