The aim of the current study was to track the movement of phosphine-resistant and -susceptible adults of the red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), which is a major pest of stored products, after brief exposures to phosphine. Exposures were followed for extended intervals to assess the recovery patterns, and how those patterns are related to known resistance to phosphine. A video-tracking procedure coupled with Ethovision software was used to assess movement after exposure. Two strains of T. castaneum were used, one susceptible and one resistant to phosphine. The susceptible T. castaneum strain had been maintained in continuous culture without any known exposure to phosphine for >30 years at the USDA-ARS Center for Grain and Animal Health Research (CGAHR), in Manhattan, KS, USA. The phosphine-resistant strain of T. castaneum was collected from wheat in Palmital, Brazil during 1988 (BRZ-5). The rearing media consisted of 95% organic, unbleached, wheat flour plus 5% brewer's yeast. Tribolium castaneum were reared under laboratory conditions of 27.5°C, and 65% relative humidity (R.H.), 14:10 L:D. Adults, of mixed sex and <1 month old, were used in the exposure bioassays. The protocol that was used in our bioassays to generate phosphine was the Phosphine Tolerance Test (Detia Degesch GmbH, Laudenbach, Germany) with some modifications, as performed by Agrafioti et al. 2021. In particular, the phosphine was generated within a plastic canister (5 L capacity) by adding 50 mL of water to two kit magnesium phosphide pellets. The concentration of phosphine gas inside the plastic canister was determined by using several dosimeter Draeger glass tubes (Draeger 25A, 0–10 000 ppm, Draeger Safety AG & Co., USA). Ten adults of each strain were placed in a plastic syringe of 100 mL with separate syringes used for each species and strain. Then, a specific gas quantity was removed from the canister with the syringe and blended with fresh air to produce a 100-mL volume with a concentration of either 1000 or 3000 ppm and compared to phosphine-free controls (0 ppm). The insects inside the syringe were held at the concentrations above for a 5 min exposure, while additional syringes containing only fresh air and insects were used as negative controls. To understand the propensity for movement after a 5 min phosphine exposure, a video-tracking procedure was used. After exposure of phosphine-resistant or phosphine-susceptible T. castaneum for 5 min, adult movement was evaluated immediately after exposure or 24 h later under the same environmental chamber conditions as the colonies (see Source Insects), but held without supplemental food. Movement was recorded for 3 h immediately after phosphine exposure but binned into 30 min intervals (e.g., 0–30, 30–60, 60–120, 120–150, and 150–180 min) in order to evaluate how movement varied over the measured time period. Movement was also recorded 24 h after exposure for periods of 1 h (binned by 30 min intervals). Movement measures of adults was tracked in six replicate Petri dishes (90 × 15 mm D:H) with a piece of filter paper (85 mm D, Grade 1, GE Healthcare, Buckinghamshire, United Kingdom) lining the bottom using a network camera (GigE, Basler AG, Ahrenburg, Germany) affixed 80 cm above the dishes. The Petri dishes were backlit using a LED light box (42 × 30 cm W:L, LPB3, Litup, Shenzhen, China) to increase contrast and affixed in place with white foam board with holes specifically cut to size for the petri dishes. Video was streamed to a nearby computer and processed in Ethovision (v. 14.0.1322, Noldus Inc., Leesburg, VA). The software was used to calculate the total distance moved (cm) and the mean instantaneous velocity (cm/s) for each adult. Each adult was considered a replicate and was never used more than once. Only adults classified as alive (normal movement speed and activity), or affected (sluggish movements or on back with legs twitching) were used in this assay. In total, 21–41 replicates were performed per treatment combination immediately after exposure, while 15–30 replicates were performed 24 h after exposure to phosphine. A total of 1525 adults were tested. There are two time periods (immediately after exposure and 24 h later), and two response variables (total distance moved in cm and mean instantaneous velocity in cm/s). There were three fixed explanatory variables: concentration of phosphine (0, 1000, or 3000 ppm), susceptibility (phosphine-susceptible or phosphine-resistant strain), and time interval (maximally 0–30, 30–60, 60–120, 120–150, and 150–180 min). Ethovision Assay morrison_ethal_ethovision_assay_fumigation_agdatacommons.csv To understand the propensity for movement after a 5 min phosphine exposure, a video-tracking procedure was used. After exposure of phosphine-resistant or phosphine-susceptible T. castaneum for 5 min, adult movement was evaluated immediately after exposure or 24 h later under the same environmental chamber conditions as the colonies (see Source Insects), but held without supplemental food. Movement was recorded for 3 h immediately after phosphine exposure, but binned into 30 min intervals (e.g., 0–30, 30–60, 60–120, 120–150, and 150–180 min) in order to evaluate how movement varied over the measured time period. Movement was also recorded 24 h after exposure for periods of 1 h (binned by 30 min intervals). Movement measures of adults was tracked in six replicate Petri dishes (90 × 15 mm D:H) with a piece of filter paper (85 mm D, Grade 1, GE Healthcare, Buckinghamshire, United Kingdom) lining the bottom using a network camera (GigE, Basler AG, Ahrenburg, Germany) affixed 80 cm above the dishes. The Petri dishes were backlit using a LED light box (42 × 30 cm W:L, LPB3, Litup, Shenzhen, China) to increase contrast and affixed in place with white foam board with holes specifically cut to size for the Petri dishes. Video was streamed to a nearby computer and processed in Ethovision (v. 14.0.1322, Noldus Inc., Leesburg, VA). The software was used to calculate the total distance moved (cm) and the mean instantaneous velocity (cm/s) for each adult. Each adult was considered a replicate and was never used more than once. Only adults classified as alive (normal movement speed and activity), or affected (sluggish movements or on back with legs twitching) were used in this assay. In total, 21–41 replicates were performed per treatment combination immediately after exposure, while 15–30 replicates were performed 24 h after exposure to phosphine. A total of 1525 adults were tested. Resources in this dataset:Resource Title: Ethovision Assay. File Name: morrison_etal_ethovision_assay_fumigation_agdatacommons.csvResource Software Recommended: Excel,url: https://www.microsoft.com/en-us/microsoft-365/excel