Development of a New Method to Track Multiple Honey Bees with Complex Behaviors on a Flat Laboratory Arena

<div><p>A computer program that tracks animal behavior, thereby revealing various features and mechanisms of social animals, is a powerful tool in ethological research. Because honeybee colonies are populated by thousands of bees, individuals co-exist in high physical densities and are difficult to track unless specifically tagged, which can affect behavior. In addition, honeybees react to light and recordings must be made under special red-light conditions, which the eyes of bees perceive as darkness. The resulting video images are scarcely distinguishable. We have developed a new algorithm, K-Track, for tracking numerous bees in a flat laboratory arena. Our program implements three main processes: (A) The object (bee's) region is detected by simple threshold processing on gray scale images, (B) Individuals are identified by size, shape and spatiotemporal positional changes, and (C) Centers of mass of identified individuals are connected through all movie frames to yield individual behavioral trajectories. The tracking performance of our software was evaluated on movies of mobile multi-artificial agents and of 16 bees walking around a circular arena. K-Track accurately traced the trajectories of both artificial agents and bees. In the latter case, K-track outperformed Ctrax, well-known software for tracking multiple animals. To investigate interaction events in detail, we manually identified five interaction categories; ‘crossing’, ‘touching’, ‘passing’, ‘overlapping’ and ‘waiting’, and examined the extent to which the models accurately identified these categories from bee's interactions. All 7 identified failures occurred near a wall at the outer edge of the arena. Finally, K-Track and Ctrax successfully tracked 77 and 60 of 84 recorded interactive events, respectively. K-Track identified multiple bees on a flat surface and tracked their speed changes and encounters with other bees, with good performance.</p>