Inducing chirality in slime mold - spontaneous alternation behavior may reveal spatial memory in Physarum polycephalum

Spontaneous alternation behavior (SAB) is a robust paradigm to investigate short-term spatial memory across diverse taxa. While extensively studied in animals, its presence in unicellular aneural organisms remains poorly understood. Here, we tested SAB in the slime mold Physarum polycephalum using 3D-printed T-mazes with forced turns at varying distances (3 mm, 7 mm, and 14 mm), as well as in a double-turn design. A total of 1274 plasmodia from a clonal line were examined under controlled laboratory conditions. Our results reveal significant alternation behavior only in the short-distance maze (3 mm), independent of turn direction. Neither medium nor long distances, nor the double-turn de-sign, yielded significant effects after correction for multiple testing. These findings suggest that SAB in Physarum depends on spatial scale, with decision-making localized to the active moving front of the plasmodium. It is yet unclear if the observed behavior is induced by the topography of the used mazes or, as in other organisms, a result of memory. Further study on possible mechanisms guiding this behavior are required.