Ant Nest Behavior Experiment Protocol

Behavior experiments were conducted in arenas constructed from cast acrylic with a plaster of Paris floor. Each arena was made of four layers; the base layer, a layer of plaster of Paris, a layer with two cut out areas separated by a tunnel, and a top layer of clear acrylic with lids. The arenas were 7 cm × 2 cm in total, with a 2 cm × 0.3 cm tunnel separating two 2.5 cm × 2 cm areas (Fig. S1). Each area contained a 0.5 cm × 2 cm “stimulus chamber” separated from a 2 cm × 2 cm “nest chamber” by a cast acrylic mesh wall. The wall was laser cut from 0.8 mm thick cast acrylic with multiple holes with a diameter of ~ 50 µm, as described previously (Chandra et al. 2021 (link)). The clear acrylic lids of the nest and stimulus chambers were separate, allowing the experimenter to open the stimulus chamber without opening the nest chamber, thereby decreasing the likelihood of alarming the ants due to increased airflow. The floor of the tunnel was covered with vapor-permeable Tyvek paper to dissuade ants from forming their nest in the tunnel, as described previously (Chandra et al. 2021 (link)).
In each arena, 30 ants were introduced without any brood. For the live ant and crushed body experiments (see below), 30 additional ants from the same stock colony were kept in a separate Petri dish with a plaster of Paris floor. These ants were used as the stimulus during experiments. Ants were fed every 1–2 days with S. invicta brood and allowed to lay eggs. About 7–10 days after introducing ants into the arenas, once ants had settled, laid eggs, and clustered into a tightly packed pile (the “nest”) in one of the two nest chambers (Fig. S1), we began behavioral experiments.