2.1 | Plant and ant materials
We used the ant species N. flavipes , which is polyphagous and
collects plant seeds in the field (Tanaka & Tokuda 2016; Hosoishi et
al., 2019). Ten N. flavipes colonies were collected from June to
July 2019 from the sites at the Yuminariyama mountain and the Hokkaido
University of Education Asahikawa Campus in Hokkaido, Japan. All the
colonies collected were queenless, consisting of approximately 60
workers and numerous eggs, pupae, and larvae. The colonies of M.
humile and N. flavipes coexist sympatrically at Yuminariyama
mountain and we also collected M. humile fruits at Yuminariyama
mountain in August 2019 and stored them in a screw tube at 4 ℃.
The ant colonies were kept in a container that consisted of an
artificial nest and a foraging arena (Figure 1). The polypropylene box
(200 mm length × 136 mm width × 68 mm height) was used as a foraging
arena. Fluon was applied to the upper 3 cm inside the feeding arena to
prevent ant escape. A vinyl tube (10 mm diam. × 120 mm length) was used
as an artificial nest and connected to the side wall of the container at
15 mm height. To prevent the artificial nest from drying out, an
absorbent piece of cotton was inserted at the wall-side of the nest and
moistened with distilled water. The absorbent cotton was replaced every
two weeks.
The feeding arena was divided into four equivalent sections to
investigate whether the ants discard seeds in areas with different
conditions. The four sections were respectively filled with dry glass
beads (1.5-2.5 mm diam.), moistened glass beads, dry cotton wool, and
moistened cotton wool.
Glass beads simulated an environment with inorganic substances like sand
or small pebbles in the forest floor, which were filled with about 10 g
of beads in each section. Cotton wool simulated an environment with
organic substances such as fallen and decomposing litter, which were
filled with about 0.25 g of cotton in each section. The entrance of the
nest was placed at the center of the arena so that the four sections
were equally aligned for the ants. The entrance of nest floats was
bridged with filter paper at the center of the feeding arena (Figure
1a). In the bioassay, the seeds or the dummy seeds were placed at the
center of the arena near where the bridge connected the different
sections (Figure 1b).
To moisten two of the sections, 5 mL of distilled water was added every
three days. All the containers were kept indoors at 25 ℃. The ants were
fed with tuna and honey.