2.4 | Bioassay using the dummy seeds of filter paper
We performed a bioassay using dummy seeds made of square bits filter
paper of 2 mm x 2 mm. The dummy seeds were soaked with the volatile
compounds of the M. humile seeds (Table 1). These compounds were
purchased from Wako Pure Chemical Industries (Osaka, Japan) or Tokyo
Kasei (Tokyo, Japan) with >98.0% purities.
In this dummy-seed assay, 20 pieces of filter paper numerically numbered
with a carbon pencil to track the outcome of the dummy seeds were used.
The odor treatment in this assay consisted of seven conditions, that is,
each of the six odors identified (six conditions) and a mixture of
equivalent amounts of the six odors (one condition). Each odor component
was diluted to a final concentration of 100 ng / µL using the 99.5%
ethanol as a solvent. Twenty pieces of filter paper were soaked with 20
µl odor solution (1 µl for each filter paper). The dummy seeds soaked
only with the solvent were used as the control. Each of the odor
treatments was replicated six times using the six different colonies,
and each colony experienced each treatment only once. The colonies were
starved (fed with water only) for 72 h before performing the assays.
The order of the odor treatments that were served to each colony was
randomized using the ’sample’ function of the base package of R 3.5.2
for Mac OSX (R core team, 2018). Dummy seeds were placed at the center
of the arena and the number of dummy seeds carried to the nest by the
ants was recorded every 15 min for 90 min. After 72 h from setting the
dummy seeds, the number of dummy seeds inside the nest and discarded
outside the nest was recorded. For discarded dummy seeds, we also
recorded their destination and the numerical number on the dummy seeds.