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.