Experimental colonies were composed of age matched, one-cycle old workers (44 and 34 days old for genotypes A and B, respectively; A colonies have slower cycles than B colonies on average) and 4-days old larvae in airtight Petri dishes (5cm in diameter, corresponding to ca. 25 ant body-lengths) with a plaster of Paris floor. All workers and larvae within an experiment—including replicate colonies of all group sizes—were clonally related and sourced from the same stock colony. All workers within an experiment were also harvested from the same cohort and had eclosed within a day of each other (due to the synchronized reproduction of O. biroi). From the time they were harvested (1–3 days post-eclosion) until the start of the experiment, workers were kept together in a box and allowed to go through a full colony cycle. Thus, all workers within an experiment experienced the same environment as larvae and adults. However, we cannot exclude that small differences in individual experience occurred even in this common environment before the start of the experiment. All workers were tagged with color marks on the thorax and gaster using oil-paint markers (uni® Paint Markers PX-20 and PX-21). Experimental colonies contained 1, 2, 4, 6, 8, 12 or 16 workers and a matching number of larvae. This 1:1 larvae-to-workers ratio corresponds to the estimated ratio found in a typical (i.e., large, healthy) laboratory stock colony in the brood care phase. The experiment was conducted in two distinct genotypes, A and B15 (link). 7–9 replicate colonies were used for each group size and genotype, for a total of 112 colonies. O. biroi is myrmecophagous and colonies were fed live pupae of fire ant (Solenopsis invicta) minor workers. These prey items are small enough to be transported by a single O. biroi worker, so small colonies were not disproportionally penalized by the feeding regime.
The experiments took place in a climate room at 25˚C and 75% relative humidity under constant light (O. biroi is blind and its behavior is not affected by light). Every 3 days we cleaned and watered the plaster, added one prey item per live larva at a random location within the Petri dish, and recorded adult survival as well as brood survival and development under a stereomicroscope in all colonies (except for eggs, which cannot be counted without substantially disturbing the colony). The experiments ended when all larvae within an experiment had either developed into adult workers or died. Two colonies (size 6 and 16, genotype B) were excluded from all analyses due to setup errors (incorrect number of workers or larvae at the beginning of the experiment). Note that although we controlled the number of workers and larvae at the beginning of the experiment, these numbers then changed throughout the experiment as workers died and reproduced, and as the brood died or developed into adults.
The experiments took place in a climate room at 25˚C and 75% relative humidity under constant light (O. biroi is blind and its behavior is not affected by light). Every 3 days we cleaned and watered the plaster, added one prey item per live larva at a random location within the Petri dish, and recorded adult survival as well as brood survival and development under a stereomicroscope in all colonies (except for eggs, which cannot be counted without substantially disturbing the colony). The experiments ended when all larvae within an experiment had either developed into adult workers or died. Two colonies (size 6 and 16, genotype B) were excluded from all analyses due to setup errors (incorrect number of workers or larvae at the beginning of the experiment). Note that although we controlled the number of workers and larvae at the beginning of the experiment, these numbers then changed throughout the experiment as workers died and reproduced, and as the brood died or developed into adults.
