Microbial load estimation
For each stock colony, microbial loads were estimated from the number of colony forming units (CFUs) cultured from individual cuticular washes of 12 alates (6 females and 6 males) and 6 workers per colony. Each alate was washed in a sterile 1.5ml tube with 300µl of a 0.1% Tween 80 solution, gently vortexed and centrifuged at 300×g at 4°C for 20 minutes (Rosengaus et al. 2003). For each sample, three 20µl replicates of the supernatant were plated on potato dextrose agar, while 20µl of the Tween 80 solution was used as a control. Plates were inverted and incubated at 37° C for three days. The number of CFUs at least 1mm in diameter was counted for each plate and averaged between triplicates. Microbial loads were quantified the same day as the alates were paired. Microbial loads were compared between colonies using a Mann-Whitney U-test. For each pairing combination, cumulative microbial load describes the sum of the microbial load across the two colonies of origin, while maximum microbial load only considers the colony of origin with the highest value.