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.