Termite collection and alate pairing
Six stock colonies (colonies A to F ) ofReticulitermes flavipes were collected in Bryan, TX, USA in March 2020, a week before the swarming flight would have naturally occurred. Colonies were extracted from their wooden logs and transferred into 20cm plastic boxes. One worker per colony was sequenced at the mitochondrial 16S gene to confirm identity of the species, following methods from Aguero et al. (Aguero et al. 2020). For each colony, male and female alates were sexed and isolated with a group of workers. They were then paired in 3-cm petri dishes with sawdust and wood pieces (Eyeret al. 2021b). The incipient colonies were kept in high humidity chambers. Only dark-pigmented alates were used to ensure they were physiologically and motivationally ready to mate.
To investigate the short-term effect of inbreeding on founding success, we set up 40 inbred pairings for each colony (only 31 for colonyD due to a lack of available alates). We also prepared 40 outbred pairings for every combination of colonies, with an equal number of each sex per colony of origin (20 queensA x kingB and 20queensB x kingA ); resulting in 231 inbred and 600 outbred incipient colonies. In addition, we estimated the long-term effect of outbreeding on incipient colony survival and productivity, as well as on pathogen resistance and microbial load of their offspring. To ensure robust sample sizes, we anticipated high mortality during colony foundation and established an additional 290 inbred and 300 outbred pairings (100 inbred pairings for three colonies with enough alates available: colonies A, B & F, only 90 inbred pairings forcolony F ; and 100 outbred pairings for all combinations of those colonies). Overall, we set up 1421 incipient colonies (521 inbred and 900 outbred), all of which were established on the same day.