ii. Survival and microbial load of the offspring produced
After 15 months, just 70 out of the 1421 incipient colonies survived, of which only 49 produced 10 or more workers. For each of the 49 colonies (24 inbred and 25 outbred colonies), a group of eight workers were isolated in 30mm petri dishes lined with filter paper (Whatman Grade 5, porosity 2.5μm). Groups were challenged with a pathogen solution containing three strains of Metarhizium fungus in equal proportions at the concentration of 1×107 conidia/ml in 0.1% Tween 80 (ITS sequences match accession numbers KU187187.1, MT374162.1 and LT220706.1, for M. anisoplae , M. brunneumand M. guizhouense , respectively). Offspring survival was monitored for 14 days following exposure by moistening the filter paper with 300μL of the fungal solution (Aguero et al. 2021b). Difference in survival between inbred and outbred offspring was determined using the Coxph model. In addition, 66 of the 70 incipient colonies had at least two workers (31 inbred and 35 outbred colonies), for which two workers (with three replicates each) were used to determine the microbial load of the offspring. Microbial loads were measured as described above, except that cuticular washes of workers were extracted in 100μL of a 0.1% Tween 80 solution.
RESULTS