We deployed leg mounted geolocators on Laysan albatrosses breeding at two colonies: Oahu in the main Hawaiian Islands, and Kure Atoll 2,150 km away in the Northwestern Hawaiian Islands (Figure 8).
At least three colonies were picked per plate and used to initiate reamplifications with primers 21M13_F and 21M13_R (Table 4).
Experiments were carried out using freshly emerged honeybees randomly mixed from at least three colonies.
For each individual, at least three colonies were analyzed to correct for possible PCR errors.
At least three colonies grown on bile-esculin plates were sub-cultured into blood agar plates to isolate Enterococcus species (Oxoid, England).
We aimed to study at least three colonies of each species, but for two species, Trigona fuscipennis and Trigona spinipes, we were only able to locate two colonies of each (Table 1).
In addition, to confirm the 4-MUC screening results, at least three colonies with a higher 4-MUC activity of each gene were diluted into OD600 = 0.1 and grown in 50 ml SC-URA medium at 30°C for 24 hours.
At least three colonies were picked from each countable plate; Gram-positive colonies (as determined by KOH method; [ 19]) and negative to the catalase test (as determined by transferring fresh colonies from agar medium to a glass slide and adding 5%H2O22) were isolated.
For these morphometric analyses we selected about 100 workers (preserved in 70% ethanol) from at least three colonies (50 workers from a single colony if insufficient samples were available) from the same study site for each species to cover the entire worker size range as much as possible.
For the most relevant combinations (R- Pl and Pl- Pl, also R-R and R- Pl), four replicates (i.e., times the experiment was repeated) were conducted, and for the rest of groups, two replicates were accomplished using honeybees from at least three colonies.
