Bee Colony Rearing and Stage-Specific Pupa Dissection

Field collected B. melanopygus queens were allowed to initiate colonies under laboratory conditions (28 °C, 60% RH, complete darkness). For transcriptome sequencing, we used dissected tissue from newly emerged male bees (also known as 0 h callows) of both red and black forms, five replicates (bees) for each color form. These were drawn from three colonies for red form and two colonies for black form individuals. 0-h callows were sampled at the point in which a bee fully emerges from its pupal brood cell and were collected between 0 and 1 h old (Tian and Hines 2018). Phenotypes were already partially apparent in the collected segments in all bees. As red coloration is the dominant phenotype, thus being displayed by both homozygous and heterozygous individuals, we confirmed individuals sampled were from colonies that are homozygous through observing queen (red only) and offspring (red males and workers produced only) phenotypes and genotyping several workers of their host colony by PCR amplification and sequencing at the previously identified color locus (primers 1F/1R, see Methods in Tian et al. [2019]). Heterozygotes are apparent by double peak patterns in resulting sequencing chromatograms.

Epidermal tissues from the second and third metasomal segments were dissected. After removal of the thorax and head, the abdomen was bissected longitudinally and the gut contents, heart, and sternites with attached tissue were removed. The sample was then immersed in ice-cold PBS buffer where the second and third tergites were separated from the remainder. These tergites were cleaned to remove trachea and muscles that were attached until just the epidermis remained, still attached to the cuticle (setal cells are broken if the epidermis is separated from cuticle). These were placed in an Eppendorf tube and flash frozen on dry ice, followed by storage at −80 °C until extraction.