Honey bee heat-shock proteomics

Honey bee colonies were maintained in an apiary at the University of British Columbia. During the summer of 2018, 40 queens were reared from a single colony and half were allowed to open mate, while the other half were kept as virgins in plastic queen cages. Two weeks after emergence, the virgin queens were given two, eight-minute carbon dioxide treatments on 2 sequential days, then re-introduced to their nucleus colonies. This process stimulates virgin queens to begin laying71, and we conducted these treatments in order to minimize the physiological differences between virgin and mated queens. Virgin and mated queens were retrieved from their nucleus colonies and half of each (10) were subjected to heat-shock (42 ͦC, 2 h), and then maintained at 30 ͦC for 2 d. The other half were held only at 30 ͦC for 2 d. Four to six weeks after mating, the queens were anesthetized with carbon dioxide, beheaded, then their spermathecae (including the tracheal net) were removed with fine forceps. Both ovaries were also removed and weighed. During the same summer, 200 drones from a different colony in the same apiary were collected and maintained in the laboratory overnight at ambient temperature with excess syrup (50% sucrose). The next day, semen was harvested with glass capillaries according to the methods described above. Because many drones were not sexually mature, 60 semen samples (out of the 200 drones) were collected. Capillaries were placed in petri dishes and half (30) were heat-shocked as described above, then kept at 25 ͦC for 2 d. The other half were only kept at 25 ͦC for 2 d. Ten samples from each experimental group were used for sperm viability assays as described above.