Sample collection
Embryos, along with worker- and queen-destined larvae, were harvested from several colonies of A. mellifera ligustica of the same genetic stock maintained in the Bee Research Lab apiary. Two groups of 50 late-stage (2-day-old) embryos were harvested from standard (worker) cells and immediately frozen at -80°C. Four groups of 20 bipotential larvae aged 24 h post-hatching also were pooled and frozen. Fourteen worker-destined larvae aged 48, 72, 96, and 120 h post-hatching were also collected, along with 13 queen-destined larvae from the same age groups, raised in natural queen-rearing (swarm) cells. The ages of these queen larvae were determined by wet weight and body size comparisons with larvae of known age raised in artificial queen cells. For both worker- and queen-destined larvae, ages were likely to be accurate within 8-12 h, on the basis of wet-weight comparisons with larvae of known age [3]. Total RNA was extracted from these samples using the RNAqueous protocol (Ambion).