Sample collection and RNA-sequencing
Colonies for this study were generated and described as in Döke M. A. et al. (2019). Sample collection was conducted as described in Döke, M. A. (2017). Briefly, four colonies headed by naturally mated honey bee queens from four different commercial breeders were established at a Pennsylvania State University apiary in central Pennsylvania. Colonies were maintained according to standard apicultural practices. More than 6 weeks after establishment (July 2013), which is sufficient time for the eggs laid by the queens to develop into adult nurses and foragers, 6 summer nurse and forager bees per colony were collected on dry ice and stored at -80oC. Bees were collected based on behavioral observations, with nurses collected as they were observed feeding young brood, while foragers were collected at the colony entrance as they returned with pollen loads. Winter bees were collected 5 months later (December 2013) from within their winter clusters.
After removal from storage at -80oC, sampled bees were submerged in InvitrogenTM AmbionTMRNAlater TM Stabilization Solution (ThermoFisher Scientific) to prevent RNA degradation, and dissected on a platform surrounded by dry ice. To collect flight muscle tissue, legs and wings were detached from the thorax and discarded, leaving flight muscles and exoskeleton. To collect fat body tissues, abdomens were allowed to thaw RNAlater TM on ice. Digestive tracts and reproductive organs were removed by gently pulling from the stinger using forceps, leaving the eviscerated abdomen with fat bodies attached to the exoskeleton. Dissected tissues for each worker were individually placed in a 1.5ml nuclease-free microcentrifuge tube (Denville Scientific Inc., Metuchen, NJ) and kept at -80oC.
For each of the 4 colonies, samples of the same tissue and phenotype were pooled (6 bees per sample). Pooled samples (4 colonies x 3 groups x 2 tissues = 24 samples, total) were homogenized using an automated homogenizer (Thermo Savant FastPrepTM FP120 Cell Disrupter System) for 3 intervals of 45 seconds at the highest speed setting, using 3-6 2mm zirconia beads (BioSpec Products, Inc.). Homogenates were stored for later use in RNA extraction via the RNeasy Plus Universal kit (QIAGEN) for RNA sequencing (RNA-seq). RNA libraries were prepared by the Sequencing and Genomic Technologies Shared Resource at the Duke Center for Genomic and Computational Biology, Durham, NC, and sequenced on an Illumina HiSeq 4000 platform (see Table S1 for a list of samples).