nurse crops and hypopharyngeal glands
We determined the composition and diversity of bacteria in the RJ, the HGs, and nurse crops. Royal jelly and nurse bees were collected from six replicate hives housed at the CHBRC in Tucson, Arizona, USA in August of 2013. All six hives were headed by A. mellifera ligustica queens and the hives were of equal size and strength compared to each other and to the hives used in the earlier experiments. For each hive, a total of 250 µl RJ was collected from multiple cells containing 1st and 2nd instar larvae and was diluted with 750 µl sterile distilled water. 100 µl of this diluted sample was sampled and spun down, the supernatant was removed, and 180 µl of lysis buffer (20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton X-100, pH 8.0; 20 mg/ml lysozyme added immediately before use) was then added. 15 nurses were collected from the same hives and were discriminated from other hive bees based on their behavior (i.e., visiting a larval cell for >5 seconds). Nurse bees were flash frozen in liquid nitrogen upon collection and kept at -20°C until they were dissected. The crops and hypopharyngeal glands from 15 nurses per hive were dissected directly into 180 µl of lysis buffer (20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton X-100, pH 8.0; 20 mg/ml lysozyme added immediately before use), pooled by hive, and were homogenized with a sterile pestle. DNA extraction followed using the GeneJET Genomic DNA Purification Kit (Fermentas) according to the manufacturer’s protocol for gram-positive bacteria. The extracted DNA was subjected to a 16S rRNA PCR amplification using universal primers (27F: 5’-AGAGTTTGATCCTGGCTCAG-3’; 338R: 5’-TGCTGCCTCCCGTAGGAGT-3’) to confirm the presence of bacterial DNA. Cycling conditions were as follows: 94°C for 2 minutes, 30 cycles of 94°C for 15 seconds, 50°C for 20 seconds, 72°C for 30 seconds, and a final extension at 72°C for 2 minutes.  For pyrosequencing, the V1-V2 region of the 16S rRNA gene of the samples were PCR amplified using universal 16S rRNA primers fitted with 454 FLX Titanium adapter sequences (27F 5’-CCATCTCATCCCTGCGTGTCTCCGACTCAG-NNNNNNNNNN-agagtttgatcctggctcag -3’; 338R: 5’- CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-tgctgcctcccgtaggagt -3’; uppercase letters denote the adapter sequences, N’s indicate library-specific barcodes (Table A1), lowercase letters indicate universal 16S rRNA primers). Amplicons were sequenced using Roche 454 GS FLX Titanium sequencing at the University of Arizona Genomics Core Facility (http://uagc.arl.arizona.edu/).