guts dissected from 3d old workers living with nurses
DNA was extracted from the entire bee digestive tract excluding the foregut (crop). We used only guts that remained intact following dissection. For each DNA library (and extraction), we pooled the guts of 5 bees per replicate. Each pooled gut sample was placed into a sterile 2 mL centrifuge tube containing 350µl of 0.5mm silica beads and 1.5ml TE/Triton X lysis buffer (20 mM Tris-HCl, 2 mM EDTA, 1.2% Triton X-100, pH 8.0) and was subjected to bead beating for 30 sec. The supernatant was transferred to a new 1.5ml centrifuge tube and centrifuged for 5 min. The supernatant was removed and 180µl TE/Triton X 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 added to the remaining pellet. Samples in lysis buffer were then subjected to genomic DNA extraction using the GeneJet Genomic DNA Purification Kit (Fermentas) following the protocol for gram-positive bacteria.     The V6-V8 region of the 16S rRNA was PCR amplified using universal 16S rRNA primers fitted with 454 FLX Titanium adapter sequences (926F 5’_-CCATCTCATCCCTGCGTGTCTCCGACTCAG -NNNNNNNNNN-aaactyaaakgaattgacgg-3’; 1392R: 5’- CCTATCCCCTGTGTGCCTTGGCAGTCTCAG-acgggcggtgtgtrc-3’; uppercase letters denote the adapter sequences, N’s indicate library-specific barcodes, lowercase letters indicate universal 16S rRNA primers). Amplicons were sequenced using Roche 454 GS FLX+ sequencing.