(B) Sample processing and DNA extraction
Before dissection, carpenter bee samples were rinsed in 70% ethanol for fifteen seconds to minimize environmental microbe contamination. They were then air dried and placed in a sterile petri plate for dissection. Dissecting tools were flame sterilized before dissection and before each organ removal. The crop and the rest of the gut (combined midgut and hindgut) were separated and stored at -20oC until DNA extraction.
Microbial DNA was extracted from crop and gut samples, extraction liquid, and kit reagents for an extraction control, using a modified procedure for the Qiagen DNeasy PowerSoil Kit (Rubin et al. 2014). Modifications include adding 4 magnetic beads per PowerBead Tube after tissue samples had been added and beating tubes in a BeadBlaster 24 Homogenizer for 3 cycles of speed 7 for 20 seconds per cycle. 60 μL of Solution C1 and 2 μL Proteinase K solution (600mAU/ML – from Qiagen Tissue and Blood) were then added to each tube and tubes were incubated overnight at 56oC. The following day tubes were beaten once more using the same cycle settings and the rest of the protocol followed the manufacturer’s protocol beginning at step 6. Briefly, full-length 16S region was amplified using 27F (Paliy et al. 2009) and 1492R (Lane 1991) and sequenced using PacBio Sequel v3 chemistry by the Dalhousie University Integrated Microbiome Resource facility (Halifax, Canada).