(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).