title: Beebread and corbicular pollen from colony 20
design: Stored and corbicular pollen were collected approximately every other month between November 2011 and October 2012 from two neighboring colonies. At each of six sampling periods, 10-12 cells of stored pollen were collected from the central brood frame of each colony using a precut 1 ml pipette tip. The same day we sampled stored pollen, we also collected eight to twelve pollen foragers per colony. Foragers were collected by blocking the colony entrance and capturing returning pollen foragers using soft forceps. Both stored pollen and entire foragers were immediately submerged in 95% ethanol. Corbicular pollen was then separated from the legs of foragers.  Newly-collected and stored pollen were pooled separately by colony and sampling event and stored in 95% ethanol until DNA extraction.     We first consolidated the microbial components of each sample according to the following protocol. Each ethanol preserved sample was vortexed to suspend the pollen grains in solution and 750 µl of this mixture was placed in a new 1.5 ml tube. This subsample of pollen was centrifuged on high for 5 minutes, and the ethanol was decanted. The process of vortexing, subsampling, and centrifuging the pollen samples was repeated 5 times until there was approximately 500 µl of pollen in the new 1.5 ml tube. The ethanol was then decanted from the pollen and 1 ml of TE buffer (20 mM Tris-HCl, 2 mM EDTA, 1% Triton-X, pH 8.0) was added. This mixture was vortexed for 5 minutes and pulse centrifuged for 5 seconds. The supernatant was removed and placed into another tube and spun down for 20 minutes (22,000 rpm, 4° C) to pellet the bacterial cells. This wash cycle was repeated 4 times. On the final (fourth) wash cycle the supernatant was retained and 350 µl of 0.5 mm silica beads was added to the mixture and bead-beaten for 30 seconds. The supernatant from these samples was then removed to a new tube and spun down for 30 minutes (22,000 rpm, 4° C) yielding a pellet used in subsequent DNA extraction. We extracted DNA using the GeneJet Genomic DNA Purification Kit (Fermentas) according to (Corby-Harris, Maes, and Anderson 2014) following the protocol for gram-positive bacteria. The extracted DNA was PCR amplified using universal 16S rRNA primers (27F: 5’-AGAGTTTGATCCTGGCTCAG-3’; 338R: 5’-TGCTGCCTCCCGTAGGAGT-3’) to confirm the presence of bacterial DNA.    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, lowercase letters indicate universal 16S rRNA primers). Amplicons were sequenced using Roche 454 GS FLX Titanium sequencing.