For dissection, individual bees (experimental microbiota− and microbiota+ bees and hive bees from the source colonies) stored in 95% ethanol at −20 °C were first dried on a clean laboratory tissue for 4–8 min, and then the gut region, including the midgut/ventriculus, the ileum, and the rectum was dissected out of each bee under sterile conditions near a flame. For DNA extraction using a cetyltrimethylammonium bromide (CTAB) bead-beating method, dissected guts were individually placed in 2 mL tubes together with 730 µL 2% CTAB-buffer (100 mL of 1 M Tris HCl adjusted to pH 8, 20 mL of 0.5 M EDTA, 81.8 g NaCl, 20 g CTAB, and ddH20 to 1 L), 250–350 µL 0.1 mm silica zirconia beads (BioSpec), and 20 µL Proteinase K (Sigma). Samples were then homogenized with a Tissue Lyser LT (Qiagen) at full speed for 2 min, placed on ice for 1 min, and bead-beaten again for 2 min before incubating the samples overnight at 56 °C. Thereafter, 750 µL phenol-chloroform-isoamyl alcohol (25:24:1) was added, tubes were mixed by inverting and placed on ice for at least 2 min before centrifugation at 7000 rpm for 15 min at 4 °C. Then, the DNA in the aqueous phase was alcohol precipitated (twice with 200 µL 2-propanol), washed (200 µL ice-cold 70% Ethanol), and air-dried prior to resuspension in 50 µL nuclease-free water. The concentration of extracted DNA was then determined with an Epoch™Microplate Spectrophotometer (BioTek), and the DNA was stored at −80 °C.
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