Amplicon of bumblebee gut: Repetition 3, 6dpi (T2), BotaniGard, Worker Replicate 2
At standardization (i.e., baseline) and at 1, 6, 13 days after starting the treatment (i.e., days post inoculation, dpi), 5 worker individuals from each hive were collected individually in a honey cup after the 12:00 bumblebee activity monitoring; the queen was retrieved at 14 days. A total of 15 workers per time-point and per treatment, and of 3 queens per treatment were collected. Each bumblebee was chilled for 10 min at -20C, then individually exposed to CO2 for 20 sec at 4 bar. The gut was extracted from each individual by pressing the insect with a finger on the thorax to make the stinger emerge, then pulling the stinger with sterile tweezers to extract the whole gut system and cutting the cuticle edge and stinger out with a sterile scalpel. The gut was then transferred to a 2 mL Eppendorf tube and stored at -70C until further processing. DNA was extracted from each gut using the DNeasy Blood & Tissue kit (Qiagen, Germany) according to the manufacturer instructions and with several modifications. Briefly, 3 glass beads (diameter of 3 mm) were added to each worker gut sample and grinded for 30 sec in a tissue lyser (30 beats/s). The incubation at 56C was performed in a water bath for 3 h, during which tubes were vortexed (5 sec, maximum speed) every 15 min. The addition of Buffer AL and of 100% ethanol was done in two steps, while the elution step was performed twice, with 75 L of Buffer AE each time to obtain an eluted volume of 150 L. Additionally, each queen gut sample required two extraction preps: the double amount of glass beads and volume of buffers were used in order to split the final mixture (approx. 600 L each) in two DNeasy Mini spin columns, which had to be centrifuged several times (6000-8000  g, 1 min) to obtain all the suspension; each column was eluted once and then pooled to obtain 150 L. Lastly, DNA was also extracted from negative and positive controls, respectively, corresponding to an empty tube and to 70 L of a microbial community standard (ZymoBIOMICS). The quality and quantity of DNA were assessed using a Nanodrop (DeNovix, DS-11, USA) and a Qubit (Thermo Fisher Scientific, USA), respectively. The fungal communities were analyzed by amplifying a 380 bp fragment of the ITS2 region using the primers ITS3F (5-GCATCGATGAAGAACGCAGC-3) and ITS4R (5-TCCTCCGCTTATTGATATGC-3). PCR amplification, purification, library preparation, Illumina MiSeq sequencing (2300 bases paired-end version), demultiplexing and barcode suppression were performed by StarSeq (Mainz, Germany).
SUB16360760
its2-B111_S257