3 mm tungsten bead

Prior to testing, ticks were washed with 70% ethanol and then rinsed with Schneider's Drosophila Medium (Lonza, Walkersville, MD, USA) containing 200 IU/ml penicillin, 200 μg/ml streptomycin and 0.5 μg/ml amphotericin B. Ticks were then cut into small pieces and snap-frozen in liquid nitrogen for 5 min before the samples were mixed with 500 μl of Dulbecco's Modified Eagle's medium (DMEM) The samples were then lysed using a Qiagen Tissue Lyser with 3 mm Tungsten beads at 25 Hz for 2 × 30 s. A further 500 μl of DMEM was added to the samples, followed by centrifugation at 600 × g for 5 min at room temperature and then supernatant was collected. One half of the supernatant was used for PCR; DNA was extracted using a previously-described DNA extraction method (Tuppurainen et al., 2011 ) followed by conventional PCR (Tuppurainen et al., 2011 ) or the real-time PCR (Bowden et al., 2008; Stubbs et al., 2012 ) as described below for testing the cell culture samples. In the real-time PCR, cycle threshold (C_t) values over 39 were considered negative. The other half of the supernatant of each sample was used for virus isolation (Lubinga et al., 2014c (link)). Tick samples collected from Egypt in 2006 did not contain sufficient material for virus isolation.

We quantified the number of Nosema spores in the midgut of bees using three randomly selected workers per treatment and colony that had survived until the end of the experiment. To do this we, dissected their midguts into a single Eppendorf tube containing 1 mL of deionized water and a single 3 mm tungsten bead (Qiagen, USA). We homogenized the sample using a mixer mill (Retsch MM301) at 25 Hz for 30 s, layered 500 µl of the homogenate onto 1.5 ml of 100% Percoll (Sigma-Aldrich) in a 2 mL Eppendorf tube and centrifuged the sample for 90 min at 20,000 × g and 4 °C. We discarded the supernatant and resuspended the pellet in 1 mL double distilled water for 10 minutes at 28,000 × g and 4 °C. This process was repeated 4 times before we resuspended the final pellet in 50 µl DDI water. We used a 10 μl subsample to quantify spores’ numbers using a Neubauer chamber and a Leica light microscope at 400× magnification. Total spore number per sample was calculated by multiplying spores counts by 50.