Ultrospec 2100 pro

The growth rate of yeast cells were determined by measuring OD₆₆₀ after appropriate dilutions using a spectrophotometer (Ultrospec 2100 Pro, GE healthcare, Fairfield, USA) every 8 h. The measurements were continued until the OD660 nm reached the plateau. This experiment was repeated 3 times. The growth curve of different strains was drawn by software of Excel Microsoft Office Professional Plus 2013 (Microsoft, Redmond, USA).

The H₂O₂ concentration in ticks was measured using the ferrous oxidation of xylenol orange assay [18 (link)]. Briefly, homogenized unfed and partially fed ticks were suspended in 200 μl of Milli-Q H₂O, while homogenized engorged ticks were suspended in 900 μl of Milli-Q H₂O. The samples were centrifuged at 500× g, and the supernatant was collected. The supernatant from the engorged ticks was further diluted 10 times in Milli-Q H₂O. Ninety microliters of the supernatant from unfed and partially fed ticks or the diluted supernatant from engorged ticks was used for a sample solution as described later. The assay reagent consisted of 125 μM xylenol orange, 250 μM ammonium iron (II) sulfate, 100 mM sorbitol, and 25 mM sulfuric acid. One hundred microliters of the sample solutions was added to a 1-ml assay reagent. The mixture was vortexed immediately, left at room temperature for 30 min, and measured at 560 nm using a spectrophotometer (Ultrospec 2100 pro; GE Healthcare, Pittsburgh, PA, USA). Finally, the ratio of the H₂O₂ concentration (μM) to the corresponding tick’s body weight (mg) was calculated.

The amount of β-glucan per gram cells were measured using aniline blue as described previously with some modifications [40 (link), 41 (link)]. Cells were grown to early log phase (2.5 × 10⁶ cells) and harvested (10,000×g, 1 min). The cells were washed twice with 1 ml TE buffer (10 mM Tris–HCl, 1 mM EDTA, pH = 8.0), resuspended to 250 μl in TE and then 6 M NaOH was added to a final concentration of 1 M. Following incubation in a water bath at 80 °C for 30 min, 1.05 ml of AB mix [0.03 % aniline blue (Sigma-Aldrich, USA), 0.18 M HCl, and 0.49 M glycine/NaOH, pH 9.5] was added. The tube was vortexed briefly and then incubated at 50 °C for 30 min. Fluorescence of β-glucan was quantified using a spectrofluorophotometer (Ultrospec 2100 Pro, GE healthcare, Fairfield, USA). Excitation wavelength was 400 nm and emission wavelength was 460 nm. This experiment was repeated three times.