Adenosine triphosphate (atp)

Deuterium oxide (99.96%) was from Euriso-Top (Saint-Aubin Cedex, France). U, UMP, uracil, K₂HPO₄, KH₂PO₄, NaHCO₃, MnCl₂, HEPES, NaCl, IPTG, imidazole, tetra-n-butylammonium bromide, glycerol, and ATP were from Carl Roth (Karlsruhe, Germany). Glc1,6diP and SYPRO orange, was from Sigma Aldrich (St. Louis, USA). Rib5P was purchased from Biosynth (Staad, Switzerland). Expression vectors (pet15b or pet28a+) containing the genes for YeiN, DeoB, UP, and Yjjg were from Genescript (Leiden, The Netherlands).

All feeding experiments were performed as previously reported (Militzer et al., 2021 (link)). Aseptically withdrawn heparinized bovine blood was supplemented with 2 g/L glucose and 0.1 M adenosine triphosphate (ATP, Carl Roth). Due to previous experiences with the artificial feeding of I. ricinus adults with blood supplemented with gentamicin, such as long feeding durations and the observation that some attached ticks turned black and died, we decided to supplement the blood meals for adults of both groups (IVG⁺ and IVG⁻) with B vitamin components (Militzer et al., 2021 (link)). For the ticks fed on blood supplemented with antibiotics (IVG⁺), 5 μg gentamicin (Cellupur, Carl Roth, Karlsruhe, Germany) was added per mL of blood.
After feeding to repletion, larvae and nymphs of all groups (IVG⁺, IVG⁻, and C) were stored at room temperature (RT) and > 90% relative humidity (RH) under a natural light–dark regime. Adult ticks were stored at 20°C, >90% RH in darkness.

Blood was collected from a calf with heparinized tubes (50 I.E./mL), and supplemented with glucose (2 g/L) (WDT, Garbsen, Germany) and gentamicin (5 µg/mL, Merck KGaA, Darmstadt, Germany). To enhance attachment to the feeding membrane, ATP (51 mg/mL, Carl Roth GmbH, Karlsruhe, Germany) was added to the blood right before usage. Blood was used for up to seven days and stored at 6 °C.