First report on the successful in vitro feeding of adult Ixodes hexagonus.

Artificial feeding systems have become essential tools for studying tick physiology, pathogen acquisition, and transmission without relying on vertebrate hosts. While in vitro feeding has been successfully established for several ixodid species, including Ixodes ricinus, Dermacentor spp., and Rhipicephalus spp., no system has yet supported complete engorgement and reproduction of Ixodes hexagonus, a widespread European tick and vector of Borrelia spp. and the tick-borne encephalitis virus. In this study, we report the first successful in vitro feeding of adult I. hexagonus using a silicone-membrane system. Two trials were conducted with ticks of different ages and starvation period. Ticks were fed on silicone membrane at 27°C and appr. 75% relative humidity. Out of 38 and 20 females, 29 and 18 successfully fed to repletion (76 and 90%, respectively) with a mean weight of 111.1 mg for the first and 132.6 mg for the second trial. The mean feeding durations were 7.2 and 8.8 days and the oviposition rates were 90 and 100% in trial 1 and 2, respectively. The mean number of eggs produced was 402±171 for trial 1 and 847±378 for trial 2, whereby all females produced fertile eggs with a mean hatching rate of 92.1±14.5% and 99.4±0.61% for the first and the second trial, respectively. These results demonstrate that I. hexagonus might be reliably maintained and reproduced under artificial conditions, establishing this species as a promising experimental model for controlled studies on pathogen acquisition, transmission dynamics, and vector competence. The development of this feeding system expands the methodological toolkit available for European tick research and provides a foundation for future work on the epidemiology of hedgehog-associated tick-borne pathogens.