In vitro feeding of nymphal Haemaphysalis longicornis supports moulting to adults and maintains Coxiella-dominated microbiome across life stages.

Artificial tick feeding systems (ATFS) enable controlled investigation of tick biology under in vitro membrane-feeding conditions. However, their application to immature stages and microbiome structure during prolonged host-free maintenance remains poorly understood. Here, we extend a recently established ATFS to the nymphal stage of Haemaphysalis longicornis, an invasive tick of global significance and the primary vector of Theileria orientalis in Australia. We provide the first integrated assessment of feeding performance, post-feeding development and trans-stadial microbiome composition under in vitro membrane-feeding conditions. Nymphs were fed in vitro across five independent feeding units, achieving high attachment (80-100%) and engorgement success (83-100%), with moulting success of 90.5%. Developmental timing was consistent, with attachment occurring within 1-5 days, detachment within 4-10 days, and moulting 14-25 days post-detachment. Variation in engorgement weight reflected individual heterogeneity while demonstrating reproducible feeding performance. Microbiome profiling revealed a low-diversity bacterial community strongly dominated by a Coxiella-like endosymbiont (CLE) across all life stages. Although alpha diversity differed significantly between unfed nymphs and adults, overall community composition remained broadly stable from unfed nymphs through in vitro-fed adults to their larval progeny. This persisted during extended maintenance without exposure to a live vertebrate host, indicating maintenance of host-symbiont interactions. Collectively, these findings demonstrate that ATFS supports nymphal feeding, development to adults and trans-stadial dominance of CLEs in H. longicornis across stages. This work represents a key step toward establishment of a complete in vitro life-cycle system for future studies of tick physiology, symbionts, microbiome-pathogen interactions and control strategies, while advancing the principles of the 3Rs in tick research.