Feeding-state is associated with shifts in the Haemaphysalis bispinosa microbiome and its potential as a sentinel for circulating livestock pathogens.

Ticks harbour microbial communities that shape their capacity to acquire and transmit host-associated bacteria. Haemaphysalis (H.) bispinosa is a tick that widely infests variety of hosts across Asia, yet how its bacterial communities are reconfigured across the transition from free-living to blood-feeding states in the field remains poorly understood. Herein, we characterise the microbiome of H. bispinosa using full-length 16 S rRNA gene sequencing. A total of 225 ticks were collected from cattle and the surrounding environment in Central Java, Indonesia. The analysis revealed a diverse array of bacterial taxa spanning 21 phyla and 443 genera. A Coxiella lineage was dominant (46.94%), was present in all samples, and constituted the sole universal core taxon. Environmental ticks exhibited near-monocultural profiles (95-99% Coxiella), whereas host-associated ticks showed increased diversity and the incorporation of additional taxa, including Staphylococcus, Mammaliicoccus, Corynebacterium, and Romboutsia. Within-sample diversity was governed by evenness rather than richness: richness (Chao1) did not differ among states (Kruskal-Wallis, p = 0.543). In contrast, the Shannon index increased significantly from environmental ticks (H = 0.23 ± 0.10) to host-associated ticks (HB, 1.62 ± 0.93; HC, 0.95 ± 0.51; p = 0.0155), a pattern mirrored by Pielou's evenness (p = 0.0155; η² = 0.60), with environmental ticks differing significantly from both host-associated states. Beta-diversity analysis confirmed significant community separation across feeding states (PERMANOVA: Bray-Curtis p = 0.0071, R² = 0.38; Aitchison p = 5 × 10⁻⁴, R² = 0.25; Jaccard p = 0.039, R² = 0.16). The environmental-to-host-attached transition represented the largest compositional shift, and engorged ticks exhibited partial convergence toward a structured microbiome characterised by co-dominance of Coxiella and Mammaliicoccus. These findings indicate that feeding state is a major factor associated with microbiome variation in H. bispinosa. The detection of Anaplasma-associated ASVs suggests that H. bispinosa has potential utility for monitoring circulating livestock-associated bacteria. Future work integrating functional approaches will be essential to resolve the pathogenic status of the dominant Coxiella lineage and clarify microbiome-mediated effects on pathogen transmission.