Integrated Metagenomic and Metatranscriptomic Analysis Reveals Microbiome Differences Between Bisexual and Parthenogenetic Populations of Haemaphysalis longicornis

The tick Haemaphysalis longicornis is an important vector of human and animal pathogens in East Asia that comprises both bisexual (Bis) and parthenogenetic (Par) populations. While previous studies have documented differences between these reproductive groups in genome ploidy, host transcription, and vector capacity, it remains unclear whether reproductive strategy is accompanied by systematic divergence in the tick-associated microbiome, including microbial community composition, transcriptional activities, and functional outputs. Here, we profiled laboratory-maintained Bis and Par populations using metagenomic (MG) and metatranscriptomic (MT) sequencing to integrate community composition, core taxa, functional potential, and RNA/DNA relative activity (RNA abundance normalized to corresponding DNA abundance). Par populations exhibited higher MG alpha diversity, whereas Bis populations showed higher MT alpha diversity, indicating a decoupling between the microbial DNA reservoir and the active RNA transcript community. MG functional analysis revealed that Par populations possessed a higher potential for environmental sensing, membrane transport, biofilm formation, porphyrin metabolism, and iron acquisition. Conversely, Bis populations exhibited enhanced potential in biotin metabolism, lipoic acid metabolism, and oxidative phosphorylation. MT analysis identified significant differences in a limited number of species and CAZy families, while selected KEGG pathways showed no significant variance, suggesting restricted rather than systemic transcriptional differentiation. By integrating DNA and RNA profiles, we classified 279 shared species into four activity states, revealing higher RNA/DNA relative activity ratios for thiamine metabolism in Par, and for biofilm formation, flagellar assembly, sulfur metabolism, and fatty acid degradation in Bis. Overall, Bis and Par populations exhibit distinct differences in their microbial reservoirs, transcript compositions, and relative transcriptional activities, providing critical microbiome insights into the symbiosis and vector adaptation associated with reproductive strategies in H. longicornis.