Environmental Low Temperatures Dynamically Reshape the Microbial Diversity and Community Structure of the Vector Tick Haemaphysalis longicornis.

Low temperature is a key abiotic factor shaping tick-associated microbial communities, which in turn influence host physiology, vector competence, and environmental adaptation. However, the impact of prolonged cold exposure, such as overwintering conditions, on the microbiome of the invasive tick Haemaphysalis longicornis remains insufficiently characterized in terms of its microbial response. In this study, unfed adult ticks were subjected to a gradient of low temperatures (8°C, 4°C, 0°C, -4°C) for 7 days, while a control group was maintained at 27°C. The bacterial communities of whole ticks were characterized using Illumina NovaSeq-based 16S rRNA gene sequencing, followed by comprehensive bioinformatics analyses to evaluate alpha diversity, beta diversity, taxonomic composition, and differentially abundant taxa. The results showed that cold exposure markedly reshaped the microbial community structure, with an overall increase in alpha diversity (Shannon index) observed in several treatment groups. Across all samples, the dominant bacterial phyla included Proteobacteria, Firmicutes, and Bacteroidota. Notable shifts were detected at the genus level, particularly in Coxiella endosymbiont and Pseudomonas, whose relative abundances changed substantially under low-temperature conditions. In addition, microbial responses exhibited clear sex-specific patterns: Escherichia-Shigella and Serratia were enriched in certain cold-treated groups, whereas Staphylococcus showed a reduction in males exposed to low temperatures. The endosymbiont Coxiella was significantly enriched in male ticks at 8°C (p = 0.009). Beta diversity analysis further demonstrated distinct clustering of the -4°C male group relative to all other groups. Collectively, these findings indicate that sustained low temperatures drive pronounced and sex-dependent restructuring of the bacterial microbiome in H. longicornis. The enrichment of specific taxa, including putative nutritional symbionts such as Coxiella, under cold stress conditions suggests a potential role for microbial communities in facilitating host adaptation to low-temperature environments, thereby providing new insights into the ecological dynamics and adaptive capacity of this invasive vector species.