Abstract: This study aimed to investigate the microflora structural characteristics of the midgut of different engorged Haemaphysalis longicornis(H. longicornis) females collected from Panthera tigris tigris. Female H. longicornis in fully engorged (FF) and half engorged (HF) were sampled, and midgut contents were collected from each group of ticks under aseptic conditions. Total bacterial DNA was extracted from each group of samples. The V3-V4 areas of 16S rRNA was amplified by PCR, and the libraries were constructed for each group. Paired-end sequencing was performed using the Illumina NovaSeq platform. Sequencing data were processed by merging, filtering, and denoising, followed by Amplicon Sequence Variant (ASV) clustering and diversity analysis. The results showed that 100 021 and 82 657 effective tags were obtained from FF and HF, respectively. The number of ASVs obtained from FF and HF after clustering was 259 and 541, respectively, of which 116 were common between the two groups. At the phylum level, the dominant common phyla were Proteobacteria, Firmicutes, Bacteroidota, and Actinobacteriota in both groups, with Proteobacteria being more abundant in FF, while the other three dominant phyla were relatively more abundant in HF. At the genus level, the dominant common genera were Coxiella, Muribaculaceae, Corynebacterium, Delftia, and Staphylococcus in both groups. Coxiella had the highest relative abundance, and the relative abundance of Coxiella was higher in FF than in HF. In FF, genera such as Schlegelella were unique and relatively abundant, while in HF, genera such as Prevotella were unique and relatively abundant. At the species level, Coxiella endosymbionts were the most abundant taxa in both groups, with distribution patterns consistent with Coxiella. The results suggested that the midgut of female H. longicornis has a complex microflora. There were certain differences in the midgut microflora of different engorged ticks, and the relative abundance of the same genus varied in different samples.

Key words: Haemaphysalis longicornis, different engorged statuses, midgut microbial community structure, ticks and tick-borne diseases, high-throughput sequencing