Hirustasin gene superfamily of the Asian buffalo leech

Leeches play an important role as medicines for the treatment of cardiovascular diseases, and a large number of antithrombotic-related genes are present in their genome. Among them, five gene families, hirustasin, guamerin, piguamerin, bdellastasin, and poecistasin, have similar functions and high sequence similarity, and are collectively referred to as the hirustasin superfamily. The comparative study of these gene families at the intraspecific level is important for revealing the antithrombotic activity of leeches. In this study, we performed genome resequencing and transcriptome sequencing on a total of 70 samples of Asian buffalo leech (Hirudinaria manillensis) collected from seven populations in Guangdong and Guangxi. We identified a total of six genes that are members of the hirustasin superfamily, and systematically analyzed the sequence variation, selective pressure, and gene expression characteristics of these genes at the intraspecific level. The results showed that all genes were rich in genetic variation, with a total of 57 and 31 variable sites detected from coding region sequences and protein sequences, respectively. Moreover, pseudogene sequences were present in a small number of individuals for the hirustasin and poecistasin2 genes. All genes as a whole were driven by purification selection pressure, with highly significant differences in selection pressure indices among the genes. A few potential positive selection sites were present for all genes, with at least 2 significant positive selection sites present for hirustasin, piguamerin, poecistasin1, and poecistasin2. The overall expression differences among the genes were highly significant, with bdellastasin being the highest, poecistasin2 the next highest, and poecistasin1 the lowest expressed genes. Except for poecistasin2, the overall differences among the seven populations were significant for the remaining five genes. The above results indicate that the genetic attributes (sequence variation, selection pressure, and expression levels) of the intraspecific level of members of the hirustasin superfamily of the Asian buffalo leech vary considerably both among genes and among individuals, providing a rich genetic resource for the study of the identification and development of antithrombotic actives in the leech.