A Comparative Study on the Expression Levels of the HMEI Gene Family in Hirudinaria manillensis and Whitmania pigra

Leeches are classic animal-derived traditional Chinese medicines for activating blood circulation and eliminating blood stasis. The hematophagous leech Hirudinaria manillensis and non-hematophagous leech Whitmania pigra are the two most widely used medicinal leeches, yet the molecular mechanisms accounting for their divergent pharmacological effects remain unclear. Current research on leeches mainly focuses on anticoagulation and thrombolysis, lacking systematic cross-species and multi-population analyses of the HMEI family, the core gene set responsible for leech anti-inflammatory activity. In this work, we collected head salivary gland tissues from five geographical populations of each leech species. We performed RNA-Seq high-throughput sequencing to obtain gene expression values. Combining phylogenetic analysis, multiple nonparametric statistical tests and data visualization, we systematically characterized the evolutionary patterns of the HMEI family, interspecies transcriptional divergence and population-level expression plasticity. Our results showed that HMEI genes from the two leech species interlaced to form eight monophyletic clades, including clades conserved in both species and clades unique to each species. Although total transcript abundance of the HMEI family exhibited no significant interspecies difference, the two species adopted obviously different transcriptional patterns. H. manillensis relies on ultra-high expression of core gene paralogs, while W. pigra exerts biological functions through coordinated moderate expression of multiple gene copies. Gene expression varied significantly across geographical populations. The Hainan Ding’an population of H. manillensis and Jiangxi Ji’an population of W. pigra presented the highest overall HMEIs expression levels. Three core highly expressed anti-inflammatory genes were identified: HMEI_Hman06 and HMEI_Hman15 from H. manillensis, and HMEI_Wpig01 from W. pigra. This study confirmed that the HMEI family undergoes adaptive evolution via retention of ancestral conserved paralogs and lineage-specific gene duplication. Feeding habit divergence drives transcriptional reprogramming within the HMEI family, which serves as the core molecular factor leading to distinct anti-inflammatory efficacy between the two leech species. Geographical environments further modulate the plasticity of gene expression. This research improves the molecular theoretical framework of leech anti-inflammatory active substances, screens high-potential medicinal functional genes, and provides transcriptomic evidence for developing novel leech-derived anti-inflammatory protein drugs, evaluating medicinal leech germplasm, and standardizing the quality of leech herbal medicines.