Comparative Genomics Unveils Unique Antithrombotic Gene Expansion in Haemadipsa yanyuanensis

Comparative Genomics Unveils Unique Antithrombotic Gene Expansion in Haemadipsa yanyuanensis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Comparative Genomics Unveils Unique Antithrombotic Gene Expansion in Haemadipsa yanyuanensis Yiquan Lin, Fang Zhao, Sijia Fan, Dezhi Yang, Xiangrong Tong, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7119690/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 31 Dec, 2025 Read the published version in BMC Genomics → Version 1 posted 9 You are reading this latest preprint version Abstract Haemadipsa yanyuanensis is a terrestrial blood-feeding leech with underexplored anticoagulant adaptations. Here, we present its first chromosome-level genome assembly (165.32 Mb, 9 chromosomes; BUSCO completeness: 97.6%) generated using Nanopore, Hi-C, and RNA-seq data. We identified 193 antithrombotic genes across 15 families, representing a 2.2- to 2.7-fold increase in gene number but a reduction in gene family diversity compared to aquatic medicinal leeches ( Hirudo medicinalis , Hirudinaria manillensis , and Hirudo nipponia ). Notably, bdellin, LDTI, and LCI gene families exhibited large-scale expansions ranging from 8.7- to 25-fold compared to aquatic leeches. while the progranulin gene exhibited a lineage-specific structure with 122 cysteine residues and nine tandem repeats. Transcriptomic profiling revealed high expression of these expanded families, suggesting their pivotal role in terrestrial blood-feeding adaptation. Our study reveals a novel gene family expansion-contraction model for antithrombotic evolution. These unique genomic data provide a new resource for the development of next-generation anticoagulant drugs. Haemadipsa yanyuanensis terrestrial leech chromosome-scale genome antithrombotic gene expansion gene family evolution anticoagulant drug discovery Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Cardiovascular diseases (CVDs) represent a major global public health challenge. According to the Global Burden of Disease study, the global CVD patient population reached 523 million in 2019, with associated mortality totaling 18.6 million cases [ 1 ]. World Health Organization reports further indicate that CVD-related fatalities accounted for 32% of all-cause mortality in 2019, with ischemic heart disease and cerebrovascular events responsible for 85% of these deaths [ 2 ]. Epidemiological projections suggest a concerning 90% surge in CVD prevalence from 2025 to 2050 [ 3 ]. Thrombosis, a central pathomechanism in CVDs, involves the activation of coagulation cascades and platelet-derived signaling pathways, ultimately leading to acute myocardial infarction and cerebral ischemia [ 4 ]. In light of this substantial clinical burden, the development of novel antithrombotic therapies is imperative. Hematophagous annelids have emerged as valuable models for developing thrombin-targeting therapeutics due to their evolutionarily optimized anticoagulant systems. Hirudotherapy, an ancient antithrombotic intervention, has been pharmacologically validated in modern research [ 5 , 6 ]. For instance, hirudin HV1, a direct thrombin inhibitor isolated from H irudo medicinalis , exhibits high-affinity anticoagulant activity by irreversibly binding to thrombin’s catalytic triad [ 7 ]. Recombinant hirudin analogs, including lepirudin, desirudin, and bivalirudin, have been approved by the FDA and EMA as first-line treatments for heparin-induced thrombocytopenia [ 8 – 10 ]. While anticoagulant mechanisms in aquatic medicinal leeches have been extensively studied, their terrestrial counterparts, such as Haemadipsa yanyuanensis , remain poorly understood, hindering comparative analyses of blood-feeding adaptations across leech species. Biochemical studies suggest that Haemadipsidae leeches possess phylogenetically distinct repertoires of hemostasis-modulating biomolecules. For example, haemadin, a thrombin inhibitor first identified in Haemadipsa sylvestris [ 11 ], exhibits antithrombotic efficacy comparable to hirudin [ 12 ]. Subsequent studies in Haemadipsa interrupta revealed that this protein demonstrates enhanced target specificity compared to classical hirudin [ 13 ]. However, the molecular basis of anticoagulation in H. yanyuanensis remains poorly understood. Although two anticoagulant proteins were detected in this species [ 14 ], their tertiary structures and amino acid sequences remain unresolved. Recent advances in genomics have elucidated the anticoagulant gene repertoires of aquatic medicinal leeches, including H. medicinalis [ 15 , 16 ], Hirudinaria manillensis [ 17 ], Whitmania pigra [ 18 ], and Hirudo nipponia [ 19 , 20 ]. In contrast, terrestrial Haemadipsidae species lack comparable genomic resolution, hindering comparative analyses of blood-feeding adaptations across leech ecotypes. This study addresses this gap by presenting the first high-quality genome assembly of H. yanyuanensis , constructed with the BRAKER-plus integrative omics approach [ 17 ]. Subsequent genomic analyses identified four evolutionarily conserved antithrombotic gene modules: (1) coagulation cascade suppressors, (2) platelet activation antagonists, (3) fibrinolysis accelerators, and (4) tissue penetration enhancers. These findings provide critical insights into the evolutionary adaptations of terrestrial leeches and highlight their potential as a source of next-generation anticoagulant and thrombolytic drugs. Materials and Methods 2.1 DNA and RNA Sequencing In this study, the specimens of H. yanyuanensis were collected from Jiaozi Snow Mountain, Luquan County, Yunnan Province, China (26.023°N, 102.872°E). Following a 7-day gut evacuation protocol with digestive tract excision, paired-end Illumina and ultralong Nanopore sequencing libraries were prepared using established methodologies [ 20 – 22 ]. The sequencing pipeline generated ultralong Oxford Nanopore Technologies (ONT) reads for de novo genome assembly alongside strand-specific RNA-seq libraries for transcriptional profiling. Genomic and transcriptomic sequencing followed previous methods, with high-quality data generated via 150 bp paired-end sequencing on the Illumina HiSeq 2000 platform (Illumina Inc., San Diego, CA, USA). 2.2 Genome Assembly and Gene Prediction The genome of H. yanyuanensis was assembled using previously described protocols [ 20 ], and genes with antithrombotic functionality were identified. Briefly, the preliminary nuclear genome assembly and mitochondrial genome assembly were performed using NextDenovo V2.5.0 in combination with a comprehensive suite of bioinformatics tools, while a HiC topological contact map was subsequently generated through visualization methods [ 23 , 24 ]. Genome completeness was assessed against the eukaryota_odb10 database using BUSCO v4.1.4, while assembly accuracy was evaluated through quality control analysis with Merqury v1.3. Then, a denovo repeat library for the repeat-masked genome was developed using RepeatModeler v2.0.3, the RepBase v20181026 database [ 25 ], and RepeatMasker v4.1.2p1. Gene annotation was performed using the BRAKERplus pipeline[ 26 ]. RNA-Seq reads were first aligned to the genome assembly, followed by de novo transcript reconstruction with Trinity v2.9.0, and coding sequences (CDSs) were predicted using GeneMarkS-T v5.1. Then, through literature review and mining of public repositories such as GenBank and UniProt, a reference library of antithrombotic genes was constructed. CDSs obtained from RNA-Seq and BRAKER predictions were queried against this library using the BLASTP algorithm to identify putative homologs. The filtered candidate genes were precisely mapped to the genome using Exonerate v2.2.0. Following manual refinement, the BRAKER prediction results were integrated with the GFF annotation file using AGAT v1.2.0, to generate a comprehensive annotation file for antithrombotic functional genes. To predict signal peptide regions within the candidate protein sequences, the online tool SignalP 6.0 [ 27 ] was employed (accessed at https://dtu.biolib.com/SignalP-6 on April 13, 2025). To investigate the phylogenetic relationships among gene family members, multiple sequence alignment was performed using MEGA v11.0.13, followed by phylogenetic tree construction with the Maximum Likelihood (ML) method using IQ-TREE v1.6.12. In addition, pairwise sequence similarity among candidate genes was assessed using the longest similarity index calculated with EMBOSS v6.6.0, providing further insights into their potential functions and evolutionary relationships. 2.3 Genetic Variation Analysis of Antithrombotic Genes The protein-coding genes identified in this study were combined with gene sequences from H. medicinalis , H. manillensis , and H. nipponia , as well as prototype gene sequences. These nucleotide sequences were translated into protein sequences using the “Translated Protein Sequences” function in MEGA with the default “standard” genetic code. The resulting protein sequences, including prototype sequences, were subjected to multiple sequence alignment using the ClustalW function in MEGA with default parameters. Pairwise sequence similarity was also calculated using the longest similarity index with EMBOSS. A phylogenetic tree based on protein sequences was constructed using IQ-TREE to investigate the evolutionary relationships among members of the HMEI gene family. A comprehensive analysis was conducted by integrating the RNA-seq dataset of H. yanyuanensis with the CDS reference sequences. Gene expression levels were quantified using Salmon v1.0.0, and TPM (transcripts per million) values were calculated. The TPM values of H. yanyuanensis were integrated and grouped with those of blood-feeding leeches ( H. nipponia and H. manillensis ) from published datasets. Following this, an in-depth analysis of gene expression variation was conducted across the three hematophagous leech species. Due to the absence of corresponding RNA-seq data for H. medicinalis , this species was excluded from the comparative analysis in this study. Results 3.1 Gene Sequencing and Assembly In this study, we generated 15.18 Gb of high-quality Oxford Nanopore (ONT) long-read data with an average read length of 11.65 kb. De novo assembly produced 37 contigs totaling 169.56 Mb, with a contig N50 of 7.92 Mb. Subsequent Hi-C sequencing using Illumina HiSeq yielded 43.48 Gb of data, enabling chromosome-scale scaffolding. The contigs were assembled into 18 scaffolds totaling 165.30 Mb, with an N50 of 18.42 Mb. Scaffold length distribution analysis (Figure. 1) revealed that the nine longest scaffolds represent nine chromosomes, comprising 164.47 Mb (99.5%) of the assembly. The remaining nine scaffolds are all < 0.3 Mb. In addition, 20.41 Gb of Illumina paired-end reads were used for mitochondrial genome assembly via GetOrganelle, resulting in a 17,851 bp circular mitochondrial genome. Altogether, the final H. yanyuanensis genome assembly spans 165.32 Mb and consists of nine chromosomes, one mitochondrial genome, and nine unplaced scaffolds. To assess assembly completeness, BUSCO (eukaryota_odb10) identified 97.6% of conserved orthologs, with 91.8% as complete single-copy genes. Merqury analysis produced a quality score of 39.03, reflecting high base-level accuracy. Repeat annotations revealed that 28.49% of the genome consists of repetitive elements, including retrotransposons and DNA transposons (Table 1 ). No small RNAs, satellite sequences, or low-complexity regions were detected in this assembly. This study detected no small RNAs, satellites, or low-complexity fragments, contrasting sharply with previous findings in H. nipponia and H. manillensis. Table 1 Different repeat sequence type percentages in genomes of Haemadipsa yanyuanensis , Hirudinaria manillensis , and Hirudo nipponia . Item H. yanyuanensis H. manillensis H. nipponia Retroelements 9.51 6.59 9.89 DNA transposons 1.98 1.76 8.40 Rolling-circles 1.06 0.20 0.18 Unclassified 13.17 8.41 10.71 Small RNA 0.00 0.00 0.00 Satellites 0.00 0.20 0.02 Simple repeats 2.77 1.80 0.58 Low complexity 0.00 0.00 0.00 Total 28.49 18.96 29.77 3.2 Gene Prediction and Annotation A systematic analysis of antithrombotic biomolecules derived from leech saliva revealed five antithrombotic gene clusters and a total of 23 antithrombotic proteins. Using the BRAKER-plus pipeline, we conducted genome-wide gene prediction and structural annotation of antithrombotic protein-coding genes in H. yanyuanensis , resulting in the identification of 15 gene families and 193 antithrombotic gene loci, including three putative pseudogenes. Further classification revealed that hirustasin, hirustasin-like, guamerin, piguamerin, bdellastasin, and poecistasin could not be clearly distinguished due to high sequence similarity in Haemadipsa species. Therefore, these were collectively grouped into the hirustasin superfamily. Ultimately, 18 distinct gene/protein families related to anticoagulation were identified. These gene families include eight coagulation inhibitors that act primarily by targeting various steps in the coagulation cascade: haemadin [ 28 ], progranulin [ 29 ], antistasin [ 30 ], lefaxin [ 31 ], hirustasin [ 32 ], eglin [ 33 ], bdellin [ 34 ], and LDTI [ 35 ]. Three fibrinolytic enhancers were also identified: destabilase [ 36 ], GGT [ 37 ], and LCI [ 38 , 39 ]. Two platelet aggregation inhibitors, apyrase [ 40 ] and lumbrokinase [ 17 , 41 ], were also detected. In addition, one tissue permeability enhancer, hyaluronidase [ 39 , 42 ], was identified. Notably, we discovered an HMEI gene family comprising 53 members, which functions as a protease inhibitor and has potential anti-inflammatory activity [ 43 , 44 ]. Conversely, no gene members belonging to the therostasin, decorsin, or saratin families were detected in H. yanyuanensis , suggesting species-specific gene loss in this lineage (Table 2 ). 3.3 Protein and Genetic Variation Analysis Among the 15 anticoagulant gene families identified in H. yanyuanensis , extensive genetic variation was detected among family members (Figure. S1–S15). Comparative analysis revealed four distinct evolutionary patterns: two expansion modes, namely copy number expansion and coding sequence elongation, and two contraction modes involving reductions in these features. The first expansion pattern involved nine gene families, including antistasin, hirustasin, eglin, bdellin, LDTI, lumbrokinase, destabilase, LCI, and HMEI. Among them, eglin (13 members), lumbrokinase (7), and HMEI (53) showed approximately twice as many members as the corresponding families in the three reference leech species. The HMEI family, in particular, included one pseudogene (HMEI_Hyan52) and another gene (HMEI_Hyan11) with multiple nonsynonymous mutations and a premature stop codon in the C-terminal domain (Figure. S1), accounting for 27.5% of all identified anticoagulant-related genes (n = 193). Notably, the LDTI, bdellin, and LCI families exhibited exceptionally large expansions. For example, 26 bdellin genes were annotated in H. yanyuanensis , including one pseudogene (bdellin_Hyan26) containing a premature stop codon in its signal peptide region (Figure. S2). In contrast, the reference species H. nipponia , H. medicinalis , and H. manillensis contained only 3, 2, and 1 bdellin genes, respectively, representing an expansion by a factor of 8.7 to 26. Similarly, the LDTI and LCI families had 16 and 25 members, whereas only a single-copy ortholog was found in each of the reference species, indicating 16-fold and 25-fold increases, respectively. Other families, such as destabilase, showed only moderate expansion and also included a pseudogene (destabilase_Hyan09). By contrast, contraction in gene copy number was observed in the lefaxin and apyrase families. The apyrase family had only two members in H. yanyuanensis , compared to six, five, and five members in the three reference leeches, respectively. Similarly, the lefaxin family contained two members in H. yanyuanensis , slightly fewer than the three found in each of the reference species (Table 2 ). Table 2 Number of functional genes of H. yanyuanensis , H. nipponia , H. manillensis , and Hirudo medicinalis. Gene family H. yanyuanensis H. nipponia H. manillensis H. medicinalis Function haemadin/ hirudin 3 3 5 3 coagulation inhibitor progranulin 1 1 1 1 coagulation inhibitor antistasin 3 2 2 2 coagulation inhibitor lefaxin 2 3 3 3 coagulation inhibitor therostasin 0 1 1 1 coagulation inhibitor hirustasin 27 19 18 13 coagulation inhibitor eglin 13 5 4 5 coagulation inhibitor bdellin 26 3 1 2 coagulation inhibitor LDTI 16 1 1 1 coagulation inhibitor decorsin 0 3 0 0 platelet aggregation inhibitor saratin 0 2 2 1 platelet aggregation inhibitor apyrase 2 6 5 5 platelet aggregation inhibitor lumbrokinase 7 3 3 2 platelet aggregation inhibitor destabilase 9 4 3 8 fibrinolysis enhancer GGT 2 4 1 2 fibrinolysis enhancer LCI 25 1 1 1 fibrinolysis enhancer hyaluronidase 4 4 3 4 tissue penetration enhancer HMEI 53 21 18 20 inflammation inhibitor total 193 86 72 74 —— The second expansion mode was characterized by significant elongation of protein-coding sequences. In several cases, the protein products were nearly twice as long as those of their orthologs in the reference medicinal leeches. This pattern was observed in four gene families: LDTI, haemadin (Figure. 2A), progranulin (Figure. 3), and antistasin (Figure. S3), yielding six antithrombotic polypeptides: LDTI_Hyan1 through LDTI_Hyan3, haemadin_Hyan3, progranulin_Hyan, and antistasin_Hyan1. For instance, haemadin_Hyan3 contained three internal tandem repeats, each consisting of six cysteine residues (Figure. 2B), and antistasin_Hyan1 included five repeats, each with ten conserved cysteines (Figure. S4). The progranulin gene was conserved as a single-copy in all four species, yet the number of cysteine residues varied dramatically: 69 in H. nipponia , 66 in H. manillensis , 72 in H. medicinalis , and as many as 122 in H. yanyuanensis (Figure. 3). The H. yanyuanensis progranulin protein also contained nine conserved tandem repeats, each comprising twelve cysteine residues, whereas only five and six repeats were found in H. nipponia and H. medicinalis [ 17 , 20 ], respectively (Figure. 4). In contrast to this elongation pattern, the bdellin gene family displayed protein-coding sequence contraction. Despite the substantial increase in gene copy number, 22 bdellin genes encoded truncated proteins that were approximately half the length of their orthologs in the reference medicinal leeches, suggesting possible functional divergence or degeneration. Table 3 The total TPM values (mean TPM ± SD) of each gene family. Gene family H. yanyuanensis H. nipponia H. manillensis haemadin/hirudin 802.3 ± 633.8 7035.9 ± 12,173.0 430.3 ± 366.7 decorsin 0.0 ± 0.0 12194.2 ± 21117.8 0.0 ± 0.0 progranulin 347.1 ± 88.1 99.8 ± 20.0 152.1 ± 47.5 antistasin 221.6 ± 222.9 1040.4 ± 183.5 820.0 ± 385.5 lefaxin 2736.2 ± 1077.5 5669.8 ± 2920.5 14865.0 ± 4011.0 therostasin 0.0 ± 0.0 98.5 ± 160.8 70.1 ± 68.2 hirustasin 18465.7 ± 15536.9 28508.9 ± 2582.0 8991.3 ± 1724.3 eglin 9565.6 ± 2142.5 6237.4 ± 2736.4 11984.0 ± 3808.0 bdellin 33174.5 ± 15723 16079.2 ± 6526.7 2550.5 ± 1346.6 LDTI 2630.4 ± 2129.6 4909.9 ± 4641.4 3244.8 ± 1808.2 HMEI 5036.7 ± 2176.8 1395.7 ± 1079.4 2859.0 ± 2282.6 saratin 0.0 ± 0.0 5700.4 ± 9870.8 1317.1 ± 2051.2 apyrase 525.7 ± 285.9 200.1 ± 286.4 448.6 ± 653.6 lumbrokinase 9450.5 ± 6577.9 5576.4 ± 7236.3 0.2 ± 0.1 destabilase 3435.4 ± 700.7 6019.5 ± 2868.7 3926.3 ± 4329 GGT 145.1 ± 32.5 78.7 ± 86.5 22.0 ± 17.1 LCI 10250.2 ± 7628.1 889.2 ± 243.2 2236.2 ± 460.4 hyaluronidase 216.0 ± 33.2 109.2 ± 94.8 313.9 ± 245.0 3.4 Gene Expression Analysis Integration of previously published data with the transcriptomic profiles of H. yanyuanensis revealed marked interspecific differences in gene expression patterns (Table 3 ). Transcriptomic profiling revealed high expression levels (mean TPM ± SD) of bdellin (33,174.5 ± 15,723.0), eglin (9,565.6 ± 2,142.5), LCI (10,250.2 ± 7,628.1), lumbrokinase (9,450.5 ± 6,577.9), HMEI (5,036.7 ± 2,176.8), and hirustasin (18,465.7 ± 15,536.9), suggesting a functional link between gene family expansion and adaptation to terrestrial blood-feeding. Among these, the bdellin family showed exceptionally high expression levels, far exceeding those of any anticoagulant gene family in the reference medicinal leeches. In contrast, the antistasin (221.6 ± 222.9) and lefaxin (2,736.2 ± 1,077.5) families displayed significantly lower expression in H. yanyuanensis compared to H. nipponia and H. manillensis . Hierarchical clustering further revealed species-specific transcriptomic profiles, with H. yanyuanensis and H. nipponia forming a closer clade in accordance with their phylogenetic relationship (Figure. 5). Discussion 4.1 Genome Assembly and Gene Mining This study used whole-genome sequencing to explore evolutionary divergence between H. yanyuanensis and three reference leeches ( H. medicinalis , H. manillensis , and H. nipponia ), uncovering conserved syntenic blocks alongside lineage-specific innovations. Employing nanopore third-generation sequencing (ONT) combined with Illumina Hi-C, survey, and RNA-Seq data enabled high-precision base calling and chromosome-level assembly, resulting in a genome of 165.31 Mb across 9 chromosomes plus a near-complete mitochondrial genome. Assembly quality was supported by 95.9% BUSCO completeness and a Merqury quality score of 39.9. Notably, 193 antithrombotic protein-coding genes were annotated—approximately 2.2 times more than in the three medicinal leech species (86 in H. nipponia , 74 in H. medicinalis , and 72 in H. manillensis ). 4.2 Gene Expansion–Contraction Model Comparative analysis revealed that H. yanyuanensis harbors 193 antithrombotic genes, approximately 2.2 to 2.7 times more than those identified in H. nipponia , H. medicinalis , and H. manillensis , yet these are distributed among only 15 gene families, compared to 17 to 18 in the reference species [17, 20]. This pattern reflects lineage-specific remodeling through selective gene expansion and contraction. However, substantial divergence was observed in gene family size and structure. The bdellin, LDTI, and LCI families experienced large-scale expansions, with up to 26 times more members than in the references, while apyrase and lefaxin showed notable reductions. Some expanded families also contained pseudogenes or truncated sequences, indicating possible functional shifts. Moreover, several proteins such as haemadin_Hyan3 and progranulin_Hyan exhibited significant elongation due to internal tandem repeats and increased cysteine content. For example, progranulin_Hyan encodes 122 cysteine residues across nine conserved repeat units, nearly twice the number found in its homologs, implying enhanced structural complexity. These findings indicate that H. yanyuanensis has adopted a distinct contraction and expansion strategy. Through gene duplication, domain expansion, and potential gene repurposing [45], this mechanism likely enhances its antithrombotic capacity and reflects an adaptive response to environmental pressures. Such a pattern represents a previously unreported evolutionary trajectory among terrestrial leeches. 4.3 Differences in Gene Family Expression Levels Due to the absence of RNA-seq data for H. medicinalis , gene expression comparisons were performed only among H. yanyuanensis , H. nipponia , and H. manillensis . In H. yanyuanensis , gene families that showed notable expansion, including eglin, bdellin, HMEI, and LCI, generally exhibited higher expression levels than in the reference species. Although LDTI and hirustasin did not follow this trend, the overall results suggest that gene family expansion often correlates with increased transcriptional activity. The observed tissue-specific expression patterns imply that divergent expression among conserved genes may contribute to ecological adaptation and niche differentiation in Haemadipsa species. 4.4 Biomedical Potential and Conservation Considerations of Haemadipsa Species Recent studies on terrestrial leeches have mainly focused on using invertebrate-derived DNA (iDNA) for monitoring vertebrate biodiversity [46, 47]. In parallel, pharmacological research has begun to highlight their antithrombotic potential, with compounds such as haemadin and the newly identified sylvestin showing significant bioactivity [12, 48]. Functional validation of molecules like decorsin and other haemadins in H. interrupta has further supported their clinical relevance [13]. These findings position Haemadipsa species, including H. yanyuanensis , as promising sources of novel antithrombotic agents. While habitat degradation has led to a decline in aquatic leech populations [49-52], forest-dwelling Haemadipsa species have remained relatively stable due to their distribution in tropical and subtropical regions. As a result, new species continue to be discovered [53]. Their ecological resilience, combined with lineage-specific expansion of antithrombotic gene families, underscores their pharmacological potential and value in future drug discovery. Conclusions This study reports the first chromosome-level genome assembly of the terrestrial leech H. yanyuanensis , uncovering a distinct evolutionary trajectory of antithrombotic gene adaptation. Despite harboring fewer antithrombotic gene families (15) compared to aquatic medicinal leeches (17–18), H. yanyuanensis possesses a markedly higher number of antithrombotic genes (193), with key families such as bdellin, LDTI, and LCI exhibiting massive copy number expansions, up to 26-fold. Additionally, elongation of coding sequences and tandem repeat structures in gene families like progranulin and haemadin further support functional innovation under terrestrial selective pressures. These findings highlight a contraction-expansion mechanism driving lineage-specific adaptation in Haemadipsa , likely reflecting ecological specialization in blood-feeding niches. By bridging the genomic gap in terrestrial leeches, this work provides both evolutionary insight and a valuable resource for antithrombotic drug discovery, offering new molecular targets for the treatment of cardiovascular and thrombotic diseases. Declarations Conflict of Interest The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Author Contributions Conceptualization, Z. L., G. L., D. K., and Y. L.; Methodology, Y. L. and G. L.; Software, Y. L. and G. L.; Validation, Formal Analysis, Investigation, Resources, Data Curation, and Visualization, Z. L., G. L., Y. L., F. Z., S. F., D. Y., X. T., and L. T.; Writing and Editing, Z. L., G. L., and Y. L.; Supervision, Project Administration, and Funding Acquisition, Z. L., G. L., and Y. L. All authors have read and agreed to the published version of the manuscript. Funding This research was funded by the National Natural Science Foundation of China (82260742 and 32260132), the Yunnan Provincial University Serving Key Industry Science and Technology Special Project (FWCY-ZD2024009), the Joint Special Project for Basic Research of Local Universities in Yunnan Province (202301BA070001-105 and 202101BA070001-164), the 2023 Frontier Research Team Program of Kunming University, the Key Laboratory of Jiangxi Province for Biological Invasion and Biosecurity (2023SSY02111), National Key R&D Program of China (2023YFF1305000), the Yunnan Ten Thousand Talents Program for Young Top Notch Talents (YNWR-QNBJ-2020-122), Special Project of Science-Technology Talent and Platform in Yunnan Province (202105AC160042), and Yunnan International Joint Laboratory with South and Southeast Asia for the Integrated Development of Animal-Derived Anti-Thrombosis Chinese Medicine (202503AP140025). Supplementary Materials Supplementary File_S1_genome. fa: Genome assembly of H. yanyuanensis ; Supplementary File_S2_all_CDS. fa: CDSs of all predicted protein-coding genes; Supplementary File_S3_antithrombotic_CDS. fa: CDSs of 193 antithrombotic genes; Supplementary Figures_S1 to S14. docx: Sequence alignments of antithrombotic protein families not shown in the main text. Supplementary data to this article can be found online at https://doi.org/10.6084/m9.figshare.29369828.v3 Data Availability Statement The raw genomic data generated in this study have been deposited at Genome Sequence Archive (GSA) portal of the China National Center for Bioinformation ( https://ngdc.cncb.ac.cn/gsa ) withaccession number of CRA025628. This repository is maintained by the Chinese Academy of Sciences, following international standards for biological data sharing. References Roth GA, Mensah GA, Johnson CO, Addolorato G, Ammirati E, Baddour LM et al . Global burden of cardiovascular diseases and risk factors, 1990–2019: update from the GBD 2019 study. Journal of the American college of cardiology 2020, 76(25):2982-3021.doi:https://doi.org/10.1016/j.jacc.2020.09.010 WHO. Cardiovascular diseases (CVDs). 2021. https://www.who.int/europe/news-room/fact-sheets/item/cardiovascular-diseases. Accessed April 26, 2025. Chong B, Jayabaskaran J, Jauhari SM, Chan SP, Goh R, Kueh MTW et al . Global burden of cardiovascular diseases: projections from 2025 to 2050. European journal of preventive cardiology 2024:zwae281.doi: https://doi.org/10.1093/eurjpc/zwac281 DeLoughery TG. Basics of coagulation. Hemostasis and thrombosis 2019:1-9.doi:https://doi.org/10.1007/978-3-030-19330-0_1 Singh AP. Medicinal leech therapy (hirudotherapy): a brief overview. Complementary therapies in clinical practice 2010, 16(4):213-215.doi:https://doi.org/10.1016/j.ctcp.2009.11.005 Nair HK, Ahmad NW, Lee HL, Ahmad N, Othamn S, Mokhtar NSHM et al . Hirudotherapy in wound healing. The international journal of lower extremity wounds 2022, 21(4):425-431.doi:https://doi.org/10.1177/1534734620948299 Sun Y, Wang D. Protein-engineered Hirudins with enhanced anticoagulant activities developed by a molecular integrative approach. Journal of medicinal chemistry 2024, 67(5):3203-3204.doi:https://doi.org/10.1017/mdh.2023.17 Patel VB, Snyder J, Shopnick RI. Successful use of low dose r-Hirudin (Refludan®) for recurrentdialysis catheter thrombosis in a patient with heparin induced thrombocytopenia. Thrombosis and haemostasis 1999, 82(09):1205-1206.doi:https://doi.org/10.1055/s-0037-1614360 Scott B, Tam CW, Moon RS. Transfemoral transcatheter aortic valve replacement in a patient with heparin-induced thrombocytopenia: intraoperative anticoagulation management with bivalirudin (Angiomax). Journal of cardiothoracic and vascular anesthesia 2015, 29(6):1603-1605.doi:https://doi.org/10.1053/j.jvca.2014.10.018 Thompson CA. New direct thrombin inhibitor becomes available. American journal of health-system pharmacy 2010, 67(8):588.doi:https://doi.org/10.2146/news100027 Strube K-H, Kröger B, Bialojan S, Otte M, Dodt J. Isolation, sequence analysis, and cloning of haemadin. an anticoagulant peptide from the Indian leech. Journal of biological chemistry 1993, 268(12):8590-8595.doi:https://doi.org/10.1016/S0021-9258(18)52916-1 Acquasaliente L, Pierangelini A, Pagotto A, Pozzi N, De Filippis V. From haemadin to haemanorm: Synthesis and characterization of full‐length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm). Protein science 2023, 32(12):e4825.doi:https://doi.org/10.1002/pro.4825 Müller C, Sponholz D, Tolksdorf C, Rauch BH, Kvist S. Identification and functional characterization of multiple haemadins and an oligomeric decorsin in the Asian land leech Haemadipsa interrupta. Parasitology research 2024, 123(11):390.doi:https://doi.org/10.1007/s00436-024-08404-w Zhao R. A new potent inhibitor of thrombin from the leech Haemendipsa yanyuanensis. Journal of shanghai university (English Edition) 2001, 5:86-88.doi:https://doi.org/10.1007/s11741-001-0035-3 Babenko VV, Podgorny OV, Manuvera VA, Kasianov AS, Manolov AI, Grafskaia EN et al . Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches. Bmc genomics 2020, 21:1-16.doi:https://doi.org/10.1186/s12864-020-6748-0 Kvist S, Manzano-Marín A, de Carle D, Trontelj P, Siddall ME. Draft genome of the European medicinal leech Hirudo medicinalis (Annelida, Clitellata, Hirudiniformes) with emphasis on anticoagulants. Scientific reports 2020, 10(1):9885.doi:https://doi.org/10.1038/s41598-020-66749-5 Liu Z, Zhao F, Huang Z, Hu Q, Meng R, Lin Y et al . Revisiting the Asian buffalo leech (Hirudinaria manillensis) genome: focus on antithrombotic genes and their corresponding proteins. Genes 2023, 14(11):2068.doi:https://doi.org/10.3390/genes14112068 Liu Z, Zhao F, Huang Z, He B, Liu K, Shi F et al . A chromosome-level genome assembly of the non-hematophagous leech Whitmania pigra (Whitman 1884): identification and expression analysis of antithrombotic genes. Genes 2024, 15(2):164.doi:https://doi.org/10.3390/genes15020164 Zheng J, Wang X, Feng T, Rehman Su, Yan X, Shan H et al . Molecular mechanisms underlying hematophagia revealed by comparative analyses of leech genomes. Gigascience 2023, 12:giad023.doi: https://doi.org/10.1093/gigascience/giad023 Zhao F, Huang Z, He B, Liu K, Li J, Liu Z et al . Comparative genomics of two Asian medicinal leeches Hirudo nipponia and Hirudo tianjinensis: with emphasis on antithrombotic genes and their corresponding proteins. International journal of biological macromolecules 2024, 270:132278.doi:https://doi.org/10.1016/j.ijbiomac.2024.132278 Zhao F, Huang Z, Tang L, Zhang W, Liu Z, Lin G. Comparative genomics of three non-hematophagous leeches (Whitmania spp.) with emphasis on antithrombotic biomolecules. Frontiers in genetics 2025, 16:1548006.doi:https://doi.org/10.3389/fgene.2025.1548006 Zhao F, Huang Z, Tang L, He B, Liu Z, Lin G. Comparative genomics of three non-hematophagous leeches (Whitmania spp.): focusing on antithrombotic genes. bioRxiv 2024:2024.2005. 2008.590400.doi:https://doi.org/10.1101/2024.05.08.590400 Zhou C, McCarthy S, Durbin R. YaHS: yet another Hi-C scaffolding tool. Bioinformatics 2023, 39(1).doi:https://doi.org/10.1093/bioinformatics/btac808 Durand NC, Robinson JT, Shamim MS, Machol I, Mesirov JP, Lander ES et al . Juicebox provides a visualization system for Hi-C contact maps with unlimited zoom. Cell systems 2016, 3(1):99-101.doi:https://doi.org/10.1016/j.cels.2015.07.012 Bao W, Kojima KK, Kohany O. Repbase update, a database of repetitive elements in eukaryotic genomes. Mobile dna 2015, 6:1-6.doi:https://doi.org/10.1186/s13100-015-0041-9 Saenko S, Hoff KJ, Stanke M. Annotation of 200 insect genomes with BRAKER for consistent comparisons across species. bioRxiv 2025:2025.2004. 2017.649312.doi: https://doi.org/10.1101/2025.04.17.649312 Teufel F, Almagro Armenteros JJ, Johansen AR, Gíslason MH, Pihl SI, Tsirigos KD et al . SignalP 6.0 predicts all five types of signal peptides using protein language models. Nature biotechnology 2022, 40(7):1023-1025.doi:https://doi.org/10.1038/s41587-021-01156-3 Kostromina MA, Tukhovskaya EA, Shaykhutdinova ER, Slashcheva GA, Ismailova AM, Palikov VA et al . Screening of the promising direct thrombin inhibitors from haematophagous organisms. Part I: recombinant analogues and their antithrombotic activity in vitro. Biomedicines 2021, 10(1):11.doi:https://doi.org/10.3390/biomedicines10010011 Tanaka Y, Suzuki G, Matsuwaki T, Hosokawa M, Serrano G, Beach TG et al . Progranulin regulates lysosomal function and biogenesis through acidification of lysosomes. Human molecular genetics 2017, 26(5):969-988.doi:https://doi.org/10.1093/hmg/ddx011 Dunwiddie C, Thornberry N, Bull H, Sardana M, Friedman P, Jacobs J et al . Antistasin, a leech-derived inhibitor of factor Xa: kinetic analysis of enzyme inhibition and identification of the reactive site. Journal of biological chemistry 1989, 264(28):16694-16699.doi:https://doi.org/10.1016/S0021-9258(19)84761-0 Faria F, Kelen EMA, Sampaio CA, Bon C, Duval N, Chudzinski-Tavassi AM. A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leech. Thrombosis and haemostasis 1999, 82(11):1469-1473.doi:https://doi.org/10.1055/s-0037-1614857 Söllner C, Mentele R, Eckerskorn C, Fritz H, Sommerhoff CP. Isolation and characterization of hirustasin, an antistasin‐type serine‐proteinase inhibitor from the medical leech Hirudo medicinalis. European journal of biochemistry 1994, 219(3):937-943.doi:https://doi.org/10.1111/j.1432-1033.1994.tb18575.x Renesto P, FERRER‐LOPEZ P, Chignard M. Eglin c and heparin inhibition of platelet activation induced by cathepsin G or human neutrophils. Annals of the New York academy of sciences 1991, 624(1):321-324.doi:https://doi.org/10.1111/j.1749-6632.1991.tb17033.x Cheng R, Tang X, Long A, Mwangi J, Lai R, Sun R et al . Purification and characterization of a novel anti-coagulant from the leech Hirudinaria manillensis. Zoological research 2019, 40(3):205.doi:https://doi.org/10.24272/j.issn.2095-8137.2019.037 Tanaka AS, Silva MM, Torquato RJ, Noguti MA, Sampaio CA, Fritz H et al . Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors. Federation of european biochemical societies letters 1999, 458(1):11-16.doi:https://doi.org/10.1016/S0014-5793(99)01106-0 Marin E, Kornilov DA, Bukhdruker SS, Aleksenko VA, Manuvera VA, Zinovev EV et al . Structural insights into thrombolytic activity of destabilase from medicinal leech. Scientific reports 2023, 13(1):6641.doi:https://doi.org/10.1038/s41598-023-32459-x Friedrich T, Kröger B, Koerwer W, Strube KH, Meyer T, Bialojan S. An isopeptide bond splitting enzyme from Hirudo medicinalis similar to γ‐glutamyl transpeptidase. European journal of biochemistry 1998, 256(2):297-302.doi:https://doi.org/10.1046/j.1432-1327.1998.2560297.x Arolas JL, Castillo V, Bronsoms S, Aviles FX, Ventura S. Designing out disulfide bonds of leech carboxypeptidase inhibitor: implications for its folding, stability and function. Journal of molecular biology 2009, 392(2):529-546.doi:https://doi.org/10.1016/j.jmb.2009.06.049 Reverter D, Fernández-Catalán C, Baumgartner R, Pfänder R, Huber R, Bode W et al . Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2. Nature structural biology 2000, 7(4):322-328.doi:https://doi.org/10.1038/74092 Pérez de León AA, Tabachnick WJ. Apyrase activity and adenosine diphosphate induced platelet aggregation inhibition by the salivary gland proteins of culicoides variipennis, the north american vector of bluetongue viruses. Veterinary parasitology 1996, 61(3-4):327-338.doi:https://doi.org/10.1016/0304-4017(95)00828-4 Agoncillo A. Meta-analysis on the safety and efficacy of lumbrokinase in peripheral arterial disease. European heart journal acute cardiovascular care 2021, 10(Supplement_1):zuab020. 219.doi:https://doi.org/10.1093/ehjacc/zuab020.219 Das BK. An overview on hirudotherapy/leech therapy. Ind Res J Pharm Sci 2014, 1(1):34, Xu K, Zhou M, Wu F, Tang X, Lu Q, Lai R et al . Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis. Chinese journal of natural medicines 2021, 19(7):540-544.doi:https://doi.org/10.1016/S1875-5364(21)60054-7 Smith TP, Alshafie TA, Cruz CP, Fan C-Y, Brown AT, Wang Y et al . Saratin, an inhibitor of collagen-platelet interaction, decreases venous anastomotic intimal hyperplasia in a canine dialysis access model. Vascular and endovascular surgery 2003, 37(4):259-269.doi:https://doi.org/10.1177/153857440303700405 Martin A, Orgogozo V. The loci of repeated evolution: a catalog of genetic hotspots of phenotypic variation. Evolution 2013, 67(5):1235-1250.doi:https://doi.org/10.1111/evo.12081 Lynggaard C, Oceguera‐Figueroa A, Kvist S, Gilbert MTP, Bohmann K. The potential of aquatic bloodfeeding and nonbloodfeeding leeches as a tool for iDNA characterisation. Molecular ecology resources 2022, 22(2):539-553.doi:https://doi.org/10.1111/1755-0998.13486 Ji Y, Baker CC, Popescu VD, Wang J, Wu C, Wang Z et al . Measuring protected-area effectiveness using vertebrate distributions from leech iDNA. Nature communications 2022, 13(1):1555.doi:https://doi.org/10.1038/s41467-022-28778-8 Zhang Z, Shen C, Fang M, Han Y, Long C, Liu W et al . Novel contact–kinin inhibitor sylvestin targets thromboinflammation and ameliorates ischemic stroke. Cellular and molecular life sciences 2022, 79(5):240.doi:https://doi.org/10.1007/s00018-022-04257-7 Copena D, Gómez-Martín M. Spanish–french leech trade and its consequences: From the increase in medical demand to resource depletion and technical innovation. Medical history 2024, 68(1):42-59.doi:https://doi.org/10.1017/mdh.2024.5 Chen A, Liu B, Zhou R, Zhang H, Zhou L, Xie X et al . Habitat suitability analysis for luehdorfia chinensis leech, 1893 (Lepidoptera: Papilionidae) in the middle and lower yangtze river: a study based on the MaxEnt Model. Insects 2025, 16(4):396.doi:https://doi.org/10.3390/insects16040396 Spitsyn VM, Spitsyna EA. New record of collita coreana (leech,[1889])(Erebidae: Arctiinae) from russia. Amurian zoological journal 2022, 14(3):413-416.doi:https://www.doi.org//10.33910/2686-9519-2022-14-3-413-416 Arabacı B. ‘Pearls’ of the nineteenth-century: from therapeutic actors to global commodities medicinal leeches in the Ottoman Empire. Medical history 2023, 67(2):128-147.doi: https://doi.org/10.1017/mdh.2023.17 Li T, Liu Y, Zhang C, Gu H, Cheng Z, Peng J et al . A new species of sinospelaeobdella from China: sinospelaeobdella jiangxiensis sp. n.(Hirudinda, Arhynchobdellida, Haemadipsidae). Animals 2025, 15(8):1079.doi:https://doi.org/10.3390/ani15081079 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 31 Dec, 2025 Read the published version in BMC Genomics → Version 1 posted Editorial decision: Revision requested 19 Aug, 2025 Reviews received at journal 18 Aug, 2025 Reviews received at journal 14 Aug, 2025 Reviewers agreed at journal 29 Jul, 2025 Reviewers agreed at journal 29 Jul, 2025 Reviewers invited by journal 27 Jul, 2025 Editor assigned by journal 16 Jul, 2025 Submission checks completed at journal 16 Jul, 2025 First submitted to journal 14 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7119690","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":492614021,"identity":"a2e6cc07-b9f7-4458-9952-04c0317567db","order_by":0,"name":"Yiquan Lin","email":"","orcid":"","institution":"Kunming University","correspondingAuthor":false,"prefix":"","firstName":"Yiquan","middleName":"","lastName":"Lin","suffix":""},{"id":492614022,"identity":"38e9db36-3387-4809-b1f2-0c7a428f97ea","order_by":1,"name":"Fang Zhao","email":"","orcid":"","institution":"Jinggangshan University","correspondingAuthor":false,"prefix":"","firstName":"Fang","middleName":"","lastName":"Zhao","suffix":""},{"id":492614023,"identity":"26f13648-095b-49e8-b02f-ad57f177ce48","order_by":2,"name":"Sijia Fan","email":"","orcid":"","institution":"Kunming University","correspondingAuthor":false,"prefix":"","firstName":"Sijia","middleName":"","lastName":"Fan","suffix":""},{"id":492614024,"identity":"89bb197f-62b0-49d4-801d-31be7757220d","order_by":3,"name":"Dezhi Yang","email":"","orcid":"","institution":"Kunming University","correspondingAuthor":false,"prefix":"","firstName":"Dezhi","middleName":"","lastName":"Yang","suffix":""},{"id":492614025,"identity":"f81262a7-855c-48e5-b8d8-479120df3489","order_by":4,"name":"Xiangrong Tong","email":"","orcid":"","institution":"Kunming University","correspondingAuthor":false,"prefix":"","firstName":"Xiangrong","middleName":"","lastName":"Tong","suffix":""},{"id":492614026,"identity":"b7119203-6eb6-4681-99fb-0c0aa22c533d","order_by":5,"name":"Lizhou Tang","email":"","orcid":"","institution":"Jiangxi Normal University","correspondingAuthor":false,"prefix":"","firstName":"Lizhou","middleName":"","lastName":"Tang","suffix":""},{"id":492614027,"identity":"33e5e56e-2d0e-471f-b9d1-2835f7ae4e27","order_by":6,"name":"Dejun Kong","email":"","orcid":"","institution":"Yunnan University","correspondingAuthor":false,"prefix":"","firstName":"Dejun","middleName":"","lastName":"Kong","suffix":""},{"id":492614028,"identity":"f64791a0-a176-4a71-9e17-db18b8b9de54","order_by":7,"name":"Gonghua Lin","email":"","orcid":"","institution":"Jinggangshan University","correspondingAuthor":false,"prefix":"","firstName":"Gonghua","middleName":"","lastName":"Lin","suffix":""},{"id":492614029,"identity":"92fd20b6-19c7-4fd6-a852-49a5942e21ec","order_by":8,"name":"Zichao Liu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAuUlEQVRIiWNgGAWjYDACCQbGAwwMNgx8zCRoYQBqSWNgI1XLYQY2onXwz25+cPBHzXl5NnYeM+kCBjs53QZCltw5ZnBA4thtwzZmoJYZDMnGZgcIWXMjh+GAYcNtRrAWHoYDidsIaZEHaUlsOGdPvBYDkJaDDQcSiddiCPTLwYZjycltzGzF1jwGRPhF7nbzw4c/auxs+/kPb7zNU2EnR9j7CMBhAHQn8cpBgP0BaepHwSgYBaNgxAAA5xU8p+G18qgAAAAASUVORK5CYII=","orcid":"","institution":"Kunming University","correspondingAuthor":true,"prefix":"","firstName":"Zichao","middleName":"","lastName":"Liu","suffix":""}],"badges":[],"createdAt":"2025-07-14 09:53:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7119690/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7119690/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12864-025-12445-5","type":"published","date":"2025-12-31T15:58:01+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88005458,"identity":"75dac915-0ea1-4fa9-a62b-27e33f62e12a","added_by":"auto","created_at":"2025-07-31 10:42:29","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":538600,"visible":true,"origin":"","legend":"\u003cp\u003eScaffold and chromosome lengths of\u003cem\u003e Haemadipsa yanyuanensis\u003c/em\u003e genome.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-7119690/v1/89abf694b64bd089f971aa40.png"},{"id":88003869,"identity":"e158f699-865c-440b-8ff5-e24e4cc2c767","added_by":"auto","created_at":"2025-07-31 10:34:29","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":807582,"visible":true,"origin":"","legend":"\u003cp\u003eComparative analysis of hirudin and haemadin protein families across leech species. (A) Multiple sequence alignment of hirudin and haemadin proteins from four leech species: \u003cem\u003eHirudo nipponia\u003c/em\u003e (Hnip), \u003cem\u003eHirudo medicinalis\u003c/em\u003e(Hmed), \u003cem\u003eHirudinaria manillensis\u003c/em\u003e (Hman), and \u003cem\u003eH. yanyuanensis\u003c/em\u003e (Hyan), alongside the prototype hirudin (GenBank: ALA22933.1). (B) Alignment of internal tandem repeats within a haemadin protein. The Red boxes denote signal peptide regions; the red triangles mark conserved cysteine residues; the black asterisks indicate stop codons.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-7119690/v1/a766f217cab8bf2b68e7c1c4.png"},{"id":88005459,"identity":"5add4bee-ea35-42b9-836e-28cb8afb57eb","added_by":"auto","created_at":"2025-07-31 10:42:29","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":2127432,"visible":true,"origin":"","legend":"\u003cp\u003eMultiple sequence alignment of progranulin proteins from \u003cem\u003eH. nipponia\u003c/em\u003e (Hnip), \u003cem\u003eH. medicinalis\u003c/em\u003e (Hmed), \u003cem\u003eH. manillensis\u003c/em\u003e (Hman), and \u003cem\u003eH. yanyuanensis\u003c/em\u003e (Hyan). The red boxes denote signal peptide regions; the red triangles mark conserved cysteine residues; the black asterisks indicate stop codons.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-7119690/v1/8fc2af932e817729f2b3e6a4.png"},{"id":88005464,"identity":"b9fe8279-c201-4e13-8df3-d4d39a67be06","added_by":"auto","created_at":"2025-07-31 10:42:30","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":902667,"visible":true,"origin":"","legend":"\u003cp\u003eAlignment of internal repeat sequences within the progranulin protein. The red triangles mark conserved cysteine residues.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-7119690/v1/f3bf0d727a76e9d021c8ff27.png"},{"id":88003871,"identity":"0a1f61e7-c1d6-41b2-86b9-7c46634983c4","added_by":"auto","created_at":"2025-07-31 10:34:29","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":560052,"visible":true,"origin":"","legend":"\u003cp\u003eHierarchical clustering heatmap of anticoagulant gene expression profiles based on RNA-Seq data from three leech species (blue indicates higher similarity). The \u003cem\u003eH. nipponia\u003c/em\u003e(Hnip1 ~ Hnip3 refers to SRA No. SRR26541742 ~ SRR26541740, respectively), the \u003cem\u003eH. manillensis \u003c/em\u003e(Hman1 ~ Hman3 refers to SRR26541746, SRR26541753, and SRR26541752, respectively) and \u003cem\u003eH. yanyuanensis \u003c/em\u003e(Hyan1 ~ Hyan3 refers to CRR1847694-CRR1847695, CRR1847696-CRR1847697, and CRR1847698-CRR1847699, respectively).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-7119690/v1/f4632deab8b764788c80f969.png"},{"id":99545305,"identity":"21a56f6f-0053-48a5-8136-664dd381cfce","added_by":"auto","created_at":"2026-01-05 16:05:39","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5323277,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7119690/v1/f85c09d1-d5f4-4276-a772-7b8144161ba2.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Comparative Genomics Unveils Unique Antithrombotic Gene Expansion in Haemadipsa yanyuanensis","fulltext":[{"header":"Introduction","content":"\u003cp\u003eCardiovascular diseases (CVDs) represent a major global public health challenge. According to the Global Burden of Disease study, the global CVD patient population reached 523\u0026nbsp;million in 2019, with associated mortality totaling 18.6\u0026nbsp;million cases [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. World Health Organization reports further indicate that CVD-related fatalities accounted for 32% of all-cause mortality in 2019, with ischemic heart disease and cerebrovascular events responsible for 85% of these deaths [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Epidemiological projections suggest a concerning 90% surge in CVD prevalence from 2025 to 2050 [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. Thrombosis, a central pathomechanism in CVDs, involves the activation of coagulation cascades and platelet-derived signaling pathways, ultimately leading to acute myocardial infarction and cerebral ischemia [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In light of this substantial clinical burden, the development of novel antithrombotic therapies is imperative.\u003c/p\u003e\u003cp\u003eHematophagous annelids have emerged as valuable models for developing thrombin-targeting therapeutics due to their evolutionarily optimized anticoagulant systems. Hirudotherapy, an ancient antithrombotic intervention, has been pharmacologically validated in modern research [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. For instance, hirudin HV1, a direct thrombin inhibitor isolated from H\u003cem\u003eirudo medicinalis\u003c/em\u003e, exhibits high-affinity anticoagulant activity by irreversibly binding to thrombin\u0026rsquo;s catalytic triad [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Recombinant hirudin analogs, including lepirudin, desirudin, and bivalirudin, have been approved by the FDA and EMA as first-line treatments for heparin-induced thrombocytopenia [\u003cspan additionalcitationids=\"CR9\" citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eWhile anticoagulant mechanisms in aquatic medicinal leeches have been extensively studied, their terrestrial counterparts, such as \u003cem\u003eHaemadipsa yanyuanensis\u003c/em\u003e, remain poorly understood, hindering comparative analyses of blood-feeding adaptations across leech species. Biochemical studies suggest that Haemadipsidae leeches possess phylogenetically distinct repertoires of hemostasis-modulating biomolecules. For example, haemadin, a thrombin inhibitor first identified in \u003cem\u003eHaemadipsa sylvestris\u003c/em\u003e [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], exhibits antithrombotic efficacy comparable to hirudin [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Subsequent studies in \u003cem\u003eHaemadipsa interrupta\u003c/em\u003e revealed that this protein demonstrates enhanced target specificity compared to classical hirudin [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. However, the molecular basis of anticoagulation in \u003cem\u003eH. yanyuanensis\u003c/em\u003e remains poorly understood. Although two anticoagulant proteins were detected in this species [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], their tertiary structures and amino acid sequences remain unresolved.\u003c/p\u003e\u003cp\u003eRecent advances in genomics have elucidated the anticoagulant gene repertoires of aquatic medicinal leeches, including \u003cem\u003eH. medicinalis\u003c/em\u003e [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], \u003cem\u003eHirudinaria manillensis\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], \u003cem\u003eWhitmania pigra\u003c/em\u003e [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], and \u003cem\u003eHirudo nipponia\u003c/em\u003e [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. In contrast, terrestrial Haemadipsidae species lack comparable genomic resolution, hindering comparative analyses of blood-feeding adaptations across leech ecotypes. This study addresses this gap by presenting the first high-quality genome assembly of \u003cem\u003eH. yanyuanensis\u003c/em\u003e, constructed with the BRAKER-plus integrative omics approach [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Subsequent genomic analyses identified four evolutionarily conserved antithrombotic gene modules: (1) coagulation cascade suppressors, (2) platelet activation antagonists, (3) fibrinolysis accelerators, and (4) tissue penetration enhancers. These findings provide critical insights into the evolutionary adaptations of terrestrial leeches and highlight their potential as a source of next-generation anticoagulant and thrombolytic drugs.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 DNA and RNA Sequencing\u003c/h2\u003e\u003cp\u003eIn this study, the specimens of \u003cem\u003eH. yanyuanensis\u003c/em\u003e were collected from Jiaozi Snow Mountain, Luquan County, Yunnan Province, China (26.023\u0026deg;N, 102.872\u0026deg;E). Following a 7-day gut evacuation protocol with digestive tract excision, paired-end Illumina and ultralong Nanopore sequencing libraries were prepared using established methodologies [\u003cspan additionalcitationids=\"CR21\" citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. The sequencing pipeline generated ultralong Oxford Nanopore Technologies (ONT) reads for de novo genome assembly alongside strand-specific RNA-seq libraries for transcriptional profiling.\u003c/p\u003e\u003cp\u003eGenomic and transcriptomic sequencing followed previous methods, with high-quality data generated via 150 bp paired-end sequencing on the Illumina HiSeq 2000 platform (Illumina Inc., San Diego, CA, USA).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Genome Assembly and Gene Prediction\u003c/h2\u003e\u003cp\u003eThe genome of \u003cem\u003eH. yanyuanensis\u003c/em\u003e was assembled using previously described protocols [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and genes with antithrombotic functionality were identified. Briefly, the preliminary nuclear genome assembly and mitochondrial genome assembly were performed using NextDenovo V2.5.0 in combination with a comprehensive suite of bioinformatics tools, while a HiC topological contact map was subsequently generated through visualization methods [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Genome completeness was assessed against the eukaryota_odb10 database using BUSCO v4.1.4, while assembly accuracy was evaluated through quality control analysis with Merqury v1.3. Then, a denovo repeat library for the repeat-masked genome was developed using RepeatModeler v2.0.3, the RepBase v20181026 database [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], and RepeatMasker v4.1.2p1.\u003c/p\u003e\u003cp\u003eGene annotation was performed using the BRAKERplus pipeline[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. RNA-Seq reads were first aligned to the genome assembly, followed by \u003cem\u003ede novo\u003c/em\u003e transcript reconstruction with Trinity v2.9.0, and coding sequences (CDSs) were predicted using GeneMarkS-T v5.1. Then, through literature review and mining of public repositories such as GenBank and UniProt, a reference library of antithrombotic genes was constructed. CDSs obtained from RNA-Seq and BRAKER predictions were queried against this library using the BLASTP algorithm to identify putative homologs. The filtered candidate genes were precisely mapped to the genome using Exonerate v2.2.0. Following manual refinement, the BRAKER prediction results were integrated with the GFF annotation file using AGAT v1.2.0, to generate a comprehensive annotation file for antithrombotic functional genes.\u003c/p\u003e\u003cp\u003eTo predict signal peptide regions within the candidate protein sequences, the online tool SignalP 6.0 [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e] was employed (accessed at \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://dtu.biolib.com/SignalP-6\u003c/span\u003e\u003cspan address=\"https://dtu.biolib.com/SignalP-6\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e on April 13, 2025). To investigate the phylogenetic relationships among gene family members, multiple sequence alignment was performed using MEGA v11.0.13, followed by phylogenetic tree construction with the Maximum Likelihood (ML) method using IQ-TREE v1.6.12. In addition, pairwise sequence similarity among candidate genes was assessed using the longest similarity index calculated with EMBOSS v6.6.0, providing further insights into their potential functions and evolutionary relationships.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Genetic Variation Analysis of Antithrombotic Genes\u003c/h2\u003e\u003cp\u003eThe protein-coding genes identified in this study were combined with gene sequences from \u003cem\u003eH. medicinalis\u003c/em\u003e, \u003cem\u003eH. manillensis\u003c/em\u003e, and \u003cem\u003eH. nipponia\u003c/em\u003e, as well as prototype gene sequences. These nucleotide sequences were translated into protein sequences using the \u0026ldquo;Translated Protein Sequences\u0026rdquo; function in MEGA with the default \u0026ldquo;standard\u0026rdquo; genetic code. The resulting protein sequences, including prototype sequences, were subjected to multiple sequence alignment using the ClustalW function in MEGA with default parameters. Pairwise sequence similarity was also calculated using the longest similarity index with EMBOSS. A phylogenetic tree based on protein sequences was constructed using IQ-TREE to investigate the evolutionary relationships among members of the HMEI gene family.\u003c/p\u003e\u003cp\u003eA comprehensive analysis was conducted by integrating the RNA-seq dataset of \u003cem\u003eH. yanyuanensis\u003c/em\u003e with the CDS reference sequences. Gene expression levels were quantified using Salmon v1.0.0, and TPM (transcripts per million) values were calculated. The TPM values of \u003cem\u003eH. yanyuanensis\u003c/em\u003e were integrated and grouped with those of blood-feeding leeches (\u003cem\u003eH. nipponia\u003c/em\u003e and \u003cem\u003eH. manillensis\u003c/em\u003e) from published datasets. Following this, an in-depth analysis of gene expression variation was conducted across the three hematophagous leech species. Due to the absence of corresponding RNA-seq data for \u003cem\u003eH. medicinalis\u003c/em\u003e, this species was excluded from the comparative analysis in this study.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e3.1 Gene Sequencing and Assembly\u003c/h2\u003e\u003cp\u003eIn this study, we generated 15.18 Gb of high-quality Oxford Nanopore (ONT) long-read data with an average read length of 11.65 kb. De novo assembly produced 37 contigs totaling 169.56 Mb, with a contig N50 of 7.92 Mb. Subsequent Hi-C sequencing using Illumina HiSeq yielded 43.48 Gb of data, enabling chromosome-scale scaffolding. The contigs were assembled into 18 scaffolds totaling 165.30 Mb, with an N50 of 18.42 Mb. Scaffold length distribution analysis (Figure. 1) revealed that the nine longest scaffolds represent nine chromosomes, comprising 164.47 Mb (99.5%) of the assembly. The remaining nine scaffolds are all \u0026lt;\u0026thinsp;0.3 Mb. In addition, 20.41 Gb of Illumina paired-end reads were used for mitochondrial genome assembly via GetOrganelle, resulting in a 17,851 bp circular mitochondrial genome. Altogether, the final \u003cem\u003eH. yanyuanensis\u003c/em\u003e genome assembly spans 165.32 Mb and consists of nine chromosomes, one mitochondrial genome, and nine unplaced scaffolds.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess assembly completeness, BUSCO (eukaryota_odb10) identified 97.6% of conserved orthologs, with 91.8% as complete single-copy genes. Merqury analysis produced a quality score of 39.03, reflecting high base-level accuracy. Repeat annotations revealed that 28.49% of the genome consists of repetitive elements, including retrotransposons and DNA transposons (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). No small RNAs, satellite sequences, or low-complexity regions were detected in this assembly. This study detected no small RNAs, satellites, or low-complexity fragments, contrasting sharply with previous findings in \u003cem\u003eH. nipponia\u003c/em\u003e and \u003cem\u003eH. manillensis.\u003c/em\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eDifferent repeat sequence type percentages in genomes of \u003cem\u003eHaemadipsa yanyuanensis\u003c/em\u003e, \u003cem\u003eHirudinaria manillensis\u003c/em\u003e, and \u003cem\u003eHirudo nipponia\u003c/em\u003e.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eItem\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eH. yanyuanensis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eH. manillensis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eH. nipponia\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRetroelements\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9.51\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6.59\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e9.89\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eDNA transposons\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.98\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.76\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e8.40\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eRolling-circles\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1.06\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.18\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eUnclassified\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13.17\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e8.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e10.71\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSmall RNA\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSatellites\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.02\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eSimple repeats\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2.77\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1.80\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.58\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLow complexity\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0.00\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eTotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e28.49\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e18.96\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e29.77\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Gene Prediction and Annotation\u003c/h2\u003e\u003cp\u003eA systematic analysis of antithrombotic biomolecules derived from leech saliva revealed five antithrombotic gene clusters and a total of 23 antithrombotic proteins. Using the BRAKER-plus pipeline, we conducted genome-wide gene prediction and structural annotation of antithrombotic protein-coding genes in \u003cem\u003eH. yanyuanensis\u003c/em\u003e, resulting in the identification of 15 gene families and 193 antithrombotic gene loci, including three putative pseudogenes. Further classification revealed that hirustasin, hirustasin-like, guamerin, piguamerin, bdellastasin, and poecistasin could not be clearly distinguished due to high sequence similarity in \u003cem\u003eHaemadipsa\u003c/em\u003e species. Therefore, these were collectively grouped into the hirustasin superfamily. Ultimately, 18 distinct gene/protein families related to anticoagulation were identified.\u003c/p\u003e\u003cp\u003eThese gene families include eight coagulation inhibitors that act primarily by targeting various steps in the coagulation cascade: haemadin [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], progranulin [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], antistasin [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], lefaxin [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], hirustasin [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], eglin [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], bdellin [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], and LDTI [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Three fibrinolytic enhancers were also identified: destabilase [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], GGT [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e], and LCI [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. Two platelet aggregation inhibitors, apyrase [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and lumbrokinase [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e], were also detected. In addition, one tissue permeability enhancer, hyaluronidase [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], was identified. Notably, we discovered an HMEI gene family comprising 53 members, which functions as a protease inhibitor and has potential anti-inflammatory activity [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Conversely, no gene members belonging to the therostasin, decorsin, or saratin families were detected in \u003cem\u003eH. yanyuanensis\u003c/em\u003e, suggesting species-specific gene loss in this lineage (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e3.3 Protein and Genetic Variation Analysis\u003c/h2\u003e\u003cp\u003eAmong the 15 anticoagulant gene families identified in \u003cem\u003eH. yanyuanensis\u003c/em\u003e, extensive genetic variation was detected among family members (Figure. S1\u0026ndash;S15). Comparative analysis revealed four distinct evolutionary patterns: two expansion modes, namely copy number expansion and coding sequence elongation, and two contraction modes involving reductions in these features.\u003c/p\u003e\u003cp\u003eThe first expansion pattern involved nine gene families, including antistasin, hirustasin, eglin, bdellin, LDTI, lumbrokinase, destabilase, LCI, and HMEI. Among them, eglin (13 members), lumbrokinase (7), and HMEI (53) showed approximately twice as many members as the corresponding families in the three reference leech species. The HMEI family, in particular, included one pseudogene (HMEI_Hyan52) and another gene (HMEI_Hyan11) with multiple nonsynonymous mutations and a premature stop codon in the C-terminal domain (Figure. S1), accounting for 27.5% of all identified anticoagulant-related genes (n\u0026thinsp;=\u0026thinsp;193). Notably, the LDTI, bdellin, and LCI families exhibited exceptionally large expansions. For example, 26 bdellin genes were annotated in \u003cem\u003eH. yanyuanensis\u003c/em\u003e, including one pseudogene (bdellin_Hyan26) containing a premature stop codon in its signal peptide region (Figure. S2). In contrast, the reference species \u003cem\u003eH. nipponia\u003c/em\u003e, \u003cem\u003eH. medicinalis\u003c/em\u003e, and \u003cem\u003eH. manillensis\u003c/em\u003e contained only 3, 2, and 1 bdellin genes, respectively, representing an expansion by a factor of 8.7 to 26. Similarly, the LDTI and LCI families had 16 and 25 members, whereas only a single-copy ortholog was found in each of the reference species, indicating 16-fold and 25-fold increases, respectively. Other families, such as destabilase, showed only moderate expansion and also included a pseudogene (destabilase_Hyan09). By contrast, contraction in gene copy number was observed in the lefaxin and apyrase families. The apyrase family had only two members in \u003cem\u003eH. yanyuanensis\u003c/em\u003e, compared to six, five, and five members in the three reference leeches, respectively. Similarly, the lefaxin family contained two members in \u003cem\u003eH. yanyuanensis\u003c/em\u003e, slightly fewer than the three found in each of the reference species (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eNumber of functional genes of \u003cem\u003eH. yanyuanensis\u003c/em\u003e, \u003cem\u003eH. nipponia\u003c/em\u003e, \u003cem\u003eH. manillensis\u003c/em\u003e, and \u003cem\u003eHirudo medicinalis.\u003c/em\u003e\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"6\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene family\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eH. yanyuanensis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eH. nipponia\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eH. manillensis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003e\u003cem\u003eH. medicinalis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003eFunction\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ehaemadin/\u003c/p\u003e\u003cp\u003ehirudin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecoagulation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eprogranulin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecoagulation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eantistasin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecoagulation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003elefaxin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecoagulation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etherostasin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecoagulation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ehirustasin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e27\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e19\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecoagulation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eeglin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e13\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecoagulation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ebdellin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e26\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecoagulation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLDTI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e16\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003ecoagulation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003edecorsin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eplatelet aggregation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esaratin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eplatelet aggregation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eapyrase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eplatelet aggregation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003elumbrokinase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003eplatelet aggregation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003edestabilase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003efibrinolysis enhancer\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGGT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003efibrinolysis enhancer\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLCI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003efibrinolysis enhancer\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ehyaluronidase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003etissue penetration enhancer\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHMEI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e53\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e21\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e18\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e20\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003einflammation inhibitor\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etotal\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e\u003cp\u003e193\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c3\"\u003e\u003cp\u003e86\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e\u003cp\u003e72\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e74\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c6\"\u003e\u003cp\u003e\u0026mdash;\u0026mdash;\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003eThe second expansion mode was characterized by significant elongation of protein-coding sequences. In several cases, the protein products were nearly twice as long as those of their orthologs in the reference medicinal leeches. This pattern was observed in four gene families: LDTI, haemadin (Figure. 2A), progranulin (Figure. 3), and antistasin (Figure. S3), yielding six antithrombotic polypeptides: LDTI_Hyan1 through LDTI_Hyan3, haemadin_Hyan3, progranulin_Hyan, and antistasin_Hyan1. For instance, haemadin_Hyan3 contained three internal tandem repeats, each consisting of six cysteine residues (Figure. 2B), and antistasin_Hyan1 included five repeats, each with ten conserved cysteines (Figure. S4). The progranulin gene was conserved as a single-copy in all four species, yet the number of cysteine residues varied dramatically: 69 in \u003cem\u003eH. nipponia\u003c/em\u003e, 66 in \u003cem\u003eH. manillensis\u003c/em\u003e, 72 in \u003cem\u003eH. medicinalis\u003c/em\u003e, and as many as 122 in \u003cem\u003eH. yanyuanensis\u003c/em\u003e (Figure. 3). The \u003cem\u003eH. yanyuanensis\u003c/em\u003e progranulin protein also contained nine conserved tandem repeats, each comprising twelve cysteine residues, whereas only five and six repeats were found in \u003cem\u003eH. nipponia\u003c/em\u003e and \u003cem\u003eH. medicinalis\u003c/em\u003e [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], respectively (Figure. 4). In contrast to this elongation pattern, the bdellin gene family displayed protein-coding sequence contraction. Despite the substantial increase in gene copy number, 22 bdellin genes encoded truncated proteins that were approximately half the length of their orthologs in the reference medicinal leeches, suggesting possible functional divergence or degeneration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab3\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 3\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eThe total TPM values (mean TPM\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of each gene family.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"4\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGene family\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003e\u003cem\u003eH. yanyuanensis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003e\u003cem\u003eH. nipponia\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003e\u003cem\u003eH. manillensis\u003c/em\u003e\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ehaemadin/hirudin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e802.3\u0026thinsp;\u0026plusmn;\u0026thinsp;633.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e7035.9\u0026thinsp;\u0026plusmn;\u0026thinsp;12,173.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e430.3\u0026thinsp;\u0026plusmn;\u0026thinsp;366.7\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003edecorsin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e12194.2\u0026thinsp;\u0026plusmn;\u0026thinsp;21117.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eprogranulin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e347.1\u0026thinsp;\u0026plusmn;\u0026thinsp;88.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e99.8\u0026thinsp;\u0026plusmn;\u0026thinsp;20.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e152.1\u0026thinsp;\u0026plusmn;\u0026thinsp;47.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eantistasin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e221.6\u0026thinsp;\u0026plusmn;\u0026thinsp;222.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1040.4\u0026thinsp;\u0026plusmn;\u0026thinsp;183.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e820.0\u0026thinsp;\u0026plusmn;\u0026thinsp;385.5\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003elefaxin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2736.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1077.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5669.8\u0026thinsp;\u0026plusmn;\u0026thinsp;2920.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e14865.0\u0026thinsp;\u0026plusmn;\u0026thinsp;4011.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003etherostasin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e98.5\u0026thinsp;\u0026plusmn;\u0026thinsp;160.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e70.1\u0026thinsp;\u0026plusmn;\u0026thinsp;68.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ehirustasin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e18465.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15536.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e28508.9\u0026thinsp;\u0026plusmn;\u0026thinsp;2582.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e8991.3\u0026thinsp;\u0026plusmn;\u0026thinsp;1724.3\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eeglin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9565.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2142.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6237.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2736.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e11984.0\u0026thinsp;\u0026plusmn;\u0026thinsp;3808.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ebdellin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e33174.5\u0026thinsp;\u0026plusmn;\u0026thinsp;15723\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e16079.2\u0026thinsp;\u0026plusmn;\u0026thinsp;6526.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2550.5\u0026thinsp;\u0026plusmn;\u0026thinsp;1346.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLDTI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e2630.4\u0026thinsp;\u0026plusmn;\u0026thinsp;2129.6\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e4909.9\u0026thinsp;\u0026plusmn;\u0026thinsp;4641.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3244.8\u0026thinsp;\u0026plusmn;\u0026thinsp;1808.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eHMEI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5036.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2176.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e1395.7\u0026thinsp;\u0026plusmn;\u0026thinsp;1079.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2859.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2282.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003esaratin\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e0.0\u0026thinsp;\u0026plusmn;\u0026thinsp;0.0\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5700.4\u0026thinsp;\u0026plusmn;\u0026thinsp;9870.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e1317.1\u0026thinsp;\u0026plusmn;\u0026thinsp;2051.2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eapyrase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e525.7\u0026thinsp;\u0026plusmn;\u0026thinsp;285.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e200.1\u0026thinsp;\u0026plusmn;\u0026thinsp;286.4\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e448.6\u0026thinsp;\u0026plusmn;\u0026thinsp;653.6\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003elumbrokinase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9450.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6577.9\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e5576.4\u0026thinsp;\u0026plusmn;\u0026thinsp;7236.3\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e0.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003edestabilase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3435.4\u0026thinsp;\u0026plusmn;\u0026thinsp;700.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e6019.5\u0026thinsp;\u0026plusmn;\u0026thinsp;2868.7\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e3926.3\u0026thinsp;\u0026plusmn;\u0026thinsp;4329\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eGGT\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e145.1\u0026thinsp;\u0026plusmn;\u0026thinsp;32.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e78.7\u0026thinsp;\u0026plusmn;\u0026thinsp;86.5\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e22.0\u0026thinsp;\u0026plusmn;\u0026thinsp;17.1\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLCI\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e10250.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7628.1\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e889.2\u0026thinsp;\u0026plusmn;\u0026thinsp;243.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e2236.2\u0026thinsp;\u0026plusmn;\u0026thinsp;460.4\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003ehyaluronidase\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e216.0\u0026thinsp;\u0026plusmn;\u0026thinsp;33.2\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e\u003cp\u003e109.2\u0026thinsp;\u0026plusmn;\u0026thinsp;94.8\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e\u003cp\u003e313.9\u0026thinsp;\u0026plusmn;\u0026thinsp;245.0\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e3.4 Gene Expression Analysis\u003c/h2\u003e\u003cp\u003eIntegration of previously published data with the transcriptomic profiles of \u003cem\u003eH. yanyuanensis\u003c/em\u003e revealed marked interspecific differences in gene expression patterns (Table\u0026nbsp;\u003cspan refid=\"Tab3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). Transcriptomic profiling revealed high expression levels (mean TPM\u0026thinsp;\u0026plusmn;\u0026thinsp;SD) of bdellin (33,174.5\u0026thinsp;\u0026plusmn;\u0026thinsp;15,723.0), eglin (9,565.6\u0026thinsp;\u0026plusmn;\u0026thinsp;2,142.5), LCI (10,250.2\u0026thinsp;\u0026plusmn;\u0026thinsp;7,628.1), lumbrokinase (9,450.5\u0026thinsp;\u0026plusmn;\u0026thinsp;6,577.9), HMEI (5,036.7\u0026thinsp;\u0026plusmn;\u0026thinsp;2,176.8), and hirustasin (18,465.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15,536.9), suggesting a functional link between gene family expansion and adaptation to terrestrial blood-feeding.\u003c/p\u003e\u003cp\u003eAmong these, the bdellin family showed exceptionally high expression levels, far exceeding those of any anticoagulant gene family in the reference medicinal leeches. In contrast, the antistasin (221.6\u0026thinsp;\u0026plusmn;\u0026thinsp;222.9) and lefaxin (2,736.2\u0026thinsp;\u0026plusmn;\u0026thinsp;1,077.5) families displayed significantly lower expression in \u003cem\u003eH. yanyuanensis\u003c/em\u003e compared to \u003cem\u003eH. nipponia\u003c/em\u003e and \u003cem\u003eH. manillensis\u003c/em\u003e. Hierarchical clustering further revealed species-specific transcriptomic profiles, with \u003cem\u003eH. yanyuanensis\u003c/em\u003e and \u003cem\u003eH. nipponia\u003c/em\u003e forming a closer clade in accordance with their phylogenetic relationship (Figure. 5).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003e4.1 \u003cstrong\u003eGenome Assembly and Gene Mining\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study used whole-genome sequencing to explore evolutionary divergence between \u003cem\u003eH. yanyuanensis\u003c/em\u003e and three reference leeches (\u003cem\u003eH. medicinalis\u003c/em\u003e, \u003cem\u003eH. manillensis\u003c/em\u003e, and \u003cem\u003eH. nipponia\u003c/em\u003e), uncovering conserved syntenic blocks alongside lineage-specific innovations. Employing nanopore third-generation sequencing (ONT) combined with Illumina Hi-C, survey, and RNA-Seq data enabled high-precision base calling and chromosome-level assembly, resulting in a genome of 165.31 Mb across 9 chromosomes plus a near-complete mitochondrial genome. Assembly quality was supported by 95.9% BUSCO completeness and a Merqury quality score of 39.9. Notably, 193 antithrombotic protein-coding genes were annotated\u0026mdash;approximately 2.2 times more than in the three medicinal leech species (86 in \u003cem\u003eH. nipponia\u003c/em\u003e, 74 in \u003cem\u003eH. medicinalis\u003c/em\u003e, and 72 in \u003cem\u003eH. manillensis\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003e4.2\u0026nbsp;Gene Expansion\u0026ndash;Contraction Model\u003c/p\u003e\n\u003cp\u003eComparative analysis revealed that \u003cem\u003eH. yanyuanensis\u003c/em\u003e harbors 193 antithrombotic genes, approximately 2.2 to 2.7 times more than those identified in \u003cem\u003eH. nipponia\u003c/em\u003e, \u003cem\u003eH. medicinalis\u003c/em\u003e, and \u003cem\u003eH. manillensis\u003c/em\u003e, yet these are distributed among only 15 gene families, compared to 17 to 18 in the reference species [17, 20]. This pattern reflects lineage-specific remodeling through selective gene expansion and contraction. However, substantial divergence was observed in gene family size and structure. The bdellin, LDTI, and LCI families experienced large-scale expansions, with up to 26 times more members than in the references, while apyrase and lefaxin showed notable reductions. Some expanded families also contained pseudogenes or truncated sequences, indicating possible functional shifts. Moreover, several proteins such as haemadin_Hyan3 and progranulin_Hyan exhibited significant elongation due to internal tandem repeats and increased cysteine content. For example, progranulin_Hyan encodes 122 cysteine residues across nine conserved repeat units, nearly twice the number found in its homologs, implying enhanced structural complexity. These findings indicate that \u003cem\u003eH. yanyuanensis\u003c/em\u003e has adopted a distinct contraction and expansion strategy. Through gene duplication, domain expansion, and potential gene repurposing [45], this mechanism likely enhances its antithrombotic capacity and reflects an adaptive response to environmental pressures. Such a pattern represents a previously unreported evolutionary trajectory among terrestrial leeches.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e4.3\u0026nbsp;Differences in Gene Family Expression Levels\u003c/p\u003e\n\u003cp\u003eDue to the absence of RNA-seq data for \u003cem\u003eH. medicinalis\u003c/em\u003e, gene expression comparisons were performed only among \u003cem\u003eH. yanyuanensis\u003c/em\u003e, \u003cem\u003eH. nipponia\u003c/em\u003e, and \u003cem\u003eH. manillensis\u003c/em\u003e. In \u003cem\u003eH. yanyuanensis\u003c/em\u003e, gene families that showed notable expansion, including eglin, bdellin, HMEI, and LCI, generally exhibited higher expression levels than in the reference species. Although LDTI and hirustasin did not follow this trend, the overall results suggest that gene family expansion often correlates with increased transcriptional activity. The observed tissue-specific expression patterns imply that divergent expression among conserved genes may contribute to ecological adaptation and niche differentiation in \u003cem\u003eHaemadipsa\u003c/em\u003e species.\u003c/p\u003e\n\u003cp\u003e4.4\u0026nbsp;Biomedical Potential and Conservation Considerations of \u003cem\u003eHaemadipsa\u003c/em\u003e Species\u003c/p\u003e\n\u003cp\u003eRecent studies on terrestrial leeches have mainly focused on using invertebrate-derived DNA (iDNA) for monitoring vertebrate biodiversity [46, 47]. In parallel, pharmacological research has begun to highlight their antithrombotic potential, with compounds such as haemadin and the newly identified sylvestin showing significant bioactivity [12, 48]. Functional validation of molecules like decorsin and other haemadins in \u003cem\u003eH. interrupta\u003c/em\u003e has further supported their clinical relevance [13]. These findings position \u003cem\u003eHaemadipsa\u003c/em\u003e species, including \u003cem\u003eH. yanyuanensis\u003c/em\u003e, as promising sources of novel antithrombotic agents.\u0026nbsp;While habitat degradation has led to a decline in aquatic leech populations [49-52], forest-dwelling \u003cem\u003eHaemadipsa\u003c/em\u003e species have remained relatively stable due to their distribution in tropical and subtropical regions. As a result, new species continue to be discovered [53]. Their ecological resilience, combined with lineage-specific expansion of antithrombotic gene families, underscores their pharmacological potential and value in future drug discovery.\u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThis study reports the first chromosome-level genome assembly of the terrestrial leech \u003cem\u003eH. yanyuanensis\u003c/em\u003e, uncovering a distinct evolutionary trajectory of antithrombotic gene adaptation. Despite harboring fewer antithrombotic gene families (15) compared to aquatic medicinal leeches (17\u0026ndash;18), \u003cem\u003eH. yanyuanensis\u003c/em\u003e possesses a markedly higher number of antithrombotic genes (193), with key families such as bdellin, LDTI, and LCI exhibiting massive copy number expansions, up to 26-fold. Additionally, elongation of coding sequences and tandem repeat structures in gene families like progranulin and haemadin further support functional innovation under terrestrial selective pressures.\u003c/p\u003e\n\u003cp\u003eThese findings highlight a contraction-expansion mechanism driving lineage-specific adaptation in \u003cem\u003eHaemadipsa\u003c/em\u003e, likely reflecting ecological specialization in blood-feeding niches. By bridging the genomic gap in terrestrial leeches, this work provides both evolutionary insight and a valuable resource for antithrombotic drug discovery, offering new molecular targets for the treatment of cardiovascular and thrombotic diseases.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eConflict of Interest\u003c/p\u003e\n\u003cp\u003eThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.\u003c/p\u003e\n\u003cp\u003eAuthor Contributions\u003c/p\u003e\n\u003cp\u003eConceptualization, Z. L., G. L., D. K., and Y. L.; Methodology, Y. L. and G. L.; Software, Y. L. and G. L.; Validation, Formal Analysis, Investigation, Resources, Data Curation, and Visualization, Z. L., G. L., Y. L., F. Z., S. F., D. Y., X. T., and L. T.; Writing and Editing, Z. L., G. L., and Y. L.; Supervision, Project Administration, and Funding Acquisition, Z. L., G. L., and Y. L. All authors have read and agreed to the published version of the manuscript. \u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (82260742 and 32260132), the Yunnan Provincial University Serving Key Industry Science and Technology Special Project (FWCY-ZD2024009), the Joint Special Project for Basic Research of Local Universities in Yunnan Province (202301BA070001-105 and 202101BA070001-164), the 2023 Frontier Research Team Program of Kunming University, the Key Laboratory of Jiangxi Province for Biological Invasion and Biosecurity (2023SSY02111), National Key R\u0026amp;D Program of China (2023YFF1305000), the Yunnan Ten Thousand Talents Program for Young Top Notch Talents (YNWR-QNBJ-2020-122), Special Project of Science-Technology Talent and Platform in Yunnan Province (202105AC160042), and Yunnan International Joint Laboratory with South and Southeast Asia for the Integrated Development of Animal-Derived Anti-Thrombosis Chinese Medicine (202503AP140025).\u003c/p\u003e\n\u003cp\u003eSupplementary Materials\u003c/p\u003e\n\u003cp\u003eSupplementary File_S1_genome. fa: Genome assembly of \u003cem\u003eH. yanyuanensis\u003c/em\u003e; Supplementary File_S2_all_CDS. fa: CDSs of all predicted protein-coding genes; Supplementary File_S3_antithrombotic_CDS. fa: CDSs of 193 antithrombotic genes; Supplementary Figures_S1 to S14. docx: Sequence alignments of antithrombotic protein families not shown in the main text. Supplementary data to this article can be found online at \u003cstrong\u003ehttps://doi.org/10.6084/m9.figshare.29369828.v3 \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eData Availability Statement\u003c/p\u003e\n\u003cp\u003eThe raw genomic data generated in this study have been deposited at Genome Sequence Archive (GSA) portal of the China National Center for Bioinformation (\u003cstrong\u003ehttps://ngdc.cncb.ac.cn/gsa\u003c/strong\u003e\u003cstrong\u003e) \u003c/strong\u003ewithaccession number of CRA025628. This repository is maintained by the Chinese Academy of Sciences, following international standards for biological data sharing.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRoth GA, Mensah GA, Johnson CO, Addolorato G, Ammirati E, Baddour LM\u003cem\u003e et al\u003c/em\u003e. Global burden of cardiovascular diseases and risk factors, 1990\u0026ndash;2019: update from the GBD 2019 study. Journal of the American college of cardiology\u003cem\u003e \u003c/em\u003e2020, 76(25):2982-3021.doi:https://doi.org/10.1016/j.jacc.2020.09.010\u003c/li\u003e\n\u003cli\u003eWHO. Cardiovascular diseases (CVDs). 2021. https://www.who.int/europe/news-room/fact-sheets/item/cardiovascular-diseases. Accessed April 26, 2025.\u003c/li\u003e\n\u003cli\u003eChong B, Jayabaskaran J, Jauhari SM, Chan SP, Goh R, Kueh MTW\u003cem\u003e et al\u003c/em\u003e. Global burden of cardiovascular diseases: projections from 2025 to 2050. European journal of preventive cardiology\u003cem\u003e \u003c/em\u003e2024:zwae281.doi: https://doi.org/10.1093/eurjpc/zwac281\u003c/li\u003e\n\u003cli\u003eDeLoughery TG. Basics of coagulation. Hemostasis and thrombosis\u003cem\u003e \u003c/em\u003e2019:1-9.doi:https://doi.org/10.1007/978-3-030-19330-0_1\u003c/li\u003e\n\u003cli\u003eSingh AP. Medicinal leech therapy (hirudotherapy): a brief overview. Complementary therapies in clinical practice\u003cem\u003e \u003c/em\u003e2010, 16(4):213-215.doi:https://doi.org/10.1016/j.ctcp.2009.11.005\u003c/li\u003e\n\u003cli\u003eNair HK, Ahmad NW, Lee HL, Ahmad N, Othamn S, Mokhtar NSHM\u003cem\u003e et al\u003c/em\u003e. Hirudotherapy in wound healing. The international journal of lower extremity wounds\u003cem\u003e \u003c/em\u003e2022, 21(4):425-431.doi:https://doi.org/10.1177/1534734620948299\u003c/li\u003e\n\u003cli\u003eSun Y, Wang D. Protein-engineered Hirudins with enhanced anticoagulant activities developed by a molecular integrative approach. Journal of medicinal chemistry\u003cem\u003e \u003c/em\u003e2024, 67(5):3203-3204.doi:https://doi.org/10.1017/mdh.2023.17\u003c/li\u003e\n\u003cli\u003ePatel VB, Snyder J, Shopnick RI. Successful use of low dose r-Hirudin (Refludan\u0026reg;) for recurrentdialysis catheter thrombosis in a patient with heparin induced thrombocytopenia. Thrombosis and haemostasis\u003cem\u003e \u003c/em\u003e1999, 82(09):1205-1206.doi:https://doi.org/10.1055/s-0037-1614360\u003c/li\u003e\n\u003cli\u003eScott B, Tam CW, Moon RS. Transfemoral transcatheter aortic valve replacement in a patient with heparin-induced thrombocytopenia: intraoperative anticoagulation management with bivalirudin (Angiomax). Journal of cardiothoracic and vascular anesthesia\u003cem\u003e \u003c/em\u003e2015, 29(6):1603-1605.doi:https://doi.org/10.1053/j.jvca.2014.10.018\u003c/li\u003e\n\u003cli\u003eThompson CA. New direct thrombin inhibitor becomes available. American journal of health-system pharmacy\u003cem\u003e \u003c/em\u003e2010, 67(8):588.doi:https://doi.org/10.2146/news100027\u003c/li\u003e\n\u003cli\u003eStrube K-H, Kr\u0026ouml;ger B, Bialojan S, Otte M, Dodt J. Isolation, sequence analysis, and cloning of haemadin. an anticoagulant peptide from the Indian leech. Journal of biological chemistry\u003cem\u003e \u003c/em\u003e1993, 268(12):8590-8595.doi:https://doi.org/10.1016/S0021-9258(18)52916-1\u003c/li\u003e\n\u003cli\u003eAcquasaliente L, Pierangelini A, Pagotto A, Pozzi N, De Filippis V. From haemadin to haemanorm: Synthesis and characterization of full‐length haemadin from the leech Haemadipsa sylvestris and of a novel bivalent, highly potent thrombin inhibitor (haemanorm). Protein science\u003cem\u003e \u003c/em\u003e2023, 32(12):e4825.doi:https://doi.org/10.1002/pro.4825\u003c/li\u003e\n\u003cli\u003eM\u0026uuml;ller C, Sponholz D, Tolksdorf C, Rauch BH, Kvist S. Identification and functional characterization of multiple haemadins and an oligomeric decorsin in the Asian land leech Haemadipsa interrupta. Parasitology research\u003cem\u003e \u003c/em\u003e2024, 123(11):390.doi:https://doi.org/10.1007/s00436-024-08404-w\u003c/li\u003e\n\u003cli\u003eZhao R. A new potent inhibitor of thrombin from the leech Haemendipsa yanyuanensis. Journal of shanghai university (English Edition)\u003cem\u003e \u003c/em\u003e2001, 5:86-88.doi:https://doi.org/10.1007/s11741-001-0035-3\u003c/li\u003e\n\u003cli\u003eBabenko VV, Podgorny OV, Manuvera VA, Kasianov AS, Manolov AI, Grafskaia EN\u003cem\u003e et al\u003c/em\u003e. Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches. Bmc genomics\u003cem\u003e \u003c/em\u003e2020, 21:1-16.doi:https://doi.org/10.1186/s12864-020-6748-0\u003c/li\u003e\n\u003cli\u003eKvist S, Manzano-Mar\u0026iacute;n A, de Carle D, Trontelj P, Siddall ME. Draft genome of the European medicinal leech Hirudo medicinalis (Annelida, Clitellata, Hirudiniformes) with emphasis on anticoagulants. Scientific reports\u003cem\u003e \u003c/em\u003e2020, 10(1):9885.doi:https://doi.org/10.1038/s41598-020-66749-5\u003c/li\u003e\n\u003cli\u003eLiu Z, Zhao F, Huang Z, Hu Q, Meng R, Lin Y\u003cem\u003e et al\u003c/em\u003e. Revisiting the Asian buffalo leech (Hirudinaria manillensis) genome: focus on antithrombotic genes and their corresponding proteins. Genes\u003cem\u003e \u003c/em\u003e2023, 14(11):2068.doi:https://doi.org/10.3390/genes14112068\u003c/li\u003e\n\u003cli\u003eLiu Z, Zhao F, Huang Z, He B, Liu K, Shi F\u003cem\u003e et al\u003c/em\u003e. A chromosome-level genome assembly of the non-hematophagous leech Whitmania pigra (Whitman 1884): identification and expression analysis of antithrombotic genes. Genes\u003cem\u003e \u003c/em\u003e2024, 15(2):164.doi:https://doi.org/10.3390/genes15020164\u003c/li\u003e\n\u003cli\u003eZheng J, Wang X, Feng T, Rehman Su, Yan X, Shan H\u003cem\u003e et al\u003c/em\u003e. Molecular mechanisms underlying hematophagia revealed by comparative analyses of leech genomes. Gigascience\u003cem\u003e \u003c/em\u003e2023, 12:giad023.doi: https://doi.org/10.1093/gigascience/giad023\u003c/li\u003e\n\u003cli\u003eZhao F, Huang Z, He B, Liu K, Li J, Liu Z\u003cem\u003e et al\u003c/em\u003e. Comparative genomics of two Asian medicinal leeches Hirudo nipponia and Hirudo tianjinensis: with emphasis on antithrombotic genes and their corresponding proteins. International journal of biological macromolecules\u003cem\u003e \u003c/em\u003e2024, 270:132278.doi:https://doi.org/10.1016/j.ijbiomac.2024.132278\u003c/li\u003e\n\u003cli\u003eZhao F, Huang Z, Tang L, Zhang W, Liu Z, Lin G. Comparative genomics of three non-hematophagous leeches (Whitmania spp.) with emphasis on antithrombotic biomolecules. Frontiers in genetics\u003cem\u003e \u003c/em\u003e2025, 16:1548006.doi:https://doi.org/10.3389/fgene.2025.1548006\u003c/li\u003e\n\u003cli\u003eZhao F, Huang Z, Tang L, He B, Liu Z, Lin G. Comparative genomics of three non-hematophagous leeches (Whitmania spp.): focusing on antithrombotic genes. bioRxiv\u003cem\u003e \u003c/em\u003e2024:2024.2005. 2008.590400.doi:https://doi.org/10.1101/2024.05.08.590400\u003c/li\u003e\n\u003cli\u003eZhou C, McCarthy S, Durbin R. YaHS: yet another Hi-C scaffolding tool. Bioinformatics\u003cem\u003e \u003c/em\u003e2023, 39(1).doi:https://doi.org/10.1093/bioinformatics/btac808\u003c/li\u003e\n\u003cli\u003eDurand NC, Robinson JT, Shamim MS, Machol I, Mesirov JP, Lander ES\u003cem\u003e et al\u003c/em\u003e. Juicebox provides a visualization system for Hi-C contact maps with unlimited zoom. Cell systems\u003cem\u003e \u003c/em\u003e2016, 3(1):99-101.doi:https://doi.org/10.1016/j.cels.2015.07.012\u003c/li\u003e\n\u003cli\u003eBao W, Kojima KK, Kohany O. Repbase update, a database of repetitive elements in eukaryotic genomes. Mobile dna\u003cem\u003e \u003c/em\u003e2015, 6:1-6.doi:https://doi.org/10.1186/s13100-015-0041-9\u003c/li\u003e\n\u003cli\u003eSaenko S, Hoff KJ, Stanke M. Annotation of 200 insect genomes with BRAKER for consistent comparisons across species. bioRxiv\u003cem\u003e \u003c/em\u003e2025:2025.2004. 2017.649312.doi: https://doi.org/10.1101/2025.04.17.649312\u003c/li\u003e\n\u003cli\u003eTeufel F, Almagro Armenteros JJ, Johansen AR, G\u0026iacute;slason MH, Pihl SI, Tsirigos KD\u003cem\u003e et al\u003c/em\u003e. SignalP 6.0 predicts all five types of signal peptides using protein language models. Nature biotechnology\u003cem\u003e \u003c/em\u003e2022, 40(7):1023-1025.doi:https://doi.org/10.1038/s41587-021-01156-3\u003c/li\u003e\n\u003cli\u003eKostromina MA, Tukhovskaya EA, Shaykhutdinova ER, Slashcheva GA, Ismailova AM, Palikov VA\u003cem\u003e et al\u003c/em\u003e. Screening of the promising direct thrombin inhibitors from haematophagous organisms. Part I: recombinant analogues and their antithrombotic activity in vitro. Biomedicines\u003cem\u003e \u003c/em\u003e2021, 10(1):11.doi:https://doi.org/10.3390/biomedicines10010011\u003c/li\u003e\n\u003cli\u003eTanaka Y, Suzuki G, Matsuwaki T, Hosokawa M, Serrano G, Beach TG\u003cem\u003e et al\u003c/em\u003e. Progranulin regulates lysosomal function and biogenesis through acidification of lysosomes. Human molecular genetics\u003cem\u003e \u003c/em\u003e2017, 26(5):969-988.doi:https://doi.org/10.1093/hmg/ddx011\u003c/li\u003e\n\u003cli\u003eDunwiddie C, Thornberry N, Bull H, Sardana M, Friedman P, Jacobs J\u003cem\u003e et al\u003c/em\u003e. Antistasin, a leech-derived inhibitor of factor Xa: kinetic analysis of enzyme inhibition and identification of the reactive site. Journal of biological chemistry\u003cem\u003e \u003c/em\u003e1989, 264(28):16694-16699.doi:https://doi.org/10.1016/S0021-9258(19)84761-0\u003c/li\u003e\n\u003cli\u003eFaria F, Kelen EMA, Sampaio CA, Bon C, Duval N, Chudzinski-Tavassi AM. A new factor Xa inhibitor (lefaxin) from the Haementeria depressa leech. Thrombosis and haemostasis\u003cem\u003e \u003c/em\u003e1999, 82(11):1469-1473.doi:https://doi.org/10.1055/s-0037-1614857\u003c/li\u003e\n\u003cli\u003eS\u0026ouml;llner C, Mentele R, Eckerskorn C, Fritz H, Sommerhoff CP. Isolation and characterization of hirustasin, an antistasin‐type serine‐proteinase inhibitor from the medical leech Hirudo medicinalis. European journal of biochemistry\u003cem\u003e \u003c/em\u003e1994, 219(3):937-943.doi:https://doi.org/10.1111/j.1432-1033.1994.tb18575.x\u003c/li\u003e\n\u003cli\u003eRenesto P, FERRER‐LOPEZ P, Chignard M. Eglin c and heparin inhibition of platelet activation induced by cathepsin G or human neutrophils. Annals of the New York academy of sciences\u003cem\u003e \u003c/em\u003e1991, 624(1):321-324.doi:https://doi.org/10.1111/j.1749-6632.1991.tb17033.x\u003c/li\u003e\n\u003cli\u003eCheng R, Tang X, Long A, Mwangi J, Lai R, Sun R\u003cem\u003e et al\u003c/em\u003e. Purification and characterization of a novel anti-coagulant from the leech Hirudinaria manillensis. Zoological research\u003cem\u003e \u003c/em\u003e2019, 40(3):205.doi:https://doi.org/10.24272/j.issn.2095-8137.2019.037\u003c/li\u003e\n\u003cli\u003eTanaka AS, Silva MM, Torquato RJ, Noguti MA, Sampaio CA, Fritz H\u003cem\u003e et al\u003c/em\u003e. Functional phage display of leech-derived tryptase inhibitor (LDTI): construction of a library and selection of thrombin inhibitors. Federation of european biochemical societies letters\u003cem\u003e \u003c/em\u003e1999, 458(1):11-16.doi:https://doi.org/10.1016/S0014-5793(99)01106-0\u003c/li\u003e\n\u003cli\u003eMarin E, Kornilov DA, Bukhdruker SS, Aleksenko VA, Manuvera VA, Zinovev EV\u003cem\u003e et al\u003c/em\u003e. Structural insights into thrombolytic activity of destabilase from medicinal leech. Scientific reports\u003cem\u003e \u003c/em\u003e2023, 13(1):6641.doi:https://doi.org/10.1038/s41598-023-32459-x\u003c/li\u003e\n\u003cli\u003eFriedrich T, Kr\u0026ouml;ger B, Koerwer W, Strube KH, Meyer T, Bialojan S. An isopeptide bond splitting enzyme from Hirudo medicinalis similar to \u0026gamma;‐glutamyl transpeptidase. European journal of biochemistry\u003cem\u003e \u003c/em\u003e1998, 256(2):297-302.doi:https://doi.org/10.1046/j.1432-1327.1998.2560297.x\u003c/li\u003e\n\u003cli\u003eArolas JL, Castillo V, Bronsoms S, Aviles FX, Ventura S. Designing out disulfide bonds of leech carboxypeptidase inhibitor: implications for its folding, stability and function. Journal of molecular biology\u003cem\u003e \u003c/em\u003e2009, 392(2):529-546.doi:https://doi.org/10.1016/j.jmb.2009.06.049\u003c/li\u003e\n\u003cli\u003eReverter D, Fern\u0026aacute;ndez-Catal\u0026aacute;n C, Baumgartner R, Pf\u0026auml;nder R, Huber R, Bode W\u003cem\u003e et al\u003c/em\u003e. Structure of a novel leech carboxypeptidase inhibitor determined free in solution and in complex with human carboxypeptidase A2. Nature structural biology\u003cem\u003e \u003c/em\u003e2000, 7(4):322-328.doi:https://doi.org/10.1038/74092\u003c/li\u003e\n\u003cli\u003eP\u0026eacute;rez de Le\u0026oacute;n AA, Tabachnick WJ. Apyrase activity and adenosine diphosphate induced platelet aggregation inhibition by the salivary gland proteins of culicoides variipennis, the north american vector of bluetongue viruses. Veterinary parasitology\u003cem\u003e \u003c/em\u003e1996, 61(3-4):327-338.doi:https://doi.org/10.1016/0304-4017(95)00828-4\u003c/li\u003e\n\u003cli\u003eAgoncillo A. Meta-analysis on the safety and efficacy of lumbrokinase in peripheral arterial disease. European heart journal acute cardiovascular care\u003cem\u003e \u003c/em\u003e2021, 10(Supplement_1):zuab020. 219.doi:https://doi.org/10.1093/ehjacc/zuab020.219\u003c/li\u003e\n\u003cli\u003eDas BK. An overview on hirudotherapy/leech therapy. Ind Res J Pharm Sci\u003cem\u003e \u003c/em\u003e2014, 1(1):34,\u003c/li\u003e\n\u003cli\u003eXu K, Zhou M, Wu F, Tang X, Lu Q, Lai R\u003cem\u003e et al\u003c/em\u003e. Identification and characterization of a novel elastase inhibitor from Hirudinaria manillensis. Chinese journal of natural medicines\u003cem\u003e \u003c/em\u003e2021, 19(7):540-544.doi:https://doi.org/10.1016/S1875-5364(21)60054-7\u003c/li\u003e\n\u003cli\u003eSmith TP, Alshafie TA, Cruz CP, Fan C-Y, Brown AT, Wang Y\u003cem\u003e et al\u003c/em\u003e. Saratin, an inhibitor of collagen-platelet interaction, decreases venous anastomotic intimal hyperplasia in a canine dialysis access model. Vascular and endovascular surgery\u003cem\u003e \u003c/em\u003e2003, 37(4):259-269.doi:https://doi.org/10.1177/153857440303700405\u003c/li\u003e\n\u003cli\u003eMartin A, Orgogozo V. The loci of repeated evolution: a catalog of genetic hotspots of phenotypic variation. Evolution\u003cem\u003e \u003c/em\u003e2013, 67(5):1235-1250.doi:https://doi.org/10.1111/evo.12081\u003c/li\u003e\n\u003cli\u003eLynggaard C, Oceguera‐Figueroa A, Kvist S, Gilbert MTP, Bohmann K. The potential of aquatic bloodfeeding and nonbloodfeeding leeches as a tool for iDNA characterisation. Molecular ecology resources\u003cem\u003e \u003c/em\u003e2022, 22(2):539-553.doi:https://doi.org/10.1111/1755-0998.13486\u003c/li\u003e\n\u003cli\u003eJi Y, Baker CC, Popescu VD, Wang J, Wu C, Wang Z\u003cem\u003e et al\u003c/em\u003e. Measuring protected-area effectiveness using vertebrate distributions from leech iDNA. Nature communications\u003cem\u003e \u003c/em\u003e2022, 13(1):1555.doi:https://doi.org/10.1038/s41467-022-28778-8\u003c/li\u003e\n\u003cli\u003eZhang Z, Shen C, Fang M, Han Y, Long C, Liu W\u003cem\u003e et al\u003c/em\u003e. Novel contact\u0026ndash;kinin inhibitor sylvestin targets thromboinflammation and ameliorates ischemic stroke. Cellular and molecular life sciences\u003cem\u003e \u003c/em\u003e2022, 79(5):240.doi:https://doi.org/10.1007/s00018-022-04257-7\u003c/li\u003e\n\u003cli\u003eCopena D, G\u0026oacute;mez-Mart\u0026iacute;n M. Spanish\u0026ndash;french leech trade and its consequences: From the increase in medical demand to resource depletion and technical innovation. Medical history\u003cem\u003e \u003c/em\u003e2024, 68(1):42-59.doi:https://doi.org/10.1017/mdh.2024.5\u003c/li\u003e\n\u003cli\u003eChen A, Liu B, Zhou R, Zhang H, Zhou L, Xie X\u003cem\u003e et al\u003c/em\u003e. Habitat suitability analysis for luehdorfia chinensis leech, 1893 (Lepidoptera: Papilionidae) in the middle and lower yangtze river: a study based on the MaxEnt Model. Insects\u003cem\u003e \u003c/em\u003e2025, 16(4):396.doi:https://doi.org/10.3390/insects16040396\u003c/li\u003e\n\u003cli\u003eSpitsyn VM, Spitsyna EA. New record of collita coreana (leech,[1889])(Erebidae: Arctiinae) from russia. Amurian zoological journal\u003cem\u003e \u003c/em\u003e2022, 14(3):413-416.doi:https://www.doi.org//10.33910/2686-9519-2022-14-3-413-416\u003c/li\u003e\n\u003cli\u003eArabacı B. \u0026lsquo;Pearls\u0026rsquo; of the nineteenth-century: from therapeutic actors to global commodities medicinal leeches in the Ottoman Empire. Medical history\u003cem\u003e \u003c/em\u003e2023, 67(2):128-147.doi: https://doi.org/10.1017/mdh.2023.17\u003c/li\u003e\n\u003cli\u003eLi T, Liu Y, Zhang C, Gu H, Cheng Z, Peng J\u003cem\u003e et al\u003c/em\u003e. A new species of sinospelaeobdella from China: sinospelaeobdella jiangxiensis sp. n.(Hirudinda, Arhynchobdellida, Haemadipsidae). Animals\u003cem\u003e \u003c/em\u003e2025, 15(8):1079.doi:https://doi.org/10.3390/ani15081079\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Haemadipsa yanyuanensis, terrestrial leech, chromosome-scale genome, antithrombotic gene expansion, gene family evolution, anticoagulant drug discovery","lastPublishedDoi":"10.21203/rs.3.rs-7119690/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7119690/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cem\u003eHaemadipsa yanyuanensis\u003c/em\u003e is a terrestrial blood-feeding leech with underexplored anticoagulant adaptations. Here, we present its first chromosome-level genome assembly (165.32 Mb, 9 chromosomes; BUSCO completeness: 97.6%) generated using Nanopore, Hi-C, and RNA-seq data. We identified 193 antithrombotic genes across 15 families, representing a 2.2- to 2.7-fold increase in gene number but a reduction in gene family diversity compared to aquatic medicinal leeches (\u003cem\u003eHirudo medicinalis\u003c/em\u003e, \u003cem\u003eHirudinaria manillensis\u003c/em\u003e, and \u003cem\u003eHirudo nipponia\u003c/em\u003e). Notably, bdellin, LDTI, and LCI gene families exhibited large-scale expansions ranging from 8.7- to 25-fold compared to aquatic leeches. while the progranulin gene exhibited a lineage-specific structure with 122 cysteine residues and nine tandem repeats. Transcriptomic profiling revealed high expression of these expanded families, suggesting their pivotal role in terrestrial blood-feeding adaptation. Our study reveals a novel gene family expansion-contraction model for antithrombotic evolution. These unique genomic data provide a new resource for the development of next-generation anticoagulant drugs.\u003c/p\u003e","manuscriptTitle":"Comparative Genomics Unveils Unique Antithrombotic Gene Expansion in Haemadipsa yanyuanensis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-07-31 10:34:24","doi":"10.21203/rs.3.rs-7119690/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-08-19T07:01:42+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-18T09:35:02+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-08-14T16:02:11+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"121916588365626112681966532207911484520","date":"2025-07-29T12:57:29+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"75090877276031508479563669309671914830","date":"2025-07-29T08:09:46+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-07-27T19:48:44+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-07-16T04:15:16+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-07-16T04:13:26+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Genomics","date":"2025-07-14T09:45:15+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-genomics","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"gics","sideBox":"Learn more about [BMC Genomics](http://bmcgenomics.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/gics","title":"BMC Genomics","twitterHandle":"#BMCGenomics","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"0d4c257d-96c5-4071-9183-e85383e0e669","owner":[],"postedDate":"July 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-01-05T16:00:54+00:00","versionOfRecord":{"articleIdentity":"rs-7119690","link":"https://doi.org/10.1186/s12864-025-12445-5","journal":{"identity":"bmc-genomics","isVorOnly":false,"title":"BMC Genomics"},"publishedOn":"2025-12-31 15:58:01","publishedOnDateReadable":"December 31st, 2025"},"versionCreatedAt":"2025-07-31 10:34:24","video":"","vorDoi":"10.1186/s12864-025-12445-5","vorDoiUrl":"https://doi.org/10.1186/s12864-025-12445-5","workflowStages":[]},"version":"v1","identity":"rs-7119690","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7119690","identity":"rs-7119690","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}