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Background Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. Results We annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis , and Hirudo verbana . Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis. Conclusions Here we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.

Medicinal leeches express a broad variety of anticoagulants and other bioactive factors that are involved in the blood feeding process. For most of the anticoagulants, several genes exist that may encode isoforms of the respective proteins, including hirudins and decorsins. Decorsins negatively affect platelet aggregation, whereas hirudins are potent thrombin inhibitors. Both factors belong to the hirudin superfamily that also includes the group of hirudin-like factors, and all hematophagous leeches analyzed so far contain several gene copies that encode representatives of at least two of the three groups of these factors. Members of the hirudin superfamily may contain only one central globular domain, but others may contain two or more copies. Here we describe the molecular identification and partial functional characterization of a broad variety of putative mono-, bi and multimeric hirudins, decorsins and hirudin-like factors in two Asian medicinal leech species, namely Hirudo nipponia and H. tianjinensis . Some of the monomeric hirudins and decorsins have already been described before, but they represent only a small part of the overall diversity. For the first time, putative monomeric and one oligomeric decorsins of H. tianjinensis were expressed as recombinant proteins, functionally characterized and successfully verified as platelet aggregation inhibitors. In addition we have conducted phylogenetic analyses based on genomic and mitochondrial markers and found convincing evidence that H. nipponia and H. tianjinensis together with members of the genus Whitmania form a monophyletic clade that is clearly distinct from clades that are formed either by European members of the genus Hirudo or by members of the genus Hirudinaria .

Haemadipsa yanyuanensis Liu & Song, 1977, is a terrestrial blood-feeding leech whose antithrombotic repertoire remains insufficiently characterized. We present its first chromosome-level genome assembly (165.32 Mb across nine chromosomes; BUSCO: 97.6%) using Nanopore long reads, Hi-C scaffolding, and RNA-seq data. We identified 193 putative antithrombotic genes in 15 putative families, representing a 2.2- to 2.7-fold increase in gene number but reduced family diversity compared with aquatic medicinal leeches, including Hirudo nipponia Whitman, 1886, Hirudinaria manillensis Lesson, 1842, and Hirudo medicinalis Linnaeus, 1758. Notably, the putative bdellin, LDTI, and LCI gene families exhibited marked lineage-specific expansions, ranging from 8.7- to 25-fold relative to those of aquatic leeches. In addition, the putative progranulin gene displayed a distinctive structural organization characterized by 122 cysteine residues and nine tandem repeats. Transcriptomic profiling revealed elevated expression of four expanded putative families, suggesting potential roles in terrestrial blood-feeding adaptation. This chromosome-scale genome provides a valuable resource for investigating anticoagulation mechanisms and may inform the development of next-generation antithrombotic therapeutics.

This study aimed to obtain and analyze the chromosome-level genome assembly of Hirudinaria bpling , a species vital for aquatic ecosystem health and medical research. Understanding its genomic information is crucial for advancing its medical applications and elucidating its ecological role. We assembled the genome of H. bpling using a combination of PacBio HiFi long reads, Illumina sequencing, and Hi-C chromosome conformation capture techniques. This approach allowed us to achieve a high-resolution genome assembly with detailed chromosomal organization. The final genome assembly of H. bpling is 144.08 Mb, with an N50 size of 11.27 Mb, anchored onto thirteen pseudo-chromosomes. BUSCO analysis indicated a genome completeness of 96.20%. We annotated a total of 20,126 protein-coding genes and identified 18.80% repetitive elements within the genome. Phylogenetic analysis included nine other leech species, positioning H. bpling as a sister taxon to Hirudo manillensis . A comparative analysis focused on the identification of putative anticoagulant proteins (e.g. Hirudin, Antistasin, Hirustasin, Therostasin, Bdellastasin, Guamerin/Piguamerin, Gelin, Bplins, Saratin, Eglin C, Bdellin B-3, LDTI, Hyaluronidase, Destabilase, Apyrase, Leech carboxypeptidase inhibitor, Gamma-glutamyl transpeptidase, Lefaxin, Progranulin), identifying conserved regions and evolutionary relationships among these proteins across different leech species. As a medically significant species, H. bpling offers promising opportunities for research into anticoagulant therapies. This study provides a comprehensive genomic and phylogenetic analysis of H. bpling , offering new insights into leech genomics and the evolution of anticoagulant genes. The findings enhance our understanding of the genetic and evolutionary mechanisms underlying anticoagulant production in leeches.

Species of Haemogregarina are blood parasites known to parasitise vertebrate hosts, including fishes ( Haemogregarina sensu lato) and freshwater turtles ( Haemogregarina sensu stricto). Their vectors, include gnathiid isopods and leeches, respectively. In turtles, Haemogregarina balli has the best-characterized life cycle in the genus. However, no studies in Brazil have suggested a possible vector for any species of Haemogregarina from freshwater turtles. Therefore, in the present study, we provide insights into a leech vector based on specimens found feeding on two species of freshwater turtles, Podocnemis unifilis and Podocnemis expansa , using morphological and molecular data . In 2017 and 2019, freshwater turtles were collected in Goiás State, Brazil. Hosts were inspected for ectoparasites and leeches were collected from two specimens of P. expansa and nine specimens of P. unifilis . Leeches were subsequently identified as members of the genus Unoculubranchiobdella. Leech histological slides revealed haemogregarine-like structures, similar to post-sporogonic merogony, found near the gills and within the posterior sucker. Molecular analysis of the haemeogregarines resulted in the identification of three species of Haemogregarina : Haemogregarina embaubali, Haemogregarina goianensis, and Haemogregarina brasiliana . Therefore, our findings, based on morphology and DNA data suggest leeches of the genus Unoculubranchiondella as vectors for at least three species of Haemogregarina from Brazilian turtles.

In Traditional Chinese Medicine (TCM), the medicinal leech is vital for treatments to promote blood circulation and eliminate blood stasis. However, the prevalence of counterfeit leech products in the market undermines the quality and efficacy of these remedies. Traditional DNA barcoding techniques, such as the COI barcode, have been limited in their application due to amplification challenges. This study identified high variability in the 16 S rRNA gene within the mitochondrial genome across five leech species, leading to the development of a novel 219 bp mini-barcode. Compared with the traditional COI barcode, our mini-barcode showed remarkable identification efficiency, classifying 142 out of 147 leech samples from fresh and processed materials. In contrast, the COI barcode could only successfully identify 79 out of the 147 samples. In the case of seven batches of leech decoction pieces, the mini-barcode identified six, whereas the COI barcode only recognized one. Additionally, the mini-barcode effectively discerned five leech species within Chinese patent medicines when combined with metabarcoding technology. These results confirm the mini-barcode’s potential as a reliable tool for rapidly and precisely identifying leech species in TCM products.

Medicinal leeches of the genus Hirudo inhabit large areas of the Palaearctic realm. The distribution range of Hirudo troctina includes the southern Iberian peninsula and the northwestern regions of Africa. H. troctina is used for medical purposes, but only very little is known about the components of its salivary gland secretion. Hirudins, bivalent inhibitors of thrombin, are probably the best known leech-derived bioactive factors. Hirudin-like factors (HLFs) represent another class of salivary gland components that share characteristic genetic and structural markers with hirudins. Hirudin is not a single entity but exists in at least four different variants. However, there are differences among the European members of the genus Hirudo with respect to the actual number of hirudin and HLF genes that are present within their genomes. Here, we describe the identification and molecular cloning of 11 genes that encode for putative hirudin and HLF variants in H. troctina . Three of the genes consist of exons and introns that originate from different “archetype” genes and are likely the result of recombination events. The diversity of hirudin and HLF genes in H. troctina surpasses that of all other European members of the genus Hirudo . The putative hirudin variants and representatives of the HLFs of H. troctina were expressed as recombinant proteins, purified and functionally characterized for their thrombin-inhibiting potencies. Phylogenetic analyses based on hirudin and HLF gene sequences of the leech genera Hirudo , Hirudinaria , and Whitmania supported the hypothesis that hirudins and HLFs diverged early in leech evolution.

Species-specific efficacy necessitates accurate identification of medicinal leeches, but standard DNA barcoding often fails with degraded DNA from traditional medicines. This deficiency highlights the need for mini-barcoding. This study aimed to develop and validate mini-barcode markers for three Chinese Pharmacopoeia-listed leech species: Whitmania pigra , Whitmania acranulata and Hirudo nipponia . Four novel mini-barcode primer sets (ND1F1/R1, 12SF1/R1, 16SF1/R1 and COX1F1/R1) were developed and validated using seven morphologically identified specimens and subsequently tested on 16 commercial leech products. DNA extractions were performed using both single-tube and column purification kits, with the latter yielding superior DNA quality and meeting the requirements for following PCR amplification. The PCR results confirmed the validation of four candidate mini-barcodes targeting specific genetic regions, which produced results in 13 out of 16 commercial leech products. Mini-barcode sequences from morphologically identified W. pigra specimens exhibit > 95% identity to the complete ND1, 12S rDNA, 16S rDNA, and COX1 sequences (EU304459), whereas sequences from H. nipponia and W. acranulata show < 85% identity, and among leech-derived products only the proprietary Chinese medicine Maxuekang exhibits lower identity. Both the optimal partition of ASAP and phylogenetic tree identified three distinct groups correlating with the morphological species: W. pigra , W. acranulata , and H. nipponia . Mislabeled species have been uncovered in proprietary Chinese medicine, notably the claimed Hirudo nipponia , which was replaced by W. pigra . The results highlight the value of mini-barcodes in enhancing product quality control and offer a reliable method for accurate species identification in traditional and commercial leech-based medicines. This advance supports safer and more effective utilization of medicinal leeches and advocates for their integration into regulatory standards.

Clitellata is a major annelid clade comprising oligochaetes ( e.g ., earthworms) and hirudineans ( e.g ., leeches). Due to their scant fossil record, the origins of clitellates, particularly Hirudinea, are poorly known. Here, we describe the first leech body fossil, Macromyzon siluricus , gen. et sp. nov., from the Brandon Bridge Formation (Waukesha Lagerstätte). This fossil, which is preserved in exceptional detail, possesses several hirudinean soft-tissue synapomorphies–including a large sucker at the posterior end and sub-divided segments–and phylogenetic analyses resolve Macromyzon siluricus as a stem leech. Its age, 437.5–436.5 Ma, is consistent with early age estimates for the origin of clitellates, and predates molecular-clock-based estimates of hirudinidan origins by at least 200 million years. These findings suggest that the earliest true leeches were marine and that, contrary to prevailing hypotheses, were unlikely to have fed on vertebrate blood.

Leech-derived invertebrate DNA (iDNA) has been successfully leveraged to conduct surveys of vertebrate host biodiversity across the Indo Pacific. However, this technique has been limited methodologically, typically only targeting mammalian 16S rDNA, or both 16S and vertebrate 12S rDNA for leech host determination. To improve the taxonomic richness of vertebrate host species in iDNA surveys, we re-analyze datasets from Bangladesh, Cambodia, China, and Madagascar through metabarcoding via next generation sequencing (NGS) of 12S, 16S (2 types, one designed to target mammals and the other, residual eDNA), nicotinamide adenine dinucleotide hydride dehydrogenase 2 (ND2), and cytochrome c oxidase subunit 1 (COI). With our 5 primer sets, we identify 41 unique vertebrate hosts to the species level, among 1,200 leeches analyzed, along with an additional 13 taxa to the family rank. Within our 41 taxa, we note that adding ND2 and COI loci increased species richness detection by 25%. NGS has emerged as more efficient than Sanger sequencing for large scale metabarcoding applications and, with the decline in cost of NGS, our pooled sample multilocus protocol is an attractive option for iDNA biodiversity surveys.