The hirudin-like factors HLF3 and HLF4-hidden hirudins of European medicinal leeches.

The hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis, Hirudo verbana, and Hirudo orientalis, respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of hirudin and HLF molecules for thrombin inhibition.

Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches.

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.

Internal and External Morphological Characteristics of the Medicinal Leech Species Hirudo sulukii and Hirudo verbana

Objective: The aim of this study was to investigate the internal and external morphological structures of two species of medicinal leeches (Hirudo sulukii and Hirudo verbana). Methods: Leech specimens were collected from various regions of Turkey between the years of 1995-2016. The leeches were first examined alive and later were stunned with 10% ethyl alcohol and fixed in 70% ethyl alcohol or 4% formaldehyde. After fixation, the internal morphology of leeches was examined by dissecting them under a stereo microscope. Results: Both species of leeches are dorso-ventrally flattened. H. verbana is larger and broader than H. sulukii. H. sulukii has black, segmentally-arranged united ellipsoid and elongated spots, and a pair of zigzagged black longitudinal stripes in the dorso-lateral area of its body. The ventral surface of H. sulukii is greenish to brown in colour and has a small number of irregular black spots. H. verbana has broad and diffuse, paramedian, orange stripes on the dorsal surface. Ventrally, H. verbana has a greenish to yellow colour, and it is characterized by a pair of black ventro-lateral stripes. Conclusion: Two of the six leech species (Hirudo sulukii and Hirudo verbana) belonging to the genus Hirudo found in the world are found in Turkey. These two leech species show significant differences from each other, morphologically.

Phylogenetic Position and Description of a New Species of Medicinal Leech from the Eastern United States.

A new species of medicinal leech, Macrobdella mimicus n. sp., is described from specimens collected in Maryland; this is the first description of a North American macrobdellid since 1975. Superficially, the new species resembles the well-known Macrobdella decora, as both species possess 4 accessory pores arranged symmetrically on the ventral surface, yet the new species is distinguished from M. decora in possessing 4-4½ annuli (rather than 3½) between the gonopores and 4 annuli (rather than 5 annuli) between the female gonopore and the first pair of accessory pores. Phylogenetic analyses, based on 2 mitochondrial and 2 nuclear loci for a set of closely related taxa, confirms the placement of the new species within the family Macrobdellidae and places it as the sister taxon to M. decora and M. diplotertia.

Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches.

Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.

[Histological and histochemical studies on mouthpart of Whitmania pigra at different months age].

Mouthpart developmental histology of Whitmania pigra at different month of age were studied by paraffin section, HE staining combined alcian blue and periodic acid schifts reaction procedure (AB-PAS). The following results was obtained: Change ranges: oral width 0.6 mm (1-3 month), 1.2 mm (34 month); oral diameter 0.3 mm (1-3 month); 1.2 mm (34 month), the oral size reached maximum during 4-6 months and unchanged thereafter. Oral lip had a thin protective film located in the front of the mouthpart. The W. pigra possessed three jaws in oral cavity, the big one was in dorsum, the other two separated on both side of abdomen respectively. Jaws and muscular pharynx were interrelated closely. The jaws were composed by cuticle, epithelial layer, muscularis and jaw cavity from outside to inside. In the front of jaws had mastoid abdomen with function of secreting acidophilic granule from 2 month age. Oral cavity was composed by mucosa, submucosa and muscularis inside and outside. Oral cavity was rich of peristomial nerves. And pharynx was composed of mucosa, muscularis, adventitia from inside to outside. The folds height and width become heighten and thicken. Mucosa epithelium from complex flat epithelium changed into columnar epithelium, muscularis gradually developed into thickened along with growing. Muscular thickness reached maximum at 4 months. Mucous cells of W. pigra were classified into I-IV types based on different staining and two mainly morphological shapes (Tubular, Pear-shaped). Jaws, oral cavity, pharynx by AB-PAS staining showed little changes at different month of age. Mucous cells were few at 1 month age, and type II cells were increased rapidly in 2-3 month age in oral lip. Oral cavity contains more mucous gland cells type I. Under the muscularis there were connective tissues which distributed a few of mucous cells type II.

[Species study on Chinese medicine leech and discussion on its resource sustainable utilization].

Through systemically sorting and studying literature of Chinese medicine, this article pointed out that leech used by the traditional Chinese medicine in ancient time has the features of small, living in water, able to suck blood of animal and people. The species of leeches having these features were Hirudo nipponia Whitman, H. pulchra Song, Poecilobdella nanjingensis sp. Nov. , P. manillensis (Lesson) and P. hubeiensis Yang, which were not fully coincidence with the species recorded in Chinese Pharmacopeia of 2010 edition. We suggests that species of leech in Chinese Pharmacopeia be revised: H. nipponica Whitman should be kept, P. manillensis (Lesson) should be added in, Whitmania pigra Whitman and W. acranulata Whitma should be temporarily reserved, and H. pulchra Song, P. nanjingensis sp. Nov. , and P. hubeiensis Yang should be considered.

[Sequence analysis of rDNA-ITS gene in medicinal leeches and their closely related species (Euhirudinea:Arhynchobdellida)].

OBJECTIVE: To explore the phylogenetic relationships among medicinal leeches and their closely related species by comparing their ITS gene fragments. METHODS: 8 different leech species, including Whitmania pigra, Whitmania acranulata, Whitmania laevis, Hirudo nipponia, Poecilobdella manillensis, Poecilobdella javanica, Poecilobdella hubeiensis and Erpobdella octoculata were collected as the materials for study. After the total DNA of all samples was extracted, amplified by PCR and cloned, their ITS sequence, AT content, variable sites and evolutionary relationships were analyzed. RESULTS: The length of ITS was 948-981 bp with 210 variable sites and 43 inserts on/deletion. The average G + C content was high up to 56.0%-57.8%. The aligned ITS sequence demonstrated that their ITS genes were relatively conserved in species. Data analysis showed that the relationship between E. octoculata and P. javanica was closer than others, their genetic distance was only 0.007. Through the phylogenetic tree analysis, three species of Haemopidae and four species of Hirudinidae were obviously clustered into two branches. CONCLUSION: The results support the taxonomic status of Haemopidae and Hirudinidae, and provide sufficient evidences that Erpobdella octoculata and Poecilobdella javanica should not be assigned to two genera, of which the taxonomic system should be further researched.

[Analysis of genetic differentiation and phylogenetic relationship between Whitmania pigra and Hirudo nipponia based on ITS sequence].

OBJECTIVE: To evaluate the genetic differentiation and phylogenetic relationship between Whitmania pigra Whitman and Hirudo nipponia Whitman. METHOD: By the sequences of the rDNA internal transcribed spacer (ITS) the molecular phylogenetic tree was constructed by MP method using software MEGA 4.0. RESULT: The average length of ITS was 857.2-861.2 bp. The A, T, G and C contents in this fragment were 25.12%, 28.28%, 17.34%, 29.29%, respectively. The GC content was higher than the AT content. Little sequence variation was observed in ITS gene fragments with in species, and transition in only 45 loci was revealed in 14 populations. 14 W. pigra and H. nipponia populations were clustered into 2 groups by MP phylogenetic tree. CONCLUSION: The results also showed that the intraspecific variation was dominated in variation types of W. pigra and H. nipponia. The classification result of W. pigra and H. nipponia and its adulterants based on DNA sequences are not totally consistent with those based on classification. It showed that a little of mutation of base in ITS sequences had occurred in the process of evolution, such as transition cites, transvertion cites among base or base gap.

Poly-paraphyly of Hirudinidae: many lineages of medicinal leeches.

BACKGROUND: Medicinal leeches became infamous for their utility in bloodletting popularized in the 19th century, and have seen a recent resurgence in post-operative treatments for flap and replantation surgeries, and in terms of characterization of salivary anticoagulants. Notorious throughout the world, the quintessential leech family Hirudinidae has been taken for granted to be monophyletic, as has the non-bloodfeeding family Haemopidae. RESULTS: This study is the first to evaluate molecular evidence from hirudinid and haemopid leeches in a manner that encompasses the global scope of their taxonomic distributions. We evaluated the presumed monophyly of the Hirudinidae and assessed previous well-accepted classification schemes. The Hirudinidae were found not to be monophyletic, falling instead into two distinct and unrelated clades. Members of the non-bloodfeeding family Haemopidae were scattered throughout the tree and among traditional hirudinid genera. A combination of nuclear 18S rDNA and 28S rDNA with mitochondrial 12S rDNA and cytochrome c oxidase I were analyzed with Parsimony and with Bayesian methods. CONCLUSION: The family Hirudinidae must be refined to include only the clade containing Hirudo medicinalis (European medicinal leech) and related leeches irrespective of bloodfeeding behavior. A second clade containing Macrobdella decora (North American medicinal leech) and its relatives may yet be recognized in Semiscolecidae in order to avoid paraphyly. The African distribution of species from each of the divergent hirudinid clades suggests that a deep divergence took place in the history of the medicinal leeches hundreds of millions of years ago.

Diverse molecular data demonstrate that commercially available medicinal leeches are not Hirudo medicinalis.

The European medicinal leech is one of vanishingly few animal species with direct application in modern medicine. In addition to the therapeutic potential held by many protease inhibitors purified from leech saliva, and notwithstanding the historical association with quackery, Hirudo medicinalis has been approved by the United States Food and Drug Administration as a prescription medical device. Accurate annotation of bioactive compounds relies on precise species determination. Interpretations of developmental and neurophysiological characteristics also presuppose uniformity within a model species used in laboratory settings. Here, we show, with mitochondrial sequences and nuclear microsatellites, that there are at least three species of European medicinal leech, and that leeches marketed as H. medicinalis are actually Hirudo verbana. Beyond the obvious need for reconsideration of decades of biomedical research on this widely used model organism, these findings impact regulatory statutes and raise concerns for the conservation status of European medicinal leeches.

A new species of the medicinal leech (Oligochaeta, Hirudinida, Hirudo) from Transcaucasia and an identification key for the genus Hirudo.

A recent molecular phylogenetic study has suggested that the genus Hirudo contains a neglected species previously known as the orientalis coloration type of the medicinal leech Hirudo medicinalis. In this paper, the new species is formally described as Hirudo orientalis sp. n. It can most readily be identified by the grass green coloration of the dorsum, segmentally arranged pairs of black quadrangular or rounded dots on its paramarginal dorsal stripes and similarly arranged, but less regular light-colored markings on the predominantly black venter. It has medium-sized epididymes and an evenly coiled vagina. H. orientalis is known from Transcaucasia, Iran, and Uzbekistan. It is widely used in medicine as the "medicinal leech." Very little is known about its exact distribution, specific habitat, and conservation status. The paper contains an identification key to all species of the genus Hirudo.

Genetic differentiation between two species of the medicinal leech, Hirudo medicinalis and the neglected H. verbana, based on random-amplified polymorphic DNA.

The medicinal leech is one of the few parasitic invertebrates widely used in medicine and as a scientific model object. Because of a dramatic decline in its natural populations, it is subject to considerable conservation effort. Despite all attention, there is confusion regarding the taxonomic status of different morphological forms. The prevailing view is that all varieties of medicinal leech in Europe represent the same species, Hirudo medicinalis. However, the present study based on RAPD molecular markers demonstrates that a second European taxon, H. verbana, forms a distinct species. Phenetic clustering and principal coordinate analysis of eight populations revealed the same basic structure, reflecting taxonomic rather than geographic subdivision. Variation between species explained 60% of the total molecular variance (phiCT=0.60, P<0.001). Both taxa displayed a significant number of specific RAPD markers. Conversely, no specific fragment supporting the geographic association of both taxa was found. Since the stronghold of commercially exploited medicinal leech populations in southeastern Europe and Turkey belongs to H. verbana, most medicinal and scientific applications probably use this species, not H. medicinalis. Appropriate taxonomic correction of international conservation conventions and legislation is essential.

[The comparison of contents of uracil, xanthine and hypoxanthine in five species of leech].

Three constituents(uracil, xanthine, hypoxanthine) were isolated from five species of leech and determined by reversed phase HPLC. The column employed was Shim-pack CLC-ODS C18(150 mm x 6 mm). The mobile phase was 0.05 mol/l ammonium phophate dibasic solution(pH = 8.4). The flow rate was 0.8 ml/min and detection was effected at 254 nm. This method is accurate, rapid and reproducible. Analytical data for five species and Whitmania pigra samples from different places were given.