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In the present study we analyzed the behavior and interactions among leeches in the same observation tank. Colored beads were glued onto their skin so that their behavior could be followed and quantified. When two or three leeches were present in the observation tank, they searched around for a maximum of 2 h and their motion and behavior were independent from those of their conspecifics. When the number of leeches in the tank was increased to 10, leeches were attracted to each other and exhibited episodes of highly correlated behavior. Solitary leeches injected with serotonin or dopamine increased the portion of time spent pseudoswimming and crawling, respectively. The behavior of three to five leeches injected with serotonin was not statistically independent, and leeches were attracted to their conspecifics and exhibited episodes of correlated behavior. Therefore, serotonin not only induces pseudoswimming in leeches but also promotes social interactions, characterized by a mutual attraction and by episodes of correlated/collective behavior. [ABSTRACT FROM AUTHOR]

The aim of the present study was to investigate how exposure to sulfate-rich surface waters affects the level of primary DNA damage in hemocytes of leech Hirudo medicinalis. Samples of surface water were collected at two sites near a gypsum factory (Knin, Croatia) and two reference sites. In the laboratory, samples were subjected to detailed chemical analysis and used in toxicity testing. For that purpose, previously acclimatized individuals of H. medicinalis were sub-chronically exposed (for 28 days) to tested water samples. Levels of primary DNA damage were evaluated using the alkaline Comet assay in hemocytes collected on days 7, 14, 21 and 28 of exposure and compared with their baseline values. Genotoxic potency of the water sample with the highest sulfate concentration was further evaluated using the alkaline, neutral and hOGG1-modified Comet assay on human peripheral blood leukocytes exposed ex vivo for 30min. The purpose was to explore which mechanisms are responsible for DNA damage. Chemical analysis revealed that sulfate concentrations in two water samples collected in Mali Kukar Lake (1630mg/L SO4) and Kosovčica River (823.3mg/L SO4) exceeded the WHO and US EPA defined limits for sulfate in drinking water. Increased levels of metals were found only in the water sample collected in Mali Kukar Lake. However, of the 65 elements analyzed, only nickel and titanium exceed the value legally accepted in Croatia for drinking water. The levels of DNA damage, estimated by the alkaline Comet assay in hemocytes of medicinal leech, increased with the duration of exposure to two sulfate-rich water samples. Since hemocytes responded sensitively to treatment, they could be used for biomonitoring purposes. As observed on treated human peripheral blood leukocytes, all versions of the Comet assay were effective in detecting DNA damage, which was measured in samples with sulfate concentrations equal to or higher than the legally accepted levels for drinking water. Based on the obtained results, it can be assumed that genotoxicity was a consequence both of direct (single- and double-strand DNA breaks) and indirect effects (oxidative damage) caused by the combined effects of all contaminants present in the tested water samples. Our results indicate the need for in situ monitoring and purification of gypsum mine water prior to its release in the natural environment. [Copyright &y& Elsevier]

Voluntary movements in animals are often episodic, with abrupt onset and termination. Elevated neuronal excitation is required to drive the neuronal circuits underlying such movements; however, the mechanisms that sustain this increased excitation are largely unknown. In the medicinal leech, an identified cascade of excitation has been traced from mechanosensory neurons to the swim oscillator circuit. Although this cascade explains the initiation of excitatory drive (and hence swim initiation), it cannot account for the prolonged excitation (10-100 s) that underlies swim episodes. We present results of physiological and theoretical investigations into the mechanisms that maintain swimming activity in the leech. Although intrasegmental mechanisms can prolong stimulus-evoked excitation for more than one second, maintained excitation and sustained swimming activity requires chains of several ganglia. Experimental and modeling studies suggest that mutually excitatory intersegmental interactions can drive bouts of swimming activity in leeches. Our model neuronal circuits, which incorporated mutually excitatory neurons whose activity was limited by impulse adaptation, also replicated the following major experimental findings: (1) swimming can be initiated and terminated by a single neuron, (2) swim duration decreases with experimental reduction in nerve cord length, and (3) swim duration decreases as the interval between swim episodes is reduced. [ABSTRACT FROM AUTHOR]

An analysis of the reproduction of three species of medicinal leeches (genus Hirudo) was carried out. The highest fecundity (34.34 ± 3.72 hatchlings per leech) was observed in H. verbana, the lowest in H. medicinalis (11.10 ± 2.56). The heaviest hatchlings were found in H. medicinalis (0.046 ± 0.0005 g), the lightest in H. verbana (0.032 ± 0.0003 g). Hirudo orientalis had an intermediate fecundity (21.63 ± 3.39 hatchlings per leech) and its hatchlings were of intermediate weight (0.038 ± 0.0005 g). The species differ in their growth rate and mortality: Hirudo verbana had the smallest hatchlings and the lowest survivorship but its growth rate was highest among the three species. The results agree with sister relationships between H. medicinalis and H. orientalis and the basal position of H. verbana with respect to them. The reproductive traits of the three species suggest that H. verbana is an r-strategist that inhabits unstable environments, such as temporary ponds in steppe landscapes, whereas its congeners are subject to K-selection and occur in more stable and predictable habitats. [ABSTRACT FROM AUTHOR]

Leeches are a class of segmented invertebrates, known for their blood-feeding habits and used in phlebotomy to treat various ailments since antiquity. In Europe, medicinal leeches have recently been rediscovered and are used by maxillofacial and other microsurgeons to aid salvage of compromised venous engorged tissue and amputations, such as digits, ears, and nasal tips. Because of their important salivary components, blood-sucking (sanguivorous) leeches, such as Hirudo medicinalis and related species, have engendered great interest from pharmaceutical companies searching for anticoagulants to prevent blood clotting during microsurgeries. Scientific research reveals that the beneficial effects of leeching, in addition to decongestion, include injection of a cocktail of several medicinally useful bioactive molecules present in their saliva. Owing to its therapeutic potential, the research is continuing as many new salivary compounds are being isolated and synthesized. (Altern Med Rev 2011;16(1):59-65) [ABSTRACT FROM AUTHOR]

Abstract: Unlike mammals, the CNS of the medicinal leech can regenerate damaged neurites, thus restoring neural functions. Our group recently demonstrated that the injured leech nerve cord is able to mount an immune response, which promotes the regenerative processes. This defense mechanism is microorganism-specific, suggesting that the leech CNS is able to discriminate among microbial components. We report here the characterization of two receptors potentially implicated in this detection: HmTLR1 and HmNLR. Interestingly, HmTLR1 presents an endosomal distribution in neurons and appears as a chimera combining the mammalian intraendosomal domain of TLR3 and the cytoplasmic section of TLR13, while HmNLR is cytosolic and has the highest homology to NLRC3 receptors. Both receptors show patterns of induction upon stimulation that suggest their involvement in the leech neuroimmune response. This work constitutes the first demonstration in an invertebrate of (i) an intracellular TLR and (ii) a cytosolic PRR related to the NLR family. [Copyright &y& Elsevier]

The article offers information on the autonomic functions and response to neural circuit controlling in medicinal leeches. It is mentioned that the leeches presents well-defined behaviors and autonomic responses and the focus of the study is on the pattern of swimming, crawling and heartbeat. Also, information on the decision making ability, and mutually exclusive behaviors that are expressed at neuronal levels is presented.

Species identifications based on DNA barcoding rely on the correct identity of previously barcoded specimens, but little attention has been given to whether deposited barcodes include correspondence to the species' name-bearing type. The information content associated with COX1 sequences in the two most commonly used repositories of barcodes, GenBank and the Barcode of Life Data System (BOLD), is often insufficient for subsequent evaluation of the robustness of the identification procedure. We argue that DNA barcoding and taxonomy alike will benefit from more information content in the annotations of barcoded specimens as this will allow for validation and re-evaluation of the initial specimen identification. The aim should be to closely connect specimens from which reference barcodes are generated with the holotype through straight-forward taxonomy, and geographical and genetic correlations. Annotated information should also include voucher specimens and collector/identifier information. We examine two case studies based on empirical data, in which barcoding and taxonomy benefit from increased information content. On the basis of data from the first case study, we designate a barcoded neotype of the European medicinal leech, Hirudo medicinalis, on morphological and geographical grounds. [ABSTRACT FROM AUTHOR]

Abstract: LAR-like receptor protein tyrosine phosphatases (RPTPs), which are abundantly expressed in the nervous systems of most if not all bilaterian animals thus far examined, have been implicated in regulating a variety of critical neuronal processes. These include neuronal pathfinding, adhesion and synaptogenesis during development and, in adult mammals, neuronal regeneration. Here we explored a possible role of a LAR-like RPTP (HmLAR1) in response to mechanical trauma in the adult nervous system of the medicinal leech. In situ hybridization and QPCR analyses of HmLAR1 expression in individual segmental ganglia revealed a significant up-regulation in receptor expression following CNS injury, both in situ and following a period in vitro. Furthermore, we observed up-regulation in the expression of the leech homologue of the Abelson tyrosine kinase, a putative signaling partner to LAR receptors, but not among other tyrosine kinases. The effects on neuronal regeneration were assayed by comparing growth across a nerve crush by projections of individual dorsal P neurons (PD) following single-cell injection of interfering RNAs against the receptor or control RNAs. Receptor RNAi led to a significant reduction in HmLAR1 expression by the injected cells and resulted in a significant decrease in sprouting and regenerative growth at the crush site relative to controls. These studies extend the role of the HmLARs from leech neuronal development to adult neuronal regeneration and provide a platform to investigate neuronal regeneration and gene regulation at the single cell level. [ABSTRACT FROM AUTHOR]

Abstract: Medicinal leeches (Hirudo spp.) swim using a metachronal, front-to-back undulation. The behavior is generated by central pattern generators (CPGs) distributed along the animal''s midbody ganglia and is coordinated by both central and peripheral mechanisms. Here we report that a component of the venom of Conus imperialis, α-conotoxin ImI, known to block nicotinic acetyl-choline receptors in other species, disrupts swimming. Leeches injected with the toxin swam in circles with exaggerated dorsoventral bends and reduced forward velocity. Fictive swimming in isolated nerve cords was even more strongly disrupted, indicating that the toxin targets the CPGs and central coordination, while peripheral coordination partially rescues the behavior in intact animals. [Copyright &y& Elsevier]

Abstract: The genomes of the mollusk Lottia gigantea, the leech Helobdella robusta and the polychaete worm Capitella teleta each have a gene encoding an ecdysone receptor homolog. Publicly available genomic and EST sequences also contain evidence for ecdysone receptors in the seahare Aplysia californica, the bobtail squid Euprymna scolopes and the medicinal leech Hirudo medicinalis. Three-dimensional models of the ligand binding domains of these predicted ecdysone receptor homologs suggest that each of them could potentially bind an ecdysone-related steroid. Thus, ecdysone receptors are not limited to arthropods and nematodes. [ABSTRACT FROM AUTHOR]

Introduction: Venous convulsion after reconstructive microsurgery procedures is one major complication a surgeon has to deal with. Today, especially in the field of reconstructive microsurgery, medicinal leech therapy enjoys a renaissance. The potential risks such as infections associated with leech therapy are generally underestimated and not sufficiently discussed in literature. Method/patients: We present five male patients with an average age of 47 years. All patients suffered from a trauma incident, which had to be treated as an emergency. Three patients showed, postoperatively, a venous congestion after the reconstructive procedures. Another two patients with flap reconstruction and flap training developed venous problems after 12 and 14 days. In all five cases, the indication was given to use medical leeches ( Hirudo medicinalis). In all the patients, a local infection of the injured extremity could be regarded after beginning with the leech treatment. The treatment duration with medical leeches for postoperative venous congestion was an average of 6 days. Results: The reconstructive procedures in all five cases were unfortunately unsuccessful as major local infections were observed. Microbiological analyses showed, in all cases, an infection with Aeromonas hydrophila. Conclusion: We recommend making a considered indication for leech therapy, to diagnose wound infections early and to think about prophylactic antibiotics in patients with leech application. [ABSTRACT FROM AUTHOR]

Preparation and purification of a recombinant protein are described along with characteristics of its specific (for ɛ-(γ-Glu)-Lys and D-dimer substrates) and nonspecific (for L-γ-Glu-pNA) isopeptidase activities; the absence of peptidase function for α-(α-Glu)-Lys substrate is noted. It is shown that the protein exhibits muramidase (cell walls of Micrococcus lysodeikticus) and specific glycosidase activities. The latter was determined towards the fluorogenic substrate 4-methylum-belliferyl-tetra-N-acetyl-β-chitotetraoxide. Antimicrobial activity of recombinant destabilase-lysozyme protein (recDest-Lys) and its 11-membered amphipathic peptide was revealed towards cells of the strict anaerobic Archaean Methanosarcina barkeri, whose cell walls contain no murein. Possible mechanisms of the effect of recDest-Lys on these cells are discussed. [ABSTRACT FROM AUTHOR]

The biological function of a cell-type-specific glycosylation of an adhesion molecule belonging to the L1CAM immunoglobulin superfamily was previously determined in the nervous system of the embryonic leech, Hirudo medicinalis. The Lan3-2 glycoepitope is a surface marker of sensory afferent neurons and is required for their appropriate developmental collateral branching and synaptogenesis in the CNS. The chemical structure of the Lan3-2 glycoepitope consists of β-(1,4)-linked mannopyranose. Here, we show the conservation of the cell-type-specific expression of this mannose polymer in Caenorhabditis elegans. The Lan3-2 glycoepitope is expressed on the cell surface of a subset of dissociated embryonic neurons and, in the adult worm, by the pharyngeal motor neuron, M5, and the chemosensory afferents, the amphids. Additionally, the vulval epithelium expresses the Lan3-2 glycoepitope in late L4 larvae and in adult hermaphrodites. To investigate proteins carrying this restrictively expressed glycoepitope, worm extract was immunoaffinity purified with Lan3-2 monoclonal antibody and Western blotted. A polyclonal antibody reactive with the cytoplasmic tail of LAD-1/SAX-7, a C. elegans member of the L1CAM family, recognizes a 270 kDa protein band while Lan3-2 antibody also recognizes a 190 kDa glycoform, its putative Lan3-2 ectodomain. Thus, in C. elegans, as in leech, the Lan3-2 epitope is located on a L1CAM homologue. The cell-type-specific expression of the Lan3-2 glycoepitope shared by leech and C. elegans will be useful for understanding how cell-type-specific glycoepitopes mediate cell–cell interactions during development. [ABSTRACT FROM AUTHOR]

Purpose: Artery-only fingertip replantation can be reliable if low-resistance flow through the replant is maintained until venous outflow is restored naturally. Injuring the tip of the replant to promote ongoing bleeding augmented with anticoagulation usually accomplishes this; however, such management results in prolonged hospitalization. In this study, we analyzed the outcomes of artery-only fingertip replantation using a standardized postoperative protocol consisting of dextran-40, heparin, and leech therapy. Methods: Between 2001 and 2008, we performed 19 artery-only fingertip replants for 17 patients. All patients had the replanted nail plate removed and received intravenous dextran-40, heparin, and aspirin to promote fingertip bleeding and vascular outflow. Anticoagulation was titrated to promote a controlled bleed until physiologic venous outflow was restored by neovascularization. We used medicinal leeches and mechanical heparin scrubbing for acute decongestion. By postoperative day 6, bleeding was no longer promoted. We initiated fluorescent dye perfusion studies to assess circulatory competence and direct further anticoagulant intervention if necessary. The absence of bleeding associated with an initial rise followed by an appropriate fall in fluorescent dye concentration would trigger a weaning of anticoagulation. Results: All of the 19 replants survived. The average length of hospital stay was 9 days (range, 7–17 d). Eleven patients received blood transfusions. The average transfusion was 1.8 units (range, 0–9 units). All patients were happy with the decision to replant, and the cosmetic result. Conclusions: A protocol that promotes temporary, controlled bleeding from the fingertip is protective of artery-only replants distal to the distal interphalangeal joint until physiologic venous outflow is restored. The protocol described is both safe and reliable. The patient should be informed that such replant attempts may result in the need for transfusions and extended hospital stays, factors that can help the physician and patient decide whether to proceed with repantation. Type of study/level of evidence: Therapeutic IV. [Copyright &y& Elsevier]

Abstract: LAR-like receptor protein tyrosine phosphatases (RPTPs), which are reported to be highly expressed in the nervous systems of most bilaterian animals, have been implicated in the regulation of critical developmental processes, such as neuronal pathfinding, cell adhesion and synaptogenesis. Here we report that two LAR-like RPTPs in the medicinal leech, HmLAR1 and HmLAR2, play roles in regulating the size and density of neuronal arbors within the developing nervous system and in the body wall. Employing single-cell RNAi knockdown and transgene expression techniques, we demonstrate that the expression level of HmLAR1 is directly correlated with the density of an identified neuron''s arborization. Knocking down HmLAR1 mRNA levels in the mechanosensory pressure (P) neurons produces a reduced central arbor and a smaller arbor in the peripheral body wall, with fewer terminal branches. By contrast, overexpression of this receptor in a P cell leads to extensive neuronal sprouting, including many supernumerary neurites and terminal branches as well as, in some instances, the normal monopolar morphology of the P cell becoming multipolar. We also report that induced neuronal sprouting requires the expression of the receptor''s membrane tethered ectodomain, including the NH2-Ig domains, but not of the intracellular phosphatase domains of the receptor. Interestingly, sprouting could be elicited upon ectopic expression of HmLAR1 and the related RPTP, HmLAR2 in the P and other neurons, including those that do not normally express either RPTP, suggesting that the substrates involved in HmLAR-induced sprouting are present in most neurons irrespective of whether they normally express these LAR-like RPTPs. Our data are consistent with the hypothesis that the receptors’ ectodomains promote an adhesive interaction that enhances the maintenance of new processes. [Copyright &y& Elsevier]

An animal's response to a stimulus depends on many factors such as age, hormonal state, experience, and its behavioral state. For example, an animal may suppress a behavior that is inappropriate or incompatible with its current state. In this study, we show that, as a medicinal leech feeds, the distention that it incurs inhibits its expression of swimming. Distention slows the swimming pattern and decreases the number of swim cycles elicited by a test electrical stimulation; large distentions inhibit swimming altogether. We have previously shown that the ingestive phase of feeding inhibits behaviors by presynaptic inhibition of mechanosensory neurons. Distention has its effects downstream (e.g., gating and central pattern generating interneurons) from these sensory neurons and thus represents a novel mechanism for choosing between conflicting behaviors during feeding. Because removing the leech's gut surgically did not eliminate the effects of body distention, we conclude that the receptors mediating the distention-induced suppression of swimming are likely to be located in the animal's body wall. Together with previous findings, these new data show that leeches rely on two different decision-making networks to ensure that a biologically important behavior is not disrupted by other behaviors. [ABSTRACT FROM AUTHOR]

Clin Microbiol Infect 2010; 16: 563–567 The use of the medicinal leech ( Hirudo medicinalis) in promoting venous drainage in tissues whose vitality is threatened by venous congestion and obstruction, especially in plastic and reconstructive surgery, has been complicated by infections caused by Aeromonas spp. These are leech endosymbionts for which patients undergoing hirudotherapy frequently receive systemic chemoprophylaxis. In order to evaluate the possibility of rendering leeches safe for use on patients, H. medicinalis were fed artificially with a 2 g/L arginine solution (used as a phagostimulant) supplemented with ciprofloxacin (100 mg/L). Aeromonads were detected in 57 out of 80 control leeches (71.3%), but in none of the 56 leeches treated with ciprofloxacin (p <0.001). Treated leeches survived for up to 4 months. Tested weekly, 61% of these leeches took human blood for at least 4 weeks after treatment and all remained negative for aeromonads. All water samples in which leeches were kept before treatment were contaminated with Aeromonas spp.; none were detected in any of the NaCl/arginine solutions with which treated animals were fed. Molecular characterization of two phenotypically distinct isolates using gyrB sequencing showed that one clustered tightly with A. veronii and the other was closely related to A. media. Other environmental bacteria and fungi were isolated from 26.5% of treated leeches that had taken a blood meal 1–4 weeks after treatment. Ciprofloxacin reduced the number of leech-associated aeromonads to undetectable levels for extended periods. Most treated leeches were ready to take a blood meal after treatment, suggesting the possibility of using ciprofloxacin-treated leeches instead of chemoprophylaxis in patients undergoing hirudotherapy. [ABSTRACT FROM AUTHOR]

The relative location of proteins and lipids in particles of medicinal leech salivary gland secretion (SGS) is revealed for the first time. Their sizes and morphology are described. Using scanning electron microscopy and transmission electron microscopy, it was determined that SGS consists of particles of different sizes and form. This picture is supported by confocal laser scanning microscopy of SGS preparations treated with fluorescein isothiocyanate. After incubation with nonionic detergents (Brij 35 and Tween 20), transmission electron microscopy revealed the dissociation of fragments composing protein-lipid particles (PLP), and in this case an increase in free protein concentration determined by a modification of the Lowry method was observed. Perylene probing of lipids in SGS preparations showed that they are concentrated mainly inside PLP and are almost absent on the surface. Cholesterol was detected during SGS probing using the cholesteryl-Bodipy (hydrophobic fluorescent analog of cholesterol) on surface sections during confocal analysis of electron microphotographs of SGS. This analysis detected PLP structures in SGS resembling caveoles full of cholesterol. SGS, preliminary frozen at −70°C, transformed into a multitude of similar small particles visualized by transmission electron microscopy, whose fixed distribution resembled water crystal structure. [ABSTRACT FROM AUTHOR]

Summary: Background: Medicinal leeches (Hirudo spp.) are simultaneous hermaphrodites. Mating occurs after a stereotyped twisting and oral exploration that result in the alignment of the male and/or female gonopores of one leech with the complementary gonopores of a partner. The neural basis of this behavior is presently unknown and currently impossible to study directly because electrophysiological recording techniques disrupt the behavior. Results: Here we report that (Arg8)-conopressin G and two other members of the oxytocin/vasopressin family of peptide hormones induce in Hirudo verbana a sequence of behaviors that closely mimic elements of spontaneous reproductive behavior. Through a series of progressively more reduced preparations, we show that one of these behaviors, a stereotyped twisting that is instrumental in aligning gonopores in preparation for copulation, is the product of a central pattern generator that consists of oscillators in ganglia M5 and M6 (the ganglia in the reproductive segments of the leech), and also in ganglion M4, which was not previously known to play a role in reproductive behavior. We find that the behavior is periodic, with a remarkably long cycle period of around five minutes, placing it among the slowest behavioral rhythms (other than diurnal and annual rhythms) yet described. Conclusion: These results establish the leech as a new model system for studying aspects of the neuronal basis of reproductive behavior. [Copyright &y& Elsevier]

Our study investigated the behavioural responses of medicinal leech and rainbow trout at different ontogenetic levels under the effect of crude oil (CO) and heavy fuel oil (HFO), performed comparative analysis of the sensitivity of animal responses, evaluated the specificity of these responses, and determined "safe" toxicant concentrations. Comparison of sensitivity of behavioural responses of leech and fish revealed that the most sensitive response to long-term exposure to CO was leech locomotor activity, while the most sensitive parameter to HFO was the coughing rate in juvenile fish. Our study showed that the sensitivity and specificity of behavioural responses of aquatic animals at different phylogenetic and ontogenetic levels can be successfully used to evaluate the toxicity of ambient water polluted with oil hydrocarbons. [ABSTRACT FROM AUTHOR]

The article focuses on the reconsideration of the distribution and population status of medicinal leeches under the new taxonomy which recognized four Western Palearctic species, including Hirudo verbena and H. troctina. It notes the three hypotheses that could explain the distribution of Hirudo species, including the impacts of anthropogenesis, ecology and history. It reveals that human impact is considered to be among the important predictions affecting the medicinal leech.

The coordination of multiple neural oscillators is key for the generation of productive locomotor movements. In the medicinal leech, we determined that activation and coordination of the segmental crawl oscillators, or unit burst generators, are dependent on signals descending from the cephalic ganglion. In nearly intact animals, removing descending input (reversibly with a sucrose block) prevented overt crawling, but not swimming. Cephalic depolarization was sufficient for coordination. To determine whether descending signals were necessary for the generation and maintenance of posterior-directed intersegmental phase delays, we induced fictive crawling in isolated whole nerve cords using dopamine (DA) and blocked descending inputs. After blockade, we observed a significant loss of intersegmental coordination. Appropriate phase delays were also absent in DA-treated chains of ganglia. In chains, when one ganglion was removed from its neighbors, crawling in that ganglion emerged robust and stable, underscoring that these oscillators operate best with either all or none of their intersegmental inputs. To study local oscillator coupling, we induced fictive crawling (with DA) in a single oscillator within a chain. Although appropriate intersegmental phase delays were always absent, when one ganglion was treated with DA, neighboring ganglia began to show crawl-like bursting, with motoneuron spikes/burst greatest in untreated posterior ganglia. We further determined that this local excitatory drive excluded the swim-gating cell, 204. In conclusion, both long-distance descending and local interoscillator coupling contribute to crawling. This dual contribution helps to explain the inherent flexibility of crawling, and provides a foundation for understanding other dynamic locomotor behaviors across animal groups. [ABSTRACT FROM AUTHOR]

A high incidence of ample diseases with a complicated etiology, including mycoses of various origins, determine the implementation of unconventional methods, including hirudotherapy, to their treatment. The study was aimed at assessing the mycological purity of medical leech body covers, their jaws and gastrointestinal systems - oesophagus, middle and posterior intestines, and the purity of water from an aquarium they were kept in. The experimental material were 20 European medical leeches (Hirudo medicinalis) and water originating from their breeding. Fungi were obtained according to our own procedures and mycological diagnostics was conducted following standard procedures applied in mycological laboratories. In total, 22 species of fungi belonging to 13 genera were isolated. The material studied was found to contain fungi classified as potential pathogens: Candida albicans, Candida guilliermondii, Candida krusei and Candida tropicalis, as well as numerous saprotrophs with a decreased pathogenicity potential. In view of the results obtained in this study, and a prospective application of hirudotherapy, a question may arise as to whether sterility of cultures and health status of leeches are sufficient and safe for patients. Isolation of a relatively high number of fungi considered pathogenic from leeches poses a potential hazard for people who could have contact with non-sterile therapeutic material, which in this case leeches are. [ABSTRACT FROM AUTHOR]

Background: The medicinal leech, Hirudo medicinalis, is an important model system for the study of nervous system structure, function, development, regeneration and repair. It is also a unique species in being presently approved for use in medical procedures, such as clearing of pooled blood following certain surgical procedures. It is a current, and potentially also future, source of medically useful molecular factors, such as anticoagulants and antibacterial peptides, which may have evolved as a result of its parasitizing large mammals, including humans. Despite the broad focus of research on this system, little has been done at the genomic or transcriptomic levels and there is a paucity of openly available sequence data. To begin to address this problem, we constructed whole embryo and adult central nervous system (CNS) EST libraries and created a clustered sequence database of the Hirudo transcriptome that is available to the scientific community. Results: A total of ∼133,000 EST clones from two directionally-cloned cDNA libraries, one constructed from mRNA derived from whole embryos at several developmental stages and the other from adult CNS cords, were sequenced in one or both directions by three different groups: Genoscope (French National Sequencing Center), the University of Iowa Sequencing Facility and the DOE Joint Genome Institute. These were assembled using the phrap software package into 31,232 unique contigs and singletons, with an average length of 827 nt. The assembled transcripts were then translated in all six frames and compared to proteins in NCBI's non-redundant (NR) and to the Gene Ontology (GO) protein sequence databases, resulting in 15,565 matches to 11,236 proteins in NR and 13,935 matches to 8,073 proteins in GO. Searching the database for transcripts of genes homologous to those thought to be involved in the innate immune responses of vertebrates and other invertebrates yielded a set of nearly one hundred evolutionarily conserved sequences, representing all known pathways involved in these important functions. Conclusions: The sequences obtained for Hirudo transcripts represent the first major database of genes expressed in this important model system. Comparison of translated open reading frames (ORFs) with the other openly available leech datasets, the genome and transcriptome of Helobdella robusta, shows an average identity at the amino acid level of 58% in matched sequences. Interestingly, comparison with other available Lophotrochozoans shows similar high levels of amino acid identity, where sequences match, for example, 64% with Capitella capitata (a polychaete) and 56% with Aplysia californica (a mollusk), as well as 58% with Schistosoma mansoni (a platyhelminth). Phylogenetic comparisons of putative Hirudo innate immune response genes present within the Hirudo transcriptome database herein described show a strong resemblance to the corresponding mammalian genes, indicating that this important physiological response may have older origins than what has been previously proposed. [ABSTRACT FROM AUTHOR]

Introduction: The use of medicinal leeches for treatment purposes is known as hirudotherapy. Hirudotherapy has long been used in traditional medicine for the treatment of chronic pain, particularly in the back and knees. However, it may cause complications such as local infections, allergy, and continued bleeding. This report discusses the effectiveness of primary sutures in controlling continued bleeding caused by hirudotherapy in three patients who presented to the emergency department. Case Report: Three patients presented to the emergency department because of bleeding in the region of where leeches were used for treating back pain. Vital signs of the patients were normal and have any diseases. Persistent bleeding stopped after primary suturing of the wounds. Conclusion: Indications and contraindications of hirudotherapy should be identified in a detail by researches. Practitioners of this therapy should be informed of the use and removal of leeches to prevent the occurrence of complications. [ABSTRACT FROM AUTHOR]

Many postoperative complications have been reported after repair of classic bladder exstrophy. We present a case of medicinal leech therapy for glans penis congestion following exstrophy repair in an infant. A 2-week-old male with classic bladder exstrophy underwent complete primary repair. On postoperative day 1, he developed rapidly worsening glans penis venous congestion. Medicinal leech therapy was instituted with antibiotics and blood transfusions to maintain a hematocrit >30%. After 24 hours, venous congestion improved and therapy was discontinued. The patient's remaining hospital course was uncomplicated. Medicinal leeches are an effective therapy to relieve glans penis venous congestion. [ABSTRACT FROM AUTHOR]

Since the 18th century, the medicinal leech Hirudo medicinalis has been thought to comprise a single species with several different color morphs, but recently some of these color morphs have been assigned to separate species based on morphology, geographical distribution, and molecular sequence data. This research was aimed at testing the ability of three of these species, H. medicinalis, Hirudo verbana, and Hirudo orientalis, to interbreed. We found that in the laboratory, all three species were able to mate with each other and produce hybrid offspring. This suggests that the reproductive isolation is not strong among these species of the genus Hirudo. However, fewer offspring were produced from interspecific crosses compared with intraspecific crosses. This decrease of fecundity (and in some cases, offspring viability) indicates some degree of reproductive isolation between H. medicinalis, H. verbana, and H. orientalis. [ABSTRACT FROM AUTHOR]

Most animals harbour symbiotic microorganisms inside their body, where intimate interactions occur between the partners. The medicinal leech, Hirudo verbana, possesses 17 pairs of excretory bladders that harbour a large number of intracellular and extracellular symbiotic bacteria. In this study, we characterized the bladder symbionts using molecular phylogenetic analyses, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Restriction fragment length polymorphism (RFLP) and sequence analyses of 16S rRNA gene clone libraries suggested that six bacterial species co-colonize the leech bladders. Phylogenetic analyses revealed that these species belong to the α- Proteobacteria ( Ochrobactrum symbiont), β- Proteobacteria ( Beta-1 and Beta-2 symbionts), δ- Proteobacteria ( Bdellovibrio symbiont) and Bacteroidetes ( Niabella and Sphingobacterium symbionts). Species-specific PCR detection and FISH confirmed the localization of the symbiotic bacteria in the bladders. The Ochrobactrum, Beta-1, Bdellovibrio and Sphingobacterium symbionts were consistently detected in 13 leeches from two populations, while infection rate of the other symbionts ranged between 20% and 100% in the two leech populations. Transmission electron microscopy observations of the bladders revealed epithelial cells harbouring a number of intracellular bacilli and an additional type of extracellular, rod-shaped bacteria in the luminal region. Fluorescence in situ hybridization with group-specific oligonucleotide probes revealed the spatial organization of the bacterial species in the bladder: the Ochrobactrum symbiont was located intracellularly inside epithelial cells; the Bacteroidetes were localized close to the epithelium in the lumen of the bladder; and the Bacteroidetes layer was covered with dense β-proteobacterial cells. These results clearly demonstrate that a simple but organized microbial community exists in the bladder of the medicinal leech. [ABSTRACT FROM AUTHOR]

Adult skeletal muscle in vertebrates contains myoendothelial cells that express both myogenic and endothelial markers, and which are able to differentiate into myogenic cells to contribute to muscle regeneration. In spite of intensive research efforts, numerous questions remain regarding the role of cytokine signalling on myoendothelial cell differentiation and muscle regeneration. Here we used Hirudo medicinalis (Annelid, leech) as an emerging new model to study myoendothelial cells and muscle regeneration. Although the leech has relative anatomical simplicity, it shows a striking similarity with vertebrate responses and is a reliable model for studying a variety of basic events, such as tissue repair. Double immunohistochemical analysis were used to characterize myoendothelial cells in leeches and, by injecting in vivo the matrigel biopolymer supplemented with the cytokine Vascular Endothelial Growth Factor (VEGF), we were able to isolate this specific cell population expressing myogenic and endothelial markers. We then evaluated the effect of VEGF on these cells in vitro. Our data indicate that, similar to that proposed for vertebrates, myoendothelial cells of the leech directly participate in myogenesis both in vivo and in vitro, and that VEGF secretion is involved in the recruitment and expansion of these muscle progenitor cells. [ABSTRACT FROM AUTHOR]

Blood-letting was a common therapeutic method in antiquity; many means were used to draw blood, including the application of leeches. In this paper, ancient Greek, Roman and Byzantine authors up to the 7th century AD were studied, a research that provided us with references that may be divided into two groups: those related to the medicinal use of leeches, and those related to cases in which leeches were swallowed and had to be removed. In the first group, detailed descriptions of the method of usage and of the diseases requiring leeching were found. In the second group, brief reference is made to the problems caused by swallowing leeches, and to the methods used to expel them from the human organism. The earliest references to the medicinal use of leeches may be found in the writings of Theocritus (3rd century BC), Nicander (2nd century BC) and Horace (1st century BC, while the phenomenon of swallowing a leech is first mentioned in one of the Epidaurian ‘iamata’ dating to the 4th century BC. [ABSTRACT FROM AUTHOR]

The medicinal leech is a useful animal model for investigating undulatory swimming in the classroom. Unlike many swimming organisms, its swimming performance can be quantified without specialized equipment. A large blood meal alters swimming behavior in a way that can be used to generate a discussion of the hydrodynamics of swimming, muscle mechanics, hydrostatic skeletons, and the physiological features that allow leeches to deal with the volume increase and osmotic load imposed by the meal. Analyses can be carried out at a range of levels tailored to suit a particular class. [ABSTRACT FROM AUTHOR]

aObjective: The implementation of leech therapy for surgical flaps is not always logistically easy or comfortable for patients or healthcare providers. We examine different methods of placing sutures in the medicinal leech, Hirudo medicinalis, to make the implementation of leech therapy easier. Methods: Sixteen leeches were randomly divided into 3 groups: a control group, a deep anchoring suture group, and a superficial anchoring suture group. The leeches were observed to determine if either of these methods had an adverse effect on survival compared with the control group. Results: No difference in survival time was observed across the different groups. Conclusion: The placement of anchoring sutures in leeches can ease the implementation of leech therapy by allowing for greater control of the leeches and thus increased patient comfort. [ABSTRACT FROM AUTHOR]

The concept of Unani medicine is based on balancing body humours, the imbalance of which causes diseases. The application of leech therapy in medical and dental science is well recognized. Although easy and non-invasive, complications also exist. The article aims to presents a brief review on the applications of leech therapy. The physiological effect, along with its therapeutic role in cancer, diabetes and dentistry have been underlined. Complications of leech therapy have also been dealt with. [ABSTRACT FROM AUTHOR]

While glycosyltransferases are restrictively expressed in invertebrate model organisms, little is known of their glycan end products. One such restrictively expressed glycoepitope was localized to sensory and epithelial cells of leech and Caenorhabditis elegans using the Lan3-2 monoclonal antibody. A biological function for the neural Lan3-2 epitope was previously determined in the leech. Here we report on the chemical structure of this mannosidic epitope harvested from whole Hirudo medicinalis. Crude glycans were liberated from glycoproteins by hydrazinolysis. Re- N-acetylated glycans were subjected to immunoaffinity purification. The affinity-purified glycans were fractioned by size chromatography into oligosaccharides and polysaccharides. Lan3-2 oligosaccharide structure was characterized by gas chromatography of alditol acetates, methylation analysis, 500 MHz 1H NMR spectroscopy, matrix-assisted laser desorption/ionization mass spectrometry, and electrospray ionization tandem MS-MS of permethylated derivatives. The predominant components of the Lan3-2 oligosaccharide fraction were a series of linear β-(1,4)-linked mannose polymers. The homologous expression of the Lan3-2 epitope in C. elegans will facilitate the exploration of its glycosylation pathway. Other invertebrates expressing the Lan3-2 epitope are Planaria dugesia, Capitella sp. I and Lumbriculus variegatus. The glycoepitope was not detected in the diploblastic animals Hydra littoralis and Aptaisia sp. or in deuterostomes. [ABSTRACT FROM AUTHOR]

Background: Leeches are commonly used in reconstructive surgery for the treatment of venous congestion in flaps. Documented mechanisms of action are (1) injection of the anticoagulant hirudin; (2) active suction of blood; and (3) passive oozing of the bite wound. Even though the benefits of leeches in venous congestion are widely accepted, little is known about their effects in mixed arterio-venous insufficiency. Methods: Thirty Wistar rats were randomized into three groups (n = 10 each), and 9 × 3 cm ischemic random skin flaps were elevated on the rat dorsum. Group 1 served as controls. In group 2, one leech was applied to the distal part of the flap and in group 3, three leeches were applied, one at a time, at 8 h intervals. Postoperatively, flap survival and perfusion were quantitated by daily planimetry and laser-Doppler imaging. Results: The application of a single leech on postoperative day 7 did not lead to any statistically significant changes in total flap survival or tissue perfusion. Triple leech application, however, caused a significant decrease in flap survival of 6.6% at day 7 (3× leech versus control: 45.8% ± 8.5% versus 52.4% ± 8.5%, respectively) (P < 0.01) and a decline in flap perfusion of 13% (3× leech versus control: 71.3% ± 16.6% versus 84.3% ± 9.3%, respectively) (P < 0.01). Conclusion: The data demonstrate that the application of leeches in the setting of mixed arterio-venous insufficiency can be hazardous to flap viability. Pure venous congestion with an adequate arterial supply remains the only indication for controlled leech application in the clinical setting. [Copyright &y& Elsevier]

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Abstract  Experimental data indicating the polyfunctionality of lysozyme destabilase from the salivary gland secretion of the medicinal leech, a unique representative of invertebrate lysozymes, were analyzed. The destabilase combines the properties of endo-ɛ-lysyl-γ-glutamyl isopeptidase (D-dimer monomerase), lysozyme, and chitinase and simultaneously is a nonenzymatic antimicrobial agent. The polypeptide sequence of lysozyme destabilase is encoded by a family of three genes (Ds1, Ds2, and Ds3). The ability of the enzyme to hydrolyze endoisopeptide bonds formed by transglutaminases, which are detected under many pathological conditions, including thrombosis, is considered from the viewpoint of its further application in practice. [ABSTRACT FROM AUTHOR]

Locomotion in segmented animals is thought to be based on the coupling of "unit burst generators," but the biological nature of the unit burst generator has been revealed in only a few animal systems. We determined that dopamine (DA), a universal modulator of motor activity, is sufficient to activate fictive crawling in the medicinal leech, and can exert its actions within the smallest division of the animal's CNS, the segmental ganglion. In the entire isolated nerve cord or in the single ganglion, DA induced slow antiphasic bursting (~15 s period) of motoneurons known to participate in the two-step elongation- contraction cycle underlying crawling behavior. During each cycle, the dorsal (DE-3) and ventral (VE-4) longitudinal excitor motoneurons fired ~180° out of phase from the ventrolateral circular excitor motoneuron (CV), which marks the elongation phase. In many isolated whole nerve cords, DE-3 bursting progressed in an anterior to posterior direction with intersegmental phase delays appropriate for crawling. In the single ganglion, the dorsal (DI-1) and ventral (VI-2) inhibitory longitudinal motoneurons fired out of phase with each DE-3 burst, further confirming that the crawl unit burst generator exists in the single ganglion. All isolated ganglia of the CNS were competent to produce DA-induced robust fictive crawling, which typically lasted uninterrupted for 5-15 min. A quantitative analysis indicated that DA-induced crawling was not significantly different from electrically evoked or spontaneous crawling. We conclude that DA is sufficient to activate the full crawl motor program and that the kernel for crawling resides within each segmental ganglion. [ABSTRACT FROM AUTHOR]

During periods of high activity neurons are expected to swell due to the uptake of Cl-. To find out whether leech Retzius neurons possess swelling-activated Cl- channels that facilitate Cl- efflux and, hence, volume recovery, we exposed the cells to hypotonic solutions. In hypotonic solutions, the cells slowly swelled but did not undergo a regulatory volume decrease. However, the cell volume increased less than predicted for an ideal osmometer, suggesting the action of a compensatory mechanism. The cell swelling was paralleled by a marked decrease in the input resistance as well as by the activation of a membrane current with a reversal potential close to the Cl- equilibrium potential. This current was substantially diminished by removing bath Cl-, by applying the Cl- channel blocker DIDS, or by treating the cells with the tubulin polymerization inhibitor colchicine. Furthermore, in the presence of colchicine or vinblastine, the cell swelling was substantially increased. It is concluded that leech Retzius neurons possess swelling-activated Cl- channels that require an intact microtubule system for activation. The channels may help to restore cell volume after periods of high neuronal activity. [ABSTRACT FROM AUTHOR]

Glial cells can be activated by neurotransmitters via metabotropic, G protein-coupled receptors. We have studied the effects of `global' G protein activation by GTP--S on the membrane potential, membrane conductance, intracellular Ca2+ and Na+ of the giant glial cell in isolated ganglia of the leech Hirudo medicinalis. Uncaging GTP--S (injected into a giant glial cell as caged compound) by moderate UV illumination hyperpolarized the membrane due to an increase in K+ conductance. Uncaging GTP--S also evoked rises in cytosolic Ca2+ and Na+, both of which were suppressed after depleting the intracellular Ca2+ stores with cyclopiazonic acid (20 μmol l-1). Uncaging inositol-trisphosphate evoked a transient rise in cytosolic Ca2+ and Na+ but no change in membrane potential. Injection of the fast Ca2+ chelator BAPTA or depletion of intracellular Ca2+ stores did not suppress the membrane hyperpolarization induced by uncaging GTP--S. Our results suggest that global activation of G proteins in the leech giant glial cell results in a rise of Ca2+-independent membrane K+ conductance, a rise of cytosolic Ca2+, due to release from intracellular stores, and a rise of cytosolic Na+, presumably due to increased Na+/Ca2+ exchange. [ABSTRACT FROM AUTHOR]

Pump activity is a homeostatic mechanism that maintains ionic gradients. Here we examined whether the slow reduction in excitability induced by sodium-pump activity that has been seen in many neuronal types is also involved in neuronal coding. We took intracellular recordings from a spike-bursting sensory neuron in the leech Hirudo medicinalis in response to naturalistic tactile stimuli with different statistical distributions. We show that regulation of excitability by sodium pumps is necessary for the neuron to make different responses depending on the statistical context of the stimuli. In particular, sodium-pump activity allowed spike-burst sizes and rates to code not for stimulus values per se, but for their ratio with the standard deviation of the stimulus distribution. Modeling further showed that sodium pumps can be a general mechanism of adaptation to statistics on the time scale of 1 min. These results implicate the ubiquitous pump activity in the adaptation of neural codes to statistics. [ABSTRACT FROM AUTHOR]

The article presents microbiological research into the cloning, sequencing, and characterization of catalase gene katA from Aeromonas veronii, a symbiont in the medicinal leech's digestive tract. Induced catalase-null mutant JG186 cells were found to be sensitive to oxidative stress. Results suggest each symbiotic system evolved specific mechanisms that affect the symbiont-host interactions.

Animals house a community of bacterial symbionts in their digestive tracts that contribute to their well being. The medicinal leech, Hirudo verbana, has a remarkably simple gut population carrying two extracellular microbes in the crop where the ingested blood is stored. This simplicity renders it attractive for studying colonization factors. Aeromonas veronii, one of the leech symbionts, can be genetically manipulated and is a pathogen of mammals. Screening transposon mutants of A. veronii for colonization defects in the leech, we found one mutant, JG752, with a transposon insertion in an ascU homolog, encoding an essential component of type III secretion systems (T3SS). Competing JG752 against the wild type revealed that JG752 was increasingly attenuated over time (10-fold at 18 h and >10,000-fold at 96 h). This colonization defect was linked to ascU by complementing JG752 with the operon containing ascU. Fluorescence in situ hybridization analysis revealed that at 42 h 38% of JG752 cells were phagocytosed by leech macrophage-like cells compared with <0.1% of the parental strain. Using mammalian macrophages, a lactate dehydrogenase release assay revealed that cytotoxicity was significantly reduced in macrophages exposed to JG752. In a mouse septicemia model, JG752 killed only 30% of mice, whereas the parent strain killed 100%, showing the importance of T3SS for both pathogenesis and mutualism. Phagocytic immune cells are important not only in defending against pathogens but also in maintaining the mutualistic symbiont community inside the leech, demonstrating that animals use similar, conserved mechanisms to control bacterial populations, even when the outcomes differ dramatically. [ABSTRACT FROM AUTHOR]

The medicinal leech, Hirudo verbana, is one of the simplest naturally occurring models for digestive-tract symbioses, where only two bacterial species, Aeromonas veronii bv. sobria (γ-Proteobacteria) and a Rikenella-like bacterium (Bacteroidetes), colonize the crop, the largest compartment of the leech digestive tract. In this study, we investigated spatial and temporal changes of the localization and microcolony structure of the native symbionts in the crop, after ingestion of a sterile blood meal, by fluorescence in situ hybridization. The population dynamics differed between the two symbiotic bacteria. A. veronii was detected mainly as individual cells inside the intraluminal fluid (ILF) during 14 days after feeding (daf) unless it was found in association with Rikenella microcolonies. The Rikenella-like bacteria were observed not only inside the ILF but also in association with the luminal surface of the crop epithelium. The sizes of Rikenella microcolonies changed dynamically through the 14-day period. From 3 daf onward, mixed microcolonies containing both species were frequently observed, with cells of both species tightly associating with each other. The sizes of the mixed microcolonies were consistently larger than the size of either single-species microcolony, suggesting a synergistic interaction of the symbionts. Lectin staining with succinylated wheat germ agglutinin revealed that the planktonic microcolonies present in the ILF were embedded in a polysaccharide matrix containing N-acetyl-glucosamine. The simplicity, symbiont-symbiont interaction, and mixed microcolonies of this naturally occurring, digestive-tract symbiosis lay the foundation for understanding the more complex communities residing in most animals. [ABSTRACT FROM AUTHOR]