Evaluation of performance characteristics of the medicinal leech (Hirudo medicinalis) for the treatment of venous congestion.

Medicinal leeches (Hirudo medicinalis) are a standard treatment for venous congestion, a complication that can occur after reconstructive surgery. If the cause of venous congestion cannot be surgically corrected, then medicinal leeches are used to temporarily increase perfusion levels and maintain physiologic requirements within the congested tissue. Leeches increase perfusion within congested tissue by actively drawing off blood as a bloodmeal. Furthermore, the leech bite continues to bleed and relieve congestion after detachment because of the anticoagulation effects of leech saliva left behind in the bite. In a porcine model, a 10 x 10 cm cutaneous flank flap was congested by clamping the venae comitantes. Four medicinal leeches were allowed to attach to the congested flap, and parameters of active feeding and passive bleeding after detachment were recorded. The average bloodmeal volume for the medicinal leeches was 2.45 ml. Average passive bleeding for the first 2 and 4 hours after leech detachment totaled 2.21 and 2.50 ml, respectively, with 90 percent of passive bleeding occurring within 5 hours after detachment. Laser Doppler imaging indicated that the spatial arrangement of surface perfusion increases were localized to a 1.6-cm-diameter circle around the leech head (bite) and corresponded well with the visual return of normal skin tones to the same area. This study provides a realistic and quantitative estimate of the spatial and volumetric characteristics of leech feeding and passive bleeding using a clinically relevant model of acute, severe congestion.     

New gammaproteobacteria associated with blood-feeding leeches and a broad phylogenetic analysis of leech endosymbionts

Many monophagous animals have coevolutionary relationships with bacteria that provide unavailable nutrients to the host. Frequently, these microbial partners are vertically inherited and reside in specialized structures or tissues. Here we report three new lineages of bacterial symbionts of blood-feeding leeches, one from the giant Amazonian leech, Haementeria ghilianii, and two others from Placobdelloides species. These hosts each possess a different mycetome or esophageal organ morphology where the bacterial cells are located. DNA sequencing of the bacterial 16S rRNA genes and fluorescent in situ hybridization placed these symbionts in two separate clades in the class Gammaproteobacteria. We also conducted a broad phylogenetic analysis of the herein-reported DNA sequences as well as others from bacterial symbionts reported elsewhere in the literature, including alphaproteobacterial symbionts from the leech genus Placobdella as well as Aeromonas veronii from the medicinal leech, Hirudo medicinalis, and a Rickettsia sp. detected in Hemiclepsis marginata. Combined, these results indicate that blood-feeding leeches have forged bacterial partnerships at least five times during their evolutionary history.     

The medicinal leech, Hirudo medicinalis (L.) (Hirudinea: Gnathobdellae), at Dungeness, Kent

WILKIN, P. J., 1989. The medicinal leech, Hirudo medicinalis (L.) (Hirudinea: Gnathobdellae), at Dungeness, Kent . An account of the ecology and conservation of the medicinal leech ( Hirudo medicinalis (L.)) at Dungeness, Kent, is presented.     

The use of a serological technique to examine host selection in a natural population of the medicinal leech, Hirudo medicinalis

SUMMARY. 1. Serological examinations were made of the bloodmeals of medicinal leeches, Hirudo medicinalis L., collected from two sites at Dungeness, Kent, in order to determine their host range.
2. A modified miniature Ouchterlony agar diffusion method was used with antisera raised in rabbits against fish, bird, frog and mammal blood sera.
3. The results suggest that the marsh frog. Rana ridibunda Pall., was the most important host with smaller contributions from birds and fishes. Mammals appeared to be very rare hosts. These results reflect the availability of hosts as determined by field studies. Many leeches showed evidence of blood meals from more than one host.
4. Field observations indicated the importance of the smooth newt, Triturus vulgaris L., as a host for the leech, particularly early in the season. Predation on the newt appeared invariably to be fatal.     

Celebrity with a neglected taxonomy: molecular systematics of the medicinal leech (genus Hirudo)

The medicinal leech is the most famous representative of the Hirudinea. It is one of few invertebrates widely used in medicine and as a scientific model object. It has recently been given considerable conservation effort. Despite all attention there is confusion regarding the taxonomic status of different morphological forms, with many different species described in the past, but only two generally accepted at present. The results of the phylogenetic analysis of a nuclear (ITS2+5.8S rRNA) and two mitochondrial gene sequences (12S rRNA, COI) suggest that the genus Hirudo is monophyletic. It consists, apart form the type Hirudo medicinalis and the East Asian Hirudo nipponia, of three other, neglected species. All of them have already been described either as species or morphological variety, and can readily be identified by their coloration pattern. The type species is in weakly supported sister relation with Hirudo sp. n. (described as variety orientalis) from Transcaucasia and Iran. Sister to them stands Hirudo verbana from southeastern Europe and Turkey, which is nowadays predominantly bred in leech farms and used as 'medicinal leech.' The North African Hirudo troctina is the sister taxon to this group of Western Eurasian species, whereas the basal split is between H. nipponia and the Western Palaearctic clade.     

Growth of the medicinal leech, Himudo medicinalis, under natural and laboratory conditions

SUMMARY 1. The growth of the medicinal leech, Hirudo medicinalis L., was studied in the wild by a mark and recapture technique, and in the laboratory. Growth in the wild, even after 2 years, was generally small and variable.
2. After being offered regular feeds of various preparations of bovine blood through a pig-ileum membrane and being allowed to feed to satiation, hatchling leeches in the laboratory showed a saltatory pattern of weight gain with increases greatly in excess of those seen in the wild. Twelve of twenty-five leeches starved from hatching survived for about 250 days. The ability to survive for long periods without feeding may well be important for the overwintering survival of late hatchlings.     

Proteins and peptides of the salivary gland secretion of medicinal leeches Hirudo verbana, H. medicinalis, and H. orientalis

The protein and peptide composition of medicinal leech salivary gland secretion (SGS) was analyzed in preparations obtained in July from three species--Hirudo verbana, H. medicinalis, and H. orientalis. Two-dimensional electrophoresis (molecular mass 10-150 kD and pI 3-10) revealed no distinctions in the distribution of over 100 silver-stained proteins. Differences were noted only in intensity of 10 protein spots at 30-90 kD and pI 4.7-7.5. Mass spectrometric profiling of SGS of the three leech species using the Zip-Tip/golden chip scheme and cation-exchanging chips CM-10 revealed over 50 components in SGS of each of the three leech species. It was noted that 30-40% of the individual masses of the SGS of each leech species fall within the masses present in SGS of at least one of the two other species. This rather small part of the total mass may be indicative of a high polymorphism of amino acid sequences or a high frequency of posttranslational modifications of the SGS proteins and peptides. Calculation of Jacquard's coefficient showed that H. medicinalis and H. orientalis are closest to each other in SGS composition, which is consistent with data in the literature on the phylogenetic relationship between these two species of medicinal leech. Comparison of detected molecular masses with those of six known biologically active compounds produced by medicinal leeches revealed their uneven distribution in SGS of each of the three medicinal leech species. This opens prospects for using certain species of medicinal leech for targeted therapy of various pathologies.     

Inhibition of kallikrein from human plasma by salivary gland secretion and extracts of the medicinal leech Hirudo medicinalis

It has been shown for the first time that the salivary gland secretion of the medicinal leech (Hirudo medicinalis) contains a human blood plasma kallikrein inhibitor which is capable of blocking the amidolytic activity of the enzyme in an irreversible manner (with D-Pro-Phe-Arg-pNA as substrate) and which also suppresses the kininogenase activity of kallikrein. The inhibition of the amidolytic activity of highly purified kallikrein preparations from human blood plasma obeys the pseudo-first order kinetics. The experimental results suggest that in the salivary-gland secretion of H. medicinalis the inhibitor concentration exceeds by one order of magnitude that in whole leech homogenate extracts, which indicates that the inhibitor biosynthesis may be localized in leech salivary glands.     

Differential effects of serotonin depletion on sensitization and dishabituation in the leech, Hirudo medicinalis.

The goal of these experiments was to test the hypothesis that serotonin (5-HT) is involved in facilitation of the shortening reflex in the leech Hirudo medicinalis. For this reason, we have used the toxin 5-hydroxytryptamine (5,7-DHT) to deplete serotonin from the nervous systems of intact leeches and have assessed the effect on early facilitation, dishabituation, and sensitization of the touch-elicited shortening reflex using behavioral procedures previously developed in our lab (Boulis and Sahley, 1988). We find that 5,7-DHT lesions completely attenuate early facilitation and sensitization but only reduce dishabituation of the touch-elicited shortening reflex. Histological analyses of the ganglia from these leeches using glyoxilic acid staining procedures revealed an absence of staining in the Retzius cell of experimental leeches. All other serotonin-containing neurons showed glyoxilic acid staining comparable to that observed in the control leeches.     

Complement resistance is essential for colonization of the digestive tract of Hirudo medicinalis by Aeromonas strains

From the crop of the medicinal leech, Hirudo medicinalis, only Aeromonas veronii bv. sobria can be cultured consistently. Serum-sensitive A. veronii mutants were unable to colonize H. medicinalis, indicating the importance of the mammalian complement system for this unusual simplicity. Complementation of one selected mutant restored its ability to colonize. Serum-sensitive mutants are the first mutant class with a colonization defect for this symbiosis.     

The effects of procaine, strychnine and penicillin on nociceptive neurons in leech segmental ganglia

Procaine, strychnine and penicillin selectively depolarized the membrane potential and prolonged the action potential recorded in the lateral but not the medial nociceptive (N) cell in the hirudinid leech Macrobdella decora. In contrast, procaine did not differentiate between medial and lateral N cells in two other hirudinid leeches Hirudo medicinalis and Haemopis marmorata. In these species, the drug equally decreased the amplitude of action potentials in both types of N cells without effecting their resting membrane properties. In the nociceptive neurons of the glossiphoniid leech Haementeria ghilianii which possesses only one type of N cell, procaine produced a depolarization and prolonged the action potential. This finding indicates that the single pair of N cells in Haementeria is of the lateral type. The results suggest that the lateral type N cell in Macrobdella and Haementeria share a unique Na+-dependent conductance which is selectively opened by the local anesthetics procaine and strychnine as well as by penicillin. This conductance is either not present or insensitive to the drugs in the homologous N cells in the two other leech species examined.     

The effects of feeding regime on the growth and reproduction of the medicinal leech Hirudo medicinalis

1. The feeding frequency, the size of meals, the number of meals required to attain reproductive maturity and the number of meals taken between iteroparous reproductive bouts were determined in the laboratory under optimal conditions for the medicinal leech Hirudo medicinalis fed exclusively on mammalian (bovine) blood. In addition the number of bouts of reproduction and the numbers of cocoons and hatchlings per cocoon produced were determined.
2. The average time for H. medicinalis to reach reproductive maturity at 20°C was 289 days, at an average wet biomass of 8143 mg with two–nine separate bouts of cocoon production. The number of meals to first reproduction was 8.9 (mean meal size of 3066.7 mg), with a significant correlation between total mass of blood ingested and the numbers of reproductive bouts and number of cocoons produced. Mean lifetime cocoon production per individual was 12.43, with 3.9 hatchlings per cocoon.
3. The significant positive relationships between ingestion, fecundity and developmental rate observed support the hypothesis that declining abundances of field populations of H. medicinalis are the result of lower available energy for growth, reflecting leeches now feeding predominantly on amphibian blood of lower energetic value than mammalian blood.     

Can different species of medicinal leeches (Hirudo spp.) interbreed?

Abstract. 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.     

The effects of feeding regime on the growth and reproduction of the medicinal leech Hirudo medicinalis

1. The feeding frequency, the size of meals, the number of meals required to attain reproductive maturity and the number of meals taken between iteroparous reproductive bouts were determined in the laboratory under optimal conditions for the medicinal leech Hirudo medicinalis fed exclusively on mammalian (bovine) blood. In addition the number of bouts of reproduction and the numbers of cocoons and hatchlings per cocoon produced were determined.
2. The average time for H. medicinalis to reach reproductive maturity at 20°C was 289 days, at an average wet biomass of 8143 mg with two–nine separate bouts of cocoon production. The number of meals to first reproduction was 8.9 (mean meal size of 3066.7 mg), with a significant correlation between total mass of blood ingested and the numbers of reproductive bouts and number of cocoons produced. Mean lifetime cocoon production per individual was 12.43, with 3.9 hatchlings per cocoon.
3. The significant positive relationships between ingestion, fecundity and developmental rate observed support the hypothesis that declining abundances of field populations of H. medicinalis are the result of lower available energy for growth, reflecting leeches now feeding predominantly on amphibian blood of lower energetic value than mammalian blood.     

Phylogeny and phylogeography of medicinal leeches (genus Hirudo): fast dispersal and shallow genetic structure

Medicinal leeches (Hirudo spp.) are among the best-studied invertebrates in many aspects of their biology. Yet, relatively little is known about their biogeography, ecology and evolution. Previous studies found vast ranges but suggested low genetic diversity for some species. To examine this apparent contradiction, the phylogeny and phylogeography of the widespread Hirudo verbana, Hirudo medicinalis and Hirudo orientalis were investigated in a comparative manner. Populations from across their ranges in Europe, Asia Minor, the Caucasus and Central Asia, were analyzed by various phylogenetic and population genetic approaches using both mitochondrial (COI and 12S) and nuclear DNA sequences (ITS1, 5.8S and ITS2). The populations showed surprisingly little genetic differentiation despite vast ranges. The only clear structure was observed in H. verbana. This species is subdivided into an Eastern (southern Ukraine, North Caucasus, Turkey and Uzbekistan) and a Western phylogroup (Balkans and Italy). The two phylogroups do not overlap, suggesting distinct postglacial colonization from separate refugia. Leeches supplied by commercial facilities belong to the Eastern phylogroup of H. verbana; they originate from Turkey and the Krasnodar Territory in Russia, two leading areas of leech export. H. verbana and H. medicinalis have experienced recent rapid population growth and range expansion, while isolation by distance has shaped the genetic setup of H. orientalis. The habitat of the latter is patchy and scattered about inhospitable arid and alpine areas of Central Asia and Transcaucasia. Centuries of leech collecting and transport across Europe seem not to have affected the natural distribution of genetic diversity, as the observed patterns can be explained by a combination of historical factors and present day climatic influences.     

The effect of preparations of Hirudo medicinalis leeches on DNA methylation in the rat liver

The effect of the salivary gland secretion and dialysable part of the homogenate of the leeches Hirudo medicinalis on the methylation of DNA in the rat liver after the intraperitoneal injection and perfusion of isolated liver has been analysed. The maximum concentration of 5-methylcytosine is observed 1 h later the injection of preparations: for the salivary gland secretion the increase is 39%, for the dialysate of leech homogenate is 28%. The 5-methylcytosine content increases on 28% after the perfusion of isolated liver with the leech saliva and after the dialysate of the leech homogenate--on 20%. No other changes in DNA content is observed. It is suggested that the DNA-methylation of the liver cells is due to the penetration of biologically active substances produced by the medical leech into the cell-targets accompanied by the forming of corresponding ligand-receptor complexes.     

Hyaluronidase activity in leeches (Hirudinea)

The leech hyaluronoglucuronidase (hyaluronidase I) was identified in Erpobdellidae (Nephelopsis obscura and Erpobdella punctata) and Glossiphoniidae (Desserobdella picta) and historically described from Hirudinidae (Hirudo medicinalis). A second leech hyaluronidase (hyaluronidase II) which hydrolyzed only a few bonds to for hyaluronan oligosaccharides larger than 6500 Da, was found in Glossiphoniidae (Helobdella stagnalis, Glossiphonia complanata, Placobdella ornata, and Theromyzon sp.) and in Haemopidae (Haemopis marmorata). The distribution of the two hyaluronidases in leech occurred in both orders (Arhynchobdellida and Rhynchobdellida) and in macrophagous and haematophagous feeding types whereas the liquidosomatophagous leeches only had hyaluronidase II.     

Immunohistochemical detection of regulatory cells in the digestive system of leeches

The digestive system of four leech species,Glossiphonia complanata, Hirudo medicinalis, Haemopis sanguisuga, andErpobdella octoculata, was studied using, as markers, antisera to biologically active peptides, neurotensin, calcitonin, FRMF-amide, and serotonin. In the epithelium and the wall of the alimentary tract, regulatory cells were revealed. They differed in shapes and detected immunoreactivities; presumably they are elements of the diffuse endocrine system as well as of the peripheral nervous system. The data obtained show that the organization of the intestinal regulatory system in leeches basically is similar morphologically to intestinal regulatory systems of representatives of other classes of this type of invertebrate animals.     

Twelve variable microsatellite loci for the North American medicinal leech,Macrobdella decora

We isolated and characterized 12 microsatellite loci for the North American medicinal leech, Macrobdella decora. Macrobdella decora is abundant in central and northern North American freshwater systems. The resulting microsatellite library, the first for any species of leech or any clitellate annelid, demonstrates the efficacy of this type of analysis on unexploited leech populations and should serve as a baseline for comparison with other species, notably the commercially important and threatened European medicinal leech, Hirudo medicinalis.