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.     

The structure of a natural population of the medicinal leech, Hirudo medicinalis, at Dungeness, Kent

SUMMARY 1. A hot branding method was used to mark medicinal leeches ( Hirudo medicinalis L.) from five samples (four in 1984, one in 1985) at agravel pit at Dungeness in Kent. Analysis of capture-recapture data by the Fisher-Ford and Jolly methods gave similar results and indicated a population in excess of 10000 (0.112 leeches per m²) in 1984 and 1985. Successive estimates were each higher than the last suggesting a thriving community.
2. The population structure was skewed towards the lower weight classes. This may indicate good recruitment of hatchlings, as also indicated by the presence of cocoons, or the failure to capture heavier satiated or gravid leeches because of their relative unresponsiveness to the sampling method involving water disturbance, or their complete absence from the water during cocoon laying. Gravid leeches were always above 2 g in weight. Recaptured marked leeches revealed that they migrated throughout the site, indicating active foraging for hosts.     

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.     

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.     

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.     

Protein Profiling of the Medicinal Leech Salivary Gland Secretion by Proteomic Analytical Methods

Protein diversity of the high molecular weight fraction (molecular mass > 500 daltons) of salivary grand secretion of the medicinal leech Hirudo medicinalis has been demonstrated using methods of proteomic analysis. One-dimensional (1D) electrophoresis revealed the presence of more than 60 bands corresponding to molecular masses ranging from 11 to 483 kD. 2D-electrophoresis revealed more than 100 specific protein spots differing in molecular masses and pI values. SELDI-mass spectrometry analysis using the ProteinChip. System based on chromatography surfaces of strong anion or weak cation exchanger detected 45 individual compounds of molecular masses ranged from 1.964 to 66.5 kD. Comparison of SELDI-MS data with protein databases revealed eight known proteins from the medicinal leech. Other masses detected by proteomic analytical methods may be related to both modifications of known proteins and unknown biologically active components of leech saliva secretion.     

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.     

Aeromonas, acclimation, and penicillin as complications when leeches are applied to skin flaps in rabbits

The medicinal leech, Hirudo medicinalis, has been recognized by plastic surgeons for its ability to reduce congestion and improve blood flow in grafted skin flaps by withdrawing excess blood. The leech's ability to digest blood is due to the presence of Aeromonas hydrophila in the gut of the leech. In this report we describe the occurrence of Aeromonas hydrophila septicaemia in rabbits used in a study to evaluate the efficacy of the medicinal leech on abdominal skin flap survival. In New Zealand White rabbits, twin 5 x 10 cm abdominal skin flaps were prepared and the epigastric vein was ligated to produce a model of venous congested skin. After 12 hours, a leech was applied to one of the congested abdominal flaps and skin survivability was assessed and compared with the unleeched flap. Five rabbits died acutely approximately 24 hours post-operatively. Gross necropsy, bacteriology and histopathology findings indicated that A. hydrophila was the causative agent of the septicaemia-toxaemia syndrome with an associated pneumonia and typhlitis. Review of the management and the experimental surgery protocol suggested that the two major disposing factors of Aeromonas septicaemia in these rabbits were stress and excessive prophylactic administration of penicillin.     

Predicting efficiency of selection on reproductive traits in the medicinal leech Hirudo medicinalis L

A set of selection measures for increasing reproduction efficiency in Hirudo medicinalis has been developed. The optimal values of reproductive traits corresponding to the highest progeny number were determined and recommended. The probability of correlated selection response in traits "number of threads in a cocoon" and "weight of threads" was estimated. Based on earlier results on phenotypic variation and heritability of reproductive traits in medicinal leech, efficiency of different selection modes was predicted.     

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.     

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.     

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.     

Ingested blood contributes to the specificity of the symbiosis of Aeromonas veronii biovar sobria and Hirudo medicinalis, the medicinal leech

Hirudo medicinalis, the medicinal leech, usually carries in its digestive tract a pure culture of Aeromonas veronii bv. sobria. Such specificity is unusual for digestive tracts that are normally colonized by a complex microbial consortium. Important questions for the symbiotic interaction and for the medical application after microvascular surgery are whether other bacteria can proliferate or at least persist in the digestive tract of H. medicinalis and what factors contribute to the reported specificity. Using a colonization assay, we were able to compare experimentally the ability of clinical isolates and of a symbiotic strain to colonize H. medicinalis. The symbiotic A. veronii bv. sobria strain proliferated well and persisted for at least 7 days inside the digestive tract. In contrast, the proliferation of Pseudomonas aeruginosa and Staphylococcus aureus was inhibited inside the animal compared to growth in the in vitro control, indicating that the ingested blood was modified within the digestive tract. However, both strains were able to persist in the digestive tract for at least 7 days. For an Escherichia coli strain, the viable counts decreased approximately 1, 000-fold within 42 h. The decrease of viable E. coli could be prevented by interfering with the activation of the membrane-attack complex of the complement system that is present in blood. This suggests that the membrane-attack complex remained active inside H. medicinalis and prevented the proliferation of sensitive bacteria. Thus, antimicrobial properties of the ingested vertebrate blood contribute to the specificity of the A. veronii-H. medicinalis symbiosis, in addition to modifications of the blood inside the digestive tract of H. medicinalis.     

Primary structure and function of novel O-glycosylated hirudins from the leech Hirudinaria manillensis.

Hirudin from the leech Hirudo medicinalis is a most powerful anticoagulant, and many isoforms have been described. In the present work, the primary structure of two hirudins from the leech Hirudinaria manillensis has been elucidated. The antithrombotic activity is similar to that of H. medicinalis hirudins although the sequence identity is below 60%. Surprisingly, the hirudins were found to be glycosylated at one site. Sugar analysis after methanolysis yielded fucose, galactose, and N-acetylgalactosamine. These results combined with data from matrix-assisted laser desorption ionization mass spectrometry, plasma desorption mass spectrometry, capillary zone electrophoresis, and lectin-binding tests indicate that the sequence is Fuc-Gal beta 1-3GalNAc-(O-threonine). This structure shows an interesting similarity to human blood group H determinants.     

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.     

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.     

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.     

Proteomic analysis methods for characterization of proteins from the salivary gland secretions of the medicinal leech during different seasons

Salivary gland secretion (SGS) of the medicinal leech Hirudo medicinalis in summer and winter was studied by proteomic analysis methods, and season-associated difference was found in the distribution of fractionated proteins with the same pattern of their positions. Differences were detected for proteins with molecular weights from 15 to 250 kD fractionated by two-dimensional SDS-PAGE and for 2-10- and 10-60-kD proteins analyzed by SELDI-MS. Thirty-two and 20 proteins were detected by MALDI-TOF-MS in the high-molecular-weight fraction of the summer and winter SGS, respectively, isolated from the corresponding two-dimensional electrophoregrams, and this was less than 20% of the total SGS protein. The N-terminal amino acid sequences were determined for 12 proteins. The peptide maps and N-terminal amino acid sequences of the proteins studied were identified, and no known proteins were revealed. These findings suggest a high content of newly revealed proteins in SGS of medicinal leech, and this correlates with multiple positive clinical effects of hirudotherapy realized through SGS, but the mechanisms of these effects remain unclear.