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.     

Proteinase inhibitors from the medicinal leech Hirudo medicinalis

The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, -chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of -chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.     

Parallel pathways coordinate crawling in the medicinal leech,Hirudo medicinalis

Changes in the behavior of crawling leeches were investigated after various kinds of manipulations, including selective transection or inactivation of body parts, as well as partial or complete transection of the central nerve cord, using a frame-by-frame analysis of video tapes of the crawling animals. From these studies, we found that: 1. Leeches made rhythmic crawling cycles even after their suckers were prevented from contacting the substrate by covering them over with glue. Hence, engagement and disengagement of the suckers are not necessary links in the crawling cycle. 2. Cutting the small, medial connective (Faivre's nerve) had no influence on crawling, but contraction during the whole-body shortening reflex was interrupted. Thus two behaviors which use the same motor output (i.e., whole-body shortening and the contraction phase of crawling) are mediated by two different pathways. 3. Cutting all the connectives between two ganglia in the middle of the leech resulted in a loss of coordination between the parts of the animal on either side of the cut. Therefore, temporally coordinated sucker activity must be mediated through these connectives. 4. Pieces of leech bodies produced by complete transection produced rhythmic crawling cycles as long as the pieces included the head or tail plus 2–4 adjacent midbody segments. In all cases, the crawling movements progressed without delays as the movements reached the cut ends. Pieces of animals that included only midbody segments did not produce crawling movements. 5. These results can be explained by a model composed of intersegmental pathways for both elongation and contraction, circuits in the head and tail brains that switch between elongation and contraction, and both ascending and descending inhibitory influences that determine when the cycle switches from elongation to contraction and back again.Abbreviations C1-C7 caudal segments 1 through 7 (comprise the tail sucker) - Circ. circular muscle(s) - CD circular element driver - CPG central pattern generator - ED elongation element driver - El elongation - El _init initiation of elongation - FN Faivre's nerve - fs + front sucker attachment - s— front sucker release - Long longitudinal muscle(s) - M1-M21 midbody segments 1 through 21 - R1-R4 rostral segments 1 through 4 (comprise the head) - rs + rear sucker attachment - rs rear sucker release - Sens sensory input - SR stretch receptors(s) - ti tonic inhibition     

A stable prostacyclin-like substance produced by the medicinal leech Hirudo medicinalis.

The medicinal leech Hirudo medicinalis produces a low-molecular mass compound with properties similar to those of prostacyclin. It extracted with organic solvent, had affinity to 6-keto-PGF1alpha antibodies, inhibited human platelet aggregation induced in vitro by thrombin (by 50% at 4 pg/ml), and caused hypotension and secretion of plasminogen (t-PA) into the blood stream of rats. A main distinction from prostacyclin is stability of the substance due to covalent binding with the polypeptide chain of destabilase. Because of the high aggregability of destabilase, the molecules of the protein-lipid complex are organized into micelles that can change their spatial orientation depending on the nature of the solvent. Incorporation of hirudin and blood plasma kallikrein inhibitor into the micelle structure causes the formation of liposomes (with a molecular mass of the structural monomer 25 kDa). This complex with polypeptides provides not only stability but also rapid transmembrane penetration. The pure prostacyclin-like substance has a molecular mass of 391 Da and can be produced on destruction of the destabilase polypeptide chain.     

Morphology of chemosensory organs required for feeding in the leech Hirudo medicinalis

Sensilla that line the upper edge of the lip in the leech Hirudo medicinalis and that contain chemoreceptors required for feeding were examined in the scanning and transmission electron microscopes. The sensilla include two size-classes of ciliated button-like mounds--one about 35 microns in diameter and another about 10 microns in diameter. The larger sensilla are at the center of unpigmented patches of skin which are visible in the light microscope, while the smaller sensilla have not been previously described as distinct structures. Electron microscopy, though not light microscopy, shows that the lip sensilla differ markedly from the segmental sensilla of the leech, which have been shown to mediate mechanoreception and photoreception. In particular, the chemosensory lip sensilla contain multiciliated cells with cilia of a uniform length, whereas the segmental sensilla contain uniciliated cells with long, whip-like cilia, as well as multiciliated cells with short, stiff cilia. Thus, the two types of sensilla differ morphologically as well as functionally. In addition to the ciliated sensilla along the upper lip, structures consisting of a short, club-like process surrounded by granular material were observed inside the mouth. These structures may also be chemosensory organs.     

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.     

Phenology of the medicinal leech, Hirudo medicinalis L., in north-western Turkey

The seasonal occurrence of medicinal leeches was studied at two lakes in the Black Sea coastland of north-western Turkey. The number of leeches attracted by a collector per hour was taken as an indicator for population density. Leeches were most abundant in May/June, and were almost absent during the cold season (November – March). Leeches with a weight of less than 1 g occurred principally between July and October with a peak in July. It is thought that these individuals represent newly hatched leeches. Although both study areas are close to each other, significant differences were found in respect to mean leech size and the seasonal frequency of leeches (both total number and certain age classes). It is suggested that leech biology is greatly dependent on factors such as microclimate and the availability of hosts, and no general conclusions on phenology can be drawn. There is therefore no scientific basis for restricting commercial leech collecting to certain months of the year.     

Distribution and status of the medicinal leech (Hirudo medicinalis L.) in Turkey

A survey of all the major potential habitats in western Turkey showed that medicinal leeches, Hirudo medicinalis L., are widely distributed over the country and are not rare. They occur in practically all suitable habitats and the only region where they were found to be absent is that of the large river deltas in the south of the country (Çukurova deltas, Göksu delta). There may be zoogeographic reasons for this (Taurus mountains barrier). The application of a semi-quantitative survey method using collecting efficiency (number of leeches collected per hour by a single person) allowed a rapid assessment to be made of its status in a large number of wetlands. Leech density varied considerably from wetland to wetland, and the results enabled a ranking of the Turkish wetlands to be made according to their importance for medicinal leeches. Taking both the leech density and the size of leech habitats into account, the largest populations were identified on the Black Sea coast (Kizilirmak delta, Yeilirmak delta and Karagöl Marshes near Sinop) and in inner and south-west Anatolia (Eber Gölü, Karamik and Sultan Marshes). Commercial exploitation for the pharmaceutical industry and for other purposes takes place at only a few places and does not appear to affect the population seriously. However, many populations are threatened by the draining of their habitats.     

Osmotic and ionic regulation during hypoxia in the medicinal leech, Hirudo medicinalis L.

The concentrations of inorganic and organic ions and osmolality in the blood of the medicinal leech, Hirudo medicinalis, were determined during normoxia and hypercapnic and hypocapnic hypoxia. In normoxic animals, the blood sodium concentration was 124.5 +/- 4.2 mmol/l and the total cation concentration was 132.2 +/- 4.3 mEq/l (mean +/- S.D.). Major anionic compounds were chloride (40.8 +/- 1.6 mmol/l), bicarbonate (8.4 +/- 1.3 mmol/l), and organic anions (42.5 +/- 2.3 mEq/l). Among the latter, malate accounts for 30.4 +/- 2.2 mEq/l. The nature of the remaining anion fraction, which balances cation and anion concentrations in leech blood, remains unknown. Within 96 h of hypercapnic hypoxia, the amount of organic osmolytes in leech tissue increased from the control level of 56.6 +/- 9.1 to 158.3 +/- 19.5 mumol/g dry weight. An even higher amount of organic acids was accumulated within 96 h of hypocapnic hypoxia (218.0 +/- 53.7 mumol/g dry weight). A possible reason for this is that lactate, which is a major end-product of hypocapnic hypoxia, cannot be excreted to the external medium as easily as propionate. The accumulation of blood organic acids generating osmotic stress in the animals was compensated by an equimolar decrease in sodium and chloride ion concentrations. In hypercapnic animals these changes resulted in a constant osmotic concentration of the blood (200 mosmol/kg H2O) during the experimental period. Between 24 and 96 h of hypocapnic hypoxia, however, the increase in the osmotic gradient between animal and medium was correlated with further net water uptake and the obvious deterioration of the volume- and ion-regulatory mechanisms in these animals.     

Kinetics of L-gamma-Glu-pNA hydrolysis by destabilase, the enzyme from the medicinal leech Hirudo medicinalis

The molecular mass of destabilase isolated from the medicinae leech Hirudo medicinalis was found to be equal to 12.3 kDa. A kinetic analysis of the sole presently known synthetic substrate, L-gamma-Glu-pNA, showed that the enzyme is relatively stable to heating (5 min, 70 degrees C); the pH optimum lies at 7.0-8.5. The enzyme has a specific activity of 0.15 x 10(-9) mol.s-1.mg-1; Km = 2.2 x 10(-4) M, kcat is 3.53 x 10(-3) s-1 (pH 8.0, 37 degrees C).     

Mechanisms of inhibition of vascular-platelet hemostasis by salivary gland secretion of the medicinal leech Hirudo medicinalis

The mechanism of inhibition of the vascular-platelet stage of hemostasis by medicinal leech salivary gland secretion was studied. It was shown that the secretion blocks platelet adhesion on the surface of collagens belonging to different genetic classes, inhibits the primary attachment of platelets and completely suppresses their spreading on collagen surface. Whatever its antithrombin activity, the leech secretion inhibits platelet aggregation stimulated by various inductors, e. g., ADP, prostaglandin endoperoxide analog U-46619, Ca2+ ionophore A23187, arachidonic acid. The secretion possessing the antithrombin activity causes a greater inhibition of the thrombin-stimulated aggregation than that devoid of this activity. Leech secretion stimulates adenylate cyclase of platelet membranes in a receptor-mediated fashion and increases the level of cAMP. The active substance is a low molecular weight, thermostable trypsin-resistant fraction of the secretion. Stimulation of adenylate cyclase is not mediated by adenosine receptors. It is supposed that the mechanism of this activating effect involves platelet prostaglandin receptors.     

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.     

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.     

Ultrastructure of an identified array of growth cones and possible substrates for guidance in the embryonic medicinal leech,Hirudo medicinalis

The oblique muscle organizer (Comb- or C-cell) in the embryonic medicinal leech, Hirudo medicinalis, provides an amenable situation to examine growth cone navigation in vivo. Each of the segmentally iterated C-cells extends an array of growth cones through the body wall along oblique trajectories. C-cell growth cones undergo an early, relatively slow period of extension followed by later, protracted and rapid directed outgrowth. During such transitions in extension, guidance might be mediated by a number of factors, including intrinsic constraints on polarity, spatially and temporally regulated cell and matrix interactions, physical constraints imposed by the environment, or guidance along particular cells in advance of the growth cones. Growth cones and their environment were examined by transmission electron microscopy to define those factors that might play a significant role in migration and guidance in this system. The ultrastructural examination has made the possibility very unlikely that simple, physical constraints play a prominent role in guiding C-cell growth cones. No anatomically defined paths or obliquely aligned channels were found in advance of these growth cones, and there were no identifiable physical boundaries, which might constrain young growth cones to a particular location in the body wall before rapid extension. There were diverse associations with many matrices and basement membranes located above, below, and within the layer in which growth cones appear to extend at the light level. Additionally, a preliminary examination of myocyte assembly upon processes proximal to the growth cones further implicates a role for matrix-associated interactions in muscle histogenesis as well as process outgrowth during embryonic development.     

Hydrolysis of the isopeptide epsilon-(gamma-glutamyl)-lysine by destabilase from the medicinal leech Hirudo medicinalis

Using amino acid analysis, the ability of destabilize to hydrolyze the epsilon-(gamma-Glu)-Lys isopeptide bond was demonstrated. Incubation of the epsilon-(gamma-Glu)-Lys isopeptide with the enzyme was accompanied by a decrease of the amount of the isopeptide and an increase of equimolar amounts of lysine and glutamic acid. Complete hydrolysis of the isopeptide was observed after 96 hour incubation with destabilize. It was supposed that the isopeptide is a less specific substrate for destabilize compared to L-gamma-Glu-pNA.