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.     

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     

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.     

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.     

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.     

Scheme for dispersion analysis for evaluating heritability of reproductive traits in medicinal leech Hirudo medicinalis L

ANOVA designs for estimating heritabilities of fecundity traits in Hirudo medicinalis including batch size, the number of juveniles per cocoon, and juvenile weight. Accounting for reproduction mode of this species, different types of kinship were identified, which were taken into account in the ANOVA designs: juveniles from one batch and one cocoon were considered respectively full sibs and polyzygotic twins. Variation components were analyzed in the following kinship groups: for batch size, in full sibs; for the number of juveniles per cocoon, between sibships, full sins, and replicates; and for juvenile weight, in full sibs and polyzygotic twins. Using these designs, heritabilities of basic reproductive traits of H. medicinalis were obtained: for batch size, h2 = 0.35-0.40, H2 = 0.40-0.45; for the number of juveniles per cocoon, h2 = 0.33-0.36, H2 = 0.36-0.39; and for juvenile weight, h2 = 0.40-0.44, H2 = 0.44-0.48.     

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).     

Recovery after anaerobic metabolism in the leech (Hirudo medicinalis L.)

Medicinal leeches (Hirudo medicinalis L.) responded to self-induced hypoxia (72 h) with typical anaerobic metabolism characterized by a decrease in adenylate energy charge, utilization of the substrates glycogen and malate, and accumulation of the main anaerobic endproducts succinate and propionate. Propionate was also excreted into the medium. Ammonia excretion was suppressed. Aerobic recovery resulted in a profound O₂ debt. Resynthesis of ATP was completed within 30 min. Disposal of succinate and restoring of malate required 2–3 h, and clearance of propionate and recharging of glycogen 6–12 h. Ammonia excretion did not exceed normoxic rates and excretion of propionate during recovery accounted for only 10% of total propionate accumulated during hypoxia. It is postulated that the clearance of succinate and propionate involves oxidation but also resynthesis of malate and glycogen. During hypoxia and recovery blood osmolality remained constant. The Na⁺ and Cl^- ion concentrations in blood, the decrease of which was nearly equimolar during hypoxia, were re-established following different time-courses. Na⁺ concentration returned to normoxic levels after 2–3 h. The delayed increase in Cl^- concentration, however, correlating with 6–12 h necessary to clear blood propionate, is interpretated as an anion regulating effect.Abbreviations AEC adenylate energy charge; fw, fresh weight - HPLC high-performance liquid chromatography - SCCA shortchain carboxylic acids     

Population size, weight distribution and food in a persistent population of the rare medicinal leech, Hirudo medicinalis

1. It is important for species recovery and conservation management projects to know the minimum viable population size for rare and endangered species, such as the medicinal leech, Hirudo medicinalis. Therefore, using a catch‐removal method, this study estimated every two years (1986, 1988, 1990, 1992) the total number of medicinal leeches in a tarn in the English Lake District, and the number of mature adults in the population. 2. Four samples were taken each year in June and July, when water temperatures exceeded 20 °C. Population size was estimated both by maximum likelihood and regression methods. All leeches were weighed alive and size groups were separated by polymodal frequency analysis. A small sample of the blood meal in each leech gut was taken before the leeches were returned to the tarn, and was used to estimate the proportion of mammalian and non‐mammalian blood in the meals. 3. Both methods of estimation produced similar values, increasing confidence in the population estimates. Values for the total population in June and July varied among years from 248 to 288, the maximum value being only 16% higher than the minimum. Values for the number of mature leeches varied from 48 to 58 (19–20% of the total population), and this was an estimate of the effective population size. 4. There were four size groups. The largest mature leeches (live weight >5 g) in group IV formed only 1% of the population, and the smallest (0.02–0.5 g) in group I 14–17%. Most leeches were in two overlapping groups of immature (64–67% of population) and mature (18%) leeches with size ranges of 0.4–3.4 g and 2.5–5 g respectively. The percentage of leeches in each size group was very consistent among years. Blood meals were found in 38–44% of the leeches in group I, 45–50% in group II, 70–75% in group III, and 100% in group IV, but mammalian blood was present only in larger mature leeches (>3.5 g). 5. Medicinal leeches were first detected in the tarn in 1980 and are still present in 2007, so the population has persisted for at least 27 years. Compared with minimum viable population sizes for other species, including many endangered species, values for this medicinal leech population are extremely low, but may be typical of some rare freshwater invertebrates in isolated habitats.