The excitatory actions of a series of piperidine dicarboxylates on central neurons of the rat, snail, leech and horseshoe crab and on crab neuromuscular junction

1. Intracellular recordings were made from Helix, Hirudo and Limulus central neurons and from Eupagurus leg muscle and extracellular recordings from rat lateral geniculate neurons. The actions of 2,3-, 2,4-, 2,5- and 2,6-piperidine dicarboxylates were tested on those preparations for both direct effects and interactions with the response to L-glutamate. 2. All four piperidine analogues were devoid of either reproducible direct or indirect effects on Helix and Limulus neurons save that the 2,5-analogue did mimic the action of L-glutamate on Limulus neurons which were inhibited by glutamate. 3. On Hirudo and rat neurons and on Eupagurus muscle, all four piperidines mimicked the action of L-glutamate, all were weak agonists on rat neurones while on Hirudo neurons the 2,5-analogue was the most potent but on Eupagurus muscle, the 2,3- and 2,6-analogues were the most potent. 4. All four piperidines potentiated the action of L-glutamate on rat and Hirudo neurons and on Eupagurus leg muscle and of muscle ejps. 5. The piperidine analogues indicate that there are differences between the glutamate receptors used in the present study and provide some supportive evidence that the preferred conformation for glutamate on crab leg muscle is partially folded while for Hirudo neurons it is more extended.     

The saliva of the medicinal leech Hirudo medicinalis--I. Biochemical characterization of the high molecular weight fraction

1. A method is described for obtaining dilute Hirudo medicinalis saliva by feeding leeches through a membrane on arginine/saline and squeezing them immediately after from the posterior end forwards. The process can be repeated at intervals. Yields are considerably higher than from salivary gland extracts. 2. Hirudo saliva contains hirudin, eglin, hyaluronidase, collagenase and apyrase. Leech collagenase and apyrase are here reported for the first time. 3. On gel filtration of lyophilized saliva, activity peaks were well defined. Approximate molecular weights were determined. Apyrase appears in two forms with optimum activity around pH 7.5. Collagenase was identified as belonging to the mammalian type.     

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.     

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 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.     

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.     

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.     

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.     

Neuropharmacological studies on the receptors mediating responses to carbachol, amino acids and octopamine on Limulus and Hirudo central neurons

Intracellular recordings were made from central neurons of Limulus polyphemus and from Retizu cells of the leech, Hirudo medicinalis. The effects of carbachol, amino acids and octopamine were examined on these neurons. Octopamine was found to have a mainly inhibitory effect on a few Limulus neurons. The effects of octopamine were mimicked by clonidine and napthazoline but not by xylazine. Both compounds were slightly more potent than octopamine. Yohimbine, metoclopramide, chlorpromazine and chlordimeform failed to antagonize this octopamine response. The excitatory effect of carbachol was blocked by alpha-bungarotoxin, 10(-7)M. Neither this concentration nor higher concentrations of alpha-bungarotoxin had any effect on L-glutamate excitation. m-Carboxyphenyl derivatives of alanine and glycine acted differentially on Limulus neurons responding to L-glutamate. m- Carboxyphenylglycine only inhibited neurones which showed a biphasic response to L-glutamate while m- carboxyphenylalanine only excited these neurons. Both compounds excited leech Retzius cells, with m- carboxyphenylalanine being about 20 times more potent than m- carboxyphenylglycine . The actions of alpha- ketokainate and allo-alpha- ketokainate were compared to kainate, dihydrokainate and L-glutamate on leech Retzius cells. The equipotent molar ratios for kainate, dihydrokainate , alpha- ketokainate and allo-alpha- ketokainate were 0.0029 +/- 0.0004, 0.021 +/- 0.047, 0.029 +/- 0.005 and 0.14 +/- 0.0093 respectively with L-glutamate as one. All the analogues were more potent than L-glutamate. Quinolinic acid had no glutamate-like activity on either Limulus or Hirudo neurons. Methyltetrahydrofolate was inactive on Limulus neurons but excited leech Retzius cells, being slightly less potent than L-glutamate. Dibutyl cAMP terminated the excitatory actions of kainate on both Limulus and Hirudo neurons. Anisatin , a putative GABA antagonist, was a potent antagonist of GABA inhibition on Limulus neurons.     

Preliminary crystallographic data on the complex of bovine alpha-chymotrypsin with the recombinant proteinase inhibitor eglin c from Hirudo medicinalis

The molecular complex built by bovine alpha-chymotrypsin and the recombinant proteinase inhibitor eglin c from Hirudo medicinalis has been crystallized from polyethylene glycol solutions, using a twofold molar excess of the inhibitor with respect to the serine proteinase. The optimum pH for crystal growth is 6.5. The crystals belong to the monoclinic space group P2(1), with unit cell constant: a = 55.3 A, b = 59.4 A, c = 42.5 A, beta = 99.0 degrees; one complex moiety is present per asymmetric unit. The crystals diffract to 2.0 A resolution and are suitable for detailed X-ray crystallographic investigations.     

Crystallization and preliminary X-ray crystallographic study of the leech protease inhibitor guamerin and its complex with bovine pancreatic chymotrypsin

Guamerin, a small peptide inhibitor of the serine protease from Hirudo nipponia, was expressed in yeast and crystallized using the vapor diffusion method, with MPD as precipitant. The crystal was found to belong to the monoclinic P2(1) space group with unit cell parameters a=136.06, b=206.59, c=227.39 A, beta=105.03 degrees. The guamerin/bovine pancreatic chymotrypsin complex was also crystallized using PEG 8K as precipitant. The space group was identified as P2(1)2(1)2(1) with unit cell parameters of a=44.01, b=44.30, c=122.47 A. The diffraction data of the complex were collected up to a resolution of 2.4 A using a synchrotron-radiation source under cryogenic condition.     

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).     

Identification of RFamide neuropeptides in the medicinal leech.

Using a four-step reverse phase HPLC separation and RIA, five RFamide peptides were purified from CNS extracts of the leech Hirudo medicinalis. YMRFamide, FMRFamide, YLRFamide, FLRFamide, and GGKYMRFamide were identified by a combination of antiserum specificity in RIA, Edman degradation, and mass spectrometry. At least three of these five endogenous peptides can modulate neuromuscular interactions in the leech (38). FMRFamide-like immunoreactivity was selectively released from neural processes on isolated heart tubes in the presence of calcium and depolarizing levels of potassium.     

Versuche über Temperatursinn an Blutegeln

Zusammenfassung Der sich mit warmem Blute ernährende Blutegel Hirudo medicinalis L. zeigt positive Reaktion auf Wärmereize. Ein Unterschied von 3° C gegenüber der Temperatur des umgebenden Wassers von 19° C, ist für Hirudo medicinalis L. ausreichend, um diese positive Reaktion auszulösen. Die untersuchten Tiere zeigten diese positive Reaktion bis zu einer Temperatur von durchschnittlich 47° C über derjenigen des umgebenden Wassers von 21,5° C.Alle ändern untersuchten Arten, die nicht von warmem Blute leben, zeigten diese Reaktionen nicht.     

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.     

Neurite-stimulating activity of secretions from the Hirudo medicinalis salivary gland in cultured sensory neurons

Components of leech (Hirudo medicinalis) salivary gland secretion (high molecular mass bdelin B, bdellastasin, eglin C, destabilize) were used to investigate their neurotrophic effect on organotypic culture of dorsal root ganglia of the 10-11-day old chicken embryos. Destabilize and high molecular mass bdelin B in concentrations of 0.01, 0.02, 0.05 and 0.1 ng/ml, bdellastasin in concentrations of 0.02 and 0.05 ng/ml, eglin C in concentrations of 0.1 ng/ml exhibit neurite-stimulating effect as compared to the control.     

Membrane responses of the leech giant glial cell to the peptide transmitter myomodulin

A myomodulin peptide has been suggested to mediate the response of the giant glial cells to stimulation of the Leydig interneuron in the central nervous system of the leech Hirudo medicinalis [Eur. J. Neurosci. 11 (1999) 3125]. We have now studied the glial response to the endogenous leech MM peptide (GMGALRL-NH(2), MMHir). The peptide evokes a membrane outward current (EC(50) approximately 2 microM), which neither desensitizes nor shows any sign of run-down, and elicits a K(+) conductance increase of the glial cell membrane. The peptidase inhibitor phenylmethylsulfonyl fluoride (PMSF) enhances the glial current response, suggesting the presence of endogenous extracellular peptidases.     

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.