Leech Hirudo Verbana Research Articles

Evaluation of per- and polyfluoroalkyl substances (PFAS) toxic effects on the acute inflammatory response in the medicinal leech Hirudoverbana

Per- and polyfluoroalkyl (PFAS) substances are a large group of chemicals with elevated water and oil-resistance properties, widely implicated in various applicative fields. Due to the extensive use and high resistance to degradative factors, these compounds pose a significant risk of environmental spreading, bioaccumulating also in living organisms. In this context, despite many researches have been performed to demonstrate “legacy” PFAS harmfulness, only few data are still available about all the emerging fluorinated molecules, industrially introduced to replace the previous ones. For this reason, we proposed the medicinal leech Hirudo verbana as consolidated invertebrate model to assess the effects of four different PFAS (HFPO-DA, PFMoBa, PFOA and PFMOPrA) following freshwater dispersion. Morphological, immunohistochemical and molecular analyses demonstrate that, despite all the compounds basically induce an acute inflammatory and oxidative stress response, a different cellular and molecular response has been observed. Whereas for PFOA and PFMOPrA an increase in the tested concentration leads to a corresponding rise in the immune response, HFPO-DA and PFMoBa trigger an entirely opposite effect. Indeed, the significant recruitment of both granulocytes and macrophage like cells, typically involved in the removal of non-self, is inhibited with increasing concentrations of these compounds. The data collected revealed a different sensitivity of the leech immune system following PFAS exposure, requiring to deepen the current knowledge on the potential toxicity of these compounds.

Effects of light on reproduction of the Mediterranean medicinal leech, Hirudo verbana Carena, 1820: do circadian rhythm disruptions positively affect reproductive performance of leeches?

ABSTRACT This study aimed to assess the impact of light on the reproductive performance of leeches for the first time using the Mediterranean medicinal leech Hirudo verbana Carena, 1820, which is important in medicinal leech aquaculture. Gravid leeches were subjected to three different light conditions: 24-h Light, 24-h Dark, and Daylight (14-h light and 10-h dark) for 11 weeks. Gravidity ended at the 8th week for the Dark (5.33 ± 0.38 weeks) and Daylight (5.41 ± 0.29 weeks) groups, whereas it persisted for 11 weeks in the Light group (6.75 ± 0.51 weeks) (p = 0.031). In comparison to the Daylight group (cocoons: 40, offspring: 538), the Light (cocoons: 50, offspring: 723) and Dark (cocoons: 45, offspring: 576) groups achieved a greater yield of cocoons (p = 0.312) and offspring (p = 0.323). We invite discussion on the notion that the enhanced reproductive capacity of leeches in full light and full dark conditions, as opposed to daylight, may be attributed to the disturbance of their circadian rhythms.

On the occurrence of the Dragon leech Hirudo troctina Johnson, 1816 (Annelida, Hirudinea) in Sardinia (Italy)

In recent decades, medicinal leeches of the genus Hirudo have experienced a sharp decline throughout their distribution range due to their overexploitation for medical use and habitat alteration. Such a phenomenon is not fully understood because of the complex taxonomy of the genus, which remained unsettled until the beginning of the XXI century, when the implementation of DNA taxonomy allowed for a better understanding of the diversity of the genus and the distribution ranges of Hirudo species. Only the Mediterranean medicinal leech Hirudo verbana Carena, 1820 is currently reported to occur in Italy and its major islands, although records of other Hirudo species have been reported in the literature. In this study, we report the occurrence of the Dragon leech Hirudo troctina Johnson, 1816 in Sardinia and provide its molecular characterization. In addition, based on a review of the existing literature and museum collections, we provide an ample synopsis of the available evidence regarding the occurrence of the species on the island. Hirudo troctina proved to be widespread and abundant in Sardinia during the XIX century but became rarer and rarer in the XX and XXI centuries possibly due to its overexploitation for medicinal use and export in mainland Europe, the abandonment of traditional grazing practices, and the decline of wetland habitats and amphibian populations. Currently, the only recently validated occurrence sites of the species are the water bodies of the “Giara di Gesturi”, a basaltic plateau in central-southern Sardinia. No corroborated evidence of the possible occurrence of other Hirudo species on the island was found. A better understanding of Hirudo troctina distribution in Sardinia will allow the amelioration of its protection and management practices, also under the provisions of the European “Habitats Directive”, where the H. medicinalis species complex is listed in Annex V.

Dynamic relationship among extracellular matrix and body wall cells in Hirudo verbana morphogenesis

A great bulk of recent experimental evidence suggests the key role of the complex crosstalk between the extracellular matrix (ECM) and the cellular component of tissues during morphogenesis and embryogenesis. In particular, remodeling of the ECM and of its physical interactions pattern with surrounding cells represent two crucial processes that might be involved in muscle development. However, little information is available on this topic, especially on invertebrate species. To obtain new insights on how tuning the ECM microenvironment might drive cellular fate during embryonic development, we used the invertebrate medicinal leech Hirudo verbana as a valuable experimental model, due to its simple anatomy and the recapitulation of many aspects of the basic biological processes of vertebrates. Our previous studies on leech post-embryonic development have already shown the pivotal role of ECM changes during the growth of the body wall and the role of Yes-associated protein 1 (YAP1) in mechanotransduction. Here, we suggest that the interactions between stromal cell telocytes and ECM might be crucial in driving the organization of muscle layers during embryogenesis. Furthermore, we propose a possible role of the pleiotropic enzyme HvRNASET2 as a possible modulator of collagen deposition and ECM remodeling not only during regenerative processes (as previously demonstrated) but also in embryogenesis.

The influence of the water-salt extract of the medicinal leech Hirudo verbana Carena, 1820 on the general course of embryogenesis in rats after intraperitoneal administration

Background. The increase in infectious and non-infectious diseases occurrence during pregnancy prompts scientists to search for substances that can fight these ailments. This especially applies to natural substances that have virtually no side effects. These include substances obtained from medicinal leeches. These substances have a wide range of therapeutic effects; as to their practical use, all preclinical studies must be completed. This particularly refers to the presence of an embryotoxic effect. The aim of this work was to investigate the effect of the water-salt extract of the medicinal leech Hirudo verbana Carena, 1820 on the general course of embryogenesis in rats when administered intraperitoneally. Materials and Methods. The work was performed on female non-linear white rats. Two experimental groups of animals were injected intraperitoneally with a water-salt extract of the medicinal leech at a concentration of 5 μg/g of the animal, in a volume of 0.5 mL, three times before mating and three times during pregnancy every 3 days. Two control groups were injected intraperitoneally with physiological saline according to the same scheme as the experimental groups. Subsequently, one of the control and experimental groups underwent euthanasia, dissection of the abdominal cavity and uterine horns on the 20th day of pregnancy. We analyzed the total number of corpora lutea of pregnancy, the number of live and dead fetuses. Fetuses were weighed, examined for the presence of skin hematomas, mutilations, and external abnormalities in the develop­ment of internal organs. The other two groups of control and experimental animals were left until the offspring were born. Then the females of both groups were weighed, and their general condition was assessed. Newborn rats were examined, weight and body length were determined, mortality was recorded from the beginning of birth to 15 days, the presence of skin hematomas, mutilations, and external anomalies of the development of internal organs were checked. Results and discussion. The study established that the intraperitoneal injection of a water-salt extract of a medicinal leech before and during pregnancy in female rats does not cause any toxic effect during embryogenesis and at the early stages of post-embryonic ontogenesis. During the external examination of fetuses and offspring in both groups, no developmental anomalies were noted, all organs in both groups were of normal topography and size. The number of subcutaneous hematomas in the experimental group of fetuses compared to the control group decreased by an average of 50%, postnatal mortality decreased by an average of more than 3 times, as well as an increase in body weight in females, an increase in body weight and body length in the fetus and offspring in the early post-embryonic period ontogenesis (p <0.05). Conclusion. Intraperitoneal administration of a therapeutic dose of a water-salt extract of the medicinal leech to female rats before and during pregnancy, after every three days, does not have any toxic effect on the course of embryogenesis and later in early post-embryonic ontogenesis.

Heterosynaptic long-term potentiation of non-nociceptive synapses requires endocannabinoids, NMDARs, CamKII, and PKCζ.

Noxious stimuli or injury can trigger long-lasting sensitization to non-nociceptive stimuli (referred to as allodynia in mammals). Long-term potentiation (LTP) of nociceptive synapses has been shown to contribute to nociceptive sensitization (hyperalgesia) and there is even evidence of heterosynaptic spread of LTP contributing to this type of sensitization. This study will focus on how activation of nociceptors elicits heterosynaptic LTP (hetLTP) in non-nociceptive synapses. Previous studies in the medicinal leech (Hirudo verbana) have demonstrated that high-frequency stimulation (HFS) of nociceptors produces both homosynaptic LTP as well as hetLTP in non-nociceptive afferent synapses. This hetLTP involves endocannabinoid-mediated disinhibition of non-nociceptive synapses at the presynaptic level, but it is not clear if there are additional processes contributing to this synaptic potentiation. In this study, we found evidence for the involvement of postsynaptic level change and observed that postsynaptic N-methyl-d-aspartate (NMDA) receptors (NMDARs) were required for this potentiation. Next, Hirudo orthologs for known LTP signaling proteins, CamKII and PKCζ, were identified based on sequences from humans, mice, and the marine mollusk Aplysia. In electrophysiological experiments, inhibitors of CamKII (AIP) and PKCζ (ZIP) were found to interfere with hetLTP. Interestingly, CamKII was found to be necessary for both induction and maintenance of hetLTP, whereas PKCζ was only necessary for maintenance. These findings show that activation of nociceptors can elicit a potentiation of non-nociceptive synapses through a process that involves both endocannabinoid-mediated disinhibition and NMDAR-initiated signaling pathways.NEW & NOTEWORTHY Pain-related sensitization involves increases in signaling by non-nociceptive sensory neurons. This can allow non-nociceptive afferents to have access to nociceptive circuitry. In this study, we examine a form of synaptic potentiation in which nociceptor activity elicits increases in non-nociceptive synapses. This process involves endocannabinoids, "gating" the activation of NMDA receptors, which in turn activate CamKII and PKCζ. This study provides an important link in how nociceptive stimuli can enhance non-nociceptive signaling related to pain.

Spatio-Temporal Changes of Extracellular Matrix (ECM) Stiffness in the Development of the Leech Hirudo verbana.

The invertebrate leech Hirudo verbana represents a powerful experimental animal model for improving the knowledge about the functional interaction between the extracellular matrix (ECM) and cells within the tissue microenvironment (TME), and the key role played by ECM stiffness during development and growth. Indeed, the medicinal leech is characterized by a simple anatomical organization reproducing many aspects of the basic biological processes of vertebrates and in which a rapid spatiotemporal development is well established and easily assessed. Our results show that ECM structural organization, as well as the amount of fibrillar and non-fibrillar collagen are deeply different from hatching leeches to adult ones. In addition, the changes in ECM remodelling occurring during the different leech developmental stages, leads to a gradient of stiffness regulating both the path of migratory cells and their fates. The ability of cells to perceive and respond to changes in ECM composition and mechanics strictly depend on nuclear or cytoplasmic expression of Yes-Associated Protein 1 (YAP1), a key mediator converting mechanical signals into transcriptional outputs, expression, and activation.

Hirudo verbana as a freshwater invertebrate model to assess the effects of polypropylene micro and nanoplastics dispersion in freshwater

Plastics are a heterogeneous class of synthetic compounds that, due to their unique characteristics find numerous applications both in industrial and civil fields. However, despite the great advantages that these materials brought in everyday life, the plastic wastes resulting from their massive use represent one of the main environmental problems at the global level. Once released, plastics persist for a long time and are subjected both to biotic and abiotic processes leading to the formation of small particles, known as micro and to nanoplastics, that interact with organisms, accumulating inside tissues and risking to enter in the trophic chain. Among the different types of plastic, polypropylene (PP) is one of the diffused, widely exploited in food and textile industries for disposable packaging and to produce surgical masks. Owing to the huge distribution and the resultant abundant presence of PP waste products, it results necessary investigate the possible toxicity on living organisms. For these reasons, here we analyzed the effects of PP micro and nanoplastics dispersed in freshwater, using the medicinal leech Hirudo verbana as invertebrate model. To better follow the plastics fate, fluorescent particles, labeled with a fluorophore, have been used. Animals were examined at various timings after plastics exposure and results were analyzed by means of microscopy, immunofluorescent and molecular biology analyses. After assessing the entrance of PP fragments into leech tissues, the activation of the innate immune response was evaluated. The results show that the presence of micro and nanoplastics induces an initial physical protection that consists in the secretion of mucus, followed by an increase of blood vessels and the recruitment of immune cells, in particular macrophages. Moreover, macrophages were directly involved in both phagocytic and encapsulation processes, as demonstrated by acid phosphatase (ACP) histoenzymatic and Thioflavin-T assays, expressing specific pro-inflammatory factors, such as HvRNASET2 and HmAIF-1, as demonstrated by immunolocalization and qPCR experiments. Finally, the expression levels of genes related to oxidative stress-induced enzymes have been investigated, in order to evaluate the possible increase in reactive oxygen species (ROS), due to the entry into the leech tissues of PP micro and nanoplastics. This work allows deepening the current knowledge of the possible harmful effects on human health deriving from micro and nanoplastics dispersion, leading new insight about freshwater ecosystems that often represent the first environments interested in plastic pollution.

Serotonin mediates stress-like effects on responses to non-nociceptive stimuli in the medicinal leech Hirudo verbana.

ABSTRACTNoxious stimuli can elicit stress in animals that produce a variety of adaptations including changes in responses to nociceptive and non-nociceptive sensory input. One example is stress-induced analgesia that may be mediated, in part, by the endocannabinoid system. However, endocannabinoids can also have pro-nociceptive effects. In this study, the effects of electroshock, one experimental approach for producing acute stress, were examined on responses to non-nociceptive mechanical stimuli and nociceptive thermal stimuli in the medicinal leech (Hirudo verbana). The electroshock stimuli did not alter the leeches’ responses to nociceptive stimuli, but did cause sensitization to non-nociceptive stimuli, characterized by a reduction in response threshold. These experiments were repeated with drugs that either blocked synthesis of the endocannabinoid transmitter 2-arachidonoylglycerol (2-AG) or transient receptor potential vanilloid (TRPV) channel, which is known to act as an endocannabinoid receptor. Surprisingly, neither treatment had any effect on responses following electroshock. However, the electroshock stimuli reliably increased serotonin (5-hydroxytryptamine or 5HT) levels in the H. verbana CNS. Injection of 5HT mimicked the effects of the electroshocks, sensitizing responses to non-nociceptive stimuli and having no effect on responses to nociceptive stimuli. Injections of the 5HT receptor antagonist methysergide reduced the sensitization effect to non-nociceptive stimuli after electroshock treatment. These results indicate that electroshocks enhance response to non-nociceptive stimuli but do not alter responses to nociceptive stimuli. Furthermore, while 5HT appears to play a critical role in this shock-induced sensitizing effect, the endocannabinoid system seems to have no effect.

The effects of substrate porosity, mechanical substrate properties and loading conditions on the attachment performance of the Mediterranean medicinal leech (Hirudo verbana).

The ectoparasitic lifestyle of the Mediterranean medicinal leech (Hirudo verbana) requires reliable functioning of its attachment organs (i.e. anterior and posterior suction discs) on multiple habitat- and host-specific surfaces under both normal and shear stresses. In addition to some intrinsic properties of the attachment devices, however, only a few extrinsic factors (e.g. substrate roughness and porosity) have been considered in previous studies on leech suckers. Using centrifugal force experiments, we analysed the attachment performance of H. verbana under different types of loading on artificial substrates differing in porosity and their mechanical properties. Whereas the substrate porosity significantly influenced leech attachment under normal and shear loading, the different mechanical properties did not noticeably affect attachment within the considered parameter limits. Furthermore, suction was confirmed to be the primary attachment mechanism independent of the prevailing loading condition. The question of whether the suction cups of H. verbana are adapted to a specific loading condition could not be answered. In any case, our results again highlight the high functional resilience of leech suckers guaranteeing a successful ectoparasitic lifestyle.

Morphological parameters of spleen and thymus of the male rats on the basis of the hirudological influence of Hirudo verbana.

The aim of this work is to study the morphological parameters of the spleen and thymus of rats along with hirudological influence from Hirudo verbana. The work was carried out on 40 male outbred laboratory rats weighing 280-300 grams, age 6.5-7 months. Animals that had passed the quarantine regime and had no external manifestations of the disease were used for the study. Manipulations with animals were performed in compliance with regulated norms and rules for the treatment of laboratory animals. For the study, two groups of animals were formed - control and experimental. After fixation of rats, the medical leech Hirudo verbana with an average weight of 0.8 ± 0.1 g was put on the animals of experimental group. The place of setting is the coccyges zone. Four times with an interval of one time per week. Then the animals were decapitated and the spleen and thymus were removed for histological examination. The white pulp of the spleen was analyzed - the area and diameter of the lymphoid follicles and central arteries, the number of lymphocytes per unit area (400 μm2) in the lymphoid follicles, in the thymus - the number of cells per unit area, both in the cortex and in the medulla. Under the influence of biologically active substances of the medicinal leech in the white pulp of the spleen, the size of the lymphoid follicles and central arteries increases, the number of lymphocytes per unit area (400 μm2) of the lymphoid follicle increases, and in the thymus, both in the cortex and in the medulla, the number of cells increases per unit area (400 μm2), the predominance of the cortical over the medullar ones. As a result of the obtained experimental study, we revealed the immune stimulating effect of Hirudo verbana on the organs of the immune system of laboratory rats: thymus and spleen.

Eğirdir Gölü (Türkiye) çevresindeki sulak alanlarda yaşayan Akdeniz tıbbi sülüğü, Hirudo verbana popülasyonlarının büyüklüğü ve yapısı

In the present study size and structure of the Mediterranean medicinal leech (Hirudo verbana Carena, 1820) populations inhabiting wetlands around Lake Eğirdir (Turkey) were investigated. Population size was estimated by removal methods, age classes were estimated for the first time in leeches (Hirudinea) using “Modal Progression Analysis” of body length frequencies using Bhattacharya's method. The population size and biomass of medicinal leeches around Lake Eğirdir were estimated to be 1,562,696 ± 805,613 leeches and 467.26 ± 172.91 kg, respectively. Three age classes were identified; the age group of 0+ was dominant with 78.6%. While 89.9% of the individuals weighed less than 1 g, whereas the broodstock was estimated to represent only 7%. Body weight, body length and condition factor were found to be 0.52 ± 1.19 g, 4.5 ± 2.4 cm and 0.620 ± 0.123, respectively. This study reveals that the population size of H. verbana in the wetlands around Lake Eğirdir is relatively well preserved and there is no significant threat to the sustainability of the exploitation of the medicinal leech populations. It is concluded that the stability of the lake's water budget, the variety and abundance of the host in the undamaged wetlands, the socioeconomic structure of the leech collectors, the sales policies, and the effective activities of conservation and control units might affect conservation status of the medicinal leech populations inhabiting wetlands around Lake Eğirdir.

Comparison of body wall histologic structure of two medicinal leeches Hirudo sulukii and Hirudo verbana (Hirudinida: Hirudinidae).

In this study, it was aimed to determine secretory cell types using histochemical properties of secretory cells and epidermis histology in the body wall of two medicinal leech species, Hirudo verbana and Hirudo sulukii. In addition, areas of epidermis epithelial cells, secretory cell types, and secretion areas of secretory cells stained with histochemical stains were statistically compared in both species. Epidermis is composed of single layer of cylindrical epithelium and secretory cells. The cuticle layer covers the epithelial layer. Some Type 1 cells within and close to the epidermis were determined as pear-shaped secretory cells. Type 2a and Type 2b secretory cells were found in large groups in the inner parts of the body wall, especially around muscles. While Type 1 cells were stained weakly with PAS and AB, Type 2b cells were stained darker than Type 2a cells. Statistical calculations showed that areas of epithelial and secretory cells were generally larger in H. sulukii than in H. verbana. Therefore, H. sulukii was thought to be a more resistant species compared to H. verbana. As secretion areas of secretory cells reacting with PAS and AB stains were generally larger in H. sulukii, it was concluded that mucus composition between the two species has different concentration.

Çinkonun Güney Tıbbi Sülüğü Hirudo verbana Carena, 1820 Üzerine Akut Toksisitesi

In the present study it was aimed to determine the acute toxicity of zinc on southern medicinal leech Hirudo verbana Carena, 1820 that being used in the ecotoxicological studies. The experiment was conducted for 96 hours using the semi-static bioassay method with replicate two times. The leeches were exposed to zinc concentrations of 6.25 mg/L, 12.50 mg/L, 25 mg/L, 50 mg/L, 100 mg/L and 200 mg/L, respectively. As a result of the experiment, the LC50 values for 24, 48, 72 and 96 hours were determined 48.30 mg/L, 19.68 mg/L, 15.48 mg/L, and 14.12 mg/L, respectively. The LT50 values for 25 mg/L, 50 mg/L, 100 mg/L and 200 mg/L zinc concentrations were determined 38.63 hours, 24.55 hours, 14.62 hours, and 12.78 hours, respectively. Defecation, intensive mucus output, uncontrolled swimming, attempt to escape from the experiment media, reducing the cling power, coiling (buckling), vomiting the digestive blood, the body deformations, penile prolapse, hemorrhage and abrasion in the wall of the body were the main physiological and behavioral responses in the leeches which were exposed to the different zinc solutions. The data obtained revealed that the zinc sensitivity of H. verbana was moderate when compared to other organisms. It is expected that the data of the present study will contribute to the management of endangered medicinal leech populations.

AGE DYNAMICS OF FREE AMINO ACIDS IN THE TISSUES OF THE MEDICINAL LEECH Hirudo verbana Carena, 1820 UNDER ARTIFICIAL REPRODUCTION IN AQUACULTURE

Medicinal leeches (Hirudo verbana Carena, 1820) used in veterinary medicine, human medicine and pharmacology, are currently bred artificially at bio-factories. The technology of leech culture as a component of aquaculture biotechnics is based on the method of accelerated growth of leeches due to intensive feeding, which allows you to get high-quality products in a fairly short time (8-10 months). Nevertheless, the problem of assessing the physiological status of raised individuals upon artificial reproduction remains not fully addressed. The findings we present herein for the first time allow us to suggest that the age-specific amino acid composition of tissues can be used to assess the physiological state and well-being of medicinal leeches in nature and in aquaculture. The aim of the work is to study the age-dependent content of the free amino acids in the tissues of medicinal leeches artificially bred in aquaculture. Studies were conducted in 2012 on the leech (Hirudo verbana Carena, 1820) individuals without signs of diseases which were purchased at the biofactory International Center for Medicinal Leeches (Moscow Province, p. Udelnaya). Leeches were kept in the laboratory in glass vessels with dechlorinated water at 20-22 oC and fed once a month with fresh bovine blood from healthy animals. The experimental specimens of H. verbana was: 5 days (newborn filaments, 0+), 1, 3, 5, 7 and 9 months old, with an average body weight of 0.029, 0.09, 0.25, 0.61, 1.33 and 1.81 g, respectively. Newborn individuals served as a control. The concentration of free amino acids (AA) in the skin and muscle tissue was determined on the analyzer AAA-339M (Mikrotechna, Czech Republic). A total of 105 leech specimens were used, 30 bioassays were prepared, and 660 element determinations were performed. The amino acid composition of the tissues of medicinal leeches H. verbana was represented by 23 AA and their derivatives. The dominant AA with an antioxidant orientation, regardless of age, were glutamine and glutamic acid, alanine, valine, leucine, and glycine, the total content of which was 65 % in filaments, and 58 % in 9-month — old individuals of the total fund AA. In the tissues of medicinal leeches, as well as in warm-blooded animals, the full composition of essential amino acids (EAA) was revealed: threonine, valine, lysine, leucine, isoleucine, histidine, arginine, methionine, phenylalanine, tryptophan. With age, the total concentrations of free amino acids decreased (r = -0.98 at p = 0.000), due to a sharp decrease in the content of arginine (24.0-fold), proline (13.4- fold), isoleucine (12.2- fold), glycine (8.5- fold), lysine (6.8- fold), histidine (6.6-fold), leucine (5.2-fold), ornithine (4.0- fold), glutamic acid and glutamine (3.8-fold), and alanine (2.9-fold). At the age of 5, 7 and 9 months, leeches had a trace content of secondary metabolites: taurine, citrulline and tryptophan (p < 0.001). However, the amino acid balance of nitrogen and protein metabolism was not disturbed, since the ratio of essential / non-essential AА did not change significantly and was 0.60 in newborn filaments, 0.73 — in 9-month-old leeches. It was found that the growth and development of H. verbana was accompanied by a significant decrease in the indicator of maturity (glycine/alanine) from 0.75 (newborn filaments) to 0.25 (9 months). The information available in the literature and the data of our research allow us to put forward the concept of using functional amino acids as biomarkers in the development of the scientific bases of technologies for the industrial breeding of these amphibionts.

ВОЗРАСТНАЯ ДИНАМИКА СВОБОДНЫХ АМИНОКИСЛОТ В ТКАНЯХ МЕДИЦИНСКОЙ ПИЯВКИ (Hirudo verbana Carena, 1820) ПРИ ИСКУССТВЕННОМ ВОСПРОИЗВОДСТВЕ В АКВА-КУЛЬТУРЕ

Medicinal leeches (Hirudo verbana Carena, 1820) used in veterinary medicine, human medicine and pharmacology, are currently bred artificially at bio-factories. The technology of leech culture as a component of aquaculture biotechnics is based on the method of accelerated growth of leeches due to intensive feeding, which allows you to get high-quality products in a fairly short time (8-10 months). Nevertheless, the problem of assessing the physiological status of raised individuals upon artificial reproduction remains not fully addressed. The findings we present herein for the first time allow us to suggest that the age-specific amino acid composition of tissues can be used to assess the physiological state and well-being of medicinal leeches in nature and in aquaculture. The aim of the work is to study the age-dependent content of the free amino acids in the tissues of medicinal leeches artificially bred in aquaculture. Studies were conducted in 2012 on the leech (Hirudo verbana Carena, 1820) individuals without signs of diseases which were purchased at the biofactory International Center for Medicinal Leeches (Moscow Province, p. Udelnaya). Leeches were kept in the laboratory in glass vessels with dechlorinated water at 20-22 oC and fed once a month with fresh bovine blood from healthy animals. The experimental specimens of H. verbana was: 5 days (newborn filaments, 0+), 1, 3, 5, 7 and 9 months old, with an average body weight of 0.029, 0.09, 0.25, 0.61, 1.33 and 1.81 g, respectively. Newborn individuals served as a control. The concentration of free amino acids (AA) in the skin and muscle tissue was determined on the analyzer AAA-339M (Mikrotechna, Czech Republic). A total of 105 leech specimens were used, 30 bioassays were prepared, and 660 element determinations were performed. The amino acid composition of the tissues of medicinal leeches H. verbana was represented by 23 AA and their derivatives. The dominant AA with an antioxidant orientation, regardless of age, were glutamine and glutamic acid, alanine, valine, leucine, and glycine, the total content of which was 65 % in filaments, and 58 % in 9-month — old individuals of the total fund AA. In the tissues of medicinal leeches, as well as in warm-blooded animals, the full composition of essential amino acids (EAA) was revealed: threonine, valine, lysine, leucine, isoleucine, histidine, arginine, methionine, phenylalanine, tryptophan. With age, the total concentrations of free amino acids decreased (r = -0.98 at p = 0.000), due to a sharp decrease in the content of arginine (24.0-fold), proline (13.4- fold), isoleucine (12.2- fold), glycine (8.5- fold), lysine (6.8- fold), histidine (6.6-fold), leucine (5.2-fold), ornithine (4.0- fold), glutamic acid and glutamine (3.8-fold), and alanine (2.9-fold). At the age of 5, 7 and 9 months, leeches had a trace content of secondary metabolites: taurine, citrulline and tryptophan (p < 0.001). However, the amino acid balance of nitrogen and protein metabolism was not disturbed, since the ratio of essential / non-essential AА did not change significantly and was 0.60 in newborn filaments, 0.73 — in 9-month-old leeches. It was found that the growth and development of H. verbana was accompanied by a significant decrease in the indicator of maturity (glycine/alanine) from 0.75 (newborn filaments) to 0.25 (9 months). The information available in the literature and the data of our research allow us to put forward the concept of using functional amino acids as biomarkers in the development of the scientific bases of technologies for the industrial breeding of these amphibionts.

New distributional data for the Mediterranean medicinal leech Hirudo verbana Carena, 1820 (Hirudinea, Hirudinidae) in Italy, with a note on its feeding on amphibians

Scarce data are currently available about the distribution of the Mediterranean medicinal leech Hirudo verbana in Italy, and most of the known occurrence localities are based on records collected in the nineteenth and the first half of the twentieth century, which were not confirmed in the last decades, mostly due to a lack of surveys. Accordingly, the available knowledge on the distribution of the species is far from being updated and representative, although a significant negative trend of H. verbana throughout the country is supposed. The lack of sound distribution data is a significant shortfall, which hinders the implementation of the monitoring of the species as required by the Article 17 of the “Habitats Directive” on the species of Union concern. To provide recent, validated distributional data for the Mediterranean medicinal leech in Italy to be used as baseline data for further surveys and monitoring, we present herein a set of unpublished observations collected in the last decades in peninsular Italy, Sicily, and Sardinia. Moreover, we report observation of H. verbana feeding on amphibians, a feeding habit to date poorly documented for the Mediterranean medicinal leech. Based on both published and novel data, H. verbana is characterised by a large but fragmented distribution in Italy. Therefore, dedicated monitoring programs and the collection of validated occasional observations are highly desirable to get a clearer picture of the real distribution of the species.

Closed Genome Sequence of Aeromonas veronii Strain Hm21, an Isolate from the Medicinal Leech Hirudo verbana.

Aeromonas veronii strain Hm21 was isolated from the medicinal leech Hirudo verbana and is used for genetic studies. We present here the 4.71-Mbp genome with a 56-kb plasmid and identify the mutations present in strains commonly used for genetic engineering.

Exploring the attachment of the Mediterranean medicinal leech (Hirudo verbana) to porous substrates.

Haematophagous ectoparasites must ensure a reliable hold to their host during blood meals and, therefore, have evolved a broad spectrum of versatile and effective attachment mechanisms. The Mediterranean medicinal leech (Hirudo verbana), for example, uses suction on both smooth and textured air-tight substrates. However, preliminary studies showed that H. verbana is also capable of attaching itself to air-permeable substrates, where suction does not work. Using high-speed videography and mechanical tests, we comparatively investigated the attachment of H. verbana on both smooth and textured air-tight as well as on porous artificial substrates, also considering the influence of mucus on sucker surfaces. In general, the leech-specific locomotion cycle did not differ between the tested surfaces, and the leeches were able to reliably attach to both air-tight and porous substrates. From our results, we conclude that suction is presumably the primary attachment mechanism of H. verbana. However, secondary mechanisms such as mechanical interlocking with surface asperities and pores or capillary forces occurring at the interface between the mucus-covered suckers and the substratum are also employed. In any case, the rich repertoire of applicable attachment principles renders the organs of H. verbana functionally highly resilient.

Draft genome sequences of Hirudo medicinalis and salivary transcriptome of three closely related medicinal leeches

BackgroundSalivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today.ResultsWe annotated the Hirudo medicinalis genome and performed RNA-seq on salivary cells isolated from three closely related leech species, H. medicinalis, Hirudo orientalis, and Hirudo verbana. Differential expression analysis verified by proteomics identified salivary cell-specific gene expression, many of which encode previously unknown salivary components. However, the genes encoding known anticoagulants have been found to be expressed not only in salivary cells. The function-related analysis of the unique salivary cell genes enabled an update of the concept of interactions between salivary proteins and components of haemostasis.ConclusionsHere we report a genome draft of Hirudo medicinalis and describe identification of novel salivary proteins and new homologs of genes encoding known anticoagulants in transcriptomes of three medicinal leech species. Our data provide new insights in genetics of blood-feeding lifestyle in leeches.