Evaluation of Methylene Blue as An Effective Antiseptic for Medicinal Leeches (Hirudo verbana).

Objective: Medicinal leeches (Hirudo spp.) have been used for therapeutic purposes in humans since ancient times. Because of their growth conditions, leeches carry certain bacteria and endosymbionts (e.g., Aeromonas spp). In both leech farms and hirudotherapy clinics, there are no reliable antiseptics that can be used with leeches. This study aimed to determine whether methylene blue (MB) is a safe antiseptic for medicinal leeches and assess its safe usage. Methods: This study evaluated the efficacy of MB by determining lethal concentrations (LC), effective concentrations (EC), and lethal times (LT) for the medicinal leech Hirudo verbena Carena, 1820. A total of 570 H. verbana specimens obtained from a local farm were used in this study. Eighteen different concentrations of MB (between 1 ppm and 512 ppm) were tested. Results: The LC50 and EC50 values for H. verbana were determined to be 60.381 (53.674-66.636) ppm and 2.013 (1.789-2.221) ppm, respectively. The LT50 durations for MB concentrations of 32 and 512 ppm were calculated as 212.92 h (138.43 h-1485.78 h) and 17.82 h (8.08 h-23.90 h), respectively. Conclusion: The results show that MB concentrations between 2 and 19 ppm can be safely used as antiseptics in hirudotherapy clinics and leech farms to address bacterial concerns caused by medicinal leeches.

Ice-inhabiting species of Bdelloidea Rotifera reveal a pre-Quaternary ancestry in the Arctic cryosphere.

Historical climate data indicate that the Earth has passed through multiple geological periods with much warmer-than-present climates, including epochs of the Miocene (23-5.3 mya BP) with temperatures 3-4°C above present, and more recent interglacial stages of the Quaternary, for example, Marine Isotope Stage 11c (approx. 425-395 ka BP) and Middle Holocene thermal maximum (7.5-4.2 ka BP), during which continental glaciers may have melted entirely. Such warm periods would have severe consequences for ice-obligate fauna in terms of their distribution, biodiversity and population structure. To determine the impacts of these climatic events in the Nordic cryosphere, we surveyed ice habitats throughout mainland Norway and Svalbard ranging from maritime glaciers to continental ice patches (i.e. non-flowing, inland ice subjected to deep freezing overwinter), finding particularly widespread populations of ice-inhabiting bdelloid rotifers. Combined mitochondrial and nuclear DNA sequencing identified approx. 16 undescribed, species-level rotifer lineages that revealed an ancestry predating the Quaternary (> 2.58 mya). These rotifers also displayed robust freeze/thaw tolerance in laboratory experiments. Collectively, these data suggest that extensive ice refugia, comparable with stable ice patches across the contemporary Norwegian landscape, persisted in the cryosphere over geological time, and may have facilitated the long-term survival of ice-obligate Metazoa before and throughout the Quaternary.

Transcriptomic Profiling at the Maternal-to-Zygotic Transition in Leech, Helobdella austinensis.

The glossiphoniid leech, Helobdella austinensis, is an experimentally tractable member of the superphylum, Lophotrochozoa. Its large embryonic cells, stereotyped asymmetric cell divisions and ex vivo development capabilities makes it a favorable model for studying the molecular and cellular events of a representative spiralian. In this study, we focused on a narrow developmental time window of ~6-8 h, comprising stages just prior to and immediately following zygote deposition. Employing RNA-Seq methodology, we identified differentially expressed transcripts at this fundamental ontogenic boundary, known as the maternal-to-zygotic transition (MZT). Gene expression changes were characterized by the massive degradation of maternal RNAs (~45%) coupled with the rapid transcription of ~5000 zygotic genes (~20% of the genome) in the first mitotic cell cycle. The latter transcripts encoded a mixture of cell maintenance and regulatory proteins that predictably influence downstream developmental events.

Biocontrol of snail-borne parasites with the glossiphoniid leech, Helobdella austinensis.

Parasites in which freshwater snails are intermediate hosts pose a serious threat to human health worldwide. We show here that freshwater snails can potentially be controlled by leech predation; in principle, this approach could significantly reduce snail-borne parasitic diseases (SBPDs). Specifically, glossiphoniid leeches, Helobdella austinensis and congener species, consume freshwater snails indiscriminately, while other common leeches do not. A single adult H. austenensis, for example, can consume up to its weight in snails, e.g. Physella acuta, per day. Our predator-prey models suggest that snail populations could be eliminated in relatively short time periods (approximately six months) using a leech biocontrol approach. This could have considerable impact on global SBPDs by breaking the intermediate host life cycle.

Hirudinea (Annelida) Fauna of Some Wetlands in Bingöl Province

Objective: Leeches are important and reliable indicators of water quality and biodiversity in the ecosystem, so the presence of specific leech species is often closely related to basic water conditions and the presence of certain animals. This study was carried out in 2017 and 2018 in order to determine the Hirudinea fauna of some wetlands in Bingöl province. The investigation was conducted on a total of 13 stations. Methods: The water parameters of the stations were measured and recorded in situ. The collected specimens were brought alive to the Zoology Laboratory of Bingöl University Biology Department and kept alive under room temperature conditions. The diagnosis of leech samples was made through the living samples, and they were identified at the level of family, genus, and species. Results: During the study, seven species, belonging to six genera and in four families were recorded. These are; Hirudo verbana Carena, 1820, Glossiphonia complanata (L. 1758), Theromyzon tessulatum (O. F. Müller, 1774), Placopdella costata (Fr. Müller, 1846), Erpobdella octoculata (L., 1758), Erpobdella testacea (Savigny, 1820), Piscicola geometra (L., 1761). Conclusion: The locations where the study was carried out are new records for the detected leech species.

Detailed ultrastructure of the Hirudo (Annelida: Hirudinea) salivary gland.

Medical leeches have been widely used in medical applications and treatments for millennia. Studies on the salivary glands of blood-sucking leeches have focused on their bioactive secretions and mechanisms of action, with little attention to ultrastructure. In this study, we examined dissected embryonic and adult Hirudo verbana salivary glands by scanning electron microscopy (SEM). Gland cells of embryos were physically separated while adults displayed highly developed cell bunches in which each cell was connected to others by fine channels. Channels from each bunch combined to form a larger canal that opened to the jaw. Secreted material from these glands prevent blood from clotting and allow the adult to feed while sucking blood.

Internal and External Morphological Characteristics of the Medicinal Leech Species Hirudo sulukii and Hirudo verbana

Objective: The aim of this study was to investigate the internal and external morphological structures of two species of medicinal leeches (Hirudo sulukii and Hirudo verbana). Methods: Leech specimens were collected from various regions of Turkey between the years of 1995-2016. The leeches were first examined alive and later were stunned with 10% ethyl alcohol and fixed in 70% ethyl alcohol or 4% formaldehyde. After fixation, the internal morphology of leeches was examined by dissecting them under a stereo microscope. Results: Both species of leeches are dorso-ventrally flattened. H. verbana is larger and broader than H. sulukii. H. sulukii has black, segmentally-arranged united ellipsoid and elongated spots, and a pair of zigzagged black longitudinal stripes in the dorso-lateral area of its body. The ventral surface of H. sulukii is greenish to brown in colour and has a small number of irregular black spots. H. verbana has broad and diffuse, paramedian, orange stripes on the dorsal surface. Ventrally, H. verbana has a greenish to yellow colour, and it is characterized by a pair of black ventro-lateral stripes. Conclusion: Two of the six leech species (Hirudo sulukii and Hirudo verbana) belonging to the genus Hirudo found in the world are found in Turkey. These two leech species show significant differences from each other, morphologically.

Correct spelling of a recently described species of Helobdella (Annelida: Clitellata: Hirudinea).

In a recently published article (Saglam et al. 2018) the name of a new species of Hirudinea (Annelida, Clitellata) was simultaneously published in three different ways, as Helobdella serendipitious (Saglam et al. 2018: 61, 70, 71, 73), Helobdella serendipitous (ibid.: 63, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74), and as Helobdella serendipidous (ibid.: 75). Acting as first reviser according to Article 24 of the International Code of Zoological Nomenclature (ICZN 1999) here we determine serendipitous as the correct spelling of the species epitheton.

Phylogenetic and morphological resolution of the Helobdella stagnalis species-complex (Annelida: Clitellata: Hirudinea).

The glossiphoniid freshwater leech, Helobdella stagnalis, was described by Linnaeus 1758 based on common European specimens. The presence of a brown, chitinous scute on the dorsal-anterior surface, as observed on leeches elsewhere in the world, has generally led to the classification of all scute-bearing members of the genus as H. stagnalis. Here we describe the morphology and behavior of the type species from Europe, and analyze H. stagnalis-like specimens collected worldwide. We present evidence for at least four distinct scute-bearing Helobdella species that can be morphologically resolved. Maximum Parsimony (MP) and Bayesian Inference (BI) analyses at the mitochondrial cytochrome c oxidase subunit I (COI) locus further supported this notion, with divergence values suggesting a mid-Miocene ancestor of this successful group of cosmopolitan hirudineans. New species, Helobdella echoensis nov. sp., Helobdella eriensis nov. sp., and Helobdella serendipitious nov. sp., are described, based on morphological, anatomical and molecular data. Current distribution patterns of Helobdella spp. suggest a robust, global dispersal mechanism, as well as local pockets of endemism.

Phylogeny and cocoon production in the parasitic leech Myzobdella lugubris Leidy, 1851 (Hirudinidae, Piscicolidae).

Myzobdella lugubris is a commensal leech on crustaceans and a parasite to fishes, surviving predominantly in brackish waters throughout North America. Specimens in this study were collected within the tidal zone of the Delaware River basin (New Jersey and Pennsylvania). To compare regional M. lugubris specimens, defined characters were scored after dorsal and ventral dissections, and phylogenetic relationships were resolved using cytochrome c oxidase subunit 1 (CO1), 12S ribosomal RNA (rDNA) and 18S rDNA gene fragments. Variance between regional populations was low, suggesting recent dispersal events and/or strong evolutionary constraints. The reproductive biology of M. lugubris was explored by quantitative analysis of secreted cocoons. Specimens produced 32.67 ± 4.50 cocoons with fertilization ratios of 88.1% and hatching times of 48 ± 7 days at 17°C under laboratory conditions. At 22°C, 46 ± 28 cocoons were produced with fertilization ratios of 70.27% and hatching times of 28 ± 5 days. Surprisingly, each cocoon supported only one embryo, which is unusual among oligochaetes.

Elaborate ultrastructure of the Hirudo (Annelida: Hirudinae) cocoon surface.

Species of medicinal leeches (Hirudo medicinalis, H. verbana, and H. sulukii) secrete hard-shelled cocoons. When initially deposited, a cocoon is surrounded by a foam. Over a short time, the foam is transformed into a three-dimensional structure. We show here that this peripheral structure likely forms by the solidification and dehydration of a moderately viscous, proteinaceous substance that surrounds bubbles of various sizes. The resulting matrix-like structure comprises a network of curved branches juxtaposed at ∼120° and taper in width as a function of distance from the outer cocoon wall. The material is proteinaceous, and traps environmental material in its composition, especially silicon. The geometry of compartments and abundance of silicon on branch surfaces suggest a mechanism for trapping water to prevent desiccation in a terrestrial environment.

Punctuated invasion of water, ice, snow and terrestrial ecozones by segmented worms (Oligochaeta: Enchytraeidae: Mesenchytraeus).

Segmented worms (Annelida) are among the most successful animal inhabitants of extreme environments worldwide. An unusual group of enchytraeid oligochaetes of genus Mesenchytraeus are abundant in the Pacific northwestern region of North America and occupy geographically proximal ecozones ranging from low elevation rainforests and waterways to high altitude glaciers. Along this altitudinal transect, Mesenchytraeus representatives from disparate habitat types were collected and subjected to deep mitochondrial and nuclear phylogenetic analyses. Our data identify significant topological discordance among gene trees, and near equivalent interspecific divergence levels indicative of a rapid radiation event. Collectively, our results identify a Mesenchytraeus 'explosion' coincident with mountain building in the Pacific northwestern region that gave rise to closely related aquatic, ice, snow and terrestrial worms.

Trichlorfon-induced haematological and biochemical changes in Cyprinus carpio: ameliorative effect of propolis.

Trichlorfon is among the most commonly used products to treat fish parasites in aquaculture. We investigated the effectiveness of propolis in alleviating the toxicity of trichlorfon on haematological and oxidant/antioxidant parameters in carp Cyprinus carpio. Fish were exposed to sublethal concentrations (11 and 22 mg l-1) of trichlorfon, and propolis (10 mg kg-1 of fish weight) was simultaneously administered. At the end of 14 d administration, blood and tissue (liver, kidney, gill) samples were collected. Haematological changes (red and white blood cell count, haemoglobin concentration, haematocrit level and erythrocyte indices: mean corpuscular volume, mean corpuscular haemoglobin and mean corpuscular haemoglobin concentration) were determined in the blood samples, while antioxidant parameters (malondialdehyde and reduced glutathione levels and superoxide dismutase, catalase and glutathione peroxidase activities) were evaluated in the liver, kidney and gill samples. Trichlorfon led to negative alterations in the haematological and antioxidant parameters investigated. The administration of propolis alleviated this effect and suggests that fish treated with trichlorfon improve their physiological status when fed a propolis-supplemented diet.

[A case report: hirudotherapy as a treatment modality in the microsurgery]. / Olgu Sunumu: Mikro Cerrahiye Yardimci Bir Metot, Hirudoterapi.

Medicinal leeches are known to inject more than 100 bioactive substances in the tissue whilst sucking blood. They are being successfully used for the treatment of diseases and symptoms, especially in plastic surgery. We report the case of a 7-year old boy, who had lost the first phalanx of the middle finger of the right hand after an accident. The detached digit was replanted in the plastic surgery clinic, and treated within a period of 15 days with medicinal leeches (Hirudo medicinalis). Soon after the initiation of the hirudotherapy, the revascularisation of the re-attached phalanx, as well as the resolving of tissue oedema and pain was observed. Turkish physicians should be aware of the advantages of hirudotherapy in plastic and reconstructive surgery, especially in cases of detached digits and flaps.

Operculum ultrastructure in leech cocoons.

Clitellate annelids (i.e., oligochaetes including leeches) secrete cocoons as part of their normal reproductive cycle. Typically, the cocoon sheath is passed over the head of the leech and sealed at both ends by opercula (i.e., glue-like material secreted by the clitellum). Both the fibrous cocoon wall (CW) and opercula are chemically-related biomaterials that share unusual physiochemical properties, including thermal and chemical resiliency. To explore the underlying morphology of the operculum, we examined cocoons from four leech species (i.e., Myzobdella lugubris, Theromyzon tessulatum, Erpobdella obscura, and Erpobdella punctata) by transmission (TEM) and scanning electron microscopy (SEM). Transmission electron micrographs of all opercula revealed a common, ultrastructural pattern comprising an electron-dense mosaic of ordered polygons that surrounded interspersed cavities. The long axes of cavities were often oriented directionally, suggesting that operculum material is pliable prior to solidification and distorted as a consequence of cocoon deposition. Concomitantly, the operculum permeates jagged edges of the cocoon sheath sealing the cocoon, which provides a mechanically strong CW/operculum boundary. SEM of leech opercula revealed globular nanoparticles comparable to that observed in bioadhesives from disparate animal phyla (e.g., mussel, barnacle, sea star), suggesting a convergent mechanism of bioadhesion among animals.

[Medicinal leeches and hirudotherapy]. / Tibbi sülükler ve hirudoterapi.

Leeches have been used in the treatment of certain diseases since ancient times. There are a few species of medicinal leeches. However, H. medicinalis and H. verbana are known in Turkey. Currently, Turkey is one of the world's most important leech-exporting countries. Secretion of the salivary glands of medical leeches contains more than 100 bioactive substances. These secretions include vasodilators, bacteriostatic, analgesic, anti-inflammatory and anticoagulants, anti-edematous, which eliminate microcirculatory disorders, restore the damaged vascular permeability of tissues and organs, eliminate hypoxia, reduce blood pressure, increase immune system activity, resolving the cause of pain and improve the bioenergetic status of the organism. U.S. Food and Drug Administration (Food and Drug Authority of USA-FDA) have allowed the sale of leeches in this country, the use for plastic surgery, the general purposes and microsurgery in 2004. Turkey is the richest source in terms of the medical leech. Better evaluation of this valuable commodity and more effective use of hirudotherapy in modern medical practice as supportive treatment is very important.

Endohelminth fauna of the marsh frog Rana ridibunda from Lake Hazar, Turkey.

In this study, 236 marsh frogs Rana ridibunda collected from Lake Hazar (Elazig, Turkey) at 15 d intervals between March 2001 and February 2002 were examined for endohelminths; of these, 148 (62.71%) frogs were found to be infected with helminths. In total, 9 helminth species (3 trematodes, 5 nematodes and 1 acanthocephalan) were identified. We observed Gorgoderina vitelliloba (prevalence 2.97%) in the urinary bladder, Haematoloechus variegatus (4.66%) and Rhabdias bufonis (8.90%) in the lung, Pleurogenoides medians (1.69%), Oswaldocruzia filiformis (3.81 %) and Acanthocephalus ranae (26.27 %) in the small intestine, Neoxysomatium brevicaudatum (16.95%) and Cosmocercoides sp. (3.39%) in the large intestine, and Eustrongylides excisus (14.41%) in the body cavity and on,the stomach. No helminth was found in the spleen, kidney, gall bladder, liver, heart or muscle. Of the 9 helminth species identified, Acanthocephalus ranae (26.27 %) had the highest prevalence and abundance and Oswaldocruzia filiformis (8.33+/-4.09) had the highest mean intensity.