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.

Characterization of a Fatty Acid Amide Hydrolase (FAAH) in Hirudo Verbana.

The endocannabinoid system plays a critical role in modulating both peripheral and central nervous system function. Despite being present throughout the animal kingdom, there has been relatively little investigation of the endocannabinoid system beyond traditional animal models. In this study, we report on the identification and characterization of a putative fatty acid amide hydrolase (FAAH) in the medicinal leech, Hirudo verbana. FAAH is the primary enzyme responsible for metabolizing the endocannabinoid signaling molecule arachidonoyl ethanolamide (anandamide or AEA) and therefore plays a critical role in regulating AEA levels in the nervous system. mRNA encoding Hirudo FAAH (HirFAAH) is expressed in the leech central nervous system (CNS) and sequence analysis suggests that this is an orthologue of FAAH-2 observed in vertebrates. Functionally, HirFAAH has serine hydrolase activity based on activity-based protein profiling (ABPP) studies using the fluorophosphonate probe TAMRA-FP. HirFAAH also hydrolyzes arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a substrate specific to FAAH. Hydrolase activity during both the ABPP and AAMCA assays was eliminated by a mutation at a conserved catalytic serine. Activity was also blocked by the known FAAH inhibitor, URB597. Treatment of Hirudo ganglia with URB597 potentiated synapses made by the pressure-sensitive mechanosensory neuron (P cell), mimicking the effects of exogenously applied AEA. The Hirudo CNS has been a useful system in which to study properties of endocannabinoid modulation of nociception relevant to vertebrates. Therefore, this characterization of HirFAAH is an important contribution to comparative studies of the endocannabinoid system.

Biomechanics of transduction by mechanosensory cilia for prey detection in aquatic organisms.

Surface-feeding aquatic animals navigate towards the source of water disturbances and must differentiate prey from other environmental stimuli. Medicinal leeches locate prey, in part, using a distribution of mechanosensory hairs along their body that deflect under fluid flow. Leech's behavioral responses to surface wave temporal frequency are well documented. However, a surface wave's temporal frequency depends on many underlying environmental and fluid properties that vary substantially in natural habitats (e.g., water depth, temperature). The impact of these variables on neural response and behavior is unknown. Here, we developed a physics-based leech mechanosensor model to examine the impact of environmental and fluid properties on neural response. Our model used the physical properties of a leech cilium and was verified against existing behavioral and electrophysiological data. The model's peak response occurred with waves where the effects of gravity and surface tension were nearly equal (i.e., the phase velocity minimum). This suggests that preferred stimuli are related to the interaction between fundamental properties of the surrounding medium and the mechanical properties of the sensor. This interaction likely tunes the sensor to detect the nondispersive components of the signal, filtering out irrelevant ambient stimuli, and may be a general property of cilia across the animal kingdom.

Comparative genomics of two Asian medicinal leeches Hirudo nipponia and Hirudo tianjinensis: With emphasis on antithrombotic genes and their corresponding proteins.

Leeches secrete various biologically active substances which have important medical and pharmaceutical values in antithrombotic treatments. Here, we provide a high quality genome of two Asian medicinal leeches Hirudo nipponia and Hirudo tianjinensis, based on which, we identified 22 antithrombotic gene families, including fourteen coagulation inhibitors, four platelet aggregation inhibitors, three fibrinolysis enhancers, and one tissue penetration enhancer. The total numbers of antithrombotic genes were similar between H. nipponia (N = 86) and H. tianjinensis (N = 83). Molecular evolution analysis showed that no significant differences were detected between the two species in any of the three selection indices (dN, dS, and dN/dS), nor in the number of sites under positive/purifying selection. RNA-Seq based gene expression analysis showed that the overall expression patterns of the antithrombotic gene families were not significantly deviated between the two species. Our results indicated that there were rather close similarities between the two leeches on genomic characteristics, especially for the molecular evolution and expression of antithrombotic genes. Our study provides the most comprehensive collection of antithrombotic biomacromolecules from the two Asian medicinal leeches to date. These results will greatly facilitate the research and application of leech derivatives for medical and pharmaceutical purposes of thrombosis.

Characterization of a Fatty Acid Amide Hydrolase (FAAH) in Hirudo verbana.

The endocannabinoid system plays a critical role in modulating both peripheral and central nervous system function. Despite being present throughout the animal kingdom, there has been relatively little investigation of the endocannabinoid system beyond the traditional animal model systems. In this study, we report on the identification and characterization of a fatty acid aminohydrolase (FAAH) in the medicinal leech, Hirudo verbana. FAAH is the primary enzyme responsible for metabolizing the endocannabinoid signaling molecule arachidonoyl ethanolamide (anandamide or AEA) and therefore plays a critical role in regulating AEA levels in the nervous system. This Hirudo FAAH (HirFAAH) is expressed in the leech central nervous system (CNS) and is an orthologue of FAAH-2 observed in vertebrates. Functionally, HirFAAH has serine hydrolase activity based on activity-based protein profiling (ABPP) studies using the fluorophosphonate probe TAMRA-FP. HirFAAH also hydrolyzes arachidonyl 7-amino, 4-methyl coumarin amide (AAMCA), a substrate specific to FAAH. Hydrolase activity during both the ABPP and AAMCA assays was eliminated by mutation at a conserved activity-binding site. Activity was also blocked by the known FAAH inhibitor, URB597. Treatment of Hirudo ganglia with URB597 potentiated synapses made by the pressure-sensitive mechanosensory neuron (P cell), mimicking the effects of exogenously applied AEA. The Hirudo CNS has been a useful system in which to study properties of endocannabinoid modulation of nociception relevant to vertebrates. Therefore, this characterization of HirFAAH is an important contribution to comparative studies of the endocannabinoid system.

Efficacy and potential mechanism of atherosclerosis prevention by the active components of leech based on network pharmacology combined with animal experiments.

Introduction: Leeches are flesh-eating and bloodsucking parasitic worms. They are being used as a traditional Chinese medicine for centuries in activating blood and dissolving statis, dreging the meridims and tick. Hirudin, an active peptide product present in leech, has blood anticoagulant property and can assist in the treatment of thrombosis and diseases related to blood circulation. The efficacy and potential mechanism of action of leeches in such diseases should be further explored. Materials and methods: First, network pharmacology was used to screen the predicted potential targets of the active constituents of leech and AS. The common targets of the active constituents of leech and AS were obtained using Venn diagram. Further, the drug-active-constituent-target network diagram, protein-protein interaction, and GO and KEGG pathway enrichment analyses were used to construct the active-constituent-AS target-pathway network diagram. Subsequently, the protein-drug molecule docking model was drawn. Finally, the results of network pharmacology were validated using a mouse model of AS. Results: In total, 34 active constituents of leech and 1172 AS-related gene targets were selected, took the drug action targets and potential disease targets to get the common targets, and took the top 10 of degree value as the main active constituents for the treatment of atherosclerosis. There were 89 common targets and 12 core targets. The main targets included MAPK, EGFR, PIK3CB, etc. Potential regulatory pathways included cancer pathways, EGFR tyrosine kinase inhibitor resistance, Rap1 signaling pathway, PPAR signaling pathway, PI3K-Akt signaling pathway, C-type lectin receptor signaling pathway, and AGE-RAGE signaling pathway in diabetic complications. Animal experiments using mouse model of AS confirmed that AS plaques were smaller after treatment with leeches. SRC level was measured using western blotting. Expression of SRC in myocardial tissue was remarkably lower in the mice treated with leech than in the mice from model group fed on high-fat chow. Conclusions: To the best of our knowledge, this is the first study to explore the mechanism of action of the active components of leech in AS prevention. The active components of leeches play a coordinated role in preventing AS through multicomponent, multitarget, and multichannel mechanism of action related to inflammatory response, oxidative stress, and lipid metabolism. This study provided a reference for subsequent cellular and animal experiments.

Research progress on the treatment of diabetic nephropathy with leech and its active ingredients.

Diabetic nephropathy (DN) is a major microvascular complication of diabetes and a common cause of chronic kidney disease. There is currently a lack of effective treatments for DN, and the prognosis for patients remains poor. Hirudin, one of the primary active components derived from leeches, demonstrates anti-coagulant, anti-fibrotic, anti-thrombotic, and anti-inflammatory properties, exhibiting significant protective effects on the kidneys. In recent years, there has been a surge of interest in studying the potential benefits of hirudin, especially in its role in the management of DN. This article delves into the mechanisms by which hirudin contributes to the treatment of DN and its clinical efficacy.

MOLECULAR PHYLOGENY OF THE LEECH GENUS PONTOBDELLA (HIRUDINIDA: PISCICOLIDAE) WITH NOTES ON PONTOBDELLA CALIFORNIANA AND PONTOBDELLA MACROTHELA.

Leech specimens of the genus Pontobdella (Hirudinida: Piscicolidae) were found off the coast of the state of Oaxaca (Pacific) as well as in Veracruz and Tabasco (Gulf of Mexico), Mexico. Based on the specimens collected in Oaxaca, a redescription of Pontobdella californiana is provided, with emphasis on the differences in the reproductive organs with the original description of the species. In addition, leech cocoons assigned to P. californiana were found attached to items hauled by gillnets and studied using scanning electron microscopy and molecular approaches. Samples of Pontobdella macrothela were found in both Pacific and Atlantic oceans, representing new geographic records. The phylogenetic position of P. californiana is investigated for the first time, and with the addition of Mexican samples of both species, the phylogenetic relationships within Pontobdella are reinvestigated. Parsimony and maximum-likelihood phylogenetic analysis were based on mitochondrial (cytochrome oxidase subunit I [COI] and 12S rRNA) and nuclear (18S rRNA and 28S rRNA) DNA sequences. Based on our results, we confirm the monophyly of Pontobdella and the pantropical distribution of P. macrothela with a new record in the Tropical Eastern Pacific.

Aeromonas hydrophila Infection following Leech Therapy for the Treatment of Nipple-Areola Complex Congestion after Breast Reduction: A Case Report.

Several strategies for the management of venous congestion of the nipple-areola complex (NAC) after reduction mammaplasty have been proposed. Among these, hirudotherapy represents an ancient but still effective method, even though the risk of infections related to leeches should be considered. We report a peculiar case of breast infection and sepsis after leech therapy in a patient who underwent a reduction mammaplasty. A prompt surgical debridement of the wounds and necrotic tissues associated with targeted antibiotic therapy led to a fast improvement of clinical conditions, and partial preservation of the NAC was obtained. Accurate knowledge of the clinical presentation of soft tissue infections related to leeching allows for an early diagnosis and would serve as a warning for surgeons who approach such breast cosmetic procedures.

Polyvinyl Butyral Loading with Combined Repellents Showed Effective Protection Against Leech Bites in Diverse Situations.

Background: Leech bites have long been a persistent problem for individuals engaged in outdoor activities, particularly in environments such as moors, jungles, and grasslands. Methods to prevent leech bites are anecdotal and individual, highlighting the need for the development of universal and effective repellent formulations. This study developed a novel approach for repelling leeches using combined repellent agents and a film-forming material (polyvinyl butyral), to enhance efficiency in multi-scenario applications. Material and methods: This study demonstrates that citronellal, icaridin and DDAC (didecyl dimethyl ammonium chloride) showcasing active avoidance and contact toxicity on leeches. the optimized repellent formulation (MSRS, containing citronellal, icaridin and DDAC as repellent agents) enables specific sustained release properties of constituents in both air and water conditions. Results: MSRS could effectively achieve the purposes of "proactive repelling", "contact repelling", and "bite detaching". The effectiveness could last for several hours. Additionally, the hydrophobic polyvinyl butyral membrane reduced the transdermal absorption of repellent agents. Moreover, the formulation is biocompatible and environmentally friendly. Conclusions: This study provides a new feasible strategy for the prevention and removal of leech bites.

Therapeutic Potentials of Medicinal Leech in Chinese Medicine.

The use of medicinal leeches in clinical therapy has been employed for a long time, as it was originally recognized for exerting antithrombin effects. These effects were due to the ability of the leech to continuously suck blood while attached to human skin. According to Chinese Pharmacopoei, leeches used in traditional Chinese medicine mainly consist of Whitmania pigra Whitman, Hirudo nipponia Whitman, and Whitmania acranulata, but the latter two species are relatively scarce. The main constituents of leeches are protein and peptide macromolecules. They can be categorized into two categories based on their pharmacological effects. One group consists of active ingredients that directly target the coagulation system, such as hirudin, heparin, and histamine, which are widely known. The other group comprises protease inhibitor components like Decorsin and Hementin. Among these, hirudin secreted by the salivary glands of the leech is the most potent thrombin inhibitor and served as the sole remedy for preventing blood clotting until the discovery of heparin. Additionally, leeches play a significant role in various traditional Chinese medicine formulations. In recent decades, medicinal leeches have been applied in fields including anti-inflammatory treatment, cardiovascular disease management, antitumor treatment, and many other medical conditions. In this review, we present a comprehensive overview of the historical journey and medicinal applications of leeches in various medical conditions, emphasizing their pharmaceutical significance within traditional Chinese medicine. This review offers valuable insights for exploring additional therapeutic opportunities involving the use of leeches in various diseases and elucidating their underlying mechanisms for future research.

Occurrence of parasitism promoted by Myzobdella lugubris Leidy, 1851 (Annelida: Piscicolidae) in Callinectes bocourti (Crustacea: Portunidae): A report of invasive leech in Brazilian Amazon province.

This study reports the presence of high parasitic load by Myzobdella lugubris Leidy, 1851 in the swimming crab Callinectes bocourti A. Milne-Edwards, 1879 from Amazon mangrove. We sampled the swimming crabs using a baited trap, between January and June 2023, in Santa Maria River, located in the municipality of Curuçá, state of Pará, Brazil (geographical coordinates 0°40'3.705"S, 047°54'43.405"W). After sampling, each swimming crab was individually placed in plastic containers for the count of leeches per individual. In the laboratory, the specimens were sexed, measured (parasite and host) and fixed in 70% alcohol. For the leech species identification, macroscopic techniques were combined with light microscopy (LM) and scanning electron microscopy (SEM). We examined 86 specimens of C. bocourti (75 males and 11 females) in a ratio of 1 M:0.14 F, all infested with leeches. In total, 186 leech specimens were collected, ranging from 1 to 21 leeches per host. Leeches oviposited the cocoons in greater quantities in ventral area of swimming crab carapace (32%), followed by dorsal area of carapace (29.09%), chelipeds (24.34%) and ambulatory legs (14.57%). The presence of M. lugubris is a risk to the health of the host, once it may transmit a range of diseases to aquatic organisms, and subsequently risk to human health.

Unraveling the structure, chemical composition, and conserved signaling in leech teeth.

Unlike vertebrates, the number of toothed taxa in invertebrates is very few, with leeches being the only tooth-bearing organisms in the phylum Annelida. Copious studies have been conducted regarding vertebrate teeth; however, studies regarding the structure and function of invertebrate teeth are limited. In this study, the tooth structure of leeches, specifically Hirudo nipponia and Haemadipsa rjukjuana, was revealed, which showed sharp and pointed teeth along the apex of three jaws. Understanding conserved signaling regulations among analogous organs is crucial for uncovering the underlying mechanisms during organogenesis. Therefore, to shed light on the evolutionary perspective of odontogenesis to some extent, we conducted de novo transcriptome analyses using embryonic mouse tooth germs, Hirudo teeth, and Helobdella proboscises to identify conserved signaling molecules involved in tooth development. The selection criteria were particularly based on the presence of tooth-related genes in mice, Hirudo teeth, and Helobdella proboscis, wherein 4113 genes were commonly expressed in all three specimens. Furthermore, the chemical nature of leech teeth was also examined via TEM-EDS to compare the chemical composition with vertebrate teeth. The examination of tissue-specific genetic information and chemical nature between leeches and mice revealed chemical similarities between leech and mice teeth, as well as conserved signaling molecules involved in tooth formation, including Ptpro, Prickle2, and Wnt16. Based on our findings, we propose that leech teeth express signaling molecules conserved in mice and these conserved tooth-specific signaling for dental hard tissue formation in mice would corresponds to the structural formation of the toothed jaw in leeches.

Exploration of leech therapy in treating gouty rats and its uric acid lowering mechanism.

BACKGROUND: Gout is a hyperuricemia (HUA)-related inflammatory reaction in the joints. Leech therapy has been effective in the gout, but the exact mechanism is unclear. OBJECTIVES: In this study, an exploration of the therapeutic mechanism of leech therapy in HUA and gouty arthritis (GA) rats was done. MATERIAL AND METHODS: HUA and GA construction utilizing sodium urate crystal, the potassium form of oxygen oxazine acid, and adenine. Serum and tissues were collected to measure uric acid (UA), creatinine (Cr), and urea nitrogen (UN). Enzyme linked immunosorbent assay was executed to evaluate the levels of xanthine oxidase (XOD), interleukin-6 (IL-6)and tumor necrosis factor α (TNF-α). The expression of glucose transporter 9 (GLUT9), organic anion transporter 3 (OAT3), adenosine triphosphate (ATP)-binding cassette efflux transporter G2 (ABCG2) and the nuclear factor kappa B (NF-kB), interleukin-1ß (IL-1ß), Toll-like Receptor 2 (TLR2) were assessed by Western blot and visualized in immunohistochemistry staining. RESULTS: Leech therapy reduces the levels of UA, Cr, and UN as well as the liver and serum levels of XOD activity, increasing the expressions of GLUT9, ABCG2, and OAT3 in the kidney. Meanwhile, it reduces joint swelling and lowers the levels of TNF-α, IL-6, IL-1ß, TLR2, and NF-kB. CONCLUSIONS: Leech therapy regulates the metabolism of uric acid and treats gouty arthritis with an anti-inflammatory effect.

Life cycle and development of the marine leech Branchellion lobata (Hirudinea: Piscicolidae), from round stingrays, Urobatis halleri, from southern California.

During captivity, round stingrays, Urobatis halleri, became infected with the marine leech Branchellion lobata. When adult leeches were deprived of blood meal, they experienced a rapid decrease in body mass and did not survive beyond 25 days. If kept in aquaria with host rays, B. lobata fed frequently and soon produced cocoons, which were discovered adhered to sand grains. A single leech emerged from each cocoon (at ~ 21 days), and was either preserved for histology or molecular analysis, or monitored for development by introduction to new hosts in aquaria. Over a 74-day observation period, leeches grew from ~ 2 to 8 mm without becoming mature. Newly hatched leeches differed from adults in lacking branchiae and apparent pulsatile vesicles. The microbiome of the hatchlings was dominated by a specific, but undescribed, member of the gammaproteobacteria, also recovered previously from the adult leech microbiome. Raising B. lobata in captivity provided an opportunity to examine their reproductive strategy and early developmental process, adding to our limited knowledge of this common group of parasites.

Antithrombotic pharmacodynamics and metabolomics study in raw and processed products of Whitmania pigra Whitman.

Objective: As a traditional Chinese medicine, leech has obvious pharmacological activities in anticoagulantion and antithrombosis. Whitmania pigra Whitman (WP) is the most commonly used leech in the Chinese market. It is often used in clinical applications after high-temperature processing by talcum powder to remove the fishy taste and facilitate crushing. The anticoagulant and thrombolytic active ingredients are protein and polypeptide, which may denaturate and lose activity after high-temperature processing. The rationality of its processing has been questioned in recent years. This study aims to investigate the effect of talcum powder scalding on the antithrombotic activity of WP in vivo and to discuss its pharmacodynamic mechanism in vivo. Methods: Raw and talcum-powdered processed WP were administered intragastrically for 14 days, and carrageenan was injected intraperitoneally to prepare a mouse model of tail vein thrombosis. The incidence rate of tail vein thrombosis and the thrombus area under pathological tissue sections were calculated to evaluate the antithrombotic effect between raw and processed WP. Non-targeted metabolomics was conducted using UPLC-Q-TOF/MS technology to analyze the changes of small molecule metabolites in the body after administration of WP. Results: After intragastric administration, both the raw product and the processed product of WP could inhibit the thrombosis induced by carrageenan, and the processed product had a more apparent antithrombotic effect than the raw product. The administration of WP could regulate the changes of some small molecular metabolites, such as amino acids, lipids, and steroids, in Sphingolipid metabolism and Glycerophospholipid metabolism. Conclusions: Based on the results of pharmacodynamics and metabolomics, processed WP will not reduce the antithrombotic activity of WP. This study provided a scientific basis for the rational use of leeches.

Discovery of a novel symbiotic lineage associated with a hematophagous leech from the genus Haementeria.

Similarly to other strict blood feeders, leeches from the Haementeria genus (Hirudinida: Glossiphoniidae) have established a symbiotic association with bacteria harbored intracellularly in esophageal bacteriomes. Previous genome sequence analyses of these endosymbionts revealed co-divergence with their hosts, a strong genome reduction, and a simplified metabolism largely dedicated to the production of B vitamins, which are nutrients lacking from a blood diet. 'Candidatus Providencia siddallii' has been identified as the obligate nutritional endosymbiont of a monophyletic clade of Mexican and South American Haementeria spp. However, the Haementeria genus includes a sister clade of congeners from Central and South America, where the presence or absence of the aforementioned symbiont taxon remains unknown. In this work, we report on a novel bacterial endosymbiont found in a representative from this Haementeria clade. We found that this symbiont lineage has evolved from within the Pluralibacter genus, known mainly from clinical but also environmental strains. Similarly to Ca. Providencia siddallii, the Haementeria-associated Pluralibacter symbiont displays clear signs of genome reduction, accompanied by an A+T-biased sequence composition. Genomic analysis of its metabolic potential revealed a retention of pathways related to B vitamin biosynthesis, supporting its role as a nutritional endosymbiont. Finally, comparative genomics of both Haementeria symbiont lineages suggests that an ancient Providencia symbiont was likely replaced by the novel Pluralibacter one, thus constituting the first reported case of nutritional symbiont replacement in a leech without morphological changes in the bacteriome. IMPORTANCE: Obligate symbiotic associations with a nutritional base have likely evolved more than once in strict blood-feeding leeches. Unlike those symbioses found in hematophagous arthropods, the nature, identity, and evolutionary history of these remains poorly studied. In this work, we further explored obligate nutritional associations between Haementeria leeches and their microbial symbionts, which led to the unexpected discovery of a novel symbiosis with a member of the Pluralibacter genus. When compared to Providencia siddallii, an obligate nutritional symbiont of other Haementeria leeches, this novel bacterial symbiont shows convergent retention of the metabolic pathways involved in B vitamin biosynthesis. Moreover, the genomic characteristics of this Pluralibacter symbiont suggest a more recent association than that of Pr. siddallii and Haementeria. We conclude that the once-thought stable associations between blood-feeding Glossiphoniidae and their symbionts (i.e., one bacteriome structure, one symbiont lineage) can break down, mirroring symbiont turnover observed in various arthropod lineages.

From bloodthirsty parasites to surgical superstars: The humble leech, a suck-cess story in surgical history.

This historical review delves into the extensive and intriguing history of leeches in surgery. The utilisation of leeches in medical practice dates back thousands of years, from ancient civilisations to the modern era. The therapeutic properties of leeches have led to their persistent presence in surgical procedures. The 'Age of Leeches' during the 18th and 19th centuries witnessed a heightened utilisation of leeches in surgical treatment, given their perceived ability to promote postoperative wound healing and alleviate blood congestion. However, advancements in medical knowledge subsequently diminished their popularity as emerging technologies overshadowed them. Nevertheless, recent years have witnessed a resurgence in the use of leeches in reconstructive surgery, driven by both a greater understanding of their biological mechanisms and their potential source of therapeutic agents. This historical review provides a comprehensive exploration of the historical timeline, societal perceptions and scientific advancements surrounding the use of leeches in surgical practice.

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.

Transcriptomic profiling and the first spatial expression analysis of candidate genes in the salivary gland of the East Asian medicinal leech, Hirudo nipponia.

Hirudo nipponia, a blood-sucking leech native to East Asia, possesses a rich repertoire of active ingredients in its saliva, showcasing significant medical potential due to its anticoagulant, anti-inflammatory, and antibacterial effects against human diseases. Despite previous studies on the transcriptomic and proteomic characteristics of leech saliva, which have identified medicinal compounds, our knowledge of tissue-specific transcriptomes and their spatial expression patterns remains incomplete. In this study, we conducted an extensive transcriptomic profiling of the salivary gland tissue in H. nipponia based on de novo assemblies of tissue-specific transcriptomes from the salivary gland, teeth, and general head region. Through gene ontology (GO) analysis and hierarchical clustering, we discovered a novel set of anti-coagulant factors-i.e., Hni-Antistasin, Hni-Ghilanten, Hni-Bdellin, Hni-Hirudin-as well as a previously unrecognized immune-related gene, Hni-GLIPR1 and uncharacterized salivary gland specific transcripts. By employing in situ hybridization, we provided the first visualization of gene expression sites within the salivary gland of H. nipponia. Our findings expand on our understanding of transcripts specifically expressed in the salivary gland of blood-sucking leeches, offering valuable resources for the exploration of previously unidentified substances with medicinal applications.