Species-specific oxylipins and the effects of ontogeny and predation on their emission from freshwater snails.

Chemical cues play important roles in mediating ecological interactions. Oxylipins, oxygenated metabolites of fatty acids, are one signalling molecule type that influences the physiology and function of species, suggesting their broader significance in chemical communication within aquatic systems. Yet, our current understanding of their function is restricted taxonomically and contextually making it difficult to infer their ecological significance. Snails and leeches are ubiquitous in freshwater ecosystems worldwide, yet little is known about their oxylipin profiles and the factors that cause their profiles to change. As snails and leeches differ taxonomically and represent different trophic groups, we postulated oxylipin profile differences. For snails, we hypothesized that ontogeny (non-reproductive vs reproductive) and predation (non-infested vs leech-infested) would affect oxylipin profiles. Oxylipins were characterized from water conditioned with the snail Planorbella duryi and leech Helobdella lineata, and included three treatment types (snails, leeches, and leech-infested snails) with the snails consisting of three size classes: small (5-6 mm, non-reproductive) and medium and large (13-14 and 19-20 mm, reproductive). The two species differed in the composition of their oxylipin profiles both in diversity and amounts. Further, ontogeny and predation affected the diversity of oxylipins emitted by snails. Our experimental profiles of oxylipins show that chemical cues within freshwater systems vary depending upon the species emitting the signals, the developmental stage of the species, as well as from ecological interactions such as predation. We also identified some candidates, like 9-HETE and PGE2, that could be explored more directly for their physiological and ecological roles in freshwater systems.

Unoculubranchiobdella sp. (Hirudinea: Ozobranchidae) as a vector for Haemogregarina spp. in freshwater turtles from Brazil.

Species of Haemogregarina are blood parasites known to parasitise vertebrate hosts, including fishes (Haemogregarina sensu lato) and freshwater turtles (Haemogregarina sensu stricto). Their vectors, include gnathiid isopods and leeches, respectively. In turtles, Haemogregarina balli has the best-characterized life cycle in the genus. However, no studies in Brazil have suggested a possible vector for any species of Haemogregarina from freshwater turtles. Therefore, in the present study, we provide insights into a leech vector based on specimens found feeding on two species of freshwater turtles, Podocnemis unifilis and Podocnemis expansa, using morphological and molecular data. In 2017 and 2019, freshwater turtles were collected in Goiás State, Brazil. Hosts were inspected for ectoparasites and leeches were collected from two specimens of P. expansa and nine specimens of P. unifilis. Leeches were subsequently identified as members of the genus Unoculubranchiobdella. Leech histological slides revealed haemogregarine-like structures, similar to post-sporogonic merogony, found near the gills and within the posterior sucker. Molecular analysis of the haemeogregarines resulted in the identification of three species of Haemogregarina: Haemogregarina embaubali, Haemogregarina goianensis, and Haemogregarina brasiliana. Therefore, our findings, based on morphology and DNA data suggest leeches of the genus Unoculubranchiondella as vectors for at least three species of Haemogregarina from Brazilian turtles.

Investigation of oxidant-antioxidant status in patients treated with hirudotherapy: an experimental study.

Objectives: To examine the influence of hirudotherapy on parameters of oxidative stress. METHODS: The cross-sectional study was conducted from March 29 to September 29, 2021, at the Alanya Research and Training Hospital's Traditional and Complementary Medicine Application Centre, Turkey, and comprised adult volunteers of either gender. The participants were subjected to two sessions of hirudotherapy 4 weeks apart. Total antioxidant status, total oxidant status, oxidative stress index values, ischaemia-modified albumin level, paraoxonase 1, disulfide, native thiol, total thiol, and arylesterase levels were assessed at baseline and after the second hirudotherapy session. Data was analysed using SPSS 15. RESULTS: Of the 50 subjects, 30(60%) were females and 20(40%) were males. The overall mean age was 47.10±15.16 years. Oxidative stress, ischaemia-modified albumin and disulfide levels decreased, but not significantly (p>0.05). The reduction in disulfide levels was significant (p=0.021). CONCLUSIONS: Hirudotherapy, within its limitations, could reduce oxidative stress.

Phylogenetic relationships and species delimitation in Haemopis (Annelida: Hirudinea: Haemopidae).

The Holarctic leech genus Haemopis currently includes 11 species, all of which are macrophagous, as opposed to their more infamous bloodfeeding counterparts among hirudiniform leeches. In spite of their ecological importance as fish food and predators of freshwater invertebrates, there is a paucity of data regarding morphology and genetic variation that might guide future identification efforts for members of the genus. The lack of detailed descriptions of distinguishing morphological features, coupled with the absence of a robust phylogenetic hypothesis for the genus, have conspired to prevent meaningful inferences on the natural history of the group. In an attempt to remedy this, we present new genetic (using COI, 12S rDNA, 28S rDNA and 18S rDNA) data for the majority of the known species diversity within the genus in order to both infer a phylogenetic hypothesis and to introduce authoritative DNA barcodes for the newly collected species. The potential of these barcodes is increased through rigorous morphological investigations of the specimens, with comparisons to the original literature. Our resulting phylogenetic hypothesis is agnostic as to the geographic origin of the genus, with equal probability afforded to both a Nearctic and Palearctic origin. Beyond this, we show that there is a strong tendency towards a barcoding gap within the genus, but that a distinct gap is lacking due to the relatively high genetic variation found within H. marmorata. Taken together, our results shed light on species delimitation within, and evolutionary history of, this often-neglected group of leeches.

Role of Antimicrobial Peptides in Immunity of Parasitic Leeches.

The review summarizes the current state of knowledge about leech immunity, with emphasis on the special role of antimicrobial peptides (AMPs), and highlights the wide variety of primary AMP structures, which seem to correlate with a variety of life strategies and the ecology of ectoparasites. Antimicrobial proteins and AMPs are a diverse class of natural molecules that are produced in all living organisms in response to an attack by a pathogen and are essential components of the immune system. AMPs can have a wide range of antibiotic activities against foreign and opportunistic bacteria, fungi, and viruses. AMPs play an important role in selection of colonizing bacterial symbionts, thus helping multicellular organisms to cope with certain environmental problems. AMPs are especially important for invertebrates, which lack an adaptive immune system. Although many AMPs are similar in physicochemical properties (a total length from 10 to 100 amino acids, a positive total charge, or a high cysteine content), their immunomodulatory activities are specific for each AMP type.

Manifestation of leech infestation as severe gastrointestinal bleeding in a 3-year-old boy: a case report and review of the literature.

BACKGROUND: Leeches are a class of hermaphroditic parasites that can attach to various body parts and start sucking blood. Gastrointestinal (GI) bleeding due to leeches is a rare phenomenon that is more common in less developed countries. Common symptoms include melena, hematemesis, pallor, weakness, and fatigue. Due to the similar symptoms of this issue to the main differential diagnoses of GI bleeding in pediatrics, such as diarrhea, constipation, diverticulitis, esophagitis, and anal fissures, it is challenging to differentiate it from the rest. CASE PRESENTATION: We present a three-year-old boy who was transferred to our center with hematemesis, tarry stool, and a drop in hemoglobin level. He finally was diagnosed with a leech in his stomach. CONCLUSIONS: In less developed counties, the inability to reach safe drinking water, swim in lakes or springs, and inadequate awareness of public health information among individuals can be risk factors for leech infestation.

Make it double: identification and characterization of a Tandem-Hirudin from the Asian medicinal leech Hirudinaria manillensis.

Haematophagous leeches express a broad variety of secretory proteins in their salivary glands, among them are hirudins and hirudin-like factors. Here, we describe the identification, molecular and initial functional characterization of Tandem-Hirudin (TH), a novel salivary gland derived factor identified in the Asian medicinal leech, Hirudinaria manillensis. In contrast to the typical structure of hirudins, TH comprises two globular domains arranged in a tandem-like orientation and lacks the elongated C-terminal tail. Similar structures of thrombin inhibitors have so far been identified only in kissing bugs and ticks. Expression of TH was performed in both cell-based and cell-free bacterial systems. A subsequent functional characterization revealed no evidence for a thrombin-inhibitory potency of TH.

Short tail stories: the hirudin-like factors HLF6 and HLF7 of the Asian medicinal leech, Hirudinaria manillensis.

The leech-derived hirudins and hirudin-like factors (HLFs) share a common molecule structure: a short N-terminus, a central globular domain, and an elongated C-terminal tail. All parts are important for function. HLF6 and HLF7 were identified in the Asian medicinal leech, Hirudinaria manillensis. The genes of both factors encode putative splice variants that differ in length and composition of their respective C-terminal tails. In either case, the tails are considerably shorter compared to hirudins. Here we describe the functional analyses of the natural splice variants and of synthetic variants that comprise an altered N-terminus and/or a modified central globular domain. All natural splice variants of HLF6 and HLF7 display no detectable thrombin-inhibitory potency. In contrast, some synthetic variants effectively inhibit thrombin, even with tails as short as six amino acid residues in length. Our data indicate that size and composition of the C-terminal tail of hirudins and HLFs can vary in a great extent, yet the full protein may still retain the ability to inhibit thrombin.

Mast Cells and Basophils in the Defense against Ectoparasites: Efficient Degradation of Parasite Anticoagulants by the Connective Tissue Mast Cell Chymases.

Ticks, lice, flees, mosquitos, leeches and vampire bats need to prevent the host's blood coagulation during their feeding process. This is primarily achieved by injecting potent anticoagulant proteins. Basophils frequently accumulate at the site of tick feeding. However, this occurs only after the second encounter with the parasite involving an adaptive immune response and IgE. To study the potential role of basophils and mast cells in the defense against ticks and other ectoparasites, we produced anticoagulant proteins from three blood-feeding animals; tick, mosquito, and leech. We tested these anticoagulant proteins for their sensitivity to inactivation by a panel of hematopoietic serine proteases. The majority of the connective tissue mast cell proteases tested, originating from humans, dogs, rats, hamsters, and opossums, efficiently cleaved these anticoagulant proteins. Interestingly, the mucosal mast cell proteases that contain closely similar cleavage specificity, had little effect on these anticoagulant proteins. Ticks have been shown to produce serpins, serine protease inhibitors, upon a blood meal that efficiently inhibit the human mast cell chymase and cathepsin G, indicating that ticks have developed a strategy to inactivate these proteases. We show here that one of these tick serpins (IRS-2) shows broad activity against the majority of the mast cell chymotryptic enzymes and the neutrophil proteases from human to opossum. However, it had no effect on the mast cell tryptases or the basophil specific protease mMCP-8. The production of anticoagulants, proteases and anti-proteases by the parasite and the host presents a fascinating example of an arms race between the blood-feeding animals and the mammalian immune system with an apparent and potent role of the connective tissue mast cell chymases in the host defense.

Antiparasitic Potential of Chromatographic Fractions of Nephrolepis biserrata and Liquid Chromatography-Quadrupole Time-of-Flight-Mass Spectrometry Analysis.

Marine aquaculture development is recently impeded by parasitic leech Zeylanicobdella arugamensis (Hirudinea, Piscicolidae) in Sabah, Malaysia. The parasitic leech infests a variety of cultured fishes in aquaculture facilities. In this study, we evaluated the antiparasitic activity of the chromatographic fractions of the medicinal plant Nephrolepis biserrata methanol extract against Z. arugamensis and highlighted the potential metabolites responsible for the antiparasitic properties through liquid chromatography (LC)-quadrupole time-of-flight (QTOF)-mass spectrometry (MS) analysis. Out of seven fractions obtained through flash column chromatography techniques, three fractions demonstrated antiparasitic properties. Significant parasitic mortality was indicated by fraction 3 at a concentration of 2.50 mg/mL, all the leeches were killed in a time limit of 1.92 ± 0.59 min. followed by fraction 4 (14 mg/mL) in 34.57 ± 3.39 and fraction 5 (15.3 mg/mL) in 36.82 ± 4.53 min. LC-QTOF-MS analysis indicated the presence of secondary metabolites including phytosphingosine (6), pyrethrosin (1), haplophytine (9), ivalin (2), warburganal (3), isodomedin (4) and pheophorbide a (16), representing sphingoid, alkaloid, terpenoid, phenolic and flavonoid groups. Thus, our study indicated that the chromatographic fractions of N. biserrata demonstrated significant antiparasitic activity against the marine parasitic leeches due to the presence of potent antiparasitic bioactive compounds.

The hirudin-like factors HLF3 and HLF4-hidden hirudins of European medicinal leeches.

The hirudin-like factors 3 (HLF3) and 4 (HLF4) belong to a new class of leech-derived factors and are present in specimens of the three European medicinal leeches, Hirudo medicinalis, Hirudo verbana, and Hirudo orientalis, respectively. Here we describe the functional analysis of natural and synthetic variants of HLF3 and HLF4. Whereas the natural variants display only very low or no detectable anti-coagulatory activities, modifications within the N-termini in combination with an exchange of the central globular domain have the potency to greatly enhance the inhibitory effects of respective HLF3 and HLF4 variants on blood coagulation. Our results support previous observations on the crucial importance of all parts (both the N- and C-termini as well as the central globular domains) of hirudin and HLF molecules for thrombin inhibition.

Leech (Myzobdella lugubris) infestations in largemouth bass (Micropterus salmoides) in Back Bay, Virginia, USA.

Back Bay is an oligohaline, coastal bay in southeast Virginia, USA. Since 2004, leeches have been observed in the oral cavities of largemouth bass (Micropterus salmoides) in this body of water. Leeches (Myzobdella lugubris) have previously been documented in the oral cavities of largemouth bass in the Currituck Sound, which is confluent with Back Bay on its southern border. Supplemental stocking of largemouth bass in Back Bay since 2009 has resulted in an increasing population; however, concern exists that leech infestation may be negatively affecting health of larger fish, which are still less abundant than expected. Despite the wide distribution of this leech, there is little available literature regarding its health impacts on hosts. In this study, we examine potential impacts of oral leech infestations on stress markers and haematological parameters of largemouth bass in Back Bay. No significant changes in plasma glucose or cortisol were observed between leech-infested and uninfested fish, and haematological parameters were not significantly different between the groups. Further, there was no evidence of systemic infections associated with leech infestation.

Spatio-temporal variation and the use of host body surface by ectoparasites of the chelonians Phrynops geoffroanus and Mesoclemmys tuberculata in areas of the Caatinga and Atlantic Forest in northeast Brazil.

Ectoparasites such as hematophagous leeches and monogeneans are common in chelonians, occupying different parts of the body. Thus, the present study aimed to identify and describe the fauna of ectoparasites that infest Phrynops geoffroanus and Mesoclemmys tuberculata to evaluate the effect of host conditions and seasonality (dry and rainy season) on the abundance and composition of ectoparasites. We verified the presence of ectoparasites in 73.2% of the examined turtles, with four species of leeches belonging to Glossiphoniidae, Haementeria brasiliensis sensu Cordero, 1937, Helobdella cf. adiastola, Haementeria sp1., and Haementeria sp2., and one monogenean Polystomatidae, Polystomoides brasiliensis. For both chelonians, we observed a significant difference in the abundance of ectoparasites in relation to sex, biome, and season, which was unrelated to length and mass. Leeches were more frequent in the cavities of the hind limbs in P. geoffroanus, and the anterior limbs of M. tuberculata. The general spatial niche overlap of ectoparasites was high, except for that of the monogenean P. brasiliensis, which did not overlap with those of other leech species. The present study is the first report of the presence of H. brasiliensis and P. brasiliensis parasitizing M. tuberculata, and Helobdella cf. adiastola in a phoretic relationship with P. geoffroanus and M. tuberculata. Finally, the differences in infestation levels may reflect ecological factors, differences in behavioral patterns of the hosts, and different anthropic alterations suffered in the Caatinga and Atlantic Forest biomes.

Hirudins of the Asian medicinal leech, Hirudinaria manillensis: same same, but different.

Blood coagulation in vertebrates is a complex mechanism that involves the precisely coordinated and regulated action of a cascade of factors in order to prevent excessive blood loss upon wounding. Any blood sucking ectoparasite, however, has to circumvent this mechanism to ensure the uptake of an adequate blood meal. Inhibitors of blood coagulation in the saliva are hence widespread among these animals. Thrombin as a key factor of blood coagulation is a prominent target of such inhibitors, and hirudin is probably the best known among the thrombin inhibitors. Hirudin was originally described in the genus Hirudo, but occurs in other leech genera like Hirudinaria and Macrobdella as well. Besides several isoforms of hirudin, a new class of putative leech saliva components, the hirudin-like factors (HLFs), was identified in both genera Hirudo and Hirudinaria. Here, we describe the expression, purification, and functional characterization of three HLFs (HLF5, 6, and 8, respectively) and two additional hirudins (HM3 and HM4) of Hirudinaria manillensis. While HLF6 lacked any inhibitory activity on thrombin, HLF5 as well as HLF8 clearly exhibited anticoagulatory properties. The inhibitory activity of HLF5 and HLF8, however, was much lower compared with both HM3 and HM4 of Hirudinaria manillensis as well as the hirudin variants 1 (HV1) and 2 (HV2) of Hirudo medicinalis. Neither an inhibition of trypsin nor a platelet aggregation was caused by HLF8. Our data indicates the presence of two classes (rather than isoforms) of hirudins in Hirudinaria manillensis with markedly different inhibitory activity on human thrombin.

The Use of Medicinal Leeches for the Treatment of Venous Congestion in Replanted or Revascularized Digits.

PURPOSE: A noteworthy cause of failure in digital replantation is venous insufficiency. External bloodletting with medicinal leeches is a common treatment for venous insufficiency after distal digital replantations. The objective of this study was to evaluate the salvage rate of digits replanted proximal to the distal interphalangeal (DIP) joint that were treated with medicinal leeches for venous congestion. METHODS: We retrospectively reviewed the charts of patients with complete or incomplete digit amputation admitted between January, 2008 and April, 2014. We included all patients with venous congestion in one or more digits replanted or revascularized at the middle or proximal phalangeal level, who were treated with medicinal leeches. Treatment initiation and duration were based on clinical judgment. Demographic, therapy, and surgical data were collected. RESULTS: Of 145 patients with 205 digits that were replanted or revascularized, 25 digits were treated with medicinal leeches for venous congestion. Venous congestion was diagnosed later than 48 hours after operation in 24 of the 25 digits. Of the 25 digits, 11 survived (44.4%) (8 of 22 replanted digits and all 3 revascularized digits). No complications were recorded except for substantial blood loss requiring transfusion in one patient. CONCLUSIONS: External bloodletting with medical leeches might be less effective with late treatment or with a higher volume of congested tissue, as is characteristic of proximal finger replantation. TYPE OF STUDY/LEVEL OF EVIDENCE: Therapeutic IV.

A Comprehensive Review of Hirudiniasis: From Historic Uses of Leeches to Modern Treatments of Their Bites.

Exposure to leeches in the wilderness setting is common. Leeches may attach themselves to exposed skin or pass through one of the body's orifices and attach internally. The condition of leech attachment is known as hirudiniasis, which can result in serious morbidity and, rarely, mortality. A comprehensive review of the literature was performed to detail the prevention of leech attachment, as well as both anecdotal and studied methods of removal. Complications from leech attachments include ongoing bleeding, wound infection, and poor wound healing. Although medicinal leeches are the most well-studied variety, this review examines all aspects of leech attachment, prevention, and management.

Leech therapy in flap salvage: Systematic review and practical recommendations.

INTRODUCTION: Medicinal leeches have been part of the therapeutic armamenterium of plastic surgeons for more than 50 years. While their use in hand surgery is a matter of course, their use in salvage of flaps with venous congestion remains facultative depending on teams. MATERIALS AND METHODS: We conducted a systematic review of leech therapy for flap salvage between 1960 and 2015, analyzing 121 articles and subsequently taking into consideration 41 studies. In parallel, we collected data from 43 patients for whom leach therapy had recently been applied in treatment of venous insufficiency in pedicled or free flaps after revision surgery had failed to improve flap vascularization, or in cases where flap revision was not appropriate. The data collected pertained to relevant indications, treatment procedure, efficacy, adjuvant therapies, side effects and complications. RESULTS: For this indication, the success rate of leech therapy ranged from 65 to 85% (83.7% in our series) according to the situations encountered. Optimal frequency of application ranged from 2 to 8hours, while average overall duration ranged from 4 to 10 days. The number of leeches to be applied can be determined depending on volume of the flap. In 50% of the cases reported in the literature, the patients required transfusion. Antibiotic prophylaxis against Aeromonas is highly advisable. A ciprofloxacin and trimethoprim-sulfametoxazole combination currently appears as the most relevant prophylactic antibiotherapy. CONCLUSION: Hirudotherapy is a reliable treatment in cases of patent venous insufficiency of pedicled or free flaps (or when revision surgery is not recommended). Even though the relevant literature is highly heterogeneous, we have attempted to put forward a specific protocol bringing together dosage, delivery route, frequency of administration and appropriate prophylactic antibiotherapy. An algorithm for treatment and management of venous congestion and a practical information sheet have been placed at the disposal of plastic surgery teams.

Generation of recombinant destabilase-lysozyme from medicinal leeches in three different expression systems.

Destabilase-lysozyme (mlDL) is an enzyme secreted by the salivary gland cells of medicinal leeches. Destabilase-lysozyme possesses lysozyme and isopeptidase activities. We generated recombinant destabilase-lysozyme isoform 2 in three expression systems, i.e., in the bacteria Escherichia coli, in the yeast Pichia pastoris, and in the human cell line Expi293F. In E. coli, we generated both polypeptide in inclusion bodies that was later undergone to the refolding and soluble protein that had been fused with the chaperone SlyD. The chaperone was later cleaved by a specific TEV-protease. In cultures of the yeast P. pastoris and the human cell line Expi293F, the soluble form of destabilase-lysozyme was accumulated in the culture media. For the generated enzymes, we determined the lysozyme, isopeptidase and fibrinolytic activities and tested their general antimicrobial effects. The comparisons of the enzymes generated in the different expression systems revealed that all of the destabilase-lysozymes obtained in the soluble forms possessed equal levels of lysozyme, isopeptidase and fibrinolytic activities that exceeded several to ten times the levels of the same activities of the destabilase-lysozyme renaturated from the inclusion bodies. A similar pattern of the differences in the levels of the general antimicrobial effects was observed for the destabilase-lysozymes generated in the soluble form and as inclusion bodies.

Ciprofloxacin-resistant Aeromonas infection following leech therapy for digit replantation: report of 2 cases.

Medicinal leeches are commonly used after finger replantation to treat surgically unsalvageable venous congestion. Infection from Aeromonas hydrophila is a recognized complication of leech therapy that can be underestimated by the medical community. Ciprofloxacin and trimethoprim-sulfamethoxazole are the most commonly recommended prophylactic antibiotics used to prevent A. hydrophila infections during leech therapy. Here, we report 2 cases of ciprofloxacin-resistant Aeromonas infections, occurring within 4 months of each other. Both cases developed after leech therapy for unsuccessful digital replantation. These infections were successfully treated with ceftriaxone. Ciprofloxacin-resistant Aeromonas should be recognized when determining prophylactic antibiotic protocols for replant centers when leech therapy is used for finger replantation.