Not that many leech species after all: Myzobdella lugubris and Myzobdella patzcuarensis (Annelida: Hirudinida) are the same species.

The genus Myzobdella groups five species of leeches parasites of fishes mainly of freshwater but with tolerance to brackish waters. Native distribution of these species includes the New World from North to South America. Myzobdella lugubris Leidy, 1851, the type species of the genus, was briefly described based on specimens from the USA, but subsequently their morphology, known distribution and host range were expanded; however, less is known about the other four species of the genus. As part of a survey focusing on characterizing the diversity of leeches from Mexico, specimens of Myzobdella patzcuarensis (Caballero, 1940), from the type locality of the species were included for the first time in a phylogenetic study. In addition, specimens assigned to Myzobdella from the southeast of Mexico as well as from Nicaragua, were also included. In the resulting phylogenetic tree, our newly generated sequences were found nested in the same clade that M. lugubris; with unresolved relationships and relatively low genetic divergence, suggesting conspecificity. In addition, the internal morphology of the specimens of Myzobdella from Mexico is consistent with the description of M. lugubris. Our morphological examination reveals high degrees of variability in the external pigmentation of the specimens. Based on our results we formally synonymize M. patzcuarensis under M. lugubris.

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.

Reproduction of Limnatis paluda (Tennent 1859) in laboratory conditions.

The reproductive functions of Limnatis paluda were studied in laboratory conditions including imposing of two thermal conditions (T1: 18 ± 2 °C and T2: 26 ± 2 °C) and with there being placement of three bedding substrates (tile, moss, and stone) in the containers specimens were confined. Furthermore, values for reproductive variables of cocoons and hatchlings were recorded. With the T2 treatment, gravid adults had cocoon depositions, whereas those when there was imposing of the T1 treatment did not have cocoon depositions. There was cocoon deposition only on the moss bedding, and it is presumed that moss could provide a robust and stable nursing microenvironment for developing embryos. Frequency (%) of cocoon depositions did not vary during the months of the deposition period. Hatching occurred after 26 ± 2 days when there was imposing of the T2 treatment. There was no correlation between the values for size of the ovigerous cocoons and number of offspring. There was a negative correlation between number and mean weight of hatchlings. There was a difference in cocoon weight for the different months and fluctuation in mortality and hatching rate which is assumed to be related to differences in feeding behaviors.

A new freshwater leech species from Asian Swamp Eel stocks in China.

Discoveries of new Hemiclepsis species (Hirudinea: Glossiphoniidae) in East and Southeast Asia were expected. A peculiar freshwater leech was found on the body surface of Asian Swamp Eel Monopterus albus (Synbranchiformes: Synbranchidae) in Jingzhou, Hubei Province, China. Here, we describe this leech species as Hemiclepsis yangtzenensis sp. nov. It could be distinguished from other congeners based on a combination of morphological features such as very small size, translucent body with dense green reticulate markings throughout dorsum, very large posterior sucker with a characteristic reticulate pattern, and two pairs of well-developed drop-like eyespots. It also represents a phylogenetic lineage that is distant from other members of the genus based on the COI and 18S rRNA sequences. This leech species seems to be a specialized fish parasite that could negatively affect farming and wild stocks of Asian Swamp Eel in China.

Is the South African leech Barbronia gwalagwalensis Westergren amp; Siddall, 2004 (Hirudinida: Erpobdelliformes: Salifidae) a Paleotropical species?

The freshwater leech family Salifidae Johansson has a Paleotropical range, with a hotspot of species richness in the Oriental Region, and a few species endemic to Africa, Madagascar, and Reunion. Barbronia gwalagwalensis Westergren Siddall, 2004 was thought to be a characteristic example of the latter group being a lineage endemic to South Africa. However, we found that this species also occurs in Asia (Myanmar and Korea). Our time-calibrated phylogeny based on the mitochondrial cytochrome c oxidase subunit I (COI) gene reveals that the split between the African and Asian populations of this species occurred in the mid-Pleistocene, approximately 1.3 Ma ago (95% HPD 0.7-2.1 Ma). The statistical biogeographic modeling indicates that a B. gwalagwalensis population in South Africa most likely originated due a long-distance dispersal event with a subsequent vicariance (probability = 88.9%). A Late Quaternary range extension towards South Africa is known to occur in some other freshwater taxa (e.g. the freshwater mussel Unio caffer Krauss species group), which agrees with our hypothesis on the ancient origin of the South African B. gwalagwalensis population. Conversely, we can assume that the African population of this species was recently introduced from Asia. If so, the high levels of genetic divergence between African and Asian populations could be a part of a more general phylogeographic pattern historically originated within the Asian subcontinent due to the isolation by orographic or marine barriers. These two alternative hypotheses need further research efforts, i.e. sampling and sequencing of other Barbronia taxa, the ranges of which are situated between South Africa and Southeast Asia, as well as of topotypes of B. weberi (Blanchard, 1897) from Indonesia. Finally, our results highlight that the salifid genus Barbronia Johansson originated in the Oriental Region and that these leeches share both recently and historically high potential for long-distance dispersal events.

Description and taxonomic analysis of a tuberculated turtle leech from the Outer Banks, North Carolina, USA, provisionally identified as Placobdella multilineata Moore, 1953 (Hirudinea: Glossiphoniidae).

A tuberculated species of turtle leech is indigenous to the Great Dismal Swamp and environs of northeastern North Carolina, and differs from other known species of Placobdella. This study of hundreds of specimens for more than a decade documents its unexpected taxonomic complexity. In fact, this seemingly innocuous leech undergoes radical transformations in terms of morphology and behaviour, each adapted to a different phase of its life cycle. Biological observations reveal a progressive darkening with age which imposes taxonomic uncertainties. Furthermore, some commonly used characters are found to be unsound for taxonomic diagnosis in that they do not occur in all individuals of this species. The primary objective of this paper is a comprehensive description of this Albemarle turtle leech. The question is then asked, what distinguishes it from allied species? Toward this end, a formal taxonomic diagnosis is proposed based on details of the proboscis complex and crop-related tuberculation. This species is allied to the northern P. rugosa (Verrill, 1874) or its southern counterpart P. multilineata Moore, 1953. However, synonymy to either of these forms is problematical due to inadequacy of type descriptions. Nonetheless, in view of habitat similarity the Albemarle leech is provisionally identified as P. multilineata pending comparable studies of variability and development of other tuberculated Placobdella.

Placobdelloides tridens sp. n., a new species of glossiphoniid leech (Hirudinea: Rhynchobdellida) found feeding on captive Orlitia borneensis in Thailand, and an update to the host distribution of P. siamensis.

A new glossiphoniid leech species, Placobdelloides tridens sp. n., is discovered on the Malayan Giant Turtle (Orlitia borneensis) at the Nakhon Ratchasima Zoo in Thailand. The morphological study of this new species revealed that it is distinguished from P. siamensis, a turtle leech species that can be found commonly in Thailand. Placobdelloides tridens presented the following diagnostic morphological characteristics: a pear-shaped and triannulate body, well-developed rod-like papillae on the dorsal surface, smooth posterior and anterior suckers with nominal pits inside, a single pair of dark contiguous eyes, light yellow-brown to greenish dorsal color, absence of median line, male and female gonopore separated by a single annulus and a unique trident shape at the tip of the crop ceca. The phylogenetic relationships of P. tridens sp. n., was clarified, and shown to be a sister clade to the P. siamensis and P. sirikanchanae clade. Furthermore, this is a new host record for P. siamensis, which was found on O. borneensis, Batagur affinis and B. borneoensis in the Khao Kheow Open Zoo, Chonburi, Thailand.

Myxobdella socotrensis sp. nov., a new parasitic leech from Socotra Island, with comments on the phylogeny of Praobdellidae (Hirudinida: Arhynchobdellida).

The Socotra Archipelago (Yemen) is remarkable for unique ecosystems and high endemism, for which it is often referred to as the "Galápagos of the Indian Ocean". Here we describe a new parasitic leech Myxobdella socotrensis sp. nov. from Socotra, the largest island of the archipelago. The new species was found in a freshwater spring attached to the endemic crab Socotrapotamon socotrensis (Hilgendorf, 1883) (Crustacea: Potamidae). Based on its morphology, ecology and a phylogenetic analysis of DNA sequence data (12S, 18S, 28S and COI gene markers), the new leech species is classified into the highly diversified family Praobdellidae (Hirudinida: Arhynchobdellida), distributed in tropical regions of Africa, Asia and Central and South America. The praobdellid leeches are known to infest mammalian mucous membranes, however, some taxa are associated with other hosts, namely crabs, amphibians and birds. By its morphology, the new species fits quite well in the current concept of the genus Myxobdella Oka, 1917. However, the monophyly of Myxobdella was not supported here by molecular data, pointing at the need of a more comprehensive systematic revision of the genus and family. As far as known, Myxobdella socotrensis sp. nov. is endemic to the Socotra Island, but more data are needed to understand its evolutionary origin, biology and distribution.

Batracobdelloides bangkhenensis sp. n. (Hirudinea: Rhynchobdellida), a new leech species parasite on freshwater snails from Thailand.

A new snail-eating leech, Batracobdelloides bangkhenensis sp. n., was discovered at Kasetsart University, Bangkhen Campus, Bangkok, Thailand. This species is found free living in the benthic zone of ponds; feeds on freshwater snails, including Bithynia siamensis siamensis, Indoplanorbis exustus, Radix rubiginosa, Physella acuta, and Pomacea canaliculata; and uses a shell as a shelter during the parental care period, with a colony of 7-15 juvenile individuals held on the venter inside the shell of host. Batracobdelloides bangkhenensis displays distinct morphological characters, including a rice-shaped body showing transparency, cephalization, two eye pairs merged on somite III, an anterior sucker twice as large as the cephalic region, a central mouth in the anterior sucker, seven light brown transverse rows in the neck region, absent dorsal papillae, rich green pigments on the dorsum, a male gonopore on XIIa2/XIIa3 (27-28), a female gonopore on XIIIa1/XIIIa2 (29-30), and diffuse aggregations of minute, spherical salivary glands in the neck region. Comparisons of the COI and COI-ND1 genes showed a monophyletic clade for Batracobdelloides, and the phylogenetic tree of the COI gene also indicated that B. bangkhenensis is distinct from other species in the genus, with strong support values.

First freshwater mussel-associated piscicolid leech from East Asia.

Parasites and symbionts of freshwater mussels are poorly understood, although a diverse assemblage of mussel-associated leeches (Glossiphoniidae) was recently described. Here, we report on the discovery of a fish leech (Piscicolidae) in the mantle cavity of the freshwater mussel Cristaria plicata (Unionidae) in the Russian Far East. It is the first member of this leech family being associated with freshwater molluscs. This leech does not match any known genus and species both morphologically and genetically, and is described here as Alexandrobdella makhrovi gen. & sp. nov. It uses mussels as shelter (and probably as a secondary host), while the Amur catfish Silurus asotus (Siluridae) seems to be the primary host. These novel findings indicate that mussel-associated leech assemblage contains at least one piscicolid species. Our fossil-calibrated phylogeny suggests that the crown group of Piscicolidae was originated in the Early Cretaceous. This primarily marine family shares at least five independent colonization events into freshwater environments.

Intraspecific Variation in Growth and Reproduction of the Marine Leech, Heptacyclus buthi.

Heptacyclus buthi was harvested from fish hosts in rocky intertidal zones of Sonoma and Marin Counties, California, in October 2008 (n = 162) and October 2010 (n = 51). The size of the leeches was quantified using a method that approximated the sagittal cross-section of each specimen. Size-frequency curves were modeled to estimate the number of size-class cohorts in each year. If H. buthi is an annual species like many of its relatives, the single cohort modeled for in 2010 and the comparable "older" cohort in 2008, both with a broad range of sizes, may represent 1 component of its reproductive life history. A second, younger, more-numerous, less-variable cohort modeled from the 2008 sample may represent a second reproductive bout during that year that was prevented in the subsequent La Niña period of 2010-2011.

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

BACKGROUND: Salivary cell secretion (SCS) plays a critical role in blood feeding by medicinal leeches, making them of use for certain medical purposes even today. RESULTS: We 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. CONCLUSIONS: Here 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.

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.

Multilocus Metabarcoding of Terrestrial Leech Bloodmeal iDNA Increases Species Richness Uncovered in Surveys of Vertebrate Host Biodiversity.

Leech-derived invertebrate DNA (iDNA) has been successfully leveraged to conduct surveys of vertebrate host biodiversity across the Indo Pacific. However, this technique has been limited methodologically, typically only targeting mammalian 16S rDNA, or both 16S and vertebrate 12S rDNA for leech host determination. To improve the taxonomic richness of vertebrate host species in iDNA surveys, we re-analyze datasets from Bangladesh, Cambodia, China, and Madagascar through metabarcoding via next generation sequencing (NGS) of 12S, 16S (2 types, one designed to target mammals and the other, residual eDNA), nicotinamide adenine dinucleotide hydride dehydrogenase 2 (ND2), and cytochrome c oxidase subunit 1 (COI). With our 5 primer sets, we identify 41 unique vertebrate hosts to the species level, among 1,200 leeches analyzed, along with an additional 13 taxa to the family rank. Within our 41 taxa, we note that adding ND2 and COI loci increased species richness detection by 25%. NGS has emerged as more efficient than Sanger sequencing for large scale metabarcoding applications and, with the decline in cost of NGS, our pooled sample multilocus protocol is an attractive option for iDNA biodiversity surveys.

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.

Freshwater mussels house a diverse mussel-associated leech assemblage.

Freshwater mussels (Unionida) are one of the most imperiled animal groups worldwide, revealing the fastest rates of extinction. Habitat degradation, river pollution and climate change are the primary causes of global decline. However, biological threats for freshwater mussels are still poorly known. Here, we describe a diverse ecological group of leeches (Hirudinea: Glossiphoniidae) inhabiting the mantle cavity of freshwater mussels. So far, examples of mussel-associated leech species are recorded from East Asia, Southeast Asia, India and Nepal, Africa, and North America. This group comprises a dozen glossiphoniid species with a hidden life style inside the mantle cavity of their hosts largely overlooked by researchers. We show that the association with freshwater mussels evolved independently in three leech clades, i.e. Batracobdelloides, Hemiclepsis, and Placobdella, at least since the Miocene. Seven mussel-associated leech species and two additional free-living taxa are described here as new to science.

Phylogenetic Position and Description of a New Species of Medicinal Leech from the Eastern United States.

A new species of medicinal leech, Macrobdella mimicus n. sp., is described from specimens collected in Maryland; this is the first description of a North American macrobdellid since 1975. Superficially, the new species resembles the well-known Macrobdella decora, as both species possess 4 accessory pores arranged symmetrically on the ventral surface, yet the new species is distinguished from M. decora in possessing 4-4½ annuli (rather than 3½) between the gonopores and 4 annuli (rather than 5 annuli) between the female gonopore and the first pair of accessory pores. Phylogenetic analyses, based on 2 mitochondrial and 2 nuclear loci for a set of closely related taxa, confirms the placement of the new species within the family Macrobdellidae and places it as the sister taxon to M. decora and M. diplotertia.

A new species of Branchellion Savigny, 1822 (Hirudinida: Piscicolidae), a marine leech parasitic on the giant electric ray Narcine entemedor Jordan & Starks (Batoidea: Narcinidae) off Oaxaca, Mexico.

Branchellion spindolaorum n. sp. (Hirudinida: Piscicolidae) is described based on specimens found parasitising the giant electric ray Narcine entemedor Jordan & Starks off the coast of Oaxaca, Mexico. The new species can be clearly distinguished from the other species of Branchellion Savigny, 1822 by the presence of 30 pairs of lateral branchiae and 10 pairs of pulsatile vesicles. The definition of the genus Branchellion is expanded to include species with either 30, 31 or 33 pairs of foliaceous (plate-like) lateral branchiae in the urosome. In addition, we provide for the first time for the genus, scanning electron micrographs of the secondary suckers located on the ventral surface of the posterior sucker. Additionally, partial DNA sequences of the mitochondrial cytochrome c oxidase subunit 1 (cox1) were generated and compared with homologous sequences of other species of the genus. Branchellion spindolaorum n. sp. represents the fourth species of the genus known in the Eastern Pacific and the first record of a leech parasitising N. entemedor.

Hirudin and Decorsins of the North American Medicinal Leech Macrobdella decora: Gene Structure Reveals Homology to Hirudins and Hirudin-like Factors of Eurasian Medicinal Leeches.

Blood-sucking leeches, some of which are referred to as medicinal leeches, have caught attention not only because of their medical purposes, but also as study organisms to conduct research within fields as diverse as neurobiology, osmoregulation, ecology, and phylogeny. Of particular interest is the question whether hemophagy in leeches is of single origin or evolved independently several times. A key component in the saliva of hematophagous leeches is hirudin, a strong natural inhibitor of thrombin and hence the blood coagulation cascade. Multiple isoforms of hirudin have been described within and among several leech species and genera, often based on sequence data only. The identification of hirudin-like factors (HLFs) illustrated the necessity to underpin such predictions by functional tests. We overexpressed and purified the hirudin of the North American medicinal leech, Macrobdella decora, and proved its thrombin-inhibiting activity. In addition, analysis of the gene structure of both hirudin and some of the decorsins of M. decora clearly indicated conserved exon and intron positions when compared to genes of hirudins and HLFs of Eurasian medicinal leeches. Our data provide evidence for the incorporation of decorsins into the hirudin superfamily and support the concept of a single origin of blood feeding in jawed leeches.

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.