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.

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.

Terrestrial predatory leeches of the genus Orobdella (Hirudinea: Erpobdelliformes: Orobdellidae) endemic to the Southern Russian Far East: a new species of the genus from Primorsky Krai, Russia.

A quadrannulate species, Orobdella ganini sp. nov., is described from the Lazovsky Nature Reserve in Primorsky Krai, the Southern Russian Far East, Russia. Morphological features of O. ghilarovi Nakano & Prozorova, 2019 from the reserve are also provided leading to an amendment of the species diagnosis. Maximum likelihood and Bayesian inference phylogenetic analyses, which were performed using nuclear 18S rRNA, 28S rRNA, and histone H3, and mitochondrial cytochrome c oxidase subunit I, tRNACys, tRNAMet, 12S rRNA, tRNAVal, 16S rRNA, tRNALeu and NADH dehydrogenase subunit 1 markers, show that O. ganini sp. nov., O. ghilarovi and two species endemic to Hokkaido, Japan form a clade, with the new species sister to a lineage composed of the two Japanese species. A partial cytochrome c oxidase subunit I sequence obtained from a cocoon found in the Lazovsky Nature Reserve reveals that Orobdella leeches deposit cocoons somewhat similar to those deposited by terrestrial blood-sucking leeches of Haemadipsidae.

Placobdella nabeulensis sp. nov. (Hirudinea: Glossiphoniidae), a new glossiphoniiform leech from Palearctic North Africa.

BACKGROUND: Herein, we describe a new species of turtle blood-feeding leech, Placobdella nabeulensis sp. nov. from Palearctic North Africa (Tunisia and Algeria). The new species is described based on detailed morphological analyses using light and scanning electron microscopes. RESULTS: Apart from the detailed morphology of the atrium, morphological features alone do not sufficiently separate the species from congeners due to the absence of distinct diagnostic characters. Therefore, we turned to molecular data to better distinguish this new species from other members of the genus and establish a basis for its genetic separation. Four DNA fragments were successfully amplified, including mitochondrial COI and 12S rDNA, as well as nuclear 28S rDNA and histone H3. We then provided the molecular descriptor of the taxon, based on redundant diagnostic nucleotide combinations in DNA sequence alignment within the Folmer region. Results of the phylogenetic analysis and species delimitation methods (ABGD, ASAP, and bPTP) based on the COI locus support the species rank of the Tunisian-Algerian Placobdella. CONCLUSIONS: The new species is most closely related to the European species Placobdella costata (Fr. Müller, 1846) and the present study indicates that Placobdella nabeulensis sp. nov. has likely been confused with the European counterpart in several previous studies. This article is registered at www.zoobank.org under urn:lsid:zoobank.org:pub:4A4B9C1D-2556-430F-8E4B-0CE99F2012F5.

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.

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.

Behavioral variation according to feeding organ diversification in glossiphoniid leeches (Phylum: Annelida).

Adaptive radiation is a phenomenon in which various organs are diversified morphologically or functionally as animals adapt to environmental inputs. Leeches exhibit a variety of ingestion behaviors and morphologically diverse ingestion organs. In this study, we investigated the correlation between behavioral pattern and feeding organ structure of leech species. Among them, we found that Alboglossiphonia sp. swallows prey whole using its proboscis, whereas other leeches exhibit typical fluid-sucking behavior. To address whether the different feeding behaviors are intrinsic, we investigated the behavioral patterns and muscle arrangements in the earlier developmental stage of glossiphoniid leeches. Juvenile Glossiphoniidae including the Alboglossiphonia sp. exhibit the fluid ingestion behavior and have the proboscis with the compartmentalized muscle layers. This study provides the characteristics of leeches with specific ingestion behaviors, and a comparison of structural differences that serves as the first evidence of the proboscis diversification.

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.

Anatomy and activity patterns in a multifunctional motor neuron and its surrounding circuits.

Dorsal Excitor motor neuron DE-3 in the medicinal leech plays three very different dynamical roles in three different behaviors. Without rewiring its anatomical connectivity, how can a motor neuron dynamically switch roles to play appropriate roles in various behaviors? We previously used voltage-sensitive dye imaging to record from DE-3 and most other neurons in the leech segmental ganglion during (fictive) swimming, crawling, and local-bend escape (Tomina and Wagenaar, 2017). Here, we repeated that experiment, then re-imaged the same ganglion using serial blockface electron microscopy and traced DE-3's processes. Further, we traced back the processes of DE-3's presynaptic partners to their respective somata. This allowed us to analyze the relationship between circuit anatomy and the activity patterns it sustains. We found that input synapses important for all the behaviors were widely distributed over DE-3's branches, yet that functional clusters were different during (fictive) swimming vs. crawling.

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.

Ovary cord micromorphology in the blood-sucking haemadipsid leech Haemadipsa japonica (Hirudinida: Arhynchobdellida: Hirudiniformes).

This is the first report that describes histological and ultrastructural details of ovary organization in haemadipsid leeches. In Haemadipsa japonica, the female reproductive system is organized similar to that of other haemadipsids. Each of the paired and oval ovaries of H. japonica is comprised of the ovary wall (ovisac), which encloses two elongated, thread-like ovarian units termed ovary cords. Ovary cords are comprised of germ-line cells and associated somatic cells. Each cord is polarized and contains germ-line cells in the consecutive developmental stages that are sequentially located along the long cord axis. There were three zones in each cord: the club-shaped apical part, the thread-like middle part, and the basal-most end, which contains degenerating germ cells. Outside of the reproductive period, the middle part of the cord in leeches is smooth, and no growing oocytes are visible; alternatively, in mature specimens, several growing oocytes protrude from the cord, and several huge vitellogenic oocytes that are completely detached from the cord occur within the ovisac. Ovary cord organization and functioning in H. japonica are very similar to the 'Hirudo' type cords that were found in several hirudiniform leeches. This conclusion supports the view that all hirudiniform leeches have conservative ovary cord organization and a similar pattern of oogenesis. Germ-line cyst composition, architecture, and functioning were also found to be evolutionarily conservative characteristics when compared with all previously examined Clitellata. In the germ-line cysts found in H. japonica each cell is connected to the central and anuclear cytoplasmic mass (cytophore) via one intercellular bridge, and, as oogenesis progresses, the fate of interconnected cell diversifies: some of them (oocytes) grow and complete oogenesis, but the majority become nurse cells and finally degenerate. Thus, oogenesis in H. japonica, similar to other clitellates, can be considered meroistic.

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.

On the origin of leeches by evolution of development.

Leeches are a unique group of annelids arising from an ancestor that would be characterized as a freshwater oligochaete worm. Comparative biology of the oligochaetes and the leeches reveals that body plan changes in the oligochaete-to-leech transition probably occurred by addition or modification of the terminal steps in embryonic development and that they were likely driven by a change in the feeding behavior in the ancestor of leeches. In this review article, developmental changes that are associated with the evolution of several leech-specific traits are discussed. These include (1) the evolution of suckers, (2) the loss of chaetae, (3) the loss of septa, and (4) a fixed number of segments. An altered developmental fate of the teloblast is further proposed to be a key factor contributing to the fixation of the segment number, and the evolutionary change in teloblast development may also account for the loss of the ability to regenerate the lost body segments in the leech.

Redescription of the Siamese shield leech Placobdelloides siamensis with new host species and geographic range.

The Siamese shield leech Placobdelloides siamensis (Oka, 1917) Sawyer, 1986 (Euhirudinea: Glossiphoniidae) was collected from five new host species, Southeastern Asian Box Turtle (Cuora amboinensis), Yellow-headed Temple Turtle (Heosemys annandalii), Malayan Snail-eating Turtle (Malayemys macrocephala), Mekong Snail-eating Turtle (M. subtrijuga), and Khorat Snail-eating Turtle (M. khoratensis) and was found for the first time in Udon Thani, Thailand. Examination of live leeches provided, for the first time, data on coloration and the combination of parental care behavior, both carrying cocoons and attaching cocoons to the substrate. This species was separated from its congeners based on the following characters: one pair of eyes; spines at proboscis subterminal; mouth terminal on oral sucker; absent plaque in neck region; gonopores located in furrow and separated by two annuli; distinctly triannulated mid-body segments; crop with seven pairs and branched caeca; caudal sucker slightly over half of maximum body width; and strongly dorsal papillae. Phylogenetic relationships based on the COI and ND1 genes were clarified and demonstrated that the species is distinct from others. The original description was amended and the taxonomic history is discussed.

New Antarctic deep-sea weird leech (Hirudinida: Piscicolidae): morphological features and phylogenetic relationships.

A new fish leech Ambulobdella shandikovi n. g., n. sp. (Hirudinida: Piscicolidae), a parasite of Whitson's grenadier Macrourus whitsoni (Regan) (Macrouridae: Gadiformes) collected in the Ross Sea at depths from 1,221 to 1,433 m, is described and compared with related taxa based on morphological and molecular characters. Ambulobdella shandikovi n. sp. is characterised by prominent segmental tubercles on the venter and dorsal segmental tubercles, an uncommon appearance of its anterior sucker with ear-like edges and an inner membrane around the mouth-pore, well-developed musculature and a unique combination of features of the reproductive and digestive systems. The presence of uncommon tubercles can be attributed, in part, to temporary associations of A. shandikovi n. sp. with its fish hosts and a need for well-developed sensory and locomotory organs. A certain locomotory function of ventrolateral tubercles of A. shandikovi n. sp. is hypothesised and discussed. Further deep-sea surveys are obviously needed to shed light on the behaviour and mode of locomotion of this species.

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.

A New Species of Orobdella (Hirudinida: Arhynchobdellida: Orobdellidae) from Japan Reveals the Function of the Orobdella Gastroporal Duct.

A quadrannulate species of Orobdella, O. kanaekoikeae sp. nov., from the mountainous region of Shikoku, Japan is described. This new species is small with a body length of mature individuals reaching only ca. 5 cm. Phylogenetic analyses using nuclear 18S, histone H3, mitochondrial cytochrome c oxidase subunit I, tRNACys, tRNAMet, 12S rRNA, tRNAVal, 16S rRNA, tRNALeu, and NADH dehydrogenase subunit 1 markers showed that O. kanaekoikeae is a sister species of the quadrannulate small O. brachyepididymis. Phylogenetic relationships among populations of this new species were also estimated using mitochondrial DNA markers. Additionally, transmission electron micrographs of a capsule-like structure obtained from a post-copulatory individual revealed that the capsule contained spermatozoa, and was thus defined as the spermatophore. The gastroporal duct of Orobdella leeches is an accessory copulatory organ that receives a spermatophore during copulation.