Alternative leech vectors for frog and turtle trypanosomes.

Trypanosoma pipientis infections were achieved by exposing laboratory-raised bullfrog tadpoles (Rana catesbeiana) to the leech Desserobdella picta that had fed on infected frogs. Likewise, a laboratory-raised snapping turtle (Chelydra serpentina) was infected with Trypanosoma chrysemydis following exposure to infected Placobdella ornata. Transmission of the trypanosomes by these leeches constitutes new vector records for the parasites. The biology of D. picta and P. ornata suggests that they are more important in transmitting these flagellates than the species of leech previously reported as vectors.     

The Life Cycle of Trypanoplasma bullocki (Zoomastigophorea: Kinetoplastida)1

The leech Calliobdella vivida (Verrill) is the vector of Trypanoplasma bullocki. At 10°C, infective-stage flagellates were first present in the leech's proboscis sheath five days after feeding. At 5°C, infective-stage flagellates were not present in the leech's proboscis sheath until 10 days after feeding, but at 20°C, flagellates were located there as early as 24 h after feeding. Infected leeches retained flagellates through three subsequent feeds on uninfected fish. When flagellates were first observed in hogchoker, Trinectes maculatus (Bloch & Schneider), they were much larger than infective stages from the leech. Average flagellate length then decreased during early acute phase, but gradually increased thereafter. Peak parasitemia was greater in a hogchoker inoculated by only one leech but held at colder temperature than in a hogchoker inoculated by 45 leeches, suggesting that temperature may be more important than inoculum in determining peak parasitemia. Cell division in the fish host is described. SEM studies of fish blood flagellates revealed a pre-oral ridge and a cytostome.     

A new lineage of trypanosomes from Australian vertebrates and terrestrial bloodsucking leeches (Haemadipsidae)

Little is known about the trypanosomes of indigenous Australian vertebrates and their vectors. We surveyed a range of vertebrates and blood-feeding invertebrates for trypanosomes by parasitological and PCR-based methods using primers specific to the small subunit ribosomal RNA (SSU rRNA) gene of genus Trypanosoma. Trypanosome isolates were obtained in culture from two common wombats, one swamp wallaby and an Australian bird (Strepera sp.). By PCR, blood samples from three wombats, one brush-tailed wallaby, three platypuses and a frog were positive for trypanosome DNA. All the blood-sucking invertebrates screened were negative for trypanosomes both by microscopy and PCR, except for specimens of terrestrial leeches (Haemadipsidae). Of the latter, two Micobdella sp. specimens from Victoria and 18 Philaemon sp. specimens from Queensland were positive by PCR. Four Haemadipsa zeylanica specimens from Sri Lanka and three Leiobdella jawarerensis specimens from Papua New Guinea were also PCR positive for trypanosome DNA. We sequenced the SSU rRNA and glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH) genes in order to determine the phylogenetic positions of the new vertebrate and terrestrial leech trypanosomes. In trees based on these genes, Australian vertebrate trypanosomes fell in several distinct clades, for the most part being more closely related to trypanosomes outside Australia than to each other. Two previously undescribed wallaby trypanosomes fell in a clade with Trypanosoma theileri, the cosmopolitan bovid trypanosome, and Trypanosoma cyclops from a Malaysian primate. The terrestrial leech trypanosomes were closely related to the wallaby trypanosomes, T. cyclops and a trypanosome from an Australian frog. We suggest that haemadipsid leeches may be significant and widespread vectors of trypanosomes in Australia and Asia.     

Molecular detection of Megatrypanum trypanosomes in tabanid flies

Trypanosomes of the subgenus Megatrypanum have been isolated from many mammalian hosts around the world. They are usually non-pathogenic, although they may confuse the parasitological diagnosis of trypanosomosis. Additionally, Trypanosoma theileri has been associated with disease in cattle. Megatrypanum trypanosomes are considered to be transmitted by different arthropods, including tabanids. However, little is known about the potential vectors of Megatrypanum trypanosomes in different parts of the world. The present study reports on the detection of Megatrypanum trypanosomes in Heamatopota pluvialis, Tabanus bromius, Tabanus maculicornis and Tabanus distinguendus in Poland. It also discusses the possible role of these tabanids in the transmission of Megatrypanum trypanosomes.     

The complete amino acid sequence of a hirudin variant from the leechHirudinaria manillensis

Unlike the European leechHirudo medicinalis, the Asian jawed leechHirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions ofHirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as withHirudo leech, various isoforms of hirudin also exist inHirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.     

Trypanosoma rangeli is phylogenetically closer to Old World trypanosomes than to Trypanosoma cruzi

Trypanosoma rangeli and Trypanosoma cruzi are generalist trypanosomes sharing a wide range of mammalian hosts; they are transmitted by triatomine bugs, and are the only trypanosomes infecting humans in the Neotropics. Their origins, phylogenetic relationships, and emergence as human parasites have long been subjects of interest. In the present study, taxon-rich analyses (20 trypanosome species from bats and terrestrial mammals) using ssrRNA, glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH), heat shock protein-70 (HSP70) and Spliced Leader RNA sequences, and multilocus phylogenetic analyses using 11 single copy genes from 15 selected trypanosomes, provide increased resolution of relationships between species and clades, strongly supporting two main sister lineages: lineage Schizotrypanum, comprising T. cruzi and bat-restricted trypanosomes, and Tra[Tve-Tco] formed by T. rangeli, Trypanosoma vespertilionis and Trypanosoma conorhini clades. Tve comprises European T. vespertilionis and African T. vespertilionis-like of bats and bat cimicids characterised in the present study and Trypanosoma sp. Hoch reported in monkeys and herein detected in bats. Tco included the triatomine-transmitted tropicopolitan T. conorhini from rats and the African NanDoum1 trypanosome of civet (carnivore). Consistent with their very close relationships, Tra[Tve-Tco] species shared highly similar Spliced Leader RNA structures that were highly divergent from those of Schizotrypanum. In a plausible evolutionary scenario, a bat trypanosome transmitted by cimicids gave origin to the deeply rooted Tra[Tve-Tco] and Schizotrypanum lineages, and bat trypanosomes of diverse genetic backgrounds jumped to new hosts. A long and independent evolutionary history of T. rangeli more related to Old World trypanosomes from bats, rats, monkeys and civets than to Schizotrypanum spp., and the adaptation of these distantly related trypanosomes to different niches of shared mammals and vectors, is consistent with the marked differences in transmission routes, life-cycles and host-parasite interactions, resulting in T. cruzi (but not T. rangeli) being pathogenic to humans.     

中国蛭类动物生态地理类群特征及其防护医用研究进展

综述中国蛭类研究如下内容:(1)生态学.山蛭生态学,包括山蛭的生态分布,海南山蛭(H. hainana)的种群动态,对温度、土壤湿度和pH值的适应,以及对温、光、湿的综合反应和人类经济活动对其种群数量的影响;山蛭行为生态学,山蛭的运动包括慢缩短、快缩短、身体摆动和转动、洗刷运动、亲吻运动、觅食行为6程序,影响山蛭行为的一些因素以及对环境因素刺激的生态学意义;淡水水蛭生态学,包括浙江水田蛭类生活习性、广州水生光润金线蛭种群数量动态与水体化学因子关系、广州水田吸血菲牛蛭生活水体化学环境;山蛭和水生吸血菲牛蛭的觅食、生长动态、生命周期和生殖生物学;(2)形态学、分类学和动物地理学.形态学包括山蛭机能组织学、山蛭器官系统解剖;分类学,中国蛭类动物有2亚纲(蛭蚓亚纲、真蛭亚纲)、3目(蛭蚓目、吻蛭目、无吻目)、9科、33属、111种,占世界蛭类物种数约1/6;动物地理学,包括世界山蛭科属动物地理,中国山蛭科动物地理、中国医蛭科动物地理.(3)蛭类的防治和驱避,淡水吸血蛭类防治所用农药种类,不同农药对海南山蛭的毒力( LD50、LD95)及使用,并比较了12种驱避剂对海南山蛭的驱避效果.(4)蛭类的医学利用,蛭素是蛭类唾液腺分泌的一种抗凝物质,蛭素有水蛭素(Hirudin)、山蛭素(Haemadin)和吻蛭素(Hementin).记述了蛭素的分离、纯化和功能以及有关分子生物学内容.     

The complete amino acid sequence of a hirudin variant from the leechHirudinaria manillensis

Unlike the European leechHirudo medicinalis, the Asian jawed leechHirudinaria manillensis is specialized for feeding on mammalian blood. In the salivary glands of both these leeches, there is a potent inhibitor of thrombin, called hirudin, which acts as an anticoagulant. We have reported previously the isolation and purification of a variant of hirudin, called bufrudin, from the head portions ofHirudinaria. In the present study, the complete amino acid sequence of bufrudin was determined by automated Edman degradation of peptide fragments generated after cleavage of protein with trypsin or thermolysin. Comparison of the primary structure of bufrudin, with hirudin HV1, show about 70% sequence identity with deletion of two amino acids, but the key amino acids at the C-terminus, involved in the inhibition of thrombin, are conserved. However, similar sequence comparison of bufrudin with hirullin P18, a hirudin variant isolated from the same leech species but from whole leech, instead of heads, reveals even less sequence identity of about 60%. From the amino acid sequence, it is suggested that the conformation of the C-terminal portion of bufrudin may be significantly different from hirullin P18, but similar to hirudin HV1, upon its interaction with thrombin. These results indicate that, as withHirudo leech, various isoforms of hirudin also exist inHirudinaria leech, with a significant change occurring in the structure of the molecule during the evolution of leeches.     

锥虫系统发育的研究进展

锥虫(trypanosome)是最早在鱼类血液中发现、随后在几乎所有的脊椎动物血液里都有发现的原生动物寄生虫。它主要通过水蛭和吸血昆虫传播,使宿主受到不同程度的危害。为了较全面地了解锥虫系统发育的研究进展,本文综述了国内外有关锥虫系统发育研究的四个方面的内容锥虫的起源(介绍了锥虫起源于脊椎动物和起源于无脊椎动物的两种不同观点);锥虫的系统发育方式(单系发育);锥虫的进化是否与寄主存在协同性及进化史上分歧时间的差异性;目前研究中存在的问题。     

Morphological and molecular characterization of a new species of leech (Glossiphoniidae,Hirudinida): Implications for the health of its imperiled amphibian host (Cryptobranchus alleganiensis)

The hellbender (Cryptobranchus alleganiensis) is among the most intriguing and imperiled amphibians in North America. Since the 1970s and 80s, western populations of the Ozark and eastern subspecies in Missouri have declined by nearly 80%. As a result of population declines, the Ozark hellbender was recently federally protected as an endangered species, and the eastern subspecies was granted protection under CITES. Although habitat degradation is probably the biggest threat to hellbender populations, recent evidence suggests that pathogens including chytrid fungus and “flesh-eating” bacteria may also contribute to declines in Ozark hellbenders. Leeches, which are very common on Ozark hellbenders, have recently been implicated as possible vectors of disease among Ozark hellbenders but have not been described in eastern hellbenders or outside of Missouri and Arkansas. We discovered a population of leeches on eastern hellbenders in southwest Virginia and confirmed that the species of leech is within the genus Placobdella, but is morphologically and genetically distinct from all previously described leech species. We named the new species Placobdella appalachiensis sp. n. Moser and Hopkins, based on the mountainous region in which it was discovered. Our surveys over a three consecutive year period suggested that this leech species may be patchily distributed and/or have a narrow geographic range. We consistently detected leeches at one site (mean prevalence in 80 hellbenders = 27.5%; median intensity = 3.0 leeches per parasitized hellbender [range 1 – >250 leeches]) during three years of surveys, but we never found leeches in any of our other seven study sites in two streams (mean prevalence in 139 hellbenders = 0%). We found a significant positive relationship between hellbender body size and the intensity of parasitism, and we suggest the possibility that the behavioral ecology of adults leading up to reproduction may increase their encounter rates with parasites. We discuss the potential conservation implications of discovery of leeches in this stream, and make recommendations for future mitigation and monitoring efforts.     

Population size, weight distribution and food in a persistent population of the rare medicinal leech, Hirudo medicinalis

1. It is important for species recovery and conservation management projects to know the minimum viable population size for rare and endangered species, such as the medicinal leech, Hirudo medicinalis. Therefore, using a catch‐removal method, this study estimated every two years (1986, 1988, 1990, 1992) the total number of medicinal leeches in a tarn in the English Lake District, and the number of mature adults in the population. 2. Four samples were taken each year in June and July, when water temperatures exceeded 20 °C. Population size was estimated both by maximum likelihood and regression methods. All leeches were weighed alive and size groups were separated by polymodal frequency analysis. A small sample of the blood meal in each leech gut was taken before the leeches were returned to the tarn, and was used to estimate the proportion of mammalian and non‐mammalian blood in the meals. 3. Both methods of estimation produced similar values, increasing confidence in the population estimates. Values for the total population in June and July varied among years from 248 to 288, the maximum value being only 16% higher than the minimum. Values for the number of mature leeches varied from 48 to 58 (19–20% of the total population), and this was an estimate of the effective population size. 4. There were four size groups. The largest mature leeches (live weight >5 g) in group IV formed only 1% of the population, and the smallest (0.02–0.5 g) in group I 14–17%. Most leeches were in two overlapping groups of immature (64–67% of population) and mature (18%) leeches with size ranges of 0.4–3.4 g and 2.5–5 g respectively. The percentage of leeches in each size group was very consistent among years. Blood meals were found in 38–44% of the leeches in group I, 45–50% in group II, 70–75% in group III, and 100% in group IV, but mammalian blood was present only in larger mature leeches (>3.5 g). 5. Medicinal leeches were first detected in the tarn in 1980 and are still present in 2007, so the population has persisted for at least 27 years. Compared with minimum viable population sizes for other species, including many endangered species, values for this medicinal leech population are extremely low, but may be typical of some rare freshwater invertebrates in isolated habitats.     

High Local Diversity of Trypanosoma in a Common Bat Species,and Implications for the Biogeography and Taxonomy of the T. cruzi Clade

The Trypanosoma cruzi clade is a group of parasites that comprises T. cruzi sensu lato and its closest relatives. Although several species have been confirmed phylogenetically to belong to this clade, it is uncertain how many more species can be expected to belong into this group. Here, we present the results of a survey of trypanosome parasites of the bat Artibeus jamaicensis from the Panamá Canal Zone, an important seed disperser. Using a genealogical species delimitation approach, the Poisson tree processes (PTP), we tentatively identified five species of trypanosomes – all belonging to the T. cruzi clade. A small monophyletic group of three putative Trypanosoma species places at the base of the clade phylogeny, providing evidence for at least five independent colonization events of these parasites into the New World. Artibeus jamaicensis presents a high diversity of these blood parasites and is the vertebrate with the highest number of putative trypanosome species reported from a single locality. Our results emphasize the need for continued efforts to survey mammalian trypanosomes.     

Experimental infections of farmed eels with different Trypanosoma granulosum life-cycle stages and investigation of pleomorphism

Trypanosoma granulosum, a flagellate protozoon commonly found in the blood of the European eel Anguilla anguilla, was injected experimentally into uninfected eels purchased from a local farm. In order to investigate the infectivity of different stages in the life cycle, trypanosomes from various sources were used for inoculation. Infectivity was greatly reduced in in vitro culture stages inoculated at 20 C. Isolated bloodstream stages injected into groups of animals held at 12 and 20 C could be detected for over 70 days but did not appear to multiply. Naturally infected Hemiclepsis marginata, a piscivorous leech known to serve as vector, produced detectable, single-peak infections in eels held at 20 C. Infections were characterized by a prepatent stage and a phase of rising parasitemia. Peak infection intensities ranged between 1 and 7 x 10(4) trypanosomes/ml. Trypanosomes in the bloodstream of eels experimentally infected with leeches, divided at a very low rate during the early stages of infection. Small morphs present during the early phase of rising parasitemia were gradually replaced by larger trypanosomes. The overall length frequency distribution of trypanosomes was unimodal.     

Patterns of co-evolution between trypanosomes and their hosts deduced from ribosomal RNA and protein-coding gene phylogenies

Trypanosomes (genus Trypanosoma) are widespread blood parasites of vertebrates, usually transmitted by arthropod or leech vectors. Most trypanosomes have lifecycles that alternate between a vertebrate host, where they exist in the bloodstream, and an invertebrate host, where they develop in the alimentary tract. This raises the question of whether one type of host has had greater influence on the evolution of the genus. Working from the generally accepted view that trypanosomes are monophyletic, here we examine relationships between trypanosomes using phylogenies based on the genes for the small subunit ribosomal RNA (SSU rRNA) and the glycosomal glyceraldehyde phosphate dehydrogenase (gGAPDH). New analysis of a combined dataset of both these genes provides strong support for many known clades of trypanosomes. It also resolves the deepest split within the genus between the Aquatic clade, which mainly contains trypanosomes of aquatic and amphibious vertebrates, and a clade of trypanosomes from terrestrial vertebrates. There is also strengthened support for two deep clades, one comprising a wide selection of mammalian trypanosomes and a tsetse fly-transmitted reptilian trypanosome, and the other combining two bird trypanosome subclades. Considering the vertebrate and invertebrate hosts of each clade, it is apparent that co-speciation played little role in trypanosome evolution. However most clades are associated with a type of vertebrate or invertebrate host, or both, indicating that 'host fitting' has been the principal mechanism for evolution of trypanosomes.     

Molecular Identification of Haemadipsa rjukjuana (Hirudiniformes: Haemadipsidae) in Gageo Island,Korea

There are 60 species of blood-feeding land leeches, 50 species belonging to the family Haemadipsidae and 10 species belonging to the family Xerobdellidae. Despite recent papers on the land leeches, their taxonomic identification is not fully understood, especially at a species level. In Korea, there have been no historical records of the terrestrial leeches, but recently an unrecorded blood-feeding land leech was discovered at Gageo-do (Island), Korea. Molecular analysis was used to identify the species of 29 leeches collected from Mt. Dock-Sil in Gageo-do. Conventional PCR was conducted using nuclear 18S rRNA and mitochondrial cytochrome c oxidase subunit 1 (CO1) genetic marker. The 18S rRNA sequences revealed that the leeches share 99.9% identity with Haemadipsa rjukjuana (inhabiting Taiwan), and the CO1 sequences revealed that the leeches are very close to H. rjukjuana (inhabiting Taiwan). The CO1 sequences were separated into 2 categories, 1 with 94.6% and the other with 94.3% similarity to the H. rjukjuana L00115A (inhabiting Taiwan). This new finding of the land leech is the first record in Korea. In addition, the north range of the distribution of the blood-feeding leech (Hirudiniformes: Haemadipisidae) should be reconsidered including Korea.     

Novel Role for Aeromonas jandaei as a Digestive Tract Symbiont of the North American Medicinal Leech

The gut bacteria of the North American medicinal leech, Macrobdella decora, were characterized. Biochemical tests and DNA sequences indicated that Aeromonas jandaei is the dominant culturable symbiont in leeches from a broad geographic area. In this work we identified a new habitat for A. jandaei, and here we suggest that there is unexpected specificity between leeches and Aeromonas species.     

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.     

Annotated checklist of the leech species diversity in the Maloe More Strait of Lake Baikal,Russia

In this paper, the very first checklist of the freshwater leeches of Maloe More Strait, a special part of Lake Baikal, is presented. It includes 14 free-living and parasitic species, of which four species belong to endemic Baikal genera – two species from Baicalobdella and one species each from Baicaloclepsis and Codonobdella. The checklist highlights six potentially new morphological species recorded for the first time in the area. The exact systematic position is stated for all leech species. Each species from the list is provided with information on taxonomic synonymy, data on its geographic distribution, and ecological characteristics. New species records are additionally provided with brief morphological characteristics and photos of their external morphology.     

The complete amino acid sequences of four globins from the land leech Haemadipsa zeylanica var. japonica

The amino acid sequences of four globins from the land leech, Haemadipsa zeylanica var. japonica, were determined using nucleotide sequencing and protein sequencing. The mature globin-molecules were composed of 146 amino acid residues for M-1 globin, 156 for M-2 globin, 143 for D-1 globin, and 149 for D-2 globin. Alignment of the four kinds of globins by Clustal X revealed 22 invariant amino acids. The four globins were 26–33% identical. A striking feature of amino acid alteration was: the replacement of the E7 distal-His of D-1 globin by phenylalanine because histidine is conserved among the rest of the globins of H. zeylanica, those of other representative species (Lumbricus and Tylorrhynchus) of Annelida and most other hemoglobins. A phylogenetic tree constructed of 18 globin structures including two species of leeches, H. zeylanica (a land leech) and Macrobdella decora (a freshwater leech), T. heterochaetus (a representative species of polychaetes), L. terrestris (a representative species of oligochaetes), and human α and β globins strongly indicated that the leech globins first separated from globin lineage of annelids.     

Morphological and Genetic Variations of the Freshwater Leech,Hirudinaria spp., in Peninsular Malaysia

In this study the genetic diversity of local freshwater leeches (Hirudinaria spp.) was inferred using mtDNA COI gene analysis and compared with the gross external variations of 26 freshwater leech specimens obtained from the wild and leech farms. Based on a neighbor-joining tree generated from 516 COI base sequences, four distinct clades of Hirudinaria were seen with interspecific genetic divergence in the range of 7.6–14.5%. The external morphological variations based on the presence of stripes, location of gonopores, and anus separated the samples into four morphologically distinct groups matching the four clades obtained from the molecular data. Two black stripes at the ventral region were observed only in specimens found clustered with clades that contained the GenBank-reported H. manillensis, whereas the brown or dark green coloration without stripes on the ventral region was seen in samples that clustered with H. javanica and H. bpling clades.