Leech mycetome endosymbionts are a new lineage of alphaproteobacteria related to the Rhizobiaceae

Mycetomal organs attached to the esophagus of hematophagous leeches which are known to harbor endosymbiotic bacteria were removed from three species in the leech family Glossiphoniidae. Anatomical observations indicated that placobdellid mycetomes are paired and caecate, inserting into the esophagus posterior to the proboscis. Light and electron microscopy demonstrated that there is a single layer of mycetome epithelial cells harboring gram-negative rods and that these epithelial cells are ultrastructurally distinct from neighboring esophageal epithelial cells. Fluorescent in situ hybridization with eubacterial and alphaproteobacterial probes localized the bacteria solely to the mycetomes both in adult and in unfed juvenile leeches whereas a gammaproteobacterial probe did not yield a bound fluorescencent signal. DNA was isolated from these tissues and subjected to PCR amplification using bacteria-specific primers for 16S and 23S rDNA. Results from sequencing the amplification products and phylogenetic analysis with other Alphaproteobacteria revealed that the bacteria resident in these organs comprise a new genus of Alphaproteobacteria, Reichenowia n. gen., closely related to the nitrogen-fixing, nodule-forming Rhizobiaceae. The three bacterial strains, though different from each other were each other's closest relatives, suggesting a history of close coevolution with their leech hosts.     

New gammaproteobacteria associated with blood-feeding leeches and a broad phylogenetic analysis of leech endosymbionts

Many monophagous animals have coevolutionary relationships with bacteria that provide unavailable nutrients to the host. Frequently, these microbial partners are vertically inherited and reside in specialized structures or tissues. Here we report three new lineages of bacterial symbionts of blood-feeding leeches, one from the giant Amazonian leech, Haementeria ghilianii, and two others from Placobdelloides species. These hosts each possess a different mycetome or esophageal organ morphology where the bacterial cells are located. DNA sequencing of the bacterial 16S rRNA genes and fluorescent in situ hybridization placed these symbionts in two separate clades in the class Gammaproteobacteria. We also conducted a broad phylogenetic analysis of the herein-reported DNA sequences as well as others from bacterial symbionts reported elsewhere in the literature, including alphaproteobacterial symbionts from the leech genus Placobdella as well as Aeromonas veronii from the medicinal leech, Hirudo medicinalis, and a Rickettsia sp. detected in Hemiclepsis marginata. Combined, these results indicate that blood-feeding leeches have forged bacterial partnerships at least five times during their evolutionary history.     

Morphology,egg cocoons,and transmission paths of the Antarctic leech Glyptonotobdella antarctica Sawyer and White, 1969 (Hirudinea: Rhynchobdelliformes: Piscicolidae)

The biology of piscicolids which move between different host species is poorly understood. Very little is known about leeches of Antarctic waters, though they are not uncommon in that region. This paper adds to the fundamental knowledge of the Antarctic piscicolid leech Glyptonotobdella antarctica Sawyer and White, 1969. According to the new findings the distribution of the species has to be extended to the geographical latitude of 76°08.1′S and to a depth of 665 m. Besides Glyptonotus antarcticus Eights 1853, sea urchins of the genus Sterechinus (Meissner 1900), and the benthic octopus Pareledone (charcoti?) (Joubin 1905) serve as hosts. Hitherto there were only two records on piscicolids from Octopus dofleini and none from sea urchins. Egg cocoons on the ventral side of the isopod Glyptonotus antarcticus were attributed to the leech. Most likely Glyptonotobdella antarctica moves between different hosts, i.e. Antarctic octopuses and their potential prey. Surface structures of the leech are studied by light and scanning electron microscope. The observations complement the basic knowledge on Antarctic leeches and provide arguments for phylogenetic discussions.     

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.     

Bacterial symbiont and salivary peptide evolution in the context of leech phylogeny

The evolutionary history of leeches is employed as a general framework for understanding more than merely the systematics of this charismatic group of annelid worms, and serves as a basis for understanding blood-feeding related correlates ranging from the specifics of gut-associated bacterial symbionts to salivary anticoagulant peptides. A variety of medicinal leech families were examined for intraluminal crop bacterial symbionts. Species of Aeromonas and Bacteroidetes were characterized with DNA gyrase B and 16S rDNA. Bacteroidetes isolates were found to be much more phylogenetically diverse and suggested stronger evidence of phylogenetic correlation than the gammaproteobacteria. Patterns that look like co-speciation with limited taxon sampling do not in the full context of phylogeny. Bioactive compounds that are expressed as gene products, like those in leech salivary glands, have 'passed the test' of evolutionary selection. We produced and bioinformatically mined salivary gland EST libraries across medicinal leech lineages to experimentally and statistically evaluate whether evolutionary selection on peptides can identify structure-function activities of known therapeutically relevant bioactive compounds like antithrombin, hirudin and antistasin. The combined information content of a well corroborated leech phylogeny and broad taxonomic coverage of expressed proteins leads to a rich understanding of evolution and function in leech history.     

Stratified bacterial community in the bladder of the medicinal leech, Hirudo verbana

Most animals harbour symbiotic microorganisms inside their body, where intimate interactions occur between the partners. The medicinal leech, Hirudo verbana, possesses 17 pairs of excretory bladders that harbour a large number of intracellular and extracellular symbiotic bacteria. In this study, we characterized the bladder symbionts using molecular phylogenetic analyses, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH). Restriction fragment length polymorphism (RFLP) and sequence analyses of 16S rRNA gene clone libraries suggested that six bacterial species co‐colonize the leech bladders. Phylogenetic analyses revealed that these species belong to the α‐Proteobacteria (Ochrobactrum symbiont), β‐Proteobacteria (Beta‐1 and Beta‐2 symbionts), δ‐Proteobacteria (Bdellovibrio symbiont) and Bacteroidetes (Niabella and Sphingobacterium symbionts). Species‐specific PCR detection and FISH confirmed the localization of the symbiotic bacteria in the bladders. The Ochrobactrum, Beta‐1, Bdellovibrio and Sphingobacterium symbionts were consistently detected in 13 leeches from two populations, while infection rate of the other symbionts ranged between 20% and 100% in the two leech populations. Transmission electron microscopy observations of the bladders revealed epithelial cells harbouring a number of intracellular bacilli and an additional type of extracellular, rod‐shaped bacteria in the luminal region. Fluorescence in situ hybridization with group‐specific oligonucleotide probes revealed the spatial organization of the bacterial species in the bladder: the Ochrobactrum symbiont was located intracellularly inside epithelial cells; the Bacteroidetes were localized close to the epithelium in the lumen of the bladder; and the Bacteroidetes layer was covered with dense β‐proteobacterial cells. These results clearly demonstrate that a simple but organized microbial community exists in the bladder of the medicinal leech.     

Molecular phylogeny supports a Northern Hemisphere origin of Golovinomyces (Ascomycota: Erysiphales)

Golovinomyces is a strictly herb-parasitic genus in the Erysiphaceae. Host–parasite co-speciation was reported recently between the genus Golovinomyces and Asteraceae from molecular phylogenetic analyses. The Asteraceae originated in South America and latterly expanded their geographic distribution into the Northern Hemisphere. If the co-speciation between Golovinomyces and Asteraceae originated in South America, the geographic origin of Golovinomyces could be assumed to be South America. To address this question, Golovinomyces species from hosts of the tribe Mutisieae, an asteraceous tribe endemic to South America, were collected and the ITS and 28S rDNA regions sequenced. Results indicate that Oidium mutisiae and Golovinomyces leuceriae isolated from the Mutisieae do not belong at the base of the Golovinomyces tree. Instead, they are situated separately within two different clades of Golovinomyces isolates from the Northern Hemisphere. Therefore, the tribe Mutisieae is not the most early host of Golovinomyces. Present results suggest that Golovinomyces originated in the Northern Hemisphere, and not in South America. The new species Oidium reginae for the previous O. mutisiae on Mutisia decurrens is proposed.     

Phylogenomics of Reichenowia parasitica, an alphaproteobacterial endosymbiont of the freshwater leech Placobdella parasitica

Although several commensal alphaproteobacteria form close relationships with plant hosts where they aid in (e.g.,) nitrogen fixation and nodulation, only a few inhabit animal hosts. Among these, Reichenowia picta, R. ornata and R. parasitica, are currently the only known mutualistic, alphaproteobacterial endosymbionts to inhabit leeches. These bacteria are harbored in the epithelial cells of the mycetomal structures of their freshwater leech hosts, Placobdella spp., and these structures have no other obvious function than housing bacterial symbionts. However, the function of the bacterial symbionts has remained unclear. Here, we focused both on exploring the genomic makeup of R. parasitica and on performing a robust phylogenetic analysis, based on more data than previous hypotheses, to test its position among related bacteria. We sequenced a combined pool of host and symbiont DNA from 36 pairs of mycetomes and performed an in silico separation of the different DNA pools through subtractive scaffolding. The bacterial contigs were compared to 50 annotated bacterial genomes and the genome of the freshwater leech Helobdella robusta using a BLASTn protocol. Further, amino acid sequences inferred from the contigs were used as queries against the 50 bacterial genomes to establish orthology. A total of 358 orthologous genes were used for the phylogenetic analyses. In part, results suggest that R. parasitica possesses genes coding for proteins related to nitrogen fixation, iron/vitamin B translocation and plasmid survival. Our results also indicate that R. parasitica interacts with its host in part by transmembrane signaling and that several of its genes show orthology across Rhizobiaceae. The phylogenetic analyses support the nesting of R. parasitica within the Rhizobiaceae, as sister to a group containing Agrobacterium and Rhizobium species.     

Bloodlines: mammals,leeches, and conservation in southern Asia

Southern Asia is a biodiversity hotspot both for terrestrial mammals and for leeches. Many small-mammal groups are under-studied in this region, while other mammals are of known conservation concern. In addition to standard methods for surveying mammals, it has recently been demonstrated that residual bloodmeals within leeches can be sequenced to find mammals in a given area. While these invertebrate-parasite-derived DNA (iDNA) methods are promising, most of the leech species utilized for this type of survey remain unevaluated, notwithstanding that their diversity varies substantially. Here we examine approximately 750 individual leech specimens in the genus Haemadipsa across a large range in southern Asia (Bangladesh, Cambodia, and China), specifically reviewing the diversity of mammals they feed on and their own genetic structuring. Leeches were found to feed on a considerable variety of mammals, corroborating prior studies. Additionally, leeches were found to have fed both on bats and on birds, neither of which has previously been recorded with this method. The genetic structuring of the leeches themselves revealed 15 distinct clades of which only two precisely corresponded to previously characterized species, indicating that much work is needed to finalize classifications in this genus. Most importantly, with regards to mammal conservation, leeches in these clades appear to feed on a broad range of mammals.     

Endosymbiotic Bacteria in the Esophageal Organ of Glossiphoniid Leeches

We characterized the intracellular symbiotic bacteria of the hematophagous glossiphoniid leeches Placobdelloides siamensis and a Parabdella sp. These leeches have a specialized structure called an “esophageal organ,” the cells of which harbor bacterial symbionts. From the esophageal organ of each species, a 1.5-kb eubacterial 16S rRNA gene segment was amplified by PCR, cloned, and sequenced. Diagnostic PCR detected the symbiont in the esophageal organ and intestine. Phylogenetic analysis of the 16S rRNA gene(s) demonstrated that the symbionts from the leeches formed a monophyletic group in a well-defined clade containing endosymbiotic bacteria of plant sap-feeding insects in the γ-subdivision of the Proteobacteria. The nucleotide compositions of the 16S rRNA gene from the leech symbionts were highly AT biased (53.7%).     

Phylogenetic position of gastrostomobdellid leeches (Hirudinida,Arhynchobdellida, Erpobdelliformes) and a new family for the genus Orobdella

Nakano, T., Ramlah, Z. & Hikida, T. (2012). Phylogenetic position of gastrostomobdellid leeches (Hirudinida, Arhynchobdellida, Erpobdelliformes) and a new family for the genus Orobdella. —Zoologica Scripta, 41, 177–185. The leech family Gastrostomobdellidae is characterized by its possession of an agnathous and euthylaematous pharynx, a ventral gastropore and a gastroporal duct. In this study, the phylogenetic position of two gastrostomobdellid genera, Gastrostomobdella and Orobdella, was investigated using partial nucleic 18S, 28S rDNA and mitochondrial 12S rDNA sequences. Our phylogenetic analyses showed that both Gastrostomobdella and Orobdella are nested within Erpobdelliformes, but Gastrostomobdellidae is not a monophyletic taxon. Orobdella is a sister taxon of the other Erpobdelliformes taxa. The phylogenetic position of Gastrostomobdella within the clade of Gastrostomobdella, Erpobdellidae and Salifidae still remains uncertain. According to the reconstruction of the ancestral state of the pharynx in Erpobdelliformes, a euthylaematous pharynx is considered to be plesiomorphic in this taxon. Examination of Gastrostomobdella and Orobdella specimens indicates that the morphology of the gastroporal duct of Orobdella is quite different from that of Gastrostomobdella. A new family, Orobdellidae fam. nov., was therefore erected for the genus Orobdella. Orobdellidae is characterized by its possession of a generally tubular gastroporal duct, lying on the female organ.     

Phylogeny of the New World medicinal leech family Macrobdellidae (Oligochaeta: Hirudinida: Arhynchobdellida)

Phylogenetic analyses of the leech family Macrobdellidae were accomplished with all nominal species in the family save one. A total of 17 specimens in nine ingroup species were analysed, along with four outgroup taxa. Twenty-two morphological characters based on jaw dentition, sexual anatomy, and external morphology failed to provide a resolution for many of the relationships in the family. DNA sequence data from nuclear 18S rDNA, nuclear 28S rDNA, mitochondrial 12S rDNA, and mitochondrial cytochrome c oxidase subunit I were examined separately and in combination with morphological characters. The resulting combined analysis strongly corroborated the placement of the genus Philobdella within the family Macrobdellidae and as sister to a monophyletic genus Macrobdella , the typical North American medicinal leeches. Furthermore, sequence divergences among these taxa confirmed the existence of two species, Philobdella gracilis and P. floridana , readily distinguishable on the basis of jaw dentition .     

Egg-feeding in the freshwater piscicolid leech Cystobranchus virginicus (Annelida, Hirudinea)

Abstract. Leeches are common terrestrial and aquatic annelids, parasitizing or feeding on a wide range of host taxa. Here we report evidence for an unusual feeding behavior of egg‐feeding in the piscicolid leech Cystobranchus virginicus. We identified distended specimens of C. virginicus in the nests of at least 4 different fish species: Campostoma anomalum, Moxostoma carinatum, Moxostoma sp. (either M. anisurum and/or M. breviceps), and Nocomis leptocephalus. We collected a total of 41 leeches from the nests of these host species and documented at least 1 leech in 19 of 55 nests (35%), with many sites containing multiple leeches. Individuals of C. virginicus were not identified feeding on any of the 41 adult specimens of Moxostoma spp. or the 635 adult specimens of Nocomis leptocephalus examined, and were never found in the absence of active host spawning (26 sites). These results are consistent with individuals of C. virginicus being an opportunistic or possibly even an obligate egg‐feeder, potentially timing their own reproductive activities with the spawning of their fish hosts. The current distribution of C. virginicus has been expanded to include North Carolina, South Carolina, and Tennessee. The potential for leech species to induce mortality in developing fish eggs could be a concern for fish conservation and merits further investigation.     

16S rDNA克隆文库方法分析太岁样品中细菌的多样性

通过构建16S rDNA克隆文库的方法,分析太岁样品中细菌的群落结构及多样性。太岁样品中的细菌归属于4个门9个目,优势类群依次是芽胞杆菌目(Bacillales,33.01%)、柄杆菌目(Caulobacterales,32.04%)和伯克霍尔德氏菌目(Burkholderiales,12.62%);优势属为短波单胞菌属(Brevundimonas,30.10%)、葡萄球菌属(Staphylococcus,29.13%)和食酸菌属(Acidovorax,7.77%)。并且其中的5个目中含有未培养的细菌,红杆菌目(Rhodobacterales)、伯克霍尔德氏菌目和红环菌目(Rhodocyclales)的11个克隆子的细菌16S rDNA序列同源性低于97%。研究表明太岁样品中细菌多样性较丰富,且蕴藏着许多未知的微生物资源。     

Phylogeny of 16S rRNA, Ribulose 1,5-Bisphosphate Carboxylase/Oxygenase, and Adenosine 5′-Phosphosulfate Reductase Genes from Gamma- and Alphaproteobacterial Symbionts in Gutless Marine Worms (Oligochaeta) from Bermuda and the Bahamas

Gutless oligochaetes are small marine worms that live in obligate associations with bacterial endosymbionts. While symbionts from several host species belonging to the genus Olavius have been described, little is known of the symbionts from the host genus Inanidrilus. In this study, the diversity of bacterial endosymbionts in Inanidrilus leukodermatus from Bermuda and Inanidrilus makropetalos from the Bahamas was investigated using comparative sequence analysis of the 16S rRNA gene and fluorescence in situ hybridization. As in all other gutless oligochaetes examined to date, I. leukodermatus and I. makropetalos harbor large, oval bacteria identified as Gamma 1 symbionts. The presence of genes coding for ribulose-1,5-bisphosphate carboxylase/oxygenase form I (cbbL) and adenosine 5′-phosphosulfate reductase (aprA) supports earlier studies indicating that these symbionts are chemoautotrophic sulfur oxidizers. Alphaproteobacteria, previously identified only in the gutless oligochaete Olavius loisae from the southwest Pacific Ocean, coexist with the Gamma 1 symbionts in both I. leukodermatus and I. makropetalos, with the former harboring four and the latter two alphaproteobacterial phylotypes. The presence of these symbionts in hosts from such geographically distant oceans as the Atlantic and Pacific suggests that symbioses with alphaproteobacterial symbionts may be widespread in gutless oligochaetes. The high phylogenetic diversity of bacterial endosymbionts in two species of the genus Inanidrilus, previously known only from members of the genus Olavius, shows that the stable coexistence of multiple symbionts is a common feature in gutless oligochaetes.     

Sexual behavior,insemination and development of the freshwater leech Helobdella stagnalis (Annelida,Hirudinea, Glossiphoniidae)

The reproductive biology of the Glossiphoniidae leech Helobdella stagnalis, under laboratory conditions, the structure of its eggs and its developmental stages were studied. Sperm transfer and insemination are made by hypodermic injection: one or numerous spermatophores were attached to the skin of the partner during copulation and sperm is injected through it. The leeches can copulate repeatedly with several partners. Each leech produces 4–8 cocoons containing 20–60 eggs whose are attached to the ventral side of the parent and carried around. At 22 °C, the developmental duration is 24 days from the oviposition until the juveniles leave the parent leech. Three (3) major stages have been distinguished: Eggs cleavage, germinal band generation, and juveniles hatching. The sexual behavior of Hellobdella stagnalis was described with a special attention given to parental care.     

Phylogenetic analysis of European species of Proteocephalus (Cestoda: Proteocephalidea): compatibility of molecular and morphological data, and parasite-host coevolution.

The phylogeny of European species of the tapeworm genus Proteocephalus was studied, based on partial 18S rDNA and morphological data. The group was found to be monophyletic. The analysis showed a low informative value of available morphological characters in comparison with molecular data. The morphological matrix resulted in a poorly resolved tree which is, however, compatible with the topology (Proteocephalus osculatus (Proteocephalus torulosus (Proteocephalus macrocephalus, Proteocephalus filicollis) (Proteocephalus tetrastomus, Proteocephalus percae, Proteocephalus longicollis))) based on the 18S rDNA data. A comparison performed by the program TreeMap showed a lack of significant congruency between parasite and host phylogenies. Therefore, the distribution of species in their hosts appears to be independent of the phylogeny and it is likely to be a result of host-switching, rather than co-speciation events.     

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.     

Phylogeny of macrophagous leeches (Hirudinea,Clitellata) based on molecular data and evaluation of the barcoding locus

Oceguera‐Figueroa, A., Phillips, A. J., Pacheco‐Chaves, B., Reeves, W. K. & Siddall, M. E. (2010). Phylogeny of macrophagous leeches (Hirudinea, Clitellata) based on molecular data and evaluation of the barcoding locus. —Zoologica Scripta, 40, 194–203. The phylogenetic relationships of macrophagous leech species are studied using two mitochondrial [cytochrome c oxidase subunit I (COI) and 12S rDNA] and two nuclear (28S rDNA and 18S rDNA) markers. The complete dataset analysed in this study included 49 terminals and 5540 aligned characters. Phylogenetic analyses were performed under two optimality criteria: Maximum Parsimony and Maximum Likelihood. The monophyly of the two currently recognized families (i.e. Erpobdellidae and Salifidae) is confirmed and well supported. The phylogenetic position of Gastrostomobdellidea is studied for the first time and found to be sister to family Salifidae nested well within Erpobdelliformes. Previously recognized taxonomic arrangements were evaluated and discarded through successive constraint analyses. Correlation between morphology and phylogeny was notable in Salifidae but not in Erpobdellidae. Variability of COI, the barcoding locus, was examined across species leading to the recognition of the invasive Barbronia weberi in Mexico, Costa Rica, Germany, South Africa and Taiwan.     

Novel Clade of Rickettsia spp. from Leeches

Intracellular rickettsia-like structures were found in the tissues of a glossiphoniid leech, Torix tagoi, by transmission electron microscopy. Diagnostic PCR analysis using specific primers suggested that of the nine glossiphoniid species examined, two species, T. tagoi and Hemicrepsis marginata, harbored bacteria of the genus Rickettsia. A 1.5-kb eubacterial 16S rRNA gene segment obtained from each of these species was amplified by PCR, cloned, and sequenced. Phylogenetic analysis of the 16S rRNA gene demonstrated that the Rickettsia species found in the leeches constituted a novel clade that is distinct from the clade of arthropod-associated Rickettsia species. In natural populations, 97.7% (43 of 44) of T. tagoi leeches and 100% (9 of 9) of H. marginata leeches carried Rickettsia, suggesting that infection with Rickettsia is prevalent in these leeches. This is the first report of Rickettsia found in annelids.