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.     

A new species of mammalian trypanosome,Trypanosoma (Megatrypanum) bubalisi sp. nov., found in the freshwater leech Hirudinaria manillensis

Leeches have long been considered potential vectors for the aquatic lineage of trypanosomes, while bloodsucking insects are generally considered as the vectors for the terrestrial lineage of trypanosomes. The freshwater leech, Hirudinaria manillensis, is a widely distributed species in southern China and could potentially act as the vector for trypanosomes. Prior to this study, no trypanosomes had been reported from this leech. However, in this study, leeches were collected from three different places in Guangdong province, China, and a large number of flagellates were isolated and successfully cultured in vitro. Based on morphology, these flagellates looked like a typical trypanosome species. Analysis was carried out on the molecular sequences of the 18S rRNA gene and the glycosomal glyceraldehyde-3-phosphate dehydrogenase (gGAPDH) gene. To our surprise, these flagellates were identified as likely to be a mammalian trypanosome belonging to the clade containing Trypanosoma (Megatrypanum) theileri but they are significantly different from the typical TthI and TthII stocks. Analyses of blood composition indicated that the source of the blood meal in these leeches was from the water buffalo (Bubalus bubalis). To further test if this flagellate from the freshwater leech was indeed a mammalian trypanosome, we transferred the trypanosomes cultured at 27–37 °C and they were able to successfully adapt to this mammalian body temperature, providing further supporting evidence. Due to the significant genetic differences from other related trypanosomes in the subgenus Megatrypanum, we propose that this flagellate, isolated from H. manillensis, is a new species and have named it Trypanosoma bubalisi. Our results indicate that freshwater leeches may be a potential vector of this new mammalian trypanosome.     

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.

     

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%).     

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.     

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.     

High prevalence of buccal ulcerations in largemouth bass, Micropterus salmoides (Centrarchidae) from Michigan inland lakes associated with Myzobdella lugubris Leidy 1851 (Annelida: Hirudinea)

Widespread mouth ulcerations were observed in largemouth bass collected from eight inland lakes in the Lower Peninsula of Michigan during the summer months of 2002 and 2003. These ulcerations were associated with, and most likely caused by, leech parasitism. Through the use of morphological dichotomous keys, it was determined that all leeches collected are of one species: Myzobdella lugubris. Among the eight lakes examined, Lake Orion and Devils Lake had the highest prevalence of leech parasitism (34% and 29%, respectively) and mouth ulcerations (53% and 68%, respectively). Statistical analyses demonstrated that leech and ulcer prevalence varied significantly from one lake to the other. Additionally, it was determined that the relationship between the prevalence of ulcers and the prevalence of leech attachment is significant, indicating that leech parasitism is most likely the cause of ulceration. The ulcers exhibited deep hemorrhagic centers and raised irregular edges. Affected areas lost their epithelial lining and submucosa, with masses of bacteria colonizing the damaged tissues. Since largemouth bass is a popular global sportfish and critical to the food web of inland lakes, there are concerns that the presence of leeches, damaged buccal mucosa, and general unsightliness may negatively affect this important sportfishery.     

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.     

Using metabarcoding to compare the suitability of two blood‐feeding leech species for sampling mammalian diversity in North Borneo

The application of high‐throughput sequencing (HTS) for metabarcoding of mixed samples offers new opportunities in conservation biology. Recently, the successful detection of prey DNA from the guts of leeches has raised the possibility that these, and other blood‐feeding invertebrates, might serve as useful samplers of mammals. Yet little is known about whether sympatric leech species differ in their feeding preferences, and whether this has a bearing on their relative suitability for monitoring local mammalian diversity. To address these questions, we collected spatially matched samples of two congeneric leech species Haemadipsa picta and Haemadipsa sumatrana from lowland rainforest in Borneo. For each species, we pooled ~500 leeches into batches of 10 individuals, performed PCR to target a section of the mammalian 16S rRNA locus and undertook sequencing of amplicon libraries using an Illumina MiSeq. In total, we identified sequences from 14 mammalian genera, spanning nine families and five orders. We found greater numbers of detections, and higher diversity of OTUs, in H. picta compared with H. sumatrana, with rodents only present in the former leech species. However, comparison of samples from across the landscape revealed no significant difference in mammal community composition between the leech species. We therefore suggest that H. picta is the more suitable iDNA sampler in this degraded Bornean forest. We conclude that the choice of invertebrate sampler can influence the detectability of different mammal groups and that this should be accounted for when designing iDNA studies.     

Two new species in the Echinoderes coulli group (Echinoderidae,Cyclorhagida, Kinorhyncha) from the Ryukyu Islands,Japan

Two new species belonging to the Echinoderes coulli group are described with their external morphologies and sequences of nuclear 18S rRNA and 28S rRNA genes, and mitochondrial COI gene. The first species, Echinoderes komatsui sp. n., is characterized by absence of acicular spines, and presence of lateroventral tubules on segments 5 and 8, laterodorsal tubules on segment 10, inverted triangle or wide oval shaped large sieve plates, lateral terminal accessory spines in female, and short tips of ventral pectinate fringe on segment 10. The second species, Echinoderes hwiizaa sp. n., is characterized by absence of acicular spines, and presence of lateroventral tubules on segments 5 and 7–9, midlateral tubules on segment 8, laterodorsal tubules on segment 10, large narrow oval shaped sieve plates on segment 9, and thick, short and blunt lateral terminal spines about 10–15% of trunk length. The diagnostic characters and key to species of E. coulli group are provided as well.     

Mitochondrial DNA data unveil highly divergent populations within the genus <Emphasis Type="Italic">Hynobius</Emphasis> (Caudata: Hynobiidae) in South Korea

Korean salamanders of the genus Hynobius are currently classified into 3 species, H. leechii, H. quelpaertensis, and H. yangi. To investigate the phylogenetic relationship of these species, we analyzed the partial sequence of mitochondrial cytochrome b gene (907 bp) of 197 specimens from 43 regions in South Korea. Of these specimens, 93 were additionally examined with 12S rRNA (799 bp). Based on the partial sequence of the mitochondrial cytochrome b gene and 12S rRNA, 89 and 36 haplotypes were defined, respectively, consisting of six subclades (H. leechii, H. quelpaertensis, H. yangi, HC1, HC2, and HC3). Among these subclades, the three subclades (HC1, HC2, and HC3) were clearly separated from the 3 previously reported species in the genus Hynobius. Pairwise sequence divergence between the six subclades ranged from 6.3 to 11.2% in cytochrome b gene and 2.0 to 4.3% in 12S rRNA. These results indicate there may be more divergent populations than the three currently described. Moreover, the estimation of divergence time revealed that the Hynobius species in South Korea diverged during the Miocene epoch, approximately 9 — 5 MYA. In addition, we confirmed the distribution of the three known species (H. leechii, H. quelpaertensis, and H. yangi) and determined the distributions of new, distinct groups (or subclades; HC1, HC1, and HC3). To more accurately establish the taxonomic status and population structure, further genetic, morphological, and ecological studies will be needed.     

Anisakis pegreffii Larvae in Sea Eels (Astroconger myriaster) from the South Sea,Republic of Korea

Anisakis simplex sensu stricto (s.s.), Anisakis pegreffii, Anisakis berlandi (=A. simplex sp. C), and Anisakis typica are the 4 major species of Anisakis type I larvae. In the Republic of Korea (Korea), A. pegreffii, A. berlandi, and A. typica larvae in fish hosts has seldom been documented. In this study, molecular analysis was performed on Anisakis larvae from the sea eels (Astroconger myriaster), the major source of human anisakiasis in Korea, collected from Tongyeong City, a southern coastal area of Korea. All 20 sea eels examined were infected with Anisakis type I larvae (160 larvae; 8 per fish). Their species were analyzed using PCR-RFLP patterns and nucleotide sequences of internal transcribed spacers (ITS1, 5.8 subunit gene, and ITS2) and mitochondrial cytochrome c oxidase 2 (cox2). Most (86.8%; 112/129) of the Anisakis type I larvae were A. pegreffii, and 7.8% (10/129) were A. typica. The remaining 5.4% (7/129) was not identified. Thus, A. pegreffii is the major species of anisakid larvae in sea eels of the southern coast of Korea.     

Genetic diversity of genus Vespa including an invaded species of V. velutina (Hymenoptera: Vespidae) in Korea inferred from DNA barcoding data

With about 5000 known species, the Vespidae is a large family belongs to order Hymenoptera. The genus Vespa with 22 species is one of the four genera of the subfamily Vespinae. In Korea, 10 species and subspecies are recognized. Because of their social behavior, their treat to human health and their impact in apiculture, the reliable and sometimes automated identification of these insects to species level are important. To test the efficacy of DNA barcoding method for identification of species of the genus Vespa in Korea, 30 samples of eight Korean species of genus Vespa were collected and mitochondrial DNAs of 658 bp fragment cytochrome oxidase subunit 1 (CO1) region were sequenced. A Bayesian Inference based on COI gene of the Korean Vespa species was constructed. The phylogenetic tree shoed that identification of all specimens is possible based on COI gene and we found strong relation between the sequences of the collected species from different localities in South Korea which clustered together with 100% support with sequences of the same species in GenBank. The results demonstrate that DNA barcoding is a useful technique for rapid and accurate species recognition in Korean Vespa species. The DNA barcode part of COI for V. binghami is provided for the first time that can help for identification of this species through DNA barcoding. Also, the genetic diversity among Korean Vespa velutina was zero suggests that the invasion might have occurred in a single event with small number of founders.     

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.     

Invasion of fish with leeches (Fam. Piscicolidae) in reservoirs of the Volga river

It is shown for the first time that during 50 years of the Rybinsk reservoir's existence the abundance of parasite leeches (Piscicolidae) has increased, and the species composition has become richer. In 70 the leech Caspiobdella fadejewi (Epstein) was registered here as a mass species. This species became a real competitor to Piscicola geometra (L.) inhabiting the Volga river in all times. In early '90s the leech Acipenserobdella volgensis (Zykoff) appeared in the Rybinsk reservoir. Till 1987 this species was not found to the north from the Kuibyschev reservoir. An increase in number of fish leeches and an extension of their specific composition occurred not only in the Rybinsk reservoir but in the other reservoirs of the Volga cascade as well.     

Does body size difference in the leeches Glossiphonia complanata (L.) and Helobdella stagnalis (L.) contribute to co-existence?

The effect of predator and prey body size on the feeding success of the British lake-dwelling leeches Glossiphonia complanata and Helobdella stagnalis was examined in the laboratory, and any involvement of size difference between the leeches in allowing coexistence in the field assessed. G. complanata breeds in advance of H. stagnalis and maintains a body size advantage throughout their annual life-cycle. In experiments, conducted at 14 °C and a photoperiod of 16 hrs L: 8 hrs D, three size classes of leeches of each species were each exposed to each of three size classes of each of five prey species, viz. Tubifex sp., Chironomus sp., Asellus aquaticus, Lymnaea peregra and Potamopyrgus jenkinsi. For each prey species, three different types of experiments were performed: one leech exposed to four prey individuals; four leeches of the same species with sixteen prey; and two leeches of each species with sixteen prey. In the first experiment, all sizes of G. complanata were capable of feeding on all sizes of the prey types offered; the same was true for H. stagnalis with exceptions of feeding on large A. aquaticus and large L. peregra. For both species, but especially for G. complanata, there was a trend within each size class of leech for decreasing proportions of fed leeches with increasing prey size, and within each size class of prey for an increasing proportion of fed leeches with increasing leech size; however there were several exceptions to these trends. Both leeches fed extensively on Tubifex sp. but there were significant differences in the proportions feeding on other prey types; G. complanata fed more on A. aquaticus and the two snail species, and less on Chironomus, than H. stagnalis. The effect of increasing the number of leech individuals from one to four individuals, of the same or mixed species, had little effect on the proportion of leeches which had fed. It is concluded that large G. complanata will have access to large individuals of certain prey taxa denied H. stagnalis, which may lessen the intensity of interspecific competition.     

A new species of Haemopis (Annelida: Hirudinea): Evolution of North American terrestrial leeches

Among the relatively few terrestrial leeches known worldwide, only two (Haemopis terrestris, Haemopis septagon) are described from North America. Here we report a third terrestrial leech collected from the southern part of New Jersey, USA. Tissue samples were obtained from 14 individuals representing three populations, and morphological characters were scored after dorsal and ventral dissections. Maximum Parsimony and Bayesian Inference analyses resolved phylogenetic relationships within the genus Haemopis using cytochrome c oxidase subunit 1 (CO1), 12S ribosomal RNA (rRNA), and 28S rRNA gene fragments, establishing the monophyly of North American haemopids and terrestrialism as a synapomorphy for some members of the group. Morphological distinctions and geographic isolation support the designation of a new species of terrestrial leech, Haemopis ottorum. Phylogeographic interpretations of the haemopid clade suggest that terrestrialism was derived from a northern, aquatic ancestor whose descendents were initially confined to Midwestern States and central Canada by the Appalachian Range. More recently, the terrestrial lineage (i.e., Haemopis terrestris) diverged near the southern extent of its range and began a northeasterly migration along coastal states giving rise to Haemopis septagon and Haemopis ottorum, the latter of which appears to define the leading edge of a northward expansion.     

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.     

Enrichment of specific protozoan populations during in situ bioremediation of uranium-contaminated groundwater

The importance of bacteria in the anaerobic bioremediation of groundwater polluted with organic and/or metal contaminants is well recognized and in some instances so well understood that modeling of the in situ metabolic activity of the relevant subsurface microorganisms in response to changes in subsurface geochemistry is feasible. However, a potentially significant factor influencing bacterial growth and activity in the subsurface that has not been adequately addressed is protozoan predation of the microorganisms responsible for bioremediation. In field experiments at a uranium-contaminated aquifer located in Rifle, CO, USA, acetate amendments initially promoted the growth of metal-reducing Geobacter species, followed by the growth of sulfate reducers, as observed previously. Analysis of 18S rRNA gene sequences revealed a broad diversity of sequences closely related to known bacteriovorous protozoa in the groundwater before the addition of acetate. The bloom of Geobacter species was accompanied by a specific enrichment of sequences most closely related to the ameboid flagellate, Breviata anathema, which at their peak accounted for over 80% of the sequences recovered. The abundance of Geobacter species declined following the rapid emergence of B. anathema. The subsequent growth of sulfate-reducing Peptococcaceae was accompanied by another specific enrichment of protozoa, but with sequences most similar to diplomonadid flagellates from the family Hexamitidae, which accounted for up to 100% of the sequences recovered during this phase of the bioremediation. These results suggest a prey–predator response with specific protozoa responding to increased availability of preferred prey bacteria. Thus, quantifying the influence of protozoan predation on the growth, activity and composition of the subsurface bacterial community is essential for predictive modeling of in situ uranium bioremediation strategies.