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.     

Molecular phylogeny of selected predaceous leeches with reference to the evolution of body size and terrestrialism

The phylogenetic relationships of erpobdellid leeches collected throughout Europe were investigated using newly obtained mitochondrial cytochrome c oxidase subunit I (CO-I) gene sequence data from 10 taxa. Monophyly of the five European Erpobdella species (sub-family Erpobdellinae) was supported, but a newly discovered leech, E. wuttkeit Kutschera, 2004 (the smallest member of its genus, discovered in an aquarium) was only distantly related to this clade. Three members of the semiaquatic Trochetinae were included in this study. The largest European leech species discovered so far, Trocheta haskonis Grosser, 2000, was found to be a terrestrial predator that feeds on earthworms. The rare species T. haskonis is the sister taxon of T. bykowskii Gedroyc, 1913, a well-known amphibious leech. Based on a comparison of body sizes and a phylogenetic tree the evolution of terrestrialism in the family Erpobdellidae is discussed.     

Biogeography and phylogenetic diversity of a cluster of exclusively marine myxobacteria

Myxobacteria are common in terrestrial habitats and well known for their formation of fruiting bodies and production of secondary metabolites. We studied a cluster of myxobacteria consisting only of sequences of marine origin (marine myxobacteria cluster, MMC) in sediments of the North Sea. Using a specific PCR, MMC sequences were detected in North Sea sediments down to 2.2 m depth, but not in the limnetic section of the Weser estuary and other freshwater habitats. In the water column, this cluster was only detected on aggregates up to a few meters above the sediment surface, but never in the fraction of free-living bacteria. A quantitative real-time PCR approach revealed that the MMC constituted up to 13% of total bacterial 16S rRNA genes in surface sediments of the North Sea. In a global survey, including sediments from the Mediterranean Sea, the Atlantic, Pacific and Indian Ocean and various climatic regions, the MMC was detected in most samples and to a water depth of 4300 m. Two fosmids of a library from sediment of the southern North Sea containing 16S rRNA genes affiliated with the MMC were sequenced. Both fosmids have a single unlinked 16S rRNA gene and no complete rRNA operon as found in most bacteria. No synteny to other myxobacterial genomes was found. The highest numbers of orthologues for both fosmids were assigned to Sorangium cellulosum and Haliangium ochraceum. Our results show that the MMC is an important and widely distributed but largely unknown component of marine sediment-associated bacterial communities.     

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.     

Multigene Phylogeography of Bactrocera caudata (Insecta: Tephritidae): Distinct Genetic Lineages in Northern and Southern Hemispheres

Bactrocera caudata is a pest of pumpkin flower. Specimens of B. caudata from the northern hemisphere (mainland Asia) and southern hemisphere (Indonesia) were analysed using the partial DNA sequences of the nuclear 28S rRNA and internal transcribed spacer region 2 (ITS-2) genes, and the mitochondrial cytochrome c oxidase subunit I (COI), cytochrome c oxidase subunit II (COII) and 16S rRNA genes. The COI, COII, 16S rDNA and concatenated COI+COII+16S and COI+COII+16S+28S+ITS-2 nucleotide sequences revealed that B. caudata from the northern hemisphere (Peninsular Malaysia, East Malaysia, Thailand) was distinctly different from the southern hemisphere (Indonesia: Java, Bali and Lombok), without common haplotype between them. Phylogenetic analysis revealed two distinct clades (northern and southern hemispheres), indicating distinct genetic lineage. The uncorrected ‘p’ distance for the concatenated COI+COII+16S nucleotide sequences between the taxa from the northern and southern hemispheres (‘p’ = 4.46-4.94%) was several folds higher than the ‘p’ distance for the taxa in the northern hemisphere (‘p’ = 0.00-0.77%) and the southern hemisphere (‘p’ = 0.00%). This distinct difference was also reflected by concatenated COI+COII+16S+28S+ITS-2 nucleotide sequences with an uncorrected ''p'' distance of 2.34-2.69% between the taxa of northern and southern hemispheres. In accordance with the type locality the Indonesian taxa belong to the nominal species. Thus the taxa from the northern hemisphere, if they were to constitute a cryptic species of the B. caudata species complex based on molecular data, need to be formally described as a new species. The Thailand and Malaysian B. caudata populations in the northern hemisphere showed distinct genetic structure and phylogeographic pattern.     

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%).     

Phenotypic and genotypic characterization of rhizobia associated with Acacia saligna (Labill.) Wendl. in nurseries from Algeria

Twenty seven rhizobial strains associated with Acacia saligna grown in northern and southern Algeria were characterized, including generation time, host-range, the 16S rRNA gene and 16S–23S rRNA intergenic spacer restriction patterns, 16S rRNA gene sequence analysis and tolerance to salinity and drought. Cross inoculation tests indicated that 11 slow-growing isolates from northern nurseries were able to nodulate introduced Australian acacias exclusively, whereas 16 fast-growing isolates, mainly from southern nurseries, were capable of also nodulating native acacias. Restriction patterns and sequence analysis of the 16S rRNA gene showed that strains of the first group belonged to Bradyrhizobium while strains of the second group were related to Sinorhizobium meliloti and Rhizobium gallicum. Interestingly, five strains of the first group formed a distinct cluster phylogenetically close to Bradyrhizobium betae, a non-nodulating species causing tumour-like deformations in sugar beet roots. Bradyrhizobium strains were in general more sensitive to NaCl and PEG than the S. meliloti and R. gallicum representatives. Among the latter, strains S. meliloti BEC1 and R. gallicum DJA2 were able to tolerate up to 1 M NaCl and 20% PEG. This, together with their wide host-range among Acacia species, make them good candidates for developing inoculants for A. saligna and other acacia trees growing in arid areas.     

Distribution of Roseobacter RCA and SAR11 lineages in the North Sea and characteristics of an abundant RCA isolate

The Roseobacter group and SAR11 clade constitute high proportions of the marine bacterioplankton, but only scarce information exists on the abundance of distinct populations of either lineage. Therefore, we quantified the abundance of the largest cluster of the Roseobacter group, the RCA (Roseobacter clade affiliated) cluster together with the SAR11 clade by quantitative PCR in the southern and eastern North Sea. The RCA cluster constituted up to 15 and 21% of total bacterial 16S ribosomal RNA (rRNA) genes in September 2005 and May 2006, respectively. At a few stations, the RCA cluster exceeded the SAR11 clade, whereas at most stations, SAR11 constituted higher fractions with maxima of 37%. In most samples, only one RCA ribotype was detected. RCA abundance was positively correlated with phaeopigments, chlorophyll, dissolved and particulate organic carbon (POC), turnover rates of dissolved free amino acids (DFAAs), temperature, and negatively correlated with salinity. The SAR11 clade was only correlated with POC (negatively, May) and with DFAA turnover rates (positively, September). An abundant RCA strain, ‘Candidatus Planktomarina temperata'', was isolated from the southern North Sea. This strain has an identical 16S rRNA gene sequence to the dominant RCA ribotype. Detection of the pufM gene, coding for a subunit of the reaction center of bacteriochlorophyll a, indicates the potential of the isolate for aerobic anoxygenic photosynthesis. Our study shows that a distinct population of the RCA cluster constitutes an abundant bacterioplankton group in a neritic sea of the temperate zone and indicates that this population has an important role during decaying phytoplankton blooms.     

Genetic diversity of Labyrinthula terrestris, a newly emergent plant pathogen, and the discovery of new Labyrinthulid organisms

Labyrinthuloid organisms are thought almost exclusively to be only associated with marine environments. However in 1995, a disease of turfgrass suddenly appeared that was eventually determined to be caused by a new Labyrinthula species (Labyrinthula terrestris). The disease is primarily thought to be caused by the use of elevated salinity irrigation water, making it a unique example of an emergent plant disease potentially induced by human activity. Our objective was to examine diversity of L. terrestris from broadly distributed isolates using AFLP, sequence analysis of two rDNA loci (SSU & LSU-ITS), and pathogenicity tests since previous research on a limited number of isolates found no variability based in ITS and SSU. In contrast to previous work, 18 unique genotypes were found out of a total of 29 analyzed based on AFLP. Sequence variability was only found in a single pathogenic isolate (Laby 31) that was isolated from the United Kingdom. The divergence based on AFLP and sequence analysis suggests that this isolate is a distinct species but closely related to the other L. terrestris isolates examined. Two putatively new nonpathogenic Labyrinthulid species were also found (Laby 13 & 32). Our results suggest that these organisms may be widely distributed in terrestrial environments based on the diversity found in this study and may have long been associated with terrestrial plants. Our results also suggest that more Labyrinthulid organisms may potentially emerge as new plant pathogens in the future if salinification of agricultural systems continues to increases worldwide.     

Isolation and Identification of Myxobacteria from Saline-Alkaline Soils in Xinjiang,China

Fifty-eight terrestrial and salt-tolerant myxobacteria were isolated from the saline-alkaline soils collected from Xinjiang, China. Based on the morphologies and the 16S rRNA gene sequences, these isolates were assigned into 6 genera, Myxococcus, Cystobacter, Corallococcus, Sorangium, Nannocystis and Polyangium. All the strains grew better with 1% NaCl than without NaCl. Some Myxococcus strains were able to grow at 2% NaCl concentration, suggesting that these strains may be particular type of terrestrial myxobacteria.     

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.     

Burmoniscus kitadaitoensis Nunomura, 2009 (Crustacea,Isopoda, Oniscidea) from southern Japan,a junior synonym of B. meeusei (Holthuis, 1947)

Re-examination of the holotype of Burmoniscus kitadaitoensis Nunomura, 2009 from Kitadaitojima Island, southern Japan reveals that this species is a junior synonym of B. meeusei (Holthuis, 1947). Partial regions of mitochondrial COI, 12S and 16S rRNA genes, and nuclear 18S and 28S rRNA genes were detected for species identification in the future.     

Bacterioplankton Community Shifts in an Arctic Lake Correlate with Seasonal Changes in Organic Matter Source

Seasonal shifts in bacterioplankton community composition in Toolik Lake, a tundra lake on the North Slope of Alaska, were related to shifts in the source (terrestrial versus phytoplankton) and lability of dissolved organic matter (DOM). A shift in community composition, measured by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes, occurred at 4°C in near-surface waters beneath seasonal ice and snow cover in spring. This shift was associated with an annual peak in bacterial productivity ([¹⁴C]leucine incorporation) driven by the large influx of labile terrestrial DOM associated with snow meltwater. A second shift occurred after the flux of terrestrial DOM had ended in early summer as ice left the lake and as the phytoplankton community developed. Bacterioplankton communities were composed of persistent populations present throughout the year and transient populations that appeared and disappeared. Most of the transient populations could be divided into those that were advected into the lake with terrestrial DOM in spring and those that grew up from low concentrations during the development of the phytoplankton community in early summer. Sequencing of DNA in DGGE bands demonstrated that most bands represented single ribotypes and that matching bands from different samples represented identical ribotypes. Bacteria were identified as members of globally distributed freshwater phylogenetic clusters within the α- and β-Proteobacteria, the Cytophaga-Flavobacteria-Bacteroides group, and the Actinobacteria.     

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.

     

Phylogenetic Analysis of Phenotypically Characterized Cryptococcus laurentii Isolates Reveals High Frequency of Cryptic Species

Background

Although Cryptococcus laurentii has been considered saprophytic and its taxonomy is still being described, several cases of human infections have already reported. This study aimed to evaluate molecular aspects of C. laurentii isolates from Brazil, Botswana, Canada, and the United States.

Methods

In this study, 100 phenotypically identified C. laurentii isolates were evaluated by sequencing the 18S nuclear ribosomal small subunit rRNA gene (18S-SSU), D1/D2 region of 28S nuclear ribosomal large subunit rRNA gene (28S-LSU), and the internal transcribed spacer (ITS) of the ribosomal region.

Results

BLAST searches using 550-bp, 650-bp, and 550-bp sequenced amplicons obtained from the 18S-SSU, 28S-LSU, and the ITS region led to the identification of 75 C. laurentii strains that shared 99–100% identity with C. laurentii CBS 139. A total of nine isolates shared 99% identity with both Bullera sp. VY-68 and C. laurentii RY1. One isolate shared 99% identity with Cryptococcus rajasthanensis CBS 10406, and eight isolates shared 100% identity with Cryptococcus sp. APSS 862 according to the 28S-LSU and ITS regions and designated as Cryptococcus aspenensis sp. nov. (CBS 13867). While 16 isolates shared 99% identity with Cryptococcus flavescens CBS 942 according to the 18S-SSU sequence, only six were confirmed using the 28S-LSU and ITS region sequences. The remaining 10 shared 99% identity with Cryptococcus terrestris CBS 10810, which was recently described in Brazil. Through concatenated sequence analyses, seven sequence types in C. laurentii, three in C. flavescens, one in C. terrestris, and one in the C. aspenensis sp. nov. were identified.

Conclusions

Sequencing permitted the characterization of 75% of the environmental C. laurentii isolates from different geographical areas and the identification of seven haplotypes of this species. Among sequenced regions, the increased variability of the ITS region in comparison to the 18S-SSU and 28S-LSU regions reinforces its applicability as a DNA barcode.     

16S rRNA Amplicon Sequencing Demonstrates that Indoor-Reared Bumblebees (Bombus terrestris) Harbor a Core Subset of Bacteria Normally Associated with the Wild Host

A MiSeq multiplexed 16S rRNA amplicon sequencing of the gut microbiota of wild and indoor-reared Bombus terrestris (bumblebees) confirmed the presence of a core set of bacteria, which consisted of Neisseriaceae (Snodgrassella), Orbaceae (Gilliamella), Lactobacillaceae (Lactobacillus), and Bifidobacteriaceae (Bifidobacterium). In wild B. terrestris we detected several non-core bacteria having a more variable prevalence. Although Enterobacteriaceae are unreported by non next-generation sequencing studies, it can become a dominant gut resident. Furthermore the presence of some non-core lactobacilli were associated with the relative abundance of bifidobacteria. This association was not observed in indoor-reared bumblebees lacking the non-core bacteria, but having a more standardized microbiota compared to their wild counterparts. The impact of the bottleneck microbiota of indoor-reared bumblebees when they are used in the field for pollination purpose is discussed.     

Bradyrhizobia from Wild Phaseolus, Desmodium, and Macroptilium Species in Northern Mexico

rRNA genetic markers were analyzed in 97 isolates of nodule bacteria from six legume species in Chihuahua, Mexico. The most common genotypes were widely shared across host species and had 16S rRNA sequences identical to those of strains from an eastern North American legume (Amphicarpaea) that are closely related to Bradyrhizobium elkanii.     

Absence of intervening sequences and point mutations in the V domain within 23S rRNA in Campylobacter lari isolates

Although the absence of intervening sequences (IVSs) within the 23S rRNA genes in Campylobacter lari isolates has been described, there are apparently no reports regarding correlations between the nucleotide sequences of 23S rRNA genes and erythromycin (Ery) susceptibility in C. lari isolates. Here, we determined the minimum inhibitory concentrations of 35 C. lari isolates [n?=?19 for urease-positive thermophilic Campylobacter (UPTC); n?=?16 urease-negative (UN) C. lari] obtained from Asia, Europe, and North America. We found that the 18 isolates were resistant to the Ery (defined as ≧8 μg/mL), and three isolates, UPTC A1, UPTC 92251, and UPTC 504, showed increased resistance (16 μg/mL). No correlations between the IVSs in the helix 45 region within the 23S rRNA gene sequences and Ery resistance were identified in the C. lari isolates examined. In addition, no point mutations occurred at any expected or putative position within the V domain in the isolates. In conclusion, antibiotic resistance against the macrolide erythromycin is mediated through an alternative pathway to that described above.     

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.     

Phylogenetic diversity of Flavobacteria isolated from the North Sea on solid media

Flavobacteria are abundant in the North Sea, an epeiric sea on the continental shelf of Europe. However, this abundance has so far not been reflected by the number of strains in culture collections. In this study, Flavobacteria were isolated from pelagic and benthic samples, such as seawater, phytoplankton, sediment and its porewater, and from surfaces of animals and seaweeds on agar plates with a variety of carbon sources. Dilution cultivation with a new medium, incubation at low temperatures and with long incubation times, and colony screening by a Flavobacteria-Cytophagia-specific PCR detecting 16S rRNA gene sequences led to a collection of phylogenetically diverse strains. Two strains affiliated with Flammeovirgaceae and seven strains affiliated with Cyclobacteriaceae, whereas within the Flavobacteriaceae 20 isolated strains presumably represented seven novel candidate genera and 355 strains affiliated with 26 of 80 validly described marine Flavobacteriaceae genera, based on a genus boundary of 95.0% 16S rRNA gene sequence identity. The majority of strains (276) affiliated with 37 known species in 16 genera (based on a boundary of 98.7% 16S rRNA gene sequence identity), whereas 79 strains likely represented 42 novel species in 22 established Flavobacteriaceae genera. Pigmentation, iridescence, gliding motility, agar lysis, and flexirubin as a chemical marker supported the taxonomy at the species level. This study demonstrated the culturability on solid medium of phylogenetically diverse Flavobacteria originating from the North Sea.