Analyses Of Small Subunit rDNA Sequences Research Articles

Morphology and SSU rDNA sequence analysis of two hypotrichous ciliates (Protozoa, Ciliophora, Hypotrichia) including the new species Metaurostylopsis parastruederkypkeae n. sp.

The morphology and phylogeny of two hypotrichous ciliates, Metaurostylopsis parastruederkypkeae n. sp. and Neourostylopsis flavicana (Wang et al., 2011) Chen et al., 2013 were investigated based on morphology, infraciliature and the small subunit (SSU) ribosomal RNA gene (rRNA) sequence. The new species, M. parastruederkypkeae n. sp. was identified according to its characteristics: body shape ellipsoidal, size about (165–200) × (45–60) μm in vivo, cell color reddish; two types of cortical granules including wheat grain-like and yellow-greenish larger ones along the marginal cirri rows and dorsal kineties and dot-like and reddish smaller ones, grouped around marginal cirri on ventral side and arranged in short lines on dorsal side; 26–41 adoral membranelles; three frontal and one parabuccal, five to seven frontoterminal, one buccal, and three to six transverse cirri; seven to thirteen midventral pairs; five to nine unpaired ventral cirri, five to seven left and three to five right marginal rows; and three complete dorsal kineties. Phylogenetic analysis based on SSU rDNA sequences showed that both Metaurostylopsis and Neourostylopsis are monophyletic. As the internal relationship between and within both genera are not clear, further studies on the species in these two genera are necessary. The key characteristics of all known twelve Metaurostylopsis-Apourostylopsis-Neourostylopsis species complex were updated.

Both SSU rDNA and RNA polymerase II data recognise that root-knot nematodes arose from migratory Pratylenchidae, but probably not from one of the economically high-impact lesion nematodes

In 2000 Siddiqi formulated a hypothesis stating that root-knot nematodes (Meloidogyne spp.) constitute a branch arising from yet another important group of plant parasites, the migratory Pratylenchidae. This hypothesis was solely based on morphological characteristics. Ribosomal DNA (rDNA) sequence analysis supports this hypothesis in its broad sense, but the more precise question about the identity of a migratory Pratylenchidae representative being closest to the most basal Meloidogyne species could not be addressed due to a lack of backbone resolution (Holterman et al., 2009). Here we present an extended small subunit rDNA sequence analysis and a data set of partial RNA polymerase II sequences from Pratylenchidae and basal Meloidogynidae. Our data point at members of the genus Pratylenchus as being closest to the common ancestor of the root-knot nematodes, but it was not possible unequivocally to identify a candidate lesion nematode species. Pratylenchus is a species-rich genus (ca 70 valid species), and we suggest that the species closest to the most basal root-knot nematode should be sought outside of the group of relatively well-characterised, agronomically relevant, species.

A new host record of Sphaerospora epinepheli (Myxosporea: Bivalvulida) occurring on orange-spotted grouper Epinephelus coioides from Thailand: Epidemiology, histopathology and phylogenetic position

In 1991, the first record of Sphaerospora epinepheli was described as a kidney parasite of wild and cultured malabar grouper, Epinephelus malabaricus, along coastlines of Thailand, the Gulf of Thailand and the Andaman Sea. However, the present study detected high infection of this parasite in kidney renal tubes of orange spotted grouper, Epinephelus coioides, collected from Andaman Sea. The highest infection rate of 36.82% was observed during the rainy season in 2009 in Phang-Nga Bay, in the north of Andaman Sea, which is an important grouper production site in Thailand. The biological and histopathological data of the parasite in this new host record are presented. Species classification is described based on morphological data of mature spore and molecular analysis of myxosporean 18S rDNA phylogeny including that of S. epinepheli which infected E. malabaricus. The genetic position of this parasite found in two host species was also studied. The phylogenetic tree analysis of small-subunit rDNA sequences of S. epinepheli from both infected hosts was constructed using two algorithms, maximum likelihood (ML) and Bayesian inference (BI). They were placed in the clustered basal sphaerosporid clade that contain four long SSU rDNA sphaerosporid species including Sphaerospora truttae, Sphaerospora elegans, Sphaerospora ranae, Sphaerospora fugu and Bipteria formosa with strong bootstrap supports. Histopathologically, renal intratubular myxosporean spores were associated with tubulonephosis, tubular necrosis, chronic interstitial nephritis and mimic membranoproliferative glomerulonephritis. This myxosporean parasite appears to be a significant pathogen on the basis of pathological changes in the renal tubules and is highly distributed in orange-spotted grouper.

Morphology and Small Subunit rDNA Phylogeny of a New Soil Ciliate, Bistichella variabilis n. sp. (Ciliophora, Stichotrichia)

We isolated a new ciliate, Bistichella variabilis n. sp., from slightly saline soil of the Yellow River Delta in China and studied it using live observation, protargol staining, and small subunit (SSU) rRNA gene sequencing. Bistichella variabilis is elongated ellipsoidal and about 220 × 70 μm in vivo, with a variable number and arrangement of cirri- ventral row VI mostly very short, but a long row observed also in one specimen, and an extra row occasionally exists besides the left or the right marginal row. Bistichella variabilis differs from all congeners except Bistichella buitkampi (Foissner, 1982) in the macronuclear pattern (four vs. two or many nodules); and from B. buitkampi by the usual oblique arrangement of transverse cirri (vs. perpendicular); and the ventral row VI mostly restricted in the posterior quarter of the cell (vs. long and stretching to the anterior quarter). Phylogenetic analyses of SSU rDNA sequences indicated that B. variabilis grouped with the clade of Orthoamphisiella breviseries and Uroleptoides magnigranulosus with high support values. Despite their distinct differences in morphology, these three species have sequence similarities of 99.5-99.7%. Nonetheless, it is still premature to assign Bistichella together with Uroleptoides to the family Orthoamphisiellidae because of the taxonomic uncertainties and undersampling.

Euplotespora binucleata n. gen., n. sp. (Protozoa: Microsporidia), a Parasite Infecting the Hypotrichous Ciliate Euplotes woodruffi, with Observations on Microsporidian Infections in Ciliophora

A new microsporidian species, Euplotespora binucleata n. gen., n. sp., from the brackish-water ciliate Euplotes woodruffi is described and defined on the basis of life history characteristics, light and electron microscopic features, and small subunit (SSU) ribosomal DNA (rDNA) sequencing. The life cycle of E. binucleata n. sp. probably has rather short merogonic and relatively long sporogonic phases. Some uninuclear meronts and sporonts, along with diplokaryotic sporoblasts and spores, were found in experimentally infected host cells. Such a peculiar life cycle has been induced experimentally in Euplotes eurystomus and constitutively microsporidian-free stocks of E. woodruffi. Spores of E. binucleata n. sp. are monomorphic, ovoid-cylindrical in shape, 3.44+/-0.17 x 1.65+/-0.22 microm in size, and characterized by a diplokaryotic condition and a large posterior vacuole. The polar tube is isofilar, 4.5-5.5 microm in length when ejected, and lacking a distinctive coiled region (half-coiled). The polaroplast is divided into two regions: the anterior part has a few lamellae close to the anchoring disc; and the posterior part is a rounded body (sack), about one-quarter of the spore length. Spores do not appear to cluster together as a group. Each spore is surrounded by a sporophorous membrane closely adjacent to the exospore layer. A phylogenetic analysis of SSU rDNA sequences by different methods placed E. binucleata n. sp. in a clade with representatives of the microsporidian genera Cystosporogenes and Vittaforma. Observations of microsporidia in several other ciliates are discussed in view of the microsporidian infection frequency in the phylum Ciliophora.

Further examination of the cyst-theca relationship of Protoperidinium thulesense (Peridiniales, Dinophyceae) and the phylogenetic significance of round brown cysts

K. Matsuoka, H. Kawami, R. Fujii and M. Iwataki. 2006. Further examination of the cyst-theca relationship of Protoperidinium thulesense (Peridiniales, Dinophyceae) and the phylogenetic significance of round brown cysts. Phycologia 45: 632–641. DOI: 10.2216/05-42.1The heterotrophic armored dinoflagellate Protoperidinium thulesense has an unusual combination of morphological characters, i.e. the thecal plate arrangement of the motile cell resembles a typical Protoperidinium, whereas the shape and archeopyle of the cyst are like the diplopsalids. We have re-examined the cyst-motile relationship of P. thulesense by cyst incubation and thecal plate analysis together with a molecular phylogenetic study based on small subunit (SSU) rDNA sequences. Five isolates of P. thulesense, including motile cells and cysts, and three Protoperidinium and three diplopsalid species were examined by using the single cell PCR method. The thecal plate arrangement of the motile cells isolated from field samples and those germinated from cysts were identical. The plate formula was: Po, X, 3′, 3a, 7″, 3c+t, 4s, 5′″, 2″″. The cysts of P. thulesense were round and brown with a theropylic archeopyle, and rather similar to the diplopsalid species, Diplopsalis lenticula, D. lebourae, Gotoius abei and others. SSU rDNA sequence analysis reveals P. thulesense is closely related to the subgenus Protoperidinium, especially species of the section Conica, and distant from the diplopsalid species. The phylogenetic and taxonomic significances of Protoperidinium and diplopsalid round brown cysts with a theropylic archeopyle are discussed.

Rapid quantitative profiling of complex microbial populations

Diverse and complex microbial ecosystems are found in virtually every environment on earth, yet we know very little about their composition and ecology. Comprehensive identification and quantification of the constituents of these microbial communities—a ‘census’—is an essential foundation for understanding their biology. To address this problem, we developed, tested and optimized a DNA oligonucleotide microarray composed of 10 462 small subunit (SSU) ribosomal DNA (rDNA) probes (7167 unique sequences) selected to provide quantitative information on the taxonomic composition of diverse microbial populations. Using our optimized experimental approach, this microarray enabled detection and quantification of individual bacterial species present at fractional abundances of <0.1% in complex synthetic mixtures. The estimates of bacterial species abundance obtained using this microarray are similar to those obtained by phylogenetic analysis of SSU rDNA sequences from the same samples—the current ‘gold standard’ method for profiling microbial communities. Furthermore, probes designed to represent higher order taxonomic groups of bacterial species reliably detected microbes for which there were no species-specific probes. This simple, rapid microarray procedure can be used to explore and systematically characterize complex microbial communities, such as those found within the human body.

High diversity of deep-sea Gromia from the Arabian Sea revealed by small subunit rDNA sequence analysis

Gromia is a large marine protist with filose pseudopodia and ovoid test, common in coastal intertidal and sublittoral waters. Although deep-water Gromia-like morphospecies were discovered in the 1990s, their relations to the shallow water species have never been established. Moreover, very little is known about the diversity within Gromia, reflecting the fact that these morphologically relatively simple protists have few characters useful for species identification. Consequently, we have analysed the SSU rDNA and ITS rDNA genes to examine gromiid diversity in two different areas located on the Oman and Pakistan margins of the Arabian Sea (water depths 1,000–2,000 m). In total, 27 deep-sea gromiid sequences of the SSU rDNA gene and six sequences of the ITS rDNA region were obtained. Our data confirm that Gromia-like protists from the bathyal deep sea are related to the shallow-water gromiids. Within Arabian Sea Gromia, we identified seven distinctive lineages, five of which form a monophyletic group branching as a sister group to shallow-water species. Six lineages of Arabian Sea Gromia can be defined morphologically, while one lineage includes specimens that look identical to specimens from two other lineages. This indicates that each Gromia lineage represents probably a separate species and suggests that deep-sea gromiid diversity is higher than indicated by their simple morphology.

Renal Myxozoanosis in Crowned River Turtles Hardella thurjii: Description of the Putative Agent Myxidium hardella n. sp. by Histopathology, Electron Microscopy, and DNA Sequencing

Chelonian myxozoanosis is rarely reported and has previously not been documented to cause disease. This report describes myxozoanosis associated with significant renal disease in two Crowned River turtles (Hardella thurjii). One turtle presented with emaciation and died. The cage mate presented with emaciation and was euthanized. Histologically, renal intratubular myxozoan spores were associated with renal tubular necrosis, tubular mineralization, and chronic interstitial nephritis, with membranoproliferative and mes-angioproliferative glomerulopathy. Both turtles also had disseminated metastatic mineralization. On the basis of these findings, chronic renal insufficiency from myxozoanosis and subsequent metastatic mineralization were considered the primary problems. By light and electron microscopy, the myxozoan spores had features of the genus Myxidium. Maximum parsimony analysis of small-subunit rDNA sequences placed the turtle myxozoan basal to a clade containing Myxidium truttae and a Myxidium sp. with strong bootstrap support. This myxozoan agent appears to be a significant pathogen in H. thurjii on the basis of morphologic changes in the kidneys of in the infected turtles.

Characterisation of the microbial diversity in a pig manure storage pit using small subunit rDNA sequence analysis

The microbial community structure of pig manure slurry (PMS) was determined with comparative analysis of 202 bacterial, 44 archaeal and 33 eukaryotic small subunit (SSU) rDNA partial sequences. Based on a criterion of 97% of sequence similarity, the phylogenetic analyses revealed a total of 108, eight and five phylotypes for the Bacteria, Archaea and Eukarya lineages, respectively. Only 36% of the bacterial phylotypes were closely related (⩾97% similarity) to any previously known sequence in databases. The bacterial groups most often represented in terms of phylotype and clone abundance were the Eubacterium (22% of total sequences), the Clostridium (15% of sequences), the Bacillus–Lactobacillus–Streptococcus subdivision (20% of sequences), the Mycoplasma and relatives (10% of sequences) and the Flexibacter–Cytophaga–Bacteroides (20% of sequences). The global microbial community structure and phylotype diversity show a close relationship to the pig gastrointestinal tract ecosystem whereas phylotypes from the Acholeplasma–Anaeroplasma and the Clostridium purinolyticum groups appear to be better represented in manure. Archaeal diversity was dominated by three phylotypes clustering with a group of uncultured microorganisms of unknown activity and only distantly related to the Thermoplasmales and relatives. Other Archaea were methanogenic H 2/CO 2 utilisers. No known acetoclastic Archaea methanogen was found. Eukaryotic diversity was represented by a pluricellular nematode, two Alveolata, a Blastocystis and an Entamoebidae. Manure slurry physico-chemical characteristics were analysed. Possible inhibitory effects of acetate, sulphide and ammonia concentrations on the microbial anaerobic ecosystem are discussed.

Characterization of a 4-methylbenzoate-degrading methanogenic consortium as determined by small-subunit rDNA sequence analysis

A methanogenic consortium that degrades 4-methylbenzoate (MBA) as the sole carbon and energy source was successfully enriched in an upflow anaerobic sludge bed bioreactor and studied. Electron microscopic observation showed that long rods with a distinct collar feature resembling Desulfomonile tiedjei rods were the predominant population, and that these rods formed a close spatial orientation with Methanobrevibacter-like bacteria. In addition, thin filaments and bamboo-shaped filaments that highly resembled the acetoclastic Methanosaeta were also frequently observed. A 16S rDNA clone library was constructed for the domain Bacteria, and 20 sequence types or operational taxonomic units (OTUs) were found out of 139 clones screened. Phylogenetic analysis classified these 20 nearly full-length OTUs into the delta (50.3% of total clones) and gamma (4.3%) subdivisions of the division Proteobacteria, the green non-sulfur bacteria subdivision I (7.2%), Cytophagales (7.2%), Planctomycetes (5.7%), gram-positive low G + C group (8.6%), candidate divisions OP8, OP10 and OP11 (9.3%), and a novel candidate division MBA1 (7.2%) that had an interdivisional sequence similarity less than 75%. However, only 3 OTUs had a sequence similarity higher than 90% to known isolates or environmental 16S rDNA clones, suggesting that the microbial community was diversified and largely unidentified. In particular, those 8 OTUs found in the delta- Proteobacteria were either clustered into novel groups or showed a low sequence similarity to closely related bacteria. It is highly possible that the delta- Proteobacteria were the long rods with a distinct collar feature observed microscopically, and together with the methanogens were mainly responsible for the syntrophic degradation of MBA. The unique and novel microbial populations identified explained the requirement of a long start-up period of up to 426 d for the MBA-degrading consortium.

Characterization of a 4-Methylbenzoate-Degrading Methanogenic Consortium as Determined by Small-Subunit rDNA Sequence Analysis.

A methanogenic consortium that degrades 4-methylbenzoate (MBA) as the sole carbon and energy source was successfully enriched in an upflow anaerobic sludge bed bioreactor and studied. Electron microscopic observation showed that long rods with a distinct collar feature resembling Desulfomonile tiedjei rods were the predominant population, and that these rods formed a close spatial orientation with Methanobrevibacter-like bacteria. In addition, thin filaments and bamboo-shaped filaments that highly resembled the acetoclastic Methanosaeta were also frequently observed. A 16S rDNA clone library was constructed for the domain Bacteria, and 20 sequence types or operational taxonomic units (OTUs) were found out of 139 clones screened. Phylogenetic analysis classified these 20 nearly full-length OTUs into the delta (50.3% of total clones) and gamma (4.3%) subdivisions of the division Proteobacteria, the green non-sulfur bacteria subdivision I (7.2%), Cytophagales (7.2%), Planctomycetes (5.7%), gram-positive low G + C group (8.6%), candidate divisions OP8, OP10 and OP11 (9.3%), and a novel candidate division MBA1 (7.2%) that had an interdivisional sequence similarity less than 75%. However, only 3 OTUs had a sequence similarity higher than 90% to known isolates or environmental 16S rDNA clones, suggesting that the microbial community was diversified and largely unidentified. In particular, those 8 OTUs found in the delta-Proteobacteria were either clustered into novel groups or showed a low sequence similarity to closely related bacteria. It is highly possible that the delta-Proteobacteria were the long rods with a distinct collar feature observed microscopically, and together with the methanogens were mainly responsible for the syntrophic degradation of MBA. The unique and novel microbial populations identified explained the requirement of a long start-up period of up to 426 d for the MBA-degrading consortium.

Molecular microbial diversity of an anaerobic digestor as determined by small-subunit rDNA sequence analysis.

The bacterial community structure of a fluidized-bed reactor fed by vinasses (wine distillation waste) was analyzed. After PCR amplification, four small-subunit (SSU) rDNA clone libraries of Bacteria, Archaea, Procarya, and Eucarya populations were established. The community structure was determined by operational taxonomic unit (OTU) phylogenetic analyses of 579 partial rDNA sequences (about 500 bp long). A total of 146 OTUs were found, comprising 133, 6, and 7 from the Bacteria, Archaea, and Eucarya domains, respectively. A total of 117 bacterial OTU were affiliated with major phyla: low-G+C gram-positive bacteria, Cytophaga-Flexibacter-Bacteroides, Proteobacteria, high-G+C gram-positive bacteria, and Spirochaetes, where the clone distribution was 34, 26, 17, 6, and 4%, respectively. The other 16 bacterial OTUs represent 13% of the clones. They were either affiliated with narrow phyla such as Planctomyces-Chlamydia, green nonsulfur bacteria, or Synergistes, or deeply branched on the phylogenetic tree. A large number of bacterial OTUs are not closely related to any other hitherto determined sequences. The most frequent bacterial OTUs represents less than 5% of the total bacterial SSU rDNA sequences. However, the 20 more frequent bacterial OTUs describe at least 50% of these sequences. Three of the six Archaea OTUs correspond to 95% of the Archaea population and are very similar to already known methanogenic species: Methanosarcina barkeri, Methanosarcina frisius, and Methanobacterium formicicum. In contrast, the three other Archaea OTUs are unusual and are related to thermophilic microorganisms such as Crenarchaea or Thermoplasma spp. Five percent of the sequences analyzed were chimeras and were removed from the analysis.

Taxonomy of Corynebacterium diphtheriae and related taxa, with recognition of Corynebacterium ulcerans sp. nov. nom. rev.

Levels of genomic DNA relatedness were determined using a SI nuclease procedure for reference bacteria identified as biotypes of Corynebacterium diphtheriae, biovars of Corynebacterium pseudotuberculosis, and ‘ Corynebacterium ulcerans’. These results showed that the three species are separate taxa at the genomospecies level whereas biotypes and biovars are closely related genomically within each species. Phylogenetic analyses of small-subunit rDNA sequences revealed that ‘ Corynebacterium ulcerans’ forms a tight cluster with Corynebacterium pseudotuberculosis within the robust branch that groups all Corynebacterium sequenced to date. Therefore, we propose that the species incertae sedis ‘ C. ulcerans’ should be conclusively recognized as a distinct species within the genus Corynebacterium with strain CCUG 2708 = NCTC 7910 as type strain. This species is characterized by urease production and fermentation of glycogen.