DNA microarray-based detection and identification of waterborne protozoan pathogens

BackgroundAmplification of small subunit (SSU) rRNA genes with universal primers is a common method used to assess microbial populations in various environmental samples. However, owing to limitations in coverage of these universal primers, some microorganisms remain unidentified. The present study aimed to establish a method for amplifying nearly full-length SSU rRNA gene sequences of previously unidentified prokaryotes, using newly designed targeted primers via primer evaluation in meta-transcriptomic datasets.MethodsPrimer binding regions of universal primer 8F/Arch21F for bacteria or archaea were used for primer evaluation of SSU rRNA sequences in meta-transcriptomic datasets. Furthermore, targeted forward primers were designed based on SSU rRNA reads from unclassified groups unmatched with the universal primer 8F/Arch21F, and these primers were used to amplify nearly full-length special SSU rRNA gene sequences along with universal reverse primer 1492R. Similarity and phylogenetic analysis were used to confirm their novel status.ResultsUsing this method, we identified unclassified SSU rRNA sequences that were not matched with universal primer 8F and Arch21F. A new group within the Asgard superphylum was amplified by the newly designed specific primer based on these unclassified SSU rRNA sequences by using mudflat samples.ConclusionWe showed that using specific primers designed based on universal primer evaluation from meta-transcriptomic datasets, identification of novel taxonomic groups from a specific environment is possible.

Phylogenetic relationships within the genus Tetrahymena inferred from the cytochrome c oxidase subunit 1 and the small subunit ribosomal RNA genes

The life cycle, spore morphology, pathogenicity, tissue specificity, mode of transmission and small subunit rRNA (SSU-rRNA) gene sequence analysis of the five new microsporidian isolates viz., NIWB-11bp, NIWB-12n, NIWB-13md, NIWB-14b and NIWB-15mb identified from the silkworm, Bombyx mori have been studied along with type species, NIK-1s_mys. The life cycle of the microsporidians identified exhibited the sequential developmental cycles that are similar to the general developmental cycle of the genus, Nosema. The spores showed considerable variations in their shape, length and width. The pathogenicity observed was dose-dependent and differed from each of the microsporidian isolates; the NIWB-15mb was found to be more virulent than other isolates. All of the microsporidians were found to infect most of the tissues examined and showed gonadal infection and transovarial transmission in the infected silkworms. SSU-rRNA sequence based phylogenetic tree placed NIWB-14b, NIWB-12n and NIWB-11bp in a separate branch along with other Nosema species and Nosema bombycis; while NIWB-15mb and NIWB-13md together formed another cluster along with other Nosema species. NIK-1s_mys revealed a signature sequence similar to standard type species, N. bombycis, indicating that NIK-1s_mys is similar to N. bombycis. Based on phylogenetic relationships, branch length information based on genetic distance and nucleotide differences, we conclude that the microsporidian isolates identified are distinctly different from the other known species and belonging to the genus, Nosema. This SSU-rRNA gene sequence analysis method is found to be more useful approach in detecting different and closely related microsporidians of this economically important domestic insect.

Oligotrichs are ciliates of great abundance, but their molecular systematics are rarely studied. In this study, nine species representing three genera (Strombidium, Novistrombidium, and Omegastrombidium) of marine oligotrich ciliates were collected from coastal waters of China. The small subunit (SSU) rRNA gene of two species and the internal transcribed spacers and 5.8S region (ITS1-5.8S-ITS2) for all nine species were sequenced for the first time. Phylogenetic trees using both the SSU rRNA gene and ITS1-5.8S-ITS2 region sequences were generated. In addition, the secondary structures of ITS2 RNA transcripts of 11 taxa representing four genera (Novistrombidium, Strombidium, Omegastrombidium, and Laboea) were investigated. The phylogenetic analyses show that (i) the family Strombidiidae is polyphyletic, (ii) the genus Novistrombidium is probably paraphyletic, containing at least two subclades, which is consistent with recent cladistic analyses based on morphological data, and (iii) the tail-less genus Laboea is separate from other genera of Strombidiidae, clustering instead with the tontoniids. Comparisons of the secondary structure of ITS2 regions also show that Laboea is clearly different from other strombidiids. These findings cast doubt on the monophyly of the family Strombidiidae.

The morphology and phylogeny of Loxodes vorax and L. striatus orientalis subsp. n. were investigated based on infraciliature and small subunit (SSU) rRNA gene sequence data. Loxodes striatus orientalis subsp. n. was separated from L. striatus striatus stat. n. by having fewer dikinetids in the intrabuccal kinety (35-55 vs. 50-70) and a variable number of macronuclei (2-4 vs. 2). In addition, the SSU rRNA gene sequence of the new subspecies differs in 13 and 11 nucleotides from that of two populations of the nominotypic subspecies. We also summarized the morphological differences between Loxodes and Remanella based on the data available. Phylogenetic analyses revealed that the genus Loxodes was monophyletic and nested within Remanella species. This study might, therefore, support the hypothesis that the freshwater genus Loxodes evolved from the marine genus Remanella.

A new myxosporean parasite, Auerbachia maamouni n. sp., infecting the gallbladder of the golden trevally Gnathanodon speciosus Forsskål, is described, based on morphology, ultrastructure, and small subunit (SSU) rRNA gene sequencing. Of 50 fish collected from the Red Sea in Jeddah, Saudi Arabia, five were found heavily infected with mature myxospores floating free in the bile. Mature spores are pyriform to club-shaped with smooth valves, and contain a single polar capsule with an S-shaped polar filament, arranged in 13-16 polar filament coils, oriented longitudinally, with an irregular distribution on the polar capsule matrix. Spores measure 15.8 (14-17) μm in total length in lateral view, 7.9 (6-9) μm in width in apical view, with spore body length of 6.2±0.4 (5-7) µm. The ellipsoidal polar capsule has two adjusted lateral folds 7.6 (6-8) µm long and 2 (2-3) µm wide. The new species is distinguished from other species of the genus based on spore morphometry and molecular data. The phylogenetic tree constructed using Maximum Likelihood and Bayesian Inference analysis of SSU rDNA sequence data supported the phylogenetic position of A. maamouni n. sp. among the species of Auerbachia Meglitsch, 1968 sequenced to date. Analysis of the SSU rDNA sequence data also supported the assumption that Auerbachia is closely related to members of the genera Coccomyxa Léger & Hesse, 1907, Zschokkella Auerbach, 1910 and Myxidium Bütschli, 1882, whose members inhabit the gallbladder of marine teleost fishes.

Nuclear small subunit (SSU) rRNA gene sequences were obtained by polymerase chain reaction from trichomonad symbionts of termites that belong to the Devescovinidae (Caduceia versatilis) and polymastigont Calonymphidae (Stephanonympha nelumbium). The unidentified SSU rRNA sequence Nk3, previously obtained from the termite Neotermes koshunensis, has also been shown to derive from a Stephanonympha sp. by in situ hybridization. These sequences were analysed in a broad phylogeny including nearly all identified parabasalid sequences available in the databases, and some as yet unidentified sequences likely deriving from the new order Cristamonadida (Devescovinidae, Calonymphidae, and hypermastigids Lophomonadida). A global phylogeny of parabasalids reveals a partial agreement between the clades identified in this work and the last classification of this phylum into four orders. However, this classification is still incongruent with our data and new taxonomic considerations are proposed. The analysis confirms the monophyly of the Cristamonadida and separates this order into two groups: the first unites nearly all the Devescovinidae including Caduceia and the Calonymphidae Coronympha and Metacoronympha, whereas the second group is composed of a few Devescovinidae, Lophomonadida, and Calonymphidae such as Stephanonympha. Caduceia is closely related to Devescovina, corroborating the marked morphological similarity between these two genera whereas Stephanonympha groups together with the Calonymphidae Snyderella and Calonympha. These data also confirm the polyphyly of the families Devescovinidae and Calonymphidae and support the arrangement of the axostyle-pelta complexes as a valuable character for taxonomic considerations within the Calonymphidae.

The Ribosomal RNA Gene Region in Acanthamoeba castellanii Mitochondrial DNA: A Case of Evolutionary Transfer of Introns between Mitochondria and Plastids?

Three trachelocercid ciliates, Kovalevaia sulcata (Kovaleva, 1966) Foissner, 1997, Trachelocerca sagitta (Müller, 1786) Ehrenberg, 1840 and Trachelocerca ditis (Wright, 1982) Foissner, 1996, isolated from two coastal habitats at Qingdao, China, were investigated using live observation and silver impregnation methods. Data on their infraciliature and morphology are supplied. The small subunit rRNA (SSU rRNA) genes of K. sulcata and Trachelocerca sagitta were sequenced for the first time. Phylogenetic analyses based on SSU rRNA gene sequence data indicate that both organisms, and the previously sequenced Trachelocerca ditis, are located within the trachelocercid assemblage and that K. sulcata is sister to an unidentified taxon forming a clade that is basal to the core trachelocercids.

A new fungal phylum, the Glomeromycota: phylogeny and evolution

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.

Two heterotrichid ciliates, Climacostomum virens (Ehrenberg, 1838) Stein, 1859 from brackish water and freshwater, and Fabrea salina Henneguy, 1890 from a solar saltern, were collected in Korea. They are novelly investigated in Korea by means of live observation, protargol staining and nuclear small subunit (SSU) rRNA gene sequencing. Climacostomum virens is characterized by pouch-like body shape, body length of <TEX>$200-370{\mu}m$</TEX> in vivo, conspicuous cytopharyngeal tube, macronuclei ribbon-like shape, and one to four in number, with or without symbiont algae in cytoplasm, 34-66 somatic kineties, 67-113 adoral zone of membranelles, 8-42 peristomial kineties, 24-37 apical membranelles. SSU rDNA sequence size is 1,591 bp and GC contents 48.52%. Fabrea salina is also characterized by scoop-like body shape with proboscis, body length of <TEX>$190-240{\mu}m$</TEX> in vivo, one to two rod-shaped macronuclei, oval micronuclei, grayish green cortical granules, 104-186 somatic kineties, 4-8 preoral kineties, 7-19 peristomial kineties and fragmented paroral membrane. SSU rDNA sequence size is 1,598 bp and GC contents 47.50%.

Morphology and SSU rRNA gene sequences of three Frontonia species, including a description of F. subtropica spec. nov. (Ciliophora, Peniculida)

Composting provides a dynamic setting for studying ecological topics such as succession, competition, and community stability in a relatively short period of time. This study used hierarchical small sub-unit-based rRNA gene probes to quantify the change in the relative abundance of phylogenetic groups common to compost in laboratory scale reactors. Bacterial 16S rRNA gene targets accounted for only 37% of all small subunit (SSU) rRNA genes initially, but increased to a maximum of 83% of the total at 84 h. The sum of rRNA genes detected using probes specific to Pseudomonas and low-G+C Gram-positive rRNA genes represented between 16% and 87% of the total. The lack of hybridization to the taxon-specific probes was most pronounced between 36 h and 60 h, when the pH was between 4.6 and 4.8. During this period the relative abundance of taxon-specific gene targets accounted for only 17-33% of the total bacterial rRNA gene targets. Pseudomonas-type 16S rRNA genes were the most abundant of the groups measured until 72 h. Those genes had their highest relative abundance at 12 h (78% of bacterial rRNA genes; 30% of all rRNA genes), after which time their relative abundance began to decline as the temperature increased. Prior to 72 h, 16S rRNA genes from low-G+C Gram-positive bacteria (LGC-GPB) represented less than 7% of the bacterial rRNA genes. However, by 84 h the relative abundance of LGC-GPB and Bacillus rRNA genes had increased to 60% and 18% of the bacterial rRNA gene targets, respectively (50% and 15% of all rRNA genes, respectively).

During an investigation of yeasts associated with insects, three strains of a d-xylose-fermenting yeast species were isolated from the gut of the host beetles Allomyrina dichotoma (Coleoptera: Scarabeidae) collected on the Baotianman National Nature Reserve, Nanyan, Henan Province, China. These strains formed two elongated ascospores, which were tapered and curved at the ends in persistent asci. Sequence analyses of the D1/D2 domains of the large subunit (LSU) and small subunit (SSU) rRNA genes showed that these new strains represent a phylogenetically distinct species in the Spathaspora clade. This novel species differed from the closest species, Candida lyxosophila NRRL Y-17539T, by a 6.7 % sequence divergence (31 substitutions and 7 gaps) in the D1/D2 LSU rRNA gene and a 1.2 % divergence (17 substitutions, 4 gaps) in the SSU rRNA gene. The novel species can also be distinguished from C. lyxosophila NRRL Y-17539T in terms of the ability to assimilate myo-inositol and to grow in the presence of 0.1 % cycloheximide, as well as the inability to assimilate citrate. The name Spathaspora allomyrinae sp. nov. is proposed for this species. The type strain is NYNU 1495T ( = CICC 33057T = CBS 13924T). The MycoBank number is MB 815071.