SSU rRNA Sequences Research Articles

Detection of Cryptosporidium sp. in Two New Seal Species, Phoca vitulina and Cystophora cristata, and a Novel Cryptosporidium Genotype in a Third Seal Species, Pagophilus groenlandicus, from the Gulf of Maine

Data on the geographic distribution and host specificity of Cryptosporidium spp. are critical for developing an understanding of likely transmission patterns in nature. During a molecular-based survey of fecal samples from 293 terrestrial and aquatic animals in Maine, USA, we detected Cryptosporidium sp. in 11 harbor seals (Phoca vitulina), 1 hooded seal (Cystophora cristata), and 1 harp seal (Pagophilus groenlandicus). None of the terrestrial or freshwater mammal fecal samples or bird samples tested positive for Cryptosporidium sp. However, the sequencing results of the small subunit (ssu) rRNA gene indicate that the seals were infected with an undescribed species of Cryptosporidium , previously isolated only from ringed seals (Phoca hispida) in northern Quebec, Canada. In addition, the Cryptosporidium sp. detected in the harp seal is significantly different from the previously observed Cryptosporidium sp. in other seals. We confirmed the genetic distinctiveness of this Cryptosporidium genotype and the identity of the other Cryptosporidium sp. seal ssu rRNA sequences by using data from the 70-kDa heat shock protein gene. Based on phylogenetic reconstructions of both genes, it seems that either Cryptosporidium canis or C. felis are sister species to the seal associated Cryptosporidium spp. Our findings extend the range of " Cryptosporidium sp. seal" well south of the 55th parallel, add other species to the list of seals affected by Cryptosporidium sp., and highlight the presence of unrecognized population and potentially species level variation in Cryptosporidium.

Microbial eukaryote life in the new hypersaline deep-sea basin Thetis

Only recently, a novel anoxic hypersaline (thalassic) basin in the eastern Mediterranean was discovered at a depth of 3,258m. The halite-saturated brine of this polyextreme basin revealed one of the highest salt concentrations ever reported for such an environment (salinity of 348‰). Using a eukaryote-specific probe and fluorescence in situ hybridization, we counted 0.6×10(4) protists per liter of anoxic brine. SSU rRNA sequence analyses, based on amplification of environmental cDNA identified fungi as the most diverse taxonomic group of eukaryotes in the brine, making deep-sea brines sources of unknown fungal diversity and hotspots for the discovery of novel metabolic pathways and for secondary metabolites. The second most diverse phylotypes are ciliates and stramenopiles (each 20%). The occurrence of closely related ciliate sequences exclusively in other Mediterranean brine basins suggests specific adaptations of the respective organisms to such habitats. Betadiversity-analyses confirm that microeukaryote communities in the brine and the interface are notably different. Several distinct morphotypes in brine samples suggest that the rRNA sequences detected in Thetis brine can be linked to indigenous polyextremophile protists. This contradicts previous assumptions that such extremely high salt concentrations are anathema to eukaryotic life. The upper salinity limits for eukaryotic life remain unidentified.

Phylogenetic Position of Lophomonas striata Bütschli (Parabasalia) from the Hindgut of the Cockroach Periplaneta americana

Lophomonas striata is a multiflagellate parabasalid commensal in the hindgut of the omnivorous cockroaches Blatta orientalis and Periplaneta americana. Its closest relatives were traditionally thought to include similar multiflagellate parabasalids with a single flagellar area that degenerates during mitosis, such as Joenia and Kofoidia. However, molecular phylogenetic analyses have shown that "lophomonads" are not monophyletic. We have determined the SSU rRNA sequence of L. striata and we find that it branches sister to the Trichonymphida with strong support. This is surprising because all other lophomonads sampled to date branch within the Cristamonadida, and the order Trichonymphida (e.g. Trichonympha, Pseudotrichonympha, and Hoplonympha) is both morphologically coherent and monophyletic in SSU rRNA phylogenies. Trichonymphida, unlike the lophomonads, share a bilateral symmetry, in which their multiple flagella occur in two (or sometimes four) regions, and instead of degenerating upon mitosis, half of the flagella are passed to each daughter cell. The single apical flagellar region characteristic of lophomonads is therefore either plesiomorphic or it has arisen multiple times in parabasalids; our phylogenetic analyses and available ultrastructural evidence suggest the latter. Our results also suggest that parabasalid gut symbionts may have been vertically transmitted in cockroaches before the common ancestor of Cryptocercus and termites.

Scanning Electron Microscopy and Molecular Characterization of A New Haplosporidium Species (Haplosporidia), A Parasite of the Marine Gastropod Siphonaria pectinata (Mollusca: Gastropoda: Siphonariidae) In the Gulf of Mexico

Based on scanning electron microscopy and the small subunit ribosomal RNA (SSU rRNA), Haplosporidium tuxtlensis n. sp. (Haplosporidia), a parasite found in the visceral tissues of the false limpet Siphonaria pectinata (Linnaeus, 1758), is described. The spores are ellipsoidal (3.61 ± 0.15 µm × 2.69 ± 0.19 µm), with a circular lid (2.94 ± 0.5 µm) representing the operculum. The spore wall bears filaments occurring singly, or in clusters, of 2 to 8, fusing distally. Phylogenetic relationships of H. tuxtlensis n. sp. were assessed with other described species using the SSU rRNA sequence. Haplosporidium tuxtlensis n. sp. is sister taxon to Haplosporidium pickfordi Barrow, 1961. The morphological characteristics (spore wall structure, shape, size, and filament structure) and the unique host identity corroborate it as a new species. Additionally, this is the first record of Haplosporidia infecting striped false limpets in the Gulf of Mexico.

Genome profiling implies high genetic diversity in microsporidia isolated from the common cutworm, Spodoptera litura (Lepidoptera: Noctuidae), in Vietnam

In order to evaluate the potential application of microsporidia as a microbial control agent against lepidopteran insect pests, microsporidian infection in a field population of the common cutworm, Spodoptera litura (Fabricius), was surveyed in vegetable crop fields in Can Tho City, Vietnam, in March 2007. The infection rate of microsporidia was 46.7% (99/212 individuals) in adult S. litura, and 16 samples of infected adults were used to characterize the microsporidia at the molecular level. Analysis of the small subunit ribosomal RNA (SSU rRNA) sequences indicated that microsporidian strains isolated from S. litura were closely related to Nosema bombycis from the silkworm, Bombyx mori (Linnaeus); however, phylogenetic analysis based on genome profiling produced a different result from the SSU rRNA sequences. Temperature gradient gel electrophoresis profiles of 12 microsporidian strains from S. litura were closely related to N. bombycis strains, while the profiles of three microsporidian strains formed a different cluster. The Vietnamese strains did not form a single group, but were classified into at least three groups. These results suggested that the microsporidia isolated from S. litura in the Mekong Delta, Vietnam, are genetically diverse.

What's new is old: resolving the identity of Leptothrix ochracea using single cell genomics, pyrosequencing and FISH.

Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms.

Morphological and molecular characterization of Pseudocohnilembus longisetus Thompson, 1965 from farmed black rockfish Sebastes schlegelii in Korea

The morphology, infraciliature, silverline system, and the small subunit ribosomal RNA (SSU rRNA) of the little-known marine scuticociliate Pseudocohnilembus longisetus Thompson, 1965 from the diseased black rockfish Sebastes schlegelii in Korea were studied. This scuticociliate possessed the typical characteristics of the genus Pseudocohnilembus, but could be discriminated from Pseudocohnilembus hargisi, and Pseudocohnilembus persalinus in terms of the body size, shape, the number of somatic kineties and kinetids in somatic kinety 1, and the number/position of contractile vacuole pores. The SSU rRNA gene of P. longisetus was sequenced in order to gain a better understanding of appropriate phylogenetic classification. The SSU rRNA was 1754 bp and the sequence was deposited in GenBank under accession number FJ899594. The SSU rRNA gene sequences of P. longisetus had an identity of 98.1%, 96.8% and 95.3% with P. hargisi, P. persalinus, and Pseudocohnilembus marinus SSU rRNA sequences, respectively. Our population of P. longisetus belonged to the genus Pseudocohnilembus and was in an isolated position based on the SSU rRNA gene tree, which was consistent with the conclusions based on the morphological studies. However, further investigation is required to determine the pathogenicity of this species.

PhylOTU: A High-Throughput Procedure Quantifies Microbial Community Diversity and Resolves Novel Taxa from Metagenomic Data

Microbial diversity is typically characterized by clustering ribosomal RNA (SSU-rRNA) sequences into operational taxonomic units (OTUs). Targeted sequencing of environmental SSU-rRNA markers via PCR may fail to detect OTUs due to biases in priming and amplification. Analysis of shotgun sequenced environmental DNA, known as metagenomics, avoids amplification bias but generates fragmentary, non-overlapping sequence reads that cannot be clustered by existing OTU-finding methods. To circumvent these limitations, we developed PhylOTU, a computational workflow that identifies OTUs from metagenomic SSU-rRNA sequence data through the use of phylogenetic principles and probabilistic sequence profiles. Using simulated metagenomic data, we quantified the accuracy with which PhylOTU clusters reads into OTUs. Comparisons of PCR and shotgun sequenced SSU-rRNA markers derived from the global open ocean revealed that while PCR libraries identify more OTUs per sequenced residue, metagenomic libraries recover a greater taxonomic diversity of OTUs. In addition, we discover novel species, genera and families in the metagenomic libraries, including OTUs from phyla missed by analysis of PCR sequences. Taken together, these results suggest that PhylOTU enables characterization of part of the biosphere currently hidden from PCR-based surveys of diversity?

ChemInform Abstract: Natural Products from Marine Derived Microorganisms

AbstractReview: 17 refs.

Multiplex PCR detection of slowly‐evolving trypanosomatids and neogregarines in bumblebees using broad‐range primers

The aims of this study were to design universal markers for different protozoan parasites of Bombus spp. based on the phylogenetic position of two important bumblebee parasites Crithidia bombi and Apicystis bombi. Standard PCR and extraction techniques were used to amplify and sequence 18S rDNA. Phylogenetic analysis of the rDNA was performed in order to predict the parasite range of the primers. Crithidia bombi phylogenetically clusters with the trypanosomatids with slowly-evolving SSU-rRNA sequences (SE), while A. bombi is the closest sister group of Mattesia. A multiplex was designed containing an internal control and two broad-range primer pairs, detecting C. bombi and other SE trypanosomatids and also A. bombi and other neogregarines. Sequence data generated will further improve the current systematics of insect trypanosomatids and gregarines that remain troublesome. Broad-range markers for bumblebee parasites are necessary tools enabling the screening of commercially imported colonies and thus controlling their worldwide distribution and to discover related emerging parasites.

海洋微生物を用いた天然物化学

Marine microbial resources have been considered as potent sources for new and potent medicines. We have constructed marine microbial library consists of 50,000 strains. Among them, 26,000 strains have phylogenetically analyzed based on SSU–rRNA sequences. Number of biologically active substances have been isolated from marine microbes in this library using the screening based on marine phenomena. One typical example is thallusin (2), isolated in 2005 after pursuing for more than 50 years, showed foliaceous morphology–inducing activity at the exceptionally low concentration. Antimicroalgal substances HML (6) and Kailuin B (9) have been isolated from marine bacteria belongs to Vibrio. These compounds showed selective toxicity only against dinofragellates. Useful microalgae such as diatom was unaffected by these substances. Interestingly, these substances also showed potent anti–malarial activity. It is not strange, because dinofragellete and malaria are phylogenetically related to each other. As shown in the isolation of above mentioned substances and unnermicins, the screening methods based on marine phenomena seemed to be useful for the efficient isolation of novel biologically active compounds.

Assessing the diversity of AM fungi in arid gypsophilous plant communities

In the present study, we used PCR-Single-Stranded Conformation Polymorphism (SSCP) techniques to analyse arbuscular mycorrhizal fungi (AMF) communities in four sites within a 10 km(2) gypsum area in Southern Spain. Four common plant species from these ecosystems were selected. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, SSCP analysis, sequencing and phylogenetic analyses. A total of 1443 SSU rRNA sequences were analysed, for 21 AM fungal types: 19 belonged to the genus Glomus, 1 to the genus Diversispora and 1 to the Scutellospora. Four sequence groups were identified, which showed high similarity to sequences of known glomalean species or isolates: Glo G18 to Glomus constrictum, Glo G1 to Glomus intraradices, Glo G16 to Glomus clarum, Scut to Scutellospora dipurpurescens and Div to one new genus in the family Diversisporaceae identified recently as Otospora bareai. There were three sequence groups that received strong support in the phylogenetic analysis, and did not seem to be related to any sequences of AM fungi in culture or previously found in the database; thus, they could be novel taxa within the genus Glomus: Glo G4, Glo G2 and Glo G14. We have detected the presence of both generalist and potential specialist AMF in gypsum ecosystems. The AMF communities were different in the plant studied suggesting some degree of preference in the interactions between these symbionts.

Sequence and Phylogenetic Analysis of SSU rRNA Gene of Five Microsporidia

The complete small subunit rRNA (SSU rRNA) gene sequences of five microsporidia including Nosema heliothidis, and four novel microsporidia isolated from Pieris rapae, Phyllobrotica armta, Hemerophila atrilineata, and Bombyx mori, respectively, were obtained by PCR amplification, cloning, and sequencing. Two phylogenetic trees based on SSU rRNA sequences had been constructed by using Neighbor-Joining of Phylip software and UPGMA of MEGA4.0 software. The taxonomic status of four novel microsporidia was determined by analysis of phylogenetic relationship, length, G+C content, identity, and divergence of the SSU rRNA sequences. The results showed that the microsporidia isolated from Pieris rapae, Phyllobrotica armta, and Hemerophila atrilineata have close phylogenetic relationship with the Nosema, while another microsporidium isolated from Bombyx mori is closely related to the Endoreticulatus. So, we temporarily classify three novel species of microsporidia to genus Nosema, as Nosema sp. PR, Nosema sp. PA, Nosema sp. HA. Another is temporarily classified into genus Endoreticulatus, as Endoreticulatus sp. Zhenjiang. The result indicated as well that it is feasible and valuable to elucidate phylogenetic relationships and taxonomic status of microsporidian species by analyzing information from SSU rRNA sequences of microsporidia.

Blastodinium galatheanum sp. nov. (Dinophyceae) a parasite of the planktonic copepod Acartia negligens (Crustacea, Calanoida) in the central Atlantic Ocean

A new dinoflagellate species, Blastodinium galatheanum sp. nov., was found parasitizing the planktonic copepods Acartia negligens and Acartia sp. in the Atlantic Ocean between the Azores and the Cape Verde Islands. These copepods have not previously been reported hosting a Blastodinium species. Characters that distinguish the new species are the shape and size of the trophic stage, its host species, and its predominantly solitary existence. Dinospores of Blastodinium galatheanum sp. nov. are peridinioid in nature and morphologically indistinguishable from dinospores of two other previously investigated Blastodinium species. SSU rRNA gene sequences from two isolates of this new species were almost identical and showed similarities to SSU rRNA sequences of other species of Blastodinium. A phylogenetic analysis based on SSU rRNA gene sequences suggested monophyly for all existing sequences of Blastodinium spp., including a sequence from the type species B. pruvoti, presented here for the first time.

Development and application of the human intestinal tract chip, a phylogenetic microarray: analysis of universally conserved phylotypes in the abundant microbiota of young and elderly adults

In this paper we present the in silico assessment of the diversity of variable regions of the small subunit ribosomal RNA (SSU rRNA) gene based on an ecosystem-specific curated database, describe a probe design procedure based on two hypervariable regions with minimal redundancy and test the potential of such probe design strategy for the design of a flexible microarray platform. This resulted in the development and application of a phylogenetic microarray for studying the human gastrointestinal microbiota – referred as the human intestinal tract chip (HITChip). Over 4800 dedicated tiling oligonucleotide probes were designed based on two hypervariable regions of the SSU rRNA gene of 1140 unique microbial phylotypes (< 98% identity) following analysis of over 16 000 human intestinal SSU rRNA sequences. These HITChip probes were hybridized to a diverse set of human intestinal samples and SSU rRNA clones to validate its fingerprinting and quantification potential. Excellent reproducibility (median Pearson's correlation of 0.99) was obtained following hybridization with T7 polymerase transcripts generated in vitro from SSU rRNA gene amplicons. A linear dose–response was observed with artificial mixtures of 40 different representative amplicons with relative abundances as low as 0.1% of total microbiota. Analysis of three consecutively collected faecal samples from ten individuals (five young and five elderly adults) revealed temporal dynamics and confirmed that the adult intestinal microbiota is an individual-specific and relatively stable ecosystem. Further analysis of the stable part allowed for the identification of a universal microbiota core at the approximate genus level (90% sequence similarity). This core consists of members of Actinobacteria, Bacteroidetes and Firmicutes. Used as a phylogenetic fingerprinting tool with the possibility for relative quantification, the HITChip has the potential to bridge the gaps in our knowledge in the quantitative and qualitative description of the human gastrointestinal microbiota composition.

Enterocytozoon hepatopenaei sp. nov. (Microsporida: Enterocytozoonidae), a parasite of the black tiger shrimp Penaeus monodon (Decapoda: Penaeidae): Fine structure and phylogenetic relationships

A new microsporidian species, Enterocytozoon hepatopenaei sp. nov., is described from the hepatopancreas of the black tiger shrimp Penaeus monodon (Crustacea: Decapoda). Different stages of the parasite are described, from early sporogonal plasmodia to mature spores in the cytoplasm of host-cells. The multinucleate sporogonal plasmodia existed in direct contact with the host-cell cytoplasm and contained numerous small blebs at the surface. Binary fission of the plasmodial nuclei occurred during early plasmodial development and numerous pre-sporoblasts were formed within the plasmodium. Electron-dense disks and precursors of the polar tubule developed in the cytoplasm of the plasmodium prior to budding of early sporoblasts from the plasmodial surface. Mature spores were oval, measuring 0.7 × 1.1 μm and contained a single nucleus, 5–6 coils of the polar filament, a posterior vacuole, an anchoring disk attached to the polar filament, and a thick electron-dense wall. The wall was composed of a plasmalemma, an electron-lucent endospore (10 nm) and an electron-dense exospore (2 nm). DNA primers designed from microsporidian SSU rRNA were used to amplify an 848 bp product from the parasite genome (GenBank FJ496356). The sequenced product had 84% identity to the matching region of SSU rRNA from Enterocytozoon bieneusi. Based upon ultrastructural features unique to the family Enterocytozoonidae, cytoplasmic location of the plasmodia and SSU rRNA sequence identity 16% different from E. bieneusi, the parasite was considered to be a new species, E. hepatopenaei, within the genus Enterocytozoon.

Increased diversity of arbuscular mycorrhizal fungi in a long-term field experiment via application of organic amendments to a semiarid degraded soil.

In this study, we tested whether communities of arbuscular mycorrhizal (AM) fungi associated with roots of plant species forming vegetative cover as well as some soil parameters (amounts of phosphatase and glomalin-related soil protein, microbial biomass C and N concentrations, amount of P available, and aggregate stability) were affected by different amounts (control, 6.5 kg m(-2), 13.0 kg m(-2), 19.5 kg m(-2), and 26.0 kg m(-2)) of an urban refuse (UR) 19 years after its application to a highly eroded, semiarid soil. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, single-stranded conformation polymorphism analysis, sequencing, and phylogenetic analyses. One hundred sixteen SSU rRNA sequences were analyzed, and nine AM fungal types belonging to Glomus groups A and B were identified: three of them were present in all the plots that had received UR, and six appeared to be specific to certain amendment doses. The community of AM fungi was more diverse after the application of the different amounts of UR. The values of all the soil parameters analyzed increased proportionally with the dose of amendment applied. In conclusion, the application of organic wastes enhanced soil microbial activities and aggregation, and the AM fungal diversity increased, particularly when a moderate dose of UR (13.0 kg m(-2)) was applied.

Ultrastructure, SSU rRNA Gene Sequences and Phylogenetic Relationships of Flamella Schaeffer, 1926 (Amoebozoa), with Description of Three New Species

We isolated and described three new freshwater amoebozoan species that could be unambiguously assigned to the genus Flamella Schaeffer, 1926 by light microscopy. The phylogenetic position of the genus Flamella within the Amoebozoa was unknown, and gene sequence data were lacking. We sequenced the SSU rRNA gene of five Flamella spp., including a previously described F. aegyptia Michel et Smirnov, 1999. The phylogenetic trees inferred from these data showed, that Flamella is monophyletic and robustly branches within Amoebozoa. It belongs to a clade comprising Filamoeba spp., "Arachnula" sp., some protostelids and several SSU rRNA sequences of unidentified or uncultured eukaryotes. This clade consistently branched close to Archamoebae, Mycetozoa, Acramoeba dendroida and Multicilia marina; in contrast to the previous hypotheses, Flamella spp. did not show any relatedness either to Leptomyxida, or to Flabellinea. The ultrastructure of trophic amoebae and especially cysts of the species studied showed considerable similarity to Comandonia operculata Pernin et Pussard, 1979. We therefore suggest that Comandonia may be a junior synonym of Flamella, although more ultrastructural data about Comandonia operculata are necessary to test this hypothesis.

Exploring Microbial Diversity and Taxonomy Using SSU rRNA Hypervariable Tag Sequencing

Massively parallel pyrosequencing of hypervariable regions from small subunit ribosomal RNA (SSU rRNA) genes can sample a microbial community two or three orders of magnitude more deeply per dollar and per hour than capillary sequencing of full-length SSU rRNA. As with full-length rRNA surveys, each sequence read is a tag surrogate for a single microbe. However, rather than assigning taxonomy by creating gene trees de novo that include all experimental sequences and certain reference taxa, we compare the hypervariable region tags to an extensive database of rRNA sequences and assign taxonomy based on the best match in a Global Alignment for Sequence Taxonomy (GAST) process. The resulting taxonomic census provides information on both composition and diversity of the microbial community. To determine the effectiveness of using only hypervariable region tags for assessing microbial community membership, we compared the taxonomy assigned to the V3 and V6 hypervariable regions with the taxonomy assigned to full-length SSU rRNA sequences isolated from both the human gut and a deep-sea hydrothermal vent. The hypervariable region tags and full-length rRNA sequences provided equivalent taxonomy and measures of relative abundance of microbial communities, even for tags up to 15% divergent from their nearest reference match. The greater sampling depth per dollar afforded by massively parallel pyrosequencing reveals many more members of the “rare biosphere” than does capillary sequencing of the full-length gene. In addition, tag sequencing eliminates cloning bias and the sequences are short enough to be completely sequenced in a single read, maximizing the number of organisms sampled in a run while minimizing chimera formation. This technique allows the cost-effective exploration of changes in microbial community structure, including the rare biosphere, over space and time and can be applied immediately to initiatives, such as the Human Microbiome Project.

High-throughput diversity and functionality analysis of the gastrointestinal tract microbiota

The human gastrointestinal (GI) tract microbiota plays a pivotal role in our health. For more than a decade a major input for describing the diversity of the GI tract microbiota...