Molecular characterization of the microbial species that colonize human ileal and colonic mucosa by using 16S rDNA sequence analysis

Aim: The aim of this study was to characterize the bacterial community adhering to the mucosa of the terminal ileum, and proximal and distal colon of the human digestive tract. Methods and Results: Pinch samples of the terminal ileum, proximal and distal colon were taken from a healthy 35-year-old, and a 68-year-old subject with mild diverticulosis. The 16S rDNA genes were amplified using a low number of PCR cycles, cloned, and sequenced. In total, 361 sequences were obtained comprising 70 operational taxonomic units (OTU), with a calculated coverage of 82.6%. Twenty-three per cent of OTU were common to the terminal ileum, proximal colon and distal colon, but 14% OTU were only found in the terminal ileum, and 43% were only associated with the proximal or distal colon. The most frequently represented clones were from the Clostridium group XIVa (24.7%), and the Bacteroidetes (Cytophaga-Flavobacteria-Bacteroides ) cluster (27.7%). Conclusion: Comparison of 16S rDNA clone libraries of the hindgut across mammalian species confirms that the distribution of phylogenetic groups is similar irrespective of the host species. Lesser site-related differences within groups or clusters of organisms, are probable. Significance and Impact: This study provides further evidence of the distribution of the bacteria on the mucosal surfaces of the human hindgut. Data contribute to the benchmarking of the microbial composition of the human digestive tract.

A preliminary phylogenetic analysis of the Capsalidae (Platyhelminthes : Monogenea : Monopisthocotylea) inferred from large subunit rDNA sequences

Phylogenetic relationships within the Capsalidae (Monogenea) were examined Using large subunit ribosomal DNA sequences from 17 capsalid species (representing 7 genera, 5 subfamilies), 2 outgroup taxa (Monocotylidae) plus Udonella caligorum (Udonellidae). Trees were constructed using maximum likelihood, minimum evolution and maximum parsimony algorithms. An initial tree, generated from sequences 315 bases long, Suggests that Capsalinae, Encotyllabinae, Entobdellinae and Trochopodinae are monophyletic, but that Benedeniinae is paraphyletic. Analyses indicate that Neobenedenia, currently in the Benedeniinae, should perhaps be placed in 2 separate subfamily. An additional analysis was made which omitted 3 capsalid taxa (for which only short sequences were available) and all outgroup taxa because of alignment difficulties. Sequence length increased to 693 bases and good branch support was achieved. The Benedeniinae was again paraphyletic. Higher-level classification of the Capsalidae, evolution of the Entobdellinae and issues of species identity in Neobenedenia are discussed.

Multiple origins of parasitism in lice: phylogenetic analysis of SSU rDNA indicates that the Phthiraptera and Psocoptera are not monophyletic

The Paraneoptera (Hemipteroid Assemblage) comprises the orders Thysanoptera (thrips), Hemiptera (bugs), Phthiraptera (lice) and Psocoptera (booklice and barklice). The phylogenetic relationships among the Psocodea (Phthiraptera and Psocoptera), Thysanoptera and Hemiptera are unresolved, as are some relationships within the Psocodea. Here, we present phylogenetic hypotheses inferred from SSU rDNA sequences; the most controversial of which is the apparent paraphyly of the Phthiraptera, which are parasites of birds and mammals, with respect to one family of Psocoptera, the Liposcelididae. The order Psocoptera and the suborder that contains the Liposcelididae, the Troctomorpha, are also paraphyletic. The two remaining psocopteran suborders, the Psocomorpha and the Trogiomorpha, are apparently monophyletic. The Liposcelididae is most closely related to lice from the suborder Amblycera. These results suggest that the taxonomy of the Psocodea needs revision. In addition, there are implications for the evolution of parasitism in insects; parasitism may have evolved twice in lice or have evolved once and been subsequently lost in the Liposcelididae.

First report of three Kudoa species from Eastern Australia: Kudoa thyrsites from Mahi mahi (Coryphaena hippurus), Kudoa amamiensis and Kudoa minithyrsites n. sp. from sweeper (Pempheris ypsilychnus)

Fish species around the world are parasitized by myxozoans of the genus Kudoa, several of which infect and cause damage of commercial importance. In particular, Kudoa thyrsites and Kudoa amamiensis infect certain cultured fish species causing damage to muscle tissue, making the fish unmarketable. Kudoa thyrsites has a broad host and geographic range infecting over 35 different fish species worldwide, while K. amamiensis has only been reported from a few species in Japanese waters. Through morphological and molecular analyses we have confirmed the presence of both of these parasites in eastern Australian waters. In addition, a novel Kudoa species was identified, having stellate spores, with one polar capsule larger than the other three. The SSU rDNA sequence of this parasite was 1.5% different from K. thyrsites and is an outlier from K. thyrsites representatives in a phylogenetic analysis. Furthermore, the spores of this parasite are distinctly smaller than those of K. thyrsites, and thus it is described as Kudoa minithyrsites n. sp. Although the potential effects of K. minithyrsites n. sp. on its fish hosts are unknown, both K. thyrsites and K. amamiensis are associated with flesh quality problems in some cultured species and may be potential threats to an expanding aquaculture industry in Australia.

Kinetic and phylogenetic characterization of an anaerobic dechlorinating microbial community

The reductive dechlorination (RD) of tetrachloroethene (PCE) to vinyl chloride (VC) and, to a lesser extent, to ethene (ETH) by an anaerobic microbial community has been investigated by studying the processes and kinetics of the main physiological components of the consortium. Molecular hydrogen, produced by methanol-utilizing acetogens, was the electron donor for the PCE RD to VC and ETH without forming any appreciable amount of other chlorinated intermediates and in the near absence of methanogenic activity. The microbial community structure of the consortium was investigated by preparing a 1 6S rDNA clone library and by fluorescence in situ hybridization (FISH). The PCR primers used in the clone library allowed the harvest of 16SrDNA from both bacterial and archaeal members in the community. A total of 616 clones were screened by RFLP analysis of the clone inserts followed by the sequencing of RFLP group representatives and phylogenetic analysis. The clone library contained sequences mostly from hitherto undescribed bacteria. No sequences similar to those of the known RD bacteria like 'Dehalococcoides ethenogenes' or Dehalobacter restrictus were found in the clone library, and none of these bacteria was present in the RD consortium according to FISH. Almost all clones fell into six previously described phyla of the bacterial domain, with the majority (56(.)6%) being deep-branching members of the Spirochaetes phylum. Other clones were in the Firmicutes phylum (18(.)5%), the Chloroflexi phylum (16(.)4%), the Bacteroidetes phylum (6(.)3%), the Synergistes genus (11(.)1%) and a lineage that could not be affiliated with existing phyla (11(.)1%). No archaeal clones were found in the clone library. Owing to the phylogenetic novelty of the microbial community with regard to previously cultured microorganisms, no specific microbial component(s) could be hypothetically affiliated with the RD phenotype. The predominance of Spirochaetes in the microbial consortium, the main group revealed by clone library analysis, was confirmed by FISH using a purposely developed probe.

Limitations of the widely used GAM42a and BET42a probes targeting bacteria in the Gammaproteobacteria radiation

The 23S rRNA-targeted probes GAM42a and BET42a provided equivocal results with the uncultured gammaproteobacterium 'Candidatus Competibacter phosphatis' where some cells bound GAM42a and other cells bound BET42a in fluorescence in situ hybridization (FISH) experiments. Probes GAM42a and BET42a span positions 1027-1043 in the 23S rRNAand differ from each other by one nucleotide at position 1033. Clone libraries were prepared from PCR products spanning the 16S rRNA genes, intergenic spacer region and 23S rRNA genes from two mixed cultures enriched in 'Candidatus C. phosphatis'. With individual clone inserts, the 16S rDNA portion was used to confirm the source organism as 'Candidatus C. phosphatis' and the 23S rDNA portion was used to determine the sequence of the GAM42a/BET42a probe target region. Of the 19 clones sequenced, 8 had the GAM42a probe target (T at position 1033) and 11 had G at position 1033, the only mismatch with GAM42a. However, none of the clones had the BET42a probe target (A at 1033). Non-canonical base-pairing between the 23S rRNA of 'Candidatus C. phosphatis' with G at position 1033 and GAM42a (G-A) or BET42a (G-T) is likely to explain the probing anomalies. A probe (GAM42_C1033) was optimized for use in FISH, targeting cells with G at position 1033, and was found to highlight not only some 'Candidatus C. phosphatis' cells, but also other bacteria. This demonstrates that there are bacteria in addition to 'Candidatus C. phosphatis' with the GAM42_C1033 probe target and not the BET42a or GAM42a probe target.

Phylogenetic and physiological characterization of a heterotrophic, chemolithoautotrophic Thiothrix strain isolated from activated sludge

The sheathed filamentous bacterium known as strain CT3, isolated by micromanipulation from an activated sludge treatment plant in Italy, is a member of the genus Thiothrix in the gamma-Proteobacteria according to 16S rDNA sequence analysis. The closest phylogenetic neighbours of strain CT3 are strains I and Q(T), which were also isolated from activated sludge and belong to the species Thiothrix fructosivorans. These strains have respectively 99.2 and 99.4 % similarity to CT3 by 16S rDNA sequence comparison. CT3 shows 63-67 % DNA-DNA hybridization with strain I, which is the only currently viable strain of T. fructosivorans. CT3 is the second strain in the genus Thiothrix that has been shown to be capable of growing autotrophically with reduced sulfur compounds as the sole energy source; autotrophy was also confirmed in strain I. The first reported chemolithoautotrophic isolate of this genus was a strain of 'Thiothrix ramosa' that was isolated from a hydrogen sulfide spring and is morphologically distinguishable from all other described strains of Thiothrix, including CT3. CT3 is an aerobic organism that is non-fermentative, not capable of denitrification and able to grow heterotrophically. Autotrophy in the genus Thiothrix should be investigated more fully to better define the taxonomy of this genus.

Symbiont diversity within the widespread Scleractinian coral Plesiastrea versipore across the northwestern Pacific

The molecular diversity of symbiotic dinoflagellates associated with the widespread western Pacific coral Plesiastrea versipora was explored in order to examine if associations between reef-building corals and symbiotic dinoflagellates change with environment. Several ribosomal DNA genes with different evolutionary rates were used.. including the large subunit (28S), the 5.8S region and the internal transcribed spacers (ITS). The phylogenetic analysis of the 28S and 5.8S rDNA regions indicated that a single endosymbiont species, highly related to one of the species of Symbiodinium in clade C (=Synbiodinium goreaui, Trench et Blank), associates with P. versipora along the Ryukyu Archipelago. The persistence of the same endosymbiont within P. versipora across this wide array of latitudes may be a result of such features as the Kuroshio Current, which brings tropical temperatures as far north as Honshu, Japan. Analysis of the faster evolving ITS rDNA region revealed significant genetic variability within endosymbionts from different populations. This variation was due to a high degree of interpopulation variability, based on the proportion of pairwise variation detected among the populations (0.95% approximately). By comparison with other studies, the results also indicate that some ITS1 haplotypes from P. versipora endosymbionts seem to be widely distributed within the western Pacific Ocean, ranging from the Great Barrier Reef to the northeast of the China Sea.

Highly organized structure in the non-coding region of the psbA minicircle from clade C Symbiodinium

The chloroplast genes of dinoflagellates are distributed among small, circular dsDNA molecules termed minicircles. In this paper, we describe the structure of the non-coding region of the psbA minicircle from Symbiodinium. DNA sequence was obtained from five Symbiodinium strains obtained from four different coral host species (Goniopora tenuidens, Heliofungia actiniformis, Leptastrea purpurea and Pocillopora damicornis), which had previously been determined to be closely related using LSU rDNA region D1/D2 sequence analysis. Eight distinct sequence blocks, consisting of four conserved cores interspersed with two metastable regions and flanked by two variable regions, occurred at similar positions in all strains. Inverted repeats (IRs) occurred in tandem or 'twin' formation within two of the four cores. The metastable regions also consisted of twin IRs and had modular behaviour, being either fully present or completely absent in the different strains. These twin IRs are similar in sequence to double-hairpin elements (DHEs) found in the mitochondrial genomes of some fungi, and may be mobile elements or may serve a functional role in recombination or replication. Within the central unit (consisting of the cores plus the metastable regions), all IRs contained perfect sequence inverses, implying they are highly evolved. IRs were also present outside the central unit but these were imperfect and possessed by individual strains only. A central adenine-rich sequence most closely resembled one in the centre of the non-coding part of Amphidinium operculatum minicircles, and is a potential origin of replication. Sequence polymorphism was extremely high in the variable regions, suggesting that these regions may be useful for distinguishing strains that cannot be differentiated using molecular markers currently available for Symbiodinium.

Phylogeny, molecular and fossil dating, and biogeographic history of Annonaceae and Myristicaceae (Magnoliales)

Annonaceae and Myristicaceae, the two largest families of Magnoliales, are pantropical groups of uncertain geographic history. The most recent morphological and molecular phylogenetic analyses identify the Asian-American genus Anaxagorea as sister to all other Annonaceae and the ambavioids, consisting of small genera endemic to South America, Africa, Madagascar, and Asia, as a second branch. However, most genera form a large clade in which the basal lines are African, and South American and Asian taxa are more deeply nested. Although it has been suggested that Anaxagorea was an ancient Laurasian line, present data indicate that this genus is basically South American. These considerations may mean that the family as a whole began its radiation in Africa and South America in the Late Cretaceous, when the South Atlantic was narrower, and several lines dispersed from Africa-Madagascar into Laurasia as the Tethys closed in the Tertiary. This scenario is consistent with the occurrence of annonaceous seeds in the latest Cretaceous of Nigeria and the Eocene of England and with molecular dating of the family. Based on distribution of putatively primitive taxa in Madagascar and derived taxa in Asia, it has been suggested that Myristicaceae had a similar history. Phylogenetic analyses of Myristicaceae, using morphology and several plastid regions, confirm that the ancestral area was Africa-Madagascar and that Asian taxa are derived. However, Myristicaceae as a whole show strikingly lower molecular divergence than Annonaceae, indicating either a much younger age or a marked slowdown in molecular evolution. The fact that the oldest diagnostic fossils of Myristicaceae are Miocene seeds might be taken as evidence that Myristicaceae are much younger than Annonaceae, but this is implausible in requiring transoceanic dispersal of their large, animal-dispersed seeds.

Ptychogyliauchen, a new genus of Gyliauchenidae (Platyhelminthes: Digenea) from siganid fishes of the Indo-West Pacific

We propose a new genus of the Gyliauchenidae Fukui, 1929 ( Digenea), Ptychogyliauchen, gen. nov., for four new species that infect Indo-West Pacific siganid fishes. Ptychogyliauchen, gen. nov. is a morphologically distinctive genus, diagnosed principally by the presence of a highly convoluted oesophagus, which generally exceeds the total body length of the worm, and by the unusual folded structure of the ejaculatory duct. Ptychogyliauchen thetidis, sp. nov. is designated as the type species, and is described from the intestine of Siganus punctatus (Siganidae) from Heron Island, Great Barrier Reef, Queensland, Australia. Ptychogyliauchen himinglaeva, sp. nov. is described from the intestine of Siganus corallinus ( Siganidae) from Lizard Island, Great Barrier Reef, Queensland, Australia. Ptychogyliauchen leucothea, sp. nov. is described from the intestine of S. argenteus, and further recorded from S. fuscescens, off Ningaloo, Western Australia, Australia. Ptychogyliauchen thistilbardi, sp. nov. is described from the intestine of S. doliatus from Noumea, New Caledonia, and is also found in S. argenteus, S. canaliculatus, S. corallinus and S. spinus from Noumea, New Caledonia, and Moorea, Tahiti, French Pacific. Ptychogyliauchen thistilbardi, sp. nov. also occurs in the intestine of Chaetodon citrinellus (Chaetodontidae) from Moorea. A key to species is provided. All species have been described following morphological examination using light microscopy, and specimens of P. thetidis, sp. nov., P. leucothea, sp. nov. and P. thistilbardi, sp. nov. have been characterised using molecular methods. Sequences were obtained for a combination of nuclear ribosomal (28S (D1-D3) and ITS2) and mitochondrial (ND1) genes. A phylogenetic analysis of sequenced specimens of Ptychogyliauchen, gen. nov. was conducted using species of Petalocotyle Ozaki, 1934 for outgroup comparison. This analysis, based on alignments of the ITS2 and 28S (D1-D3) rDNA genes, supports monophyly of the geographically widespread P. thistilbardi, sp. nov., which is known from both siganid and chaetodontid hosts. We discuss the taxonomy of the genus and the host associations of each species and the group.

The life cycle of Paracardicoloides yamagutii Martin, 1974 (Digenea: Sanguinicolidae)

The sanguinicolids Paracardicoloides yamagutii Martin, 1974 and Plethorchis acanthus Martin, 1975 were obtained from their definitive hosts, Anguilla reinhardtii Steindachner and Mugil cephalus Linnaeus (respectively) in the tributaries of the Brisbane River, Queensland, Australia. Two putative sanguinicolid cercariae were collected from a hydrobiid gastropod, Posticobia brazieri Smith, in the same waters. The two cercariae differ markedly in size and the form of their sporocysts. Both putative cercariae develop in the digestive gland of Po. brazieri. The ITS2 rDNA region from these sanguinicolids and a Clinostomum species (utilised as an outgroup due to the close morphological similarities between the cercarial stages of the Clinostomidae and the Sanguinicolidae) were sequenced and aligned. Comparison of the ITS2 sequences showed one cercaria to be that of P. yamagutii. This is the first sanguinicolid life history determined by a molecular method. P. yamagutii is the fourth sanguinicolid known to utilise a freshwater hydrobiid gastropod as its intermediate host. ITS2 rDNA is effective in distinguishing sanguinicolids at the species level.