Molecular characterization and phylogenetic analyses of Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970 based on 18S rRNA gene sequence data.

In the present study, we provided the first 18S rRNA gene sequence data of two Tripartiella species, Tripartiella macrosoma Basson and Van As, 1987 and Tripartiella obtusa Ergens and Lom, 1970, which were isolated from Tachysurus fulvidraco (Richardson, 1846) and Hemibarbus maculatus Bleeker, 1871 in Chongqing, China, respectively. Morphologically, both species fall within the morphometry range of the original descriptions and are very similar to the original populations in the overall appearance of the adhesive disc. Tripartiella macrosoma can be easily distinguished from the other Tripartiella species by possessing the denticle with a long strip and conspicuously inclined backward blade and a robust and short ray. Tripartiella obtusa is mainly characterized by a broad blade and a relatively long ray. Phylogenetically, T. macrosoma clustered with Trichodinella myakkae (Mueller, 1937) Raabe, 1950 and further with Trichodinella sp., which was sister to a group that includes four populations of Trichodinella epizootica (Raabe, 1950) Srámek-Husek, 1953; finally, they formed a small clade with T. obtusa. This result suggested that T. macrosoma had a closer relationship with Trichodinella spp. than with T. obtusa and T. obtusa diverged earlier than T. macrosoma and Trichodinella spp. By combining morphological and molecular data, the polyphyletic characteristics of Tripartiella and Trichodinella were further analyzed, and the results revealed that the validity of the genus Tripartiella is doubtful.

Morphological Redescription and Molecular Identification of Trichodina reticulata Hirschmann & Partsch, 1955 (Ciliophora, Mobilida, Trichodinidae) with the Supplemental New Data of SSU rDNA and ITS-5.8S rDNA.

During a survey of trichodinids in Chongqing, China, two populations of Trichodina reticulata Hirschmann & Partsch, 1955 were isolated from gills of the freshwater fishes, Carassius auratus and Ctenopharyngodon idellus, and 11 molecular samples containing small subunit (SSU) rDNA and internal transcribed spacer (ITS)-5.8S rDNA were newly sequenced. Both populations fell within the range of morphometry and corresponded well with the diagnostic morphological features of Trichodina reticulata Hirschmann & Partsch, 1955. It should be noted that one population possessed obvious central granules in the adhesive disc, while the other one did not. The detailed morphological redescription for these two populations is given in this work. Although some intraspecific differences were found, both populations were confirmed as Trichodina reticulata based on the morphological study and molecular data, including the sequence analysis of the primary and secondary structures of SSU rDNA, genetic distances and phylogenetic tree. Our study supports the assertion that central granules in the adhesive disc might be an auxiliary feature rather than a heritable character for determining taxonomic affiliations among trichodinids.

A New Molecular Approach Based on the Secondary Structure of Ribosomal RNA for Phylogenetic Analysis of Mobilid Ciliates.

We analyzed the secondary structure of the small subunit (SSU) rRNA genes of Mobilida (Ciliophora, Peritrichia) and found that the secondary structures of some regions within the SSU-rRNA gene are distinct between the families Trichodinidae and Urceolariidae. Therefore, some of these important regions including H10, H11, H17, H47, H29, H30, H37, E10-1, H45-H46, and V4 (E23-4, E23-7) could be used as the barcodes for classification of these two families. In contrast, V4 (E23-1, E23-2) belongs to a hypervariable region and is not a good barcode at the genus level because of its great inter-specific variation. Our results indicated that the comprehensive analysis of the secondary structure of SSU-rRNA genes is a reliable auxiliary approach for phylogenic study of mobilid ciliates. It was further found that the coevolution between hosts or habitats and the Mobilida ciliates was existent, because the host types and their habitats were critical ecological factors that influenced the evolution of Mobilida ciliates.

Morphological and Molecular Identification of the New Species, Trichodina pseudoheterodentata sp. n. (Ciliophora, Mobilida, Trichodinidae) from the Channel Catfish, Ictalurus punctatus, in Chongqing China.

It is difficult to differentiate similar trichodinids solely based on morphological examination, thus other identification methods, such as molecular identification, are necessary for identification. One mobilid ciliate named Trichodina pseudoheterodentata sp. n. was isolated from the gills of channel catfish, Ictalurus punctatus, in Chongqing, China. In the present study, its SSU rDNA was sequenced for the first time. Based on the results from both morphological identification and SSU rDNA sequencing, the new species was identified and compared with similar species. The morphological analysis revealed that T. pseudoheterodentata is a large Trichodina species (cell diameter 73.0-82.5 µm) and possesses robust denticles with broad blades and well-developed blade connections. Characterization of its primary and secondary SSU rDNA structures indicated that T. pseudoheterodentata was distinctly different from congeneric species in H12, H15, E10_1, and V4 regions. Phylogenetic analysis revealed that the genetic distances among the new species and similar species reached interspecific levels, furthermore, the phylogenetic study also validated the identification of T. pseudoheterodentata and its placement in the genus Trichodina.

New Comparative Analysis Based on the Secondary Structure of SSU-rRNA Gene Reveals the Evolutionary Trend and the Family-Genus Characters of Mobilida (Ciliophora, Peritrichia).

In order to reveal the structural evolutionary trend of Mobilida ciliates, twenty-six SSU-rRNA sequences of mobilid species, including seven ones newly sequenced in the present work, were used for comparative phylogenic analysis based on the RNA secondary structure. The research results indicate that all the secondary structures except domains Helix 10, Helix 12, and Helix 37 could be regarded as the criterions in classification between the family Trichodinidae and Urceolariida, and four regions including Helix E10-1, Helix 29, Helix 43, and Helix 45-Helix 46 could be as criterions in classification between the genus Trichodinella and Trichodina in family Trichodinidae. After the analysis of common structural feature within the Mobilida, it was found that the secondary structure of V6 could prove the family Urceolariidae primitive status. This research has further suggested that the genus Trichodina could be divergent earlier than Trichodinella in the family Trichodinidae. In addition, the relationship between the secondary structure and topology of phylogenic tree that the branching order of most clades corresponds with the secondary structure of species within each clade of phylogenetic tree was first uncovered and discussed in the present study.

Phylogenetic analyses of Trichodinids (Ciliophora, Oligohymenophora) inferred from 18S rRNA gene sequence data.

Partial 18S rRNA gene sequences of the three trichodinids, namely Trichodina modesta Lom, 1970, Trichodina paraheterodentata Tang and Zhao 2012. and Trichodinella epizootica (Raabe 1950) Srámek-Husek, 1953, were acquired and used to construct phylogenetic trees. The results revealed that Trichodinella epizootica clustered with Trichodinella sp.; Trichodina paraheterodentata Tang and Zhao 2012 was sister to the clade composed of Trichodina heterodentata Duncan, 1977 and Trichodina nobilis Chen, 1963; Trichodina modesta Lom, 1970 clustered with Trichodina reticulata Hirschman and Partsch, 1955. The branching order of species within the Mobilia clade was closely correlated with GC content. Furthermore, blade morphology was also found to be the primary morphological character in determining the phylogenetic relationships among members of the genus Trichodina. The present findings suggest that the genus Trichodina is paraphyletic when species of Trichodinella are included in the analyses.

First records of three Tripartiella species (Ciliophora, Oligohymenophora, Peritrichida) from freshwater fishes along Yangtze River in China.

During parasitological surveys along Yangtze River in China, three species of trichodinid ectoparasites (Ciliophora, Oligohymenophora, Peritrichia) belonging to the genus Tripartiella Lom, 1959, were found from freshwater fishes. Tripartiella obtusa Ergens & Lom, 1970 and Tripartiella macrosoma Basson & Van As, 1987 were found from the same fish host, Rhinogobio typus; and Tripartiella orthodens Basson & Van As, 1987 was collected from another fish host, Pelteobagrus nitidus. These three Tripartiella species all fall within the range of morphometry and agree closely in the overall appearance of the adhesive disc with the original populations. In the present work, both Tripartiella obtusa and Tripartiella orthodens are recorded for the first time in Asia. The host fish, Rhinogobio typus was recorded as a new host for Tripartiella obtusa and Tripartiella macrosoma. For each Tripartiella species, detailed descriptions are provided based the examinations of specimens prepared using the dry silver nitrate impregnation in the present study.

Molecular characterization and phylogenetic analysis of Paratrichodina africana Kazubski and El-Tantawy, 1986 based on 18S rRNA gene data with the evolutionary hypothesis of trichodinids.

In this study, we provided the morphological data and the first 18S rRNA gene data of Paratrichodina africana Kazubski and El-Tantawy, 1986, isolated from hybrids of Oreochromis niloticus × Oreochromis mossambicus in Chongqing, China. Morphologically, P. africana is mainly characterized by the triangular blade and prominent anterior projection. The present population is consistent with the original populations in the overall appearance of the adhesive disc, and falls within the morphometry range of the original descriptions. Phylogenetically, P. africana was clustered into one large clade with Trichodinella and Tripartiella species, which was nested within Trichodina ones with strong support. By combining morphological and molecular data, our results revealed that the validity of the genus Paratrichodina was doubtful, and suggested that the three genera Trichodinella, Tripartiella, and Paratrichodina should be incorporated into one independent genus. In addition, we provided morphological and molecular data of additional eight trichodinids, and further performed the phylogenetic analysis and traced the evolution history of trichodinids' five morphological and bionomical characters for the first time by taking advantage of the current GenBank data. According to the present results, one evolutionary hypothesis of trichodinids was proposed as follows. The most recent common ancestor of trichodinids inhabiting the freshwater environment as a symbiont of vertebrates should evolve from the ancestor with a long-spiral adoral ciliary turn. The first differentiated Trichodina species should be parasitic on one vertebrate distributed in the freshwater environment. During their evolution, some trichodinids expanded to the marine environment, and some switched to invertebrates in the freshwater environment. The denticle of some freshwater Trichodina species became narrower, and the adoral ciliary spiral turn got shorter, forming the ancestor-oid organism with a short-spiral adoral ciliary turn. Then, those Trichodinella, Tripartiella, and Paratrichodina species might evolve from those ancestor-oid organisms with short-spiral adoral ciliary turn.

The nuclear apparatus and chromosomal DNA of the microsporidian Nosema antheraeae.

The microsporidian Nosema antheraeae is a pathogen of the Chinese oak silkmoth Antheraea pernyi, the molecular karyotype of which is still poorly understood. Here the diplokaryon of N. antheraeae strain NP-YY has been visualized both by fluorescence and electron microscopy. In addition, pulsed-field gel electrophoresis (PFGE) showed that the haploid genome of N. antheraeae is approximately 9.3-9.5 million base pairs organized into 15 chromosomal bands. The mean fluorescence intensity of N. antheraeae and Nosema bombycis DNA measured by flow cytometry confirmed that the genome size of these two species was congruent with measurements obtained by PFGE. These initial results on the chromosome organization of N. antheraeae provide a foundation for the comparative genomics of N. antheraeae with other species of Nosema.

Morphological redescription and molecular characterization of Trichodina matsu Basson amp; Van As, 1994 (Ciliophora, Mobilida, Trichodinidae) infecting Tachysurus fulvidraco (Richardson, 1846) from Chongqing, China.

During a survey of parasitic ciliates in Chongqing, China, Trichodina matsu Basson Van As, 1994 was isolated from gills of Tachysurus fulvidraco. Furthermore, the 18S rRNA gene and ITS-5.8S rRNA region of T. matsu were sequenced for the first time and applied for the species identification and comparison with similar species in the present study. Based on the morphological and molecular comparisons, the results indicate that T. matsu is an ectoparasite specific for the Siluriformes catfish. Based on the analyses of genetic distance, multiple sequence alignments, and phylogenetic analyses, no obvious differentiation within populations of T. matsu was found. In addition, the 'Trichodina hyperparasitis (KX904933) in GenBank is a misidentification and appears to be conspecific with T. matsu according to the comparison of morphological and molecular data.

Identification of NbME MITE families: potential molecular markers in the microsporidia Nosema bombycis.

Six novel families of miniature inverted-repeat transposable elements (MITEs) were characterized in the microsporidia Nosema bombycis and were named NbMEs. The structural characteristics and the distribution of NbME copies in the N. bombycis genome were investigated, and it was found that portions of NbMEs are associated with gene sections. Potential molecular markers for various N. bombycis strains were identified in this study through utilization of the MITE-AFLP technique. Three distinct pathogenic isolates collected from different areas were distinguished, and polymorphisms were detected using the NbME5 marker, thereby establishing this NbME as a potential marker for studying isolate variation in N. bombycis.

Trichodinid ectoparasites (Ciliophora: Peritricha) from Misgurnus anguillicaudatus (Cantor) and Anodonta woodiana (Lea) in China, with descriptions of two new species of Trichodina Ehrenberg, 1838.

During parasitological surveys in Chongqing, China, three trichodinids infecting a freshwater fish, Misgurnus anguillicaudatus (Cantor), and a mollusc, Anodonta woodiana (Lea), were investigated. Two of these species were found to be new. Trichodina lechriodentata n. sp., from the fish, was identified by its small-sized body and irregularly rectangular blade, while T. rara n. sp., from the mollusc, is characterised by its overall dimensions, denticle morphology and the presence of a white speckle in some specimens. T. modesta Lom, 1979 is also described from the fish. Photomicrographs and morphometric data are presented for all of the species.