Redescription and molecular characterization of two Trichodina species (Ciliophora, Peritrichia, Mobilida) from freshwater fish in China.

During an investigation of parasitic ciliates in northern China, two Trichodina species, T. acuta Lom, 1970 and T. nigra Lom, 1960, were isolated from the freshwater fish Cyprinus carpio Linnaeus, 1758. The morphology of each species was investigated based on dry silver nitrate-stained specimens. In addition, the molecular phylogeny of each was analyzed based on small subunit ribosomal DNA (SSU rDNA) sequence data. Trichodina acuta can be distinguished from its congeners by the undefined periphery of the central circle, the distinct gap between the rays and the central circle, and the distinctly sickle-shaped blades. Trichodina nigra is a cosmopolitan ciliate and is characterized by its densely linked denticles, broad, rounded spatula-shaped blades, robust central parts, and well developed rays. Phylogenetic analyses revealed that T. acuta and T. nigra nest within different clades, supporting the assertion that the GC content of SSU rDNA sequences could reflect evolutionary relationships among Trichodina species.

Taxonomy and molecular phylogeny of a new freshwater ciliate Frontonia apoacuminata sp. nov. (Protista, Ciliophora, Oligohymenophorea) from Qingdao, PR China.

The morphology and ciliature of a new freshwater ciliate, Frontonia apoacuminata sp. nov., isolated from an artificial pond in Qingdao, PR China, were investigated using live observation and silver staining methods. The main features separating F. apoacuminata sp. nov. from its congeners are as follows: a broad elliptical body that is slightly pointed at the posterior end, four ophryokineties, one or two spherical micronuclei of a 'compact' type, a dorsally positioned contractile vacuole, and peniculi 1-3 each with five kinetosome rows though the left-most two rows in peniculus 3 are extremely shortened (with only two or three kinetosomes each). Additionally, an improved diagnosis of F. acuminata is provided. Phylogenetic analyses based on the small subunit ribosomal RNA (SSU rRNA) gene show that F. apoacuminata sp. nov. clusters with F. atra, F. minuta, F. acuminata and F. terricola. These five species group with Disematostoma, Marituja and Stokesia rather than with other Frontonia species, causing polyphyly of the genus Frontonia.

Morphology, morphogenesis and phylogeny of Anteglaucoma orientalis n. sp. (Ciliophora, Oligohymenophorea), a freshwater hymenostomatid from northeastern China.

A new hymenostomatid ciliate, Anteglaucoma orientalis n. sp., isolated from a freshwater pond in Harbin, northeastern China, was investigated using live observation and silver staining methods. Anteglaucoma orientalis is characterized as follows: size in vivo about 50-60 × 30-35 µm; oval body shape; buccal area occupies about 25% of body length; 28-36 somatic kineties; membranelle 1 having six or seven basal body rows, membranelle 2 five to seven rows, and membranelle 3 three rows; single macronucleus with one micronucleus attached. Morphogenesis of the genus Anteglaucoma is revealed for the first time. The main events during binary fission are as follows: morphogenesis begins with proliferation of kinetosomes in the middle part of postoral kinety 1, and kinetosomes of this primordial field multiply and organize to finally form the paroral membrane and membranelles 1-3 of the opisthe; the parental apparatus in the proter does not take part in the stomatogenetic process. Phylogenetic analyses based on SSU rRNA gene sequences show that Anteglaucoma orientalis n. sp. clusters with the type species, A. harbinensis Pan et al., 2017, with full support.

A checklist of species of the family Zoothamniidae (Ciliophora: Peritrichia), symbionts of crustaceans.

Eight genera are included in the family Zoothamniidae, and at the present the data of the epibiotic species on crustaceans is not updated. Thus, the main goal of the present work is to provide the checklist of zoothamniids worldwide reported as associated to several groups of crustaceans, including some data of their geographic distribution, and also to provide the complete list of species of this peritrich group. We obtained all available data of family Zoothamniidae and checked their taxonomic status. Then we provided the complete list of species including those reported as epibionts on crustacean hosts. We obtained 156 species included in eight genera of Zoothamniidae, being 85 species recorded as symbionts on 86 species of hosts. Genus Zoothamnium contained the higher number (56) of epibiotic species on crustaceans, and genus Pseudohaplocaulus has not been reported for any crustacean. The reviewed data highlights the concentration of records in Europe, and stands out the need to amplify sampling and studies in the Southern Hemisphere. The main groups of crustaceans infested by zoothamniids are the amphipods, isopods and decapods.

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.

Morphology and molecular phylogeny of three Epistylis species found in freshwater habitats in China, including the description of E. foissneri n. sp. (Ciliophora, Peritrichia).

The peritrich genus Epistylis is speciose, however many species lack complete morphological description based on modern criteria and/or molecular data. In the present study, one new species, i.e., E. foissneri n. sp., and two morphologically similar species, i.e., E. hentscheli Kahl, 1935 and E. vaginula Stokes, 1884, collected from freshwater habitats in China, were studied. Epistylis foissneri n. sp. is characterized by its extremely slender zooids encased in a gelatinous sheath, symmetrically dichotomously branched stalk, trochal band located at mid-body, contractile vacuole located on dorsal wall of infundibulum, infundibular polykinety 3 (P3) composed of three equal-length rows that terminate above infundibular polykinety 1 (P1), 105-110 silverlines between the peristome and the trochal band, and about 110 silverlines between the trochal band and the scopula. Epistylis hentscheli is characterized by its asymmetric pyriform zooids (average length ca. 160 µm in vivo), dichotomously branched stalk with transverse striations on the surface of the upper portion, P3 three-rowed and terminating slightly above P1, 60-75 silverlines between the peristome and the trochal band, and 55-90 silverlines between the trochal band and the scopula. Epistylis vaginula is characterized by its elongated body shape (about 100 µm in length in vivo), dichotomously branched and smooth stalk, P3 three-rowed and terminating above P1, 80-100 silverlines between the peristome and the trochal band, and 45-80 silverlines between the trochal band and the scopula. The small subunit ribosomal DNA gene (SSU rDNA) of these three species was sequenced and supported the validity of each. Phylogenetic analyses based on SSU rDNA sequence data revealed that all three morphospecies group with other congeners within the major clade of Epistylis.

Large-scale phylogenomic analysis provides new insights into the phylogeny of the class Oligohymenophorea (Protista, Ciliophora) with establishment of a new subclass Urocentria nov. subcl.

The class Oligohymenophorea is one of the most diverse assemblage of ciliated protists, which are particularly important in fundamental biological studies including understanding the evolutionary relationships among the lineages. Phylogenetic relationships within the class remain largely elusive, especially within the subclass Peniculia, which contains the long-standing problematic taxa Urocentrum and Paranassula. In the present study, we sequenced the genomes and/or transcriptomes of six non-culturable oligohymenophoreans using single-cell sequencing techniques. Phylogenomic analysis was performed based on expanded taxon sampling of 85 taxa, including 157 nuclear genes encoding 36,953 amino acids. The results indicate that: (1) urocentrids form an independent branch that is sister to the clade formed by Scuticociliatia and Hymenostomatia, which, together with the morphological data, supports the establishment of a new subclass, Urocentria n. subcl., within Oligohymenophorea; (2) phylogenomic analysis and ortholog comparison reveal a close relationship between Paranassula and peniculines, providing corroborative evidence for removing Paranassula from Nassulida and elevating it as an order, Paranassulida, within the subclass Peniculia; (3) based on the phylogenomic analyses and morphological data, we hypothesize that Peritrichia is the earliest diverging clade within Oligohymenophorea while Scuticociliatia and Hymenostomatia share the most common ancestor, followed successively by Urocentria and Peniculia. In addition, stop codon analyses indicate that oligohymenophoreans widely use UGA as the stop codon, while UAR are reassigned to glutamate (peritrichs) or glutamine (others), supporting the evolutionary hypothesis.

Incorporating mitogenome sequencing into integrative taxonomy: The multidisciplinary redescription of the ciliate Thuricola similis (Peritrichia, Vaginicolidae) provides new insights into the evolutionary relationships among Oligohymenophorea subclasses.

The evolutionary relationships among Oligohymenophorea subclasses are under debate as the phylogenomic analysis using a large dataset of nuclear coding genes is significantly different to the 18S rDNA phylogeny, and it is unfortunately not stable within and across different published studies. In addition to nuclear genes, the faster-evolving mitochondrial genes have also shown the ability to solve phylogenetic problems in many ciliated taxa. However, due to the paucity of mitochondrial data, the corresponding work is scarce, let alone the phylogenomic analysis based on mitochondrial gene dataset. In this work, we presented the characterization on Thuricola similis Bock, 1963, a loricate peritrich (Oligohymenophorea), incorporating mitogenome sequencing into integrative taxonomy. As the first mitogenome for the subclass Peritrichia, it is linear, 38,802 bp long, and contains two rRNAs, 12 tRNAs, and 43 open reading frames (ORFs). As a peculiarity, it includes a central repeated region composed of tandemly repeated A-T rich units working as a bi-transcriptional start. Moreover, taking this opportunity, the phylogenomic analyses based on a set of mitochondrial genes were also performed, revealing that T. similis, as a representative of Peritrichia subclass, branches basally to other three Oligohymenophorea subclasses, namely Hymenostomatia, Peniculia, and Scuticociliatia. Evolutionary relationships among those Oligohymenophorea subclasses were discussed, also in the light of recent phylogenomic reconstructions based on a set of nuclear genes. Besides, as a little-known species, T. similis was also redescribed and neotypified based on data from two populations collected from wastewater treatment plants (WWTPs) in Brazil and Italy, by means of integrative methods (i.e., living observation, silver staining methods, scanning and transmission electron microscopy, and 18S rDNA phylogeny). After emended diagnosis, it is characterized by: (1) the sewage habitat; (2) the lorica with a single valve and small undulations; (3) the 7-22 µm-long inner stalk; and (4) the presence of only a single postciliary microtubule on the left side of the aciliferous row in the haplokinety. Among Vaginicolidae family, our 18S rRNA gene-based phylogenetic analysis revealed that Thuricola and Cothurnia are monophyletic genera, and Vaginicola could be a polyphyletic genus.

Functional Ecology of Two Contrasting Freshwater Ciliated Protists in Relation to Temperature.

We conducted microcosm experiments with two contrasting freshwater ciliates on functional traits (FTs) related to their growth rate (numerical response, NR) and ingestion rate (functional response, FR) over a range of ecologically relevant temperatures. Histiobalantium bodamicum and Vorticella natans are common planktonic ciliates but their abundance, swimming behavior, and temperature tolerance are different. In contrast to most sessile peritrich species, the motile V. natans is not strictly bacterivorous but also voraciously feeds upon small algae. We observed three main alterations in the shape of NR of both species with temperature, that is, change in the maximum growth rate, in the initial slope and in the threshold food level needed to sustain the population. Similarly, maximum ingestion rate, gross growth efficiency (GGE), and cell size varied with temperature and species. These findings caution against generalizing ciliate performance in relation to the ongoing global warming. Our results suggest that V. natans is the superior competitor to H. bodamicum in terms of temperature tolerance and bottom-up control. However, the abundance of V. natans is usually low compared to H. bodamicum and other common freshwater ciliates, suggesting that V. natans is more strongly top-down controlled via predation than H. bodamicum. The taxonomic position of V. natans has been debated. Therefore, to confirm species and genus affiliation of our study objects, we sequenced their small subunit ribosomal RNA (SSU rDNA) gene.

Further insights into the phylogeny of peniculid ciliates (Ciliophora, Oligohymenophorea) based on multigene data.

Peniculids comprise a large order of ciliated protists in Class Oligohymenophorea having many unresolved evolutionary relationships. Herein, we report 27 new sequences, including 18S rRNA, ITS1-5.8S- ITS2 rRNA, 28S rRNA and the mitochondrial cox1 genes of eight peniculids. We conducted phylogenetic analyses based on each these markers and on a four-gene concatenated data set (18S rRNA, ITS1-5.8S- ITS2 rRNA, 28S rRNA, and cox1 gene). The main findings are: 1) subclass Peniculia and family Parameciidae are monophyletic, with genus Frontonia remaining non-monophyletic; 2) Urocentrids have traditionally been regarded as a family, multi-gene analyses support the rank of Urocentrida and consistently recovers this order as sister to Peniculida, and Urocentrida and Peniculida comprise subclass Peniculia in agreement with Lynn's (2008) classification; 3) discrepancies between multiple-gene phylogenies, and conflicts with morphologic data regarding genus Frontonia necessitate expansion and revision of species diagnoses and we propose consideration of Group III of Frontonia (including F. didieri, F. ocularis, F. anatolica, F. pusilla and F. elegans) as incertae sedis in Peniculida; 4) multi-gene analyses of Parameciidae support five previously established subgenera. Paramecium buetschlii is placed in subgenus Chloroparamecium, and P. chlorelligerum into subgenus Viridoparamecium.

Morphological, Phylogenetic and Ecophysiological Characterization of a New Ciliate, Platynematum rossellomorai n. sp. (Oligohymenophorea, Scuticociliatia), Detected in a Hypersaline Pond on Mallorca, Spain.

With highly specialized morphology and unexplored functional capacities, ciliates from extreme habitats are drawing increasing attention. During a microbial investigation of a solar saltern pond (salinity 240‰) on Mallorca, Spain, a previously unknown scuticociliate, Platynematum rossellomorai n. sp. was isolated, cultured and studied using a tripartite approach consisting of a morphological description, a molecular analysis and an ecophysiological characterization. The ciliate has distinct morphological characteristics and its main diagnostic features include a large anteriorly positioned oral area (occupying almost half of the body length), two caudal cilia and a small number of somatic kineties. However, due to the most important generic feature of Cinetochilidae, the consistency of the arrangement of the adoral membranes, the ciliate is classified as a new member of the genus Platynematum. Its 18S rRNA gene sequence shows a sequence similarity of 91.0% to the closest deposited relative, Platynematum salinarum, and a phylogenetic analysis reveals a close relationship to other members of the family Cinetochilidae Perty, 1852. Growth experiments identify the ciliate as a borderline halophile, with a tolerance range between 180 and 280‰ salinity. The ciliate apparently accumulates the compatible solutes glycine betaine and ectoine to counterbalance osmotic stress, however, other osmoregulatory mechanisms are not excluded.

Isolation, characterization and virulence of Mesanophrys sp. (Ciliophora: Orchitophryidae) in farmed swimming crab (Portunus trituberculatus) in eastern China.

A disease outbreak occurred in swimming crab (Portunus trituberculatus) farmed in eastern China, with a mortality rate of more than 80%. To further investigate the characteristics and pathogenesis, we reported isolation, characterization and virulence of the causative agent of this disease from 10 sick crabs. Histopathological observation found that multiple tissues, especially haemolymph, contained lots of ciliates. The ciliate was isolated and cultured in vitro, and molecular and morphological studies were done. The results showed that SSU rDNA and LSU rDNA sequences of the ciliate were similar to Mesanophrys ciliates (>96.81%), while ITS1-5.8s-ITS2 sequence was similar to Mesanophrys pugettensis (95.37%) and identical to Orchitophrya stellarum (100%). Furthermore, the results of the morphological study confirmed that the ciliate was similar to Mesanophrys ciliates and O. stellarum cultured in supportive media, but different from O. stellarum cultured in living sperm cells of starfish (Leptasterias spp.). Also, the growth of the ciliate did not interfere with light, which was different from O. stellarum. Accordingly, the ciliate was classified as genus Mesanophrys and temporarily named as Mesanophrys sp. In addition, experimental infection confirmed that Mesanophrys sp. was the pathogen that infected farmed crabs. In summary, Mesanophrys sp. was first isolated and characterized in P. trituberculatus.

Morphology, molecular phylogeny, and taxonomy of trichodinids (Ciliophora, Mobilida) from Calanoid copepods.

Trichodinid ciliates were isolated from calanoid copepods and were characterized by morphological and molecular techniques. The trichodinids found in this study were morphologically similar to Trichodina acuta, but morphometric differences were observed between our specimens and T. acuta. The T. acuta specimens, isolated from fish in several geographical locations around the world, were slightly larger than specimens in our population. In fact, our morphological and morphometric data are more similar to Trichodina diaptomi populations, also found on copepods. Moreover, our phylogenetic analyses revealed that the 18S rRNA gene sequence from our samples emerged in a clade with two T. acuta sequences, infesting fish hosts in China. These three sequences showed 100% identity. Our data, along with a taxonomic review, suggest that the species T. diaptomi, described recently, is actually a synonym of T. acuta and that this species may be related to fish and copepod hosts. In conclusion, we highlight the urgent need to better investigate the polymorphism and host specificity within the Mobilida, as this data may represent important characters to better understand the evolution into this order.

Molecular characterization and gene expression modulation of the alternative oxidase in a scuticociliate parasite by hypoxia and mitochondrial respiration inhibitors.

Philasterides dicentrarchi is a marine benthic microaerophilic scuticociliate and an opportunistic endoparasite that can infect and cause high mortalities in cultured turbot (Scophthalmus maximus). In addition to a cytochrome pathway (CP), the ciliate can use a cyanide-insensitive respiratory pathway, which indicates the existence of an alternative oxidase (AOX) in the mitochondrion. Although AOX activity has been described in P. dicentrarchi, based on functional assay results, genetic evidence of the presence of AOX in the ciliate has not previously been reported. In this study, we conducted genomic and transcriptomic analysis of the ciliate and identified the AOX gene and its corresponding mRNA. The AOX gene (size 1,106 bp) contains four exons and three introns that generate an open reading frame of 915 bp and a protein with a predicted molecular weight of 35.6 kDa. The amino acid (aa) sequence of the AOX includes an import signal peptide targeting the mitochondria and the protein is associated with the inner membrane of the mitochondria. Bioinformatic analysis predicted that the peptide is a homodimeric glycoprotein, although monomeric forms may also appear under native conditions, with EXXH motifs associated with the diiron active centers. The aa sequences of the AOX of different P. dicentrarchi isolates are highly conserved and phylogenetically closely related to AOXs of other ciliate species, especially scuticociliates. AOX expression increased significantly during infection in the host and after the addition of CP inhibitors. This confirms the important physiological roles of AOX in respiration under conditions of low levels of O2 and in protecting against oxidative stress generated during infection in the host.

Two new scuticociliates from southern China: Uronema apomarinum sp. nov. and Homalogastra parasetosa sp. nov., with improved diagnoses of the genus Homalogastra and its type species Homalogastra setosa (Ciliophora, Oligohymenophorea).

The morphology of two new scuticociliates, Uronema apomarinum sp. nov. and Homalogastra parasetosa sp. nov., isolated from a mangrove wetland in Shenzhen, PR China, was studied using live observation and the protargol impregnation method. Uronema apomarinum is characterized by a body size of about 20-35×10-15 µm in vivo, a partly two-rowed membranelle 1, and 12 or 13 somatic kineties. Homalogastra parasetosa is distinguished by a membranelle 1 comprising two longitudinal rows of basal bodies. Three Homalogastra setosa populations are suggested as subjective synonyms of the new species. Improved diagnoses of the genus Homalogastra Kahl, 1926 and its type species Homalogastra setosa Kahl, 1926 are provided. Results of phylogenetic analyses based on 18S rRNA gene and ITS1-5.8S-ITS2 region sequences indicate that U. apomarinum is most closely related to U. marinum, while the closest relative of H. parasetosa is H. setosa.

Taxonomy and molecular phylogeny of three freshwater scuticociliates, with descriptions of one new genus and two new species (Protista, Ciliophora, Oligohymenophorea).

Three freshwater scuticociliates, Apouronema harbinensis gen. nov. spec. nov., Cyclidium vorax spec. nov., and C. glaucomaMüller, 1773, collected from rivers in Hulan District, Harbin, northeastern China, were investigated using morphological and phylogenetic criteria. Apouronema gen. nov., assigned to the family Uronematidae, is mainly distinguished from the other genera of the family by its paroral membrane extending anteriorly to the middle of membranelle 1. Apouronema harbinensis spec. nov. is defined by body size in vivo about 45-55 × 20-25 µm, buccal field about 70-80% of cell length; 12 or 13 somatic kineties; membranelle 1 having two rows, with 16-18 basal bodies in each kinety; membranelle 2 and membranelle 3 both having two rows each; scutica X-shaped with five pairs of basal bodies. Cyclidium vorax spec. nov. is characterized by the following features: body size 35-40 × 18-20 µm in vivo; 9 or 10 somatic kineties; membranelle 1 having two longitudinal rows, much shorter than M2; M2 triangle-shaped. The phylogenetic analyses show that: (1) Apouronema clustered in the Uronematidae clade, and grouped with genera Uronemita and Uronema; (2) Cyclidium vorax spec. nov. grouped with C. glaucoma and C. sinicum, which supports the assignment of the new species to the genus Cyclidium; (3) Cyclidium remains non-monophyletic with the addition of the new sequence.

Hyalophysa lynni n. sp. (Ciliophora, Apostomatida), a new pathogenic ciliate and causative agent of shrimp black gill in penaeid shrimp.

The parasitic ciliate causing shrimp black gill (sBG) infections in penaeid shrimp has been identified. The sBG ciliate has a unique life cycle that includes an encysted divisional stage on the host's gills. The ciliature of the encysted trophont stage has been determined and is quite similar to that of the closely related apostomes Hyalophysa bradburyae and H. chattoni. Hyalophysa bradburyae is a commensal ciliate associated with freshwater caridean shrimp and crayfish, while H. chattoni is a common commensal found on North American marine decapods. Based on 18S rRNA gene sequence comparisons, the sBG ciliate is more closely related to the marine species H. chattoni than to the freshwater species H. bradburyae. The unique life cycle, morphology, 18S rRNA gene sequence, hosts, location, and pathology of the sBG ciliate distinguish this organism as a new species, Hyalophysa lynni n. sp.

Morphology and molecular analyses of four epibiotic peritrichs on crustacean and polychaete hosts, including descriptions of two new species (Ciliophora, Peritrichia).

Four epibiotic sessilid peritrichs, i.e., Zoothamnium wilberti n. sp., Baikalonis microdiscus n. sp., Epistylis anastatica (Linnaeus, 1767) Ehrenberg, 1830, and Rhabdostyla commensalisMöbius, 1888, were isolated from one syllid polychaete and three crustacean hosts in Qingdao, China. For each species, specimens were observed both in vivo and following silver staining. Their SSU rDNA was also sequenced for phylogenetic analyses. Zoothamnium wilberti n. sp. is characterized by the appearance of its colony, which is up to 350 µm high, and usually has fewer than 16 zooids, and the dichotomously branched stalk with transverse wrinkles, the conspicuously conical peristomial disc, and infundibular polykinety 3 comprising three isometric ciliary rows. Baikalonis microdiscus n. sp. can be recognized by its barrel-shaped zooid, small peristomial disc, smooth and short stalk, and its unusual infundibular polykinety 3 comprising a long inner row and a short outer row. Two poorly known species, i.e., Epistylis anastatica and Rhabdostyla commensalis, are redescribed and redefined. Phylogenetic analyses reveal that: (i) R. commensalis is closely related to the family Astylozoidae rather than to the morphologically similar Epistylididae; (ii) B. microdiscus n. sp. is sister to the family Scyphidiidae; (iii) E. anastatica groups with vorticellids and ophrydiids, which further supports the polyphyly of the genus Epistylis; and (iv) Z. wilberti n. sp. is nested within the Zoothamniidae, as expected.

Morphological and molecular characterization of two Trichodina (Ciliophora, Peritrichia) species from freshwater fishes in China.

In the present study, we provide morphological and molecular characterization of two Trichodina species, T. acuta Lom, 1970 and T. funduliWellborn, 1967, isolated from koi (Cyprinus carpio) and loach (Paramisgurnus dabryanus), respectively. Morphological characters of the two Trichodina species were mainly investigated on the basis of dry silver nitrate-impregnated specimens. Both species are medium-sized and possess well-developed denticles comprising strongly sickle-shaped blades, well-developed central parts, and straight rays. Trichodina acuta can be easily distinguished from the other Trichodina species that possess a clear central circle by the well-developed sharp blade apophysis, and the gap between ray tip and central circle. Trichodina funduli is a poorly known species that is easily confused with T. heterodentata Duncan, 1977, however the latter species has thinner denticles. The small subunit ribosomal RNA gene sequences of Trichodina acuta and T. funduli were incorporated into phylogenetic analyses. Our findings suggest that the phylogenetic lineage of trichodinids might not correspond with their living environments, host species or even some morphological characteristics.

Taxonomy and phylogeny of Pseudovorticella littoralis sp. n. and P. alani sp. n. (Ciliophora: Peritrichia) from coastal waters of southern China.

The morphology, infraciliature, and silverline system of two peritrich ciliates, Pseudovorticella littoralis sp. n. and P. alani sp. n., isolated from coastal waters of southern China, were investigated based on both living and silver-stained specimens. Pseudovorticella littoralis sp. n. is characterized by the following combination of characters: cell inverted cone-shaped; contractile vacuole ventral; J-shaped macronucleus; infundibular polykinety 3 with two kinetosome rows of equal length; 19-26 silverlines from peristome to trochal band and 5-14 from trochal band to scopula. Pseudovorticella alani sp. n. is characterized by: cell inverted bell-shaped; contractile vacuole ventral; J-shaped macronucleus recurved almost forming a loop; infundibular polykinety 3 with three kinetosome rows, outer two rows longer than inner one; 48-61 silverlines between peristome and aboral trochal band, and 12-20 between aboral trochal band and scopula. The SSU rDNA sequences of both new species are reported and their genetic distances with congeners and phylogenetic relationships are investigated. Pseudovorticella and Epicarchesium cluster into two subclades with low support values. One subclade contains nearly all the available sequences of Pseudovorticella and Epicarchesium. Another one contains P. monilata and E. pectinatum. This calls on the need of a generic re-classification of Pseudovorticella and Epicarchesium based on more morphological and molecular data.