Morphological and Molecular Identification of a New Ciliate, Zoothamnium palmphlatum nov. spec. (Ciliophora, Peritrichia) from North China.

A new peritrichous ciliate, Zoothamnium palmphlatum nov. spec., was collected from an estuary in Yantai, China. It was investigated, using both live observation and silver staining. The new species can be identified by a palm-shaped colony consisting of highly developed and alternately arranged secondary branches, a double-layered peristomial lip, and an infundibular polykinety 3 composed of two parallel kinetosomal rows. Phylogenetic analyses of the small subunit (SSU) rRNA gene sequence show that Z. palmphlatum clusters with other members of the family Zoothamniidae. Furthermore, the comparison of primary and secondary SSU rDNA structures indicates that Z. palmphlatum is distinctly different from its morphologically similar species (93.2-97.0% in sequence similarity) in combination of H10 and H31 regions. Although Z. palmphlatum shares highest sequence similarity with Zoothamnium mucedo (98.9%), the new species has distinctly different structures in the H11, H12, H18, and H31 regions compared to Z. mucedo, which indicates that sequence similarity may not determine the similarity of the secondary structure.

Morphology and Phylogeny of Four New Vorticella Species (Ciliophora: Peritrichia) from Coastal Waters of Southern China.

Four new species of Vorticella, V. parachiangi sp. n., V. scapiformis sp. n., V. sphaeroidalis sp. n., and V. paralima sp. n., were isolated from coastal brackish waters of southern China. Their morphology, infraciliature, and silverline system were investigated based on observations of specimens both in vivo and following silver staining. Vorticella parachiangi sp. n. is distinguished by: a J-shaped macronucleus; a single dorsally located contractile vacuole; a two-rowed infundibular polykinetid 3, in which row 1 is shorter than row 2; 21-31 silverlines between peristome and aboral trochal band, 6-11 between aboral trochal band and scopula. Vorticella scapiformis sp. n. is characterized by its conspicuously thin and irregularly edged peristomial lip; a J-shaped macronucleus; a single, ventrally located contractile vacuole; row 1 of the infundibular polykinetid 3 proximally shortened; 18-25 silverlines between peristome and aboral trochal band, 8-12 between aboral trochal band and scopula. Vorticella sphaeroidalis sp. n. can be identified by its small, sub-spherical zooid; a C-shaped macronucleus; a ventrally located contractile vacuole; an aboral trochal band adjacent to the scopula; 16-18 silverlines between persitome and aboral trochal band, two between aboral trochal band and scopula. Vorticella paralima sp. n. can be identified by its ovoidal zooid; a J-shaped macronucleus; a dorsally positioned contractile vacuole; rows 1 and 2 of the infundibular polykinetid 3 proximally shortened; 26-35 silverlines from peristome to aboral trochal band, and 7-13 from aboral trochal band to scopula. The SSU rDNA genes of these four species were sequenced and their phylogeny was analyzed.

Phylogeny of the families Zoothamniidae and Epistylididae (Protozoa: Ciliophora: Peritrichia) based on analyses of three rRNA-coding regions.

Peritrichs are a major group of ciliates with worldwide distribution, and they play important roles in many habitats. Intrafamilial phylogeny of some peritrichs was investigated using information from three genes, which provided more robust interpretations than single-gene analyses. Sixty-seven new sequences including SSU rDNA, ITS1-5.8S-ITS2 and LSU rDNA were aligned with available sequences in GenBank to infer phylogenetic relationships within the families Zoothamniidae and Epistylididae. Results reveal the following relationships: (1) Epistylididae is polyphyletic, consisting of two clades that nest within the Zoothamniidae as part of the crown clade of peritrichs (order Vorticellida) and a third one that is part of the basal clade of peritrichs (order Opercularida); (2) Epistylis elongata falls within one of the clades of Zoothamnium rather than with congeners; (3) Zoothamnium is probably paraphyletic, consisting of three divergent clades, with the genera Myoschiston and Zoothamnopsis intermingled with species of Zoothamnium. The following evolutionary hypotheses can be inferred from these results: (1) the contractile stalk of Zoothamnium is plesiomorphic. (2) Myoschiston, Zoothamnopsis and clade II of Epistylididae are derived from the Zoothamnium morphotype by partial or incomplete development of the spasmoneme that forms the contractile center of the stalk around which the rigid cortex is secreted. (3) Clade I of the Epistylididae, which are primarily colonial forms that appear never to have evolved a spasmoneme of any sort, may represent the ancestral morphotype of peritrichs.

An Optimized Protocol of Protargol Staining for Ciliated Protozoa.

Protargol staining is a crucial method to reveal the infraciliature of ciliates, which is the most important morphological character for species identification. In the present study, Wilbert's protocol of protargol staining was emended mainly toward the highly happened improper bleaching. Through reciprocal treatments, both insufficient and excessive bleachings were much eliminated from the protargol protocol and the tests performed with four different species of ciliates established that the stainings were considerably improved and more reliable with optimized bleaching. Compared to the original protocol, the optimized method was proved to be more suitable for the groups difficult to stain, and it is also friendlier for the beginners and researchers in related fields.

Morphology and Phylogenetic Placement of Three New Zoothamnium species (Ciliophora: Peritrichia) from Coastal Waters of Southern China.

The morphology, infraciliature, and silverline system of three peritrichous ciliates, Zoothamnium bucciniiformum sp. n., Zoothamnium florens sp. n., and Zoothamnium zhanjiangense sp. n., were investigated based on both living and silver-stained specimens. Zoothamnium bucciniiformum sp. n., collected from coastal waters (salinity 30‰) off Zhanjiang, southern China, can be distinguished by the following characters: dichotomously branched stalk, peristomial lip with medial circumferential infolding, contractile vacuole apically positioned, 32-49 silverlines between the anterior end and the aboral trochal band, 15-26 between the aboral trochal band and the scopula; two kineties in peniculus 3, not parallel to each other. Zoothamnium florens sp. n., collected from a mangrove wetland (salinity 13‰) off Zhanjiang, is characterized by its large conical zooid, tuberculate peristomial lip, asymmetrical dichotomously branched colony, 59-81 silverlines between the anterior end and the aboral trochal band and 29-36 between the aboral trochal band and the scopula. Zoothamnium zhanjiangense, collected from a mangrove wetland (salinity about 9.5‰) off Zhanjiang, differs from its congeners by the alternately branched stalk, peristomial lip with medial circumferential infolding, 40-63 silverlines from the peristomial area to the aboral trochal band and 13-24 from the aboral trochal band to the scopula. The comparison and analysis of SSU rDNA sequences also support present identifications.

Two New Species of Zoothamnium (Ciliophora, Peritrichia) from Korea, with New Observations of Z. parahentscheli Sun et al., 2009.

Three peritrichous ciliates, Zoothamnium arcuatum n. sp., Z. grossi n. sp., and Z. parahentscheli Sun et al., 2009, were collected from an estuary of the Taehwagang River, Korea. All these species were investigated based on live observations and silver staining, and their small subunit (SSU) rRNA gene was also sequenced. Zoothamnium arcuatum can be identified by a goblet-shaped colony, double-layered peristomial lip, and abstomally shortened row 3 of infundibular polykinety 3 (P3). Zoothamnium grossi is morphologically characterized by an alternately branched stalk with the lowest secondary stalk diverging from the main part of colony, asymmetrically bell-shaped zooids, and three short, parallel ciliary rows in P3. Phylogenetic analysis revealed that the three Zoothamnium species described in this paper clustered with other members of the family Zoothamniidae, as expected.

N2O emission in temperate seagrass meadows: Fluxes, pathway and molecular mechanism.

Seagrass meadows act as filters for nitrogen in coastal areas, but whether they are a source or sink for N2O has been still controversy. Additionally, the production pathways of N2O as well as the microbial driving mechanism in seagrass meadows are seldom reported. In this study, the air-sea fluxes, sediment release potential, and production pathway of N2O in a temperate Zostera marina and Z. japonica mixed meadow were investigated by using gas chromatography and 15N isotopic tracing methods. The qPCR and metagenome sequencing were used to compare the difference in functional gene abundance and expression between seagrass vegetated and non-grass sediments. The results showed that the N2O air-sea fluxes in the meadow ranged from -1.97 to -1.77 nmol m⁻2 h⁻1, which was slightly lower in the seagrass region than in the adjacent bare region. Seagrass sediment N2O release potential dramatically increased after warming and nitrogen enrichment treatments. Heterotrophic nitrification was firstly investigated in seagrass meadows, and the process (26.80%-62.41%) and denitrification (37.55%-72.83%) contributed significantly to N2O production in the meadow, affected deeply by sediment organic content, while the contribution of autotrophic nitrification can be neglected. Compared with the bare sediments, the ammonia monooxygenase genes amoA, amoB and amoC, and nitrite oxidoreductase genes nxrA and nxrB, as well as nitrite reductase gene nirS and nitric oxide reductase gene norB were down-regulated, while the nitrous oxide reductase gene nosZ was up-regulated in the seagrass sediments, explaining less N2O emission in seagrass regions from the perspective of molecular. The nosZII-bearing bacteria like Bacteroidia, Polyangia, Anaerolineae, and Verrucomicrobiae could play important roles in N2O reduction in the seagrass meadow. The result is of great significance for highlighting the ability of seagrass meadows to mitigate climate changes.

Morphology and small subunit (SSU) rRNA gene sequence of the new brackish water ciliate Neobakuella flava n. g., n. sp. (Ciliophora, Spirotricha, Bakuellidae) and SSU rRNA gene sequences of six additional hypotrichs from Korea.

The morphology and the small subunit (SSU) rRNA gene sequence of the hypotrich Neobakuella flava n. g., n. sp. from the estuary of the Taehwagang River (Ulsan, South Korea) were investigated. The three frontal cirri, the composition of the midventral complex of cirral pairs and rows, and the simple dorsal kinety pattern of three bipolar kineties assign it to the urostyloid taxon Bakuellidae. The increased number of buccal and parabuccal cirri, the presence of transverse cirri, and more than one left marginal row, as well as the lack of caudal cirri separate Neobakuella n. g. from the other bakuellids. Neobakuella flava n. sp. has many 0.3 µm sized green and/or yellow usually dark-green cortical granules and some sparsely distributed, 2 × 1 µm sized grass green with yellowish shimmer granules. The gene sequence data indicate a close relationship with Diaxonella and a distinct separation from the bakuellid Metaurostylopsis and parabirojimid Parabirojimia. The SSU rRNA gene sequences of four further urostyloids (i.e. Diaxonella pseudorubra, Anteholosticha monilata, Metaurostylopsis struederkypkeae, Pseudourostyla cristata) and two stylonychines (i.e. Sterkiella cavicola, Sterkiella histriomuscorum) from Korea were analyzed. Anteholosticha monilata, type of the genus, is clearly separated from the Holosticha clade, supporting the morphological separation from Holosticha. Sterkiella cavicola, type of Sterkiella, clusters within the stylonychines and is obviously closely related with S. histriomuscorum.

Morphology and morphogenesis of a freshwater ciliate, Epistylis chlorelligerum Shen, 1980 (Ciliophora, Peritrichia).

An oligohalobic peritrichous ciliate, Epistylis chlorelligerum Shen, 1980, was collected from a ditch in Hangzhou, China. The morphology, oral infraciliature, and morphogenesis of the species were studied using living and protargol-impregnated specimens. Zooids of E. chlorelligerum are 160-230 × 50-60 µm in vivo, and characterized by green-colored endoplasm containing symbiotic algae. The oral infraciliature presents a well-developed filamentous reticulum linked to the circular fiber of the cytostome; the outer two rows of P3 extend adstomally over P1 and usually enfold it. During binary fission, one daughter cell inherits most part of the old buccal apparatus and the reorganized haplokinety and germinal kinety (Hk' and G'), and new buccal apparatus of the other daughter cell is mostly developed from the original germinal kinety (G) and haplokinety (Hk): new peniculi 2, 3 (2P2, 2P3), new haplokinety (2Hk), and new germinal kinety (2G) are formed from G, while the new peniculus 1 (2P1) and its peristomial extention (2Pk) originate from Hk. The epistomial membrane can be observed until the two sets of buccal apparatus begin to separate from each other.

Morphology and phylogeny of two Phialina species (Ciliophora, Haptoria) from northern China.

The morphology and infraciliature of two haptorian ciliates, Phialina clampi nov. spec. and P. caudata (Kahl, 1933) nov. comb. (original combination: Lacrymaria caudataKahl, 1933), isolated from sandy sediments of an estuary in Yantai, northern China, were investigated using live observations and protargol preparations. Phialina clampi nov. spec. is distinguished from its congeners by the following traits: extended cells about 80-300 × 18-50 µm in vivo; a single oval-shaped macronucleus; caudally located contractile vacuole; two types of extrusomes: type I about 20-35 × 1-4 µm in size, type II 1.5-3.0 × 0.7-1.0 µm in size and attached to somatic cortex; 27-31 somatic kineties. Phialina caudata is characterized as follows: extended cells about 80-170 × 20-45 µm in vivo; body radish-shaped, with a sharp posterior end; a single globular to oval macronucleus and micronucleus; subterminal contractile vacuole; 20-24 somatic kineties. Phylogenetic analyses based on SSU rRNA gene sequences indicate that P. caudata clusters with an unidentified Phialina before grouping with Phialina clampi, which form a basal clade of the family Lacrymariidae.

Reconsideration of the phylogenetic positions of five peritrich genera, Vorticella, Pseudovorticella, Zoothamnopsis, Zoothamnium, and Epicarchesium (Ciliophora, Peritrichia, Sessilida), based on small subunit rRNA gene sequences.

In order to re-evaluate the systematics of sessilid peritrich ciliates, small subunit (SSU) rRNA gene sequences were determined for 12 species belonging to five genera: Vorticella, Pseudovorticella, Epicarchesium, Zoothamnium, and Zoothamnopsis. Phylogenetic trees were deduced using Bayesian inference, maximum parsimony, and maximum likelihood methods. The phylogenetic analyses suggest that (1) sessilids which have stalks with continuous myonemes that contract in a zig-zag fashion form a separate clade from those which have stalks that contract independently and in a spiral fashion, supporting the separation of the family Zoothamniidae from the family Vorticellidae and (2) Epicarchesium and Pseudovorticella, both of which have reticulate silverline systems, are more closely related to each other than to other vorticellids, suggesting that differences in the silverline system (i.e. transverse vs. reticulate) may be the result of genuine evolutionary divergence among sessilid peritrichs. However, the newly sequenced Zoothamnopsis sinica, which has a reticulate silverline pattern, nests within the unresolved Zoothamnium species that have transverse silverline patterns. Thus, there were at least two evolutions of the reticulate silverline pattern character state from a plesiomorphic transverse state in the peritrichid ciliates. The molecular work demonstrates the genus Zoothamnium to be paraphyletic in relation to morphological studies, and suggests that Astylozoon, Opisthonecta, and Vorticella microstoma possibly share a SSU rRNA secondary structure in the helix E10-1 region.

Pseudovorticella zhengae n. sp., P. difficilis (Kahl, 1933) Jankowski, 1976, and P. punctata (Dons, 1918) Warren, 1987, three marine peritrichous ciliates from north China.

The morphology, infraciliature and silverline system of three marine peritrichous ciliates, Pseudovorticella zhengae n. sp., P. difficilis (Kahl, 1933) Jankowski, 1976 and P. punctata (Dons, 1918) Warren, 1987, were investigated by observation in vivo and silver-staining methods. The new species is characterized by its body shape, the appearance of the peristomial lip, the number of transverse silverlines, and the configuration of infundibular polykinety 3; P. difficilis by its body shape, the configuration of infundibular polykinety 3, and unusual annular swellings on the stalk; and P. punctata by the appearance of the pellicle, the configuration of infundibular polykinety 3, and the number of transverse silverlines.

A new Peritrich Ciliate from a Hypersaline Habitat in Northern China.

A new peritrichous ciliate, Cothurnia salina n. sp., collected from a brine pond of a salt factory in Yantai, China, was investigated based on live observations, silver staining method and molecular phylogenetic analysis. The diagnosis for this new taxon: body elongated columnar, in vivo 80-98 × 12-19 µm; lorica barrel-shaped, with aboral part heavily thickened; stalk extremely short, with approximately ½ of its length within the lorica; macronucleus wormlike, longitudinally oriented; single contractile vacuole ventrally located; pellicle with conspicuous parallel transverse striations, 62-73 from aboral trochal band to peristome and 32-38 from aboral trochal band to scopula; infundibular polykinety 3 (P3) consisting of two ciliary rows, which are equal length, parallel to each other and terminate adstomally between P1 and P2. Small subunit (SSU) rRNA gene trees revealed that the new species clustered with other members of the family Vaginicolidae as expected.

Description of two marine amphisiellid ciliates, Amphisiella milnei (Kahl, 1932) Horváth, 1950 and A. sinica sp. nov. (Ciliophora: Hypotrichia), with notes on their ontogenesis and SSU rDNA-based phylogeny.

The morphology and taxonomy of two marine Amphisiella species, isolated from mariculture waters in northern China, were investigated using standard techniques. One species corresponds well with the original description of the poorly known Amphisiella milnei (Kahl, 1932) Horváth, 1950 by remarkable characteristics, inter alia, (i) the additional cirri between the left frontal cirrus and the buccal cirrus, and (ii) ring-shaped structure in the anterior and posterior body portion (posterior one sometimes lacking). The detailed description of the cirral pattern and an informative ontogenetic stage first reveal that the additional cirri, which are uniformly absent in all congeners, originate from the frontal-ventral transverse cirral anlage I. The other species represents a new species of Amphisiella, A. sinica sp. nov. It is distinguished from its most closely related congener A. annulata (Kahl, 1928) Borror, 1972 by having numerous ring-shaped structures with a shallow brim more or less densely centralised at both ends of the body. Brief notes on the cell division and phylogenetic analyses based on small subunit (SSU) rRNA gene sequence for both organisms are also supplied in order to get further understanding of their systematic positions. The molecular information indicates that both organisms belong to two separate clades and confirms that the genus Amphisiella might be polyphyletic.

Description and phylogeny of a new prostomatid, Metacystis similis nov. spec. (Protista, Ciliophora) from the East China Sea.

The living morphology and infraciliature of a new marine prostomatid, Metacystis similis nov. spec., have been observed and investigated. Based on both morphological and molecular biological data, several closely-related and easily confused taxa were compared. The diagnosis for this new taxon: cylindrical marine Metacystis with a slightly blunt anterior end, in vivo 50-70 x 18-23 µm; body not loricate, uniformly cinctured by 16-18 transverse rings of cilia, which are also arranged in 32-36 longitudinal kineties; oral apparatus consisting of one circumoral dikinety and six girdles of densely packed monokinetids; single spherical macronucleus located in the center of the body; caudal region possessing one transparent, protruding hemicycle terminal vacuole. Information concerning the small-subunit rRNA gene sequence of the new species was also provided.

A description of Apocarchesium rosettum n. gen., n. sp. and a redescription of Ophrydium eichornii Ehrenberg, 1838, two freshwater peritrichous ciliates from Japan.

This paper includes the proposal of a new genus for a new species of peritrichous ciliate, Apocarchesium rosettum n. gen., n. sp., as well as the first description by modern techniques of another peritrich, Ophrydium eichornii Ehrenberg, 1838. The genus Apocarchesium is separated from the related genus Carchesium by the appearance of the stalk and the arrangement of zooids, and diagnosed as follows: colonial vorticellid, with all zooids attached in rosette fashion to the tip of their common stalk, which is unbranched and contracts spirally; pellicle with parallel silverlines. The morphology, infraciliature, and silverline system of both species were investigated by observation in vivo and silver-staining methods.

Taxonomic characterization of two marine peritrichous ciliates, Pseudovorticella clampi n. sp. and Zoothamnium pararbuscula n. sp. (Ciliophora: Peritrichia), from North China.

The morphology, infraciliature, and silverline system of two new peritrichous ciliates, Pseudovorticella clampi n. sp. and Zoothamnium pararbuscula n. sp., have been investigated based on both living and silver-impregnated specimens. Partial sequence of 18S-ITS1-5.8S rDNA of Z. pararbuscula is also determined in order to compare it with the closely related congener, Zoothamnium arbuscula. Zoothamnium pararbuscula can be distinguished from its close form Z. arbuscula by the different habitats, the appearance of the main stalk, the position of the contractile vacuole, and the information derived from 18S-ITS1-5.8S rDNA sequence analysis. Pseudovorticella clampi n. sp. is distinguished from its congeners by its body shape and size, pellicle granules, habitat, and number of transverse silverlines.

Description of a marine peritrichous ciliate, Pseudovorticella sinensis n. sp. (Ciliophora, Peritrichia) from China.

A new marine peritrich ciliate, Pseudovorticella sinensis n. sp. was isolated from a shrimp-farming pond in the littoral area of Qingdao, China. The morphology, infraciliature, and silverline system were studied based on living and silver-impregnated specimens. This species is characterized by (1) an elongated bell-shaped body that measures 50-60 x 35-45 microm in vivo, (2) one large, ventrally located contractile vacuole, and (3) a pellicle covered by a layer of transparent, cortical vesicles. The number of transverse silverlines from the peristomial area to the aboral ciliary wreath is 26-32, and from the aboral ciliary wreath to the scopula is 12-15. The stalk measures about 160-250 microm long x 5-6 microm wide. The spasmoneme has one row of conspicuous thecoplasmic granules, which are about 0.8 microm in diameter.

New contributions to two heterotrichous ciliates, Folliculina simplex (Dons, 1917), Condylostoma curva Burkovsky, 1970 and one licnophorid, Licnophora lyngbycola Fauré-Fremiet, 1937 (Protozoa, Ciliophora): descriptions of morphology and infraciliature.

The living morphology and infraciliature of two heterotrichous and one licnophorid ciliates from the north China seas, i.e. Folliculina simplex (Dons 1917), Condylostoma curva Burkovsky, 1970, and Licnophora lyngbycola Fauré-Fremiet, 1937, were investigated using protargol impregnation and in vivo observations. Updated diagnoses and redescriptions of each species are provided. The improved diagnosis of Folliculina simplex is as follows: blue-greyish to blue-greenish Folliculina with broad and unequal-sized peristomial lobes; adoral zone of membranelles comprises about 400 membranelles and forms two-and-half turns within buccal cavity; paroral membrane extends entire length of adoral zone of membranelles; about 50 somatic kineties in mid-body; single ovoid to ellipsoid macronucleus and several micronuclei; lorica vase-shaped, about 150-250 microm long, often with jelly-like covering and inconspicuous neck; marine habitat. Improved diagnosis for Licnophora lyngbycola: marine Licnophora with cup-shaped body and glabrous adhesive disc with which the organism attaches to the filaments of the colourless cyanobacterium Lyngbya; in vivo 80-130 microm long; about 140 adoral membranelles, one micronucleus and a single C-shaped, moniliform macronucleus consisting of about 14 nodules; two specialized dorsal kineties; five peripheral rings on adhesive disc, the 2nd, 3rd, and 5th (outermost) which are incomplete. Based on the Qingdao population, an improved diagnosis for Condylostoma curva is supplied: yellow-brownish Condylostoma with oval to elongated body shape; about 150-350 microm long in vivo; about 68-108 membranelles in adoral zone of membranelles, 4-8 frontal cirri, 22-32 somatic kineties; macronucleus moniliform with 5-13 nodules; with cortical granules; marine habitat.