Morphogenesis and Molecular Phylogeny of the Soil Ciliate Holostichides chardezi (Ciliophora,Hypotrichia, Bakuellidae), with Redefinition of Holostichides Foissner, 1987 and Establishment of a New Genus Anteholostichides

Morphogenesis of the soil hypotrich ciliate, Holostichides chardezi Foissner, 1987, collected from southeastern China, was investigated using the protargol staining method. The main morphogenetic events follow a similar process with that of its congeners. Phylogenetic analyses based on the SSU rDNA sequence data indicate that Holostichides is nonmonophyletic; H. chardezi, the type species of Holostichides, clusters with H. heterotypicus, while H. terrae is distinctly separate from these species. H. terrae can be distinguished from H. chardezi (type species of Holostichides) and H. heterotypicus by undulating membranes relatively long and distinctly curved (vs. relatively short and straight), pharynx with (vs. without) rod‐shaped structure, and two (vs. more than two) frontoterminal cirri. Therefore, a new genus, Anteholostichides nov. gen., has been proposed for H. terrae. Further, the diagnosis of the genus Holostichides is improved.     

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.     

Caenimonas terrae sp. nov., isolated from a soil sample in Korea, and emended description of the genus Caenimonas Ryu et al. 2008

A white-coloured bacterium, SGM1-15^T, was isolated from a paddy soil sample from Suwon, Republic of Korea. The cells were strictly aerobic, Gram-negative and curved rod-shaped. A phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SGM1-15^T was closely related to Curvibacter delicatus LMG 4328^T (97.6% similarity) and Caenimonas koreensis EMB320^T (97.5% similarity). The major respiratory quinone system was Q-8 and the predominant cellular fatty acids were C_16:0 (39.9%), summed feature 3 (C_16:1 ω7c and/or iso-C_15:0 2-OH; 24.3%) and C_17:0 cyclo (22.7%). The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphatidylethanolamine. The major polyamines were 2-hydroxypurescine, purescine and spermidine. The DNA G+C content was 68.7 mol%. On the basis of the phylogenetic, physiologicl and chemotaxonomic data, stain SGM1-15^T represents a novel species of the genus Caenimonas, for which the name Caenimonas terrae sp. nov. is proposed. The type strain of Caenimonas terrae is SGM1-15^T (=KACC 13365^T =NBRC 106341^T).     

Identification of New Genomospecies in the Mycobacterium terrae Complex

Members of the Mycobacterium terrae complex are slow-growing, non-chromogenic acid-fast bacilli found in the natural environment and occasionally in clinical material. These genetically closely-related members are difficult to differentiate by conventional phenotypic and molecular tests. In this paper we describe the use of whole genome data for the identification of four strains genetically similar to Mycobacterium sp. JDM601, a newly identified member of the M. terrae complex. Phylogenetic information from the alignment of genome-wide orthologous genes and single nucleotide polymorphisms show consistent clustering of the four strains together with M. sp. JDM601 into a distinct clade separate from other rapid and slow growing mycobacterial species. More detailed inter-strain comparisons using average nucleotide identity, tetra-nucleotide frequencies and analysis of synteny indicate that our strains are closely related to but not of the same species as M. sp. JDM601. Besides the 16S rRNA signature described previously for the M. terrae complex, five more hypothetical proteins were found that are potentially useful for the rapid identification of mycobacterial species belonging to the M. terrae complex. This paper illustrates the versatile utilization of whole genome data for the delineation of new bacterial species and introduces four new genomospecies to add to current members in the M. terrae complex.     

Establishment of a new hypotrichous genus,Heterotachysoma n. gen. and notes on the morphogenesis of Hemigastrostyla enigmatica (Ciliophora,Hypotrichia)

A marine hypotrich ciliate, Heterotachysoma multinucleatum (Gong and Choi, 2007) n. comb., found in coastal waters near Qingdao, China, was investigated. Heterotachysoma multinucleatum is characterized by its dorsal ciliature arranged in Gonostomum-pattern. Additionally, a new genus, Heterotachysoma n. gen., is established which is mainly characterized by: 18-cirri pattern; flexible body; three dorsal kineties with no dorsomarginal kineties nor kinety fragmentation; one right and one left row of marginal cirri; caudal cirri absent. The genus Tachysoma is redefined, and three new combinations, T. multinucleatum, T. ovatum and T. dragescoi, are proposed. The morphogenesis of Hemigastrostyla enigmatica (Dragesco and Dragesco-Kernéis, 1986) Song and Wilbert, 1997, is also described. Compared with that of its congeners, the differences are mainly in the dorsal ciliature: (1) the dorsal kinety anlagen are formed de novo in H. enigmatica (vs. intrakinetally in H. paraenigmatica and H. elongata); (2) the dorsal kineties anlagen develop in secondary mode in H. enigmatica (vs. primary mode in H. paraenigmatica); (3) the kinetal fragment anterior to the right marginal row in both filial product is absent in both H. enigmatica and H. elongata (vs. present in H. paraenigmatica). These findings suggest that morphogenesis is not uniform among members of the genus Hemigastrostyla.     

<Emphasis Type="Italic">Hymenobacter daeguensis</Emphasis> sp. nov. isolated from river water

A Gram-stain-negative, non-motile, non-spore-forming, rod-shaped, aerobic bacterial strain, designated 16F3Y-2^T, was isolated from the Han River, South Korea, and was characterized taxonomically using a polyphasic approach. Comparative 16S rRNA gene sequence analysis showed that strain 16F3Y-2^T belonged to the family Cytophagaceae in the phylum Bacteroidetes and was most closely related to ‘Hymenobacter terrae’ DG7A (98.01%), H. soli PB17^T (97.26%), H. glaciei VUG-A130^T (96.78%), H. antarcticus VUG-A42aa^T (96.72%), H. ruber PB156^T (96.61%), and H. saemangeumensis GSR0100^T (95.77%). The G+C content of the genomic DNA of strain 16F3Y-2^T was 62.9 mol%. The isolate contained MK-7 as the predominant respiratory quinone, and summed feature 3 (C_16:1 ω7c/C_16:1 ω6c; 35.5%), C_15:0 iso (16.9%), C_16:1 ω5c (10.9%), and C_15:0 anteiso (9.9%) as major fatty acids. The major polar lipid was phosphatidylethanolamine. Phenotypic and chemotaxonomic data supported the affiliation of strain 16F3Y-2^T with the genus Hymenobacter. However, strain 16F3Y-2^T exhibited relatively low levels of DNA-DNA relatedness with ‘H. terrae’ KCTC 32554 (44.1%) and H. soli KCTC 12607^T (24.3%), clearly indicating that the isolate constitutes a new genospecies. Strain 16F3Y-2^T could be differentiated from its phylogenetic neighbors on the basis of several phenotypic, genotypic, and chemotaxonomic features. Therefore, strain 16F3Y-2^T represents a novel species in the genus Hymenobacter, for which the name Hymenobacter daeguensis sp. nov. is proposed. The type strain is 16F3Y-2^T (=KCTC 52537^T =JCM 31654^T).     

Morphology and molecular phylogeny of a new hypotrich ciliate,Anteholosticha songi nov. spec., and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991 (Ciliophora,Hypotrichia)

A new urostylid ciliate, Anteholosticha songi nov. spec., isolated from forest soil in Tibet, and an American population of Holosticha pullaster (Müller, 1773) Foissner et al., 1991, isolated from a freshwater pond in the USA, are investigated in terms of their morphology, ontogenesis, and molecular biology. Anteholosticha songi nov. spec. is characterized by a slender to ellipsoidal body measuring 160–205 × 40–55 μm in vivo; rod-shaped yellowish cortical granules arranged in irregular short rows; four dorsal kineties; adoral zone consisting of 35–40 membranelles; three frontal, one buccal, one parabuccal, two frontoterminal, two pretransverse, and four to six transverse cirri and 14–25 midventral pairs; 12–22 ellipsoidal macronuclear nodules longitudinally arranged in pairs left of cell mid-line. Supplemental information on morphogenesis in Holosticha pullaster is also presented. The phylogenetic relationship of Anteholosticha and Holosticha inferred from SSU rDNA sequence data are concordant with previous studies and showing that Holosticha is monophyletic whereas Anteholosticha is polyphyletic and should be split into two or more genera.     

Differential Immune Responses and Protective Effects in Avirulent Mycobacterial Strains Vaccinated BALB/c Mice

Screening live mycobacterial vaccine candidates is the important strategy to develop new vaccines against adult tuberculosis (TB). In this study, the immunogenicity and protective efficacy of several avirulent mycobacterial strains including Mycobacterium smegmatis, M. vaccae, M. terrae, M. phlei, M. trivial, and M. tuberculosis H37Ra were compared with M. bovis BCG in BALB/c mice. Our results demonstrated that differential immune responses were induced in different mycobacterial species vaccinated mice. As BCG-vaccinated mice did, M. terrae immunization resulted in Th1-type responses in the lung, as well as splenocytes secreting IFN-γ against a highly conserved mycobacterial antigen Ag85A. M. smegmatis also induced the same splenocytes secreting IFN-γ as BCG and M. terrae did. In addition, M. terrae and M. smegmatis-immunized mice predominantly increased expression of IL-10 and TGF-β in the lung. Most importantly, mice vaccinated with H37Ra and M. vaccae could provide the same protection in the lung against virulent M. tuberculosis challenge as BCG. The result may have important implications in developing adult TB vaccine.     

Systematics and species-specific response to pH of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora,Hypotricha) from acid mining lakes

We investigated the morphology, phylogeny of the 18S rDNA, and pH response of Oxytricha acidotolerans sp. nov. and Urosomoida sp. (Ciliophora, Hypotricha) isolated from two chemically similar acid mining lakes (pH ～ 2.6) located at Langau, Austria, and in Lusatia, Germany. Oxytricha acidotolerans sp. nov. from Langau has 18 frontal-ventral-transverse cirri but a very indistinct kinety 3 fragmentation so that the assignment to Oxytricha is uncertain. The somewhat smaller species from Lusatia has a highly variable cirral pattern and the dorsal kineties arranged in the Urosomoida pattern and is, therefore, preliminary designated as Urosomoida sp. The pH response was measured as ciliate growth rates in laboratory experiments at pH ranging from 2.5 to 7.0. Our hypothesis was that the shape of the pH reaction norm would not differ between these closely related (3% difference in their SSU rDNA) species. Results revealed a broad pH niche for O. acidotolerans, with growth rates peaking at moderately acidic conditions (pH 5.2). Cyst formation was positively and linearly related to pH. Urosomoida sp. was more sensitive to pH and did not survive at circumneutral pH. Accordingly, we reject our hypothesis that similar habitats would harbour ciliate species with virtually identical pH reaction norm.     

A Comparative Analysis of the Morphology of Holosticha corlissi N. Sp. (Ciliophora,Hypotrichida) and Related Species1

ABSTRACT The morphology of the hypotrichous ciliate, Holosticha corlissi n. sp., found in the moss Calliergonella cuspidata taken from the beech wood of Montejo de la Sierra (Madrid, Spain) is described. Some characteristics (organization of the mid-ventral, frontal and fronto-terminal cirri, presence of a buccal cirrus and the number of transverse cirri) are sufficiently different from the closely related species H. intermedia, H. sigmoidea and H. xanthichroma to suggest that it is a separate species, although its body shape, nucleus and buccal apparatus are very similar.     

Morphology and ontogenesis of two new Hemiholosticha species (Ciliophora,Hypotrichia, Hemiholostichidae nov. fam.)

The morphology and ontogenesis of two new hypotrich ciliates, Hemiholosticha solitaria and Hemiholosticha germanica, were studied using live observation, protargol impregnation, and scanning electron microscopy. Both species share a medium-sized, almost globular body with a short anterior projection; two macronuclear nodules with a single micronucleus in between; a central contractile vacuole; three or four ventral, one postoral, one right and one left marginal cirral row; and three dorsal kineties extending along ribs. However, H. germanica is distinguished from congeners by a higher number of cirri in ventral rows R1 and R2 (3–6 vs. 2 cirri in each row). Hemiholosticha solitaria differs from congeners by having four (vs. three) ventral cirral rows and by the lack (vs. presence) of intracellular green algae. The ontogenesis of H. solitaria follows the H. pantanalensis mode in that (i) the oral primordium develops in a deep pouch and generates the first two cirral streaks in addition to adoral membranelles and undulating membranes, (ii) the undulating membrane anlage does not produce any cirri, and (iii) the longitudinal ventral cirral row R3 originates from two anlagen. The ontogenetic peculiarities along with the 18S rRNA gene phylogenies suggest classification of Hemiholosticha, Psilotrichides, and Urospinula into a new family, Hemiholostichidae.     

“Candidatus Haloectosymbiotes riaformosensis” (Halobacteriaceae), an archaeal ectosymbiont of the hypersaline ciliate Platynematum salinarum

The novel ciliate Platynematum salinarum (Scuticociliatia) was isolated only recently from a thalassohaline solar saltern pond (12%) in Portugal. Scanning electron microscopy showed numerous bacterial-shaped cells covering the complete surface of the ciliate. The rod-shaped epibionts were identified and characterized following the “Full-Cycle rRNA Approach”. The almost full-length 16S rRNA gene sequence was obtained using archaeal-specific primers and two species-specific probes were designed for fluorescence in situ hybridization. The 16S rRNA gene sequence of the epibiotic cells showed 87% sequence identity with the type strain sequence of the closest characterized species Halolamina pelagica. Phylogenetic reconstructions affiliated the novel organism to the genus Halolamina (Halobacteria, Archaea). Attempts to isolate the epibionts failed and, therefore, growth experiments incorporating the antibiotic anisomycin were conducted in order to investigate the potential symbiotic relationship between P. salinarum and the epibionts. The results suggested an obligate symbiosis between the two organisms and revealed the first symbiotic representative of the Halobacteria. Based on the phylogenetic analyses and growth experiments we propose the classification of this novel organism in a new genus, with the taxon name “Candidatus Haloectosymbiotes riaformosensis”.     

Morphology and phylogeny of Bryophryoides ocellatus n. g., n. sp. (Ciliophora,Colpodea) from in situ soil percolates of Idaho,U.S.A.

We describe the morphology and 18S rDNA phylogeny of Bryophryoides ocellatus n. g., n. sp., a bryophryid ciliate inhabiting in situ soil percolates from Idaho, U.S.A. The new genus is distinguished from other bryophryid genera by a combination of the following features: (1) kreyellid (irregularly meshed) silverline pattern, (2) polymorphic adoral organelles in the preoral suture, (3) absence of vestibular kineties. In phylogenetic analyses, Bryophryoides ocellatus is most closely related to Bryophrya gemmea. The 18S rDNA sequence pairwise distance of 2% between these genera, while similar to that between many colpodidan species, exceeds that between some colpodidan genera (e.g. Mykophagophrys and Pseudoplatyophrya, 1.1%), further supporting establishment of the new genus. Topology hypothesis testing strongly supports the monophyly of the Colpodida including the bryophryids. Despite weak nodal support, tests of topology constraints narrowly reject the non-monophyly of the sequenced Bryophryidae (Bryophrya + Bryophryoides + Notoxoma). Likewise, the monophyletic origin of the sequenced Bryophryidae is indicated in the phylogenetic networks though with low support.     

Ontogeny and molecular phylogeny of a new marine ciliate genus,Heterokeronopsis g. n. (Protozoa,Ciliophora, Hypotricha), with description of a new species

The morphology and morphogenesis of a new ciliate, Heterokeronopsis pulchra g. n., sp. n., isolated from a mangrove wetland near Shenzhen, southern China, were investigated using live observation and protargol impregnation methods. Heterokeronopsis g. n. is characterized by having a bipartite adoral zone, an extremely shortened paroral membrane, frontal cirri arranged in an indistinct bicorona, a midventral complex composed of midventral pairs and midventral row(s), one left and one right marginal row, bipolar dorsal kineties, and buccal cirri; frontoterminal, transverse, and caudal cirri are absent. The single species H. pulchra sp. n. has a long, slender body that is brownish in colour, two kinds of cortical granules, one posteriorly located contractile vacuole, one buccal cirrus, usually six frontal cirri, three dorsal kineties, and one midventral row. The main morphogenetic features are: (i) the old oral apparatus is completely replaced by new structures derived from the oral primordium of the proter which originates de novo on the dorsal wall of the buccal cavity, (ii) the posteriormost frontal-midventral-transverse cirral anlage generates a midventral row, (iii) no frontoterminal and transverse cirri are formed, (iv) the anlagen for the marginal rows and the dorsal kineties are formed intrakinetally, and (v) the macronuclear nodules fuse into a mass at the middle stage. Phylogenetic analyses based on SSU rRNA gene sequence data reveal that Heterokeronopsis pulchra is a member of the family Pseudokeronopsidae and is most closely related to Nothoholosticha fasciola.     

Morphology and molecular phylogeny of Oxytricha seokmoensis sp. nov. (Hypotrichia: Oxytrichidae), with notes on its morphogenesis

A new hypotrichous ciliate, Oxytricha seokmoensis sp. nov., was discovered in a soil from a forest in South Korea and described based on the observations of living and stained specimens. In addition, phylogenetic analyses were performed using the small subunit ribosomal RNA (18S rRNA) gene sequence. Morphologically, the new species is similar to the O. granulifera-complex in terms of ciliary structure and arrangement of cortical granules, but dorsal kineties 3 and 4 (not completely separated vs. separated) and macronuclear nodules in the cyst (separated vs. fused) differ. Oxytricha seokmoensis is most similar to O. pulvillus, but can be distinguished by the number of adoral membranelles (30–40 vs. 23–27), contractile vacuole (present vs. absent), number of left (27–37 vs. 17–25) and right (27–35 vs. 18–23) marginal cirri, and lepidosomes on the cyst surface (present vs. absent). In a phylogenetic tree, O. seokmoensis is distinctly separated from the O. granulifera clade, but is sister to the Paroxytricha clade. In addition, O. seokmoensis and P. longigranulosa have the smallest genetic difference (d = 0.015, 23 of 1579 nt difference). This close relationship is supported by incomplete dorsal kinety 3 fragmentation and separated macronuclear nodules in resting cysts.     

Morphology and morphogenesis of a new oxytrichid ciliate,Notohymena limus n. sp. (Ciliophora,Oxytrichidae) from Delhi,India

The morphology and morphogenesis of a new oxytrichid ciliate, Notohymena limus n. sp. were studied in vivo and after protargol impregnation. The new ciliate was isolated from the sewage sludge at Delhi Jal Board Sewage Treatment Plant located at Rithala, Delhi, India, using the non-flooded Petri dish method. N. limus n. sp. is characterized as follows: flexible dorsoventrally flattened ellipsoidal body; Notohymena-pattern undulating membranes; adoral zone of membranelles (AZM) occupied about 39% of the body length, and consists of around 26 membranelles; large and deep buccal cavity; colorless subpellicular granules present in groups and arranged around the bases of dorsal bristles; 4 macronuclear nodules; 2 micronuclei; 18 fronto-ventral-transverse (FVT) cirri in typical Oxytricha-pattern; 6 dorsal rows of bristles; 3 caudal cirri; about 16 right and 15 left marginal cirri; N. limus n. sp. is a new species on the basis of the combination of morphological, morphometric and morphogenetic characteristic features.     

Morphology,morphogenesis and molecular phylogeny of a novel soil ciliate,Afrokahliella paramacrostoma n. sp. (Ciliophora,Hypotrichia)

A soil hypotrich ciliate, Afrokahliella paramacrostoma n. sp., was discovered in China. Its morphology, morphogenesis and molecular phylogeny were investigated using standard methods. The new species is characterized as follows: body about 140–180 × 60–70 μm in vivo, cortical granules absent, contractile vacuole positioned about 40% down length of body, 5–9 macronuclear nodules, 34–49 adoral membranelles, 3–5 buccal and 3–6 parabuccal cirri, usually two frontoventral rows, three or four left and two or three right marginal rows, three dorsal kineties and one dorsomarginal kinety; 1–3 and one or two caudal cirri located at the ends of dorsal kineties 1 and 2, respectively. The ontogenetic process is characterized by: (1) the marginal anlagen on each side develop in the outer right and the inner left marginal rows, respectively; (2) five frontoventral-transverse cirral anlagen, anlagen II–IV develop in secondary mode; (3) dorsal morphogenesis follows a typical Urosomoida-pattern, no parental dorsal kineties are retained; (4) caudal cirri are generated at the ends of dorsal kineties 1 and 2. Phylogenetic analyses based on SSU rDNA sequence data reveals that Afrokahliella paramacrostoma n. sp. is closely related to Parakahliella macrostoma and Hemiurosomoida longa.     

Morphology,morphogenesis, and molecular phylogeny of a novel saline soil ciliate,Heterourosomoida sinica n. sp. (Ciliophora,Hypotrichia)

The living morphology, infraciliature, morphogenesis and phylogenetic position of a new hypotrichous ciliate, Heterourosomoida sinica n. sp., discovered in saline soil in Northwest of China, were investigated. Heterourosomoida sinica n. sp. is characterized as follows: body size about 70–95 × 20–30 μm in vivo, ellipsoid or elongate; cortical granules dark green, 0.5 μm across, arranged around dorsal cilia and in short irregular rows on ventral side; consistently 18 frontoventral-transverse cirri; left and right marginal rows composed of 17–24 and 13–21 cirri, respectively; two macronuclear nodules and one or two micronuclei. The detailed morphogenesis of a species of Heterourosomoida is reported for the first time. The ontogenesis on dorsal side is basically similar to that in Urosomoida-species and the process on ventral side is characterized by: 18 frontoventral-transverse cirri develop from six anlagen with the anlagen V and VI formed in the primary mode. Phylogenetic analyses based on SSU rDNA sequence data reveal that Heterourosomoida sinica n. sp. clusters with Kleinstyla dorsicirrata and forms a clade with the type species H. lanceolata.