Morphology and Phylogeny of Three Pleuronema Species (Ciliophora, Scuticociliatia) from Hangzhou Bay, China, with Description of Two New Species, P. binucleatum n. sp. and P. parawiackowskii n. sp.

The morphology and phylogeny of Pleuronema binucleatum n. sp., P. parawiackowskii n. sp., and P. marinum Dujardin 1841, collected from Hangzhou Bay estuary, China, were investigated using standard methods. Pleuronema binucleatum n. sp. can be identified by possessing about 90-120 × 35-50 µm cell size in vivo, reniform body outline, two macronuclei, six to eight preoral kineties, 32-41 somatic kineties, and posterior end of the anterior fragment of membranelle 2 (M2a) hook-like. Pleuronema parawiackowskii n. sp. is characterized by the combination of the following characters: body size about 60-90 × 20-40 µm in vivo, elliptical in outline, four to eight preoral kineties, 20-29 somatic kineties, posterior portion of the M2a slightly curved but nonhooked, and single macronucleus sausage-like. After comparison with other populations of P. marinum, it is suggested that many misidentifications exist in previous studies. And an improved diagnosis of P. marinum was supplied: cell about 95-180 µm long, elliptical in outline; 2-4 preoral kineties and 53-70 somatic kineties; both membranelle 1 and membranelle 3 three-rowed; posterior end of the M2a straight; single contractile vacuole characteristically positioned near mid-body. The small subunit rRNA genes of three forms were sequenced. Phylogenetic analyses indicate that the monophyly of the genus Pleuronema is still not supported.

Morphology and phylogeny of two species of Loxodes (Ciliophora, Karyorelictea), with description of a new subspecies, Loxodes striatus orientalis subsp. n.

The morphology and phylogeny of Loxodes vorax and L. striatus orientalis subsp. n. were investigated based on infraciliature and small subunit (SSU) rRNA gene sequence data. Loxodes striatus orientalis subsp. n. was separated from L. striatus striatus stat. n. by having fewer dikinetids in the intrabuccal kinety (35-55 vs. 50-70) and a variable number of macronuclei (2-4 vs. 2). In addition, the SSU rRNA gene sequence of the new subspecies differs in 13 and 11 nucleotides from that of two populations of the nominotypic subspecies. We also summarized the morphological differences between Loxodes and Remanella based on the data available. Phylogenetic analyses revealed that the genus Loxodes was monophyletic and nested within Remanella species. This study might, therefore, support the hypothesis that the freshwater genus Loxodes evolved from the marine genus Remanella.

Characterization of the life cycle and heteromeric nature of the macronucleus of the ciliate Chilodonella uncinata using fluorescence microscopy.

Only a limited number of studies exist on the life cycles of nonmodel ciliates such as Chilodonella uncinata (Cl: Phyllopharyngea). The handful of papers on this taxon indicate the presence of a heteromeric macronucleus, marked by separate DNA-rich and DNA-poor regions. Here, we study the life cycle of C. uncinata using confocal laser scanning microscopy with 4',6-diamidino-2-phenylindole staining, which allows us to differentiate nuclear dynamics of the micronucleus and the macronucleus during life-cycle stages. We photo-documented various stages and confirmed aspects of the development of the new macronucleus previously characterized by electron microscopy. We further reveal the heteromeric structure of the macronucleus with Z-stacks and three-dimensional (3D) reconstructions. We find no evidence for the presence of an endosome at the center of the macronucleus during vegetative growth. In addition to illustrating the life cycle of this ciliate, the approaches developed for this study will enable additional comparative analyses of nuclear dynamics using fluorescence microscopy.

[Fibrillar actin in the nuclear apparatus of the ciliate Paramecium caudatum].

Due to their nuclear dualism, ciliates provide a good model for studying the role of actin in spatial organization and transcription activity of the nucleus. The actin in the nuclear apparatus of the ciliate Paramecium caudatum was studied using fluorescently labeled phalloiodin and indirect immunocytochemistry. Fibrillar actin was demonstrated in both of the nuclei. Actin was revealed in the chromatin areas, and was often associated with the periphery of the amplified nucleoli in the macronucleus. Redistribution of actin was observed depending on different physiological state of the cells. Stable infection of the macronulear with the intranuclear endobionts Holospora obtuse led to the loss of nuclear actin accompanied by significant nuclear fragility and redistribution of the phosphorylated form of the actin-binding protein cofilin. Spherical bodies resembling karyosphere were found in the macronuclear anlagen.

[Morphological variations of the nuclear apparatus of astome ciliates Almophrya bivacuolata and A. maediovacuolata (protozoa: ciliophora) endocommensal of terricolous oligochaetes in Cameroon]. / Variations morphologiques de l'appareil nucléaire chez les ciliés Astomes Almophrya bivacuolata et A. mediovacuolata (protozoa: ciliophora) endocommensaux d'oligochites terricoles du Cameroun.

The silver impregnation supplemented by DAPI and Feulgen nuclear coloration enabled us to study the morphological variations of the nuclear apparatus of two species of endocommensal Astome ciliates, Almophrya bivacuoloata (de Puytorac & Dragesco, 1968) and A. mediovocuolata (Ngassam, 1983). We highlighted important digitations and the presence of dark bands in the structure of the "H" macronucleus of the small cellular types as well as the presence of intermediate forms between "H" and "X" in these two species.

A new genus of marine scuticociliate (Protozoa, Ciliophora) from northern China, with a brief note on its phylogenetic position inferred from small subunit ribosomal DNA sequence data.

The morphology, infraciliature, and silverline system of a new marine scuticociliate, Wilbertia typica n. g., n. sp., collected from coastal waters off northern China, were investigated. The new genus Wilbertia is characterized as follows: sculptured and dorso-ventrally flattened body; dominant buccal field that is almost completely surrounded by the paroral membrane; three apically positioned long membranelles, arranged in parallel; membranelle (M)1 and M2 prominent, M3 small; reticulate silverline system. The type species W. typica n. sp. is defined by having a conspicuous anterior beak-like protrusion; five to eight caudal cilia; M1 four-rowed, M2 two-rowed; M3, single-rowed, bipartite; 15 or 16 somatic kineties; contractile vacuole positioned just posterior to the buccal field; globular macronucleus. The small subunit ribosomal DNA sequence of W. typica is 98.5% similar to the similar morphotype, Eurystomatella sinica. Phylogenetic analyses indicate that Wilbertia groups with Eurystomatella sinica forming a branch that diverges at a deep level from all other pleuronematid scuticociliates. The molecular and morphological data indicate that Wilbertia should be placed within the family Eurystomatidae, which is closely related to the well-known Cyclidiidae.

Taxonomic studies on three marine pleurostomatid ciliates, Litonotus bergeri nov. spec., L. blattereri nov. spec. and L. petzi nov. spec. (Ciliophora, Pleurostomatida) from North China Sea.

The morphology and infraciliature of three pleurostomatid ciliates, Litonotus bergeri nov. spec., L. blattereri nov. spec. and L. petzi nov. spec., collected from mariculture ponds near Qingdao (Tsingtao), China, were investigated using live observations and the protargol impregnation method. These new species are distinguished from their congeners by a combination of characters including the typical distribution of extrusomes, i.e., along entire ventral margin, the number of macronuclear nodules, features and number of somatic kineties, living morphology, number and position of contractile vacuoles and their marine biotopes. Considering the distribution of extrusomes and general morphology, five new combinations are suggested, Litonotus vermiforme (Sauerbrey, 1928) nov. comb. [basionym: Loxophyllum vermiforme Sauerbrey, 1928], Litonotus levigatum (Sauerbrey, 1928) nov. comb. [basionym: Loxophyllum levigatum Sauerbrey, 1928], Litonotus undulatum (Sauerbrey, 1928) nov. comb. [basionym: Loxophyllum undulatum Sauerbrey, 1928], Loxophyllum pictus (Gruber, 1884) nov. comb. [basionym: Litonotus pictus Gruber, 1884] and Loxophyllum trichocystiferus (Foissner, 1984) nov. comb. [basionym: Litonotus trichocystiferus Foissner, 1984].

[Stylonychia lemnae as a model organism for studies of macronuclear minichromosomes of the ciliates].

The unique structural and functional organization of macronuclear (somatic nucleus) genome of the spirotrichous ciliates, exemplified by Stylonychia lemnae, has been reviewed. Data on the architecture of S. lemnae nuclear apparatus at interphase and during vegetative cell division, conjugation or autogamy are summarized. Special attention being paid to the structural and functional peculiarities of short macronuclear minichromosomes known to contain protein-coding regions, 5'- and 3'-flanking nontranslated regions, and telomeres. A hypothesis, previously put forward, according to which in the spirotrichous ciliates the telomeres themselves may serve as starting points of replication in minichromosomes, has now received its further substantiation. The recent experimental data, which confirm that 5'-nontranscribed DNA leader sequence of alpha1- and alpha2-tubulin-encoding minichromosomes display at least several regulatory elements typical for eukaryote promoter (TATA-box, CAAT-box, transcriptional initiator), are discussed. Up to now, there is no confirmation with regard to a possible existence in the spirotrichous minichromosomes of specific regulatory sequences capable of controlling both replication and transcription processes.

Lysosomal enzymes in the macronucleus of Tetrahymena during its apoptosis-like degradation.

A key characteristic of apoptosis is its regulated nuclear degradation. Apoptosis-like nuclear degradation also occurs in the ciliated unicellular organism, Tetrahymena thermophila. Chromatin of the macronucleus undergoes massive condensation, a process that can be blocked by caspase inhibitors. The nucleus becomes TUNEL-positive, and its DNA is cleaved into nucleosome-sized fragments. In a matter of hours the macronucleus is completely degraded, and disappears. The condensed nucleus sequesters acridine orange, which means that it might become an acidic compartment. We therefore asked whether lysosomal bodies fuse with the condensed macronucleus to form an autophagosome. We monitored acid phosphatase (AP) activity, which is associated with lysosomal bodies but is not found in normal nuclei. We find that after the macronucleus condenses AP activity is localized in cap-like structures at its cortex. Later, after the degrading macronucleus loses much of its DNA, acid phosphatase deposits appear deeper within the nucleus. We conclude that although macronuclear elimination is initiated by an apoptosis-like mechanism, its final degradation may be achieved through autophagosomy.

A novel mutagen, 2-(5-hydroxy-4,6-dinitroindolyl) ethanol, formed in the reaction between 5-hydroxytryptamine and nitrite under acid conditions, especially in the presence of L-cysteine.

We examined the mutagenic activity of each of 29 amino acids mixed under acidic conditions with 5-hydroxytryptamine (5-HT) and nitrite using Salmonella typhimurium strain TA 100 with or without a metabolic activation system (S9 mix). The reaction mixture containing L-cysteine was strongly mutagenic without S9 mix. We subjected an ethyl acetate extract of the reaction mixture to HPLC, isolated a mutagenic component, and investigated its chemical structure by LC-mass spectrometry (MS), high-resolution fast atom bombardment (HRFAB)-MS, and 1H and 13C NMR. We identified the mutagen as 2-(5-hydroxy-4,6-dinitro-3-indolyl) ethanol (2HDIE). We injected 8 mg/kg 2HDIE i.p. into male ICR mice and found that the compound increased the frequency of micronuclei in peripheral reticulocytes. Our results suggest that 2HDIE might be formed in vivo by consumption of 5-HT, nitrite and L-cysteine in foods, and might act as a mutagen.

Multivariate morphometric analyses of the predatory ciliate genus Semispathidium (Ciliophora: Litostomatea), with description of S. longiarmatum nov. spec.

We studied morphometrical variation, species boundaries, and importance of morphometric features for a reliable separation of five African Semispathidium taxa. Altogether, 20 features traditionally used in alpha-taxonomy of the predatory genus Semispathidium were measured or scored on 85 protargol-impregnated interphase specimens, and were analyzed using hierarchical clustering as well as principal component and canonical discriminant analyses. This multivariate approach confirmed that a population found in Botswanan floodplain soil represents a distinct taxon. The new species is described here as S. longiarmatum, using live observation, protargol impregnation, and scanning electron microscopy. Semispathidium longiarmatum strongly resembles S. armatum and S. breviarmatum but it is clearly distinguished from these species by the extrusome pattern. The reliability of S. longiarmatum is also strengthened, according to the canonical discriminant analysis, by several quantitative features, viz., the number of ciliary rows, the length:width ratio of the macronucleus, and the number of dikinetids in brush row 1. Moreover, the present study documents the distinctness of all African Semispathidium species which can be separated by a combination of both qualitative and quantitative (morphometric) features. Consequently, Semispathidium species do not form a continuous complex but fairly discrete clusters in the phenotypic space.

Morphological and molecular information of a new species of Geleia (Ciliophora, Karyorelictea), with redescriptions of two Kentrophoros species from China.

The morphology and infraciliature of three karyorelictean ciliates, Geleia sinica spec. nov. and two poorly known Kentrophoros species, K. flavus and K. gracilis, isolated from the intertidal zone of a beach at Qingdao, China, were investigated. Geleia sinica spec. nov. is distinguished from its congeners by the following combination of characters: body medium-sized and slender-cylindrical; with a conspicuous prebuccal fossa; 28-34 somatic kineties; about 40 short adoral polykineties; intrabuccal kinety composed of 25-34 dikinetids; paroral kineties composed of closely spaced dikinetids. The comparison with similar congeners clearly supports the validity of this new species based on morphological and small subunit (SSU) rRNA gene sequence data. In light of these new data the "well-known" morphotype, Geleia simplex (Fauré-Fremiet, 1951), is redefined. Two Kentrophoros species are redescribed and improved diagnoses are supplied. Kentrophoros flavus Raikov and Kovaleva, 1968 is mainly characterized by having about 33 macronuclei and 12 micronuclei forming a row that extends along the cell meridian, and 12-19 ciliary rows on the right side of the cell. Kentrophoros gracilis Raikov, 1963 is characterized by having about 14 macronuclei, 13 micronuclei and 10-13 kineties on the right side of the cell.

Gigantic macroautophagy in programmed nuclear death of Tetrahymena thermophila.

Programmed nuclear death (PND) in Tetrahymena is a unique process during conjugation, in which only the parental macronucleus is degraded and then eliminated from the progeny cytoplasm, but other co-existing nuclei such as new micro- and macronuclei are unaffected. PND through autophagic elimination is expected to be strictly controlled, considering the significant roles in ciliates such as turnover of disused organelles and production of the next generation. Here we demonstrate that PND in Tetrahymena involves peculiar aspects of autophagy, which differ from mammalian or yeast macroautophagy. Drastic change of the parental macronucleus occurs when differentiation of new macronuclei is initiated. Combined use of monodansylcadaverine and a lysosome indicator LysoTracker Red showed that prior to nuclear condensation, the envelope of the parental macronucleus changed its nature as if it is an autophagic membrane, without the accumulation of a pre-autophagosomal structure from the cytoplasm. Subsequently, lysosomes approached only to the parental macronucleus and localized at the envelope until a final resorption stage. In addition, we found that the parental macronucleus exhibits certain sugars and phosphatidylserine on the envelope, which are possible "attack me" signals, that are not found on other types of nuclei. These findings suggest that PND is a highly elaborated process, different from the typical macroautophagy seen in other systems, and is executed through interaction between specific molecular signals on the parental macronuclear envelope and autophagic/lysosomal machineries.

Nuclear dimorphism: two peas in a pod.

The macro- and micronuclei of Tetrahymena reside in the same cytoplasm but are about as different as night and day. This extreme case of nuclear dimorphism can now be partially attributed to differences in the subunit compositions of their nuclear pore complexes.

Two distinct repeat sequences of Nup98 nucleoporins characterize dual nuclei in the binucleated ciliate tetrahymena.

Ciliated protozoa have two functionally distinct nuclei, a micronucleus (MIC) and a macronucleus (MAC) [1]. These two nuclei are distinct in size, transcriptional activity, and division cycle control, proceeding with cycles of DNA replication and nuclear division at different times within the same cell [2, 3]. The structural basis generating functionally distinct nuclei remains unknown. Here, we show that, in Tetrahymena thermophila, the nuclear pore complexes (NPCs) of MIC and MAC are composed of different sets of nucleoporins. Among the 13 nucleoporins identified, Nup98 homologs were of interest because two out of the four homologs were localized exclusively in the MAC and the other two were localized exclusively in the MIC. The two MAC-localizing Nup98s contain repeats of GLFG [4]. In contrast, the two MIC-localizing Nup98s lack the GLFG repeats and instead contain a novel repeat signature of NIFN. Ectopic expression of a chimeric MIC-localizing Nup98 homolog bearing GLFG repeats obstructed the nuclear accumulation of MIC-specific nuclear proteins, and expression of a chimeric MAC-localizing Nup98 homolog bearing NIFN repeats obstructed the nuclear accumulation of MAC-specific nuclear proteins. These results suggest that Nup98s act as a barrier to misdirected localization of nucleus-specific proteins. Our findings provide the first evidence that the NPC contributes to nucleus-selective transport in ciliates.

Class I histone deacetylase Thd1p promotes global chromatin condensation in Tetrahymena thermophila.

Class I histone deacetylases (HDACs) regulate DNA-templated processes such as transcription. They act both at specific loci and more generally across global chromatin, contributing to acetylation patterns that may underlie large-scale chromatin dynamics. Although hypoacetylation is correlated with highly condensed chromatin, little is known about the contribution of individual HDACs to chromatin condensation mechanisms. Using the ciliated protozoan Tetrahymena thermophila, we investigated the role of a specific class I HDAC, Tauhd1p, in the reversible condensation of global chromatin. In this system, the normal physiological response to cell starvation includes the widespread condensation of the macronuclear chromatin and general repression of gene transcription. We show that the chromatin in Thd1p-deficient cells failed to condense during starvation. The condensation failure correlated with aberrant hyperphosphorylation of histone H1 and the overexpression of CDC2, encoding the major histone H1 kinase. Changes in the rate of acetate turnover on core histones and in the distribution of acetylated lysines 9 and 23/27 on histone H3 isoforms that were found to correlate with normal chromatin condensation were absent from Thd1p mutant cells. These results point to a role for a class I HDAC in the formation of reversible higher-order chromatin structures and global genome compaction through mechanisms involving the regulation of H1 phosphorylation and core histone acetylation/deacetylation kinetics.

Pelagostrobilidium wilberti n. sp. (Oligotrichea, Choreotrichida): morphology and morphogenesis.

Morphology, infraciliature, morphogenetic features, and some ecological data for Pelagostrobilidium wilberti n. sp. are described. This new species was collected from a temporary pond in Magdalena, Buenos Aires province, Argentina, which was sampled monthly from August 2003 to July 2005. The species was found in autumn and winter. Observations were made in vivo and after staining with protargol. Pelagostrobilidium wilberti n. sp. measures 63-84 x 42-49 microm in vivo and is conical in shape, with a posterior spine-like cytoplasmic process. It possesses 6 somatic kineties, with kinety 2 sinistrally curved and shorter than the others. The oral apparatus is composed of 25-32 external and two internal membranelles. The macronucleus is horseshoe-shaped and located beneath the oral apparatus; two or three spherical micronuclei lie dorsally. There is a posterior contractile vacuole. Morphogenesis is hypo-apokinetal and begins dorsally between the curved kinety 2 and kinety 3. After the discovery of this new species, the diagnosis of the genus Pelagostrobilidium was amended.

Class I histone deacetylase Thd1p affects nuclear integrity in Tetrahymena thermophila.

Class I histone deacetylases (HDACs) participate in the regulation of DNA-templated processes such as transcription and replication. Members of this class can act locally at specific sites, or they can act more globally, contributing to a baseline acetylation state, both of which actions may be important for genome maintenance and organization. We previously identified a macronuclear-specific class I HDAC in Tetrahymena thermophila called Thd1p, which is expressed early in the development of the macronucleus when it initially becomes transcriptionally active. To test the idea that Thd1p is important for global chromatin integrity in an active macronucleus, Tetrahymena cells reduced in expression of Thd1p were generated. We observed phenotypes that indicated loss of chromatin integrity in the mutant cells, including DNA fragmentation and extrusion of chromatin from the macronucleus, variable macronuclear size and shape, enlarged nucleoli, and reduced phosphorylation of histone H1 from bulk chromatin. Macronuclei in mutant cells also contained more DNA. This observation suggests a role for Thd1p in the control of nuclear DNA content, a previously undescribed role for class I HDACs. Together, these phenotypes implicate Thd1p in the maintenance of macronuclear integrity in multiple ways, probably through site-specific changes in histone acetylation since no change in the acetylation levels of bulk histones was detected in mutant cells.

Nocodazole inhibits macronuclear infection with Holospora obtusa in Paramecium caudatum.

Holospora obtusa is a Gram-negative bacterium inhabiting the macronucleus of the ciliate Paramecium caudatum. Experimental infection with H. obtusa was carried out under nocodazole treatment. Nocodazole has been shown to cause disassembly of the cytoplasmic microtubules radiating from the cytopharynx and postoral fibers in P. caudatum. Treatment with this drug did not prevent the ingestion of both prey bacteria and H. obtusa, but it reduced the phagosome number and affected cyclosis. In situ hybridization revealed infectious forms of this endobiont very close to the macronucleus, but never inside it. These results indicate that disassembly of microtubules does not impair transportation of the infectious forms of H. obtusa in the cytoplasm, but that it completely blocks the invasion of the nucleus by the bacteria.

Delayed degradation of parental macronuclear DNA in programmed nuclear death of Paramecium caudatum.

In the ciliated protozoan Paramecium caudatum, a parental macronucleus that is fragmented into some 40-50 pieces during conjugation does not degenerate immediately, but persists until the eighth cell cycle after conjugation. Here we demonstrate that the initiation of the parental macronuclear degeneration occurs at about the fifth cell cycle. The size of parental macronuclear fragments continued to increase between the first and fourth cell cycle, but gradually decreased thereafter. By contrast, a new macronucleus grew and reached a maximum size by the fourth cell cycle, suggesting that the new macronucleus matured by that stage. Southern blot analysis revealed that parental macronuclear DNA was degraded at about the fifth cell cycle. The degradation was supported by acridine orange staining, indicating degeneration of the macronuclear fragments. Prior to the degradation, the fragments once attached to the new macronucleus were subsequently liberated from it. These observations lead us to conclude that once a new macronucleus has been fully formed by the fourth cell cycle, the parental macronuclear fragments are destined to degenerate, probably through direction by new macronucleus. Considering the long persistence of the parental macronucleus during the early cell cycles after conjugation, the macronuclear fragments might function in the maturation of the imperfect new macronucleus. Two possible functions, a gene dosage compensation and adjustment of ploidy level, are discussed.