Evolutionary Origin and Host Range of Plagiotoma lumbrici (Ciliophora, Hypotrichia), an Obligate Gut Symbiont of Lumbricid Earthworms.

Four common earthworm species, the anecic Lumbricus terrestris, the endogeic Octolasion tyrteum as well as the epigeic Eisenia fetida and Dendrobaena veneta, were examined for the presence of the microbial gut symbiont Plagiotoma lumbrici. The evolutionary origin of this endobiotic microbe was reconstructed, using the 18S rRNA gene, the ITS1-5.8S-ITS2 region, and the first two domains of the 28S rRNA gene. Plagiotoma lumbrici was exclusively detected in the anecic Lumbricus terrestris. Multigene analyses and the ITS2 secondary structure robustly determined the phylogenetic home of Plagiotoma lumbrici populations within the oxytrichid Dorsomarginalia (Spirotrichea: Hypotrichia) as a sister taxon of the free-living Hemiurosomoida longa. This indicates that earthworms obtained their gut endosymbiont by ingesting soil/leaf litter containing oxytrichine ciliates that became adapted to the intestinal tract of earthworms. Interestingly, according to the literature data, Plagiotoma lumbrici was detected in multiple anecic and some epigeic but never in endogeic earthworms. These observations suggest that Plagiotoma lumbrici might be adapted to certain gut conditions and the lifestyle of anecic Lumbricidae, such as Lumbricus, Aporrectodea, and Scherotheca, as well as of some co-occurring epigeic Lumbricus species.

Interaction Between a Bacterivorous Ciliate Aspidisca cicada and a Rotifer Lecane inermis: Doozers and Fraggles in Aquatic Flocs.

Activated sludge is a semi-natural habitat composed of macroaggregates made by flocculating bacteria and inhabited by numerous protozoans and metazoans, creating a complicated interactome. The activated sludge resembles the biological formation of naturally occurring floc habitats, such as "marine snow." So far, these two types of habitat have been analyzed separately, despite their similarities. We examined the effect of a bacterivorous ciliate, Aspidisca cicada, on the quality of the macroaggregate ecosystem by estimating (i) the floc characteristics, (ii) the proliferation of other bacterivores (rotifers), and (iii) the chemical processes. We found that A. cicada (i) positively affected floc quality by creating flocs of larger size; (ii) promoted the population growth of the rotifer Lecane inermis, an important biological agent in activated sludge systems; and (iii) increased the efficiency of ammonia removal while at the same time improving the oxygen conditions. The effect of A. cicada was detectable long after its disappearance from the system. We therefore claim that A. cicada is a very specialized scavenger of flocs with a key role in floc ecosystem functioning. These results may be relevant to the ecology of any natural and engineered aggregates.

Resting cysts of Parentocirrus hortualis Voß, 1997 (Ciliophora, Hypotrichia), with preliminary notes on encystation and various types of excystation.

Resting cysts of Parentocirrus hortualis were investigated, using live observation, SEM and TEM. Processes during encystation and excystation were observed in vivo under the light microscope. During encystation, the trophic body becomes globular, the ciliature is resorbed in an anterior direction, the macronuclear nodules fuse into an elongated mass, and finally a cyst wall develops. As typical for oxytrichids, the resting cysts of P. hortualis are of the kinetosome-resorbing type and their wall is made of four layers: ectocyst, mesocyst, endocyst, and metacyst. The beginning of excystation is indicated by the formation of an excystation vacuole that helps the regenerating specimen to break the cyst wall. The excysting specimen leaves the resting cyst in a thin membrane that is gradually resorbed in the outer environment. Also two other excystation modes were observed. During the rare mode, the excystation vacuole breaks the thin membrane instead of the cyst wall that ruptures under the pressure of the body of the regenerating specimen. During the reproduction mode, the regenerating specimen divides within the resting cyst, producing two to four tomites. This is the first report of division in resting cysts of oxytrichids, but reproduction in division cysts was already described in keronopsids.

Morphology and SSU rRNA gene-based phylogeny of two Diophrys-like ciliates from northern China, with notes on morphogenesis of Pseudodiophrys nigricans (Protozoa, Ciliophora).

Two free-living marine euplotid ciliates, Pseudodiophrys nigricans and Diophrys japonica, collected from the coastal waters off Qingdao, northern China, were investigated using live observations and protargol impregnation methods. The cortical development of P. nigricans was observed during binary division. Although its general pattern of morphogenesis is similar to that of other Diophrys-like species, three unusual features are noteworthy: 1) the frontoventral transverse cirral anlagen develop in the secondary mode, similar to that of Euplotes; 2) the undulating membrane anlage migrates far from the cytostome and does not split into two membranes; and 3) the parental adoral zone of membranelles remains nearly intact throughout the entire morphogenetic process. Diophrys japonica is redescribed based on a Chinese population and can be recognized by having one left marginal cirrus, densely arranged cortical granules, and a fragment kinety with three dikinetids. Phylogenetic analyses based on the small subunit rRNA (SSU rRNA) gene sequence data indicate that D. japonica is placed within the Diophrys clade and is most closely related to the well-known D. apoligothrix.

Some ultrastructural observations of the vegetative, resting and excysting ciliate, urostyla grandis (urostylidae, hypotrichida).

In order to reveal the differentiation characteristics of organelles of ciliates under different physiological status, the cellular ultrastructure of Urostyla grandis was studied by transmission electron microscopy. In the resting cells most ciliary shafts, kinetosomes and sub-pellicle microtubules were resorbed, and the endoplasmic reticulum (ER) disappeared with the autophagocytosis taking place within the cytoplasm. As well, the nuclear matrix of the macronucleus was extruded into the cytoplasm, forming pseudopodia-like structures with large quantities of heterochromatin (CH) attached to the inner nuclear membrane. During excystment, membraneous structures developed and gradually increased in number to form the ER. Autophagic vacuoles (AVs) appeared containing mitochondria, paraglycogen particles (PGP), membranous structures, etc. Moreover, the number of nucleoli decreased with the chromatin, condensing in parallel with the process of recombination. Based on these observations, it could be concluded that the de-differentiation of microtubular organelles and the changes occurring in macronuclei in the resting Urostyla grandis, as well as the differentiation of cytoplasmic organelles, digestion by AVs, and the recombination of chromatin during excystment, are not similar to events that occur in non-kinetosome-resorbing cysts (NKR).

Some ultrastructural observations of the vegetative, resting and excysting ciliate, urostyla grandis (urostylidae, hypotrichida)

In order to reveal the differentiation characteristics of organelles of ciliates under different physiological status, the cellular ultrastructure of Urostyla grandis was studied by transmission electron microscopy. In the resting cells most ciliary shafts, kinetosomes and sub-pellicle microtubules were resorbed, and the endoplasmic reticulum (ER) disappeared with the autophagocytosis taking place within the cytoplasm. As well, the nuclear matrix of the macronucleus was extruded into the cytoplasm, forming pseudopodia-like structures with large quantities of heterochromatin (CH) attached to the inner nuclear membrane. During excystment, membraneous structures developed and gradually increased in number to form the ER. Autophagic vacuoles (AVs) appeared containing mitochondria, paraglycogen particles (PGP), membranous structures, etc. Moreover, the number of nucleoli decreased with the chromatin, condensing in parallel with the process of recombination. Based on these observations, it could be concluded that the de-differentiation of microtubular organelles and the changes occurring in macronuclei in the resting Urostyla grandis, as well as the differentiation of cytoplasmic organelles, digestion by AVs, and the recombination of chromatin during excystment, are not similar to events that occur in non-kinetosome-resorbing cysts (NKR).

snRNA and heterochromatin formation are involved in DNA excision during macronuclear development in stichotrichous ciliates.

Several models for specific excision of micronucleus-specific DNA sequences during macronuclear development in ciliates exist. While the template-guided recombination model suggests recombination events resulting in specific DNA excision and reordering of macronucleus-destined sequences (MDS) guided by a template, there is evidence that an RNA interference-related mechanism is involved in DNA elimination in holotrichous ciliates. We describe that in the stichotrichous ciliate Stylonychia, snRNAs homologous to micronucleus-specific sequences are synthesized during macronuclear differentiation. Western and in situ analyses demonstrate that histone H3 becomes methylated at K9 de novo during macronuclear differentiation, and chromatin immunoprecipitation revealed that micronucleus-specific sequences are associated with methylated H3. To link both observations, expression of a PIWI homolog, member of the RNA-induced silencing complex, was silenced. In these cells, the methylated micronucleus-specific histone H3 variant "X" is still present in macronuclear anlagen and no K9 methylation of histone H3 is observed. We suggest that snRNA recruits chromatin-modifying enzymes to sequences to be excised. Based on our and earlier observations, we believe that this mechanism is not sufficient for specific excision of sequences and reordering of MDS in the developing macronucleus and propose a model for internal eliminated sequence excision and MDS reordering in stichotrichous ciliates.

Organization of the macronuclear gene-sized pieces of stichotrichous ciliates into a higher order structure via telomere-matrix interactions.

Macronuclear DNA of stichotrichous ciliates occurs in small 'gene-sized' molecules with sizes of about 0.5 to 40 kb. Each of these molecules is terminated by telomeric sequences of defined length. A single macronucleus contains up to 10(8) DNA molecules; due to the high concentration of telomeric sequences in this nucleus it is an attractive model to study telomere behaviour. We recently provided evidence that macronuclear telomeres are attached to the nuclear matrix and that this interaction is mediated by the telomere binding protein (TeBP). Using various experimental approaches, we now demonstrate that telomeres as well as both subunits of the telomere binding protein are associated with the nuclear matrix. However, there is no direct binding of telomeric DNA to the matrix but telomere matrix interaction is exclusively mediated by the TeBP. In addition, we show that telomeric sequences adopt in vivo the antiparallel G-quartet structure when bound to the nuclear matrix. These data not only allow us to propose a model for macronuclear architecture but may also be relevant for further analysis of telomere-matrix interactions in higher eukaryotes.

Minichromosomal DNA replication in the macronucleus of the hypotrichous ciliate Stylonychia lemnae is independent of chromosome-internal sequences.

The origins of DNA replication in prokaryotes and eukaryotes are typically defined by cis-acting sequences. However, in ciliates, evidence suggests that the replication of short macronuclear minichromosomes may not require such determinants. In hypotrichous ciliates, macronuclei contain millions of gene-sized minichromosomes, which generally have a single protein-coding region, two short noncoding flanks and, on each end, a short telomere consisting of a double-stranded repeat region and a single-stranded 3' overhang. Electron microscopic studies that showed that replication of minichromosomes initiates at or near telomeres and the discovery of a primase activity synthesizing RNA primers over the whole 3' telomeric overhang in vitro suggested that minichromosome replication starts directly at telomeres. Conversely, many minichromosomes contain an AT-rich, semi-conserved, palindromic sequence motif in their subtelomeric regions and it has been proposed that this motif is involved in regulating minichromosomal replication. To analyze what sequences or structures of the minichromosomes are essential for DNA replication, we stably transfected genetically modified alpha1-tubulin-encoding minichromosomes into the hypotrichous ciliate Stylonychia lemnae. Cotransfection of mutated and control minichromosomes revealed that noncoding regions can be deleted or replaced with unrelated sequences without affecting minichromosome replication efficiency in vegetatively growing cells. Similarly, replacement of the coding region resulted in a minichromosome that was stably maintained in transfected cells at the same high copy number for many months. In contrast, alpha1-tubulin-encoding minichromosomes without telomeres were rapidly lost after transfection. Hence, DNA replication of the alpha1-tubulin-encoding minichromosome does not depend on chromosome-internal sequences but may depend on telomeres.

Association of the telomere-telomere-binding protein complex of hypotrichous ciliates with the nuclear matrix and dissociation during replication.

Telomeric interactions with the nuclear matrix have been described in a variety of eukaryotic cells and seem to be essential for specific nuclear localization. Macronuclear DNA of hypotrichous ciliates occurs in small gene-sized DNA molecules, each being terminated by telomeres. Each macronucleus contains over 10(8 )individual DNA molecules. Owing to the high number of telomeres present in this nucleus it provides an excellent model to study telomere behaviour throughout the cell cycle. In this study we provide experimental evidence that the telomere-telomere-binding protein (TEBP) complex specifically interacts with components of the nuclear matrix in vivo. In the course of replication the specific interaction of the TEBP with components of the nuclear matrix is resolved and an attachment of the telomeres to the matrix no longer occurs.

Messenger RNA in dormant cells of Sterkiella histriomuscorum (Oxytrichiade): indentification of putative regulatory gene transcripts.

In the absence of food, the oxytrichid Sterkiella histriomuscorum, like many ciliates, enters into dormancy and transforms into a round and walled encysted cell. When transferred back into a feeding medium, the cyst re-transforms into a vegetative cell in a few hours. This encystment-excystment pathway, which is common to many free-living and parasitic protists, is still poorly understood at the molecular level. In order to identify potential dormant transcripts in the cysts of Sterkiella, we have constructed cDNA libraries from mature cysts. Transcripts have been isolated confirming the presence of a mRNA pool in the dormant cells. The sequence analysis of two cDNA indicates open reading frames which show significant similarities to known proteins involved in mechanisms of regulation: 1) nifR3, an element of the nitrogen regulatory system in bacteria and 2) CROC-1, a newly identified human transcription factor. The two corresponding macronuclear genes represent the first putative regulatory genes isolated in ciliates. From a differential screening of the cDNA library against vegetative cDNA, one cyst-specific (and very abundant) transcript has been isolated but the product has not yet been identified. The possible involvment of these new ciliate genes in the excystment process is discussed.