Abundant and diverse Tetrahymena species living in the bladder traps of aquatic carnivorous Utricularia plants.

Ciliates are unicellular eukaryotes known for their cellular complexity and wide range of natural habitats. How they adapt to their niches and what roles they play in ecology remain largely unknown. The genus Tetrahymena is among the best-studied groups of ciliates and one particular species, Tetrahymena thermophila, is a well-known laboratory model organism in cell and molecular biology, making it an excellent candidate for study in protist ecology. Here, based on cytochrome c oxidase subunit I (COX1) gene barcoding, we identify a total of 19 different putative Tetrahymena species and two closely related Glaucoma lineages isolated from distinct natural habitats, of which 13 are new species. These latter include 11 Tetrahymena species found in the bladder traps of Utricularia plants, the most species-rich and widely distributed aquatic carnivorous plant, thus revealing a previously unknown but significant symbiosis of Tetrahymena species living among the microbial community of Utricularia bladder traps. Additional species were collected using an artificial trap method we have developed. We show that diverse Tetrahymena species may live even within the same habitat and that their populations are highly dynamic, suggesting that the diversity and biomass of species worldwide is far greater than currently appreciated.

The Green Tetrahymena utriculariae n. sp. (Ciliophora, Oligohymenophorea) with Its Endosymbiotic Algae (Micractinium sp.), Living in Traps of a Carnivorous Aquatic Plant.

The genus Tetrahymena (Ciliophora, Oligohymenophorea) probably represents the best studied ciliate genus. At present, more than forty species have been described. All are colorless, i.e. they do not harbor symbiotic algae, and as aerobes they need at least microaerobic habitats. Here, we present the morphological and molecular description of the first green representative, Tetrahymena utriculariae n. sp., living in symbiosis with endosymbiotic algae identified as Micractinium sp. (Chlorophyta). The full life cycle of the ciliate species is documented, including trophonts and theronts, conjugating cells, resting cysts and dividers. This species has been discovered in an exotic habitat, namely in traps of the carnivorous aquatic plant Utricularia reflexa (originating from Okavango Delta, Botswana). Green ciliates live as commensals of the plant in this anoxic habitat. Ciliates are bacterivorous, however, symbiosis with algae is needed to satisfy cell metabolism but also to gain oxygen from symbionts. When ciliates are cultivated outside their natural habitat under aerobic conditions and fed with saturating bacterial food, they gradually become aposymbiotic. Based on phylogenetic analyses of 18S rRNA and mitochondrial cox1 genes T. utriculariae forms a sister group to Tetrahymena thermophila.

Tetrahymena australis (Protozoa, Ciliophora): A Well-Known But "Non-Existing" Taxon - Consideration of Its Identification, Definition and Systematic Position.

A cryptic species of the Tetrahymena pyriformis complex, Tetrahymena australis, has been known for a long time but never properly diagnosed based on taxonomic methods. The species name is thus invalid according to the International Code of Zoological Nomenclature. Recently, a population isolated from a freshwater lake in Wuhan, China was investigated using live observations, silver staining methods and gene sequence data. This organism can be separated from other described species of the T. pyriformis complex by its relatively small body size, the number of somatic kineties and differences in sequences of two genes, namely the small subunit ribosomal RNA (SSU rRNA) and the mitochondrial cytochrome c oxidase subunit I (cox1). We compared the SSU rRNA gene sequences of all available Tetrahymena species to reveal the nucleotide differences within this genus. The sequence of the Wuhan population is identical to two sequences of a previously isolated strain of T. australis (ATCC #30831). Phylogenetic analyses indicate that these three sequences (X56167, M98015, KT334373) cluster with Tetrahymena shanghaiensis (EF070256) in a polytomy. However, sequence divergence of the cox1 gene between the Wuhan population and another strain of T. australis (ATCC #30271) is 1.4%, suggesting that these may represent different subspecies.

Mortality in northern corroboree frog tadpoles (Pseudophryne pengilleyi) associated with Tetrahymena-like infection.

CASE REPORT: Mortality of northern corroboree frog tadpoles and eggs occurred in association with Tetrahymena-like ciliates. The predominant lesions in the tadpoles were inflammation and necrosis of the dermis and skeletal muscle. Some of the egg capsules also contained ciliates, but were overgrown with bacteria and fungi. CONCLUSION: Disease occurred, secondary to underlying husbandry issues, and resolved following their correction.

Environmental and physiological conditions affecting Tetrahymena sp. infection in guppies, Poecilia reticulata Peters.

Parasitic infections caused by Tetrahymena sp. constitute a serious problem in guppies, Poecilia reticulata. Tetrahymena was isolated from skin lesions of naturally infected guppies in a commercial aquaculture farm, cultured in vitro and used in subsequent experimental infections. In addition to guppies, angelfish, Pterophyllum scalare, platyfish, Xiphophorus maculates, and neontetra, Paracheirodon innesi, were susceptible, whereas tilapia (Oreochromis niloticus xO. aureus) was resistant. The ciliate had a high affinity for dead fish. Skin abrasion did not affect the infection, but fish with gas bubble disease exhibited a significantly higher infection than non-affected fish. Infection was significantly higher when fish were exposed to high levels of ammonia, high organic load and low water temperatures. Under shipment conditions, infection was significantly elevated. Full recovery was achieved at a low fish density. Results suggest that poor environmental and physiological conditions enhance infection with Tetrahymena sp.

Enhanced survival of Salmonella enterica in vesicles released by a soilborne Tetrahymena species.

Nondestructive ingestion by soilborne protozoa may enhance the environmental resiliency of important bacterial pathogens and may model how such bacteria evade destruction in human macrophages. Here, the interaction of Salmonella enterica serovar Thompson with a soilborne Tetrahymena sp. isolate was examined using serovar Thompson cells labeled with the green fluorescent protein. The bacteria were mixed in solution with cells of Tetrahymena at several ratios. During incubation with serovar Thompson, Tetrahymena cells released a large number of vesicles containing green fluorescent serovar Thompson cells. In comparison, grazing on Listeria monocytogenes cells resulted in their digestion and thus the infrequent release of this pathogen in vesicles. The number of serovar Thompson cells per vesicle increased significantly as the initial ratio of serovar Thompson to Tetrahymena cells increased from 500:1 to 5,000:1. The density of serovar Thompson was as high as 50 cells per vesicle. Staining with propidium iodide revealed that a significantly higher proportion of serovar Thompson cells remained viable when enclosed in vesicles than when free in solution. Enhanced survival rates were observed in vesicles that were secreted by both starved (F = 28.3, P < 0.001) and unstarved (F = 14.09, P < 0.005) Tetrahymena cells. Sequestration in vesicles also provided greater protection from low concentrations of calcium hypochlorite. Thus, the release of this human pathogen from Tetrahymena cells in high-density clusters enclosed in a membrane may have important implications for public health.

Core formation and the acquisition of fusion competence are linked during secretory granule maturation in Tetrahymena.

The formation of dense core secretory granules is a multistage process beginning in the trans Golgi network and continuing during a period of granule maturation. Direct interactions between proteins in the membrane and those in the forming dense core may be important for sorting during this process, as well as for organizing membrane proteins in mature granules. We have isolated two mutants in dense core granule formation in the ciliate Tetrahymena thermophila, an organism in which this pathway is genetically accessible. The mutants lie in two distinct genes but have similar phenotypes, marked by accumulation of a set of granule cargo markers in intracellular vesicles resembling immature secretory granules. Sorting to these vesicles appears specific, since they do not contain detectable levels of an extraneous secretory marker. The mutants were initially identified on the basis of aberrant proprotein processing, but also showed defects in the docking of the immature granules. These defects, in core assembly and docking, were similarly conditional with respect to growth conditions, and therefore are likely to be tightly linked. In starved cells, the processing defect was less severe, and the immature granules could dock but still did not undergo stimulated exocytosis. We identified a lumenal protein that localizes to the docking-competent end of wildtype granules, but which is delocalized in the mutants. Our results suggest that dense cores have functionally distinct domains that may be important for organizing membrane proteins involved in docking and fusion.

Novel cytoskeletal proteins in the cortex of Tetrahymena.

An important unsolved problem lies in the mechanisms that determine overall size, shape, and the localization of subcellular structures in eukaryotic cells. The membrane skeleton must play a central role in these processes in many cell types, and the ciliate membrane skeleton, or epiplasm, offers favorable opportunities for exploring the molecular determinants of cortical organization. Among the ciliates, Tetrahymena is well suited for the application of a wide range of molecular and cellular approaches. Progress has been made in the identification and sequencing of genes and proteins that encode epiplasmic and cortical proteins. The amino acid sequences of these proteins suggest that they define new classes of cytoskeletal proteins, distinct from the articulin and epiplasmin proteins. We will also discuss recent in vivo and in vitro studies of the regulation of assembly of these cortical proteins. This will include information regarding the down-regulation of epiplasmic proteins during cleavage, their topographic regulation in the cell cycle, and the results of in vitro assembly and binding studies of the epiplasmic C protein.

High mortality due to Tetrahymena sp. infection in laboratory-maintained zebrafish (Brachydanio rerio).

A large colony of laboratory zebrafish (Brachydanio rerio) used in the study of early vertebrate embryogenesis began experiencing acute, unexplained mortality that approached 100% among approximately 30-day-old resident fry. The initial differential diagnosis included ammonia, nitrite, or chlorine toxicosis, as well as iatrogenically induced toxicosis associated with improper sanitation procedures of laboratory equipment. Necropsy of dead and moribund fry prior to fixation revealed swarms of ovoid-shaped, motile, ciliated protozoa with a "spiraling football" motion. Wet mount preparations of various water samples also contained high numbers of similar protozoa. Histologic examination of affected fry revealed numerous, periodic acid-Schiff-positive forms within the body coelom, and epithelial and muscle tissues. The protozoa were consistent morphologically with members of the genus Tetrahymena, which is usually a free-living, nonpathogenic ciliated protozoa in fresh and saltwater environments. Relevant disease associated with Tetrahymena spp. in viviparous fish has been reported as a result of concurrent disease, immunosuppression, or poor water quality conditions. To the authors' knowledge, this is the first report of an epizootic involving laboratory maintained zebrafish, and the diagnostic course and therapeutic interventions undertaken to alleviate Tetrahymena species-associated clinical disease.

Improvement of ciliate identification and quantification: a new protocol for fluorescence in situ hybridization (FISH) in combination with silver stain techniques.

A new protocol for taxon specific probe based fluorescent in situ hybridization was developed for the identification and quantification of ciliates in microbial communities. Various fixatives and experimental parameters were evaluated and optimized with respect to cell permeability and morphological preservation. Optimum results were adaption by obatined of a modified fixation method using Bouin's solution. Furthermore, conventional staining procedures such as different Protargol stain techniques and a silver nitrate impregnation method were modified and can now be applied in combination with fluorescence in situ hybridization. The new protocol allows a rapid and reliable identification as well as quantification of ciliates based upon classical morphological aspects and rRNA based phylogenetic relationships performed in one experiment. Furthermore, a set of specific probes targeting different regions of the 18S rRNA was designed for Glaucoma scintillans Ehrenberg, 1830 and tested by applying this new approach of combining in situ cell hybridization with conventional staining techniques.

A conditional mutant having paralyzed cilia and a block in cytokinesis is rescued by cytoplasmic exchange in Tetrahymena thermophila.

Nineteen mutants that are conditional for both the ability to regain motility following deciliation and the ability to grow were isolated. The mutations causing slow growth were placed into five complementation groups. None of the mutations appears to affect energy production as all mutants remained motile at the restrictive temperature. In three complementation groups protein synthesis and the levels of mRNA encoding alpha-tubulin or actin were largely unaffected at the restrictive temperature, consistent with the hypothesis that mutations in these three groups directly affect the assembly of functional cilia and growth. Complementation group 1 was chosen for further characterization. Both phenotypes were shown to be linked, suggesting they are caused by a single mutation. Group 1 mutants regenerated cilia at the restrictive temperature, but the cilia were nonmotile. This mutation also caused a block in cytokinesis at the restrictive temperature but did not affect nuclear divisions or DNA synthesis. The block in cell division was transiently rescued by wild-type cytoplasm exchanged when mutants were paired with wild-type cells during conjugation (round 1 of genomic exclusion). Thus, at least one mutation has been isolated that affects assembly of some microtubule-based structures in Tetrahymena (cilia during regeneration) but not others (nuclei divide at 38 degrees), and the product of this gene is likely to play a role in both ciliary function and in cytokinesis.

Isolation and characterization of a mutant of Tetrahymena thermophila blocked in secretion of lysosomal enzymes.

The development of a sensitive screening procedure for mutants of Tetrahymena thermophila blocked in secretion of lysosomal enzymes is described. By means of this procedure a mutant blocked in secretion of lysosomal enzymes has been isolated. This sec- mutant, MS-1, is constitutively blocked in release of at least six lysosomal enzymes, under both nutrient and non-nutrient conditions. MS-1 possesses, bound within the cell, the same amount of active lysosomal enzymes as the wild type. During starvation in media of low ionic strength MS-1 develops a highly vacuolated phenotype. This phenotype is caused by the sec- allele. It is reversed to a normal cell shape when the mutant is transferred to isotonic medium. The sec- mutant MS-1 contains mucocysts and is capable of inducing exocytosis of these secretory organelles, suggesting that Tetrahymena possesses at least two independent protein-secreting organelles.

[Ciliated protozoa and thanatology]. / Protozoaires ciliés et thanatologie.

Different ciliated protozoa are observed by the immersion of dead bodies in soft water. In death from drowning a ciliated Tetrahymena kind was discovered whose size and hemotactism facilitate in a priviliged way the penetration in the internal medium through pulmonary channels. Diagnosis in blood was easy by putting this organism in culture. This method had an astonishing power of reproduction. This search fortunately completes the series about diatoms by means of corroboration on the diagnosis of drowning.

New wild Tetrahymena from Southeast Asia, China, and North America, including T. malaccensis, T. asiatica, T. nanneyi, T. caudata, and T. silvana n. spp.

Tetrahymena of the T. pyriformis complex collected from varied habitats in Malaysia, Thailand, and The People's Republic of China include strains of the micronucleate species T. americanis and T. canadensis and the amicronucleate T. pyriformis and T. elliotti. Two new breeding species are described-T. malaccensis from Malaysia and T. asiatica from China and Thailand. Two wild selfers from China and some of the amicronucleate strains from all three countries fall into isozymic groups similar to named micronucleate and amicronucleate species. The T. patula complex is represented by two groups of clones from Malaysia that fit the morphological description of T. vorax. They, however, have radically different isozymic electrophoretic patterns and both groups differ from those of previously described T. vorax. As their molecules indicate relationships to other "T. vorax" strains as distant as that between T. vorax and T. leucophrys, they are considered to be new species, T. caudata and T. silvana. A third new breeding species, T. nanneyi, was identified among strains previously collected in North America. Viable immature progeny were obtained from the new strains of the five breeding species. Maximum temperature tolerances were determined for the new strains of four of the breeding species.

Isolation and phenotypic characterization of Tetrahymena thermophila size mutants: the relationship between cell size and regulation of DNA content.

Temperature-sensitive size mutants of the ciliate Tetrahymena thermophila were selected following chemical mutagenesis. Phenotypical characteristics are given for seven cell lines, which have a range of average cell volumes from 8000 microns 3 to more than 100 000 microns 3. wild-type Tetrahymena cells have an average cell volume of 15 000 microns 3. Two of the mutagenized cell lines have comparatively small cells at 29 degrees C but normal cells at 37 degrees C; whereas the other five lines are normal at 29 degrees C but large at 37 degrees C. While the small cells are poor growers, the large cells grow excellently at 37 degrees C. Measurements of DNA, RNA and protein contents indicate a significant correlation between all parameters and cell size. However, since the cells tolerate considerably different concentrations of each class of macromolecules, the amount of any of these macromolecules cannot be tightly controlled by cell size.

Mass isolation and fertility testing of temperature-sensitive mutants in Tetrahymena.

A set of 239 heat-sensitive (38 degrees) and 16 cold-sensitive (18 degrees) conditional mutants of Tetrahymena has been generated by combining techniques to manipulate large numbers of clones with a method for the selection of self-fertilized cells after mutagen treatment. A simple technique is presented for determining the fertility of individual clones; 179 of the clones in this set (71%) are fertile. The fertile conditional mutants have already been shown to have lesions in a number of diverse functions, including nucleic acid metabolism, mobility, cell cycle, and cortical pattern.

Protozoa and the decline of Rhizobium populations added to soil.

A fall in Rhizobium abundance occurred in nonsterile soil inoculated with large numbers of the root-nodule bacteria, but many of the rhizobia still survived. No such decline was evident in sterile soil. Protozoa feeding on these bacteria were isolated from soil and other environments. As the abundance of Rhizobium meliloti and a cowpea Rhizobium strain in soil decreased, the protozoan density increased. The inability of the predators to eliminate their prey from soil was not the result of the presence of organisms feeding on the protozoa because many rhizobia survived in sterile soil inoculated with the prey and cultures of individual protozoa, nor was it the result of the rapid multiplication of the bacteria to replace those consumed because survivors were still numerous in essentially organic matter free soil in which the bacteria did not grow appreciably. The lack of elimination also was not associated with a protective effect of soil particles because survivors were still abundant in solutions inoculated with protozoa and bacteria. It is suggested that the size of the prey population diminishes until a density is attained at which the energy used by the predator in hunting for the survivors equals that obtained from the feeding.