Activation of an Aquareovirus,Chum Salmon Reovirus (CSV), by the Ciliates Tetrahymena thermophila and T. canadensis

For the first time, ciliates have been found to activate rather than inactivate a virus, chum salmon reovirus (CSV). Activation was seen as an increase in viral titre upon incubation of CSV at 22 °C with Tetrahymena canadenesis and two strains of T. thermophila: wild type (B1975) and a temperature conditional mutant for phagocytosis (NP1). The titre increase was not likely due to replication because CSV had no visible effects on the ciliates and no vertebrate virus has ever been shown unequivocally to replicate in ciliates. When incubated with B1975 and NP1 at 30 °C, CSV was activated only by B1975. Therefore, activation required CSV internalization because at 30 °C only B1975 exhibited phagocytosis. CSV replicated in fish cells at 18 to 26 °C but not at 30 °C. Collectively, these observations point to CSV activation being distinct from replication. Activation is attributed to the CSV capsid being modified in the ciliate phagosomal‐lysosomal system and released in a more infectious form. When allowed to swim in CSV‐infected fish cell cultures, collected, washed, and transferred to uninfected cultures, T. canadensis caused a CSV infection. Overall the results suggest that ciliates could have roles in the environmental dissemination of some fish viral diseases.     

Climacostol inhibits <Emphasis Type="Italic">Tetrahymena</Emphasis> motility and mitochondrial respiration

Climacostol is a resorcinol derivative that is produced by the ciliate Climacostomum virens. Exposure to purified climacostol results in lethal damage to the predatory ciliate Dileptus margaritifer and several other ciliates. To elucidate the mechanism of climacostol toxic action, we have investigated the effects of this compound on the swimming behavior of Tetrahymena thermophila and the respiratory system of isolated rat liver mitochondria. When added to living T. thermophila cells, climacostol markedly increased the turning frequency that was accompanied by a decrease in swimming velocity and subsequently by cell death. Observations by DIC and fluorescence microscopy showed morphological alterations in climacostol treated T. thermophila, indicating that climacostol might exert cytotoxic action on this organism. In the experiment with isolated rat liver mitochondria, climacostol was found to inhibit the NAD-linked respiration, but had no apparent effect on succinate-linked respiration. This finding indicates that climacostol specifically inhibits respiratory chain complex I in mitochondria. The combination of results suggest that the inhibition of mitochondrial respiration may be the cytotoxic mechanism of climacostol’s defenses against predatory protozoa.     

Survival and Growth of Tetrahymena thermophila in Media that are Conventionally Used for Piscine and Mammalian Cells

The transfer of Tetrahymena thermophila from normosmotic solutions (~20–80 mOsm/kg H₂O) to hyperosmotic solutions (> 290 mOsm/kg H₂O) was investigated. During the first 24 h of transfer from proteose peptone yeast extract (PPYE) to either 10 mM HEPES or PPYE with added NaCl to give ~300 mOsm/kg H₂O, most ciliates died in HEPES but survived in PPYE. Supplementing hyperosmotic HEPES or PPYE with fetal bovine serum (FBS) enhanced survival. When ciliates were transferred from PPYE to a basal medium for vertebrate cells, L‐15 (~320 mOsm/kg H₂O), only a few survived the first 24 h but many survived when the starting cell density at transfer was high (100,000 cells/ml) or FBS was present. These results suggest that nutrients and/or osmolytes in either PPYE or FBS helped ciliates survive the switch to hyperosmotic solutions. FBS also stimulated T. thermophila growth in normosmotic HEPES and PPYE and in hyperosmotic L‐15. In L‐15 with 10% FBS, the ciliates proliferated for several months and could undergo phagocytosis and bacterivory. These cell culture systems and results can be used to explore how some Tetrahymena species function in hyperosmotic hosts and act as opportunistic pathogens of vertebrates.     

Influence of extremely low frequency electromagnetic fields on the swimming behavior of ciliates

Different species of ciliates (Paramecium biaurelia, Loxodes striatus, Tetrahymena thermophila) have been taken as model systems to study the effects of extremely low-frequency electromagnetic fields (50 Hz, 0.5–2.0 mT) on the cellular level. A dose-dependent increase in the mean swimming velocity and a decrease in the linearity of cell tracks were observed in all wild-type cells. In contrast, field-exposure did not increase the number of directional turns of the Paramecium tetraurelia pawn mutant (d4–500r), which is characterized by defective Ca²⁺-channels. The described changes indicate a direct effect of low frequency electromagnetic fields on the transport mechanisms of the cell membrane for ions controlling the motile activity of cilia. Bioelectromagnetics 18:491–498, 1997. © 1997 Wiley-Liss, Inc.     

Chemical stimulation of phagocytosis in Amoeba proteus and the influence of external calcium

Summary The process of phagocytosis in Amoeba proteus was examined by following the uptake of Tetrahymena pyriformis and agarose beads. The ciliates are taken up in a time dependent and saturable manner. T. pyriformis apparently emits a water-soluble substance that acts as a chemoattractant to the amoebae. Plain agarose beads are not engulfed by A. proteus, but those beads having reducedglutathione with the -SH group exposed are taken up almost to the same extent as T. pyriformis. Phagocytosis of the glutathione beads is calcium-dependent with maximum bead uptake at 10^-4M Ca⁺⁺. Glutathione applied to A. proteus brings about pseudopod formation, increased phagocytosis and displacement of surface-associated calcium.     

The Relation of Concanavalin A Receptor Distribution to the Conjugation Process in Tetrahymena thermophila

By using fluorescent isothiocyanate-conjugated concanavalin A (FITC-Con A), cell surface events were examined under a light microscope during the early period of the conjugation process in Tetrahymena thermophila. Until the two complementary mating types (D-III and IV) were mixed, Con A-binding activities were hardly detected on the cell surface of ciliates. After mixing, however, the FITC-Con A (25 μg/ml) bound especially to the anterior cell surface at the early stage of conjugation, followed by characteristic changes of the Con A-binding pattern and, subsequently, by formation of a bright fluorescent ring around the area of contact between conjugants. Such alterations of FITC-Con A-binding pattern were found to be interrupted or eliminated by cycloheximide (2 μg/ml). These findings are related to the onset and subsequent conjugation in T. thermophila.     

Genome analysis of sphingolipid metabolism-related genes in Tetrahymena thermophila and identification of a fatty acid 2-hydroxylase involved in the sexual stage of conjugation

Sphingolipids are bioactive lipids present in all eukaryotes. Tetrahymena thermophila is a ciliate that displays remarkable sphingolipid moieties, that is, the unusual phosphonate-linked headgroup ceramides, present in membranes. To date, no identification has been made in this organism of the functions or related genes implicated in sphingolipid metabolism. By gathering information from the T. thermophila genome database together with sphingolipid moieties and enzymatic activities reported in other Tetrahymena species, we were able to reconstruct the putative de novo sphingolipid metabolic pathway in T. thermophila. Orthologous genes of 11 enzymatic steps involved in the biosynthesis and degradation pathways were retrieved. No genes related to glycosphingolipid or phosphonosphingolipid headgroup transfer were found, suggesting that both conserved and innovative mechanisms are used in ciliate. The knockout of gene TTHERM_00463850 allowed to identify the gene encoding a putative fatty acid 2-hydroxylase, which is involved in the biosynthesis pathway. Knockout cells have shown several impairments in the sexual stage of conjugation since different mating types of knockout strains failed to form cell pairs and complete the conjugation process. This fatty acid 2-hydroxylase gene is the first gene of a sphingolipid metabolic pathway to be identified in ciliates and have a critical role in their sexual stage.     

Effect of <Emphasis Type="Italic">Tetrahymena</Emphasis> on the occurrence of achlyosis in the guppy

Tetrahymena infection has become the most problematic parasitic disease of the guppy Poecilia reticulata in Southeast Asia. Tetrahymena corlissi was isolated from guppies with a fungal infection in Thailand, and the fungus was identified as Achlya bisexualis. Male and female guppies were artificially infected with both organisms. The results showed that guppies could easily be artificially infected with a culture of Tetrahymena corlissi and that female guppies were more sensitive than male guppies. Achlya bisexualis infection was shown to be a secondary infection after the Tetrahymena infection. Received: April 29, 2001 / Accepted: September 6, 2001     

Tetrahymena Expresses More than a Hundred Proteins with Lipid‐binding MORN Motifs that can Differ in their Subcellular Localisations

Proteins with membrane occupation and recognition nexus (MORN) motifs are associated with cell fission in apicomplexan parasites, chloroplast division in Arabidopsis and the motility of sperm cells. We found that ciliates are among those that encode the largest variety of MORN proteins. Tetrahymena thermophila expresses 129 MORN protein‐encoding genes, some of which are specifically up‐regulated during conjugation. A lipid‐binding assay underpins the assumption that the predominant function of MORN motifs themselves is to confer the ability of lipid binding. The localisation of four MORN candidate proteins with similar characteristics highlights the functional diversity of this group especially in ciliates.     

Wild-type and food-vacuole-less Tetrahymena thermophila : Model for iron uptake and utilization in animal cells

Wild-type Tetrahymena thermophila and a conditional lethal mutant defective in food-vacuole formation constitute a useful model system for studies of iron uptake and utilization in animal cells.     

Marked Amplification and Diversification of Products of ras Genes from Rat Brain,Rab GTPases,in the Ciliates Tetrahymena thermophila and Paramecium tetraurelia

ABSTRACT. Small GTPase Rab (products of ras genes from rat brain) is a widely conserved molecular switch among eukaryotes and regulates membrane trafficking pathways. It is generally considered that the number of Rab encoded in the genome correlates with multicellularity; however, we found that unicellular ciliates Tetrahymena thermophila (Tt) and Paramecium tetraurelia (Pt) possess many more Rab genes in their genome than the 64 HsRab genes in the human genome. We succeeded in isolating 86 cDNA clones of 88 TtRab genes in the Tetrahymena genome. By comparing the amino acid sequence of Rab in humans and the budding yeast Saccharomyces cerevisiae, 42 TtRab belonged to subfamilies functionally characterized and designated as conventional Rab, while the remaining 44 TtRab were considered to be species‐specific. To examine the diversity of Rab in ciliates, we searched for Rab genes in the genome database of P. tetraurelia. Overall, 229 PtRab genes were found and categorized as 157 conventional and 72 species‐specific PtRab, respectively. Among them, nine PtRab genes showed high homology to seven TtRab, suggesting the conservation of ciliate‐specific Rab. These data suggested that the range of Rab is markedly amplified and diversified in ciliates, which may support the elaborate cellular structures and vigorous phagocytosis of those organisms.     

Bioenergetic Investigation of the Effects of La(III) and Ca(II) on the Metabolic Activity of Tetrahymena thermophila BF5

The heat flux of Tetrahymena thermophila BF5 during growth and the effects of La³⁺ and Ca²⁺ on them were investigated with microcalorimetry; simultaneously, morphological changes of T. thermophila were obtained by light microscope. La³⁺ in low concentration (0–5.0 × 10^–4 mol/l) remarkably stimulated T. thermophila metabolism, but high dose of La³⁺ (5.8–8.6 × 10^–4 mol/l) restrained it in a linear manner with IC₅₀ being 7.2 × 10^–4 mol/l. In contrast, low concentration of Ca²⁺ did not manifest obvious stimulation on T. thermophila metabolism; moreover, the IC₅₀ of Ca²⁺ was much higher than that of La³⁺. Low concentration of La³⁺ did not lead to changes in appearance of T. thermophila, but low dose of Ca²⁺ clearly promoted the cell proliferation. In addition, the morphological changes of T. thermophila evoked by high concentrations of La³⁺ and Ca²⁺ were consistent with relevant microcalorimetric results. It is concluded that La and Ca influence T. thermophila via different pathways, and La represents toxic action rather than Ca analogy.     

Efficiency of Filter Feeding in Two Species of Tetrahymena*

SYNOPSIS. Rates of removal of suspended India ink particles from the surrounding medium by 2 ciliates, Tetrahymena pyriformis strain GL and Tetrahymena vorax strain V₂S, have been measured. It is evident from the results that the food vacuoles concentrate the suspended particles, clearing a volume of the surrounding suspension fluid 500 × greater than the total volume of food vacuoles made during the same period of time.     

Expression,secretion and surface display of a human alkaline phosphatase by the ciliate <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Tetrahymena thermophila possesses many attributes that render it an attractive host for the expression of recombinant proteins. Surface proteins from the parasites Ichthyophthirius multifiliis and Plasmodium falciparum and avian influenza virus antigen H5N1 were displayed on the cell membrane of this ciliate. Furthermore, it has been demonstrated that T. thermophila is also able to produce a functional human DNase I. The present study investigates the heterologous expression of the functional human intestinal alkaline phosphatase (hiAP) using T. thermophila and thereby presents a powerful tool for the optimization of the ciliate-based expression system.     

Role of apoptosis-inducing factor (AIF) in programmed nuclear death during conjugation in <Emphasis Type="Italic">Tetrahymena thermophila</Emphasis>

Background  

Programmed nuclear death (PND), which is also referred to as nuclear apoptosis, is a remarkable process that occurs in ciliates during sexual reproduction (conjugation). In Tetrahymena thermophila, when the new macronucleus differentiates, the parental macronucleus is selectively eliminated from the cytoplasm of the progeny, concomitant with apoptotic nuclear events. However, the molecular mechanisms underlying these events are not well understood. The parental macronucleus is engulfed by a large autophagosome, which contains numerous mitochondria that have lost their membrane potential. In animals, mitochondrial depolarization precedes apoptotic cell death, which involves DNA fragmentation and subsequent nuclear degradation.     

The Comparative Systematics of Species Comprising the Hymenostome Ciliate Genus Tetrahymena*

SYNOPSIS. It has been 10 years since the taxonomic composition of the important hymenostome genus Tetrahymena has been given overall consideration, and even then the treatment was not extensive. New data of significance have been accumulated and a fresh analysis is clearly in order. Also today we recognize that entire assemblages or combinations of characteristics must be considered in understanding the species-composition of a protozoan genus, and such an approach has never been uniformly applied in a comparative systematic study of all ciliates belonging to the Tetrahymena group. Three complexes within the genus may now be identified. In the 1st, the pyriformis complex, are placed T. pyriformis (the type-species), T. setifera, and T. chironomi. In the 2nd, the rostrata group, are assigned T. rostrata, T. limacis, T. corlissi, and T. stegomyiae. In the 3rd, the patula complex, are found T. patula, T. vorax, and T. paravorax. Three additional, formerly independent species are here recognized as doubtful forms: T. faurei, T. glaucomaeformis, and T. parasitica. In spite of some overlapping in certain characters, such as total number of kineties or ciliary meridians, the 3 complexes may be considered distinctive on the basis of constellations of features of taxonomic value, including physiologic and morphogenetic as well as structural characteristics. Yet within each complex it is possible to differentiate clearly a number of separate species. The present analysis in no way closes the door to discovery of still additional, new species of Tetrahymena in the future, but it attempts to lay the groundwork for a uniform usage of combinatiomnas of criteria in comparative taxonomic studies of these and other relatively undifferentiated hymenostome species which possibly will be of some value in the whole area of ciliate systematics.     

Growth Kinetics of Three Species of Tetrahymena On Solid Agar*

A nutrient-agar method without liquid overlay has been developed for cultivation of ciliates. Three species of Tetrahymena-T. pyriformis strain W, T. rostrata strain UNI, and T. vorax strain V₂S, representing the 3 main groups of Tetrahymena species, were used; however the method should apply to other ciliates. Growth on the surface of the agar was facilitated by an optimal surface-to-volume ratio yielding a high density of ciliates (5.8 × 10₅ cells/cm² for T. pyriformis at 25 C) and short generation times (3 h for T. pyriformis at 30 C). At the highest density achieved, the cells became irregularly hexagonal and formed a monolayer “tissue” on the agar. Ciliates grown on agar were like those in liquid culture, typical oral ciliature, food-vacuole formation, and typical cortical patterns being retained. Advantages of this method include high cell density, easy recovery, and optimal O₂ supply. the organisms can also be cultivated on the surface of sterile cellulose-nitrate filters, facilitating in situ fixation and staining as well as transfer into different media by transfer of filters with cells, without prior centrifugation and resuspension.     

Adaptive characteristics of a ciliateTetrahymena thermophila in endosymbiotic association with a green algaChlorella vulgaris derived in a long-term microcosm culture

We investigated directly an early stage of the development of an endosymbiosis between an alga and a ciliate, beginning from a non-associated stage by conducting a long-term microcosm culture composed of a green alga (Chlorella vulgaris), a bacterium (Escherichia coli) and a ciliate (Tetrahymena thermophila) for three years. During this culture,Chlorella-harboringTetrahymena appeared and increased in frequency. We examined the adaptive characteristics of theT. thermophila in association with theC. vulgaris derived from a well-established microcosm culture maintained for 1164–1400 days, and compared it with the original culture ofT. thermophila. TheT. thermophila with the associated, intracellularC. vulgaris grew at a lower density ofE. coli than the originalT. thermophila with the originalC. vulgaris, and the former survived longer than the latter in the absence ofE. coli. These results suggest that this induced algal-ciliate association confers some fitness advantage at least to the host under the conditions of less or no available food such asE. coli. Although the algal-ciliate association is not completely stable, this quasi-stable association may enable the host to exploit a new niche through the advantages of mixotrophy.