A discovery of two new Tetrahymena species parasitizing slugs and mussels: morphology and multi-gene phylogeny of T. foissneri sp. n. and T. unionis sp. n.

The presence of parasitic ciliates of the hymenostome genus Tetrahymena was examined in 150 mollusks belonging to six bivalve and 13 gastropod species in Slovakia, Central Europe. Tetrahymenids were detected only in two species, viz., in the invasive Lusitanian slug (Arion vulgaris) and in the native swollen river mussel (Unio tumidus). Although only 10.52% of the examined mollusk taxa were positive, their Tetrahymena infections were very intensive accounting for several hundreds of ciliates per host. Phylogenetic analyses of the 16S and 18S rRNA genes as well as of the barcoding region of the gene encoding for cytochrome c oxidase subunit I revealed that both isolates represent new taxa, T. foissneri sp. n. and T. unionis sp. n. The former species belongs to the 'borealis' clade and its nearest relative is T. limacis, a well-known parasite of slugs and snails. Besides molecular data, T. foissneri can be distinguished from T. limacis also morphologically by the body shape of the parasitic-phase form, dimensions of micronuclei, and the silverline system. On the other hand, T. unionis was classified within the 'paravorax' clade along with T. pennsylvaniensis, T. glochidiophila, and T. nigricans. Although these four species are genetically distinct, T. unionis could be morphologically separated only from T. nigricans by body shape and size. The present study suggests that both aquatic and terrestrial mollusks represent interesting hosts for the discovery of novel Tetrahymena lineages.

Uni-cellular integration of complex spatial information in slime moulds and ciliates.

Single-celled organisms show a fascinating faculty for integrating spatial information and adapting their behaviour accordingly. As such they are of potential interest for elucidating fundamental mechanisms of developmental biology. In this mini-review we highlight current research on two organisms, the true slime mould Physarum polycephalum and the ciliates Paramecium and Tetrahymena. For each of these, we present a case study how applying physical principles to explain behaviour can lead to the understanding of general principles possibly relevant to developmental biology.

The effect of tertiary treated wastewater on fish growth and health: Laboratory-scale experiment with Poecilia reticulata (guppy).

Treated wastewater (TWW) constitutes a sustainable water resource and has been used for fish culture in some countries around the world, although there are no comprehensive data on the effect of TWW on fish growth and health in the context of aquaculture production. Our objectives were to examine how fish culture in TWW affected fish growth and fitness, as well as compliance with the international standards for safe consumption. Guppy (Poecilia reticulata) fingerlings were reared in 0%, 50% and 100% tertiary TWW (TTWW), from the age of five days, for a period of four months. In water analyses, 33 out of 67 tested organic micropollutants (OMPs) were detected in the TTWW samples at least once, at concentrations that are typically reported in domestic TTWW. Fish survival ranged between 77-80% and did not differ between treatment groups. Fish growth and mortality following challenge infection with Tetrahymena sp. (which ranged between 64-68%), were similar among treatment groups. Of tested immunological parameters, lysozyme and anti-protease was similar among treatments while complement activity was highest in the 50% TTWW-reared fish. No abnormalities were observed in the histopathological analysis. Levels of heavy metals, polychlorinated-biphenyls (PCBs) and organochlorines (OCs) in fish were below the detection limit and below the Food and Agriculture Organization of the United Nations (FAO) and the European Union EU maximal permitted levels in food fish. Results suggest that the yield of fish grown in TTWW is potentially similar to that in freshwater, and the produced fish comply with the standards of consumer safety. The results are in line with previous studies that examined the feasibility of TWW-fed aquaculture.

Genetics and Epigenetics of Mating Type Determination in Paramecium and Tetrahymena.

While sex is an ancient and highly conserved eukaryotic invention, self-incompatibility systems such as mating types or sexes appear to be derived limitations that show considerable evolutionary plasticity. Within a single class of ciliates, Paramecium and Tetrahymena species have long been known to present a wide variety of mating type numbers and modes of inheritance, but only recently have the genes involved been identified. Although similar transmembrane proteins mediate self/nonself recognition in both ciliates, the mechanisms of mating type determination differ widely, ranging from Mendelian systems to developmental nuclear differentiation, either stochastic or maternally inherited. The non-Mendelian systems rely on programmed editing of the germline genome that occurs during differentiation of the somatic nucleus, and they have co-opted different DNA recombination mechanisms-some previously unknown. Here we review the recent molecular advances and some remaining unsolved questions and discuss the possible implications of these diverse mechanisms for inbreeding/outbreeding balance regulation.

Transgenerational function of Tetrahymena Piwi protein Twi8p at distinctive noncoding RNA loci.

Transgenerational transmission of genome-regulatory epigenetic information can determine phenotypes in the progeny of sexual reproduction. Sequence specificity of transgenerational regulation derives from small RNAs assembled into Piwi-protein complexes. Known targets of transgenerational regulation are primarily transposons and transposon-derived sequences. Here, we extend the scope of Piwi-mediated transgenerational regulation to include unique noncoding RNA loci. Ciliates such as Tetrahymena have a phenotypically silent germline micronucleus and an expressed somatic macronucleus, which is differentiated anew from a germline genome copy in sexual reproduction. We show that the nuclear-localized Tetrahymena Piwi protein Twi8p shuttles from parental to zygotic macronuclei. Genetic elimination of Twi8p has no phenotype for cells in asexual growth. On the other hand, cells lacking Twi8p arrest in sexual reproduction with zygotic nuclei that retain the germline genome structure, without the DNA elimination and fragmentation required to generate a functional macronucleus. Twi8p-bound small RNAs originate from long-noncoding RNAs with a terminal hairpin, which become detectable in the absence of Twi8p. Curiously, the loci that generate Twi8p-bound small RNAs are essential for asexual cell growth, even though Twi8 RNPs are essential only in sexual reproduction. Our findings suggest the model that Twi8 RNPs act on silent germline chromosomes to permit their conversion to expressed macronuclear chromosomes. Overall this work reveals that a Piwi protein carrying small RNAs from long-noncoding RNA loci has transgenerational function in establishing zygotic nucleus competence for gene expression.

Ecological Traits of the Algae-Bearing Tetrahymena utriculariae (Ciliophora) from Traps of the Aquatic Carnivorous Plant Utricularia reflexa.

Trap fluid of aquatic carnivorous plants of the genus Utricularia hosts specific microbiomes consisting of commensal pro- and eukaryotes of largely unknown ecology. We examined the characteristics and dynamics of bacteria and the three dominant eukaryotes, i.e. the algae-bearing ciliate Tetrahymena utriculariae (Ciliophora), a green flagellate Euglena agilis (Euglenophyta), and the alga Scenedesmus alternans (Chlorophyta), associated with the traps of Utricularia reflexa. Our study focused on ecological traits and life strategies of the highly abundant ciliate whose biomass by far exceeds that of other eukaryotes and bacteria independent of the trap age. The ciliate was the only bacterivore in the traps, driving rapid turnover of bacterial standing stock. However, given the large size of the ciliate and the cell-specific uptake rates of bacteria we estimated that bacterivory alone would likely be insufficient to support its apparent rapid growth in traps. We suggest that mixotrophy based on algal symbionts contributes significantly to the diet and survival strategy of the ciliate in the extreme (anaerobic, low pH) trap-fluid environment. We propose a revised concept of major microbial interactions in the trap fluid where ciliate bacterivory plays a central role in regeneration of nutrients bound in rapidly growing bacterial biomass.

Tetrahymena as a Unicellular Model Eukaryote: Genetic and Genomic Tools.

Tetrahymena thermophila is a ciliate model organism whose study has led to important discoveries and insights into both conserved and divergent biological processes. In this review, we describe the tools for the use of Tetrahymena as a model eukaryote, including an overview of its life cycle, orientation to its evolutionary roots, and methodological approaches to forward and reverse genetics. Recent genomic tools have expanded Tetrahymena's utility as a genetic model system. With the unique advantages that Tetrahymena provide, we argue that it will continue to be a model organism of choice.

Biogenic amines at a low level of evolution: Production, functions and regulation in the unicellular Tetrahymena.

The unicellular eukaryote Tetrahymena synthesize, store and secrete biogenic amines (histamine, serotonin, epinephrine, dopamine, melatonin) and also can take up amines from the milieu. It also has (G-protein-coupled) receptors (binding sites) for these amines as well, as second messengers. The factors infuencing the mentioned processes are shown. For certain amines the genes and the coded enzymes are demonstrated. The amines influence phagocytosis, cell division, ciliary regeneration, glucose metabolism and chemotaxis. There are interhormone actions between the amines, and between the amines and other hormones produced by Tetrahymena. The critical review discusses the role of amines in the early stages of evolution and compares this to their functions in mammals. It tries to give answer how and why biogenic amines were selected to hormones, and why new functions formed for them in higher ranked animals, preserving also the ancient ones.

In vivo evolution of a catalytic RNA couples trans-splicing to translation.

How does a non-coding RNA evolve in cells? To address this question experimentally we evolved a trans-splicing variant of the group I intron ribozyme from Tetrahymena over 21 cycles of evolution in E.coli cells. Sequence variation was introduced during the evolution by mutagenic and recombinative PCR, and increasingly active ribozymes were selected by their repair of an mRNA mediating antibiotic resistance. The most efficient ribozyme contained four clustered mutations that were necessary and sufficient for maximum activity in cells. Surprisingly, these mutations did not increase the trans-splicing activity of the ribozyme. Instead, they appear to have recruited a cellular protein, the transcription termination factor Rho, and facilitated more efficient translation of the ribozyme's trans-splicing product. In addition, these mutations affected the expression of several other, unrelated genes. These results suggest that during RNA evolution in cells, four mutations can be sufficient to evolve new protein interactions, and four mutations in an RNA molecule can generate a large effect on gene regulation in the cell.

Method for toxicity test of titanium dioxide nanoparticles in ciliate protozoan Tetrahymena.

Titanium dioxide (TiO2) nanoparticles have a high surface-to-mass ratio and rapidly aggregate in water causing great difficulties for toxicity test exposed to aquatic organisms or other cell lines. This study uses a cell viability kit for routine toxicity test of TiO2 as well as other nanoparticles which accumulate in the aquatic environment. Tetrahymena immediately endocytoses TiO2 nanoparticles and stores them in food vacuoles until the particles undergo exocytosis as larger aggregates. However, during the process of endocytosis and exocytosis, TiO2 particles interfere with cell growth and consequently induce acute toxicity. It exerted high cell growth inhibition at 20 h incubation and induces significant cytotoxic effects. Surprisingly, the effect of TiO2 decreases at 40 h incubation, due to the recovery of cell growth and reduction of the cytotoxicity of the particles.

Acute toxicity of heavy metals to Tetrahymena in an in vitro experiment and envelope damage study.

The toxicity of Cr³âº, Cu²âº and Cd²âº to Tetrahymena growth metabolism was studied by microcalorimetry at 28°C, and the growth constant (k), peak time (T) and generation times (T(G)) were calculated. The metal ion concentrations that resulted in 50% inhibition (IC50) of population growth were obtained through the dynamic parameters. The results indicated that the order of toxicity was Cd²âº> Cr³âº> Cu²âº. Inductively coupled plasma-atomic emission spectrometry results suggested that the metal ions affected the permeability of the cell membrane. Observations of the Cd-exposed organisms by scanning electron microscopy revealed damage to the cell membrane in the form of an altered surface appearance. The cells suffered serious damage after sufficient acting time. Attenuated total reflection Fourier transform infrared spectra revealed that amide groups and PO2⁻ of the phospholipid phospho-diester, both located in the hydrophobic end of the outer layer of the cell membrane, were most readily affected.

Electrophysiological properties of the microstome and macrostome morph of the polymorphic ciliate Tetrahymena vorax.

Polymorphic ciliates, like Tetrahymena vorax, optimize food utilization by altering between different body shapes and behaviours. Microstome T. vorax feeds on bacteria, organic particles, and solutes, whereas the larger macrostome cells are predators consuming other ciliates. We have used current clamp and discontinuous single electrode voltage clamp to compare electrophysiological properties of these morphs. The resting membrane potential was approximately -30 mV in both morphs. The input resistance and capacitance of microstomes were approximately 350 MΩ and 105 pF, whereas the corresponding values for the macrostomes were 210 MΩ and 230 pF, reflecting the larger cell size. Depolarizing current injections elicited regenerative Ca(2+) spikes with a maximum rate of rise of 7.5 Vs(-1) in microstome and 4.7 Vs(-1) in macrostome cells. Depolarizing voltage steps from a holding potential of -40 mV induced an inward Ca(2+) -current (I(ca) ) peaking at -10 mV, reaching approximately the same value in microstome (-1.4 nA) and macrostome cells (-1.2 nA). Because the number of ciliary rows is the same in microstome and macrostome cells, the similar size of I(Ca) in these morphs supports the notion that the voltage-gated Ca(2+) channels in ciliates are located in the ciliary membrane. In both morphs, hyperpolarizing voltage steps revealed inward membrane rectification that persisted in Na(+) -free solution and was only partially inhibited by extracellular Cs(+) . The inward rectification was completely blocked by replacing Ca(2+) with Co(2+) or Ba(2+) in the recording solution, and is probably due to Ca(2+) -activated inward K(+) current secondary to Ca(2+) influx through channels activated by hyperpolarization.

Assessment of the efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and Tetrahymena spp.

The efficacy of benzalkonium chloride and sodium hypochlorite against Acanthamoeba polyphaga and two Tetrahymena spp. was determined based on the European Standard EN 1276:2009 suspension test. Trophozoite viability was assessed by determination of the membrane integrity using flow cytometry as a fast screening technique. Bovine serum albumin was added to simulate clean (0.3 g/liter) and dirty (3 g/liter) conditions. Benzalkonium chloride caused cell lysis at concentrations above 50 mg/liter under clean and dirty conditions. A concentration of 50 mg of free chlorine per liter had a strong biocidal effect on acanthamoebae and tetrahymenae after 15 min under clean and dirty conditions. Our results suggest that benzalkonium chloride and sodium hypochlorite were effective against the three microorganisms at concentrations commonly applied in the food industry.

Acute toxicity of chlorobenzenes in tetrahymena: estimated by microcalorimetry and mechanism.

The toxicity of chlorobenzenes to Tetrahymena growth metabolism was studied by microcalorimetry. The growth constant (k), peak time (T) and generation times (T(G)) were calculated. IC(50) of chlorobenzenes was obtained through the kinetic parameters. The results suggested that the order of toxicity was 1,2,4-trichlorobenzene>o-dichlorobenzene>p-dichlorobenzene>m-dichlorobenzene>chlorobenzene. ATR-FTIR spectra revealed that amide groups and PO(2)(-) of the phospholipid phospho-diester, both in the hydrophobic end exposed to the outer layer, were the easiest to be damaged. The relationship between IC(50) and chemicals structure parameters (E(LUMO), E(HOMO), logK(OW), ∑Q(R), ΔQ(πR) and ΔE), indicated that the more chlorine atoms were substituted, the greater the toxicity was. Chlorobenzenes have toxicity of non-polar narcosis. Their toxicity is proportional to their concentrations at the site of action, and caused by membrane perturbation.

The life and times of Tetrahymena.

The genus Tetrahymena is defined on the basis of a four-part oral structure composed of an undulating membrane and three membranelles. It is a monophyletic genus with 41 named species and numerous unnamed species, many of which are morphologically indistinguishable. Nuclear small subunit rRNA and mitochondrial cytochrome c oxidase subunit 1 sequences indicate two major clades, a "borealis" clade of less closely related species and an "australis" clade of more closely related species that correlate to differences in mating-type determination and frequency of amicronucleates. Members of both clades show convergence for histophagy (primarily facultative), macrostome transformation, and (rare) cyst formation. Life cycle parameters of species are presented and problematic species discussed.

Tetrahymena in the laboratory: strain resources, methods for culture, maintenance, and storage.

The ciliated protozoan Tetrahymena thermophila has been an important model system for biological research for many years. During that time, a variety of useful strains, including highly inbred stocks, a collection of diverse mutant strains, and wild cultivars from a variety of geographical locations have been identified. In addition, thanks to the efforts of many different laboratories, optimal conditions for growth, maintenance, and storage of Tetrahymena have been worked out. To facilitate the efficient use of Tetrahymena, especially by those new to the system, this chapter presents a brief description of many available Tetrahymena strains and lists possible resources for obtaining viable cultures of T. thermophila and other Tetrahymena species. Descriptions of commonly used media, methods for cell culture and maintenance, and protocols for short- and long-term storage are also presented.

Transformation and strain engineering of Tetrahymena.

Transformation of Tetrahymena by microinjection of DNA was established 25 years ago. This rather labor-intensive technique has since been shelved, replaced by less time consuming and more efficient methods, electroporation and biolistics. Conjugative electroporation is the method of choice for introducing autonomously replicating, rDNA-based vectors into Tetrahymena. These are maintained as high-copy linear mini-chromosomes. Versatile expression cassettes in these vectors facilitate expression of most genes. Transformation efficiencies are sufficiently high to permit screens using expression libraries. Biolistic transformation is primarily used to introduce DNA for integration into the genome by homologous recombination. This technique has greatly enhanced strain engineering of Tetrahymena through facilitating the disruption of genes (creating targeted knockout cell lines) or epitope-tagging coding regions, allowing researchers to take full advantage of the sequenced genome. The presence of both germline and somatic nuclei in these cells requires different strategies to target DNA to the desired compartment. This presents challenges, including the need to engineer the polygenic macronuclear genome, which has nearly 50 copies of each gene. However, separate manipulation of functionally distinct genomes provides experimental opportunities, especially for the analysis of essential genes, by modifying the silent micronucleus then subsequently examining phenotypes in the next sexual generation. The flexibility to engineer strains as needed makes Tetrahymena a facile system with which to answer many biological questions.

Behavioral bioassays and their uses in Tetrahymena.

The swimming behaviors of Tetrahymena can be used in sensitive behavioral bioassays for estimating the effects of drugs, mutations, and other conditions on the physiological state of the cell. These assays can be used in both forward and reverse genetic approaches to help understand cellular functions from genotype to phenotype.

Chemotaxis induced by SXWS tetrapeptides in Tetrahymena--overlapping chemotactic effects of SXWS sequences and their identical amino acids.

The chemotactic potential of SXWS peptides and the components of the extracellular domain of cytokine receptors were investigated in Tetrahymena as a functional index of substitution with different amino acids in the position 'X' of the tetrapeptide. Data obtained demonstrate that position X plays a special determining role in the ligand, SEWS and STWS possess extremely strong chemoattractant ability, and aromatic amino acids result in chemorepellent ligands. Diverse effects of structurally related molecules, for example, SNWS-SDWS, demonstrate a highly sensitive discrimination potential in the applied model system. Physicochemical characteristics (hydropathy, residue size, and solvent-exposed area) of the amino acids were correlated with the chemotactic activity. Data obtained by computer-assisted conformation analysis of SXWS peptides and the highly overlapping chemotactic effects of the investigated SXWS peptides as well as the presence of the amino acids in the 'X' position indicate that member 'X' of the SXWS sequence performs a special role in interactions with the chemotaxis receptors in the membrane.

Adaptive regulation of membrane lipids and fluidity during thermal acclimation in Tetrahymena.

The free-living eukaryotic protozoan Tetrahymena is a potentially useful model for the thermoadaptive membrane regulation because of easy growth in the axenic culture, systematic isolation of subcellular organelles, and quick response to temperature stress. Exposure of Tetrahymena cells to the cold temperature induces marked alterations in the lipid composition and the physical properties (fluidity) of various membranes. The increase in fatty acid unsaturation of membrane phospholipids is required to preserve the proper fluidity. In this homeoviscous adaptive response, acyl-CoA desaturase plays a pivotal role and its activity is regulated by induction of the enzyme via transcriptional activation.