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Phenotypic plasticity is increasingly recognized as a key element of eco‐evolutionary dynamics, but it remains challenging to assess because of its multidimensional nature. Indeed, organisms live in complex environments where numerous factors can impact the phenotypic expression of traits (inter‐environment axis), possess multiple traits that can influence each other's expression (inter‐trait axis) and differ in their genetic background (inter‐genotype axis), which can not only impact the traits' values but also their plasticity.We addressed six questions related to phenotypic plasticity: (a) do different environmental gradients show similar effects on a given trait? (b) Are the effects of two environmental gradients on a trait additive? (c) Do different traits show similar plastic response to a given environmental gradient? (d) Do the (co)variances between traits vary across environmental gradients? (e) Do genotypes differ in their plastic response to a given environmental gradient? (f) Are some genotypes more plastic than others across all traits?We designed a microcosm experiment using the protist Tetrahymena thermophila aimed at encompassing all these aspects of phenotypic plasticity. We exposed 15 distinct genotypes to 25 combinations of temperature and nutrient availability and assessed the plasticity of five phenotypic traits.Our results show strong differences in the plastic response depending on the environmental gradient, not only regarding the shape of the reaction norm of the different traits tested, but also in the overall plasticity of the organisms. We did not find any covariance between traits that was consistent across all environments.Overall, our results suggest independent impacts of the environmental dimension considered on the observed plastic response. These results underline potential difficulties in generalizing findings about plasticity to all environments and all traits. A free Plain Language Summary can be found within the Supporting Information of this article. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The host specificity of the recently described ciliate species Tetrahymena utriculariae was tested in a greenhouse growth experiment, which included 14 different species of aquatic Utricularia as potential host plants. We confirmed the high specificity of the interaction between U. reflexa and T. utriculariae, the former being the only tested host species able to maintain colonization for prolonged time periods. We conclude that this plant–microbe relationship is a unique and specialized form of digestive mutualism and the plant–microbe unit a suitable experimental system for future ecophysiological studies. [ABSTRACT FROM AUTHOR]

Recognition of self and nonself is important for outcrossing organisms, and different mating types establish the barrier against self-mating. In the unicellular ciliate T. thermophila, mating type determination requires complex DNA rearrangements at a single mat locus during conjugation to produce a type-specific gene pair (MTA and MTB) for 1 of 7 possible mating types. Surprisingly, we found that decreased expression of the DNA breakage-repair protein Ku80 at late stages of conjugation generated persistent selfing phenotype in the progeny. DNA analysis revealed multiple mating-type gene pairs as well as a variety of mis-paired, unusually arranged mating-type genes in these selfers that resemble some proposed rearrangement intermediates. They are found also in normal cells during conjugation and are lost after 10 fissions but are retained in Ku mutants. Silencing of TKU80 or TKU70-2 immediately after conjugation also generated selfing phenotype, revealing a hidden DNA rearrangement process beyond conjugation. Mating reactions between the mutant and normal cells suggest a 2-component system for self–nonself-recognition through MTA and MTB genes. In the unicellular ciliate Tetrahymena thermophila, mating type determination requires complex DNA rearrangements to produce a type-specific MAT gene pair for each of seven possible mating types. This study shows that DNA recombination produces a large number of unusual MAT gene pairs, but through a KU-dependent process only one normal pair is retained in a mature cell; retention of unusual gene pairs leads to self-mating and suggests a system for self/non-self recognition. [ABSTRACT FROM AUTHOR]

• Molecular basis of rust virulence follows the classic gene-for-gene concept. • Knockout mutations and amino acid sequence variation of effectors enable evolution. • Prevalence of sexual and asexual reproduction impacts on virulence diversity. • Genomic advances have recently allowed phasing of dikaryotic rust genomes. • Somatic hybridization can allow exchange of whole nuclei between strains. Rust fungi are major pathogens that negatively affect crops and ecosystems. Recent rust disease epidemics driven by the emergence of strains with novel virulence profiles demand a better understanding of the evolutionary mechanisms of these organisms. Here, we review research advances in genome-scale analysis coupled with functional validation of effector candidate genes that have been instrumental to elucidate processes that contribute to changes in virulence phenotypes. We highlight how haplotype-phased genome references have paved the road to link these processes to the reproductive phases of rust fungi and have provided evidence for somatic exchange between strains as an important mechanism for generating diversity in asexual populations. With increasing data availability, we envision the future development of molecular virulence diagnostic tools. [ABSTRACT FROM AUTHOR]

Understanding the effects of temperature on ecological and evolutionary processes is crucial for generating future climate adaptation scenarios. Using experimental evolution, we evolved the model ciliate Tetrahymena thermophila in an initially novel high temperature environment for more than 35 generations, closely monitoring population dynamics and morphological changes. We observed initially long lag phases in the high temperature environment that over about 26 generations reduced to no lag phase, a strong reduction in cell size and modifications in cell shape at high temperature. When exposing the adapted populations to their original temperature, most phenotypic traits returned to the observed levels in the ancestral populations, indicating phenotypic plasticity is an important component of this species thermal stress response. However, persistent changes in cell size were detected, indicating possible costs related to the adaptation process. Exploring the molecular basis of thermal adaptation will help clarify the mechanisms driving these phenotypic responses. [ABSTRACT FROM AUTHOR]

The behaviours of micro-organisms at interfaces play important roles in various biological, medical and engineering phenomena. Despite its widely recognized importance, our understanding of swimming micro-organisms at interfaces is limited. Ferracci et al. (PLoS One, vol. 8, 2013, e75238) reported that the ciliate, Tetrahymena, was entrapped at a water–air interface, while it escaped from a solid wall. Although the entrapment was speculated to be induced by physical processes, the mechanism is still unclear. To clarify the entrapment phenomenon, we focus on cell shape and numerically investigate the behaviour of a swimming micro-organism at interfaces from a hydrodynamic point of view. The model cell is assumed to propel itself by generating homogeneous tangential stress above the cell body. The results reveal that two major shape parameters, i.e. fore-and-aft asymmetry and a constriction, are dominant in the entrapment phenomenon. The mechanism can be explained by the balance of two opposite rotational velocities: repelling velocity due to the ciliary beat and attracting velocity due to the collision at the interface. In other words, the mechanism can be understood by hydrodynamic and steric effects. Moreover, cells tend to be entrapped more by the water–air interface than by the solid wall, which agrees with experimental observations reported previously (Ferracci et al. 2013). Finally, we experimentally observe Tetrahymena thermophila entrapped on the surface of an air bubble, and qualitatively discuss the shape of entrapped cells. The knowledge obtained provides a basis for understanding the behaviours of swimming micro-organisms at various interfaces, both in nature and in industrial applications. [ABSTRACT FROM AUTHOR]

Our previous studies have verified that moroxydine hydrochloride (Mor) could inhibit replication of grass carp reovirus (GCRV) and suppress apoptosis of Ctenopharyngodon idella kidney cells, but be lack of information whether exists on antiviral activity in vivo. The paper was undertaken to explore the antiviral response of Mor against GCRV in grass carp and investigate the safety of drug for aquatic organisms. The results showed that injection treatment of Mor could more effectively inhibit GCRV replication than immersion administration. All the RNA systheses of vp3 and vp6 on day 7 in head kidney, gill, hepatopancreas and dorsal muscle in the Mor injection group were lesser than 0.07‐fold than that of in control group. And the GCRV‐inducing grass carp mortality was effectively controlled within 7 days post Mor injection therapy. Additionally, the reduction of superoxide dismutase activity, total antioxidant capacity and catalase activity in serum was effectively controlled by Mor. Moreover, drug safety assessment results showed that 500 mg/L of Mor was safe to C. idella, Bacillus subtilis, Chlorella vulgaris and Tetrahymena thermophila, which was far higher than the therapeutic concentration. The present study proved Mor as harmless formulations or products had potential value in the control of GCRV in aquaculture, with the advantage of super in vivo antiviral activity and environmental safety. [ABSTRACT FROM AUTHOR]

Abiotic stress is a major force of selection that organisms are constantly facing. While the evolutionary effects of various stressors have been broadly studied, it is only more recently that the relevance of interactions between evolution and underlying ecological conditions, that is, eco‐evolutionary feedbacks, have been highlighted. Here, we experimentally investigated how populations adapt to pH‐stress under high population densities. Using the protist species Tetrahymena thermophila, we studied how four different genotypes evolved in response to stressfully low pH conditions and high population densities. We found that genotypes underwent evolutionary changes, some shifting up and others shifting down their intrinsic rates of increase (r0). Overall, evolution at low pH led to the convergence of r0 and intraspecific competitive ability (α) across the four genotypes. Given the strong correlation between r0 and α, we argue that this convergence was a consequence of selection for increased density‐dependent fitness at low pH under the experienced high density conditions. Increased density‐dependent fitness was either attained through increase in r0, or decrease of α, depending on the genetic background. In conclusion, we show that demography can influence the direction of evolution under abiotic stress. [ABSTRACT FROM AUTHOR]

The ciliate Tetrahymena thermophila does not require sterols for growth and synthesizes pentacyclic triterpenoid alcohols, mainly tetrahymanol, as sterol surrogates. However, when sterols are present in the environment, T. thermophila efficiently incorporates and modifies them. These modifications consist of desaturation reactions at positions C5(6), C7(8), and C22(23), and de‐ethylation at C24 of 29‐carbon sterols (i.e. phytosterols). Three out of four of the enzymes involved in the sterol modification pathway have been previously identified. However, identification of the sterol C22 desaturase remained elusive, as did other basic aspects of this metabolism. To get more insights into this peculiar metabolism, we here perform a whole transcriptome analysis of T. thermophila in response to exogenous cholesterol. We found 356 T. thermophila genes to be differentially expressed after supplementation with cholesterol for 2 h. Among those that were upregulated, we found two genes belonging to the long spacing family of desaturases that we tentatively identified by RNAi analysis as sterol C22 desaturases. Additionally, we determined that the inhibition of tetrahymanol synthesis after supplementation with cholesterol occurs by a transcriptional downregulation of genes involved in squalene synthesis and cyclization. Finally, we identified several uncharacterized genes that are likely involved in sterols transport and signaling. [ABSTRACT FROM AUTHOR]

Hormones, characteristic to higher ranked animals, are synthesized, stored, and secreted by unicellular eukaryote animals. The unicells also have receptors for recognizing these materials and transmit the message into the cells for provoking response. The hormones are effective in very low concentrations (down to 10–21 M) and opposite effects of lower and higher concentrations can be observed. However, sometimes linear concentration effects can be found, which means that hormesis exists, nevertheless uncertain, as it is in the phase of formation (evolutionary experimentation). Hormesis, by transformation (fixation) of cytoplasmic receptor-like membrane components to receptors in the presence of the given hormone, likely helps the development of unicellular endocrine character and by this the evolution of endocrine system. The effect by extremely low concentrations of hormones had been forced by the watery way of unicellular life, which could establish the physiological concentrations of hormones in the blood of higher ranked animals. This means that hormetic low doses are the normal, effective concentrations and the high concentrations are artificial, consequently could be dangerous. [ABSTRACT FROM AUTHOR]

Diverse inner arm dyneins cooperate with outer arm dyneins to produce ciliary beating. This study demonstrates an expression system for inner arm dyneins in Tetrahymena. The motor domain of inner arm dynein (Dyh8p or Dyh12p) was fused with the tail of outer arm dynein (Dyh3p) and expressed in viable DYH3-knockout (vKO-DYH3) cells. The chimeric dyneins were observed in the oral apparatus and cilia on the cell bodies, and did not change the swimming speed of vKO-DYH3 cells. In a gliding assay, the motor domains of Dyh8p and Dyh12p moved toward the minus ends of microtubules at 0.8 and 0.3 μm/s, respectively. The gliding velocities of Dyh8p and Dyh12p were decreased in 5 mM ATP but not increased in 0.1 or 0.5 mM ADP. This expression system will be useful for molecular studies on diverse inner arm dyneins. • This study developed an expression system for Tetrahymena inner arm dyneins. • Fusion with the Dyh3p tail enabled the Dyh8p and Dyh12p motor domains to be purified. • Dyh8p moved toward the minus end of microtubules at 0.8 μm/s. • Dyh12p moved toward the minus end of microtubules at 0.3 μm/s. • The motilities of Dyh8p and Dyh12p were changed by ATP but not ADP. [ABSTRACT FROM AUTHOR]

In endolysosomal networks, two hetero-hexameric tethers called HOPS and CORVET are found widely throughout eukaryotes. The unicellular ciliate Tetrahymena thermophila possesses elaborate endolysosomal structures, but curiously both it and related protozoa lack the HOPS tether and several other trafficking proteins, while retaining the related CORVET complex. Here, we show that Tetrahymena encodes multiple paralogs of most CORVET subunits, which assemble into six distinct complexes. Each complex has a unique subunit composition and, significantly, shows unique localization, indicating participation in distinct pathways. One pair of complexes differ by a single subunit (Vps8), but have late endosomal versus recycling endosome locations. While Vps8 subunits are thus prime determinants for targeting and functional specificity, determinants exist on all subunits except Vps11. This unprecedented expansion and diversification of CORVET provides a potent example of tether flexibility, and illustrates how 'backfilling' following secondary losses of trafficking genes can provide a mechanism for evolution of new pathways. [ABSTRACT FROM AUTHOR]

Chromatin organization influences most aspects of gene expression regulation. The linker histone H1, along with the core histones, is a key component of eukaryotic chromatin. Despite its critical roles in chromatin structure and function and gene regulation, studies regarding the H1 protein-protein interaction networks, particularly outside of Opisthokonts, are limited. The nuclear dimorphic ciliate protozoan Tetrahymena thermophila encodes two distinct nucleus-specific linker histones, macronuclear Hho1 and micronuclear Mlh1. We used a comparative proteomics approach to identify the Hho1 and Mlh1 protein-protein interaction networks in Tetrahymena during growth, starvation, and sexual development. Affinity purification followed by mass spectrometry analysis of the Hho1 and Mlh1 proteins revealed a non-overlapping set of co-purifying proteins suggesting that Tetrahymena nucleus-specific linker histones are subject to distinct regulatory pathways. Furthermore, we found that linker histones interact with distinct proteins under the different stages of the Tetrahymena life cycle. Hho1 and Mlh1 co-purified with several Tetrahymena-specific as well as conserved interacting partners involved in chromatin structure and function and other important cellular pathways. Our results suggest that nucleus-specific linker histones might be subject to nucleus-specific regulatory pathways and are dynamically regulated under different stages of the Tetrahymena life cycle. [ABSTRACT FROM AUTHOR]

Multi-ciliary arrays promote fluid flow and cellular motility using the polarized and coordinated beating of hundreds of motile cilia. Tetrahymena basal bodies (BBs) nucleate and position cilia, whereby BB-associated striated fibers (SFs) promote BB anchorage and orientation into ciliary rows. Mutants that shorten SFs cause disoriented BBs. In contrast to the cytotaxis model, we show that disoriented BBs with short SFs can regain normal orientation if SF length is restored. In addition, SFs adopt unique lengths by their shrinkage and growth to establish and maintain BB connections and cortical interactions in a ciliary force-dependent mechanism. Tetrahymena SFs comprise at least eight uniquely localizing proteins belonging to the SFassemblin family. Loss of different proteins that localize to the SF base disrupts either SF steady-state length or ciliary forceinduced SF elongation. Thus, the dynamic regulation of SFs promotes BB connections and cortical interactions to organize ciliary arrays. [ABSTRACT FROM AUTHOR]

Triadinium was created to include Triadinium caudatum. Further, four other species were included, T. minimum, T. galea, T. elongatum, and T. magnum, all sharing a characteristic helmet‐shaped body. Wolska and Grain argued that the inclusion of T. minimum and T. galea into Triadinium was done based on superficial morphological aspects, and established two new genera to accommodate these species: Circodinium and Gassovskiella. Although the phylogenetic relationships within Entodiniomorphida have been investigated by multiple authors, none of them discussed the evolutionary relationship of helmet‐shaped entodiniomorphids. We performed molecular phylogenetics and revisited old literature digging for morphological data to explain our results. According to our analyses, the helmet‐shaped body is homoplastic and may have evolved from at least three different entodiniomorphid ancestors. Circodinium minimum is phylogenetically related to members of Blepharocorythidae, T. caudatum emerged within Spirodiniidae and G. galea within Polydiniellidae. This phylogenetic hypothesis is partially supported by information on infraciliature and ultrastructure of C. minimum and T. caudatum. However, such morphological information is not available for polydiniellids. In order to shed some light into the evolution of the helmet‐shaped ciliates, future works should focus to collect information on the infraciliature and the ultrastructure of Polydiniella mysorea and of other Triadinium species. [ABSTRACT FROM AUTHOR]

Many species exhibit high‐amplitude, extinction‐prone cycles, for instance due to resource enrichment (called the paradox of enrichment). How do such species manage to persist? One possibility is metapopulation dynamics, but it is unclear if these can mitigate the paradox of enrichment. The paradox of enrichment might increase local population extinction rates, and might also increase the spatial synchrony of population fluctuations because population cycles are easily synchronized by even low rates of dispersal. Spatially‐synchronous population fluctuations would leave no scope for rescue effects, and spatially‐synchronous local extinctions would leave no source for recolonization. We conducted a protist microcosm experiment to test how dispersal and enrichment affect the persistence of predator–prey metapopulations by altering spatial synchrony. We assembled 54, 4‐patch metapopulations of the protist predator Euplotes patella and its protist prey Tetrahymena pyriformis. Each metapopulation experienced one of nine dispersal rates, crossed with one of six enrichment levels. We tested the effects of enrichment and dispersal on predator–prey cycle amplitude, predator population extinction risk, spatial synchrony of predator population fluctuations, and predator metapopulation persistence time. Enrichment increased predator–prey cycle amplitude but had no detectable effect on population extinction risk. Dispersal had no effect on predator–prey cycle amplitude. Spatial synchrony increased with enrichment and dispersal. Metapopulation persistence decreased with enrichment and spatial synchrony. We conclude that increasing enrichment reduced metapopulation persistence primarily by increasing spatial synchrony of local extinctions, rather than by increasing their frequency. These results highlight the importance of synchrony‐preventing mechanisms for metapopulation persistence. [ABSTRACT FROM AUTHOR]

Oxidative stress results from an imbalance between the intracellular antioxidants and the radicals ROS or RNS, in favor of the latter. Oxidative stress is associated with several human diseases and causes significant cellular damage to the structure, nucleic acids (DNA, RNA), proteins and lipids. These consequences can compromise the health and viability of the living cell by causing cell death by necrosis or apoptosis. A variety of antioxidant supplements have been developed in recent years to enrich the endogenous defense systems of living organisms. However, the prevention of chronic diseases through the use of antioxidant supplements remains controversial until now. However, natural products with regard to essential oils are very much in demand in biology research because of their antimicrobial and antioxidant properties. We investigated the protective effect of oil of oregano and sage against the oxidative stress induced by hydrogen peroxide (H 2 O 2) and sodium nitroprusside (SNP) in two cellular models Tetrahymena thermophila and Tetrahymena pyriformis. The results of our study showed that oxidative stress inhibits growth, mobility and changes in cell shape of both species. The application of essential oils has shown that both oils protect cells treated with H 2 O 2 and SNP. However, the intracellular mechanism of SNP inhibition is longer than that of H 2 O 2. Therefore, the use of these essential oils could be important to the prevention of chronic diseases related to oxidative stress in higher organisms. [ABSTRACT FROM AUTHOR]

Peroxiredoxins are antioxidant enzymes that use redox active Cys residues to reduce H2O2 and various organic hydroperoxides to less reactive products, and thereby protect cells against oxidative stress. In yeasts and mammals, the Prx1 proteins are sensitive to hyperoxidation and consequent loss of their peroxidase activity whereas in most bacteria they are not. In this paper we report the characterization of the Prx1 family in the non-parasitic protist Tetrahymena thermophila. In this organism, four genes potentially encoding Prx1 have been identified. In particular, we show that the mitochondrial Prx1 protein (Prx1m) from T. thermophila is relatively robust to hyperoxidation. This is surprising given that T. thermophila is a eukaryote like yeasts and mammals. In addition, the proliferation of the T. thermophila cells was relatively robust to inhibition by H2O2, cumene hydroperoxide and plant natural products that are known to promote the production of H2O2. In the presence of these agents, the abundance of the T. thermophila Prx1m protein was shown to increase. This suggested that the Prx1m protein may be protecting the cells against oxidative stress. There was no evidence for any increase in Prx1m gene expression in the stressed cells. Thus, increasing protein stability rather than increasing gene expression may explain the increasing Prx1m protein abundance we observed. [ABSTRACT FROM AUTHOR]

Understanding how and why individual movement translates into dispersal between populations is a long-term goal in ecology. Movement is broadly defined as ‘any change in the spatial location of an individual’, whereas dispersal is more narrowly defined as a movement that may lead to gene flow. Because the former may create the condition for the latter, behavioural decisions that lead to dispersal may be detectable in underlying movement behaviour. In addition, dispersing individuals also have specific sets of morphological and behavioural traits that help them coping with the costs of movement and dispersal, and traits that mitigate costs should be under selection and evolve if they have a genetic basis. Here, we experimentally study the relationships between movement behaviour, morphology and dispersal across 44 genotypes of the actively dispersing unicellular, aquatic model organism Tetrahymena thermophila. We used two-patch populations to quantify individual movement trajectories, as well as activity, morphology and dispersal rate. First, we studied variation in movement behaviour among and within genotypes (i.e. between dispersers and residents) and tested whether this variation can be explained by morphology. Then, we addressed how much the dispersal rate is driven by differences in the underlying movement behaviour. Genotypes revealed clear differences in terms of movement speed and linearity. We also detected marked movement differences between resident and dispersing individuals, mediated by the genotype. Movement variation was partly explained by morphological properties such as cell size and shape, with larger cells consistently showing higher movement speed and higher linearity. Genetic differences in activity and movement were positively related to the observed dispersal and jointly explained 47% of the variation in dispersal rate. Our study shows that a detailed understanding of the interplay between morphology, movement and dispersal may have potential to improve dispersal predictions over broader spatio-temporal scales. [ABSTRACT FROM AUTHOR]

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[Display omitted] • T. thermophila has 121 papain-family cysteine protease encoding genes. • TtPFCPs were divided into four distinct subgroups CATL, CATB, CATC, and CATX. • The majority of true TtPFCPs (96 out of the total) were in TtCATL subgroup. • Phylogenetic analysis showed that ciliate's PFCPs have ancestral features. • The results may help researchers to construct TtPFCP's KO mutants in T. thermophila. Tetrahymena thermophila is a promising host for recombinant protein production, but its utilization in biotechnology is mostly limited due to the presence of intracellular and extracellular papain-family cysteine proteases (PFCPs). In this study, we employed bioinformatics approaches to investigate the T. thermophila PFCP genes and their encoded proteases (TtPFCPs), the most prominent protease family in the genome. Results from the multiple sequence alignment, protein modeling, and conserved motif analyses revealed that all TtPFCPs showed considerably high homology with mammalian cysteine cathepsins and contained conserved amino acid motifs. The total of 121 TtPFCP-encoding genes, 14 of which were classified as non-peptidase homologs, were found. Remaining 107 true TtPFCPs were divided into four distinct subgroups depending on their homology with mammalian lysosomal cathepsins: cathepsin L-like (TtCATLs), cathepsin B-like (TtCATBs), cathepsin C-like (TtCATCs), and cathepsin X-like (TtCATXs) PFCPs. The majority of true TtPFCPs (96 out of the total) were in TtCATL-like peptidase subgroup. Both phylogenetic and chromosomal localization analyses of TtPFCPs supported the hypothesis that TtPFCPs likely evolved through tandem gene duplication events and predominantly accumulated on micronuclear chromosome 5. Additionally, more than half of the identified TtPFCP genes are expressed in considerably low quantities compared to the rest of the TtPFCP genes, which are expressed at a higher level. However, their expression patterns fluctuate based on the stage of the life cycle. In conclusion, this study provides the first comprehensive in-silico analysis of TtPFCP genes and encoded proteases. The results would help designing an effective strategy for protease knockout mutant cell lines to discover biological function and to improve the recombinant protein production in T. thermophila. [ABSTRACT FROM AUTHOR]

Ciliates are single cell eukaryotic organisms with two nuclei and unusual genome biology. The purpose of this project is to study the molecular evolution of macronuclear (MAC) and mitochondrial (Mt) genomes of ciliate protist Tetrahymena (T.) to decipher and interpret their evolutionary processes. Tetrahymena species contain rapidly evolving mitochondrial (Mt) genomes, which have apparently diverged significantly from the ancestral pattern leaving half of their 44 genes without an assigned function. Thus I sequenced three Mt genomes of Tetrahymena paravorax, Tetrahymena pigmentosa, and Tetrahymena malaccensis via shotgun sequencing. I found unique features in these linear 47kb long genomes including long telomeric repeats, long noncoding sequences, a pseudo-tRNA gene, unusual DNA secondary structure for T. paravorax, and the transcription initiation site, a GC box. More analyses were carried out using programs that calculate distance, nucleotide substitution (dn/ds), and their rate ratios (oo) to discover mutation hotspots and relaxed selective constraint in Ymf genes. Hence I concluded that the presence of mutation hot spots in Tetrahymena Mt genomes is a major reason and obstacle in identifying their function.To study the MAC evolution I annotated a Tetrahymena thermophila chromosome, 1Mb in size, based on the translation of its possible open reading frames (ORFs) and their homologues in the GenBank. An objective of this study was to examine the TIGR automated annotation programs capability in identifying all of the biologically meaningful ORFs. Through searching the databases using BLAST I found proteins and ORFs with homologous domains that were not present in the TIGR automated annotation. Thus, I suggest phylogenetic footprinting to complement automated annotations. I also sought to reveal the extent of chromosome rearrangements between T. thermophila and the largest chromosome (LC) of Paramecium (P.) tetraurelia. Mapping all 463 putative proteins from the LC of P. tetraurelia to the chromosomes in T. thermophila genome revealed no orthologous chromosome. Interestingly, gene order between the Mt genome of these two ciliates was almost perfectly conserved, which suggests that the most plausible explanation for such an extensive gene order loss and rearrangement could be the developmentally programmed elimination of germ line sequences and randomization of MAC gene order.

Although general mechanisms of RNA folding and catalysis have been elucidated, little is known about how ribozymes achieve structural stability at high temperature. A previous in vitro evolution experiment identified a small number of mutations that significantly increase the thermostability of the tertiary structure of the Tetrahymena ribozyme. Because we also determined the crystal structure of this thermostable ribozyme, we have for the first time the opportunity to compare the structural interactions and thermodynamic contributions of individual nucleotides in a ribozyme. We investigated the contribution of five mutations to thermostability by using temperature gradient gel electrophoresis. Unlike the case with several well-studied proteins, the effects of individual mutations on thermostability of this RNA were highly context dependent. The three most important mutations for thermostability were actually destabilizing in the wild-type background. A269G and A304G contributed to stability only when present as a pair, consistent with their proximity in the ribozyme structure. In an evolutionary context, this work supports and extends the idea that one advantage of protein enzyme systems over an RNA world is the ability of proteins to accumulate stabilizing single-site mutations, whereas RNA may often require much rarer double mutations to improve the stability of both its tertiary and secondary structures.

TEP1 is a protein component of two ribonucleoprotein complexes: vaults and telomerase. The vault-associated small RNA, termed vault RNA (VR), is dependent upon TEP1 for its stable association with vaults, while the association of telomerase RNA with the telomerase complex is independent of TEP1. Both of these small RNAs have been shown to interact with amino acids 1-871 of TEP1 in an indirect yeast three-hybrid assay. To understand the determinants of TEP1-RNA binding, we generated a series of TEP1 deletions and show by yeast three-hybrid assay that the entire Tetrahymena p80 homology region of TEP1 is required for its interaction with both telomerase and VRs. This region is also sufficient to target the protein to the vault particle. Electrophoretic mobility shift assays using the recombinant TEP1 RNA-binding domain (TEP1-RBD) demonstrate that it binds RNA directly, and that telomerase and VRs compete for binding. VR binds weakly to TEP1-RBD in vitro, but mutation of VR sequences predicted to disrupt helices near its central loop enhances binding. Antisense oligonucleotide-directed RNase H digestion of endogenous VR indicates that this region is largely single stranded, suggesting that TEP1 may require access to the VR central loop for efficient binding.

RNAi-mediated positive feedback loops are pivotal for the maintenance of heterochromatin, but how they are downregulated at heterochromatin-euchromatin borders is not well understood. In the ciliated protozoan Tetrahymena, heterochromatin is formed exclusively on the sequences that are removed from the somatic genome by programmed DNA elimination, and an RNAi-mediated feedback loop is important for assembling heterochromatin on the eliminated sequences. In this study, we show that the heterochromatin protein 1 (HP1)-like protein Coi6p, its interaction partners Coi7p and Lia5p, and the histone demethylase Jmj1p are crucial for confining the production of small RNAs and the formation of heterochromatin to the eliminated sequences. The loss of Coi6p, Coi7p, or Jmj1p causes ectopic DNA elimination. The results provide direct evidence for the existence of a dedicated mechanism that counteracts a positive feedback loop between RNAi and heterochromatin at heterochromatin-euchromatin borders to maintain the integrity of the somatic genome.

Intraspecific trait variation (ITV), potentially driven by genetic and non-genetic mechanisms, can underlie variability in resource acquisition, individual fitness and ecological interactions. Impacts of ITV at higher levels of biological organizations are hence likely, but up-scaling our knowledge about ITV importance to communities and comparing its relative effects at population and community levels has rarely been investigated. Here, we tested the effects of genetic and non-genetic ITV on morphological traits in microcosms of protist communities by contrasting the effects of strains showing different ITV levels (i.e. trait averages and variance) on population growth, community composition and biomass production. We found that genetic and non-genetic ITV can lead to different effects on populations and communities across several generations. Furthermore, the effects of ITV declined across levels of biological organization: ITV directly altered population performance, with cascading but indirect consequences for community composition and biomass productivity. Overall, these results show that the drivers of ITV can have distinct effects on populations and communities, with cascading impacts on higher levels of biological organization that might mediate biodiversity-ecosystem functioning relationships., (© 2024 The Authors. Journal of Animal Ecology © 2024 British Ecological Society.)

Small RNAs target their complementary chromatin regions for gene silencing through nascent long non-coding RNAs (lncRNAs). In the ciliated protozoan Tetrahymena , the interaction between Piwi-associated small RNAs (scnRNAs) and the nascent lncRNA transcripts from the somatic genome has been proposed to induce target-directed small RNA degradation (TDSD), and scnRNAs not targeted for TDSD later target the germline-limited sequences for programmed DNA elimination. In this study, we show that the SUMO E3 ligase Ema2 is required for the accumulation of lncRNAs from the somatic genome and thus for TDSD and completing DNA elimination to make viable sexual progeny. Ema2 interacts with the SUMO E2 conjugating enzyme Ubc9 and enhances SUMOylation of the transcription regulator Spt6. We further show that Ema2 promotes the association of Spt6 and RNA polymerase II with chromatin. These results suggest that Ema2-directed SUMOylation actively promotes lncRNA transcription, which is a prerequisite for communication between the genome and small RNAs., Competing Interests: SS, TN, JS, KM No competing interests declared, (© 2024, Shehzada et al.)

Bacterivorous protists are thought to serve as training grounds for bacterial pathogens by subjecting them to the same hostile conditions that they will encounter in the human host. Bacteria that survive intracellular digestion exhibit enhanced virulence and stress resistance after successful passage through protozoa but the underlying mechanisms are unknown. Here we show that the opportunistic pathogen Burkholderia cenocepacia survives phagocytosis by ciliates found in domestic and hospital sink drains, and viable bacteria are expelled packaged in respirable membrane vesicles with enhanced resistance to oxidative stress, desiccation, and antibiotics, thereby contributing to pathogen dissemination in the environment. Reactive oxygen species generated within the protozoan phagosome promote the formation of persisters tolerant to ciprofloxacin by activating the bacterial SOS response. In addition, we show that genes encoding antioxidant enzymes are upregulated during passage through ciliates increasing bacterial resistance to oxidative radicals. We prove that suppression of the SOS response impairs bacterial intracellular survival and persister formation within protists. This study highlights the significance of protozoan food vacuoles as niches that foster bacterial adaptation in natural and built environments and suggests that persister switch within phagosomes may be a widespread phenomenon in bacteria surviving intracellular digestion., (© The Author(s) 2024. Published by Oxford University Press on behalf of the International Society for Microbial Ecology.)

SUMMARYCiliated protozoa undergo large-scale developmental rearrangement of their somatic genomes when forming a new transcriptionally active macronucleus during conjugation. This process includes the fragmentation of chromosomes derived from the germline, coupled with the efficient healing of the broken ends by de novo telomere addition. Here, we review what is known of developmental chromosome fragmentation in ciliates that have been well-studied at the molecular level ( Tetrahymena , Paramecium , Euplotes , Stylonychia , and Oxytricha ). These organisms differ substantially in the fidelity and precision of their fragmentation systems, as well as in the presence or absence of well-defined sequence elements that direct excision, suggesting that chromosome fragmentation systems have evolved multiple times and/or have been significantly altered during ciliate evolution. We propose a two-stage model for the evolution of the current ciliate systems, with both stages involving repetitive or transposable elements in the genome. The ancestral form of chromosome fragmentation is proposed to have been derived from the ciliate small RNA/chromatin modification process that removes transposons and other repetitive elements from the macronuclear genome during development. The evolution of this ancestral system is suggested to have potentiated its replacement in some ciliate lineages by subsequent fragmentation systems derived from mobile genetic elements., Competing Interests: The authors declare no conflict of interest.

Importantly, these methods produced "generative models" that distinguish unique BB populations, assign BBs into distinct rows of cilia, distinguish new from mature BBs, and predict cortical BB spatial organization with high fidelity. The microtubule-based cilia of I Tetrahymena thermophila i are anchored in the cell cortex by an intricate array of organized basal bodies (BBs) which template cilia biogenesis. [Extracted from the article]

Current influenza vaccines manufactured using eggs have considerable limitations, both in terms of scale up production and the potential impact passaging through eggs can have on the antigenicity of the vaccine virus strains. Alternative methods of manufacture are required, particularly in the context of an emerging pandemic strain. Here we explore the production of recombinant influenza haemagglutinin using the ciliated protozoan Tetrahymena thermophila. For the first time we were able to produce haemagglutinin from both seasonal influenza A and B strains. This ciliate derived material was immunogenic, inducing an antibody response in both mice and non-human primates. Mice immunized with ciliate derived haemagglutinin were protected against challenge with homologous influenza A or B viruses. The antigen could also be combined with submicron particles containing a Nod2 ligand, significantly boosting the immune response and reducing the dose of antigen required. Thus, we show that Tetrahymena can be used as a manufacturing platform for viral vaccine antigens. [ABSTRACT FROM AUTHOR]

Ciliates are unicellular eukaryotes with separate germline and somatic genomes and diverse life cycles, which make them a unique model to improve our understanding of population genetics through the detection of genetic variations. However, traditional sequencing methods cannot be directly applied to ciliates because the majority are uncultivated. Single‐cell whole‐genome sequencing (WGS) is a powerful tool for studying genetic variation in microbes, but no studies have been performed in ciliates. We compared the use of single‐cell WGS and bulk DNA WGS to detect genetic variation, specifically single nucleotide polymorphisms (SNPs), in the model ciliate Tetrahymena thermophila. Our analyses showed that (i) single‐cell WGS has excellent performance regarding mapping rate and genome coverage but lower sequencing uniformity compared with bulk DNA WGS due to amplification bias (which was reproducible); (ii) false‐positive SNP sites detected by single‐cell WGS tend to occur in genomic regions with particularly high sequencing depth and high rate of C:G to T:A base changes; (iii) SNPs detected in three or more cells should be reliable (an detection efficiency of 83.4–97.4% was obtained for combined data from three cells). This analytical method could be adapted to measure genetic variation in other ciliates and broaden research into ciliate population genetics. [ABSTRACT FROM AUTHOR]

The herbicide amicarbazone (AMZ), which appeared as a possible alternative to atrazine, presents moderate environmental persistence and is unlikely to be removed by conventional water treatment techniques. Advanced oxidation processes (AOPs) driven by •OH and/or SO4•− radicals are then promising alternatives to AMZ-contaminated waters remediation, even though, in some cases, they can originate more toxic degradation products than the parent-compound. Therefore, assessing treated solutions toxicity prior to disposal is of extreme importance. In this study, the toxicity of AMZ solutions, before and after treatment with different •OH-driven and SO4•−-driven AOPs, was evaluated for five different microorganisms: Vibrio fischeri, Chlorella vulgaris, Tetrahymena thermophila, Escherichia coli, and Bacillus subtilis. In general, the toxic response of AMZ was greatly affected by the addition of reactants, especially when persulfate (PS) and/or Fe(III)-carboxylate complexes were added. The modifications of this response after treatment were correlated with AMZ intermediates, which were identified by mass spectrometry. Thus, low molecular weight by-products, resulting from fast degradation kinetics, were associated with increased toxicity to bacteria and trophic effects to microalgae. These observations were compared with toxicological predictions given by a Structure-Activity Relationships software, which revealed to be fairly compatible with our empirical findings. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

There has been an increasing incidence rate of rice false smut in global rice cultivation areas. However, there is a dearth of studies on the environmental concentrations and hazards of ustiloxin A (UA), which is the major mycotoxin produced by a pathogenic fungus of the rice false smut. Here, the concentrations of UA in the surface waters of two paddy fields located in Enshi city, Hubei province, China, were measured, and its toxicity in T. Thermophila was evaluated. This is the first study to detect UA in the surface waters of the two paddy fields, and the measured mean concentrations were 2.82 and 0.26 μg/L, respectively. Exposure to 2.19, 19.01 or 187.13 μg/L UA for 5 days significantly reduced the theoretical population and cell size of T. thermophila. Furthermore, treatment with 187.13 μg/L UA changed the percentages of T. thermophila cells in different cell-cycle stages, and with an increased malformation rate compared with the control, suggesting the disruption of the cell cycle. The expressions of 30 genes involved in the enriched proteasome pathway, 7 cyclin genes (cyc9 , cyc10 , cyc16 , cyc22 , cyc23, cyc26, cyc33) and 2 histone genes (mlh1 and hho1) were significantly down-regulated, which might be the modes of action responsible for the disruption of cell cycling due to UA exposure. Image 1 • For the first time, ustiloxin A was detected in the surface water of paddy fields. • Exposure to ustiloxin A reduced the theoretical population of T. Thermophila and caused cell malformation. • Cell cycle was disrupted by ustiloxin A. • Expressions of genes related to cell cycling were changed due to ustiloxin A exposure. For the first time, it was founded that ustiloxin A (UA) was detected in surface waters of paddy fields, and exposure to UA in T. Thermophila caused toxicity by disrupting cell cycle. [ABSTRACT FROM AUTHOR]

The length of cilia is controlled by a poorly understood mechanism that involves members of the conserved RCK kinase group, and among them, the LF4/MOK kinases. The multiciliated protist model, Tetrahymena, carries two types of cilia (oral and locomotory) and the length of the locomotory cilia is dependent on their position with the cell. In Tetrahymena, loss of an LF4/MOK ortholog, LF4A, lengthened the locomotory cilia, but also reduced their number. Without LF4A, cilia assembled faster and showed signs of increased intraflagellar transport (IFT). Consistently, overproduced LF4A shortened cilia and downregulated IFT. GFP-tagged LF4A, expressed in the native locus and imaged by total internal reflection microscopy, was enriched at the basal bodies and distributed along the shafts of cilia. Within cilia, most LF4A-GFP particles were immobile and a few either diffused or moved by IFT. We suggest that the distribution of LF4/MOK along the cilium delivers a uniform dose of inhibition to IFT trains that travel from the base to the tip. In a longer cilium, the IFT machinery may experience a higher cumulative dose of inhibition by LF4/MOK. Thus, LF4/MOK activity could be a readout of cilium length that helps to balance the rate of IFT-driven assembly with the rate of disassembly at steady state. We used a forward genetic screen to identify a CDK-related kinase, CDKR1, whose loss-of-function suppressed the shortening of cilia caused by overexpression of LF4A, by reducing its kinase activity. Loss of CDKR1 alone lengthened both the locomotory and oral cilia. CDKR1 resembles other known ciliary CDK-related kinases: LF2 of Chlamydomonas, mammalian CCRK and DYF-18 of C. elegans, in lacking the cyclin-binding motif and acting upstream of RCKs. The new genetic tools we developed here for Tetrahymena have potential for further dissection of the principles of cilia length regulation in multiciliated cells. [ABSTRACT FROM AUTHOR]

The silencing of repetitive transposable elements (TEs) is ensured by signal amplification of the initial small RNA trigger, which occurs at distinct steps of TE silencing in different eukaryotes. How such a variety of secondary small RNA biogenesis mechanisms has evolved has not been thoroughly elucidated. Ciliated protozoa perform small RNA-directed programmed DNA elimination of thousands of TE-related internal eliminated sequences (IESs) in the newly developed somatic nucleus. In the ciliate Paramecium, secondary small RNAs are produced after the excision of IESs. In this study, we show that in another ciliate, Tetrahymena, secondary small RNAs accumulate at least a few hours before their derived IESs are excised. We also demonstrate that DNA excision is dispensable for their biogenesis in this ciliate. Therefore, unlike in Paramecium, small RNA amplification occurs before IES excision in Tetrahymena. This study reveals the remarkable diversity of secondary small RNA biogenesis mechanisms, even among ciliates with similar DNA elimination processes, and thus raises the possibility that the evolution of TE-targeting small RNA amplification can be traced by investigating the DNA elimination mechanisms of ciliates. [ABSTRACT FROM AUTHOR]

• C. parvum gp60 was efficiently expressed in the ciliate T. thermophila. • Gp60 was localized at the ciliate cell surface and oral apparatus. • Sera of calves infected with C. parvum reacted with gp60 expressed in the ciliate. Cryptosporidium parvum is a protozoan parasite of the phylum Apicomplexa responsible for cryptosporidiosis in calves, a disease that causes significant diarrhea and impairs gain of body weight, generating important production losses. As to now, no effective drugs or vaccines are available for the treatment or prevention of bovine cryptosporidiosis. Several reports suggest that development of a vaccine to prevent cryptosporidiosis is feasible, but relatively few vaccine candidates have been characterized and tested. The most prominent C. parvum antigen is gp60, an O- glycosylated mucin-like protein tethered to the parasite membrane by a glycosylphosphatidylinositol (GPI) anchor. Gp60 has been shown to be involved in essential mechanisms for the survival of C. parvum , such as recognition, adhesion to, and invasion of host cells. This work was aimed at expressing gp60 in Tetrahymena thermophila , a ciliated protozoon with numerous advantages for the heterologous expression of eukaryotic proteins, as a first approach for the development of a recombinant vaccine for bovine cryptosporidiosis. T. thermophila -expressed gp60 localized to the protozoon cell surface and oral apparatus, and partitioned into the Triton X-114 detergent phase. This indicates that the protein entered the reticuloendothelial system of the ciliate, and suggests it contains a GPI-anchor. Homogenates of gp60-expressing T. thermophila cells were recognized by sera from calves naturally infected with C. parvum demonstrating their immunoreactivity. In summary, the heterologous expression of gp60, a C. parvum -encoded GPI-anchored protein, has been successfully demonstrated in the ciliate T. thermophila. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A morphospecies is defined as a taxonomic species based wholly on morphology, but often morphospecies consist of clusters of cryptic species that can be identified genetically or molecularly. The nature of the evolutionary novelty that accompanies speciation in a morphospecies is an intriguing question. Morphospecies are particularly common among ciliates, a group of unicellular eukaryotes that separates 2 kinds of nuclei—the silenced germline nucleus (micronucleus [MIC]) and the actively expressed somatic nucleus (macronucleus [MAC])—within a common cytoplasm. Because of their very similar morphologies, members of the Tetrahymena genus are considered a morphospecies. We explored the hidden genomic evolution within this genus by performing a comprehensive comparative analysis of the somatic genomes of 10 species and the germline genomes of 2 species of Tetrahymena. These species show high genetic divergence; phylogenomic analysis suggests that the genus originated about 300 million years ago (Mya). Seven universal protein domains are preferentially included among the species-specific (i.e., the youngest) Tetrahymena genes. In particular, leucine-rich repeat (LRR) genes make the largest contribution to the high level of genome divergence of the 10 species. LRR genes can be sorted into 3 different age groups. Parallel evolutionary trajectories have independently occurred among LRR genes in the different Tetrahymena species. Thousands of young LRR genes contain tandem arrays of exactly 90-bp exons. The introns separating these exons show a unique, extreme phase 2 bias, suggesting a clonal origin and successive expansions of 90-bp–exon LRR genes. Identifying LRR gene age groups allowed us to document a Tetrahymena intron length cycle. The youngest 90-bp exon LRR genes in T. thermophila are concentrated in pericentromeric and subtelomeric regions of the 5 micronuclear chromosomes, suggesting that these regions act as genome innovation centers. Copies of a Tetrahymena Long interspersed element (LINE)-like retrotransposon are very frequently found physically adjacent to 90-bp exon/intron repeat units of the youngest LRR genes. We propose that Tetrahymena species have used a massive exon-shuffling mechanism, involving unequal crossing over possibly in concert with retrotransposition, to create the unique 90-bp exon array LRR genes. [ABSTRACT FROM AUTHOR]

Oil supplementation in the ruminant diet has been proven to reduce the rumen protozoa population and maintain their low amount during supplementation. Majority of oil that contains medium chain fatty acids are also known to reduce cellulolytic bacteria. This experiment was conducted to study the effect of different types of oil supplementation on the protozoa and fiber-degrading bacteria population in goats. Sixteen male goats equipped with rumen cannula were assigned to four experimental dietary treatments. The first group acted as the control group (CON) and received the basal diet. The second group (OL) received the basal diet with the supplementation of olive oil, whereas the third group (SO) and fourth group (PL) received the basal diet supplemented with sunflower and palm olein oils, respectively. The rumen content of each animal was collected for pH measurement as well as enumeration of the rumen protozoa and fiber-degrading bacteria. The rumen pH level was affected by sampling days (P<0.05). The total protozoa counts were higher in the CON compared to the other treatments, which the OL group had the lowest rumen protozoa counts and were significantly affected (P<0.05) by the diet and day of sampling. Treatment groups did not affect Fibrobacter succinogenes, Ruminococcus albus and Ruminococcus flavefaciens populations, but there were significantly affected (P<0.05) by day of sampling and the interaction of treatment × day. In conclusion, dietary oils supplementation affects the rumen protozoa population, but the effects on bacteria population are not extensive. [ABSTRACT FROM AUTHOR]

In recent years, Tetrahymena species have been used as model organisms for research in a wide range of fields, highlighting the need for a fuller understanding of the taxonomy of this group. It is in this context that this paper uses living observation and silver staining methods to investigate the morphology and infraciliature of one Tetrahymena species, T. setosa (Schewiakoff 1892 Verh. Naturh. Med. Ver. Heidelb., 4:544) McCoy (1975) Acta Protozool., 14:253; the senior subjective synonym of T. setifera Holz and Corliss (1956) J. Protozool., 3:112; isolated from a freshwater pond in Harbin, north‐eastern China. This organism can be distinguished from other described Tetrahymena species mainly by its single caudal cilium, which is about twice the length of the somatic ciliature. While the Harbin isolate appears similar to the population described by Holz and Corliss (1956) J. Protozool., 3:112, an improved diagnosis for T. setosa is given based on the previous descriptions and the Harbin population. In summary, this species can be recognized mainly by the combination of the following characters: body in vivo approximately 40 μm × 25 μm, 21–26 somatic kineties, one to four contractile vacuole pores associated with meridians 6–11 and a single caudal cilium. The small subunit ribosomal (SSU) rRNA gene and the cox1 gene sequences of Harbin population are also characterized in order to corroborate that the isolated species branches in phylogenetic trees as a T. setosa species. The phylogenetic analysis also indicated that sequences of populations of Tetrahymena species should be published with detailed morphological identifications. [ABSTRACT FROM AUTHOR]

Meiotic bouquet formation (known as crescent formation in Tetrahymena thermophila) is indispensable for homologous pairing and recombination, but the regulatory mechanism of bouquet formation remains largely unknown. As a conjugation specific cyclin gene, CYC2 knockout mutants failed to form an elongated crescent structure and aborted meiosis progress in T. thermophila. γ-H2A.X staining revealed fewer micronuclear DNA double-strand breaks (DSBs) in cyc2Δ cells than in wild-type cells. Furthermore, cyc2Δ cells still failed to form a crescent structure even though DSBs were induced by exogenous agents, indicating that a lack of DSBs was not completely responsible for failure to enter the crescent stage. Tubulin staining showed that impaired perinuclear microtubule structure may contribute to the blockage in micronuclear elongation. At the same time, expression of microtubule-associated kinesin genes, KIN11 and KIN141, was significantly downregulated in cyc2Δ cells. Moreover, micronuclear specific accumulation of heterochromatin marker trimethylated H3K23 abnormally increased in the cyc2Δ mutants. Together, these results show that cyclin Cyc2p is required for micronuclear bouquet formation via controlling microtubule-directed nuclear elongation in Tetrahymena. [ABSTRACT FROM AUTHOR]

During programmed nuclear death (PND), apoptosis‐inducing factor (AIF) translocates from mitochondria to the parental macronucleus (MAC) in Tetrahymena thermophila. In the degenerating parental MAC, AIF induces chromatin condensation and large‐scale DNA fragmentation in a caspase‐independent manner. However, the regulation of AIF nuclear translocation and molecular mechanism of PND are less clear. In this study, we demonstrated that the asymmetric distribution of nuclear GDP‐bound Ran1‐mimetic mutant Ran1T25N and cytoplasmic GTP‐bound Ran1‐mimetic mutant Ran1Q70L exists across the parental macronuclear‐cytoplasmic barrier during PND. Knockdown of RAN1 led to defects in PND progression and failure of parental macronuclear accumulation of AIF. Moreover, AIF parental macronuclear import occurred in Ran1T25N mutants, while it was inhibited in Ran1Q70L mutants. Importantly, artificial accumulation of AIF in the parental MAC rescued PND progression defects in RAN1 knockdown mutants. These data suggest that Ran1 is essential for parental macronuclear import of AIF and PND in T. thermophila. During programmed nuclear death (PND), apoptosis‐inducing factor (AIF) translocates from mitochondria to the parental macronucleus (MAC) in Tetrahymena thermophila. Knockdown of RAN1 (i∆RAN1) inhibited import of AIF to the degenerating parental MAC during PND. The asymmetric distribution of Ran1GTP and Ran1GDP was involved in parental macronuclear import of AIF, which occurred in Ran1T25N (GDP‐bound Ran1‐mimetic) mutants but was inhibited in Ran1Q70L (GTP‐bound Ran1‐mimetic) mutants. [ABSTRACT FROM AUTHOR]

Koumine is a component of the Chinese medicinal herb Gelsemium elegans and is toxic to vertebrates. We used the ciliate Tetrahymena thermophila as a model to evaluate the toxic effects of this indole alkaloid in eukaryotic microorganisms. Koumine inhibited T. thermophila growth and viability in a dose-dependent manner. Moreover, this drug produced oxidative stress in T. thermophila cells and expressions of antioxidant enzymes were significantly elevated at high koumine levels (p < 0.05). Koumine also caused significant levels of apoptosis (p < 0.05) and induced DNA damage in a dose-dependent manner. Mitophagic vacuoles were present in cells indicating induction of autophagy by this drug. Expression of ATG7, MTT2/4, CYP1 and HSP70 as well as the MAP kinase pathway gene MPK1 and MPK3 were significantly altered after exposed to koumine. This study represents a preliminary toxicological evaluation of koumine in the single celled eukaryote T. thermophila. [ABSTRACT FROM AUTHOR]