RebL1 is required for macronuclear structure stability and gametogenesis in Tetrahymena thermophila.

Histone modification and nucleosome assembly play important roles in chromatin-related processes. Histone chaperones form different complexes and coordinate histone transportation and assembly. Various histone chaperone complexes have been identified in different organisms. The ciliate protozoa (ciliates) have various chromatin structures and different nuclear morphology. However, histone chaperone components and functions of different subunits remain unclear in ciliates. Tetrahymema thermophila contains a transcriptionally active macronucleus (MAC) and a transcriptionally inactive micronucleus (MIC) which exhibit multiple replication and various chromatin remodeling progresses during vegetative growth and sexual developmental stages. Here, we found histone chaperone RebL1 not only localized evenly in the transcriptionally active MAC but also dynamically changed in the MIC during vegetative growth and sexual developmental stages. REBL1 knockdown inhibited cellular proliferation. The macronuclear morphology became bigger in growing mutants. The abnormal macronuclear structure also occurred in the starvation stage. Furthermore, micronuclear meiosis was disturbed during sexual development, leading to a failure to generate new gametic nuclei. RebL1 potentially interacted with various factors involved in histone-modifying complexes and chromatin remodeling complexes in different developmental stages. REBL1 knockdown affected expression levels of the genes involved in chromatin organization and transcription. Taken together, RebL1 plays a vital role in maintaining macronuclear structure stability and gametogenesis in T. thermophila. Supplementary Information: The online version contains supplementary material available at 10.1007/s42995-024-00219-z.

Cellular Response of Adapted and Non-Adapted Tetrahymena thermophila Strains to Europium Eu(III) Compounds.

Europium is one of the most reactive lanthanides and humans use it in many different applications, but we still know little about its potential toxicity and cellular response to its exposure. Two strains of the eukaryotic microorganism model Tetrahymena thermophila were adapted to high concentrations of two Eu(III) compounds (EuCl3 or Eu2O3) and compared to a control strain and cultures treated with both compounds. In this ciliate, EuCl3 is more toxic than Eu2O3. LC50 values show that this microorganism is more resistant to these Eu(III) compounds than other microorganisms. Oxidative stress originated mainly by Eu2O3 is minimized by overexpression of genes encoding important antioxidant enzymes. The overexpression of metallothionein genes under treatment with Eu(III) compounds supports the possibility that this lanthanide may interact with the -SH groups of the cysteine residues from metallothioneins and/or displace essential cations of these proteins during their homeostatic function. Both lipid metabolism (lipid droplets fusing with europium-containing vacuoles) and autophagy are involved in the cellular response to europium stress. Bioaccumulation, together with a possible biomineralization to europium phosphate, seems to be the main mechanism of Eu(III) detoxification in these cells.

Molecular Cloning, Expression and Enzymatic Characterization of Tetrahymena thermophila Glutathione-S-Transferase Mu 34.

Glutathione-S-transferase enzymes (GSTs) are essential components of the phase II detoxification system and protect organisms from oxidative stress induced by xenobiotics and harmful toxins such as 1-chloro-2,4-dinitrobenzene (CDNB). In Tetrahymena thermophila, the TtGSTm34 gene was previously reported to be one of the most responsive GST genes to CDNB treatment (LD50 = 0.079 mM). This study aimed to determine the kinetic features of recombinantly expressed and purified TtGSTm34 with CDNB and glutathione (GSH). TtGSTm34-8xHis was recombinantly produced in T. thermophila as a 25-kDa protein after the cloning of the 660-bp full-length ORF of TtGSTm34 into the pIGF-1 vector. A three-dimensional model of the TtGSTm34 protein constructed by the AlphaFold and PyMOL programs confirmed that it has structurally conserved and folded GST domains. The recombinant production of TtGSTm34-8xHis was confirmed by SDS‒PAGE and Western blot analysis. A dual-affinity chromatography strategy helped to purify TtGSTm34-8xHis approximately 3166-fold. The purified recombinant TtGSTm34-8xHis exhibited significantly high enzyme activity with CDNB (190 µmol/min/mg) as substrate. Enzyme kinetic analysis revealed Km values of 0.68 mM with GSH and 0.40 mM with CDNB as substrates, confirming its expected high affinity for CDNB. The optimum pH and temperature were determined to be 7.0 and 25 °C, respectively. Ethacrynic acid inhibited fully TtGSTm34-8xHis enzyme activity. These results imply that TtGSTm34 of T. thermophila plays a major role in the detoxification of xenobiotics, such as CDNB, as a first line of defense in aquatic protists against oxidative damage.

Demography and movement patterns of a freshwater ciliate: The influence of oxygen availability.

In freshwater habitats, aerobic animals and microorganisms can react to oxygen deprivation by a series of behavioural and physiological changes, either as a direct consequence of hindered performance or as adaptive responses towards hypoxic conditions. Since oxygen availability can vary throughout the water column, different strategies exist to avoid hypoxia, including that of active 'flight' from low-oxygen sites. Alternatively, some organisms may invest in slower movement, saving energy until conditions return to more favourable levels, which may be described as a 'sit-and-wait' strategy. Here, we aimed to determine which, if any, of these strategies could be used by the freshwater ciliate Tetrahymena thermophila when faced with decreasing levels of oxygen availability in the culture medium. We manipulated oxygen flux into clonal cultures of six strains (i.e. genotypes) and followed their growth kinetics for several weeks using automated image analysis, allowing to precisely quantify changes in density, morphology and movement patterns. Oxygen effects on demography and morphology were comparable across strains: reducing oxygen flux decreased the growth rate and maximal density of experimental cultures, while greatly expanding the duration of their stationary phase. Cells sampled during their exponential growth phase were larger and had a more elongated shape under hypoxic conditions, likely mirroring a shift in resource investment towards individual development rather than frequent divisions. In addition to these general patterns, we found evidence for intraspecific variability in movement responses to oxygen limitation. Some strains showed a reduction in swimming speed, potentially associated with a 'sit-and-wait' strategy; however, the frequent alteration of movement paths towards more linear trajectories also suggests the existence of an inducible 'flight response' in this species. Considering the inherent costs of turns associated with non-linear movement, such a strategy may allow ciliates to escape suboptimal environments at a low energetic cost.

Effect of α-tubulin acetylation on the doublet microtubule structure.

Acetylation of α-tubulin at the lysine 40 residue (αK40) by αTAT1/MEC-17 acetyltransferase modulates microtubule properties and occurs in most eukaryotic cells. Previous literatures suggest that acetylated microtubules are more stable and damage resistant. αK40 acetylation is the only known microtubule luminal post-translational modification site. The luminal location suggests that the modification tunes the lateral interaction of protofilaments inside the microtubule. In this study, we examined the effect of tubulin acetylation on the doublet microtubule (DMT) in the cilia of Tetrahymena thermophila using a combination of cryo-electron microscopy, molecular dynamics, and mass spectrometry. We found that αK40 acetylation exerts a small-scale effect on the DMT structure and stability by influencing the lateral rotational angle. In addition, comparative mass spectrometry revealed a link between αK40 acetylation and phosphorylation in cilia.

A seven-sex species recognizes self and non-self mating-type via a novel protein complex.

Although most species have two sexes, multisexual (or multi-mating type) species are also widespread. However, it is unclear how mating-type recognition is achieved at the molecular level in multisexual species. The unicellular ciliate Tetrahymena thermophila has seven mating types, which are determined by the MTA and MTB proteins. In this study, we found that both proteins are essential for cells to send or receive complete mating-type information, and transmission of the mating-type signal requires both proteins to be expressed in the same cell. We found that MTA and MTB form a mating-type recognition complex that localizes to the plasma membrane, but not to the cilia. Stimulation experiments showed that the mating-type-specific regions of MTA and MTB mediate both self- and non-self-recognition, indicating that T. thermophila uses a dual approach to achieve mating-type recognition. Our results suggest that MTA and MTB form an elaborate multifunctional protein complex that can identify cells of both self and non-self mating types in order to inhibit or activate mating, respectively.

Noncanonical usage of stop codons in ciliates expands proteins with structurally flexible Q-rich motifs.

Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 26 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in 10 ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g. D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.

MicroRNAs in Tetrahymena thermophila: An epigenetic regulatory mechanism in the response to cadmium stress.

Among the epigenetic mechanisms based on non-coding RNA are microRNAs (miRNAs) that are involved in the post-transcriptional regulation of mRNAs. In many organisms, the expression of genes involved in the cellular response to biotic or abiotic stress depends on the regulation, generally inhibitory, performed by miRNAs. For the first time in the eukaryotic microorganism (ciliate-model) Tetrahymena thermophila, miRNAs involved in the post-transcriptional regulation of transcripts linked to the response to cadmium have been isolated and analyzed. Forty de novo miRNAs (we named tte-miRNAs) have been isolated from control and Cd-treated populations (1 or 24 h exposures). An exhaustive comparative analysis of the features of these mature tte-miRNAs and their precursor sequences (pre-tte-miRNAs) confirms that they are true miRNAs. In addition to the three types of miRNA isoforms previously described in other organisms, two new types are also described among the tte-miRNAs studied. A certain percentage of the pre-tte-miRNA sequences are in introns from genes with many introns, and have been defined as 5', 3'-tailed mirtrons. A qRT-PCR analysis of selected tte-miRNAs together with some of their targets has validated them. Cd is one of the most toxic metals for the cell, which must defend itself against its toxicity by various mechanisms, such as expulsion by membrane pumps, chelation by metallothioneins, among others. Like other toxic metals, Cd also causes a well-known series of cellular effects such as intense proteotoxicity. Many of the targets that are regulated by the tte-miRNAs are transcripts encoding proteins that fit into these defense mechanisms and toxic metal effects.

Independent and Complementary Functions of Caf1b and Hir1 for Chromatin Assembly in Tetrahymena thermophila.

Histones and DNA associate to form the nucleosomes of eukaryotic chromatin. Chromatin assembly factor 1 (CAF-1) complex and histone regulatory protein A (HIRA) complex mediate replication-couple (RC) and replication-independent (RI) nucleosome assembly, respectively. CHAF1B and HIRA share a similar domain but play different roles in nucleosome assembly by binding to the different interactors. At present, there is limited understanding for the similarities and differences in their respective functions. Tetrahymena thermophila contains transcriptionally active polyploid macronuclei (MAC) and transcriptionally silent diploid micronuclei (MIC). Here, the distribution patterns of Caf1b and Hir1 exhibited both similarities and distinctions. Both proteins localized to the MAC and MIC during growth, and to the MIC during conjugation. However, Hir1 exhibited additional signaling on parental MAC and new MAC during sexual reproduction and displayed a punctate signal on developing anlagen. Caf1b and Hir1 only co-localized in the MIC with Pcna1 during conjugation. Knockdown of CAF1B impeded cellular growth and arrested sexual reproductive development. Loss of HIR1 led to MIC chromosome defects and aborted sexual development. Co-interference of CAF1B and HIR1 led to a more severe phenotype. Moreover, CAF1B knockdown led to the up-regulation of HIR1 expression, while knockdown of HIR1 also led to an increase in CAF1B expression. Furthermore, Caf1b and Hir1 interacted with different interactors. These results showed that CAF-1 and Hir1 have independent and complementary functions for chromatin assembly in T. thermophila.

Mismatch Repair Protein Msh6Tt Is Necessary for Nuclear Division and Gametogenesis in Tetrahymena thermophila.

DNA mismatch repair (MMR) improves replication accuracy by up to three orders of magnitude. The MutS protein in E. coli or its eukaryotic homolog, the MutSα (Msh2-Msh6) complex, recognizes base mismatches and initiates the mismatch repair mechanism. Msh6 is an essential protein for assembling the heterodimeric complex. However, the function of the Msh6 subunit remains elusive. Tetrahymena undergoes multiple DNA replication and nuclear division processes, including mitosis, amitosis, and meiosis. Here, we found that Msh6Tt localized in the macronucleus (MAC) and the micronucleus (MIC) during the vegetative growth stage and starvation. During the conjugation stage, Msh6Tt only localized in MICs and newly developing MACs. MSH6Tt knockout led to aberrant nuclear division during vegetative growth. The MSH6TtKO mutants were resistant to treatment with the DNA alkylating agent methyl methanesulfonate (MMS) compared to wild type cells. MSH6Tt knockout affected micronuclear meiosis and gametogenesis during the conjugation stage. Furthermore, Msh6Tt interacted with Msh2Tt and MMR-independent factors. Downregulation of MSH2Tt expression affected the stability of Msh6Tt. In addition, MSH6Tt knockout led to the upregulated expression of several MSH6Tt homologs at different developmental stages. Msh6Tt is involved in macronuclear amitosis, micronuclear mitosis, micronuclear meiosis, and gametogenesis in Tetrahymena.

Iron-loading N and S heteroatom doped porous carbon derived from chitosan and CdS-Tetrahymena thermophila for peroxymonosulfate activation.

Transforming waste into resources is an important strategy to enhance the economic efficiency and reduce the waste entering the environment. In this work, iron-loading N and S co-doped porous carbon materials, as peroxymonosulfate (PMS) activator for pollutants degradation, were prepared by pyrolysis of the mixture of iron loading chitosan and CdS-Tetrahymena thermophila under N2 flow. Chitosan is mainly derived from the shell waste of shrimp and crab, and CdS-Tetrahymena thermophila is produced in the removing process of Cd2+ pollution bioremediation using Tetrahymena thermophila. The synergistic effects of iron related species and heteroatoms (S/N) co-doped porous carbon in the obtained carbon materials improved the performance for activating PMS. The prepared Fe-S-CS-1-900 exhibited high performance for the degradation of Rhodamine B (RhB) by activating PMS. Radical quenching tests and electron paramagnetic resonance measurements suggested that superoxide radical (O2-) and singlet oxygen (1O2) were the primary reactive oxygen species in RhB degradation. These results propose new insights of using biomass waste to derive Fe-loading N and S heteroatom co-doping carbon as PMS activator applied in the removal of organic pollutants.

Target Protein Expression on Tetrahymena thermophila Cell Surface Using the Signal Peptide and GPI Anchor Sequences of the Immobilization Antigen of Cryptocaryon irritans.

Cryptocaryoniasis, caused by Cryptocaryon irritans, is a significant threat to marine fish cultures in tropical and subtropical waters. However, controlling this disease remains a challenge. Fish infected with C. irritans acquires immunity; however, C. irritans is difficult to culture in large quantities, obstructing vaccine development using parasite cells. In this study, we established a method for expressing an arbitrary protein on the surface of Tetrahymena thermophila, a culturable ciliate, to develop a mimetic C. irritans. Fusing the signal peptide (SP) and glycosylphosphatidylinositol (GPI) anchor sequences of the immobilization antigen, a surface protein of C. irritans, to the fluorescent protein, monomeric Azami-green 1 (mAG1) of the stony coral Galaxea fascicularis, allowed protein expression on the surface and cilia of transgenic Tetrahymena cells. This technique may help develop transgenic Tetrahymena displaying parasite antigens on their cell surface, potentially contributing to the development of vaccines using "mimetic parasites".

Mismatch Repair Protein Msh2 Is Necessary for Macronuclear Stability and Micronuclear Division in Tetrahymena thermophila.

Mismatch repair (MMR) is a conserved mechanism that is primarily responsible for the repair of DNA mismatches during DNA replication. Msh2 forms MutS heterodimer complexes that initiate the MMR in eukaryotes. The function of Msh2 is less clear under different chromatin structures. Tetrahymena thermophila contains a transcriptionally active macronucleus (MAC) and a transcriptionally silent micronucleus (MIC) in the same cytoplasm. Msh2 is localized in the MAC and MIC during vegetative growth. Msh2 is localized in the perinuclear region around the MIC and forms a spindle-like structure as the MIC divides. During the early conjugation stage, Msh2 is localized in the MIC and disappears from the parental MAC. Msh2 is localized in the new MAC and new MIC during the late conjugation stage. Msh2 also forms a spindle-like structure with a meiotic MIC and mitotic gametic nucleus. MSH2 knockdown inhibits the division of MAC and MIC during vegetative growth and affects cellular proliferation. MSH2 knockdown mutants are sensitive to cisplatin treatment. MSH2 knockdown also affects micronuclear meiosis and gametogenesis during sexual development. Furthermore, Msh2 interacts with MMR-dependent and MMR-independent factors. Therefore, Msh2 is necessary for macronuclear stability, as well as micronuclear mitosis and meiosis in Tetrahymena.

Tubulysins are Essential for the Preying of Ciliates by Myxobacteria.

Tubulysins are bioactive secondary metabolites produced by myxobacteria that promote microtubule disassembly. Microtubules are required for protozoa such as Tetrahymena to form cilia and flagella. To study the role of tubulysins in myxobacteria, we co-cultured myxobacteria and Tetrahymena. When 4000 Tetrahymena thermophila and 5.0 × 108 myxobacteria were added to 1 ml of CYSE medium and co-cultured for 48 h, the population of T. thermophila increased to more than 75,000. However, co-culturing tubulysin-producing myxobacteria, including Archangium gephyra KYC5002, with T. thermophila caused the population of T. thermophila to decrease from 4000 to less than 83 within 48 h. Almost no dead bodies of T. thermophila were observed in the culture medium. Co-culturing of T. thermophila and the A. gephyra KYC5002 strain with inactivation of the tubulysin biosynthesis gene led to the population of T. thermophila increasing to 46,667. These results show that in nature, most myxobacteria are preyed upon by T. thermophila, but some myxobacteria prey on and kill T. thermophila using tubulysins. Adding purified tubulysin A to T. thermophila changed the cell shape from ovoid to spherical and caused cell surface cilia to disappear.

Quantitative proteomic analyses of a Pb-adapted Tetrahymena thermophila strain reveal the cellular strategy to Pb(II) stress including lead biomineralization to chloropyromorphite.

A strain of the protozoan ciliate Tetrahymena thermophila adapted to increasing Pb(II) concentrations over two years has shown that one of the resistance mechanisms to this extreme metal stress is the lead biomineralization to chloropyromorphite, one of the most stable minerals in the earth's crust. Several techniques such as microanalysis coupled to transmission and scanning electron microscopy (X-Ray Energy Disperse Spectroscopy), fluorescence microscopy and X-ray power diffraction analysis have revealed the presence of chloropyromorphite as crystalline aggregates of nano-globular structure, together with the presence of other secondary lead minerals. This is the first time that the existence of this type of biomineralization in a ciliate protozoan is described. The Pb(II) bioremediation capacity of this strain has shown that it can remove >90 % of the toxic soluble lead from the medium. A quantitative proteomic analysis of this strain has revealed the main molecular-physiological elements involved in adaptation to Pb(II) stress: increased activity of proteolytic systems against lead proteotoxicity, occurrence of metallothioneins to immobilize Pb(II) ions, antioxidant enzymes to mitigate oxidative stress, and an intense vesicular trafficking presumably involved in the formation of vacuoles where pyromorphite accumulates and is subsequently excreted, together with an enhanced energy metabolism. As a conclusion, all these results have been compiled into an integrated model that could explain the eukaryotic cellular response to extreme lead stress.

Population dynamics hide phenotypic changes driven by subtle chemical exposures: implications for risk assessments.

Ecological risk assessment of chemicals focuses on the response of different taxa in isolation not taking ecological and evolutionary interplay in communities into account. Its consideration would, however, allow for an improved assessment by testing for implications within and across trophic levels and changes in the phenotypic and genotypic diversity within populations. We present a simple experimental system that can be used to evaluate the ecological and evolutionary responses to chemical exposure at microbial community levels. We exposed a microbial model system of the ciliate Tetrahymena thermophila (predator) and the bacterium Pseudomonas fluorescens (prey) to iron released from Magnetic Particles (MP-Fedis), which are Phosphorus (P) adsorbents used in lake restoration. Our results show that while the responses of predator single population size differed across concentrations of MP-Fedis and the responses of prey from communities differed also across concentration of MP-Fedis, the community responses (species ratio) were similar for the different MP-Fedis concentrations. Looking further at an evolutionary change in the bacterial preys' defence, we found that MP-Fedis drove different patterns and dynamics of defence evolution. Overall, our study shows how similar community dynamics mask changes at evolutionary levels that would be overlooked in the design of current risk assessment protocols where evolutionary approaches are not considered.

Transcriptome analysis of Tetrahymena thermophila response to exposure with dihydroartemisinin.

Dihydroartemisinin (DHA) is a derivative of artemisinin and is toxic to parasites. We used the Tetrahymena thermophila (T. thermophila) as a model to explore DHA toxicity. Results showed that low concentration of DHA (20 µmol/L) promoted cell proliferation, whereas high concentrations of DHA (40-1280 µmol/L) inhibited that. Appearance of nucleus was pycnosis by laser scanning confocal microscope. DHA significantly elevated activities of SOD and GSH-Px (P < 0.01) and MDA was markedly increased at high level but decreased at low level (P < 0.01). Further results of transcriptome in T. thermophila treated with different concentration DHA group (0, 20, 160 µmol/L) showed that differentially expressed genes (DEGs) were involved in oxidation-reduction and metabolism of exogenous substances indicated oxidative stress stimulation. Kyoto Encyclopedia of Genes and Genomes showed that DEGs were involved in the cytochrome P450-mediated metabolism of exogenous substances, glutathione metabolism and ABC transport. Remarkably, DNA replication was significantly enriched in low concentration DHA, energy metabolism related pathways and necrotic process were considerably enriched in high concentration DHA. The results of RT-qPCR of 13 DEGs were the same as that of transcriptome, in which the expression of GST and GPx family genes were significantly altered after exposed to high-DHA group. DHA induced oxidative stress damage through disturbing with energy. However, detoxification pathways in T. thermophila to resist oxidative damage and cell alleviated low concentration DHA stress by regulating antioxidant enzyme. This study provides good practice on pharmacological mechanism of artemisinin-based drugs in antiparasitic.

Developmentally Programmed Switches in DNA Replication: Gene Amplification and Genome-Wide Endoreplication in Tetrahymena.

Locus-specific gene amplification and genome-wide endoreplication generate the elevated copy number of ribosomal DNA (rDNA, 9000 C) and non-rDNA (90 C) chromosomes in the developing macronucleus of Tetrahymena thermophila. Subsequently, all macronuclear chromosomes replicate once per cell cycle during vegetative growth. Here, we describe an unanticipated, programmed switch in the regulation of replication initiation in the rDNA minichromosome. Early in development, the 21 kb rDNA minichromosome is preferentially amplified from 2 C to ~800 C from well-defined origins, concurrent with genome-wide endoreplication (2 C to 8-16 C) in starved mating Tetrahymena (endoreplication (ER) Phase 1). Upon refeeding, rDNA and non-rDNA chromosomes achieve their final copy number through resumption of just the endoreplication program (ER Phase 2). Unconventional rDNA replication intermediates are generated primarily during ER phase 2, consistent with delocalized replication initiation and possible formation of persistent RNA-DNA hybrids. Origin usage and replication fork elongation are affected in non-rDNA chromosomes as well. Despite the developmentally programmed 10-fold reduction in the ubiquitous eukaryotic initiator, the Origin Recognition Complex (ORC), active initiation sites are more closely spaced in ER phases 1 and 2 compared to vegetative growing cells. We propose that initiation site selection is relaxed in endoreplicating macronuclear chromosomes and may be less dependent on ORC.

Complexity vs linearity: relations between functional traits in a heterotrophic protist.

BACKGROUND: Functional traits are phenotypic traits that affect an organism's performance and shape ecosystem-level processes. The main challenge when using functional traits to quantify biodiversity is to choose which ones to measure since effort and money are limited. As one way of dealing with this, Hodgson et al. (Oikos 85:282, 1999) introduced the idea of two types of traits, with soft traits that are easy and quick to quantify, and hard traits that are directly linked to ecosystem functioning but difficult to measure. If a link exists between the two types of traits, then one could use soft traits as a proxy for hard traits for a quick but meaningful assessment of biodiversity. However, this framework is based on two assumptions: (1) hard and soft traits must be tightly connected to allow reliable prediction of one using the other; (2) the relationship between traits must be monotonic and linear to be detected by the most common statistical techniques (e.g. linear model, PCA). RESULTS: Here we addressed those two assumptions by focusing on six functional traits of the protist species Tetrahymena thermophila, which vary both in their measurement difficulty and functional meaningfulness. They were classified as: easy traits (morphological traits), intermediate traits (movement traits) and hard traits (oxygen consumption and population growth rate). We detected a high number (> 60%) of non-linear relations between the traits, which can explain the low number of significant relations found using linear models and PCA analysis. Overall, these analyses did not detect any relationship strong enough to predict one trait using another, but that does not imply there are none. CONCLUSIONS: Our results highlighted the need to critically assess the relations among the functional traits used as proxies and those functional traits which they aim to reflect. A thorough assessment of whether such relations exist across species and communities is a necessary next step to evaluate whether it is possible to take a shortcut in quantifying functional diversity by collecting the data on easily measurable traits.

Glyphosate metabolism in Tetrahymena thermophila: A shotgun proteomic analysis approach.

Glyphosate is one of the most widely used herbicides in the world. However, because of its overuse and resistance to degradation, high levels of glyphosate residues in the environment are reported. Therefore, this study aimed to investigate the effects of glyphosate on proteomic aspects of Tetrahymena thermophila and their uses as bioindicators of freshwater ecosystem. First, an acute toxicity test was performed to determine the median inhibition concentration (IC50 ). The toxicity test results showed that glyphosate inhibited the growth (proliferation) of T. thermophila. The 96 h-IC50 value of glyphosate was 171 mg L-1 . No visible changes in aggregation behavior and cell morphology were observed under glyphosate exposure. In addition, the effects of low and high dose glyphosate concentrations (77.5 mg L-1 , 171 mg L-1 ) on the proteomic changes of T. thermophila was investigated using a label-free shotgun proteomic approach. A total of 3191 proteins were identified, 2791 proteins were expressed in the control, 2651 proteins were expressed in 77.5 mg L-1 glyphosates, and 3012 proteins were expressed in 171 mg L-1 glyphosates. Under glyphosate exposure at both low and high dose glyphosate, 400 unique proteins were upregulated. The majority of these proteins was classified as proteins associated with oxidative stress response and intracellular transport indicating the shifts in the internal metabolism. Proteomics revealed that the glyphosate metabolism by T. thermophila is a multi-step process involving several enzymes, which can be divided into four phases, including modification (phase I), conjugation (phase II), transport (phase III), and degradation (phase IV). The accumulation of various biochemical reactions contributes to overall glyphosate resistance. With the proteomics approach, we have found that T. thermophila was equipped with glyphosate detoxification and degradation mechanisms.