Construction of Tetrahymena strains with highly active arsenic methyltransferase genes for arsenic detoxification in aquatic environments.

Biomethylation is an effective means of arsenic detoxification by organisms living in aquatic environments. Ciliated protozoa (including Tetrahymena species) play an important role in the biochemical cycles of aquatic ecosystems and have a potential application in arsenic biotransformation. This study compared arsenic tolerance, accumulation, methylation, and efflux in 11 Tetrahymena species. Nineteen arsenite (As(III)) S-adenosylmethionine (SAM) methyltransferase (arsM) genes, of which 12 are new discoveries, were identified, and protein sequences were studied. We then constructed recombinant cell lines based on the Tetrahymena thermophila (T. thermophila) wild-type SB210 strain and expressed each of the 19 arsM genes under the control of the metal-responsive the MTT1 promoter. In the presence of Cd2+ and As(V), expression of the arsM genes in the recombinant cell lines was much higher than in the donor species. Evaluation of the recombinant cell line identified one with ultra-high arsenic methylation enzyme activity, significantly higher arsenic methylation capacity and much faster methylation rate than other reported arsenic methylated organisms, which methylated 89% of arsenic within 6.5 h. It also had an excellent capacity for the arsenic detoxification of lake water containing As(V), 56% of arsenic was methylated at 250 µg/L As(V) in 48 h. This study has made a significant contribution to our knowledge on arsenic metabolism in protozoa and demonstrates the great potential to use Tetrahymena species in the arsenic biotransformation of aquatic environments.

The three-dimensional structure of the Vint domain from Tetrahymena thermophila suggests a ligand-regulated cleavage mechanism by the HINT fold.

Vint proteins have been identified in unicellular metazoans as a novel hedgehog-related gene family, merging the von Willebrand factor type A domain and the Hedgehog/INTein (HINT) domains. We present the first three-dimensional structure of the Vint domain from Tetrahymena thermophila corresponding to the auto-processing domain of hedgehog proteins, shedding light on the unique features, including an adduct recognition region (ARR). Our results suggest a potential binding between the ARR and sulfated glycosaminoglycans like heparin sulfate. Moreover, we uncover a possible regulatory role of the ARR in the auto-processing by Vint domains, expanding our understanding of the HINT domain evolution and their use in biotechnological applications. Vint domains might have played a crucial role in the transition from unicellular to multicellular organisms.

The macronuclear genomic landscape within Tetrahymena thermophila.

The extent of intraspecific genomic variation is key to understanding species evolutionary history, including recent adaptive shifts. Intraspecific genomic variation remains poorly explored in eukaryotic micro-organisms, especially in the nuclear dimorphic ciliates, despite their fundamental role as laboratory model systems and their ecological importance in many ecosystems. We sequenced the macronuclear genome of 22 laboratory strains of the oligohymenophoran Tetrahymena thermophila, a model species in both cellular biology and evolutionary ecology. We explored polymorphisms at the junctions of programmed eliminated sequences, and reveal their utility to barcode very closely related cells. As for other species of the genus Tetrahymena, we confirm micronuclear centromeres as gene diversification centres in T. thermophila, but also reveal a two-speed evolution in these regions. In the rest of the genome, we highlight recent diversification of genes coding for extracellular proteins and cell adhesion. We discuss all these findings in relation to this ciliate's ecology and cellular characteristics.

Genome-wide identification and in-silico analysis of papain-family cysteine protease encoding genes in Tetrahymena 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.

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.

Global and local functions of the Fused kinase ortholog CdaH in intracellular patterning in Tetrahymena.

Ciliates assemble numerous microtubular structures into complex cortical patterns. During ciliate division, the pattern is duplicated by intracellular segmentation that produces a tandem of daughter cells. In Tetrahymena thermophila, the induction and positioning of the division boundary involves two mutually antagonistic factors: posterior CdaA (cyclin E) and anterior CdaI (Hippo kinase). Here, we characterized the related cdaH-1 allele, which confers a pleiotropic patterning phenotype including an absence of the division boundary and an anterior-posterior mispositioning of the new oral apparatus. CdaH is a Fused or Stk36 kinase ortholog that localizes to multiple sites that correlate with the effects of its loss, including the division boundary and the new oral apparatus. CdaH acts downstream of CdaA to induce the division boundary and drives asymmetric cytokinesis at the tip of the posterior daughter. CdaH both maintains the anterior-posterior position of the new oral apparatus and interacts with CdaI to pattern ciliary rows within the oral apparatus. Thus, CdaH acts at multiple scales, from induction and positioning of structures on the cell-wide polarity axis to local organelle-level patterning.

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.

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.

Efflux transport proteins of Tetrahymena thermophila play important roles in resistance to perfluorooctane sulfonate exposure.

The biotoxicity of perfluorooctane sulfonate (PFOS) has been a concern. However, the effects of PFOS on Tetrahymena thermophila, a unicellular model organism, remain unclear. This study aimed to investigate the toxicity and detoxification mechanism of PFOS in this protozoan. PFOS did not show prominent toxic effects on T. thermophila. Cell viability of T. thermophila can be concentration-dependently increased by PFOS. PFOS also increased the stability of cell membranes and the activity of lysosomes. However, PFOS inhibited efflux transporter activities. Most of the PFOS amount remained in the culture medium during the culture periods. Only a low amount of PFOS was absorbed by cells, where PFOS molecules were mainly combined with membrane proteins. The expressions of four membrane protein genes involved in transporting xenobiotics were analyzed by real time-PCR. The gene abcg25 was significantly up-regulated. The growth of abcg25 gene knockout protozoans under PFOS treatment was slightly inhibited. However, the amount of PFOS adsorbed by the knockout protozoans showed no significant difference from the Wild-type protozoans. We concluded that the ABCG25 protein might play a key role in preventing PFOS from entering the cell or being exported from the cells to protect T. thermophila against PFOS. However, ABCG25 was not the only membrane protein able to bind with PFOS.

Recombinant production of hormonally active human insulin from pre-proinsulin by Tetrahymena thermophila.

Alternative cell factories, such as the unicellular ciliate eukaryotic Tetrahymena thermophila, may be required for the production of protein therapeutics that are challenging to produce in conventional expression systems. T. thermophila (Tt) can secrete proteins with the post-translational modifications necessary for their function in humans. In this study, we tested if T. thermophila could process the human pre-proinsulin to produce hormonally active human insulin (hINS) with correct modifications. Flask and bioreactor culture of T. thermophila were used to produce the recombinant Tt-hINS either with or without an affinity tag from a codon-adapted pre-proinsulin sequence. Our results indicate that T. thermophila can produce a 6 kDa Tt-hINS monomer with the appropriate disulfide bonds after removal of the human insulin signal sequence or endogenous phospholipase A signal sequence, and the C-peptide of the human insulin. Additionally, Tt-hINS can form 12 kDa dimeric, 24 kDa tetrameric, and 36 kDa hexameric complexes. Tt-hINS-sfGFP fusion protein was localized to the vesicles within the cytoplasm and was secreted extracellularly. Assessing the affinity-purified Tt-hINS activity using the in vivo T. thermophila extracellular glucose drop assay, we observed that Tt-hINS induced a significant reduction (approximately 21 %) in extracellular glucose levels, indicative of its functional insulin activity. Our results demonstrate that T. thermophila is a promising candidate for the pharmaceutical and biotechnology industries as a host organism for the production of human protein drugs.

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.

A precise and efficient circular RNA synthesis system based on a ribozyme derived from Tetrahymena thermophila.

Classic strategies for circular RNA (circRNA) preparation always introduce large numbers of linear transcripts or extra nucleotides to the circularized product. In this study, we aimed to develop an efficient system for circRNA preparation based on a self-splicing ribozyme derived from an optimized Tetrahymena thermophila group Ⅰ intron. The target RNA sequence was inserted downstream of the ribozyme and a complementary antisense region was added upstream of the ribozyme to assist cyclization. Then, we compared the circularization efficiency of ribozyme or flanking intronic complementary sequence (ICS)-mediated methods through the DNMT1, CDR1as, FOXO3, and HIPK3 genes and found that the efficiency of our system was remarkably higher than that of flanking ICS-mediated method. Consequently, the circularized products mediated by ribozyme are not introduced with additional nucleotides. Meanwhile, the overexpressed circFOXO3 maintained its biological functions in regulating cell proliferation, migration, and apoptosis. Finally, a ribozyme-based circular mRNA expression system was demonstrated with a split green fluorescent protein (GFP) using an optimized Coxsackievirus B3 (CVB3) internal ribosome entry site (IRES) sequence, and this system achieved successful translation of circularized mRNA. Therefore, this novel, convenient, and rapid engineering RNA circularization system can be applied for the functional study and large-scale preparation of circular RNA in the future.

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.

Structures of Tetrahymena thermophila respiratory megacomplexes on the tubular mitochondrial cristae.

Tetrahymena thermophila, a classic ciliate model organism, has been shown to possess tubular mitochondrial cristae and highly divergent electron transport chain involving four transmembrane protein complexes (I-IV). Here we report cryo-EM structures of its ~8 MDa megacomplex IV2 + (I + III2 + II)2, as well as a ~ 10.6 MDa megacomplex (IV2 + I + III2 + II)2 at lower resolution. In megacomplex IV2 + (I + III2 + II)2, each CIV2 protomer associates one copy of supercomplex I + III2 and one copy of CII, forming a half ring-shaped architecture that adapts to the membrane curvature of mitochondrial cristae. Megacomplex (IV2 + I + III2 + II)2 defines the relative position between neighbouring half rings and maintains the proximity between CIV2 and CIII2 cytochrome c binding sites. Our findings expand the current understanding of divergence in eukaryotic electron transport chain organization and how it is related to mitochondrial morphology.

A split ribozyme that links detection of a native RNA to orthogonal protein outputs.

Individual RNA remains a challenging signal to synthetically transduce into different types of cellular information. Here, we describe Ribozyme-ENabled Detection of RNA (RENDR), a plug-and-play strategy that uses cellular transcripts to template the assembly of split ribozymes, triggering splicing reactions that generate orthogonal protein outputs. To identify split ribozymes that require templating for splicing, we use laboratory evolution to evaluate the activities of different split variants of the Tetrahymena thermophila ribozyme. The best design delivers a 93-fold dynamic range of splicing with RENDR controlling fluorescent protein production in response to an RNA input. We further resolve a thermodynamic model to guide RENDR design, show how input signals can be transduced into diverse outputs, demonstrate portability across different bacteria, and use RENDR to detect antibiotic-resistant bacteria. This work shows how transcriptional signals can be monitored in situ and converted into different types of biochemical information using RNA synthetic biology.

Sex, amitosis, and evolvability in the ciliate Tetrahymena thermophila.

Understanding the mechanisms that generate genetic variation, and thus contribute to the process of adaptation, is a major goal of evolutionary biology. Mutation and genetic exchange have been well studied as mechanisms to generate genetic variation. However, there are additional factors, such as genome architecture, that may also impact the amount of genetic variation in some populations, and the extent to which these variation generating mechanisms are themselves shaped by natural selection is still an open question. To test the effect of genome architecture on the generation of genetic variation, and hence evolvability, we studied Tetrahymena thermophila, a ciliate with an unusual genome structure and mechanism of nuclear division, called amitosis, whereby homologous chromosomes are randomly distributed to daughter cells. Amitosis leads to genetic variation among the asexual descendants of a newly produced sexual progeny because different progeny cells will contain different combinations of parental alleles. We hypothesize that amitosis thus increases the evolvability of newly produced sexual progeny relative to their unmated parents and species that undergo mitosis. To test this hypothesis, we used experimental evolution and simulations to compare the rate of adaptation in T. thermophila populations founded by a single sexual progeny to parental populations that had not had sex in many generations. The populations founded by a sexual progeny adapted more quickly than parental populations in both laboratory populations and simulated populations. This suggests that the additional genetic variation generated by amitosis of a heterozygote can increase the rate of adaptation following sex and may help explain the evolutionary success of the unusual genetic architecture of Tetrahymena and ciliates more generally.

Transcriptome analysis of the binucleate ciliate Tetrahymena thermophila with asynchronous nuclear cell cycles.

Tetrahymena thermophila harbors two functionally and physically distinct nuclei within a shared cytoplasm. During vegetative growth, the "cell cycles" of the diploid micronucleus and polyploid macronucleus are offset. Micronuclear S phase initiates just before cytokinesis and is completed in daughter cells before onset of macronuclear DNA replication. Mitotic micronuclear division occurs mid-cell cycle, while macronuclear amitosis is coupled to cell division. Here we report the first RNA-seq cell cycle analysis of a binucleated ciliated protozoan. RNA was isolated across 1.5 vegetative cell cycles, starting with a macronuclear G1 population synchronized by centrifugal elutriation. Using MetaCycle, 3244 of the 26,000+ predicted genes were shown to be cell cycle regulated. Proteins present in both nuclei exhibit a single mRNA peak that always precedes their macronuclear function. Nucleus-limited genes, including nucleoporins and importins, are expressed before their respective nucleus-specific role. Cyclin D and A/B gene family members exhibit different expression patterns that suggest nucleus-restricted roles. Periodically expressed genes cluster into seven cyclic patterns. Four clusters have known PANTHER gene ontology terms associated with G1/S and G2/M phase. We propose that these clusters encode known and novel factors that coordinate micro- and macronuclear-specific events such as mitosis, amitosis, DNA replication, and cell division.

Altered tRNA processing is linked to a distinct and unusual La protein in Tetrahymena thermophila.

Nascent pre-tRNAs are transcribed by RNA polymerase III and immediately bound by La proteins on the UUU-3'OH sequence, using a tandem arrangement of the La motif and an adjacent RNA recognition motif-1 (RRM1), resulting in protection from 3'-exonucleases and promotion of pre-tRNA folding. The Tetrahymena thermophila protein Mlp1 has been previously classified as a genuine La protein, despite the predicted absence of the RRM1. We find that Mlp1 functions as a La protein through binding of pre-tRNAs, and affects pre-tRNA processing in Tetrahymena thermophila and when expressed in fission yeast. However, unlike in other examined eukaryotes, depletion of Mlp1 results in 3'-trailer stabilization. The 3'-trailers in Tetrahymena thermophila are uniquely short relative to other examined eukaryotes, and 5'-leaders have evolved to disfavour pre-tRNA leader/trailer pairing. Our data indicate that this variant Mlp1 architecture is linked to an altered, novel mechanism of tRNA processing in Tetrahymena thermophila.

Comparative transcriptome combined with morphophysiological analyses revealed the molecular mechanism underlying Tetrahymena thermophila predation-induced antiphage defense in Aeromonas hydrophila.

Protozoan predation has been demonstrated to be a strong driving force for bacterial defence strategies in the environment. Our previous study demonstrated that Aeromonas hydrophila NJ-35, which evolved small-colony variants (SCVs), displayed various adaptive traits in response to Tetrahymena thermophila predation, such as enhanced phage resistance. However, the evolutionary mechanisms are largely unknown. In this study, we performed a genome- and transcriptome-wide analysis of the SCV1, representing one strain of the SCVs, for identification of the genes of mutation and altered expression underlying this phage resistance phenotype. Our study demonstrated that phage resistance caused by T. thermophila predation was due to the downregulation of a flagellar biosynthesis regulator, flhF, in SCV1. Interestingly, we confirmed that phage resistance in SCV1 was not straightforwardly attributable to the absence of flagella but to FlhF-mediated secretion of extracellular protein that hinders phage adsorption. This finding improves our understanding of the mechanisms by which A. hydrophila lowers the susceptibility to phage infection under predation pressure, and highlights an important contribution of bacterium-protozoan interactions in driving the adaptive evolution of pathogens in complex environments.

Establishing a High-Throughput Locomotion Tracking Method for Multiple Biological Assessments in Tetrahymena.

Protozoa are eukaryotic, unicellular microorganisms that have an important ecological role, are easy to handle, and grow rapidly, which makes them suitable for ecotoxicity assessment. Previous methods for locomotion tracking in protozoa are largely based on software with the drawback of high cost and/or low operation throughput. This study aimed to develop an automated pipeline to measure the locomotion activity of the ciliated protozoan Tetrahymena thermophila using a machine learning-based software, TRex, to conduct tracking. Behavioral endpoints, including the total distance, velocity, burst movement, angular velocity, meandering, and rotation movement, were derived from the coordinates of individual cells. To validate the utility, we measured the locomotor activity in either the knockout mutant of the dynein subunit DYH7 or under starvation. Significant reduction of locomotion and alteration of behavior was detected in either the dynein mutant or in the starvation condition. We also analyzed how Tetrahymena locomotion was affected by the exposure to copper sulfate and showed that our method indeed can be used to conduct a toxicity assessment in a high-throughput manner. Finally, we performed a principal component analysis and hierarchy clustering to demonstrate that our analysis could potentially differentiate altered behaviors affected by different factors. Taken together, this study offers a robust methodology for Tetrahymena locomotion tracking in a high-throughput manner for the first time.