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.

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.

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.

Metabolic alteration of Tetrahymena thermophila exposed to CdSe/ZnS quantum dots to respond to oxidative stress and lipid damage.

CdSe/ZnS Quantum dots (QDs) are possibly released to surface water due to their extensive application. Based on their high reactivity, even small amounts of toxicant QDs will disturb water microbes and pose a risk to aquatic ecology. Here, we evaluated CdSe/ZnS QDs toxicity to Tetrahymena thermophila (T. thermophila), a model organism of the aquatic environment, and performed metabolomics experiments. Before the omics experiment was conducted, QDs were found to induce inhibition of cell proliferation, and reactive oxygen species (ROS) production along with Propidium iodide labeled cell membrane damage indicated oxidative stress stimulation. In addition, mitochondrial ultrastructure alteration of T. thermophila was also confirmed by Transmission Electron Microscope results after 48 h of exposure to QDs. Further results of metabolomics detection showed that 0.1 µg/mL QDs could disturb cell physiological and metabolic metabolism characterized by 18 significant metabolite changes, of which twelve metabolites improved and three decreased significantly compared to the control. Kyoto Encyclopedia of Genes and Genomes analysis showed that these metabolites were involved in the ATP-binding cassette transporter and purine metabolism pathways, both of which respond to ROS-induced cell membrane damage. In addition, purine metabolism weakness might also reflect mitochondrial dysfunction associated with energy metabolism and transport abnormalities. This research provides deep insight into the potential risks of quantum dots in aquatic ecosystems.

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.

Structural and functional insights into CST tethering in Tetrahymena thermophila telomerase.

Telomerase is crucial for telomere maintenance and genome integrity. The most salient feature of Tetrahymena telomerase is that its CST subcomplex (p75-p45-p19) is tethered to the telomerase catalytic core by interacting with the hub p50. Although the cryoelectron microscopy (cryo-EM) structures of Tetrahymena telomerase have recently been reported, the mechanisms of how and why p50 bridges the CST subcomplex to the telomerase catalytic core remain unclear. Here, we present the nuclear magnetic resonance (NMR) structure of the p75OB1-p50PBM complex. Loss of the interaction between p75 and p50 detaches the CST subcomplex from the telomerase catalytic core in Tetrahymena. The tethering of the CST subcomplex to telomerase is required for telomere homeostasis. However, the detached CST subcomplex is still capable of facilitating the telomeric complementary-strand (C-strand) fill in similar to the non-tethered CST complexes in other organisms. These results expand our understanding of telomere synthesis in Tetrahymena.

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.

Optimisation and characterization of α-D-glucan produced by Bacillus velezensis RSDM1 and evaluation of its protective effect on oxidative stress in Tetrahymena thermophila induced by H2O2.

A novel exopolysaccharide was isolated from Bacillus velezensis RSDM1, purified and characterized. The optimization was carried out by Box-Behnken design and the highest yield of EPS-RSDM1 was 1969.53 mg/L. The EPS was composed by glucose with a molecular weight of 1.78 × 104 Da and connected by α-1,6 and α-1,3. The EPS was thermally stable and showed pseudoplastic rheology. The SEM have demonstrated a porous structure. Furthermore, EPS-RSDM1 showed a protective effect against the oxidative stress induced by H2O2 in Tetrahymena thermophila. The exposure of the cells to H2O2 reduced significantly the cell survival and CAT, SOD, GR, SDH and GAPDH activities, and increased the MDA production. However, pretreatment of the cells with EPS-RSDM1 prior to H2O2 exposure elevated the cell survival and enzyme activities, and decreased the level of MDA activity. Our study suggest that EPS-RSDM1 could be useful for preventing cellular oxidation in pharmaceutical and food industries.

Construction of CdS-Tetrahymena thermophila hybrid system by efficient cadmium adsorption for dye removal under light irradiation.

The water pollution caused by heavy metals and dyes emitted by industries has become a worldwide problem. These pollutants are difficult to be biodegraded. Even at low concentrations, they are toxic and at last threaten human health. Herein, while using Tetrahymena thermophila, a single-celled ciliate protozoa, to enrich and remove the heavy metal Cd2+ from water, CdS nanoparticle-Tetrahymena thermophila hybrid system (CdS-T. thermophila) for dye pollution remediation under light irradiation was developed. The conditions of Cd2+ enrichment and removal by T. thermophila, construction of efficient CdS-T. thermophila, and decolorization of Congo red using CdS-T. thermophila were investigated. In the presence of cysteine ethyl ester, the removal rate of Cd2+ by T. thermophila was 94% at low Cd2+ concentration of 1 mg L-1. The adsorption capacity of T. thermophila to Cd2+ reached 43 mg g-1 at Cd2+ concentration of 80 mg L-1. Using 0.1 g L-1 constructed CdS-T. thermophila, the decolorization rate of 50 mg L-1 Congo red solution reached 95% in 60 min under light irradiation. This study provides a new insight to effective removing Cd2+ from water by T. thermophila to construct the CdS-T. thermophila and using it to remediate dye pollution in the environment.

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.

A novel Tetrahymena thermophila sterol C-22 desaturase belongs to the fatty acid hydroxylase/desaturase superfamily.

Sterols in eukaryotic cells play important roles in modulating membrane fluidity and in cell signaling and trafficking. During evolution, a combination of gene losses and acquisitions gave rise to an extraordinary diversity of sterols in different organisms. The sterol C-22 desaturase identified in plants and fungi as a cytochrome P-450 monooxygenase evolved from the first eukaryotic cytochrome P450 and was lost in many lineages. Although the ciliate Tetrahymena thermophila desaturates sterols at the C-22 position, no cytochrome P-450 orthologs are present in the genome. Here, we aim to identify the genes responsible for the desaturation as well as their probable origin. We used gene knockout and yeast heterologous expression approaches to identify two putative genes, retrieved from a previous transcriptomic analysis, as sterol C-22 desaturases. Furthermore, we demonstrate using bioinformatics and evolutionary analyses that both genes encode a novel type of sterol C-22 desaturase that belongs to the large fatty acid hydroxylase/desaturase superfamily and the genes originated by genetic duplication prior to functional diversification. These results stress the widespread existence of nonhomologous isofunctional enzymes among different lineages of the tree of life as well as the suitability for the use of T. thermophila as a valuable model to investigate the evolutionary process of large enzyme families.

Protection of Grouper Against Cryptocaryon irritans by Immunization With Tetrahymena thermophila and Protective Cross-Reactive Antigen Identification.

Vaccination is an effective method to prevent Cryptocaryon irritans infection. Although some vaccines have been developed, large-scale production of these vaccines is costly. Development of a heterogenous vaccine generated by low-cost antigens is an alternative method. In the present study, grouper immunized with Tetrahymena thermophila, a free-living ciliate that easily grows in inexpensive culture media at high density, showed protective immunity against C. irritans infection. Higher immobilization against C. irritans theronts was detected in T. thermophila-immunized grouper serum, which suggested the existence of a cross-reactive antibody in the serum. By immunoprecipitation and mass spectrometry analyses, tubulin was identified as a potential cross-reactive antigen between C. irritans and T. thermophila. Recombinant T. thermophila tubulin protein (rTt-tubulin) and its antibody were prepared, and immunofluorescence showed that both C. irritans and T. thermophila cilia were stained by the anti-rTt-tubulin antibody. Grouper immunized with rTt-tubulin showed a reduced infective rate after the C. irritans challenge. An enhanced level of C. irritans-binding immunoglobulin M (IgM) antibody was detected in serum from rTt-tubulin-immunized grouper. Moreover, specific antibodies were also found in the mucus and tissue culture medium from rTt-tubulin-immunized grouper. Overall, these findings suggested that vaccination with T. thermophila elicits cross-reactive protective immunity in grouper against C. irritans, and T. thermophila may be a potential heterologous antigen for vaccine development.

Autophagy and lipid droplets are a defense mechanism against toxic copper oxide nanotubes in the eukaryotic microbial model Tetrahymena thermophila.

The widespread use of inorganic nanomaterials of anthropogenic origin has significantly increased in the last decade, being now considered as emerging pollutants. This makes it necessary to carry out studies to further understand their toxicity and interactions with cells. In the present work we analyzed the toxicity of CuO nanotubes (CuONT) in the ciliate Tetrahymena thermophila, a eukaryotic unicellular model with animal biology. CuONT exposure rapidly induced ROS generation in the cell leading to oxidative stress and upregulation of genes encoding antioxidant enzymes (catalase, superoxide dismutase, glutathione peroxidase), metal-chelating metallothioneins and cytochrome P450 monooxygenases. Comet assays and overexpression of genes involved in DNA repair confirmed oxidative DNA damage in CuONT-treated cells. Remarkably, both electron and fluorescent microscopy revealed numerous lipid droplets and autophagosomes containing CuONT aggregates and damaged mitochondria, indicating activation of macroautophagy, which was further confirmed by a dramatic upregulation of ATG (AuTophaGy related) genes. Treatment with autophagy inhibitors significantly increased CuONT toxicity, evidencing the protective role of autophagy towards CuONT-induced damage. Moreover, increased formation of lipid droplets appears as an additional mechanism of CuONT detoxification. Based on these results, we present a hypothetical scenario summarizing how T. thermophila responds to CuONT toxicity. This study corroborates the use of this ciliate as an excellent eukaryotic microbial model for analyzing the cellular response to stress caused by toxic metal nanoparticles.

Intracellular connections between basal bodies promote the coordinated behavior of motile cilia.

Hydrodynamic flow produced by multiciliated cells is critical for fluid circulation and cell motility. Hundreds of cilia beat with metachronal synchrony for fluid flow. Cilia-driven fluid flow produces extracellular hydrodynamic forces that cause neighboring cilia to beat in a synchronized manner. However, hydrodynamic coupling between neighboring cilia is not the sole mechanism that drives cilia synchrony. Cilia are nucleated by basal bodies (BBs) that link to each other and to the cell's cortex via BB-associated appendages. The intracellular BB and cortical network is hypothesized to synchronize ciliary beating by transmitting cilia coordination cues. The extent of intracellular ciliary connections and the nature of these stimuli remain unclear. Moreover, how BB connections influence the dynamics of individual cilia has not been established. We show by focused ion beam scanning electron microscopy imaging that cilia are coupled both longitudinally and laterally in the ciliate Tetrahymena thermophila by the underlying BB and cortical cytoskeletal network. To visualize the behavior of individual cilia in live, immobilized Tetrahymena cells, we developed Delivered Iron Particle Ubiety Live Light (DIPULL) microscopy. Quantitative and computer analyses of ciliary dynamics reveal that BB connections control ciliary waveform and coordinate ciliary beating. Loss of BB connections reduces cilia-dependent fluid flow forces.

Structural basis for MTA1c-mediated DNA N6-adenine methylation.

DNA N6-adenine methylation (6 mA) has recently been found to play a crucial role in epigenetic regulation in eukaryotes. MTA1c, a newly discovered 6 mA methyltransferase complex in ciliates, is composed of MTA1, MTA9, p1 and p2 subunits and specifically methylates ApT dinucleotides, yet its mechanism of action remains unknown. Here, we report the structures of Tetrahymena thermophila MTA1 (TthMTA1), Paramecium tetraurelia MTA9 (PteMTA9)-TthMTA1 binary complex, as well as the structures of TthMTA1-p1-p2 and TthMTA1-p2 complexes in apo, S-adenosyl methionine-bound and S-adenosyl homocysteine-bound states. We show that MTA1 is the catalytically active subunit, p1 and p2 are involved in the formation of substrate DNA-binding channel, and MTA9 plays a structural role in the stabilization of substrate binding. We identify that MTA1 is a cofactor-dependent catalytically active subunit, which exhibits stable SAM-binding activity only after assembly with p2. Our structures and corresponding functional studies provide a more detailed mechanistic understanding of 6 mA methylation.

A novel membrane complex is required for docking and regulated exocytosis of lysosome-related organelles in Tetrahymena thermophila.

In the ciliate Tetrahymena thermophila, lysosome-related organelles called mucocysts accumulate at the cell periphery where they secrete their contents in response to extracellular events, a phenomenon called regulated exocytosis. The molecular bases underlying regulated exocytosis have been extensively described in animals but it is not clear whether similar mechanisms exist in ciliates or their sister lineage, the Apicomplexan parasites, which together belong to the ecologically and medically important superphylum Alveolata. Beginning with a T. thermophila mutant in mucocyst exocytosis, we used a forward genetic approach to uncover MDL1 (Mucocyst Discharge with a LamG domain), a novel gene that is essential for regulated exocytosis of mucocysts. Mdl1p is a 40 kDa membrane glycoprotein that localizes to mucocysts, and specifically to a tip domain that contacts the plasma membrane when the mucocyst is docked. This sub-localization of Mdl1p, which occurs prior to docking, underscores a functional asymmetry in mucocysts that is strikingly similar to that of highly polarized secretory organelles in other Alveolates. A mis-sense mutation in the LamG domain results in mucocysts that dock but only undergo inefficient exocytosis. In contrast, complete knockout of MDL1 largely prevents mucocyst docking itself. Mdl1p is physically associated with 9 other proteins, all of them novel and largely restricted to Alveolates, and sedimentation analysis supports the idea that they form a large complex. Analysis of three other members of this putative complex, called MDD (for Mucocyst Docking and Discharge), shows that they also localize to mucocysts. Negative staining of purified MDD complexes revealed distinct particles with a central channel. Our results uncover a novel macromolecular complex whose subunits are conserved within alveolates but not in other lineages, that is essential for regulated exocytosis in T. thermophila.

Mitochondrial complex complexification.

Variation in complex composition provides clues about the function of individual subunits.

Molecular evolutionary analysis of the SM and SNARE vesicle fusion machinery in ciliates shows concurrent expansions in late secretory machinery.

Protists in the phylum Ciliophora possess a complex membrane-trafficking system, including osmoregulatory Contractile Vacuoles and specialized secretory organelles. Molecular cell biological investigations in Tetrahymena thermophila have identified components of the protein machinery associated with the secretory organelles, mucocysts. The Qa-SNARE Syn7lp plays a role in mucocyst biogenesis as do subunits of the CORVET tethering complex (specifically Vps8). Indeed, Tetrahymena thermophila possesses expanded gene complements of several CORVET components, including Vps33 which is also a Sec1/Munc18 (SM) protein that binds Qa-SNAREs. Moreover, the Qa-SNAREs in Paramecium tetraurelia have been localized to various endomembrane organelles. Here, we use comparative genomics and phylogenetics to determine the evolutionary history of the SM and Qa-SNARE proteins across the Ciliophora. We identify that the last ciliate common ancestor possessed the four SM proteins and six Qa-SNAREs common to eukaryotes, including the uncommonly retained Syntaxin 17. We furthermore identify independent expansion of these protein families in several ciliate classes, including concurrent expansions of the SM protein-Qa SNARE partners Sec1:SynPM in the oligohymenophorean ciliates lineage, consistent with novel Contractile Vacuole specific innovations. Overall, these data are consistent with SM proteins and Qa-SNAREs being a common set of components for endomembrane modulation in the ciliates.