Characterization of Crystals in Ciliate Paramecium bursaria Harboring Endosymbiotic Chlorella variabilis.

Protists, including ciliates retain crystals in their cytoplasm. However, their functions and properties remain unclear. To comparatively analyze the crystals of Paramecium bursaria, a ciliate, associated with and without the endosymbiotic Chlorella variabilis, we investigated the isolated crystals using a light microscope and analyzed their length and solubility. A negligible number of crystals was found in P. bursaria cells harboring symbiotic algae. The average crystal length in alga-free and algae-reduced cells was about 6.8 µm and 14.4 µm, respectively. The crystals of alga-free cells were spherical, whereas those of algae-reduced cells were angular in shape. The crystals of alga-free cells immediately dissolved in acids and bases, but not in water or organic solvents, and were stable at - 20 °C for more than 3 weeks. This study, for the first time, reveals that the characteristics of crystals present in the cytoplasm of P. bursaria vary greatly depending on the amount of symbiotic algae.

Defense against Paramecium predation via long filament morphology favors the survival of Raphidiopsis raciborskii populations.

Raphidiopsis blooms are notorious for cyanotoxin formation and strong invasiveness, threatening the stability of aquatic ecosystems and human health. The protozoa Paramecium can potentially serve as an organism for controlling Raphidiopsis blooms owing to its grazing effect. However, the grazing ability of Paramecium is largely determined by the size of the prey, and the population of Raphidiopsis consists of filaments of varying lengths and sizes. The selective grazing behavior of Paramecium toward short-length or small-sized filaments in the Raphidiopsis population, as opposed to long filaments, remains unclear. Therefore, in this study, we co-cultured the predator Paramecium sp. with different initial abundances and the prey Raphidiopsis raciborskii to explore this knowledge gap. Our results suggested that: (1) the population of R. raciborskii declined under the selective grazing effect of Paramecium sp. on short filaments, whereas R. raciborskii with long filaments survived; (2) the growth of Paramecium sp. feeding on the same abundance of R. raciborskii was reduced at higher initial abundances, whereas its carrying capacity exhibited an opposite trend; (3) under ingestion by Paramecium sp., the morphology of R. raciborskii developed in the direction of becoming larger, and higher initial abundances of Paramecium sp. intensified this process; (4) increasing initial abundance of Paramecium sp. aggravated the decline of R. raciborskii photosynthetic activity. Therefore, the grazing effect of Paramecium sp. on R. raciborskii mainly affects filaments of short length or small size. Collectively, these results clarify the inter-species interaction between the protozoa Paramecium and filamentous cyanobacteria Raphidiopsis, including population dynamics and morphological and physiological changes in the predator and prey. Such insights into the interactions between Paramecium and R. raciborskii may have implications for the biological control of blooms caused by filamentous cyanobacteria.

Anoctamin-like protein 1 regulates repolarization in Paramecium behavioral responses.

Paramecium exhibits responsive behavior to environmental changes, moving either closer to or further away from stimuli. Electrophysiological experiments have revealed that these behavioral responses are controlled by membrane potentials. Anoctamin, a Ca2+-activated Cl- channel, is involved in the regulation of membrane potential in mammals. However, it remains uncertain whether Cl- channels like anoctamin regulate Paramecium behavior. Herein, replacement of external Cl- ions with acetate ion and application of Cl- channel blocker niflumic acid (NFA, 0.1 µM) increased spontaneous avoiding reactions (sARs). Hence, we hypothesized that anoctamin is involved in the stabilization of membrane potential fluctuation. Paramecium cells in which the anoctamin-like protein 1 gene was knocked down displayed frequent sARs in the culture medium without external stimulation. Treatment of anoctamin-like protein 1-knockdown cells with the Ca2+ chelator BAPTA or Ca-channel blocker nicardipine reversed the increase in sARs. Electrophysiological experiments revealed extension of membrane depolarization when positive currents were applied to anoctamin-like protein 1-knockdown cells. We concluded that anoctamin-like protein 1 works as a Cl-channel and stabilizes the membrane potential oscillation, reducing sARs.

Mutualism on the edge: Understanding the Paramecium-Chlorella symbiosis.

Exploring the mechanisms that underpin symbiosis requires an understanding of how these complex interactions are maintained in diverse model systems. The ciliate protist, Paramecium bursaria, offers a valuable insight into how emergent endosymbiotic interactions have evolved.

Insights into the differential proteome landscape of a newly isolated Paramecium multimicronucleatum in response to cadmium stress.

Employing microbial systems for the bioremediation of contaminated waters represent a potential option, however, limited understanding of the underlying mechanisms hampers the implication of microbial-mediated bioremediation. The omics tools offer a promising approach to explore the molecular basis of the bioremediation process. Here, a mass spectrometry-based quantitative proteome profiling approach was conducted to explore the differential protein levels in cadmium-treated Paramecium multimicronucleatum. The Proteome Discoverer software was used to identify and quantify differentially abundant proteins. The proteome profiling generated 7,416 peptide spectral matches, yielding 2824 total peptides, corresponding to 989 proteins. The analysis revealed that 29 proteins exhibited significant (p ≤ 0.05) differential levels, including a higher abundance of 6 proteins and reduced levels of 23 proteins in Cd2+ treated samples. These differentially abundant proteins were associated with stress response, energy metabolism, protein degradation, cell growth, and hormone processing. Briefly, a comprehensive proteome profile in response to cadmium stress of a newly isolated Paramecium has been established that will be useful in future studies identifying critical proteins involved in the bioremediation of metals in ciliates. SIGNIFICANCE: Ciliates are considered a good biological indicator of chemical pollution and relatively sensitive to heavy metal contamination. A prominent ciliate, Paramecium is a promising candidate for the bioremediation of polluted water. The proteins related to metal resistance in Paramecium species are still largely unknown and need further exploration. In order to identify and reveal the proteins related to metal resistance in Paramecia, we have reported differential protein abundance in Paramecium multimicronucleatum in response to cadmium stress. The proteins found in our study play essential roles during stress response, hormone processing, protein degradation, energy metabolism, and cell growth. It seems likely that Paramecia are not a simple sponge for metals but they could also transform them into less toxic derivatives or by detoxification by protein binding. This data will be helpful in future studies to identify critical proteins along with their detailed mechanisms involved in the bioremediation and detoxification of metal ions in Paramecium species.

Uncoupling programmed DNA cleavage and repair scrambles the Paramecium somatic genome.

In the ciliate Paramecium, precise excision of numerous internal eliminated sequences (IESs) from the somatic genome is essential at each sexual cycle. DNA double-strands breaks (DSBs) introduced by the PiggyMac endonuclease are repaired in a highly concerted manner by the non-homologous end joining (NHEJ) pathway, illustrated by complete inhibition of DNA cleavage when Ku70/80 proteins are missing. We show that expression of a DNA-binding-deficient Ku70 mutant (Ku70-6E) permits DNA cleavage but leads to the accumulation of unrepaired DSBs. We uncoupled DNA cleavage and repair by co-expressing wild-type and mutant Ku70. High-throughput sequencing of the developing macronucleus genome in these conditions identifies the presence of extremities healed by de novo telomere addition and numerous translocations between IES-flanking sequences. Coupling the two steps of IES excision ensures that both extremities are held together throughout the process, suggesting that DSB repair proteins are essential for assembly of a synaptic precleavage complex.

Novel evolutionary insights on the interactions of the Holosporales (Alphaproteobacteria) with eukaryotic hosts from comparative genomics.

Holosporales are an alphaproteobacterial order engaging in obligate and complex associations with eukaryotes, in particular protists. The functional and evolutionary features of those interactions are still largely undisclosed. Here, we sequenced the genomes of two members of the species Bealeia paramacronuclearis (Holosporales, Holosporaceae) intracellularly associated with the ciliate protist Paramecium, which resulted in high correspondence. Consistent with the short-branched early-divergent phylogenetic position, Bealeia presents a larger functional repertoire than other Holosporaceae, comparable to those of other Holosporales families, particularly for energy metabolism and motility. Our analyses indicate that different Holosporales likely experienced at least partly autonomous genome reduction and adaptation to host interactions, for example regarding dependence on host biotin driven by multiple independent horizontal acquisitions of transporters. Among Alphaproteobacteria, this is reminiscent of the convergently evolved Rickettsiales, which however appear more diverse, possibly due to a probably more ancient origin. We identified in Bealeia and other Holosporales the plasmid-encoded putative genetic determinants of R-bodies, which may be involved in a killer trait towards symbiont-free hosts. While it is not clear whether these genes are ancestral or recently horizontally acquired, an intriguing and peculiar role of R-bodies is suggested in the evolution of the interactions of multiple Holosporales with their hosts.

Comparative Analyses of the Symbiotic Associations of the Host Paramecium bursaria with Free-Living and Native Symbiotic Species of Chlorella.

Each symbiotic Chlorella variabilis associated with the ciliate Paramecium bursaria is enclosed in a symbiosome called the perialgal vacuole. Various potential symbionts, such as bacteria, yeasts, other algae, and free-living Chlorella spp., can infect P. bursaria. However, the detailed infection process of each of them in algae-free P. bursaria is unknown. Here, we aimed to elucidate the difference of the infection process between the free-living C. sorokiniana strain NIES-2169 and native symbiotic C. variabilis strain 1N. We investigated the fate of ingested algae using algae-free P. bursaria exposed separately to three types of algal inocula: NIES-2169 only, 1N only, or a mixture of NIES-2169 and 1N. We found that (1) only one algal species, preferably the native one, was retained in host cells, indicating a type of host compatibility and (2) the algal localization style beneath the host cell cortex varied between different Chlorella spp. showing various levels of host compatibilities, which was prospectively attributable to the difference in the formation of the perialgal vacuole membrane.

Paramecium (Oligohymenophorea, Ciliophora) diversity in Thailand sheds light on the genus biogeography and reveals new phylogenetic lineages.

Paramecium (Ciliophora, Oligohymenophorea) is a good model to study ciliate biogeography. Extensive sampling mainly in northern hemisphere has led to 16 valid morphological species description thus far. However, a majority of hard-to-reach regions, including South East Asia, are underinvestigated. Our study combined traditional morphological and molecular approaches to reveal the biodiversity of Paramecium in Thailand from more than 110 samples collected in 10 provinces. Representatives of seven morphological species were identified from our collection, including the rare species, such as P. gigas and P. jenningsi. Additionally, we detected five different sibling species of the P. aurelia complex, described a new cryptic species P. hiwatashii n. sp. phylogenetically related to P. caudatum, and discovered a potentially new genetic species of the P. bursaria species complex. We also documented a variety of bacterial cytoplasmic symbionts from at least nine monoclonal cultures of Paramecium.

Structural maintenance of chromosomes (SMC) proteins are required for DNA elimination in Paramecium.

Chromosome (SMC) proteins are a large family of ATPases that play important roles in the organization and dynamics of chromatin. They are central regulators of chromosome dynamics and the core component of condensin. DNA elimination during zygotic somatic genome development is a characteristic feature of ciliated protozoa such as Paramecium This process occurs after meiosis, mitosis, karyogamy, and another mitosis, which result in the formation of a new germline and somatic nuclei. The series of nuclear divisions implies an important role of SMC proteins in Paramecium sexual development. The relationship between DNA elimination and SMC has not yet been described. Here, we applied RNA interference, genome sequencing, mRNA sequencing, immunofluorescence, and mass spectrometry to investigate the roles of SMC components in DNA elimination. Our results show that SMC4-2 is required for genome rearrangement, whereas SMC4-1 is not. Functional diversification of SMC4 in Paramecium led to a formation of two paralogues where SMC4-2 acquired a novel, development-specific function and differs from SMC4-1. Moreover, our study suggests a competitive relationship between these two proteins.

Paramecium Genetics, Genomics, and Evolution.

The ciliate genus Paramecium served as one of the first model systems in microbial eukaryotic genetics, contributing much to the early understanding of phenomena as diverse as genome rearrangement, cryptic speciation, cytoplasmic inheritance, and endosymbiosis, as well as more recently to the evolution of mating types, introns, and roles of small RNAs in DNA processing. Substantial progress has recently been made in the area of comparative and population genomics. Paramecium species combine some of the lowest known mutation rates with some of the largest known effective populations, along with likely very high recombination rates, thereby harboring a population-genetic environment that promotes an exceptionally efficient capacity for selection. As a consequence, the genomes are extraordinarily streamlined, with very small intergenic regions combined with small numbers of tiny introns. The subject of the bulk of Paramecium research, the ancient Paramecium aurelia species complex, is descended from two whole-genome duplication events that retain high degrees of synteny, thereby providing an exceptional platform for studying the fates of duplicate genes. Despite having a common ancestor dating to several hundred million years ago, the known descendant species are morphologically indistinguishable, raising significant questions about the common view that gene duplications lead to the origins of evolutionary novelties.

Microbial impact to environmental toxicants Ni(II) and Co(II): Joint toxicity and cellular response in Paramecium.

Cobalt (Co) and Nickel (Ni) are increasingly found in our environment. We analysed their combined toxicity and uptake mechanisms in the early food chain by studying bacteria and the bacterivorous ciliate Paramecium as a primary consumer. We exposed both species to these metals to measure the toxicity, uptake and transfer of metals from bacteria to Paramecium. We found that Ni is more toxic than Co, and that toxicity increases for both metals when (i) food bacteria are absent and (ii) both metals are applied in combination. The cellular content in bacteria after exposure shows a concentration dependent bias for either Ni or Co. Comparing single treatment and joint exposure, bacteria show increased levels of both metals when these are both exposed. To imitate the basic level of the food chain, we fed these bacteria to paramecia. The cellular content shows a similar ratio of Nickel and Cobalt as in food bacteria. This is different to the direct application of both metals to paramecia, where Cobalt is enriched over Nickel. This indicates that bacteria can selectively pre-accumulate metals for introduction into the food chain. We also analysed the transcriptomic response of Paramecium to sublethal doses of Nickel and Cobalt to gain insight into their toxicity mechanisms. Gene ontology (GO) analysis indicates common deregulated pathways, such as ammonium transmembrane transport and ubiquitine-associated protein degradation. Many redox-related genes also show deregulation of gene expression, indicating cellular adaptation to increased RONS stress. This suggests that both metals may also target the same cellular pathways and this is consistent with the increased toxicity of both metals when used together. Our data reveal complex ecotoxicological pathways for these metals and highlights the different parameters for their fate in the ecosystem, in the food chain and their ecotoxicological risk after environmental contamination.

Role of host ciliate Paramecium bursaria mitochondria and trichocysts for symbiotic Chlorella variabilis attachment beneath the host cell cortex.

Symbiotic Chlorella variabilis is encased in the perialgal vacuole (PV) membrane of ciliate Paramecium bursaria. The PV membrane is stably anchored below the host cell cortex by adhesion to host mitochondria. Host trichocysts, which are defensive organelles against predators, are present in the mitochondria and PV membrane vicinity. The mechanism by which PV attaches beneath the host cell cortex remains unknown. When P. bursaria is centrifuged at high speed, the symbiotic algae are displaced from the host cell cortex and concentrate at the posterior end. When centrifugation is stopped, the dislocated algae reattach beneath the host cell cortex with fast cytoplasmic streaming. The densities of mitochondria and trichocysts before and after centrifugation were compared using indirect immunofluorescence microscopy with monoclonal antibodies. Almost all trichocysts were shed by high-speed centrifugation, but dislocated algae could reattach even in the absence of trichocysts. In contrast, host mitochondria were unaffected in localization and number, and the dislocated algae also reattached. These findings suggest trichocysts are unnecessary for algal relocalization and that mitochondria are colocalized with the algae. However, many mitochondria were also present in the cell's anterior region without symbiotic algae. Therefore, not all areas with mitochondria contained algae, but there was an algal localization bias within the host cell.

Viral Chemotaxis of Paramecium Bursaria Altered by Algal Endosymbionts.

Chemotaxis is widespread across many taxa and often aids resource acquisition or predator avoidance. Species interactions can modify the degree of movement facilitated by chemotaxis. In this study, we investigated the influence of symbionts on Paramecium bursaria's chemotactic behavior toward chloroviruses. To achieve this, we performed choice experiments using chlorovirus and control candidate attractors (virus stabilization buffer and pond water). We quantified the movement of Paramecia grown with or without algal and viral symbionts toward each attractor. All Paramecia showed some chemotaxis toward viruses, but cells without algae and viruses showed the most movement toward viruses. Thus, the endosymbiotic algae (zoochlorellae) appeared to alter the movement of Paramecia toward chloroviruses, but it was not clear that ectosymbiotic viruses (chlorovirus) also had this effect. The change in behavior was consistent with a change in swimming speed, but a change in attraction remains possible. The potential costs and benefits of chemotactic movement toward chloroviruses for either the Paramecia hosts or its symbionts remain unclear.

Viral DNA Accumulation Regulates Replication Efficiency of Chlorovirus OSy-NE5 in Two Closely Related Chlorella variabilis Strains.

Many chloroviruses replicate in Chlorella variabilis algal strains that are ex-endosymbionts isolated from the protozoan Paramecium bursaria, including the NC64A and Syngen 2-3 strains. We noticed that indigenous water samples produced a higher number of plaque-forming viruses on C. variabilis Syngen 2-3 lawns than on C. variabilis NC64A lawns. These observed differences led to the discovery of viruses that replicate exclusively in Syngen 2-3 cells, named Only Syngen (OSy) viruses. Here, we demonstrate that OSy viruses initiate infection in the restricted host NC64A by synthesizing some early virus gene products and that approximately 20% of the cells produce a small number of empty virus capsids. However, the infected cells did not produce infectious viruses because the cells were unable to replicate the viral genome. This is interesting because all previous attempts to isolate host cells resistant to chlorovirus infection were due to changes in the host receptor for the virus.

Holospora-like bacteria "Candidatus Gortzia yakutica" and Preeria caryophila: Ultrastructure, promiscuity, and biogeography of the symbionts.

Two already known representatives of Holospora-like bacteria, "Candidatus Gortzia yakutica" from Paramecium putrinum and Preeria caryophila, originally retrieved from the Paramecium aurelia complex, were found in new hosts: Paramecium nephridiatum and Paramecium polycaryum, respectively. In the present study, these bacteria were investigated using morphological and molecular methods. For "Ca. G. yakutica", the first details of the electron microscopic structure in the main and new hosts were provided. Regarding Pr. caryophila, the ultrastructural description of this species was implemented by several features previously unknown, such as the so called "membrane cluster" dividing periplasm from cytoplasm and fine composition of infectious forms before and during its releasing from the infected macronucleus. The new combinations of these Holospora-like bacteria with ciliate hosts were discussed from biogeographical and ecological points of view. Host specificity of symbionts as a general paradigm was critically reviewed as well.

Dynamics of Gene Loss following Ancient Whole-Genome Duplication in the Cryptic Paramecium Complex.

Whole-genome duplications (WGDs) have shaped the gene repertoire of many eukaryotic lineages. The redundancy created by WGDs typically results in a phase of massive gene loss. However, some WGD-derived paralogs are maintained over long evolutionary periods, and the relative contributions of different selective pressures to their maintenance are still debated. Previous studies have revealed a history of three successive WGDs in the lineage of the ciliate Paramecium tetraurelia and two of its sister species from the Paramecium aurelia complex. Here, we report the genome sequence and analysis of 10 additional P. aurelia species and 1 additional out group, revealing aspects of post-WGD evolution in 13 species sharing a common ancestral WGD. Contrary to the morphological radiation of vertebrates that putatively followed two WGD events, members of the cryptic P. aurelia complex have remained morphologically indistinguishable after hundreds of millions of years. Biases in gene retention compatible with dosage constraints appear to play a major role opposing post-WGD gene loss across all 13 species. In addition, post-WGD gene loss has been slower in Paramecium than in other species having experienced genome duplication, suggesting that the selective pressures against post-WGD gene loss are especially strong in Paramecium. A near complete lack of recent single-gene duplications in Paramecium provides additional evidence for strong selective pressures against gene dosage changes. This exceptional data set of 13 species sharing an ancestral WGD and 2 closely related out group species will be a useful resource for future studies on Paramecium as a major model organism in the evolutionary cell biology.

Paramecium: RNA sequence-structure phylogenetics.

Organisms classified as members of the genus Paramecium belong to the best-known group of single-celled eukaryotes. Nevertheless, the phylogeny within the genus Paramecium has been discussed and revisited in recent decades and remains partly unresolved. By applying an RNA sequence-structure approach, we attempt to increase accuracy and robustness of phylogenetic trees. For each individual 18S and internal transcribed spacer 2 (ITS2) sequence, a putative secondary structure was predicted through homology modelling. While searching for a structural template, we found, in contrast to the available literature, that the ITS2 molecule consists of three helices in members of the genus Paramecium and four helices in members of the genus Tetrahymena. Two sequencestructure neighbor-joining overall trees were reconstructed with (1) more than 400 taxa (ITS2) and (2) more than 200 taxa (18S). For smaller subsets, neighbor-joining, maximum-parsimony, and maximum-likelihood analyses were executed using sequence-structure information simultaneously. Based on a combined data set (ITS2+18S rDNA) a well-supported tree was reconstructed with bootstrap values over 50 in at least one of the applied analyses. Our results are in general agreement with those published in the available literature based on multi-gene analyses. Our study supports the simultaneous use of sequence-structure data to reconstruct accurate and robust phylogenetic trees.

Comparative transcriptome and antioxidant biomarker response reveal molecular mechanisms to cope with zinc ion exposure in the unicellular eukaryote Paramecium.

The development of industry has resulted in excessive environmental zinc exposure which has caused various health problems in a wide range of organisms including humans. The mechanisms by which aquatic microorganisms respond to environmental zinc stress are still poorly understood. Paramecium, a well-known ciliated protozoan and a popular cell model in heavy metal stress response studies, was chosen as the test unicellular eukaryotic organism in the present research. In this work, Paramecium cf. multimicronucleatum cells were exposed in different levels of zinc ion (0.1 and 1.0 mg/L) for different periods of exposure (1 and 4 days), and then analyzed population growth, transcriptomic profiles and physiological changes in antioxidant enzymes to explore the toxicity and detoxification mechanisms during the zinc stress response. Results demonstrated that long-term zinc exposure could have restrained population growth in ciliates, however, the response mechanism to zinc exposure in ciliates is likely to show a dosage-dependent and time-dependent manner. The differentially expressed genes (DEGs) were identified the characters by high-throughput sequencing, which remarkably enriched in the phagosome, indicating that the phagosome pathway might mediate the uptake of zinc, while the pathways of ABC transporters and Na+/K+-transporting ATPase contributed to the efflux transport of excessive zinc ions and the maintenance of osmotic balance, respectively. The accumulation of zinc ions triggered a series of adverse effects, including damage to DNA and proteins, disturbance of mitochondrial function, and oxidative stress. In addition, we found that gene expression changed significantly for metal ion binding, energy metabolism, and oxidation-reduction processes. RT-qPCR of ten genes involved in important biological functions further validated the results of the transcriptome analysis. We also continuously monitored changes in activity of four antioxidant enzymes (SOD, CAT, POD and GSH-PX), all of which peaked on day 4 in cells subjected to zinc stress. Collectively, our results indicate that excessive environmental zinc exposure initially causes damage to cellular structure and function and then initiates detoxification mechanisms to maintain homeostasis in P. cf. multimicronucleatum cells.

Single-cell transcriptome reveals cell division-regulated hub genes in the unicellular eukaryote Paramecium.

The transition from growth to division during the cell cycle encompasses numerous conserved processes such as large-scale DNA replication and protein synthesis. In ciliate cells, asexual cell division is accompanied by additional cellular changes including amitotic nuclear division, extensive ciliogenesis, and trichocyst replication. However, the molecular mechanisms underlying these processes remain elusive. In this study, we present single-cell gene expression profiles of Paramecium cf. multimicronucleatum cells undergoing cell division. Our results reveal that the most up-regulated genes in dividing cells compared to growing cells are associated with 1) cell cycle signaling pathways including transcription, DNA replication, chromosome segregation and protein degradation; 2) microtubule proteins and tubulin glycylases which are essential for ciliogenesis, nuclei separation and structural differentiation signaling; and 3) trichocyst matrix proteins involved in trichocyst synthesis and reproduction. Furthermore, weighted gene co-expression network analysis identified hub genes that may play crucial roles during cell division. Our findings provide insights into cell cycle regulators, microtubules and trichocyst matrix proteins that may exert influence on this process in ciliates.