The Paramecium histone chaperone Spt16-1 is required for Pgm endonuclease function in programmed genome rearrangements.

In Paramecium tetraurelia, a large proportion of the germline genome is reproducibly removed from the somatic genome after sexual events via a process involving small (s)RNA-directed heterochromatin formation and DNA excision and repair. How germline limited DNA sequences are specifically recognized in the context of chromatin remains elusive. Here, we use a reverse genetics approach to identify factors involved in programmed genome rearrangements. We have identified a P. tetraurelia homolog of the highly conserved histone chaperone Spt16 subunit of the FACT complex, Spt16-1, and show its expression is developmentally regulated. A functional GFP-Spt16-1 fusion protein localized exclusively in the nuclei where genome rearrangements take place. Gene silencing of Spt16-1 showed it is required for the elimination of all germline-limited sequences, for the survival of sexual progeny, and for the accumulation of internal eliminated sequence (ies)RNAs, an sRNA population produced when elimination occurs. Normal accumulation of 25 nt scanRNAs and deposition of silent histone marks H3K9me3 and H3K27me3 indicated that Spt16-1 does not regulate the scanRNA-directed heterochromatin pathway involved in the early steps of DNA elimination. We further show that Spt16-1 is required for the correct nuclear localization of the PiggyMac (Pgm) endonuclease, which generates the DNA double-strand breaks required for DNA elimination. Thus, Spt16-1 is essential for Pgm function during programmed genome rearrangements. We propose a model in which Spt16-1 mediates interactions between the excision machinery and chromatin, facilitating endonuclease access to DNA cleavage sites during genome rearrangements.

Noninvasive and safe cell viability assay for Paramecium using natural pigment extracted from food.

Noninvasive, safe and cost-effective cell viability assay is important in many fields of biological research such as cell culture and counting. We examined ten typical natural pigments extracted from food to find that Monascus pigment (MP) or anthocyanin pigment (AP: purple sweet potato and purple cabbage) with Tris (Trimethylolaminomethane) works as a good indicator of viability assay for dye exclusion test (DET) of Paramecium. This was confirmed spectrally by scan-free, non-invasive absorbance spectral imaging A (x, y, λ) microscopy. We developed a new method of cell capture using a metal mesh to confine live Paramecium in a restricted space. This has the advantage that a low-cost and robust capture can be fabricated without using special equipment, compared to a conventional lab-on-a-chip. As a result, MP and AP stained dead cells as quick as methylene blue (MB), a synthetic dye conventionally used in DET within 1 min when treated with microwave and benzalkonium chloride. The natural pigments with Tris had little effect on inhibiting the growth of Paramecium, but MB killed all the cells within 1 h. MP is most useful because it allows non-invasive DET without Tris. This approach provides less invasive and safe DET.

What's Genetic Variation Got to Do with It? Starvation-Induced Self-Fertilization Enhances Survival in Paramecium.

The pervasiveness of sex despite its well-known costs is a long-standing puzzle in evolutionary biology. Current explanations for the success of sex in nature largely rely on the adaptive significance of the new or rare genotypes that sex may generate. Less explored is the possibility that sex-underlying molecular mechanisms can enhance fitness and convey benefits to the individuals that bear the immediate costs of sex. Here, we show that the molecular environment associated with self-fertilization can increase stress resistance in the ciliate Paramecium tetraurelia. This advantage is independent of new genetic variation, coupled with a reduced nutritional input, and offers fresh insights into the mechanistic origin of sex. In addition to providing evidence that the molecular underpinnings of sexual reproduction and the stress response are linked in P. tetraurelia, these findings supply an integrative explanation for the persistence of self-fertilization in this ciliate.

Uni-cellular integration of complex spatial information in slime moulds and ciliates.

Single-celled organisms show a fascinating faculty for integrating spatial information and adapting their behaviour accordingly. As such they are of potential interest for elucidating fundamental mechanisms of developmental biology. In this mini-review we highlight current research on two organisms, the true slime mould Physarum polycephalum and the ciliates Paramecium and Tetrahymena. For each of these, we present a case study how applying physical principles to explain behaviour can lead to the understanding of general principles possibly relevant to developmental biology.

Evaluation of phenol-induced ecotoxicity in two model ciliate species: Population growth dynamics and antioxidant enzyme activity.

The application of identical exposure dosages in different species generally leads to a limited understanding of dose-response patterns because of species-specific factors. To evaluate phenol-induced ecotoxicity, antioxidant enzyme activity and population growth dynamics were compared in two model ciliates, the marine species Euplotes vannus and the freshwater species Paramecium multimicronucleatum. Dosage ranges of phenol exposure were based on tolerance limits of test ciliates as determined by their carrying capacity (K) and growth rate (r). When the exposure duration of phenol increased from 48 h to 96 h, the median effective dose (ED50) for P. multimicronucleatum decreased faster than that for E. vannus, and the ratio of the former to the latter declined from 2.75 to 0.30. When E. vannus was exposed to increasing concentrations of phenol (0-140 mg l-1), r rose initially and then dropped significantly at concentrations higher than 40 mg l-1, whereas K decreased linearly over the entire range. For P. multimicronucleatum, both r and K declined gradually over the range 0-200 mg l-1 phenol. Dose-response patterns of activities of three individual antioxidant enzymes, and the integrative index of the three enzymes, presented a biphasic (inverse U-shaped) curve at each of four durations of exposure, i.e. 12 h, 24 h, 36 h and 48 h. Cluster analyses and multidimensional scaling analyses of antioxidant enzyme activities revealed differences in the temporal succession of physiological states between the two model ciliates. In brief, combining ED50 with growth dynamic parameters is helpful for designing exposure dosages of toxicants in ecotoxicity tests.

Variation and asymmetry in host-symbiont dependence in a microbial symbiosis.

BACKGROUND: Symbiosis is a major source of evolutionary innovation and, by allowing species to exploit new ecological niches, underpins the functioning of ecosystems. The transition from free-living to obligate symbiosis requires the alignment of the partners' fitness interests and the evolution of mutual dependence. While symbiotic taxa are known to vary widely in the extent of host-symbiont dependence, rather less is known about variation within symbiotic associations. RESULTS: Using experiments with the microbial symbiosis between the protist Paramecium bursaria and the alga Chlorella, we show variation between pairings in host-symbiont dependence, encompassing facultative associations, mutual dependence and host dependence upon the symbiont. Facultative associations, that is where both the host and the symbiont were capable of free-living growth, displayed higher symbiotic growth rates and higher per host symbiont loads than those with greater degrees of dependence. CONCLUSIONS: These data show that the Paramecium-Chlorella interaction exists at the boundary between facultative and obligate symbiosis, and further suggest that the host is more likely to evolve dependence than the algal symbiont.

The Impact of UV Radiation on Paramecium Populations from Alpine Lakes.

Paramecium populations from a clear and a glacier-fed turbid alpine lake were exposed to solar simulated ultraviolet (UVR) and photosynthetically active radiation (PAR) at 8 and 15 °C. The ciliates were tested for DNA damage (comet assay), behavioral changes, and mortality after UVR + PAR exposure. High DNA damage levels (~58% tail DNA) and abnormal swimming behavior were observed, although no significant changes in cell numbers were found irrespective of the lake origin (clear, turbid), and temperatures. We conclude that environmental stressors such as UVR and their effects may influence the adaptation of ciliates living in alpine lakes.

Genetics and Epigenetics of Mating Type Determination in Paramecium and Tetrahymena.

While sex is an ancient and highly conserved eukaryotic invention, self-incompatibility systems such as mating types or sexes appear to be derived limitations that show considerable evolutionary plasticity. Within a single class of ciliates, Paramecium and Tetrahymena species have long been known to present a wide variety of mating type numbers and modes of inheritance, but only recently have the genes involved been identified. Although similar transmembrane proteins mediate self/nonself recognition in both ciliates, the mechanisms of mating type determination differ widely, ranging from Mendelian systems to developmental nuclear differentiation, either stochastic or maternally inherited. The non-Mendelian systems rely on programmed editing of the germline genome that occurs during differentiation of the somatic nucleus, and they have co-opted different DNA recombination mechanisms-some previously unknown. Here we review the recent molecular advances and some remaining unsolved questions and discuss the possible implications of these diverse mechanisms for inbreeding/outbreeding balance regulation.

Assessing phagotrophy in the mixotrophic ciliate Paramecium bursaria using GFP-expressing yeast cells.

We used cells of the yeast Saccharomyces cerevisiae expressing green fluorescent protein (GFP) as fluorescently labelled prey to assess the phagocytic activities of the mixotrophic ciliate Paramecium bursaria, which harbours symbiotic Chlorella-like algae. Because of different fluorescence spectra of GFP and algal chlorophyll, ingested GFP-expressing yeast cells can be distinguished from endosymbiotic algal cells and directly counted in individual P. bursaria cells using fluorescence microscopy. By using GFP-expressing yeast cells, we found that P. bursaria altered ingestion activities under different physiological conditions, such as different growth phases or the presence/absence of endosymbionts. Use of GFP-expressing yeast cells allowed us to estimate the digestion rates of live prey of the ciliate. In contrast to the ingestion activities, the digestion rate within food vacuoles was not affected by the presence of endosymbionts, consistent with previous findings that food and perialgal vacuoles are spatially and functionally separated in P. bursaria. Thus, GFP-expressing yeast may provide a valuable tool to assess both ingestion and digestion activities of ciliates that feed on eukaryotic organisms.

Did Gause Have a Yeast Infection?

We planned to develop predator-prey models using Paramecium and yeast, but they have not been empirically examined since work by Gause in the 1930s. Therefore, we evaluated if Paramecium aurelia ingests and grows on eight yeasts. Recognising that it ingested yeasts but could not grow, we assessed if it might grow on other yeasts, by empirically parameterising a predator-prey model that relies on ingestion, not growth. Simulations were compared to P. aurelia-yeast time-series data, from Gause. We hypothesised that if the model simulated predator-prey dynamics that mimicked the original data, then possibly P. aurelia could grow on yeast; simulations did not mimic the original data. Reviewing works by Gause exposed two issues: experiments were undoubtedly contaminated with bacteria, allowing growth on bacteria, not yeast; and the population cycle data cannot be considered a self-sustaining time series, as they were manipulated by adding yeast and ciliates. We conclude that past and future work should not rely on this system, for either empirical or theoretical evaluations. Finally, although we show that P. aurelia, P. caudatum, Euplotes patella, and Blepharisma sp. cannot grow on yeast, Tetrahymena pyriformis and Colpidium striatum can; these may provide models to explore predator-prey dynamics.

Simultaneous Evaluation of Life Cycle Dynamics between a Host Paramecium and the Endosymbionts of Paramecium bursaria Using Capillary Flow Cytometry.

Endosymbioses are driving forces underlying cell evolution. The endosymbiosis exhibited by Paramecium bursaria is an excellent model with which to study symbiosis. A single-cell microscopic analysis of P. bursaria reveals that endosymbiont numbers double when the host is in the division phase. Consequently, endosymbionts must arrange their cell cycle schedule if the culture-condition-dependent change delays the generation time of P. bursaria. However, it remains poorly understood whether endosymbionts keep pace with the culture-condition-dependent behaviors of P. bursaria, or not. Using microscopy and flow cytometry, this study investigated the life cycle behaviors occurring between endosymbionts and the host. To establish a connection between the host cell cycle and endosymbionts comprehensively, multivariate analysis was applied. The multivariate analysis revealed important information related to regulation between the host and endosymbionts. Results show that dividing endosymbionts underwent transition smoothly from the division phase to interphase, when the host was in the logarithmic phase. In contrast, endosymbiont division stagnated when the host was in the stationary phase. This paper explains that endosymbionts fine-tune their cell cycle pace with their host and that a synchronous life cycle between the endosymbionts and the host is guaranteed in the symbiosis of P. bursaria.

Host control and nutrient trading in a photosynthetic symbiosis.

Photosymbiosis is one of the most important evolutionary trajectories, resulting in the chloroplast and the subsequent development of all complex photosynthetic organisms. The ciliate Paramecium bursaria and the alga Chlorella have a well established and well studied light dependent endosymbiotic relationship. Despite its prominence, there remain many unanswered questions regarding the exact mechanisms of the photosymbiosis. Of particular interest is how a host maintains and manages its symbiont load in response to the allocation of nutrients between itself and its symbionts. Here we construct a detailed mathematical model, parameterised from the literature, that explicitly incorporates nutrient trading within a deterministic model of both partners. The model demonstrates how the symbiotic relationship can manifest as parasitism of the host by the symbionts, mutualism, wherein both partners benefit, or exploitation of the symbionts by the hosts. We show that the precise nature of the photosymbiosis is determined by both environmental conditions (how much light is available for photosynthesis) and the level of control a host has over its symbiont load. Our model provides a framework within which it is possible to pose detailed questions regarding the evolutionary behaviour of this important example of an established light dependent endosymbiosis; we focus on one question in particular, namely the evolution of host control, and show using an adaptive dynamics approach that a moderate level of host control may evolve provided the associated costs are not prohibitive.

Response of the bacterial symbiont Holospora caryophila to different growth conditions of its host.

Previous studies on bacterial symbionts of ciliates have shown that some symbionts can be maintained relatively well under standard laboratory conditions whereas others are frequently lost, especially when the host is cultivated at a high division rate. In this study, the variation in infection level by the endosymbiont Holospora caryophila within its host population Paramecium octaurelia was investigated in response to three alimentary treatments and a subsequent starvation phase. The response of the ciliates was determined as a nearly exponential growth rate with different slopes in each treatment, proportional to the amount of food received. The initial infection level was higher than 90%. After 24 days of exponential host's growth, the prevalence remained stable at approximately 90% in all treatments, even after a subsequent starvation phase of 20 days. However, at intermediate time-points in both the feeding and the starvation phase, fluctuations in the presence of the intracellular bacteria were observed. These results show that H. caryophila is able to maintain its infection under the tested range of host growth conditions, also due to the possibility of an effective re-infection in case of partial loss.

Pdsg1 and Pdsg2, novel proteins involved in developmental genome remodelling in Paramecium.

The epigenetic influence of maternal cells on the development of their progeny has long been studied in various eukaryotes. Multicellular organisms usually provide their zygotes not only with nutrients but also with functional elements required for proper development, such as coding and non-coding RNAs. These maternally deposited RNAs exhibit a variety of functions, from regulating gene expression to assuring genome integrity. In ciliates, such as Paramecium these RNAs participate in the programming of large-scale genome reorganization during development, distinguishing germline-limited DNA, which is excised, from somatic-destined DNA. Only a handful of proteins playing roles in this process have been identified so far, including typical RNAi-derived factors such as Dicer-like and Piwi proteins. Here we report and characterize two novel proteins, Pdsg1 and Pdsg2 (Paramecium protein involved in Development of the Somatic Genome 1 and 2), involved in Paramecium genome reorganization. We show that these proteins are necessary for the excision of germline-limited DNA during development and the survival of sexual progeny. Knockdown of PDSG1 and PDSG2 genes affects the populations of small RNAs known to be involved in the programming of DNA elimination (scanRNAs and iesRNAs) and chromatin modification patterns during development. Our results suggest an association between RNA-mediated trans-generational epigenetic signal and chromatin modifications in the process of Paramecium genome reorganization.

Modelling the dynamics of an experimental host-pathogen microcosm within a hierarchical Bayesian framework.

The advantages of Bayesian statistical approaches, such as flexibility and the ability to acknowledge uncertainty in all parameters, have made them the prevailing method for analysing the spread of infectious diseases in human or animal populations. We introduce a Bayesian approach to experimental host-pathogen systems that shares these attractive features. Since uncertainty in all parameters is acknowledged, existing information can be accounted for through prior distributions, rather than through fixing some parameter values. The non-linear dynamics, multi-factorial design, multiple measurements of responses over time and sampling error that are typical features of experimental host-pathogen systems can also be naturally incorporated. We analyse the dynamics of the free-living protozoan Paramecium caudatum and its specialist bacterial parasite Holospora undulata. Our analysis provides strong evidence for a saturable infection function, and we were able to reproduce the two waves of infection apparent in the data by separating the initial inoculum from the parasites released after the first cycle of infection. In addition, the parameter estimates from the hierarchical model can be combined to infer variations in the parasite's basic reproductive ratio across experimental groups, enabling us to make predictions about the effect of resources and host genotype on the ability of the parasite to spread. Even though the high level of variability between replicates limited the resolution of the results, this Bayesian framework has strong potential to be used more widely in experimental ecology.

Epiplasmins and epiplasm in paramecium: the building of a submembraneous cytoskeleton.

In ciliates, basal bodies and associated appendages are bound to a submembrane cytoskeleton. In Paramecium, this cytoskeleton takes the form of a thin dense layer, the epiplasm, segmented into regular territories, the units where basal bodies are inserted. Epiplasmins, the main component of the epiplasm, constitute a large family of 51 proteins distributed in 5 phylogenetic groups, each characterized by a specific molecular design. By GFP-tagging, we analyzed their differential localisation and role in epiplasm building and demonstrated that: 1) The epiplasmins display a low turnover, in agreement with the maintenance of an epiplasm layer throughout the cell cycle; 2) Regionalisation of proteins from different groups allows us to define rim, core, ring and basal body epiplasmins in the interphase cell; 3) Their dynamics allows definition of early and late epiplasmins, detected early versus late in the duplication process of the units. Epiplasmins from each group exhibit a specific combination of properties. Core and rim epiplasmins are required to build a unit; ring and basal body epiplasmins seem more dispensable, suggesting that they are not required for basal body docking. We propose a model of epiplasm unit assembly highlighting its implication in structural heredity in agreement with the evolutionary history of epiplasmins.

Low frequency ultrasound and UV-C for elimination of pathogens in recirculating aquaculture systems.

Low frequency ultrasound (LFUS) was evaluated as a novel disinfection technique within recirculating aquaculture systems both individually and combined with UV-C. Dose-dependent inactivation rates were determined for the total viable counts and model organisms representing different taxa of common fish parasites: the ciliate Paramecium sp., second larval stage (L2) of the nematode Anguillicola crassus and metanauplii of Artemia sp. Application of LFUS up to 19 kJ/L did not reduce the number of colony forming units (CFU), whilst UV-C irradiation was highly effective. Pre-treatment with LFUS reduced the mean size of suspended solids in aquaculture water and thus increased the germicidal effect of UV-C by up to 0.6 log units. LFUS was effective against the eukaryotic organisms, and the dose-dependent inactivation could be well described by functions of an exponential decay. However, the efficiency of LFUS differed greatly between species. A LFUS dose of 1.9 kJ/L (consumed energy) was sufficient to inactivate Artemia by 99%, but a ten times higher dose was necessary to inactivate 95% and 81% of Paramecium and Anguillicola larvae, respectively. In clear water, the energetic efficiency of UV-C (emitted by a low pressure lamp) against Paramecium and Anguillicola larvae was higher compared to LFUS, but LFUS was more efficient against Artemia. However, the efficiency of LFUS against ciliates or nematode larvae would be similar or even higher than UV-C in highly turbid water or if less efficient medium pressure lamps are used. This study shows that LFUS can be applied safely at energy densities that are effective against a wide range of parasites like ciliates, nematodes and crustaceans. The combination of LFUS and UV-C could provide an appropriate water treatment with regards to all relevant pathogens in recirculating aquaculture systems.

Quorum sensing and density-dependent dispersal in an aquatic model system.

Many organisms use cues to decide whether to disperse or not, especially those related to the composition of their environment. Dispersal hence sometimes depends on population density, which can be important for the dynamics and evolution of sub-divided populations. But very little is known about the factors that organisms use to inform their dispersal decision. We investigated the cues underlying density-dependent dispersal in inter-connected microcosms of the freshwater protozoan Paramecium caudatum. In two experiments, we manipulated (i) the number of cells per microcosm and (ii) the origin of their culture medium (supernatant from high- or low-density populations). We found a negative relationship between population density and rates of dispersal, suggesting the use of physical cues. There was no significant effect of culture medium origin on dispersal and thus no support for chemical cues usage. These results suggest that the perception of density - and as a result, the decision to disperse - in this organism can be based on physical factors. This type of quorum sensing may be an adaptation optimizing small scale monitoring of the environment and swarm formation in open water.

Highly precise and developmentally programmed genome assembly in Paramecium requires ligase IV-dependent end joining.

During the sexual cycle of the ciliate Paramecium, assembly of the somatic genome includes the precise excision of tens of thousands of short, non-coding germline sequences (Internal Eliminated Sequences or IESs), each one flanked by two TA dinucleotides. It has been reported previously that these genome rearrangements are initiated by the introduction of developmentally programmed DNA double-strand breaks (DSBs), which depend on the domesticated transposase PiggyMac. These DSBs all exhibit a characteristic geometry, with 4-base 5' overhangs centered on the conserved TA, and may readily align and undergo ligation with minimal processing. However, the molecular steps and actors involved in the final and precise assembly of somatic genes have remained unknown. We demonstrate here that Ligase IV and Xrcc4p, core components of the non-homologous end-joining pathway (NHEJ), are required both for the repair of IES excision sites and for the circularization of excised IESs. The transcription of LIG4 and XRCC4 is induced early during the sexual cycle and a Lig4p-GFP fusion protein accumulates in the developing somatic nucleus by the time IES excision takes place. RNAi-mediated silencing of either gene results in the persistence of free broken DNA ends, apparently protected against extensive resection. At the nucleotide level, controlled removal of the 5'-terminal nucleotide occurs normally in LIG4-silenced cells, while nucleotide addition to the 3' ends of the breaks is blocked, together with the final joining step, indicative of a coupling between NHEJ polymerase and ligase activities. Taken together, our data indicate that IES excision is a "cut-and-close" mechanism, which involves the introduction of initiating double-strand cleavages at both ends of each IES, followed by DSB repair via highly precise end joining. This work broadens our current view on how the cellular NHEJ pathway has cooperated with domesticated transposases for the emergence of new mechanisms involved in genome dynamics.

Remembrance of things past retrieved from the Paramecium genome.

Paramecium and other ciliates are the only unicellular eukaryotes that separate germinal and somatic functions. A germline micronucleus transmits the genetic information to sexual progeny, while a somatic macronucleus expresses the genetic information during vegetative growth to determine the phenotype. At each sexual generation, a new macronucleus develops from the zygotic nucleus through programmed rearrangements of the germline genome. Paramecium tetraurelia somatic genome sequencing, reviewed here, has provided insight into the organization and evolution of the genome. A series of at least 3 whole genome duplications was detected in the Paramecium lineage and selective pressures that determine the fate of the gene duplicates analyzed. Variability in the somatic DNA was characterized and could be attributed to the genome rearrangement processes. Since, in Paramecium, alternative genome rearrangement patterns can be inherited across sexual generations by homology-dependent epigenetic mechanisms and can affect phenotype, I discuss the possibility that ciliate nuclear dimorphism buffers genetic variation hidden in the germline.