Recent Advances in Ciliate Biology.

Ciliates are a diverse group of unicellular eukaryotes that vary widely in size, shape, body plan, and ecological niche. Here, we review recent research advances achieved with ciliate models. Studies on patterning and regeneration have been revived in the giant ciliate Stentor, facilitated by modern omics methods. Cryo-electron microscopy and tomography have revolutionized the structural study of complex macromolecules such as telomerase, ribozymes, and axonemes. DNA elimination, gene scrambling, and mating type determination have been deciphered, revealing interesting adaptations of processes that have parallels in other kingdoms of life. Studies of common eukaryotic processes, such as intracellular trafficking, meiosis, and histone modification, reveal conservation as well as unique adaptations in these organisms that are evolutionarily distant from other models. Continual improvement of genetic and molecular tools makes ciliates accessible models for all levels of education and research. Such advances open new avenues of research and highlight the importance of ciliate research.

Circular Concatemers of Ultra-Short DNA Segments Produce Regulatory RNAs.

In the ciliated protozoan Paramecium tetraurelia, Piwi-associated small RNAs are generated upon the elimination of tens of thousands of short transposon-derived DNA segments as part of development. These RNAs then target complementary DNA for elimination in a positive feedback process, contributing to germline defense and genome stability. In this work, we investigate the formation of these RNAs, which we show to be transcribed directly from the short (length mode 27 bp) excised DNA segments. Our data support a mechanism whereby the concatenation and circularization of excised DNA segments provides a template for RNA production. This process allows the generation of a double-stranded RNA for Dicer-like protein cleavage to give rise to a population of small regulatory RNAs that precisely match the excised DNA sequences. VIDEO ABSTRACT.

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.

Two paralogous PHD finger proteins participate in natural genome editing in Paramecium tetraurelia.

The unicellular eukaryote Paramecium tetraurelia contains functionally distinct nuclei: germline micronuclei (MICs) and a somatic macronucleus (MAC). During sex, the MIC genome is reorganized into a new MAC genome and the old MAC is lost. Almost 45,000 unique internal eliminated sequences (IESs) distributed throughout the genome require precise excision to guarantee a functional new MAC genome. Here, we characterize a pair of paralogous PHD finger proteins involved in DNA elimination. DevPF1, the early-expressed paralog, is present in only some of the gametic and post-zygotic nuclei during meiosis. Both DevPF1 and DevPF2 localize in the new developing MACs, where IES excision occurs. Upon DevPF2 knockdown (KD), long IESs are preferentially retained and late-expressed small RNAs decrease; no length preference for retained IESs was observed in DevPF1-KD and development-specific small RNAs were abolished. The expression of at least two genes from the new MAC with roles in genome reorganization seems to be influenced by DevPF1- and DevPF2-KD. Thus, both PHD fingers are crucial for new MAC genome development, with distinct functions, potentially via regulation of non-coding and coding transcription in the MICs and new MACs.

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.

A Population-Genetic Lens into the Process of Gene Loss Following Whole-Genome Duplication.

Whole-genome duplications (WGDs) have occurred in many eukaryotic lineages. However, the underlying evolutionary forces and molecular mechanisms responsible for the long-term retention of gene duplicates created by WGDs are not well understood. We employ a population-genomic approach to understand the selective forces acting on paralogs and investigate ongoing duplicate-gene loss in multiple species of Paramecium that share an ancient WGD. We show that mutations that abolish protein function are more likely to be segregating in retained WGD paralogs than in single-copy genes, most likely because of ongoing nonfunctionalization post-WGD. This relaxation of purifying selection occurs in only one WGD paralog, accompanied by the gradual fixation of nonsynonymous mutations and reduction in levels of expression, and occurs over a long period of evolutionary time, "marking" one locus for future loss. Concordantly, the fitness effects of new nonsynonymous mutations and frameshift-causing indels are significantly more deleterious in the highly expressed copy compared with their paralogs with lower expression. Our results provide a novel mechanistic model of gene duplicate loss following WGDs, wherein selection acts on the sum of functional activity of both duplicate genes, allowing the two to wander in expression and functional space, until one duplicate locus eventually degenerates enough in functional efficiency or expression that its contribution to total activity is too insignificant to be retained by purifying selection. Retention of duplicates by such mechanisms predicts long times to duplicate-gene loss, which should not be falsely attributed to retention due to gain/change in function.

From representations to servomechanisms to oscillators: my journey in the study of cognition.

The study of comparative cognition bloomed in the 1970s and 1980s with a focus on representations in the heads of animals that undergird what animals can achieve. Even in action-packed domains such as navigation and spatial cognition, a focus on representations prevailed. In the 1990s, I suggested a conception of navigation in terms of navigational servomechanisms. A servomechanism can be said to aim for a goal, with deviations from the goal-directed path registering as an error. The error drives action to reduce the error in a negative-feedback loop. This loop, with the action reducing the very signal that drove action in the first place, is key to defining a servomechanism. Even though actions are crucial components of servomechanisms, my focus was on the representational component that encodes signals and evaluates errors. Recently, I modified and amplified this view in claiming that, in navigation, servomechanisms operate by modulating the performance of oscillators, endogenous units that produce periodic action. The pattern is found from bacteria travelling micrometres to sea turtles travelling thousands of kilometres. This pattern of servomechanisms working with oscillators is found in other realms of cognition and of life. I think that oscillators provide an effective way to organise an organism's own activities while servomechanisms provide an effective means to adjust to the organism's environment, including that of its own body.

Why an animal needs a brain.

In Principles of Neural Design (2015, MIT Press), inspired by Charles Darwin, Sterling and Laughlin undertook the unfashionable task of distilling principles from facts in the technique-driven, data-saturated domain of neuroscience. Their starting point for deriving the organizing principles of brains are two brainless single-celled organisms, Escherichia coli and Paramecium, and the 302-neuron brain of the nematode Caenorhabditis elegans. The book is an exemplar in how to connect the dots between simpler and (much) more complex organisms in a particular area. Here, they have generously agreed to republish an abridged version of Chapter 2 (Why an Animal Needs a Brain), in which many of their principles are first described.

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.

"Candidatus Intestinibacterium parameciiphilum"-member of the "Candidatus Paracaedibacteraceae" family (Alphaproteobacteria, Holosporales) inhabiting the ciliated protist Paramecium.

Protists frequently host diverse bacterial symbionts, in particular those affiliated with the order Holosporales (Alphaproteobacteria). All characterised members of this bacterial lineage have been retrieved in obligate association with a wide range of eukaryotes, especially multiple protist lineages (e.g. amoebozoans, ciliates, cercozoans, euglenids, and nucleariids), as well as some metazoans (especially arthropods and related ecdysozoans). While the genus Paramecium and other ciliates have been deeply investigated for the presence of symbionts, known members of the family "Candidatus Paracaedibacteraceae" (Holosporales) are currently underrepresented in such hosts. Herein, we report the description of "Candidatus Intestinibacterium parameciiphilum" within the family "Candidatus Paracaedibacteraceae", inhabiting the cytoplasm of Paramecium biaurelia. This novel bacterium is almost twice as big as its relative "Candidatus Intestinibacterium nucleariae" from the opisthokont Nuclearia and does not present a surrounding halo. Based on phylogenetic analyses of 16S rRNA gene sequences, we identified six further potential species-level lineages within the genus. Based on the provenance of the respective samples, we investigated the environmental distribution of the representatives of "Candidatus Intestinibacterium" species. Obtained results are consistent with an obligate endosymbiotic lifestyle, with protists, in particular freshwater ones, as hosts. Thus, available data suggest that association with freshwater protists could be the ancestral condition for the members of the "Candidatus Intestinibacterium" genus.

Two Predators, One Prey - the Interaction Between Bacteriophage, Bacterivorous Ciliates, and Escherichia coli.

Bacterivorous ciliates and lytic bacteriophages are two major predators in aquatic environments, competing for the same type of prey. This study investigated the possible interaction of these different microorganisms and their influence on the activity of each other. Therefore, two bacterivorous ciliates, Paramecium sp. RB1 and Tetrahymena sp. RB2, were used as representative ciliates; a T4-like Escherichia coli targeting lytic bacteriophage as a model virus; and E. coli ATCC 25922 as a susceptible bacterial host and prey. The growth of the two ciliates with E. coli ATCC 25922 as prey was affected by the presence of phage particles. The grazing activity of the two ciliates resulted in more than a 99% reduction of the phage titer and bacterial cell numbers. However, viable phage particles were recovered from individual washed cells of the two ciliates after membrane filtration. Therefore, ciliates such as Paramecium sp. RB1 and Tetrahymena sp. RB2 can remove bacteriophages present in natural and artificial waters by ingesting the viral particles and eliminating bacterial host cells required for viral replication. The ingestion of phage particles may marginally contribute to the nutrient supply of the ciliates. However, the interaction of phage particles with ciliate cells may contribute to the transmission of bacteriophages in aquatic environments.

The Effect of the Agonist Cholecystokinin-4 D-GB-115 On the Character of Motor Activity of Paramecium caudatum.

The study investigated the effect of GABA in various concentrations and D-GB-115 at a concentration of 10-7 M on the behavior of Paramecium caudatum. It was shown that GABA increases motor activity and changes the movement strategy of these protozoans, and the dose-effect relationship is domed, which can be explained by the presence of two types of GABA receptors in the outer membrane of paramecia: GABA-A and GABA-B. The active concentrations of GABA range from 10-3 to 10-13 M. The effect of pharmacological agents interacting with the GABA system on the behavior of ciliates (nembutal and D-GB-115) was studied.

N-glycans from Paramecium bursaria chlorella virus MA-1D: Re-evaluation of the oligosaccharide common core structure.

Paramecium bursaria chlorella virus MA-1D is a chlorovirus that infects Chlorella variabilis strain NC64A, a symbiont of the protozoan Paramecium bursaria. MA-1D has a 339-kb genome encoding ca. 366 proteins and 11 tRNAs. Like other chloroviruses, its major capsid protein (MCP) is decorated with N-glycans, whose structures have been solved in this work by using nuclear magnetic spectroscopy and matrix-assisted laser desorption ionization-time of flight mass spectrometry along with MS/MS experiments. This analysis identified three N-linked oligosaccharides that differ in the nonstoichiometric presence of three monosaccharides, with the largest oligosaccharide composed of eight residues organized in a highly branched fashion. The N-glycans described here share several features with those of the other chloroviruses except that they lack a distal xylose unit that was believed to be part of a conserved core region for all the chloroviruses. Examination of the MA-1D genome detected a gene with strong homology to the putative xylosyltransferase in the reference chlorovirus PBCV-1 and in virus NY-2A, albeit mutated with a premature stop codon. This discovery means that we need to reconsider the essential features of the common core glycan region in the chloroviruses.

Oscillators and servomechanisms in orientation and navigation, and sometimes in cognition.

Navigational mechanisms have been characterized as servomechanisms. A navigational servomechanism specifies a goal state to strive for. Discrepancies between the perceived current state and the goal state specify error. Servomechanisms adjust the course of travel to reduce the error. I now add that navigational servomechanisms work with oscillators, periodic movements of effectors that drive locomotion. I illustrate this concept selectively over a vast range of scales of travel from micrometres in bacteria to thousands of kilometres in sea turtles. The servomechanisms differ in sophistication, with some interrupting forward motion occasionally or changing travel speed in kineses and others adjusting the direction of travel in taxes. I suggest that in other realms of life as well, especially in cognition, servomechanisms work with oscillators.

Ion channels of cilia: Paramecium as a model.

Holotrichous ciliates, like Paramecium, swim through their aqueous environment by beating their many cilia. They can alter swimming speed and direction, which seems to have mesmerized early microscopists of the 1600s. We know from extensive and elegant physiological studies and generation of mutants that these cells can be considered little swimming neurons because their ciliary beating is under bioelectric control of ion channels in the cilia. This chapter will focus on the ionic control of swimming behavior by ciliary ion channels, primarily in the holotrichous ciliate Paramecium. Voltage-gated and calcium-activated channels for calcium, magnesium, sodium, and potassium are regulated in a closely orchestrated manner that allows cilia to bend and propel the cell forward or backward. Sensory input that generates receptor potentials feeds into the control of this channel activity and allows the cell to turn or speed up. This in turn helps the cell to avoid predators or toxic conditions. While the focus is on P. tetraurelia and P. caudatum, the principles of ciliary ion channel activity and control are easily extendable to other ciliates and protists. The high conservation of channel and ion pump structures also extends the lessons from Paramecium to higher organisms.

Evolutionary bioenergetics of ciliates.

Understanding why various organisms evolve alternative ways of living requires information on both the fitness advantages of phenotypic modifications and the costs of constructing and operating cellular features. Although the former has been the subject of a myriad of ecological studies, almost no attention has been given to how organisms allocate resources to alternative structures and functions. We address these matters by capitalizing on an array of observations on diverse ciliate species and from the emerging field of evolutionary bioenergetics. A relatively robust and general estimator for the total cost of a cell per cell cycle (in units of ATP equivalents) is provided, and this is then used to understand how the magnitudes of various investments scale with cell size. Among other things, we examine the costs associated with the large macronuclear genomes of ciliates, as well as ribosomes, various internal membranes, osmoregulation, cilia, and swimming activities. Although a number of uncertainties remain, the general approach taken may serve as blueprint for expanding this line of work to additional traits and phylogenetic lineages.

Bacterial symbiosis in ciliates (Alveolata, Ciliophora): Roads traveled and those still to be taken.

The diversity of prokaryotic symbionts in Ciliophora and other protists is fascinatingly rich; they may even include some potentially pathogenic bacteria. In this review, we summarize currently available data on biodiversity and some morphological and biological peculiarities of prokaryotic symbionts mainly within the genera Paramecium and Euplotes. Another direction of ciliate symbiology, neglected for a long time and now re-discovered, is the study of epibionts of ciliates. This promises a variety of interesting outcomes. Last, but not least, we stress the new technologies, such as next generation sequencing and the use of genomics data, which all can clarify many new aspects of relevance. For this reason, a brief overview of achievements in genomic studies on ciliate's symbionts is provided. Summing up the results of numerous scientific contributions, we systematically update current knowledge and outline the prospects as to how symbiology of Ciliophora may develop in the near future.

Comparative Analysis Between Paramecium Strains with Different Syngens Using the RAPD Method.

Paramecium spp. are a genus of free-living protists that live mainly in freshwater environments. They are ciliates with high motility and phagocytosis and have been used to analyze cell motility and as a host model for pathogens. Besides such biological characteristics, apart from the usual morphological and genetic classification of species, the existence of taxonomies (such as syngens) and mating types related to Paramecium's unique reproduction is known. In this study, we attempted to develop a simple method to identify Paramecium strains, which are difficult to distinguish morphologically, using random amplified polymorphic DNA (RAPD) analysis. Consequently, we can observe strain-specific band patterns. We also confirm that the presence of endosymbiotic Chlorella cells affects the band pattern of P. bursaria. Furthermore, the results of the RAPD analysis using several P. caudatum strains with different syngens show that it is possible to detect a band specific to a certain syngen. By improving the reaction conditions and random primers, based on the results of this study, RAPD analysis can be applied to the identification of Paramecium strains and their syngen confirmation tests.

Uptake and accumulation of microplastic particles by two freshwater ciliates isolated from a local river in South Africa.

Microplastics are considered environmental pollutants of serious concern. In freshwater environments, they can affect aquatic biota and accumulate along the food web. Therefore, this study investigated the capacity of bacterivorous freshwater ciliates, essential members of the aquatic food chain, to ingest plain and fluorescently-labeled polystyrene microspheres. Two holotrich ciliates were isolated from a stream in KwaZulu-Natal (South Africa) and identified as members of the genera Paramecium and Tetrahymena based on morphological characteristics and 18S rRNA gene sequence analysis. While the larger bacterivorous ciliate Paramecium sp. strain RB1 ingested all three sizes of plain polystyrene microbeads tested (2,5,10 µm), the smaller sized Tetrahymena sp. strain RB2 only ingested microbeads of 2 and 5 µm. The two ciliates ingested polystyrene microbeads at rates ranging from 1650 to 3870 particles x ciliate-1 x hour-1 for all particle sizes ingested, matching rates determined for selected microbial prey (E. coli, S. cerevisiae) of similar size. The ability to ingest non-nutritious microplastic particles was confirmed for both ciliates using fluorescently-labeled microbeads as these were detected in food vacuoles by fluorescence microscopy. Therefore, ciliates such as Paramecium sp. strain RB1 and Tetrahymena sp. strain RB2 can contribute to the transfer and bioaccumulation of microplastics in freshwater food webs in South Africa.

Comparative and Evolutionary Aspects of the Digestive System and Its Enteric Nervous System Control.

All life forms must gain nutrients from the environment and from single cell organisms to mammals a digestive system is present. Components of the digestive system that are recognized in mammals can be seen in the sea squirt that has had its current form for around 500my. Nevertheless, in mammals, the organ system that is most varied is the digestive system, its architecture being related to the dietary niche of each species. Forms include those of foregut or hindgut fermenters, single or multicompartment stomachs and short or capacious large intestines. Dietary niches include nectarivores, folivores, carnivores, etc. The human is exceptional in that, through food preparation (>80% of human consumption is prepared food in modern societies), humans can utilize a wider range of foods than other species. They are cucinivores, food preparers. In direct descendants of simple organisms, such as sponges, there is no ENS, but as the digestive tract becomes more complex, it requires integrated control of the movement and assimilation of its content. This is achieved by the nervous system, notably the enteric nervous system (ENS) and an array of gut hormones. An ENS is first observed in the phylum cnidaria, exemplified by hydra. But hydra has no collections of neurons that could in any way be regarded as a central nervous system. All animals more complex than hydra have an ENS, but not all have a CNS. In mammals, the ENS is extensive and is necessary for control of movement, enteric secretions and local blood flow, and regulation of the gut immune system. In animals with a CNS, the ENS and CNS have reciprocal connections. From hydra to human, an ENS is essential to life.