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.

An apical membrane complex for triggering rhoptry exocytosis and invasion in Toxoplasma.

Apicomplexan parasites possess secretory organelles called rhoptries that undergo regulated exocytosis upon contact with the host. This process is essential for the parasitic lifestyle of these pathogens and relies on an exocytic machinery sharing structural features and molecular components with free-living ciliates. However, how the parasites coordinate exocytosis with host interaction is unknown. Here, we performed a Tetrahymena-based transcriptomic screen to uncover novel exocytic factors in Ciliata and conserved in Apicomplexa. We identified membrane-bound proteins, named CRMPs, forming part of a large complex essential for rhoptry secretion and invasion in Toxoplasma. Using cutting-edge imaging tools, including expansion microscopy and cryo-electron tomography, we show that, unlike previously described rhoptry exocytic factors, TgCRMPs are not required for the assembly of the rhoptry secretion machinery and only transiently associate with the exocytic site-prior to the invasion. CRMPs and their partners contain putative host cell-binding domains, and CRMPa shares similarities with GPCR proteins. Collectively our data imply that the CRMP complex acts as a host-molecular sensor to ensure that rhoptry exocytosis occurs when the parasite contacts the host cell.

Nontriplet feature of genetic code in Euplotes ciliates is a result of neutral evolution.

The triplet nature of the genetic code is considered a universal feature of known organisms. However, frequent stop codons at internal mRNA positions in Euplotes ciliates ultimately specify ribosomal frameshifting by one or two nucleotides depending on the context, thus posing a nontriplet feature of the genetic code of these organisms. Here, we sequenced transcriptomes of eight Euplotes species and assessed evolutionary patterns arising at frameshift sites. We show that frameshift sites are currently accumulating more rapidly by genetic drift than they are removed by weak selection. The time needed to reach the mutational equilibrium is several times longer than the age of Euplotes and is expected to occur after a several-fold increase in the frequency of frameshift sites. This suggests that Euplotes are at an early stage of the spread of frameshifting in expression of their genome. In addition, we find the net fitness burden of frameshift sites to be noncritical for the survival of Euplotes. Our results suggest that fundamental genome-wide changes such as a violation of the triplet character of genetic code can be introduced and maintained solely by neutral evolution.

Genomic insights into the cellular specialization of predation in raptorial protists.

BACKGROUND: Predation is a fundamental mechanism for organisms to acquire energy, and various species have evolved diverse tools to enhance their hunting abilities. Among protozoan predators, raptorial Haptorian ciliates are particularly fascinating as they possess offensive extrusomes known as toxicysts, which are rapidly discharged upon prey contact. However, our understanding of the genetic processes and specific toxins involved in toxicyst formation and discharge is still limited. RESULTS: In this study, we investigated the predation strategies and subcellular structures of seven Haptoria ciliate species and obtained their genome sequences using single-cell sequencing technology. Comparative genomic analysis revealed distinct gene duplications related to membrane transport proteins and hydrolytic enzymes in Haptoria, which play a crucial role in the production and discharge of toxicysts. Transcriptomic analysis further confirmed the abundant expression of genes related to membrane transporters and cellular toxins in Haptoria compared to Trichostomatia. Notably, polyketide synthases (PKS) and L-amino acid oxidases (LAAO) were identified as potentially toxin genes that underwent extensive duplication events in Haptoria. CONCLUSIONS: Our results shed light on the evolutionary and genomic adaptations of Haptorian ciliates for their predation strategies in evolution and provide insights into their toxic mechanisms.

Stop or Not: Genome-Wide Profiling of Reassigned Stop Codons in Ciliates.

Bifunctional stop codons that have both translation and termination functions in the same species are important for understanding the evolution and function of genetic codes in living organisms. Considering the high frequency of bifunctional codons but limited number of available genomes in ciliates, we de novo sequenced seven representative ciliate genomes to explore the evolutionary history of stop codons. We further propose a stop codon reassignment quantification method (stopCR) that can identify bifunctional codons and measure their frequencies in various eukaryotic organisms. Using our newly developed method, we found two previously undescribed genetic codes, illustrating the prevalence of bifunctional stop codons in ciliates. Overall, evolutionary genomic analyses suggest that gain or loss of reassigned stop codons in ciliates is shaped by their living environment, the eukaryotic release factor 1, and suppressor tRNAs. This study provides novel clues about the functional diversity and evolutionary history of stop codons in eukaryotic organisms.

Single cell transcriptomics reveals UAR codon reassignment in Palmarella salina (Metopida, Armophorea) and confirms Armophorida belongs to APM clade.

Anaerobes have emerged in several major lineages of ciliates, but the number of independent transitions to anaerobiosis among ciliates is unknown. The APM clade (Armophorea, Muranotrichea, Parablepharismea) represents the largest clade of obligate anaerobes among ciliates and contains free-living marine and freshwater representatives as well as gut endobionts of animals. The evolution of APM group has only recently started getting attention, and our knowledge on its phylogeny and genetics is still limited to a fraction of taxa. While ciliates portray a wide array of alternatives to the standard genetic code across numerous classes, the APM ciliates were considered to be the largest group using exclusively standard nuclear genetic code. In this study, we present a pan-ciliate phylogenomic analysis with emphasis on the APM clade, bringing the first phylogenomic analysis of the family Tropidoatractidae (Armophorea) and confirming the position of Armophorida within Armophorea. We include five newly sequenced single cell transcriptomes from marine, freshwater, and endobiotic APM ciliates - Palmarella salina, Anteclevelandella constricta, Nyctotherus sp., Caenomorpha medusula, and Thigmothrix strigosa. We report the first discovery of an alternative nuclear genetic code among APM ciliates, used by Palmarella salina (Tropidoatractidae, Armophorea), but not by its close relative, Tropidoatractus sp., and provide a comparative analysis of stop codon identity and frequency indicating the precedency to the UAG codon loss/reassignment over the UAA codon reassignment in the specific ancestor of Palmarella. Comparative genomic and proteomic studies of this group may help explain the constraints that underlie UAR stop-to-sense reassignment, the most frequent type of alternative nuclear genetic code, not only in ciliates, but eukaryotes in general.

Morphology, morphogenesis, and molecular characterization of Castula specialis sp. nov. (Ciliophora, Armophorea, Metopida).

The morphology, morphogenesis, and molecular phylogeny of a new metopid ciliate, Castula specialis sp. nov., comprising three strains from geographically distant (China, Mexico, Czech Republic) anoxic freshwater habitats, were studied based on microscopic observation of live and protargol-stained specimens as well as SSU rRNA gene sequence data. The new species is characterized as follows: size in vivo 105-220 × 25-70 µm, body oblong to elongated ellipsoidal and asymmetrical; preoral dome distinctly projecting beyond the body; 32-46 adoral membranelles; 31-52 somatic kineties; and 4-7 setae. This study brings the first morphogenetic investigation of a member of the genus Castula. The morphogenesis of the type population (China) of the new species proceeds as in Metopus spp. comprising drastic changes in body shape and a pleurotelokinetal stomatogenesis; however, the main difference is the origin of the opisthe's paroral membrane that derives from all perizonal rows and some adjacent dome kineties. Phylogenetically, the genus Castula is paraphyletic.

Highly divergent morphology but a close molecular phylogenetic relationship between two little-known ciliate genera Actinobolina and Papillorhabdos (Protozoa: Ciliophora: Litostomatea) with description of two new species.

Free-living litostomatean ciliates, prominent microeukaryote predators commonly encountered in freshwater and marine habitats, play vital roles in maintaining energy flow and nutrient cycles. Nevertheless, understanding their biodiversity and phylogenetic relationships remains challenging due to insufficient morphological information and molecular data. As a new contribution to this group, three haptorian ciliates, including two new species (Actinobolina bivacuolata sp. nov. and Papillorhabdos foissneri sp. nov.) and the insufficiently described type species, Actinobolina radians, were isolated from wetlands around Lake Weishan, China and investigated by a combination of living morphology, stained preparations, and 18S rRNA gene sequence data. An illustrated key of the valid species within the two genera is provided. In addition, we reveal the phylogenetic positions of these two genera for the first time. Although they differ in all key morphologic characters such as general appearance (ellipsoidal with numerous tentacles vs. cylindrical), extrusomes (stored in tentacles vs. anchored to pellicle), circumoral kinety (present vs. absent), composition of somatic kineties (kinetosome clusters vs. monokinetids), and number of dorsal brush rows (1 vs. 4), they both cluster in a fully supported clade in the phylogenetic tree, which indicates that the biodiversity and additional molecular markers of this group need further exploration.

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.

Past-President address: My journey in microbial ecology-footprints in the sand, island hopping, supply chains, and technology bridges.

This paper highlights and honors the connectivity among protistan researchers, using my own research journey as a backdrop, with attention to the supply chain of ideas, supporters, and other influencers who helped to shape and guide my career by sharing their ideas, protocols, skills, and enthusiasm. In looking back at the journey, the supply chain in my career has also included changes in the conceptual framework for my research studies, converging with a continuous flow of ideas and support from colleagues and mentors. To illustrate the complex map of ideas and supporters, this paper will examine technological advances, paradigm shifts in ecological constructs, geographical considerations, breakthroughs in peritrich biology, and the importance of an integrated perspective as we navigate the changing realities of today's scientific challenges.

Numerical and Thermal Response of the Bacterivorous Ciliate Colpidium kleini, a Species Potentially at Risk of Extinction by Rising Water Temperatures.

We investigated the food-dependent growth and thermal response of the freshwater ciliate Colpidium kleini using numerical response (NR) experiments. This bacterivorous ciliate occurs in lotic water and the pelagial of lakes and ponds. The C. kleini strain used in this work was isolated from a small alpine lake and identified by combining detailed morphological inspections with molecular phylogeny. Specific growth rates (rmax) were measured from 5 to 21 °C. The ciliate did not survive at 22 °C. The threshold bacterial food levels (0.3 - 2.2 × 106 bacterial cells mL-1) matched the bacterial abundance in the alpine lake from which C. kleini was isolated. The food threshold was notably lower than previously reported for C. kleini and two other Colpidium species. The threshold was similar to levels reported for oligotrich and choreotrich ciliates if expressed in terms of bacterial biomass (0.05 - 0.43 mg C L-1). From the NR results, we calculated physiological mortality rates at zero food concentration. The mean mortality (0.55 ± 0.17 d-1) of C. kleini was close to the mean estimate obtained for other planktonic ciliates that do not encyst. We used the data obtained by the NR experiments to fit a thermal performance curve (TPC). The TPC yielded a temperature optimum at 17.3 °C for C. kleini, a maximum upper thermal tolerance limit of 21.9 °C, and a thermal safety margin of 4.6 °C. We demonstrated that combining NR with TPC analysis is a powerful tool to predict better a species' fitness in response to temperature and food.

Diatom bloom trigger notable variations in microzooplanktonic ciliate composition, body-size spectrum and biotic-abiotic interaction in the Arctic Ocean.

How microzooplanktonic ciliate adaptative strategies differ across diatom bloom and non-diatom bloom areas in the Arctic Ocean remains poorly documented. To address this gap, two different situations were categorized in the Arctic Ocean at summer 2023: diatom bloom stations (DBS) (genus Thalassiosira, chain-like) and non-diatom bloom stations (nDBS). Total abundance of ciliate at 3 m and 25 m in DBS was 2.8 and 1.8 folds higher than in nDBS, respectively. Aloricate ciliates were singled out in both DBS and nDBS, whilst their average abundance and biomass of large size-fraction (>50 µm) in former were 4.5-5.6 folds higher than in latter. Regarding tintinnids, high abundance of Ptychocylis acuta (Bering Strait species) mainly occurred at DBS, coupled with distribution of co-occurring Pacific-origin species Salpingella sp.1, collectively suggested a strong intrusion of Pacific Inflow during summer 2023. Additionally, presence of high abundance of Acanthostomella norvegica and genus Parafavella in nDBS might indicate the trajectory of the Transpolar Drift. Alternatively, tintinnids can serve as credible bioindicators for either monitoring currents or evaluating microzooplankton Borealization. Average abundance of total ciliate within 15-135 µm body-size spectrum in DBS was higher than nDBS. Moreover, spearman's rank correlation between biotic and abiotic analysis revealed that temperature and dissolved oxygen at DBS determined tintinnid species richness and ciliate total abundance, respectively. The results clearly demonstrate that remarkable divergences in large size-fraction of ciliate abundance between DBS and nDBS validate their irreplaceable role in controlling phytoplankton outbreak and associated biological processes in polar seas.

Antimicrobial activity of coral-associated beneficial bacteria against coral disease-causing microbial pathogens.

Microbial infection of immune-compromised corals influences disease severity, resulting in coral mortality. However, coral-associated beneficial bacteria are known to produce antimicrobial compounds that prevent the growth of potential pathogens and invading microbes. Hence, beneficial bacteria associated with coral Porites lutea were isolated and antimicrobial protein and bioactive secondary metabolites were extracted and tested for their antimicrobial activity against putative prokaryotic and eukaryotic coral pathogens. Bioactive secondary metabolites exhibited remarkable antagonism against various coral pathogens such as Serratia marcescens, Vibrio species, and Aspergillus sydowii. Besides, the metabolites of Cobetia marina, Cobetia amphilecti, Pseudoalteromonas neustonica, and Virgibacillus halodenitrificans manifested notable inhibition against the protozoan ciliates (Uronema marinum, Holosticha diademata, Cohnilembus verminus, and Euplotes vannus) and zooplankton that are known to be involved in the secondary pathogenesis in coral diseased lesion progression. Thus, the present study may benefit in understanding coral-associated beneficial bacteria for their antagonistic interactions with microbial pathogens, as well as their potential involvement in reducing coral disease severity.

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.

Possible beneficial interactions of ciliated protozoans with coral health and resilience.

Microbial interactions contribute significantly to coral health in the marine environment. Most beneficial associations have been described with their bacterial communities, but knowledge of beneficial associations between protozoan ciliates and corals is still lacking. Ciliates are important bacterial predators and provide nutrition to higher trophic-level organisms. The mucus secreted by corals and the microenvironment of the coral surface layer attract ciliates based on their food preferences. The mixotrophic and heterotrophic ciliates play a major role in nutrient cycling by increasing nitrogen, phosphorus, and extractable sulfur, which can enhance the proliferation of coral beneficial microbe. Besides, bacterial predator ciliates reduce the pathogenic bacterial population that infects the coral and also act as bioindicators for assessing the toxicity of the reef ecosystem. Thus, these ciliates can be used as a beneficial partner in influencing coral health and resilience under various stress conditions. Herein, we explore the urgent need to understand the complex beneficial interactions of ciliates that may occur in the coral reef ecosystem.

Comparative genomics provides insights into the phylogeny and environmental adaptations of Peritrichia (Protista, Ciliophora) - A potential resource for environmental pollution control and bioremediation.

Peritrichs are one of the largest groups within the class Oligohymenophorea. They have a worldwide distribution and a high degree of species diversity. Using the single-cell genome sequencing technique, we obtained the genomes of five sessilid peritrichs. Combining both genomic and transcriptomic data of other publicly available oligohymenophorean ciliates (including the genomes of three sessilid peritrichs from our team's previous study), we conducted a comparative genomics study. Our phylogenomic analyses using both maximum likelihood and Bayesian inference methods recovered the subclass Peritrichia and each of its two orders (Sessilida and Mobilida) as being monophyletic. The non-monophyly of two families (Vorticellidae and Zoothamniidae) was also well supported in both trees. Molecular clock analysis showed that the origin of the subclass Peritrichia was estimated to be during the late Proterozoic. We also analyzed the stop codon usage of 44 oligohymenophoreans. The results showed that most of these species used TGA as the biased stop codon and reassigned the other two stop codons (TAA and TAG) to code amino acids. In addition, we found that the presence of a typical peritrich lorica is a plesiomorphic character of the family Vaginicolidae. Through GO enrichment analysis for group-specific orthogroups of Vaginicolidae, we successfully identified the biological process and molecular function GO terms that were linked with the typical peritrich lorica, including three glycosaminoglycan-related and two chitin-related GO terms. Finally, our enrichment analyses of significantly expanded gene families in Peritrichia found that sessilids were more tolerant to environmental stress (mainly organic matter) than mobilids, suggesting that peritrich lineages (especially sessilids) may have the potential for application in environmental pollution control and bioremediation. Together, the results presented in this study will facilitate wider genome-scale phylogenetic analyses of Peritrichia and deepen the understanding of their unique advantages for environmental pollution control bioremediation.

Four ciliate-specific expansion events occurred during actin gene family evolution of eukaryotes.

Actin gene family is a divergent and ancient eukaryotic cellular cytoskeletal gene family, and participates in many essential cellular processes. Ciliated protists offer us an excellent opportunity to investigate gene family evolution, since their gene families evolved faster in ciliates than in other eukaryotes. Nonetheless, actin gene family is well studied in few model ciliate species but little is known about its evolutionary patterns in ciliates. Here, we analyzed the evolutionary pattern of eukaryotic actin gene family based on genomes/transcriptomes of 36 species covering ten ciliate classes, as well as those of nine non-ciliate eukaryotic species. Results showed: (1) Except for conventional actins and actin-related proteins (Arps) shared by various eukaryotes, at least four ciliate-specific subfamilies occurred during evolution of actin gene family. Expansions of Act2 and ArpC were supposed to have happened in the ciliate common ancestor, while expansions of ActI and ActII may have occurred in the ancestor of Armophorea, Muranotrichea, and Spirotrichea. (2) The number of actin isoforms varied greatly among ciliate species. Environmental adaptability, whole genome duplication (WGD) or segmental duplication events, distinct spatial and temporal patterns of expression might play driving forces for the variation of isoform numbers. (3) The 'birth and death' model of evolution could explain the evolution of actin gene family in ciliates. And actin genes have been generally under strong negative selection to maintain protein structures and physiological functions. Collectively, we provided meaningful information for understanding the evolution of eukaryotic actin gene family.

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.