Free-living Ciliates Research Articles

Comparative genomics reveals the evolutionary history of the unicellular eukaryote class Litostomatea and its adaptive evolution based on biochemical metabolic capacity

Abstract Ciliated protists are unicellular eukaryotic organisms characterized by their morphological diversity, ubiquitous distribution, and the important roles they play in a wide range of biological studies. The class Litostomatea is a morphologically diverse ciliate group that comprises hundreds of free-living and endosymbiotic species. Here, we sequenced 14 predatory litostomateans, i.e. 12 haptorians and two rhynchostomatians. A comparative analysis was performed with other published omics’ data on litostomateans. Our first phylogenomic analysis of litostomateans showed the monophyly of the subclasses Trichostomatia and Rhynchostomatia, the non-monophyly of the subclass Haptoria, and the monophyly of all orders and families that were analysed. Evolutionary history analysis suggested that Litostomatea diverged during the Late Neoproterozoic, the family Chaeneidae was the earliest diverging haptorian lineage, and the Rhynchostomatia probably separated from the order Lacrymariida (subclass Haptoria) during the Early Palaeozoic. Stop codon usage analysis of 28 litostomateans showed that they use TAA as the biased stop codon and reassign the other two stop codons (TAG and TGA) to code for amino acids. In addition, the preferred codons in the 14 newly sequenced litostomateans are strongly biased towards A/U bases in the third position, very probably due to the comparatively low GC content. Genes encoding carbohydrate-active enzymes (CAZymes) are more diversified in the endosymbiotic Trichostomatia than in the free-living predatory Rhynchostomatia and Haptoria, suggesting that trichostomes have the strongest capability of carbohydrate utilization. Notably, we found that three free-living litostomateans (Didinium sp.1, Myriokaryon sp., and Apodileptus visscheri) exhibit substantial differences from other free-living ciliates in terms of their number of CAZymes. Considering the potency and versatility of CAZymes in the degradation and biotransformation of carbohydates, we propose that the multifarious CAZymes in these three ciliates could be a survival strategy for nutrient acquisition and niche adaptation. Finally, the functional annotation of significantly expanded gene families in these three ciliates revealed their vigorous potency in biochemical metabolism. These findings will facilitate wider omic-scale phylogenetic analyses of Litostomatea and deepen our understanding of this group from an evolutionary standpoint.

Bit by bit toward the diversity of metopids: Description of the genus Pidimetopus n. gen. (Ciliophora: Armophorea).

While metopids (Armophorea: Metopida) represent the most species-rich group of free-living anaerobic ciliates thriving in hypoxic environments, our understanding of their true diversity remains incomplete. Most metopid species are still characterized only morphologically. Particularly, the so-called IAC clade (named in the past after some of the taxa included, Idiometopus, Atopospira, and Clevelandellida), comprising free-living members as well as the endosymbiotic ones (order Clevelandellida), is in serious need of revision. In our study, we establish a new free-living genus in the IAC clade, Pidimetopus n. gen., with descriptions of two new species, P. nanus n. sp., and P. permonicus n. sp., using up-to-date molecular and morphologic methods. The genus is characterized by small cells (up to 75 μm long), not more than 10 adoral membranelles and eight somatic kineties, and usually, four long caudal cilia that can stiffen. In addition to morphologic and molecular characterizations, we also conducted a statistical morphotype analysis of the polymorphic species P. nanus n. sp. We discuss the relevance of the earlier morphologically described species Metopus minor as a putative collective taxon for several small metopids less than 50 μm long.

Methanogenic symbionts of anaerobic ciliates are host and habitat specific.

The association between anaerobic ciliates and methanogenic archaea has been recognized for over a century. Nevertheless, knowledge of these associations is limited to a few ciliate species, and so the identification of patterns of host-symbiont specificity has been largely speculative. In this study, we integrated microscopy and genetic identification to survey the methanogenic symbionts of 32 free-living anaerobic ciliate species, mainly from the order Metopida. Based on Sanger and Illumina sequencing of the 16S rRNA gene, our results show that a single methanogenic symbiont population, belonging to Methanobacterium, Methanoregula, or Methanocorpusculum, is dominant in each host strain. Moreover, the host's taxonomy (genus and above) and environment (i.e. endobiotic, marine/brackish, or freshwater) are linked with the methanogen identity at the genus level, demonstrating a strong specificity and fidelity in the association. We also established cultures containing artificially co-occurring anaerobic ciliate species harboring different methanogenic symbionts. This revealed that the host-methanogen relationship is stable over short timescales in cultures without evidence of methanogenic symbiont exchanges, although our intraspecific survey indicated that metopids also tend to replace their methanogens over longer evolutionary timescales. Therefore, anaerobic ciliates have adapted a mixed transmission mode to maintain and replace their methanogenic symbionts, allowing them to thrive in oxygen-depleted environments.

Molecular basis of phenotypic plasticity in a marine ciliate.

Phenotypic plasticity, which involves phenotypic transformation in the absence of genetic change, may serve as a strategy for organisms to survive in complex and highly fluctuating environments. However, its reaction norm, molecular basis, and evolution remain unclear in most organisms, especially microbial eukaryotes. In this study, we explored these questions by investigating the reaction norm, regulation, and evolution of phenotypic plasticity in the cosmopolitan marine free-living ciliates Glauconema spp., which undergo significant phenotypic changes in response to food shortages. This study led to the de novo assembly of macronuclear genomes using long-read sequencing, identified hundreds of differentially expressed genes associated with phenotypic plasticity in different life stages, validated the function of two of these genes, and revealed that the reaction norm of body shape in response to food density follows a power-law distribution. Purifying selection may be the dominant evolutionary force acting on the genes associated with phenotypic plasticity, and the overall data support the hypothesis that phenotypic plasticity is a trait maintained by natural selection. This study provides novel insight into the developmental genetics of phenotypic plasticity in non-model unicellular eukaryotes and sheds light on the complexity and long evolutionary history of this important survival strategy.

Study of Free-living Protozoa in Azerbaijan

The article provides data on the biological significance of two groups of free-living Protozoa: testate amoebae and free-living ciliates. Their importance and role in the main biological processes of production and destruction of organic matter in both water and soil ecosystems are indicated. A review of the knowledge of these two groups of Protozoa in Azerbaijan is given in chronological order, indicating the most important publications of the early authors as well as modern ones. A review of modern works on the ecology of free-living Protozoa was carried out, including practical importance for humans (The use of Protozoa as indicators of the organic pollution degree and in toxicological studies).

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.

Cilioplankton Communities as an Indicator of the Pollution Degree in the Caspian Sea

During 2014-2021 years a study of the species diversity of planktonic ciliates of the Caspian coast of the Absheron Peninsula was conducted. As a result of research in planktonic communities of free-living ciliates, 62 species, belonging to 22 families were observed. Among the recorded families, Strombidiidae takes first place in terms of the number of species. The families Uronychiidae, Nassulidae, Epistylidae, and Zoothamniidae were represented by only one species. A correlation between similarity of the planktonic ciliates species diversity and the degree of organic pollution of coastal area marine water has been established. The results of the cluster analysis showed that the highest similarity of the species diversity (95.5%) was noted for two collection points on the northern coast of Absheron, near the settlements of Novkhani and Bilgah. This is due to the fact that the northern coast is less polluted by oil products and domestic sewage. Thus, according to the similarity of species diversity of planktonic communities of ciliates on the coast of the Absheron Peninsula, the two clusters are clearly distinguished. One unites the cleanest sites (Novkhani and Bilgah — 95.5%), and all the other 4 stationary points of the collection (Sumgait, Turkan, Shikh, Sangachal — more than 50%) are combined into the second cluster.

Rediscovery of Remarkably Rare Anaerobic Tentaculiferous Ciliate Genera Legendrea and Dactylochlamys (Ciliophora: Litostomatea).

Free-living anaerobic ciliates are of considerable interest from an ecological and an evolutionary standpoint. Extraordinary tentacle-bearing predatory lineages have evolved independently several times within the phylum Ciliophora, including two rarely encountered anaerobic litostomatean genera, Legendrea and Dactylochlamys. In this study, we significantly extend the morphological and phylogenetic characterization of these two poorly known groups of predatory ciliates. We provide the first phylogenetic analysis of the monotypic genus Dactylochlamys and the three valid species of Legendrea based on the 18S rRNA gene and ITS-28S rRNA gene sequences. Prior to this study, neither group had been studied using silver impregnation methods. We provide the first protargol-stained material and also a unique video material including documentation, for the first time, of the hunting and feeding behavior of a Legendrea species. We briefly discuss the identity of methanogenic archaeal and bacterial endosymbionts of both genera based on 16S rRNA gene sequences, and the importance of citizen science for ciliatology from a historical and contemporary perspective.

Species-Specific Duplication of Surface Antigen Genes in Paramecium.

Paramecium is a free-living ciliate that undergoes antigenic variation and still the functions of these variable surface antigen coats in this non-pathogenic ciliate remain elusive. Only a few surface antigen genes have been described, mainly in the two model species P. tetraurelia strain 51 and P. primaurelia strain 156. Given the lack of suitable sequence data to allow for phylogenetics and deeper sequence comparisons, we screened the genomes of six different Paramecium species for serotype genes and isolated 548 candidates. Our approach identified the subfamilies of the isogenes of individual serotypes that were mostly represented by intrachromosomal gene duplicates. These showed different duplication levels, and chromosome synteny suggested rather young duplication events after the emergence of the P. aurelia species complex, indicating a rapid evolution of surface antigen genes. We were able to identify the different subfamilies of the surface antigen genes with internal tandem repeats, which showed consensus motifs across species. The individual isogene families showed additional consensus motifs, indicating that the selection pressure holds individual amino acids constant in these repeats. This may be a hint of the receptor function of these antigens rather than a presentation of random epitopes, generating the variability of these surface molecules.

Biodiversity of Benthic Ciliates of Fresh Waters of Lankaran Natural Area

The article presents data on the species diversity of benthic ciliates in various water bodies of the Lankaran natural area. The conducted studies have shown that the greatest species diversity of free-living ciliates was observed on silty soils prevailing in the fresh waters of the natural area. Among them, the biotopes of gray oily and algal oozes should be noted, on which more than 60% of all species were found. When studying ciliates in relation to the active reaction of the environment, it was revealed that, although in most cases, the maximum number of species is observed in a slightly alkaline environment, sometimes there are exceptions. Since ciliates belonging to the euryionic group have been noted several times at pH above 8.5 and at pH 4.7 with a relatively high number. The diversity of conditions in freshwater bodies is the main for the rich species diversity of freshwater benthic ciliafauna. In addition, observations of the food relationships of free-living ciliates with other groups of benthic inhabitants are presented. The noted ciliates were divided into groups, such as phytophages, bacteriophages, predators, and their diet was studied. Small diatoms have been found to be of great importance in the diet of many species. And the free-living ciliates themselves are food objects of large predatory species. Thus, we observed that representatives of the Trachelidae family actively feed on many bacteriophages ciliates.

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

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

Targeting the Contractile Vacuole of Parasites

The contractile vacuole (CV) is a poorly studied organelle involved in osmotic regulation. In every contraction cycle, the CV expels excess liquid to maintain osmoregulation. CVs are found in single‐celled eukaryotes including parasitic and free‐living ciliates. Parasitic examples include Trypanosoma cruzi, amoebas, and Leishmaniaparasites. We use the free‐living ciliate Vorticella convallaria as a model for CV cycling, which can be observed directly under a microscope. The CV contraction rate in Vorticella convallaria,increases with temperature within physiologically relevant non‐lethal temperature ranges. We performed a historical literature review of CV cycling rates and temperature across ciliates and amoeba. The Arrhenius equation was used to calculate the activation energy barrier for CV contraction. The slope of the Arrhenius graph was similar across all organisms ranging from 6000 to 17000 calories (per mol CV cycle), suggesting that conserved chemical or physical processes control CV cycling across biology. Future research will investigate whether using drugs to inhibit the CV will change the slope of the Arrhenius graph in Vorticella convallaria,and whether those same drugs will exacerbate osmotic cell death in the parasite Crithidia fasciculata. Ultimately, our results may contribute to the development of anti‐parasitic drugs targeting parasites’ unique CV organelle.

The Codon Usage Bias Analysis of Free-Living Ciliates' Macronuclear Genomes and Clustered Regularly Interspaced Short Palindromic Repeats/Cas9 Vector Construction of Stylonychia lemnae.

Ciliates represent higher unicellular animals, and several species are also important model organisms for molecular biology research. Analyses of codon usage bias (CUB) of the macronuclear (MAC) genome in ciliates can not only promote a better understanding of the genetic mode and evolution history of these organisms but also help optimize codons to improve the gene editing efficiency of model ciliates. In this study, macronuclear genome sequences of nine free-living ciliates were analyzed with CodonW software to calculate the following indices: the guanine-cytosine content (GC); the frequency of the nucleotides U, C, A, and G at the third position of codons (U3s, C3s, A3s, G3s); the effective number of codons (ENC); the correlation between GC at the first and second positions (GC12); the frequency of the nucleotides G + C at the third position of synonymous codons (GC3s); the relative synonymous codon usage (RSCU). Parity rule 2 plot analysis, neutrality plot analysis, and correlation analysis were performed to explore the factors that influence codon preference. The results showed that the GC contents in nine ciliates’ MAC genomes were lower than 50% and appeared AT-rich. The base compositions of GC12 and GC3s are markedly distinct and the codon usage pattern and evolution of ciliates are affected by genetic mutation and natural selection. According to the synonymous codon analysis, the codons of most ciliates ended with A or U and eight codons were the general optimal codons of nine ciliates. A clustered regularly interspaced short palindromic repeats/Cas9 (CRISPR/Cas9) expression vector of Stylonychia lemnae was constructed by optimizing the macronuclear genome codon and was successfully used to knock out the Adss gene. This is the first such extensive investigation of the MAC genome CUB of ciliates and the initial successful application of the CRISPR/Cas9 technique in free-living ciliates.

A Case Building Ciliate in the Genus Pseudoblepharisma Found in Subtropical Fresh Water

The genus Pseudoblepharisma is currently comprised of only one species, P. tenue, and one variant, P. tenue var. viride, both described as free-living ciliates thriving in oxygen depleted freshwater habitats of Europe. Here we report on this genus being discovered from subtropical freshwaters of Florida, USA. The Florida strain diverges from the P. tenue by being much larger, as well as having a far higher density of intracellular symbiotic green algae, resembling P. tenue var. viride. Unlike its European counterparts, the North American strain was observed to build a lorica, likely useful for protection and feeding; this has not been previously described for this genus. In contrast to P. tenue, the Florida strain does not have endosymbiotic purple bacteria. Despite large distances between sampling zones, the species is a close match at a morphological level to Pseudoblepharisma tenue var. viride. We provide the first 18S rRNA gene sequence for this species, allowing future investigations into the biogeography of this genus. As intensive sampling efforts continue to increase, cryptic microbial species will continue to be recorded from diverse freshwater habitats at a global scale.

PCD Genes-From Patients to Model Organisms and Back to Humans.

Primary ciliary dyskinesia (PCD) is a hereditary genetic disorder caused by the lack of motile cilia or the assembxly of dysfunctional ones. This rare human disease affects 1 out of 10,000–20,000 individuals and is caused by mutations in at least 50 genes. The past twenty years brought significant progress in the identification of PCD-causative genes and in our understanding of the connections between causative mutations and ciliary defects observed in affected individuals. These scientific advances have been achieved, among others, due to the extensive motile cilia-related research conducted using several model organisms, ranging from protists to mammals. These are unicellular organisms such as the green alga Chlamydomonas, the parasitic protist Trypanosoma, and free-living ciliates, Tetrahymena and Paramecium, the invertebrate Schmidtea, and vertebrates such as zebrafish, Xenopus, and mouse. Establishing such evolutionarily distant experimental models with different levels of cell or body complexity was possible because both basic motile cilia ultrastructure and protein composition are highly conserved throughout evolution. Here, we characterize model organisms commonly used to study PCD-related genes, highlight their pros and cons, and summarize experimental data collected using these models.

HAP2-Mediated Gamete Fusion: Lessons From the World of Unicellular Eukaryotes.

Most, if not all the cellular requirements for fertilization and sexual reproduction arose early in evolution and are retained in extant lineages of single-celled organisms including a number of important model organism species. In recent years, work in two such species, the green alga, Chlamydomonas reinhardtii, and the free-living ciliate, Tetrahymena thermophila, have lent important new insights into the role of HAP2/GCS1 as a catalyst for gamete fusion in organisms ranging from protists to flowering plants and insects. Here we summarize the current state of knowledge around how mating types from these algal and ciliate systems recognize, adhere and fuse to one another, current gaps in our understanding of HAP2-mediated gamete fusion, and opportunities for applying what we know in practical terms, especially for the control of protozoan parasites.

Overview of the Diversity, Phylogeny and Biogeography of Strombidiid Oligotrich Ciliates (Protista, Ciliophora), With a Brief Revision and a Key to the Known Genera.

Strombidiids are common free-living ciliates that have colonized coastal and open oceanic waters across the world. In recent years, numerous new taxa and gene sequences of strombidiids have been reported, revealing a large diversity of both their morphologic and genetic features. Here, we compare the taxonomic characters of all genera in the family Strombidiidae, provide a key to their identification, and investigate their molecular phylogeny. In addition, we analyze their regional distribution based on faunal data accumulated in China and attempt to infer their global distribution based on SSU rRNA gene sequence data. The current work revises the systematics of strombidiids based on morphologic, phylogenetic, and biogeographic evidence and provides a genus-level review of marine strombidiids.

PRIMER REGISTRO DE CILIADOS DE VIDA LIBRE (ALVEOLATA) DE LA CASCADA DE CACALOTENANGO, GUERRERO, MEXICO

Eleven morphospecies belonging to seven different genera (Coleps, Vorticella, Halteria, Spirostomum, Loxodes, Cyclidium, and Euplotes) of free-living ciliates were recorded for the first time in Cacalotenango fall in Guerrero state, Mexico. Coleps hirtus and Vorticella microstoma the most abundant species in the samples.

Spatiotemporal distribution of planktonic ciliates in the Kuybyshev water reservoir in the area of Klimovskaya narrowing

The paper attempts to link changes in quantitative characteristics of the community of free-living planktonic ciliates and the nature of their vertical distribution with the operation mode of the hydroelectric power station in the reservoir. Low values of the abundance (8–1,560 thousand cells / m3) and biomass (0.1–66.9 mg / m3) of ciliates in the water column, and a fairly wide range of their variation in the reservoir are probably due to the oscillatory nature of the hydrological regime in the regulated river conditions. The vertical distribution of free-living planktonic ciliates of the near-dam section of the Kuibyshev reservoir was studied at all times of the day on all days of the week. The species were distributed throughout the water column to the bottom with some preference for the surface (0–2 m) and medium (4–10 m) layers. There was no clear attachment of individual species to certain layers in the conditions of homothermy. The preference for certain horizons is due to some biological characteristics of the species (for example, peritriches on the coenobia of algae in the surface layers), departure from “blooming spots”, etc. No data in favor of vertical migrations or “turbulent transport” has yet been revealed. The influence of the weekly regime of regulation of the Zhigulevskaya HPS, in which at the end of the working week, the fluctuation amplitude of many hydrological parameters probably decreases, is manifested in an increase in the abundance and biomass of planktonic ciliates at this time.The nature of the daily change in the average ciliates abundance in the water column was the same for a variant of the community in working days and was in antiphase with the change in the community abundance on weekends, namely: the maximum number on working days and on weekends was recorded in the evening (20.00) and in the daytime (14.00), respectively.

Macroevolutionary analyses of ciliates associated with hosts support high diversification rates

Ciliophora is a phylum that is comprised of extremely diverse microorganisms with regard to their morphology and ecology. They may be found in various environments, as free-living organisms or associated with metazoans. Such associations range from relationships with low metabolic dependence such as epibiosis, to more intimate relationships such as mutualism and parasitism. We know that symbiotic relationships occur along the whole phylogeny of the group, however, little is known about their evolution. Theoretical studies show that there are two routes for the development of parasitism, yet few authors have investigated the evolution of these characteristics using molecular tools. In the present study, we inferred a wide dated molecular phylogeny, based on the 18S rDNA gene, for the entire Ciliophora phylum, mapped life habits throughout the evolutionary time, and evaluated whether symbiotic relationships were linked to the variation in diversification rates and to the mode of evolution of ciliates. Our results showed that the last common ancestor for Ciliophora was likely a free-living organism, and that parasitism is a recent adaptation in ciliates, emerging more than once and independently via two distinct routes: (i) a free-living ciliate evolved into a mutualistic organism and, later, into a parasitic organism, and (ii) a free-living ciliate evolved directly into a parasitic organism. Furthermore, we have found a significant increase in the diversification rate of parasitic and mutualistic ciliates compared with their free-living conspecifics. The evolutionary success in different lineages of symbiont ciliates may be associated with many factors including type and colonization placement on their host, as well as physical and physiological conditions made available by the hosts.