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Members of the order Rickettsiales are often found in association with ciliated protists. An interesting case is the bacterial endosymbiont “ Candidatus Megaira”, which is phylogenetically closely related to the pathogen Rickettsia . “ Candidatus Megaira” was first described as an intracellular bacterium in several ciliate species. Since then it has been found in association with diverse evolutionary distantly-related hosts, among them other unicellular eukaryotes, and also algae, and metazoa, such as cnidarians. We provide the characterization of several new strains of the type species “ Candidatus Megaira polyxenophila”, and the multidisciplinary description of a novel species, “ Candidatus Megaira venefica”, presenting peculiar features, which highlight the diversity and variability of these widespread bacterial endosymbionts. Screening of the 16S rRNA gene short amplicon database and phylogenetic analysis of 16S rRNA gene hypervariable regions revealed the presence of further hidden lineages, and provided hints on the possibility that these bacteria may be horizontally transmitted among aquatic protists and metazoa. The phylogenetic reconstruction supports the existence of at least five different separate species-level clades of “ Candidatus Megaira”, and we designed a set of specific probes allowing easy recognition of the four major clades of the genus.

The order Rickettsiales ( Alphaproteobacteria ) encompasses multiple diverse lineages of host-associated bacteria, including pathogens, reproductive manipulators, and mutualists. Here, in order to understand how intracellularity and host association originated in this order, and whether they are ancestral or convergently evolved characteristics, we built a large and phylogenetically-balanced dataset that includes de novo sequenced genomes and a selection of published genomic and metagenomic assemblies. We perform detailed functional reconstructions that clearly indicates “late” and parallel evolution of obligate host-association in different Rickettsiales lineages. According to the depicted scenario, multiple independent horizontal acquisitions of transporters led to the progressive loss of biosynthesis of nucleotides, amino acids and other metabolites, producing distinct conditions of host-dependence. Each clade experienced a different pattern of evolution of the ancestral arsenal of interaction apparatuses, including development of specialised effectors involved in the lineage-specific mechanisms of host cell adhesion and/or invasion. Rickettsiales encompass diverse host-associated bacteria, including pathogens, parasites, and mutualists. This study shows that obligate associations with their hosts likely evolved multiple times independently, thus providing an alternative, generalisable view, on evolution of intracellularity.

Rickettsiales are a lineage of obligate intracellular Alphaproteobacteria , encompassing important human pathogens, manipulators of host reproduction, and mutualists. Here we report the discovery of a novel Rickettsiales bacterium associated with Paramecium , displaying a unique extracellular lifestyle, including the ability to replicate outside host cells. Genomic analyses show that the bacterium possesses a higher capability to synthesise amino acids, compared to all investigated Rickettsiales . Considering these observations, phylogenetic and phylogenomic reconstructions, and re-evaluating the different means of interaction of Rickettsiales bacteria with eukaryotic cells, we propose an alternative scenario for the evolution of intracellularity in Rickettsiales . According to our reconstruction, the Rickettsiales ancestor would have been an extracellular and metabolically versatile bacterium, while obligate intracellularity would have evolved later, in parallel and independently, in different sub-lineages. The proposed new scenario could impact on the open debate on the lifestyle of the last common ancestor of mitochondria within Alphaproteobacteria .

Paramecium (Ciliophora) is an ideal model organism to study the biogeography of protists. However, many regions of the world, such as Central America, are still neglected in understanding Paramecium diversity. We combined morphological and molecular approaches to identify paramecia isolated from more than 130 samples collected from different waterbodies in several states of Mexico. We found representatives of six Paramecium morphospecies, including the rare species Paramecium jenningsi, and Paramecium putrinum, which is the first report of this species in tropical regions. We also retrieved five species of the Paramecium aurelia complex, and describe one new member of the complex, Paramecium quindecaurelia n. sp., which appears to be a sister species of Paramecium biaurelia. We discuss criteria currently applied for differentiating between sibling species in Paramecium. Additionally, we detected diverse bacterial symbionts in some of the collected ciliates.

Ciliated protists often form symbioses with many diverse microorganisms. In particular, symbiotic associations between ciliates and green algae, as well as between ciliates and intracellular bacteria, are rather wide-spread in nature. In this study, we describe the complex symbiotic system between a very rare ciliate, Paramecium chlorelligerum, unicellular algae inhabiting its cytoplasm, and novel bacteria colonizing the host macronucleus. Paramecium chlorelligerum, previously found only twice in Germany, was retrieved from a novel location in vicinity of St. Petersburg in Russia. Species identification was based on both classical morphological methods and analysis of the small subunit rDNA. Numerous algae occupying the cytoplasm of this ciliate were identified with ultrastructural and molecular methods as representatives of the Meyerella genus, which before was not considered among symbiotic algae. In the same locality at least fifteen other species of \"green\" ciliates were found, thus it is indeed a biodiversity hot-spot for such protists. A novel species of bacterial symbionts living in the macronucleus of Paramecium chlorelligerum cells was morphologically and ultrastructurally investigated in detail with the description of its life cycle and infection capabilities. The new endosymbiont was molecularly characterized following the full-cycle rRNA approach. Furthermore, phylogenetic analysis confirmed that the novel bacterium is a member of Holospora genus branching basally but sharing all characteristics of the genus except inducing connecting piece formation during the infected host nucleus division. We propose the name \"Candidatus Holospora parva\" for this newly described species. The described complex system raises new questions on how these microorganisms evolve and interact in symbiosis.

Holospora and related bacteria are a group of obligate Paramecium symbionts. Characteristic features are their infectivity, the presence of two distinct morphotypes, and usually a strict specialization for a single Paramecium species as host and for a nuclear compartment (either somatic or generative nucleus) for reproduction. Holospora caryophila steps out of line, naturally occurring in Paramecium biaurelia and Paramecium caudatum. This study addresses the phylogenetic relationship among H. caryophila and other Holospora species based on 16S rRNA gene sequence comparison analyzing the type strain and seven new macronuclear symbionts. Key aspects of Holospora physiology such as infectivity, symbiosis establishment and host range were determined by comprehensive infection assays. Detailed morphological investigations and sequence-based phylogeny confirmed a high similarity between the type strain of H. caryophila and the novel strains. Surprisingly, they are only distantly related to other Holospora species suggesting that they belong to a new genus within the family Holosporaceae, here described as Preeria caryophila comb. nov. Adding to this phylogenetic distance, we also observed a much broader host range, comprising at least eleven Paramecium species. As these potential host species exhibit substantial differences in frequency of sexual processes, P. caryophila demonstrates which adaptations are crucial for macronuclear symbionts facing regular destruction of their habitat.

Ciliates are the largest group of ubiquitous aquatic bacterivorous protists, and many species are easily cultivated. However, only few studies reported prokaryotic communities naturally associated with ciliate cells. Herein, we analyzed the microbiome composition of several strains of Paramecium (Ciliophora) originating from different locations and belonging to two morphospecies by high-throughput sequencing (HTS) of the 16S rRNA gene. Possible reasons of HTS results bias were addressed comparing DNA libraries obtained using different primers and different number of ciliate cells. Microbiomes associated with ciliates and their environments were always significantly different by prokaryotic taxonomic composition and bacterial richness. There were also pronounced differences between Paramecium strains. Interestingly, potentially pathogenic bacteria were revealed in Paramecium microbiomes.

Hot springs are known as highly adverse extreme environments where thermophilic and hyperthermophilic microorganisms can survive. We describe taxonomic diversity of several microbial biofilms collected along water temperature gradient in hot streams in the aquatic system of the Stolbovskie hot springs on Kunashir Island, Kurils, Russia. The taxonomic composition of the studied microbial communities was assessed by the 16S rRNA gene metabarcoding for bacteria and archaea, and by the 18S rRNA gene metabarcoding for protists. Richness and diversity of bacteria in the geothermal microbial communities decreased with the increase of temperature, while for archaea, the tendency was the opposite. Ciliophora was the most represented taxon of protists. The biofilms of various kinds that we found in a very local area of the geothermal system were different from each other by taxonomic composition, and the level of their taxonomic diversity was significantly influenced by water temperature.

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.

Current understanding of the population genetics of free-living unicellular eukaryotes is limited, and the amount of genetic variability in these organisms is still a matter of debate. We characterized--reproductively and genetically--worldwide samples of multiple Paramecium species belonging to a cryptic species complex, Paramecium aurelia, whose species have been shown to be reproductively isolated. We found that levels of genetic diversity both in the nucleus and in the mitochondrion are substantial within groups of reproductively compatible P. aurelia strains but drop considerably when strains are partitioned according to their phylogenetic groupings. Our study reveals the existence of discrepancies between the mating behavior of a number of P. aurelia strains and their multilocus genetic profile, a controversial finding that has major consequences for both the current methods of species assignment and the species problem in the P. aurelia complex. [PUBLICATION ABSTRACT]