Host association and intracellularity evolved multiple times independently in the Rickettsiales.

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.

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.

Holospora-like bacteria "Candidatus Gortzia yakutica" and Preeria caryophila: Ultrastructure, promiscuity, and biogeography of the symbionts.

Two already known representatives of Holospora-like bacteria, "Candidatus Gortzia yakutica" from Paramecium putrinum and Preeria caryophila, originally retrieved from the Paramecium aurelia complex, were found in new hosts: Paramecium nephridiatum and Paramecium polycaryum, respectively. In the present study, these bacteria were investigated using morphological and molecular methods. For "Ca. G. yakutica", the first details of the electron microscopic structure in the main and new hosts were provided. Regarding Pr. caryophila, the ultrastructural description of this species was implemented by several features previously unknown, such as the so called "membrane cluster" dividing periplasm from cytoplasm and fine composition of infectious forms before and during its releasing from the infected macronucleus. The new combinations of these Holospora-like bacteria with ciliate hosts were discussed from biogeographical and ecological points of view. Host specificity of symbionts as a general paradigm was critically reviewed as well.

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.

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.

Cryptic Diversity in Paramecium multimicronucleatum Revealed with a Polyphasic Approach.

Paramecium (Ciliophora) systematics is well studied, and about twenty morphological species have been described. The morphological species may include several genetic species. However, molecular phylogenetic analyses revealed that the species diversity within Paramecium could be even higher and has raised a problem of cryptic species whose statuses remain uncertain. In the present study, we provide the morphological and molecular characterization of two novel Paramecium species. While Paramecium lynni n. sp., although morphologically similar to P. multimicronucleatum, is phylogenetically well separated from all other Paramecium species, Paramecium fokini n. sp. appears to be a cryptic sister species to P. multimicronucleatum. The latter two species can be distinguished only by molecular methods. The number and structure of micronuclei, traditionally utilized to discriminate species in Paramecium, vary not only between but also within each of the three studied species and, thus, cannot be considered a reliable feature for species identification. The geographic distribution of the P. multimicronucleatum and P. fokini n. sp. strains do not show defined patterns, still leaving space for a role of the geographic factor in initial speciation in Paramecium. Future findings of new Paramecium species can be predicted from the molecular data, while morphological characteristics appear to be unstable and overlapping at least in some species.

'Candidatus Gromoviella agglomerans', a novel intracellular Holosporaceae parasite of the ciliate Paramecium showing marked genome reduction.

Holosporales are an alphaproteobacterial lineage encompassing bacteria obligatorily associated with multiple diverse eukaryotes. For most representatives, little is known on the interactions with their hosts. In this study, we characterized a novel Holosporales symbiont of the ciliate Paramecium polycaryum. This bacterium inhabits the host cytoplasm, frequently forming quite large aggregates. Possibly due to such aggregates, host cells sometimes displayed lethal division defects. The symbiont was also able to experimentally stably infect another Paramecium polycaryum strain. The bacterium is phylogenetically related with symbionts of other ciliates and diplonemids, forming a putatively fast-evolving clade within the family Holosporaceae. Similarly to many close relatives, it presents a very small genome (<600 kbp), and, accordingly, a limited predicted metabolism, implying a heavy dependence on Paramecium, thanks also to some specialized membrane transporters. Characterized features, including the presence of specific secretion systems, are overall suggestive of a mild parasitic effect on the host. From an evolutionary perspective, a potential ancestral trend towards pronounced genome reduction and possibly linked to parasitism could be inferred, at least among fast-evolving Holosporaceae, with some lineage-specific traits. Interestingly, similar convergent features could be observed in other host-associated lineages, in particular Rickettsiales among Alphaproteobacteria.

'Candidatus Sarmatiella mevalonica' endosymbiont of the ciliate Paramecium provides insights on evolutionary plasticity among Rickettsiales.

Members of the bacterial order Rickettsiales are obligatorily associated with a wide range of eukaryotic hosts. Their evolutionary trajectories, in particular concerning the origin of shared or differential traits among distant sub-lineages, are still poorly understood. Here, we characterized a novel Rickettsiales bacterium associated with the ciliate Paramecium tredecaurelia and phylogenetically related to the Rickettsia genus. Its genome encodes significant lineage-specific features, chiefly the mevalonate pathway gene repertoire, involved in isoprenoid precursor biosynthesis. Not only this pathway has never been described in Rickettsiales, it also is very rare among bacteria, though typical in eukaryotes, thus likely representing a horizontally acquired trait. The presence of these genes could enable an efficient exploitation of host-derived intermediates for isoprenoid synthesis. Moreover, we hypothesize the reversed reactions could have replaced canonical pathways for producing acetyl-CoA, essential for phospholipid biosynthesis. Additionally, we detected phylogenetically unrelated mevalonate pathway genes in metagenome-derived Rickettsiales sequences, likely indicating evolutionary convergent effects of independent horizontal gene transfer events. Accordingly, convergence, involving both gene acquisitions and losses, is highlighted as a relevant evolutionary phenomenon in Rickettsiales, possibly favoured by plasticity and comparable lifestyles, representing a potentially hidden origin of other more nuanced similarities among sub-lineages.

Evolutionary Plasticity of Mating-Type Determination Mechanisms in Paramecium aurelia Sibling Species.

The Paramecium aurelia complex, a group of morphologically similar but sexually incompatible sibling species, is a unique example of the evolutionary plasticity of mating-type systems. Each species has two mating types, O (Odd) and E (Even). Although O and E types are homologous in all species, three different modes of determination and inheritance have been described: genetic determination by Mendelian alleles, stochastic developmental determination, and maternally inherited developmental determination. Previous work in three species of the latter kind has revealed the key roles of the E-specific transmembrane protein mtA and its highly specific transcription factor mtB: type O clones are produced by maternally inherited genome rearrangements that inactivate either mtA or mtB during development. Here we show, through transcriptome analyses in five additional species representing the three determination systems, that mtA expression specifies type E in all cases. We further show that the Mendelian system depends on functional and nonfunctional mtA alleles, and identify novel developmental rearrangements in mtA and mtB which now explain all cases of maternally inherited mating-type determination. Epistasis between these genes likely evolved from less specific interactions between paralogs in the P. aurelia common ancestor, after a whole-genome duplication, but the mtB gene was subsequently lost in three P. aurelia species which appear to have returned to an ancestral regulation mechanism. These results suggest a model accounting for evolutionary transitions between determination systems, and highlight the diversity of molecular solutions explored among sibling species to maintain an essential mating-type polymorphism in cell populations.

Epidemiology of Nucleus-Dwelling Holospora: Infection, Transmission, Adaptation, and Interaction with Paramecium.

The chapter describes the exceptional symbiotic associations formed between the ciliate Paramecium and Holospora, highly infectious bacteria residing in the host nuclei. Holospora and Holospora-like bacteria (Alphaproteobacteria) are characterized by their ability for vertical and horizontal transmission in host populations, a complex biphasic life cycle, and pronounced preference for host species and colonized cell compartment. These bacteria are obligate intracellular parasites; thus, their metabolic repertoire is dramatically reduced. Nevertheless, they perform complex interactions with the host ciliate. We review ongoing efforts to unravel the molecular adaptations of these bacteria to their unusual lifestyle and the host's employment in the symbiosis. Furthermore, we summarize current knowledge on the genetic and genomic background of Paramecium-Holospora symbiosis and provide insights into the ecological and evolutionary consequences of this interaction. The diversity and occurrence of symbioses between ciliates and Holospora-like bacteria in nature is discussed in connection with transmission modes of symbionts, host specificity and compatibility of the partners. We aim to summarize 50 years of research devoted to these symbiotic systems and conclude trying to predict some perspectives for further studies.

The first case of microsporidiosis in Paramecium.

A new microsporidian species, Globosporidium paramecii gen. nov., sp. nov., from Paramecium primaurelia is described on the basis of morphology, fine structure, and SSU rRNA gene sequence. This is the first case of microsporidiosis in Paramecium reported so far. All observed stages of the life cycle are monokaryotic. The parasites develop in the cytoplasm, at least some part of the population in endoplasmic reticulum and its derivates. Meronts divide by binary fission. Sporogonial plasmodium divides by rosette-like budding. Early sporoblasts demonstrate a well-developed exospore forming blister-like structures. Spores with distinctive spherical shape are dimorphic in size (3.7 ± 0.2 and 1.9 ± 0.2 µm). Both types of spores are characterized by a thin endospore, a short isofilar polar tube making one incomplete coil, a bipartite polaroplast, and a large posterior vacuole. Experimental infection was successful for 5 of 10 tested strains of the Paramecium aurelia species complex. All susceptible strains belong to closely related P. primaurelia and P. pentaurelia species. Phylogenetic analysis placed the new species in the Clade 4 of Microsporidia and revealed its close relationship to Euplotespora binucleata (a microsporidium from the ciliate Euplotes woodruffi), to Helmichia lacustris and Mrazekia macrocyclopis, microsporidia from aquatic invertebrates.

Role of bacterivorous organisms on fungal-based systems for natural tannin degradation.

Tannery wastewater presents high concentrations of organic load and pollutant recalcitrant molecules (e.g. tannins), which reduce the efficiency of biological treatment processes. Recent studies showed that several fungal species and strains are effective in the degradation of tannins. However, high bacterial load can negatively affect fungal growth, reducing system stability and degradation performances. The aim of the present study was to evaluate the effects of the introduction of bacterivorous grazers (ciliates and/or rotifers) in batch scale experiments using fungi to remove Tara tannin, i.e. to check the potential synergistic effect between fungi and bacterivorous grazers in the degradation of recalcitrant compounds. In this context, the ciliated grazers Paramecium calkinsi, Tetrahymena sp., Pseudovorticella sp., and the rotifer Lecane inermis, preliminary selected according to their ability to grow in a solution prepared with Tara tannin, were separately tested. Activated sludge, including a complex mixture of native grazers, was used as experimental control. The following parameters were monitored: bacterial load, number of grazers/mL and Soluble Chemical Oxygen Demand (SCOD). Colony Forming Unit (CFU)/grazers ratio was also calculated. Particular attention was paid to: i) bacterial load reduction and ii) enhancement of recalcitrant compounds degradation, and we observed that in all experimental conditions where grazers occurred bacterial load was significantly reduced and the system achieved a higher SCOD removal in a shorter time. Our findings provide useful insights for the stabilization of fungal-based systems in non-sterile conditions.

Genetic diversity of Paramecium species on the basis of multiple loci analysis and ITS secondary structure models.

Among ciliates, Paramecium has become a privileged model for the study of "species problem" particularly in the case of the "Paramecium aurelia complex" that has been intensely investigated. Despite extensive studies, the taxonomy of Paramecium is still challenging. The major problem is an uneven sampling of Paramecium with relatively few representatives of each species. To investigate species from the less discovered region (Pakistan), 10 isolates of Paramecium species including a standing-alone FT8 strain previously isolated by some of us were subjected to molecular characterization. Fragments of 18S recombinant DNA (rDNA), ITS1-5.8S-ITS2-5'LSU rDNA, cytochrome c oxidase subunit II, and hsp70 genes were used as molecular markers for phylogenetic analysis of particular isolates. The nucleotide sequences of polymerase chain reaction products of all markers were compared with the available sequences of relevant markers of other Paramecium species from GenBank. Phylogenetic trees based on all molecular markers showed that all the nine strains had a very close relationship with Paramecium primaurelia except for the FT8 strain. FT8 consistently showed its unique position in comparison to all other species in the phylogenetic trees. Available sequences of internal transcribed spacer 1 (ITS1) and ITS2 and some other ciliate sequences from GenBank were used for the construction of secondary models. Two highly conserved helices supported by compensatory base changes among all ciliates of ITS2 secondary structures were found similar to other eukaryotes. Therefore, the most conserved 120 to 180 base pairs regions were identified for their comparative studies. We found that out of the three helices in ITS1 structure, helix B was more conserved in Paramecium species. Despite various substitutions in the primary sequence, it was observed that secondary structures of ITS1 and ITS2 could be helpful in interpreting the phylogenetic relationships both at species as well as at generic level.

The core microbiome of sessile ciliate Stentor coeruleus is not shaped by the environment.

Microbiomes of multicellular organisms are one of the hottest topics in microbiology and physiology, while only few studies addressed bacterial communities associated with protists. Protists are widespread in all environments and can be colonized by plethora of different bacteria, including also human pathogens. The aim of this study was to characterize the prokaryotic community associated with the sessile ciliate Stentor coeruleus. 16S rRNA gene metabarcoding was performed on single cells of S. coeruleus and on their environment, water from the sewage stream. Our results showed that the prokaryotic community composition differed significantly between Stentor cells and their environment. The core microbiome common for all ciliate specimens analyzed could be defined, and it was composed mainly by representatives of bacterial genera which include also potential human pathogens and commensals, such as Neisseria, Streptococcus, Capnocytophaga, Porphyromonas. Numerous 16S rRNA gene contigs belonged to endosymbiont "Candidatus Megaira polyxenophila". Our data suggest that each ciliate cell can be considered as an ecological microniche harboring diverse prokaryotic organisms. Possible benefits for persistence and transmission in nature for bacteria associated with protists are discussed. Our results support the hypothesis that ciliates attract potentially pathogenic bacteria and play the role of natural reservoirs for them.

Loss of a Fragile Chromosome Region leads to the Screwy Phenotype in Paramecium tetraurelia.

A conspicuous cell-shape phenotype known as "screwy" was reported to result from mutations at two or three uncharacterized loci in the ciliate Paramecium tetraurelia. Here, we describe a new screwy mutation, Spinning Top, which appeared spontaneously in the cross of an unrelated mutant with reference strain 51. The macronuclear (MAC) genome of the Spinning Top mutant is shown to lack a ~28.5-kb segment containing 18 genes at the end of one chromosome, which appears to result from a collinear deletion in the micronuclear (MIC) genome. We tested several candidate genes from the deleted locus by dsRNA-induced silencing in wild-type cells, and identified a single gene responsible for the phenotype. This gene, named Spade, encodes a 566-aa glutamine-rich protein with a C2HC zinc finger. Its silencing leads to a fast phenotype switch during vegetative growth, but cells recover a wild-type phenotype only 5-6 divisions after silencing is stopped. We analyzed 5 independently-obtained mutant alleles of the Sc1 locus, and concluded that all of them also lack the Spade gene and a number of neighboring genes in the MAC and MIC genomes. Mapping of the MAC deletion breakpoints revealed two different positions among the 5 alleles, both of which differ from the Spinning Top breakpoint. These results suggest that this MIC chromosome region is intrinsically unstable in strain 51.

Deianiraea, an extracellular bacterium associated with the ciliate Paramecium, suggests an alternative scenario for the evolution of Rickettsiales.

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.

Diversity and environmental distribution of the cosmopolitan endosymbiont "Candidatus Megaira".

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.

High-Throughput Sequencing of the 16S rRNA Gene as a Survey to Analyze the Microbiomes of Free-Living Ciliates Paramecium.

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 morpho-species 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.

Complex life cycle, broad host range and adaptation strategy of the intranuclear Paramecium symbiont Preeria caryophila comb. nov.

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.