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.

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

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

'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.

The neotypification of Frontonia vernalis (Ehrenberg, 1833) Ehrenberg, 1838 and the description of Frontonia paravernalis sp. nov. trigger a critical revision of frontoniid systematics.

BACKGROUND: Among Oligohymenophorea (Ciliophora, Alveolata) the subclass Peniculia stands as one of the most well-known groups. Frontonia is the largest genus of Peniculia, and its representatives are spread in any type of water bodies as well as in soil. At a first glance, Frontonia species exhibit an overall similar morphology, and form a well-recognizable taxon of ciliates. Despite the general morphological homogeneity, the phylogenetic analysis based on the 18S rDNA sequencing showed that Frontonia is a non-monophyletic group. The systematics of this genus should be deeply reviewed, although additional issues complicate the task solving. First, type species of the genus is not yet clearly established, and no type material is available. In this context, the situation of F. vernalis, one of the first Frontonia ever described, is somehow puzzled: the description of this ciliate made by Ehrenberg (in 1833 and 1838) contains several inaccuracies and subsequent misidentifications by other authors occurred. Moreover, the 18S rDNA sequence of a putative F. vernalis is available on GenBank, but no morphological description of the correspondent specimens is provided; thus, in our opinion, it should be only prudently associated with F. vernalis or at least indicated as "F. vernalis". RESULTS: In the present work, we provide the neotypification of F. vernalis newly found in Italy, presenting its multidisciplinary description and its neotype material. Similarly, we describe a novel species bearing Chlorella-like endosymbionts, Frontonia paravernalis sp. nov., retrieved in two far distant locations (Italy, Russia). A critical discussion on the status of Frontonia taxonomy and phylogeny is also presented, based on the 18S rDNA sequencing of both these two newly collected species and other 14 frontoniids isolated in different parts of the world. Finally, in the present study F. leucas was neotypified and proposed as the type species of the genus. CONCLUSIONS: Green frontoniids form a monophyletic clade of freshwater organisms characterized by having a single contractile vacuole and bearing intracytoplasmatic Chlorella-like symbionts. With the neotypification of F. vernalis and F. leucas a fundamental step in Frontonia systematics was taken, and the bases for further taxonomic studies were laid.

Prof. H.-D. Görtz and his contribution to our knowledge of protozoan symbiosis.

Since the beginning of the 1980s, the Paramecium-Holospora system has become an important model and a subject of international scientific cooperation in symbiosis studies, established by Prof. H.-D. Görtz. In the article, a brief sketch of the scientist's life, his areas of interest, expertise and his contribution to our knowledge of protozoan symbiosis are discussed.

"Candidatus Trichorickettsia mobilis", a Rickettsiales bacterium, can be transiently transferred from the unicellular eukaryote Paramecium to the planarian Dugesia japonica.

Most of the microorganisms responsible for vector-borne diseases (VBD) have hematophagous arthropods as vector/reservoir. Recently, many new species of microorganisms phylogenetically related to agents of VBD were found in a variety of aquatic eukaryotic hosts; in particular, numerous new bacterial species related to the genus Rickettsia (Alphaproteobacteria, Rickettsiales) were discovered in protist ciliates and other unicellular eukaryotes. Although their pathogenicity for humans and terrestrial animals is not known, several indirect indications exist that these bacteria might act as etiological agents of possible VBD of aquatic organisms, with protists as vectors. In the present study, a novel strain of the Rickettsia-Like Organism (RLO) endosymbiont "Candidatus (Ca.) Trichorickettsia mobilis" was identified in the macronucleus of the ciliate Paramecium multimicronucleatum. We performed transfection experiments of this RLO to planarians (Dugesia japonica) per os. Indeed, the latter is a widely used model system for studying bacteria pathogenic to humans and other Metazoa. In transfection experiments, homogenized paramecia were added to food of antibiotic-treated planarians. Treated and non-treated (i.e. control) planarians were investigated at day 1, 3, and 7 after feeding for endosymbiont presence by means of PCR and ultrastructural analyses. Obtained results were fully concordant and suggest that this RLO endosymbiont can be transiently transferred from ciliates to metazoans, being detected up to day 7 in treated planarians' enterocytes. Our findings might offer insights into the potential role of ciliates or other protists as putative vectors for diseases caused by Rickettsiales or other RLOs and occurring in fish farms or in the wild.

Parablepharisma (Ciliophora) is not a Heterotrich: A Phylogenetic and Morphological Study with the Proposal of New Taxa.

The genus Parablepharisma Jankowski, 2007 at present includes five species, most of which have not been studied in detail, therefore phylogenetic affinities remained uninvestigated up to now. Parablepharisma is traditionally placed within Heterotrichea based on insufficient existing morphological data, and there are no available Parablepharisma gene sequences in molecular databases to support this placement. This work presents an 18S rDNA-based phylogeny of P. bacteriophora (Kahl, 1932) Jankowski, 2007 and P. brasiliensis sp. nov. We also provide a redescription of P. bacteriophora and P. chlamydophorum (Kahl, 1932) Jankowski, 2007 based on live morphological observations combined with silver impregnation and scanning and transmission electron microscopy. According to characters such as macro- and micronucleus number, the position of the ectosymbiotic bacteria, and the presence/absence of caudal cilia, two new species are described, i.e. P. granulata sp. nov. and P. brasiliensis sp. nov. In addition, we establish Kahlium gen. nov. to include P. chlamydophorum, which has a segmented anterior paroral portion and a twisted posterior paroral section as diagnostic features. To include Parablepharisma and Kahlium gen. nov., we propose Parablepharismidae fam. nov. According to our phylogenetic analyses, Parablepharisma belongs to SAL (Spirotrichea, Armophorea, Litostomatea), being a sister group of Cariacotrichea.

"Candidatus Hafkinia simulans" gen. nov., sp. nov., a Novel Holospora-Like Bacterium from the Macronucleus of the Rare Brackish Water Ciliate Frontonia salmastra (Oligohymenophorea, Ciliophora): Multidisciplinary Characterization of the New Endosymbiont and Its Host.

We characterized a novel Holospora-like bacterium (HLB) (Alphaproteobacteria, Holosporales) living in the macronucleus of the brackish water ciliate Frontonia salmastra. This bacterium was morphologically and ultrastructurally investigated, and its life cycle and infection capabilities were described. We also obtained its 16S rRNA gene sequence and performed in situ hybridization experiments with a specifically-designed probe. A new taxon, "Candidatus Hafkinia simulans", was established for this HLB. The phylogeny of the family Holosporaceae based on 16S rRNA gene sequences was inferred, adding to the already available data both the sequence of the novel bacterium and those of other Holospora and HLB species recently characterized. Our phylogenetic analysis provided molecular support for the monophyly of HLBs and placed the new endosymbiont as the sister genus of Holospora. Additionally, the host ciliate F. salmastra, recorded in Europe for the first time, was concurrently described through a multidisciplinary study. Frontonia salmastra's phylogenetic position in the subclass Peniculia and the genus Frontonia was assessed according to 18S rRNA gene sequencing. Comments on the biodiversity of this genus were added according to past and recent literature.

The Hidden World of Rickettsiales Symbionts: "Candidatus Spectririckettsia obscura," a Novel Bacterium Found in Brazilian and Indian Paramecium caudatum.

Symbioses between bacteria and eukaryotes are widespread and may have significant impact on the evolutionary history of symbiotic partners. The order Rickettsiales is a lineage of intracellular Alphaproteobacteria characterized by an obligate association with a wide range of eukaryotic hosts, including several unicellular organisms, such as ciliates and amoebas. In this work, we characterized the Rickettsiales symbionts associated with two different genotypes of the freshwater ciliate Paramecium caudatum originated from freshwater environments in distant geographical areas. Phylogenetic analyses based on 16S rRNA gene showed that the two symbionts are closely related to each other (99.4% identity), belong to the family Rickettsiaceae, but are far-related with respect to previously characterized Rickettsiales. Consequently, they were assigned to a new species of a novel genus, namely "Candidatus Spectririckettsia obscura." Screening on a database of short reads from 16S rRNA gene amplicon-based profiling studies confirmed that bacterial sequences related to the new symbiont are preferentially retrieved from freshwater environments, apparently with extremely scarce occurrence (< 0.1% positive samples). The present work provides new information on the still under-explored biodiversity of Rickettsiales, in particular those associated to ciliate host cells.

Detecting Associations Between Ciliated Protists and Prokaryotes with Culture-Independent Single-Cell Microbiomics: a Proof-of-Concept Study.

Symbioses between prokaryotes and microbial eukaryotes, particularly ciliated protists, have been studied for a long time. Nevertheless, researchers have focused only on a few host genera and species, mainly due to difficulties in cultivating the hosts, and usually have considered a single symbiont at a time. Here, we present a pilot study using a single-cell microbiomic approach to circumvent these issues. Unicellular ciliate isolation followed by simultaneous amplification of eukaryotic and prokaryotic markers was used. Our preliminary test gave reliable and satisfactory results both on samples collected from different habitats (marine and freshwater) and on ciliates belonging to different taxonomic groups. Results suggest that, as already assessed for many macro-organisms like plants and metazoans, ciliated protists harbor distinct microbiomes. The applied approach detected new potential symbionts as well as new hosts for previously described ones, with relatively low time and cost effort and without culturing. When further developed, single-cell microbiomics for ciliates could be applied to a large number of studies aiming to unravel the evolutionary and ecological meaning of these symbiotic systems.

Symbiont replacement between bacteria of different classes reveals additional layers of complexity in the evolution of symbiosis in the ciliate Euplotes.

Symbiosis is a diverse and complex phenomenon requiring diverse model systems. The obligate relationship between a monophyletic group of Euplotes species ("clade B") and the betaproteobacteria Polynucleobacter and "Candidatus Protistobacter" is among the best-studied in ciliates, and provides a framework to investigate symbiont replacements. Several other Euplotes-bacteria relationships exist but are less understood, such as the co-dependent symbiosis between Euplotes magnicirratus (which belongs to "clade A") and the alphaproteobacterium "Candidatus Devosia euplotis". Here we describe a new Devosia inhabiting the cytoplasm of a strain of Euplotes harpa, a clade B species that usually depends on Polynucleobacter for survival. The novel bacterial species, "Candidatus Devosia symbiotica", is closely related to the symbiont of E. magnicirratus, casting a different light on the history of bacteria colonizing ciliates of this genus. The two Devosia species may have become symbionts independently or as the result of a symbiont exchange between hosts, in either case replacing a previous essential bacterium in E. harpa. Alternatively, both may be remnants of an ancient symbiotic relationship between Euplotes and Devosia, in which case Polynucleobacter and "Ca. Protistobacter" are recent invaders. Either way, symbiont replacement between bacteria belonging to different classes must be evoked to explain this fascinating system.

Strengths and Biases of High-Throughput Sequencing Data in the Characterization of Freshwater Ciliate Microbiomes.

Molecular surveys of eukaryotic microbial communities employing high-throughput sequencing (HTS) techniques are rapidly supplanting traditional morphological approaches due to their larger data output and reduced bench work time. Here, we directly compare morphological and Illumina data obtained from the same samples, in an effort to characterize ciliate faunas from sediments in freshwater environments. We show how in silico processing affects the final outcome of our HTS analysis, providing evidence that quality filtering protocols strongly impact the number of predicted taxa, but not downstream conclusions such as biogeography patterns. We determine the abundance distribution of ciliates, showing that a small fraction of abundant taxa dominates read counts. At the same time, we advance reasons to believe that biases affecting HTS abundances may be significant enough to blur part of the underlying biological picture. We confirmed that the HTS approach detects many more taxa than morphological inspections, and highlight how the difference varies among taxonomic groups. Finally, we hypothesize that the two datasets actually correspond to different conceptions of "diversity," and consequently that neither is entirely superior to the other when investigating environmental protists.

Rare Freshwater Ciliate Paramecium chlorelligerum Kahl, 1935 and Its Macronuclear Symbiotic Bacterium "Candidatus Holospora parva".

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.

"Candidatus Gortzia shahrazadis", a Novel Endosymbiont of Paramecium multimicronucleatum and a Revision of the Biogeographical Distribution of Holospora-Like Bacteria.

Holospora spp. and "Candidatus Gortzia infectiva", known as Holospora-like bacteria (HLB), are commonly found as nuclear endosymbionts of ciliates, especially the Paramecium genus. HLB are related by phylogenetic relationships, morphological features, and life-cycles, which involve two alternating morphotypes: reproductive and infectious forms (RF, IF). In this paper we describe a novel species belonging to the "Ca. Gortzia" genus, detected in P. multimicronucleatum, a ciliate for which infection by an HLB has not been reported, discovered in India. This novel endosymbiont shows unusual and surprising features with respect to other HLB, such as large variations in IF morphology and the occasional ability to reproduce in the host cytoplasm. We propose the name of "Candidatus Gortzia shahrazadis" for this novel HLB. Moreover, we report two additional species of HLB from Indian Paramecium populations: "Ca. Gortzia infectiva" (from P. jenningsi), and H. obtusa (from P. caudatum); the latter is the first record of Holospora from a tropical country. Although tropical, we retrieved H. obtusa at an elevation of 706 m corresponding to a moderate climate not unlike conditions where Holospora are normally found, suggesting the genus Holospora does exist in tropical countries, but restricted to higher elevations.

Ciliate communities and hidden biodiversity in freshwater biotopes of the Pistoia province (Tuscany, Italy).

Ciliates are essential components of aquatic environments, playing a pivotal role in microbial loops. Thus, the composition and dynamics of ciliate communities have been subjected to intense studying. Morphological methods have been traditionally employed, until the development of next-generation sequencing recently allowed to explore the topic with exclusively molecular techniques. However, the results of the two approaches are hardly comparable, and the pictures they offer can be quite different. This may be due, among other reasons, to two factors: (1) morphological descriptions may miss a large portion of "hidden biodiversity" (including rare species and resistance forms) that is detected instead by molecular methods; (2) identification errors may arise due to difficulties in recognizing microbial taxa without in-depth analyses. In this survey of freshwater systems of the Pistoia province (Tuscany, Italy) we address both issues, trying to quantify the hidden diversity through prolonged observations of differentially treated sample aliquots, combining morphological identification with Sanger sequencing. We provide the first insights into the ciliate fauna of this area presenting results that are suitable for future comparisons thanks to their multidisciplinary origin, and supply the first molecular data on well-known taxa such as Linostomella and Disematostoma.

Response of the bacterial symbiont Holospora caryophila to different growth conditions of its host.

Previous studies on bacterial symbionts of ciliates have shown that some symbionts can be maintained relatively well under standard laboratory conditions whereas others are frequently lost, especially when the host is cultivated at a high division rate. In this study, the variation in infection level by the endosymbiont Holospora caryophila within its host population Paramecium octaurelia was investigated in response to three alimentary treatments and a subsequent starvation phase. The response of the ciliates was determined as a nearly exponential growth rate with different slopes in each treatment, proportional to the amount of food received. The initial infection level was higher than 90%. After 24 days of exponential host's growth, the prevalence remained stable at approximately 90% in all treatments, even after a subsequent starvation phase of 20 days. However, at intermediate time-points in both the feeding and the starvation phase, fluctuations in the presence of the intracellular bacteria were observed. These results show that H. caryophila is able to maintain its infection under the tested range of host growth conditions, also due to the possibility of an effective re-infection in case of partial loss.

Focusing on genera to improve species identification: revised systematics of the ciliate Spirostomum.

Although many papers dealing with the description of new ciliate taxa are published each year, species taxonomy and identification in most groups of the phylum Ciliophora remain confused. This is largely due to a scarcity of surveys on the systematics of immediately higher levels (genera and families) providing data for old and new species together. Spirostomum is a common and distinctive inhabitant of fresh- and brackish water environments, including artificial and eutrophic ones, and is a good model for applied ecology and symbiosis research. Despite this, only 3 of the numerous species are commonly cited, and no studies have yet confirmed their monophyly, with the consequence that reproducibility of the results may be flawed. In this paper we present morphological and molecular data for 30 Spirostomum populations representing 6 different morphospecies, some of which were collected in previously unreported countries. We performed a detailed revision of Spirostomum systematics combining literature surveys, new data on hundreds of organisms and statistical and phylogenetic analyses; our results provide insights on the evolution, ecology and distribution of known morphospecies and a novel one: Spirostomum subtilis sp. n. We also offer tools for quick species identification.

Free-living ciliates as potential reservoirs for eukaryotic parasites: occurrence of a trypanosomatid in the macronucleus of Euplotes encysticus.

BACKGROUND: Flagellates of the family Trypanosomatidae are obligate endoparasites, which can be found in various hosts. Several genera infect insects and occur as monoxenous parasites especially in representatives of Diptera and Hemiptera. These trypanosomatid flagellates probably share the worldwide distribution of their hosts, which are often infested by large numbers of endoparasites. Traditionally, their taxonomy was based on morphology, host origin, and life cycle. Here we report the characterization of a trypanosomatid infection detected in a protozoan, a ciliate collected from a polluted freshwater pond in a suburb of New Delhi (India). METHODS: Live observations and morphological studies applying light, fluorescence and transmission electron microscopy were conducted. Molecular analyses of host and parasite were performed and used for phylogenetic reconstructions and species (host) or genus level (parasite) identification. RESULTS: Although the morphological characteristics were not revealing, a high similarity of the trypanosomatids 18S rRNA gene sequence to Herpetomonas ztiplika and Herpetomonas trimorpha (Kinetoplastida, Trypanosomatidae), both parasites of biting midges (Culicoides kibunensis and Culicoides truncorum, respectively) allowed the assignment to this genus. The majority of the host population displayed a heavy infection that significantly affected the shape of the host macronucleus, which was the main site of parasite localization. In addition, the growth rate of host cultures, identified as Euplotes encysticus according to cell morphology and 18S rRNA gene sequence, was severely impacted by the infection. CONCLUSIONS: The host-parasite system described here represents a recent example of free-living protists acting as environmental reservoirs for parasitic eukaryotic microorganisms.

Flagellar movement in two bacteria of the family rickettsiaceae: a re-evaluation of motility in an evolutionary perspective.

Bacteria of the family Rickettsiaceae have always been largely studied not only for their importance in the medical field, but also as model systems in evolutionary biology. In fact, they share a recent common ancestor with mitochondria. The most studied species, belonging to genera Rickettsia and Orientia, are hosted by terrestrial arthropods and include many human pathogens. Nevertheless, recent findings show that a large part of Rickettsiaceae biodiversity actually resides outside the group of well-known pathogenic bacteria. Collecting data on these recently described non-conventional members of the family is crucial in order to gain information on ancestral features of the whole group. Although bacteria of the family Rickettsiaceae, and of the whole order Rickettsiales, are formally described as non-flagellated prokaryotes, some recent findings renewed the debate about this feature. In this paper we report the first finding of members of the family displaying numerous flagella and active movement inside their host cells. These two new taxa are hosted in aquatic environments by protist ciliates and are described here by means of ultrastructural and molecular characterization. Data here reported suggest that the ancestor of Rickettsiales displayed flagellar movement and re-evaluate the hypothesis that motility played a key-role in the origin of mitochondria. Moreover, our study highlights that the aquatic environment represents a well exploited habitat for bacteria of the family Rickettsiaceae. Our results encourage a deep re-consideration of ecological and morphological traits of the family and of the whole order.

Morphology, ultrastructure, and molecular phylogeny of the ciliate Sonderia vorax with insights into the systematics of order Plagiopylida.

BACKGROUND: Ciliates of the family Sonderiidae are common members of the eukaryotic communities in various anoxic environments. They host both ecto- and endosymbiotic prokaryotes (the latter associated with hydrogenosomes) and possess peculiar morpho-ultrastructural features, whose functions and homologies are not known. Their phylogenetic relationships with other ciliates are not completely resolved and the available literature, especially concerning electron microscopy and molecular studies, is quite scarce. RESULTS: Sonderia vorax Kahl, 1928 is redescribed from an oxygen-deficient, brackish-water pond along the Ligurian Sea coastlines of Italy. Data on morphology, morphometry, and ultrastructure are reported. S. vorax is ovoid-ellipsoid in shape, dorsoventrally flattened, 130 x 69 µm (mean in vivo); it shows an almost spherical macronucleus, and one relatively large micronucleus. The ventral kinetom has a "secant system" including fronto-ventral and fronto-lateral kineties. A distinctive layer of bacteria laying between kineties covers the ciliate surface. Two types of extrusomes and hydrogenosomes-endosymbiotic bacteria assemblages are present in the cytoplasm. The phylogeny based on 18S rRNA gene sequences places S. vorax among Plagiopylida; Sonderiidae clusters with Plagiopylidae, although lower-level relationships remain uncertain. The studied population is fixed as neotype and the ciliate is established as type species of the genus, currently lacking. CONCLUSIONS: This is the first description of a representative of Sonderiidae performed with both morphological and molecular data. To sum up, many previous hypotheses on this interesting, poorly known taxon are confirmed but confusion and contradictory data are as well highlighted.