Novel evolutionary insights on the interactions of the Holosporales (Alphaproteobacteria) with eukaryotic hosts from comparative genomics.

Holosporales are an alphaproteobacterial order engaging in obligate and complex associations with eukaryotes, in particular protists. The functional and evolutionary features of those interactions are still largely undisclosed. Here, we sequenced the genomes of two members of the species Bealeia paramacronuclearis (Holosporales, Holosporaceae) intracellularly associated with the ciliate protist Paramecium, which resulted in high correspondence. Consistent with the short-branched early-divergent phylogenetic position, Bealeia presents a larger functional repertoire than other Holosporaceae, comparable to those of other Holosporales families, particularly for energy metabolism and motility. Our analyses indicate that different Holosporales likely experienced at least partly autonomous genome reduction and adaptation to host interactions, for example regarding dependence on host biotin driven by multiple independent horizontal acquisitions of transporters. Among Alphaproteobacteria, this is reminiscent of the convergently evolved Rickettsiales, which however appear more diverse, possibly due to a probably more ancient origin. We identified in Bealeia and other Holosporales the plasmid-encoded putative genetic determinants of R-bodies, which may be involved in a killer trait towards symbiont-free hosts. While it is not clear whether these genes are ancestral or recently horizontally acquired, an intriguing and peculiar role of R-bodies is suggested in the evolution of the interactions of multiple Holosporales with their hosts.

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.

'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.

Francisella novicida can utilize Paramecium bursaria as its potential host.

Francisella novicida is a facultative intracellular pathogen and the causative agent of tularemia. Although cases of infection caused by exposure to contaminated water have been reported, its natural host and ecology in the environment remain unclear. In this study, we investigated in vitro the possibility that Paramecium bursaria may be a useful tool as a protist host model of F. novicida. Experimental infection with F. novicida resulted in a stable intracellular relationship within P. bursaria. This symbiotic intracellular relationship was not observed in experimental infections with other Francisella species and Legionella pneumophila. We found that F. novicida showed similar behaviour to that of the eukaryotic endosymbiont of P. bursaria, the green algae Chlorella, in the internalization process. In addition, stable intracellular localization of F. novicida was possible only when Chlorella was not present. Although we investigated the type VI secretion system of F. novicida as a candidate for the bacterial factor, we found that it was not involved in the establishment of an intracellular relationship with P. bursaria. These results suggested that P. bursaria is potentially a protist host model for F. novicida and may be a useful tool for understanding the relationship between protist hosts and their symbionts.

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.

Dual-Seq reveals genome and transcriptome of Caedibacter taeniospiralis, obligate endosymbiont of Paramecium.

Interest in host-symbiont interactions is continuously increasing, not only due to the growing recognition of the importance of microbiomes. Starting with the detection and description of novel symbionts, attention moves to the molecular consequences and innovations of symbioses. However, molecular analysis requires genomic data which is difficult to obtain from obligate intracellular and uncultivated bacteria. We report the identification of the Caedibacter genome, an obligate symbiont of the ciliate Paramecium. The infection does not only confer the host with the ability to kill other cells but also renders them immune against this effect. We obtained the C. taeniospiralis genome and transcriptome by dual-Seq of DNA and RNA from infected paramecia. Comparison of codon usage and expression level indicates that genes necessary for a specific trait of this symbiosis, i.e. the delivery of an unknown toxin, result from horizontal gene transfer hinting to the relevance of DNA transfer for acquiring new characters. Prediction of secreted proteins of Caedibacter as major agents of contact with the host implies, next to several toxin candidates, a rather uncharacterized secretome which appears to be highly adapted to this symbiosis. Our data provides new insights into the molecular establishment and evolution of this obligate symbiosis and for the pathway characterization of toxicity and immunity.

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.

Towards an ecological understanding of the killer trait - A reproducible protocol for testing its impact on freshwater ciliates.

Paramecium strains with the ability to kill other paramecia often harbour intracellular bacteria belonging to the genera Caedibacter or Caedimonas. Central structures of this killer trait are refractile bodies (R-bodies) produced by the endosymbionts. Once ingested by a sensitive Paramecium, R-bodies presumably act as delivery system for an unidentified toxin which causes the death of endosymbiont-free paramecia while those infected gain resistance from their symbionts. The killer trait is therefore considered as competitive advantage for the hosts of R-body producers. While its effectiveness against paramecia is well documented, the effects on other aquatic ciliates are much less studied. In order to address the broadness of the killer trait, a reproducible killer test assay considering the effects on predatory ciliates (Climacostomum virens and Dileptus jonesi) as well as potential bacterivorous Paramecium competitors (Dexiostoma campyla, Euplotes aediculatus, Euplotes woodruffi, and Spirostomum teres) as possibly susceptible species was established. All used organisms were molecularly characterized to increase traceability and reproducibility. The absence of any lethal effects in both predators and competitors after exposure to killer paramecia strongly suggests a narrow action range for the killer trait. Thus, R-body producing bacteria provide their host with a complex, costly strategy to outcompete symbiont-free congeners only.

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.

Identification of novel Legionella genes required for endosymbiosis in Paramecium based on comparative genome analysis with Holospora spp.

The relationship between Legionella and protist hosts has a huge impact when considering the infectious risk in humans because it facilitates the long-term replication and survival of Legionella in the environment. The ciliate Paramecium is considered to be a protist host for Legionella in natural environments, but the details of their endosymbiosis are largely unknown. In this study, we determined candidate Legionella pneumophila genes that are likely to be involved in the establishment of endosymbiosis in Paramecium caudatum by comparing the genomes of Legionella spp. and Holospora spp. that are obligate endosymbiotic bacteria in Paramecium spp. Among the candidate genes, each single deletion mutant for five genes (lpg0492, lpg0522, lpg0523, lpg2141 and lpg2398) failed to establish endosymbiosis in P. caudatum despite showing intracellular growth in human macrophages. The mutants exhibited no characteristic changes in terms of their morphology, multiplication rate or capacity for modulating the phagosomes in which they were contained, but their resistance to lysozyme decreased significantly. This study provides insights into novel factors required by L. pneumophila for endosymbiosis in P. caudatum, and suggests that endosymbiotic organisms within conspecific hosts may have shared genes related to effective endosymbiosis establishment.

Variation and asymmetry in host-symbiont dependence in a microbial symbiosis.

BACKGROUND: Symbiosis is a major source of evolutionary innovation and, by allowing species to exploit new ecological niches, underpins the functioning of ecosystems. The transition from free-living to obligate symbiosis requires the alignment of the partners' fitness interests and the evolution of mutual dependence. While symbiotic taxa are known to vary widely in the extent of host-symbiont dependence, rather less is known about variation within symbiotic associations. RESULTS: Using experiments with the microbial symbiosis between the protist Paramecium bursaria and the alga Chlorella, we show variation between pairings in host-symbiont dependence, encompassing facultative associations, mutual dependence and host dependence upon the symbiont. Facultative associations, that is where both the host and the symbiont were capable of free-living growth, displayed higher symbiotic growth rates and higher per host symbiont loads than those with greater degrees of dependence. CONCLUSIONS: These data show that the Paramecium-Chlorella interaction exists at the boundary between facultative and obligate symbiosis, and further suggest that the host is more likely to evolve dependence than the algal symbiont.

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.

The Genome Sequence of "Candidatus Fokinia solitaria": Insights on Reductive Evolution in Rickettsiales.

"Candidatus Fokinia solitaria" is an obligate intracellular endosymbiont of a unicellular eukaryote, a ciliate of the genus Paramecium. Here, we present the genome sequence of this bacterium and subsequent analysis. Phylogenomic analysis confirmed the previously reported positioning of the symbiont within the "Candidatus Midichloriaceae" family (order Rickettsiales), as well as its high sequence divergence from other members of the family, indicative of fast sequence evolution. Consistently with this high evolutionary rate, a comparative genomic analysis revealed that the genome of this symbiont is the smallest of the Rickettsiales to date. The reduced genome does not present flagellar genes, nor the pathway for the biosynthesis of lipopolysaccharides (present in all the other so far sequenced members of the family "Candidatus Midichloriaceae") or genes for the Krebs cycle (present, although not always complete, in Rickettsiales). These results indicate an evolutionary trend toward a stronger dependence on the host, in comparison with other members of the family. Two alternative scenarios are compatible with our results; "Candidatus Fokinia solitaria" could be either a recently evolved, vertically transmitted mutualist, or a parasite with a high host-specificity.

Cultivation Conditions Can Cause a Shift from Mutualistic to Parasitic Behavior in the Symbiosis Between Paramecium and Its Bacterial Symbiont Caedibacter taeniospiralis.

Caedibacter taeniospiralis is an obligate bacterial symbiont living in the cytoplasm of the ciliate Paramecium tetraurelia. Different studies analyzing the effect of this symbiont on its host's growth and maximal cell density arrive at contradicting conclusions, labeling it as either a parasite or a mutualist. We address the question whether extrinsic factors such as medium and food organism are responsible for the opposing results. Thus, we performed fitness assays comparing previously applied cultivation conditions. By confirming the dependency of the parasitic and mutualistic behavior of C. taeniospiralis on the cultivation conditions of its host P. tetraurelia, we demonstrate the context-dependent impact on host fitness of this bacterium.

Phylogenetic relationships among endosymbiotic R-body producer: Bacteria providing their host the killer trait.

R-body producing bacterial endosymbionts of Paramecium spp. transform their hosts into "killer" paramecia and provide them a selective advantage. This killer trait is connected to the presence of R-bodies, which are peculiar, tightly coiled protein ribbons capable of rapid unrolling. Based mainly on those two characteristics the respective obligate intracellular bacteria have been comprised in the genus Caedibacter and additional traits such as host species, subcellular localization, and R-body dimensions and mode of unrolling were used for species discrimination. Previous studies applying the full-cycle rRNA approach demonstrated the polyphyly of this assemblage. Following this approach, we obtained new sequences and in situ hybridizations for five strains of Caedibacter taeniospiralis and four strains associated to Caedibacter varicaedens and Caedibacter caryophilus. Detailed phylogenetic reconstructions confirm the association of C. taeniospiralis to Fastidiosibacteraceae and to Holosporales in case of the others. Therefore, we critically revise the taxonomy of the latter group. The high 16S rRNA gene sequence similarity among the type strains of Caedibacter varicaedens and C. caryophilus indicate that they should be classified within a single species for which we propose Caedimonas varicaedens comb. nov. owing to the priority of Caedibacter varicaedens. Moreover, we propose to establish the new family Caedimonadaceae fam. nov. to encompass Caedimonas varicaedens, "Ca. Paracaedimonas acanthamoebae" comb. nov. and "Ca. Nucleicultrix amoebiphila" within the order Holosporales.

More than the "Killer Trait": Infection with the Bacterial Endosymbiont Caedibacter taeniospiralis Causes Transcriptomic Modulation in Paramecium Host.

Endosymbiosis is a widespread phenomenon and hosts of bacterial endosymbionts can be found all-over the eukaryotic tree of life. Likely, this evolutionary success is connected to the altered phenotype arising from a symbiotic association. The potential variety of symbiont's contributions to new characteristics or abilities of host organisms are largely unstudied. Addressing this aspect, we focused on an obligate bacterial endosymbiont that confers an intraspecific killer phenotype to its host. The symbiosis between Paramecium tetraurelia and Caedibacter taeniospiralis, living in the host's cytoplasm, enables the infected paramecia to release Caedibacter symbionts, which can simultaneously produce a peculiar protein structure and a toxin. The ingestion of bacteria that harbor both components leads to the death of symbiont-free congeners. Thus, the symbiosis provides Caedibacter-infected cells a competitive advantage, the "killer trait." We characterized the adaptive gene expression patterns in symbiont-harboring Paramecium as a second symbiosis-derived aspect next to the killer phenotype. Comparative transcriptomics of infected P. tetraurelia and genetically identical symbiont-free cells confirmed altered gene expression in the symbiont-bearing line. Our results show up-regulation of specific metabolic and heat shock genes whereas down-regulated genes were involved in signaling pathways and cell cycle regulation. Functional analyses to validate the transcriptomics results demonstrated that the symbiont increases host density hence providing a fitness advantage. Comparative transcriptomics shows gene expression modulation of a ciliate caused by its bacterial endosymbiont thus revealing new adaptive advantages of the symbiosis. Caedibacter taeniospiralis apparently increases its host fitness via manipulation of metabolic pathways and cell cycle control.

Host and symbiont intraspecific variability: The case of Paramecium calkinsi and "Candidatus Trichorickettsia mobilis".

Newly isolated strains of the ciliate Paramecium calkinsi and their cytoplasmic bacterial endosymbionts were characterized by a multidisciplinary approach, including live observation, ultrastructural investigation, and molecular analysis. Despite morphological resemblance, the characterized P. calkinsi strains showed a significant molecular divergence compared to conspecifics, possibly hinting for a cryptic speciation. The endosymbionts were clearly found to be affiliated to the species "Candidatus Trichorickettsia mobilis" (Rickettsiales, Rickettsiaceae), currently encompassing only bacteria retrieved in an obligate intracellular association with other ciliates. However, a relatively high degree of intraspecific divergence was observed as well, thus it was possible to split "Candidatus Trichorickettsia" into three subspecies, one of which represented so far only by the newly characterized endosymbionts of P. calkinsi. Other features distinguished the members of each different subspecies. In particular, the endosymbionts of P. calkinsi resided in the cytoplasm and possessed numerous peritrichous flagella, although no motility was evidenced, whereas their conspecifics in other hosts were either cytoplasmic and devoid of flagella, or macronuclear, displaying flagellar-driven motility. Moreover, contrarily to previously analyzed "Candidatus Trichorickettsia" hosts, infected P. calkinsi cells frequently became amicronucleate and demonstrated abnormal cell division, eventually leading to decline of the laboratory culture.

Stringent Expression Control of Pathogenic R-body Production in Legume Symbiont Azorhizobium caulinodans.

R bodies are insoluble large polymers consisting of small proteins encoded by reb genes and are coiled into cylindrical structures in bacterial cells. They were first discovered in Caedibacter species, which are obligate endosymbionts of paramecia. Caedibacter confers a killer trait on the host paramecia. R-body-producing symbionts are released from their host paramecia and kill symbiont-free paramecia after ingestion. The roles of R bodies have not been explained in bacteria other than CaedibacterAzorhizobium caulinodans ORS571, a microsymbiont of the legume Sesbania rostrata, carries a reb operon containing four reb genes that are regulated by the repressor PraR. Herein, deletion of the praR gene resulted in R-body formation and death of host plant cells. The rebR gene in the reb operon encodes an activator. Three PraR binding sites and a RebR binding site are present in the promoter region of the reb operon. Expression analyses using strains with mutations within the PraR binding site and/or the RebR binding site revealed that PraR and RebR directly control the expression of the reb operon and that PraR dominantly represses reb expression. Furthermore, we found that the reb operon is highly expressed at low temperatures and that 2-oxoglutarate induces the expression of the reb operon by inhibiting PraR binding to the reb promoter. We conclude that R bodies are toxic not only in paramecium symbiosis but also in relationships between other bacteria and eukaryotic cells and that R-body formation is controlled by environmental factors.IMPORTANCECaedibacter species, which are obligate endosymbiotic bacteria of paramecia, produce R bodies, and R-body-producing endosymbionts that are released from their hosts are pathogenic to symbiont-free paramecia. Besides Caedibacter species, R bodies have also been observed in a few free-living bacteria, but the significance of R-body production in these bacteria is still unknown. Recent advances in genome sequencing technologies revealed that many Gram-negative bacteria possess reb genes encoding R-body components, and interestingly, many of them are animal and plant pathogens. Azorhizobium caulinodans, a microsymbiont of the tropical legume Sesbania rostrata, also possesses reb genes. In this study, we demonstrate that A. caulinodans has ability to kill the host plant cells by producing R bodies, suggesting that pathogenicity conferred by an R body might be universal in bacteria possessing reb genes. Furthermore, we provide the first insight into the molecular mechanism underlying the expression of R-body production in response to environmental factors, such as temperature and 2-oxoglutarate.

Intracellular symbiosis of algae with possible involvement of mitochondrial dynamics.

Algal endosymbiosis is widely present among eukaryotes including many protists and metazoans. However, the mechanisms involved in their interactions between host and symbiont remain unclear. Here, we used electron microscopy and three-dimensional reconstruction analyses to examine the ultrastructural interactions between the symbiotic zoochlorella and the organelles in the host Paramecium bursaria, which is a model system of endosymbiosis. Although in chemically fixed samples the symbiotic algae show no direct structural interactions with the host organelles and the perialgal vacuole membrane (PVM), in cryofixed P. bursaria samples the intimate connections were identified between the host mitochondria and the symbiotic algae via the PVM. The PVM was closely apposed to the cell wall of the symbiotic algae and in some places it showed direct contacts to the host mitochondrial membrane and the cell wall of the symbiotic algae. Further, the PVM-associated mitochondria formed a mitochondrial network and were also connected to host ER. Our observations propose a new endosymbiotic systems between the host eukaryotes and the symbionts where the benefiting symbiosis is performed through intimate interactions and an active structural modification in the host organelles.

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.