Rediscovering the genus Lyticum, multiflagellated symbionts of the order Rickettsiales

Among the bacterial symbionts harbored by the model organism Paramecium, many still lack a recent investigation that includes a molecular characterization. The genus Lyticum consists of two species of large-sized bacteria displaying numerous flagella, despite their inability to move inside their hosts’ cytoplasm. We present a multidisciplinary redescription of both species, using the deposited type strains as well as newly collected material. On the basis of 16S rRNA gene sequences, we assigned Lyticum to the order Rickettsiales, that is intensely studied because of its pathogenic representatives and its position as the extant group most closely related to the mitochondrial ancestor. We provide conclusive proofs that at least some Rickettsiales possess actual flagella, a feature that has been recently predicted from genomic data but never confirmed. We give support to the hypothesis that the mitochondrial ancestor could have been flagellated, and provide the basis for further studies on these ciliate endosymbionts

"Candidatus Fokinia solitaria", a novel "stand-Alone" symbiotic lineage of Midichloriaceae (Rickettsiales)

Recently, the family Midichloriaceae has been described within the bacterial order Rickettsiales. It includes a variety of bacterial endosymbionts detected in different metazoan host species belonging to Placozoa, Cnidaria, Arthropoda and Vertebrata. Representatives of Midichloriaceae are also considered possible etiological agents of certain animal diseases. Midichloriaceae have been found also in protists like ciliates and amoebae. The present work describes a new bacterial endosymbiont, "Candidatus Fokinia solitaria", retrieved from three different strains of a novel Paramecium species isolated from a wastewater treatment plant in Rio de Janeiro (Brazil). Symbionts were characterized through the full-cycle rRNA approach: SSU rRNA gene sequencing and fluorescence in situ hybridization (FISH) with three species-specific oligonucleotide probes. In electron micrographs, the tiny rod-shaped endosymbionts (1.2 × 0.25-0.35 μm in size) were not surrounded by a symbiontophorous vacuole and were located in the peripheral host cytoplasm, stratified in the host cortex in between the trichocysts or just below them. Frequently, they occurred inside autolysosomes. Phylogenetic analyses of Midichloriaceae apparently show different evolutionary pathways within the family. Some genera, such as "Ca. Midichloria" and "Ca. Lariskella", have been retrieved frequently and independently in different hosts and environmental surveys. On the contrary, others, such as Lyticum, "Ca. Anadelfobacter", "Ca. Defluviella" and the presently described "Ca. Fokinia solitaria", have been found only occasionally and associated to specific host species. These last are the only representatives in their own branches thus far. Present data do not allow to infer whether these genera, which we named "stand-Alone lineages", are an indication of poorly sampled organisms, thus underrepresented in GenBank, or represent fast evolving, highly adapted evolutionary lineages. Copyright

“Candidatus Mystax nordicus” Aggregates with Mitochondria of Its Host, the Ciliate Paramecium nephridiatum

Extensive search for new endosymbiotic systems in ciliates occasionally reverts us to the endosymbiotic bacteria described in the pre-molecular biology era and, hence, lacking molecular characterization. A pool of these endosymbionts has been referred to as a hidden bacterial biodiversity from the past. Here, we provide a description of one of such endosymbionts, retrieved from the ciliate Paramecium nephridiatum. This curve-shaped endosymbiont (CS), which shared the host cytoplasm with recently described “Candidatus Megaira venefica”, was found in the same host and in the same geographic location as one of the formerly reported endosymbiotic bacteria and demonstrated similar morphology. Based on morphological data obtained with DIC, TEM and AFM and molecular characterization by means of sequencing 16S rRNA gene, we propose a novel genus, “Candidatus Mystax”, with a single species “Ca. Mystax nordicus”. Phylogenetic analysis placed this species in Holosporales, among Holospora-like bacteria. Contrary to all Holospora species and many other Holospora-like bacteria, such as “Candidatus Gortzia”, “Candidatus Paraholospora” or “Candidatus Hafkinia”, “Ca. Mystax nordicus” was never observed inside the host nucleus. “Ca. Mystax nordicus” lacked infectivity and killer effect. The striking peculiarity of this endosymbiont was its ability to form aggregates with the host mitochondria, which distinguishes it from Holospora and Holospora-like bacteria inhabiting paramecia

Phenotypic Polymorphism in Two Endosymbiotic Bacteria of the Ciliate <i>Paramecium</i>: <i>Pseudolyticum multiflagellatum</i> and “<i>Ca</i>. Megaira venefica”

Here, we report a comprehensive description of the stable associations between two Paramecium species (P. nephridiatum and P. caudatum) and their cytoplasmic bacterial endosymbiont Pseudolyticum multiflagellatum. These spindle-like, rod-shaped, non-motile peritrichous bacteria demonstrate significant phenotypic polymorphism. Considering the differences in bacterial morphology and host species, several scientific groups have previously described these endosymbionts as distinct species. Our study provides brand-new molecular data, which allows us to unify earlier descriptions and determine the phylogenetic position of this endosymbiont as a member of the family “Ca. Midichloriaceae” (Rickettsiales). The distinguishing feature of this bacterium is the presence of a highly refractive granule in its cytoplasm, well detectable with differential interference contrast (DIC) microscopy. The protein nature of these peculiar inclusion bodies is considered. The other endosymbiont, “Ca. Megaira venefica”, co-inhabiting the cytoplasm of the studied P. nephridiatum strains, also displayed polymorphism, rounded forms being infected with phages

“MITOCHONDRION, SWEET MITOCHONDRION”: THE ENDOSYMBIONT “CANDIDATUS BANDIELLA WOODRUFFII” AND ITS POSSIBLE LOCALIZATION INSIDE EUPLOTES WOODRUFFI RESPIRATORY ORGANELLE

Euplotes species (Ciliophora, Spirotrichea) display a certain attitude to harbor microbial consortia, especially if they present the obligate symbiosis with Polynucleobacter necessarius (Heckmann et al., 1983; Boscaro et al., 2012, 2013; Schrallhammer et al., 2013; Vannini et al, 2010, 2012, 2014). Herein we describe the case of Euplotes woodruffi strain NDG2, sampled in an inner channel of Visakhapatnam harbor (Andhra Pradesh, India), in which three endosymbiotic bacteria have been detected by means of FISH analysis: Polynucleobacter necessarius (Burkholderiales, Burkholderiaceae), “Candidatus (Ca.) Megaira polyxenophyla” (Rickettsiales, Rickettsiaceae), and “Ca. Bandiella woodruffii” (Rickettsiales, Midichloriaceae). Among those three endosymbionts, P. necessarius, is known to be an obligate endosymbiont of several Euplotes species (Vannini et al., 2012) and “Ca. Megaira polyxenophyla” has been repeatedly found in several ciliate hosts, belonging to different genera and even classes (Schrallhammer et al., 2013) and in metazoans as well (Penn et al., 2006; Fraune & Bosch, 2007; Sunagawa et al., 2009; Murakami et al., 2017). “Ca. Bandiella woodruffii”, on the contrary, has been described from an E. woodruffi strain from Brazil (Senra et al., 2015) and, so far, detected exclusively in this ciliate species. P. necessarius and “Ca. Megaira polyxenophyla” are cytoplasmic endosymbionts, and, similarly, “Ca. Bandiella woodruffii” has been supposed to be harbored in the host cytoplasm according to FISH analysis in the original description of the species (Senra et al., 2015); however, to date, no data concerning its morphology and ultrastructure are available. Preliminary TEM analysis carried out in the present work on E. woodruffi strain NDG2 consortium, suggest that “Ca. Bandiella woodruffii” could occupy the mitochondrion of the ciliate. Interestingly, this feature has been previously described in other Midichloriaceae, such as “Ca. Midichloria mitochondrii”. Indeed, this close relative of “Ca. Bandiella woodruffii”, is specifically adapted for colonizing these cellular compartments in the ovarian cells of the tick Ixodes ricinus (Sassera et al., 2006). Further analyses with imaging techniques are currently undergoing with the aim to validate these ultrastructural observations

Diversity, environmental distribution, and molecular variability of the bacterial endosymbiont "Candidatus Megaira" widespread in ciliates and other protists

In the last years, the recently described bacterial genus “Candidatus Megaira” (Rickettsiales) caught the attention of many researchers from different fields. This genus comprises obligatory intracellular bacteria, which are closely phylogenetically related to the pathogen Rickettsia. Many diverse organisms have been colonized by bacteria of the genus “Candidatus Megaira”, both unicellular (e.g. ciliates, amoebae, and algae), but also multicellular (cnidarians, porifers, plants, and worms). Herein, we report the multidisciplinary description of a novel “Candidatus Megaira” species inhabiting the cytoplasm of Paramecium bursaria and Paramecium nephridiatum, together with the characterization of several new isolates of the type species “Candidatus Megaira polyxenophila” in different ciliate species. Our ultrastructural investigations showed that these bacteria display variations in dimensions like other members of Rickettsiaceae; and the novel species presents polymorphic features such as the presence of flagella, and the predilection of being closely associated to endosymbiontic algae of Paramecium bursaria. Moreover, we performed several trans-infection experiments in order to understand how this bacterium is spread in the aquatic environment, how it influences the host fitness within the same population of ciliates, and how it interacts with other symbiotic bacteria already present or invading the same host cell. In addition, we also screened 16S rDNA amplicon databases to see host and environmental distribution of members of the “Candidatus Megaira” genus. We propose an updated phylogeny of the genus based both on phylogenetic analysis and 16S rRNA gene diversity, thus subdividing the genus in four distinct and separated species. All together our findings give support to the hypothesis that this poorly investigated bacterium may be transmitted by protists in aquatic ecosystems

Host association and intracellularity evolved multiple times independently in the Rickettsiales

Abstract 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

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

“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 antibiotictreated 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