Disentangling the taxonomy of Rickettsiales and description of two novel symbionts ("Candidatus Bealeia paramacronuclearis" and "Candidatus Fokinia cryptica") sharing the cytoplasm of the ciliate protist Paramecium biaurelia

In the past 10 years, the number of endosymbionts described within the bacterial order Rickettsiales has constantly grown. Since 2006, 18 novel Rickettsiales genera inhabiting protists, such as ciliates and amoebae, have been described. In this work, we characterize two novel bacterial endosymbionts from Paramecium collected near Bloomington, IN. Both endosymbiotic species inhabit the cytoplasm of the same host. The Gram-negative bacterium "Candidatus Bealeia paramacronuclearis" occurs in clumps and is frequently associated with the host macronucleus. With its electron-dense cytoplasm and a distinct halo surrounding the cell, it is easily distinguishable from the second smaller symbiont, "Candidatus Fokinia cryptica," whose cytoplasm is electron lucid, lacks a halo, and is always surrounded by a symbiontophorous vacuole. For molecular characterization, the small-subunit rRNA genes were sequenced and used for taxonomic assignment as well as the design of species-specific oligonucleotide probes. Phylogenetic analyses revealed that "Candidatus Bealeia paramacronuclearis" clusters with the so-called "basal" Rickettsiales, and "Candidatus Fokinia cryptica" belongs to "Candidatus Midichloriaceae." We obtained tree topologies showing a separation of Rickettsiales into at least two groups: one represented by the families Rickettsiaceae, Anaplasmataceae, and "Candidatus Midichloriaceae" (RAM clade), and the other represented by "basal Rickettsiales," including "Candidatus Bealeia paramacronuclearis." Therefore, and in accordance with recent publications, we propose to limit the order Rickettsiales to the RAM clade and to raise "basal Rickettsiales" to an independent order, Holosporales ord. nov., inside Alphaproteobacteria, which presently includes four family-level clades. Additionally, we define the family "Candidatus Hepatincolaceae" and redefine the family Holosporaceae

The Obligate Symbiont “Candidatus Megaira polyxenophila” Has Variable Effects on the Growth of Different Host Species

“Candidatus Megaira polyxenophila” is a recently described member of Rickettsiaceae which comprises exclusively obligate intracellular bacteria. Interestingly, these bacteria can be found in a huge diversity of eukaryotic hosts (protist, green algae, metazoa) living in marine, brackish or freshwater habitats. Screening of amplicon datasets revealed a high frequency of these bacteria especially in freshwater environments, most likely associated to eukaryotic hosts. The relationship of “Ca. Megaira polyxenophila” with their hosts and their impact on host fitness have not been studied so far. Even less is known regarding the responses of these intracellular bacteria to potential stressors. In this study, we used two phylogenetically close species of the freshwater ciliate Paramecium, Paramecium primaurelia and Paramecium pentaurelia (Ciliophora, Oligohymenophorea) naturally infected by “Ca. Megaira polyxenophila”. In order to analyze the effect of the symbiont on the fitness of these two species, we compared the growth performance of both infected and aposymbiotic paramecia at different salinity levels in the range of freshwater and oligohaline brackish water i.e., at 0, 2, and 4.5 ppt. For the elimination of “Ca. Megaira polyxenophila” we established an antibiotic treatment to obtain symbiont-free lines and confirmed its success by fluorescence in situ hybridization (FISH). The population and infection dynamics during the growth experiment were observed by cell density counts and FISH. Paramecia fitness was compared applying generalized additive mixed models. Surprisingly, both infected Paramecium species showed higher densities under all salinity concentrations. The tested salinity concentrations did not significantly affect the growth of any of the two species directly, but we observed the loss of the endosymbiont after prolonged exposure to higher salinity levels. This experimental data might explain the higher frequency of “Ca. M. polyxenophila” in freshwater habitats as observed from amplicon data

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 â\u80\u9cCandidatus Trichorickettsia mobilisâ\u80\u9d (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 â\u80\u9cCandidatus Trichorickettsiaâ\u80\u9d 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 â\u80\u9cCandidatus Trichorickettsiaâ\u80\u9d hosts, infected P. calkinsi cells frequently became amicronucleate and demonstrated abnormal cell division, eventually leading to decline of the laboratory culture

Transmission of Members of the “Candidatus Midichloriaceae” Family to Vertebrates and Possible Involvement in Disease Pathogenesis

“Candidatus Midichloriaceae” (order Rickettsiales) is a family that encompasses obligate intracellular bacteria present in a wide range of hosts, from parasitic arthropods (ticks, fleas, bedbugs) to aquatic animals (e.g. sponges and corals) and protists, including pathogenic amoebae (genus Acanthamoeba). In addition, multiple studies have detected evidence of the presence of “Candidatus Midichloriaceae” in vertebrates, suggesting a capacity to cause infections, with possible pathogenic effects. Two members of the family that are described as symbionts of ticks, i.e. “Candidatus Midichloria mitochondrii” and “Candidatus Lariskella arthropodarum” have also been found in the blood of humans and other mammals, using molecular and serological methods. Consequently, it has been hypothesized that these arthropods could transmit the microorganisms during the blood meal, as is known to happen with pathogenic members of the order Rickettsiales; for example, ticks can be vectors of rickettsiosis or ehrlichiosis. Indeed, clinical cases compatible with these pathologies in humans or animals parasitized by ticks, but characterized by the absence of antibody titers for known pathogenic rickettsiae, could be reinvestigated for the presence of “Candidatus Midichloriaceae”. A third member of the family, a “Candidatus Midichloria”-related organism, has repeatedly been found in association with red mark syndrome in the rainbow trout Oncorhynchus mykiss. Although the etiology of this economically relevant disease is currently unknown, multiple independent studies suggest that this member of “Candidatus Midichloriaceae” is the most probable causative agent

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