Tripartite Symbiosis of an Anaerobic Scuticociliate with two Hydrogenosome-Associated Endosymbionts, a Holospora-related Alphaproteobacterium and a Methanogenic Archaeon

A number of anaerobic ciliates, unicellular eukaryote, intracellularly possess methanogenic archaea and bacteria as symbiotic partners. Although this tripartite relationship is of interest in terms of the fact that each participant is from three different domains, the difficulty in culture and maintenance of those host species with symbiotic partners has disturbed both ecological and functional studies so far. In this study, we obtained a stable culture of a small anaerobic scuticociliate, strain GW7. By transmission electron microscopic observation and fluorescent in situ hybridization with domain-specific probes, we demonstrated that GW7 possessed both archaeal and bacterial endosymbionts in its cytoplasm. These endosymbionts were independently associated with hydrogenosomes, which are organelle producing hydrogen and ATP in anaerobic condition. Clone library analyses targeting prokaryotic 16S rRNA genes, fluorescent in situ hybridization with endosymbiont-specific probes, and molecular phylogenetic analyses revealed phylogenetic affiliation and intracellular localization of these endosymbionts. The endosymbiotic archaeon is a methanogen belonging to the genus Methanoregula (order Methanomicrobiales); a member of this genus has been previously described as the endosymbiont of an anaerobic ciliate from the genus Metopus (class Armophorea), which is only distantly related to strain GW7 (class Oligohymenophorea). The endosymbiotic bacterium belongs to the family Holosporaceae of the class Alphaproteobacteria, which also comprises several endosymbionts of various aerobic ciliates. For this endosymbiotic bacterium, we proposed the novel candidate species in the novel candidate genus “Candidatus Hydrogenosomobacter endosymbioticus.” Importance Tripartite symbioses between anaerobic ciliated protists and their intracellular archaeal and bacterial symbionts are not uncommon, but most reports have been based mainly on microscopic observations. Deeper insights into the function, ecology, and evolution of these fascinating symbioses involving partners from all three domains of life have been hampered by the difficulties of culturing anaerobic ciliates in the laboratory and the frequent loss of their prokaryotic partners during long-term cultivation. In the present study, we report the isolation of an anaerobic scuticociliate, strain GW7, which has been stably maintained in our laboratory for more than three years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that bacterial partner of GW7 is phylogenetically related to intranuclear endosymbionts of aerobic ciliates. This strain will enable future genomic, transcriptomic, and proteomic analyses of the interactions in this tripartite symbiosis and a comparison with the endosymbioses in aerobic ciliates.