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

Abstract

A number of anaerobic ciliates, unicellular eukaryotes, 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 a different domain, 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 demonstrate that GW7 possesses both archaeal and bacterial endosymbionts in its cytoplasm. These endosymbionts are in dependently associated with hydrogenosomes, which are organelle producing hydrogen and ATP under anaerobic conditions. Clone library analyses targeting prokaryotic 16S rRNA genes, fluorescent in situ hybridization with endosymbiont-specific probes, and molecular phylogenetic analyses revealed the phylogenetic affiliations and intracellular localizations of these endosymbionts. The endosymbiotic archaeon is a methanogen belonging to the genus Methanoregula (order Methanomicrobiales); a member of this genus has previously been 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 propose a novel candidate genus and species, “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 3 years without losing either of its endosymbionts. Unexpectedly, molecular characterization of the endosymbionts revealed that the 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 endosymbioses in aerobic ciliates.

Genomics of New Ciliate Lineages Provides Insight into the Evolution of Obligate Anaerobiosis

“…For the host, an advantage of maintaining prokaryotic symbionts is the faster removal of undesirable hydrogen and other metabolic waste, such as acetate, from the cell, improving the rate and efficiency of the host's energetic metabolism, as confirmed, besides anaerobic ciliates, also in breviates [19,62]. Both Muranotrichea and Parablepharismea, like some other anaerobic ciliates [51,52], host multiple symbionts that might not only interact with their hosts but also among themselves. Competition for hydrogen molecules and other metabolic byproducts likely further increases the rate of hydrogen removal from the host cells and improves the efficiency of the host's energetic metabolism.…”

“…More often than not, symbiotic relationships do not represent an exclusive, stable mutualism but a dynamic and competitive interaction for resources between several players, as shown in the scuticociliate sp. GW7 (Oligohymenophorea) [51]. On the other hand, two anaerobic symbiotic groups hosted by Parduczia sp.…”

A dual endosymbiosis drives nutritional adaptation to hematophagy in the invasive tick Hyalomma marginatum

“…This degeneration-replacement model has been proposed in sap-feeding insects (Campbell et al, 2015;Łukasik et al, 2017;Manzano-Marı ń et al, 2020;Matsuura et al, 2018), but is difficult to document as such replacements are expected to be transient (McCutcheon et al, 2019). In parallel, other forms of multi-partite interactions have been reported, where a novel bacterium with similar function to a pre-existing symbiont enters a bipartite system (Husnik and Mccutcheon, 2016;Santos-Garcia et al, 2018;Takeshita et al, 2019). This can lead to relaxation of conservative selection pressure on the original symbionts, which can lose genes that were previously fundamental, ultimately leading to a tripartite stable symbiosis (Husnik and Mccutcheon, 2016;Takeshita et al, 2019).…”

New Intranuclear Symbiotic Bacteria from Macronucleus of Paramecium putrinum—“Candidatus Gortzia Yakutica”

“…Hydrogenosomobacter endosymbioticus" [39]. This endosymbiont has an uncertain position on the phylogenetic trees based on 16S rRNA genes: according to Takeshita et al it forms a sister taxon to HLB, but with the low branch support (less than 70%) [39]. In our analysis this species appears within the HLB clade (Figure 6), but the branch support is quite low as well.…”

“…Takeshita et al recently described an endosymbiont from an anaerobic Scuticociliate-"Ca. Hydrogenosomobacter endosymbioticus" [39]. This endosymbiont has an uncertain position on the phylogenetic trees based on 16S rRNA genes: according to Takeshita et al it forms a sister taxon to HLB, but with the low branch support (less than 70%) [39].…”