**4. Discussion**

#### *4.1. RS- and CS-Bacteria Are Endosymbionts from the Past*

Here we provide morphological and molecular description of the two bacterial endosymbionts from the cytoplasm of *P. nephridiatum*, RS- and CS-bacteria. These endosymbionts show morphological and ecological similarity to symbionts from the past, first identified by Fokin in 1989 as endobionts 1 and 3 (Eb1 and Eb3 respectively) [33], retrieved in the same species in the same sampling spot. 16S rRNA gene of RS-bacteria showed 99–100% sequence similarity to that of "*Ca.* Megaira venefica", which has been described recently from the same ciliate species from the same location [49]. In the latter

study, "*Ca.* Megaira venefica" from *P. nephridiatum* has not been shown to possess any flagella, though in other hosts, such as *Paramecium bursaria*, structures resembling flagella have been occasionally revealed. Until recently, bacteria of order *Rickettsiales* have been thought to be devoid of flagella and flagella-mediated movement [56]. However, new findings have appeared, showing the inaccuracy of that statement [18,20,30]. Initially, flagella traces were found as a set of *fli*-genes in the genome of "*Ca.* Midichloria mitochondrii" [20], and later heavily flagellated endosymbionts *Lyticum* sp. [30], "*Ca.* Trichorickettsia mobilis" and "*Ca.* Gigarickettsia flagellate" [18] were identified in ciliates *Paramecium* spp. Here we provide direct evidence showing the presence of flagella in another paramecia endosymbiont "*Ca.* Megaira venefica" [49]. Although "*Ca.* Megaira venefica" has been shown to have the structures resembling flagella in one of the host species, the data were not conclusive. We demonstrated the presence of rare flagella (up to seven flagella) distributed over the whole bacterial surface. Similarly to Lanzoni and colleagues [49], we were not able to notice any substantial signs of flagella in TEM images, which could be explained by the low thickness of fine sections and a few flagella presented on the surface of bacteria.

By its size and morphology, the curved endosymbiont (CS) retrieved from the cytoplasm of *P. nephridiatum* resembles one of the endobionts (Eb3) isolated from the same place about 30 years ago [33]. Both of them are curved, and their dimensions are 4–7 × 0.6–0.8 μm for CS in our study, and 3.5–7 × 1.2–1.3 μm for Eb3 according to the study of 1989 [33]. In both studies, bacteria did not show any motility. The appearance of living bacteria in DIC images is very similar. The only significant di fference is the presence of the host cell membrane encircling the bacterium in the TEM image provided by Fokin [33]. However, the vast space of the vacuole, the thickness of its membrane, and the appearance of the bacterium enclosed cause some doubts concerning the function of the vacuole. It might be considered an autophagosome, rather than a symbiontophorous vacuole. In support of this idea is the TEM image presented by Fokin [33], which shows the vacuole to contain the fragment of the host cell cytoplasm alongside the bacterium, suggesting disposal of parts of the cytoplasm. In addition, in Fokin's study, high endosymbiont load (up to 100 bacteria) has been shown to be detrimental for the host cells, while our observations demonstrated that ciliates were viable even in the case of much higher bacterial load, which might be explained as a result of endosymbiont adaptation to the host or more favorable cultivation conditions. Despite the presence of these discrepancies, taking into account the dimensions, the shape, the general appearance and the identical host species, we sugges<sup>t</sup> the identity of Eb3 and CS-symbiont.

#### *4.2. Phylogeny and Taxonomy*

The sequence of 16S rRNA gene of the CS-symbiont demonstrated the highest proximity to the endosymbiont of *Paramecium sexaurelia* "*Ca.* Paraholospora nucleivisitans" [54]. According to Yarza et al. the minimum and median identity values to delineate bacterial genera are 94.5% and 96.4%, correspondingly, though it is 94.5% similarity of 16S rRNA genes that is most commonly used to delineate representatives of one or two di fferent genera [57]. Along these lines of reasoning, CS-symbionts are at the border with the genus "*Ca.* Paraholospora" as their sequence similarity is 95%. Recent analysis has shown that at least in some taxa (e.g., Bacillaceae [*Bacillus*]), the 16S rRNA gene identity minimum threshold value of 94.5% could underestimate genera diversity [58]. Thus, we are inclined to propose a new genus, "*Ca.* Mystax" with a single species "*Ca.* Mystax nordicus," taking into account the following considerations and basing on the bacterial shape and geographic origin.

"*Ca.* Paraholospora nucleivisitans" is a curve-shaped endosymbiont of *Paramecium sexaurelia* standing apart in the phylogenetic tree, which demonstrates a unique trait of shuttling between two host cell compartments: cytoplasm and macronucleus [54]. Although "*Ca.* Mystax nordicus" has the same curved shape, the cell length is much shorter and endosymbionts exceeding 7 μm were never observed. Throughout two years of observations, CS-symbionts always resided in one host compartment, in the cytoplasm, and were never observed in the host nucleus, which distinguishes it from the most closely related *Holospora* and *Holospora*-like bacteria, except

"*Ca.* Bealeia paramacronuclearis", which demonstrates a ffinity to the host macronucleus, but is never found inside it [55]. An important peculiarity of "*Ca.* Mystax nordicus" is its ability to aggregate with the host cell mitochondria. Besides that, in contrast to "*Ca.* Paraholospora nucleivisitans" residing in *P. sexaurelia*, which inhabits predominantly tropical and subtropical areas [59], "*Ca.* Mystax nordicus" was identified in *P. nephridiatum*, dwelling about 30 km from the Arctic Circle. As the endosymbionts incapable of living outside their host are restricted to the host habitat, di fferences in the latter sugges<sup>t</sup> that "*Ca.* Paraholospora nucleivisitans" and "*Ca.* Mystax nordicus" might have di fferent temperature optimums.

#### *4.3. Biological Peculiarities of "Ca. Mystax nordicus"*

"*Ca.* Mystax nordicus" lacks a life cycle typical of *Holospora* and "*Ca.* Gortzia", comprising a reproductive and an infectious form. The infectious form responsible for the horizontal spread of these endosymbionts is equipped with an infectious tip—a morphological structure on a bacterial pole that is thought to play a crucial role in the infection. The absence of the infectious tip, demonstrated by both TEM and AFM, supports the results of our experimental infections. Contrary to "*Ca.* Paraholospora nucleivisitans" shuttling between the cytoplasm and the nucleus [54] and "*Ca.* Gortzia shahrazadis" occasionally found in the cytoplasm [9], "*Ca.* Mystax nordicus" never occurred in the nucleus throughout two years of cultivation. Nuclear location is believed to be a prerequisite for di fferentiation of infectious forms in *Holospora* and *Holospora*-like bacteria [51]. The absence of the infectious tip and the infectious capacity seems to correlate with the cytoplasmic location of the endosymbiont. To our knowledge, it is the intranuclear endosymbionts that are highly infectious.

One of the most remarkable features of "*Ca.* Mystax nordicus" distinguishing it from many other cytoplasmic endosymbionts, such as "*Ca.* Fokinia" [35], "*Ca.* Bealeia" [55], "*Ca.* Megaira" [49] and *Lyticum* [30], is the formation of aggregates that are potentially formed around mitochondria (Figure 1C, Figure 2B, Figure 5C and Figure S1). Since one of the main functions of mitochondria in a eukaryotic cell is the synthesis of energy supply in the form of ATP [60], it is tempting to sugges<sup>t</sup> that "*Ca.* Mystax nordicus" might be directly using host mitochondria as energy factories. To our knowledge, this is the first report of putative interaction of mitochondria and endosymbionts observed in Paramecia spp. in situ. Our finding is in good agreemen<sup>t</sup> with the in silico studies [61]. Indeed, comparative genomic analysis of *Holospora* spp.—relatives of "*Ca.* Mystax nordicus" and another genus of endosymbionts from the family Holosporaceae infecting the ciliates of the genus *Paramecium*—has shown the absence of many biochemical pathways, e.g., citric acid cycle and glycolysis, and endosymbiont dependence on host energy supplies [61]. Interestingly, several other cases of more intimate mitochondria–endosymbiont interaction, when the bacterium resides in the host mitochondrion, have been reported for endosymbionts of another ciliate, *Spirostomum* [62], and tick *Ixodes* [63]. Additionally, "*Ca.* Cytomitobacter primus" has been recently registered invading mitochondria of the flagellate *Diplonema japonicum* [64]. Another example of endosymbiotic bacteria of ciliates associated with energy-producing organelles is "*Ca*. Hydrogenosomobacter endosymbioticus", found adjacent to hydrogenosomes of its host, anaerobic scuticociliate *Trimyema compressum*, together with methanogenic archea [65]. Besides the endosymbiont consumption of the host energy organelles' metabolites, the host–endosymbiont interaction might also involve communication between the intracellular bacteria and the host mitochondria or hydrogenosomes. Recently, it has been hypothesized "that microbiome may a ffect the host by directly interacting with mitochondria through bacterial metabolites and specific signaling mechanisms" [66].

Endosymbiont number control and host sparing way of their egress from the host cell are believed to be characteristic of the evolutionary old endosymbiotic systems, while hyperinfection and rupture of the host cell membrane are typical for evolutionary young systems with unstable host–endosymbiont relations [67]. Along these lines of reasoning, profusion of "*Ca.* Mystax nordicus", leading to the rupture of the host cell pellicle and, finally, to the host death, provides grounds for considering "*Ca.* Mystax nordicus" a recently acquired endosymbiont, as it poorly controls its number at least in the

laboratory conditions. However, this is rather surprising, taking into account the apparently vertical transmission of "*Ca.* Mystax nordicus", since vertical transmission usually implies co-adaptation of the partners [68].

As was mentioned before, BMS16-23 and BMS17-1 cells represent the triple symbiotic association (CS + RS + host). Seemingly simultaneous occurrence of several endosymbionts in one host is much more common than was thought before [55]. It is assumed that the coexistence of two di fferent endosymbionts in one host compartment is unstable and leads to survival of only one symbiont [28]. Nevertheless, these symbiotic systems could be maintained for almost 1.5 years. Only by the end of the second year "*Ca.* Mystax nordicus" was lost in both strains, while "*Ca.* Megaira venefica" remained in the host cytoplasm, which provides evidence for the more balanced interaction between the host and "*Ca.* Megaira venefica" than with "*Ca.* Mystax nordicus". Our observations of non-viable host cells with "*Ca.* Mystax nordicus" sticking out of the host cortex support this suggestion. Thus, given all the morphological and biological peculiarities of the CS-endosymbionts, we propose to define the new genus "*Ca.* Mystax" with "nordicus" as the only species described so far.

#### *4.4. Description of "Candidatus Mystax nordicus"*

Mystax nordicus (Mys.tax nor.di.cus; L. masc. n. mystax, moustache—allusion to the symbiont shape, which resembles the gentleman's mustache (Figure 3); L. adj. nordicus, northern, meaning that the symbiont and the host were found in northern latitudes). A large nonmotile curve-shaped bacterium; 0.6–0.8 μm wide and up to 7 μm long. Cytoplasmic endosymbiont of *P. nephridiatum*, forming conglomerations. Belongs to family Holosporaceae, order Holosporales. Basis of assignment: 16S rRNA gene sequence (accession number MK673804) and positive match with the specific FISH oligonucleotide probe 16S\_Myst965 (5-CCT GTA CTA AAT CGG CCG AAC CG-3). Uncultured thus far.
