Plagiopyla, STEIN, 1860

SYMBIONTS OF PLAGIOPYLA View in CoL View at ENA

Some groups of ciliates are known to host methanogenic Archaea in their cytoplasm (for review see: Hackstein, 2010); this association appears common, even obligate, for those living in oxygen-depleted environments, such as wet sediments poor in oxygen or animal digestive systems (gut, rumens, etc.). Mutual benefits, in this kind of symbiosis, are mostly related to physiological processes in the anaerobic lifestyle. Indeed, methanogenic Archaea are able to use H 2 and CO 2 to produce CH 4, thus reducing H 2 concentration and partial pressure inside the host cell, optimizing the chemo-physical condition for anaerobic respiration mediated by ciliate hydrogenosomes (van Brugen et al., 1984; Embley et al., 1995). However, other authors suggested that the main role of methanogens in symbiosis with ciliates is the production of organic material, quickly available for the host cell, rather than the control of H

2

partial pressure ( Fenchel & Finlay, 1991).

Several different methanogenic endosymbionts have been identified so far from anaerobic, free-living ciliates ( Fenchel et al., 1977; van Brugen et al., 1983, 1984, 1986; Wagener et al., 1990; Embley et al., 1992a, b; Finlay et al., 1993; Embley & Finlay, 1994; Narayanan et al., 2009; Filker et al., 2014). In plagiopylids, for example, P. nasuta has been infected by Methanobacterium formicicum ( Goosen et al., 1988) and Methanocorpusculum sp. ( Embley & Finlay, 1994), P. frontata hosted a species of Methanolobus ( Embley & Finlay, 1994) , whereas in Trimyema compressum , Methanobacterium formicicum and Methanobrevibacter arboriphilicus have been detected ( Wagener et al., 1990; Shinzato et al. 2007).

Our Plagiopyla populations were not an exception, showing the presence of at least one Archaea endosymbiont (putative for P. nasuta TP 2, since unfortunately the strain became extinct before FISH experiments with Archaea probe) showing one or two morphotypes in close association with their hydrogenosomes. Alternatively, the second endosymbiotic organism could be another Archaea or, perhaps, a bacterium not detected by EUB338 probe at applied experimental conditions.

In most of the cases, hydrogenosomes seemed to adapt their shape to maximize the surface in contact with these microorganisms, as already reported by other studies ( Fenchel et al., 1977; Embley & Finlay, 1994; Modeo et al., 2013). This indicates a close, mutualistic association among Plagiopyla hosts and their endosymbionts.

The rod-shaped morphology of these endosymbionts closely resembled those described by previous authors, with comparable size and without flagella ( Fenchel et al., 1977; Goosen et al., 1988). In P. ramani , P. narasimhamurtii and P. nasuta we observed two forms of endosymbionts, either indicating the presence of multiple symbionts, as already described for other ciliate species ( Görtz, 1987; Boscaro et al., 2012; Senra et al., 2016), or a single species of symbiont with different polymorphic life cycle, as supposed by previous studies on anaerobic ciliates ( Fenchel & Finlay, 1991; Finlay et al., 1993) and observed in some bacterial endosymbiont such as Caedibacter ( Anderson et al., 1964; Schrallhammer, 2010), Holospora (reviewed in: Fokin & Görtz, 2009) and Gortzia ( Boscaro et al., 2013; Serra et al., 2016), or ectosymbionts such as epixenosomes ( Petroni et al., 2000). In line with this, we could suppose that the more electron-dense type of endosymbiont detected in both P. ramani and P. narasimhamurtii ( Figs 5C–E, 11D, E, G), and P. nasuta ( Fig. 16A, C) could represent a sort of reproductive form, since it was the only one observed during binary fission. However, further analyses are required to solve this interesting issue.

In addition, sulphate-reducing ectosymbiotic Archaea have been previously described from anaerobic ciliates such as Metopus contortus , Caenomorpha lavanderi , Parablepharisma sp. and Sonderia sp. ( Fenchel et al., 1977; Fenchel & Ramsing, 1992). We detected ectosymbionts as well on P. ramani’ s and P. nasuta’ s surface with a shape and a size comparable to those of the above mentioned sulphate-reducing organisms ( Fenchel & Ramsing, 1992); they could be potentially assigned to this group of microorganisms able to gain energy from secondary metabolites excreted by the host such as acetate, lactate, ethanol, hydrogen or methane (the latter if endosymbiotic methanogens are present) ( Fenchel & Ramsing, 1992). A detailed molecular characterization of these ecto- and endosymbionts from Plagiopyla is ongoing.