Outwitting planarian’s antibacterial defence mechanisms: Rickettsiales bacterial trans-infection from Paramecium multimicronucleatum to planarians

Most of the microorganisms belonging to genera responsible for vector-borne diseases (VBD) have hematophagous arthropods as vector/reservoir. Recently, many new species of microorganisms phylogenetically related to agents of VBD were found in a variety of aquatic eukaryotic hosts, in particular, numerous new bacterial species related to the genus Rickettsia (Alphaproteobacteria, Rickettsiales) were discovered in protist ciliates and other unicellular eukaryotes. Although their pathogenicity for humans and terrestrial animals is not known, these bacteria might act as etiological agents of possible VBD of aquatic organisms, with protist as vectors. In the present study, we characterized a novel strain of the Rickettsia-Like Organism (RLO) endosymbiont “Candidatus (Ca.) Trichorickettsia mobilis” in the macronucleus of the ciliate Paramecium multimicronucleatum through Fluorescence In Situ Hybridization (FISH) and molecular analyses. Ultrastructural investigations on the presence of flagella confirmed previous studies on the same bacterial species. The potential trans-infection per os of this bacterium to planarians (Dugesia japonica), a widely used model system able to eliminate a wide range of bacteria pathogenic to humans and other Metazoa, was further verified. Ciliate mass cultures were set up, and trans-infection experiments were performed by adding homogenized paramecia to food of antibiotic-treated planarians, performed. Treated and non-treated (i.e. control) planarians were investigated at day 1, 3, and 7 after feeding for endosymbiont presence by means of PCR and ultrastructural analyses. Obtained results were fully concordant and suggest that this RLO endosymbiont can be transferred from ciliates to metazoans, being detected up to day 7 in treated planarian enterocytes inside and, possibly, outside phagosomes.

comfortably feed for a period of 2h under regular culturing conditions (see above). Attention was 243 paid to planarian feeding behaviour during this period. As no differences were noted concerning 244 feeding behaviour between treated and control planarians, and feeding procedure was exerted by 245 all planarians as expected according to regular planarian culturing, we proceeded with the next 246 steps. Two washing steps were then carried out removing the medium and adding fresh planarian 247 culturing water. Finally, the two groups of animals were left in their fresh culturing water and in 248 regular cultivation conditions in the two Petri dishes until the collection of specimens for the next  (Fig 1c) and the species-specific probe Trichorick_142 (Fig 1d) 303 confirmed this result but disclosed a different, novel bacterial localization, i.e. the ciliate 304 macronucleus (roughly with a presence of about 100 endosymbionts). Additionally, the full 305 overlapping of eubacterial universal probe EUB338 and "Ca. Trichorickettsia"-specific probe signals 14 306 indicated that this symbiont constituted the total set of intracellular bacteria in host P.

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In TEM-processed ciliate cells, the endosymbionts were confirmed to be hosted in the 309 macronucleus; they showed a two-membrane cell wall typical for Gram-negative bacteria and 310 appeared rod-shaped, with rounded to narrower ends (Fig 2). They were surrounded by a clear halo

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One day after feeding, several bacteria were recognizable in planarian enterocyte phagosomes.
377 Some of them were still intact (Figs 4a and 4b), even performing cell division. Others appeared 378 degraded, being subjected to digestion (data not shown). In several cases, membrane of bacteria-379 including phagosomes was damaged and interrupted (Figs 4a and 4b). Bacteria free in the cytoplasm 380 of planarian intestinal cells, i.e. in direct contact with their cytoplasm, were detected as well; they 381 showed a two-membrane cell wall and a surrounding clear halo (Figs 4c-4f), with electron-lucid 382 "holes" sometimes visible inside their cytoplasm (Fig 4e). Flagella (diameter: ~ 0.009 µm) could also 383 be detected all around bacteria (Figs 4b-4d and 4f). Additionally, extruded trichocysts (extrusive 384 organelles typical of Paramecium), presumably included in Paramecium homogenate and ingested 385 by treated planarians, were easily recognizable inside intestinal cell phagosomes (Fig 4g).

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A similar scenario was observed 7 days after feeding, when most of the bacteria occurred 387 enclosed inside planarian enterocyte phagosomes (Figs 5a and 5b); some appeared degraded (not 388 shown), while many others did not show degraded conditions and could survive in the phagosomes 389 (Figs 5a and 5b). In few cases, bacterial-like circular-ovoid shapes were also observed in the 390 cytoplasm of intestinal cells, outside from phagosomes (Fig 5c).

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We believe that our findings may offer intriguing insights when considered from several 502 points of view, such as concerning the pathologies caused by Rickettsiales or RLOs occurring in fish 503 farms or in the wild, which might have ciliates or other protists as putative vectors. Although there is 504 still a need for further investigations on this topic to expand its implications, we think that our study 505 can serve as basis for conceiving long-lasting experiments aiming to better understand whether "Ca.
506 Trichorickettsia mobilis", as well as other Rickettsiales symbionts of protists, can be able to survive 507 longer and potentially replicate in tissues of planarians and other aquatic Metazoa, and whether these 508 RLOs may have some impact on the recipient host health.