Outwitting planarian’s antibacterial defence mechanisms:Rickettsialesbacterial trans-infection fromParamecium multimicronucleatumto planarians

Abstract

AS) 22 23 RUNNING PAGE HEAD: Rickettsiales macronuclear endosymbiont of a ciliate can transiently 24 enter planarian tissues 25 Abstract 38 Most of the microorganisms belonging to genera responsible for vector-borne diseases (VBD) have 39 hematophagous arthropods as vector/reservoir. Recently, many new species of microorganisms 40 phylogenetically related to agents of VBD were found in a variety of aquatic eukaryotic hosts, in 41 particular, numerous new bacterial species related to the genus Rickettsia (Alphaproteobacteria,42 Rickettsiales) were discovered in protist ciliates and other unicellular eukaryotes. Although their 43 pathogenicity for humans and terrestrial animals is not known, these bacteria might act as 44 etiological agents of possible VBD of aquatic organisms, with protist as vectors. In the present 45 study, we characterized a novel strain of the Rickettsia-Like Organism (RLO) endosymbiont 46 "Candidatus (Ca.) Trichorickettsia mobilis" in the macronucleus of the ciliate Paramecium 47 multimicronucleatum through Fluorescence In Situ Hybridization (FISH) and molecular analyses.48 Ultrastructural investigations on the presence of flagella confirmed previous studies on the same 49 bacterial species. The potential trans-infection per os of this bacterium to planarians (Dugesia 50 japonica), a widely used model system able to eliminate a wide range of bacteria pathogenic to 51 humans and other Metazoa, was further verified. Ciliate mass cultures were set up, and trans-52 infection experiments were performed by adding homogenized paramecia to food of antibiotic-53 treated planarians, performed. Treated and non-treated (i.e. control) planarians were investigated at 54 day 1, 3, and 7 after feeding for endosymbiont presence by means of PCR and ultrastructural 55 analyses. Obtained results were fully concordant and suggest that this RLO endosymbiont can be 56 transferred from ciliates to metazoans, being detected up to day 7 in treated planarian enterocytes 57 inside and, possibly, outside phagosomes. 58 59 KEY WORDS Planarians, molecular and ultrastructural investigations, Rickettsiaceae, RLO 60 endosymbiont, trans-infection experiments, vector-borne disease 61 4 62 Introduction63 Bacteria of order Rickettsiales (Alphaproteobacteria) live in an obligate association with a wide range 64 of eukaryotes [1-8], and, for their vast majority, are localized intracellularly, although the case of an 65 extracellular Rickettsiales bacterium was recently documented [9]. Rickettsiales are widely studied 66 for their involvement in medical and veterinary fields. Indeed, many of them (e.g. Rickettsia spp., 67 Anaplasma spp., Ehrlichia spp., and Orientia tsutsugamushi) are vectored by lice, ticks and mites, 68 and cause mild to severe disease [1, 10], such as epidemic typhus, Rocky Mountain spotted fever [11-69 13], anaplasmosis, ehrlichiosis [14, 15], heartwater [16], and scrub typhus [17]. 70 In the last two decades, many new genera and species of Rickettsiales were found as symbionts 71 in a variety of other non-vecto...