Protist ciliates as natural reservoir for bacteria potentially pathogenic for metazoan: preliminary results of the experimental infection of the planarian Dugesia.

Most of the microorganisms belonging to genera responsible for vector-borne diseases (VBD) has hematophagous arthropods as vector/reservoir. Recently, many new species of such microorganisms were found in a variety of terrestrial and aquatic eukaryotic hosts: in particular, numerous new bacterial species belonging/related to the genus Rickettsia, Alphaproteobacteria of the order Rickettsiales with an obligate intracellular lifestyle (e.g. the etiological agents of epidemic typhus and spotted fever), were discovered in protist ciliates. Although their pathogenicity for either humans or animals is still under study, these bacteria could actually act as etiological agents of possible VBD of aquatic environment. Indeed, an increasing number of massive fish death was recorded in intensive aquaculture facilities during the lasts years due to epidemics caused by Rickettsia-like bacteria with unknown natural vector. As ciliates could vector pathogenic organisms possibly responsible for zoonosis, we intended to verify the transmission of the Rickettsiales endosymbionts hosted by two species of ciliates to a metazoan model, the planarian Dugesia japonica. The ciliates were Euplotes woodruffi and Paramecium multimicronucleatum; the first hosts in the cytoplasm two different Rickettsiales endosymbionts, “Candidatus (Ca) Megaira polyxenophila” and “Ca. Bandiella woodruffii”, in addition to the betaproteobacterium Polynucleobacter necessarius; the second hosts in the macronucleus the Rickettsiales endosymbiont “Ca. Trichorickettsia mobilis”. Ciliate monoclonal mass cultures were set up to perform the following infection experiments: 1. Ciliate washing, concentrating, homogenizing, and addition to regular food for planarians. 2. Antibiotic-treated planarians feeding on ciliate-enriched food or regular food (as control), then washing and letting digest for 24, 48, and 72 h; 3. Comparative multidisciplinary investigation: molecular analyses, whole mount in situ hybridization, and TEM observation. Preliminary results are encouraging: we recovered endosymbionts up to 72 h after feeding, and identified them in planarians’ intestine and digestive vacuoles in TEM material. Endosymbionts were frequently observed near vacuole edge, with vacuole membrane sometimes somehow interrupted. TEM results were compared with literature data where endosymbionts were demonstrated to escape vacuoles invading the cytoplasm. Financial support: PRA 2016, University of Pisa.