Detection of endocytobionts inhabiting the macronucleus of Frontonia paramagna (Ciliophora, Peniculida)

Bacterial endosymbionts of Frontonia, a widely distributed ciliate genus, remain poorly characterized. Here, we investigated the endosymbiotic microbiota of a Shanghai population of Frontonia paramagna using an integrated morphological and molecular approach. Fluorescence in situ hybridization (FISH) targeting the 16S rRNA gene, coupled with V3-V4 high-throughput sequencing, consistently identified Caedimonas as the bacterial symbiont, specifically localized within the host macronucleus. FISH and transmission electron microscopy confirmed this intramacronuclear colonization with high prevalence and revealed that the symbionts lack flagella and R-bodies. Phylogenetic analysis of full-length 16S rRNA gene sequences placed the F. paramagna symbionts within a well-supported clade containing Caedimonas from divergent hosts. Comparative analysis of the 16S rRNA internal excised element (IEE) showed substantial sequence and secondary structural divergence between the Frontonia-associated lineage and other Caedimonas strains from different ciliates. We conservatively designate this lineage as Caedimonas varicaedens Fpa. These distinct molecular features suggest that the diversity and host distribution of Caedimonas are far from fully described, and genomic approaches will be necessary to evaluate species delimitation within the genus and the possible presence, distribution, and horizontal transfer of R-body genetic determinants.