Endosymbiotic Bacteria in the Parasitic Ciliate Ichthyophthirius multifiliis

Endosymbiotic bacteria were identified in the parasitic ciliate Ichthyophthirius multifiliis, a common pathogen of freshwater fish. PCR amplification of DNA prepared from two isolates of I. multifiliis, using primers that bind conserved sequences in bacterial 16S rRNA genes, generated an ∼1,460-bp DNA product, which was cloned and sequenced. Sequence analysis demonstrated that 16S rRNA gene sequences from three classes of bacteria were present in the PCR product. These included Alphaproteobacteria (Rickettsiales), Sphingobacteria, and Flavobacterium columnare. DAPI (4′,6-diamidino-2-phenylindole) staining showed endosymbionts dispersed throughout the cytoplasm of trophonts and, in most, but not all theronts. Endosymbionts were observed by transmission electron microscopy in the cytoplasm, surrounded by a prominent, electron-translucent halo characteristic of Rickettsia. Fluorescence in situ hybridization demonstrated that bacteria from the Rickettsiales and Sphingobacteriales classes are endosymbionts of I. multifiliis, found in the cytoplasm, but not in the macronucleus or micronucleus. In contrast, F. columnare was not detected by fluorescence in situ hybridization. It likely adheres to I. multifiliis through association with cilia. The role that endosymbiotic bacteria play in the life history of I. multifiliis is not known.The ciliate Ichthyophthirius multifiliis is an obligate parasite of freshwater fish that infects epithelia of the skin and gills. The life cycle of I. multifiliis consists of three stages: an infective theront, a parasitic trophont, and a reproductive tomont. Infection is initiated by invasion of the skin and gills by free-swimming, 40-μm-long, pyriform-shaped theronts that burrow several cell layers deep into epithelial tissue of the skin and gills and rapidly differentiate into trophonts. Trophonts feed on epithelial cells and grow into 500- to 800-μm-diameter cells, causing extensive damage to skin and gills, which in severe infections results in mortality (10-12). After feeding for 5 to 7 days, trophonts leave the host, form encysted tomonts, and undergo up to 10 cell divisions over 18 to 24 h, producing as many as 10³ daughter cells, which exit the cyst as infective theronts to reinitiate the life cycle. I. multifiliis is ciliated at all stages (9).DNA sequencing of the I. multifiliis genome at the J. Craig Venter Institute unexpectedly revealed that bacterial DNA sequences, including sequences with homology to Rickettsia, were present in the DNA preparations (R. S. Coyne, 2009 [http://www.jcvi.org/cms/research/projects/ich/overview]). The origin of these sequences was unclear, but they represented evidence for either horizontal gene transfer into the I. multifiliis genome (17, 27) or the presence of intracellular bacteria. No previous evidence suggested the presence of intracellular bacteria in I. multifiliis, even though the fine structure of I. multifiliis theronts and trophonts has been examined by transmission electron microscopy (10-12). Intracellular or endosymbiotic bacteria, however, are commonly found in protists, and about 200 ciliate species are known to harbor intracellular bacteria (13, 15). Sonneborn and Preer in their classic studies on endosymbionts in Paramecium characterized a number of different endosymbionts, including “killers,” named for their ability to kill uninfected strains of Paramecium. Cytoplasmic endosymbionts in Paramecium now include Caedibacter taeniospiralis (Gammaproteobacteria), and Pseudocaedibacter conjugates, Tectibacter vulgaris, and Lyticum flagellatum (Alphaproteobacteria). Macronuclear endosymbionts include the Alphaproteobacteria, Holospora caryophila, and Caedibacter caryophila, which can also infect the cytoplasm (4, 16, 22, 26). The roles these endosymbionts play in protists are not well understood.The presence of sequences with homology to bacterial genomes prompted us to determine if I. multifiliis contained endosymbionts, or if these sequences represented evidence for horizontal gene transfer into the I. multifiliis genome. Our identification of the same two endosymbionts, in two different isolates of I. multifiliis, suggests that endosymbionts are common in I. multifiliis. However, the physiological relationships between I. multifiliis and its resident endosymbionts are unclear. It is not known if the endosymbionts contribute to the growth of I. multifiliis, if they contribute to the severity or pathogenicity of infection, or if they provide their host with any selective advantage, as occurs with Paramecium containing killer particles (4). It has not been determined if they influence the immune response of fish infected with I. multifiliis. It is possible that they may simply be parasites of this parasitic ciliate.