Fig 1.
Sampling location in Peterhof (Russia) in different seasons: (A) August; (B) January; (C) April.
Table 1.
In silico matching of the species-specific probe HoloParv_645 against bacterial 16S rRNA gene sequences available from RDP (release 11, update 4) and SILVA (release 123) databases.
The number of sequences (“hits”) which hybridize with the designed probe are reported for 0 and 1 mismatches.
Fig 2.
Morphological features of P. chlorelligerum.
(A) General view of living cell; (B) nuclear apparatus: macronucleus (Ma) and micronucleus (black arrowhead) are indicated; (C) squashed cell: macronucleus (Ma) and micronucleus (black arrowhead) are indicated; (D) pore of contractile vacuole (white arrowhead); (E) contractile vacuole with satellite vesicles; (F) cytoplasmic symbiotic algae released from the squashed ciliate cell; (G) tuft of long caudal cilia. Scale bars: 17 μm (A); 6 μm (B, C); 10 μm (D-F); 20 μm (G).
Fig 3.
Silver nitrate impregnation (A-C) and Feulgen staining (G-J) of P. chlorelligerum. (A) ventral and (B, C) dorsal sides of the ciliate; position of oral aperture and pores of contractile vacuoles (black arrows) well visible; (D) oral ciliature: two peniculi and quadrulus (white arrow); (E) nuclear apparatus: macronucleus (Ma) and micronucleus (black arrowhead) are indicated; (F) macronucleus (Ma) infected with bacteria; (G) general view of a ciliate after Feulgen staining; nuclear apparatus with two (H) and one (I) micronuclei; (J) symbiotic algae in the cytoplasm of ciliate. Scale bars: 13 μm (A-C); 20 μm (D, G); 8 μm (E, F); 10 μm (H, I); 7 μm (J).
Fig 4.
Ultrastructure of cytoplasmic symbiotic algae.
(A) longitudinal section of alga cell; (B) cross section of alga cell; (C) part of the cell with nucleus. Nucleus (N), chloroplast (Ch) and mitochondria (Mt) are indicated. Scale bars: 0.5 μm (A), 0.4 μm (C); 3 μm (B).
Table 2.
18S rDNA gene sequences identities among members of Meyerella genus.
Fig 5.
Diversity of cells in the culture of presumably ex-symbiotic algae obtained from P. chlorelligerum.
(A) The young vegetative cells (vc), the mature cells (mc), and the cyst-like cells (cl); (B) division into four cells as tetrads or (C) chains; (D) production of autospores; (E) mature cell with a lipid droplet in cytoplasm. Scale bars: 7 μm (A); 5 μm (B-E).
Fig 6.
Bayesian inference phylogenetic tree of genus Paramecium based on 18S rDNA gene sequences.
Numbers associated to each node represent bootstraps values inferred after 1000 pseudoreplicates and Bayesian poster probabilities (values below 70 | 0.70 are not shown). Sequences in bold were characterized in this study. The bar stands for an estimated genetic distance of 0.05.
Fig 7.
Bayesian inference phylogenetic tree of genus Paramecium based on ITS1-5.8S-ITS2 sequences.
Numbers associated to each node represent bootstraps values inferred after 1000 pseudoreplicates and Bayesian poster probabilities (values below 70 | 0.70 are not shown). Sequences in bold were characterized in this study. The bar stands for an estimated genetic distance of 0.09.
Fig 8.
P. chlorelligerum infected with Holospora-like bacterium.
(A) general view of infected cell: macronucleus (Ma) is indicated; (B) highly infected macronucleus; (C) slightly infected macronucleus; (D) bacteria releasing from the squashed macronucleus; (E) infectious (IF) and reproductive (RF) forms of the bacterium; (F) spindle-like IFs under high magnification. Scale bars: 13 μm (A); 10 μm (B); 8 μm (C); 5 μm (E, F).
Fig 9.
Micrograph of P. chlorelligerum macronucleus infected with Holospora-like bacterium.
In karyoplasm both infectious (IF) and reproductive (RF) forms of the bacterium are visible. IF manifest double composition of periplasm (white asterisks). Attached to the macronucleus (Ma) a part of the micronucleus (Mi) with distinctive a hyaline “achromatic cap” (black asterisk). Scale bar: 2 μm.
Fig 10.
Peculiarities of Holospora-like bacterium.
(A) part of infected macronucleus; (B) composition of infectious form: cytoplasm (c), periplasm regions with different density (white asterisks) and recognition tip (t); (C) cross sections of infectious (IF) and reproductive (RF) forms; (D) fine fibrous material can be presented on the surface of some of IFs (black arrows). Scale bars: 2 μm (A); 1.5 μm (B); 1 μm (C); 0.6 μm (D).
Table 3.
16S rDNA gene sequences identities among members of Holospora genus and within the same species, when more than one sequence is present.
Fig 11.
Fluorescence in situ hybridization results on fixed P. chlorelligerum cell infected with Holospora-like bacterium.
(A) The ciliate macronucleus stained by DAPI; bacteria in the macronucleus are visualized both with eubacterial probe EUB338 (B) labeled with AlexaFluor® (green signal) and with the probe specific for Holospora-like bacterium HoloParv_645 labeled with Cy3 (red signal) (C). Scale bars: 20 μm.
Fig 12.
Bayesian inference phylogenetic tree of order Rickettsiales based on 16S rDNA gene sequences.
Numbers associated to each node represent bootstraps values inferred after 1000 pseudoreplicates and Bayesian poster probabilities (values below 70 | 0.70 are not shown). Sequences in bold were characterized in this study. The bar stands for an estimated genetic distance of 0.07. “Ca.” stands for “Candidatus”.