Simultaneous Evaluation of Life Cycle Dynamics between a Host Paramecium and the Endosymbionts of Paramecium bursaria Using Capillary Flow Cytometry

Abstract

Endosymbioses are driving forces underlying cell evolution. The endosymbiosis exhibited by Paramecium bursaria is an excellent model with which to study symbiosis. A single-cell microscopic analysis of P. bursaria reveals that endosymbiont numbers double when the host is in the division phase. Consequently, endosymbionts must arrange their cell cycle schedule if the culture-condition-dependent change delays the generation time of P. bursaria. However, it remains poorly understood whether endosymbionts keep pace with the culture-condition-dependent behaviors of P. bursaria, or not. Using microscopy and flow cytometry, this study investigated the life cycle behaviors occurring between endosymbionts and the host. To establish a connection between the host cell cycle and endosymbionts comprehensively, multivariate analysis was applied. The multivariate analysis revealed important information related to regulation between the host and endosymbionts. Results show that dividing endosymbionts underwent transition smoothly from the division phase to interphase, when the host was in the logarithmic phase. In contrast, endosymbiont division stagnated when the host was in the stationary phase. This paper explains that endosymbionts fine-tune their cell cycle pace with their host and that a synchronous life cycle between the endosymbionts and the host is guaranteed in the symbiosis of P. bursaria.

Figure 1. Examples of P. bursaria in a natural state and some experimental states.

(A) P. bursaria in a natural state. (B) Algae-free Paramecium produced by treatment with paraquat herbicide, as described previously. (C) Sexual reproduction and conjugation of natural P. bursaria with algae-free Paramecium produced by treatment with acrylamide, as described previously. (D) A fluorescence image of panel C is shown. Red and blue fluorescence derive, respectively, from endogenous chlorophyll of endosymbiotic algae and from DAPI-staining Paramecium nuclei.

Figure 2. Growth dynamics of P. bursaria host cells.

(A,B) Time-dependent population dynamics of P. bursaria were tracked over time. Then, the generation time and division times were estimated. (C–F) Microscopic images of P. bursaria at interphase and division phase. (G) Data obtained with P. bursaria possessing endosymbiotic algae were reconstructed using external software for FSS vs. count (% of total signals for intact P. bursaria cells). Dotted lines in the panel G represent each detectable and distinguishable peak size of P. bursaria. Here, the population of small cells of P. bursaria immediately after cell division (dotted line, A_D), that of cells at cytokinesis as in Fig. 2F (dotted line, B_D), and that of the intermediate cell sizes at the interphase or nuclear division phase (dotted line, Int) were detected using FCM.

Figure 3. Cell cycle dynamics of endosymbionts in P. bursaria by PCA method.

(A) The PCA reduces multiple-dimensional information to arbitrary one-dimensional information and produces new components such as PC1-PC3. Each contribution rate of each component was expressed as a stacked bar graph. (B) Factor loading plots of each parameter for PC1 and PC2, respectively. (C) Score plots of PC1 vs. PC2 were produced using data from different culture durations.

Figure 4. Population distribution of endosymbionts in P. bursaria using FCM.

The obtained data were reconstructed to produce a dot plot for FSS vs. red fluorescence intensity (% of total signals for endosymbiotic algae).

Figure 5. Analysis of population distribution dynamics of endosymbiotic algae in P. bursaria.

(A) Population distribution of exosymbiotic algae (SA-1) as a reference. St. 1, “growth” stage; St. 2, “ripening” stage; and St. 3, “division and autospore liberation” stages. (B) Comparison among distribution patterns of endosymbiotic algae was made culture term by culture term. Based on algal optical properties that have been related to the algal cell cycle, the distribution patterns of endosymbionts were categorized into three populations (regions I-III). (C) Time-dependent changes of algal distribution patterns were estimated as the stacked bar graph at each incubation time of P. bursaria.

Figure 6. Synchronization of culture time-dependent behaviors between endosymbiotic algae and their host paramecia.

Paramecium generation time from Fig. 2A and the time-dependent population changes of endosymbionts in P. bursaria from Fig. 5C were compared. Yellow and white areas respectively represent the duration for the logarithmic phase and for the early stationary phase of P. bursaria.

Figure 7. Evaluation of endosymbiosis status for the transition from photosymbionts to organelle-like structures vs. transmission of photosynthetic factories as plastids or intact algae to daughter cells.

The graph was quartered: Organisms use organelle-like structures such as plastids in upper areas, whereas they use eukaryotic algae as photosynthetic factories but not plastids in lower areas. Organisms can transmit their photosynthetic factories to daughter cells as shown right areas, although not in the left areas. Consequently, a lower left area presents heterotrophs only, whereas the upper right shows those using an authorized strategy like photosynthetic organisms from primary, secondary, and tertiary endosymbioses. Areas other than secondary and tertiary endosymbioses are multiple endosymbioses. The abbreviated words N_H, N_S, KP and CP in Fig. 7 respectively indicate each host nucleus, each nucleus of their symbionts, each kleptoplast derived from their algal prey and each chloroplast.