Life cycle analysis of unicellular algae.

Unicellular green alga is a very convenient object for flow cytometric characterization. Flow cytometry has been proposed as a quick and reliable tool for studying life cycle and growth of unicellular algae. Cell size of vegetating algae can be monitored in association with their DNA and endogenous chlorophyll content. Cells of interest (e.g., group of cells of a certain stage of the life cycle) in an asynchronously proliferating cell population can be sorted out for further microscopical or molecular biology studies. This methodological approach can be helpful for researchers who are interested in algal proliferation.

Arrest of cytoplasmic streaming induces algal proliferation in green paramecia.

A green ciliate Paramecium bursaria, bearing several hundreds of endosymbiotic algae, demonstrates rotational microtubule-based cytoplasmic streaming, in which cytoplasmic granules and endosymbiotic algae flow in a constant direction. However, its physiological significance is still unknown. We investigated physiological roles of cytoplasmic streaming in P. bursaria through host cell cycle using video-microscopy. Here, we found that cytoplasmic streaming was arrested in dividing green paramecia and the endosymbiotic algae proliferated only during the arrest of cytoplasmic streaming. Interestingly, arrest of cytoplasmic streaming with pressure or a microtubule drug also induced proliferation of endosymbiotic algae independently of host cell cycle. Thus, cytoplasmic streaming may control the algal proliferation in P. bursaria. Furthermore, confocal microscopic observation revealed that a division septum was formed in the constricted area of a dividing paramecium, producing arrest of cytoplasmic streaming. This is a first report to suggest that cytoplasmic streaming controls proliferation of eukaryotic cells.

Myosin II activity is not essential for recruitment of myosin II to the furrow in dividing HeLa cells.

To elucidate whether phosphorylation of myosin II regulatory light chain (MRLC) is essential for myosin II recruitment to the furrow during cytokinesis, HeLa cells transfected with three types of GFP-tagged recombinant MRLCs, wild-type MRLC, non-phosphorylated form of MRLC, and phosphorylated form of MRLC, were examined. Living cell-imaging showed that both phosphorylated and non-phosphorylated form of MRLCs were recruited to the equator at the same time after anaphase onset, suggesting that phosphorylation of MRLC is not responsible for recruitment of myosin II to the equator. Moreover, the treatment with an inhibitor of myosin II activity, blebbistatin, induced no effect on recruitment of those three recombinant MRLCs. During cytokinesis, phosphorylated but not non-phosphorylated form of MRLC was retained in the equator. These results suggest that phosphorylation of MRLC is essential for retainment of myosin II in the furrow but not for initial recruitment of myosin II to the furrow in dividing HeLa cells.

Altered toxicities of fatty acid salts in green paramecia cultured in different waters.

Detergents including fatty acid salts act as surface-active agents and thus possibly damage the plasma membrane structures of aquatic organisms. Therefore, when excess, the house-used and industrial outflows of such detergents into aquatic environments may have considerable impacts on the ecosystem. In this study, we propose the use of green paramecia (Paramecium bursaria) for assessing the acute toxicity of eight fatty acid salts (Na and K salts of oleate, palmitate, laurate and myristate) under various water conditions. The Paramecium in the stationary phase were used for a toxicity assay carried out on 12-well microplates and the median lethal concentration (LC50) was determined for each fatty acid salt. In the low mineral culture medium prepared with ultra-pure water, the LC50 for each fatty acid ranged from 5.8 to 144 ppm (w/v). The toxic levels of fatty acid salts differed in the following order: laurate, myristate > or = oleate, palmitate. The toxic levels of oleate and palmitate salts were ca. 10-fold lower than those of laurate and myristate salts. When river water and local tap water instead of ultra-pure water were used for culturing, the toxic levels of all fatty acid salts were drastically lowered compared to the low mineral condition by 30- to 100-fold (198-660 ppm, w/v). Similar detoxification effect was observed when Ca or Mg was added to the low mineral culture media, indicating that the toxicity of fatty acid salts can be notably lowered as the mineral content increases. As we demonstrated that toxicities of fatty acid salts can be lowered in river water and tap water compared to the low mineral condition, some chemical substances behave differently in the different water conditions. Therefore, the use of natural waters reflecting the real environmental conditions in further collection of data on the ecotoxicity impacts of variety of chemicals is highly encouraged.

A new approach for the assessment of acrylamide toxicity using a green paramecium.

Exposure to acrylamide induces neurotoxic effects in humans. In addition, it induces genotoxic, reproductive and carcinogenic effects in laboratory animals. However, no convenient bioassay system for assessing acrylamide toxicity to animal and plant cells has been proposed to date. The present study aims to evaluate acrylamide toxicity to a green paramecium, Paramecium bursaria, bearing many endosymbiotic algae, because some chemicals are highly toxic to paramecia or microalgae, and some protozoa are already used for evaluation of environmental contaminations. Results showed that high acrylamide concentrations (> or = 1500 mg/l) have a lethal effect on P. bursaria. Although low acrylamide concentrations (< or = 150 mg/l) induced less change on the paramecium growth, the number of endosymbiotically growing algal cells drastically decreased. The acrylamide concentration required to induce a 50% decrease in the cell number (IC(50)) was determined to be 7.8 mg/l for endosymbiotic algae, indicating that the algal sensitivity to acrylamide was 7 and 15 times higher than that of Syrian hamster embryo (SHE) cells and the host cells, respectively. Here, we propose the use of P. bursaria being a convenient and sensitive bioindicator as a new approach for the assessment of acrylamide toxicity.

Green paramecia as an evolutionary winner of oxidative symbiosis: a hypothesis and supportive data.

A single cell of the green paramecia (Paramecium bursaria) harbors several hundreds of endo-symbiotic Chlorella-like algae in its cytoplasm. Removal of algae from the host organism and re-association of ex-symbiotic host paramecia with ex-symbiotic algae can be experimentally demonstrated in the laboratory. However, the mechanism precisely governing the alga-protozoan association is not fully understood, and the origin of symbiosis in the evolutionary view has not been given. Here, we propose the possible biochemical models (models 1 and 2) explaining the co-evolution between Paramecium species and algal symbionts by pointing out that algal photosynthesis in the host paramecia plays a dual role providing the energy source and the risk of oxidative damage to the host. Model 1 lays stress on the correlation between the (re)greening ability of the paramecia and the tolerance to oxidative stress whereas model 2 emphasizes the cause of evolutionary selection leading to the emergence of Paramecium species tolerant against reactive oxygen species.

Optical compartmentation of vegetating algae species as a basis for their growth-specific characterization.

BACKGROUND: The number of microalgal strains known to date is enormous and continuously growing, and their characterization accordingly requires quick and reliable methodologies. METHODS: Asynchronously growing logarithmic (3- and 6-day cultures) and stationary (9-day cultures) phase cell populations of two algae species that are difficult to distinguish microscopically (one Chlorella sp., C. vulgaris [c-27], and another that might belong to the same genus, SA-3 algae exsymbiotic from Paramecium bursaria) were characterized by means of flow cytometry (FCM). Forward light scatter (FSC) of algae was monitored in association with their 90 degrees side light scatter (SSC) and fluorescence of endogenous chlorophyll (FL3-height). RESULTS: Two-parameter FSC versus SSC and FSC versus FL3-height plots distinctly showed growth-specific compartmentation of algae into discrete cell subpopulations staying at a particular stage of the life cycle, and numbers of cells constituting these subpopulations could be quantitated. The growth pattern of C. vulgaris (c-27) differed substantially from that of SA-3 algae, particularly in the late-logarithmic (6-day) cultures. At this phase of growth, C. vulgaris (c-27) cells compartmentalized into three subpopulations, whereas SA-3 cells compartmentalized into two subpopulations. Different compartmentations of optical signals from late-logarithmic phase SA-3 algae and C. vulgaris (c-27) likely were caused by the differences in timing of the life cycle stages of these types of cells. CONCLUSIONS: Growth-specific compartmentation of vegetating microalgae by FCM provides a good basis for characterization of morphologically similar algae species. Because algae are also present in symbiotic relationships with other organisms, this tool might be of potential interest for the study of symbiosis mechanisms.

Stimulatory effect of aerosil on algal growth.

Unicellular green alga represents not only a convenient model for its biochemical and physiological studies but also a sensitive system to test the effects of various environmental factors. Algae cells of two strains, SA-3 strain (exsymbiotic from Paramecium bursaria) and Chlorella vulgaris c-27, were asynchronously cultured in the presence of 0.01% Aerosil A-300. Aerosil effects on algae were monitored at logarithmic and stationary phases of their growth by flow cytometry and microscopic counting of algal numbers. The growth patterns of algae were evaluated by their forward light scatter versus fluorescence of endogenous chlorophyll (FL3-height) signal distributions. Although aerosil itself did not cause any direct effects on algal morphology, it affected the growth patterns and the numbers of algae of both strains. Their growth patterns were remarkably altered in the late logarithmic phase cultures (6-day cultures). However, a significant increase of cell numbers was found in the stationary phase cultures (9- and 12-day cultures). While C. vulgaris c-27 demonstrated an increase of cell numbers by approximately 11% in the 9- and 12-day cultures, the amounts of SA-3 cells in the 9- and 12-days cultures were increased by 16% and 35%, respectively. Our study shows aerosil in its colloidal form stimulates proliferation of algae mainly via an acceleration of their life cycles. The stimulatory effect of silica on the growth of algae, the mechanism of which remains to be clarified, might have a practical (e.g., ecological) interest for regulation of algal expansion.

Presence of a Tetrahymena growth promoting activity in fetal bovine serum.

Treatment of cultures of the ciliate Tetrahymena with fetal bovine serum (FBS) enhanced the rate of cell proliferation. The growth promoting activity was partially purified from FBS as a high Mr complex including four components with apparent Mr values of 180 kDa, 68 kDa, 60 kDa and 30 kDa by a 4-step procedure. The 180 kDa component was identified by amino acid sequencing as α2-macroglobulin. The addition of purified α2-macroglobulin from bovine plasma to cultures of Tetrahymena was also found to enhance the rate of cell proliferation. This report is the first dealing with the direct identification of a mammalian factor which promotes the growth of free-living protozoa.