Ca2+-induced encystment of Colpoda sp. was found to be cancelled by bacteria suspended in the surrounding medium. Continuation of the uptake of polystyrene latex particles into the food vacuoles slightly suppressed encystment. When the cells of Colpoda containing a large number of food vacuoles filled with Congo-red stained bacteria were transferred into an encystment-inducing medium, they encysted after promptly expelling the contents of the food vacuoles. Supernatant obtained from bacterial suspension, commercial albumin or polypeptone markedly prevented encystment, although free amino acid did not have much of an affect. Boiling or dialysis (MWCO: 10,000) of the supernatant reduced the encystment-suppression effect. These results suggest that the essential factors for the suppression of encystment are components such as peptides or proteins that are released from bacteria suspended in the surrounding medium.
Excystment of Colpoda sp. was found to be triggered by a brownish fraction obtained by high performance liquid chromatography (HPLC) of cereal infusion that was expected to contain porphyrins. A commercial porphyrin, chlorophyllin (coppered, sodium salt) induced excystment and prevented encystment, while protoporphyrin IX without metal, protoporphyrin IX bound to zinc, and riboflavin did not have any excystment-inducing effect. These results suggest that the porphyrins contained in cereal leaves might trigger excystment and prevent encystment. Green-colored chlorophyllin is expected to be useful as a molecular probe to visualize and isolate receptors for triggering the encystment and excystment processes.
The behavior of the social amoeba Dictyostelium discoideum following UV irradiation was analyzed. Cells cultured in a liquid medium migrated at 4.5 ± 0.7 μm/min, while their motility increased to 19.4 ± 1.7 μm/min following UV irradiation (50 J/m2). A conditioned medium prepared from the irradiated cells, CM (UV), was used in the following experiments. Motility of cells that had not been exposed to UV increased to 29.7 ± 2.8 μm/min when the medium was changed to CM (UV). In addition, the expression levels of recA and rad51 were increased and sensitivity to UV was decreased in these cells. These results indicate that UV light-irradiated D. discoideum cells release a factor that enhances motility and induces expression of DNA repair enzymes in unirradiated cells. The factor would be important for D. discoideum to alleviate damage.
Polycystinea (Radiolaria) is a class of planktonic protists widely distributed in tropical, subtropical, and even polar marine environments. Various types of algae occur as intracellular symbionts in the cells of the polycystines (e.g., Anderson, 1976) which causes significant cross-contamination in polycystine DNA-extractions, complicating DNA-based molecular analyses. We designed potential primers; Spu 191F and Spu 1731R, specifically for spumellarian polycystines, which are solitary and have spongy or latticed siliceous shells. These primers amplified 18S rDNA sequences directly from the specimens containing symbiotic algae. They will be used to accelerate phylogenetic analyses of polycystine radiolarians, which have not been established in culture and possess symbiotic algae.
A bacteria-free monoxenic culture of Paramecium bursaria has been established. Chlorogonium elongatum was used as the sole food source and large numbers of easily maintained P. bursaria were obtained. Additional enhancement of the growth of P. bursaria was achieved by supplementing the culture medium with soybean lecithin. The ability of monoxenically cultured P. bursaria to re-establish its symbiotic relationship with Chlorella in bacteria-free conditions was examined. Bacteria-free, monoxenic, Chlorella-free (white) P. bursaria, and axenic isolated symbiotic Chlorella cultures were established from bacteria-free monoxenic cultures of Chlorella-bearing (green) P. bursaria. When the white paramecia and isolated Chlorella were mixed in bacteria-free conditions, they re-established a symbiotic relationship, and normal green paramecia were reconstructed. This newly developed bacteria-free monoxenic culturing of P. bursaria will be useful in the study of protozoan symbiosis, as this method provides us with large numbers of cells that possess the ability to reestablish a symbiotic relationship with Chlorella.
A Paramecium tetraurelia ND7 gene homolog was cloned and sequenced in P. caudatum. The predicted protein possessed well-conserved regions, a transmembrane helix and two coiled-coil domains possibly involved in protein-protein interaction, as previously suggested for P. tetraurelia. Southern hybridization revealed that the ND7 gene homolog was also present in P. multimicronucleatum, suggesting that the ND7p is well-conserved and plays an important role in regulated exocytosis in Paramecium species. We treated cells with lysozyme to discharge most of the trichocysts and incubated cells to regenerate them. The ND7 mRNA was present only in a trace amount in cells with abundant trichocysts, while, it drastically increased in trichocyst-regenerating cells. This work also provides a useful system to isolate genes/proteins which are involved in trichocyst discharge in Paramecium.