Cloning and characterization of endosymbiotic algae isolated from Paramecium bursaria

The green paramecium Paramecium bursaria has many endosymbiotic algae in its cytoplasm. Here, we cloned and characterized endosymbiotic algae from P. bursaria and examined in detail the interaction between the cloned algae and algae-free paramecia. Homogenates of P. bursaria were cultured on agar plates containing various kinds of media to establish clones of the endosymbiotic algae. Many algal colonies were obtained from poorly nutritious medium (CA medium) after one month in culture. Algae were picked up from these colonies and inoculations were repeated 9 times on agar plates containing CA medium. On enriched media including bacto-peptone, glucose, proteose-peptone and/or yeast extract, however, bacteria and mold grew rapidly and no algal colonies were formed. When the cloned algae were cultured in liquid CA medium, they grew faster than on agar plates and the numbers stayed constant at 1 × 107 algae/ml after 7 days in culture. They revealed high infectivity to algae-free paramecia, and an incubation period of 24 h and at least 1 × 103 algae/paramecium were required to achieve successful infection (80–90%). The growth and infection rate did not change through 74 repeated inoculations of algae in liquid CA medium. Optical microscopic observations revealed marked morphological similarity between endosymbiotic algae and free-living Chlorella, but the latter showed no infectivity to algae-free paramecia. The cloned endosymbiotic algae presented here will provide an excellent opportunity to examine the mechanism of symbiont-host interaction.