Endosymbiotic Green Algae inParamecium bursaria: A New Isolation Method and a Simple Diagnostic PCR Approach for the Identification

Paramecium bursariais a single-celled model organism for studying endosymbiosis among ciliates and green algae. Most strains ofP. bursariabear eitherChlorella variabilisorMicractinium conductrixas endosymbionts. Both algal genera are unicellular green algae characterized by cup-shaped chloroplasts containing a single pyrenoid and reproduction by autospores. Due to their size and only few morphological characteristics, these green algae are very difficult to discriminate by microscopy only. Their cultivation is laborious and often unsuccessful, but we developed a three-step isolation method, which provided axenic cultures of endosymbionts. In addition to the time-consuming isolation, we developed a simple diagnostic PCR identification method using specific primers forC. variabilisandM. conductrixthat provided reliable results. One advantage of this approach was that the algae do not have to be isolated from their host. For a comparative study, we investigated 19 strains ofP. bursariafrom all over the world (new isolates and available laboratory strains) belonging to the five known syngens (R1-R5). Six European ciliate strains belonging to syngens R1 and R2 boreM. conductrixas endosymbiont whereasC. variabiliswas discovered in syngens R1-R5 having worldwide origins. Our results reveal the first evidence ofC. variabilisas endosymbiont inP. bursariain Europe.