A defined synthetic algal medium enables lettuce-free culturing of unfed Paramecium bursaria while preserving host-associated microbiome composition

Paramecium bursaria is widely cultured using undefined plant-based infusions such as lettuce extract, yet the variable composition of these media remains a major obstacle to experimental reproducibility and microbiome research. Here, we tested whether a chemically defined synthetic algal medium (AF-6) can replace conventional lettuce infusion while maintaining host physiology and associated microbial communities. An unfed clonal strain of P. bursaria , established in 2023 and capable of growth without external nutrient supplementation, proliferated comparably in AF-6 and lettuce media. To confirm that these results were not specific to unfed conditions, we additionally examined a publicly maintained algae-fed strain (NIES-2891), which exhibited similar growth patterns across both media. Cell size, compression-induced extension, and symbiotic algal abundance showed no significant differences between culture conditions. rbc L metataxonomic analysis revealed that Chlorella variabilis was the sole algal endosymbiont detected in all samples. Furthermore, 16S rRNA gene sequencing demonstrated that host-associated bacterial community composition remained largely conserved after replacement of lettuce infusion with AF-6 within each strain, although clear differences were observed between strains. Together, these findings establish an “unfed strain + defined algal medium” framework as a reproducible experimental platform for investigating tripartite interactions among ciliate hosts, symbiotic algae, and associated bacteria.