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. rbcL 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.