Effects of Ammonium Chloride-Mediated Control of Digestive Vacuole Acidification on Endosymbiosis Between Paramecium tritobursaria and Chlorella variabilis

Paramecium tritobursaria is a ciliate that harbors intracellular Chlorella sp. symbionts and serves as a model organism for studying endosymbiosis. After ingestion by P. tritobursaria, some algal cells are digested within digestive vacuoles (DVs), whereas others escape digestion and become enclosed by a perialgal vacuole (PV) membrane, establishing a stable symbiosis. We investigated whether inhibiting DV acidification with ammonium chloride (NH4Cl) modulates algal digestion and the establishment of endosymbiosis. Congo red-stained yeast assays showed that treatment with 20 mM NH4Cl for 40 min effectively suppressed DV acidification. Under these conditions, algal intracellular behavior was altered; escape from DVs appeared to be delayed, particularly at 6 h after uptake, and many cells remained within the DVs. Despite this delay, symbiosis was established at 24 h in both groups. Notably, NH4Cl treatment significantly increased the symbiosis establishment rate, with an average 1.5-fold increase and up to a 3-fold increase compared with the control. These results demonstrate that transient inhibition of DV acidification alters digestion dynamics and promotes the establishment of symbiosis. This study provides a novel strategy for dissecting P. tritobursaria–Chlorella sp. endosymbiosis through controlled modulation of DV maturation.