First Direct Evidence for a Structurally Stable Adhesion Between the Perialgal Vacuole Membrane and Host Mitochondria in the <em>Paramecium</em>-<em>Chlorella</em> Endosymbiosis

Physical integration between endosymbiotic algae and host mitochondria is a recurring feature across photosynthetic symbioses, yet the structural nature of this association has remained unresolved. In the ciliate Paramecium bursaria, each endosymbiotic Chlorella cell is enclosed by a perialgal vacuole (PV) membrane consistently surrounded by host mitochondria, suggesting a conserved architecture for metabolic interaction. Although transmission electron microscopy has shown close membrane apposition, it has remained unclear whether this reflects incidental proximity or a reinforced adhesion. Here, we provide direct evidence that the PV membrane and host mitochondrial membrane form a stable physical association. Using discontinuous Percoll density-gradient centrifugation, we isolated intact units in which Chlorella and mitochondria co-sedimented, indicating that their association withstands mechanical disruption. By fluorescently labeling the PV and mitochondrial membranes with BODIPY FL C5-ceramide complexed to BSA, together with a mitochondria-specific monoclonal antibody and DAPI, we visualized the PV membrane under light microscopy and demonstrated that the mitochondrial–PV membrane complex persists after homogenization and centrifugation. As expected from the membrane-insertion behavior of BC5C, this fluorescent labeling revealed that the PV–mitochondrial membrane association is structurally reinforced rather than incidental, providing a mechanistic framework for understanding how Chlorella cells are stably positioned beneath the host cortex.