Calcium Ions as Conjugation-Specific Regulators in Paramecium caudatum

The unicellular ciliate Paramecium caudatum undergoes a developmental transition from asexual binary fission to sexual reproduction during its mature stage. This transition is triggered by mating interactions between cells of complementary mating types, leading to aggregate formation, mating pairs, and the meiotic division of micronuclei. Although calcium-driven EF-hand kinases have been implicated as mating type proteins, the spatiotemporal dynamics of calcium signaling during conjugation have not been comprehensively characterized. In this study, we established a behavioral assay to isolate committed cells from aggregates immediately after mating onset, and developed an experimental system to monitor intracellular calcium fluctuations specifically expressed in these cells. By combining Ca²⁺/EGTA buffering and microinjection approaches, we manipulated extracellular and intracellular calcium levels and confirmed the continuous requirement of calcium ions for conjugation-specific functions. Two significant findings emerged. First, we identified, for the first time, a calcium atlas covering the entire cell, with ascending centers localized in the anterior, oral apparatus, and posterior regions. The calcium/Indo-1-AM fluorescence peaked at 6 h after mating initiation and declined gradually, but persisted until conjugation was completed at ~48 h. Second, we demonstrated that distinct intracellular calcium thresholds are required for each stage of mating, including maintenance of mating activity, commitment of micronuclei to meiosis, and two-stepwise formation of mating pairs. These thresholds function as regulatory checkpoints that coordinate subcellular localization and stage synchronization. Collectively, our findings highlight calcium ions as pivotal regulators of conjugation in Paramecium and propose a novel framework, the Paramecium calcium atlas, for understanding the cellular and molecular mechanisms underlying sexual reproduction in ciliates.