Abstract

Euryhaline species exhibit remarkable osmoregulatory adaptability, yet their underlying molecular basis of osmoregulation—especially those governing short-term versus long-term adaptation—remains poorly understood. This study investigates these issues using the euryhaline unicellular eukaryote Paramecium calkinsi. Based on a de novo assembled high-quality genome, comparative genomic analysis across 17 species revealed an expansion of osmoregulatory genes, identifying 195 expanded gene families implicated in oxidoreductase activity, ion binding, and transmembrane transport. Transcriptomic analysis under varying salinity treatments revealed distinct molecular foundations that underpin transient and sustained adaptation to hyperosmotic and hypoosmotic stress. Under hyperosmotic conditions, cells exhibit a rapid activation of the membrane transport system in the short term, whereas long-term adaptation features enhanced ribosome biogenesis, chromatin remodeling, activation of transport systems, and metabolic downregulation to optimize energy use. For hypoosmotic stress, short-term adaptation is characterized by specific hydrolysis of intracellular substances, while long-term adaptation involves sustained oxidoreductase activation and enhanced vesicular/proton transport to maintain oxidative balance and cellular homeostasis. Moreover, we found that genes involved in signaling, transport, and protein stability pathways exhibit high rates of alternative splicing, suggesting a potential role of mRNA splicing in osmoregulation. Our findings provide molecular insights into osmoregulation in unicellular eukaryotes, highlighting their unique genetic responses to transient and sustained salinity stress and offering a valuable starting point for future functional studies.