Ammonia Can Damage Hemocytes in Portunus trituberculatus by Disrupting MCU-Mediated Mitochondrial Calcium Homeostasis

Calcium (Ca²⁺)-based signaling is vital for responses to environmental stress, with mitochondrial Ca homeostasis being extremely sensitive to changes in cell status. However, the mechanisms by which waterborne ammonia affects mitochondrial Ca dynamics in crustaceans remain unclear. This study examined the impacts of ammonia exposure on mitochondrial Ca²⁺ homeostasis in the hemocytes of the swimming crabs, Portunus trituberculatus. Crabs were exposed to varying ammonia concentrations (0, 1, 5, 15, and 45 mg/L) for 24 h and different durations (0, 6, 12, 24, and 48 h) with 15 mg/L ammonia to assess the dose- and time-dependent impacts. Hemocytes were analyzed for Ca²⁺ status and the role of the mitochondrial Ca²⁺ uniporter (MCU) in maintaining it. Ammonia significantly disrupted mitochondrial Ca²⁺ homeostasis, inducing Ca²⁺ overload in the mitochondria and endoplasmic reticulum (ER), reducing cytosolic Ca²⁺ levels, and increasing MCU and sarcoplasmic/ER Ca²⁺-ATPase (SERCA) expression. These changes were coincident with impaired ATP production, enhanced opening of the mitochondrial permeability transition pore (mPTP), ROS-induced oxidative stress, and loss of membrane potential. MCU inhibition with ruthenium red markedly reduced mitochondrial Ca overload, mPTP opening, autophagic cell fraction, and apoptosis induced by ammonia. These findings suggest that MCU-mediated Ca²⁺ homeostasis is crucial for maintaining mitochondrial function in crustaceans under ammonia-induced stress. The study underscores the importance of understanding environmental adaptive mechanisms in aquatic organisms and proposes potential strategies to mitigate ammonia toxicity by targeting the MCU pathway.