Gradual mitochondrial collapse underlies cytotoxicity induced by a B-type prymnesin-containing Prymnesium parvum extract in rainbow trout gill cells

Bioactive secondary metabolites produced by the haptophyte microalga Prymnesium parvum are key mediators of ichthyotoxicity during harmful algal bloom events. In the present study, we investigated the effects of a P. parvum extract containing B-type prymnesins, from an isolate obtained during the 2022 Oder River bloom, on mitochondrial health and cellular homeostasis in the rainbow trout gill epithelial cell line RTgill-W1, a widely used model in aquatic toxicology. Mitochondrial function and structure were assessed using multiparameter flow cytometry and high-resolution confocal microscopy (Airyscan). Exposure to prymnesin-containing extract (PRYMCE) induced concentration-dependent mitochondrial dysfunction, characterized by loss of mitochondrial membrane potential (ΔΨm), changes in mitochondrial mass-associated fluorescence, and reduced cardiolipin-associated 10-N-nonyl acridine orange (NAO) fluorescence. Structural analysis revealed mitochondrial network remodeling across PRYMCE concentrations, including perinuclear clustering and a transition from elongated filamentous structures to condensed spherical forms. Time-resolved imaging further demonstrated that mitochondrial structural alterations preceded overt plasma membrane permeabilization. Single-cell analysis demonstrated pronounced cellular heterogeneity, with distinct injury-associated subpopulations exhibiting increased side scatter and a ΔΨm low /mitochondrial mass low phenotype. These findings identify mitochondrial impairment and network remodeling as early, quantifiable hallmarks of PRYMCE-induced cytotoxicity and provide mechanistic insight into how P. parvum blooms may damage aquatic organisms.