Dose-dependent genetic diversity and biochemical adaptation in H. lacustris and D. salina under chemical mutagen

Microalgae H. lacustris and D. salina are biotechnologically valuable for astaxanthin, carotenoid, and lipid biofuel production, yet their responses to xenobiotic contaminants remain poorly understood. This study investigated the effects of ethidium bromide (EtBr), a persistent aromatic intercalating agent, on growth, genomic stability, photosynthetic pigments, and metabolomic profiles of both species across concentrations of 0, 5, 10, and 20 µg/mL. H. lacustris exhibited non-linear, threshold-based tolerance, with adaptive stress responses evident at 10 µg/mL. In contrast, D. salina showed dose-dependent growth inhibition, reaching a 20% reduction at the highest EtBr concentration, indicating higher cytotoxic sensitivity. Random Amplified Polymorphic DNA–Polymerase Chain Reaction (RAPD-PCR) analysis revealed pronounced species-specific genomic effects. H. lacustris displayed extensive polymorphisms and genetic divergence among treatments (genetic similarity = 0.32), whereas D. salina demonstrated heightened mutagenic sensitivity at lower EtBr doses. Both species activated antioxidant defenses, with significant increases in chlorophyll a (112.7-144.9%) and carotenoids (147.8-370.7%), reflecting compensatory responses to oxidative stress. Metabolomic analysis showed dose-dependent reprogramming of carbohydrate, amino acid, organic acid, and lipid metabolism. Low EtBr exposure (5 µg/mL) induced hormetic lipid accumulation (67.9% in H. lacustris and 76.8% in D. salina ), comparable to responses elicited by chemical mutagens. However, high EtBr levels (20 µg/mL) caused metabolic collapse, including substantial depletion of amino acids, organic acids, and fatty acids. The greater vulnerability of D. salina was associated with its permeable plasma membrane and lack of a rigid cell wall. Overall, EtBr induces dose-dependent mutagenic and metabolic effects, defining adaptive low-dose windows versus maladaptive high-dose toxicity, with pronounced species-specific differences relevant to environmental risk assessment and biotechnological applications.