Empirical screening of sesamol, melatonin, and spermidine combinations on growth, biochemical composition, and antioxidant responses of Nitzschia closterium under low-salinity stress

Low-salinity stress, frequently caused by spring rainfall and estuarine fluctuations, is a major constraint on the large-scale cultivation of marine diatoms and can disrupt their growth, photosynthesis, and carbon metabolism. Nitzschia closterium is a marine diatom with high nutritional value and strong potential for producing PUFAs such as EPA and DHA, and it is also widely used as a live feed in aquaculture. However, effective mitigation strategies for low-salinity stress in this species remain limited, and the effects of multiple exogenous regulators remain poorly understood. In this study, an L 9 (3 4 ) orthogonal design was used to evaluate nine combinations of sesamol (SL), melatonin (MT), and spermidine (SPD) on growth, biochemical composition, and antioxidant responses of N. closterium at salinity 5. Responses varied among the tested combinations and endpoints. Among the nine combinations, 30 mg·L −1 SL + 20 mg·L −1 MT + 150 mg·L −1 SPD (A 2 B 2 C 3 ) produced favorable results for several measured parameters, including higher total lipid (26.8%) and PUFA (55.28%) relative to the low-salinity control, as well as elevated SOD and CAT activities. The lowest MDA content among the additive treatments was observed under a different combination (A 2 B 1 C 2 ). Range analysis indicated that SPD had the strongest influence on growth rate, chlorophyll a, total lipid, EPA, PUFA, soluble sugar, and antioxidant enzyme activities within the tested matrix, whereas SL showed the largest range for MDA reduction and DHA. Because the orthogonal design contained only non-zero levels and did not include additive-free or single-additive treatments, the results are interpreted as an empirical comparison of the nine tested combinations rather than evidence of synergistic or coordinated action. Further dose-range expansion, independent verification, and cultivation-scale assessment are required before practical application can be recommended.