Dietary Ferrous Sulfate Enhances Resistance to Vibrio splendidus-Induced Skin Ulceration in Apostichopus japonicus via Immune and Antioxidant Modulation

The sea cucumber (<i>Apostichopus japonicus</i>) is a commercially important marine species. However, its survival is increasingly threatened by frequent outbreaks of Skin Ulceration Syndrome caused by <i>Vibrio splendidus</i>. This study evaluated the effects of dietary supplementation with ferrous sulfate (FeSO₄) at two concentrations (0.5% and 1%) over short-term (21 days) and long-term (56 days) feeding periods on immune defense, antioxidant capacity, and resistance to <i>V. splendidus</i> infection. Key parameters measured included survival rate, cellular immune activity, antioxidant enzyme levels, and expression of immune-related genes. Long-term (56 days) supplementation with 1% FeSO₄ significantly improved survival after infection (90 ± 4.7%). Phagocytic activity and respiratory burst were enhanced by approximately ~1.9-fold and ~1.8-fold, respectively (<i>p</i> < 0.05). The expression of <i>sod</i>, <i>ferritin</i>, and <i>hsp70</i> genes was upregulated by ~2.1-fold, ~2.0-fold, and ~1.6-fold, respectively (<i>p</i> < 0.05). These results indicate strengthened cellular immunity and antioxidant capacity. Long-term (56 days) supplementation with 0.5% FeSO₄ increased lysozyme activity (~1.3-fold) and <i>c3</i> expression (~4-fold) (<i>p</i> < 0.05), thereby enhancing humoral immunity. In contrast, short-term (21 days) supplementation increased ACP and AKP activities by approximately ~2-fold each, and LZM activity by ~1.2-fold (<i>p</i> < 0.05). However, it did not significantly improve survival, indicating limited protective effects. Overall, 56-day dietary supplementation with FeSO₄, particularly at 1%, effectively enhances immune and antioxidant responses in <i>A. japonicus.</i> This supplementation represents a promising strategy for preventing <i>V. splendidus</i>-induced skin ulceration in aquaculture.