Immune and Redox Responses of Galleria mellonella to Caffeic Acid: Concentration- and Time-Dependent Patterns

Background/Objectives: Caffeic acid is a naturally occurring phenolic compound with antioxidant and immunomodulatory properties, but its integrated effects on innate immune and redox-related physiology in Galleria mellonella remain unclear. This study evaluated concentration- and time-related effects of caffeic acid on immune and biochemical endpoints. Methods: Last-instar larvae were exposed to 0.1-5 mg/mL caffeic acid, with 20% ethanol as the vehicle comparator. Total hemocyte count (THC), Sephadex A-25 bead encapsulation, and melanization were assessed as immune endpoints; bead responses were evaluated at 4 and 24 h using independent cohorts. Total protein, superoxide dismutase (SOD), catalase (CAT), and malondialdehyde (MDA) were measured at 24 h. Experimental run was included as a fixed blocking factor in the primary inferential models; THC and biochemical endpoints were analyzed using endpoint-appropriate run-adjusted linear or rank-based models, whereas encapsulation and melanization were analyzed using ordinal and binomial generalized estimating equations (GEEs), respectively; n = 16 larvae per treatment for each endpoint. Results: THC increased significantly only at 0.5 mg/mL relative to the vehicle. Encapsulation showed a significant treatment × time interaction; all concentrations shifted encapsulation toward higher categories at 4 h, whereas 2.5 and 5 mg/mL shifted toward lower categories at 24 h. Melanization showed no significant treatment × time interaction; only 2.5 mg/mL increased melanization odds at 4 h, with no treatment-vehicle differences at 24 h. CAT increased at 2.5 and 5 mg/mL, and SOD at 2.5 mg/mL. MDA and total protein showed overall heterogeneity without significant individual treatment-vehicle differences. Conclusions: Caffeic acid produced non-uniform, endpoint-specific modulation of immune and antioxidant responses, with temporal dependence most evident in encapsulation.