Dietary Lyophilized Bee Venom from Apis mellifera Enhances Growth Performance, Antioxidant Capacity, Innate Immunity, and Resistance to Vibrio harveyi in Juvenile Pacific White Shrimp (Litopenaeus vannamei).

Bee venom, a complex mixture of bioactive peptides and enzymes, is a promising natural immunostimulant for aquaculture applications, particularly as escalating antibiotic resistance necessitates alternative disease-prevention strategies. This study evaluated the dose-dependent effects of dietary lyophilized bee venom (LBV) from Apis mellifera on growth performance, antioxidant capacity, innate immunity, and resistance to Vibrio harveyi in juvenile Pacific white shrimp (Litopenaeus vannamei). LBV was chemically characterized by LC-DAD-ESI/MSⁿ, with melittin identified as the predominant component (67.16 ± 12.62µg/mL), followed by phospholipase A₂ and apamin. In vitro disk-diffusion assays showed dose-dependent inhibition of Vibrio harveyi (ATCC 35084), producing zones of up to 19.67mm at 50µg/mL, approaching those of ampicillin. Juveniles (initial weight 1.25 ± 0.15g) were fed isonitrogenous diets supplemented with 0, 0.1, 0.5, 0.75, or 1.0mg LBV /kg diet for 10 weeks (n=600/treatment, four replicates). Growth parameters (final weight, specific growth rate, and feed conversion ratio) improved progressively with LBV inclusion (P < 0.05), with digestive enzymes (amylase, protease, lipase) increasing up to threefold at 1.0mg/kg. Innate immune parameters (total hemocyte count, total protein, respiratory burst, lysozyme, phagocytic activity, and phenoloxidase) were significantly elevated (P < 0.05), concurrent with higher superoxide dismutase and catalase activities and lower malondialdehyde levels. qPCR revealed dose-dependent upregulation of antioxidant genes (cMn-SOD, CAT) and immune genes (Hc, a2M, proPO, pen3a). After intramuscular challenge with V. harveyi (10⁸ CFU/mL), LBV-supplemented groups exhibited reduced mortality (37.8% at 1.0mg/kg) and relative percent survival (RPS) up to 54%. Histopathological and immunohistochemical analyses demonstrated restored intestinal architecture, increased goblet cell density, and reduced caspase-3-mediated apoptosis. Heatmap analysis of restoration scores indicated that higher LBV doses not only restored baseline health metrics but also exceeded them. These findings establish dietary LBV as an effective, eco-friendly additive that enhances shrimp resilience to vibriosis and offers a sustainable alternative to antibiotics in aquaculture.