Immune response in honey bees (Apis mellifera) following humic substance feeding

Humic substances (HS) are increasingly investigated as natural additives to support health in humans and animals, yet their effects on honey bees remain insufficiently characterised. This study evaluated the impact of a commercial HS-based product on immunity and oxidative status of Apis mellifera. Newly emerged worker bees were kept under laboratory conditions and fed sugar syrup supplemented with 0.5% HS for seven days, while the control group received unsupplemented syrup. Effects of HS were evaluated by analysing the expression of selected immune-related genes, quantifying antimicrobial peptides (AMPs), measuring the activity of antioxidant enzymes, evaluating lipid peroxidation, and characterising hemocyte populations. HS supplementation significantly increased relative gene expression of superoxide dismutase 1 and prophenoloxidase, while expression of genes for apidaecin and defensin 1 decreased. HS supplementation significantly increased relative gene expression of superoxide dismutase 1 and prophenoloxidase, while expression of genes for apidaecin and defensin 1 decreased. Similarly, catalase enzymatic activity was significantly decreased in the HS group. No significant changes were detected in gene expression for other AMPs and relative abundance of AMPs, or other antioxidant parameters. HS supplementation significantly increased the proportion of medium granular hemocytes (p < 0.05) at the expense of high granular cells (p < 0.01). In conclusion, the 7-day administration of HS at 0.5% did not adversely affect bee health and modulated selected immune parameters. The biological significance of these immunomodulatory effects requires confirmation through challenge studies and field trials under natural conditions.