Developmental exposure to amitraz impairs reproductive development and metabolic homeostasis in honey bee (Apis mellifera) queens

Chemicals used within managed pollinator systems can become developmental stressors for non-target organisms, yet their effects on reproductively specialized individuals remain insufficiently understood. Here, we examined the developmental ecotoxicity of the in-hive acaricide amitraz in honey bee ( Apis mellifera ) queens by exposing fourth-instar larvae to amitraz in acute-toxicity and sublethal-exposure assays and assessing responses from organismal to metabolic levels. The 72-h LD 50 of amitraz was 4.428 μg/queen. Residue-informed sub-LD 50 exposure (0.01–1 μg/queen) impaired queen development, as shown by reduced capping and emergence rates, prolonged developmental duration, and decreased body and abdomen weights. Notably, ovary mass was reduced in a dose-dependent manner even at 0.01 μg/queen, indicating high sensitivity of reproductive development to larval amitraz exposure. These phenotypic effects were accompanied by elevated catalase, superoxide dismutase, and glutathione S-transferase activities, together with increased malondialdehyde content, indicating redox disturbance and lipid peroxidation. Amitraz exposure also altered the expression of antimicrobial peptide genes, including Abaecin , Defensin1 , Defensin2 , Hymenoptaecin , and Apidaecin , suggesting disruption of immune homeostasis. Untargeted hemolymph metabolomics identified 502 differentially abundant metabolite annotations. Upregulated features were mainly associated with glycerophospholipid and alpha-linolenic acid metabolism, whereas downregulated features were associated with tryptophan and porphyrin metabolism. Collectively, these findings demonstrate that larval amitraz exposure compromises queen development and reproductive quality through oxidative, immune, and metabolic perturbations. This study highlights the need to incorporate developmentally sensitive measures in the environmental safety assessment of in-hive acaricides.