Metabolomic Profiling Reveals Heat-Responsive Metabolic Markers in Honey Bee Workers Under Contrasting Diets

Acute heat exposure challenges honey bee (Apis mellifera) workers, yet the associated small-molecule response and its relationship to nutritional status remain incompletely resolved. Workers were conditioned for 7 days on bee bread plus sucrose (B+S) or sucrose alone (S), maintained at 37 °C or exposed to 45 °C for 3 h, and thorax–abdomen metabolites were profiled by untargeted Ultra-performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-QTOF/MS). Three independent cages were used per diet × temperature group, with 15 workers per cage pooled to generate one cage-level biological replicate (n = 3 per group). Putative metabolite annotation used accurate-mass (10 ppm) and isotope-pattern matching against METLIN and the Agilent Personal Compound Database and Library (PCDL), followed by manual curation, without tandem mass spectrometry (MS/MS) o or authentic-standard confirmation. After statistical filtering and biological curation, 149 heat-responsive metabolite annotations were retained under B+S and 123 under S. Targeted re-extraction of proteinogenic amino acids from the same raw files recovered 16 common peaks across all samples. Fifteen increased at 45 °C in both diets, whereas proline decreased; 14 of 16 showed a larger fold change under B+S. Phenylalanine-related metabolism showed the strongest convergence across targeted amino-acid analysis, untargeted phenylpyruvate detection, and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment. Lipid-class responses also differed descriptively between diets, with a bias toward decreased free fatty acyls under B+S and decreased phospholipids under S. These findings define coordinated metabolic markers of acute heat exposure in worker bees and show that, within the targeted amino-acid dataset, response magnitudes were generally larger under B+S while retaining the same overall direction.