The division of labor among honeybee workers contributes to efficient pollination activities. Lipids play a crucial role in behavioral regulation, with pollen serving as a primary source of these lipids. However, the regulatory effect of pollen lipids on the division of labor of honeybee workers remains to be studied. In this study, an in-depth lipidomic analysis based on HPLC-QTOF-MS and UPLC-IM-QTOF-MS was applied. We conducted the first comprehensive lipidomic profiling of the abdomen, brain, and intestine of emerging, nursing, and foraging bees, as well as pollen, thereby establishing a lipid library comprising 646 lipid species. The lipid composition of pollen was found to exhibit characteristics similar to those of honeybee workers, with alpha linolenic acid (ALA) validated as a key labor characteristic lipid. Moderate dietary ALA supplementation reshaped lipid levels and facilitated metabolism related to the labor of division. These findings advance the field of lipidomic analysis in honeybee workers, revealing a novel behavioral regulator and extending the understanding of the nutritional regulation of ALA in the division of labor among honeybee workers.
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