Comparative Proteomics of Adult Honeybee Brains Reveals Differential Lysine Crotonylation Associated with Cognitive Impairment from Prepupal Cold Exposure

Honeybee larvae and pupae are extremely stenothermic. Cold exposure during development causes mortality and developmental arrest and impairs adult cognitive functions. Lysine crotonylation (Kcr) regulates neural activity, yet its role in cold-impaired honeybee cognition is unknown. Adult honeybees with 8 h/12 h/16 h cold exposure during the prepupae stage (T8/T12/T16) were compared to controls with optimal brood-rearing temperature (CK). The learning and memory assay revealed graded levels of cognitive impairment in the adult honeybee cohorts. Western blot analysis revealed that as learning and memory capacity declined, the pan-Kcr levels in adult bee brains also decreased. Further brain LC-MS/MS identified 3017 proteins with 15,233 Kcr sites. Relative to CK, T12 and T16 exhibited 402 differentially modified sites (DMSs) on 304 proteins and 216 DMSs on 325 proteins, respectively. Enrichment analysis of proteins with differentially modified Kcr suggested that Kcr may regulate cognitive function by modulating protein quality control and energy metabolism. Key pathway proteins were validated by parallel reaction monitoring. This first comprehensive profiling of honeybee brain Kcr elucidates its metabolism-linked neural regulation and provides a novel, accessible model for neurological health research.