Seasonal shifts in mitochondrial and reactive oxygen species metabolism are linked to ultrastructural remodelling in honey bees ( Apis mellifera )

Domesticated honey bees ( Apis mellifera ) remain highly active year‐round, even in northern climates, requiring a tight regulation of their energetic metabolism. However, the overwintering process, particularly the mitochondrial mechanisms sustaining it, remains poorly understood. Here we investigated honey bee energetic metabolism across four seasons by measuring mitochondrial oxygen consumption, ATP production, site‐specific reactive oxygen species (ROS) production, markers of oxidative stress and supercomplex (SC) assembly in thorax‐isolated mitochondria. Complex I (CI)‐linked respiration decreased by half in winter compared to summer, yet ATP production remained stable, indicating tighter energetic efficiency. This came at a cost, as winter bees exhibited higher ROS emission across multiple sites, which was somehow buffered as no clear evidence of oxidative damage to proteins was detected. Strikingly spring bees displayed elevated CI respiration together with greater ROS generation and protein carbonyl accumulation, identifying this stage as a critical window of oxidative vulnerability. Further in‐gel activity revealed marked remodelling of mitochondrial complexes and SC. CI assembled into multiple SC, but their activity remained largely stable across seasons and did not reflect respiration shifts. By contrast CII and mG3PDH in‐gel activities increased in winter, consistent with enhanced succinate and G3P oxidation, whereas CIV‐containing SCs were also more abundant. Finally CV dimers and oligomers were only detected in fall and winter, structural features associated with higher oxidative phosphorylation (OXPHOS) efficiency. Overall this study provides the first integrative view of seasonal mitochondrial remodelling in honey bees and identifies winter and spring as critical physiological windows for colony survival. image Key points Honey bees remain active and generate heat by shivering throughout winter, but how their mitochondria adapt across seasons is still poorly understood. Previous work showed that winter bee mitochondria rely less on one major metabolic pathway and more on alternative fuels, but the consequences on energetic efficiency and oxidative stress were unknown. We show that mitochondria from winter bees maintain energy output despite major changes in fuel use, while producing more reactive oxygen species, a byproduct of metabolism that can cause oxidative damage. Spring bees displayed the highest levels of mitochondrial activity and oxidative damage, suggesting that this season may represent a critical period of physiological vulnerability for colonies. These results reveal that honey bee mitochondria are seasonally remodelled to balance energy homoeostasis, heat generation and oxidative stress, providing new insight into how colonies survive winter and transition into spring.