Endogenous circannual cycles drive seasonal cold hardening in temperate ants

The ability to acquire cold tolerance and cope with seasonality underlies the broad geographical distribution of ectotherms. Among insects, cold-adapted ants, which are successful terrestrial insects in temperate and boreal ecosystems, rely on complex circannual life cycles to survive multiple consecutive winters. Following winter reactivation, colonies enter a fixed developmental phase whose duration is controlled by "Kipyatkovs sand-glass device", an endogenous programmed timer that ultimately enforces a new dormancy period for colonies after a set period, even under permissive light or temperature cues. Here, using five temperate ant species, we leveraged this system to tease apart the contribution of intrinsic seasonal programming and extrinsic thermal cues on worker cold tolerance, metabolic rates, and metabolomic profiles. We show that obligate colony-level dormancy alone is sufficient to modulate both the critical thermal minimum (CTmin) and the temperature causing 50% mortality during acute cold exposure (LTe50), and further interacts with cold acclimation to shape worker phenotypic cold tolerance. While colony-level dormancy was only associated with limited metabolomic reorganization, cold acclimation triggered the accumulation of metabolites potentially involved in osmotic balance and membrane reorganization. Our results highlight a circannual cycle of endogenous cold hardening that operates independently of environmental exposure in temperate ants, providing new insight into the physiological adaptations underpinning the evolutionary success of this insect family in highly seasonal climates.