Cold hardiness and deacclimation of overwintering Papilio zelicaon pupae

Seasonally-acquired cold tolerance can be reversed at warm temperatures, leaving temperate ectotherms vulnerable to cold snaps. However, deacclimation, and its underlying mechanisms, has not been well-explored in insects. Swallowtail butterflies are widely distributed but in some cases their range is limited by low temperature and their cold tolerance is seasonally acquired, implying that they experience mortality resulting from deacclimation. We investigated cold tolerance and hemolymph composition of Anise swallowtail (Papilio zelicaon) pupae during overwintering in the laboratory, and after four days exposure to warm temperatures in spring. Overwintering pupae had supercooling points around -20.5°C and survived brief exposures to -30°C, suggesting partial freeze tolerance. Overwintering pupae had hemolymph osmolality of approximately 920 mOsm, imparted by high concentrations of glycerol, K⁺ and Na⁺. After exposure to spring warming, supercooling points increased to approximately -17°C, and survival of a 1h exposure to -20°C decreased from 100% to 0%. This deacclimation was associated with decreased hemolymph osmolality and reduced glycerol, trehalose, Na⁺ and Ca²⁺ concentrations. We compared cold tolerance of pupae to weather conditions at and beyond the species' northern range boundary. Minimum temperatures at the range boundary approached the lower lethal temperature of pupae, and were colder north of the range, suggesting that cold hardiness may set northern range limits. Minimum temperatures following warm snaps were likely to cause mortality in at least one of the past three years. Cold snaps in the spring are increasing in frequency as a result of global climate change, so are likely to be a significant source of mortality for this species, and other temperate ectotherms.