The freeze-avoiding mountain pine beetle (Dendroctonus ponderosae) survives prolonged exposure to stressful cold by mitigating ionoregulatory collapse.

Insect performance is linked to environmental temperature, and surviving through winter represents a key challenge for temperate, alpine and polar species. To overwinter, insects have adapted a range of strategies to become truly cold hardy. However, although the mechanisms underlying the ability to avoid or tolerate freezing have been well studied, little attention has been given to the challenge of maintaining ion homeostasis at frigid temperatures in these species, despite this limiting cold tolerance for insects susceptible to mild chilling. Here, we investigated how prolonged exposure to temperatures just above the supercooling point affects ion balance in freeze-avoidant mountain pine beetle (Dendroctonus ponderosae) larvae in autumn, mid-winter and spring, and related it to organismal recovery times and survival. Hemolymph ion balance was gradually disrupted during the first day of exposure, characterized by hyperkalemia and hyponatremia, after which a plateau was reached and maintained for the rest of the 7-day experiment. The degree of ionoregulatory collapse correlated strongly with recovery times, which followed a similar asymptotical progression. Mortality increased slightly during extensive cold exposures, where hemolymph K+ concentration was highest, and a sigmoidal relationship was found between survival and hyperkalemia. Thus, the cold tolerance of the freeze-avoiding larvae of D. ponderosae appears limited by the ability to prevent ionoregulatory collapse in a manner similar to that of chill-susceptible insects, albeit at much lower temperatures. Based on these results, we propose that a prerequisite for the evolution of insect freeze avoidance may be a convergent or ancestral ability to maintain ion homeostasis during extreme cold stress.