Thermal hysteresis antifreeze proteins in the midgut fluid of overwintering larvae of the beetle Dendroides canadensis

Freeze‐susceptible insects (those that die if frozen) must lower the freezing and supercooling points of their body fluids in winter. Although it is well known that many overwintering insects concentrate antifreezes such as polyols (glycerol, sorbitol, etc.) and/or thermal hysteresis proteins in the hemolymph, antifreezes have not previously been found in gut fluid. Overwintering freeze‐susceptible larvae of the Pyrochroid beetle Dendroides canadensis accumulated glycerol, sorbitol, and thermal hysteresis proteins (THPs) in the hemolymph. Although polyols were not found in the midgut fluid, THPs began to be accumulated in the autumn, and in midwinter THP levels were the same in midgut fluid and hemolymph. Midgut THP concentrations decreased in late April, and THPs were gone from the midgut by June. This seasonal increase in THPs is thought to be partially responsible for the decrease in the supercooling point of midgut fluid from ∼ −5°C in summer to ∼ −25°C in winter, although cessation of feeding in winter is also likely to be important. Proteolytic enzyme activities were at least as high in January as in June, and the THPs appear to be somewhat susceptible to these midgut enzymes, thus raising the question of how THPs are concentrated and maintained in their presence. One possible answer is that the enzymes are extremely temperature sensitive and that temperatures above 25°C are required for high levels of activity.