Insect cell plasma membranes do, while soluble enzymes do not, need stabilization by accumulated cryoprotectant molecules during freezing stress

Ability to survive freezing of extracellular body fluids evolved in several species of vertebrate ectotherms, many plants, and occurs relatively often in freeze-tolerant insects. Most of the multicellular organisms, however, are freeze-sensitive. Here we test coupled hypotheses postulating that: (i) irreversible denaturation of proteins and loss of integrity of biological membranes are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects; and (ii) seasonally accumulated small cryoprotective molecules (CPs) protect the proteins and membranes against the injury in freeze-tolerant insects. We show that seven different enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larva of drosophilid fly, Chymomyza costata . In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding to buffer relatively low concentrations of a wide array of different compounds including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even biologically inert artificial compounds Histodenz and Ficoll. Next, we show that the plasma membranes of fat body cells lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro , however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities (from none to high) to protect membrane integrity when added to in vitro freezing medium. A complete rescue of membrane integrity was observed for a mixture of proline, trehalose and BSA. Significance statement Here we suggest that insect soluble enzymes are not primary targets of freezing injury. They are not inactivated in freeze-sensitive insects exposed to lethal freezing stress as they are sufficiently protected from loss of activity by complex composition of native biological solutions. Next we show that cell plasma membranes are likely targets of freezing injury. The membranes lose integrity in freeze-sensitive insects exposed to freezing stress, while their integrity is protected by accumulated small cryoprotective molecules, and also by proteins, in freeze-tolerant insects. ### Competing Interest Statement The authors have declared no competing interest.