Freeze-tolerant insect protects its plasma membranes with accumulated proline, trehalose, protein.

What is it about?

Insects (i.e. small ectotherm animals) must cope with low body temperatures during temperate or polar winter, and some even survive when their body water partially turns to ice. In preparation for overwintering, many insects accumulate mixtures of different small molecules in their hemolymph – most often sugars, amino acids and their derivatives, and also proteins. It is a common belief among insect environmental physiologists that these accumulated compounds stabilize proteins and biological membranes, protecting them against loss of viable structure during the freezing process – a harsh combination of stresses caused by growing extracellular ice crystals, osmotic outflow of water from cells, cell shrinkage, and concentration of potentially toxic compounds in the cytosol. Strong support for the hypothesis on protein and membrane protection during freeze-dehydration by select sugars and amino acids comes from numerous in vitro assays with enzymes and lipid bilayers and also from theoretical physical chemistry. It has always been a challenge, however, to demonstrate that these in vitro–observed stabilizing effects of such molecules also participate in cryoprotection in vivo, during the extracellular freezing of a freeze-tolerant insect. We tested this hypothesis using larvae of drosophilid fly, Chymomyza costata, which has two distinct seasonal phenotypes: summer active larvae that die upon freezing, and winter dormant (diapausing) larvae that survive freezing (when 68% of body water turns to ice crystals) and even after long-term cryopreservation in liquid nitrogen. The winter phenotype larvae are known to accumulate high concentrations of proline (313 mM), trehalose (108 mM) and plasma protein (160 mg.ml-1).

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Photo by Marek Okon on Unsplash

Photo by Marek Okon on Unsplash

Why is it important?

In this work we found that seven different soluble enzymes in summer-type C. costata larvae are well protected against freeze-dehydration injury in vivo, i.e. when residing in their native biological solutions crowded by different microsolutes and macromolecules. The same enzymes were highly sensitive to freezing stress in diluted aqueous solutions in vitro, but were strongly protected by adding even relatively low concentrations of proline, trehalose (and many other small molecules including biologically inert HistoDenz), protein (bovine serum albumin), and the biologically inert macromolecule Ficoll, to the in vitro solution. These results suggest that the enzymes are not primary targets of freezing injury in insects and that seasonally accumulated compounds are not needed to stabilize them. In contrast, we found that the fat body cell plasma membrane is highly sensitive to freezing injury in both summer- and winter-type larvae, but its integrity in winter larvae is protected by relatively high concentrations of specifically proline and trehalose (working in synergy) but not by other small molecules or HistoDenz. By adding a mixture of proline (313 mM), trehalose (108 mM), and bovine serum albumin (160 mg.ml-1) to the in vitro freezing solution, we completely rescued the plasma membranes of winter-type larval cells from freeze-dehydration induced loss of integrity. These results confirm the hypothesis that plasma cell membranes are the primary targets of freezing injury, and supports seasonally accumulated compounds as protectants of plasma membrane integrity in vivo.

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