Proline protects frozen malt fly larvae from damage

Abstract

Frostbite victims know the excruciating damage inflicted when ice crystals begin shattering and destroying delicate cellular structures within. Yet, freezing is just a natural part of the yearly life cycle for some animals, which emerge unscathed when conditions thaw. Some species are so tolerant of freezing that they can even withstand being plunged into liquid nitrogen at almost −200°C without any ill effects. Vladimir Koštál from the Czech Academy of Sciences is fascinated by the ability of some insects to endure freezing and how the process of ice formation in these resilient animals differs from that in vulnerable species. Knowing that vinegar fly (Drosophila melanogaster) larvae do not survive freezing, while malt fly (Chymomyza costata) larvae do, Koštál and Jan Rozsypal painstakingly measured the heat released by the insects as they gradually froze to find out whether differences in the way that ice forms in the larvae's bodies could account for their different survival abilities.‘We wished to assess the ice formation process under conditions simulating the situation in nature’, says Koštál. However, he explains that malt flies enter a form of suspended animation – known as diapause – when temperatures drop and the days shorten, during which metabolic processes that are not essential for survival are suspended, so first he triggered the process by shortening the day length in the lab. Koštál then placed these diapausing malt flies and chilled vinegar flies into individual sealed chambers and measured the heat released and absorbed by their bodies as they froze and thawed in order to calculate how much of the water in their bodies was turned to ice. However, when Koštál and Rozsypal compared the amount of ice that formed in the frozen bodies of the vinegar and malt flies, it was essentially the same, ranging from 68% to 78%, with all of the free water in the insects’ bodies forming ice between –3.9 and –10.5°C. The robust malt flies accumulated as much damaging ice in their bodies as the doomed vinegar flies.But when Martin Moos and Petr Šimek analysed the concentration of various compounds – such as amino acids and sugars – in the larvae's bodies, Koštál was amazed to see that the concentration of one amino acid, proline, rocketed to almost 500 mmol l−1 kg−1 in the bodies of the malt flies, while the vinegar flies accumulated less than a tenth of this. Warning that proline is toxic at levels exceeding ∼50 mmol l−1 kg−1, Koštál suspects that the malt flies are protected from the lethal effects of the amino acid by their state of suspended animation. He suggests that the amino acid might protect delicate proteins from unravelling and losing function when the temperature falls too low, in addition to protecting the cells from toxic oxygen compounds, known as free radicals, which damage proteins, lipids and DNA. In addition, Koštál noticed a tell-tale bump in the plot showing the amount of heat given out by the malt fly larvae as they froze down to temperatures of –30°C, which told him that the remaining water in the insect's body that had not turned to ice solidified to form a glassy solid, which locks protein structures and locations in place, protecting them from damage.Koštál says, ‘The accumulated proline exerts its protective roles by a combination of mechanisms and the importance of individual mechanisms gradually changes during the course of the insect's entry into dormancy, cold preparedness, cooling and freezing’. And he is keen to learn more about the protective properties of proline and how the body manages this wonder compound, which allows insects to emerge Lazarus-like from their winter slumber.