Do ice nucleating agents limit the supercooling ability of the land snail Cornu aspersum?

The supercooling ability of adults and eggs of the partially freezing tolerant land snail Cornu aspersum remains limited to high subzero temperatures (ca. -5 degree C) whatever the conditions, suggesting the presence of ice nucleating agents (INAs). In this study, we investigated the nucleation activity of the digestive tract of adult snails, eggs and their direct environment: food, faeces and soil. The mucous ribbon always present in the distal intestine of adults exhibited a heat-sensitive (i.e. organic) nucleation activity, close to that of the entire snails during dormant states (aestivation and hibernation). However, a microbial nature of these INAs could not be established in inactive snails. The food provided to active snails contained ice nucleating bacteria, which followed the digestive tract to be found in the intestine and in the faeces, but with a decreasing concentration along the transit. Eggshells also presented a heat-sensitive nucleation activity, which could be related to its structure. Moreover, eggs are laid directly in the soil which contained both organic and mineral INAs. This study is the first to demonstrate the implication of organic INAs in the cold hardiness of a terrestrial gastropod.