Given that each cryoprotectant differentially impacts survival in the frozen state, we conclude that small cryoprotectants are not interchangeable and likely function non-colligatively in insect freeze tolerance. No cryoprotectant (alone or in combination) is sufficient to confer freeze tolerance on non-acclimated, freeze-intolerant G. Similarly, exogenous myo-inositol and trehalose increase ex vivo freezing survival of fat body cells from freeze-tolerant crickets. Injecting freeze-tolerant crickets with proline and trehalose increases survival of freezing to lower temperatures or for longer times. veletis are freeze-tolerant and accumulate myo-inositol, proline and trehalose in their haemolymph and fat body. Here, we use a combination of metabolomics and manipulative experiments in vivo and ex vivo to examine the function of multiple cryoprotectants in the spring field cricket Gryllus veletis. Low-molecular-weight cryoprotectants such as sugars, polyols and amino acids are hypothesized to facilitate freeze tolerance, but their in vivo function is poorly understood. Freeze tolerance, the ability to survive internal ice formation, facilitates survival of some insects in cold habitats.
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