Pitfalls in insect cryoprotectant functional studies: A case study of myo-inositol in Drosopila lummei

The correlation between the accumulation of a potentially cryoprotective substance and the increase in cold hardiness has been documented in many cold-acclimated insects. Nevertheless, the literature offers scant direct evidence for the cryoprotective function of the accumulated substances. Here, we sought to obtain direct evidence of non-colligative cryoprotective function of myo-inositol in adults of boreal fly, Drosophila lummei. The diapause flies accumulated myo-inositol in concentration of up to 500 mmol·kg^-1 fresh mass during several weeks of gradual cold acclimation simulating winter onset. Concurrently, their cold hardiness, measured by five different metrics, substantially increased. We found that the primary source of myo-inositol is glycogen breakdown to glucose units, followed by their subsequent conversion through the activity of myo-inositol phosphate synthase, which is encoded by the Inos gene. The relative expression of Inos increased 86-fold during cold acclimation. We successfully augmented the levels of myo-inositol in the bodies of pre-acclimated flies, achieving levels comparable to those attained through cold acclimation. However, the study demonstrated that both methods of exogenous myo-inositol delivery (microinjection into the hemolymph and feeding enriched diets) were unsuccessful in achieving proper tissue localization of myo-inositol, which naturally accumulates primarily in the thoracic flight muscles and CNS. At the same time, the artificial increase in myo-inositol concentration did not affect any of the five measured cold hardiness metrics. We conclude by discussing various pitfalls of functional studies of insect cryoprotectants and identify ways to overcome them.