Research on insect cold hardiness has a more than a century of tradition (Bachmetjew, 1901; Payne, 1927), and has culminated in a consensus that it is a broad adaptive complex based on a combination of developmental, physiological and biochemical mechanisms (Lee, 2010, Teets and Denlinger, 2013, Teets et al., 2023, Toxopeus and Sinclair, 2018). Glycerol was the first biochemical component of the adaptive complex to be recognized in the late 1950 s (Chino, 1958, Salt, 1958, Wyatt and Kalf, 1957). Soon, the range of other accumulated low molecular weight compounds – possible cryoprotectants (CPs) – was expanded to include sorbitol and trehalose (Asahina and Tanno, 1964), and, later, a number of other sugars, polyols and also free amino acids such as proline and alanine (Sømme, 1982, Storey and Storey, 1988, Storey and Storey, 1991, Zachariassen, 1985). To date, the literature contains hundreds of observations on the accumulation of various putative CPs in insects overwintering in nature or exposed to various cold stimuli in the laboratory. The temporal coincidence of CP accumulation with an increase in insect cold tolerance has often been interpreted as correlative evidence for their cryoprotective role.

Correlative evidence is relatively strong when extremely high concentrations of a substance are achieved through accumulation. Thus, in early studies, the cryoprotection exerted by CPs accumulated in higher than molar concentrations were mainly associated with their strong colligative (non-specific, osmolality-dependent) effects on the phase behavior of water in insect body solutions. The CPs were seen either as antifreezes (extending the supercooling capacity of a solution far below zero) in freeze-sensitive insects (Berman et al., 2010, Gehrken, 1984, Salt, 1959); or as significantly reducing the amount of ice formed in freeze-tolerant insects (Asahina, 1970; Zachariassen, 1979). However, in many cases the observed CP concentrations were moderate (in the range of tens to low hundreds of millimolar), which means that their colligative effects were too small, if not biologically negligible. In the 1980 s, a theory of non-colligative cryoprotection was developed based on thermodynamic modeling of molecular interactions between CPs, water (or ice), and the protected macromolecular structures – functional conformations of proteins and phospholipid bilayers. The theory was well supported by in vitro experiments on thermal stabilization of proteins and bilayers by different CPs (Anchordoguy et al., 1987, Arakawa and Timasheff, 1985, Arakawa and Timasheff, 1982, Carpenter and Crowe, 1988, Rudolph et al., 1986, Timasheff, 1993, Timasheff, 2002), leading to the widespread acceptance of the theory of the non-colligative action of CPs in the insect cold hardiness research community. However, direct pieces of evidence for in vivo non-colligative effects of CPs on insect cold hardiness are scarce in the literature (Grgac et al., 2022, Koštál et al., 2001, Kučera et al., 2022, Sømme, 1968, Toxopeus et al., 2019).

In this study, we sought to directly assess a non-colligative cryoprotective role of myo-inositol in a Finnish population of Drosophila lummei, one of the cold-hardy boreal drosophilids with a Palearctic distribution between about 40⁰N and 65⁰N (Throckmorton, 1982). D. lummei belongs to the virilis group of Drosophila subgenus and is closely related to three other boreal species, D. montana, D. ezoana, and D. littoralis, which occur sympatrically in northern Europe (Aspi et al., 1993) where they overwinter as adults in photoperiodically-mediated reproductive diapause (Lumme, 1978, Lumme and Lakovaara, 1983). Working previously with D. montana, we found that it responds to long-term cold acclimation (simulating autumn temperature drop) by accumulation of myo-inositol (Vesala et al., 2012). The concentrations of myo-inositol increased from < 1 nmol mg−1 in late summer to 147 nmol mg−1 fresh mass in winter-acclimated flies, which was accompanied by an increase in cold tolerance of the flies, leading us to interpretation that myo-inositol may act as a non-colligative CP (Vesala et al., 2012). In a more recent study (Moos et al., 2025), we confirmed that many species of virilis group (i.e. montana, ezoana, littoralis, virilis, and novamexicana) respond to cold acclimation by a massive accumulation of myo-inositol and also by significant increase of cold tolerance. In addition, the cryoprotective role of myo-inositol in D. montana was assessed by systemic RNAi silencing of the expression of a gene Inos, coding for myo-inositol phosphate synthase (MIP synthase), a key enzyme responsible for conversion of glucose to myo-inositol (Vigoder et al., 2016). Although the latter study was not very conclusive (see more in the Discussion), the body of correlative evidence for cryoprotective role of myo-inositol was relatively strong and our goal in this study was to bring direct evidence. We tested the relatively simple hypothesis that artificial delivery of myo-inositol into the body of diapause flies prior to cold acclimation would lead to an increase in the flies’ cold tolerance.

Towards this goal, we first detailed changes in cold hardiness in D. lummei (measured by five different metrics) in response to entry into photoperiodic diapause and subsequent long-term cold acclimation of adult flies. We further correlated these changes with changes in metabolomic profile, glycogen content and gene expression involved in myo-inositol metabolism. We have shown that cold-acclimated D. lummei accumulates large amounts of myo-inositol, which is at least partly derived from glycogen depots via Glycogen phosphorylase (GlyP) and MIP synthase activities. We were able to artificially increase the levels of myo-inositol in the flies, but we did not see any effect on the cold tolerance of the flies. Using MALDI mass spectrometry imaging, we found that while flies naturally accumulate myo-inositol in their tissues, primarily in flight muscle, our artificial enhancement methods (feeding enriched diet and microinjection to hemolymph) resulted in accumulation in the gut and hemolymph. The correct tissue localization of myo-inositol seemed to be a major pitfall, which led us to discuss the possibilities of how to overcome it and proceed further in the verification of the cryoprotective role not only of myo-inositol in D. lummei, but of insect cryoprotectants in general.