The accumulation of trehalose has been suggested as a mechanism underlying insect cross-tolerance to cold/freezing and drought. Here we show that exposing diapausing larvae of the drosophilid fly, Chymomyza costata to dry conditions significantly stimulates their freeze tolerance. It does not, however, improve their tolerance to desiccation, nor does it significantly affect trehalose concentrations. Next, we use metabolomics to compare the complex alterations to intermediary metabolism pathways in response to three environmental factors with different ecological meanings: environmental drought (an environmental stressor causing mortality), decreasing ambient temperatures (an acclimation stimulus for improvement of cold hardiness), and short days (an environmental signal inducing diapause). We show that all three factors trigger qualitatively similar metabolic rearrangement and a similar phenotypic outcome-improved larval freeze tolerance. The similarities in metabolic response include (but are not restricted to) the accumulation of typical compatible solutes and the accumulation of energy-rich molecules (phosphagens). Based on these results, we suggest that transition to metabolic suppression (a state in which chemical energy demand is relatively low but need for stabilization of macromolecules is high) represents a common axis of metabolic pathway reorganization towards accumulation of non-toxic cytoprotective compounds, which in turn stimulates larval freeze tolerance.

• Klíčová slova
• cold, cross-tolerance, cytoprotectants, drought, freezing, metabolism,
• MeSH
• aklimatizace fyziologie   MeSH
• Drosophilidae * MeSH
• hmyz MeSH
• larva fyziologie   MeSH
• nízká teplota MeSH
• období sucha * MeSH
• trehalosa MeSH
• zmrazování MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• trehalosa MeSH

Many cold-acclimated insects accumulate high concentrations of low molecular weight cryoprotectants (CPs) in order to tolerate low subzero temperatures or internal freezing. The sources from which carbon skeletons for CP biosynthesis are driven, and the metabolic reprogramming linked to cold acclimation, are not sufficiently understood. Here we aim to resolve the metabolism of putative CPs by mapping relative changes in concentration of 56 metabolites and expression of 95 relevant genes as larvae of the drosophilid fly, Chymomyza costata transition from a freeze sensitive to a freeze tolerant phenotype during gradual cold acclimation. We found that C. costata larvae may directly assimilate amino acids proline and glutamate from diet to acquire at least half of their large proline stocks (up to 55 µg per average 2 mg larva). Metabolic conversion of internal glutamine reserves that build up in early diapause may explain the second half of proline accumulation, while the metabolic conversion of ornithine and the degradation of larval collagens and other proteins might be two additional minor sources. Next, we confirm that glycogen reserves represent the major source of glucose units for trehalose synthesis and accumulation (up to 27 µg per larva), while the diet may serve as an additional source. Finally, we suggest that interconversions of phospholipids may release accumulated glycero-phosphocholine (GPC) and -ethanolamine (GPE). Choline is a source of accumulated methylamines: glycine-betaine and sarcosine. The sum of methylamines together with GPE and GPC represents approximately 2 µg per larva. In conclusion, we found that food ingestion may be an important source of carbon skeletons for direct assimilation of, and/or metabolic conversions to, CPs in a diapausing and cold-acclimated insect. So far, the cold-acclimation- linked accumulation of CPs in insects was considered to be sourced mainly from internal macromolecular reserves.

• Klíčová slova
• betaine, cryoprotectant metabolites, metabolic pathways, metabolomics, proline, transcriptomics, trehalose,
• Publikační typ
• časopisecké články MeSH

Freeze tolerance, the ability to survive internal ice formation, facilitates survival of some insects in cold habitats. 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. 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. Cold-acclimated G. veletis are freeze-tolerant and accumulate myo-inositol, proline and trehalose in their haemolymph and fat body. Injecting freeze-tolerant crickets with proline and trehalose increases survival of freezing to lower temperatures or for longer times. Similarly, exogenous myo-inositol and trehalose increase ex vivo freezing survival of fat body cells from freeze-tolerant crickets. No cryoprotectant (alone or in combination) is sufficient to confer freeze tolerance on non-acclimated, freeze-intolerant G. veletis. 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. Our study is the first to experimentally demonstrate the importance of non-colligative cryoprotectant function for insect freeze tolerance both in vivo and ex vivo, with implications for choosing new molecules for cryopreservation.

• Klíčová slova
• Gryllus veletis, acclimation, cold tolerance, cryopreservation, cryoprotectants, freeze tolerance,
• MeSH
• aklimatizace * MeSH
• dlouhověkost MeSH
• Gryllidae růst a vývoj   fyziologie   MeSH
• hemolymfa fyziologie   MeSH
• kryoprotektivní látky metabolismus   MeSH
• metabolomika MeSH
• nízká teplota * MeSH
• nymfa růst a vývoj   fyziologie   MeSH
• prolin metabolismus   MeSH
• trehalosa metabolismus   MeSH
• tukové těleso fyziologie   MeSH
• zmrazování MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• kryoprotektivní látky MeSH
• prolin MeSH
• trehalosa MeSH