Background: Many insect species have evolved the ability to survive extracellular freezing. The search for the underlying principles of their natural freeze tolerance remains hampered by our poor understanding of the mechanistic nature of freezing damage itself. Objectives: Here, in search of potential primary cellular targets of freezing damage, we compared mitochondrial responses (changes in morphology and physical integrity, respiratory chain protein functionality, and mitochondrial inner membrane (IMM) permeability) in freeze-sensitive vs. freeze-tolerant phenotypes of the larvae of the drosophilid fly, Chymomyza costata. Methods: Larvae were exposed to freezing stress at -30°C for 1 h, which is invariably lethal for the freeze-sensitive phenotype but readily survived by the freeze-tolerant phenotype. Immediately after melting, the metabolic activity of muscle cells was assessed by the Alamar Blue assay, the morphology of muscle mitochondria was examined by transmission electron microscopy, and the functionality of the oxidative phosphorylation system was measured by Oxygraph-2K microrespirometry. Results: The muscle mitochondria of freeze-tolerant phenotype larvae remained morphologically and functionally intact after freezing stress. In contrast, most mitochondria of the freeze-sensitive phenotype were swollen, their matrix was diluted and enlarged in volume, and the structure of the IMM cristae was lost. Despite this morphological damage, the electron transfer chain proteins remained partially functional in lethally frozen larvae, still exhibiting strong responses to specific respiratory substrates and transferring electrons to oxygen. However, the coupling of electron transfer to ATP synthesis was severely impaired. Based on these results, we formulated a hypothesis linking the observed mitochondrial swelling to a sudden loss of barrier function of the IMM.

• Keywords
• freeze tolerance, insects, mitochondrial swelling, oxidative phosphorylation, permeability transition, respiration,
• Publication type
• Journal Article MeSH

Most multicellular organisms are freeze sensitive, but the ability to survive freezing of the extracellular fluids evolved in several vertebrate ectotherms, some plants, and many insects. Here, we test the coupled hypotheses that are perpetuated in the literature: that irreversible denaturation of proteins and loss of biological membrane integrity are two ultimate molecular mechanisms of freezing injury in freeze-sensitive insects and that seasonally accumulated small cryoprotective molecules (CPs) stabilize proteins and membranes against injury in freeze-tolerant insects. Using the drosophilid fly, Chymomyza costata, we show that seven different soluble enzymes exhibit no or only partial loss of activity upon lethal freezing stress applied in vivo to whole freeze-sensitive larvae. In contrast, the enzymes lost activity when extracted and frozen in vitro in a diluted buffer solution. This loss of activity was fully prevented by adding low concentrations of a wide array of different compounds to the buffer, including C. costata native CPs, other metabolites, bovine serum albumin (BSA), and even the biologically inert artificial compounds HistoDenz and Ficoll. Next, we show that fat body plasma membranes lose integrity when frozen in vivo in freeze-sensitive but not in freeze-tolerant larvae. Freezing fat body cells in vitro, however, resulted in loss of membrane integrity in both freeze-sensitive and freeze-tolerant larvae. Different additives showed widely different capacities to protect membrane integrity when added to in vitro freezing media. A complete rescue of membrane integrity in freeze-tolerant larvae was observed with a mixture of proline, trehalose, and BSA.

• Keywords
• biological membrane integrity, cryopreservation, enzyme activity, freeze tolerance, protein stabilization,
• MeSH
• Acclimatization MeSH
• Cell Membrane metabolism   MeSH
• Ficoll MeSH
• Insecta metabolism   MeSH
• Cryoprotective Agents pharmacology   MeSH
• Larva metabolism   MeSH
• Proline metabolism   MeSH
• Serum Albumin, Bovine * MeSH
• Trehalose * MeSH
• Freezing MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Ficoll MeSH
• Cryoprotective Agents MeSH
• Proline MeSH
• Serum Albumin, Bovine * MeSH
• Trehalose * MeSH

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.

• Keywords
• cold, cross-tolerance, cytoprotectants, drought, freezing, metabolism,
• MeSH
• Acclimatization physiology   MeSH
• Drosophilidae * MeSH
• Insecta MeSH
• Larva physiology   MeSH
• Cold Temperature MeSH
• Droughts * MeSH
• Trehalose MeSH
• Freezing MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Trehalose 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.

• Keywords
• betaine, cryoprotectant metabolites, metabolic pathways, metabolomics, proline, transcriptomics, trehalose,
• Publication type
• Journal Article MeSH

Many insects survive internal freezing, but the great complexity of freezing stress hinders progress in understanding the ultimate nature of freezing-induced injury. Here, we use larvae of the drosophilid fly, Chymomyza costata to assess the role of mitochondrial responses to freezing stress. Respiration analysis revealed that fat body mitochondria of the freeze-sensitive (non-diapause) phenotype significantly decrease oxygen consumption upon lethal freezing stress, while mitochondria of the freeze-tolerant (diapausing, cold-acclimated) phenotype do not lose respiratory capacity upon the same stress. Using transmission electron microscopy, we show that fat body and hindgut mitochondria swell, and occasionally burst, upon exposure of the freeze-sensitive phenotype to lethal freezing stress. By contrast, mitochondrial swelling is not observed in the freeze-tolerant phenotype exposed to the same stress. We hypothesize that mitochondrial swelling results from permeability transition of the inner mitochondrial membrane and loss of its barrier function, which causes osmotic influx of cytosolic water into the matrix. We therefore suggest that the phenotypic transition to diapause and cold acclimation could be associated with adaptive changes that include the protection of the inner mitochondrial membrane against permeability transition and subsequent mitochondrial swelling. Accumulation of high concentrations of proline and other cryoprotective substances might be a part of such adaptive changes as we have shown that freezing-induced mitochondrial swelling was abolished by feeding the freeze-sensitive phenotype larvae on a proline-augmented diet.

• Keywords
• freeze tolerance, insects, mitochondrial morphology,
• MeSH
• Acclimatization MeSH
• Drosophilidae MeSH
• Insecta physiology   MeSH
• Larva physiology   MeSH
• Mitochondria * MeSH
• Freezing * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Few invertebrates can survive cryopreservation in liquid nitrogen, and the mechanisms by which some species do survive are underexplored, despite high application potential. Here, we turn to the drosophilid Chymomyza costata to strengthen our fundamental understanding of extreme freeze tolerance and gain insights about potential avenues for cryopreservation of biological materials. We first use RNAseq to generate transcriptomes of three C. costata larval phenotypic variants: those warm-acclimated in early or late diapause (weak capacity to survive cryopreservation), and those undergoing cold acclimation after diapause entry (extremely freeze tolerant, surviving cryopreservation). We identify mRNA transcripts representing genes and processes that accompany the physiological transition to extreme freeze tolerance and relate cryopreservation survival to the transcriptional profiles of select candidate genes using extended sampling of phenotypic variants. Enhanced capacity for protein folding, refolding and processing appears to be a central theme of extreme freeze tolerance and may allow cold-acclimated larvae to repair or eliminate proteins damaged by freezing (thus mitigating the toxicity of denatured proteins, endoplasmic reticulum stress and subsequent apoptosis). We also find a number of candidate genes (including both known and potentially novel, unannotated sequences) whose expression profiles tightly mirror the change in extreme freeze tolerance status among phenotypic variants.

• Keywords
• cold acclimation, cryopreservation, cryoprotectant, insect, transcriptome,
• MeSH
• Acclimatization genetics   MeSH
• Drosophilidae genetics   MeSH
• Insecta genetics   MeSH
• Transcriptome MeSH
• Freezing * MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't 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.

• Keywords
• Gryllus veletis, acclimation, cold tolerance, cryopreservation, cryoprotectants, freeze tolerance,
• MeSH
• Acclimatization * MeSH
• Longevity MeSH
• Gryllidae growth & development   physiology   MeSH
• Hemolymph physiology   MeSH
• Cryoprotective Agents metabolism   MeSH
• Metabolomics MeSH
• Cold Temperature * MeSH
• Nymph growth & development   physiology   MeSH
• Proline metabolism   MeSH
• Trehalose metabolism   MeSH
• Fat Body physiology   MeSH
• Freezing MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Cryoprotective Agents MeSH
• Proline MeSH
• Trehalose MeSH