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

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