Insect cross-tolerance to freezing and drought stress: role of metabolic rearrangement.

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.

A mixture of innate cryoprotectants is key for freeze tolerance and cryopreservation of a drosophilid fly larva.

Insects that naturally tolerate internal freezing produce complex mixtures of multiple cryoprotectants (CPs). Better knowledge on composition of these mixtures, and on the mechanisms of individual CP interactions, could inspire development of laboratory CP formulations optimized for cryopreservation of cells and other biological material. Here, we identify and quantify (using high resolution mass spectrometry) a range of putative CPs in larval tissues of a subarctic fly, Chymomyza costata, which survives long-term cryopreservation in liquid nitrogen. The CPs proline, trehalose, glutamine, asparagine, glycine betaine, glycerophosphoethanolamine, glycerophosphocholine and sarcosine accumulate in hemolymph in a ratio of 313:108:55:26:6:4:2.9:0.5 mmol l-1. Using calorimetry, we show that artificial mixtures, mimicking the concentrations of major CPs in hemolymph of freeze-tolerant larvae, suppress the melting point of water and significantly reduce the ice fraction. We demonstrate in a bioassay that mixtures of CPs administered through the diet act synergistically rather than additively to enable cryopreservation of otherwise freeze-sensitive larvae. Using matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI-MSI), we show that during slow extracellular freezing trehalose becomes concentrated in partially dehydrated hemolymph where it stimulates transition to the amorphous glass phase. In contrast, proline moves to the boundary between extracellular ice and dehydrated hemolymph and tissues where it probably forms a layer of dense viscoelastic liquid. We propose that amorphous glass and viscoelastic liquids may protect macromolecules and cells from thermomechanical shocks associated with freezing and transfer into and out of liquid nitrogen.

Transcriptional analysis of insect extreme freeze tolerance.

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.

Recovery from supercooling, freezing, and cryopreservation stress in larvae of the drosophilid fly, Chymomyza costata.

Physiological adjustments accompanying insect cold acclimation prior to cold stress have been relatively well explored. In contrast, recovery from cold stress received much less attention. Here we report on recovery of drosophilid fly larvae (Chymomyza costata) from three different levels of cold stress: supercooling to -10 °C, freezing at -30 °C, and cryopreservation at -196 °C. Analysis of larval CO2 production suggested that recovery from all three cold stresses requires access to additional energy reserves to support cold-injury repair processes. Metabolomic profiling (targeting 41 metabolites using mass spectrometry) and custom microarray analysis (targeting 1,124 candidate mRNA sequences) indicated that additional energy was needed to: clear by-products of anaerobic metabolism, deal with oxidative stress, re-fold partially denatured proteins, and remove damaged proteins, complexes and/or organelles. Metabolomic and transcriptomic recovery profiles were closely similar in supercooled and frozen larvae, most of which successfully repaired the cold injury and metamorphosed into adults. In contrast, the majority of cryopreseved larvae failed to proceed in ontogenesis, showed specific metabolic perturbations suggesting impaired mitochondrial function, and failed to up-regulate a set of 116 specific genes potentially linked to repair of cold injury.