Synchrotron X-Ray Visualisation of Ice Formation in Insects during Lethal and Non-Lethal Freezing

John J. Socha,Allen G. Gibbs,Alejandro Lucia,Arun Rajamohan,Wah-Keat Lee,Brent J. Sinclair,Stephen P. Roberts

doi:10.1371/journal.pone.0008259

John J. Socha, Allen G. Gibbs + Show 5 more

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https://doi.org/10.1371/journal.pone.0008259

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Abstract

Although the biochemical correlates of freeze tolerance in insects are becoming well-known, the process of ice formation in vivo is subject to speculation. We used synchrotron x-rays to directly visualise real-time ice formation at 3.3 Hz in intact insects. We observed freezing in diapausing 3rd instar larvae of Chymomyza amoena (Diptera: Drosophilidae), which survive freezing if it occurs above −14°C, and non-diapausing 3rd instar larvae of C. amoena and Drosophila melanogaster (Diptera: Drosophilidae), neither of which survive freezing. Freezing was readily observed in all larvae, and on one occasion the gut was seen to freeze separately from the haemocoel. There were no apparent qualitative differences in ice formation between freeze tolerant and non-freeze tolerant larvae. The time to complete freezing was positively related to temperature of nucleation (supercooling point, SCP), and SCP declined with decreasing body size, although this relationship was less strong in diapausing C. amoena. Nucleation generally occurred at a contact point with the thermocouple or chamber wall in non-diapausing larvae, but at random in diapausing larvae, suggesting that the latter have some control over ice nucleation. There were no apparent differences between freeze tolerant and non-freeze tolerant larvae in tracheal displacement or distension of the body during freezing, although there was markedly more distension in D. melanogaster than in C. amoena regardless of diapause state. We conclude that although control of ice nucleation appears to be important in freeze tolerant individuals, the physical ice formation process itself does not differ among larvae that can and cannot survive freezing. This suggests that a focus on cellular and biochemical mechanisms is appropriate and may reveal the primary adaptations allowing freeze tolerance in insects.

Highlights

The ability of some insects to survive internal ice formation has been known since the 18th century [1]
Ice formation did not appear to be initiated at points of contact in diapausing C. amoena, whereas it was in non-diapausing and D. melanogaster
There is high variability in the freezing process, but we have confirmed the basic relationship between ice formation and body size, and between supercooling point (SCP) and ice formation processes

Summary

Introduction

The ability of some insects to survive internal ice formation has been known since the 18th century [1]. A general model of freeze tolerance suggests that ice formation is restricted to extracellular spaces, resulting in osmotic dehydration of cells [3]. Carbohydrate cryoprotectants are thought to protect membranes and proteins during this dehydration as well as to change the aqueous properties of the cell [4]. Aquaporins have been implicated as key in freeze tolerance, perhaps by facilitating osmotic equilibration of cells during the freezing process [7,8]. None of these cellular-level adaptations appear to be both necessary and sufficient for freeze tolerance, and it is unclear whether organismal-level ice formation processes are important in determining freezing survival

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