Department of Biology, The University of Western Ontario, London, Ontario, Canada. mailto:
PLoS One. 2009 Dec 14;4(12):e8259. doi: 10.1371/journal.pone.0008259.
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 3(rd) instar larvae of Chymomyza amoena (Diptera: Drosophilidae), which survive freezing if it occurs above -14 degrees C, and non-diapausing 3(rd) 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.
虽然昆虫抗冻性的生化相关性已广为人知，但体内冰形成的过程仍存在推测。我们使用同步加速器 X 射线以 3.3Hz 的频率直接实时观察完整昆虫体内的冰形成。我们观察了休眠 3 龄幼虫 Chymomyza amoena（双翅目：果蝇科）的冻结过程，如果温度在-14°C 以上，它们可以在冻结中存活，而非休眠的 3 龄幼虫和果蝇 Drosophila melanogaster（双翅目：果蝇科）则不能在冻结中存活。所有幼虫都很容易被冻结，有一次还观察到肠道与血腔分开冻结。在抗冻和非抗冻幼虫之间，冰形成似乎没有明显的定性差异。完全冻结的时间与成核温度（过冷点，SCP）呈正相关，并且 SCP 随体型减小而降低，尽管休眠的 Chymomyza amoena 中这种关系较弱。在非休眠幼虫中，成核通常发生在与热电偶或腔室壁的接触点处，但在休眠幼虫中则随机发生，这表明后者可以控制冰核的形成。在抗冻和非抗冻幼虫中，冻结过程中气管位移或身体膨胀没有明显差异，尽管果蝇的膨胀明显大于 Chymomyza amoena，而与休眠状态无关。我们的结论是，尽管控制冰核形成似乎对抗冻个体很重要，但在可以和不能在冻结中存活的幼虫中，物理冰形成过程本身没有差异。这表明关注细胞和生化机制是合适的，并且可能揭示允许昆虫抗冻的主要适应机制。
