Like other cold-blooded animals, insects constantly adjust their behaviour and physiology to cope with diurnal and seasonal temperature changes. Many studies have focused on the acclimatization that insects undergo to deal with seasonal temperature changes, but some rapid acclimatory responses have also been shown to enhance insects' cold tolerance following acute drops in temperature. One of these rapid responses is termed rapid cold hardening(RCH), where a short pre-exposure to cold markedly improves insects' tolerance during a subsequent severe cold insult. The RCH response occurs in many insect orders and, in addition to enhanced survival after cold shock, it enables them to remain active at low but sub-lethal temperatures (typically above 0°C). Even though RCH has been found in many insects, little is known about the physiological basis of this response. To improve our understanding of this phenomenon, Yi and Lee investigated the effects of RCH on in vivo and in vitro cellular viability following cold shock in flesh flies(Sarcophaga crassipalpis).
To see if RCH protects fly tissues from cold shock, Yi and Lee decided to compare cellular survival in cold-hardened flies (pre-exposed to cold for two hours) and non-hardened flies after exposure to a cold shock. If RCH offers cold protection, they expected the tissues of cold-hardened flies to fare better than the non-hardened flies. The authors exposed both groups to a cold shock, and dissected the insects immediately after the shock. To see which tissues are protected by RCH, they assessed the cellular survival of four different tissue types (fat body, gut tissue, salivary gland, and Malpighian tubules) using a modified live/dead sperm assay that allowed them to distinguish live cells from dead with fluorescent dyes. As hypothesised, they found that the cellular viability of the hardened flies was markedly improved compared with the unhardened flies. This effect was significant in all four tissue types examined, demonstrating that RCH offers protection to several different tissue types, rather than being exclusive to a particular tissue type.
So just two hours of cold-hardening can provide protection from subsequent cold. But how is the RCH response activated so quickly? The team wondered if the RCH response can function in vitro, independently of central nervous or hormonal regulation, which could explain how the RCH response is transmitted so quickly. To test this, the authors used the same fluorescent dyes to investigate cellular viability, but this time the four tissues were dissected from the flies before the RCH and cold shock treatment. Sure enough,they found that in vitro cold-hardened tissues had a higher cellular survival rate after a cold shock than unhardened tissues, so RCH does protect cells in vitro. In fact, in vitro cold-hardened tissues responded much like tissues that had been hardened and cold shocked in vivo. These results are the first to demonstrate that RCH occurs independently of central regulation.
Even though this study does not resolve the basic physiological mechanisms underlying RCH, it provides guidance for future studies on RCH by emphasising that the nature of RCH is likely to be found among the basic cellular responses to lowered temperature.