Teets NM, Elnitsky MA, Benoit JB, Lopez-Martinez G, Denlinger DL, Lee RE, Jr. Rapid cold-hardening in larvae of the Antarctic midge Belgica antarctica: cellular cold-sensing and a role for calcium. In many insects, the rapid cold-hardening (RCH) response significantly enhances cold tolerance in minutes to hours. Larvae of the Antarctic midge, Belgica antarctica, exhibit a novel form of RCH, by which they increase their freezing tolerance. In this study, we examined whether cold-sensing and RCH

more »

... n B. antarctica occur in vitro and whether calcium is required to generate RCH. As demonstrated previously, 1 h at Ϫ5°C significantly increased organismal freezing tolerance at both Ϫ15°C and Ϫ20°C. Likewise, RCH enhanced cell survival of fat body, Malpighian tubules, and midgut tissue of larvae frozen at Ϫ20°C. Furthermore, isolated tissues retained the capacity for RCH in vitro, as demonstrated with both a dye exclusion assay and a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)-based viability assay, thus indicating that coldsensing and RCH in B. antarctica occur at the cellular level. Interestingly, there was no difference in survival between tissues that were supercooled at Ϫ5°C and those frozen at Ϫ5°C, suggesting that temperature mediates the RCH response independent of the freezing of body fluids. Finally, we demonstrated that calcium is required for RCH to occur. Removing calcium from the incubating solution slightly decreased cell survival after RCH treatments, while blocking calcium with the intracellular chelator BAPTA-AM significantly reduced survival in the RCH treatments. The calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7) also significantly reduced cell survival in the RCH treatments, thus supporting a role for calcium in RCH. This is the first report implicating calcium as an important second messenger in the RCH response. insect cold-hardening; rapid acclimation; calcium signaling; freezetolerant insects RAPID COLD-HARDENING (RCH), which allows many insects to significantly enhance their cold tolerance, was first described in freeze-susceptible insects more than 20 years ago (8, 29). While most cold acclimation processes occur over days, weeks, or even months, RCH drastically enhances cold tolerance after only a brief exposure to moderately low temperatures. In addition to protecting insects against chilling injury, RCH also improves courtship and reproduction (50, 52). Furthermore, RCH occurs during ecologically relevant diurnal temperature cycles, allowing insects to track daily changes in temperature (24, 25) . Thus it appears that RCH is part of a generalized acclimatory response that allows insects to adjust rapidly to changes in their environment. While the ecological relevance of the RCH response is established, its underlying mechanism is poorly understood.