Some insects are capable of dramatically adjusting their cold tolerance over an extremely short period of time. Early studies in fruit flies showed that a brief pre-treatment of an hour or two to a sub-lethal temperature allowed these individuals to survive what would have otherwise been a lethal cold exposure. The result of this work was the discovery of a groundbreaking physiological response, termed `rapid cold-hardening'. Later work showed that isolated cell and tissue samples also possessed the capacity for rapid cold-hardening and discounted the role of the central nervous system as a major regulator of the speedy response. Subsequently, many researchers have sought to explain the mechanisms involved in this swift alteration of lethal low temperatures. Are there common cellular stress pathways responsible for rapid cold-hardening? What are the cellular triggers responsible for generating this response?
Nicholas Teets and co-workers from David Denlinger's laboratory at Ohio State University and Rick Lee's research group at Miami University teamed up to tackle some of these challenging questions. For this work, the team used larvae of a midge, Belgica antarctica, collected from Antarctica during a summer field season and shipped back to the lab at Miami University. Next, the team performed a series of whole animal, in vivo and in vitro experiments to elucidate several questions related to the rapid cold-hardening (RCH) response. Specifically, the team asked whether RCH occurs in vitro at the cellular and tissue levels and whether calcium is necessary to generate this response.
First, the team reconfirmed that B. antarctica produces a typical RCH response: 1 h at –5°C increased the insects' survival from less than 10% after 24 h at –20°C to 70%, while survival at–15°C increased from ∼55% to ∼85%. Second, the isolated tissues from the fat body, midgut and Malpighian tubules retained the RCH response and cell survival improved under potentially lethal conditions. Using viability assays that discriminately stain damaged and normally functioning cells, Teets and his co-workers showed that cold sensing and RCH occur at the cellular level in this insect.
Next, the team demonstrated that calcium played an important role in the RCH responses. When calcium was blocked the RCH response was significantly suppressed. Finally, a calmodulin inhibitor reduced cell survival during cold treatments, further supporting the functional importance of calcium as a secondary messenger in the RCH response.
Calcium has already been demonstrated to be an important cellular messenger in cold stress responses over long time scales in other organisms, as well as functioning in a number of important downstream pathways including the regulation of gene expression and subsequent protein synthesis. The results of this study extend this information into insects and clearly show that calcium plays an important role in RCH of insects. Furthermore, it shows that calcium probably acts as a key first step in a line of complex responses. With the results of this neat study the precise cellular responses and how these are fine-tuned to meet the animal's needs are beginning to be mapped out in detail. These results have significant implications for understanding how insects can rapidly adjust thermal tolerances in their ever-changing environment.