Rapid cold hardening protects against sublethal freezing injury in an Antarctic insect

Abstract

Rapid cold hardening (RCH) is a type of beneficial phenotypic plasticity that occurs on extremely short time scales (minutes to hours) to enhance insects' ability to cope with cold snaps and diurnal temperature fluctuations. RCH has a well-established role in extending lower lethal limits, but its ability to prevent sublethal cold injury has received less attention. The Antarctic midge, Belgica antarctica, is Antarctica's only endemic insect and has a well-studied RCH response that extends freeze tolerance in laboratory conditions. However, the discriminating temperatures used in previous studies of RCH are far below those ever experienced in the field. Here, we tested the hypothesis that RCH protects against non-lethal freezing injury. Larvae of B. antarctica were exposed to control (2°C), direct freezing (−9°C for 24 h) or RCH (−5°C for 2 h followed by −9°C for 24 h). All larvae survived both freezing treatments, but RCH larvae recovered more quickly from freezing stress and had a significantly higher metabolic rate during recovery. RCH larvae also sustained less damage to fat body and midgut tissue and had lower expression of two heat shock protein transcripts (hsp60 and hsp90), which is consistent with RCH protecting against protein denaturation. The protection afforded by RCH resulted in energy savings; directly frozen larvae experienced a significant depletion in glycogen energy stores that was not observed in RCH larvae. Together, these results provide strong evidence that RCH protects against a variety of sublethal freezing injuries and allows insects to rapidly finetune their performance in thermally variable environments.

Changes in Energy Reserves and Gene Expression Elicited by Freezing and Supercooling in the Antarctic Midge, Belgica antarctica

“…Our objectives were to (1) assess survival and tissue damage in winter-acclimated larvae that are frozen and supercooled at the same temperature, (2) quantify the energetic costs of freezing and supercooling by measuring levels of key energy stores, and (3) test the hypothesis that freezing and supercooling elicit distinct cell stress responses by measuring transcript levels of heat shock proteins from each of the five major families. Heat shock proteins are a class of molecular chaperones that are upregulated in direct response to protein denaturation [23], and expression of these genes has been used as a biomarker for sublethal stress in B. antarctica [24]. Our experiments indicate that while winter-acclimated larvae survive freezing and supercooling equally well, freezing results in slightly greater energetic costs and elevated expression of certain heat shock proteins.…”

“…The slight glycogen reduction in frozen larvae may be explained by mobilization of glucose, as frozen larvae had the highest glucose levels after 14 days of cold exposure (Figure 2c). Glucose mobilization during stress is frequently observed in B. antarctica, and in previous studies, treatments that lead to higher mortality or higher sublethal injury yield higher rates of glucose mobilization [21,24,28]. During stress, glucose can function as a cryoprotectant or serve as a substrate for the synthesis of cryoprotective sugar alcohols [34], but we did not assess cryoprotectant loads in this study.…”

“…In the case of hsp90, there was a significant effect of treatment in our full factorial model, once again, with frozen larvae tending to have higher expression. Interestingly, these two transcripts were also elevated in frozen larvae relative to those that had enhanced freezing tolerance via rapid cold hardening [24], suggesting that hsp60 and hsp90 may be biomarkers of freezing stress. While there was no general treatment effect for hsp70, transcript levels were higher in frozen larvae following 12 h recovery from a 24 h cold exposure.…”

“…Glucose was measured using the Glucose Assay Kit (Sigma-Aldrich, St. Louis, MO, USA) according to the manufacturer's instructions but scaled down for microplates. While glucose was not a primary energy store for B. antarctica, in previous work, mobilization of glucose in response to stress was observed, with the degree of mobilization proportionate to the stressfulness of the treatment [21,24,28]. Glycogen was measured by first treating samples with amyloglucosidase from Aspergillus niger (Sigma-Aldrich) at 55 • C for 2 h and then measuring glucose concentration.…”

Rapid cold hardening: ecological relevance, physiological mechanisms and new perspectives

“…(C) Phenotypic outcomes of RCH. The graphs include example data illustrating the general phenomena; from Teets and Denlinger 2016, Kawarasaki et al (2013), Kelty and Lee (1999), Teets et al (2019) and Rinehart et al (2000), respectively. All figures were adapted with permission from the authors.…”