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Insects that experience sub-zero temperatures either succumb to ice formation (i.e., known as freeze-intolerant or freeze-avoiding) or tolerate ice formation (i.e., known as freeze-tolerant, Bale 1993). For freeze-tolerant insects, the ability to repair injury and restore homeostasis (i.e., recovery from freezing) is critical for successfully resuming development and/or other essential functions (e.g., reproduction).
Underlying mechanisms of recovery from freezing
As we conducted some work on recovery from freezing vs. supercooling in the lab, we noticed that B. antarctica maintained high transcriptional activity after several hours of restoring neuromuscular function, which led us to hypothesize that recovery from freezing would be taking place even though animals were "superficially" recovered. If transcriptional activity was increased for a prolonged period after a freezing event, it was possible that recovery from freezing was energetically costly thereby leading to deleterious consequences in the long term.
In this project, I investigated long-term effects of exposure to acute freezing stress in this species by measuring changes in gene expression and energy stores during recovery from sub-lethal freezing. I hypothesized that recovery from sub-lethal freezing would require large-scale changes in gene expression, resulting in long-term energetic costs to B. antarctica.
Experimental design
(Maintaining this species through an entire generation to fully capture fitness costs is not currently possible, so I monitored survival, gene expression, and energy stores for a 15-day recovery period as a proxy for potential fitness consequences of a freezing event)
Hypothesis and predictions
Since the freeze tolerance of B. antarctica (see bibliography) and the physicochemical effects of freezing have been extensively studied on insects in general (see Storey and Storey, 2013; Toxopeus and Sinclair, 2018; Roszyspal 2022 for extended reviews), we had a solid basis to start from when thinking about predictions. In general, I predicted that early recovery would be characterized by the activation of an "emergency stress-response" system to mitigate the cascade of negative effects of ice formation. This stress-response would, for example,
a) repair macromolecules by upregulating chaperones to aid in refolding if damage was reversible;
b) remove/clear irreversibly damaged proteins (e.g., via ubiquitin-proteasome degradation) and organelles (e.g., via autophagy)
c) prevent further damage by upregulating antioxidants, which would mitigate oxidative damage upon rapid reoxygenation.
This stress-respond would also be accompanied by a developmental arrest until freezing injury was addressed. Moreover, energy stores would be depleted throughout recovery, as a result of fueling repair processes and replenishment of macromolecules, organelles, and cells.
Results!!
This work will be published soon and I will have to remove this, but I am very excited about the conceptual model of recovery from freezing that I developed and so I will leave it here until the paper gets peer-reviewed!!
Conceptual model of long-term recovery from freezing in Belgica antarctica. The figure shows major physiological responses hypothesized to occur in response to a freezing event immediately after thawing and at the 1st, 3rd, 7th and 15th day of recovery. Blue boxes represent physiological responses that were activated and/or genes that had increased activity, and grey boxes represent processes that had decreased activity and/or were suppressed. Dashed boxes contain supporting evidence from gene expression or enrichment analyses. Processes in bold had supporting evidence that agreed in all approaches (i.e., differential expression, GO and KEGG).
Genome sequencing
Another cool part of this project was having the opportunity to annotate Belgica antarctica's new genome assembly, which was sequenced last year and improved the first assembly (Kelley et al., 2014) due to the more modern HiFi approach.
Communication
This project is completed, written up and ready for peer-review. We are planning on submitting to the Journal of Experimental Biology.
References
Bale, J. S. (1993). Classes of insect cold hardiness. Functional Ecology, 751-753.
Storey, K. B., & Storey, J. M. (2013). Molecular biology of freezing tolerance. Comprehensive Physiology, 3(3), 1283-1308
Toxopeus, J., & Sinclair, B. J. (2018). Mechanisms underlying insect freeze tolerance. Biological Reviews, 93(4), 1891–1914.
Rozsypal, J. (2022). Cold and freezing injury in insects: An overview of molecular mechanisms. EJE, 119(1), 43-57.
Kelley, J. L., Peyton, J. T., Fiston-Lavier, A. S., Teets, N. M., Yee, M. C., Johnston, J. S., ... & Denlinger, D. L. (2014). Compact genome of the Antarctic midge is likely an adaptation to an extreme environment. Nature communications, 5(1), 4611.
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