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. 2020 Aug 10;10:13466. doi: 10.1038/s41598-020-70311-8

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© The Author(s) 2020

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Glycerol upregulation upon osmotic preconditioning is essential for freezing tolerance. (A) Schematic diagram illustrating the gluconeogenic mode of dauer larvae. (B) Dauer larvae utilize glyoxylate shunt for elevation of glycerol upon osmotic preconditioning. Error bars indicate standard deviation from three independent experiments with three technical replicates. Statistical comparison was performed with unpaired t test with Welch correction. n.s. p > 0.05, *p < 0.05. (C) Glyoxylate shunt is essential for osmotic preconditioned dauer larvae to survive freezing. Survival rates of daf-2(1370), daf-2; icl-1(ok 531) dauer larvae to freezing. For daf-2(e1370) water n = 783, ChaP n = 1,331, OsP n = 1,802, daf-2; icl-1(ok 531) water n = 937, ChaP n = 2,082, OsP n = 2,684. Error bars indicate standard deviation of two biological replicates with two technical replicates. Statistical comparison was performed with two-way ANOVA with Sidak’s multiple comparisons test. *p < 0.05. (D) Osmotic preconditioned daf-2(e1370) dauer larvae survive to freezing for longer periods. For OsP n = 17,305, ChaP n = 7,763. Error bars indicate standard error of mean of three biological replicates with two technical replicates. (E) Schematic diagram illustrating that osmotic preconditioning enhances survival of C. elegans dauer larvae to desiccation and freezing.