Gough, A. L. | bioRxiv Science

bioRxiv2025

Tissue- and temperature-dependent expression, enzyme activity, and RNAi knockdown of Catalase in a freeze-tolerant insect

Organisms that overwinter in temperate climates may experience freezing and freezing-induced oxidative stress during winter. While many insect species can survive freezing, molecular tools such as RNA interference (RNAi) or CRISPR have not been used to understand the physiological mechanisms underlying freeze tolerance. The spring field cricket Gryllus veletis can survive freezing following a 6-week fall-like acclimation. We used RNAi of an antioxidant enzyme in G. veletis to test the hypothesis that minimizing oxidative stress is important for freeze tolerance. In fat body tissue, Catalase mRNA abundance and enzyme activity increased during the acclimation that induces freeze tolerance. Other tissues such as midgut and Malpighian tubules had more stable or lower Catalase expression and activity during acclimation. In unacclimated (freeze-intolerant) crickets, RNA interference (RNAi) effectively knocked down production of the Catalase mRNA and protein in fat body and midgut, but not Malpighian tubules. In acclimated (freeze-tolerant) crickets, RNAi efficacy was temperature-dependent, functioning well at warm (c. 22{degrees}C) but not cool (15{degrees}C or lower) temperatures. This highlights a challenge of using RNAi in cold-acclimated organisms, as they may need to be warmed up for RNAi to work, potentially affecting their stress physiology. Knockdown of Catalase via RNAi in acclimated crickets also had no effect on the ability of the crickets to survive a mild freeze treatment, suggesting that Catalase may not be necessary for freeze tolerance. Our study is the first to demonstrate that RNAi is possible in a freeze-tolerant insect, but further research is needed to examine whether other genes and antioxidant molecules are important in freeze tolerance of G. veletis. HighlightsO_LICatalase expression and activity are elevated in freeze-tolerant cricket fat body C_LIO_LIRNAi knocks down Catalase in fat body and midgut at a warm temperature (22{degrees}C) C_LIO_LIRNAi is not effective at a cool temperature (15{degrees}C) that preserves freeze tolerance C_LIO_LICatalase knockdown has no impact on survival of a mild freeze treatment C_LIO_LIThe role of antioxidants in freeze tolerance warrants further study C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/637938v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@1c4d9b8org.highwire.dtl.DTLVardef@b1c304org.highwire.dtl.DTLVardef@7a421eorg.highwire.dtl.DTLVardef@8af62f_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Invasive adult jumping worms in Atlantic Canada are chill-susceptible

The jumping worm Amynthas tokioensis is invasive in North America, and it has been expanding its range northward in recent years. Because low temperatures typically restrict the geographic distribution of organisms, our goal was to characterize the cold tolerance physiology of adult jumping worms from a site in New Brunswick, Canada (c. 45{degrees}N), with the intent of better understanding their geographic range limits. Most of our experiments supported the conclusion that these worms are chill-susceptible: they die during or after exposure to relatively mild low temperatures. When gradually cooled, adult worms lost neuromuscular coordination at approximately 0 {degrees}C and froze at a mean temperature of -4.5 {degrees}C. They did not survive freezing and showed poor survival following 1 h exposures to 0 {degrees}C and subzero temperatures. At higher mild temperatures (5 {degrees}C), the worms could survive short (up to 6 h) but not long (e.g., 48 h) chilling durations. We attempted to induce improved cold tolerance via a five-week gradual acclimation to fall-like temperatures, but fall-acclimated worms showed poor survival during and after this acclimation. Acclimation also did not induce accumulation of glucose, a typical cryoprotectant in earthworms. We suggest that A. tokioensis can likely persist wherever the growing season is sufficiently warm and long enough for the adults to mature, reproduce, and lay cocoons prior to the chilling temperatures associated with early fall. Future work examining the cold tolerance of the overwintering cocoons will be important for fully understanding the northern range limits of these jumping worms.

zoology↗