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