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

Organisms that overwinter in temperate climates may experience freezing and freezing-induced oxidative stress during winter. While many insect species can survive freezing, reverse genetics techniques such as RNA interference (RNAi) 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 to knock down expression 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 fall-like acclimation that induces freeze tolerance. Other tissues such as midgut and Malpighian tubules had more stable or lower Catalase expression and activity during this acclimation. In summer-acclimated (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 fall-acclimated (freeze-tolerant) crickets, RNAi efficacy was temperature-dependent, functioning well at warm (c. 22 °C) but not cool (15 °C or lower) temperatures. This highlights a challenge of using RNAi in organisms acclimated to low temperatures, as they may need to be warmed up for RNAi to work, potentially affecting their stress physiology. Knockdown of Catalase via RNAi in fall-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 antioxidants are needed for G. veletis freeze tolerance.