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Dryophytes versicolor is one of the most extreme freeze-tolerant frogs from eastern North America. In this study, the mitochondrial genome of D. versicolor was sequenced to analyze the phylogenetic relationships among Hylidae and investigate mitochondrial gene expression in response to freezing and anoxia. The total length of the D. versicolor mitogenome is the longest known to date among the available family members of Hylidae. Both maximum likelihood (ML) and Bayesian inference (BI) analyses strongly supported D. versicolor as a sister clade to (D. japonica + D. ussuriensis) + (D. suweonensis + D. immaculata (KP212702)), and indicated that Dryophytes is monophyletic. Using the mitochondrial genome, gene expression analysis was performed using RT-qPCR in skeletal muscle samples, and determined that relative levels of D. versicolor COX2 increased by 2.40 ± 0.23 fold in response to anoxia, but did not change with exposure to freezing. In addition, ND3 transcript levels decreased in response to anoxia but remained constant during freezing. By contrast, COX1 transcript levels decreased with exposure to freezing, but did not change under anoxic conditions. These results suggest that modulations of protein-coding mitochondrial genes of D. versicolor may play a role in the molecular response to freezing and anoxia tolerance. [ABSTRACT FROM AUTHOR]

Background: The North American wood frog, Rana sylvatica, is able to overcome subzero conditions through overwintering in a frozen state. Freezing imposes ischemic and oxidative stress on cells as a result of cessation of blood flow. Superoxide dismutases (SODs) catalyze the redox reaction involving the dismutation of superoxide (O(2)(-)) to molecular oxygen and hydrogen peroxide., Methods: The present study investigated the regulation of CuZnSOD and MnSOD kinetics as well as the transcript, protein and phosphorylation levels of purified enzyme from the muscle of control and frozen R. sylvatica., Results: CuZnSOD from frozen muscle showed a significantly higher V(max) (1.52 fold) in comparison to CuZnSOD from the muscle of control frogs. MnSOD from frozen muscle showed a significantly lower Km for O(2)(-) (0.66 fold) in comparison to CuZnSOD from control frogs. MnSOD from frozen frogs showed higher phosphorylation of serine (2.36 fold) and tyrosine (1.27 fold) residues in comparison to MnSOD from control animals. Susceptibility to digestion via thermolysin after incubation with increasing amount of urea (C(m)) was tested, resulting in no significant changes for CuZnSOD, whereas a significant change in MnSOD stability was observed between control (2.53 M urea) and frozen (2.92 M urea) frogs. Expressions of CuZnSOD and MnSOD were quantified at both mRNA and protein levels in frog muscle, but were not significantly different., Conclusion: The physiological consequence of freeze-induced SOD modification appears to adjust SOD function in freezing frogs., General Significance: Augmented SOD activity may increase the ability of R. sylvatica to overcome oxidative stress associated with ischemia., (Copyright © 2013 Elsevier B.V. All rights reserved.)