Activation of the Hippo Pathway in Rana sylvatica: Yapping Stops in Response to Anoxia

Wood frogs (Rana sylvatica) display well-developed anoxia tolerance as one component of their capacity to endure prolonged whole-body freezing during the winter months. Under anoxic conditions, multiple cellular responses are triggered to efficiently cope with stress by suppressing gene transcription and promoting activation of mechanisms that support cell survival. Activation of the Hippo signaling pathway initiates a cascade of protein kinase reactions that end with phosphorylation of YAP protein. Multiple pathway components of the Hippo pathway were analyzed via immunoblotting, qPCR or DNA-binding ELISAs to assess the effects of 24 h anoxia and 4 h aerobic recovery, compared with controls, on liver and heart metabolism of wood frogs. Immunoblot results showed significant increases in the relative levels of multiple proteins of the Hippo pathway representing an overall activation of the pathway in both organs under anoxia stress. Upregulation of transcript levels further confirmed this. A decrease in YAP and TEAD protein levels in the nuclear fraction also indicated reduced translocation of these proteins. Decreased DNA-binding activity of TEAD at the promoter region also suggested repression of gene transcription of its downstream targets such as SOX2 and OCT4. Furthermore, changes in the protein levels of two downstream targets of TEAD, OCT4 and SOX2, established regulated transcriptional activity and could possibly be associated with the activation of the Hippo pathway. Increased levels of TAZ in anoxic hearts also suggested its involvement in the repair mechanism for damage caused to cardiac muscles during anoxia. In summary, this study provides the first insights into the role of the Hippo pathway in maintaining cellular homeostasis in response to anoxia in amphibians.

Effects of Road De-Icing Salts on Two Amphibian Species, Rana Sylvatica and Rana Pipiens, and Their Trematode Parasites

With ongoing amphibian declines, it is essential to determine possible contributors such as diseases and environmental contaminants that may increase susceptibility. A potential contaminant is road salt (mainly NaCl), which leaches into aquatic environments. I examined whether road salts make larval amphibians (tadpoles) more susceptible to trematode parasite infection, and also how these affect free-living trematode infectious stages (cercariae). I exposed Rana sylvatica (wood frogs) and R. pipiens (northern leopard frogs) to control, medium (400 mg/L), and high salt (800 mg/L) treatments, and then to trematodes. High salt tended to reduce wood frog anti-parasite behaviour and resistance to infection but the opposite was seen for R. pipiens, although these tadpoles had elevated lymphocyte counts in high salinity. Trematodes were differentially affected by increased salinities. The results suggest that host-parasite-environment interactions are complex, with species differentially affected by contaminants, which may lead to community shifts in predominant hosts and parasite species.

Effects of Road De-Icing Salts on Two Amphibian Species, Rana Sylvatica and Rana Pipiens, and Their Trematode Parasites

With ongoing amphibian declines, it is essential to determine possible contributors such as diseases and environmental contaminants that may increase susceptibility. A potential contaminant is road salt (mainly NaCl), which leaches into aquatic environments. I examined whether road salts make larval amphibians (tadpoles) more susceptible to trematode parasite infection, and also how these affect free-living trematode infectious stages (cercariae). I exposed Rana sylvatica (wood frogs) and R. pipiens (northern leopard frogs) to control, medium (400 mg/L), and high salt (800 mg/L) treatments, and then to trematodes. High salt tended to reduce wood frog anti-parasite behaviour and resistance to infection but the opposite was seen for R. pipiens, although these tadpoles had elevated lymphocyte counts in high salinity. Trematodes were differentially affected by increased salinities. The results suggest that host-parasite-environment interactions are complex, with species differentially affected by contaminants, which may lead to community shifts in predominant hosts and parasite species.

Mitochondria and the Frozen Frog

The wood frog, Rana sylvatica, is the best-studied of a small group of amphibian species that survive whole body freezing during the winter months. These frogs endure the freezing of 65–70% of their total body water in extracellular ice masses. They have implemented multiple adaptations that manage ice formation, deal with freeze-induced ischemia/reperfusion stress, limit cell volume reduction with the production of small molecule cryoprotectants (glucose, urea) and adjust a wide variety of metabolic pathways for prolonged life in a frozen state. All organs, tissues, cells and intracellular organelles are affected by freeze/thaw and its consequences. This article explores mitochondria in the frozen frog with a focus on both the consequences of freezing (e.g., anoxia/ischemia, cell volume reduction) and mitigating defenses (e.g., antioxidants, chaperone proteins, upregulation of mitochondria-encoded genes, enzyme regulation, etc.) in order to identify adaptive strategies that defend and adapt mitochondria in animals that can be frozen for six months or more every year. A particular focus is placed on freeze-responsive genes in wood frogs that are encoded on the mitochondrial genome including ATP6/8, ND4 and 16S RNA. These were strongly up-regulated during whole body freezing (24 h at −2.5 °C) in the liver and brain but showed opposing responses to two component stresses: strong upregulation in response to anoxia but no response to dehydration stress. This indicates that freeze-responsive upregulation of mitochondria-encoded genes is triggered by declining oxygen and likely has an adaptive function in supporting cellular energetics under indeterminate lengths of whole body freezing.

Salted roads lead to edema and reduced locomotor function in wood frogs

Human activities have caused massive losses of natural populations across the globe. Like many groups, amphibians have experienced substantial declines worldwide, driven by environmental changes such as habitat conversion, pollution, and disease emergence. Each of these drivers is often found in close association with the presence of roads. Here we report a novel consequence of roads affecting an amphibian native to much of North America, the wood frog (Rana sylvatica). Across 38 populations distributed from southern to central New England, we found that adult wood frogs living adjacent to roads had higher incidence and severity of edema (bloating caused by fluid accumulation) during the breeding season than frogs living away from the influence of roads. This effect was best explained by increased conductivity of breeding ponds, caused by runoff pollution from road salt used for de-icing. Edema severity was negatively correlated with locomotor performance in more northerly populations. Interestingly, northern populations experience more intense winters, which tends to result in more de-icing salt runoff and increased energetic demands associated with overwintering cryoprotection needs. Thus, this emerging consequence of roads appears to impose potential fitness costs associated with locomotion, and these effects might be most impactful on populations living in regions where de-icing is most intense.