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.

Amphibian breeding phenology influences offspring size and response to a common wetland contaminant

Background Increases in temperature variability associated with climate change have critical implications for the phenology of wildlife across the globe. For example, warmer winter temperatures can induce forward shifts in breeding phenology across taxa (“false springs”), which can put organisms at risk of freezing conditions during reproduction or vulnerable early life stages. As human activities continue to encroach on natural ecosystems, it is also important to consider how breeding phenology interacts with other anthropogenic stressors (e.g., pollutants). Using 14 populations of a widespread amphibian (wood frog; Rana sylvatica), we compared 1) growth; 2) tolerance to a common wetland contaminant (NaCl); and 3) the ability of tadpoles to acclimate to lethal NaCl exposure following sublethal exposure earlier in life. We evaluated these metrics across two breeding seasons (2018 and 2019) and across populations of tadpoles whose parents differed in breeding phenology (earlier- versus later-breeding cohorts). In both years, the earlier-breeding cohorts completed breeding activity prior to a winter storm and later-breeding cohorts completed breeding activities after a winter storm. The freezing conditions that later-breeding cohorts were exposed to in 2018 were more severe in both magnitude and duration than those in 2019. Results In 2018, offspring of the later-breeding cohort were larger but less tolerant of NaCl compared to offspring of the earlier-breeding cohort. The offspring of the earlier-breeding cohort additionally were able to acclimate to a lethal concentration of NaCl following sublethal exposure earlier in life, while the later-breeding cohort became less tolerant of NaCl following acclimation. Interestingly, in 2019, the warmer of the two breeding seasons, we did not detect the negative effects of later breeding phenology on responses to NaCl. Conclusions These results suggest that phenological shifts that expose breeding amphibians to freezing conditions can have cascading consequences on offspring mass and ability to tolerate future stressors but likely depends on the severity of the freeze event.

Habitat suitability index models for the wood frog (Rana sylvatica) and boreal chorus frog (Pseudacris triseriata maculata) in the foothills parkland natural sub-region and Bow River sub-basin, Alberta

Habitat suitability index models for the wood frog (Rana sylvatica) and boreal chorus frog (Pseudacris triseriata maculata) in the foothills parkland natural sub-region and Bow River sub-basin, Alberta

Habitat suitability index models for the wood frog (Rana sylvatica) and boreal chorus frog (Pseudacris triseriata maculata) in the foothills parkland natural sub-region and Bow River sub-basin, Alberta

Habitat suitability index models for the wood frog (Rana sylvatica) and boreal chorus frog (Pseudacris triseriata maculata) in the foothills parkland natural sub-region and Bow River sub-basin, Alberta

Size-assortative mating in explosive breeders: a case study of adaptive male mate choice in anurans

Exploration of size-assortative mating (SAM) in animals has led to a near consensus that it arises through constraints in choice, such as preference for large females combined with a large male advantage during intrasexual competition. Although such ‘apparent’ SAM is well explored, whether SAM arises because of specific preferences for size-matched mates has been less thoroughly considered. We tested for ‘preference-based’ SAM in an explosively breeding frog (Rana sylvatica), quantifying how male and female sizes affected fertilization and if males preferred size-matched females. We found that size mismatch severely reduced fertilization. Furthermore, males preferred size-matched, not larger, females in mate choice trials. Because males that mated with much larger females fertilized fewer eggs overall than they would have with size-matched females, male preference for size-matched females may be adaptive. Our results expand understanding of the mechanisms underlying SAM, suggesting that multiple mechanisms may simultaneously cause size-assortative mating patterns to emerge.

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.