Wood Frog Rana Sylvatica Research Articles

Isolation and Characterization of a Frog Virus 3 Strain from a Wood Frog (Rana sylvatica) in Wood Buffalo National Park.

Members of the Iridoviridae family, genus Ranavirus, represent a group of globally emerging pathogens of ecological and economic importance. In 2017, an amphibian die-off of wood frogs (Rana sylvatica) and boreal chorus frogs (Pseudacris maculata) was reported in Wood Buffalo National Park, Canada. Isolation and complete genomic sequencing of the tissues of a wood frog revealed the presence of a frog virus 3 (FV3)-like isolate, Rana sylvatica ranavirus (RSR), with a genome size of 105,895 base pairs, 97 predicted open reading frames (ORFs) bearing sequence similarity to FV3 (99.98%) and a FV3-like isolate from a spotted salamander in Maine (SSME; 99.64%). Despite high sequence similarity, RSR had a unique genomic composition containing ORFs specific to either FV3 or SSME. In addition, RSR had a unique 13 amino acid insertion in ORF 49/50L. No differences were found in the in vitro growth kinetics of FV3, SSME, and RSR; however, genomic differences between these isolates were in non-core genes, implicated in nucleic acid metabolism and immune evasion. This study highlights the importance of viral isolation and complete genomic analysis as these not only provide information on ranavirus spatial distribution but may elucidate genomic factors contributing to host tropism and pathogenicity.

Freshwater salinization and the evolved tolerance of amphibians.

The increasing salinization of freshwaters is a growing environmental issue as a result of mining, agriculture, climate change, and the application of de-icing salts in regions that experience ice and snow. Due to narrow osmotic limits, many freshwater species are particularly susceptible to salinization, but it is possible that repeated exposures over time could favor the evolution of increased salt tolerance. Using collected nine populations of larval wood frogs (Rana sylvatica) as eggs from ponds and wetlands with close proximity to roads and spanning a wide gradient of salt concentrations. In the first experiment, we used a time-to-death experiment to examine the salt tolerance. In a second experiment, we examined whether population differences in salt tolerance were associated with trade-offs in growth, development, or behavior in the presence of control water or a sublethal salt concentration. We found that populations collected from ponds with low and intermediate salt concentrations exhibited similar tolerance curves over a 96-h exposure. However, the population from a pond with the highest salt concentration exhibited a much higher tolerance. We also found population differences in growth, development, and activity level among the populations, but these were not associated with population differences in tolerance. In addition, the sublethal concentration of salt had no impact on growth and development, but it did cause a reduction in tadpole activity across the populations. Collectively, these results provide further evidence that some species of freshwater organisms can evolve tolerance to increasing salinization, although it may only occur under relatively high concentrations and without trade-offs in growth, development, or behavior.

Establishment and characterization of a continuous cell culture derived from wood frog (Rana sylvatica) skin

Establishment and characterization of a continuous cell culture derived from wood frog (Rana sylvatica) skin

The wood frog (Rana sylvatica): An emerging comparative model for anuran immunity and host-ranavirus interactions

The wood frog (Rana sylvatica) is widely distributed across North America and is the only amphibian found north of the Arctic Circle due to its remarkable ability to tolerate whole-body freezing. Recent mass mortalities attributable to Ranavirus spp. (family Iridoviridae) in wild juvenile wood frogs, coupled with the apparent high susceptibility of wood frogs to experimental infection with frog virus 3 (FV3), the type species of the Ranavirus genus, or FV3-like isolates underscore the serious threat ranaviruses poses to wood frog populations. Despite the ecological relevance and unique life history of wood frogs, our understanding of the wood frog immune system and antiviral response to ranaviral infections is in its infancy. Here we aim to (1) synthesize the limited knowledge of wood frog immune defences, (2) review recent progress in establishing the wood frog as a study system for ranavirus infection, and (3) highlight the future use of wood frogs as a model anuran to provide insight into the evolution of anuran immune systems and antiviral responses.

Diet- and salinity-induced modifications of the gut microbiota are associated with differential physiological responses to ranavirus infection in Rana sylvatica.

Greater knowledge of how host-microbiome interactions vary with anthropogenic environmental change and influence pathogenic infections is needed to better understand stress-mediated disease outcomes. We investigated how increasing salinization in freshwaters (e.g. due to road de-icing salt runoff) and associated increases in growth of nutritional algae influenced gut bacterial assembly, host physiology and responses to ranavirus exposure in larval wood frogs (Rana sylvatica). Elevating salinity and supplementing a basic larval diet with algae increased larval growth and also increased ranavirus loads. However, larvae given algae did not exhibit elevated kidney corticosterone levels, accelerated development or weight loss post-infection, whereas larvae fed a basic diet did. Thus, algal supplementation reversed a potentially maladaptive stress response to infection observed in prior studies in this system. Algae supplementation also reduced gut bacterial diversity. Notably, we observed higher relative abundances of Firmicutes in treatments with algae-a pattern consistent with increased growth and fat deposition in mammals-that may contribute to the diminished stress responses to infection via regulation of host metabolism and endocrine function. Our study informs mechanistic hypotheses about the role of microbiome mediation of host responses to infection that can be tested in future experiments in this host-pathogen system. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Freeze-induced suppression of pyruvate kinase in liver of the wood frog (Rana sylvatica).

The wood frog (Rana sylvatica) undergoes physiological and metabolic changes to withstand subzero temperatures and whole body freezing during the winter months. Along with metabolic rate depression, high concentrations of glucose are produced as a cryoprotectant by liver and distributed to all other tissues. Pyruvate kinase (PK; EC:2.7.1.40), the final enzyme of glycolysis, plays an important role in the modulation of glucose metabolism and, therefore, overall metabolic regulation. The present study investigated the functional and kinetic properties of purified PK from liver of control (5°C acclimated) and frozen (-2.5°C for 24h) wood frogs. Liver PK was purified to homogeneity by a two-step chromatographic process, followed by analysis of enzyme properties. A significant decrease in the affinity of PK for its substrates, phosphoenolpyruvate (PEP) and adenosine diphosphate (ADP) at 22°C and 5°C was noted in liver from frozen frogs, as compared with controls. Immunoblotting also revealed freeze-responsive changes in posttranslational modifications with a significant increase in serine and threonine phosphorylation by 1.46-fold and 1.73- fold for PK from frozen frogs as compared with controls. Furthermore, a test of thermal stability showed that PK from liver of frozen wood frogs showed greater stability as compared with PK from control animals. Taken together, these results suggest that PK is negatively regulated, and glycolysis is suppressed, during freezing. This response acts as an important survival strategy for maintaining continuously elevated levels of cryoprotectant in frogs while they remain in a hypometabolic frozen state.

Chemical communication in wood frog (Rana sylvatica) tadpoles is influenced by early-life exposure to naphthenic acid fraction compounds

Environmental pollutants can disrupt chemical communication between aquatic organisms by interfering with the production, transmission, and/or detection of, as well as responses to, chemical cues. Here, we test the hypothesis that early-life exposure to naphthenic acid fraction compounds (NAFCs) from oil sands tailings disrupts antipredator-associated chemical communication in larval amphibians. Wild adult wood frogs (Rana sylvatica) captured during their natural breeding period were combined (1 female:2 males) in six replicate mesocosms filled with either uncontaminated lakewater or with NAFCs isolated from an active tailings pond in Alberta, Canada, at nominal 5 mg/L concentrations. Egg clutches were incubated and tadpoles maintained in their respective mesocosms for ∼40 days post-hatch. Tadpoles (Gosner stage 25-31) were then transferred individually to trial arenas filled with uncontaminated water and exposed to one of six chemical alarm cue (AC) stimuli solutions following a 3 × 2 × 2 design (3 AC types × 2 stimulus carriers × 2 rearing exposure groups). Relative to control tadpoles, NAFC-exposed tadpoles demonstrated higher baseline activity levels (line crosses and direction changes) when introduced to uncontaminated water. Antipredator responses differed in graded fashion with AC type, with control ACs eliciting the greatest latency to resume activity, water the least, and NAFC-exposed ACs intermediate. Pre- to post-stimulus difference scores were non-significant in control tadpoles, while NAFC-exposed tadpoles demonstrated significantly greater variation. While this suggests that exposure to NAFCs from fertilization through hatching may have interfered with AC production, it is unclear whether the quality or quantity of cues was affected. There was also no clear evidence that NAFC carrier water interfered with ACs or the alarm response in unexposed control tadpoles. These results emphasize the importance of understanding how behavioral and physiological effects of early-life NAFC exposure on critical antipredator responses may persist across life history stages.

Environmental Drivers of Amphibian Breeding Phenology across Multiple Sites

A mechanistic understanding of phenology, the seasonal timing of life history events, is important for understanding species’ interactions and the potential responses of ecological communities to a rapidly changing climate. We present analysis of a seven-year dataset on the breeding phenology of wood frogs (Rana sylvatica), tiger salamanders (Ambystoma tigrinum), blue-spotted salamanders (Ambystoma laterale), and associated unisexual Ambystoma salamanders from six wetlands in Southeast Michigan, USA. We assess whether the ordinal date of breeding migrations varies among species, sexes, and individual wetlands, and we describe the specific environmental conditions associated with breeding migrations for each species/sex. Breeding date was significantly affected by species/sex identity, year, wetland, and the interactions between species/sex and year as well as wetland and year. There was a great deal of variation among years, with breeding occurring nearly synchronously among groups in some years but widely spaced between groups in other years. Specific environmental triggers for movement varied for each species and sex and changed as the breeding season progressed. In general, salamanders responded to longer temperature lags (more warmer days in a row) than wood frogs, whereas wood frogs required longer precipitation lags (more rainy days in a row) than salamanders. Wood frogs were more likely to migrate around the time of a new moon, whereas in contrast, Ambystoma salamander migration was not associated with a moon phase. Ordinal day was an important factor in all models, suggesting that these amphibians require a latency period or similar mechanism to avoid breeding too early in the year, even when weather conditions appear favorable. Male wood frogs migrated earlier than female wood frogs, and male blue-spotted salamanders migrated earlier than female A. laterale and associated unisexual females. Larger unisexual salamanders migrated earlier than smaller individuals. Differences in species’ responses to environmental cues led to wood frogs and A. laterale breeding later than tiger salamanders in colder years but not in warmer years. This suggests that, as the climate warms, wood frog and A. laterale larvae may experience less predation from tiger salamander larvae due to reduced size differences when they breed simultaneously. Our study is one of few to describe the proximate drivers of amphibian breeding migrations across multiple species, wetlands, and years, and it can inform models predicting how climate change may shift ecological interactions among pond-breeding amphibian species.

MRNA m6 A methylation in wood frog brain is maintained during freezing and anoxia.

Freeze tolerance is an adaptive strategy that wood frogs (Rana sylvatica) use to survive the subzero temperatures of winter. It is characterized by a variety of metabolic and physiological changes that facilitate successful freezing and anoxia. As both mRNA regulation and posttranslation protein modification have been implicated in freeze tolerance, we hypothesized that posttranslational RNA regulation is also involved in coordinating freeze-thaw cycles and metabolic rate depression. As such, we investigated the most abundant RNA modification, adenosine methylation (N6 -methyladenosine; m6 A) in wood frog brains during 24 h periods of freezing and anoxia. This was followed by an examination of levels of RNA methyltransferases, demethyltransferases, and the readers of RNA methylation. Despite relative levels of methylation on mRNA remaining constant throughout freezing and anoxia, a significant increase in relative abundance of m6 A methyltransferases METTL3 and METTL14 was observed. In addition, we investigated the effect of m6 A RNA methylation on mRNA triaging to stress granules and report a significant increase in stress granule markers TIAR and TIA-1 in both freezing and anoxia. Our findings are the first report of RNA posttranslational regulation during metabolic rate depression in the wood frog brain and suggest that the dynamic RNA methylation observed is not directly linked to mRNA regulation during periods of extreme metabolic reorganization, warranting future investigations.

Role of FOXO transcription factors in the tolerance of whole-body freezing in the wood frog, Rana sylvatica

The wood frog (Rana Sylvatica) can endure the sub-zero temperatures of winter by freezing up to 65% of total body water as extracellular ice and retreating into a prolonged hypometabolic state. Freeze survival requires the coordination of various adaptations, including a global suppression of metabolic functions and select activation of pro-survival genes. Transcription factors playing roles in metabolism, stress tolerance, and cell proliferation may assist in making survival in a frozen state possible. In this study, the role of Forkhead box ‘other’ (FOXO) transcription factors in freeze tolerance, and related changes to the insulin pathway, are investigated. Immunoblotting was used to assess total and phosphorylated amounts of FOXO proteins in wood frogs subjected to freezing for 24 h and thawed recovery for 8 h. Levels of active FOXO3 increased in brain, kidney, and liver during freezing and thawing, suggesting a need to maintain or enhance antioxidant defenses under these stresses. Results implicate FOXO involvement in the metabolic regulation of natural freeze tolerance.

Naphthenic acid fraction compounds reduce the reproductive success of wood frogs (Rana sylvatica) by affecting offspring viability

Understanding the toxicity of organic compounds in oil sands process-affected water (OSPW) is necessary to inform the development of environmental guidelines related to wastewater management in Canada's oil sands region. In the present study, we investigated the effects of naphthenic acid fraction compounds (NAFCs), one of the most toxic components of OSPW, on mating behaviour, fertility, and offspring viability in the wood frog (Rana sylvatica). Wild adult wood frogs were exposed separately from the opposite sex to 0, 5, or 10 mg/L of OSPW-derived NAFCs for 24 h and then combined in outdoor lake water mesocosms containing the same NAFC concentrations (n = 2 males and 1 female per mesocosm, n = 3 mesocosms per treatment). Mating events were recorded for 48 h and egg masses were measured to determine adult fertility. NAFC exposure had no significant effect on mating behaviour (probability of amplexus and oviposition, amplexus and oviposition latency, total duration of amplexus and number of amplectic events) or fertility (fertilization success and clutch size). Tadpoles (50 individuals per mesocosm at hatching, and 15 individuals per mesocosm from 42 d post-hatch) were reared in the same mesocosms under chronic NAFC exposure until metamorphic climax (61–85 d after hatching). Offspring exposed to 10 mg/L NAFCs during development were less likely to survive and complete metamorphosis, grew at a reduced rate, and displayed more frequent morphological abnormalities. These abnormalities included limb anomalies at metamorphosis, described for the first time after NAFC exposure. The results of this study suggest that NAFCs reduce wood frog reproductive success through declines in offspring viability and therefore raise the concern that exposure to NAFCs during reproduction and development may affect the recruitment of native amphibian populations in the oil sands region.

Effects of a Neonicotinoid Insecticide and Population Density on Behavior and Development of Wood Frogs (Rana sylvatica).

Amphibians have been facing global declines over the last decades from direct and indirect effects of anthropogenic activities. A contributor to declines is waterway contamination from agricultural runoffs of pesticides such as neonicotinoids. Beyond direct and indirect effects of the pesticide, few studies have investigated the possible interactions between neonicotinoids and natural environmental stressors across larval development, which could alter the strength and direction of observed neonicotinoid effects. The present study used a fully crossed design to investigate how a concentration of imidacloprid (a neonicotinoid; 10 µg/L) measured in surface waters interacted with low and high population densities (0.33 and 1 tadpole/L, respectively), an important environmental stressor, to influence behavior and development across metamorphosis in wood frogs (Rana sylvatica), known to breed in agricultural landscapes. Behaviors were measured in the absence and presence of predation cues using open-field tests at three distinct developmental stages, up to the metamorph stage. We found that imidacloprid did not interact with population density or independently affect behaviors in the absence of predation cues. However, individuals raised at high density compared with low density were more active at an early developmental stage but less active at metamorphic climax. Furthermore, both density and imidacloprid independently decreased the natural freezing response of tadpoles to predation cues. Finally, we found that distance traveled in the open-field test was weakly repeatable between aquatic stages but not repeatable across metamorphosis, a pattern that was not affected by treatments. The present study provides novel insights on the ecotoxicology of imidacloprid in the presence of a natural stressor, highlighting the importance of including behavioral assays and natural stressors in studies of amphibian ecotoxicology. Environ Toxicol Chem 2022;41:2968-2980. © 2022 SETAC.

Ranavirus infection-induced avoidance behaviour in wood frog juveniles: do amphibians socially distance?

Hosts may limit exposure to pathogens through changes in behaviour, such as avoiding infected individuals or contaminated areas. Here, we tested for a behavioural response to ranavirus infection in juvenile wood frogs (<i>Rana sylvatica</i>) because the majority of dispersal between populations occurs during this life stage. We hypothesized that if infections are transmissible and detectable at this life stage, then susceptibles would display avoidance behaviours when introduced to an infected conspecific. Despite no apparent signs of infection, we observed a greater distance between susceptible-infected pairs, compared to pairs of either two infected or two susceptible animals. Further, distances between susceptible-infected pairs were positively related to the infection intensity of the focal exposed frog, suggesting the cue to avoid infected conspecifics may become more detectable with more intense infections. Although we did not quantify whether the transmission was affected by their distancing, our findings suggest that juvenile frogs have the potential to reduce terrestrial transmission of ranaviruses through avoidance behaviours.

Status of the Janus Kinase-Signal Transducer and Activator of Transcription (JAK-STAT) pathway in liver and skin of the freeze tolerant wood frog

The wood frog (Rana sylvatica) has adapted full-body freezing and thawing as a means of sub-zero winter survival and early-breeding in ephemeral pools. One such protective process implicated recently in freeze-thaw tolerance is that of anti-apoptotic signaling, which has been proposed to play a cytoprotective role by modulating stress-induced death signals. This study employed the use of immunoblotting to examine response of a potent cell cycle and apoptosis regulator, known as the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling pathway, to freezing and thawing in the liver and skin of the wood frog. This pathway demonstrably exhibits factor- and tissue-specific changes between non-frozen, 24 h-frozen, and 8 h-thawed conditions. There were few changes in JAK-STAT proteins in frozen frogs, but protective changes were observed upon thaw: Elevated levels of pJAK3 and nuclear localization of pSTAT3 and pSTAT5 suggested an increase in anti-apoptotic signaling after thaw. By contrast, both STAT1 and STAT3 signaling appeared to increase in frozen skin, suggesting frogs use homeostatic regulation of apoptotic- and anti-apoptotic signals, in an antagonistic and compensatory manner. As such, these findings support that JAK-STAT pathway signaling modulation is a plausible adaptation that contributes to fast and reversible manipulation of anti-apoptotic signals, thus assisting in freeze survival of the wood frog.

Sunny-side up: ontogenetic variation in egg mass temperatures of the wood frog Rana sylvatica

The efficacy of most biological processes is temperature dependent and, within physiological limits, on average, warmer is better. This axiom of biology has led to a wide range of adaptations for dealing with temperatures that are outside of an organism’s preferred temperature. Many pond-breeding amphibians lay their eggs during early spring, when water temperatures are near freezing. Communal nest-site selection has been proposed as a mechanism to increase developmental temperatures, and temperatures near the center of egg-mass aggregations are elevated relative to egg-masses on the aggregation’s periphery. It is unclear whether this spatial variation in temperature is due to concentration of metabolic heat, absorption of solar radiation, or both. Here, we explore finer scale spatial variation within egg masses of the wood frog Rana sylvatica, one of the earliest amphibians to breed during the North American spring. We compared peripheral and core temperatures of egg masses that were exposed either to 1) ambient sunlight from above, or 2) sunlight reflected by a mirror from below. We found that differences between core and peripheral temperatures were higher in the control than in the mirror treatment, but core and peripheral temperatures were statistically indistinguishable in both cases. Moreover, the difference in peripheral and internal temperatures increased significantly over the course of development. However, these trends were only significant in ambient sunlight and actually decreased in the mirror group. Our results suggest that the benefits of communal nesting are also experienced by individual egg masses, albeit to a lesser extent. In addition, the lack of effect in shaded egg masses suggests that the thermal advantage is tied to sun exposure and not due to concentration of metabolic heat.

RAGE management: ETS1- EGR1 mediated transcriptional networks regulate angiogenic factors in wood frogs

Freeze-tolerant species, such as wood frogs (Rana sylvatica), are susceptible to multiple co-occurring stresses that they must overcome to survive. Freezing is accompanied by mechanical stress and dehydration due to ice crystal formation in the extracellular space, ischemia/anoxia due to interruption in blood flood, and hyperglycemia due to cryoprotective measures. Wood frogs can survive dehydration, anoxia, and high glucose stress independently of freezing, thereby creating a multifactorial model for studying freeze-tolerance. Oxidative stress and high glucose levels favors the production of pro-oxidant molecules and advanced glycation end product (AGE) adducts that could cause substantial cellular damage. In this study, the involvement of the high mobility group box 1 (HMGB1)-AGE/RAGE (receptor for AGE) axis and the regulation of ETS1 and EGR1-mediated angiogenic responses were investigated in liver of wood frogs expose to freeze/thaw, anoxia/reoxygenation and dehydration/rehydration treatments. HMGB1 and not AGE-adducts are likely to induce the activation of ETS1 and EGR1 via the RAGE pathway. The increase in nuclear localization of both ETS1 and EGR1, but not DNA binding activity in response to stress hints to a potential spatial and temporal regulation in inducing angiogenic factors. Freeze/thaw and dehydration/rehydration treatments increase the levels of both pro- and anti-angiogenic factors, perhaps to prepare for the distribution of cryoprotectants or enable the repair of damaged capillaries and wounds when needed. Overall, wood frogs appear to anticipate the need for angiogenesis in response to freezing and dehydration but not anoxic treatments, probably due to mechanical stress associated with the two former conditions.

DNA Hypomethylation May Contribute to Metabolic Recovery of Frozen Wood Frog Brains.

Transcriptional suppression is characteristic of extreme stress responses, speculated to preserve energetic resources in the maintenance of hypometabolism. In recent years, epigenetic regulation has become heavily implicated in stress adaptation of many animals, including supporting freeze tolerance of the wood frog (Rana sylvatica). However, nervous tissues are frequently lacking in these multi-tissue analyses which warrants investigation. The present study examines the role of DNA methylation, a core epigenetic mechanism, in the response of wood frog brains to freezing. We use immunoblot analysis to track the relative expression of DNA methyltransferases (DNMT), methyl-CpG-binding domain (MBD) proteins and ten-eleven-translocation (TET) demethylases across the freeze-thaw cycle in R. sylvatica brain, including selected comparisons to freeze-associated sub-stresses (anoxia and dehydration). Global methyltransferase activities and 5-hmC content were also assessed. The data show coordinated evidence for DNA hypomethylation in wood frog brains during freeze-recovery through the combined roles of depressed DNMT3A/3L expression driving lowered DNMT activity and increased TET2/3 levels leading to elevated 5-hmC genomic content (p < 0.05). Raised levels of DNMT1 during high dehydration were also noteworthy. The above suggest that alleviation of transcriptionally repressive 5-mC DNA methylation is a necessary component of the wood frog freeze-thaw cycle, potentially facilitating the resumption of a normoxic transcriptional state as frogs thaw and resume normal metabolic activities.

Epigenetics of the frozen brain: roles for lysine methylation in hypometabolism.

Wood frog (Rana sylvatica) freeze tolerance necessitates metabolic rate depression, where costly processes such as gene transcription are commonly suppressed. Epigenetic mechanisms, such as histone lysine methylation, have recently been implicated in hypometabolic states of various animals, although they are underreported in nervous tissues. In the present study, we track the expression of eight lysine methyltransferases, as well as the activity on, and abundance of putative histone products across the freeze-thaw cycle and freeze-associated substresses (anoxia, dehydration) of wood frog brains. Our results suggest that hypomethylation of transcriptionally repressive H3K9 may be a key facet of metabolic recovery during the thawing of nervous tissue, which we speculate may have a positive effect on global gene transcription. Some nonhistone roles for lysine methylation are also proposed.

Testing the effectiveness of environmental DNA (eDNA) to quantify larval amphibian abundance

AbstractEnvironmental DNA (eDNA) monitoring is rapidly becoming an established approach for detecting the presence of aquatic organisms and may also be useful for indexing or estimating species abundance. However, the link between eDNA concentration and abundance of individuals (i.e., density or biomass) remains tenuous and may vary widely across species and environmental conditions. We investigated the relationship between eDNA concentration and abundance in two common and closely related amphibians in eastern North America, the wood frog (Rana sylvatica), and northern leopard frog (R. pipiens). We manipulated tadpole density in 80‐L mesocosms and documented the relationship between tadpole density and biomass and eDNA concentration through time. The two species differed in the amount of detectible genetic material produced, despite having comparable biomass. Concentration of eDNA increased with tadpole numbers and was primarily correlated with tadpole density in wood frogs and biomass in leopard frogs. eDNA degradation rates were rapid and comparable between species, with tadpoles becoming indetectable within 5 days post‐removal from the mesocosm, irrespective of tadpole density. Overall, our findings support that eDNA concentration has potential for tracking amphibian abundance in wetlands, but that indices of abundance are likely to be coarse and species‐specific calibration will be required. Future research should address how biotic and abiotic factors influence eDNA production, degradation, and recovery across species and through time before relying on eDNA for monitoring amphibian abundance in nature.

One-step purification and regulation of fructose 1,6-bisphosphatase from the liver of the freeze-tolerant wood frog, Rana sylvatica.

The wood frog (Rana sylvatica) undergoes numerous changes to its physiology and metabolic processes to survive the winter months, including adaptations that let them endure whole-body freezing. The regulation of key enzymes of central carbohydrate metabolism in the liver plays a crucial role in mediating the synthesis and maintenance of high concentrations of glucose as a cryoprotectant during freezing as well as glucose reconversion to glycogen after thawing. The present study characterized the regulation of fructose-1,6-bisphosphatase (FBPase; EC 3.1.3.11) from wood frog liver during freezing, FBPase being a crucial enzyme regulating gluconeogenesis. Liver FBPase was purified to homogeneity from control and frozen wood frogs by a one-step chromatographic process. Kinetic and regulatory parameters of the enzyme were investigated and demonstrated a significant decrease in sensitivity to its substrate fructose-1,6-bisphosphatein the liver of frozen frogs, as compared with controls. Immunoblotting also revealed freeze-responsive changes in posttranslational modifications with a significant decrease in serine phosphorylation (by 53%) for FBPase from frozen frogs. Taken together, these results suggest that FBPase is suppressed, and gluconeogenesis is inhibited during freezing. This response acts as an important component of the metabolic survival strategy of the wood frog.