Rana Sylvatica Research Articles

Characterization of the Complete Mitochondrial Genome of Pleurogenoides japonicus (Digenea, Pleurogenidae): Comparison With the Members of Microphalloidea and Phylogenetic Implications.

Pleurogenoides japonicus (Trematoda: Microphalloidea) is an important parasite in wood frogs with high infection rates and significant ecological, economic, and societal importance. The scarcity of molecular data for these parasites severely limits population genetics and phylogenetic studies. In the present study, for the first time, we determined and described the entire mitochondrial (mt) genome of P. japonicus as the first representative of the family Pleurogenidae. The entire mt genome of P. japonicus was circular, with 15,043 bp (GenBank accession number OR900118), containing 36 genes, comprising 12 protein-coding genes (cox1-3, nad1-6, nad4L, cytb, and atp6), two ribosomal RNA genes, 22 transfer RNA genes, and two non-coding regions. There were 23 intergenic spacers, ranging from 2 to 162 bp, and only one 40 bp overlap between nad4L and nad4 genes in the P. japonicus mt genome. The nucleotide composition of P. japonicus mt genome exhibited a strong AT bias with a 63.75% A + T content, while the AT- and GC-skews were - 0.435 and 0.407, respectively. Comparative analysis demonstrated that the P. japonicus mt genome shared the most common characteristics with Microphalloidea trematodes, and the cox1 gene was the longest and most conserved gene in Microphalloidea trematodes. The gene arrangements of Xiphidiata trematodes were of the same order based on protein-coding genes and rRNA genes, except for tRNA. More than two gene arrangement types exist in Echinostomata and Xiphidiata, and the gene rearrangement events mainly occurred in "trnE-trnG" and "trnG-trnE". Phylogenetic analysis suggested that trematodes of the family Pleurogenidae clustered more with Prosthogonimidae than Eucotylidae. The mt genome data of P. japonicus provide an accurate genetic marker for further studies of Xiphidiata trematodes.

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.

First detection probability, comparative analysis of ecological disparities and population dynamics of the Iranian long-legged wood frog in the Hyrcanian forests

First detection probability, comparative analysis of ecological disparities and population dynamics of the Iranian long-legged wood frog in the Hyrcanian forests

Effects of amphibian genetic diversity on ecological communities.

The amount of genetic diversity within a population can affect ecological processes at population, community, and ecosystem levels. However, the magnitude, consistency, and scope of these effects are largely unknown. To investigate these issues, we conducted two experiments manipulating the amount of genetic diversity and environmental factors in larval amphibians. The first experiment manipulated wood frog genetic diversity, the presence or absence of caged predators, and competition from leopard frogs to test whether these factors affected survival, growth, and morphology of wood frogs and leopard frogs. The second experiment manipulated wood frog genetic diversity, the presence or absence of uncaged predators, and resource abundance to test whether these factors affected wood frog traits (survival, morphology, growth, development, and behavior) and other components of the ecological community (zooplankton abundance, phytoplankton, periphyton, and bacterial community structure). Genetic diversity did not affect wood frog survival, growth, and development in either experiment. However, genetic diversity did affect the mean morphology of wood frog tadpoles in the first experiment and the abundance and distribution of zooplankton in the second experiment. It did not affect phytoplankton abundance, periphyton abundance, or bacterial community structure. While effect sizes (Cohen's d) of genetic diversity were approximately half those of environment treatments, the greatest effect sizes were for interaction effects between genetic diversity and environment. Our results indicate that genetic diversity can have a large effect on ecological processes, but the direction of those effects is highly dependent upon environmental conditions, and not easily predicted from simple measures of traits.

Winter break? The effect of overwintering on immune gene expression in wood frogs

Among terrestrial ectotherms, hibernation is a common response to extreme cold temperatures and is associated with reduced physiological rates, including immunity. When winter wanes and temperatures increase, so too do vital rates of both ectothermic hosts and their parasites. Due to metabolic scaling, if parasite activity springs back faster than host immune functions then cold seasons and transitions between cold and warm seasons may represent periods of vulnerability for ectothermic hosts. Understanding host regulation of physiological rates at seasonal junctions is a first step toward identifying thermal mismatches between hosts and parasites. Here we show that immune gene expression is responsive to transitions into and out of the cold season in a winter-adapted amphibian, the wood frog (Lithobates sylvaticus), and that frogs experienced parasitism by at least two nematode species throughout the entirety of the cold season. In both splenic and skin tissues, we observed a decrease in immune gene expression going from fall to winter, observed no changes between winter and emergence from hibernation, and observed increases in immune gene expression after hibernation ended. At all timepoints, differentially expressed genes from spleens were more highly enriched for immune system processes than those from ventral skin, especially with respect to terms related to adaptive immune processes. Infection with nematode lungworms was also associated with upregulation of immune processes in the spleen. We suggest that rather than being a period of stagnation, during which physiological processes and infection potential cease, the cold season is immunologically dynamic, requiring coordinated regulation of many biological processes, and that the reemergence period may be an important time during which hosts invest in preparatory immunity.

Reversible Histone Modifications Contribute to the Frozen and Thawed Recovery States of Wood Frog Brains.

Epigenetic regulation, notably histone post-translational modification (PTM), has emerged as a major transcriptional control of gene expression during cellular stress adaptation. In the present study, we use an acid extraction method to isolate total histone protein and investigate dynamic changes in 23 well-characterized histone methylations/acetylations in the brains of wood frogs subject to 24-h freezing and subsequent 8-h thawed recovery conditions. Our results identify four histone PTMs (H2BK5ac, H3K14ac, H3K4me3, H3K9me2) and three histone proteins (H1.0, H2B, H4) that were significantly (p < 0.05) responsive to freeze-thaw in freeze-tolerant R. sylvatica brains. Two other permissive modifications (H3R8me2a, H3K9ac) also trended downwards following freezing stress. Together, these data are strongly supportive of the proposed global transcriptional states of hypometabolic freeze tolerance and rebounded thawed recovery. Our findings shed light on the intricate interplay between epigenetic regulation, gene transcription and energy metabolism in wood frogs' adaptive response to freezing stress.

Proteomic analysis of Rana sylvatica reveals differentially expressed proteins in liver in response to anoxia, dehydration or freezing stress

Ectothermic animals that live in seasonally cold regions must adapt to seasonal variation and specific environmental conditions. During the winter, some amphibians hibernate on land and encounter limited environmental water, deficient oxygen, and extremely low temperatures that can cause the whole body freezing. These stresses trigger physiological and biochemical adaptations in amphibians that allow them to survive. Rana sylvatica, commonly known as the wood frog, shows excellent freeze tolerance. They can slow their metabolic activity to a near halt and endure freezing of 65–70% of their total body water as extracellular ice during hibernation, returning to normal when the temperatures rise again. To investigate the molecular adaptations of freeze-tolerant wood frogs, a comprehensive proteomic analysis was performed on frog liver tissue after anoxia, dehydration, or freezing exposures using a label-free LC–MS/MS proteomic approach. Quantitative proteomic analysis revealed that 87, 118, and 86 proteins were significantly upregulated in dehydrated, anoxic, and frozen groups, suggesting potential protective functions. The presence of three upregulated enzymes, glutathione S-transferase (GST), aldolase (ALDOA), and sorbitol dehydrogenase (SORD), was also validated. For all enzymes, the specific enzymatic activity was significantly higher in the livers of frozen and anoxic groups than in the controls. This study reveals that GST, ALDOA, and SORD might participate in the freeze tolerance mechanism by contributing to regulating cellular detoxification and energy metabolism.

DNA hypomethylation in wood frog liver under anoxia and dehydration stresses

Wood frogs are freeze-tolerant vertebrates that can endure weeks to months frozen during the winter without breathing and with as much as 65% of total body water frozen as extracellular ice. Underlying tolerances of anoxia and of cellular dehydration support whole body freezing. One pro-survival mechanism employed by these frogs is epigenetic modifications via DNA hypomethylation processes facilitating transcriptional repression or activation. These processes involve proteins such as DNA Methyltransferases (DNMTs), Methyl Binding Domain proteins (MBDs), Ten-Eleven Translocases (TETs), and Thymine Deglycosylase (TDG). The present study evaluates the responses of these proteins to dehydration and anoxia stresses in wood frog liver. DNMT relative protein expression was reduced in liver, but nuclear DNMT activity did not change significantly under anoxia stress. By contrast, liver DNMTs and nuclear DNMT activity were upregulated under dehydration stress. These stress-specific differences were speculated to arise from Post-Translational Modifications (PTMs). DNMT3A and DNMT3B showed increased relative protein expression during recovery from dehydration and anoxia. Further, MBD1 was elevated during both conditions suggesting transcriptional repression. TET proteins showed varying responses to anoxia likely due to the absence of oxygen, a main substrate required by TETs. Similarly, TDG, an enzyme that corrects DNA damage, was downregulated under anoxia potentially due to lower levels of reactive oxygen species that damage DNA, but levels returned to normal during reperfusion of oxygen. Our results indicate differential stress-specific responses that indicate the need for more research in the DNA hypomethylation mechanisms employed by the wood frog during stress.

Effects of road salt, egg predation and alterations to the egg-mass jelly layers on the embryos of spotted salamander Ambystoma maculatum

Road de-icing salt has been used to make winter driving safer. However, its eventual drainage into adjacent water bodies has caused detrimental effects on organisms in those aquatic communities, including amphibians. We used the spotted salamander Ambystoma maculatum as a model species and examined whether there would be additive or interactive effects of road salt, egg predation from wood frog Lithobates sylvaticus tadpoles, and alteration to egg-mass jelly layers on the hatching rate, incubation period, snout-vent length (SVL) and developmental stage of hatchlings. We found neither main nor interactive effects of salt on the measured traits, which contradicts some of the previous studies. The other body of previous studies, however, showed observational and experimental evidence that amphibian populations became locally adapted to saline water. Given the roadside origin of our study animals, our negative results provide insight into the ubiquity of local adaptation to saline water among amphibian populations. We also found that egg predation and the presence of egg-mass jelly layers negatively affected the incubation period, SVL, or development in the predicted manners based on the O2 demand-supply balance within egg masses. These responses might be altered by dissolved salt if populations naive to freshwater salinisation were exposed to saline water. Keywords: aquatic pollution, community ecology, conservation, larval amphibians, Lithobates sylvaticus

Naïve and induced tolerance of 15 amphibian populations to three commonly applied insecticides

Human impacts on ecological communities are pervasive and species must either move or adapt to changing environmental conditions. For environments polluted by contaminants, researchers have found hundreds of target pest species evolving increased tolerance, but we have substantially fewer cases of evolved tolerance in non-target species. When species do evolve increased tolerance, inducible tolerance can provide immediate protection and favor the evolution of increased tolerance over generations via genetic assimilation. Using a model larval amphibian (wood frogs, Rana sylvatica), we examined the tolerance of 15 populations from western Pennsylvania and eastern New York (USA), when first exposed to no pesticide or sublethal concentrations and subsequently exposed to lethal concentrations of three common insecticides (carbaryl, chlorpyrifos, and diazinon). We found high variation in naïve tolerance among the populations for all three insecticides. We also discovered that nearly half of the populations exhibited inducible tolerance, though the degree of inducible tolerance (magnitude of tolerance plasticity; MoTP) varied. We observed a cross-tolerance pattern of the populations between chlorpyrifos and diazinon, but no pattern of similar MoTP among the pesticides. With populations combined from two regions, increased tolerance was not associated with proximity to agricultural fields, but there were correlations between proximity to agriculture and MoTP. Collectively, these results suggests that amphibian populations possess a wide range of naïve tolerance to common pesticides, with many also being able to rapidly induce increased tolerance. Future research should examine inducible tolerance in a wide variety of other taxa and contaminants to determine the ubiquity of these responses to anthropogenic factors.

Wetland Creation and Reforestation of Legacy Surface Mines in the Central Appalachian Region (USA): A Potential Climate-Adaptation Approach for Pond-Breeding Amphibians?

Habitat restoration and creation within human-altered landscapes can buffer the impacts of climate change on wildlife. The Forestry Reclamation Approach (FRA) is a coal surface mine reclamation practice that enhances reforestation through soil decompaction and the planting of native trees. Recently, wetland creation has been coupled with FRA to increase habitat available for wildlife, including amphibians. Our objective was to evaluate the response of pond-breeding amphibians to the FRA by comparing species occupancy, richness, and abundance across two FRA age-classes (2–5-year and 8–11-year reclaimed forests), traditionally reclaimed sites that were left to naturally regenerate after mining, and in mature, unmined forests in the Monongahela National Forest (West Virginia, USA). We found that species richness and occupancy estimates did not differ across treatment types. Spotted Salamanders (Ambystoma maculatum) and Eastern Newts (Notophthalmus viridescens) had the greatest estimated abundances in wetlands in the older FRA treatment. Additionally, larger wetlands had greater abundances of Eastern Newts, Wood Frogs (Lithobates sylvaticus), and Green Frogs (L. clamitans) compared to smaller wetlands. Our results suggest that wetland creation and reforestation increases the number of breeding sites and promotes microhabitat and microclimate conditions that likely maximize the resilience of pond-breeding amphibians to anticipated climate changes in the study area.

Population origin and heritable effects mediate road salt toxicity and thermal stress in an amphibian

Human impacts on wild populations are numerous and extensive, degrading habitats and causing population declines across taxa. Though these impacts are often studied individually, wild populations typically face suites of stressors acting concomitantly, compromising the fitness of individuals and populations in ways poorly understood and not easily predicted by the effects of any single stressor. Developing understanding of the effects of multiple stressors and their potential interactions remains a critical challenge in environmental biology. Here, we focus on assessing the impacts of two prominent stressors associated with anthropogenic activities that affect many organisms across the planet – elevated salinity (e.g., from road de-icing salt) and temperature (e.g. from climate change). We examined a suite of physiological traits and components of fitness across populations of wood frogs originating from ponds that differ in their proximity to roads and thus their legacy of exposure to pollution from road salt. When experimentally exposed to road salt, wood frogs showed reduced survival (especially those from ponds adjacent to roads), divergent developmental rates, and reduced longevity. Family-level effects mediated these outcomes, but high salinity generally eroded family-level variance. When combined, exposure to both temperature and salt resulted in very low survival, and this effect was strongest in roadside populations. Taken together, these results suggest that temperature is an important stressor capable of exacerbating impacts from a prominent contaminant confronting many freshwater organisms in salinized habitats. More broadly, it appears likely that toxicity might often be underestimated in the absence of multi-stressor approaches.

Accumulation of Microplastics by the Siberian Wood Frog Rana amurensis (Anura, Amphibia) in the Western Baikal Region

Accumulation of Microplastics by the Siberian Wood Frog Rana amurensis (Anura, Amphibia) in the Western Baikal Region

Predator metamorphosis and its consequence for prey risk assessment.

Living with a diverse array of predators provides a significant challenge for prey to learn and retain information about each predator they encounter. Consequently, some prey respond to novel predators because they have previous experience with a perceptually similar predator species, a phenomenon known as generalization of predator recognition. However, it remains unknown whether prey can generalize learned responses across ontogenetic stages of predators. Using wood frog tadpole (Lithobates sylvaticus) prey, we conducted two experiments to explore the extent of predator generalization of different life stages of two different predators: (1) predacious diving beetles (Dytiscus sp.) and (2) tiger salamanders (Ambystoma mavortium). In both experiments, we used chemical alarm cues (i.e., injured conspecific cues) to condition tadpoles to recognize the odor of either the larval or adult stage of the predator as risky. One day later, we tested tadpoles with either the larval or adult predator odor to determine whether they generalized their learned responses to the other life stages of the predator. Tadpoles generalized between larval and adult beetle odors but failed to generalize between larval and adult salamander odors. These results suggest that the odor of some predator species changes during metamorphosis to an extent that reduces their recognisability by prey. This "predator identity reset" increases the number of threats to which prey need to attend.

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.

Occurrence of Lymphedema in Wild-Caught Anurans

Lymphedema is a condition in which the lymph hearts fail to pump fluid from the lymph sacs of anurans and other amphibians. This causes the sacs to fill with fluid and provide the frog with balloon-like swellings or over-all appearance. The condition has previously been connected with various diseases including tadpole edema virus and chytrids. I observed lymphedema in six anuran species (Acris blanchadi*, Anaxyrus fowleri*, Hyla squirrela*, Pseudacris streckeri illinoensis*, Rana sylvatica, Rana sphenocephala* [species with * are species records for lymphedema]).

Cross-species gut microbiota transplantation predictably affects host heat tolerance.

The gut microbiome is known to influence and have regulatory effects in diverse physiological functions of host animals, but only recently has the relationship between host thermal biology and gut microbiota been explored. Here, we examined how early-life manipulations of the gut microbiota in larval amphibians influenced their critical thermal maximum (CTmax) at different acclimation temperatures. We stripped the resident microbiome from egg masses of wild-caught wood frogs (Lithobates sylvaticus) via an antibiotic wash, and then inoculated the eggs with pond water (control), no inoculation, or the intestinal microbiota of another species that has a wider thermal tolerance - green frogs (Lithobates clamitans). We predicted that this cross-species transplant would increase the CTmax of the recipient wood frog larvae relative to the other treatments. In line with this prediction, green frog microbiome-recipient larvae had the highest CTmax while those with no inoculum had the lowest CTmax. Both the microbiome treatment and acclimation temperature significantly influenced the larval gut microbiota communities and α-diversity indices. Green frog microbiome-inoculated larvae were enriched in Rikenellaceae relative to the other treatments, which produce short-chain fatty acids and could contribute to greater energy availability and enhanced heat tolerance. Larvae that received no inoculation had a higher relative abundance of potentially pathogenic Aeromonas spp., which negatively affects host health and performance. Our results are the first to show that cross-species gut microbiota transplants alter heat tolerance in a predictable manner. This finding has repercussions for the conservation of species that are threatened by climate change and demonstrates a need to further explore the mechanisms by which the gut microbiota modulate host thermal tolerance.

Cytotoxicity of the field lampricide 3-trifluoromethyl-4-nitrophenol (TFM) in tadpole cell lines from North American frogs.

The common field lampricide, 3-trifluoromethyl-4-nitrophenol (TFM), is used to treat streams and creeks infested with highly invasive and destructive sea lamprey (Petromyzon marinus) in the tributaries of the Great Lakes. Unfortunately, amphibian deaths have been reported following stream treatments with TFM. Larval amphibians (tadpoles) are more susceptible to TFM toxicity than adult amphibians. The aim of this study was to test the toxicity of TFM in eight new tadpole cell lines from the green frog (Lithobates clamitans), wood frog (Lithobates sylvaticus), and American toad (Anaxyrus americanus). A cell viability bioassay using two fluorescent dyes, Alamar Blue and CFDA-AM, was performed following 24-h and 72-h exposures to a range of TFM concentrations. In general, TFM exposure reduced Alamar Blue fluorescence more rapidly than CFDA-AM fluorescence in tadpole cells, suggesting that Alamar Blue is perhaps a better diagnostic indicator of cell health for acute TFM cytotoxicity. At present, the in vivo 96-h LC50s of TFM are only available for L. clamitans and they correlated well with the in vitro EC50 values for the green frog tadpole cell lines in this study. The eight tadpole cell lines with different relative sensitivities to TFM cytotoxicity could prove to be useful tools in assessing next-generation lampricides in high-throughput bioassays to ensure safety in frogs before their sea lamprey-targeted application in the field.

Warmer temperature overrides the effects of antidepressants on amphibian metamorphosis and behavior.

Climate change can exacerbate the effects of environmental pollutants on aquatic organisms. Pollutants such as human antidepressants released from wastewater treatment plants have been shown to impact life-history traits of amphibians. We exposed tadpoles of the wood frog Lithobates sylvaticus to two temperatures (20°C and 25°C) and two antidepressants (fluoxetine and venlafaxine), and measured timing of metamorphosis, mass at metamorphosis, and two behaviors (startle response and percent motionless). Antidepressants significantly shortened time to metamorphosis at 20°C, but not at 25°C. At 25°C, tadpoles metamorphosed significantly faster than those at 20°C independent of antidepressant exposure. Venlafaxine reduced body mass at 25°C, but not at 20°C. Temperature and antidepressant exposure affected the percent of tadpoles showing a startle response. Tadpoles at 20°C displayed significantly more responses than at 25°C. Exposure to fluoxetine also increased the percent of tadpoles showing a startle response. Venlafaxine reduced the percent of motionless tadpoles at 25°C but not at 20°C. While our results showed that antidepressants can affect the timing of metamorphosis in tadpoles, warmer temperatures overrode these effects and caused a reduction in an important reaction behavior (startle response). Future studies should address how warmer global temperatures may exacerbate or negate the effects of environmental pollutants.

Phenotypically plastic responses to freshwater salinization in larval amphibians: Induced tolerance and induced sensitivity

Contamination of aquatic ecosystems is pervasive around the world and there has been a growing interest in understanding the ecological and evolutionary impacts. For contaminants such as pesticides, researchers are discovering widespread evolution of increased tolerance in target and non-target species and the role of phenotypic plasticity in facilitating this evolution. In contrast, we know much less about the evolution of tolerance in response to the increasing problem of freshwater salinization. In amphibians, recent studies have discovered that some populations from ponds with high salt pollution (from deicing road salts) have evolved higher tolerance. In this study, we examined whether populations of wood frog tadpoles (Rana sylvatica) possess rapid, inducible tolerance to salinity in a manner similar to their inducible tolerance to pesticides. Using newly hatched tadpoles from nine populations, we discovered that eight of the populations were able to alter their tolerance to salt. However, seven of the eight inducible populations experienced a higher sensitivity to salt while the eighth population experienced a higher tolerance to salt. Such inducible responses likely reflect the interplay of salt dynamics in the ponds, combined with the available genetic variation and selection intensity of each pond. This appears to be the first example of inducible salt tolerance in any animal and future studies should examine the generality of the response and how it may affect the evolution of tolerance to the global issue of freshwater salinization.