Rana Sylvatica Research Articles

Amphibian chytrid prevalence on boreal toads in SE Alaska and NW British Columbia: tests of habitat, life stages, and temporal trends.

Tracking and understanding variation in pathogens such as Batrachochytrium dendrobatidis (Bd), the agent of amphibian chytridiomycosis which has caused population declines globally, is a priority for many land managers. However, relatively little sampling of amphibian communities has occurred at high latitudes. We used skin swabs collected during 2005-2017 from boreal toads Anaxyrus boreas (n = 248), in southeast Alaska (USA; primarily in and near Klondike Gold Rush National Historical Park [KLGO]) and northwest British Columbia (Canada) to determine how Bd prevalence varied across life stages, habitat characteristics, local species richness, and time. Across all years, Bd prevalence peaked in June and was >3 times greater for adult toads (37.5%) vs. juveniles and metamorphs (11.2%). Bd prevalence for toads in the KLGO area, where other amphibian species are rare or absent, was highest from river habitats (55.0%), followed by human-modified upland wetlands (32.3%) and natural upland wetlands (12.7%)-the same rank-order these habitats are used for toad breeding. None of the 12 Columbia spotted frogs Rana luteiventris or 2 wood frogs R. sylvatica from the study area tested Bd-positive, although all were from an area of low host density where Bd has not been detected. Prevalence of Bd on toads in the KLGO area decreased during 2005-2015. This trend from a largely single-species system may be encouraging or concerning, depending on how Bd is affecting vital rates, and emphasizes the need to understand effects of pathogens before translating disease prevalence into management actions.

Detecting Amphibians in Agricultural Landscapes Using Environmental DNA Reveals the Importance of Wetland Condition.

Amphibians are declining worldwide, in part because of large-scale degradation of habitat from agriculture and pervasive pathogens. Yet a common North American amphibian, the wood frog (Lithobates sylvaticus), ranges widely and persists in agricultural landscapes. Conventional survey techniques rely on visual encounters and dip-netting efforts, but detectability limits the ability to test for the effects of environmental variables on amphibian habitat suitability. We used environmental DNA to determine the presence of wood frogs and an amphibian pathogen (ranavirus) in Prairie Pothole wetlands and investigated the effects of 32 water quality, wetland habitat, and landscape-level variables on frog presence at sites representing different degrees of agricultural intensity. Several wetland variables influenced wood frog presence, the most influential being those associated with wetland productivity (i.e., nutrients), vegetation buffer width, and proportion of the surrounding landscape that is comprised of other water bodies. Wood frog presence was positively associated with higher dissolved phosphorus (>0.4 mg/L), moderate dissolved nitrogen (0.1-0.2 mg/L), lower chlorophyll a (≤15 µg/L), wider vegetation buffers (≥10 m), and more water on the landscape (≥0.25). These results highlight the effects of environmental factors at multiple scales on the presence of amphibians in this highly modified landscape-namely the importance of maintaining wetland water quality, vegetation buffers, and surrounding habitat heterogeneity. Environ Toxicol Chem 2019;38:2750-2763. © 2019 SETAC.

Landscape genetics reveals unique and shared effects of urbanization for two sympatric pool-breeding amphibians.

Metapopulation‐structured species can be negatively affected when landscape fragmentation impairs connectivity. We investigated the effects of urbanization on genetic diversity and gene flow for two sympatric amphibian species, spotted salamanders (Ambystoma maculatum) and wood frogs (Lithobates sylvaticus), across a large (>35,000 km2) landscape in Maine, USA, containing numerous natural and anthropogenic gradients. Isolation‐by‐distance (IBD) patterns differed between the species. Spotted salamanders showed a linear and relatively high variance relationship between genetic and geographic distances (r = .057, p < .001), whereas wood frogs exhibited a strongly nonlinear and lower variance relationship (r = 0.429, p < .001). Scale dependence analysis of IBD found gene flow has its most predictable influence (strongest IBD correlations) at distances up to 9 km for spotted salamanders and up to 6 km for wood frogs. Estimated effective migration surfaces revealed contrasting patterns of high and low genetic diversity and gene flow between the two species. Population isolation, quantified as the mean IBD residuals for each population, was associated with local urbanization and less genetic diversity in both species. The influence of geographic proximity and urbanization on population connectivity was further supported by distance‐based redundancy analysis and multiple matrix regression with randomization. Resistance surface modeling found interpopulation connectivity to be influenced by developed land cover, light roads, interstates, and topography for both species, plus secondary roads and rivers for wood frogs. Our results highlight the influence of anthropogenic landscape features within the context of natural features and broad spatial genetic patterns, in turn supporting the premise that while urbanization significantly restricts interpopulation connectivity for wood frogs and spotted salamanders, specific landscape elements have unique effects on these two sympatric species.

Indicators of wood frog (Lithobates sylvaticus) condition in a suburbanizing landscape

AbstractWood frogs (Lithobates sylvaticus) are threatened by habitat degradation associated with suburbanization. Suburban development near pools may affect larvae with ramifications for population persistence and vitality. We studied larval development and documented key vernal pool and terrestrial characteristics within 1000 m of 43 pools across the suburban development gradient near Bangor, Maine, USA. Specifically, we examined how survival and morphological characteristics (e.g., developmental phenology, condition, body length, and tail length and shape) varied with characteristics at pool and landscape scales. Secondarily, we explored associations between morphology and survival. Differences in tadpole morphology were associated with suburban land development, hydrology, within‐pool vegetation indicative of light availability at the water's surface, and density of pool‐breeding amphibian egg masses. Across all pools, tadpoles had delayed development and were larger in pools with longer hydroperiods, while tadpoles in suburban pools developed earlier and were larger than those in rural pools with comparable hydroperiods. Tadpoles developed later and had longer tails in pools with greater canopy cover. Morphology profiles also differed between rural and suburban sites and among years. Survival in suburban pools was predicted to be 15 times greater than in rural pools, but across all pools (including those at intermediate intensities of suburbanization), survival was not predicted to vary with either morphology or site characteristics. No strong relationship existed between developmental phenology and any condition or size metric. Because rural and suburban tadpoles responded similarly to within‐pool conditions, our results support the need to maintain natural hydrology and vegetation conditions in pools even in developing areas. Although we detected benefits to tadpoles with increasing suburbanization, suburbanization is well known to extirpate breeding populations; thus, it is likely that wood frog population declines associated with suburbanization are responding to stressors beyond the pool at terrestrial life stages.

A highly invasive chimeric ranavirus can decimate tadpole populations rapidly through multiple transmission pathways

A consequence of genetic recombination can be the evolution of highly virulent pathogen strains. Virulence can manifest through various mechanisms of host–pathogen interaction that facilitate transmission. We discovered a highly virulent chimeric ranavirus in Georgia, USA, estimated transmission parameters using controlled experiments, and developed compartmental disease models to examine potential consequences on tadpoles of a susceptible host species (Lithobates sylvaticus). Our models included three transmission pathways: direct transmission via host contact, environmental transmission via shed virions in water, and transmission via necrophagy of morbid individuals. Unlike previous models, we categorized individuals into multiple stages of infection (susceptible, latency, and infectious), where the probability of disease-induced mortality increased throughout the duration of infection following a gamma distribution with an integer shape parameter. Our simulations showed that accounting for pathogen incubation improved model predictions when compared to survival data from controlled experiments. We found that transmission due to direct contact of tadpoles was the dominant transmission pathway; however, environmental transmission played a larger role as tadpole density increased and the epidemic progressed. Estimated R0 (basic reproduction number) values >570 for all transmission pathways indicate that targeting only one transmission pathway is unlikely to thwart invasion. Additionally, the high invasion potential and diseased-induced mortality associated with this chimeric ranavirus indicate that this pathogen is a substantial threat to amphibian biodiversity in the United States.

Assessment of Sublethal Effects of Neonicotinoid Insecticides on the Life-History Traits of 2 Frog Species.

Neonicotinoid insecticides are used extensively in agriculture and, as a consequence, are now detectable in nearby aquatic environments. Few studies have evaluated the effects of neonicotinoids on amphibians in these aquatic environments. In the present study, we examined the effects of 2 commercial formulations of neonicotinoids (active ingredients clothianidin and thiamethoxam) on survival and life‐history traits of wood frogs (Lithobates sylvaticus) and northern leopard frogs (Lithobates pipiens). We used artificial pond mesocosms to assess the effects of these neonicotinoids, at nominal concentrations of 2.5 and 250 µg/L, on amphibian larval development through metamorphosis. We found no differences between controls and neonicotinoid exposure for any of the endpoints assessed for either wood frogs or leopard frogs. The present study suggests that concentrations meeting or exceeding observed levels of clothianidin and thiamethoxam in surface waters will not directly affect metamorphosis in 2 amphibians. Environ Toxicol Chem 2019;38:1967–1977. © 2019 Her Majesty the Queen in Right of Canada. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.

Characterization of ice recrystallization inhibition activity in the novel freeze-responsive protein Fr10 from freeze-tolerant wood frogs, Rana sylvatica.

Fr10 is a secreted freeze-responsive protein found in the wood frog (Rana sylvatica). This protein has gained notable research attention for its highly dynamic expression in response to seasonal freezing stress, while its over-expression has been documented to enhance freeze tolerance in cold-susceptible cultured cells. This study further characterizes the properties of this novel protein with regards to thermal stability and ice recrystallization inhibition (i.e. IRI) activity. Thermal stability was assessed using differential scanning fluorimetry, with an experimental Tm value of 50.8 ± 0.1 °C. Potential IRI activity of Fr10 was evaluated using a recently developed nanoparticle-based colorimetric assay, where Fr10 displayed the ability to prevent freeze-induced aggregation of gold nanoparticles. Based upon this assay, Fr10 protein appeared to have a low level of IRI activity and it was therefore predicted that one of Fr10's biological functions may be to inhibit ice crystal growth via recrystallization. A SPLAT cooling assay was then employed to directly characterize the IRI properties of Fr10 and provide further insight into this hypothesis. In the presence of 30 μM of Fr10, a 40% reduction in the mean grain size of ice crystals relative to the control samples was observed, thus introducing the possibility of Fr10 to inhibit ice recrystallization. Collectively, the results from this study provide new insight into the potential of further exploring the potential of this vertebrate freeze-responsive protein in cryoprotection.

Replicated Landscape Genomics Identifies Evidence of Local Adaptation to Urbanization in Wood Frogs.

Native species that persist in urban environments may benefit from local adaptation to novel selection factors. We used double-digest restriction-side associated DNA (RAD) sequencing to evaluate shifts in genome-wide genetic diversity and investigate the presence of parallel evolution associated with urban-specific selection factors in wood frogs (Lithobates sylvaticus). Our replicated paired study design involved 12 individuals from each of 4 rural and urban populations to improve our confidence that detected signals of selection are indeed associated with urbanization. Genetic diversity measures were less for urban populations; however, the effect size was small, suggesting little biological consequence. Using an FST outlier approach, we identified 37 of 8344 genotyped single nucleotide polymorphisms with consistent evidence of directional selection across replicates. A genome-wide association study analysis detected modest support for an association between environment type and 12 of the 37 FST outlier loci. Discriminant analysis of principal components using the 37 FST outlier loci produced correct reassignment for 87.5% of rural samples and 93.8% of urban samples. Eighteen of the 37 FST outlier loci mapped to the American bullfrog (Rana [Lithobates] catesbeiana) genome, although none were in coding regions. This evidence of parallel evolution to urban environments provides a powerful example of the ability of urban landscapes to direct evolutionary processes.

In defense of proteins: Chaperones respond to freezing, anoxia, or dehydration stress in tissues of freeze tolerant wood frogs.

Wood frogs (Rana sylvatica LeConte) are the major model for studies of natural freeze tolerance by ectothermic vertebrates. Multiple biochemical adaptations support winter freezing survival but, to date, the protective role of chaperone proteins has received little attention. The present study analyzes responses to freezing, anoxia or dehydration exposures and recovery from these stresses by chaperone proteins in six wood frog organs: Five heat shock proteins (Hsc70, Hsp110, Hsp60, Hsp40, and Hsp10) and two glucose-regulated proteins (Grp78 and Grp94) were assessed. Hsc70 was upregulated in liver, muscle, heart and kidney (1.5-2.0 fold) during freezing and levels of its partner proteins also rose (Hsp110 in three tissues and Hsp40 in four tissues), these responses aligning most closely with comparable responses to anoxia rather than to dehydration. The resident chaperones of the endoplasmic reticulum (Grp78 and Grp94) also rose during freezing in liver and muscle (1.4-1.8 fold) but were suppressed in heart and skin, patterns that generally differed from responses to anoxia or dehydration. Elevated GRPs in liver may support the production and secretion of novel freeze responsive proteins. Increased levels of mitochondrial Hsp60 and Hsp10 (1.5-2.2 fold) occurred in most tissues during freezing and generally mimicked responses to anoxia. Overall, this study indicates that increased levels of chaperone proteins resident in multiple subcellular compartments contribute to stabilizing the cellular proteome during whole body freezing of wood frogs. These responses are probably derived from pre-existing amphibian defenses for stabilizing the proteome under environmental low oxygen or dehydration stresses.

Modeling the distribution of niche space and risk for a freeze‐tolerant ectotherm, Lithobates sylvaticus

AbstractMany animals depend on stable below‐the‐snow (subnivium) conditions to survive winter in seasonally cold regions. Freeze‐tolerant ectotherms may experience increased ice content and/or energy expenditure in suboptimal subnivium conditions, with implications for overwinter survival and body reserves available for spring reproduction. We used a novel mechanistic modeling approach to explore effects of winter climate on the microclimate conditions, energy expenditure, and ice dynamics of the freeze‐tolerant, subnivium‐dwelling wood frog (Lithobates sylvaticus) in the Upper Midwest and Great Lakes Basin region of the United States. We hypothesized that (1) frogs would experience the greatest energy cost to survive winter in southern regions of our study area, where air temperatures are warmer and shallower snow could allow for increased numbers of freeze–thaw cycles, and (2) frogs would be most vulnerable to lethal freezing in the cold, dry northwest portion of our study region. We found that total winter energy expenditure changed little with latitude because the effect of warmer soil temperatures (higher metabolic rates) to the south was offset by a shorter winter duration. Energy expenditures were greatest in the snowbelts of the Great Lakes, characterized by more persistent snow cover and relatively warm soil temperatures. In contrast, highest ice contents occurred in the northwest of the study region where air temperatures were coldest and snow was shallow. Thus, it appears that wood frogs experience a trade‐off between risk of lethal ice content and extensive use of body reserves across geographic space. Simulations showed that interpopulation differences in burrow depth and cryoprotectant concentration can influence risk of lethal ice content and overuse of body reserves prior to spring breeding, and those risks vary in relation to winter climate. Our mechanistic modeling approach is a novel tool for predicting risk and shifting niche space for cold‐adapted and subnivium‐dependent species.

Experimental Evidence That Metamorphosis Alleviates Genomic Conflict.

Whenever genetically correlated traits experience antagonistic selection, an adaptive response in one trait can lead to a maladaptive response in the correlated trait. This is a form of genome-level conflict that can have important evolutionary consequences by impeding organisms from reaching their adaptive optima. Antagonistic selection should be pervasive in organisms with complex life histories because larval and adult life stages specialize in dramatically different environments. Since individuals express larval and adult morphologies from a single genome, genomic conflict across ontogenetic stages should also be prevalent. Using wood frogs as a study system, we measured natural selection on larval and postmetamorphic life stages and estimated genetic correlations among traits. Alternative life stages experienced a mix of both antagonistic and congruent viability selection. The integration between traits changed over the course of metamorphosis, reducing genetic correlations that cause conflict. Our results provide novel experimental evidence that metamorphosis can alleviate genomic conflict by partitioning life-history stages into modules that can more readily respond to selection. These results highlight the adaptive potential of metamorphosis as a means to avoid ecological specialization trade-offs. Moreover, they provide insights into the prevalence and evolutionary maintenance of complex life cycles.

Bioaccumulation of methylmercury in wood frogs and spotted salamanders in Vermont vernal pools.

Mercury (Hg) has accumulated in forested landscapes in the Northeastern U.S., and hotspots with enhanced deposition have been identified throughout the region. Due to a variety of favorable landscape characteristics, including relatively high dissolved organic carbon (DOC), fluctuating water levels, and low pH and dissolved oxygen, vernal pools provide ideal conditions for the conversion of Hg to its more toxic and bioavailable form, methylmercury (MeHg). Yet little is known about the concentrations, speciation, and bioavailability of Hg in vernal pools, or its bioaccumulation in vernal pool fauna and potential export into terrestrial systems. We investigated the role of forest cover type on the bioaccumulation of MeHg in wood frog (Lithobates sylvatica) and spotted salamander (Ambystoma maculatum) eggs, larvae, and adults, and investigated relationships among MeHg and water chemistry (pH, DOC). Water samples from pools located in coniferous stands had greater concentrations of THg and MeHg compared to deciduous pool water, and showed significant positive correlation to DOC (r = 0.683, P < 0.001) and correlated negatively with pH (r = -0.613, P < 0.001). Methylmercury levels in amphibian embryos were similar between the two species (L. sylvatica mean = 5.4 ng/g dw; A. maculatum mean = 3.5 ng/g dw). Concentrations of MeHg increased substantially in larvae, and were significantly greater in A. maculatum (mean = 237.6 ng/g ± 18.5 SE) than L. sylvatica larvae (62.5 ng/g ± 5.7 SE). Forest cover type did not explain variation in MeHg concentration among amphibian embryos or larvae. Methylmercury levels in adult tissue samples were significantly greater in A. maculatum (mean = 79.9 ng/g ± 8.9 SE) compared to L. sylvatica (mean = 47.7 ng/g ± 9.7 SE). This research demonstrates that vernal pools are important hotspots where amphibians bioaccumulate MeHg, which may then be transferred to terrestrial ecosystems. The abundance of amphibian larvae suggests they could be important bioindicators for monitoring MeHg loading and bioavailability.

Multi-tool diagnosis of an outbreak of ranavirosis in amphibian tadpoles in the Canadian boreal forest.

Investigation of mortalities in isolated wild amphibian populations presents diagnostic difficulties that can hinder reaching a definitive diagnosis for the cause of death. Disease can only be diagnosed when pathogen presence (e.g. detection by PCR) is linked to tissue lesions (histopathology) in the host. We report a 2-site outbreak of ranavirosis in wild anuran tadpoles in the boreal forest of Wood Buffalo National Park, Canada, diagnosed by histologic and molecular techniques. Mortalities occurred in wood frog Rana sylvatica tadpoles and boreal chorus frog Pseudacris maculata tadpoles. Lack of mortality in sympatric Canadian toad Bufo (Anaxyrus) hemiophrys tadpoles suggested lower disease susceptibility in this species. In the former 2 species, ranavirosis was diagnosed based on consistent histopathology, immunohistochemistry (IHC), in situ hybridization (ISH), and quantitative PCR results. The most common histopathologic lesion present in wood and boreal chorus frog tadpoles was necrosis of the skin, oral mucosa, renal tubular epithelium, renal hematopoietic tissue, and branchial epithelium. Mild hepatic and pancreatic necrosis and rare intracytoplasmic inclusion bodies in hepatocytes were less common. Skeletal and connective tissues in budding limbs often had multifocal to coalescing necrosis and were intensely positive for ranavirus, with IHC staining even in areas where no obvious necrosis could be observed. Abundant IHC and ISH staining in actively growing tissues support a link between disease emergence and amphibian developmental stage. Our findings provide a definitive diagnosis of ranavirosis in free-living amphibians and highlight the effectiveness of multi-tool approaches to mortality investigation and elucidation of pathogenesis of ranavirosis in wild amphibians.

Forest land-use history affects outcomes of habitat augmentation for amphibian conservation

Historical land use (especially agriculture) has seldom been considered in the development of conservation programs for amphibians, a group in global decline. Habitat augmentation (e.g., wetland creation) is a growing conservation strategy to stem population declines in regions affected by habitat loss, but such actions are often implemented indiscriminately of landscape contexts that may mediate conservation outcomes for species with complex life cycles (i.e., distinct larval, juvenile, and adult stages). We examined the contribution of variation in quantity of larval (aquatic) and quality of juvenile/adult (terrestrial) habitat to the distribution of two pool-breeding and forest-dependent amphibian species in upland forest patches with histories that ranged from former agricultural lands (secondary forests) to never having been deforested (legacy forests). Using area-constrained visual encounter surveys in two consecutive years with contrasting precipitation conditions (extreme drought and extreme rainfall), we estimated forest patch occupancy probabilities of wood frogs (Lithobates sylvaticus) and spotted salamanders (Ambystoma maculatum) at a study area in central New York State, USA, and characterized terrestrial microhabitats in 29 population “neighborhoods” (9.3 ha) that varied in larval habitat availability (0–10 constructed vernal pools) and extent of secondary versus legacy forest. Terrestrial amphibian habitat in secondary forests featured shallower leaf litter, denser understory vegetation, and fewer cover object refuges than in legacy forests. The presence of wood frogs was solely affected by breeding pool densities whereas that of the longer-lived and less dispersive spotted salamanders depended on both quantity of larval habitat and quality of terrestrial habitat, with higher levels of occurrence in legacy forests and, during the drought year, where pool densities were greater. These results suggest that larval habitat augmentation can boost populations of amphibians with complex life cycles, but climate variation and land-use legacies mediate conservation outcomes for long-lived habitat specialists.

Isolation by resistance analysis reveals major barrier effect imposed by the Tsinling Mountains on the Chinese wood frog

AbstractAmphibians are excellent model systems for studying how heterogeneous landscapes drive population differentiation. Though mountain ridges have been widely implicated as barriers to amphibian dispersal, a landscape genetic study by Zhan et al. (BMC Genet, 10, 2009, 17) indicated that the Tsinling Mountains of northwestern China did not significantly impede gene flow of the Chinese wood frog (Rana chensinensis). Using their published genetic data, we re‐assessed the impact of elevation, land cover, and roads on the Chinese wood frog using an isolation by resistance (IBR) approach, which allows for the modeling of complex and heterogeneous landscapes unavailable in classical population genetic analysis. We developed a novel method for optimization of IBR analysis using Circuitscape involving initial permutation‐based optimization of the correlation coefficient obtained through partial Mantel tests, followed by subsequent sensitivity analysis to improve upon the model’s biological relevance. Our results indicated that mountain ridges indeed functioned as barriers to dispersal of Chinese wood frogs, and that previous conclusions were based on methodological limitations. Furthermore, a prominent threshold effect at elevations from 1500–2000 m was evident, with elevations below this range minimally impeding gene flow and higher elevations having a significant barrier effect. We suggest that a combination of expert opinion, sensitivity analysis, and permutation‐based optimization procedures in landscape genetic studies will likely generate models that are both highly explanatory and biologically relevant.

Class A scavenger receptor expression and function in eight novel tadpole cell lines from the green frog (Lithobates clamitans) and the wood frog (Lithobates sylvatica).

A total of eight tadpole cell lines were established from green frogs (Lithobates clamitans) and wood frogs (Lithobates sylvatica). The five green frog cell lines were named GreenTad-HF1, GreenTad-HF2, GreenTad-HF3, GreenTad-HE4, and GreenTad-gill. The three wood frog cell lines were named WoodTad-HE1, WoodTad-Bone, and WoodTad-rpe. DNA barcoding confirmed the cell lines to be from the correct species and the growth characteristics (optimal temperature and FBS requirement) were elucidated. In order to begin studying the innate immune capacity for each cell line, class A scavenger receptor expression and function were next explored. All cell lines expressed genes for at least 3 of the 5 class A scavenger receptor (SR-A) family members, but the gene expression patterns varied between cell lines. MARCO was only expressed in GreenTad-HE4 and WoodTad-Bone, while only GreenTad-HF3 did not express SCARA5 and only WoodTad-rpe did not express SR-AI. Acetylated low density lipoprotein (AcLDL) is a well-defined ligand for SR-As and WoodTad-rpe was the only cell line to which it was unable to bind. In the other seven tadpole cell lines, the SR-A competitive ligands (dextran sulfate, fucoidan, polyinosinic acid) blocked AcLDL binding whereas the SR-A non-competitive ligand counterparts (chondroitin sulfate, fetuin, polycytidylic acid, respectively) did not. Overall, these new eight cell lines can become important tools in the study of innate immunity in general and SR-A functions in particular in green frogs and wood frogs.

Managing the trifecta of disease, climate, and contaminants: Searching for robust choices under multiple sources of uncertainty

Wood frogs, like other amphibian species worldwide, are experiencing population declines due to multiple stressors. In the northeastern United States, wood frog declines are thought to result from a reduction in successful metamorphosis in part due to climate change, disease (specifically ranavirus), and contaminant exposure. The presence of multiple stressors can increase uncertainty in characterizing the main effects of each stressor, as well as understanding the degree to which their effects interact (additively or synergistically) to impact populations. This uncertainty adds inherent challenges to selecting appropriate management actions for conserving populations. Finding solutions that are robust to these uncertainties can improve management amid absent or equivocal knowledge. We used a Bayesian Belief Network (BBN), a quantitative tool that allowed us to evaluate how potential management actions might mitigate the effects of increasingly frequent and severe droughts, ranavirus exposure, and methylmercury on wood frog populations in the northeastern U.S. In our system, successful wood frog recruitment was largely driven by hydroperiod regardless of other stressors. Our modelling indicated that increased hydroperiod lowered the probability of complete metamorphosis failure from 0.6 to 0.37, suggesting that under the conditions tested in the model, pond hydrology is more important for successful recruitment than either methylmercury or ranavirus exposure. As more information becomes available on stressor interactions, model scenarios could be re-evaluated, and management options reconsidered.

Survival, behaviour, and morphology of larval wood frogs, Lithobates sylvaticus, under threat from an exotic crayfish predator, Orconectes virilis

There are numerous examples of species introductions that have caused declines in native populations. In many cases, exotic species are predators of native prey which do not respond correctly to these new and often much different threats. Amphibians, as a group, have been strongly affected by introductions of fish and other aquatic predators such as crayfish. Our goal in this study was to explore the potential impacts of exotic crayfish on the behaviour, morphology, and survival of naive wood frog tadpoles, Lithobates sylvaticus. In mesocosms, groups of tadpoles were exposed to either a native predator (larval beetle or dragonfly nymph) or an exotic crayfish, Orconectes virilis. Tadpoles were the largest following exposure to dragonflies, indicating that dragonflies were selecting smaller tadpoles. Vertical space use of tadpoles was highest in the presence of crayfish, suggesting that tadpoles were learning to avoid crayfish in the benthos. Mortality was highest in the presence of beetles and lowest with crayfish, and hence in isolation, exotic crayfish were poorer predators of wood frog tadpoles. However, half way through the experiment, we replaced each predator with a new predator of either the same species or a different species to assess how the impact of the new predator was affected by experience with the first predator. When crayfish were added following beetles, the mortality due to crayfish increased significantly, possibly due to differences in predator space use and foraging mode.

Effects of 2 Neonicotinoid Insecticides on Blood Cell Profiles and Corticosterone Concentrations of Wood Frogs (Lithobates sylvaticus).

Neonicotinoids are widely used insecticides that are detectable in agricultural waterways. These insecticides are of concern due to their potential impacts on nontarget organisms. Pesticides can affect development of amphibians and suppress the immune system, which could impact disease susceptibility and tolerance. No previous studies on amphibians have examined the effects of these insecticides on differential blood cell proportions or concentrations of corticosterone (a general stress hormone). We investigated the effects of chronic exposure to 2 neonicotinoids, thiamethoxam and clothianidin, on immunometrics of wood frogs (Lithobates sylvaticus). Frogs were exposed to single, chronic treatments of 2.5 or 250 µg/L of clothianidin or thiamethoxam for 7 wk from Gosner stages 25 to 46. The juvenile frogs were then maintained for 3 wk post metamorphosis without exposure to neonicotinoids. We measured water-borne corticosterone twice: at 6 d and 8 wk after exposure in larval and juvenile frogs, respectively. We assessed differential blood cell profiles from juvenile frogs. Corticosterone was significantly lower in tadpoles exposed to 250 µg/L of thiamethoxam compared with other tadpole treatments, but no significant differences in corticosterone concentrations were found in treatments using juvenile frogs. Anemia was detected in all treatments compared with controls with the exception of tadpoles exposed to 2.5 µg/L of clothianidin. Neutrophil-to-leukocyte and neutrophil-to-lymphocyte ratios were elevated in frogs exposed to 250 µg/L of thiamethoxam. Collectively, these results indicate that chronic exposure to neonicotinoids has varied impacts on blood cell profiles and corticosterone concentrations of developing wood frogs, which are indicative of stress. Future studies should investigate whether exposure to neonicotinoids increases susceptibility to infection by parasites in both larval and adult wood frogs. Environ Toxicol Chem 2019;38:1273-1284. © 2019 Crown in the right of Canada. Published by Wiley Periodicals Inc. on behalf of SETAC.

DNA methylation and regulation of DNA methyltransferases in a freeze-tolerant vertebrate.

The wood frog is one of the few freeze-tolerance vertebrates. This is accomplished in part by the accumulation of cryoprotectant glucose, metabolic rate depression, and stress response activation. These may be achieved by mechanisms such as DNA methylation, which is typically associated with transcriptional repression. Hyperglycemia is also associated with modifications to epigenetic profiles, indicating an additional role that the high levels of glucose play in freeze tolerance. We sought to determine whether DNA methylation is affected during freezing exposure, and whether this is due to the wood frog's response to hyperglycemia. We examined global DNA methylation and DNA methyltransferases (DNMTs) in the liver and muscle of frozen and glucose-loaded wood frogs. The results showed that levels of 5-methylcytosine (5mC) increased in the muscle, suggesting elevated DNA methylation during freezing. DNMT activities also decreased in muscle during thawing, glucose loading, and in vitro glucose experiments. Liver DNMT activities were similar to muscle; however, a varied response to DNMT levels and a decrease in 5mC highlight the metabolic role the liver plays during freezing. Glucose was also shown to decrease DNMT activity levels in the wood frog, in vitro, elucidating a potentially novel regulatory mechanism. Together these results suggest an interplay between freeze tolerance and hyperglycemic regulation of DNA methylation.