Ambystoma Maculatum Research Articles

Micromanagement: conditions influencing antipathogen function of the skin microbiome in spotted salamanders, Ambystoma maculatum

The emerging fungal pathogen Batrachochytrium salamandrivorans (Bsal) threatens the diversity of amphibians, particularly in North America where it is projected to invade. Amphibian skin defenses include a mucosal layer containing microorganisms that can potentially modulate host response to pathogens such as Bsal. In this study, we focused on the composition of the skin microbiome across life stages of spotted salamanders (Ambystoma maculatum). We also evaluated the stress hormone corticosterone and skin microbiome response to inoculations with Bsal and probiotics at both the larval and juvenile developmental stages, and the response to different environmental conditions. Results indicated that both bacterial and fungal communities found on the skin significantly differed in structure and diversity between life stages of A. maculatum. Exposure to three different probiotics (Bacillus thuringiensis, Chryseobacterium rhizoplanae, and Penicillium sp.) and Bsal evoked shifts in the microbiome of larvae and juveniles, and the metabolite profile of the larval mucosal layer of A. maculatum. Despite changes in the microbiome, all tested probiotics and Bsal were unable to persist on the skin. Larval bacterial microbiomes shifted in response to Bsal and B. thuringiensis with no significant impacts on antifungal function or bacteria richness, however fungi strongly responded to Bsal and B. thuringiensis application. This indicates that developmental shifts in the microbiome can be initiated by microbial applications such as B. thuringiensis, a widely used mosquito larvicide. Overall, experimental results indicate that life stage, growth and development, and environmental conditions appeared to be the main factors driving changes in the amphibian skin microbiome and potential anti-Batrachochytrium function.

Frequency of adult amphibian abnormalities and consequences for fitness-related traits in an uncontaminated environment

Morphological abnormalities in amphibians are commonly associated with anthropogenic activity, although little baseline information on the prevalence of abnormalities in uncontaminated environments exist. Here, we leverage a 12-year study of spotted salamanders (Ambystoma maculatum Shaw, 1802) in an uncontaminated ecosystem in Algonquin Provincial Park, Canada, to estimate abnormality rates and explore how abnormalities affect fitness-related traits. Annual abnormality rates estimated from drift fence data, ranged from 4.3% to 5.8% of individuals sampled. Abnormality rates from aquatic trapping between 2008–2019 varied from 1.2% to 16.7%, where temporal increases in abnormality rate were observed. We also performed a targeted but systematic literature survey and found that Caudata exhibited a slightly higher abnormality prevalence than Anura, and that the baseline frequency of abnormalities described at our drift fence site is slightly lower than rates reported in the literature (8.1%, 95% CI, 4.76–13.3%). Salamanders with abnormalities exhibited a slightly, but not significantly, higher body condition and a significantly earlier arrival date at the breeding site, both of which are traits typically associated with high-fitness individuals. Our study suggests that abnormalities have detectable phenotypic consequences and underlines the need for temporal sampling efforts to provide ranges of baseline abnormality rates, rather than a point estimate.

Ups and downs of fossorial life: migration restlessness and geotaxis may explain overwintering emergence in the spotted salamander.

To decide whether to remain underground or to emerge from overwintering, fossorial ectotherms simultaneously process environmental, gravitational and circannual migratory cues. Here, we provide an experimental framework to study the behaviour of fossorial ectotherms during soil temperature inversion - a phenomenon that marks the transition between winter and spring - based on three non-mutually exclusive hypotheses (thermoregulation, negative geotaxis and migration restlessness). Using a vertical thermal gradient, we evaluated how temperature selection (Tsel), activity and vertical position selection differed under simulated soil temperature inversion (contrasting the active versus overwintering thermal gradients) in the spotted salamander (Ambystoma maculatum). Salamanders had different Tsel and activity levels between gradients, but selected similar heights regardless of thermal gradient orientation. Negative geotaxis may explain responses to changes in vertical thermal gradient orientation, with migratory restlessness contributing to differences in activity levels. Ultimately, our work should benefit those who aim to better understand the biology of fossorial ectotherms.

Seasonal variation of behavioural thermoregulation in a fossorial salamander (Ambystoma maculatum).

Temperature seasonality plays a pivotal role in shaping the thermal biology of ectotherms. However, we still have a limited understanding of how ectotherms maintain thermal balance in the face of varying temperatures, especially in fossorial species. Due to thermal buffering underground, thermal ecology theory predicts relaxed selection pressure over thermoregulation in fossorial ectotherms. As a result, fossorial ectotherms typically show low thermoregulatory precision and low evidence of thermotactic behaviours in laboratory thermal gradients. Here, we evaluated how temperature selection (T sel) and associated behaviours differed between seasons in a fossorial amphibian, the spotted salamander (Ambystoma maculatum). By comparing thermoregulatory parameters between the active and overwintering seasons, we show that A. maculatum engages in active behavioural thermoregulation despite being fossorial. In both seasons, T sel was consistently offset higher than acclimatization temperatures. Thermoregulation differed between seasons, with salamanders having higher T sel and showing greater evidence of thermophilic behaviours in the active compared with the overwintering season. Additionally, our work lends support to experimental assumptions commonly made but seldom tested in thermal biology studies. Ultimately, our study demonstrates that the combination of careful behavioural and thermal biology measurements is a necessary step to better understand the mechanisms that underlie body temperature control in amphibians.

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

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.

Trait variation in patchy landscapes: Morphology of spotted salamanders (Ambystoma maculatum) varies more within ponds than between ponds.

The influence of intraspecific trait variation on species interactions makes trait-based approaches critical to understanding eco-evolutionary processes. Because species occupy habitats that are patchily distributed in space, species interactions are influenced not just by the degree of intraspecific trait variation but also the relative proportion of trait variation that occurs within- versus between-patches. Advancement in trait-based ecology hinges on understanding how trait variation is distributed within and between habitat patches across the landscape. We sampled larval spotted salamanders (Ambystoma maculatum) across six spatially discrete ponds to quantify within- and between-pond variation in mass, length, and various metrics associated with their relationship (scaling, body condition, shape). Across all traits, within-pond variation contributed more to total observed morphological variation than between-pond variation. Between-pond variation was not negligible, however, and explained 20-41% of total observed variation in measured traits. Between-pond variation was more pronounced in salamander tail morphology compared to head or body morphology, suggesting that pond-level factors more strongly influence tails than other body parts. We also observed differences in mass-length relationships across ponds, both in terms of scaling slopes and intercepts, though differences in the intercepts were much stronger. Preliminary evidence hinted that newly constructed ponds were a driver of the observed differences in mass-length relationships and morphometrics. General pond-level difference in salamander trait covariation suggest that allometric scaling of morphological traits is context dependent in patchy landscapes. Effects of pond age offer the hypothesis that habitat restoration through pond construction is a driver of variation in trait scaling, which managers may leverage to bolster trait diversity.

Ecosystem effects of intraspecific variation in a colour polymorphic amphibian.

An emerging consensus suggests that evolved intraspecific variation can be ecologically important. However, evidence that evolved trait variation within vertebrates can influence fundamental ecosystem-level processes remains sparse. In this study, we sought to assess the potential for evolved variation in the spotted salamander (Ambystoma maculatum) to affect aquatic ecosystem properties. Spotted salamanders exhibit a conspicuous polymorphism in the colour of jelly encasing their eggs-some females produce clear jelly, while others produce white jelly. Although the functional significance of jelly colour variation remains largely speculative, evidence for differences in fecundity and the morphology of larvae suggests that the colour morphs might differ in the strength or identity of ecological effects. Here, we assessed the potential for frequency variation in spotted salamander colour morphs to influence fundamental physiochemical and ecosystem properties-dissolved organic carbon, conductivity, acidity and primary production-with a mesocosm experiment. By manipulating colour morph frequency across a range of larval densities, we were able to demonstrate that larva density and colour morph variation were ecologically relevant: population density reduced dissolved organic carbon and increased primary production while mesocosms stocked with white morph larvae tended to have higher dissolved organic carbon and conductivity. Thus, while an adaptive significance of jelly coloration remains hypothetical, our results show that colour morphs differentially influence key ecosystem properties-dissolved organic carbon and conductivity.

Validation of Salamander Dermal Mucus Swabs as a Novel, Nonlethal Approach for Amphibian Metabolomics and Glutathione Analysis Following Pesticide Exposure.

Evaluating biomarkers of stress in amphibians is critical to conservation, yet current techniques are often destructive and/or time-consuming, which limits ease of use. In the present study, we validate the use of dermal swabs in spotted salamanders (Ambystoma maculatum) for biochemical profiling, as well as glutathione (GSH) stress response following pesticide exposure. Thirty-three purchased spotted salamanders were acclimated to laboratory conditions at Washington College (Chestertown, MD, USA) for 4 weeks. Following acclimation, salamanders were randomly sorted into three groups for an 8-h pesticide exposure on soil: control with no pesticide, 2,4-dichlorophenoxyacetic acid (2,4-D), or chlorpyrifos. Before and after exposure, mucus samples were obtained by gently rubbing a polyester-tipped swab 50 times across the ventral and dorsal surfaces. Salamanders were humanely euthanized and dissected to remove the brain for acetylcholinesterase and liver for GSH and hepatic metabolome analyses, and a whole-body tissue homogenate was used for pesticide quantification. Levels of GSH were present in lower quantities on dermal swabs relative to liver tissues for chlorpyrifos, 2,4-D, and control treatments. However, 2,4-D exposures demonstrated a large effect size increase for GSH levels in livers (Cohen's d = 0.925, p = 0.036). Other GSH increases were statistically insignificant, and effect sizes were characterized as small for 2,4-D mucosal swabs (d = 0.36), medium for chlorpyrifos mucosal swabs (d = 0.713), and negligible for chlorpyrifos liver levels (d = 0.012). The metabolomics analyses indicated that the urea cycle, alanine, and glutamate metabolism biological pathways were perturbed by both sets of pesticide exposures. Obtaining mucus samples through dermal swabbing in amphibians is a viable technique for evaluating health in these imperiled taxa. Environ Toxicol Chem 2024;43:1126-1137. © 2024 SETAC.

Long-Term Decline of the Spotted Salamander (Ambystoma maculatum) in an Undeveloped Landscape

Long-Term Decline of the Spotted Salamander (Ambystoma maculatum) in an Undeveloped Landscape

Taxonomy and nomenclature of Oophila amblystomatis (Chlorophyceae, Chlamydomonadales).

The unicellular green alga Oophila amblystomatis was named by Lambert in 1905 based upon its association with egg masses of the spotted salamander Ambystoma maculatum. We collected algal cells from Lambert's original egg capsule preparations that were contributed to Phycotheca Boreali-Americana (PBA) in 1905 and subjected them to DNA extraction and PCR with O. amblystomatis-specific 18S rRNA gene primers. DNA amplified from these preparations was cloned and nine clones were sequenced. Along with representative sequences from the Oophila clade and Chlorophyceae, a phylogenetic tree was inferred. Seven sequences clustered within the Oophila clade and two clustered with Chlamydomonas moewusii, which is included in a sister clade to Oophila. By sequencing algal material from the egg capsules of representative type material we can unambiguously characterize O. amblystomatis and define a monophyletic clade centered on this type material. Accordingly, we reject a recent proposal that this species be transferred to Chlorococcum.

Local scale population risk determines the adaptive responses of larval salamanders to predator kairomones

Abstract Organisms can exhibit plasticity in phenotypic responses to environmental stimuli but the response can vary based on the evolutionary history of a population. Numerous studies have found that prey will express adaptive responses when subjected to various predators in experimental settings. For species with a large distribution, such as Ambystoma maculatum (spotted salamander), it is not surprising that phenotypic variance can be high across their range. This variance has been hypothesized to be because of different predator regimes of populations. While A. maculatum preferentially oviposit eggs in low-risk ponds (temporary fishless) to decrease egg and larval mortality, Missouri populations still use high-risk ponds (permanent with fish) for reproduction. Using a series of experiments, we investigated how A. maculatum population risk influenced adaptive responses to both native and novel predator kairomones. For natural predators, we used larval A. opacum (marbled salamanders) and Lepomis macrochirus (bluegill) and for novel predators we used Siren intermedia (lesser siren). We found that larval salamanders generally responded with no differences in morphological traits to all three predators. However, head width was larger for low-risk populations. One hypothesized benefit of larger heads (increased foraging efficiency) was not fully supported. All larval salamanders also increased refuge use with predators but this decreased over time. Our results suggest that predation risk of a population may influence the degree of phenotypic expression in response to larval predators. Overall, local adaptation may dictate the ability of prey to respond to environmental conditions within a life stage.

Egg predators improve the hatching success of salamander eggs.

A common challenge that oviparous animals face is securing survivorship during the vulnerable embryonic stage. One of the parental investment strategies to improve survivorship is providing physical structures to protect the embryos. In amphibians, there is a notable diversity in jelly-layer structures surrounding eggs. Previous studies show that these jelly layers provide eggs with protection against egg predators, egg pathogens, and desiccation. However, few studies examined the cost-benefit relationship of the jelly-layer structures. By using the predator-prey interaction between wood frog (Lithobates sylvaticus) tadpoles and spotted salamander (Ambystoma maculatum) eggs as a model system, we tested three hypotheses: (1) having the outer jelly layers would be costly to the embryos, (2) the relative benefit of the structural egg defense would become apparent and increase as the intensity of egg predation increases, and (3) a certain degree of predation would increase the hatching success of salamander embryos by mechanically thinning the thick outer jelly layers and increasing oxygen diffusion throughout an egg mass. To test these hypotheses, we conducted a factorial experiment in which we crossed four egg-predation levels with two jelly-layer conditions, intact or removed. We found that the jelly layers were essential in protecting spotted salamander embryos from wood frog tadpoles but that the associated cost was apparent in no-predation treatments. The differential survivorship between intact eggs and eggs without jelly layers showed that the fitness advantage of jelly layers increased as the level of predation increased. Finally, the hatching success of intact egg masses was highest under the high predation conditions. These results imply that the evolution of the jelly-layer thickness occurred under constant egg-predation pressure. Given this predator-prey coevolution, egg predators may play a critical role in improving the hatching success of salamander embryos under certain conditions.

Environmental drivers of juvenile dispersal and adult non-breeding movements in Ambystoma salamanders

Understanding the environmental drivers of species’ dispersal and migration patterns is needed to accurately predict climate change impacts on populations. For pond-breeding amphibians, adult movements associated with the breeding period are well studied but major gaps exist in our knowledge of the drivers of adult and juvenile non-breeding movements. Here, we assess environmental drivers of adult and juvenile Ambystoma maculatum (Shaw, 1802), Ambystoma jeffersonianum (Green, 1827), and their unisexual dependants’ ( Ambystoma laterale–jeffersonianum (Uzzell, 1964)) summer and fall non-breeding movements using data from pitfall trapping and radio telemetry tracking. We used generalized linear models to assess the influence of precipitation, minimum temperature, day of year, and days since last precipitation on salamander movements. Juvenile summer dispersal movements were related to days since last precipitation in Jefferson Salamander complex individuals and 24 h precipitation in Spotted Salamanders. Adult and juvenile fall movements were driven by minimum temperature, 24 h precipitation, and days since last precipitation, and the effect of these environmental factors varied slightly between species and between Jefferson bisexuals and unisexuals. Our work indicates that changes in both temperature and precipitation will likely impact non-breeding dispersal and migration in these species and, overall, improves our understanding of ecological patterns throughout their entire life cycle.

Salinity Characteristics of Rocky Cliff Pools Used as Breeding Habitat by Spotted Salamanders (Ambystoma maculatum)

Salinity Characteristics of Rocky Cliff Pools Used as Breeding Habitat by Spotted Salamanders (Ambystoma maculatum)

In Vitro Investigation of the Antibacterial Activity of Salamander Skin Peptides.

Given the current and future costs of antibiotic-resistant bacteria to human health and economic productivity, there is an urgent need to develop new antimicrobial compounds. Antimicrobial peptides are a promising alternative to conventional antibiotics and other antimicrobials. Amphibian skin is a rich source of bioactive compounds, but the antibacterial properties of salamander skin peptides have been neglected. Here, we examined the in vitro ability of skin peptides from 9 species of salamander representing 6 salamander families to inhibit the growth of ESKAPE pathogens, which are bacteria that have developed resistance to conventional antibiotics. We also examined whether the skin peptides caused lysis of human red blood cells. Skin peptides from Amphiuma tridactylum had the greatest antimicrobial properties, completely inhibiting the growth of all bacterial strains except for Enterococcus faecium. Likewise, skin peptides from Cryptobranchus alleganiensis completely inhibited the growth of several of the bacterial strains. In contrast, skin peptide mixtures from Ambystoma maculatum, Desmognathus fuscus, Eurycea bislineata, E. longicauda, Necturus beyeri, N. maculosus, and Siren intermedia did not completely inhibit bacterial growth even at the highest concentrations. Finally, none of the skin peptide mixtures caused lysis of human red blood cells. Together, we demonstrate that salamander skin produces peptides with potent antibacterial properties. It remains to elucidate the peptide sequences and their antibacterial mechanisms.

Bacterial Diversity in Egg Capsular Fluid of the Spotted Salamander Ambystoma maculatum Decreases with Embryonic Development.

Egg capsules within egg masses of the spotted salamander Ambystoma maculatum host a symbiosis with the unicellular green alga Oophila amblystomatis. However, this alga is not the only microbe to inhabit those capsules, and the significance of these additional taxa for the symbiosis is unknown. Spatial and temporal patterns of bacterial diversity in egg capsules of A. maculatum have recently begun to be characterized, but patterns of bacterial diversity as a function of embryonic development are unknown. We sampled fluid from individual capsules in egg masses over a large range of host embryonic development in 2019 and 2020. We used 16S rRNA gene amplicon sequencing to examine how diversity and relative abundance of bacteria changed with embryonic development. In general, bacterial diversity decreased as embryos developed; significant differences were observed (depending on the metric) by embryonic development, pond, and year, and there were interaction effects. The function of bacteria in what is thought of as a bipartite symbiosis calls for further research.

PREVALENCE OF RANAVIRUS IN SPOTTED SALAMANDER (AMBYSTOMA MACULATUM) LARVAE FROM CREATED VERNAL POOLS IN WEST VIRGINIA, USA.

Ranavirosis is a disease of high concern for amphibians due to widespread documentation of its lethal and sublethal impacts and its high transmission potential across populations and species. We investigated whether spotted salamander (Ambystoma maculatum) ranavirus prevalence and viral load were associated with habitat characteristics, genetic diversity, corticosterone levels, and body size. In 2015 and 2016, we sampled 34 recently created vernal pools in the Monongahela National Forest, West Virginia, USA. We collected tail clippings from 1,128 spotted salamander larvae and waterborne hormone samples from 436 of those larvae, along with eight environmental characteristics of the pools. Over the 2-yr period, we detected ranavirus in 62% of pools, with prevalence ranging from 0% to 63% (mean, 7.68%). Spotted salamander size was positively correlated with ranavirus presence and viral load; however, we did not find associations between ranavirus prevalence or viral load and habitat characteristics, spotted salamander genetic diversity, relatedness, effective number of breeders, or corticosterone levels. The widespread occurrence of ranavirus in the vernal pools illustrates the potential for rapid natural introduction of the pathogen to created wetlands. Managers could consider monitoring local distributions of ranavirus before creation of new vernal pools to guide strategic placement of the wetlands to minimize occurrence and prevalence of this pathogen.

Pool Age and Local Habitat Are Associated with Effective Number of Breeders in Spotted Salamander Populations Colonizing Created Vernal Pools

Population genetics can reveal whether colonization of created habitats has been successful and inform future strategies for habitat creation. We used genetic analysis to investigate spotted salamander (Ambystoma maculatum) colonization of created vernal pools and explored the impact of habitat characteristics on the genetic diversity and connectivity of the pools. Our first objective was to examine genetic structure, differentiation, diversity, and potential for a founder effect. Our second objective was to determine if habitat characteristics were associated with effective number of breeders, relatedness, or genetic diversity. We sampled spotted salamander larvae in 31 created vernal pools (1–5 years old) in Monongahela National Forest (WV) in May and June 2015 and 2016. The youngest pools exhibited genetic differentiation, a founder effect, and low effective number of breeders. Effective number of breeders was positively associated with pool age, vegetation cover, pool diameter, and sample size. Vegetation cover was also negatively associated with relatedness. Genetic diversity did not have strong environmental predictors. Our results indicated the effective number of breeders increased and genetic differentiation decreased within 4–5 years of pool creation, a sign of rapid colonization and potential population establishment. Our research also showed that higher vegetative cover within the pool and larger pool diameters could impact habitat quality and should be incorporated into future pool creation.

Multistate model to estimate sex‐specific dispersal rates and distances for a wetland‐breeding amphibian population

AbstractHow animals move across space and time in heterogeneous landscapes has important implications for the conservation of imperiled and dispersal‐limited taxa, such as amphibians. Wetland‐breeding amphibians are thought to exhibit strong site fidelity, but there is growing evidence that many species are more vagile than previously assumed. Few studies have quantified breeding dispersal probabilities or distances while also accounting for observational uncertainty, which means that resultant estimates could be biased or confounded with sampling intensity and other demographic rates (e.g., site‐ and time‐specific detection and survival probabilities). Here, we analyze data from a 6‐year capture–mark–recapture study on adult spotted salamanders (Ambystoma maculatum) conducted at 12 wetlands in central Pennsylvania to estimate population dynamics and population structure at multiple scales. We used a multistate, hidden Markov estimator to quantify sex‐specific site fidelity and breeding dispersal as a function of Euclidean distance between wetlands while accounting for imperfect detection. We estimated short‐timescale movements (i.e., those that occur within a single breeding season) and longer timescale movements (i.e., those that occur among breeding seasons) to determine if dispersal rates and distances differed. We found that interannual site fidelity of males varied among wetlands and was positively associated with population density. Females exhibited higher interannual site fidelity and dispersed further than males between breeding seasons. Within breeding seasons, we found that up to 6% of males dispersed to a new wetland each day. Our study is the first to directly compare amphibian breeding dispersal probabilities and distances at multiple scales and provides a robust framework for improving inference on the spatial and temporal patterns of amphibian movement.