Intraspecific priority effects in response to egg hatching delay in a pond-breeding salamander.

As reproduction phenologies shift with climate change, populations can experience intraspecific priority effects, wherein early hatching cohorts experience an advantage over late-hatching cohorts, resulting in altered demography. Our study objective was to identify how variation in egg hatching phenology alters intraspecific interactions in small-mouthed salamanders, Ambystoma texanum. We addressed two research questions: (Q1) How are demographic responses altered by variation in the temporal duration of hatching between cohorts, and (Q2) How does the seasonality of hatching delays affect demographic responses? We manipulated hatching phenologies of A. texanum eggs and reared larvae in outdoor mesocosms to metamorphosis. For Q1, hatching delay exhibited non-linear relationships with survival and body size, with the greatest asynchrony in cohort additions resulting in the highest mortality and largest body sizes. For Q2, hatching delay effects were stronger (i.e., survival was lower and body sizes larger) when they occurred later in the season, potentially due to temperature differences that larvae experienced. Overall, our results demonstrate that changes in intraspecific interactions due to phenological shifts can be context-dependent, depending on the strength (i.e., temporal duration) and seasonality of such processes. Identifying context-dependencies of phenological shifts will be critical for predicting changes in organismal demographics with climatic shifts.

Oral administration of GnRH via a cricket vehicle stimulates spermiation in tiger salamanders (Ambystoma tigrinum).

More than 50% of caudates are threatened with extinction and are in need of ex-situ breeding programs to support conservation efforts and species recovery. Unfortunately, many salamander populations under human care can experience reproductive failure, primarily due to missing environmental cues necessary for breeding. Assisted reproductive technologies (ARTs) are a useful suite of techniques for overcoming or bypassing these missing environmental cues to promote breeding. Exogenous hormones are used to stimulate natural breeding behaviors or gamete expression for in-vitro fertilization or biobanking and are typically administered intramuscularly in caudates. While effective, intramuscular injection is risky to perform in smaller-bodied animals, resulting in health and welfare risks. This research investigated the spermiation response to hormone administration through a non-invasive oral bioencapsulation route using the tiger salamander (Ambystoma tigrinum) as a model species. Male salamanders were randomly rotated six weeks apart through four treatments (n = 11 males/treatment) in which animals received a resolving dose of gonadotropin-releasing hormone (GnRH) as follows: (1) Prime-Only (0.0 µg/g); (2) Low (0.25 µg/g); (3) Medium (1.0 µg/g); and (4) High (2.0 µg/g). All males were given a GnRH priming dose (0.25 µg/g) 24 hours prior to the resolving dose. Exogenous hormone was delivered inside of a cricket (Gryllodes sigillatus) that was presented as a food item by tweezers. Sperm samples were collected at 1, 3, 6, 9, 12, and 24 hours after the resolving dose and analyzed for quantity and quality. For all treatments, sperm concentration was produced in an episodic pattern over time. The Prime-Only treatment had a lower (p < 0.05) percent of sperm exhibiting normal morphology compared to treatments utilizing a resolving dose of GnRH. Overall, oral administration of GnRH is a feasible route of inducing spermiation in salamanders, yielding sperm of sufficient quantity and quality for in-vitro fertilization and biobanking efforts.

Isolation of Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea) from the feces of Ambystoma annulatum (Ringed Salamander) in Northwest Arkansas.

The salamander, Ambystoma annulatum, is considered a "species of special concern" in the state of Arkansas, USA, due to its limited geographic range, specialized habitat requirements and low population size. Although metazoan parasites have been documented in this salamander species, neither its native protists nor microbiome have yet been evaluated. This is likely due to the elusive nature and under-sampling of the animal. Here, we initiate the cataloguing of microbial associates with the identification of a new heterlobosean species, Naegleria lustrarea n. sp. (Excavata, Discoba, Heterolobosea), isolated from feces of an adult A. annulatum.

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.

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.

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.

Gene functions of the Ambystoma altamirani skin microbiome vary across space and time but potential antifungal genes are widespread and prevalent.

Amphibian skin microbiomes can play a critical role in host survival against emerging diseases by protecting their host against pathogens. While a plethora of biotic and abiotic factors have been shown to influence the taxonomic diversity of amphibian skin microbiomes it remains unclear whether functional genomic diversity varies in response to temporal and environmental factors. Here we applied a metagenomic approach to evaluate whether seasonality, distinct elevations/sites, and pathogen presence influenced the functional genomic diversity of the A. altamirani skin microbiome. We obtained a gene catalogue of 92 107 nonredundant annotated genes and a set of 50 unique metagenome assembled genomes (MAGs). Our analysis showed that genes linked to general and potential antifungal traits significantly differed across seasons and sampling locations at different elevations. Moreover, we found that the functional genomic diversity of A. altamirani skin microbiome differed between B. dendrobatidis infected and not infected axolotls only during winter, suggesting an interaction between seasonality and pathogen infection. In addition, we identified the presence of genes and biosynthetic gene clusters (BGCs) linked to potential antifungal functions such as biofilm formation, quorum sensing, secretion systems, secondary metabolite biosynthesis, and chitin degradation. Interestingly genes linked to these potential antifungal traits were mainly identified in Burkholderiales and Chitinophagales MAGs. Overall, our results identified functional traits linked to potential antifungal functions in the A. altamirani skin microbiome regardless of variation in the functional diversity across seasons, elevations/sites, and pathogen presence. Our findings suggest that potential antifungal traits found in Burkholderiales and Chitinophagales taxa could be related to the capacity of A. altamirani to survive in the presence of Bd, although further experimental analyses are required to test this hypothesis.

Polyphenism predicts actuarial senescence and lifespan in tiger salamanders.

Actuarial senescence (called 'senescence' hereafter) often shows broad variation at the intraspecific level. Phenotypic plasticity likely plays a central role in among-individual heterogeneity in senescence rate (i.e. the rate of increase in mortality with age), although our knowledge on this subject is still very fragmentary. Polyphenism-the unique sub-type of phenotypic plasticity where several discrete phenotypes are produced by the same genotype-may provide excellent study systems to investigate if and how plasticity affects the rate of senescence in nature. In this study, we investigated whether facultative paedomorphosis influences the rate of senescence in a salamander, Ambystoma mavortium nebulosum. Facultative paedomorphosis, a unique form of polyphenism found in dozens of urodele species worldwide, leads to the production of two discrete, environmentally induced phenotypes: metamorphic and paedomorphic individuals. We leveraged an extensive set of capture-recapture data (8948 individuals, 24 years of monitoring) that were analysed using multistate capture-recapture models and Bayesian age-dependent survival models. Multistate models revealed that paedomorphosis was the most common developmental pathway used by salamanders in our study system. Bayesian age-dependent survival models then showed that paedomorphs have accelerated senescence in both sexes and shorter adult lifespan (in females only) compared to metamorphs. In paedomorphs, senescence rate and adult lifespan also varied among ponds and individuals. Females with good body condition and high lifetime reproductive success had slower senescence and longer lifespan. Late-breeding females also lived longer but showed a senescence rate similar to that of early-breeding females. Moreover, males with good condition had longer lifespan than males with poor body condition, although they had similar senescence rates. In addition, late-breeding males lived longer but, unexpectedly, had higher senescence than early-breeding males. Overall, our work provides one of the few empirical cases suggesting that environmentally cued polyphenism could affect the senescence of a vertebrate in nature, thus providing insights on the ecological and evolutionary consequences of developmental plasticity on ageing.

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.

Environmental RNA can distinguish life stages in amphibian populations.

Applications of environmental DNA (eDNA) analysis methods for biomonitoring have grown exponentially over the last decade and provide a wealth of new information on the distribution of species. However, eDNA methods have limited application for estimating population-level metrics. Environmental RNA (eRNA) has the potential to address ecological questions by gathering population demographic information from environmental media but may be challenging to detect and analyze. We developed gene-specific eRNA assays targeting keratin-associated genes in two focal species, American bullfrogs (Lithobates catesbeianus) and tiger salamanders (Ambystoma mavortium) to answer an important question in amphibian management: whether species detections represent breeding populations versus transitory adults. We performed an extensive laboratory validation with amphibians housed across development stages, where we collected 95 and 127 environmental samples for bullfrogs and salamanders, respectively. Both assays were highly specific to the larval stage and amplified with high sensitivity (90% in bullfrog and 88.4% in tiger salamander samples). We then applied our validated assays to multiple natural systems. When larvae were present, we found 74.1% overall detection in bullfrog field samples and 70.8% and 48.5% overall detection in field samples from ponds with A. macrodactylum and A. californiense larvae, correlating with eDNA detection rates. When only adults were present, we did not detect larvae-specific eRNA in A. macrodactylum ponds, despite high eDNA detection rates. Although much work is ahead for optimizing assay design, sampling and filtering methods, we demonstrate that eRNA can successfully be used to discern life stages with direct application for ecology and conservation management.

Managing invasive hybrids with pond hydroperiod manipulation in an endangered salamander system.

When invasive and endangered native taxa hybridize, the resulting admixture introduces novel conservation challenges. Across a large region of central California, a hybrid swarm consisting of admixed endangered California tiger salamanders (CTS) (Ambystoma californiense) and introduced barred tiger salamanders (BTS) (Ambystoma mavortium) has replaced native populations, threatening the genetic integrity of CTS and the vernal pool systems they inhabit. We employed a large-scale, genomically informed field experiment to test whether shortening breeding pond hydroperiod would favor native CTS genotypes. We constructed 14 large, seminatural ponds to evaluate the effect of hydroperiod duration on larval survival and mass at metamorphosis. We tracked changes in non-native allele frequencies with a 5237-gene exon capture array and employed a combination of custom Bayesian and generalized linear models to quantify the effect of pond duration on salamander fitness. Earlier work on this system showed hybrid superiority under many conditions and suggested that hybrids are favored in human-modified ponds with artificially long hydroperiods. Consistent with these earlier studies, we found overwhelming evidence for hybrid superiority. Very short hydroperiods substantially reduced the mass (1.1-1.5 fold) and survival probability (10-13 fold) of both native and hybrid larvae, confirming that hydroperiod likely exerts a strong selective pressure in the wild. We identified 86 genes, representing 1.8% of 4723 screened loci, that significantly responded to this hydroperiod-driven selection. In contrast to earlier work, under our more natural experimental conditions, native CTS survival and size at metamorphosis were always less than hybrids, suggesting that hydroperiod management alone will not shift selection to favor native larval genotypes. However, shortening pond hydroperiod may limit productivity of hybrid ponds, complementing other strategies to remove hybrids while maintaining vernal pool ecosystems. This study confirms and expands on previous work that highlights the importance of hydroperiod management to control invasive aquatic species.

Manejo de híbridos invasores mediante la manipulación del hidroperiodo de los estanques en el sistema de una salamandra en peligro de extinción Resumen La hibridación entre un taxón nativo en peligro y uno invasor introduce nuevos retos para la conservación. Una plaga híbrida de salamandras tigre de California (STC) (Ambystoma californiense), especie en peligro, y salamandras tigre barradas (STB) (Ambystoma mavortium) introducidas ha reemplazado a las poblaciones nativas en una región amplia del centro de California, lo que amenaza la integridad genética de las STC y el sistema de estanques vernales que habitan. Realizamos un experimento de campo a gran escala y con información genética para probar si la reducción del hidroperiodo reproductivo del estanque favorecería al genotipo de las STC nativas. Construimos 14 estanques seminaturales grandes para analizar el efecto de la duración del hidroperiodo sobre la supervivencia y masa larval durante la metamorfosis. Monitoreamos los cambios en la frecuencia de alelos no nativos con una matriz de captura de exones de 5,237 genes y utilizamos una combinación de modelos lineales generalizados y bayesianos a medida para cuantificar los efectos de la duración del estanque sobre la adaptabilidad de las salamandras. Los primeros trabajos en este sistema mostraron la superioridad híbrida bajo varias condiciones y sugirieron que los híbridos están favorecidos en los estanques con modificaciones antropogénicas e hidroperiodos de larga duración artificial. En coherencia con estos primeros resultados, encontramos evidencia abrumadora de la superioridad híbrida. Los hidroperiodos muy cortos redujeron sustancialmente la masa (1.1­1.5 más veces) y la probabilidad de supervivencia (10­13 más veces) de las larvas nativas e híbridas, lo que confirma que el hidroperiodo probablemente ejerce una fuerte presión selectiva en vida silvestre. Identificamos 86 genes, que representan el 1.8% de los 4,723 loci examinados, que respondieron significativamente a la selección basada en el hidroperiodo. Con las condiciones más naturales de nuestro experimento, y en contraste a nuestros primeros trabajos, la supervivencia y el tamaño de las STC nativas durante la metamorfosis siempre fueron menores a las de los híbridos, lo que sugiere que el manejo del hidroperiodo por sí solo no cambiará la selección a favor de los genotipos larvales nativos. Sin embargo, la reducción del hidroperiodo del estanque puede limitar la productividad de los estanques híbridos y complementar otras estrategias para extirpar a los híbridos mientras que mantiene el ecosistema del estanque vernal. Este estudio confirma y amplía los trabajos anteriores que resaltan la importancia del manejo del hidroperiodo para controlar las especies acuáticas invasoras.

Age- and dose-dependent susceptibility of axolotls (Ambystoma mexicanum) by bath exposure to Ambystoma tigrinum virus (ATV).

Ranaviruses are large, dsDNA viruses that have significant ecological and economic impact on cold-blooded vertebrates. However, our understanding of the viral proteins and subsequent host immune response(s) that impact susceptibility to infection and disease is not clear. The ranavirus Ambystoma tigrinum virus (ATV), originally isolated from the Sonoran tiger salamander (Ambystoma mavortium stebbinsi), is highly pathogenic at low doses of ATV at all tiger salamander life stages and this model has been used to explore the host-pathogen interactions of ATV infection. However, inconsistencies in the availability of laboratory reared larval tiger salamanders required us to look at the well characterized axolotl (A. mexicanum) as a model for ATV infection. Data obtained from five infection experiments over different developmental timepoints suggest that axolotls are susceptible to ATV in an age- and dose-dependent manner. These data support the use of the ATV-axolotl model to further explore the host-pathogen interactions of ranavirus infections.

Forecasting the flooding dynamics of flatwoods salamander breeding wetlands under future climate change scenarios.

Ephemeral wetlands are globally important systems that are regulated by regular cycles of wetting and drying, which are primarily controlled by responses to relatively short-term weather events (e.g., precipitation and evapotranspiration). Climate change is predicted to have significant effects on many ephemeral wetland systems and the organisms that depend on them through altered filling or drying dates that impact hydroperiod. To examine the potential effects of climate change on pine flatwoods wetlands in the southeastern United States, we created statistical models describing wetland hydrologic regime using an approximately 8-year history of water level monitoring and a variety of climate data inputs. We then assessed how hydrology may change in the future by projecting models forward (2025-2100) under six future climate scenarios (three climate models each with two emission scenarios). We used the model results to assess future breeding conditions for the imperiled Reticulated Flatwoods Salamander (Ambystoma bishopi), which breeds in many of the study wetlands. We found that models generally fit the data well and had good predictability across both training and testing data. Across all models and climate scenarios, there was substantial variation in the predicted suitability for flatwoods salamander reproduction. However, wetlands with longer hydroperiods tended to have fewer model iterations that predicted at least five consecutive years of reproductive failure (an important metric for population persistence). Understanding potential future risk to flatwoods salamander populations can be used to guide conservation and management actions for this imperiled species.

Diversity and composition of mixed-ploidy unisexual salamander assemblages reflect the key influence of host species.

Understanding processes that govern and sustain biological diversity is a central goal of community ecology. Unisexual complexes, where reproduction depends on sperm from males of one or more bisexual host species, are rare and the processes driving their diversity and structure remain poorly understood. Unisexual Ambystoma salamanders produce distinct biotypes ('genomotypes') depending on which bisexual species they 'steal' sperm from. This reproductive mode should generate distinct assemblages depending on the locally available bisexual host species. Yet, how availability and relative abundance of multiple bisexual hosts influences composition and diversity of natural unisexual assemblages at local or regional scales remains unknown. We hypothesize that host identity most directly drives local assemblage composition, with host variation associated with increased beta and gamma diversity within unisexuals. We collected genetic samples from Ambystoma salamanders across Pelee Island, Ontario, Canada (2015-2022). Two host species were identified (A. texanum and A. laterale) with nine sites having a single host and one site having both. Unisexual assemblages were grouped into four clusters by similarity, with host identity being a key determinant. Gamma diversity increased as a result of distinct host-specific assemblages forming at different sites on the island (i.e., high beta diversity). Assemblage composition, but not diversity, was correlated with relative host abundance, which may reflect matching niche requirements between host and unisexual forms they produce. Our results demonstrate that diversity and structure of unisexual assemblages are clearly shaped by their host(s) and such systems may serve as models for studying how biotic interactions shape ecological communities.

The role of environmental variation in mediating fitness trade-offs for an amphibian polyphenism.

Fitness trade-offs are a foundation of ecological and evolutionary theory because trade-offs can explain life history variation, phenotypic plasticity, and the existence of polyphenisms. Using a 32-year mark-recapture dataset on lifetime fitness for 1093 adult Arizona tiger salamanders (Ambystoma mavortium nebulosum) from a high elevation, polyphenic population, we evaluated the extent to which two life history morphs (aquatic paedomorphs vs. terrestrial metamorphs) exhibited fitness trade-offs in breeding and body condition with respect to environmental variation (e.g. climate) and internal state-based variables (e.g. age). Both morphs displayed a similar response to higher probabilities of breeding during years of high spring precipitation (i.e. not indicative of a morph-specific fitness trade-off). There were likely no climate-induced fitness trade-offs on breeding state for the two life history morphs because precipitation and water availability are vital to amphibian reproduction. Body condition displayed a contrasting response for the two morphs that was indicative of a climate-induced fitness trade-off. While metamorphs exhibited a positive relationship with summer snowpack conditions, paedomorphs were unaffected. Fitness trade-offs from summer snowpack are likely due to extended hydroperiods in temporary ponds, where metamorphs gain a fitness advantage during the summer growing season by exploiting resources that are unavailable to paeodomorphs. However, paedomorphs appear to have the overwintering fitness advantage because they consistently had higher body condition than metamorphs at the start of the summer growing season. Our results reveal that climate and habitat type (metamorphs as predominately terrestrial, paedomorphs as fully aquatic) interact to confer different advantages for each morph. These results advance our current understanding of fitness trade-offs in this well-studied polyphenic amphibian by integrating climate-based mechanisms. Our conclusions prompt future studies to explore how climatic variation can maintain polyphenisms and promote life history diversity, as well as the implications of climate change for polyphenisms.

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.

Testing whether adrenal steroids mediate phenotypic and physiologic effects of elevated salinity on larval tiger salamanders.

Salinity (sodium chloride, NaCl) from anthropogenic sources is a persistent contaminant that negatively affects freshwater taxa. Amphibians can be susceptible to salinity, but some species are innately or adaptively tolerant. Physiological mechanisms mediating tolerance to salinity are still unclear, but changes in osmoregulatory hormones such as corticosterone (CORT) and aldosterone (ALDO) are prime candidates. We exposed larval barred tiger salamanders (Ambystoma mavortium) to environmentally relevant NaCl treatments (<32-4000 mg·L-1 ) for 24 days to test effects on growth, survival, and waterborne CORT responses. Of those sampled, we also quantified waterborne ALDO from a subset. Using a glucocorticoid antagonist (RU486), we also experimentally suppressed CORT signaling of some larvae to determine if CORT mediates effects of salinity. There were no strong differences in survival among salinity treatments, but salinity reduced dry mass, snout-vent length, and body condition while increasing water content of larvae. High survival and sublethal effects demonstrated that salamanders were physiologically challenged but could tolerate the experimental concentrations. CORT signaling did not attenuate sublethal effects of salinity. Baseline and stress-induced (after an acute stressor, shaking) CORT were not influenced by salinity. ALDO was correlated with baseline CORT, suggesting it could be difficult to decouple the roles of CORT and ALDO. Future studies comparing ALDO and CORT responses of adaptively tolerant and previously unexposed populations could be beneficial to understand the roles of these hormones in tolerance to salinity. Nevertheless, our study enhances our understanding of the roles of corticosteroid hormones in mediating effects of a prominent anthropogenic stressor.

Assessing Leukocyte Profiles of Salamanders and Other Amphibians: A Herpetologists' Guide.

Assessing numbers of leukocytes in salamanders and other amphibians can be useful metrics for understanding health or stress levels of individuals in a population. In this chapter we describe the procedures for obtaining blood samples from amphibians, preparing blood films for microscopy, counting, and identifying cells. We also provide reference values for amphibian leukocytes for use in interpreting leukocyte data. From our assessment of the published and unpublished literature, "non-stressed" salamanders would have a leukocyte profile where 60-70% of cells are lymphocytes, 17-30% are neutrophils, 1-4% are eosinophils, 4-12% are basophils, and 2-6% are monocytes. In Ambystoma spp., the eosinophil abundance can be notably higher (30% of all white blood cells), for reasons unknown. Finally, the neutrophil-lymphocyte ratio of most non-stressed salamanders tends to be between 0.3 and 0.4 (sometimes less), while the ratios of stressed salamanders tend to be over 1.0.

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.

The effects of road salt (NaCl), predation, and competition on the growth and community interactions of spotted salamanders (Ambystoma maculatum) and wood frogs (Lithobates sylvaticus).

Road deicing salts are frequently used in northern regions of the world during the winter and early spring months. As a result, a significant portion of road runoff into surrounding aquatic habitats contains road deicing salts. Previous studies found road salt contaminations in vernal pools that pond-breeding amphibians commonly use, including spotted salamanders (Ambystoma maculatum) and wood frogs (Lithobates sylvaticus). Studies have examined the impact of road salt on both amphibian species, but to our knowledge no previous studies have examined how road salt impacts the interspecific competition between both amphibians. We hypothesized that road salt would negatively impact growth and survivorship of both amphibian species. During the spring and summer of 2017, we conducted an outdoor mesocosm experiment in which we created eight experimental conditions with three main factors: presence/absence of NaCl (1000 mg/L Cl-), presence/absence of interspecific competition between the two amphibian species (A. maculatum and L. sylvaticus), and presence/absence of predatory dragonfly larvae (Family Libellulidae). Our experiment revealed that salt delayed hatching and increased deformity in spotted salamander hatchlings. Additionally, salt reduced salamander survivorship by 62% and frog survivorship by 30%. Wood frog tadpoles and road salt interacted to diminish salamander survivorship a further 80% beyond salt alone, likely through an increase in interspecific competition. Road salt increased the larval period of salamanders and decreased the proportion metamorphosed by the end of the experiment. Dragonfly larvae reduced salamander survivorship by 35%, whereas they increased wood frog tadpole development rates. Dragonfly larvae and salt interacted to alter tadpole denticle size, with salt negating the impact of dragonfly larvae. Thus, we found that salt interfered with aquatic predatory chemical cues. Overall, the results of this study suggest that management strategies should be implemented in order to reduce the impact of road salts on freshwater aquatic ecosystems.