Notophthalmus Viridescens Research Articles

Getting to the Root of the Newt: Larval Occurrence Patterns of a Newly Discovered Eastern Newt Population in Southeast Kansas

Getting to the Root of the Newt: Larval Occurrence Patterns of a Newly Discovered Eastern Newt Population in Southeast Kansas

Macrophages modulate fibrosis during newt lens regeneration

BackgroundPrevious studies have suggested that macrophages are present during lens regeneration in newts, but their role in the process is yet to be elucidated.MethodsHere we generated a transgenic reporter line using the newt, Pleurodeles waltl, that traces macrophages during lens regeneration. Furthermore, we assessed early changes in gene expression during lens regeneration using two newt species, Notophthalmus viridescens and Pleurodeles waltl. Finally, we used clodronate liposomes to deplete macrophages during lens regeneration in both species and tested the effect of a subsequent secondary injury after macrophage recovery.ResultsMacrophage depletion abrogated lens regeneration, induced the formation of scar-like tissue, led to inflammation, decreased iris pigment epithelial cell (iPEC) proliferation, and increased rates of apoptosis in the eye. Some of these phenotypes persisted throughout the last observation period of 100 days and could be attenuated by exogenous FGF2 administration. A distinct transcript profile encoding acute inflammatory effectors was established for the dorsal iris. Reinjury of the newt eye alleviated the effects of macrophage depletion, including the resolution of scar-like tissue, and re-initiated the regeneration process.ConclusionsTogether, our findings highlight the importance of macrophages for facilitating a pro-regenerative environment in the newt eye by regulating fibrotic responses, modulating the overall inflammatory landscape, and maintaining the proper balance of early proliferation and late apoptosis of the iPECs.

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.

Predicted Occurrence of Eastern Newts (Notophthalmus viridescens viridescens) across the Northeastern United States

Predicted Occurrence of Eastern Newts (Notophthalmus viridescens viridescens) across the Northeastern United States

A Pilot Study Investigating Plasma Protein Electrophoresis in One Anuran and Six Urodelan Species.

As threats to amphibian health increase, there is a growing need for diagnostic tools to assess and monitor their health status. Plasma protein electrophoresis has proven to be useful in other nonmammalian species. It enables quantification of protein fractions in plasma that may be altered in various disease processes, and is therefore useful in narrowing down differential diagnoses and detecting inflammation, in combination with other modalities such as biochemical and hematologic testing. The amphibian electrophoretogram must be defined before baseline reference intervals are obtained across species. Agarose gel electrophoresis was performed on plasma samples collected from presumed clinically normal individuals of one anuran and six urodelans: Osteopilus septentrionalis (n=2), Gyrinophilus porphyriticus (n=1), Notophthalmus viridescens (n=1), Eurycea guttolineata (n=2), Amphiuma tridactylum (n=2), Cryptobranchus alleganiensis (n=5), and Siren lacertina (n=6). The electrophoretograms varied in number of fractions between each species; however, the number of fractions was consistent within a species. An albumin migrating fraction was consistently observed in all species. A prealbumin migrating fraction was identified in species that primarily use organs other than skin for respiration. This study provides preliminary examples of a normal plasma protein electrophoretogram for seven amphibian species. Further studies quantifying reference intervals and identification of protein fractions will help establish protein electrophoresis as a useful tool in amphibian health investigations.

Effects of exogenous elevation of corticosterone on immunity and the skin microbiome of eastern newts (Notophthalmus viridescens).

The amphibian chytrid fungus, Batrachochytrium salamandrivorans (Bsal) threatens salamander biodiversity. The factors underlying Bsal susceptibility may include glucocorticoid hormones (GCs). The effects of GCs on immunity and disease susceptibility are well studied in mammals, but less is known in other groups, including salamanders. We used Notophthalmus viridescens (eastern newts) to test the hypothesis that GCs modulate salamander immunity. We first determined the dose required to elevate corticosterone (CORT; primary GC in amphibians) to physiologically relevant levels. We then measured immunity (neutrophil lymphocyte ratios, plasma bacterial killing ability (BKA), skin microbiome, splenocytes, melanomacrophage centres (MMCs)) and overall health in newts following treatment with CORT or an oil vehicle control. Treatments were repeated for a short (two treatments over 5 days) or long (18 treatments over 26 days) time period. Contrary to our predictions, most immune and health parameters were similar for CORT and oil-treated newts. Surprisingly, differences in BKA, skin microbiome and MMCs were observed between newts subjected to short- and long-term treatments, regardless of treatment type (CORT, oil vehicle). Taken together, CORT does not appear to be a major factor contributing to immunity in eastern newts, although more studies examining additional immune factors are necessary. This article is part of the theme issue 'Amphibian immunity: stress, disease and ecoimmunology'.

Community Structure of Terrestrial Vertebrates in a Restored Meadow Habitat in Pennsylvania: Assessing the Potential Prey Base for Eastern Massasauga Rattlesnakes

We sampled the vertebrate community of a 20-ha conservation easement in Venango County, PA, from April through August 2021. The restoration and management efforts on this easement were designed to improve the meadow habitat for one of the few remaining populations of Sistrurus catenatus (Eastern Massasauga Rattlesnake) in Pennsylvania, and our sampling efforts were focused on evaluating the potential prey base available to the rattlesnakes at this site. We identified 9 amphibian species, 10 reptile species, and 11 small mammal species. For amphibians, Anaxyrus americanus (American Toad) was the most abundant species, accounting for 32.6% of the total, followed by Notophthalmus viridescens (Red-Spotted Newt) with 30.6% of total abundance. Thamnophis brachystoma (Short-Headed Gartersnake) comprised more than half (53.7%) of all reptiles sampled, followed by Thamnophis sirtalis (Eastern Gartersnake; 22.7%). Peromyscus leucopus (White-Footed Mouse) was the most abundant mammal species, accounting for 37.4% of all mammals surveyed, followed by Microtus pennsylvanicus (Eastern Meadow Vole; 24.8%). Mammals had the greatest species richness (S = 11), while reptiles had the greatest total abundance (TA = 361), and amphibians had the greatest species evenness (E = 0.799). Species accounts for Venango County indicate that we sampled 40.9% (9/22), 37.0% (10/27), and 21.2% (11/52) of the known amphibian, reptilian, and mammalian species, respectively, within this restored meadow habitat. Our results suggest that the restoration efforts on this easement study site have been extremely effective in creating meadow habitat that supports a diversity of prey species for the Eastern Massasauga Rattlesnake.

The morphological effects of artificial light at night on amphibian predators and prey are masked at the community level

Artificial light at night (ALAN) is a pervasive pollutant that influences wildlife at both the individual and community level. In this study, we tested the individual-level effects of ALAN on three species of tadpole prey and their newt predators by measuring prey pigmentation and predator and prey mass. Then we evaluated whether the individual-level effects of ALAN on pigmentation and mass had cascading community-level effects by assessing the outcome of predator-prey interactions. We found that spring peepers exposed to ALAN were significantly darker than those reared under control conditions. Additionally, wood frogs reared in ALAN conditions were significantly smaller than those reared in control conditions. In contrast, Eastern newts collected earlier in the spring that were exposed to ALAN were significantly larger than controls while those collected later in the spring were not affected by ALAN, suggesting phenological differences in the effect of ALAN. To understand how changes in pigmentation and size due to ALAN influence predation rates, we ran predation assays in both ALAN-polluted and ALAN-free outdoor environments. After the predation assay, the size disparity in wood frogs reared in ALAN was eliminated such that there was no longer a treatment difference in wood frog size, likely due to size-selective predation. This demonstrates the beneficial nature of predators’ selective pressure on prey populations. Lastly, despite individual-level effects of ALAN on pigmentation and mass, we did not detect cascading community-level effects on predation rates. Overall, this study highlights important species-level distinctions in the effects of ALAN. It also emphasizes the need to incorporate ecological complexity to understand the net impact of ALAN.

Molecular Identification of an Avian Predator of Mimetic Salamanders

Natural field observations of salamander predation are uncommon, in some cases limiting the ability of scientists to study the evolution of toxicity, aposematism, and mimicry in these amphibians. Here, we document field observations of recently depredated salamanders: 1 Pseudotriton ruber (Red Salamander) and 2 Gyrinophilus porphyriticus (Spring Salamander), members of a putative Müllerian mimetic complex involving Notophthalmus viridescens (Eastern Newt). We used molecular methods to identify the likely avian predator of the Spring Salamanders and underscore the opportunities for future research enabled by the pairing of traditional natural history with modern molecular techniques.

Comparative transcriptional profiling of regenerating damaged knee joints in two animal models of the newt Notophthalmus viridescens strengthens the role of candidate genes involved in osteoarthritis

ObjectivesTo compare joint regeneration in adult newts (N. viridescens) upon both newly established surgical removal and previously reported enzymatic destruction of articular cartilage to identify molecular factors and functionally analyze potentially important regulators involved in osteoarthritis (OA). MethodsDamage of knee cartilage was induced by either intra-articular injection of collagenase or by surgical removal of articular cartilage as a novel additional approach. Changes over time were clinically and histologically analyzed and studied by cDNA microarray analysis, real-time quantitative PCR, immunohistochemistry and functional assays to identify relevant candidate genes and determine their impact on regeneration. ResultsSeveral genes were found to be up-regulated during regeneration, including extracellular matrix components and mediators of cell-matrix interactions, genes encoding for cellular components, for cell and tissue homeostasis and tissue remodelling, for cellular processes as well as signalling molecules. A high activity and diversity of transcription was detected on days 10 and 20, especially in the surgical model. 10 candidate genes were further analyzed. The matricellular protein tenascin C (TN-C) attracted our particular attention due to its prominent up-regulation during regeneration in both models and at different time points. ConclusionsNewts are able to regenerate OA-like articular cartilage damage ad integrum both after enzymatic and mechanical injury. Most of the genes involved in amphibians are also known to be operative in humans and other mammals, especially matricellular factors interfering with optimized matrix remodelling. Our results stress the necessity to elucidate mechanistic differences in different species potentially using identical molecules but with different functional results.

Life stage and proximity to roads shape the skin microbiota of eastern newts (Notophthalmus viridescens).

Host-associated microbiomes play an essential role in the health of organisms, including immune system activation, metabolism and energy uptake. It is well established that microbial communities differ depending on the life stage and natural history of the organism. However, the effects of life stage and natural history on microbial communities may also be influenced by human activities. We investigated the effects of amphibian life stage (terrestrial eft vs. aquatic adult) and proximity to roadways on newt skin bacterial communities. We found that the eft and adult life stages differed in bacterial community composition; however, the effects of roads on community composition were more evident in the terrestrial eft stage compared to the aquatic adult stage. Terrestrial efts sampled close to roads possessed richer communities than those living further away from the influence of roads. When accounting for amplicon sequence variants with predicted antifungal capabilities, in the adult life stage, we observed a decrease in anti-fungal bacteria with distance to roads. In contrast, in the eft stage, we found an increase in anti-fungal bacteria with distance to roads. Our results highlight the need to consider the effects of human activities when evaluating how host-associated microbiomes differ across life stages of wildlife.

Batrachochytrium salamandrivorans Can Devour More than Salamanders.

Batrachochytrium salamandrivorans is an emerging fungus that is causing salamander declines in Europe. We evaluated whether an invasive frog species (Cuban treefrog, Osteopilus septentrionalis) that is found in international trade could be an asymptomatic carrier when exposed to zoospore doses known to infect salamanders. We discovered that Cuban treefrogs could be infected with B. salamandrivorans and, surprisingly, that chytridiomycosis developed in animals at the two highest zoospore doses. To fulfill Koch's postulates, we isolated B. salamandrivorans from infected frogs, exposed eastern newts (Notophthalmus viridescens) to the isolate, and verified infection and disease by histopathology. This experiment represents the first documentation of B. salamandrivorans chytridiomycosis in a frog species and substantially expands the conservation threat and possible mobilization of this pathogen in trade.

An Evaluation of Immersive and Handling Methods for Collecting Salamander Skin Peptides

Amphibian skin secretions are a rich source of antimicrobial peptides that have long interested both the biomedical and herpetological research communities. While anuran skin peptides are well studied, little is known about skin peptides in salamanders, in part because methods for collecting them are poorly developed. In anurans, pharmacological agents are commonly used to stimulate skin peptide responses, thus increasing the amount of peptide collected. It was unclear whether similar methods would work for salamanders. We conducted a series of experiments to test the effects of immersion in pharmaceutical agents (norepinephrine hydrochloride [NE]; acetylcholine chloride [Ach]), handling, and the interaction of both on peptide collection from the skin of Red-Spotted Newts (Notophthalmus viridescens) and Allegheny Mountain Dusky Salamanders (Desmognathus ochrophaeus). Our results suggest that vigorous handling, but not immersion in NE or Ach, is a better method for collecting salamander skin peptides. Despite the widespread use of pharmaceuticals to collect anuran skin peptides, we found little evidence that NE or Ach induces peptide release in salamanders. In fact, we show that inclusion of NE in collection solutions interferes with peptide quantification in protein assays.

Newt regeneration genes regulate Wingless signaling to restore patterning in Drosophila eye

SummaryNewts utilize their unique genes to restore missing parts by strategic regulation of conserved signaling pathways. Lack of genetic tools poses challenges to determine the function of such genes. Therefore, we used the Drosophila eye model to demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s), viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L2 mutant and GMR-hid, GMR-GAL4 eye. L2 exhibits the loss of ventral half of early eye and head involution defective (hid) triggers cell-death during later eye development. Surprisingly, newt genes significantly restore missing photoreceptor cells both in L2 and GMR>hid background by upregulating cell-proliferation and blocking cell-death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts downstream of newt genes. Our data highlights that unique newt proteins can regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells in Drosophila eye model with weak restoration capability.

Evaluation of created wetlands as amphibian habitat on a reforested surface mine

Wetlands created within disturbed landscapes may be an important key to restoring lost ecosystem functions. Reclaimed mines provide an opportunity to create wetlands and restore natural features within a disturbed landscape while benefiting amphibians, a taxa affected by habitat loss. A large-scale restoration effort within the Monongahela National Forest, West Virginia, USA has resulted in the creation of over 1400 small wetlands. We sampled 39 of these wetlands among four ages (2, 4, 6, and 8 years) to: 1) characterize differences in wetland habitat, 2) estimate amphibian occupancy and abundance, 3) identify wetland characteristics most important for amphibian utilization of wetlands. Mean wetland size, percent canopy cover, percent vegetation cover, distance to the nearest stream, and distance to the nearest forest varied significantly among wetland age classes. Water quality was within normal parameters for natural and created wetlands within our study region. Amphibian occupancy and abundance varied by species, with occupancy predicted by wetland age (Green Frog, Lithobates clamitans) and lower percent vegetation cover (Spotted Salamander, Ambystoma maculatum, L. clamitans) while abundance was predicted by wetland age (A. maculatum), higher percent vegetation cover (Eastern Newt, Notophthalmus viridescens), larger wetland surface area (A. maculatum, N. viridescens), and shorter distance to forest (N. viridescens). Our results indicate, with adequate site preparation, created wetlands on reforested surface mines provide suitable breeding habitat for pond breeding amphibians.

Frequency-dependent transmission of Batrachochytrium salamandrivorans in eastern newts.

Transmission is the fundamental process whereby pathogens infect their hosts and spread through populations, and can be characterized using mathematical functions. The functional form of transmission for emerging pathogens can determine pathogen impacts on host populations and can inform the efficacy of disease management strategies. By directly measuring transmission between infected and susceptible adult eastern newts (Notophthalmus viridescens) in aquatic mesocosms, we identified the most plausible transmission function for the emerging amphibian fungal pathogen Batrachochytrium salamandrivorans (Bsal). Although we considered a range of possible transmission functions, we found that Bsal transmission was best explained by pure frequency dependence. We observed that >90% of susceptible newts became infected within 17 days post‐exposure to an infected newt across a range of host densities and initial infection prevalence treatments. Under these conditions, we estimated R0 = 4.9 for Bsal in an eastern newt population. Our results suggest that Bsal has the capability of driving eastern newt populations to extinction and that managing host density may not be an effective management strategy. Intervention strategies that prevent Bsal introduction or increase host resistance or tolerance to infection may be more effective. Our results add to the growing empirical evidence that transmission of wildlife pathogens can saturate and be functionally frequency‐dependent.

Changes in the gut microbial community of the eastern newt (Notophthalmus viridescens) across its three distinct life stages.

Understanding the forces that shape vertebrate gut microbial community assembly and composition throughout development is a major focus of the microbiome field. Here, we utilize the complex life cycle of the eastern newt (Notophthalmus viridescens) as a natural wild model to compare the effects of host and environmental factors on gut microbiome development. We compared bacterial inventories of each of the newt's three physiologically distinct developmental stages to determine if each hosted a unique community, or if the two stages which share an aquatic habitat (larvae and adults) harbored more similar communities than those of the third stage, the terrestrial juvenile eft. Additionally, we assessed how the contribution of selective processes to gut microbial assembly changed through development. We found that structurally, each life stage harbored a distinct community, which may be attributable to host factors. Further, across development, we found that community assembly processes shifted from a predominance of neutral to selective forces. However, habitat may also be important in determining community membership and diversity due the uniqueness of eft communities based on these metrics. Our results are similar to those in other vertebrate taxa, suggesting that gut microbiota assembly processes may be conserved across diverse lineages.

Novel Newt Regeneration Genes Regulate Wingless Signaling to Restore Patterning in <i>Drosophila</i> Eye

A fundamental process of regeneration, which varies among animals, recruits conserved signaling pathways to restore missing parts. Only a few animals like newts can repeatedly regenerate lost body parts throughout their lifespan that can be attributed to strategic regulation of conserved signaling pathways by newt’s regeneration tool-kit genes. Lack of genetic tools pose challenges to understand the molecular genetic mechanism of regeneration response of these genes. Here we report use of genetically tractable Drosophila eye model to demonstrate the regeneration potential of a group of unique protein(s) from newt (Notophthalmus viridescens), which when ectopically expressed can significantly rescue missing photoreceptor cells in a Drosophila eye mutant. These newt proteins with signal peptides motifs exhibit non-cell-autonomous rescue properties and their regeneration potential even extends into later stages of fly development. Ectopic expression of these newt genes can rescue eye mutant phenotype by promoting cell proliferation and blocking cell death. These novel newt genes downregulate evolutionarily conserved Wingless (Wg)/Wnt signaling pathway to promote rescue. Modulation of Wg/Wnt signaling levels by using antagonists or agonists of Wg/Wnt signaling pathway in eye mutant background where newt gene(s) is ectopically expressed suggests that Wg signaling acts downstream of newt genes. Our data highlights the regeneration potential of novel newt proteins that regulate conserved pathways to trigger a robust regeneration response in Drosophila model with weak regeneration capability.

Seasonal changes and the unexpected impact of environmental disturbance on skin bacteria of individual amphibians in a natural habitat.

Amphibians host diverse skin bacteria that have a role in pathogen defense, but these skin communities could change over time and impact this function. Here, we monitored individual Eastern red-spotted newts (Notophthalmus viridescens; N= 17) for 2 years in a field pond enclosure and assessed the effects of season and disturbance on skin bacterial community dynamics. We created disturbances by adding additional pond substrate to the enclosure at two timepoints. We planned to sample the skin bacterial community and metabolite profiles of each newt every 6 weeks; we ultimately sampled eight individuals at least six times. We used 16S rRNA gene amplicon sequencing to characterize the bacterial communities and HPLC-MS for metabolite profiling. We found that disturbance had a dramatic effect on skin bacterial communities and metabolite profiles, while season had an effect only using select metrics. There were seven core bacterial taxa (97% OTUs) that were found on all newts in all seasons, pre- and post-disturbance. Lastly, there was a correlation between bacterial and metabolite profiles post-disturbance, which was not observed pre-disturbance. This longitudinal study suggests that environmental disturbances can have lasting effects on skin bacterial communities that overwhelm seasonal changes, although the core bacteria remain relatively consistent over time.

Regeneration and development. An amphibian call to arms.

Unlike axolotls, the urodele Notophthalmus viridescens completes two metamorphoses and emerges from its aquatic environment to mate as a fully terrestrial adult. Larval and adult limb regeneration are commonly treated as roughly equivalent processes and, at least in part, as a recapitulation of embryonic development. We compared larval limb development to regeneration of both larval and adult forelimbs and found that there are substantial differences in developmental pattern among larvae and adults. The larval pattern of preaxial dominance is absent in adult regenerates: adult regenerates instead develop digits synchronously, and they do so before proximal autopodial elements have formed discrete aggregation zones. By contrast, larval regenerates follow a pattern of sequential digit formation from anterior to posterior, like their embryonic limb buds. Based upon these morphological clues, we conclude that larval regenerates are unlikely to exhibit features of epimorphic regeneration seen in adults, but are more likely to represent a form of developmental regulation. Furthermore, we confirm that post-metamorphic limb regeneration is not a simple recapitulation of ontology at the morphological level. These distinctions may help to explain and interpret some experiments and observations of regeneration in neotenic or paedomorphic urodeles.