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'.

Coccidiosis in Wild Eastern Newts (Notophthalmus viridescens): An Unexpected Finding in Consecutive Mortality Events in Tennessee, USA.

Wildlife diseases are a major threat for species conservation, and there is a growing need to implement more comprehensive disease response programs to better identify these diseases of concern. During March 2017, we observed moribund and dead eastern newts, Notophthalmus viridescens, in a single pond in middle Tennessee. All moribund individuals were emaciated. We euthanized and processed all individuals immediately on-site and later performed histopathology and quantitative PCR for ranavirus, the protist Perkinsea, and chytrid fungi Batrachochytrium dendrobatidis and Batrachochytrium salamandrivorans. One newt was positive for ranavirus. Histopathology showed no evidence of ranavirosis but did reveal overwhelming coccidiosis. Overlapping partial sequences of coccidian 18S subunit DNA showed a 96.4% match with Eimeria steinhausi, suggesting that lesions were due to a previously undescribed Eimeria sp. In 2019, two more moribund newts were encountered at the same pond. Histopathology revealed the same suspicious parasitic organisms, and one individual was positive for B. dendrobatidis. Further research on how seasonal and other environmental parameters may influence coccidia-associated morbidity and mortality is warranted. These events highlight the importance of histopathologic evaluation of mortality events and provide guidance for investigation of future outbreaks.

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.

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.

Winter is coming-Temperature affects immune defenses and susceptibility to Batrachochytrium salamandrivorans.

Environmental temperature is a key factor driving various biological processes, including immune defenses and host-pathogen interactions. Here, we evaluated the effects of environmental temperature on the pathogenicity of the emerging fungal pathogen, Batrachochytrium salamandrivorans (Bsal), using controlled laboratory experiments, and measured components of host immune defense to identify regulating mechanisms. We found that adult and juvenile Notophthalmus viridescens died faster due to Bsal chytridiomycosis at 14°C than at 6 and 22°C. Pathogen replication rates, total available proteins on the skin, and microbiome composition likely drove these relationships. Temperature-dependent skin microbiome composition in our laboratory experiments matched seasonal trends in wild N. viridescens, adding validity to these results. We also found that hydrophobic peptide production after two months post-exposure to Bsal was reduced in infected animals compared to controls, perhaps due to peptide release earlier in infection or impaired granular gland function in diseased animals. Using our temperature-dependent susceptibility results, we performed a geographic analysis that revealed N. viridescens populations in the northeastern United States and southeastern Canada are at greatest risk for Bsal invasion, which shifted risk north compared to previous assessments. Our results indicate that environmental temperature will play a key role in the epidemiology of Bsal and provide evidence that temperature manipulations may be a viable disease management strategy.

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.

Regeneration and development. An amphibian call to arms.

BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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.

Revisited - Failure of tetrodotoxin to protect red-spotted newts, Notophthalmus viridescens, from endoparasites.

Red-spotted newts, Notophthalmus viridescens, contain tetrodotoxin (TTX) and its analogue 6-epiTTX in variable concentrations. In a follow-up study, newts were sampled from a pond in Pennsylvania, USA, in 2010, 2014, and 2018. Their toxin levels were assayed by liquid-chromatography-fluorescence detection (LC-FLD), and assessment of their infection with endoparasites such as nematodes and helminths was performed by histological examination of internal organs. In the 2010 and 2014 samples, average prevalence of parasite infection was 53 and 60%, respectively, but reached 100% in the 2018 sample, where metacercaria stages of the digenean trematode genus Australapatemon/Apatemon (family: Strigeidae) were predominant causing severe tissue damage in liver and kidney. Mean values of TTX and 6-epiTTX were not significantly different in parasitized or parasite-free newts over the study period, confirming previous findings that host toxicity and parasite load are not negatively correlated. Whereas the role of TTX in defence against predators is undisputed, its efficacy to prevent parasitic infections is less obvious. Toxin-resistance of various metazoan parasites may promote their widespread occurrence in poisonous newts.

Comparative transcriptomic analysis and structure prediction of novel Newt proteins.

Notophthalmus viridescens (Red-spotted Newt) possess amazing capabilities to regenerate their organs and other tissues. Previously, using a de novo assembly of the newt transcriptome combined with proteomic validation, our group identified a novel family of five protein members expressed in adult tissues during regeneration in Notophthalmus viridescens. The presence of a putative signal peptide suggests that all these proteins are secretory in nature. Here we employed iterative threading assembly refinement (I-TASSER) server to generate three-dimensional structure of these novel Newt proteins and predicted their function. Our data suggests that these proteins could act as ion transporters, and be involved in redox reaction(s). Due to absence of transgenic approaches in N. viridescens, and conservation of genetic machinery across species, we generated transgenic Drosophila melanogaster to misexpress these genes. Expression of 2775 transcripts were compared between these five newly identified Newt genes. We found that genes involved in the developmental process, cell cycle, apoptosis, and immune response are among those that are highly enriched. To validate the RNA Seq. data, expression of six highly regulated genes were verified using real time Quantitative Polymerase Chain Reaction (RT-qPCR). These graded gene expression patterns provide insight into the function of novel protein family identified in Newt, and layout a map for future studies in the field.

Differential plasticity of membrane fatty acids in northern and southern populations of the eastern newt (Notophthalmus viridescens).

Seasonal changes in membrane composition and metabolic activity allow many temperate ectotherms to contend with changes in body temperature, but few studies have investigated whether the plasticity of these traits has diverged within a single species. Therefore, we studied the effects of thermal acclimation on the membrane fatty acid composition and the activities of cytochrome c oxidase (CCO) and citrate synthase (CS) in the skeletal muscle and liver of eastern newts from Maine and Florida. Newts were acclimated to either 6 °C or 28 °C for 12 weeks prior to experiments. Cold acclimation resulted in a lower saturated fatty acid (SFA) content in the muscle membranes of both populations. SFA content in liver was lower in cold compared to warm-acclimated newts from Florida, but acclimation did not affect SFA content in liver membranes of the Maine population. In liver, cold acclimation resulted in a higher monounsaturated fatty acid (MUFA) content in the Florida population and a higher polyunsaturated fatty acid (PUFA) content in the Maine population. Regardless of acclimation conditions, the muscle and liver membranes of the Maine population had higher SFA and PUFA contents compared to those of the Florida population. MUFA content of muscle and liver membranes was higher in the Florida population compared to the Maine population. The effect of acclimation on CCO and CS activity was tissue-specific. In muscle, CCO and CS activities were higher in cold compared to warm-acclimated newts in both populations, and CS and CCO activities were higher in the Maine compared to the Florida population. In liver, CCO and CS activity were unaffected by acclimation in the Florida population, but activity was lower in cold compared to warm-acclimated Maine newts. These results demonstrate that the phenotypic plasticity of these traits in response to seasonal change has diverged between northern and southern populations.

Sexual dimorphism in a top predator (Notophthalmus viridescens) drives aquatic prey community assembly.

Intraspecific variation can have important consequences for the structure and function of ecological communities, and serves to link community ecology to evolutionary processes. Differences between the sexes are an overwhelmingly common form of intraspecific variation, but its community-level consequences have never been experimentally investigated. Here, we manipulate the sex ratio of a sexually dimorphic predacious newt in aquatic mesocosms, then track their impact on prey communities. Female and male newts preferentially forage in the benthic and pelagic zones, respectively, causing corresponding reductions in prey abundances in those habitats. Sex ratio differences also explained a large proportion (33%) of differences in the composition of entire pond communities. Ultimately, we demonstrate the impact of known patterns of sexual dimorphism in a predator on its prey, uncovering overlooked links between evolutionary adaptation and the structure of contemporary communities. Given the extreme prevalence of sexual dimorphism, we argue that the independent evolution of the sexes will often have important consequences for ecological communities.

Habitat partitioning during character displacement between the sexes.

Ecological differences between the sexes are often interpreted as evidence of within-species ecological character displacement (ECD), a hypothesis with almost no direct tests. Here, we experimentally test two predictions that are direct corollaries of ECD between the sexes, in a salamander. First, we find support for the prediction that each sex has a growth rate advantage in the aquatic microhabitat where it is most commonly found. Second, we test the prediction that selection for ECD in the breeding environment may affect partial migration out of this environment. We found that phenotype-dependent migration resulted in a shift in the phenotypic distribution across treatments, with the highest sexual dimorphism occurring among residents at high founding density, suggesting that migration and ECD can both be driven by competition. Our work illustrates how complex patterns of habitat partitioning evolve during ECD between the sexes and suggest ECD and partial migration can interact to effect both ecological dynamics and evolution of sexual dimorphism.

The association between male-biased sex ratio and indicators of stress in red-spotted newts.

In populations with a male-biased operational sex ratio, coercive mating by males can have fitness consequences for females. One component of reduced fitness for females in populations with a male-biased OSR may be greater activation of the stress response, resulting in higher corticosterone release rates (CORT; a glucocorticoid stress hormone in amphibians). We test the hypothesis that a male-biased sex ratio affects female activity and release rates of CORT and testosterone (T) in male and female red-spotted newts (Notophthalmus viridescens). First, we evaluated if chemical cues from a male-biased sex ratio affect activity and CORT release rates in females. We predicted that females exposed to chemical cues of three males would be less active and have higher CORT release rates than those exposed to chemical cues of one male. Second, we measured CORT release rates of red-spotted newts in field enclosures with either a male-biased or a female-biased sex ratio. We predicted that females in the male-biased treatment would have higher CORT and T release rates than those in a female-biased treatment, owing to higher levels of male harassment. We also predicted that males would have higher CORT and T release rates in male-biased treatments due to higher levels of male-male competition. Females were not less active in response to chemical cues from more males over fewer males, but there was a positive relationship between female activity and CORT when they were exposed to the cues of three males. We also found that females, but not males, in the male-biased sex ratio treatment had higher CORT and T release rates than those in the female-biased treatment. Our results support the hypothesis that a male-biased sex ratio leads to a higher stress response, which may underlie the observed decrease in immune function and body condition in previous work exposing female red-spotted newts to a male-biased sex ratio. This study furthers our understanding of the mechanistic basis for costs associated with a male-biased sex ratio in a pond-breeding amphibian.

A linkage map for the Newt Notophthalmus viridescens: Insights in vertebrate genome and chromosome evolution.

Genetic linkage maps are fundamental resources that enable diverse genetic and genomic approaches, including quantitative trait locus (QTL) analyses and comparative studies of genome evolution. It is straightforward to build linkage maps for species that are amenable to laboratory culture and genetic crossing designs, and that have relatively small genomes and few chromosomes. It is more difficult to generate linkage maps for species that do not meet these criteria. Here, we introduce a method to rapidly build linkage maps for salamanders, which are known for their enormous genome sizes. As proof of principle, we developed a linkage map with thousands of molecular markers (N=2349) for the Eastern newt (Notophthalmus viridescens). The map contains 12 linkage groups (152.3-934.7cM), only one more than the number of chromosome pairs. Importantly, this map was generated using RNA isolated from a single wild caught female and her 28 offspring. We used the map to reveal chromosome-scale conservation of synteny among N. viridescens, A. mexicanum (Urodela), and chicken (Amniota), and to identify large conserved segments between N. viridescens and Xenopus tropicalis (Anura). We also show that met1, a major effect QTL that regulates the expression of alternate metamorphic and paedomorphic modes of development in Ambystoma, associates with a chromosomal fusion that is not found in the N. viridescens map. Our results shed new light on the ancestral amphibian karyotype and reveal specific fusion and translocation events that shaped the genomes of three amphibian model taxa. The ability to rapidly build linkage maps for large salamander genomes will enable genetic and genomic analyses within this important vertebrate group, and more generally, empower comparative studies of vertebrate biology and evolution.

Comparative and developmental patterns of amphibious auditory function in salamanders.

Early amphibious tetrapods may have detected aquatic sound pressure using sound-induced lung vibrations, but their lack of tympanic middle ears would have restricted aerial sensitivity. Sharing these characteristics, salamanders could be models for the carryover of auditory function across an aquatic-terrestrial boundary without tympanic middle ears. We measured amphibious auditory evoked potential audiograms in five phylogenetically and ecologically distinct salamanders (Amphiuma means, Notophthalmus viridescens, Ambystoma talpoideum, Eurycea spp., and Plethodon glutinosus) and tested whether metamorphosis and terrestrial niche were linked to aerial sensitivity. Threshold differences between media varied between species. A. means' relative aerial sensitivity was greatest at 100 Hz and decreased with increasing frequency. In contrast, all other salamanders retained greater sensitivity up to 500 Hz, and in A. talpoideum and Eurycea, relative sensitivity at 500 Hz was higher than at 100 Hz. Aerial thresholds of terrestrial P. glutinosus above 200 Hz were similar to A. talpoideum and Eurycea, but lower than N. viridescens and A. means. Metamorphosis did not affect aerial sensitivity in N. viridescens or A. talpoideum. These results fail to support a hypothesis of terrestrial hearing specialization across ontogeny or phylogeny. We discuss methodological limitations to our amphibious comparisons and factors affecting variation in amphibious performance.

Dependence of the structure and mechanics of metaphase chromosomes on oxidized cysteines.

We have found that reagents that reduce oxidized cysteines lead to destabilization of metaphase chromosome folding, suggesting that chemically linked cysteine residues may play a structural role in mitotic chromosome organization, in accord with classical studies by Dounce et al. (J Theor Biol 42:275-285, 1973) and Sumner (J Cell Sci 70:177-188, 1984a). Human chromosomes isolated into buffer unfold when exposed to dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine (TCEP). In micromanipulation experiments which allow us to examine the mechanics of individual metaphase chromosomes, we have found that the gel-like elastic stiffness of native metaphase chromosomes is dramatically suppressed by DTT and TCEP, even before the chromosomes become appreciably unfolded. We also report protein labeling experiments on human metaphase chromosomes which allow us to tag oxidized and reduction-sensitive cysteine residues. PAGE analysis using fluorescent labels shows a small number of labeled bands. Mass spectrometry analysis of similarly labeled proteins provides a list of candidates for proteins with oxidized cysteines involved in chromosome organization, notably including components of condensin I, cohesin, the nucleosome-interacting proteins RCC1 and RCC2, as well as the RNA/DNA-binding protein NONO/p54NRB.

MARCKS-like protein is an initiating molecule in axolotl appendage regeneration.

Identifying key molecules that launch regeneration has been a long-sought goal. Multiple regenerative animals show an initial wound-associated proliferative response that transits into sustained proliferation if a considerable portion of the body part has been removed. In the axolotl, appendage amputation initiates a round of wound-associated cell cycle induction followed by continued proliferation that is dependent on nerve-derived signals. A wound-associated molecule that triggers the initial proliferative response to launch regeneration has remained obscure. Here, using an expression cloning strategy followed by in vivo gain- and loss-of-function assays, we identified axolotl MARCKS-like protein (MLP) as an extracellularly released factor that induces the initial cell cycle response during axolotl appendage regeneration. The identification of a regeneration-initiating molecule opens the possibility of understanding how to elicit regeneration in other animals.

Ecological Character Displacement between the Sexes.

Theory suggests that the evolution of sexual dimorphism in ecologically relevant traits can evolve purely through competition between the sexes for a shared resource. Although more parsimonious hypotheses exist for the evolution of ecological sexual dimorphisms, there are some underappreciated reasons to expect that competition may often play some role in the evolution of sexual dimorphism. Here, we build on past work to outline a set of sufficient criteria to demonstrate a role for resource competition in the evolution of sexual dimorphism, the most critical of which is that resource competition can be directly linked to sexual divergence along the axis of ecologically relevant dimorphism. We then compare the geometry of fitness surfaces across experimental manipulations of density and sex ratio in a semiaquatic salamander (Notophthalmus viridescens). We find consistent disruptive selection on multivariate sexual dimorphism in feeding morphology, which increases in strength with density. Fitness and the strength of divergent selection are negative-frequency dependent in the manner expected under competition-driven divergence between the sexes. Our results constitute direct evidence of resource competition as a driver of sexually antagonist selection and consequently the evolution of sexual dimorphism, providing an illustration of how cause and effect can be separated in studies of sexual divergence in morphology and ecology. We suggest that resource competition may often contribute to sexual divergence jointly with other sources of sex-biased selection, especially when ecological opportunity is sex specific.

Turning terminally differentiated skeletal muscle cells into regenerative progenitors.

The ability to repeatedly regenerate limbs during the entire lifespan of an animal is restricted to certain salamander species among vertebrates. This ability involves dedifferentiation of post-mitotic cells into progenitors that in turn form new structures. A long-term enigma has been how injury leads to dedifferentiation. Here we show that skeletal muscle dedifferentiation during newt limb regeneration depends on a programmed cell death response by myofibres. We find that programmed cell death-induced muscle fragmentation produces a population of 'undead' intermediate cells, which have the capacity to resume proliferation and contribute to muscle regeneration. We demonstrate the derivation of proliferating progeny from differentiated, multinucleated muscle cells by first inducing and subsequently intercepting a programmed cell death response. We conclude that cell survival may be manifested by the production of a dedifferentiated cell with broader potential and that the diversion of a programmed cell death response is an instrument to achieve dedifferentiation.

High pheromone diversity in the male cheek gland of the red-spotted newt Notophthalmus viridescens (Salamandridae).

BACKGROUND: Male salamanders (Urodela) often make use of pheromones that are produced in sexually dimorphic glands to persuade the female into courtship and mating. The mental gland of lungless salamanders (Plethodontidae) and dorsal cloacal glands (or abdominal glands) of newts (Salamandridae) have been particularly well studied in that respect. In both families, sodefrin precursor-like factor (SPF) proteins have been identified as major components of the courtship pheromone system. However, similar to plethodontids, some newts also make use of subtle head glands during courtship, but few pheromones have been characterized from such structures. Males of red-spotted newts (Notophthalmus viridescens, Salamandridae) have both cloacal and cheek (genial) glands, and are known to apply secretions to the female's nose by both tail-fanning and cheek-rubbing. Here we combined transcriptomic and phylogenetic analyses to investigate the presence, diversity and evolution of SPF proteins in the cloacal and cheek glands of this species. RESULTS: Our analyses indicate that the cheek glands of male N. viridescens produce a similar amount and diversity of SPF isoforms as the cloacal glands in this species. Expression in other tissues was much lower, suggesting that both male-specific courtship glands secrete SPF pheromones during courtship. Our phylogenetic analyses show that N. viridescens expresses a combination of isoforms that stem from four highly diverged evolutionary lineages of SPF variants, that together form a basis for the broad diversity of SPF precursors in the breeding glands. CONCLUSIONS: The similar SPF expression of cheek and cloacal glands suggests that this protein family is used for pheromone signalling through cheek rubbing in the red-spotted newt. Since several male salamandrids in other genera have comparable head glands, SPF application via other glands than the cloacal glands may be more widespread than currently appreciated in salamandrids.