Newt Notophthalmus Viridescens Research Articles

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.

Host density and habitat structure influence host contact rates and Batrachochytrium salamandrivorans transmission

Batrachochytrium salamandrivorans (Bsal) is an emerging invasive pathogen that is highly pathogenic to salamander species. Modeling infection dynamics in this system can facilitate proactive efforts to mitigate this pathogen's impact on North American species. Given its widespread distribution and high abundance, the eastern newt (Notophthalmus viridescens) has the potential to significantly influence Bsal epidemiology. We designed experiments to 1) estimate contact rates given different host densities and habitat structure and 2) estimate the probability of transmission from infected to susceptible individuals. Using parameter estimates from data generated during these experiments, we modeled infection and disease outcomes for a population of newts using a system of differential equations. We found that host contact rates were density-dependent, and that adding habitat structure reduced contacts. The probability of Bsal transmission given contact between newts was very high (>90%) even at early stages of infection. Our simulations show rapid transmission of Bsal among individuals following pathogen introduction, with infection prevalence exceeding 90% within one month and >80% mortality of newts in three months. Estimates of basic reproductive rate (R0) of Bsal for eastern newts were 1.9 and 3.2 for complex and simple habitats, respectively. Although reducing host density and increasing habitat complexity might decrease transmission, these management strategies may be ineffective at stopping Bsal invasion in eastern newt populations due to this species’ hyper-susceptibility.

Palatability and Defense of Eastern Newt (Notophthalmus viridescens) Larvae and Metamorphic Juveniles against Predatory Dragonfly Nymphs

We examined the predator–prey relationship between nymphs of the predatory dragonfly Anax junius (Common Green Darner) and larval and metamorphic Notophthalmus viridescens (Eastern Newt), some of which may contain the potent neurotoxin, tetrodotoxin. First, we conducted a palatability study to determine which life-history stages were palatable to dragonflies. We also tested the metamorphosis and survival rates of larval newts when exposed to predatory dragonflies in small microcosms. Finally, we tested the predator avoidance behavior of larval newts in response to chemical cues from a control, food stimulus, and predatory dragonflies. All life-history stages (small and large larvae, and recent metamorphs) were palatable to dragonflies. In microcosm trials, we found that newt larvae had a lower chance of surviving and transforming when dragonflies were present compared to a control. Finally, newt larvae decreased movement significantly when exposed to predatory dragonfly stimulus compared to either a control or food stimulus. These results suggest dragonflies are effective predators of newts from hatching through metamorphosis. However, the larvae do possess behavioral avoidance mechanisms that likely reduce the risk of predation by dragonflies.

"Lost and Found": snoRNA Annotation in the Xenopus Genome and Implications for Evolutionary Studies.

Small nucleolar RNAs (snoRNAs) function primarily as guide RNAs for posttranscriptional modification of rRNAs and spliceosomal snRNAs, both of which are functionally important and evolutionarily conserved molecules. It is commonly believed that snoRNAs and the modifications they mediate are highly conserved across species. However, most relevant data on snoRNA annotation and RNA modification are limited to studies on human and yeast. Here, we used RNA-sequencing data from the giant oocyte nucleus of the frog Xenopus tropicalis to annotate a nearly complete set of snoRNAs. We compared the frog data with snoRNA sets from human and other vertebrate genomes, including mammals, birds, reptiles, and fish. We identified many Xenopus-specific (or nonhuman) snoRNAs and Xenopus-specific domains in snoRNAs from conserved RNA families. We predicted that some of these nonhuman snoRNAs and domains mediate modifications at unexpected positions in rRNAs and snRNAs. These modifications were mapped as predicted when RNA modification assays were applied to RNA from nine vertebrate species: frogs X. tropicalis and X. laevis, newt Notophthalmus viridescens, axolotl Ambystoma mexicanum, whiptail lizard Aspidoscelis neomexicana, zebrafish Danio rerio, chicken, mouse, and human. This analysis revealed that only a subset of RNA modifications is evolutionarily conserved and that modification patterns may vary even between closely related species. We speculate that each functional domain in snoRNAs (half of an snoRNA) may evolve independently and shuffle between different snoRNAs.

The influence of environmental factors on pond activity of aquatic red-spotted newts Notophthalmus viridescens

ABSTRACTMany important components of animal behavior, growth, and reproduction are tightly linked to environmental conditions, particularly for ectothermic freshwater organisms. For amphibian species, factors such as temperature and rainfall can be physiologically limiting and alter activity levels. The effects of environmental conditions on terrestrial amphibian movement have been well characterized, but less is known about the importance of these factors in aquatic habitats. Here we investigate the impact of temperature and rainfall on the activity of a pond-dwelling amphibian using capture patterns of aquatic adult red-spotted newts (Notophthalmus viridescens). Data on newt captures, air temperature, and rainfall were collected for 6 years (2009, 2011, 2013–2016) during the winter breeding season at a wetland in the Piedmont region of North Carolina, USA. In 2016, we collected more detailed data on the size and sex of captured newts, as well as recording water temperature. Overall, temperature played a significant role in determining newt activity, while rainfall had little effect. As expected, and consistent with findings on amphibian activity in terrestrial systems, newt captures increased with increasing air temperature. During the 2016 breeding season we found sex-based changes in activity in response to temperature, with a higher proportion of males captured during warmer temperatures and a reduction in the male capture bias during colder temperatures. We found no evidence of size-based shifts in activity in response to temperature. This study increases our knowledge of amphibian responses to temperature while in aquatic habitats. Although the duration of time spent in aquatic habitats can be highly variable between species, these breeding locations are critical for the persistence of populations and activity levels, and reproductive success in these wetlands can be highly impacted by future changes in environmental conditions.

Absence of visual discrimination by female eastern newts during mate selection

Male eastern newts (Notophthalmus viridescens) exhibit tremendous variation in several characteristics, including the number of dorsal spots, the brightness of those spots, tail depth and tail length. These traits could be used by females for mate selection decisions. We conducted two experiments to determine whether females use visual signals to assess these characteristics in their mate selection decisions by simultaneously exposing a female to males of opposing characteristics. We observed female preference for the number of spots on a male’s dorsum, the brightness of male dorsal spots, male body mass, and male tail depth and length. We then recorded the amount of time the female spent in a preference zone adjacent to each of the males. Female newts did not show a significant preference for any of the characteristics observed, spending roughly equal amounts of time in both preference zones. These results, combined with the results of several previous studies, suggest that female eastern newts do not use visual signals to assess these traits in their mate selection decisions. Given that most of these traits are sexually dimorphic, this is likely due to the reproductive advantage created by the intense male–male competition present and their amplexus-based mating system.

The Role of Prey in Microgeographic Variation in Red-Spotted Newt (Notophthalmus viridescens viridescens) Head Width

Abstract For gape-limited predators, investment in larger heads should occur when larger prey items are more profitable or are the only prey option available. This may result in evolutionary or within-lifetime (plastic) increases in gape size in animals exposed to larger prey. This phenomenon has been well documented in larval but not adult caudates (salamanders and newts). We report here evidence of greater gape size in populations of a newt that co-occur with energetically profitable, large prey. We collected morphological data from populations of Red-Spotted Newts (Notophthalmus viridescens viridescens) that occupied nearby ponds that varied in the presence of eggs and tadpoles of Wood Frogs (Lithobates sylvaticus). Newts were larger in ponds occupied by Wood Frogs, suggesting a potential energetic advantage to this prey. Newts syntopic with Wood Frogs had relatively wider heads, which we interpret as a potential adaptation for consuming seasonally abundant Wood Frog eggs and tadpoles. Male newts had w...

Both novelty and conspicuousness influence selection by mammalian predators on the colour pattern ofPlethodon cinereus(Urodela: Plethodontidae)

Predators influence the evolution of colour pattern in prey species, yet how these selective forces might differ among predators is rarely considered. In particular, prey colour patterns that indicate unpalatability to some predator species may not carry the same signal for other predators. We test several hypotheses of selection on patterning between mammal predators and the polymorphic salamander Plethodon cinereus, which, under an avian visual system appears as a mimic of the toxic newt Notophthalmus viridescens. We fit each hypothesis against field observations of mammalian attacks on salamander clay replicas. We then develop a novel analytical procedure that enables the combination of multiple non-exclusive models in a likelihood framework. We find that mammals do not follow any single hypothesis proposed, including the hypothesis of mimicry. Instead, mammals in this system use visual cues while foraging to avoid unfamiliar, novel prey and attack conspicuous prey. We propose that mammals may help to maintain colour pattern polymorphism within populations of P. cinereus by avoiding novel, unfamiliar colour morphs. Additionally, selective pressures from multiple predators and variation in predator communities among sites may contribute to the maintenance of colour polymorphism within and among localities in this salamander species.

Only fear the fatal foe: predation risk assessment by eastern newts (Notophthalmus viridescens) in response to common snapping turtles and other potential predators

Many organisms utilize toxic or noxious compounds as a means of deterring predation. Eastern newts (Notopthalmus viridescens), along with other species in the family Salamandridae, possess a potent neurotoxin called tetrodotoxin (TTX). Although TTX can serve as an effective antipredator mechanism in species of newts with high concentrations (e.g., Taricha), eastern newts have relatively low levels of toxicity in comparison to those species, and it may not serve as an effective antipredator mechanism against all threats. In this case, they may benefit rather by utilizing behavioral changes to avoid initial contact with predators. We tested for predator-avoidance behavior in newts by exposing individuals to kairomones from various predators. We recorded activity patterns of newts when they were exposed to cues from potential predators including bullfrogs (Lithobates catesbeiana), water snakes (Nerodia sipedon) and snapping turtles (Chelydra serpentina), as well as a non-predator (bullfrog tadpoles), and a control (deionized water). Newts reduced activity when exposed to snapping turtle stimuli, but did not change activity when exposed to any other chemical cues. We verified that newts interact with this predator by trapping snapping turtles found in ponds from which newts were collected. Finally, we used turtles caught during this sampling to test whether they are an actual predator of newts and whether newts shift microhabitat use when exposed to this predator. In each replicate, turtles consumed newts, and newts spatially avoided the snapping turtle, relative to a control. The results of these experiments indicate newts rely on predator-avoidance behavior to reduce the probability of being consumed by snapping turtles, but do not reduce activity in response to other potential predators that may only consume them rarely.

Phylogeography and evolution of the Red Salamander (Pseudotriton ruber)

Phylogeographic studies frequently result in the elevation of subspecific taxa to species given monophyly, or the synonymy of subspecies that are not monophyletic. However, given limited or incongruent datasets, retention of subspecies can be useful to describe hypothesized incipient species or to illustrate interesting biological phenomena driving morphological diversity. Four subspecific taxa have been used to describe largely allopatric geographic variation within the species Pseudotriton ruber, a plethodontid salamander occupying stream and spring habitats across eastern North America: P. r. vioscai occurs in lowland Coastal Plain habitats, while P. r. ruber, P. r. nitidus, and P. r. schencki occupy upland regions in and around the Appalachian Mountains. Pseudotriton ruber co-occurs through its distribution with the aposematic newt Notophthalmus viridescens, and both species are hypothesized to be part of a Müllerian mimicry complex. In this study, we sequenced regions of two mitochondrial (cytochrome b, NADH dehydrogenase subunit 2) and one single copy nuclear protein-coding gene (pro-opiomelanocortin) from individuals sampled across much of the distribution of P. ruber and then used maximum-likelihood and Bayesian phylogenetic inference to test the monophyly of subspecies, reconstruct biogeographic history, and make inferences about morphological evolution. Phylogeographic hypotheses from mitochondrial and nuclear datasets described structure among populations of P. ruber which separated Coastal Plain and upland Appalachian populations, but subspecies were not monophyletic. Biogeographic reconstruction estimated the ancestor of all populations to have occupied and initially diverged in the Coastal Plain during the Pliocene (∼3.6mya), before one lineage subsequently invaded upland areas of Appalachia. Bold bright coloration of high elevation subspecies P. r. nitidus and P. r. schencki appears to have evolved twice. We hypothesize that the Müllerian mimicry complex with N. viridescens and P. ruber may provide a selective mechanism driving the co-evolution of striking bright and dull morphological variation among populations of both species. While P. ruber subspecies were not consistent with our criteria for diagnosing species (monophyly) and therefore could not be elevated to species, we advocate for the retention of subspecies because they describe hypotheses about an incipient species (P. r. vioscai) and how Müllerian mimicry may shape morphological diversity of species.

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

BackgroundMale 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.ResultsOur 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.ConclusionsThe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12862-015-0333-1) contains supplementary material, which is available to authorized users.

Data mining in newt-omics, the repository for omics data from the newt.

Salamanders are an excellent model organism to study regenerative processes due to their unique ability to regenerate lost appendages or organs. Straightforward bioinformatics tools to analyze and take advantage of the growing number of "omics" studies performed in salamanders were lacking so far. To overcome this limitation, we have generated a comprehensive data repository for the red-spotted newt Notophthalmus viridescens, named newt-omics, merging omics style datasets on the transcriptome and proteome level including expression values and annotations. The resource is freely available via a user-friendly Web-based graphical user interface ( http://newt-omics.mpi-bn.mpg.de) that allows access and queries to the database without prior bioinformatical expertise. The repository is updated regularly, incorporating new published datasets from omics technologies.

Studying newt brain regeneration following subtype specific neuronal ablation.

The realization that neuronal injury does not result in permanent functional or cellular loss in all vertebrates has fascinated regenerative biologists. Neuronal regeneration occurs in a subset of species, including lizards, teleost fish, axolotls, and newts. One tool for studying neuronal regeneration in the adult brain is intraventricular injection of selective neuronal toxins, which leads to loss of subpopulations of neurons. To trace cells involved in the regeneration process, plasmids encoding reporter proteins can be electroporated in vivo into the cells of interest. This protocol describes methods to label the ependymoglial cells of the brain of the red spotted newt Notophthalmus viridescens and follow their response after ablation of dopaminergic neurons.

The thermal plasticity of locomotor performance has diverged between northern and southern populations of the eastern newt (Notophthalmus viridescens).

Many temperate ectotherms undergo thermal acclimation to remain functional over a wide range of body temperatures, but few studies have investigated whether populations of a single species have evolved differences in the thermal plasticity of locomotor performance. Therefore, we asked whether the thermal plasticity of locomotor performance has diverged between northern and southern populations of eastern newts (Notophthalmus viridescens). We acclimated eastern newts from Florida and Maine to cold (6 °C) or warm (28 °C) conditions for 12 weeks. Following acclimation, we measured the burst speed of newts at 6, 11.5, 17, 22.5, 28, and 33.5 °C. We also measured the activities of creatine kinase (CK) and lactate dehydrogenase (LDH) in skeletal muscle of newts. The newts from Maine were better able to acclimate to low temperature compared to newts from Florida. Regardless of acclimation, the thermal sensitivity of burst speed was higher in the Florida compared to the Maine population. In general, newts from Maine performed better at low temperatures, whereas newts from Florida performed better at high temperatures. The activities of CK and LDH were lower in cold compared to warm-acclimated newts in the Florida population, but acclimation did not affect the activities of these enzymes in the Maine population. The activities of CK and LDH do not explain differences in the thermal plasticity of locomotor performance between populations. Our results demonstrate that the thermal sensitivity and plasticity of locomotor performance differ between northern and southern populations of eastern newts, suggesting that these traits readily adapt to the thermal environment.

Eastern Newt (Notophthalmus viridescens) larvae alter morphological but not chemical defenses in response to predator cues

Prey traits are often modified in response to exposure to predators, a phenomenon known as predator-induced phenotypic plasticity. Morphological plasticity in response to predator cues is well documented in amphibians; however, predator-induced chemical defenses have received relatively little attention. The Eastern Newt (Notophthalmus viridescens (Rafinesque, 1820)), which possesses tetrodotoxin—a toxin for chemical defense, is most vulnerable to predation during its larval stage. We assessed whether exposing Eastern Newt larvae to predator scent cues (from dragonfly larvae) would elicit change in their morphological and chemical defenses. Newt larvae exposed to scent cues of predatory dragonfly larvae exhibited significantly greater tail depths, which should increase survival of attempted predation by allowing them to swim faster, but did not differ in mass, snout–vent length, or tail length. Toxin concentrations in newt larvae were not significantly affected by exposure to these predator cues. Larval toxicity may be derived maternally or environmentally and is inflexible, or induced toxicity may only be detectable later in development. Predator-induced phenotypic plasticity, especially of chemical defenses, warrants greater attention, as potentially important outcomes of species interactions remain unclear.

A9.2 Tenascin-C expression during knee joint regeneration in the red-spotted NEWT notophthalmus viridescens

BackgroundIn mammals, tissue or organ damage often results in fibrotic scar formation, which in turn may impair proper organ function. Many urodele amphibians are capable of regenerating different tissues including...

AB0140 Investigation of joint regeneration mechanisms in NEWTS (notophthalmus viridescens)

BackgroundThe adult red-spotted newt (notophthalmus viridescens) is able to regenerate damaged knee joints after local injury involving surgically induced defects, and collagenase induced joint instability. The mechanism behind this capacity...

A de novo assembly of the newt transcriptome combined with proteomic validation identifies new protein families expressed during tissue regeneration

BackgroundNotophthalmus viridescens, an urodelian amphibian, represents an excellent model organism to study regenerative processes, but mechanistic insights into molecular processes driving regeneration have been hindered by a paucity and poor annotation of coding nucleotide sequences. The enormous genome size and the lack of a closely related reference genome have so far prevented assembly of the urodelian genome.ResultsWe describe the de novo assembly of the transcriptome of the newt Notophthalmus viridescens and its experimental validation. RNA pools covering embryonic and larval development, different stages of heart, appendage and lens regeneration, as well as a collection of different undamaged tissues were used to generate sequencing datasets on Sanger, Illumina and 454 platforms. Through a sequential de novo assembly strategy, hybrid datasets were converged into one comprehensive transcriptome comprising 120,922 non-redundant transcripts with a N50 of 975. From this, 38,384 putative transcripts were annotated and around 15,000 transcripts were experimentally validated as protein coding by mass spectrometry-based proteomics. Bioinformatical analysis of coding transcripts identified 826 proteins specific for urodeles. Several newly identified proteins establish novel protein families based on the presence of new sequence motifs without counterparts in public databases, while others containing known protein domains extend already existing families and also constitute new ones.ConclusionsWe demonstrate that our multistep assembly approach allows de novo assembly of the newt transcriptome with an annotation grade comparable to well characterized organisms. Our data provide the groundwork for mechanistic experiments to answer the question whether urodeles utilize proprietary sets of genes for tissue regeneration.

Multi-tissue microarray analysis identifies a molecular signature of regeneration.

The inability to functionally repair tissues that are lost as a consequence of disease or injury remains a significant challenge for regenerative medicine. The molecular and cellular processes involved in complete restoration of tissue architecture and function are expected to be complex and remain largely unknown. Unlike humans, certain salamanders can completely regenerate injured tissues and lost appendages without scar formation. A parsimonious hypothesis would predict that all of these regenerative activities are regulated, at least in part, by a common set of genes. To test this hypothesis and identify genes that might control conserved regenerative processes, we performed a comprehensive microarray analysis of the early regenerative response in five regeneration-competent tissues from the newt Notophthalmus viridescens. Consistent with this hypothesis, we established a molecular signature for regeneration that consists of common genes or gene family members that exhibit dynamic differential regulation during regeneration in multiple tissue types. These genes include members of the matrix metalloproteinase family and its regulators, extracellular matrix components, genes involved in controlling cytoskeleton dynamics, and a variety of immune response factors. Gene Ontology term enrichment analysis validated and supported their functional activities in conserved regenerative processes. Surprisingly, dendrogram clustering and RadViz classification also revealed that each regenerative tissue had its own unique temporal expression profile, pointing to an inherent tissue-specific regenerative gene program. These new findings demand a reconsideration of how we conceptualize regenerative processes and how we devise new strategies for regenerative medicine.

Induction of human lampbrush chromosomes

We previously demonstrated that sperm heads from amphibians (Xenopus and Rana) and zebrafish (Danio) could form giant lampbrush chromosomes when injected into the nucleus of amphibian oocytes. However, similar experiments with mammalian sperm heads were unsuccessful. Here, we describe a slightly modified procedure and demonstrate that human sperm heads can form giant lampbrush chromosomes when injected into the oocyte nucleus of the frog Xenopus laevis or the newt Notophthalmus viridescens. Human and other mammalian chromosomes do not form recognizable lampbrush chromosomes in their own oocytes or in any somatic cells. These experiments thus demonstrate that the lampbrush condition is an inducible state and that the amphibian oocyte nucleus contains all factors required to remodel the inactive chromatin of a mammalian sperm into a transcriptionally active state. They also demonstrate that absence of lampbrush chromosomes from human oocytes must relate to specific features of mammalian oogenesis, not to permanent genetic or epigenetic changes in the chromatin.