Iberian Ribbed Newt Research Articles

Effective to census population size ratios in two Near Threatened Mediterranean amphibians: Pleurodeles waltl and Pelobates cultripes

Efforts to mitigate amphibian declines are hindered by a lack of information about basic aspects of their biology and demography. The effective to census population size ratio (Ne/Nc) is one of the most important parameters for the management of wildlife populations because it combines information on population abundance and genetic diversity and helps predict population viability in the long term. Few studies have calculated this ratio in amphibians, which sometimes show low ratios, associated with a higher extinction risk. Here we integrate field-based (capture-mark-recapture studies, egg string counts) and molecular approaches (estimation of the effective number of breeders (Nb) and the effective population size (Ne) based on genotypes from larval cohorts and candidate parents) to produce the first estimates of the Ne/Nc and Nb/Nc ratios in two amphibians, the Iberian ribbed newt Pleurodeles waltl and the western spadefoot Pelobates cultripes. Additionally, we investigate sex-biased dispersal in both species based on direct (field observations) and indirect (genetic) evidence. Both species showed similar ratios, slightly lower in Pleurodeles (0.21–0.24) than in Pelobates (0.25–0.30). Observed displacement rates were low in both species (P. waltl = 0.51%; P. cultripes = 1.23%). We found no evidence for sex-biased dispersal in P. cultripes, but both direct and indirect evidences suggest a tendency for female-biased dispersal in P. waltl. We discuss differences in the genetic estimates of Ne and Nb provided by three inference methods and the implications of our findings for the management of these species, characteristic of Mediterranean wetlands in the Iberian Peninsula and listed as Near Threatened.

Effects of cadmium exposure on Iberian ribbed newt (<i>Pleurodeles waltl</i>) testes

To characterize the histomorphologic effects of cadmium on adult newt testes, male Iberian ribbed newts (6 months post-hatching) were intraperitoneally exposed to a single dose of 50 mg/kg of cadmium, with histologic analysis of the testes at 24, 48, 72, and 96 h. Beginning 24 h after cadmium exposure, apoptosis of spermatogonia and spermatocytes was observed, and congestion was observed in the interstitial vessels of the testes. Throughout the experimental period, the rates of pyknotic cells and TUNEL and cleaved caspase-3 positivity were significantly higher in the spermatogonia and spermatocytes of cadmium-treated newts compared with control newts. There were no significant differences between cadmium-treated and control newts in phospho-histone H3 positivity in the spermatogonia and spermatocytes. These results suggest that spermatogonia and spermatocytes in adult Iberian ribbed newts are highly sensitive to cadmium. This is the first report of the histomorphologic characteristics of cadmium-induced testicular dysfunction in newts.

Male Courtship Pheromones Induce Cloacal Gaping in Female Newts (Salamandridae).

Pheromones are an important component of sexual communication in courting salamanders, but the number of species in which their use has been demonstrated with behavioral evidence remains limited. Here we developed a behavioral assay for demonstrating courtship pheromone use in the aquatically courting Iberian ribbed newt Pleurodeles waltl. By performing an in-depth study of the courtship behavior, we show that females invariably open their cloaca (cloacal gaping) before engaging in pinwheel behavior, the circling movement that is the prelude to spermatophore uptake. In contrast, cloacal gaping was not observed in failed courtships, where females escaped or displayed thanatosis. Since gaping mainly occurred during male amplexus and cloacal imposition, which is the obvious period of pheromone transfer, we next investigated whether male courtship water (i.e., water holding courtship pheromones) alone was able to induce this reaction in females. These tests showed that courtship water induced cloacal gaping significantly more than water, even in the absence of a male. Cloacal gaping thus provides a simple and robust test for demonstrating courtship pheromone use in the Iberian ribbed newt. Since opening the cloaca is an essential prerequisite for spermatophore pick-up in all internally fertilizing salamanders, we hypothesize that variations on this assay will also be useful in several other species.

Mutagenesis in Newts: Protocol for Iberian Ribbed Newts.

Newts have the remarkable capability of organ/tissue regeneration, and have been used as a unique experimental model for regenerative biology. The Iberian ribbed newt (Pleurodeles waltl) is suitable as a model animal. We have established methods for artificial insemination and efficient transgenesis using P. waltl newts. In addition to the transgenic technique, development of TALENs enables targeting mutagenesis in the newts. We have reported that TALENs efficiently disrupted targeted genes in newt embryos. In this chapter, we introduce a protocol for TALEN-mediated gene targeting in Iberian ribbed newts.

Gene manipulation for regenerative studies using the Iberian ribbed newt, Pleurodeles waltl.

Newts provide a unique model animal for regenerative studies. An experimental model system for molecular genetics in newts is needed in order to clarify the mechanisms of regeneration from the perspective of gene functions. We have identified that Iberian ribbed newt (Pleurodeles waltl) is a suitable model animal for such studies. Here we describe protocols for gene manipulation using Pleurodeles waltl.

Molecular cloning and expression analysis of Pleurodeles waltl complement component C3 under normal physiological conditions and environmental stresses

C3 is a component of the complement system that plays a central role in immunity, development and tissue regeneration. In this study, we isolated the C3 cDNA of the Iberian ribbed newt Pleurodeles waltl. This cDNA encodes a 1637 amino acid protein with an estimated molecular mass of 212.5kDa. The deduced amino acid sequence showed that P. waltl C3 contains all the conserved domains known to be critical for C3 function. Quantitative real-time PCR (qRT-PCR) demonstrated that under normal physiological conditions, P. waltl C3 mRNA is expressed early during development because it is likely required for neurulation. Then, its expression increased as the immune system developed. In adults, the liver is the richest source of C3, though other tissues can also contribute. Further analysis of C3 expression demonstrated that C3 transcription increased when P. waltl larvae were exposed to pH or temperature stress, suggesting that environmental modifications might affect this animal’s defenses against pathogens.

Development and characterization of twelve new polymorphic microsatellite loci in the Iberian ribbed newt, Pleurodeles waltl (Caudata: Salamandridae), with data on cross-amplification in P. nebulosus

Twelve novel polymorphic microsatellite loci were isolated and characterized for the Iberian ribbed newt, Pleurodeles waltl (Caudata, Salamandridae). The distribution of this newt ranges from central and southern Iberia to northwestern Morocco. Polymorphism of these novel loci was tested in 40 individuals from two Iberian populations and compared with previously published markers. The number of alleles per locus ranged from two to eight. Observed and expected heterozygosity ranged from 0.13 to 0.57 and from 0.21 to 0.64, respectively. Cross-species amplification was tested in Pleurodeles nebulosus, which is listed as Vulnerable by the IUCN. Eight new and seven previously published loci amplified successfully in that species and thus represent a valuable conservation tool. The novel microsatellites will be useful for a better understanding of the population dynamics, demography, genetic structure, and evolutionary history of Pleurodeles waltl and P. nebulosus.

Transcription activator‐like effector nucleases efficiently disrupt the target gene in Iberian ribbed newts (Pleurodeles waltl), an experimental model animal for regeneration

Regeneration of a lost tissue in an animal is an important issue. Although regenerative studies have a history of research spanning more than a century, the gene functions underlying regulation of the regeneration are mostly unclear. Analysis of knockout animals is a very powerful tool with which to elucidate gene function. Recently, transcription activator-like effector nucleases (TALENs) have been developed as an effective technique for genome editing. This technique enables gene targeting in amphibians such as newts that were previously impossible. Here we show that newts microinjected with TALEN mRNAs designed for targeting the tyrosinase gene in single-cell stage embryos revealed an albino phenotype. Sequence analysis revealed that the tyrosinase genes were effectively disrupted in these albino newts. Moreover, precise genome alteration was achieved using TALENs and single strand oligodeoxyribonucleotides. Our results suggest that TALENs are powerful tools for genome editing for regenerative research in newts.

Modulation of Pleurodeles waltl DNA Polymerase mu Expression by Extreme Conditions Encountered during Spaceflight

DNA polymerase µ is involved in DNA repair, V(D)J recombination and likely somatic hypermutation of immunoglobulin genes. Our previous studies demonstrated that spaceflight conditions affect immunoglobulin gene expression and somatic hypermutation frequency. Consequently, we questioned whether Polμ expression could also be affected. To address this question, we characterized Polμ of the Iberian ribbed newt Pleurodeles waltl and exposed embryos of that species to spaceflight conditions or to environmental modifications corresponding to those encountered in the International Space Station. We noted a robust expression of Polμ mRNA during early ontogenesis and in the testis, suggesting that Polμ is involved in genomic stability. Full-length Polμ transcripts are 8–9 times more abundant in P. waltl than in humans and mice, thereby providing an explanation for the somatic hypermutation predilection of G and C bases in amphibians. Polμ transcription decreases after 10 days of development in space and radiation seem primarily involved in this down-regulation. However, space radiation, alone or in combination with a perturbation of the circadian rhythm, did not affect Polμ protein levels and did not induce protein oxidation, showing the limited impact of radiation encountered during a 10-day stay in the International Space Station.

Molecular genetic system for regenerative studies using newts

Urodele newts have the remarkable capability of organ regeneration, and have been used as a unique experimental model for more than a century. However, the mechanisms underlying regulation of the regeneration are not well understood, and gene functions in particular remain largely unknown. To elucidate gene function in regeneration, molecular genetic analyses are very powerful. In particular, it is important to establish transgenic or knockout (mutant) lines, and systematically cross these lines to study the functions of the genes. In fact, such systems have been developed for other vertebrate models. However, there is currently no experimental model system using molecular genetics for newt regenerative research due to difficulties with respect to breeding newts in the laboratory. Here, we show that the Iberian ribbed newt (Pleurodeles waltl) has outstanding properties as a laboratory newt. We developed conditions under which we can obtain a sufficient number and quality of eggs throughout the year, and shortened the period required for sexual maturation from 18 months to 6 months. In addition, P. waltl newts are known for their ability, like other newts, to regenerate various tissues. We revealed that their ability to regenerate various organs is equivalent to that of Japanese common newts. We also developed a method for efficient transgenesis. These studies demonstrate that P. waltl newts are a suitable model animal for analysis of regeneration using molecular genetics. Establishment of this experimental model will enable us to perform comparable studies using these newts and other vertebrate models.

Gravity changes during animal development affect IgM heavy‐chain transcription and probably lymphopoiesis

Our previous research demonstrated that spaceflight conditions affect antibody production in response to an antigenic stimulation in adult amphibians. Here, we investigated whether antibody synthesis is affected when animal development occurs onboard a space station. To answer this question, embryos of the Iberian ribbed newt, Pleurodeles waltl, were sent to the International Space Station (ISS) before the initiation of immunoglobulin heavy-chain expression. Thus, antibody synthesis began in space. On landing, we determined the effects of spaceflight on P. waltl development and IgM heavy-chain transcription. Results were compared with those obtained using embryos that developed on Earth. We find that IgM heavy-chain transcription is doubled at landing and that spaceflight does not affect P. waltl development and does not induce inflammation. We also recreated the environmental modifications encountered by the embryos during their development onboard the ISS. This strategy allowed us to demonstrate that gravity change is the factor responsible for antibody heavy-chain transcription modifications that are associated with NF-κB mRNA level variations. Taken together, and given that the larvae were not immunized, these data suggest a modification of lymphopoiesis when gravity changes occur during ontogeny.

Dynamics of body mass and oxygen consumption in the ribbed newt Pleurodeles waltl ontogenesis: 1. Endotrophic development

The correlation between parameters of growth and energy metabolism in the example of embryonic and larval development of the Iberian ribbed newt Pleurodeles waltl has been studied. The wet body mass increases five times during this period due to water absorption by developing tissues and the yolk, and the dry mass decreases 1.18 times. The highest specific growth rate of developing tissues was noticed at the 33rd stage of embryogenesis (13th-14th day of development). These indexes decreased after the hatching, but increased after larvae switched to external nutrition. Comparison of the studied parameters has identified a similar features in alteration of the specific growth rate, intensity of oxygen consumption, and watering of developing tissues in early development of the Iberian ribbed newt.

The nasal bone of the Iberian ribbed newt (Pleurodeles waltl; Salamandridae, Urodela): Development and regulatory mechanisms of its ontogeny

The nasal bone of the Iberian ribbed newt (Pleurodeles waltl; Salamandridae, Urodela): Development and regulatory mechanisms of its ontogeny

Lifelong testicular differentiation in Pleurodeles waltl (Amphibia, Caudata).

BackgroundIn numerous Caudata, the testis is known to differentiate new lobes at adulthood, leading to a multiple testis. The Iberian ribbed newt Pleurodeles waltl has been studied extensively as a model for sex determination and differentiation. However, the evolution of its testis after metamorphosis is poorly documented.MethodsTestes were obtained from Pleurodeles waltl of different ages reared in our laboratory. Testis evolution was studied by several approaches: morphology, histology, immunohistochemistry and RT-PCR. Surgery was also employed to study testis regeneration.ResultsIn this species, the testis is linked to the lung. This association consists of connective tissue derived from the mesorchium and the coelomic epithelium surrounding the lung and takes place at the end of larval life. This tissue contains lobules including primordial germ cells with a typical large and polylobular nucleus. The anterior part of the testis remains thin and undifferentiated while the posterior part differentiates in a large first testis lobe where spermatogenesis occurs during the first year of life. The undifferentiated status of the anterior part is attested by the lack of expression of the testis marker Dmrt1 and the meiosis entry marker Dmc1. Three-year-old Pleurodeles waltl possess multiple testes made up of two lobes. The second lobe appears at the caudal extremity of the first one from residual primordial germ cells located near or even inside efferent ducts in the glandular tissue that usually appears following spermatozoa extrusion. Surprisingly, in the case of surgical elimination of the anterior part of the testis, de novo spermatogenesis is stopped in the first lobe which becomes restricted to the glandular tissue. Following first testis lobe removal, the anterior part of the testis regenerates a new testis lobe, a process stimulated in the presence of DHT.ConclusionPleurodeles waltl constitute an original gonochoristic vertebrate model in which testis differentiation is observed up to adulthood.

IgX antibodies in the urodele amphibian Ambystoma mexicanum

Until recently, it was believed that urodele amphibians are able to synthesize only two immunoglobulin isotypes, IgM and IgY. We reinvestigated this issue in the Iberian ribbed newt Pleurodeles waltl and reported recently that this urodele expresses at least three isotypes: IgM, IgP and IgY. In this study, we demonstrate that another urodele, Ambystoma mexicanum, has also a third isotype whose amino acid sequence presents the highest homology with the amino acid sequence of Xenopus IgX. This isotype has typical Ig H-chain characteristics, could form multimers and is mainly expressed in mucosal tissues thereby indicating that it is likely the physiological counterpart of Xenopus IgX and mammalian IgA. Interestingly, no IgP could be found in A. mexicanum, in contrast to P. waltl, in which IgX was not found in previous investigations. These data indicate, for the first time, that different families of urodeles can express different immunoglobulin isotypes.