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We studied a Triops population from a Mediterranean temporary pond located in Guadalcázar, a township in the western part of Córdoba Province. There are four morphologically similar species of Triops occurring in the SW Iberian Peninsula. In particular, the two species found in southern Andalusia, T. baeticus and T. gadensis, cannot reliably be told apart based on known morphological characters. We thus used sequences of 12S rDNA as a molecular marker to soundly identify the population to species rank. Our results demonstrate that the Triops population studied belongs to T. gadensis. This result is rather unexpected given that the species was thought to be restricted to a small distribution range along the Atlantic coast in Cádiz Province. The population is located at approximately 170 km from previously known sites of T. gadensis and shows a previously unknown haplotype, the sixth 12S haplotype reported for the species. Based on a molecular clock designed for dating divergence events among conspecific samples of Triops, the haplotype is estimated to have diverged at 10.3–29.4 ka BP. We thus suggest that the population has high conservation value, and its habitat, including the surroundings, should be legally protected. We further propose that an open habitat type as pastureland should be retained for the surroundings of the site in order to support its ecological function in the frame of meta-populations dynamics, and as a possible source population from which new localities could be populated via passive dispersal.

Wetland ecosystems worldwide are threatened by habitat alteration, climate change and the introduction of invasive species, even within protected areas. Unravelling the reliance of sensitive wetland‐dwelling species, such as amphibians, on habitat characteristics is thus essential to identify conservation targets.Here we assess the distribution of genetic diversity of two strongly aquatic amphibians (Pelophylax perezi and Pleurodeles waltl) in association with habitat features across the most extensive, protected wetland of the Iberian Peninsula: Doñana National Park.Despite inhabiting a protected area free from anthropogenic barriers, the genetic diversity of P. perezi and P. waltl is not homogeneously distributed across the wetland, but instead concentrates in core areas, mainly in the northern zone. Both genetic diversity and connectivity (as opposed to genetic differentiation) showed significant positive associations with the area of the breeding sites and the flooded area surrounding the breeding sites within the dispersal potential of either species, that is nearby pond availability.Large water bodies connected to abundant temporary ponds are key for the maintenance of amphibian genetic diversity. Nevertheless, the core populations of our target species, which show markedly aquatic habits, are concentrated in areas colonised by invasive species, which could compromise their long‐term viability.Our results highlight that maintaining widely connected arrays of ponds of different hydroperiods, including large breeding sites free from invasive predators and competitors, is paramount for amphibian conservation in Mediterranean wetlands. [ABSTRACT FROM AUTHOR]

Turbidity represents an obstacle to visual acuity underwater that might entail considerable consequences for multiple faunal traits. Morphology, especially eye size, could be adjusted to cope with reduced visibility in turbid water. Moreover, turbidity could interfere with colour signals or crypsis. Turbid water may simultaneously impair the ability of prey and predators to detect each other, which may cause reduced speed, flight initiation distance, activity time, boldness, and exploratory behaviour by prey.In this work, we compared these traits in larvae of the newt Pleurodeles waltl from transparent and turbid ponds (around 100 and 300 FTU, respectively). Body size did not differ between pond types, but larvae from turbid ponds had smaller eyes, probably because visual acuity is structurally hindered by the environment. Colouration did not differ between transparent and turbid ponds on average, but it was more variable in larvae from transparent than turbid ponds, probably because transparent ponds trigger crypsis towards diverse background colours, whereas turbid ponds promote reduced pigmentation in all cases.In addition, behavioural traits were gauged in larvae from both types of ponds, in tests conducted in either transparent or turbid water. Speed was unaffected by turbidity, but larvae from turbid ponds increased their flight initiation distance when tested in transparent water. In all cases, larvae from transparent ponds were more active, and bolder in transparent water, and all larvae were more exploratory when tested in transparent water (regardless of which pond type they came from).Turbidity could trigger more cautious behaviours, probably because it makes predators harder to track. Alternatively, transparent water could stimulate a more expansive use of space through improved visibility, or it might encourage a more active search for shelter.These results shed light on the role of turbidity as a promotor of morphological and behavioural variability in amphibian larvae. These ecological consequences of water turbidity can be framed in terms of energy allocation and detectability in the context of predator–prey interactions. [ABSTRACT FROM AUTHOR]

Pleurodeles waltl is coming to light as a model animal, especially in regeneration studies, but deep studies on the molecular mechanisms have been limited due to the absence of primary tissue cells for wide usage. Therefore, we aimed to grow primary cells from limb tissue of P. waltl for in vitro experiments. Limb tissues were cut into small pieces and seeded as "explants" on culture dishes coated with fibronectin and gelatin. Compared to the control without coating, both fibronectin and gelatin supported quicker outgrowth of cells from explants and faster cell adhesion, and fibronectin showed significantly better performance than gelatin. Interestingly, the doubling time of cells on fibronectin‐ and gelatin‐coated surfaces was almost the same (42.39 ± 2.79 h vs. 42.91 ± 3.69 h) and was not significantly different from that on non‐coated plates (49.64 ± 3.63 h). The cryopreserved cells were successfully recovered and showed a multiplication capacity that was similar to that of fresh cells. Senescent cells were barely detected even after long‐term sub‐culture (>15 passages). Moreover, enhanced fluorescence of MitoSOX™ Red in cells under H2O2 exposure confirmed the respondence to chemical stimuli. Collectively, our results show that we are able to grow enough good‐quality cells from P. waltl limb tissue for in vitro experiments, and fibronectin coating provides the best biocompatible environment for cell outgrowth and attachment. [ABSTRACT FROM AUTHOR]

Amphibians and fish often regenerate lost parts of their appendages (tail, limb, and fin) after amputation. Limb regeneration in adult amphibians provides an excellent model for appendage (limb) regeneration through 3D morphogenesis along the proximodistal, dorsoventral, and anteroposterior axes in mammals, because the limb is a homologous organ among amphibians and mammals. However, manipulating gene expression in specific appendages of adult amphibians remains difficult; this in turn hinders elucidation of the molecular mechanisms underlying appendage regeneration. To address this problem, we devised a system for appendage‐specific gene induction using a simplified protocol named the "agarose‐embedded heat shock (AeHS) method" involving the combination of a heat‐shock‐inducible system and insertion of an appendage in a temperature‐controlled agarose gel. Gene expression was then induced specifically and ubiquitously in the regenerating limbs of metamorphosed amphibians, including a frog (Xenopus laevis) and newt (Pleurodeles waltl). We also induced gene expression in the regenerating tail of a metamorphosed P. waltl newt using the same method. This method can be applied to adult amphibians with large body sizes. Furthermore, this method enables simultaneous induction of gene expression in multiple individuals; further, the data are obtained in a reproducible manner, enabling the analysis of gene functions in limb and tail regeneration. Therefore, this method will facilitate elucidation of the molecular mechanisms underlying appendage regeneration in amphibians, which can support the development of regenerative therapies for organs, such as the limbs and spinal cord. [ABSTRACT FROM AUTHOR]

Naturalized quarries can host rich amphibian communities, potentially playing an important role in local and regional population dynamics. Despite their importance for conservation, few studies have evaluated their potential to host large, viable populations and to connect breeding nuclei in neighboring areas, promoting long-term demographic resilience at the regional scale. We used photo-identification as a capture-mark-recapture method to estimate the population sizes and dispersal patterns of two amphibians of regional conservation concern (Pleurodeles waltl and Pelodytes punctatus), which breed in temporary ponds in an abandoned, naturalized quarry in SE Madrid (central Spain). We also surveyed all water bodies potentially harboring amphibian populations in an 80 km2 area centered in our study site to detect additional breeding nuclei of the two species. Both species are abundant in the core study site, with estimated population sizes of around 300 individuals in P. waltl and about 800 in P. punctatus. We recorded 23 accumulated displacements, ranging from 80 to 840 m, performed by 20 individuals of P. waltl captured on multiple occasions, but were unable to detect spatial displacements of P. punctatus during the study. Regional surveys confirmed the presence of P. waltl and/or P. punctatus in 7 additional locations, mostly quarries, at distances of 0.6-4.4 km from the study site. Our results show that temporary ponds in naturalized quarries can represent key assets for the long-term resilience of amphibian populations in areas where human action has resulted in extensive habitat fragmentation and loss. [ABSTRACT FROM AUTHOR]

Background: Cardiac regeneration in the adult mouse is not substantial. Some vertebrates, such as newts and zebrafish, regenerate the heart throughout their lives. To understand how regenerative abilities differ among animal species, comparative research has been conducted in animals like mouse, zebrafish, and newt. For those purposes, cryo‐injury is suitable as an experimental model for the pathological condition of human myocardial infarction. In fact, cryo‐injury procedures are common in mouse and zebrafish. Results: In the present study, we induced cryo‐damage on the ventricle in Iberian ribbed newts using a liquid nitrogen‐chilled probe. We observed that the injured area recovered within 8 weeks, with remodeling of scar tissue and proliferation of cardiomyocytes. We investigated the subsequent recovery of cryo‐injured and amputated tissues by comparative analysis of the gene expression profiles following these two procedures. Conclusions: Notably, we established a cryo‐injury procedure for the newt and confirmed that regeneration of the cryo‐damaged myocardial tissue is achieved by changes in gene expression that are milder than those observed in the amputation model. Our results suggest that the cryo‐injury method is suitable for comparing the process of cardiac regeneration in the newt with that in other animal models. Key Findings: Urodele amphibian newts can regenerate their hearts.Established a cryo‐injury model for cardiac regeneration for the newt.Genes with common expression changes in the cryo‐injury and amputation models are shown. [ABSTRACT FROM AUTHOR]

Understanding demographic dynamics and functional connectivity among demes is essential to prevent, identify and reverse amphibian population declines. Attempts to characterize connectivity among amphibian populations have largely relied on the use of molecular markers to assess patterns of genetic structure at the landscape scale and identify factors that promote or restrict gene flow. However, few studies have integrated population size estimates and direct records of dispersal rates (e.g. based on capture-mark-recapture data) to help interpret molecular-based inferences. We conducted such an integrative, long-term monitoring program of a metapopulation of the sharp-ribbed newt (Pleurodeles waltl), a pond-breeding salamander endemic to the Iberian Peninsula and North Africa. Over 12 breeding seasons, we compiled individual capture histories to estimate population sizes and document patterns of dispersal, and inferred migration rates and pairwise relatedness based on 22 microsatellite loci. Direct (based on capture-mark-recapture) estimates revealed low levels of contemporary dispersal between the two major breeding sites in the study area, whereas indirect (molecular) estimates provided evidence for asymmetric gene flow, more intense from the larger to the smaller population, without consistent differences between sexes. Direct and indirect estimates can be reconciled based on the species' generation time, and support a scenario of density-dependent dispersal, with recent colonization of the smaller pond inferred from differences in the relative proportions of ancestry categories across ponds. Our integrative approach can be applied to design conservation actions aiming to improve functional connectivity among amphibian populations based on empirical evidence. [ABSTRACT FROM AUTHOR]

Abstract One of the greatest enigmas of modern biology is how the geometry of muscular and skeletal structures are created and how their development is controlled during growth and regeneration. Scaling and shaping of vertebrate muscles and skeletal elements has always been enigmatic and required an advanced technical level in order to analyse the cell distribution in 3D. In this work, synchrotron X-ray computed microtomography (µCT) and chemical contrasting has been exploited for a quantitative analysis of the 3D-cell distribution in tissues of a developing salamander (Pleurodeles waltl) limb – a key model organism for vertebrate regeneration studies. We mapped the limb muscles, their size and shape as well as the number and density of cells within the extracellular matrix of the developing cartilage. By using tomographic approach, we explored the polarity of the cells in 3D, in relation to the structure of developing joints. We found that the polarity of chondrocytes correlates with the planes in joint surfaces and also changes along the length of the cartilaginous elements. Our approach generates data for the precise computer simulations of muscle-skeletal regeneration using cell dynamics models, which is necessary for the understanding how anisotropic growth results in the precise shapes of skeletal structures.

Animals may face trade-offs when several simultaneous functions or processes compete for limiting resources. In that context, allocation of resources to the most fitness-enhancing competing function can be prioritized. Immune response and locomotor performance are relevant fitness components, mainly due to their important roles as defence against pathogens and predators, respectively. However, both promote oxidative stress and consume energy and metabolites. Consistently, trade-offs between immune response and locomotion have been reported in some taxa. In this work, we examine whether an immune challenge hinders short-distance swimming speed of adult Iberian ribbed newts, Pleurodeles waltl. To that end, we inoculated Escherichia coli cell wall lipopolysaccharide (LPS), an innocuous antigen that triggers an immune response, in the sole pads of experimental newts, and phosphate buffer saline, with no physiological effect, in those of control newts. We estimated immune response as sole pad swelling and calculated swimming speed from videos of newts swimming towards a refuge nearby, 24 h and 7 days after inoculation. Sole pad swelling was greater in LPS-inoculated newts, but short-distance swimming speed was not affected by the immune challenge. These results could indicate priority allocation of resources to locomotion, which might be a consequence of compensatory mechanisms capable of avoiding locomotion impairment due to immune challenge. In any case, these findings could highlight the importance of short-distance escape as an antipredator strategy. • We examined potential immune system–locomotion trade-offs in adult newts. • We used Escherichia coli cell wall lipopolysaccharide (LPS) to trigger an immune response. • An immune challenge by LPS did not affect newt swimming speed. • The results were consistent 24 h and 7 days following the challenge. • Locomotion could be prioritized due to its important antipredator role. [ABSTRACT FROM AUTHOR]

The cranial ossification sequence in Pleurodeles waltl is widely used in phylogenetic analyses of amphibian origin and evolution. However, the patterns published to date are far from completely resolved and contain certain discrepancies. Based on a large sample of P. waltl specimens ranging from early post‐hatching larvae to post‐metamorphic newts, we determined the most common cranial ossification sequence and revealed its intraspecific variations. Since thyroid hormones (THs) are involved in the mediation of skull development in salamanders, we studied the role of THs in the cranial development of P. waltl. The normal sequence and timing of bone appearance were compared with those in larvae reared under conditions of high (in 1 and 2 ng mL‐1 triiodothyronine) and low [in 0.02% thiourea (TU), which inhibits thyroid gland activity] TH levels. Metamorphosis was greatly accelerated in the TH‐treated larvae and was arrested in the TU‐treated larvae, which retained the larval pattern of the palate and rudimentary external gills even after 2 years of the experiment. Early‐appearing bones (the coronoid, vomer, palatine, dentary, squamosal, premaxilla, parasphenoid, pterygoid, prearticular, vomer, frontal, parietal, exoccipital, in this order) arise at the same stages and ages, and follow the same ossification sequence under different TH levels. The timing of the appearance of bones normally arising in the late larval and metamorphic periods (the quadratojugal, orbitosphenoid, prootic, maxilla, nasal, os thyroideum, prefrontal, quadrate, in this order) changes depending on the TH level. The maxilla and nasal display the most pronounced reaction to changes in the TH level: they appear precociously in TH‐treated animals, while their appearance is postponed and they remain rudimentary in TU‐treated animals. Because of different responses to THs, the order in which late‐arising bones appear changes depending on the TH level. Although bones appearing early in larval ontogeny (e.g. the premaxilla, vomer, squamosal, palatine) display no TH‐induced reaction when they start to develop, their further differentiation shows dependence on THs, and these bones become TH‐inducible closer to metamorphosis. These findings indicate that TH involvement in the mediation of cranial development changes from minimal (if at all) in its early stages to maximal during metamorphosis. It is likely that the appearance of bones early in development is mediated by factors other than THs. Their further development is accompanied by changes in the mechanisms mediating their morphological differentiation. That is, likely non‐hormonal mediation becomes replaced or/and complemented by hormonal mediation. The constituent parts of the same bone may exhibit differences in their reactions to changes in TH levels. Although in normal development, the overall cranial ossification sequence is constant, there was variation in the order in which late‐appearing bones was recorded. These observations suggest that this variation results from individual variability in the internal TH level. Comparison with other salamanders suggests that (a) the pattern of TH mediation described in P. waltl is common for cranial development of metamorphosing urodeles and (b) the same bone may differ in its TH dependence in different salamanders, e.g. there are interspecific variations in the degree of TH dependence of individual cranial bones. [ABSTRACT FROM AUTHOR]

Foraging is key for most animals, because their energy and matter income depend on food ingestion. However, animals may respond adaptively to perceived competitor and/or predator proximity by modifying their foraging rates. Increased foraging activity is expected to improve the chances of succeeding in competition. However, the benefits of increased foraging activity could be offset by the risks of increased conspicuousness to predators. Plus, starvation may have counteracting or additive effects on foraging behaviour. Here, we test these hypotheses in larval Iberian ribbed newts (Pleurodeles waltl). We estimated foraging rates of individuals haphazardly assigned to either a restricted or an unrestricted-feeding regime, in water with (1) self-odour as control, (2) predator Dytiscus sp. chemical cues, and (3) competitor conspecific larva chemical cues. Treatments were presented in a random sequence. Newt larvae responded to either predator or conspecific chemical cues by increasing foraging rates relative to the control. Conspecific chemical cues increased foraging rates over the other two treatments. Feeding regime did not modify larva response to perceived predator and competitor proximity. Increased foraging rates in the presence of conspecific chemical cues could simultaneously improve larval energetic status to face competition and reduce prey available for the competitor. Intermediate foraging rates in the presence of predator chemical cues suggest a trade-off between the benefits (likely improved energetic status or accelerated metamorphosis) and the costs (likely increased conspicuousness) of increased food ingestion. These results prove that larval newts adjust foraging rates to perceived predator and competitor proximity. [ABSTRACT FROM AUTHOR]

As an evolutionary force, predation increases predator fitness, whereas prey future fitness is reduced to zero. Mesopredators are subjected to pressures as both predator and prey, which could generate trade-offs between predatory and anti-predatory behaviours. We subjected larvae of the mesopredator Pleurodeles waltl newt to a potential conflict of interest between reducing predation risk and fulfilling nutritional requirements. For this, we performed an experiment with a factorial design: (1) to induce different urges as predators, individuals underwent either restricted or unrestricted feeding; and (2) to induce different urges as prey, individuals were exposed to environments with and without predator snake kairomones. We recorded activity rate, swim rate, flight initiation distance (FID) and escape speed. FID increased in the presence of kairomones, as predicted by escape theory when the costs of not fleeing are greater than those of fleeing. Escape speed increased in the presence of kairomones, but only in restricted feeding larvae, probably due to lower body weight because of reduced food intake. We detected no effect of treatment on activity or swim rates. Predation risk apparently outweighed the costs of feeding opportunities lost rather than generated a behavioural trade-off. Collectively, the data suggested a greater effect of predator avoidance than of prey acquisition. [ABSTRACT FROM AUTHOR]

Background: Urodele animals can regenerate their limbs from the blastemas. The previous results of grafting proximal blastemas to distal limb levels (P to D transplantation) led to serial duplication of limb segments. However, it is unknown whether grafting to any distal levels in P to D transplantation causes serial duplication. In other words, it is unknown whether or not newt limbs can normalize such a kind of duplicated type of positional disorder in the proximal–distal axis. Therefore, we grafted the most proximal blastemas to various distal levels of the proximal–distal axis using newts (Pleurodeles waltl). The transgenic newts expressing green fluorescent protein or mCherry were used to clearly distinguish between donor and host tissues. Results: Normal segmental formation without duplication occurred in P to D transplantation within the stylopod. In addition, donor blastemas lost the fates of the stylopods, and the missing portion in the stylopod by amputation was restored by the insertion of host cells. In contrast, the blastemas from the stylopod formed whole limbs after transplantation to the tail. Conclusions: These results showed that urodele limbs can normalize the duplicated type of positional disorder within the stylopod by erasing a part of the fate in the blastemas. Developmental Dynamics 247:1276–1285, 2018. © 2018 Wiley Periodicals, Inc. Key Findings: Normal segmental formation without duplication occurred in grafting proximal blastemas to distal limb levels within the stylopod. Donor blastemas lost the fates of the stylopods. Urodele limbs can normalize the duplicated type of positional disorder within the stylopod by erasing a part of the fate in the blastemas. [ABSTRACT FROM AUTHOR]

Abstract Newts have remarkable ability to regenerate their organs and have been used in research for centuries. However, the laborious work of breeding has hampered reverse genetics strategies in newt. Here, we present simple and efficient gene knockout using Cas9 ribonucleoprotein complex (RNP) in Pleurodeles waltl , a species suitable for regenerative biology studies using reverse genetics. Most of the founders exhibited severe phenotypes against each target gene (tyrosinase , pax6 , tbx5); notably, all tyrosinase Cas9 RNP-injected embryos showed complete albinism. Moreover, amplicon sequencing analysis of Cas9 RNP-injected embryos revealed virtually complete biallelic disruption at target loci in founders, allowing direct phenotype analysis in the F 0 generation. In addition, we demonstrated the generation of tyrosinase null F 1 offspring within a year. Finally, we expanded this approach to the analysis of noncoding regulatory elements by targeting limb-specific enhancer of sonic hedgehog, known as the zone of polarizing activity regulatory sequence (ZRS; also called MFCS1). Disruption of ZRS led to digit deformation in limb regeneration. From these results, we are confident that this highly efficient gene knockout method will accelerate gene functional analysis in the post-genome era of salamanders. Highlights • Cas9-RNP targeted to tyr , pax6 , and tbx5 caused severe phenotypes in founder newts. • Cas9-RNP injected founders are designated as crispants. • Crispants exhibited virtually complete biallelic disruption in targeted genes. • The first homozygous F 1 offspring were obtained from tyr crispants within a year. • Disruption of shh limb-specific enhancer led to digit deformation in regeneration. [ABSTRACT FROM AUTHOR]

Comparative landscape genetics studies can provide key information to implement cost-effective conservation measures favouring a broad set of taxa. These studies are scarce, particularly in Mediterranean areas, which include diverse but threatened biological communities. Here, we focus on Mediterranean wetlands in central Iberia and perform a multi-level, comparative study of two endemic pond-breeding amphibians, a salamander ( Pleurodeles waltl) and a toad ( Pelobates cultripes). We genotyped 411 salamanders from 20 populations and 306 toads from 16 populations at 18 and 16 microsatellite loci, respectively, and identified major factors associated with population connectivity through the analysis of three sets of variables potentially affecting gene flow at increasingly finer levels of spatial resolution. Topographic, land use/cover, and remotely sensed vegetation/moisture indices were used to derive optimized resistance surfaces for the two species. We found contrasting patterns of genetic structure, with stronger, finer scale genetic differentiation in Pleurodeles waltl, and notable differences in the role of fine-scale patterns of heterogeneity in vegetation cover and water content in shaping patterns of regional genetic structure in the two species. Overall, our results suggest a positive role of structural heterogeneity in population connectivity in pond-breeding amphibians, with habitat patches of Mediterranean scrubland and open oak woodlands (' dehesas') facilitating gene flow. Our study highlights the usefulness of remotely sensed continuous variables of land cover, vegetation and water content (e.g., NDVI, NDMI) in conservation-oriented studies aimed at identifying major drivers of population connectivity. [ABSTRACT FROM AUTHOR]

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 ( N / N ) 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 ( N ) and the effective population size ( N ) based on genotypes from larval cohorts and candidate parents) to produce the first estimates of the N / N and N / N 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 N and N 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. [ABSTRACT FROM AUTHOR]

The relationship between animal growth and oxygen consumption was studied by the example of the postlarval period in ontogenesis of the newt Pleurodeles waltl. These parameters increased during the first 1.5 years after metamorphosis and then did not change. Gender differences were found for the body mass, growth rate, and rate of oxygen consumption, which in males were less than in females. The mass-specific rates of oxygen consumption in males and females were the same. The relationship between the body mass and the rate of oxygen consumption in immature and mature newts can be expressed by a common allometric equation. [ABSTRACT FROM AUTHOR]

Inference of population histories from the molecular signatures of past demographic processes is challenging, but recent methodological advances in species distribution models and their integration in time-calibrated phylogeographic studies allow detailed reconstruction of complex biogeographic scenarios. We apply an integrative approach to infer the evolutionary history of the Iberian ribbed newt ( Pleurodeles waltl ), an Ibero-Maghrebian endemic with populations north and south of the Strait of Gibraltar. We analyzed an extensive multilocus dataset (mitochondrial and nuclear DNA sequences and ten polymorphic microsatellite loci) and found a deep east-west phylogeographic break in Iberian populations dating back to the Plio-Pleistocene. This break is inferred to result from vicariance associated with the formation of the Guadalquivir river basin. In contrast with previous studies, North African populations showed exclusive mtDNA haplotypes, and formed a monophyletic clade within the Eastern Iberian lineage in the mtDNA genealogy. On the other hand, microsatellites failed to recover Moroccan populations as a differentiated genetic cluster. This is interpreted to result from post-divergence gene flow based on the results of IMA2 and Migrate analyses. Thus, Moroccan populations would have originated after overseas dispersal from the Iberian Peninsula in the Pleistocene, with subsequent gene flow in more recent times, implying at least two trans-marine dispersal events. We modeled the distribution of the species and of each lineage, and projected these models back in time to infer climatically favourable areas during the mid-Holocene, the last glacial maximum (LGM) and the last interglacial (LIG), to reconstruct more recent population dynamics. We found minor differences in climatic favourability across lineages, suggesting intraspecific niche conservatism. Genetic diversity was significantly correlated with the intersection of environmental favourability in the LIG and LGM, indicating that populations of P. waltl are genetically more diverse in regions that have remained environmentally favourable through the last glacial cycle, particularly southern Iberia and northern Morocco. This study provides novel insights into the relative roles of geology and climate on the biogeography of a biodiversity hotspot. [ABSTRACT FROM AUTHOR]

The reproduction of captive Pleurodeles nebulosus was followed during five breeding seasons. The breeding season lasted from the onset of autumn to early spring. Most eggs were laid in October, although oviposition occurred throughout the breeding season. Each season a female would produce one or more clutches of 19-224 eggs. Diameter of the ova was 1.63-1.65 mm. Eggs hatched after 8-12 days, and at hatching the larvae were in Harrison stages 37-38. It took 73-108 days for the larvae to reach metamorphosis and the metamorphs measured 25.6-32.1 mm in standard length. A modest correlation was found between standard length at metamorphosis and larval development time. Juveniles, which were kept aquatic, reached maturity after 9-10 months, whereas juveniles, which were reared on land, did not reach maturity until approx. 20-22 months after metamorphosis. [ABSTRACT FROM AUTHOR]

Short-range endemics are high-priority conservation targets, and valuable information for their management can be obtained from assessments of genetic diversity and structure, including potential hybridization with related species, and the investigation of factors shaping their patterns of occurrence. The Edough ribbed newt, Pleurodeles poireti, is restricted to the Edough Peninsula in Algeria, and listed as Endangered in IUCN's Red List. Its range is completely surrounded by the more widespread, closely related, Algerian ribbed newt, Pleurodeles nebulosus. Here we examined patterns of genetic diversity and structure in P. poireti using mtDNA sequences of the ND4 gene and a suite of 12 polymorphic microsatellites in a sample of 193 individuals from 46 populations covering the whole range of P. poireti and neighboring populations of P. nebulosus. We also investigated the influence of environmental variables (climate, topography, soil texture and plant cover) on the patterns of occurrence of both species. Genetic data allow fine scale delineation of the ranges of the two species and provide evidence for hybridization in several localities in a broad area of overlap. Niche analyses indicate that P. nebulosus and P. poireti tend to occupy similar niches and that the hybrid zone is not associated to an ecotone. This suggests that the range boundaries of the two species may be constrained by reciprocal exclusion, although in the case of P. poireti there are additional environmental constraints. These distributional patterns could be explained by high occupation of habitats by P. poireti, hindering the penetration of P. nebulosus by a 'density blocking' effect. [ABSTRACT FROM AUTHOR]

Urodele amphibians are an interesting model because although they possess the cardinal elements of the vertebrate immune system, their immune response is apparently subdued. This phenomenon, sometimes regarded as a state of immunodeficiency, has been attributed by some authors to limited antibody diversity. We reinvestigated this issue in Pleurodeles waltl , a metamorphosing urodele, and noted that upsilon transcripts of its IgY repertoire were as diverse as alpha transcripts of the mammalian IgA repertoire. Mu transcripts encoding the IgM repertoire were less diverse, but could confer more plasticity. Both isotypes present potential polyreactive features that may confer urodele antibodies with the ability to bind to a variety of antigens. Finally, we observed additional cysteines in CDR1 and 2 of the IGHV5 and IGHV6 domains, some of which specific to urodeles, that could allow the establishment of a disulfide bond between these CDRs. Together, these data suggest that urodele antibody diversity is not as low as previously thought. [ABSTRACT FROM AUTHOR]

Urodele amphibians (newts and salamanders) and anuran amphibians (frogs) are excellent research models to reveal mechanisms of three-dimensional organ regeneration since they have exceptionally high regenerative capacity among tetrapods. However, the difficulty in manipulating gene expression in cells in a spatially restricted manner has so far hindered elucidation of the molecular mechanisms of organ regeneration in amphibians. Recently, local heat shock by laser irradiation has enabled local gene induction even at the singlecell level in teleost fishes, nematodes, fruit flies and plants. In this study, local heat shock was made with infrared laser irradiation (IR-LEGO) by using a gene expression inducible system in transgenic animals containing a heat shock promoter, and gene expression was successfully induced only in the target region of two amphibian species, Xenopus laevis and Pleurodeles waltl (a newt), at postembryonic stages. Furthermore, we induced spatially restricted but wider gene expression in Xenopus laevis tadpoles and froglets by applying local heat shock by a temperature-controlled metal probe (temperature stimulator). The local gene manipulation systems, the IRLEGO and the temperature stimulator, enable us to do a rigorous cell lineage trace with the combination of the Cre-LoxP system as well as to analyze gene function in a target region or cells with less off-target effects in the study of amphibian regeneration. [ABSTRACT FROM AUTHOR]

Substances of a peptide nature isolated from the hepatopancreas of the king crab Paralithodes camtschaticus exhibited physicochemical properties and membranotropic and specific activities similar to those of membranotropic homeostatic tissue-specific bioregulators previously found in different mammalian and plant tissues. Their biological effect on vertebrate tissues was demonstrated on a model of roller organotypic cultivation of Pleurodeles waltl newt liver tissue. [ABSTRACT FROM AUTHOR]

Nucleotide sequences were identified in the eye tissues (lens, retina, and retinal pigment epithelium) of the adult newt Pleurodeles waltl by the polymerase chain reaction with primers for the Ns gene. Sequencing showed that these nucleotide sequences belong to the Ns gene of the newt P. waltl, which encodes the nucleolar protein nucleostemin. Structural analysis revealed a high homology of Ns nucleotide sequences of P. waltl with those of newts Cynops pyrrhogaster and Notophthalmus viridescens. The expression of the Ns gene of P. waltl, identified in the specialized eye cells of adult newts of the studied species, indicates that these differentiated cells retain some of the molecular characteristics inherent to the undifferentiated cells. [ABSTRACT FROM AUTHOR]

Following spinal lesion, connections between the supra-spinal centers and spinal neuronal networks can be disturbed, which causes the deterioration or even the complete absence of sublesional locomotor activity. In mammals, possibilities of locomotion restoration are much reduced since descending tracts either have very poor regenerative ability or do not regenerate at all. However, in lower vertebrates, there is spontaneous locomotion recuperation after complete spinal cord transection at the mid-trunk level. This phenomenon depends on a translesional descending axon re-growth originating from the brainstem. On the other hand, cellular and molecular mechanisms underlying spinal cord regeneration and in parallel, locomotion restoration of the animal, are not well known. Fibroblast growth factor 2 (FGF-2) plays an important role in different processes such as neural induction, neuronal progenitor proliferation and their differentiation. Studies had shown an over expression of this growth factor after tail amputation. Nestin, a protein specific for intermediate filaments, is considered an early marker for neuronal precursors. It has been recently shown that its expression increases after tail transection in urodeles. Using this marker and western blots, our results show that the number of FGF-2 and FGFR2 mRNAs increases and is correlated with an increase in neurogenesis especially in the central canal lining cells immediately after lesion. This study also confirms that spinal cord re-growth through the lesion site initially follows a rostrocaudal direction. In addition to its role known in neuronal differentiation, FGF-2 could be implicated in the differentiation of ependymal cells into neuronal progenitors. [ABSTRACT FROM AUTHOR]

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 212.5 kDa. 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. [ABSTRACT FROM AUTHOR]

The FGF2 signaling pathway components in tissues of the posterior wall in the normal and regenerating eye of the adult Pleurodeles waltl newt were detected for the first time. The fgf2 gene expression was found in the retina, retinal pigment epithelium, and choroid using polymerase chain reaction (PCR). A high homology of the mRNA nucleotide sequence of the most conservative fgf2 gene region in the P. waltl with the fgf2 orthologs in other vertebrates was proved. The Fgf2 protein amino acid sequence of the P. waltl newt demonstrates even more homology with this growth factor in other vertebrates. The Fgf2 protein with a molecular weight 35 kDa was found in the studied eye tissues using Western blot hybridization. Localization of the Fgf2 protein and its Fgfr receptors was immunohistochemically studied in the pigment epithelium, choroid, central and growth retina regions of the newt native eye, and in the connective cilium of photoreceptors. Using real-time PCR and immunohistochemistry methods, it was found that the fgf2 gene down-regulation and a decrease in the intensity of the immunochemical reaction of its protein product (Fgf2) occur in the early period after the retina removal (in 4-8 days) (as compared with those in the same department of the unoperated eye). [ABSTRACT FROM AUTHOR]

Enamel and enameloid, the highly mineralized tooth-covering tissues in living vertebrates, are different in their matrix composition. Enamel, a unique product of ameloblasts, principally contains enamel matrix proteins (EMPs), while enameloid possesses collagen fibrils and probably receives contributions from both odontoblasts and ameloblasts. Here we focused on type I collagen (COL1A1) and amelogenin (AMEL) gene expression during enameloid and enamel formation throughout ontogeny in the caudate amphibian, Pleurodeles waltl. In this model, pre-metamorphic teeth possess enameloid and enamel, while post-metamorphic teeth possess enamel only. In first-generation teeth, qPCR and in situ hybridization (ISH) on sections revealed that ameloblasts weakly expressed AMEL during late-stage enameloid formation, while expression strongly increased during enamel deposition. Using ISH, we identified COL1A1 transcripts in ameloblasts and odontoblasts during enameloid formation. COL1A1 expression in ameloblasts gradually decreased and was no longer detected after metamorphosis. The transition from enameloid-rich to enamel-rich teeth could be related to a switch in ameloblast activity from COL1A1 to AMEL synthesis. P. waltl therefore appears to be an appropriate animal model for the study of the processes involved during enameloid-to-enamel transition, especially because similar events probably occurred in various lineages during vertebrate evolution. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

Dispersal and vicariance are the principal mechanisms responsible for the formation of biogeographic patterns, driven or maintained by the role of past and current barriers to dispersal. Southwest Portugal harbors a rich endemic fauna and flora where strongly differentiated taxa can be observed, suggesting ancient isolating mechanisms acted in this region. In this study, we used information from two partial mitochondrial sequences, combined with information from microsatellite loci and amplification success, to investigate biogeographic patterns of genetic divergence in populations of Pleurodeles waltl in Portugal. Our results demonstrate genetic differentiation at different time frames creating distinct populations in southwest Portugal, which might have arisen following isolation by a large river estuary and mountainous barriers. Habitat destruction associated with agricultural intensification raises conservation concerns over the unique biodiversity in this region. [ABSTRACT FROM AUTHOR]

Highlights: [•] Microgravity modifies microbial physiology, pathogenesis and virulence. [•] Space travel has immunosuppressive effects that increase vulnerability to infections. [•] Pleurodeles waltl is an adequate model for studying immunity under space conditions. [•] Spaceflight is a model of stress with potential societal impact. [ABSTRACT FROM AUTHOR]

Expression of genes and heat shock proteins in normal intact retina of the Spanish Ribbed Newt Pleurodeles waltl was studied using polymerase chain reaction, Western blot hybridization, and immunohis-tochemistry. It was shown that the proteins HSP70 and HSP90, as well as their encoding transcripts of relevant genes, are constitutively expressed in eye tissues. These proteins were distributed differentially, and they were characterized by expression of different levels in the retina: HSP70 dominated in the external retina, while HSP90 dominated in the internal one, in particular, in Muller glial cells and the optic nerve. Transcripts and heat shock proteins HSP70 and HSP90 were also found in the retinal pigment epithelium and eye growth zone. [ABSTRACT FROM AUTHOR]

The sequence of appearance of calretinin and calbindin-D28k immunoreactive (CRir and CBir, respectively) cells and fibers has been studied in the brain of the urodele amphibian Pleurodeles waltl. Embryonic, larval and juvenile stages were studied. The early expression and the dynamics of the distribution of CBir and CRir structures have been used as markers for developmental aspects of distinct neuronal populations, highlighting the accurate extent of many regions in the developing brain, not observed on the basis of cytoarchitecture alone. CR and, to a lesser extent, CB are expressed early in the central nervous system and show a progressively increasing expression from the embryonic stages throughout the larval life and, in general, the labeled structures in the developing brain retain their ability to express these proteins in the adult brain. The onset of CRir cells primarily served to follow the development of the olfactory bulbs, subpallium, thalamus, alar hypothalamus, mesencephalic tegmentum, and distinct cell populations in the rhombencephalic reticular formation. CBir cells highlighted the development of, among others, the pallidum, hypothalamus, dorsal habenula, midbrain tegmentum, cerebellum, and central gray of the rostral rhombencephalon. However, it was the relative and mostly segregated distribution of both proteins in distinct cell populations which evidenced the developing regionalization of the brain. The results have shown the usefulness in neuroanatomy of the analysis during development of the onset of CBir and CRir structures, but the comparison with previous data has shown extensive variability across vertebrate classes. Therefore, one should be cautious when comparing possible homologue structures across species only on the basis of the expression of these proteins, due to the variation of the content of calcium-binding proteins observed in well-established homologous regions in the brain of different vertebrates. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Vernal pools are preferred breeding habitats for numerous amphibian species. The analysis of prey composition during the amphibian larval stages is necessary to understand diet composition and food choice in aquatic communities. We studied the diet of larvae of Pleurodeles waltl when exogenous feeding begins and throughout the flooding period in an episodic Mediterranean pond. The stomach contents were analyzed and the diet was compared to the availability of prey in the pond. The diet analysis revealed an ample diversity of prey, the most abundant being cyclopoid and calanoid copepods and podocopid ostracods. Changes in the composition of the diet corresponded principally to the variations in the availability of different prey as a consequence of the succession of the zooplankton community throughout the flooding period. However, certain taxa such as large anostracans, small anomopods, and turbellarias were not consumed although they were available and abundant. In contrast, prey scarcely represented in the pond such as coleopteran, dipteran, and hemipteran larvae were efficiently captured, suggesting their accessibility to newt larvae. The proportion of prey found in stomachs with respect to their abundance in the pond increased with larval size, coinciding with a reduction in number and bio-volume of available prey. The results suggest that newt larvae are opportunistic feeders and that breeding success of newts may be highly dependent on early egg-laying to match the abundant food supply found at early flooding stages of vernal pools. [ABSTRACT FROM AUTHOR]

Subterminal lampbrush loops of one of the 12 bivalents of the oocyte karyotype of Pleurodeles waltl (Amphibian, Urodele) underwent prominent morphological changes upon in vitro culture. These loops exhibited a fine ribonucleoprotein (RNP) granular matrix, which evolved during culture into huge structures that we have named 'chaussons' (slippers). This phenomenon involved progressive accumulation of proteins in the RNP matrix without protein neosynthesis. One of these proteins, which translocated into the nucleus during the culture, was identified as a homolog of the human Ro52 E3 ubiquitin ligase. RNA polymerase III was also found to accumulate on the same loops. These results suggest that the subterminal loops of bivalent XII act as a storage site for the components of a nuclear machinery involved in the quality control of RNA synthesis and maturation in response to cellular stress. They also emphasise the considerable value of the lampbrush chromosome system for a direct visualisation of modifications in gene expression and open the question of a nuclear accumulation of Ro52 in human or animal oocytes cultured in vitro for assisted reproductive technologies (ART). [ABSTRACT FROM AUTHOR]

Gravity-dependent shape alterations in newt tail regenerates are described, which were previously noticed in experiments onboard satellites Foton M2, M3 and in corresponding laboratory controls. Laboratory conditions were developed that allow reproducing this phenomenon persistently in the adult newts Pleurodeles waltl (Michahelles, 1830). The newts kept in an aquarium (in partial weightlessness) after 1/3 tail amputation developed normal lanceolate regenerates, while those that stayed on a moist mat (exposed to greater gravity than in aquarium) developed curved tail regenerates. Dynamics of the shape alterations were described using computer morphometric analysis. The curve was shown to develop at stage III of regeneration and to be caused by bending of the developing axial structures: the ependymal tube and the cartilage rode. Cellular processes were described that accompany the tail shape changes, such as cell migration and formation of dense aggregates. Unequal proliferation throughout the wound epidermis and blastema was revealed using BrdU assay. Proliferation increased within dorsal and apical regions of the regenerates in the newts kept on the mat cell compared with the aquarian animals. [ABSTRACT FROM AUTHOR]

ABSTRACT Stimulus deprivation or stimulus augmentation can induce long-lasting modifications to sensory and motor systems. If deprivation is effective only during a limited period of life this phase is called 'critical period.' A critical period was described for the development of the roll-induced vestibuloocular reflex (r VOR) of Xenopus laevis using spaceflights. Spaceflight durations and basic conditions of Xenopus' development did not make it possible to answer the question whether exposure of the immature vestibular organ to weightlessness affects r VOR development. The embryonic development of Pleurodeles waltl is slow enough to solve this problem because the r VOR cannot be induced before 15 dpf. Stage 20-21 embryos (4 dpf) were exposed to microgravity during a 10-day spaceflight, or to 3 g hypergravity following the same time schedule. After termination of altered gravity, the r VOR was recorded twice in most animals. The main observations were as follows: (1) after the first r VOR appearance at stage 37 (16 dpf), both r VOR gain and amplitude increased steadily up to saturation levels of 0.22 and 20°, respectively. (2) Three days after termination of microgravity, flight and ground larvae showed no r VOR; 1 day later, the r VOR could be induced only in ground larvae. Differences disappeared after 3 weeks. (3) For 10 days after 3 g exposure, r VOR development was similar to that of 1 g-controls but 3 weeks later, 3 g-larvae showed a larger r VOR than 1 g-controls. These observations indicate that the immature vestibular system is transiently sensitive to microgravity exposure and that exposure of the immature vestibular system to hypergravity leads to a slowly growing vestibular sensitization. J. Exp. Zool. 317A:333-346, 2012. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The correlation between characteristics of growth and energy metabolism during the larval stage of development of the Spanish ribbed newt ( Pleurodeles waltl) has been studied. During this period, its body mass is found to increase 140 times and the oxygen consumption rate, 77 times. The highest rate of specific body mass increase and oxygen consumption rate are noted in the early larval stage. Later, these characteristics decrease except for a brief period before completion of metamorphosis when the rate specific body mass increase rises. Comparison of the studied characteristics allows us to note a similar pattern in changes of the specific growth rate and the oxygen consumption rate during the premetamorphic development of the Spanish ribbed newt. [ABSTRACT FROM AUTHOR]

The article presents a study on Pleurodeles waltl, a species that displays a ZZ/ZW genetic mode of sex determination and a temperature-dependent mechanism of female-to-male sex reversal. It was found that the deduced protein segregates from the group of anuran and could be more closely associated with amniote. The results indicate that in Pleurodeles waltl, the SOX family of transcription factors could play a role during the late phase of gonad differentiation instead of sex determination.