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.
Using an original protocol, we are able to grow primary cells from limb tissue of Pleurodeles waltl. The cells can be maintained by long‐term sub‐culturing without senescence induction and can be recovered from cryopreservation. High‐quality primary tissue cells from P. waltl may become useful for future studies.
Muellerian inhibiting substance (MIS, also known as anti-Muellerian hormone), is a key factor of male sex differentiation in vertebrates. In amniotes, it is responsible for Muellerian duct regression ...in male embryos. In fish, despite the absence of Muellerian ducts, MIS is produced and controls germ cell proliferation during gonad differentiation. Here we show for the first time the presence of MIS in an amphibian species, Pleurodeles waltl. This is very astonishing because in caudate amphibians, Muellerian ducts do not regress in males. Phylogenetic analysis of MIS P. waltl ortholog revealed that the deduced protein segregates with MIS from other vertebrates and is clearly separated from other TGF- beta family members. In larvae, MIS mRNA was expressed at higher levels in the developing testes than in the ovaries. In the testis, MIS mRNA expression was located within the lobules that contain Sertoli cells. Besides, expression of MIS was modified in the case of sex reversal: it increased after masculinizing heat treatment and decreased after estradiol feminizing exposure. In addition to the data obtained recently in the fish medaka, our results suggest that the role of MIS on Muellerian ducts occurred secondarily during the course of evolution.
Salamanders exhibit an extraordinary ability among vertebrates to regenerate complex body parts. However, scarce genomic resources have limited our understanding of regeneration in adult salamanders. ...Here, we present the ~20 Gb genome and transcriptome of the Iberian ribbed newt Pleurodeles waltl, a tractable species suitable for laboratory research. We find that embryonic stem cell-specific miRNAs mir-93b and mir-427/430/302, as well as Harbinger DNA transposons carrying the Myb-like proto-oncogene have expanded dramatically in the Pleurodeles waltl genome and are co-expressed during limb regeneration. Moreover, we find that a family of salamander methyltransferases is expressed specifically in adult appendages. Using CRISPR/Cas9 technology to perturb transcription factors, we demonstrate that, unlike the axolotl, Pax3 is present and necessary for development and that contrary to mammals, muscle regeneration is normal without functional Pax7 gene. Our data provide a foundation for comparative genomic studies that generate models for the uneven distribution of regenerative capacities among vertebrates.
Robots are increasingly used as scientific tools to investigate animal locomotion. However, designing a robot that properly emulates the kinematic and dynamic properties of an animal is difficult ...because of the complexity of musculoskeletal systems and the limitations of current robotics technology. Here, we propose a design process that combines high-speed cineradiography, optimization, dynamic scaling, three-dimensional printing, high-end servomotors and a tailored dry-suit to construct Pleurobot: a salamander-like robot that closely mimics its biological counterpart, Pleurodeles waltl. Our previous robots helped us test and confirm hypotheses on the interaction between the locomotor neuronal networks of the limbs and the spine to generate basic swimming and walking gaits. With Pleurobot, we demonstrate a design process that will enable studies of richer motor skills in salamanders. In particular, we are interested in how these richer motor skills can be obtained by extending our spinal cord models with the addition of more descending pathways and more detailed limb central pattern generator networks. Pleurobot is a dynamically scaled amphibious salamander robot with a large number of actuated degrees of freedom (DOFs: 27 in total). Because of our design process, the robot can capture most of the animal's DOFs and range of motion, especially at the limbs. We demonstrate the robot's abilities by imposing raw kinematic data, extracted from X-ray videos, to the robot's joints for basic locomotor behaviours in water and on land. The robot closely matches the behaviour of the animal in terms of relative forward speeds and lateral displacements. Ground reaction forces during walking also resemble those of the animal. Based on our results, we anticipate that future studies on richer motor skills in salamanders will highly benefit from Pleurobot's design.
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, 24h 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 24h and 7 days following the challenge.•Locomotion could be prioritized due to its important antipredator role.
The evolution of advanced cognition in vertebrates is associated with two independent innovations in the forebrain: the six-layered neocortex in mammals and the dorsal ventricular ridge (DVR) in ...sauropsids (reptiles and birds). How these innovations arose in vertebrate ancestors remains unclear. To reconstruct forebrain evolution in tetrapods, we built a cell-type atlas of the telencephalon of the salamander
. Our molecular, developmental, and connectivity data indicate that parts of the sauropsid DVR trace back to tetrapod ancestors. By contrast, the salamander dorsal pallium is devoid of cellular and molecular characteristics of the mammalian neocortex yet shares similarities with the entorhinal cortex and subiculum. Our findings chart the series of innovations that resulted in the emergence of the mammalian six-layered neocortex and the sauropsid DVR.
Abstract
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.
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.
Numerous prey traits are the result of the pervasive evolutionary pressure imposed by predators. Mirroring their fundamental role in the fitness of prey, some antipredator defences can evolve into ...elaborate strategies. Such is the case of the Iberian ribbed newt (Pleurodeles waltl), a semiaquatic urodele that inhabits multiple types of inland waterbodies in central and southern Iberian Peninsula and north‐western Africa. It possesses sharp, protractile ribs, which can thwart predators by emerging through skin ‘warts’ they pierce, becoming impregnated with the toxicants these warts produce, and making the newts difficult to swallow. Plus, the orange colour of these warts contrasts with the predominantly dull dorsum.
In this work, I studied the interactions between the number of these warts, the saliency of wart colour against dorsum colour of each individual (i.e., the difference in colour between the warts and the dorsum), rib response time (i.e., the time needed for the ribs to emerge in response to a standardised stimulus), body size, and locomotor performance in males and females in either their aquatic or terrestrial stage. I also conducted a field experiment to determine whether predators more frequently attacked plasticine models with conspicuous or inconspicuous warts.
I found that wart colour saliency functions as a decoy, attracting predator attacks towards this well‐protected body part. Plus, many of the antipredator defences studied, namely the number of warts, their colour saliency, rib response time, body size, and locomotor performance, were interrelated.
Accordingly, more salient warts indicated a faster rib response, suggesting that these defences are coordinated with each other. Plus, locomotion was considerably slower in the terrestrial than in the aquatic stage. Moreover, warts were more salient, and thus more efficient attracting predator attacks, in newts in the slower terrestrial stage. However, rib response was faster in the aquatic stage, which coincides with the newt's activity peak, and therefore with the height of its conspicuousness to predators. Remarkably, faster newts display less salient warts and a slower rib response, which suggests a trade‐off between both defences. Nonetheless, some of these trends are reversed in females, among which faster individuals also display a faster rib response. This could be a likely consequence of reduced predation pressure.
These results further the understanding of how prey integrate and tune multiple antipredator defences according to factors that influence predation pressure, such as sex and life stage.
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