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PLEURODELES MICHAHELLES, 1830 Species: Pleurodeles waltl * Michahelles, 1830. Otic–occipitum complex (Fig. 5F) The otic–occipitum complex is generally sharp, because the prominentiae semicircularis anterioris and posterioris are usually covered by numerous crests (except for MNCM 16165, in which the crests are low). The prominentia semicircularis lateralis is more rounded and clearly recognizable. The middle depression is not visible between the prominentiae semicircularis. The circular fenestra ovalis is visible in posterior and ventral views. Its dorsal edge is also visible in dorsal view. Its anterior edge is located at the mid-length of the complex, and it is connected to the cotyles with a horizontal lamina. The parietal crest is low and limited to parts of the tectum synoticum (e.g. in HNHM 2004.90.1 and MNHN 2013-27) or, alternatively, it starts from the mid-length of the distal edge of the tectum synoticum, and it reaches the mid-length of the prominentia semicircularis anterioris (e.g. in MNHN 2013-26 and HNHM 2004.91.1). The parotic crest and process generally are not present. Instead, anterior and lateral to the anterior half of the prominentia semicircularis lateralis and parallel to the otic process, there are several small, anteriorly pointing crests or a single crest that ends in a sharp and anteriorly directed process. The anterior edge of the tectum synoticum is posterior to the mid-length of the complex. The tectum is developed medially as the prefacial and hypochordal commissures, and no concavity is visible between the prefacial commissure and the tectum. Posteriorly, the cotyle surrounds the circular or kidney-shaped postoticum foramen. The cylindrical otic process is much more anteriorly projecting than the processus basalis and bears a subcircular articular surface and sometimes an anterodorsal spine. The articular surface of the processus basalis is kidney-shaped, with the foramen faciale posterodorsal to it. A deep sulcus petrosus separates the otic and basalis processes. The foramen prooticum is entirely surrounded by bone. The auditory cavity is not deep. The basicapsular commissure bears an anteromedian ventral spine, also visible in dorsal view. The crista retrosellaris between the hypochordal and basicapsular commissures covers the whole fenestra basicranialis, which, therefore, is absent. The middle of the ventral side of the complex is depressed and strongly perforated, especially next to the ventral spine, and no medial crest is visible. Remarks: In MNCM 16167, the parietal crest contacts a low parotic crest in the mid-length of the prominentia semicircularis anterioris. In MNCM 16165, the prooticum foramen is not entirely surrounded by bone. Atlas (Fig. 8D) The neural canal is circular in anterior view and is as high as each occipital joint or slightly higher. In the same view, the dorsal edge of the neural canal is V-shaped. In posterior view, the neural canal is as wide as the circular cotyle. The occipital joints are circular or elliptical, with the major axis being horizontal. The articular facets of the odontoid process are separated by a narrow groove visible in ventral view. In this view, the base of the odontoid process is as wide as or narrower than each occipital joint. The low neural crest starts posterior to the anterior edge of the neural arch, and it is posteriorly broadened or forked (in this case, forming an incisura dorsalis visible in dorsal view and rarely in posterior view). The secondary crests are as inclined as the neural crest or ventrally concave. They reach the posterior edge of the neural arch. The neural spine is absent or low. The lateral surface of the atlas bears several foramina per side. The incisura vertebralis cranialis is small and V-shaped. In lateral view, the dorsal edge of the neural arch is strongly inclined. The deep incisura caudalis reaches the atlantal centrum, and its maximum concavity lies in the horizontal plane containing the maximum concavity of the incisura cranialis. The low lateral crests generally reach the postzygapophyses. The inferior crests are low, from the ventral edge of the occipital joints, forming a deep lateral concavity, and extending straight until the end of the centrum. In lateral view, the postzygapophyses extend posteriorly beyond the cotyle for more than half of their length. The posterodorsal edge of the atlas is flat in lateral view, and its posterior surface shows several small foramina. In dorsal view, the neural arch is concave anteriorly (U- or V-shaped concavity), and the cotyle is not visible posteriorly. The ventral surface bears two or more foramina. Remarks: In MDHC 253, the neural crest is equally developed throughout its length. The anterior edge of the lateral walls of the neural arch is not convex in lateral view in MNHN 2013-27, hence the incisura vertebralis cranialis is only slightly visible. The lateral crests are not visible dorsal to the first spinal nerve foramen in MDHC 253. In HNHM 2004.90.1 and in the right side of MDHC 253, a third crest is present between the lateral and inferior crests of the atlas. In MNCN 161165, the incisura cranialis is roundly concave, and the incisura caudalis is not deep (neural arch slightly concave), whereas the anterior edge of the neural arch is flat in dorsal view. The incisura dorsalis is not visible in this specimen, in which the neural crest is clearly thicker posteriorly, forming a perforated bony triangle. Precaudal vertebrae (Fig. 11E) The precaudal vertebrae are opisthocoelous. The neural canal is triangular and, in general, lower than the condyle. The condyle is elliptical, with a horizontal major axis. In lateral view, the neural arch between the condyle and the elliptical prezygapophyses is weakly inclined. Diapophyses and parapophyses are distinguishable, connected by a smooth lamina that reaches their distal ends and is distally indented. The lamina is smooth anteriorly and bears some foramina posteriorly. The transverse processes are subvertical or with a slightly posterior orientation, covering the incisura caudalis only marginally in lateral view or, in some cases, not covering it. In lateral view, the neural arch dorsal to the prezygapophyses is visible and vertical or anteriorly concave. Half of the height of the vertebra is formed by the neural crest and neural arch dorsal to the postzygapophyses. The neural crest is blade-like and high, starting posterior to the line connecting the posterior edge of the prezygapophyses. The neural crest gently broadens posteriorly, being high in lateral view, and is pierced by several small foramina. The neural spine is absent. The anterior zygapophyseal crests contact the dorsal part of the parapophyses or the lamina connecting the diapophyses and parapophyses (the anterior zygapophyseal crest never contacts the dorsal part of the diapophyses; see also Stoetzel et al., 2011). The posterior zygapophyseal crests are well developed and horizontal. The small anterior ventral crest starts from the anteroventral edge of the parapophyses and contacts the prezygapophyses in their ventral part, thus being dorsally concave. The posterior ventral crests are absent or small. The ventral lamina is trapezoidal. Generally, the lateral surface of the vertebrae is perforated. In lateral view, the dorsal edge of the neural arch is anterodorsally concave. The incisura vertebralis caudalis is deep and generally reaches the centrum. In posterior view, the dorsal edge of the neural arch is horizontal or dorsally convex (inverted U-shaped). The neural arch dorsal to the postzygapophyses is sigmoid in lateral view. In lateral view, the postzygapophyses extend posteriorly beyond the cotyle for more than half of their length. In dorsal view, the neural arch is flat or anteriorly concave (U-shaped). Part of the condyle is visible in dorsal view, whereas the cotyle is not visible. The ventral surface is generally perforated. Caudal vertebrae (Fig. 14B, C) The caudal vertebrae are not particularly high (height/ length ratio 130°, almost reaching 180° in some cases. In lateral view, the anterior margin of the haemal arch is concave (or posteriorly inclined) between the centrum and the anterior ventral crest, and convex (or anteriorly inclined) ventral to it. The haemal crest projects anteriorly beyond the haemal arch, forming a sharp or rounded tip in lateral view. The haemal arch and crest form a sharp tip posteriorly. Remarks: In MDHC 253, the transverse processes a r e V- s h a p e d, f o r m i n g a n a n t e r i o r c o n c av i t y. MNCN 16165 has procoelous caudal vertebrae and small anterior middle crests not in contact with either the prezygapophyses or the anterior edge of the cotyle., Published as part of Macaluso, Loredana, Wencker, Lukardis C M, Castrovilli, Maria, Carnevale, Giorgio & Delfino, Massimo, 2023, A comparative atlas of selected skeletal elements of European urodeles (Amphibia: Urodela) for palaeontological investigations, pp. 569-619 in Zoological Journal of the Linnean Society 197 (3) on pages 604-605, DOI: 10.1093/zoolinnean/zlac063, http://zenodo.org/record/7690954, {"references":["Stoetzel E, Bailon S, Rage JC. 2011. First fossil representative of the salamander crown-group from a Gondwanan continent: Pleurodeles cf. aealtl from the Quaternary of Morocco. Amphibia-Reptilia 32: 245 - 252."]}

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.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.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.

Salamanders are unique among tetrapods in their ability to regenerate their limbs throughout life. Like other poikilothermic amphibians, salamanders also show a remarkable capacity to survive long periods of starvation. Whether the physiological reserves necessary for tissue regeneration are preserved or sacrificed in starved salamanders is unknown. In the current study, we maintained Iberian ribbed newts ( Pleurodeles waltl) under extreme physiological stress to assess the extent of regeneration and identify the molecular and cellular changes that may occur under such conditions. After 19 months of complete food deprivation, the animals exhibited extensive morphological and physiological adaptations but remained behaviorally active and vigilant. Autophagy was elevated in different tissues and the transformed gut microbiota indicated remodeling of the intestinal tract related to autophagy. Upon limb amputation in animals starved for 21 months, regeneration proceeded with progenitor cell proliferation and migration, leading to limb blastema formation. However, limb outgrowth and patterning were substantially attenuated. Blockage of autophagy inhibited cell proliferation and blastema formation in starved animals, but not in fed animals. Hence, tissue autophagy and the regenerative response were tightly coupled only when animals were under stress. Our results demonstrate that under adverse conditions, salamanders can exploit alternative strategies to secure blastema formation for limb regeneration.

Salamanders have been used as research models for centuries. While they exhibit a wide range of biological features not seen in mammals, none has captivated scientists like their ability to regenerate. Interestingly, axolotl macrophages have emerged as an essential cell population for tissue regeneration. Whether the same is true in other salamanders such as newt species Notophthalmus viridescens, Cynops pyrrhogaster, or Pleurodeles waltl remains to be seen. Unfortunately, regardless of the species, molecular tools to study macrophage function in salamanders are lacking. We propose that the readily available, terminally differentiated peritoneal macrophages from newts or axolotls could be used to validate molecular reagents in the study of macrophage function during tissue regeneration in salamanders.

Urodele amphibian newts have unique biological properties in male gametogenesis, in addition to their extreme regenerative capacity. Male newts are able to regenerate new testes even after reaching sexual maturity and can possess multiple testes. Notably, these animals maintain primordial germ cell-like cells in a tissue adjacent to the testis. Spermatogenesis proceeds while synchronizing in a region-specific manner in the testis. However, the newt species that have been used most commonly require 2-3 years to achieve sexual maturity, and spermatogenesis in these species shows seasonality. These traits have restricted the use of newts for studies on testicular development and spermatogenesis, and testis development in newts remains poorly characterized. Recently, the Iberian ribbed newt Pleurodeles waltl has been established as an emerging model organism. P. waltl reaches sexual maturity more quick after birth than do other newts and is capable of breeding year-round. Thus, P. waltl is expected to serve as an appealing experimental model for studying the mechanisms of male gametogenesis in the urodeles. In the present study, we use P. waltl to describe the entire developmental process of the newt testis from primordial gonad to maturity. Notably, the mature testes show synchronized progression of spermatogenesis along the anteroposterior axis. Additionally, we demonstrate that the process of spermatogenesis in P. waltl proceeds irrespective of day length. Our results show that P. waltl newts are a suitable model for investigating the process of testicular development. We also expect that these results will be useful for the maintenance of P. waltl bioresources.

Pleurodeles waltl (Figs 3–5) Smirnov et al. (2020) described cranial skeletogenesis of P. waltl and reported that ossification began soon after hatching. The results of Smirnov et al. (2020) and our results show a similar pattern of ossification. In the study by Smirnov et al. (2020), specimen body size was 5–7 mm at the hatching stage and 28–36 mm at the stage when metamorphosis occurs. However, in our study, these measurements were 8.5–9.2 mm and 48–54 mm, respectively. Given that the growth rate of salamanders is influenced strongly by external factors, such as temperature and food, this difference in the timing of ossification onset could be attributable to the different rearing environments (Stewart, 1956). In phases I–V, ossification has not yet started. In the skull, the sphenethmoid, otic capsule and exoccipital are observed as cartilaginous elements. In the mandible and pharynx, the Meckel’s cartilage, the first and second basibranchials, ceratohyal, hypohyal, the first and second hypobranchials and the first to fourth ceratobranchials are detected as cartilaginous elements. Ossification begins in phase VI. The premaxilla, vomer, prootic, squamosal, exoccipital, parasphenoid, dentary and angular begin to ossify. The palatine and pterygoid bones appear fused into a single bone. In phase VII, ossification of the skull progresses rapidly, and all bones except the nasal and orbitosphenoid begin to ossify. In the mandible, the coronoid is ossified from the medial side. In phase VIII, the nasal begins to ossify, meaning that all bones of the skull are ossified. In the hyobranchial skeleton, the second basibranchial starts to ossify. In phase IX, the posterolateral projection of the frontal and anteromedial projection of the squamosal are visible. The pterygoid is elongated in the anterior– posterior direction, forming a bony bar in the ventral side of the skull. In phases X and XI, the third to fourth ceratobranchials degenerate. In phases XII and XIII, the tips of the posterolateral projection of the frontal and the anteromedial projection of the squamosal fuse to form the frontosquamosal arch, a characteristic skull feature of the family Salamandridae. The maxilla is elongated posterolaterally and its posterior end is now bifurcated, possessing short lateral and medial projections. The bony bar formed by the formerly continuous anterior palatine and posterior pterygoid is now separated in the middle. In the mandible, the second basibranchial degenerates, and the previously cartilaginous ceratohyal, second ceratobranchial and second hypobranchial ossify., Published as part of Ishikawa, Kaoru, Taguchi, Yuki, Kobayashi, Ryomei, Anzai, Wataru, Hayashi, Toshinori & Tokita, Masayoshi, 2022, Cranial skeletogenesis of one of the largest amphibians, Andrias japonicus, provides insight into ontogenetic adaptations for feeding in salamanders, pp. 299-314 in Zoological Journal of the Linnean Society 195 (1) on page 305, DOI: 10.1093/zoolinnean/zlab038, http://zenodo.org/record/6530541, {"references":["Smirnov SV, Merkulova KM, Vassilieva AB. 2020. Skull development in the Iberian newt, Pleurodeles waltl (Salamandridae: Caudata: Amphibia): timing, sequence, variations, and thyroid hormone mediation of bone appearance. Journal of Anatomy 237: 543 - 555.","Stewart MM. 1956. The separate effects of food and temperature differences on development of marbled salamander larvae. Journal of the Mitchell Society 72: 47 - 56."]}

Dear Editor,A 12.5-year-old, previously healthy male presented at his general practitioner’s 30 min after a sting of his pet Iberian Ribbed Newt (Figure 1). The animal had struggled while it was be...

Marine tetrapods quickly diversified and were established as marine top predators after the end-Permian Mass extinction (EPME). Ichthyosaurs were the forerunner of this rapid radiation but the main drivers of the diversification are poorly understood. Cartorhynchus lenticarpus is a basal ichthyosauriform with the least degree of aquatic adaptation, holding a key to identifying such a driver. The unique specimen appeared edentulous based on what was exposed but a CT scanning revealed that the species indeed had rounded teeth that are nearly perpendicular to the jaw rami, and thus completely concealed in lateral view. There are three dental rows per jaw ramus, and the root lacks infoldings of the dentine typical of ichthyopterygians. The well-developed and worn molariform dentition with three tooth rows supports the previous inference that the specimen is not of a juvenile. The premaxilla and the corresponding part of the dentary are edentulous. Molariform dentition evolved three to five times independently within Ichthyosauriformes in the Early and Middle Triassic. Convergent exploitation of hard-shelled invertebrates by different subclades of ichthyosauriforms likely fueled the rapid taxonomic diversification of the group after EPME.

The objective of this investigation was to assess the usefulness of three different doses of alfaxalone administered intramuscularly in amphibian species. Thirty-two healthy, adult (5–9 years old) captive Spanish ribbed newts (Pleurodeles waltl) were used. Alfaxalone at a dose of 15, 20, and 30 mg/kg was administered into the muscles of the front legs of each newt, with a 4-week washout period between trials. Heart rate and depth of sedation were assessed for each animal prior to the administration of anesthetic (T0), and at 2-minute intervals over the initial 20 minutes, then at 5-minute intervals over 60 minutes, and at 15-minute intervals until a full duration of 120 minutes for each trial. The average induction time was reduced with increasing doses. The recovery period was prolonged with increasing doses. Intramuscular administration of alfaxalone proved a feasible method for immobilization and sedation in Spanish ribbed newts and resulted in loss of both the escape reflex and righting reflex.

Urodelan amphibian larvae develop three pairs of branching external gills on both sides of the pharyngeal region, and this study focuses on motile cilia on the gill surface. High-speed camera was used to observe ciliary strokes on the surface of the external gills of Pleurodeles waltl larvae. We found that the directionality of ciliary beating is position-dependent on the gill surface, and this rule is applicable to all the samples examined. For visualizing water flow around the external gills, we used bead suspensions. We revealed continuous anterior-to-posterior water flow generated by coordinated ciliary beating. Around the frontal surface of the gill stem (gill rachis), water flows countercurrent to the bloodstream beneath the gill epidermis. These results suggest that ciliary beating in each ciliated cell is coordinated, which cooperatively generates continuous and directional ciliary flow. We next visualized the overall distribution of ciliated cells on the gill surface by immunostaining of acetylated alpha-tubulin. Our results showed that the fine branches of external gills (fimbriae) have a circumferential distribution of cilia aligned orthogonal to the longitudinal axes of fimbriae, which facilitates water flow from proximal to the distal part of the fimbriae through the gills. This ciliary distribution pattern and directionality of cilia-driven flow are shared among five urodelan and two anuran species. Taken together, our findings suggest that the distribution and motility of ciliated cells on the surface of external gills is finely controlled, and this might support efficient respiration by the gills in urodeles.

Wide-pore proteinaceous freeze&ndash, thaw spongy gels were synthesized via the cryotropic gelation technique using the bovine blood serum or its diluted solutions as the protein-containing precursors. The feed systems also included the denaturant (urea) and the thiol-reductant (cysteine). The gel-fraction yield decreased and the swelling degree of the walls of macropores in such heterophase matrices increased with decreasing the initial protein concentration. The optimum freezing temperature was found to be within a rather narrow range from &minus, 15 to &minus, 20 &deg, C. In this case, the average size of the macropores in the resultant cryogels was 90&ndash, 110 &mu, m. The suitability of such soft wide-pore gel materials for the application as the carriers of peptide bioregulators was demonstrated in the in vitro experiments, when the posterior segments of the Pleurodeles waltl adult newts&rsquo, eyes were used as a model biological target. It was shown that a statistically reliable protective effect on the state of the sclera, vascular membrane and retinal pigment epithelium, as well as on the viability of fibroblasts, was inherent in the proteinaceous cryogels loaded with the peptide bioregulator (Viophtan-5&trade, ) isolated from the bovine eye sclera.

Until recently, different families of urodele amphibians were thought to express distinct subsets of immunoglobulin (Ig) isotypes. In this study, we explored cDNAs encoding Ig heavy-chains (H-chains) in three species of urodele amphibians. We found that Cynops pyrrhogaster, Pleurodeles waltl, and Ambystoma mexicanum each carry genes encoding four Ig H-chain isotypes, including IgM, IgY, IgD, and IgX, similar to those found in anuran amphibians. We also found that urodele IgDs have a long constant region similar to those found in anuran, reptiles, and bony fishes. We also found several putative IgD splice variants. Our findings indicated that P. waltl IgP is not a novel isotype but an IgD splice variant. Altogether, our findings indicate that IgD splice variants may be universally expressed among amphibian species.

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.

Prevalence of morphological abnormalities usually is less than 5% in most amphibian populations (Ouellet et al. 1997, Vandenlangenberg et al. 2003, Mester et al. 2015). Morphology is closely related to whole-organism performance in amphibians (Zamora-Camacho 2018, Zamora- Camacho and Aragón 2019a), and thus, is under strong selection (Watkins 1996). Therefore, this low frequency likely reflects the negative effects of abnormalities on whole-organism performance (Zamora-Camacho and Aragón 2019b). However, amphibian populations are experiencing an alarming increase in morphological abnormality rates worldwide (Lanoo 2008, Johnson and Bowerman 2010, Laurentino et al. 2016). Amongst these, the most common are limb malformations, such as misshapen or fused limbs, and missing, or presence of extra limbs and toes (Harris et al. 2008, Johnson and Bowerman 2010, Reeves et al. 2013)., The author was partially supported by a Juan de la Cierva-Formación postdoctoral fellowship from the Spanish Ministerio de Economía, Industria y Competitividad.

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.

The peptide–protein complex from bovine sclera was studied. It is shown that it contained a protein with a molecular weight of 66387 Da with the partial N-terminal amino acid sequence DTHKSEIAHRFKDLG-, which is homologous to the mature molecule of bovine serum albumin, and polypeptides with molecular weights of 1300–5080 Da. With a model of the organotypic cultivation of posterior eye tissues of the newt Pleurodeles waltl in vitro, it was shown that the effect of this peptide–protein complex in low doses increased the viability of scleral fibroblasts.

During aquatic feeding salamanders use the hyobranchial apparatus to capture prey. The hyobranchial apparatus depresses the floor of the mouth, effectively expanding the oropharyngeal cavity and generating suction. Within the family Salamandridae, there is a wide range of ecological diversity, with salamanders being terrestrial, semi-aquatic, or aquatic as adults. The purpose of this research was to quantify the diverse morphology and suction feeding performance of aquatically feeding salamandrids. We hypothesized that a more robust hyobranchial apparatus morphology would yield increased aquatic feeding performance. When compared to semi-aquatic newts, the fully aquatic species Paramesotriton labiatus had greater mineralization of the hyobranchial apparatus, as well as relatively more narrow basibranchial and wider ceratobranchial I+II complexes. These morphological differences coincide with greater aquatic feeding performance. Kinematics from high-speed videography revealed that maximum mouth opening velocity and acceleration were approximately two and five times greater, respectively, in Paramesotriton, and hyobranchial depression acceleration was found to be approximately three times greater than in the semi-aquatic species Pleurodeles waltl, Notophthalmus viridescens, Triturus dobrogicus, and Cynops cyanurus. Using digital particle image velocimetry, peak and average fluid velocity generated in Paramesotriton during suction feeding events were found to be 0.5ms-1 and 0.2ms-1, respectively, doubling that of all semi-aquatic species. These findings reveal that specialized morphology increases aquatic feeding performance in a fully aquatic newt.

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.

The diversity of the vertebrate cranial shape of phylogenetically related taxa allows conclusions on ecology and life history. As pleurodeline newts (the genera Echinotriton, Pleurodeles and Tylototriton) have polymorphic reproductive modes, they are highly suitable for following cranial shape evolution in relation to reproduction and environment. We investigated interspecific differences externally and differences in the cranial shape of pleurodeline newts via two‐dimensional geometric morphometrics. Our analyses also included the closely related but extinct genus Chelotriton to better follow the evolutionary history of cranial shape. Pleurodeles was morphologically distinct in relation to other phylogenetically basal salamanders. The subgenera within Tylototriton (Tylototriton and Yaotriton) were well separated in morphospace, whereas Echinotriton resembled the subgenus Yaotriton more than Tylototriton. Oviposition site choice correlated with phylogeny and morphology. Only the mating mode, with a random distribution along the phylogenetic tree, separated crocodile newts into two morphologically distinct groups. Extinct Chelotriton likely represented several species and were morphologically and ecologically more similar to Echinotriton and Yaotriton than to Tylototriton subgenera. Our data also provide the first comprehensive morphological support for the molecular phylogeny of pleurodeline newts., We applied geometric morphometrics to cranial morphology in extant and extinct crocodile newts. Our data provides the first comprehensive morphological support for the molecular phylogeny of these newts. Further, we could show that life history traits correlate with head shape and that fossil Chelotriton show similarities in shape with some extant taxa allowing conclusions on its ecology.

The thermosensation mechanism plays critical roles in various animals living in different thermal environment. We focused on an axolotl, which is a tailed amphibian originally from Lake Xochimilco area in the Vally of Mexico, and examined its behavior response to heat stimulation. Mild heat at 33 °C induced noxious locomotive activity to axolotls, but the noxious response of another tailed amphibian, Iberian ribbed newt, was not observed at 33 °C. To explore the mechanism for the temperature sensitivity of axolotls, we isolated a cDNA of TRPV1. Using the degenerate primer PCR method, we identified the DNA fragment encoding axolotl TRPV1 (axTRPV1), and then cloned a full-length cDNA. We studied the chemical and thermal sensitivities of axTRPV1 by two-electrode voltage clamp method using Xenopus oocyte expression system. Capsaicin, acid, and 2-aminoethoxydiphenylborane apparently activated axTRPV1 channels in a dose-dependent manner. The analysis of thermal sensitivity showed that axTRPV1 was significantly activated by heat but not by cold. The average temperature threshold for heat-activation was 30.95 ± 0.12 °C. This thermal activation threshold of axTRPV1 is unique and significantly low, when compared with the known thresholds of TRPV1s from various animals. Further, this threshold of axTRPV1 is well consistent with the observation of heat-induced behavior of axolotls at 33 °C, demonstrating that axolotl shows noxious response to mild heat mediated through axTRPV1.

PosteriorHoxgenes play crucial roles in limb development and specify regions in the proximal-distal (PD) axis of limbs. However, there is no direct genetic evidence thatHoxgenes are essential for limb regeneration. Moreover, if essential, it is totally unknown whichHoxgenes have the same or distinct functions between development and regeneration. Here, we mutatedhox13using an efficient CRISPR/Cas9 system in newts (Pleurodeles waltl), which have strong regenerative capacities in various tissues. Triple or double mutants ofhox13paralogs lost their digit and metacarpal/metatarsal bones. Limb regeneration progressed but regenerates lacked the same autopod region. These results showed thathox13paralogs have the same functions in limb development and regeneration.

Urodele amphibian newts have an outstanding history as experimental animals in various research fields such as developmental biology and regeneration biology. We have reported a model experimental system using the Spanish newt, Pleurodeles waltl, and it enables reverse/molecular genetics through gene manipulation. Microinjection is one of the core techniques in gene manipulation in newts. In the present study, we examined the conditions of the microinjection method, such as egg preparation, de-jelly solution, and formulation of injection medium. We have successfully optimized the injection protocol for P. waltl newts, and our improved protocol is more efficient and lower in cost than previous methods. This protocol can be used for the microinjection of plasmid DNA with I-SceI or mRNA, as well as genome editing using the CRISPR-Cas9 system. This protocol will facilitate research through gene manipulation in newts.

Heat-shock protein 90 (HSP90) is an ubiquitous chaperone in eukaryotes that facilitates folding of proteins both in normal conditions and in response to environmental stress. In addition, HSP90 is able to buffer genetic variation resulting from random mutations by regulating intracellular protein degradation mechanisms. It has been shown that experimental HSP90 inhibition produces anatomical variability during embryonic development in Arabidopsis, Drosophila and zebrafish. It is not clear whether these anatomical changes are caused by the expression of genomic cryptic mutations or by modifications of HSP90 dependent molecular pathways. However, several results suggest that HSP90 modulation is a relevant mechanism underlying the de novo appearance of character states. In order to test whether HSP90 is involved in the generation of apomorphic phenotypes in tetrapods, we pharmacologically inhibited HSP90 in Pleurodeles waltl (Urodela) embryos (n=141), by exposition to 10 µM Radicicol during the first 24 hours of development. Control specimens (n=20) received no treatment. The 54 surviving larvae were euthanatized at stages 46 (n=25) and 50 (n=29), and subjected to whole-mount double staining with alcian blue and alizarin red. All the control and 7 (13%) of the treated specimens were phenotypically normal. The remaining 47 (87%) larvae showed 20 abnormal phenotypes best described as heterochronic skull ossification sequences. Interestingly, these heterochronies fit the ossification sequences described in other urodele families. We propose that modulation of HSP90 function during the embryonic development, caused naturally by environmental stress, may underlay the evolutive modifications of skull ossification sequences in urodeles. New studies on the inheritance of the phenotypes obtained in untreated embryos from treated parents may confirm the plausibility of this hypothesis. Universidad de Málaga. Campus de Excelencia Internacional Andalucía Tech. FPU15/03209, EST17/00194 and University of Málaga.

Urodele amphibians have remarkable organ regeneration ability. They can regenerate not only limbs but also a tail throughout their life. It has been demonstrated that the regeneration of some organs are governed by the presence of neural tissues. For instance, limb regeneration cannot be induced without nerves. Thus, identifying the nerve factors has been the primary focus in amphibian organ regeneration research. Recently, substitute molecules for nerves in limb regeneration, Bmp and Fgfs, were identified. Cooperative inputs of Bmp and Fgfs can induce limb regeneration in the absence of nerves. In the present study, we investigated whether similar or same regeneration mechanisms control another neural tissue governed organ regeneration, i.e., tail regeneration, in Ambystoma mexicanum. Neural tissues in a tail, which is the spinal cord, could transform wound healing responses into organ regeneration responses, similar to nerves in limb regeneration. Furthermore, the identified regeneration inducer Fgf2+Fgf8+Bmp7 showed similar inductive effects. However, further analysis revealed that the blastema cells induced by Fgf2+Fgf8+Bmp7 could participate in the regeneration of several tissues, but could not organize a patterned tail. Regeneration inductive ability of Fgf2+Fgf8+Bmp7 was confirmed in another urodele, Pleurodeles waltl. These results suggest that the organ regeneration ability in urodele amphibians is controlled by a common mechanism.

Urodele amphibian newts have unique biological properties, notably including prominent regeneration ability. Iberian ribbed newt,Pleurodeles waltl, is a promising model newt along with the successful development of the easy breeding system and efficient transgenic and genome editing methods. However, genetic information ofP. waltlwas limited. In the present study, we conducted an intensive transcriptome analysis ofP. waltlusing RNA-sequencing to build gene models and annotate them. We generated 1.2 billion Illumina reads from a wide variety of samples across 11 different tissues and 9 time points during embryogenesis. They were assembled into 202,788 non-redundant contigs that appear to cover nearly complete (~98%)P. waltlprotein-coding genes. Using the gene set as a reference, our gene network analysis identified regeneration-, developmental-stage-, and tissue-specific co-expressed gene modules. Ortholog analyses with other vertebrates revealed the gene repertoire evolution of amphibians which includes urodele-specific loss ofbmp4and duplications ofwnt11b. Our transcriptome resource will enhance future research employing this emerging model animal for regeneration research as well as other areas such as developmental biology, stem cell biology, cancer research, ethology and toxico-genomics. These data are available via our portal website, iNewt (http://www.nibb.ac.jp/imori/main/).

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.

Adult newts can regenerate large parts of their brain from adult neural stem cells (NSCs), but how adult NSCs reorganize brain structures during regeneration remains unclear. In development, elaborate brain structures are produced under broadly coordinated regulations of embryonic NSCs in the neural tube, whereas brain regeneration entails exquisite control of the re-establishment of certain brain parts, suggesting that a yet-unknown mechanism directs NSCs upon partial brain excision. Here we report that upon excision of a quarter of the adult newt (Pleurodeles waltl) mesencephalon, active participation of local NSCs around specific brain subregions’ boundaries leads to some imperfect and some perfect brain regeneration along an individual's rostrocaudal axis. Regeneration phenotypes depend on how wound closing occurs using local NSCs, and perfect regeneration replicates development-like processes, but takes more than 1 year. Our findings indicate that newt brain regeneration is supported by modularity of boundary-domain NSCs with self-organizing ability in neighboring fields. This article has an associated First Person interview with the first author of the paper., Summary: Upon large-scale brain excision in adult newts, the outcome of structural regeneration of the brain cytoarchitecture depends on ways of closing the wound by NSCs residing between brain subregions.

Expression patterns of Nkx2.1 and Islet-1 (Isl1), which encode transcription factors that are key in the regionalization of the forebrain, were analyzed by combined immunohistochemical methods in young adult specimens of two lungfishes (Neoceratodus forsteri and Protopterus dolloi) and a urodele amphibian (Pleurodeles waltl). We aimed to get insights into the possible organization of the forebrain in the common ancestor of all tetrapods because of the pivotal phylogenetic significance of these two groups, being lungfishes the closest living relatives of tetrapods, and representing urodeles a model of simple brain organization with most shared features with amniotes. These transcription factors display regionally restricted expression domains in adult (juvenile) brains that are best interpreted according to the current prosomeric model. The regional patterns observed serve to identify regions and compare between the three species studied, and with previous data reported mainly for amniotes. We corroborate that Nkx2.1 and Isl1 expressions have very similar topologies in the forebrain. Common features in all sarcopterygians (lungfishes and tetrapods) have been observed, such as the Isl1 expression in most striatal neurons, whereas Nkx2.1 is restricted to migrated interneurons that reach the ventral pallium (VP). In the pallidal derivatives, the combination of both markers allows the identification of the boundaries between the ventral septum, the bed nucleus of the stria terminalis (BST) and the preoptic commissural region. In addition, the high Isl1 expression in the central amygdala (CeA), its boundary with the lateral amygdala (LA), and the scattered Nkx2.1 expression in the medial amygdala (MeA) are also shared features. The alar and basal hypothalamic territories, and the prethalamus and posterior tubercle (TP) in the diencephalon, have maintained a common pattern of expression. This regional distribution of Isl1 and Nkx2.1 observed in the forebrain of urodeles and lungfishes contributes further to our understanding of the first terrestrial vertebrates and their ancestors.

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 F0 generation. In addition, we demonstrated the generation of tyrosinase null F1 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.

The overall bauplan of the tetrapod brain is highly conserved, yet significant variations exist among species in terms of brain size, structural composition and cellular diversity. Understanding processes underlying neural and behavioral development in a wide range of species is important both from an evolutionary developmental perspective as well as for the identification of cell sources with post-developmental neurogenic potential. Here we characterize germinal processes in the brain of Notophthalmus viridescens and Pleurodeles waltl during both development and adulthood. Using a combination of cell tracking tools, including clonal analyses in new transgenic salamander lines we examine the origin of neural stem and progenitor cells found in the adult brain, determine regional variability in cell cycle length of progenitor cells, and show spatio-temporally orchestrated neurogenesis. We analyze how maturation of different brain regions and neuronal subpopulations are linked to the acquisition of complex behaviors, and how these behaviors are altered upon chemical ablation of dopamine neurons. Our data analyzed from an evolutionary perspective reveal both common and species-specific processes in tetrapod brain formation and function.

International audience; Sox9 is a member of the gene family of SOX transcription factors, which is highly conserved among vertebrates. It is involved in different developmental processes including gonadogenesis. In all amniote species examined thus far, Sox9 is expressed in the Sertoli cells of the male gonad, suggesting an evolutionarily conserved role in testis development. However, in the anamniotes, fishes and amphibians, it is also expressed in the oocyte but the significance of such an expression remains to be elucidated. Here, we have investigated the nuclear localization of the SOX9 protein in the oocyte of three amphibian species, the urodelan Pleurodeles waltl, and two anurans, Xenopus laevis and Xenopus tropicalis. We demonstrate that SOX9 is associated with ribonucleoprotein (RNP) transcripts of lampbrush chromosomes in an RNA-dependent manner. This association can be visualized by Super-resolution Structured Illumination Microscopy (SIM). Our results suggest that SOX9, known to bind DNA, also carries an additional function in the posttranscriptional processes. We also discuss the significance of the acquisition or loss of Sox9 expression in the oocyte during evolution at the transition between anamniotes and amniotes.

We investigated the concentration of tetrodotoxin (TTX) in sections of skin containing and lacking red dorsal spots in both Eastern newt (Notophthalmus viridescens) efts and adults. Several other species, such as Pleurodeles waltl and Echinotriton andersoni, have granular glands concentrated in brightly pigmented regions on the dorsum, and thus we hypothesized that the red dorsal spots of Eastern newts may also possess higher levels of TTX than the surrounding skin. We found no difference between the concentrations of TTX in the red spots as compared to neighboring skin lacking these spots in either efts or adults. However, efts with more red dorsal spots had elevated TTX levels relative to efts with fewer spots.

The Ribbed newts (genus Pleurodeles) are endemic to the Iberian Peninsula and northwest Africa (Raffaëlli 2013), and comprise three species: Pleurodeles nebulosus (Guichenot 1850), Pleurodeles poireti (Gervais 1836) and Pleurodeles waltl Michahelles 1830. Pleurodeles nebulosus is found in northern Algeria and Tunisia with an average size of 18 cm, while P. poireti is endemic to the Edough Massif, in northeastern Algeria and is the smallest species, with 12.9 cm in total length (Escoriza et al. 2016; Carranza & Wade 2004). Pleurodeles waltl appears on the Iberian Peninsula and Morocco (García-París et al. 2004). The eastern and western populations of P. waltl although they are not differentiated taxonomically show high levels of genetic divergence (estimated to have occurred 2.0–3.2 Ma; Carranza & Arnold 2003). The individuals of P. waltl attain large sizes (30 cm of total length) and have 8−10 cutaneous glands on both sides (Pasteur 1958), which are absent in other species of the genus.

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.

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. walt, which encodes the nucleolar protein nucleostemin. Structural analysis revealed a high homology of Ns nucleotide sequences of P. walt! with those of newts. Cynops pyrrhogaster and Notophthalmus viridescens. The expression of the Ns gene of P. walt, 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.