Morphological variation, modularity and integration in the scapula and humerus of Lissotriton newts.

The modular organization of tetrapod paired limbs and girdles, influenced by the expression of Hox genes is one of the primary driving forces of the evolution of animal locomotion. The increased morphological diversification of the paired limbs is correlated with reduced between-limb covariation, while correlation within the elements is usually higher than between the elements. The tailed amphibians, such as Lissotriton newts, have a biphasic lifestyle with aquatic and terrestrial environments imposing different constraints on limb skeleton. By employing the methods of computerized microtomography and 3D geometric morphometrics, we explored the pattern of morphological variation, disparity, modularity and morphological integration in the proximal parts of the anterior limbs of six species of Eurasian small bodied newts. Although the species significantly differ in limb shape, there is a great overlap in morphology of scapula and humerus, and there are no differences in morphological disparity. For the scapula, the shape differences related to the duration of the aquatic period are in length, depth and curvature. The shape of the humerus is not affected by the length of aquatic period, and shape differences between the species are related to robustness of the body. The length of aquatic period has statistically supported phylogenetic signal. The scapula and humerus are structures of varying modularity. For the humerus, the strongest support on the phylogenetic level was for the capitulum/shaft hypothesis, which can also be interpreted as functional modularity. For the scapula, the greatest support was for the antero-posterior hypothesis of modularity in case of Lissotriton vulgaris, which can be explained by different functional roles and muscle insertion patterns, while there was no phylogenetic modularity. The modularity patterns seem to correspond with the general tetrapod pattern, with modularity being more pronounced in the distal structure. The future research should include more salamandrid taxa with different habitat preferences and both adult and larval stages, in order to explore how size, phylogeny and ecology affect the morphology and covariation patterns of limbs.

Learning the native pond odor as one of the mechanisms of olfactory orientation in juvenile smooth newt Lissotriton vulgaris.

Olfaction is an important mechanism of orientation in amphibians toward the breeding site. It is known that anurans can memorize the odor of the native pond during larval development and prefer this odor prior to the beginning of dispersion. However, such a mechanism in urodeles has not been studied yet. We conducted experiments on recognition of the odor of a native water body in juveniles of the smooth newt Lissotriton vulgaris. One group of larvae were reared in pure water (control), the other group in water with morpholine (10-7 mol/L). A few days after metamorphosis, the newts were tested under paired-choice conditions in a T-maze. A total of 73 newts from the experimental group and 47 newts from the control group were tested. The results of the experiment show that the newts in the experimental group preferred the morpholine solution, whereas the individuals of the control group made the choice randomly. We conclude that newts can learn the odor of the environment in which they developed and use this memory for orientation in later stages of life.

A CatSper-Uninvolved Mechanism to Induce Forward Sperm Motility in the Internal Fertilization.

Sperm-specific cation channel (CatSper), sperm-specific Na + /H + exchanger (sNHE), and soluble adenylyl cyclase (sAC) are necessary in the signaling pathways to control sperm motility in many animals, whereas some animals have lost some or all of them. In the present study, we examined CatSper-uninvolved signaling for vigorous undulation of the undulating membrane that is attached to the sperm tail and gives thrust for forward motility in the internally fertilizing newt Cynops pyrrhogaster. Reverse-transcription PCR failed to detect sNHE in the newt sperm. However, the pH of sperm cytoplasm was raised under a high extracellular pH equivalent to that of egg jelly, where sperm motility is initiated by sperm motility-initiating substance (SMIS). Carbonic anhydrase XII/ XVI and SLC4A4/8 were suggested to be present in the sperm, and transported bicarbonates raised the intracellular pH. In egg jelly extract that contained SMIS, the anion transporter inhibitor DIDS weakened the undulation of the undulating membrane, while bicarbonates enhanced it. The cyclic AMP concentration was found to increase in sperm cytoplasm in the egg-jelly extract. An inhibitor of sAC (KH7) weakened the undulation of the undulating membrane, and dibutyryl cyclic AMP blocked the inhibitory effect. Inhibitor of transmembrane AC (DDA) limitedly affected the undulation. The undulation was weakened by an inhibitor of protein kinase A (H89), and by an inhibitor of transient receptor potential (TRP) channels (RN1747). Our results support the conclusions that the high pH of the egg jelly triggers a signaling pathway through sAC, PKA, and TRP channels, and coacts with SMIS to induce forward sperm motility.

Terrestrial amphibians respond to rapidly changing temperatures with individual plasticity of exploratory behaviour.

Terrestrial ectotherms react to acute changes in environmental temperatures by adjusting their behaviour. Evaluating the adaptive potential of these behavioural adjustments requires information on their repeatability and plasticity. We examined behavioural response (exploration) to acute temperature change in two amphibian taxa, alpine (Ichthyosaura alpestris) and smooth (Lissotriton vulgaris) newts. These responses were investigated at both population and individual levels under multiple thermal contexts (dimensions), represented by the direction and range of changing temperature and rearing thermal regimes. Population-level analyses showed species-specific, non-additive effects of direction and range of temperature change on acute thermal reaction norms for exploration, but explained only a low amount (7-23%) of total variation in exploration. In contrast, within- and among-individual variation in acute thermal reaction norm parameters explained 42-50% of total variation in the examined trait. Although immediate thermal responses varied among individuals (repeatability = 0.07 to 0.53), they were largely shaped by environmental contexts during repeated trials. We conclude that these amphibians respond to acute temperature change through individual plasticity of behavioural traits. A repeated-measures approach under multiple thermal contexts will be needed to identify the selective and plastic potential of behavioural responses used by juvenile newts and perhaps other ectotherm taxa to cope with rapidly changing environmental temperatures.

Gene Conversion Facilitates the Adaptive Evolution of Self-Resistance in Highly Toxic Newts.

Reconstructing the histories of complex adaptations and identifying the evolutionary mechanisms underlying their origins are two of the primary goals of evolutionary biology. Taricha newts, which contain high concentrations of the deadly toxin tetrodotoxin (TTX) as an antipredator defense, have evolved resistance to self-intoxication, which is a complex adaptation requiring changes in six paralogs of the voltage-gated sodium channel (Nav) gene family, the physiological target of TTX. Here, we reconstruct the origins of TTX self-resistance by sequencing the entire Nav gene family in newts and related salamanders. We show that moderate TTX resistance evolved early in the salamander lineage in three of the six Nav paralogs, preceding the proposed appearance of tetrodotoxic newts by ∼100 My. TTX-bearing newts possess additional unique substitutions across the entire Nav gene family that provide physiological TTX resistance. These substitutions coincide with signatures of positive selection and relaxed purifying selection, as well as gene conversion events, that together likely facilitated their evolution. We also identify a novel exon duplication within Nav1.4 encoding an expressed TTX-binding site. Two resistance-conferring changes within newts appear to have spread via nonallelic gene conversion: in one case, one codon was copied between paralogs, and in the second, multiple substitutions were homogenized between the duplicate exons of Nav1.4. Our results demonstrate that gene conversion can accelerate the coordinated evolution of gene families in response to a common selection pressure.

The road not taken: Evolution of tetrodotoxin resistance in the Sierra garter snake (Thamnophis couchii) by a path less travelled.

The repeated evolution of tetrodotoxin (TTX) resistance provides a model for testing hypotheses about the mechanisms of convergent evolution. This poison is broadly employed as a potent antipredator defence, blocking voltage-gated sodium channels (Nav ) in muscles and nerves, paralysing and sometimes killing predators. Resistance in taxa bearing this neurotoxin and a few predators appears to come from convergent replacements in specific Nav residues that interact with TTX. This stereotyped genetic response suggests molecular and phenotypic evolution may be constrained and predictable. Here, we investigate the extent of mechanistic convergence in garter snakes (Thamnophis) that prey on TTX-bearing newts (Taricha) by examining the physiological and genetic basis of TTX resistance in the Sierra garter snake (Th. couchii). We characterize variation in this predatory adaptation across populations at several biological scales: whole-animal TTX resistance; skeletal muscle resistance; functional genetic variation in three Nav encoding loci; and levels of gene expression for one of these loci. We found Th. couchii possess extensive geographical variation in resistance at the whole-animal and skeletal muscle levels. As in other Thamnophis, resistance at both levels is highly correlated, suggesting convergence across the biological levels linking organism to organ. However, Th. couchii shows no functional variation in Nav loci among populations or difference in candidate gene expression. Local variation in TTX resistance in Th. couchii cannot be explained by the same relationship between genotype and phenotype seen in other taxa. Thus, historical contingencies may lead different species of Thamnophis down alternative routes to local adaptation.

Impacts of temperature on O2 consumption of the Pyrenean brook newt (Calotriton asper) from populations along an elevational gradient.

Global warming impacts biodiversity worldwide, leading to species' adaptation, migration, or extinction. The population's persistence depends on the maintenance of essential activities, which is notably driven by phenotypic adaptation to local environments. Metabolic rate - that increases with temperature in ectotherms - is a key physiological proxy for the energy available to fuel individuals' activities. Cold-adapted ectotherms can exhibit a higher resting metabolism than warm-adapted ones to maintain functionality at higher elevations or latitudes, known as the metabolic cold-adaptation hypothesis. How climate change will affect metabolism in species inhabiting contrasting climates (cold or warm) is still a debate. Therefore, it is of high interest to assess the pace of metabolic responses to global warming among populations adapted to highly different baseline climatic conditions. Here, we conducted a physiological experiment in the endemic Pyrenean brook newt (Calotriton asper). We measured a proxy of standard metabolic rate (SMR) along a temperature gradient in individuals sampled among 6 populations located from 550 to 2189 m a.s.l. We demonstrated that SMR increased with temperature, but significantly diverged depending on populations' origins. The baseline and the slope of the relationship between SMR and temperature were both higher for high-elevation populations than for low-elevation populations. We discussed the stronger metabolic response observed in high-elevation populations suggesting a drop of performance in essential life activities for these individuals under current climate change. With the increase of metabolism as the climate warms, the metabolic-cold adaptation strategy selected in the past could compromise the sustainability of cold-adapted populations if short-term evolutionary responses do not allow to offset this evolutionary legacy.

Repeatability and heritability of resting metabolic rate in a long-lived amphibian.

Resting metabolic rate (RMR), i.e. spent energy necessary to maintain basic life functions, is a basic component of energy budget in ectotherms. The evolution of RMR through natural selection rests on the premise of its non-zero repeatability and heritability, i.e. consistent variation within individual lifetimes and resemblance between parents and their offspring, respectively. Joint estimates of RMR repeatability and heritability are missing in ectotherms, however, which precludes estimations of the evolutionary potential of this trait. We examined RMR repeatability and heritability in a long-lived ectotherm, the alpine newt (Ichthyosaura alpestris). Individual RMR was repeatable over both six-month (0.28 ±â€¯0.09 [SE]) and five-year (0.16 ±â€¯0.07) periods. While there was no resemblance between parent and offspring RMR (0.21 ±â€¯0.34), the trait showed similarity among offspring within families (broad-sense heritability; 0.25 ±â€¯0.09). Similar repeatability and broad-sense heritability values in parental and offspring generations, respectively, and non-conclusive narrow-sense heritability suggest the contribution of non-additive genetic factors to total phenotypic variance in this trait. We conclude that RMR evolutionary trajectories are shaped by other processes than natural selection in this long-lived ectotherm.

Molecular cloning, the characterization of metallothionein and catalase, and the evaluation of testicular toxicity of Cd in the Chinese fire-bellied newt (Cynops orientalis).

Cadmium (Cd) is an environmental toxicant and a nonessential metal. Cd can attack a wide range of organs, such as the liver, kidney, lung, ovary, testis, brain, and muscle in vertebrates. Among these organs, the testis might be the most sensitive organ to Cd toxicity. Metallothionein (MT) is a cysteine-rich protein with a low molecular weight, that can bind with Cd and eliminate reactive oxygen species (ROSs). Hydrogen peroxide, which as a crucial type of ROS that is induced by Cd, can be eliminated by catalase (CAT) in the self-protection of cells and to realize Cd toxicity resistance. To investigate the functions of MT and CAT in the testis of Cynops orientalis, we cloned the full-length MT and CAT genes of C. orientalis for the first time. Immunofluorescence results demonstrated that MT and CAT were expressed in Sertoli cells and all spermatogenic cells in the testis of C. orientalis. The results of the ultrastructural damage assay demonstrated that there were various impairments, which included organelle vacuolization, abnormal chromatin distribution, and apoptotic bodies, in somatic cells that were exposed to Cd. However, the anomalies of spermatozoa were located mainly in the mid-piece and head, many of which showed severely impaired structures. The results demonstrated that MT and CAT expression had distinct patterns in response to various Cd concentrations: an increase in MT mRNA levels with elevated Cd levels and a persistent increase in CAT mRNA levels with elevated Cd levels. These results suggested that MT and CAT play roles in Cd toxicity resistance in the testis and that the expression of CAT may be a better biomarker than the expression of MT for assessing Cd pollution.

Thermal dependence and individual variation in tonic immobility varies between sympatric amphibians.

Tonic immobility (TI) is an important antipredator response employed by prey in the last stages of a predation sequence. Evolution by natural selection assumes consistent individual variation (repeatability) in this trait. In ectotherms, which experience variable body temperatures, TI should be repeatable over a thermal gradient to be targeted by natural selection; however, information on thermal repeatability of this trait is missing. We examined thermal repeatability of TI in juveniles of two sympatric amphibians, smooth (Lissotriton vulgaris) and alpine (Ichthyosaura alpestris) newts. Both species showed disparate TI responses to body temperature variation (13-28 °C). While the proportion of TI response was repeatable in both taxa, it increased with body temperature in alpine newts but was temperature independent in smooth newts. Duration of TI decreased with body temperature in both taxa but was only repeatable in smooth newts. Our results suggest that a warming climate may affect population dynamics of sympatric ectotherms through asymmetry in thermal reaction norms for antipredator responses.

Resistance and tolerance: A hierarchical framework to compare individual versus family-level host contributions in an experimental amphibian-trematode system.

Hosts have two general strategies for mitigating the fitness costs of parasite exposure and infection: resistance and tolerance. The resistance-tolerance framework has been well developed in plant systems, but only recently has it been applied to animal-parasite interactions. However, difficulties associated with estimating fitness, controlling parasite exposure, and quantifying parasite burden have limited application of this framework to animal systems. Here, we used an experimental approach to quantify the relative influence of variation among host individuals and genetic families in determining resistance and tolerance within an amphibian-trematode system. Importantly, we used multiple, alternative metrics to assess each strategy, and employed a Bayesian analytical framework to compare among responses while incorporating uncertainty. Relative to unexposed hosts, exposure to the pathogenic trematode (Ribeiroia ondatrae) reduced the survival and growth of California newts (Taricha torosa) (survival: 93% vs. 74%; growth: 0.29 vs. -0.5 vs mm day -1). Similarly, parasite infection success (the inverse of resistance) ranged from 8% to 100%. Yet despite this broad variation in host resistance and tolerance among individual newts, we found no evidence for transmissable, among-family variation in any of the resistance or tolerance metrics. This suggests that opportunities for evolution of these traits is limited, likely requiring significant increases in mutation, gene flow, or environmental heterogeneity. Our study provides a quantitative framework for evaluating the importance of alternative metrics of resistance and tolerance across multiple time points in the study of host-parasite interactions in animal systems.

Identification and characterization of POU class V family genes in Japanese red bellied newt, Cynops pyrrhogaster.

Mammalian Pou5f1 encodes the POU family class V (POU-V) transcription factor which is essential for the pluripotency of embryonic cells and germ cells. In vertebrates, various POU-V family genes have been identified and classified into the POU5F1 family or its paralogous POU5F3 family. In this study, we cloned two cDNAs named CpPou5f1 and CpPou5f3, which encode POU-V family proteins of the Japanese red bellied newt Cynops pyrrhogaster. In the predicted amino acid sequence encoded by CpPou5f1, the typical MAGH sequence at the N-terminus and deletion of arginine at the fifth position of POU-homeodomain were recognized, but not in the sequence encoded by CpPou5f3. Phylogenetic analysis using Clustal Omega software indicated that CpPou5f1 and CpPou5f3 are classified into the clade of the POU5F1 and POU5F3 families, respectively. In a real-time polymerase chain reaction (RT-PCR) analysis, the marked gene expression of CpPou5f1 was observed during oogenesis and early development up to the tail-bud stage, whereas weak gene expression of CpPou5f3 was detected only in the early stages of oogenesis and gastrula. In adult organs, CpPou5f1 was expressed only in the ovary, while gene expression of CpPou5f3 was recognized in various organs. A regeneration experiment using larval forelimb revealed that transient gene expression of CpPou5f1 occurred at the time of wound healing, followed by gene activation of CpPou5f3 during the period of blastema formation. These results suggest that CpPou5f1 and CpPou5f3 might play different roles in embryogenesis and limb regeneration.

Repeatability of thermal reaction norms for spontaneous locomotor activity in juvenile newts.

Locomotor activity is a major attribute of animals. Although this trait determines important ecological processes, such as dispersal and species interactions, the sources of its variation are not fully understood. We examined the influence of body temperature (13, 18, 23, and 28 °C) and individual identity on spontaneous locomotor activity in juvenile alpine newts, Ichthyosaura alpestris, over three consecutive weeks. Locomotor activity was characterized by four parameters: distance covered, mean velocity, frequency of movements, and total activity rate (all directional and non-directional movements). Apart from total activity rate, thermal reaction norms for locomotor parameters had convex or concave curvilinear shapes. During the first trial series, i.e. across the four body temperatures that were tested, individual identity explained less variation in thermal reaction norms than during the second series. Individual means, i.e. the vertical positions of individual thermal reaction norms, were repeatable between trial series in all locomotor activity parameters but the frequency of movements. We conclude that spontaneous locomotor activity is a complex trait, which can be characterized by several parameters with varying individual repeatability and thermal dependency. This information should be considered for planning further locomotor activity experiments, conservation strategies, and modeling ectotherm responses to climate change.

Divergence of species-specific protein sex pheromone blends in two related, nonhybridizing newts (Salamandridae).

In animals that use chemical communication during courtship and reproduction, speciation is often associated with divergence of their sex pheromones. In multicomponent pheromone systems, divergence can be obtained either by adding or deleting components, or by altering the relative contribution of individual components to the mixture. Protein pheromone systems can additionally evolve by amino acid sequence divergence to produce pheromones with a species-specific effect. The sodefrin precursor-like factor (SPF) pheromone system, a blend of proteins that essentially enhances receptivity in salamanders, has had a long and dynamic evolution of gene duplications, but the mechanisms that govern interspecific divergence and the role they play in reproductive isolation remain elusive. Here, we use transcriptomics and proteomics to characterize the SPF protein repertoire of the alpine newt (Ichthyosaura alpestris), and compare it to the previously identified repertoire of SPF proteins of the palmate newt (Lissotriton helveticus), a related but nonhybridizing species. Subsequent phylogenetic analyses indicate that, despite the availability of multiple SPF gene copies, both species predominantly express the same subset of orthologs. Our study demonstrates that species specificity in the SPF protein pheromone system can be established by gradual sequence divergence of the same set of proteins alone.

A biogeographic and ecological perspective to the evolution of reproductive behaviour in the family Salamandridae.

Amphibians have a complex reproductive behaviour, which shows the highest diversity among tetrapodes. The family Salamandridae, distributed across the entire Holarctic, is one of the most diverse groups of extant salamanders comprising 114 species in 21 genera. The family has a remarkable diversity of courtship modes, amplexus and sperm transfer. It is often hypothesised that this diversity has evolved in adaptation to a specific mating and/or breeding habitat. We test this hypothesis based upon a phylogenetic reconstruction using the complete mitochondrial genome sequences of 45 Salamandridae species, representing all existing genera. We used ancestral character state reconstruction methods and geographic range models and applied relaxed Bayesian molecular clock models to discuss the results in a temporal framework of Salamandridae evolution. Our results show that the family Salamandridae started to diversify in the Late Cretaceous (ca. 87 mya) and is of Western Palearctic origin. Ancestral character state reconstruction predicts that its common ancestor was oviparous, mated on land without amplexus and probably showed a pin wheel spermatophore transfer, which is still found in the Italian endemic Salamandrina terdigidata. Our results suggest that several colonization of continents with subsequent radiations took place, once to the Nearctic and twice into Eastern Asian realms. However, these events were only in one case associated with a change in mating behaviour (dorsal amplexus in Nearctic newts). Around the Cretaceous-Paleogene boundary (K-Pg boundary) several Salamandridae lineages further diverged, again with no obvious changes in mating behaviour. Overall, there is no significant signal for mating character evolution being caused by changes in habitat type, with only a slight tendency that changes in mating habitat might have led to changes in the type of sperm transfer which in turn was associated with changes in the presence or absence of amplexus.

Egg-laying environment modulates offspring responses to predation risk in an amphibian.

Predator-induced plasticity has been in the focus of evolutionary ecological research in the last decades, but the consequences of temporal variation in the presence of cues predicting offspring environment have remained controversial. This is partly due to the fact that the role of early environmental effects has scarcely been scrutinized in this context while also controlling for potential maternal effects. In this study, we investigated how past environmental conditions, that is different combinations of risky or safe adult (prenatal) and oviposition (early post-natal) environments, affected offspring's plastic responses in hatching time and locomotor activity to predation risk during development in the smooth newt (Lissotriton vulgaris). We found that females did not adjust their reproductive investment to the perceived level of risk in the adult environment, and this prenatal environment had generally negligible effect on offspring phenotype. However, when predator cues were absent during oviposition, larvae raised in the presence of predator cues delayed their hatching and exhibited a decreased activity compared to control larvae developing without predator cues, which responses are advantageous when predators pose a threat to hatched larvae. In the presence of predator cues during oviposition, the difference in hatching time persisted, but the difference in general locomotor activity disappeared between risk-exposed and control larvae. Our findings provide clear experimental evidence that fine-scale temporal variation in a predictive cue during and after egg-laying interactively affects offspring phenotype, and highlight the importance of the early post-natal environment, which may exert a substantial influence on progeny's phenotype also under natural conditions.

Heat shock response and shape regulation during newt tail regeneration.

Regenerating newt tail has recently been found to react to hypergravity in a stable and reproducible way - by curving downwards. Such morphogenetic effect of non-specific physical factor applied to a complex structure of an adult animal is a rare phenomenon with unknown molecular basis. For the first steps of unraveling this basis we've chosen heat shock proteins (HSPs) as promising candidates. Morphometrical analysis of tail regeneration was performed in aquarium (control), on substrate (relative hypergravity) and in aquarium under weekly application of heat shock. HSPs were inhibited pharmacologically during regeneration in aquarium and on substrate. Hsp70, 90 gene expression and protein localization were analyzed in the studied conditions. Weekly application of heat shock to newts regenerating tails in otherwise normal conditions led to development of curved tails (both upwards and downwards), suggesting that similar mechanisms are at play in both hypergravity-altered and heat shock-altered morphogenesis. Heat shock protein inhibitor KNK437 didn't affect tail shape during normal regeneration, but prevented the formation of tail curve in appropriate conditions. It was shown that HSP70 and HSP90 proteins are present in muscle and connective tissue of intact tails as well as regenerates, but only appear in epidermis in hypergravity-altered regenerates and heated tails. Based on our data, we hypothesize, that different external factors (e.g. hypergravity and heat shock) are received, analyzed and transmitted further to affect morphogenesis by similar mechanisms that utilize a set of HSP in epidermal cells.

Suppression of ciliary movements by a hypertonic stress in the newt olfactory receptor neuron.

Olfactory receptor neurons isolated from the newt maintain a high activity of the ciliary beat. A cilium of neuron is so unique that only little is known about regulatory factors for its beat frequency. We examined the olfactory receptor neuron immersed in various extracellular media under the video-enhanced differential interference contrast microscope. The activation of voltage-gated Ca2+ channels by K+ depolarization or by application of Ca2+ to membrane-permeabilized olfactory cells did not affect the ciliary movement, suggesting that Ca2+ influx through the cell membrane has no direct effect on the movement. However, when an extracellular medium contained NaCl or sucrose at concentrations only 30% higher than normal levels, ciliary movement was greatly and reversibly suppressed. In contrast, a hypotonic solution of such a solute did not change the ciliary movement. The hypertonic solutions had no effect when applied to permeabilized cells. Suction of the cell membrane with a patch pipette easily suppressed the ciliary movement in an isotonic medium. Application of positive pressure inside the cell through the same patch pipette eliminated the suppressive effect. From these findings, we concluded that the hypertonic stress suppressed the ciliary movement not by disabling the motor proteins, microtubules, or their associates in the cilia, but rather by modifying the chemical environment for the motor proteins. The ciliary motility of the olfactory receptor cell is directly sensitive to the external environment, namely, the air or water on the nasal epithelium, depending on lifestyle of the animal.

ITRAQ-based quantitative proteomic analysis of Cynops orientalis limb regeneration.

BACKGROUND: Salamanders regenerate their limbs after amputation. However, the molecular mechanism of this unique regeneration remains unclear. In this study, isobaric tags for relative and absolute quantification (iTRAQ) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) was employed to quantitatively identify differentially expressed proteins in regenerating limbs 3, 7, 14, 30 and 42 days post amputation (dpa). RESULTS: Of 2636 proteins detected in total, 253 proteins were differentially expressed during different regeneration stages. Among these proteins, Asporin, Cadherin-13, Keratin, Collagen alpha-1(XI) and Titin were down-regulated. CAPG, Coronin-1A, AnnexinA1, Cathepsin B were up-regulated compared with the control. The identified proteins were further analyzed to obtain information about their expression patterns and functions in limb regeneration. Functional analysis indicated that the differentially expressed proteins were associated with wound healing, immune response, cellular process, metabolism and binding. CONCLUSIONS: This work indicated that significant proteome alternations occurred during salamander limb regeneration. The results may provide fundamental knowledge to understand the mechanism of limb regeneration.

The 'male escape hypothesis': sex-biased metamorphosis in response to climatic drivers in a facultatively paedomorphic amphibian.

Paedomorphosis is a major evolutionary process that bypasses metamorphosis and allows reproduction in larvae. In newts and salamanders, it can be facultative with paedomorphs retaining gills and metamorphs dispersing. The evolution of these developmental processes is thought to have been driven by the costs and benefits of inhabiting aquatic versus terrestrial habitats. In this context, we aimed at testing the hypothesis that climatic drivers affect phenotypic transition and the difference across sexes because sex-ratio is biased in natural populations. Through a replicated laboratory experiment, we showed that paedomorphic palmate newts (Lissotriton helveticus) metamorphosed at a higher frequency when water availability decreased and metamorphosed earlier when temperature increased in these conditions. All responses were sex-biased, and males were more prone to change phenotype than females. Our work shows how climatic variables can affect facultative paedomorphosis and support theoretical models predicting life on land instead of in water. Moreover, because males metamorphose and leave water more often and earlier than females, these results, for the first time, give an experimental explanation for the rarity of male paedomorphosis (the 'male escape hypothesis') and suggest the importance of sex in the evolution of paedomorphosis versus metamorphosis.