An experimental assessment of detection dog ability to locate great crested newts (Triturus cristatus) at distance and through soil.

Detection dogs are increasingly used to locate cryptic wildlife species, but their use for amphibians is still rather underexplored. In the present paper we focus on the great crested newt (Triturus cristatus), a European species which is experiencing high conservation concerns across its range, and assess the ability of a trained detection dog to locate individuals during their terrestrial phase. More specifically, we used a series of experiments to document whether a range of distances between target newts and the detection dog (odour channelled through pipes 68 mm in diameter) affects the localisation, and to assess the ability and efficiency of target newt detection in simulated subterranean refugia through 200 mm of two common soil types (clay and sandy soil, both with and without air vents to mimic mammal burrows, a common refuge used by T. cristatus). The detection dog accurately located all individual T. cristatus across the entire range of tested distances (0.25 m- 2.0 m). The substrate trials revealed that the detection dog could locate individuals also through soil. Contrary to existing studies with detection dogs in human forensic contexts, however, detection was generally slower for T. cristatus under sandy soil compared to clay soil, particularly when a vent was absent. Our study provides a general baseline for the use of detection dogs in locating T. cristatus and similar amphibian species during their terrestrial phase.

Histological changes of the skin during postembryonic development of the crested newt Triturus ivanbureschi (Urodela, Salamandridae).

BACKGROUND: Amphibian skin has been studied for many decades, especially the metamorphic changes in the skin of frogs. Less attention has been paid to salamander skin. Here, we describe changes in the skin structure during postembryonic development in a salamandrid species, the Balkan crested newt Triturus ivanbureschi. METHOD: Using traditional histological techniques we examined the skin in the trunk region of three premetamorphic larval stages (hatchling, mid larval and late larval) and two postmetamorphic stages (juvenile, just after metamorphosis, and adult). RESULTS: In larval stages, skin consists only of the epidermis, which gradually develops from the single epithelial cell layer in hatchlings, to a stratified epidermis with gland nests and characteristic Leydig cells at the late larval stage. During metamorphosis, Leydig cells disappear, and the dermal layer develops. In postmetamorphic stages, skin is differentiated on stratified epidermis and the dermis with well-developed glands. Three types of glands were observed in the skin of the postmetamorphic stages: mucous, granular and mixed. Gland composition appears to be stage- and sex-specific, with juveniles and adult female being more similar to each other. In juveniles and adult female, there are a similar proportion of glands in both dorsal and ventral skin, whereas in adult male granular glands dominated the dorsal skin, while mixed glands dominated the ventral skin. CONCLUSION: Our results provide a baseline for future comparative research of skin anatomy in salamanders.

Higher temperature induces oxidative stress in hybrids but not in parental species: A case study of crested newts.

Ectotherms are particularly sensitive to global warming due to their limited capacity to thermoregulate, which can impact their performance and fitness. From a physiological standpoint, higher temperatures often enhance biological processes that can induce the production of reactive oxygen species and result in a state of cellular oxidative stress. Temperature alters interspecific interactions, including species hybridization. Hybridization under different thermal conditions could amplify parental (genetic) incompatibilities, thus affecting a hybrid's development and distribution. Understanding the impact of global warming on the physiology of hybrids and particularly their oxidative status could help in predicting future scenarios in ecosystems and in hybrids. In the present study, we investigated the effect of water temperature on the development, growth and oxidative stress of two crested newt species and their reciprocal hybrids. Larvae of Triturus macedonicus and T. ivanbureschi, and their T. macedonicus-mothered and T. ivanbureschi-mothered hybrids were exposed for 30 days to temperatures of 19°C and 24°C. Under the higher temperature, the hybrids experienced increases in both growth and developmental rates, while parental species exhibited accelerated growth (T. macedonicus) or development (T. ivanbureschi). Warm conditions also had different effects on the oxidative status of hybrid and parental species. Parental species had enhanced antioxidant responses (catalase, glutathione peroxidase, glutathione S-transferase and SH groups), which allowed them to alleviate temperature-induced stress (revealed by the absence of oxidative damage). However, warming induced an antioxidant response in the hybrids, including oxidative damage in the form of lipid peroxidation. These findings point to a greater disruption of redox regulation and metabolic machinery in hybrid newts, which can be interpreted as the cost of hybridization that is likely linked to parental incompatibilities expressed under a higher temperature. Our study aims to improve mechanistic understanding of the resilience and distribution of hybrid species that cope with climate-driven changes.

Interspecific introgression of MHC genes in Triturus newts: Evidence from multiple contact zones.

The major histocompatibility complex (MHC) genes are central to the adaptive immune response in vertebrates. Selection generally maintains high MHC variation because the spectrum of recognized pathogens depends on MHC polymorphism. Novel alleles favoured by selection originate by interallelic recombination or de novo mutations but may also be acquired by introgression from related species. However, the extent and prevalence of MHC introgression remain an open question. In this study, we tested for MHC introgression in six hybrid zones formed by six Triturus newt species. We sequenced and genotyped the polymorphic second exons of the MHC class I and II genes and compared their interspecific similarity at various distances from the centre of the hybrid zone. We found evidence for introgression of both MHC classes in the majority of examined hybrid zones, with support for a more substantial class I introgression. Furthermore, the overall MHC allele sharing outside of hybrid zones was elevated between pairs of Triturus species with abutting ranges, regardless of the phylogenetic distance between them. No effect of past hybrid zone movement on MHC allele sharing was found. Finally, using previously published genome-wide data, we demonstrated that MHC introgression was more extensive than genome-wide introgression, supporting its adaptive potential. Our study thus provides evidence for the prevalence of MHC introgression across multiple Triturus hybrid zones, indicating that MHC introgression between divergent hybridizing species may be widespread and adaptive.

Changes in thyroid histomorphology and thyroglobulin immunostaining upon exposure to thiourea in Triturus newts.

Amphibians are useful bioindicators for monitoring aquatic health and the influence of xenobiotics such as endocrine disrupting chemicals. Because aquatic ecosystems experience the majority of global pollution, aquatic organisms are most exposed and vulnerable to endocrine disruptors. Furthermore, penetration of endocrine disruptors into aquatic organisms especially in amphibians is even easier because of more permeable skin, resulting in high bioavailability and bioaccumulation of chemicals. One of the most potent endocrine disruptors is thiourea, which chemically blocks the synthesis of thyroid hormones and prevents metamorphosis in amphibians. We investigated the influence of thiourea on histomorphology of the thyroid gland in Triturus newts at the metamorphic stage, when thyroid hormone concentrations should reach their maximum level. Chronic exposure to thiourea induced hypertrophy and hyperplasia of follicular cells as well as a significant reduction of interstitial tissue. The intensity of the thyroglobulin immunostaining signal significantly decreases upon chronic exposure to thiourea. Successful cross-reactivity of human primary antibody in immunochemical detection of thyroglobulin in Urodela confirms potential homology in thyroglobulin structure throughout the vertebrates.

Reliability of environmental DNA surveys to detect pond occupancy by newts at a national scale.

The distribution assessment and monitoring of species is key to reliable environmental impact assessments and conservation interventions. Considerable effort is directed towards survey and monitoring of great crested newts (Triturus cristatus) in England. Surveys are increasingly undertaken using indirect methodologies, such as environmental DNA (eDNA). We used a large data set to estimate national pond occupancy rate, as well as false negative and false positive error rates, for commercial eDNA protocols. Additionally, we explored a range of habitat, landscape and climatic variables as predictors of pond occupancy. In England, 20% of ponds were estimated to be occupied by great crested newts. Pond sample collection error rates were estimated as 5.2% false negative and 1.5% false positive. Laboratory error indicated a negligible false negative rate when 12 qPCR replicates were used. Laboratory false positive error was estimated at 2% per qPCR replicate and is therefore exaggerated by high levels of laboratory replication. Including simple habitat suitability variables into the model revealed the importance of fish, plants and shading as predictors of newt presence. However, variables traditionally considered as important for newt presence may need more precise and consistent measurement if they are to be employed as reliable predictors in modelling exercises.

Oxidative Stress Parameters in Goitrogen-Exposed Crested Newt Larvae (Triturus spp.): Arrested Metamorphosis.

Thiourea is an established disruptor of thyroid hormone synthesis and is frequently used as an inhibitor of metamorphosis. The changes caused by thiourea can affect processes associated with the oxidative status of individuals (metabolic rate, the HPI axis, antioxidant system). We investigated the parameters of oxidative stress in crested newt (Triturus spp.) larvae during normal development in late larval stage 62 and newly metamorphosed individuals, and during thiourea-stimulated metamorphosis arrest in individuals exposed to low (0.05%) and high (0.1%) concentrations of thiourea. Both groups of crested newts exposed to thiourea retained their larval characteristics until the end of the experiment. The low activities of antioxidant enzymes and the high lipid peroxidation level pointed to increased oxidative stress in larvae at the beginning of stage 62 as compared to fully metamorphosed individuals. The activities of catalase (CAT) and glutathione-S-transferase (GST) and the concentration of sulfhydryl (SH) groups were significantly lower in larvae reared in aqueous solutions containing thiourea than in newly metamorphosed individuals. The high thiourea concentration (0.1%) affected the antioxidative parameters to the extent that oxidative damage could not be avoided, contrary to a lower concentration. Our results provide a first insight into the physiological adaptations of crested newts during normal development and simulated metamorphosis arrest.

Molecular determinants of protein reabsorption in the amphibian kidneys.

Participation of molecular determinants of endocytosis in the processes of glomerular filtration and tubular reabsorption of albumin and lysozyme in the mesonephros of grass frogs (Rana temporaria L.), lake frogs (Rana ridibunda P.), and newts (Triturus vulgaris L.) is investigated. In all studied species, the constitutive expression of endocytic receptors in proximal tubule (PT) cells is established using immunofluorescence microscopy and immunoblotting. The certain stages of lysozyme and albumin endocytosis involving megalin/LRP2, cubilin, clathrin and protein Rab11 are detailed, and the central role of ligand-induced megalin/LRP2 activity in this process is shown. Increased ligand-induced expression for clathrin and Rab11was also found. In grass frogs, the different patterns of endocytic receptors and both absorbed proteins in the initial parts of proximal tubules suggest the proximo-distal specialization of absorptive processes along these tubule segments, similar to this in more complex mammalian nephrons. This data, as well as the revealed peculiarities of ligand-receptor interactions during intracellular trafficking of proteins prove that megalin is mainly involved in the absorption of lysozyme. At the same time, albumin absorption is mediated by both receptors, or cubilin contributes the most. The detection of endocytic receptor in glomerular structural elements in frogs and newts suggests the participation of filtration barrier components in endocytosis of filterable proteins. The results represent a new contribution to the study of the fundamental mechanisms of renal protein uptake in the amphibian mesonephros as a more primitive kidney compared to mammalian metanephros.

Evidence for the use of a high-resolution magnetic map by a short-distance migrant, the Alpine newt (Ichthyosaura alpestris).

Newts can use spatial variation in the magnetic field (MF) to derive geographic position, but it is unclear how they detect the 'spatial signal', which, over the distances that newts move in a day, is an order of magnitude lower than temporal variation in the MF. Previous work has shown that newts take map readings using their light-dependent magnetic compass to align a magnetite-based 'map detector' relative to the MF. In this study, time of day, location and light exposure (required by the magnetic compass) were varied to determine when newts obtain map information. Newts were displaced from breeding ponds without access to route-based cues to sites where they were held and/or tested under diffuse natural illumination. We found that: (1) newts held overnight at the testing site exhibited accurate homing orientation, but not if transported to the testing site on the day of testing; (2) newts held overnight under diffuse lighting at a 'false testing site' and then tested at a site located in a different direction from their home pond oriented in the home direction from the holding site, not from the site where they were tested; and (3) newts held overnight in total darkness (except for light exposure for specific periods) only exhibited homing orientation the following day if exposed to diffuse illumination during the preceding evening twilight in the ambient MF. These findings demonstrate that, to determine the home direction, newts require access to light and the ambient MF during evening twilight when temporal variation in the MF is minimal.

The herpetological legacy of Jacob Green and the nomenclature of some North American lizards and salamanders.

Jacob Green was born in 1790 to a prominent New Jersey family of scholars and theologians. He taught at the College of New Jersey (now Princeton University) from 1818 to 1822 before co-founding Jefferson Medical College (now Thomas Jefferson University) in 1825, where he taught Chemistry until his death in 1841. Between 1818 and 1831, he published a series of nine papers on lizards, salamanders, and snakes, authoring the original description of several well-known species of salamanders from the eastern United States. Many of his names are ambiguous; some have been adjudicated by the ICZN, while others are currently treated as nomina dubia. Here, we review all of Green's publications, report on newly re-discovered or re-interpreted material from several major natural history collections, and resolve most if not all remaining issues through a series of taxonomic actions. In particular, we first designate a neotype for Salamandra nigra Green, 1818. We then place S. sinciput-albida Green, 1818 and S. frontalis Gray in Cuvier, 1831 in synonymy with S. scutata Temminck in Temminck Schlegel, 1838 and invoke Reversal of Precedence under Article 23.9 to designate them nomina oblita. We also designate a lectotype for S. bislineata Green, 1818. Finally, we resurrect the name S. fusca Green, 1818 as the valid name for the species Desmognathus fuscus, assuming priority over Triturus fuscus Rafinesque, 1820, designating S. fusca Laurenti, 1768 a nomen oblitum, and placing S. nigra Green, 1818 in synonymy. While Green's herpetological legacy is not as expansive as that of some of his successors such as Holbrook, he is nonetheless a foundational early worker in salamanders, having described some of the most-studied species in the world.

Flexibility of intraoral food processing in the salamandrid newt Triturus carnifex: effects of environment and prey type.

Intraoral food processing mechanisms are known for all major vertebrate groups, but the form and function of systems used to crush, grind or puncture food items can differ substantially between and within groups. Most vertebrates display flexible mechanisms of intraoral food processing with respect to different environmental conditions or food types. It has recently been shown that newts use cyclical loop-motions of the tongue to rasp prey against the palatal dentition. However, it remains unknown whether newts can adjust their food processing behavior in response to different food types or environmental conditions. Newts are interesting models for studying the functional adaptation to different conditions because of their unique and flexible lifestyle: they seasonally change between aquatic and terrestrial habitats, adapt their prey-capture mode to the respective environment, and consume diverse food types with different mechanical properties. Using X-ray high-speed recordings, anatomical investigations, behavioral analyses and mechanical property measurements, we tested the effects of the medium in which feeding occurs (water/air) and the food type (maggot, earthworm, cricket) on the processing behavior in Triturus carnifex We discovered that food processing, by contrast to prey capture, differed only slightly between aquatic and terrestrial habitats. However, newts adjusted the number of processing cycles to different prey types: while maggots were processed extensively, earthworm pieces were barely processed at all. We conclude that, in addition to food mechanical properties, sensory feedback such as smell and taste appear to induce flexible processing responses, while the medium in which feeding occurs appears to have less of an effect.

Testing the applicability of tagging the Great crested newt (Triturus cristatus) using passive integrated transponders.

Tracking individual animals with small-sized passive integrated transponder tags (PIT tags) has become a popular and widespread method, one which can be used for investigating life history traits, including dispersal patterns of small protected animals such as newts. In this study, we tested the applicability of PIT tag usage for individual marking with the Great crested newt (Triturus cristatus) as a model amphibian species, and to test the detection of the newts in nature using a passive telemetry system. Clove oil was used as an anaesthetic before surgery. We implanted PIT tags under the skin of 140 newts. The survival rate of newts was 98.57%. X-ray images were taken to check the exact positions of the PIT tags. Since approximately 15.71% of the newts were capable of expelling the tag from their bodies, tag loss has to be accounted for in future behavioural studies dealing with newts and other amphibians potentially capable of frequent tag expulsion. Lastly, we detected by passive telemetry 97 individuals out of 100 released into a natural breeding pond. Males had higher activity (13 detected males vs 7 females per hour) than females, thus males could be detected if present with more certainty. The result of the movement behaviour showed that e.g. the male of T. cristatus in a breeding pond can travel up to 20 m in 78 seconds. In summary, this promising method could allow the automatic data collection of marked newts in aquatic as well as in terrestrial biotopes, providing data on their dispersal, diurnal activity and movement behaviour.

Testing the evolutionary constraints of metamorphosis: The ontogeny of head shape in Triturus newts.

In vertebrates with complex, biphasic, life cycles, larvae have a distinct morphology and ecological preferences compared to metamorphosed juveniles and adults. In amphibians, abrupt and rapid metamorphic changes transform aquatic larvae to terrestrial juveniles. The main aim of this study is to test whether, relative to larval stages, metamorphosis (1) resets the pattern of variation between ontogenetic stages and species, (2) constrains intraspecific morphological variability, and (3) similar to the "hour-glass" model reduces morphological disparity. We explore postembryonic ontogenetic trajectories of head shape (from hatching to completed metamorphosis) of two well-defined, morphologically distinct Triturus newts species and their F1 hybrids. Variation in head shape is quantified and compared on two levels: dynamic (across ontogenetic stages) and static (at a particular stage). Our results show that the ontogenetic trajectories diverge early during development and continue to diverge throughout larval stages and metamorphosis. The high within-group variance and the largest disparity level (between-group variance) characterize the metamorphosed stage. Hence, our results indicate that metamorphosis does not canalize head shape variation generated during larval development and that metamorphosed phenotype is not more constrained relative to larval ones. Therefore, metamorphosis cannot be regarded as a developmental constraint, at least not for salamander head shape.

Chewing or not? Intraoral food processing in a salamandrid newt.

Food processing refers to any form of mechanical breakdown of food prior to swallowing. Variations of this behaviour are found within all major gnathostome groups. Chewing is by far the most commonly used intraoral processing mechanism and involves rhythmic mandibular jaw and hyobranchial (tongue) movements. Chewing occurs in chondrichthyans (sharks and rays), actinopterygians (ray-finned fishes), dipnoi (lungfishes) as well as amniotes and involves similarities in the patterns of muscle activity and movement of the feeding apparatus. It has been suggested that amniote chewing, which involves the interaction of movements of the mandibular jaw and the muscular tongue, has evolved as part of the tetrapod land invasion. However, little is known about food-processing mechanisms in lissamphibians, which might have retained many ancestral tetrapod features. Here, we identified a processing mechanism in the salamandrid newt, Triturus carnifex, which after prey capture displays cyclic head bobbing in concert with rhythmic jaw and tongue movements. We used high-speed fluoroscopy, anatomical reconstructions and analyses of stomach contents to show that newts, although not using their mandibular jaws, deploy a derived processing mechanism where prey items are rasped rhythmically against the dentition on the mouth roof, driven by a loop motion of the tongue. We then compared patterns and coordination of jaw and tongue movements across gnathostomes to conclude that food processing in this newt species shares traits with processing mechanisms in fish as well as amniotes. This discovery casts salamanders as promising models for reconstructing the evolution of intraoral processing mechanisms at the fish-tetrapod split.

Triturus newts.

Ben Wielstra introduces Eurasian newts of the genus Triturus.

Phylogenomics of the adaptive radiation of Triturus newts supports gradual ecological niche expansion towards an incrementally aquatic lifestyle.

Newts of the genus Triturus (marbled and crested newts) exhibit substantial variation in the number of trunk vertebrae (NTV) and a higher NTV corresponds to a longer annual aquatic period. Because the Triturus phylogeny has thwarted resolution to date, the evolutionary history of NTV, annual aquatic period, and their potential coevolution has remained unclear. To resolve the phylogeny of Triturus, we generated a c. 6000 transcriptome-derived marker data set using a custom target enrichment probe set, and conducted phylogenetic analyses using: (1) data concatenation with RAxML, (2) gene-tree summary with ASTRAL, and (3) species-tree estimation with SNAPP. All analyses produce the same, highly supported topology, despite cladogenesis having occurred over a short timeframe, resulting in short internal branch lengths. Our new phylogenetic hypothesis is consistent with the minimal number of inferred changes in NTV count necessary to explain the diversity in NTV observed today. Although a causal relationship between NTV, body form, and aquatic ecology has yet to be experimentally established, our phylogeny indicates that these features have evolved together, and suggest that they may underlie the adaptive radiation that characterizes Triturus.

Investigation of axonal regeneration of Triturus ivanbureschi by using physiological and proteomic strategies.

Peripheral nerve injuries are frequently observed and successful treatment depends mainly on the injury type, location of the damage, and the elapsed time prior to treatment. The regenerative capacity is limited only to the embryonic period in many mammalian tissues, but urodele amphibians do not lose this feature during adulthood. The main purpose of this study is to define the recovery period after serious sciatic nerve damage of a urodele amphibian, Triturus ivanbureschi. Experimental transection damage was performed on the sciatic nerves of T. ivanbureschi specimens. The recovery period of sciatic nerves were investigated by walking track analysis, electrophysiological recordings, and bottom-up proteomic strategies at different time points during a 35-day period. A total of 34 proteins were identified related to the nerve regeneration process. This study showed that the expression levels of certain proteins differ between distal and proximal nerve endings during the regeneration period. In distal nerve stumps, transport proteins, growth factors, signal, and regulatory molecules are highly expressed, whereas in proximal nerve stumps, neurite elongation proteins, and cytoskeletal proteins are highly expressed.

Resilience to climate variation in a spatially structured amphibian population.

Understanding the impact of weather fluctuations on demographic parameters is of crucial interest to biodiversity research in a context of global climate change. Amphibians are valuable candidates for investigating this topic due to their strong physiological dependence on water availability and temperature. In this study, we took advantage of data from a long-term capture-mark-recapture (CMR) monitoring program of a great crested newt (Triturus cristatus) population inhabiting a 12-pond archipelago in southeastern France. We investigated the interactions between vital rates (survival and recruitment), the internal structure of the population, and climatic variables both at a local and a regional (North Atlantic Oscillation: NAO) scale. Overall, we found a weak relationship between climatic variables and the survival of large-bodied newts. The only strong relationship was found to be a high NAO index during the post-breeding period, suggesting that dry, hot summers negatively impact survival. In terms of recruitment, the results indicated that hot weather during the activity period had delayed deleterious effects on adult recruitment two years later, suggesting high larval and juvenile mortality due to unsuitable growing conditions. Recruitment was also impacted by a high NAO index during the overwintering period preceding recruitment, suggesting that mild weather increases the mortality of juveniles, probably by enhancing the depletion of energy reserves without any possibility of refueling.

Oxidative cost of interspecific hybridization: a case study of two Triturus species and their hybrids.

Oxidative stress has most recently been suggested as one of the possible mechanisms responsible for reduced fitness of hybrids. To explore possible oxidative cost of hybridization, we examined anti-oxidant defence system parameters (superoxide dismutase, catalase, glutathione peroxidase, glutathione S-transferase, glutathione reductase, glutathione, SH groups), their interconnectedness (index of integration) and levels of oxidative damage [concentrations of lipid peroxides, TBARS (thiobarbituric acid reactive substances)] in laboratory-reared newt species, Triturus macedonicus and T. ivanbureschi, and their hybrid. Our results showed that parental species differed in anti-oxidant defence system parameters, but not in the levels of integration of the whole system and oxidative damage. Individuals of T. ivanbureschi had higher activities of superoxide dismutase, glutathione S-transferase and concentrations of glutathione. Hybrid individuals of crested newts displayed higher levels of the anti-oxidant defence system (higher superoxide dismutase, catalase, glutathione peroxidase activities and concentrations of SH groups), and a lower overall correlation of anti-oxidant system (lower index of integration) in comparison with both parental species, suggesting that they may possess a less efficient anti-oxidant defence system and a higher investment in maintaining oxidative balance. The higher investment in the anti-oxidant system could divert limited resources away from other functions and affect further hybrid fitness. The presented findings contribute to a better understanding of the anti-oxidant defence system of crested newts and their interspecies differences, and support the hypothesis that oxidative stress is one of the costs of interspecific hybridization.