Regionalization and morphological integration in the vertebral column of Eurasian small-bodied newts (Salamandridae: Lissotriton).

Serially homologous structures may have complex patterns of regionalization and morphological integration, influenced by developmental Hox gene expression and functional constraints. The vertebral column, consisting of a number of repeated, developmentally constrained, and highly integrated units-vertebrae-is such a complex serially homologous structure. Functional diversification increases regionalization and modularity of the vertebral column, particularly in mammals. For salamanders, three concepts of regionalization of the vertebral column have been proposed, recognizing one, two, or three presacral regions. Using three-dimensional geometric morphometrics on vertebra models acquired with microcomputerized tomography scanning, we explored the covariation of vertebrae in four closely related taxa of small-bodied newts in the genus Lissotriton. The data were analyzed by segmented linear regression to explore patterns of vertebral regionalization and by a two-block partial least squares method to test for morphological integration. All taxa show a morphological shift posterior to the fifth trunk vertebra, which corresponds to the two-region concept. However, morphological integration is found to be strongest in the mid-trunk. Taken jointly, these results indicate a highly integrated presacral vertebral column with a subtle two-region differentiation. The results are discussed in relation to specific functional requirements, developmental and phylogenetic constraints, and specific requirements posed by a biphasic life cycle and different locomotor modes (swimming vs. walking). Further research should be conducted on different ontogenetic stages and closely related but ecologically differentiated species.

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.

Ladies in armor: A micro-computed tomographic study of skin calcification in European toads (genus Bufo).

Micro-computed tomography is a powerful tool toward the detailed reconstruction of internal and external morphology, in particular for ossified and other dense tissues. Here, we document and compare the level of calcification in the skin of the head and the parotoids (the external skin glands) in males and females of common and spined toads, Bufo bufo and B. spinosus. In some anurans, including Bufo species, a specific acellular calcified tissue layer within the dermis has been documented (the Eberth-Katschenko, or EK-layer). By a combination of micro-computed tomography and classical histology, we detected additional calcium deposits located in the dermal layer stratum spongiosum, positioned above the EK-layer. We showed that the level of calcification and the presence of additional calcium deposits are size and sex related, increasing in the order B. bufo males, B. spinosus males, B. bufo females to B. spinosus females. The last of these groups is the least variable. Bufo spinosus females have dense calcium deposits in the parotoids and the dorsal and ventral skin. Three-dimensional volume renderings and cross-sectional slices obtained by micro-CT scanning indicate that this approach is a promising technique for further studies on bufonid skin anatomy and geographic variation in skin calcification.

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.

The Reproductive Success of Triturus ivanbureschi × T. macedonicus F1 Hybrid Females (Amphibia: Salamandridae).

Two large-bodied newt species, Triturus ivanbureschi and T. macedonicus, hybridize in nature across the Balkan Peninsula. Consequences of hybridization upon secondary contact of two species include species displacement and asymmetrical introgression of T. ivanbureschi mtDNA. We set an experimental reciprocal cross of parental species and obtained two genotypes of F1 hybrids (with T. ivanbureschi or T. macedonicus mtDNA). When hybrids attained sexual maturity, they were engaged in mutual crossings and backcrossing with parental species. We followed reproductive traits over two successive years. Our main aim was to explore the reproductive success of F1 females carrying different parental mtDNA. Additionally, we tested for differences in reproductive success within female genotypes depending on the crossing with various male genotypes (hybrids or parental species). Both female genotypes had similar oviposition periods, number of laid eggs and hatched larvae but different body and egg sizes. Overall reproductive success (percentage of egg-laying females and viability of embryos) was similar for both genotypes. The type of crossing led to some differences in reproductive success within female genotypes. The obtained results suggest that processes that led to exclusion of T. macedonicus mtDNA in natural populations may be related to the survival at postembryonic stages of F2 generation or reproductive barriers that emerged in subsequent hybrid generations.

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.

Effects of thiourea on the skull of Triturus newts during ontogeny.

BACKGROUND: In amphibians, thyroid hormone (TH) has a profound role in cranial development, especially in ossification of the late-appearing bones and remodeling of the skull. In the present study, we explored the influence of TH deficiency on bone ossification and resulting skull shape during the ontogeny of Triturus newt hybrid larvae obtained from interspecific crosses between T. ivanbureschi and T. macedonicus. METHODS: Larvae were treated with two concentrations of thiourea (an endocrine disruptor that chemically inhibits synthesis of TH) during the midlarval and late larval periods. Morphological differences of the cranium were assessed at the end of the midlarval period (ontogenetic stage 62) and the metamorphic stage after treatment during the late larval period. RESULTS: There was no difference in the ossification level and shape of the skull between the experimental groups (control and two treatment concentrations) at stage 62. During the late larval period and metamorphosis, TH deficit had a significant impact on the level of bone ossification and skull shape with no differences between the two treatment concentrations of thiourea. The most pronounced differences in bone development were: the palatopterygoid failed to disintegrate into the palatal and pterygoid portions, retardation was observed in development of the maxilla, nasal and prefrontal bones and larval organization of the vomer was retained in thiourea-treated larvae. CONCLUSIONS: This implies that deficiency of TH caused retardation in development and arrested metamorphic cranium skeletal reorganization, which resulted in divergent cranial shape compared to the control group. Our results confirmed that skull remodeling and ossification of late-appearing bones is TH-dependent, as in other studied Urodela species. Also, our results indicate that TH plays an important role in the establishment of skull shape during the ontogeny of Triturus newts, especially during the late larval period and metamorphosis, when TH concentrations reach their maximum.

The effect of short-term fasting on the oxidative status of larvae of crested newt species and their hybrids.

In nature, animals often face periods without food caused by seasonal fluctuations and/or prey scarcity. An organism's physiological response to imposed energetic limitations is followed by changes in mitochondrial functioning (adjustment of energy metabolism) and a reduction of non-essential processes. However, this energy-saving strategy can have its costs. In this study, we examined oxidative stress as one of the possible physiological costs of short-term, two-week-long food deprivation on developing amphibian larvae of the crested newts Triturus macedonicus and Triturus ivanbureschi and their hybrids. We investigated whether this exogenous factor additionally affected the oxidative status (fitness-related trait) of hybrid individuals. The fasting treatment led to lower growth and a lower body mass and body condition index of individuals. The results revealed that the antioxidant system (AOS) of food-deprived larvae could not cope in a proper manner with reactive oxygen species production under limited energy availability, leading to higher lipid oxidative damage. The lowest AOS response was observed for H2O2 scavenging parameters (catalase, glutathione peroxidase, and total glutathione), which together with the elevated activity of superoxide dismutase suggested increased H2O2 concentrations. Comparison between parental species and their hybrids showed that hybrid individuals suffered greater oxidative damage (as demonstrated by higher concentrations of lipid peroxides), indicating that they were more susceptible to fasting-induced oxidative stress. Overall, this study illustrates that: (i) an oxidative event is one of the costs amphibian larvae face during short-term periods of fasting, (ii) hybrids are less capable of dealing with this stressful condition, which can lower their chances of survival in a changing environment.

The Effect of Shelter on Oxidative Stress and Aggressive Behavior in Crested Newt Larvae (Triturus spp.).

Shelters are important for animal survival. Provision of adequate hiding places allow animals to express their natural sheltering behavior and it can have different positive effects on cortisol levels, physiological processes and mental performance. Although the absence of a refuge activates some stress response, its effect on oxidative stress has not been adequately examined. This study investigated whether the presence/absence of a shelter modifies the oxidative status (the antioxidant system and oxidative damage) and aggressive behavior of crested newt larvae (Triturus macedonicus and its hybrid with T. ivanbureschi). Our results show that individuals reared with shelters had lower values of the tested antioxidant parameters (catalase, glutathione peroxidase, glutathione S-transferase and glutathione), indicating a lower production of reactive species than individuals reared without shelter. The same pattern was observed in both T. macedonicus and its hybrid. Contrary to the activation of some physiological pathways, shelter availability did not significantly affect the rate of intraspecific aggressive behavior. The physiological benefits of shelter use can be manifested as a lower requirement for investment in the energy necessary for the maintenance of the upregulated antioxidant defenses, activation of repair systems and synthesis of endogenous antioxidants. This study highlights the importance of shelter provision, which may be valuable in habitat restoration and animal conservation studies.

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.

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.