First Evidence of Anti-Steatotic Action of Macrotympanain A1, an Amphibian Skin Peptide from Odorrana macrotympana.

Many different amphibian skin peptides have been characterized and proven to exert various biological actions, such as wound-healing, immunomodulatory, anti-oxidant, anti-inflammatory and anti-diabetic effects. In this work, the possible anti-steatotic effect of macrotympanain A1 (MA1) (FLPGLECVW), a skin peptide isolated from the Chinese odorous frog Odorrana macrotympana, was investigated. We used a well-established in vitro model of hepatic steatosis, consisting of lipid-loaded rat hepatoma FaO cells. In this model, a 24 h treatment with 10 µg/mL MA1 exerted a significant anti-steatotic action, being able to reduce intracellular triglyceride content. Accordingly, the number and diameter of cytosolic lipid droplets (LDs) were reduced by peptide treatment. The expression of key genes of hepatic lipid metabolism, such as PPARs and PLINs, was measured by real-time qPCR. MA1 counteracted the fatty acid-induced upregulation of PPARγ expression and increased PLIN3 expression, suggesting a role in promoting lipophagy. The present data demonstrate for the first time a direct anti-steatotic effect of a peptide from amphibian skin secretion and pave the way to further studies on the use of amphibian peptides for beneficial actions against metabolic diseases.

Energy storage in salamanders' tails: the role of sex and ecology.

In vertebrates, the main tissue devoted to energy storage is the adipose tissue. In salamanders, energy reserves can also be stored in the adipose tissues of the tail. Therefore, we evaluated if energy storage in salamanders' tails is related to individual body condition, life cycle and environmental constraints. We calculated a scaled measure of tail width for 345 salamanders belonging to six Mediterranean taxa exhibiting wide phylogenetic, behavioural and ecological variation. We related this measure to the Scaled Mass Index (SMI), a body condition index which reliably predicts body fat. We found significant relationships between the SMI and scaled tail width in the terrestrial Spectacled salamander and Alpine salamanders, independently of sex. At the same time, we found that energy storage in the tail is maximum in Alpine Salamanders, which experience reduced activity periods and restricted access to resources. Conversely, we found a significant effect of sex in Imperial cave salamanders, where females store reserves in the tail to counterbalance resource investment in parental care, and in Corsican Brook Newts, where the reproductive function of males' tails may imply a greater tail width. Finally, in the biphasic Great Crested Newt, tail width was not related to SMI in both sexes.

Peptides for Skin Protection and Healing in Amphibians.

Amphibian skin is not to be considered a mere tegument; it has a multitude of functions related to respiration, osmoregulation, and thermoregulation, thus allowing the individuals to survive and thrive in the terrestrial environment. Moreover, amphibian skin secretions are enriched with several peptides, which defend the skin from environmental and pathogenic insults and exert many other biological effects. In this work, the beneficial effects of amphibian skin peptides are reviewed, in particular their role in speeding up wound healing and in protection from oxidative stress and UV irradiation. A better understanding of why some species seem to resist several environmental insults can help to limit the ongoing amphibian decline through the development of appropriate strategies, particularly against pathologies such as viral and fungal infections.

Safe caves and dangerous forests? Predation risk may contribute to salamander colonization of subterranean habitats.

Recent studies suggest that many organisms actively colonize the subterranean environment to avoid climatic stress, exploit new ecological opportunities and reduce competition and predation. Terrestrial salamanders are known to colonize the more stable subterranean habitats mainly to escape external climatic extremes, while the role of predation avoidance remains untested. To better understand the importance of predation, we used clay models of the cave salamander Speleomantes strinatii to compare the predation occurring in woodland and subterranean habitats. Models were positioned in three forests and in three caves in NW Italy. One-hundred eighty-four models were retrieved from the field and 59 (32%) were attacked by predators. Models were attacked on their head more often than expected by chance and, therefore, were perceived by predators as real prey items. In the woodlands, clay models showed a four-time higher probability of being attacked in comparison to caves, suggesting a different level of potential predation risk in these surface habitats. These findings are one of the first experimental evidences that, in terrestrial ecosystems, predation avoidance may contribute to the salamander underground colonization process.

Seasonality and microhabitat selection in a forest-dwelling salamander.

Many small terrestrial vertebrates exhibit limited spatial movement and are considerably exposed to changes in local environmental variables. Among such vertebrates, amphibians at present experience a dramatic decline due to their limited resilience to environmental change. Since the local survival and abundance of amphibians is intrinsically related to the availability of shelters, conservation plans need to take microhabitat requirements into account. In order to gain insight into the terrestrial ecology of the spectacled salamander Salamandrina perspicillata and to identify appropriate forest management strategies, we investigated the salamander's seasonal variability in habitat use of trees as shelters in relation to tree features (size, buttresses, basal holes) and environmental variables in a beech forest in Italy. We used the occupancy approach to assess tree suitability on a non-conventional spatial scale. Our approach provides fine-grained parameters of microhabitat suitability and elucidates many aspects of the salamander's terrestrial ecology. Occupancy changed with the annual life cycle and was higher in autumn than in spring, when females were found closer to the stream in the study area. Salamanders showed a seasonal pattern regarding the trees they occupied and a clear preference for trees with a larger diameter and more burrows. With respect to forest management, we suggest maintaining a suitable number of trees with a trunk diameter exceeding 30 cm. A practice of selective logging along the banks of streams could help maintain an adequate quantity of the appropriate microhabitat. Furthermore, in areas with a presence of salamanders, a good forest management plan requires leaving an adequate buffer zone around streams, which should be wider in autumn than in spring.

Lagged influence of North Atlantic Oscillation on population dynamics of a Mediterranean terrestrial salamander.

The North Atlantic Oscillation (NAO) is a large-scale climatic pattern that strongly influences the atmospheric circulation in the northern Hemisphere and by consequence the long-term variability of marine and terrestrial ecosystem over great part of northern Europe and western Mediterranean. In the Mediterranean, the effects of the NAO on vertebrates has been studied mainly on bird populations but was rarely analysed in ectothermic animals, and in particular in amphibians. In this study, we investigated the relationships between winter, spring and summer NAO indexes and the long-term population dynamics of the plethodontid salamander Speleomantes strinatii. This terrestrial salamander was monitored inside an artificial cave in NW Italy for 24 consecutive years. The relationships between seasonal NAO indexes and the salamander dynamics were assessed by cross-correlation function (CCF) analysis, after prewhitening the time series by autoregressive moving average statistical modelling. Results of CCF analyses indicated that the salamander abundance varied in relation to the one-year ahead winter NAO (P = 0.018), while no relationships were found with spring and summer indexes. These results strengthen some previous findings that suggested a high sensitivity of temperate terrestrial amphibians to wintertime climatic conditions.

Disentangling Exploitative and Interference Competition on Forest Dwelling Salamanders.

Exploitative competition and interference competition differ in the way access to resources is modulated by a competitor. Exploitative competition implies resource depletion and usually produces spatial segregation, while interference competition is independent from resource availability and can result in temporal niche partitioning. Our aim is to infer the presence of spatial or temporal niche partitioning on a two-species system of terrestrial salamanders in Northern Italy: Speleomantes strinatii and Salamandrina perspicillata. We conducted 3 repeated surveys on 26 plots in spring 2018, on a sampling site where both species are present. We modelled count data with N-mixture models accounting for directional interactions on both abundance and detection process. In this way we were able to disentangle the effect of competitive interaction on the spatial scale, i.e., local abundance, and from the temporal scale, i.e., surface activity. We found strong evidence supporting the presence of temporal niche partitioning, consistent with interference competition. At the same time, no evidence of spatial segregation has been observed.

Size-Mediated Trophic Interactions in Two Syntopic Forest Salamanders.

Exploitative competition and interference competition differ in the way they affect re-source availability for competitors: in the former, organisms reduce resource availability for the competitors; in the latter, one organism actively prevents the competitor from accessing resources, independently of their availability. Our aim is to test for the presence of foraging competition in two forest-dwelling salamanders in Italy: Speleomantes strinatii and Salamandrina perspicillata. We also aim at testing for size-mediated competition. We obtained stomach contents from 191 sampled individuals by means of stomach flushing at 8 sampling sites where both species occur. We focused our analysis on the core prey taxa shared by both species: Collembola and Acarina. We found that the foraging activity of S. perspicillata is positively affected by body size and negatively affected by potential competitor's activity on the forest floor during the sampling, which also significantly weakened the positive relationship with body size. These results suggest the presence of an interference/interaction occurring between the two species and affecting the foraging activity of S. perspicillata. This competitive interaction is size mediated and configured as interference competition rather than exploitative competition.

Recent Advances in the Behavioral Ecology of European Plethodontid Salamanders.

There is a recent growing interest in the study of evolutionary and behavioral ecology of amphibians. Among salamanders, Plethodontidae is the most speciose family, with more than 500 species, while in Europe, there are only 8 species, all belonging to the genus Speleomantes. European plethodontids recently received increasing attention with regard to the study of their natural history, ecology and behavior; however, the lack of standardized data, especially for the latter, hampers comparative analysis with the species from the New World. We here synthetized the recent advances in Speleomantes behavioral ecology, considering as a starting point the comprehensive monography of Lanza and colleagues published in 2006. We identified the behavioral categories that were investigated the most, but we also highlighted knowledge gaps and provided directions for future studies. By reviewing the scientific literature published within the period 2006-2022, we observed a significant increase in the number of published articles on Speleomantes behavior, overall obtaining 36 articles. Behavioral studies on Speleomantes focused mainly on trophic behavior (42%), and on intraspecific behavior (33%), while studies on pheromonal communication and interspecific behavioral interactions were lacking. In addition, most of the studies were observational (83%), while the experimental method was rarely used. After providing a synthesis of the current knowledge, we suggest some relevant topics that need to be considered in future research on the behavioral ecology of European plethodontids, highlighting the importance of a more integrative approach in which both field observations and planned experiments are used.

Weighted individual-resource networks in prey-predator systems: the role of prey availability on the emergence of modular structures.

Ecological networks, usually depicting interactions among species, have been recently down-scaled to the individual level, permitting description of patterns of inter-individual resource variation that are usually hindered at the species level. Optimal diet theory (ODT) models, applied to prey-predator systems, predict different patterns of nestedness and modularity in the network, depending on the available resources and intra-specific competition. The effect of resource availability on the emergence of networks structures, and ODT framework, has not yet fully been clarified. Here, we analyzed the structural patterns of individual-resource networks in 3 species of Mediterranean salamanders, in relation to changes in prey availability. We used weighted individual-resource network metrics to interpret the observed patterns, according to 3 ODT models. We found significant nestedness recurring in our study system, indicating that both selective and opportunistic individuals occur in the same population. Prey diversity, rather than abundance, was apparently related to inter-individual resource variation and promoted the emergence of significant modularity within all networks. The observed patterns of nestedness and modularity, together with the variation in resource diversity and intra-specific competition, are in agreement with the distinct preferences model of ODT. These findings suggest that in the focal prey-predator systems, individuals were able to perceive changes in prey diversity and to exploit in different ways the variations in composition of available resources, shifting their diet assembly rules accordingly. Our findings also confirm that the use of weighted individual-resource networks, in prey-predator systems, allows to disclose dynamics that are masked at the species or population level.

Time-for-space substitution in N-mixture models for estimating population trends: a simulation-based evaluation.

N-mixture models usually rely on a meta-population design, in which repeated counts of individuals in multiple sampling locations are obtained over time. The time-for-space substitution (TSS) in N-mixture models allows to estimate population abundance and trend of a single population, without spatial replication. This application could be of great interest in ecological studies and conservation programs; however, its reliability has only been evaluated on a single case study. Here we perform a simulation-based evaluation of this particular application of N-mixture modelling. We generated count data, under 144 simulated scenarios, from a single population surveyed several times per year and subject to different dynamics. We compared simulated abundance and trend values with TSS estimates. TSS estimates are overall in good agreement with real abundance. Trend and abundance estimation is mainly affected by detection probability and population size. After evaluating the reliability of TSS, both against real world data, and simulations, we suggest that this particular application of N-mixture model could be reliable for monitoring abundance in single populations of rare or difficult to study species, in particular in cases of species with very narrow geographic ranges, or known only for few localities.

Bayesian salamanders: analysing the demography of an underground population of the European plethodontid Speleomantes strinatii with state-space modelling.

BACKGROUND: It has been suggested that Plethodontid salamanders are excellent candidates for indicating ecosystem health. However, detailed, long-term data sets of their populations are rare, limiting our understanding of the demographic processes underlying their population fluctuations. Here we present a demographic analysis based on a 1996-2008 data set on an underground population of Speleomantes strinatii (Aellen) in NW Italy. We utilised a Bayesian state-space approach allowing us to parameterise a stage-structured Lefkovitch model. We used all the available population data from annual temporary removal experiments to provide us with the baseline data on the numbers of juveniles, subadults and adult males and females present at any given time. RESULTS: Sampling the posterior chains of the converged state-space model gives us the likelihood distributions of the state-specific demographic rates and the associated uncertainty of these estimates. Analysing the resulting parameterised Lefkovitch matrices shows that the population growth is very close to 1, and that at population equilibrium we expect half of the individuals present to be adults of reproductive age which is what we also observe in the data. Elasticity analysis shows that adult survival is the key determinant for population growth. CONCLUSION: This analysis demonstrates how an understanding of population demography can be gained from structured population data even in a case where following marked individuals over their whole lifespan is not practical.

Generalisation within specialization: inter-individual diet variation in the only specialized salamander in the world.

Specialization is typically inferred at population and species level but in the last decade many authors highlighted this trait at the individual level, finding that generalist populations can be composed by both generalist and specialist individual. Despite hundreds of reported cases of individual specialization there is a complete lack of information on inter-individual diet variation in specialist species. We studied the diet of the Italian endemic Spectacled Salamander (Salamandrina perspicillata), in a temperate forest ecosystem, to disclose the realised trophic niche, prey selection strategy in function of phenotypic variation and inter-individual diet variation. Our results showed that Salamandrina is highly specialized on Collembola and the more specialized individuals are the better performing ones. Analyses of inter-individual diet variation showed that a subset of animals exhibited a broader trophic niche, adopting different foraging strategies. Our findings reflects the optimal foraging theory both at population and individual level, since animals in better physiological conditions are able to exploit the most profitable prey, suggesting that the two coexisting strategies are not equivalent. At last this species, feeding on decomposers of litter detritus, could play a key role determining litter retention rate, nutrient cycle and carbon sequestration.

Resistance to chytridiomycosis in European plethodontid salamanders of the genus Speleomantes.

North America and the neotropics harbor nearly all species of plethodontid salamanders. In contrast, this family of caudate amphibians is represented in Europe and Asia by two genera, Speleomantes and Karsenia, which are confined to small geographic ranges. Compared to neotropical and North American plethodontids, mortality attributed to chytridiomycosis caused by Batrachochytrium dendrobatidis (Bd) has not been reported for European plethodontids, despite the established presence of Bd in their geographic distribution. We determined the extent to which Bd is present in populations of all eight species of European Speleomantes and show that Bd was undetectable in 921 skin swabs. We then compared the susceptibility of one of these species, Speleomantes strinatii, to experimental infection with a highly virulent isolate of Bd (BdGPL), and compared this to the susceptible species Alytes muletensis. Whereas the inoculated A. muletensis developed increasing Bd-loads over a 4-week period, none of five exposed S. strinatii were colonized by Bd beyond 2 weeks post inoculation. Finally, we determined the extent to which skin secretions of Speleomantes species are capable of killing Bd. Skin secretions of seven Speleomantes species showed pronounced killing activity against Bd over 24 hours. In conclusion, the absence of Bd in Speleomantes combined with resistance to experimental chytridiomycosis and highly efficient skin defenses indicate that the genus Speleomantes is a taxon unlikely to decline due to Bd.

Population dynamics and regulation in the cave salamander Speleomantes strinatii.

Time series analysis has been used to evaluate the mechanisms regulating population dynamics of mammals and insects, but has been rarely applied to amphibian populations. In this study, the influence of endogenous (density-dependent) and exogenous (density-independent) factors regulating population dynamics of the terrestrial plethodontid salamander Speleomantes strinatii was analysed by means of time series and multiple regression analyses. During the period 1993-2005, S. strinatii population abundance, estimated by a standardised temporary removal method, displayed relatively low fluctuations, and the autocorrelation function (ACF) analysis showed that the time series had a noncyclic structure. The partial rate correlation function (PRCF) indicated that a strong first-order negative feedback dominated the endogenous dynamics. Stepwise multiple regression analysis showed that the only climatic factor influencing population growth rate was the minimum winter temperature. Thus, at least during the study period, endogenous, density-dependent negative feedback was the main factor affecting the growth rate of the salamander population, whereas stochastic environmental variables, such as temperature and rainfall, seemed to play a minor role in regulation. These results stress the importance of considering both exogenous and endogenous factors when analysing amphibian long-term population dynamics.