Performance of different automatic photographic identification software for larvae and adults of the European fire salamander.

For many species, population sizes are unknown despite their importance for conservation. For population size estimation, capture-mark-recapture (CMR) studies are often used, which include the necessity to identify each individual, mostly through individual markings or genetic characters. Invasive marking techniques, however, can negatively affect the individual fitness. Alternatives are low-impact techniques such as the use of photos for individual identification, for species with stable distinctive phenotypic traits. For the individual identification of photos, a variety of different software, with different requirements, is available. The European fire salamander (Salamandra salamandra) is a species in which individuals, both at the larval stage and as adults, have individual specific patterns that allow for individual identification. In this study, we compared the performance of five different software for the use of photographic identification for the European fire salamander: Amphibian & Reptile Wildbook (ARW), AmphIdent, I3S pattern+, ManderMatcher and Wild-ID. While adults can be identified by all five software, European fire salamander larvae can currently only be identified by two of the five (ARW and Wild-ID). We used one dataset of European fire salamander larval pictures taken in the laboratory and tested this dataset in two of the five software (ARW and Wild-ID). We used another dataset of European fire salamander adult pictures taken in the field and tested this using all five software. We compared the requirements of all software on the pictures used and calculated the False Rejection Rate (FRR) and the Recognition Rate (RR). For the larval dataset (421 pictures) we found that the ARW and Wild-ID performed equally well for individual identification (99.6% and 100% Recognition Rate, respectively). For the adult dataset (377 pictures), we found the best False Rejection Rate in ManderMatcher and the highest Recognition Rate in the ARW. Additionally, the ARW is the only program that requires no image pre-processing. In times of amphibian declines, non-invasive photo identification software allowing capture-mark-recapture studies help to gain knowledge on population sizes, distribution, movement and demography of a population and can thus help to support species conservation.

Food and light availability induce plastic responses in fire salamander larvae from contrasting environments.

Phenotypic plasticity has been proposed as a mechanism facilitating the colonisation and adaptation to novel environments, such as caves. However, phenotypic plasticity in subterranean environments remains largely unexplored. Here, we test for plasticity in growth and development of fire salamander larvae (Salamandra salamandra) from subterranean and surface habitats, in response to contrasting food availability and light conditions. We hypothesized that: (i) low food availability and absence of light decrease larval growth and delay metamorphosis, (ii) light conditions mediate the effects of food availability on growth and time to metamorphosis, and (iii) larval response to contrasting light and food conditions is shaped by the habitat of origin. Our study showed that reduced food availability significantly delayed metamorphosis and slowed total length and body mass growth rates, while exposure to constant darkness slowed body mass growth rate. However, larvae slowed growth rates and increased time to metamorphosis without compromising size at metamorphosis. The effect of food availability on growth and time to metamorphosis did not change under different light conditions. Fire salamanders from subterranean and surface habitats responded differently only in relation to contrasting food availability conditions. Specifically, larvae from the surface habitat grew faster in high food conditions, while growth in larvae from the subterranean habitat was not influenced by food availability. Initial size also appeared to be an influential factor, since larger and heavier larvae grew slower, metamorphosed faster, and the size advantage was maintained in newly-metamorphosed juveniles. Overall, the results of our experiment suggest that plasticity and local adaptation favor the exploitation of aquatic subterranean habitats for breeding by fire salamanders, allowing successful development even under food shortage and day-length constraints, without compromising metamorphic size. Our findings have implications for conservation because they confirm that phenotypic plasticity plays a critical role in allowing fire salamanders to overcome altered environmental conditions.

De novo transcriptome assembly and annotation for gene discovery in Salamandra salamandra at the larval stage.

Dispersal is a key process in ecology and evolutionary biology, as it shapes biodiversity patterns over space and time. Attitude to disperse is unevenly distributed among individuals within populations, and that individual personality can have pivotal roles in the shaping of this attitude. Here, we assembled and annotated the first de novo transcriptome of the head tissues of Salamandra salamandra from individuals, representative of distinct behavioral profiles. We obtained 1,153,432,918 reads, which were successfully assembled and annotated. The high-quality of the assembly was confirmed by three assembly validators. The alignment of contigs against the de novo transcriptome led to a mapping percentage higher than 94%. The homology annotation with DIAMOND led to 153,048 (blastx) and 95,942 (blastp) shared contigs, annotated on NR, Swiss-Prot and TrEMBL. The domain and site protein prediction led to 9850 GO-annotated contigs. This de novo transcriptome represents reliable reference for comparative gene expression studies between alternative behavioral types, for comparative gene expression studies within Salamandra, and for whole transcriptome and proteome studies in amphibians.

Factors determining the dorsal coloration pattern of aposematic salamanders.

Aposematic bright colors have a key role for animal defense and can be expressed through metabolic production or by acquiring pigments from diet. Aposematic coloration can be related to both local adaptations and availability of trophic resources. The European fire salamander (Salamandra salamandra) shows significant color variability and occurs across a broad range of habitats. Here we combined field observations with common rearing experiments to disentangle the role of environmental conditions and local adaptations in determining aposematic coloration of salamander populations. We assessed color variation and measured habitat features and food availability in adults from 25 populations. Furthermore, we reared newborn larvae from 10 populations under different food availability and analyzed color of metamorphs. To assess color pattern, we measured the percentage of yellow covering the body, and the Hue, Saturation and Value of yellow coloration. Adult showed strong variation of color pattern; variation was strongly related to the individual's size, to habitat productivity and to food availability. Under common garden conditions, differences between populations were not anymore evident, and coloration was only affected by resource availability during larval development. Our results suggest that environmental conditions and food availability are more important than local adaptations in determining differences in aposematic color pattern.

In vivo and ex vivo range of motion in the fire salamander Salamandra salamandra.

Joint range of motion (RoM) analyses are fundamental to our understanding of how an animal moves throughout its ecosystem. Recent technological advances allow for more detailed quantification of this RoM (e.g. including interaction of degrees of freedom) both in ex vivo joints and in vivo experiments. Both types of data have been used to draw comparisons with fossils to reconstruct locomotion. Salamanders are often used as analogues for early tetrapod locomotion; testing such hypotheses requires an in-depth analysis of salamander joint RoM. Here, we provide a detailed dataset of the ex vivo ligamentous rotational joint RoM in the hindlimb of the fire salamander Salamandra salamandra, using a new method for collecting and visualising joint RoM. We also characterise in vivo joint RoM used during walking, via scientific rotoscoping and compare the in vivo and ex vivo data. In summary, we provide (1) a new method for joint RoM data experiments and (2) a detailed analysis of both in vivo and ex vivo data of salamander hindlimbs, which can be used for comparative studies.

A Protocol for Prolonged Surgical Anaesthesia with Recovery in Fire Salamanders Using Tricaine Mesylate (MS-222): A Case Series.

The objective of this study was to describe prolonged surgical anaesthesia and recovery in fire salamanders (Salamandra salamandra) using tricaine methanesulfonate (MS-222). A total of 14 salamanders were anaesthetised for electromyography wire implantation. Sodium bicarbonate buffered solutions (0.5-4 g l-1) of MS-222 were prepared (adjusted to pH 7.0). Anaesthesia was induced by partial immersion in pre-oxygenated 3 g l-1 solution for 20 min. Buprenorphine (0.5 mg kg-1) was administered subcutaneously. During microsurgery, heart rate (HR), solution pH and temperature were recorded. Reflectance pulse oximeter (SpO2) (Masimo Rad-57) was recorded in two salamanders. Anaesthetic plane and MS-222 pH stability (pH 7.6) were maintained by renewing administration of oxygenated MS-222 solution (0.5-3 g l-1) onto swabs that partially covered the body. Recovery started at the end of surgery (MS-222 0 g l-1). Postoperatively, salamanders were given oral meloxicam (0.2 mg kg-1). Mean time for loss of righting reflex during induction was 13.7 ± 2.2 min. Duration of anaesthesia and time to recovery were 111 ± 24.2 and 31 ± 10.3 min, respectively. Due to complications, two salamanders did not recover. Baseline HR was 67.4 ± 34.5 beats/min, and it decreased significantly until recovery (p ≤ 0.0001). In two salamanders, baseline SpO2 was 85.5% ± 14.5, SpO2 during surgery was 61% ± 6.4, improving to 80.5% ± 2.1 on recovery.In conclusion, prolonged recovery anaesthesia is achievable with MS-222 dilutions in salamander. Reflectance SpO2 could prove valuable during immersion anaesthesia.

Effects of Pine and Eucalypt ashes on bacterial isolates from the skin microbiome of the fire salamander (Salamandra salamandra).

Environmental contamination influences the diversity of the resident skin microbial community of amphibians, ultimately affecting the individual's immune system. Wildfires are expected to impact the skin microbiome, since post-fire runoff typically transports hazardous substances, that can affect terrestrial and aquatic ecosystems. The present study is the first to assess the effects of Eucalypt and Pine wildfire ash on cultivable bacterial isolates from the skin microbiome of amphibians, in particular the fire salamander (Salamandra salamandra), a common species in fire-prone Mediterranean ecosystems. To achieve this goal, samples of skin bacteria of adult individuals of S. salamandra were collected at a site without influence of wildfires. The bacterial isolates were tested against the pathogenic agent Aeromonas salmonicida for assessing their antimicrobial activity, before exposing them to a series of dilutions of aqueous extracts of Eucalypt and Pine ashes (AAEs) from high severity wildfires. From the 80 bacterial isolates collected, 48 (mostly Pseudomonas spp.) showed antimicrobial activity. Exposure of bacteria with antimicrobial activity to the Eucalypt and Pine AAEs at concentrations of 0, 6.25, 12.5, 25, 50, 75, and 100%, revealed that bacterial growth could be significantly inhibited, stimulated or unaffected by ash. Growth inhibition was found for Pine and Eucalypt AAEs at concentrations as low as 6.25% and 12.5%, respectively, but were more expressive at concentrations equal or above 50%. Eucalypt AAEs had a higher negative impact on bacterial growth than Pine AAEs, likely due to differences in metal concentrations between ash types. These findings raise concern about the future of amphibians in fire-prone regions since the foreseen increase in fire frequency and severity owing to climate changes are likely to alter the skin microbiome of amphibians, weaken the immune system and consequently increasing the incidence of infections or diseases, further contributing to the decline of the populations.

A near-natural experiment on factors influencing larval drift in Salamandra salamandra.

The larval stage of the European fire salamander (Salamandra salamandra) inhabits both lentic and lotic habitats. In the latter, they are constantly exposed to unidirectional water flow, which has been shown to cause downstream drift in a variety of taxa. In this study, a closed artificial creek, which allowed us to keep the water flow constant over time and, at the same time, to simulates with predefined water quantities and durations, was used to examine the individual movement patterns of marked larval fire salamanders exposed to unidirectional flow. Movements were tracked by marking the larvae with VIAlpha tags individually and by using downstream and upstream traps. Most individuals showed stationarity, while downstream drift dominated the overall movement pattern. Upstream movements were rare and occurred only on small distances of about 30 cm; downstream drift distances exceeded 10 m (until next downstream trap). The simulated flood events increased drift rates significantly, even several days after the flood simulation experiments. Drift probability increased with decreasing body size and decreasing nutritional status. Our results support the production hypothesis as an explanation for the movements of European fire salamander larvae within creeks.

Allocation of Salamandra auriculata Holbrook, 1838, with a new species of swamp-dwelling dusky salamander (Plethodontidae: Desmognathus) from the Atlantic Coastal Plain.

Most swamp-dwelling dusky salamanders of the genus Desmognathus from the Coastal Plain were long treated as a single species (Desmognathus auriculatus) ranging from east Texas to southeastern Virginia. This taxon concept was based on the name Salamandra auriculata Holbrook, 1838 with type locality Riceboro, Liberty County, Georgia and a type series that could not be located by later authors. Recent workers have been unable to locate or verify swamp-dwelling populations from east Texas and western Louisiana, which appear to be extirpated and may not have represented a distinct taxon from co-occurring lineages of D. conanti. Recent molecular phylogenies have supported at least four distinct species-level taxa within D. auriculatus. Populations from the Gulf Coastal Plain in eastern Louisiana, Mississippi, and southwestern Alabama were recently described as D. valentinei Means, Lamb, and Bernardo, 2017 and D. pascagoula Pyron, O'Connell, Lamb, and Beamer, 2022. This leaves two remaining species-level lineages with uncertain taxonomy and nomenclature: D. auriculatus A (Alabama, Florida, and Georgia), and D. auriculatus B/C (Georgia, South Carolina, and North Carolina), both of which occur near the type locality. We recently located a specimen at the Muséum national d'Histoire naturelle in Paris (MNHN 0.4675) that we concluded is one of Holbrook's syntypes and designated it as the lectotype, but without allocation. Here, we use linear morphometrics to confidently allocate it to D. auriculatus A, bolstered by examination of three historical topotypic collections. This requires a new name for D. auriculatus B/C, which we describe as D. valtos sp. nov. (suggested common name: Carolina Swamp Dusky Salamander) from Otter Creek, Craven County, North Carolina. Other related and sympatric species of Desmognathus remain to be described from the Atlantic Coastal Plain and adjacent Piedmont of the southeastern United States.

Independent evolutionary transitions to pueriparity across multiple timescales in the viviparous genus Salamandra.

The ability to bear live offspring, viviparity, has evolved multiple times across the tree of life and is a remarkable adaptation with profound life-history and ecological implications. Within amphibians the ancestral reproductive mode is oviparity followed by a larval life stage, but viviparity has evolved independently in all three amphibian orders. Two types of viviparous reproduction can be distinguished in amphibians; larviparity and pueriparity. Larviparous amphibians deliver larvae into nearby ponds and streams, while pueriparous amphibians deliver fully developed juveniles and thus do not require waterbodies for reproduction. Among amphibians, the salamander genus Salamandra is remarkable as it exhibits both inter- and intraspecific variation in the occurrence of larviparity and pueriparity. While the evolutionary relationships among Salamandra lineages have been the focus of several recent studies, our understanding of how often and when transitions between modes occurred is still incomplete. Furthermore, in species with intraspecific variation, the reproductive mode of a given population can only be confirmed by direct observation of births and thus the prevalence of pueriparous populations is also incompletely documented. We used sequence capture to obtain 1,326 loci from 94 individuals from across the geographic range of the genus, focusing on potential reproductive mode transition zones. We also report additional direct observations of pueriparous births for 20 new locations and multiple lineages. We identify at least five independent transitions from the ancestral mode of larviparity to pueriparity among and within species, occurring at different evolutionary timescales ranging from the Pliocene to the Holocene. Four of these transitions occurred within species. Based on a distinct set of markers and analyses, we also confirm previous findings of introgression between species and the need for taxonomic revisions in the genus. We discuss the implications of our findings with respect to the evolution of this complex trait, and the potential of using five independent convergent transitions for further studies on the ecological context in which pueriparity evolves and the genetic architecture of this specialized reproductive mode.

Microplastics increase susceptibility of amphibian larvae to the chytrid fungus Batrachochytrium dendrobatidis.

Microplastics (MPs), a new class of pollutants that pose a threat to aquatic biodiversity, are of increasing global concern. In tandem, the amphibian chytrid fungus Batrachochytrium dendrobatidis (Bd) causing the disease chytridiomycosis is emerging worldwide as a major stressor to amphibians. We here assess whether synergies exist between this infectious disease and MP pollution by mimicking natural contact of a highly susceptible species (midwife toads, Alytes obstetricans) with a Bd-infected reservoir species (fire salamanders, Salamandra salamandra) in the presence and absence of MPs. We found that MP ingestion increases the burden of infection by Bd in a dose-dependent manner. However, MPs accumulated to a greater extent in amphibians that were not exposed to Bd, likely due to Bd-damaged tadpole mouthparts interfering with MP ingestion. Our experimental approach showed compelling interactions between two emergent processes, chytridiomycosis and MP pollution, necessitating further research into potential synergies between these biotic and abiotic threats to amphibians.

Measuring athletic performance in post-metamorphic fire salamanders.

OBJECTIVE: Athletic performances are dynamic movements that are physically challenging and often predict individual success in ecological contexts. They stem from a complex integration of multiple phenotypic traits-e.g., morphological, physiological and behavioural-that dictate animal survival and individual fitness. However, directly quantifying athletic performances can be particularly challenging in cryptic, slow-moving species or not very reactive in attitude. Here we present and describe a rapid, simple, and low-cost method to measure athletic performance in post-metamorphic individuals of the fire salamander Salamandra salamandra. While extremely reactive during the larval stage, adult salamanders are, in fact, cryptic and relatively slow-moving. RESULTS: Forcing terrestrial juveniles to swim under standard, albeit ecologically plausible, laboratory conditions, and using an automatic point-mass tracking tool, we were able to measure maximal and average performance indicators of post-metamorphic individuals. This method avoids inter-individual variation in motivation, as it forces individuals to perform at their best. Moreover, with this method, measures of athletic performance will be directly comparable between larval and terrestrial stages, allowing to study the contribution of carryover effects to the wide range of processes implicated in the eco-evo-devo of athletic performance in salamanders.

Diet diversity and environment determine the intestinal microbiome and bacterial pathogen load of fire salamanders.

Diverse communities of symbiotic microbes inhabit the digestive systems of vertebrates and play a crucial role in animal health, and host diet plays a major role in shaping the composition and diversity of these communities. Here, we characterized diet and gut microbiome of fire salamander populations from three Belgian forests. We carried out DNA metabarcoding on fecal samples, targeting eukaryotic 18S rRNA of potential dietary prey items, and bacterial 16S rRNA of the concomitant gut microbiome. Our results demonstrated an abundance of soft-bodied prey in the diet of fire salamanders, and a significant difference in the diet composition between males and females. This sex-dependent effect on diet was also reflected in the gut microbiome diversity, which is higher in males than female animals. Proximity to human activities was associated with increased intestinal pathogen loads. Collectively, the data supports a relationship between diet, environment and intestinal microbiome in fire salamanders, with potential health implications.

Physical and ecological isolation contribute to maintain genetic differentiation between fire salamander subspecies.

Landscape features shape patterns of gene flow among populations, ultimately determining where taxa lay along the continuum between panmixia to complete reproductive isolation. Gene flow can be restricted, leading to population differentiation in two non-exclusive ways: "physical isolation", in which geographic distance in combination with the landscape features restricts movement of individuals promoting genetic drift, and "ecological isolation", in which adaptive mechanisms constrain gene flow between different environments via divergent natural selection. In central Iberia, two fire salamander subspecies occur in parapatry across elevation gradients along the Iberian Central System mountains, while in the adjacent Montes de Toledo Region only one of them occurs. By integrating population and landscape genetic analyses, we show a ubiquitous role of physical isolation between and within mountain ranges, with unsuitable landscapes increasing differentiation between populations. However, across the Iberian Central System, we found strong support for a significant contribution of ecological isolation, with low genetic differentiation in environmentally homogeneous areas, but high differentiation across sharp transitions in precipitation seasonality. These patterns are consistent with a significant contribution of ecological isolation in restricting gene flow among subspecies. Overall, our results suggest that ecological divergence contributes to reduce genetic admixture, creating an opportunity for lineages to follow distinct evolutionary trajectories.

Celiotomia e gastrotomia para remoção cirúrgica de corpos estranhos em axolote (Ambystoma mexicanum): relato de caso / Celiotomy and gastrotomy for surgical removal of foreign bodies in axolotl (Ambystoma mexicanum): case report

A ingestão de corpos estranhos em aquários artificiais é uma ocorrência frequente observada na clínica cirúrgica de anfíbios criados como pets, o que inclui os axolotes (Ambystoma mexicanum). O presente relato descreve um caso de ingestão de sete corpos estranhos em um axolote macho, de cinco meses de idade, com histórico de abaulamento irregular e irredutível da superfície corpórea ventral, de consistência firme. Na anamnese foi informado que o animal habitava um aquário com substrato de cascalhos. Ao exame físico, o axolote apresentou bom estado geral e parâmetros vitais dentro da normalidade para a espécie. Os materiais deglutidos foram identificados em região gástrica por meio de exame radiográfico corpóreo total, e suas características condiziam com o substrato utilizado no aquário do paciente. Como protocolo anestésico, foi priorizada a imersão em Isofluorano e gás oxigênio, com o objetivo de atingir a via branquial e, ocasionalmente, transdérmica. A remoção cirúrgica foi feita através de celiotomia e gastrotomia em ambiente aquático com temperatura, pH e coleção bacteriana controlados, conforme literatura disponível e tendo em consideração a natureza, diâmetro e localização dos corpos estranhos. Após cinco e quinze dias do procedimento, acompanhou-se a cicatrização da ferida cirúrgica, sendo possível constatar bom restabelecimento da continuidade dos tecidos e bom estado geral do paciente.

The ingestion of foreign bodies in artificial aquariums is a frequent occurrence observed in the surgical clinic of amphibians raised as pets, which includes axolotls (Ambystoma mexicanum). The present report describes a case of ingestion of seven foreign bodies in a male axolotl, five months old, with a history of irregular and irreducible bulging of the ventral body surface, with firm consistency. In the anamnesis, it was reported that the animal inhabited an aquariums with gravel substrate. On physical examination, the axolotl showed good general condition and vital parameters within the normal range for the species. The swallowed materials were identified in the gastric region by means of total body radiographic examination, and their characteristics were consistent with the substrate used in the patient's aquarium. As anesthetic protocol, immersion in Isofluorane and oxygen gas was prioritized, in order to reach the branchial and, occasionally, transdermal route. Surgical removal was performed through celiotomy and gastrotomy in an aquatic environment with controlled temperature, pH and bacterial collection, according to available literature and taking into account the nature, diameter and location of foreign bodies. After five and fifteen days of the procedure, the healing of the surgical wound was monitored, showing a good restoration of tissue continuity and a good general condition of the patient.

Toxin variation among salamander populations: discussing potential causes and future directions.

Amphibians produce defensive chemicals which provide protection against both predators and infections. Within species, populations can differ considerably in the composition and amount of these chemical defenses. Studying intraspecific variation in toxins and linking it to environmental variables may help us to identify the selective drivers of toxin evolution, such as predation pressure and infection risk. Recently, there has been a renewed interest in the unique toxins produced by salamanders from the genus Salamandra: the samandarines. Despite this attention, intraspecific variation has largely been ignored within Salamandra-species. The aim of this study was to investigate whether geographic variation in profiles of samandarines exists, by sampling 4 populations of Salamandra atra over its range in the Dinaric Alps. In addition, we preliminary explored whether potential variation could be explained by predation (counting the number of snake species) and infection risk (cultivation and genomic analyses of collected soil samples). Salamanders from the 4 populations differed in toxin composition and in the size of their poison glands, although not in overall toxin quantity. Nor predation nor infection risk could explain this variation, as populations barely differed in these variables. Sampling over a much broader geographic range, using better estimators for predation and infection risk, will contribute to an improved understanding of how environment may shape variation in chemical defenses. Nevertheless, as the 4 populations of S. atra did differ in their toxin profiles, we propose that this species provides an interesting opportunity for further ecological and evolutionary studies on amphibian toxins.

Macro- and micromorphological remodeling of olfactory organs throughout the ontogeny of the fire salamander Salamandra salamandra (Linnaeus, 1758).

This article studies the morphological remodeling of olfactory organs in the fire salamander (Salamandridae, Caudata), from the larval stages of ontogeny to the adult and throughout the course of the annual cycle. The fire salamander exhibits adaptations to the aquatic environment during premetamorphic life and terrestrial adaptations after metamorphosis. During adulthood, the annual activity of this species is divided into three seasonal periods: a breeding period, a nonbreeding period, and hibernation. We observed significant differences in morphology of olfactory organs between developmental stages as well as between each period within the annual cycle. For the first time in caudates, we examined the morphology of olfactory organs during the winter period (wintering larvae, hibernating adults). The results show that the remodeling of olfactory organs during the life of the fire salamander occurs both on macro- and micromorphological levels. Macromorphological ontogenetic variability includes the shape of the main olfactory chamber (MOC) and the distribution of olfactory epithelium (OE) in the MOC and in the vomeronasal organ (VNO). In larvae, the vomeronasal epithelium (VNE) is in a separate cavity, while in the post-metamorphic stages of ontogeny, the VNE occurs in the diverticulum of the MOC. In adult fire salamanders, both olfactory organs are most developed during the breeding season and reduced during hibernation. The VNE and OE in the MOC are also reduced during hibernation. Micro-morphological changes included different types/subtypes of olfactory receptor neurons (ORNs) in the OE in particular stages of ontogeny and periods within the annual cycle, for example, ciliate ORNs are present in the VNE only in the larval stages and giant ORNs occur only in nonbreeding adults. Also, there was a variable set of types of olfactory supporting cells in the VNO of the fire salamander during pre- and postmetamorphic life stages.

The Antioxidant Peptide Salamandrin-I: First Bioactive Peptide Identified from Skin Secretion of Salamandra Genus (Salamandra salamandra).

Amphibian skin is a multifunctional organ that plays key roles in defense, breathing, and water balance. In this study, skin secretion samples of the fire salamander (Salamandra salamandra) were separated using RP-HPLC and de novo sequenced using MALDI-TOF MS/MS. Next, we used an in silico platform to screen antioxidant molecules in the framework of density functional theory. One of the identified peptides, salamandrin-I, [M + H]+ = 1406.6 Da, was selected for solid-phase synthesis; it showed free radical scavenging activity against DPPH and ABTS radicals. Salamandrin-I did not show antimicrobial activity against Gram-positive and -negative bacteria. In vitro assays using human microglia and red blood cells showed that salamandrin-I has no cytotoxicity up to the concentration of 100 µM. In addition, in vivo toxicity tests on Galleria mellonella larvae resulted in no mortality at 20 and 40 mg/kg. Antioxidant peptides derived from natural sources are increasingly attracting interest. Among several applications, these peptides, such as salamandrin-I, can be used as templates in the design of novel antioxidant molecules that may contribute to devising strategies for more effective control of neurological disease.

Comparative landscape genetics reveals the evolution of viviparity reduces genetic connectivity in fire salamanders.

Evolutionary changes in reproductive mode may affect co-evolving traits, such as dispersal, although this subject remains largely underexplored. The shift from aquatic oviparous or larviparous reproduction to terrestrial viviparous reproduction in some amphibians entails skipping the aquatic larval stage and, thus, greater independence from water. Accordingly, amphibians exhibiting terrestrial viviparous reproduction may potentially disperse across a wider variety of suboptimal habitats and increase population connectivity in fragmented landscapes compared to aquatic-breeding species. We investigated this hypothesis in the fire salamander (Salamandra salamandra), which exhibits both aquatic- (larviparity) and terrestrial-breeding (viviparity) strategies. We genotyped 426 larviparous and 360 viviparous adult salamanders for 13 microsatellite loci and sequenced a mitochondrial marker for 133 larviparous and 119 viviparous individuals to compare population connectivity and landscape resistance to gene flow within a landscape genetics framework. Contrary to our predictions, viviparous populations exhibited greater differentiation and reduced genetic connectivity compared to larviparous populations. Landscape genetic analyses indicate viviparity may be partially responsible for this pattern, as water courses comprised a significant barrier only in viviparous salamanders, probably due to their fully terrestrial life cycle. Agricultural areas and, to a lesser extent, topography also decreased genetic connectivity in both larviparous and viviparous populations. This study is one of very few to explicitly demonstrate the evolution of a derived reproductive mode affects patterns of genetic connectivity. Our findings open avenues for future research to better understand the eco-evolutionary implications underlying the emergence of terrestrial reproduction in amphibians.

Weighing density and kinship: Aggressive behavior and time allocation in fire salamander (Salamandra infraimmaculata).

Kin-biased behavior (that is responding differentially to kin and non-kin) is thought to be adaptive in many social interactions. One example of this kin bias is behaving less aggressively toward a relative than a non-relative, a behavior which yields inclusive fitness benefits. However, data are lacking about the ability of animals to weigh their preference for kinship and the density of conspecifics simultaneously and to respond accordingly. Fire salamanders (Salamandra infraimmaculata) larviposit in high densities in ponds. Thus, larvae of different females confront competition and predation by other larvae. We studied whether larvae prefer their kin over particular density or vice versa. We experimentally used a transparent glass aquarium with inner chambers to test the responses of a focal larva toward its siblings and non-siblings. Specifically, we quantified the time a focal larva spent near its siblings or non-siblings, presented in varying densities, and the aggression level it demonstrated. We found that focal larvae spent more time near non-siblings if non-sibling and sibling groups were of equal density. The focal larvae were also more aggressive toward non-siblings. The results may be explained by the cannibalistic nature of these larvae: high density may provide more opportunities for food, especially when non-siblings are present. Further explanations for these findings may include other advantages of staying in a larger group and/or the stronger olfactory and visual stimulation offered by groups compared to a single individual. These findings suggest that larvae make differential responses toward conspecifics depending simultaneously on the level of relatedness and the density of the group. Such responses have important implications for social-aggregation decisions and may especially affect the fitness of cannibalistic species.