Triturus Carnifex Research Articles

Salamanders on the bench – A biocompatibility study of salamander skin secretions in cell cultures

Salamanders have evolved a wide variety of antipredator mechanisms and behavior patterns, including toxins and noxious or adhesive skin secretions. The high bonding strength of the natural bioadhesives makes these substances interesting for biomimetic research and applications in industrial and medical sectors. Secretions of toxic species may help to understand the direct effect of harmful substances on the cellular level.In the present study, the biocompatibility of adhesive secretions from four salamander species (Plethodon shermani, Plethodon glutinosus, Ambystoma maculatum, Ambystoma opacum) were analyzed using the MTT assay in cell culture and evaluated against toxic secretions of Pleurodeles waltl, Triturus carnifex, Pseudotriton ruber, Tylototriton verrucosus, and Salamandra salamandra. Their effect on cells was tested in direct contact (direct culture) or under the influence of the extract (indirect exposure) in accordance with the protocol of the international standard norm ISO 10993-5. Human dermal fibroblasts (NHDF), umbilical vein endothelial cells (HUVEC), and articular chondrocytes (HAC), as well as the cell lines C2C12 and L929 were used in both culture types.While the adhesive secretions from Plethodon shermani are cytocompatible and those of Ambystoma opacum are even advantageous, those of Plethodon glutinosus and Ambystoma maculatum appear to be cytotoxic to NDHF and HUVEC. Toxic secretions from Salamandra salamandra exhibited harmful effects on all cell types. Pseudotriton ruber and Triturus carnifex secretions affected certain cell types marginally; those from Pleurodeles waltl and Tylototriton verrucosus were generally well tolerated.The study shows for the first time the effect of salamander secretions on the viability of different cell types in culture. Two adhesive secretions appeared to be cell compatible and are therefore promising candidates for future investigations in the field of medical bioadhesives. Among the toxic secretions tested, only two of the five had a harmful effect on cells, indicating different cell toxicity mechanisms.

Factors driving semi-aquatic predator occurrence in traditional cattle drinking pools: conservation issues

<p>In several cases, human impact on water bodies and on their freshwater communities is detrimental, but in some cases the human activity may favour and enhance the biodiversity of small water bodies, as traditional cattle drinking pools. Despite their small size, small water bodies may constitute hot spot of biodiversity often representing the only lentic aquatic biotope in landscapes where superficial water lacks or flows in lotic environments like creeks and streams. Predators are good indicators of biodiversity in ponds and give information of food chain web complexity. In particular, semi-aquatic predators like amphibians and dragonflies may account for a substantial percentage of energy flow between aquatic and terrestrial ecosystems. In this study, we evaluated the conservation value of traditional cattle drinking pools building by assessing the factors determining the occurrence and distribution of the semi-aquatic predators. From April to August 2015, we investigated 30 distinct pools recording several abiotic and biotic environmental variables. We detected 4 semi-aquatic predators: <em>Salamandra salamandra</em> larvae, <em>Triturus carnifex</em>, <em>Aeshna</em> sp. larvae and <em>Libellula</em> sp. larvae. Abiotic features played a major role in shaping the predator community that resulted linked to stable, with no dryness period, and large drinking pools. Invertebrate prey biomass was not particularly important, while vegetation cover and occurrence of unpalatable tadpoles were the most important biotic features of the pools. Our study provides novel evidence on the importance of cattle drinking pools management to preserve biodiversity especially in areas where traditional pastoral activity is disappearing.</p>

Photo-identification in amphibian studies: a test of I3S Pattern

Photo-identification is used for individual recognition of several animal species. It gives the possibility to take photos of large species from a distance or to avoid invasive marking techniques in small animals. For amphibians, the use of non-invasive marking methods is even more relevant in the light of their global decline. Here we use the photo-identification data from a population of Triturus carnifex to validate the photo-identification software I 3 S Pattern. This recently developed utility has never been applied to amphibians. The software proved to be efficient and accurate for individual recognition for this species. Contrarily to the previous releases of the I 3 S family, I 3 S Pattern is particularly suitable for amphibians characterized by a complex individual pattern of large blotches or irregular spots, which are not readily identified by eye.

Histological changes, apoptosis and metallothionein levels in Triturus carnifex (Amphibia, Urodela) exposed to environmental cadmium concentrations

The aim of this study was to verify if the freshwater safety values established from the European Community (1998) and the Italian Ministry of Health (2001) for cadmium (44.5nM/L in drinking water and 178nM/L in sewage waters) were safe for amphibians, since at these same concentrations cadmium induced endocrine disruption in the newt Triturus carnifex. Adult male specimens of T. carnifex were exposed daily to cadmium (44.5nM/L and 178nM/L as CdCl2, nominal concentrations), respectively, during 3- and 9-months; at the same time, control newts were exposed to tap water only. The accumulation of cadmium in the skin, liver and kidney, the levels of metallothioneins in the skin and the liver, the expression of metallothionein mRNA in the liver, as well as the presence of histological alterations and of apoptosis in the target organs were evaluated. The 9-months exposure induced cadmium accumulation in all the tissues examined; moreover, histological changes were observed in all the tissues examined, irrespective of the dose or the time of exposure. Apoptosis was only detected in the kidney, whereas metallothioneins and metallothionein mRNA did not increase. This study demonstrates that the existing chronic water quality criterion established for cadmium induces in the newt T. carnifex cadmium accumulation and histological alterations in the target organs examined. Together with our previous results, showing that, at these same concentrations, cadmium induced endocrine disruption, the present results suggest that the existing chronic water quality criterion for cadmium appears to be not protective of amphibians.

Genetic pollution of a threatened native crested newt species through hybridization with an invasive congener in the Netherlands

Genetic pollution of a native species through hybridization with an invasive species poses an insidious conservation threat. To expose genetic pollution, molecular methods employing multilocus data are required. We present a case study of genetic pollution via hybridization of a native crested newt species, Triturus cristatus, by the invasive Triturus carnifex on the Veluwe in the Netherlands. We sequenced 50 nuclear markers by next generation sequencing and one mitochondrial marker by Sanger sequencing for four populations from the native range of both parent species and eleven ponds on the Veluwe. We use three population genetic approaches (HIest, BAPS and Structure) to determine the genetic composition of the Veluwe newts based on all nuclear markers, a subset of 18 diagnostic markers and the complementary 32 non-diagnostic markers, with and without parental populations. BAPS underestimates genetic pollution, whereas Structure is comparatively accurate compared to HIest, although Structure’s relative advantage decreases with the diagnosticity of the markers. Data simulation confirms these findings. Genetic composition of the Veluwe ponds ranges from completely native, via different degrees of genetic admixture, to completely invasive. The observed hybrid zone appears to be bimodal, suggesting negative selection against hybrids. A genetic footprint of the native species is present in invasive populations, evidencing that the invasive locally replaced the native species. Genetic pollution is currently confined to a small area, but the possibility of further expansion cannot be excluded. Removal of genetic pollution will not be easy. We emphasize the need for legal guidance to manage genetic pollution.

Beam Brook revisited: a molecular study of a historically introduced non-native amphibian (Triturus carnifex) and its limited introgression into native UK Triturus cristatus populations

Human mediated introductions of non-native species can pose a major threat to global biodiversity on several accounts i.e. through competition, the introduction of novel pathogens, and genetic pollution. Where hybridization occurs between two closely related species the F1 offspring are usually phenotypically discernible whereas F2 hybrid, backcrossed or admixed individuals become more difficult to separate. At this point the utilization of molecular methods is required in conservation efforts to differentiate and manage populations. This study demonstrated how a possible threat of hybridization from an introduced non-native (T. carnifex) with a protected native newt species (T. cristatus) could be investigated with molecular tools, and examined the current extent of its genetic introgression over an 80 years period. The results confirmed that hybridization had taken place at the site of introduction (and continues to do so), and that historically limited local dispersal of both non-natives and/or hybrids had occurred sometime in the past. However, the data suggests that although dispersal of hybrids into a local satellite site may still be occuring, hybridization with native species appears limited.

Micronucleus test on Triturus carnifex as a tool for environmental biomonitoring.

The amphibian micronucleus test has been widely used during the last 30 years to test the genotoxic properties of several chemicals and as a tool for ecogenotoxic monitoring. The vast majority of these studies were performed on peripheral blood of urodelan larvae and anuran tadpoles and to a lesser extent adults were also used. In this study, we developed protocols for measuring micronuclei in adult shed skin cells and larval gill cells of the Italian crested newt (Triturus carnifex). Amphibians were collected from ponds in two protected areas in Italy that differed in their radon content. Twenty-three adult newts and 31 larvae were captured from the radon-rich pond, while 20 adults and 27 larvae were taken from the radon-free site. The animals were brought to the laboratory and the micronucleus test was performed on peripheral blood and shed skins taken from the adults and on larval gills. Samples from the radon-rich site showed micronucleus frequencies higher than those from the radon-free site and the difference was statistically significant in gill cells (P < 0.00001). Moreover, the larval gills seem to be more sensitive than the adult tissues. This method represents an easy (and noninvasive in the case of the shed skin) application of the micronucleus assay that can be useful for environmental studies in situ.

THE DISTRIBUTION AND HABITATS OF THE NEWT (TRITURUS, AMPHIBIA) IN CALABRIA (SOUTHERN ITALY)

SUMMARY We provide data about the present day distribution and ecology of the Italian southern-most newt populations triturus, Amphibia). In 1981–1986 the Calabrian wetlands were studied and classified. The presence/absence of the species were recorded as well as their main morphometric parameters. We observed and described 69 Triturus italicus (Peracca), 15 Triturus camifex (Laurenti) and 4 Triturus alpestris (Laurenti) breeding sites. Triturus italicus shows a high ecological plasticity and preferably colonizes man-made tanks or small seasonal pools below 800 m. In contrast, Triturus carnifex and Triturus alpestris are mostly found above 800 m and in ponds located in the beech woods. Ecological analysis highlighted how species density depends on climate and on the presence of predators.

Metacercariae of Clinostomum complanatum (Trematoda: Digenea) in European newts Triturus carnifex and Lissotriton vulgaris (Caudata: Salamandridae)

Adults of Clinostomum spp. are digenetic trematodes found in fish-eating birds, reptiles and occasionally mammals, including humans. Freshwater snails serve as first intermediate hosts and many fish species and amphibians as second intermediate hosts. To date, amphibian hosts of Clinostomum metacercariae include members of urodele and anuran families in North America, but no data are available on infections of European amphibians, including newts. In this study, we characterize infections of Clinostomum complanatum metacercariae in four smooth (Lissotriton vulgaris) and 18 Italian crested newts (Triturus carnifex) from an artificial pond located in a protected area in Tuscany, Italy. Parasites were surgically removed from the infected newts and identified both morphologically and using sequences of a mitochondrial gene, cytochrome c oxidase I, and the ribosomal markers, internal transcribed spacers. This is the first record of C. complanatum in European newts and, more generally, in amphibians in Europe.

Diet of the newt, Triturus carnifex (Laurenti, 1768), in the flooded karst sinkhole Pozzo del Merro, central Italy

Karst habitats host a high number of specialized organisms that contribute to complex and peculiar food webs. In underground aquatic habitats, vertebrates are the top predators that strongly influence and regulate prey communities. In this study, the diet of the Italian crested newt, Triturus carnifex, in the world's deepest karst phreatic sinkhole, the Pozzo del Merro in Latium, central Italy, was analyzed. We obtained both stomach and fecal contents from twenty adult newts (ten females and ten males) sampled in summer 2010. Availability of prey in the sinkhole also was determined. Prey items were identified and classified into ten ecological groups. At Pozzo del Merro, during the summer, the aquatic stage of T. carnifex was specialized on the pre-imaginal stages of the small China-mark, Cataclysta lemnata. The recently described endemic stygobitic crustacean Niphargus cornicolanus was not found in stomach contents. Finally, our results showed that analyses of stomach and fecal contents may provide different information on the diet of newts in their aquatic phase.

A Review of a Rare Type of Anomaly in Amphibians, Tail Duplication and Bifurcation, with Description of Three New Cases in European Species (Triturus dobrogicus,Triturus carnifex, andHyla arborea)

Abstract We review the natural occurrence of tail duplications and tail bifurcations in amphibians as well as experimental studies that induced tail duplications. For natural populations, we found 10 publications that mention tail duplications or bifurcations in a total of 24 individual larval amphibians belonging to 15 species, mainly from Europe and South America. Nineteen publications describe the incidence for 34 individual postlarval urodeles from 12 species from North America, Europe, and Asia. Here we add three new observations: a subadult female Triturus dobrogicus with a duplicated tail and an adult female Triturus carnifex with a bifurcated tail from Hungary, and a tadpole of Hyla arborea with a bifurcated tail tip from Greece. The causes of tail duplications in wild amphibians remain unknown. Similar tail anomalies have been obtained in the laboratory when parts of the chorda dorsalis were destroyed, when tar is injected into the tail, or when the lumbar region is irradiated.

Metabolic costs of hybridization in Newts

Abstract. Heterospecific hybrids often suffer from a lowered fitness relative to parental species. Contextdependent intrinsic costs of hybridization are partially due to a malfunction in cell biochemical machinery that affects metabolic rates at the organismal level. This study examines whether heterospecific hybridization influences the metabolic costs of maintenance in F1 hybrids between closely related newts, Triturus carnifex and T. dobrogicus. When controlled for body size, oxygen consumption in hybrid newts was 59–76 % higher than in the parental species. This suggests that high standard metabolic rates in hybrids may contribute to the costs of hybridization in newts.

One species, three Pleistocene evolutionary histories: phylogeography of the Italian crested newt, Triturus carnifex.

Phylogeographic patterns of temperate species from the Mediterranean peninsulas have been investigated intensively. Nevertheless, as more phylogeographies become available, either unique patterns or new lines of concordance continue to emerge, providing new insights on the evolution of regional biotas. Here, we investigated the phylogeography and evolutionary history of the Italian crested newt, Triturus carnifex, through phylogenetic, molecular dating and population structure analyses of two mitochondrial gene fragments (ND2 and ND4; overall 1273 bp). We found three main mtDNA lineages having parapatric distribution and estimated divergence times between Late Pliocene and Early Pleistocene. One lineage (S) was widespread south of the northern Apennine chain and was further geographically structured into five sublineages, likely of Middle Pleistocene origin. The second lineage (C) was widespread throughout the Padano–Venetian plain and did not show a clear phylogeographic structure. The third lineage (N) was observed in only two populations located on western Croatia/Slovenia. Results of analysis of molecular variance suggested that partitioning populations according to the geographic distribution of these lineages and sublineages explains 76% of the observed genetic variation. The phylogeographic structure observed within T. carnifex and divergence time estimates among its lineages, suggest that responses to Pleistocene environmental changes in this single species have been as diverse as those found previously among several codistributed temperate species combined. Consistent with the landscape heterogeneity, physiographic features, and palaeogeographical evolution of its distribution range, these responses encompass multiple refugia along the Apennine chain, lowland refugia in large peri-coastal plains, and a ‘cryptic’ northern refugium.

Can macrophytes be a surrogate for amphibians and physico-chemical features in pond classifications?

Our primary goal was to test how consistently macrophytes, physico-chemical features and amphibians classify pond sites, by applying a measure of classification strength based on a set of cross-tests performed with randomisation protocols. Finally, we used ordination methods to identify the major environmental factors correlated with each biotic group. Significant results of concordance and higher values of relative classification strength were obtained at two (or more) cut levels, when the plant classification was performed on amphibians and on physico-chemical characteristics. Significant results and higher values of relative classification strength were also obtained at a cut level when the amphibian classification was performed on physico-chemical features. The ordination analyses revealed that plants and amphibians were affected by the same pond features, mainly conductivity, size and depth. Ponds with high conductivity were dominated by tall emergent plants of the genus Typha and were the preferential sites for Bufo bufo. Smaller shallow ponds with small emergent plants seemed instead to favour Rana dalmatina. Deep ponds with low conductivity were mostly occupied by floating and submerged plants, such as Potamogeton natans and Chara hispida, and hosted newts (Triturus carnifex and T. vulgaris), probably because the latter depend on well structured vegetation with submerged plants for egg deposition. These results suggest that pond ecosystems have “two levels of influence”, and that plants are the “middle level” between environmental features and amphibian assemblages, since they are directly influenced by the former and directly influence the latter. It is probably by virtue of this intermediate position that the classification of ponds based on plant assemblages can be used as a surrogate for predicting environmental features and the presence of amphibian species of conservation interest, in order to preserve their habitat through preliminary and cost-effective assessments. Given the ongoing threats to ponds, these findings are important for their protection, and better understanding of the ecological preferences of various plant and amphibian species is useful for planning management and conservation strategies.

Endocrine-disrupting effects of nonylphenol in the newt, Triturus carnifex (Amphibia, Urodela)

The aim of our study was to verify whether environmental concentrations of nonylphenol influenced the adrenal gland of Triturus carnifex. Newts were exposed to 19μg/L nominal concentration of nonylphenol throughout the periods of December–January and March–April, corresponding to different stages of the chromaffin cell functional cycle. The morphological features of the steroidogenic and chromaffin tissues, and the serum levels of ACTH, aldosterone, corticosterone, norepinephrine and epinephrine were evaluated. Nonylphenol did not influence ACTH serum levels. During the two periods examined, the steroidogenic tissue had the same reaction: the quantity of cytoplasmic lipids, and the corticosteroid serum levels, decreased, suggesting the inhibition of synthesis and release of corticosteroids. During the two periods examined, the chromaffin tissue reacted differently to nonylphenol. During December–January, the numeric ratio of norepinephrine granules to epinephrine granules, and the epinephrine serum levels, increased, suggesting the stimulation of epinephrine release. During March–April, the numeric ratio of norepinephrine granules to epinephrine granules did not change, and the norepinephrine serum levels decreased, suggesting the inhibition of norepinephrine release. Our results show that nonylphenol influences the activity of the newt adrenal gland; considering the physiological role of this gland, our results suggest that nonylphenol may contribute to amphibian decline.

The evolution of the adult body form of the crested newt (Triturus cristatussuperspecies, Caudata, Salamandridae)

We characterized the adult body form of the crested newt (Triturus cristatus superspecies) and explored its evolution. From seven morphometric traits, we determined that body size, interlimb distance and head width define the body form. None of the morphometric traits showed a phylogenetic signal. Three body-shape morphotypes (Triturus dobrogicus + T. cristatus, Triturus carnifex + Triturus macedonicus and Triturus karelinii + Triturus arntzeni) and three body-size morphotypes (T. dobrogicus, T. cristatus and all other crested newts) could be recognized. The ancestral phenotype (a large body with a short trunk and a wide head) characterized T. karelinii and T. arntzeni. Triturus carnifex and T. macedonicus had a somewhat different phenotype (large body and wide head, accompanied by mild body elongation). The most derived phenotype included body size reduction and more pronounced body elongation in T. cristatus and, especially, in T. dobrogicus. Body elongation occurred by trunk lengthening but not head and tail lengthening. Additionally, contrary to other tetrapods, evolutionary axis elongation in crested newts was followed by a decrease in body size. We advocate the hypothesis that ecology drives the evolution of body form in crested newts.

Annual and daily serum aldosterone and catecholamine patterns in males of the Italian crested newt, Triturus carnifex (Amphibia, Urodela)

The present work studied the annual and daily aldosterone, norepinephrine and epinephrine patterns in the newt Triturus carnifex. The annual variations of the serum aldosterone were evaluated every month by radioimmunoassay, from September to July; the daily variations of this hormone were evaluated in the months of January, April, July and November. Aldosterone levels were low from September to November (lowest value 187.23 ± 19.7 pg/ml in November), increased in December, remained high until March and peaked in April (587.25 ± 58.2 pg/ml). In May, the levels decreased and remained steady until July. Considering the daily aldosterone pattern, in all the examined months, serum aldosterone levels were lowest at 5:00 am (lowest value 162.51 ± 17.4 pg/ml in November) and peaked at 5:00 pm (maximal value 2075.00 ± 213.00 pg/ml in April). As regards the annual catecholamine pattern as measured by HPLC, serum norepinephrine levels were low from September to November (lowest value 541.13 ± 39.8 pg/ml in September), increased in December, peaked in January (1457.03 ± 106.97 pg/ml) and remained high in February. In March, the levels decreased and remained steady until June, then decreased in July. Serum epinephrine levels increased from September to November, decreased in December and remained low until February. In March, the levels increased and peaked in April (721.55 ± 49.91 pg/ml), then decreased (lowest value 140.00 ± 10.0 pg/ml in June) and remained steady until July. Considering the daily catecholamines levels, in all the examined months, there were no daily variations among serum norepinephrine levels. Otherwise, serum epinephrine levels were always lowest at 5:00 am (lowest value 100.77 ± 9.9 pg/ml in January) and peaked at 11:00 pm (maximal value 3831.05 ± 211.5 pg/ml in November). The annual and daily aldosterone and catecholamine patterns appear correlated to the metabolic activity of this species.

Amphibians of the “Cilento e Vallo di Diano” National Park (Campania, Southern Italy): updated check list, distribution and conservation notes

In this study, we present the results of our field and bibliographic survey on the amphibians of the “Cilento and Vallo di Diano” National Park (Southern Italy). Two hundred and thirty three spawning sites (167 original and 66 derived from literature), and 11 amphibian species were found. Reproductive activity was recorded for Salamandra salamandra , Salamandrina terdigitata , Triturus carnifex , Lissotriton italicus , Bufo bufo , Hyla intermedia , Rana italica , Rana dalmatina and Pelophylax synkl. hispanica . The distribution record of many species is widely improved with respect to bibliographic data. Our results also suggested that preservation and restoration of small aquatic sites, in particular of the artificial ones, such as stony wells and drinking-troughs, are fundamental for an appropriate conservation management of amphibians in the “Cilento and Vallo di Diano” National Park.

Amphibians of the Simbruini Mountains (Latium, Central Italy)

Little attention has been paid to the herpetological fauna of the Simbruini Mountains Regional Park, Latium (Central Italy). In this study, we surveyed 50 sites in the course of about ten years of field research, especially during the period 2005-2008. Nine amphibian species, four Caudata and five Anura, 60.0% out of the 15 amphibian species so far observed in Latium, were discovered in the protected area: Salamandra salamandra , Salamandrina perspicillata , Lissotriton vulgaris , Triturus carnifex , Bombina pachypus , Bufo balearicus , Bufo bufo , Rana dalmatina , Rana italica . Physiography of sites has been detailed together with potential threatening patterns. For each species the following topics have been discussed; ecology of sites, altitudinal distribution, phenology, sintopy. Salamandra salamandra and Bombina pachypus are at higher risk. The importance of the maintenance of artificial/natural water bodies for the conservation management of amphibian population of this territory is discussed.

Pituitary adenylate cyclase-activating polypeptide and its receptor PAC 1 in the testis of Triturus carnifex and Podarcis sicula

The pituitary adenylate cyclase-activating polypeptide (PACAP) is a member of the glucagon-related family that occurs in two amidated forms with 38 (PACAP38) and 27 (PACAP27) amino acids. First discovered in the brain, it was then localized in several peripheral tissues of mammals, including the testis. However, current knowledge of the expression and function of PACAP and its receptor PAC 1 in the reproductive system of non-mammalian vertebrates, and particularly in the testis, is still limited. The aim of this work was to study the presence of PACAP and its receptor PAC 1 in the testis of two non-mammalian vertebrates during the breeding season: the crested newt Triturus carnifex and the wall lizard Podarcis sicula. The expression and distribution of this neuropeptide and its receptor PAC 1 were investigated by using in situ hybridization and immunohistochemistry techniques. Our results demonstrated that PACAP and its receptor PAC 1 were highly represented in the testis of these two species. In particular, we showed that they are present within some germ cells and that PACAP, unlike in mammals, is expressed also in the somatic cells (Sertoli and Leydig cells) of the testis of these two non-mammalian vertebrates, suggesting that this neuropeptide is involved in the hormonal control of spermatogenesis and steroidogenesis.