Triturus Italicus Research Articles

Good new(t)s: Rapid recolonization of a restored fish‐invaded habitat by two newt species in southern Italy

Abstract Habitat modification and invasive species are considered some of the most impelling causes of the ongoing sixth mass extinction, especially in freshwater ecosystems. Artificial water troughs are, in this regard, particularly vulnerable environments, while being pivotal breeding habitats for many amphibian species. However, despite their well‐known importance and their conservation concern, detailed information on the resilience of such ecosystems is lacking. This note describes the events following the introduction of invasive fish species into a water trough in southern Italy and reconstructs the recolonization times of two newt species (Lissotriton italicus, Triturus carnifex) after disappearance of the fish a few months later. As expected, both newt species were no longer observed in the water as soon as the fish were introduced in the trough. However, in contrast to previous reports, they recolonized it within 2 months of the last observation of fish and, in the case of L. italicus, bred shortly after. Although the short permanence of fish in the trough may have facilitated the recolonization process, this event represents the fastest recolonization by newts of a restored habitat ever reported in the wild and sheds further insights on the resilience of amphibian species.

First record of leucism in the Italian newt Lissotriton italicus

First record of leucism in the Italian newt Lissotriton italicus

The differential role of Leydig cells in the skin and gills of Lissotriton italicus larvae.

Larval urodeles are provided with external gills involved, along with the skin, in gas exchange and osmoregulation. Gills and skin epithelia are different, each showing a peculiar set of specialized cells but both provided with Leydig cells (LCs). Information on LCs in the gills is lacking as the literature has focused primarily on the epidermis. Contradictory and fragmentary results highlight that LCs origin, fate, and functions remain not fully understood. Here, we investigated the morpho‐functional differences of LCs in the skin and gills of Lissotriton italicus larvae for the first time. LCs showed the same morphological and ultrastructural features in both tissues, even if LCs were significantly larger in the epidermis. Despite the uniform morphology within the LCs population, the proliferative ability was different. The putative diversity in the mucus composition was evaluated using a panel of 4 lectins as markers of specific carbohydrate moieties, revealing that sites of specific glycoconjugates were comparable in two tissues. To disclose the involvement of LCs in water storage and transport, immunofluorescence assay for aquaporin‐3 has also been performed, demonstrating the expression of this protein only in gills epithelium. By demonstrating that LCs can multiply by cell division in gills, our results will also contribute to the discussion about their proliferative ability. Finally, we found that the LCs cytoplasm is rich in glycoconjugates, which are involved in many diverse and essential functions in vertebrates.Research Highlights In gills LCs can multiply by cell division and express aquaporin‐3 demonstrating a tissue‐specific role of LCs.LCs cytoplasm is rich in glycoconjugates.LCs population show a uniform morphology in both gills and skin.

Can We Share? Feeding Strategy in Three Syntopic Newts in Artificial Habitats

Natural aquatic sites are disappearing worldwide, especially in the Mediterranean region where amphibians are frequently forced to move for reproduction to artificial sites designed for irrigation and cattle watering (i.e., wells, tanks and drinking troughs). In artificial aquatic sites, where resources (space and food) are usually limited, trophic niche information can be particularly useful to infer the suitability of habitats for amphibian conservation especially when more than one species co-occurs. In this paper, we focused on three newt species: The Italian newt (Lissotriton italicus), the Italian smooth newt (Lissotriton vulgaris meridionalis) and the Italian crested newt (Triturus carnifex) inhabiting man-made wells widespread in an area in Central Italy characterized by few available natural aquatic sites. Specifically, we analyzed the trophic spectrum of the species, their interactions and overlap, and discussed the potential role of wells in amphibian conservation. Overall, 550 newt individuals occurring in 16 distinct wells were sampled. The study species consumed similar resources, mainly of aquatic origin, with Diptera larvae and Cladocera representing the most important preys. The high degree of diet overlap observed may be due to site oligotrophy and high availability of small-sized prey, and it does not necessarily lead to competition. Newts had similar narrow niche width values and a generalist feeding pattern with high diversity among individuals. Lissotriton italicus and T. carnifex showed wider niche width in isolation than in syntopy condition, probably as a result of interspecific competition and/or intraguild predation. We showed that artificial aquatic sites are important for newt ecology and conservation since they allow up to three species to cohabit, thus representing a good surrogate of natural habitats. The study wells apparently provided suitable trophic conditions for newts in terms of prey availability and catchability. To date, just a few studies have contributed to a greater understanding of newts’ diet in artificial aquatic sites and this gap of knowledge has to be filled to clarify their role in amphibian ecology and conservation.

Comparative morphological analysis during larval development of three syntopic newt species (Urodela: Salamandridae)

We provide comparative and updated staging tables for the larvae of three syntopic newt species – Triturus carnifex, Lissotriton italicus and Ichthyosaura alpestris inexpectatus –living in Calabria. A detailed description, stage by stage, of the full larval period until completion of metamorphosis is furnished, accompanied by photographic tables of the most important stages. The appearance of the most prominent and easily identifiable morphological features (i.e. development of gills, balancers and limbs; and body pigmentation), and length, have been used as staging criteria. Ontogenic development in the three considered species is compared with that of other Caudata species. For the three newt species, we also performed a comparative analysis of the developmental larval phases, emphasising the shared and species-specific features. The present descriptive study adds comparative information on distinctive morphological traits that may be useful for the recognition of newts during larval ontogeny and, in some cases, when sampling in the field.

Histological and ultrastructural alterations of the Italian newt (Lissotriton italicus) skin after exposure to ecologically relevant concentrations of nonylphenol ethoxylates

Nonylphenol ethoxylates (NPEs) are well known endocrine disruptors. Widespread environmental contamination from NPEs is an issue of great concern. Despite amphibians are often exposed to such contaminants, very little attention has been dedicated to this vertebrate group. No information is available on the effects of NPEs onto the amphibian skin and only few reports have been conducted on fish. Here, histological and ultrastructural modifications of the skin have been evaluated in the Italian newt Lissotriton italicus. After a short-term exposure to two ecologically relevant concentrations of NP, severe pathological alterations, both dose and time-dependent, have been observed. The main effects were an increased mucous secretion, the dilation of the endomembrane, the wrinkling of the epidermal surface, the appearance of tubercles, the increased cellular turnover, continuous shedding processes. Some of the described skin alterations can easily interfere with physiological functions, such as osmoregulation and body protection, with detrimental consequences for the amphibian populations.

Morphological and immunohistochemical reactions of the larval epidermis in the Italian newt (Lissotriton italicus) after exposure to low pH

Mounting evidence suggests that amphibians are globally and currently the most threatened group of vertebrates and different causes might be responsible for this phenomenon. Acidification of water bodies is a global environmental issue that has been proposed as a possible cause for amphibian populations decline. Indeed, it has been widely demonstrated that low pH may exert harmful effects on amphibians, either directly or by increasing the adverse effects of other stressors. Surprisingly only few studies documented the response of amphibian integument to acidic pH conditions and no data are available on the effects of a non-lethal level of pH onto the amphibian larval epidermis. The present study showed that acidic pH (4.5) condition has severe effects on the epidermis of the Italian newt (Lissotriton italicus, formerly Triturus italicus) inducing both morphological and functional alterations. The increase of mucus is the first evident effect of acid injury, followed by the flattening of the epithelium and the appearance of a keratinized shedding layer. The immunolabeling of cytokeratins substantially changes acquiring an adult-like pattern. Also aquaporin 3 and iNOS expression modify their distribution according to a change of the histological features of the epidermis. These results clearly indicate that a short-term exposure to a sub-lethal pH disrupts the epidermis morphology and function in L. italicus larvae. Since the skin exerts a prominent role in both respiration and osmoregulation, the described alterations may adversely affect the overall ionic balance, with a long chain of cascading effects significantly decreasing newts survival probabilities when environmental pH lowering occurs.

Liver histology and ultrastructure of the Italian newt (Lissotriton italicus): Normal structure and modifications after acute exposure to nonylphenol ethoxylates

We examined, from a morphological and ultrastructural point of view, the liver of the Italian newt (Lissotriton italicus), under basal conditions and after exposure to nonylphenol ethoxylates (NPEs). Nonylphenol ethoxylates are surfactants widely used in a variety of industrial and agricultural processes that may pose a significant risk to aquatic fauna. NPEs, and their degradation intermediates, are known to affect reproductive biology acting as endocrine disruptors; besides estrogenic effects, nonylphenolic compounds may induce organ toxicity, particularly in liver and gonads. We investigated the effects of a nonylphenol ethoxylate (NPE10-ETO) on L. italicus liver using two low concentrations, consistent with the environmental concentrations. For this purpose, animals were exposed to nominal concentrations of 50 and 100μg/L in a short-term experiment (96h). A morpho-functional analysis was performed in order to investigate the amphibian responses to NPEs thus contributing to elucidate other potential mode of action of these compounds; indeed very little attention has been dedicated to amphibians though they are often exposed to such contaminants in aquatic ecosystems. Pathological alterations on liver histology and ultrastructure were observed at both tested concentrations; the main effects recorded were: increase of intercellular spaces, accumulation of large lipid droplets, increase in melanin content, and a degeneration phenomenon. We also detected, through confocal analysis, the induction of caspase-3, a key mediator of apoptosis, and an up-regulation of cytochrome P450-1A. By using both ultrastructural and a morpho-functional approach, we found that a short-term exposure to NPEs negatively affected the amphibian liver.

Remodelling of the skin during metamorphosis in the Italian newt (Lissotriton italicus) (Amphibia, Urodela): localization pattern of keratins, stromelysin-3 (MMP-11), and pan-cadherin

During amphibian metamorphosis, modifications of several important body systems take place involving different molecular mechanisms driving, among other, cell differentiation, cell–cell adhesion, and extracellular matrix turnover. Amphibians have been used as a classic model to investigate organs remodelling, including the skin; however, previous studies on metamorphosis have examined, for the most part, anurans, whereas only a few reports have considered urodeles. The main goal of this study was to evaluate the presence of keratins in the epidermis of the Italian newt (Lissotriton italicus). In order to assess the putative differential expression of keratins, we immunodetected cytokeratin type I, type II, and high molecular weight (HMW) during the larval, pre-metamorphic, and adult phase. Our results clearly indicated that keratins expression changes as the epidermis modifies during the functional maturation; interestingly, HMW keratins showed a strong immunopositivity in adult, thus suggesting that, in urodeles, these keratins become expressed in adult epidermis. Moreover, by using a monoclonal antibody against pan-cadherin, we demonstrated that in L. italicus epidermis during pre-metamorphic stages, the conversion from larval to adult type is not completed in whole epidermis; we also showed the strong spatiotemporal correlation of stromelysin-3 expression with tissue remodelling in L. italicus skin. Considering that skin remodelling in urodeles is a topic relatively neglected, our findings will contribute to fill a gap in the existing research literature; moreover, these data emphasize that unexpected results could be obtained when different model systems were used.

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.

The shift from aquatic to terrestrial phenotype in Lissotriton italicus: larval and adult remodelling of the skin

Morphology and ultrastructure of the skin of Lissotriton italicus (previously named Triturus italicus) have been described in different phases of its biological cycle: larval stage, metamorphic stage and adult stage with emphasis on modifications occurring between aquatic and terrestrial adults. In the present study, light microscopy and both scanning and transmission electron microscopy were employed to analyze the histological and cytological remodelling that occurs in the skin of L. italicus during metamorphosis. The ultrastructure of the larval epidermis is arranged into three principal layers comprising an external layer of pavement cells, a basal layer and 1–3 intermediate layers consisting of Leydig cells along with accessory cells and mitochondria-rich cells. By the onset of metamorphosis, morphological changes of the skin include stratification and flattening of epidermal layers and disappearance of typical larval cells. In both aquatic and terrestrial adult phases the thin, cornified epidermis shows the same general arrangement as found in other vertebrates with an external stratum corneum and a variable number of intermediate cell layers. During the terrestrial adult phase, the skin is characterized by the presence of numerous tubercles; moreover, the lower epithelium is thicker than in the aquatic phase. Ultrastructural analysis revealed no substantial differences in the cellular composition of the skin between aquatic and terrestrial phases.

Ultrastructural alterations in the ventricular myocardium of the adult italian newt ( Lissotriton italicus) following exposure to nonylphenol ethoxylate

Nonylphenols ethoxylates (NPEs) are surface active agents (surfactants) commonly used in cleaning products, in industrial processes, agricultural formulations and paints. They are found in sewage, municipal wastewaters and industrial effluents, and as contaminants in water bodies. Accumulating data suggest that exposure to NPEs can adversely affects functional properties of the neurologic, reproductive, immune, and endocrine systems. In order to examine whether NPEs exert similar damaging effects on the cardiovascular system, we used an amphibian model to examine the ultrastructural alterations of the ventricular myocardium following exposure to NPEs. Adult Lissotriton italicus in the aquatic phase were exposed to NPE10 (100 μg/L, ppb) for 96 h. Heart specimens were collected from a total of 10 individuals and processed for scanning and transmission electron microscopy. Our ultrastructural examinations demonstrated that amphibian ventricle is susceptible to the effects of NPEs. The most pronounced alterations were observed in the membrane compartments of both myocardial and endothelial cells as demonstrated by the presence of swollen mitochondria with disrupted cristae and dilated rough endoplasmic reticulum. We suggest that destabilization of the lipid milieu within membranes might represent one of the potential mechanisms by which NPEs exert their toxic effects on amphibian heart.

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.

Male reproductive system in the Italian newt Lissotriton italicus (Peracca 1898) (Amphibia, Urodela): ultrastructural and morphological study with description of spermiogenesis, spermatozoon and spermatophore

The first morphological and ultrastructural description of testis, Wolffian duct, spermatophore, and spermatozoon of Lissotriton italicus is provided. The male reproductive system of this species consists of a pair of monolobed testes, fat bodies, efferent ductules, paired Wolffian duct, and the cloaca. The Wolffian duct is pseudostratified, consisting of an epithelium composed of an alternation of ciliated cells and non-ciliated secretory cells, with scattered basal cells. Melanocytes are found in the basal lamina of the Wolffian duct. Spermiogenesis is of the discontinuous type. In the early spermatid nuclear elongation, flagellar extrusion and development of a large acrosomal vesicle occur. Soon the perforatorium develops and the flagellum elongates. In the late spermatid gradual chromatin condensation and nuclear elongation is noticeable. At the end of spermiogenesis the flagellum develops marginal filament and undulating membrane. The mature sperm of L. italicus consists of a distinct head (acrosome, perforatorium, nucleus and nuclear ridge, plus the apical acrosomal barb) and a tail with axoneme, undulating membrane, and axial fiber. Nuclear ridge and apical acrosomal barb are two autapomorphic characters found in the Urodela. Several apomorphic characters for Salamandroidea are also present: elongation of the connecting piece, an apically modified acrosome vesicle, trifoliate axial fiber within the principal piece, elongate annulus, elongate midpiece, and the gentle merging of the axial fiber/principal piece into the endpiece. The spermatophore of L. italicus is formed by a cap consisting of randomly distributed spermatozoa surmounting a short pedicel. A membranelike structure involving the spermatophore is absent.

Phylogeny and biogeography of the family Salamandridae (Amphibia: Caudata) inferred from complete mitochondrial genomes

Phylogenetic relationships of members of the salamander family Salamandridae were examined using complete mitochondrial genomes collected from 42 species representing all 20 salamandrid genera and five outgroup taxa. Weighted maximum parsimony, partitioned maximum likelihood, and partitioned Bayesian approaches all produce an identical, well-resolved phylogeny; most branches are strongly supported with greater than 90% bootstrap values and 1.0 Bayesian posterior probabilities. Our results support recent taxonomic changes in finding the traditional genera Mertensiella, Euproctus, and Triturus to be non-monophyletic species assemblages. We successfully resolved the current polytomy at the base of the salamandrid tree: the Italian newt genus Salamandrina is sister to all remaining salamandrids. Beyond Salamandrina, a clade comprising all remaining newts is separated from a clade containing the true salamanders. Among these newts, the branching orders of well-supported clades are: primitive newts ( Echinotriton, Pleurodeles, and Tylototriton), New World newts ( Notophthalmus-Taricha), Corsica-Sardinia newts ( Euproctus), and modern European newts ( Calotriton, Lissotriton, Mesotriton, Neurergus, Ommatotriton, and Triturus) plus modern Asian newts ( Cynops, Pachytriton, and Paramesotriton).Two alternative sets of calibration points and two Bayesian dating methods (BEAST and MultiDivTime) were used to estimate timescales for salamandrid evolution. The estimation difference by dating methods is slight and we propose two sets of timescales based on different calibration choices. The two timescales suggest that the initial diversification of extant salamandrids took place in Europe about 97 or 69 Ma. North American salamandrids were derived from their European ancestors by dispersal through North Atlantic Land Bridges in the Late Cretaceous (∼69 Ma) or Middle Eocene (∼43 Ma). Ancestors of Asian salamandrids most probably dispersed to the eastern Asia from Europe, after withdrawal of the Turgai Sea (∼29 Ma).

The balancers of Triturus italicus: an ultrastructural approach

Balancer morphology and ultrastructure, with emphasis on changes occurring during development, are described for Triturus italicus, using stereomicroscopy and light microscopy, as well as scanning and transmission electron microscopy. Morphological differentiation of balancers involves elongation, swelling of the distal region of the organ and appearance of secretory cones. The extensive granular endoplasmic reticulum and the well‐developed Golgi apparatus characterize the inner cell layer of the mature balancer. Other characteristics include the presence of numerous rounded lipid‐like granules and extensive innervation. In the cytoplasm, numerous bundles of tonofilaments can be found, and their amount increases as development progresses. As already stated in previous studies, the general morphology of balancers shows great constancy.

A molecular assessment of phylogenetic relationships and lineage accumulation rates within the family Salamandridae (Amphibia, Caudata)

We examine phylogenetic relationships among salamanders of the family Salamandridae using approximately 2700 bases of new mtDNA sequence data (the tRNA Leu, ND1, tRNA Ile, tRNA Gln, tRNA Met, ND2, tRNA Trp, tRNA Ala, tRNA Asn, tRNA Cys, tRNA Tyr, and COI genes and the origin for light-strand replication) collected from 96 individuals representing 61 of the 66 recognized salamandrid species and outgroups. Phylogenetic analyses using maximum parsimony and Bayesian analysis are performed on the new data alone and combined with previously reported sequences from other parts of the mitochondrial genome. The basal phylogenetic split is a polytomy of lineages ancestral to (1) the Italian newt Salamandrina terdigitata, (2) a strongly supported clade comprising the “true” salamanders (genera Chioglossa, Mertensiella, Lyciasalamandra, and Salamandra), and (3) a strongly supported clade comprising all newts except S. terdigitata. Strongly supported clades within the true salamanders include monophyly of each genus and grouping Chioglossa and Mertensiella as the sister taxon to a clade comprising Lyciasalamandra and Salamandra. Among newts, genera Echinotriton, Pleurodeles, and Tylototriton form a strongly supported clade whose sister taxon comprises the genera Calotriton, Cynops, Euproctus, Neurergus, Notophthalmus, Pachytriton, Paramesotriton, Taricha, and Triturus. Our results strongly support monophyly of all polytypic newt genera except Paramesotriton and Triturus, which appear paraphyletic, and Calotriton, for which only one of the two species is sampled. Other well-supported clades within newts include (1) Asian genera Cynops, Pachytriton, and Paramesotriton, (2) North American genera Notophthalmus and Taricha, (3) the Triturus vulgaris species group, and (4) the Triturus cristatus species group; some additional groupings appear strong in Bayesian but not parsimony analyses. Rates of lineage accumulation through time are evaluated using this nearly comprehensive sampling of salamandrid species-level lineages. Rate of lineage accumulation appears constant throughout salamandrid evolutionary history with no obvious fluctuations associated with origins of morphological or ecological novelties.

Triturus italicus: Analysis of lateralisation in the courtship behaviour

At the population level, male smooth newts (Triturus vulgaris) have a marked bias for left turning during spermatophore transfer behaviour. In a much more basal species of the genus, the Alpine newt, less than 50% of individuals show a significant left‐ or right‐side preference during courtship, whereas no lateral bias is found at population level. To verify whether the smooth newt's lateralised behaviour is a recent (apomorphic) acquisition, the degree of lateralisation during courtship displays occurring in another relatively more basal species (although less basal than T. alpestris), the Italian newt (T. italicus) was investigated. In the latter species, the analysis of the temporal structure of the sequences showed that males significantly utilised the same side during a single courtship sequence. However, 31% of the Italian newts showed a significant left‐ and 46% a right‐side preference. The presence of lateralisation in two relatively ‘primitive’ species (Alpine and Italian newt) at individual level only, with no evidence at population level, and the presence of a bias in a more derived species of the genus (T. vulgaris), support the hypothesis that population‐level lateralisation, in newts, is an independent autoapomorphic evolutionary event that took place in the smooth newt.

Embryonic development of the newt Triturus italicus in relation to temperature

AbstractThe embryonic development of Triturus italicus was observed at different temperatures. Normal development occurred between 10°C and 22.5°C. In this range of temperatures an hyperbolic relationship between development time and temperature was confirmed.

On the Sexual Behaviour of the Italian Newt, Triturus Italicus (Caudata: Salamandridae)

The sexual behaviour of the Italian Newt Triturus italicus is characterized by an extended phase of static display. Tail fanning is the predominant movement during this stage. Shortly before creeping, a flicking tail movement is inserted in the display. Occasionally the male retreats in front of the female before turning to the spermatophore transfer phase. This behaviour is comparable to the retreat display of the T. vulgaris-helveticus-montandoni group. The long phase of static display resembles the behaviour of another species, T. boscai. In T. italicus, behaviours like retreat and tail touch by the female appear to be less stereotyped than in the T. vulgaris-helveticus-montandoni group.