Karyotype Diversification and Chromosome Rearrangements in Squamate Reptiles.

Karyotype diversification represents an important, yet poorly understood, driver of evolution. Squamate reptiles are characterized by a high taxonomic diversity which is reflected at the karyotype level in terms of general structure, chromosome number and morphology, and insurgence of differentiated simple or multiple-sex-chromosome systems with either male or female heterogamety. The potential of squamate reptiles as unique model organisms in evolutionary cytogenetics has been recognised in recent years in several studies, which have provided novel insights into the chromosome evolutionary dynamics of different taxonomic groups. Here, we review and summarize the resulting complex, but promising, general picture from a systematic perspective, mapping some of the main squamate karyological characteristics onto their phylogenetic relationships. We highlight how all the major categories of balanced chromosome rearrangements contributed to the karyotype evolution in different taxonomic groups. We show that distinct karyotype evolutionary trends may occur, and coexist, with different frequencies in different clades. Finally, in light of the known squamate chromosome diversity and recent research advances, we discuss traditional and novel hypotheses on karyotype evolution and propose a scenario of circular karyotype evolution.

The Ephrin tyrosine kinase a3 (EphA3) is a novel mediator of RAGE-prompted motility of breast cancer cells.

BACKGROUND: The receptor for advanced glycation-end products (RAGE) and its ligands have been implicated in obesity and associated inflammatory processes as well as in metabolic alterations like diabetes. In addition, RAGE-mediated signaling has been reported to contribute to the metastatic progression of breast cancer (BC), although mechanistic insights are still required. Here, we provide novel findings regarding the transcriptomic landscape and the molecular events through which RAGE may prompt aggressive features in estrogen receptor (ER)-positive BC. METHODS: MCF7 and T47D BC cells stably overexpressing human RAGE were used as a model system to evaluate important changes like cell protrusions, migration, invasion and colony formation both in vitro through scanning electron microscopy, clonogenic, migration and invasion assays and in vivo through zebrafish xenografts experiments. The whole transcriptome of RAGE-overexpressing BC cells was screened by high-throughput RNA sequencing. Thereafter, Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses allowed the prediction of potential functions of differentially expressed genes (DEGs). Flow cytometry, real time-PCR, chromatin immunoprecipitation, immunofluorescence and western blot assays were performed to investigate the molecular network involved in the regulation of a novel RAGE target gene namely EphA3. The clinical significance of EphA3 was explored in the TCGA cohort of patients through the survivALL package, whereas the pro-migratory role of EphA3 signaling was ascertained in both BC cells and cancer-associated fibroblasts (CAFs). Statistical analysis was performed by t-tests. RESULTS: RNA-seq findings and GSEA analysis revealed that RAGE overexpression leads to a motility-related gene signature in ER-positive BC cells. Accordingly, we found that RAGE-overexpressing BC cells exhibit long filopodia-like membrane protrusions as well as an enhanced dissemination potential, as determined by the diverse experimental assays. Mechanistically, we established for the first time that EphA3 signaling may act as a physical mediator of BC cells and CAFs motility through both homotypic and heterotypic interactions. CONCLUSIONS: Our data demonstrate that RAGE up-regulation leads to migratory ability in ER-positive BC cells. Noteworthy, our findings suggest that EphA3 may be considered as a novel RAGE target gene facilitating BC invasion and scattering from the primary tumor mass. Overall, the current results may provide useful insights for more comprehensive therapeutic approaches in BC, particularly in obese and diabetic patients that are characterized by high RAGE levels.

Chromosome Diversity and Evolution of the Endemic Malagasy Velvet Geckos of the Genus Blaesodactylus (Reptilia, Gekkonidae).

We performed a molecular and phylogenetic analysis and a comparative cytogenetic study with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, + fluorochromes on several Blaesodactylus samples. The phylogenetic inference retrieved two main clades, the first comprises B. victori, B. microtuberculatus and B. boivini, while the second includes B. sakalava, B. antongilensis and B. ambonihazo. The available samples of B. sakalava form two different clades (here named B. sakalava clade A and clade B), which probably deserve a taxonomic re-evaluation. We found a karyological variability in Blaesodactylus in terms of chromosome number (2n = 40-42), morphology, location of NORs, and heterochromatin distribution pattern. Blaesodactylus antongilensis and B. sakalava clade A and B showed a karyotype of 2n = 40 mostly telocentric chromosomes. Pairs 1 and 6 were metacentric in B. sakalava clade A and B, while pair 1 was composed of subtelocentric/submetacentric elements in B. antongilensis. In contrast, B. boivini displayed a karyotype with 2n = 42 only telocentric chromosomes. NORs were on the first chromosome pair in B. boivini, and on the second pair in B. antongilensis. Adding our data to those available from the literature on evolutionarily related species, we highlight that the chromosome diversification in the genus probably proceeded towards a progressive reduction in the chromosome number and the formation of metacentric elements.

Short-term effects of the strobilurin fungicide dimoxystrobin on zebrafish gills: A morpho-functional study.

Strobilurins represent the most widely used class of fungicides nowadays andare considered relatively non-toxic to mammals and birds but highly toxic to aquatic biota. Dimoxystrobin is one of the novel strobilurins, recently included in the 3rd Watch List of the European Commission as available data indicate that it could pose a significant risk to aquatic species. As yet, the number of studies explicitly assessing the impact of this fungicide on terrestrial and aquatic species is extremely low, and the toxic effects of dimoxystrobin on fish have not been reported. Here we investigate for the first time the alterations induced by two environmentally relevant and very low concentrations of dimoxystrobin (6.56 and 13.13 µg/L) in the fish gills. morphological, morphometric, ultrastructural, and functional alterations have been evaluated using zebrafish as a model species. We demonstrated that even short-term exposure (96 h) to dimoxystrobin alters fish gills reducing the surface available for gas exchange and inducing severe alterations encompassing three reaction patterns: circulatory disturbance and both regressive and progressive changes. Furthermore, we revealed that this fungicide impairs the expression of key enzymes involved in osmotic and acid-base regulation (Na+/K+-ATPase and AQP3) and the defensive response against oxidative stress (SOD and CAT). The information presented here highlights the importance of combining data from different analytical methods for evaluating the toxic potential of currently used and new agrochemical compounds. Our results will also contribute to the discussion on the suitability of mandatory ecotoxicological tests on vertebrates before the introduction on the market of new compounds.

Evolutionary and Genomic Diversity of True Polyploidy in Tetrapods.

True polyploid organisms have more than two chromosome sets in their somatic and germline cells. Polyploidy is a major evolutionary force and has played a significant role in the early genomic evolution of plants, different invertebrate taxa, chordates, and teleosts. However, the contribution of polyploidy to the generation of new genomic, ecological, and species diversity in tetrapods has traditionally been underestimated. Indeed, polyploidy represents an important pathway of genomic evolution, occurring in most higher-taxa tetrapods and displaying a variety of different forms, genomic configurations, and biological implications. Herein, we report and discuss the available information on the different origins and evolutionary and ecological significance of true polyploidy in tetrapods. Among the main tetrapod lineages, modern amphibians have an unparalleled diversity of polyploids and, until recently, they were considered to be the only vertebrates with closely related diploid and polyploid bisexual species or populations. In reptiles, polyploidy was thought to be restricted to squamates and associated with parthenogenesis. In birds and mammals, true polyploidy has generally been considered absent (non-tolerated). These views are being changed due to an accumulation of new data, and the impact as well as the different evolutionary and ecological implications of polyploidy in tetrapods, deserve a broader evaluation.

First Insights on the Karyotype Diversification of the Endemic Malagasy Leaf-Toed Geckos (Squamata: Gekkonidae: Uroplatus).

We provide here the first karyotype description of eight Uroplatus species and a characterization of their chromosomal diversity. We performed a molecular taxonomic assessment of several Uroplatus samples using the mitochondrial 12S marker and a comparative cytogenetic analysis with standard karyotyping, silver staining (Ag-NOR) and sequential C-banding + Giemsa, +Chromomycin A3 (CMA3), +4',6-diamidino-2-phenylindole (DAPI). We found chromosomal variability in terms of chromosome number (2n = 34-38), heterochromatin composition and number and localization of loci or Nucleolar Organizer Regions (NORs) (alternatively on the 2nd, 6th, 10th or 16th pair). Chromosome morphology is almost constant, with karyotypes composed of acrocentric chromosomes, gradually decreasing in length. C-banding evidenced a general low content of heterochromatin, mostly localized on pericentromeric and telomeric regions. Centromeric bands varied among the species studied, resulting in CMA3 positive and DAPI negative or positive to both fluorochromes. We also provide evidence of a first putative heteromorphic sex chromosome system in the genus. In fact, in U. alluaudi the 10th pair was highly heteromorphic, with a metacentric, largely heterochromatic W chromosome, which was much bigger than the Z. We propose an evolutionary scenario of chromosome reduction from 2n = 38 to 2n = 34, by means of translocations of microchromosomes on larger chromosomes (often involving the NOR-bearing microchromosomes). Adding our data to those available from the literature, we show that similar processes characterized the evolutionary radiation of a larger gecko clade. Finally, we hypothesize that sex chromosome diversification occurred independently in different genera.

The role of exposure window and dose in determining lead toxicity in developing Zebrafish.

Heavy metal contamination is recognized worldwide as a serious threat to human health and wildlife, and reducing their emissions is a priority of international and EU actions. Due to its persistence, high bioaccumulation tendency, and toxicity properties, lead (Pb) is one of the heavy metals of greatest concern. Even at low concentrations, lead induces various clinical and subclinical conditions in both humans and animals, and it has been included in the priority list of hazardous substances. In the present study, we used zebrafish's early stages as a model, given their well-acknowledged predictive value in the risk assessment of chemicals. This study was designed to investigate the morphological and morphometric alterations induced by Pb during zebrafish's early development and disclose the putative effects stage- and/or dose-dependent. We examined injuries induced by two environmentally relevant and extremely low concentrations of Pb (2.5 µg/L and 5 µg/L) during two exposure windows: early (between 1 and 7 dpf) and late (between 2 and 8 dpf). We clearly demonstrated that the incidence and severity of morphological abnormalities increased with increasing Pb dose and exposure time in both early and late-exposed groups. Furthermore, we revealed that malformation severity was significantly higher in the early exposed group than in the late exposure group at all exposure times and for both tested doses, thus highlighting the high sensitivity of zebrafish during the initial stages of development. The information presented in this paper emphasizes the effectiveness of morphological biomarkers in unveiling threatening situations and supports the role of zebrafish embryos and larvae in risk assessment and environmental monitoring.

Morphological and Functional Alterations Induced by Two Ecologically Relevant Concentrations of Lead on Danio rerio Gills.

Lead (Pb), due to its high toxicity and bioaccumulation tendency, is one of the top three pollutants of concern for both humans and wildlife and occupies second place in the Priority List of Hazardous Substances. In freshwater fish, Pb is mainly absorbed through the gills, where the greatest accumulation occurs. Despite the crucial role of gills in several physiological functions such as gas exchange, water balance, and osmoregulation, no studies evaluated the effects of environmentally relevant concentrations of Pb on this organ, and existing literature only refers to high levels of exposure. Herein we investigated for the first time the molecular and morphological effects induced by two low and environmentally relevant concentrations of Pb (2.5 and 5 µg/L) on the gills of Danio rerio, a model species with a high translational value for human toxicity. It was demonstrated that Pb administration at even low doses induces osmoregulatory dysfunctions by affecting Na+/K+-ATPase and AQP3 expression. It was also shown that Pb upregulates MTs as a protective response to prevent cell damage. Modulation of SOD confirms that the production of reactive oxygen species is an important toxicity mechanism of Pb. Histological and morphometric analysis revealed conspicuous pathological changes, both dose- and time-dependent.

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.

Antarctic Fish as a Global Pollution Sensor: Metals Biomonitoring in a Twelve-Year Period.

Antarctica represents a unique natural laboratory for ecotoxicological studies as it is characterized by low internal pollutants emissions but high external contamination levels. Indeed, warm temperatures promote pollutant evaporation (low latitudes), while cool temperatures (high latitudes) promote its deposition from the atmosphere on land/water. Metals are the most important pollutants in ecosystems and represent a serious and global threat to aquatic and terrestrial organisms. Since 2000, the risks posed by metals have led many States to ratify protocols aimed at reducing their emissions. Endemic Antarctic organisms represent excellent bioindicators in order to evaluate the efficacy of global measures adopted to mitigate pollutants release into the environment. In this study (supported by PNRA18-00133), we estimated the metals contamination levels and the metallothionein-1 expression in liver samples of two Antarctic fish species, the icefish Chionodraco hamatus and the red-blooded Trematomus bernacchii, collected in the same area during 2002 and 2014. The chosen area is located in the Ross Sea, a unique area as it is also isolated from the rest of the Southern Ocean. The analysis of contamination trends throughout this period showed, in both species, a significant increase over time of metals bioaccumulation and metallothionein-1 expression. In addition, our result clearly indicated that the detoxifying ability of the two organisms analyzed greatly differs, probably due to haemoglobin presence/absence. Our work represents an important early step to obtain valuable information in conservation strategies for both Antarctic and non-Antarctic ecosystems.

Exploring the critical points of teaching STEM subjects in the time of COVID 19: the experience of the course "Microscopy Techniques for Forensic Biology".

Background: The University was among the first structures to be hit by the health emergency, transferring all its teaching and research activities remotely. It was not easy for teachers and students to find themselves suddenly shifted into different teaching and socializing context. Results: This article describes and analyzes the online teaching experience carried out for the course of Microscopy Techniques for Forensic Biology offered as a part of the Master's degree program in Biology at the University of Calabria (Italy). A cross-sectional survey (pilot study) was designed to investigate the accessibility of distance learning along with an evaluation of adjustments needed for the conversion from offline to online instruction. Particular attention has been paid to learning material and lesson duration, with specific emphasis on practical activities. Conclusions: The author's intent is that of opening a comparison between the strengths and weaknesses that emerged in this experience, highlighting, in particular, how the educational relationship between teacher and student has changed.

Morpho-Functional Alterations in the Gills of a Seawater Teleost, the Ornate Wrasse (Thalassoma pavo L.), after Short-Term Exposure to Chlorpyrifos.

Chlorpyrifos (CPF) is an organophosphorus insecticide commonly used for domestic and agricultural purposes. The risk posed by environmental contamination from CPF is well acknowledged, and it has been detected worldwide in aquatic habitats and coastal areas. In addition, due to its slower degradation in seawater compared to freshwater, CPF is of particular concern for marine environments. Here, we investigated for the first time the morpho-functional alterations induced by CPF on the gills of Thalassoma pavo, a widespread species in the Mediterranean Sea. We tested the effects of two sublethal concentrations (4 and 8 µg/L) after 48 and 96 h. Our study demonstrates that the alterations induced by CPF are dose and time-dependent and highlight the harmful properties of this insecticide. After exposure to the low tested concentration, the more frequent alteration is an intense proliferation of the primary epithelium, whereas after exposure to the high concentration, the primary epithelium proliferation is less extensive, and the most evident effects are the thinning of secondary lamellae and the ectopia of chloride and goblet cells. CPF also modulated the expression of Na+/K+-ATPase. Dilation of lamellar apical tips, pillar cell degeneration, and appearance of aneurysms are often observed.

Tissues injury and pathological changes in Hyla intermedia juveniles after chronic larval exposure to tebuconazole.

Tebuconazole (TBZ), an azole pesticide, is one of the most frequently detected fungicides in surface water. Despite its harmful effects, mainly related to endocrine disturbance, the consequences of TBZ exposure in amphibians remain poorly understood. Here, we investigated the adverse and delayed effects of TBZ chronic exposure on a native anuran species, often inhabiting cultivated areas, the Italian tree frog (Hyla intermedia). To disclose the multiple mechanisms of action through which TBZ exerts its toxicity we exposed tadpoles over the whole larval period to two sublethal TBZ concentrations (5 and 50 µg/L), and we evaluated histological alterations in three target organs highly susceptible to xenobiotics: liver, kidney, and gonads. We also assessed morphometric and gravimetric parameters: snout-vent length (SVL), body mass (BM), liver somatic index (LSI), and gonad-mesonephros complex index (GMCI) and determined sex ratio, gonadal development, and differentiation. Our results show that TBZ induces irreversible effects on multiple target organs in H. intermedia, exerting its harmful effects through several pathological pathways, including a massive inflammatory response. Moreover, TBZ markedly affects sexual differentiation also by inducing the appearance of sexually undetermined individuals and a general delay of germ cell maturation. Given the paucity of data on the effects of TBZ in amphibians, our results will contribute to a better understanding of the environmental risk posed by this fungicide to the most endangered group of vertebrates.

Exposure and post-exposure effects of chlorpyrifos on Carassius auratus gills: An ultrastructural and morphofunctional investigation.

Widespread environmental contamination from chlorpyrifos (CPF) is well acknowledged and has led to the proposal to ban or limit its use in agricultural and domestic, within the regulatory context of both America and Europe. Furthermore, great concerns arise as to whether exposure to CPF represents a potential risk to human health. In the present study, by subjecting the goldfish model to three environmentally realistic concentrations of CPF (1, 4, and 8 µg/L) for 96 h, we demonstrated that this pesticide has the potential to induce severe morphological, ultrastructural and functional alterations in gills, even at very low concentrations. The degree of pathological effects was dose-dependent, and the main morphological alterations recorded were: regression of interlamellar cellular mass (ILCM), hypertrophy, and hyperplasia of epithelial cells, degeneration of both chloride cells and pillar cells. CPF exposure resulted in a decrease of Na+/K+-ATPase expression and the induction of iNOS, as revealed by immunohistochemical analysis. In order to determine the overall toxicity of CPF, we also investigated the recovery capability of goldfish gills following a period of 7 days in pesticide-free water. Our results clearly showed that there exists a threshold of CPF dose below which the effects on gills are reversible and beyond which the ability of gills to recover their typical features is completely lost. The information presented in this paper emphasises the importance of evaluating the recovery ability of organisms after chemical input and enhances our knowledge of the potential hazard of organophosphorus pesticides (OPs) on freshwater ecosystems.

Lead toxicity in seawater teleosts: A morphofunctional and ultrastructural study on the gills of the Ornate wrasse (Thalassoma pavo L.).

Lead is one of the most alarming toxic pollutants for the environment due to its acute toxicity and high bioaccumulation tendency. Despite legislative efforts, world lead production has more than doubled since the early 1970s to 2016. Due to extensive exploitation and human activity, the coastal and estuarine regions belong to marine environments that are mostly and more rapidly deteriorated by pollutants including lead. A limited number of studies examined the effects of lead in fishes, compared to other aquatic models and even fewer studies have been dedicated to seawater fishes especially regarding Pb adsorption and accumulation in specific organs. Fish gills, key compartments involved in several crucial functions such as gas exchange, osmoregulation, and excretion, are also the organs in which Pb is mainly accumulated. Herein, we investigated the morphofunctional and ultrastructural modifications in the gills of a marine teleost (Thalassoma pavo) after acute exposure (48, 96, 192 h) to three lead concentrations. We showed that pathological alterations can be detected in all the examined samples. The most common modifications observed were: the curling of the lamellae and the dilation of their apical tips, the lamellar edema, the proliferation and the hypertrophy of CCs, the progressive epithelial disorganization with detachment of the epithelium from connective tissue. This study also demonstrates that there is a weak influence on the expression pattern of Na+/K+-ATPase and AQP3 biomarker enzymes while high metallothioneins expression has been observed. The described alterations may adversely affect gas exchange and ionic balance, with a long chain of cascading effects. This is the first evidence of the effects exerted by lead on gills of seawater fishes that highlights the harmful properties of this metal, even at low concentration.

Morphological and Immunohistochemical Modifications in Zebrafish (Danio rerio) Gills After Short-Term Exposure to the Fungicide Tebuconazole.

In agroecosystems, a variety of pesticides may enter aquatic habitats exerting a number of toxic effects on living organisms. Due to their wide use and the repeated application, fungicides can be found at a much greater concentration than other agrochemicals in all environmental compartments. One of these fungicides, tebuconazole (TBZ), has been recognized as an endocrine-disrupting chemical, and the available literature focuses mainly on this mechanism of action. In the present study by subjecting the zebrafish model to an environmentally realistic concentration of TBZ, we clearly demonstrate that this fungicide has the potential to induce severe morphological and ultrastructural alterations in gills. The main morphological effects recorded in this study were as follows: the proliferation of the main epithelium, ectopia of chloride cells, lamellar shortening, necrosis, and apoptosis. TBZ exposure also resulted in the overtime decrease in the expression of both Na+/K+-ATPase and aquaporin 3 and in the induction of oxidative stress enzyme (superoxide dismutase-1) as revealed by immunohistochemical analysis. Our results clearly indicate that respiratory and osmoregulatory disorders represent an important mechanism of TBZ toxicity on Danio rerio gills. This is the first evidence of sublethal effects exerted by TBZ on fish gills and highlights the harmful properties of this fungicide, even at very low concentration.

Bergamot Polyphenols Boost Therapeutic Effects of the Diet on Non-Alcoholic Steatohepatitis (NASH) Induced by "Junk Food": Evidence for Anti-Inflammatory Activity.

Wrong alimentary behaviors and so-called "junk food" are a driving force for the rising incidence of non-alcoholic fatty liver disease (NAFLD) among children and adults. The "junk food" toxicity can be studied in "cafeteria" (CAF) diet animal model. Young rats exposed to CAF diet become obese and rapidly develop NAFLD. We have previously showed that bergamot (Citrus bergamia Risso et Poiteau) flavonoids, in the form of bergamot polyphenol fraction (BPF), effectively prevent CAF diet-induced NAFLD in rats. Here, we addressed if BPF can accelerate therapeutic effects of weight loss induced by a normocaloric standard chow (SC) diet. 21 rats fed with CAF diet for 16 weeks to induce NAFLD with inflammatory features (NASH) were divided into three groups. Two groups were switched to SC diet supplemented or not with BPF (CAF/SC±BPF), while one group continued with CAF diet (CAF/CAF) for 10 weeks. BPF had no effect on SC diet-induced weight loss, but it accelerated hepatic lipid droplets clearance and reduced blood triglycerides. Accordingly, BPF improved insulin sensitivity, but had little effect on leptin levels. Interestingly, the inflammatory parameters were still elevated in CAF/SC livers compared to CAF/CAF group after 10 weeks of dietary intervention, despite over 90% hepatic fat reduction. In contrast, BPF supplementation decreased hepatic inflammation by reducing interleukin 6 (Il6) mRNA expression and increasing anti-inflammatory Il10, which correlated with fewer Kupffer cells and lower inflammatory foci score in CAF/SC+BPF livers compared to CAF/SC group. These data indicate that BPF mediates a specific anti-inflammatory activity in livers recovering from NASH, while it boosts lipid-lowering and anti-diabetic effects of the dietary intervention.

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.