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Stable isotopes (δ13C and δ15N) were analysed in vertebrae of Scyliorhinus canicula (n=40; 13 males, 27 females) from the central Tyrrhenian Sea to explore a potential trophic shift in relation to sex and maturity condition. Isotopic values were obtained from the centre and the periphery of the vertebra of each individual to test for the effect of maturity condition. The values of δ13C and δ15N showed significant differences between the immature and mature state of the individuals analysed (U’=1392, P

Aim: Determine the evolutionary origin of the heretofore poorly characterized contemporary Great White Shark (GWS; Carcharodon carcharias) of the Mediterranean Sea, using phylogenetic and dispersal vicariance analyses to trace back its global palaeo-migration pattern. Location: Mediterranean Sea. Taxon: Carcharodon carcharias. Methods: We have built the largest mitochondrial DNA control region (CR) sequence dataset for the Mediterranean GWS from referenced historical jaws spanning the 19th and 20th centuries. Mediterranean and global GWS CR sequences were analysed for genetic diversity, phylogenetic relationships and divergence time. A Bayes factor approach was used to assess two scenarios of GWS lineage divergence and emergence of the Mediterranean GWS line using fossil records and palaeo-geographical events for calibration of the molecular clock. Results: The results confirmed a closer evolutionary relationship between Mediterranean GWS and populations from Australia–New Zealand and the North-eastern Pacific coast rather than populations from South African and North-western Atlantic. The Mediterranean GWS lineage showed the lowest genetic diversity at the global level, indicating its recent evolutionary origin. An evaluation of various divergence scenarios determined the Mediterranean GWS lineage most likely appeared some 3.23 million years ago by way dispersal/vicariance from Australian/Pacific palaeo-populations. Main conclusion: Based on the fossil records, phylogeographic patterns and divergence time, we revealed that the Mediterranean GWS population originated in the Pliocene following the Messinian Salinity Crisis. Colonization of the Mediterranean by GWS likely occurred via an eastward palaeo-migration of Australian/eastern Pacific elements through the Central American Seaway, before the complete closure of the Isthmus of Panama. This Pliocene origin scenario contrasts with a previously proposed scenario in which Australian GWS colonized the Mediterranean via antipodean northward migration resulting from navigational errors from South Africa during Quaternary climatic oscillations.

The first record of Tetragonurus cuvieri Risso, 1810 off Calabrian coast (Southern Italy, Central Mediterranean) is reported. The specimen, a male of 361 mm total length, was found at a depth of 7 m during a scientific visual census research activity, in March 2017. The sighting was located off the Tyrrhenian coast of Calabria (Paola: 39.355453N, 16.029192E). The present finding represents the 1st documented record for the Tyrrhenian coast of the Calabria region.

The lesser spotted dogfish Scyliorhinus canicula has an important role in the trophic web of demersal fish communities, representing an important trophic link between invertebrates and larger predatory fish. Despite its scientific interest, there is still scarce knowledge about many biological and ecological aspects of this shark. Parasitological, hematological and ecotoxicological analyses were carried out on a population of lesser spotted dogfish S. canicula from central Mediterranean. Parasitological analyses highlighted a poor helminthic community, highly dominated by a single taxon represented by the cestode Nybelinia sp. and no differences between females and males were observed. Hematological analyses showed that the number of leukocytes was significantly lower in the sharks that resulted parasitized: this was probably due to the ability of some trace elements (like As) in weakening of the immune system, exposing the animals to a higher risk of parasite infection. Trace elements analyses were performed in vertebrae, skin, and liver tissues and the most abundant and potentially toxic elements were: Pb, As, Cd, Mn, Zn, Ni and Fe. Pb, As and Mn showed the highest concentrations in vertebrae due to the diet of the sharks; Fe and Ni in the skin, due to their presence in the water column where animals were collected, and Cd and Zn in the liver, linked to their ability to induce trace elements binding metallothionein proteins (MT), which can reduce the concentration of Cd. These results confirm that elasmobranchs, being predators at the apex of the marine food chain, act as final receptors for a series of polluting elements regularly discharged into the sea that could weak their immune system increasing the parasitic load. For that reason, they could be considered as bioindicator of the health status of marine ecosystems.

The white shark Carcharodon carcharias has been present in the Mediterranean Sea since 3.2 million years ago. Nevertheless, the current population shows a low genetic variability suggesting an endangered small population, on which there is scarce information regarding ecotoxicology or trophic ecology. Given that white shark’s sightings are rare in the Mediterranean and the possibility of obtaining samples is highly limited, the aim of this research was to provide general information regarding the concentration of trace elements and stable isotopes (δ15N and δ13C). Laboratory analyses were performed on 18 and 12 subsamples from two different white sharks’ vertebrae obtained from two adult specimens caught in 1987, in Favignana Island, Italy. Perforations were made along the vertebrae to describe both trace elements and stable isotopes at different life stages. A total of 38 trace elements were analysed, in which the highest concentrations were found in Fe, Sr, U, Pb, and Zn. The fluctuations of these elements during the ontogeny of both individuals could have been related to changes in diet and environment, although the specific origin remains unknown. Regarding stable isotopes, the vertebrae from the male showed an isotopic range from 9.6‰ to 10.8‰ (δ15N) and from −16.5‰ to −13.0‰ (δ13C) with a mean ± SD value of 10.3 ± 0.4‰ for δ15N and −14.6 ± 1.3‰ for δ13C; whereas the female vertebrae had an isotopic range from 9.8‰ to 11.1‰ (δ15N) and from −16.9‰ to −15.0‰ (δ13C), with a mean ± SD value of 10.8 ± 0.6‰ for δ15N and −15.8 ± 0.8‰ for δ13C. There were no significant δ15N differences (U = 6, p = 0.07346) between the two individuals. However, there were just significant differences in δ13C (t = −1.8, p = 0.049256), which could suggest sexual segregation in terms of habitat use and feeding habits.

In this contribution the checklist of chondrichthyans of Calabria (Central Mediterranean, southern Italy) is reported. Data presented is derived from twenty years of opportunistic and active surveys from 2000 to 2020. A total of 55 species of chondrichthyans is present in Calabrian seas: 33 sharks, 20 rays, and 2 chimaeras. These species represent approximately 62% of the total reported for the Mediterranean. Approximately 71% of Calabrian species have been reported in the Tyrrhenian Sea, 49% in the Ionian Sea, and 33% in the Strait of Messina. According to IUCN criteria, new records of Endangered and Critically Endangered species (i.e., Carcahrodon carcharias [Linnaeus, 1758], Lamna nasus [Bonnaterre, 1788], Cetorhinus maximus [Gunnerus, 1765], Mobula mobular [Bonnaterre, 1788], Sphyrna zygaena [Linnaeus, 1758]) are reported, together with the first record of Raya brachyura Lafont, 1873 for the Ionian coasts and probably third confirmed record of the rare chimaera Hydrolagus mirabilis (Collett, 1904) for the Mediterranean.

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.

Bioaccumulation of 13 trace elements in the livers of 38 Pelophylax sinkl. hispanicus (Ranidae) and its helminth communities were studied and compared among three sites, each with a different degree of pollution along River Neto (south Italy) during September, 2014. Trace element concentrations in water and liver were measured using inductively coupled plasma mass spectrometry. For most elements, the highest concentration was recorded in the frogs inhabiting the third site, the one with the highest degree of pollution. The trend of trace element concentration in the liver can be represented as follows: Cu > Zn > Mn > Se > Cr. Concentrations of some elements in water and liver samples were significantly different among the three sites and this is evidenced by the bioaccumulation in the frogs. Four species of helminths, all belonging to Nematoda, were found: Rhabdias sp., Oswaldocruzia filiformis (Goeze, 1782), Cosmocerca ornata (Dujarden, 1845), Seuratascaris numidica (Seurat, 1917). The parasite survey presents an important difference of prevalence and average number of helminths in frogs between the three sites. Correlating parasitological and ecotoxicological data showed a strong positive correlation between prevalence and number of parasites with some trace elements such as Mn, Co, Ni, As, Se, and Cd.

DURING 4 STUDY EXPEDITIONS IN SOUTHAFRICA, PERFORMED IN 2000, 2003, 2004 AND 2005, OBSERVATIONS ABOUT SURFACE PREDATORY BEHAVIOUR OF THE GREAT WHITE SHARK IN PRESENCE OF PASSIVE PREYS WERE MADE. OBSERVATIONS WERE CARRIED OUT BY UNLIMITED SHARK DIVING BOATS AND BY CAGE DIVING AROUND DYER ISLAND, ABOUT 5 MILES FAR FROM GANSBAAI. DURING THE 27 OBSERVATION DAYS WERE IDENTIFIED 78 DIFFERENT SPECIMENS, THAT EXHIBITED 8 DIFFERENT BEHAVIOURS. THE GREAT WHITE SHARK POPULATION OBSERVED AROUND DYER ISLAND SHOWS A GREATER VARIETY OF BEHAVIOURS THAN THE POPULATION STUDIED ALONG THE CALIFORNIA COASTS.

The common chameleon Chamaeleo chamaeleon is present in many Mediterranean countries. Its occurrence in Italy is historically reported for Sicily, where it appears to be no longer present. While many contemporary findings across the Italian Penisula refer to single observations, likely due to introduced or released animals, the species turned out to be established in two southern regions (Apulia and Calabria) with acclimatised populations. To investigate their provenance, we analysed tissue samples of individuals from these localities. A fragment of the mitochondrial 16S rRNA gene was amplified and compared with sequences available in Genbank. Our results showed that individuals from Calabria share haplotypes with those from Tunisia (N. Africa), while those from Apulia are coincident to samples from Israel. This suggests that individuals were most likely introduced in Italy.

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 100 μg/L in a short-term experiment (96 h). 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.

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 nesting ecology of the slider turtle Trachemys scripta elegans was studied in Angitola Lake, a natural protected area in Calabria (Southern Italy). During 2008-2010, data were collected based on 1081 egg shells from 250 nests. The average number of egg shells per nest was 4.3 (min-max = 1-16) and the clutch size increased from 2008 to 2010. Two main hatching periods were identified: one in July and another in September. There were significant correlations between nest diameter and nest depth, between nest diameter and egg shell number, and between nest depth and egg shell number.

Endosulfan is a persistent organic pollutant (POP) that has lethal and sublethal effects on non-target organisms, including amphibians. In a laboratory study, we investigated direct and post-exposure effects of endosulfan on Bufo bufo tadpoles. For this purpose we exposed the tadpoles to a single short-term contamination event (96 h) at an environmentally-realistic concentration (200 μg endosulfan/L). This was followed by a recovery period of 10 days when the experimental animals were kept in pesticide-free water. The endpoints were assessed in terms of mortality, incidence of deformity, effects on behavior, and the morpho-functional features of the epidermis. We found that a short-term exposure to the tested concentration of endosulfan did not cause mortality but induced severe sublethal effects, such as hyperactivity, convulsions, and axis malformations. Following relocation to a pesticide-free environment, we noted two types of response within the experimental sample, in terms of morphological and behavioral traits. Moreover, by using both ultrastructural and a morpho-functional approach, we found that a short-term exposure to endosulfan negatively affected the amphibian epidermis. We also observed several histo-pathological alterations: increased mucous secretion, an increase in intercellular spaces and extensive cell degeneration, together with the induction of an inducible isoform of nitric oxide synthase (iNOS). Following the post-exposure period, we found large areas of epidermis in which degeneration phenomena were moderate or absent, as well as a further increase in iNOS immunoreactivity. Thus, after 10 days in a free-pesticide environment, the larval epidermis was able to partially replace elements that had been compromised due to a physiological and/or a pathological response to the pesticide. These results highlight the need for both exposure and post-exposure experiments, when attempting to assess pollutant effects.

Mercury (Hg) is a highly hazardous pollutant widely used in industrial, pharmaceutical and agricultural fields. Mercury is found in the environment in several forms, elemental, inorganic (iHg) and organic, all of which are toxic. Considering that the liver is the organ primarily involved in the regulation of metabolic pathways, homeostasis and detoxification we investigated the morphological and ultrastructural effects in Danio rerio liver after 96 h exposure to two low HgCl2 concentrations (7.7 and 38.5 μg/L). We showed that a short-term exposure to very low concentrations of iHg severely affects liver morphology and ultrastructure. The main effects recorded in this work were: cytoplasm vacuolization, decrease in both lipid droplets and glycogen granules, increase in number of mitochondria, increase of rough endoplasmic reticulum and pyknotic nuclei. Pathological alterations observed were dose dependent. Trough immunohistochemistry, in situ hybridization and real-time PCR analysis, the induction of metallothionein (MT) under stressor conditions was also evaluated. Some of observed alterations could be considered as a general response of tissue to heavy metals, whereas others (such as increased number of mitochondria and increase of RER) may be considered as an adaptive response to mercury.

Six research expeditions were conducted at Dyer Island to observe the surface behaviour of white sharks in the presence of bait. Observations were made from a commercial cage-diving boat. We observed 140 white sharks that exhibited 9 different types of behaviour: parading, bait following, visual inspection, breach, tail slap, tail stand, spy hop, repetitive aerial gaping, and head-up vertical emerging. Breach and tail slap were most often performed by male sharks, and tail slap and tail stand were more often performed by mature animals. The ethogram consisted of an average of 20 behavioural units, with a significant transitional pattern from bait follow to parading and from parading to bait follow. The individual surface behaviour of white sharks is not a simple stimulus–response reflex, but rather a complex tactical situation in which animals show plastic responses. Many aspects of white shark surface behaviour resemble those of other predator species that spend time at the surface.

Rising rates of varicocele and diabetes mellitus (DM) pose a significant problem to human fertility. Recent studies have pointed out the impact of cyclooxygenase (COX) in the regulation of testicular function and male fertility. Prominent COX-2 expression has been described recently in the testes of infertile patients, but little is known about the role and identity of COX isoforms in human sperm under certain disease states such as varicocele and DM. We therefore examined the expression profile and ultrastructural localization of COX-1 and COX-2 concomitantly in semen samples from healthy donors, and patients with varicocele and DM. Using Western blotting assay, ‘varicocele’ and ‘diabetic’ sperm showed enhanced COX isoforms expression with respect to the ‘healthy’ sperm. Immunogold labeling revealed human sperm anatomical regions containing COX-1 and COX-2, confirming their increased expression in pathological samples. Our data demonstrate that both COX isoforms are upregulated in the spermatozoa of varicocele and diabetic patients, suggesting the harmful effect of the diseases also at the sperm molecular level, going beyond the abnormal morphology described to date. In conclusion, COX enzymes may possess a biological relevance in the pathogenesis and/or maintenance of male factor infertility associated with varicocele and DM, and may be considered additional molecular markers for the diagnosis of male infertility disorders.

The present study was conducted to assess the toxicity of Foroozan (Iranian crude oil) on the ornate wrasse Thalassoma pavo (Labridae) using three sub-lethal crude oil concentrations. Gills, selected as target organ being the major route of hydrocarbon uptake, were excised after 48, 96 and 192 hours and analyzed by light and both scanning and transmission electron microscopy. Cellular biomarkers of exposure and/or effect were measured in branchial tissues of T. pavo, and severe lesions such as necrosis, aneurysms and disorganization of the lamellae proportional to the Foroozan exposure periods and concentrations were observed. The main alteration was on the lamellar epithelium, which displayed an irregular profile characterized by pavement cell cytoplasmic protrusion and an alteration of the oxygen chemosensing cells, resulting in impairment of various biological activities. Nevertheless, the ability of specimens to regulate calcium homeostasis and neurotransmission, as well as balance cell turno...

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.

Perrotta I, Russo E, Camastra C, Filice G, Di Mizio G, Colosimo F, Ricci P, Tripepi S, Amorosi A, Triumbari F & Donato G (2011) Histopathology59, 504–513 New evidence for a critical role of elastin in calcification of native heart valves: immunohistochemical and ultrastructural study with literature review Aims: Calcific aortic stenosis is a progressive disease characterized by massive fibrosis andmineralization of the valve leaflets. The aim of this study was to determine whether the onset of native calcific aortic stenosis is associated primarily with matrix remodelling events, and particularly with elastin degradation. Methods and results: The immunohistochemical expression profile of matrix degradating enzymes and tenascin-C was investigated in both healthy and native calcified aortic valves. Collagen and elastic tissue were studied by light microscopy and electron microscopy. Immunophenotypic analysis of inflammatory cells was carried out by using monoclonal antibodies to macrophages, T and B lymphocytes. Immunoreactivity for tenascin-C and matrix metalloproteinase-12 (MMP-12) was associated with areas of dense mineralization, which were characterized by fibrosis, fragmentation and calcification of elastic fibres a positive reaction was also found around small islands of calcification. MMP-11 was not detected in the diseased valves. Osteopontin and osteonectin were also found at sites of mineralization. All calcified valves examined showed inflammatory cell infiltration. Conclusions: Our results demonstrate the direct involvement of MMP-12 in native aortic valve stenosis. MMP-mediated degradation of elastic fibres might contribute actively to valve mineralization by inducing calcium deposition onto fragmented elastin.

Background Several lines of clinical and experimental evidence have demonstrated that reactive oxygen species and nitrogen species are generated in unregulated amounts during diverse cardiovascular disorders. It has been previously reported by our group and others that augmented expression of nitric oxide synthase isoforms is associated with human atherogenesis and that the activity of the enzymes in an atherosclerotic environment may promote the formation of peroxynitrite. Among the downstream mechanisms triggered by oxidants, poly(ADP-ribose) polymerase-1 activation has recently been implicated in the pathogenesis of acute and chronic myocardial dysfunction, diabetes, hypertension, aging, and various forms of shock. Methods Based on these observations, we performed immunohistochemical and immunogold labeling analyses to evaluate the expression profile and the subcellular localization of inducible nitric oxide synthase and poly(ADP-ribose) polymerase-1 in healthy and atherosclerotic human aortae. Results We have demonstrated that inducible nitric oxide synthase colocalizes with poly(ADP-ribose) polymerase-1 within vascular cells of atherosclerotic human aortae. We have reported for the first time, to our knowledge, the ultrastructural localization of poly(ADP-ribose) polymerase-1 within the nuclei of lesional smooth muscle cells. Finally, we have evidenced that poly(ADP-ribose) polymerase-1 induction within cells of the diseased aorta strongly correlates with alterations in mitochondrial morphology. Conclusions Our data imply the possibility of a significant role for cross-talk between inducible nitric oxide synthase and poly(ADP-ribose) polymerase-1 in human atherosclerotic lesions. We conclude that the prooxidant milieu of the plaque might exert damaging effects on mitochondria via a poly(ADP-ribose) polymerase-1-mediated mechanism since the absence of the enzyme results in a corresponding lack of changes in mitochondrial morphology. The present report may open avenues for further researches that could have important therapeutic consequences for the treatment of atherosclerosis and its clinical sequelae.

The term aneurysm is used to indicate a permanent and irreversible localized vascular dilatation that involves all three layers of the blood vessel wall. It may develop in any part of the circulatory system, including veins, and its history, presentation, and management differ depending on the location. A venous aneurysm is defined as a solitary area of fusiform or saccular dilatation occurring in the course of a major vein or connected by a single channel to a major venous structure. The lower extremities are the most frequently affected, with the popliteal vein being the most common site. Although different theories have been advanced, the etiology of the disease remains uncertain. Mechanical stress and/or degenerative changes within the vein wall are believed to represent the most likely causes of venous aneurysm. To date, there are only a few publications dealing with the histological appearance of popliteal vein aneurysm, and no studies that specifically describe its ultrastructural details have been published to our knowledge. In an attempt to fill this gap and to provide better insights into the pathophysiological mechanisms possibly involved in aneurysmal venous disease, we describe the fine structure of popliteal vein wall and valve in health and disease using both scanning and transmission electron microscopy.

Chlorpyrifos is an organophosphate pesticide among the most widely used in the world, which is suspected to be an endocrine-disrupting compound. To determine the capacity of chlorpyrifos to affect gonadal differentiation in Rana dalmatina, tadpoles were exposed to this pesticide during larval development until completion of metamorphosis at ecologically relevant concentrations (0.025 and 0.05mg/L). No effects of chlorpyrifos exposure on survival, development, or meta morphosis were observed. After a 1 month metamorphosis, the gonadal phenotype was determined by gross morphology and histological examination. Morphological and histological analysis revealed normal ovaries or testes in froglets belonging to control group, whereas testes from several froglets exposed to chlorpyrifos were interspersed with testicular oocytes in histological sections. Chlorpyrifos exposure during the entire larval period did not affect sex ratio, but reduced the percentage of males with histologically normal testes. The findings suggest that chlorpyrifos exposure has significant effects on gonadal differentiation in some animals by inducing an intersex condition and alterations to testicular morphology, and that R. dalmatina is sensitive to endocrine disruption. Thereby, this study provides evidence that the ecologically relevant concentrations of chlorpyrifos, although not adversely affect the survival, development, or metamorphosis, may interfere with sex differentiation and reproductive development of R. dalmatina via endocrinedisrupting mechanisms. J. Exp. Zool. 313A, 2011. & 2011 Wiley-Liss, Inc.

Chlorpyrifos [O,O-diethyl-O-(3,5,6-trichloro-2-pyridyl)phosphorothioate] is a widely used non-systemic organophosphorus insecticide frequently detected in surface waters around the world. The goal of this study is to evaluate the acute and chronic effects of this insecticide on Rana dalmatina tadpoles. To assess the sensitivity of this species, the LC50 value (i.e. the concentration at which 50% of tadpoles die) was determined after 96 h. Our results showed that 5.174 mg L(-1) chlorpyrifos caused 50% mortality in tadpoles at Gosner stage 25. Chronic toxicity tests were also conducted to evaluate the sublethal effects of chlorpyrifos; tadpoles were exposed to three ecologically relevant concentrations (0.025, 0.05 and 0.1 mg L(-1)) in static renewal tests from Gosner stage 25 (tadpoles shortly after hatching) until completed metamorphosis (Gosner stage 46). No significant reduction was observed in survival, larval growth (mass), snout-vent length, stage development or number metamorphosed. In contrast, chlorpyrifos exhibited significant chronic toxic effects on larval development, manifested as the appearance of abnormalities, including tail flexure, skeletal and muscle defects in later stages of development in tadpoles exposed to all tested concentrations. We also evaluated the chronic effects of chlorpyrifos on gill morphology and ultrastructure. Tadpoles were sacrificed after 8 days and 30 days of exposure. Observations by both scanning (SEM) and transmission electron microscopy (TEM) showed considerable morphological and ultrastructural changes. The main gill effects recorded were mucous secretion, epithelium detachment and a degeneration phenomenon. Comparing these results with our previous findings, we demonstrate that the first effect of chlorpyrifos on R. dalmatina is gill alteration, thus supporting the role of a morphological approach in toxicological studies.

The goal of this preliminary research was to provide an ethogram of social interactions among white sharks at Dyer Island's Nature Reserve (Gansbaai, South Africa) in the presence of bait. Eighty-three social interactions were observed, both from a boat and from a cage. Seven different interaction types were recognized: swim by; follow give way; follow; parallel swimming; give way; splash fights; and piggybacking. The preliminary data analysis did not reveal any significant relationship between the behavioural patterns and the sex of the sharks, but a strong correlation between behavioural patterns and the size of the animals was found. The observed displays were more often performed by animals of the same length than by animals of different size.

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.

Endosulfan is a widely used organochlorine pesticide with well-documented neurotoxic effects in both humans and laboratory animals (mammals and fish). Neurotoxicity has been implied also in amphibians after short-term exposure to endosulfan. Little is known about effects of chronic exposure of endosulfan in amphibians. Previously, we examined the short-term toxicity of endosulfan in common toad (Bufo bufo) tadpoles and determined the LC50 value to 0.43 mg/L. In the present study, we investigated the effects of endosulfan on B. bufo tadpoles after chronic exposure to ecologically relevant concentrations. Tadpoles were exposed in a static renewal test, from shortly after hatching (Gosner stage 25) to completed metamorphosis, to 0.01, 0.05 and 0.1mg endosulfan/L (nominal). The exposure period lasted 43-52 days. Mortality, larval growth (mass), development (reached Gosner stage at various times and deformities presence), metamorphosis and behaviour (swimming activity) were monitored regularly over the entire course of larval development. Our results show that 0.05 and 0.1mg endosulfan/L caused impaired behaviour, prolonged time to metamorphosis, increased incidences of mouth and skeletal malformations as well as mortality, and reduced body weight (observed also at 0.01 mg/L) in B. bufo tadpoles. Behavioural effects occurred at exposure day 4, before any other effects occurred, indicating a neurotoxic effect. Endosulfan levels found in groundwater and surface water range from 0.1 to 100 microg/L and after extraordinary runoff events, concentrations exceed 0.5 mg/L in surface water. Our results indicate that endosulfan may negatively affect wild frog populations in agricultural areas.

Previous studies have suggested that 1alpha,25-dihydroxyvitamin D(3)[1,25(OH)(2)D(3)] has a role in reproductive function. Gonadal insufficiencies were observed as a result of 1,25(OH)(2)D(3) deficiency and in 1,25(OH)(2)D(3) receptor (VDR) null mutant mice. To study human sperm anatomy at the molecular level, we first evaluated the ultrastructural localization of VDR by immunogold electron microscopy using a monoclonal antibody against amino acids 344-424 of human VDR, in normozoospermic samples. Intriguingly, VDR was associated predominantly with the cell nucleus. In fact, it is known that VDR is a transcription factor, and that in vitamin-D-depleted animals, VDR is largely localized in the cell nucleus. To assess the significance of VDR in the male gamete, we investigated the role of 1,25(OH)(2)D(3)/VDR in sperm survival and capacitation. Our results revealed that the action of 1,25(OH)(2)D(3) depended on its concentration because although lower doses induced cholesterol efflux, protein phosphorylation and sperm survival, a higher concentration seemed to be ineffective or did not show an increased effect. These results increase our knowledge of human sperm anatomy at the molecular level and suggest that 1,25(OH)(2)D(3)/VDR may have an important role in sperm survival and the acquisition of fertilizing ability.

We analysed the morphology and ultrastructure of the gill apparatus of the ornate wrasse, Thalassoma pavo , under normal conditions and after exposure to a sublethal concentration of sodium lauryl sulphate (3.5 mg/l, which is one-third of the 96LC99 value). To identify the biochemical mechanisms affected by this pollutant, we evaluated and compared the localisation of Na + /K + ATPase in normal and experimental conditions. Immunocytochemical analysis revealed that this enzyme was active in the chloride cells (CCs), which were distributed in clusters in the interlamellar region of the filament. Ultrastructural analysis revealed conspicuous alterations on the epithelium after 96 and 192 h of exposure to sodium lauryl sulphate: structural features of the surface cells were lost, the appearance of intercellular lacunae changed, and cellular degeneration occurred. Statistical analysis comparing the number and dimensions of CCs in normal conditions and after 96 h of exposure showed that the CC area decreased after exposure to the detergent.

The morphology and development of the larval oral apparatus of Rana dalmatina , Bombina variegata , Bufo bufo , and Bufo viridis are described and compared using scanning electron microscopy. The species show different arrangements of the mouthparts. The small oral apparatus of R. dalmatina larvae has three labial tooth rows on the upper labium, while there are four tooth rows on the lower labium with a medial gap in row proximal to the mouth. The margins of the oral apparatus are defined by papillae that encircle the lower labium. B. variegata tadpoles have two upper labial tooth rows and three lower labial tooth rows that are uninterrupted, unlike the ones of R. dalmatina . The mouth is encircled by papillae that are larger than those of R. dalmatina . The oral discs of tadpoles of both B. bufo and B. viridis are similar. They are defined by two upper labial tooth rows (the second of which is interrupted by a medial gap) and by three lower tooth rows that differ in lengths in the two Bufo species. Both species develop papillae on the mouth angles and in two rows on the upper labium. Some morphological differences among the oral discs of R. dalmatina , B. variegata , B. bufo , and B. viridis tadpoles can be attributed to phylogenetic differences, but most can be related to their varying feeding habits and/or to their dietary specializations.

Endosulfan is an organochlorine pesticide used in agriculture for a wide range of crops. Endosulfan concentrations of up to 0.7 mg/L can be found in ponds and streams near sprayed agricultural fields. We investigated the short-term toxicity of endosulfan in common toad (Bufo bufo) tadpoles after 24, 48, and 96 h of exposure. Acute toxicity was evaluated at nominal concentrations ranging from 0.01 to 0.6 mg/L: concentrations that could be found after the application of pesticide. Our results show that 0.43 mg/L of endosulfan caused 50% mortality (LC(50)). The effects of a sublethal endosulfan concentration (0.2mg/L) on gill apparatus morphology were evaluated by scanning and transmission electron microscopy. Immunohistochemical methods were also applied to detect the expression pattern of the inducible isoform of nitric oxide synthase (iNOS) in the gills using the confocal laser scanner microscope. Exposure to 0.2mg/L of endosulfan caused an apparent increase in mucus production, the occurrence of secretory vesicles and lamellar bodies, a widening of intercellular spaces and additionally there was evidence of an inflammatory response in the gill apparatus. The morphological alterations occurred after 24h and were more pronounced after 48 and 96 h of exposure. Altered morphology and increased mucus secretion indicate impaired gas exchange and osmoregulation in the gills. In addition, there was an increase of iNOS expression after 24 and 48 h which may reflect hypoxia and inflammation in the gill epithelium. Our results clearly indicate that short-term exposure to a sublethal concentration of endosulfan, near the high end of the environmental range, disrupts gill morphology and function in B. bufo tadpoles.

Ultrastructural data about large cell variant ovarian small cell carcinoma (LCV-SCC) are scarce and contradictory and the role of transmission electronmicroscopy (TEM) is not clear in the assessment of such tumors. The authors present a case of LCV-SCC without hypercalcemia in a 30-year-old woman. The diagnosis was confirmed by histopathological and immunohistochemical studies. Cytopathological examination of peritoneal washing showed a population of large neoplastic cells. TEM demonstrated that the neoplasia comprised two types of cells: one type showed many coarse secretory granules without dense core, and the other type was without granules and showed dilated endoplasmic reticulum and sometimes indented nuclei. The present case indicates that different underlying ultrastructural patterns, not yet well known, exist in connection with the pathological and clinical behaviour of LCV-SCC. TEM might play a role in the identification of subtypes of LCV-SCC with different prognostic and therapeutic impact.

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.

Comparisons within and among populations offer important insights into variation in life-history traits and possible adaptive patterns to environmental conditions. We present the results of observed differences in body size, body shape and patterns of reproduction in four separate populations of the European pond turtle Emys orbicularis in central and southern Italy – coastal (n=3) and mountainous (n=1) sites and pond (n=2) and canal (n=2) habitats – to determine whether phenotypic plasticity affects reproductive output. Although we did not find any significant latitudinal variation in body size, we observed significant differences in body shape between canal (rounded body shape) and pond (elongated body shape) systems and smaller size with rounded shape in the mountainous population. Reproductive output is similar among populations (median=5 eggs per clutch), whereas reproductive investment (relative clutch mass to maternal body mass) is higher in the mountain population (one clutch per year) than in coastal populations (two clutches per year), suggesting differential trade-offs between geographic locality, elevation and habitat type. Turtle shell shape and geographic location together affect reproductive output in E. orbicularis in Italy.

The sperm of Caprimulgus europaeus is typical of other nonpasserines in many respects. Features shared with Paleognathae and Galloanserae are the conical acrosome, shorter than the nucleus; the presence of a perforatorium and endonuclear canal; the presence of a proximal as well as distal centriole; the elongate midpiece with mitochondria grouped around a central axis (here maximally six mitochondria in approximately 10 tiers); and the presence of a fibrous or amorphous sheath around the principal piece of the axoneme. A major (apomorphic) difference from paleognaths and galloanserans is the short distal centriole, the midpiece being penetrated for most of its length by the axoneme and for only a very short proximal portion by the centriole. Nonpasserines differ from paleognaths in that the latter have a transversely ribbed fibrous sheath, whereas in nonpasserines it is amorphous, as in Caprimulgus, or absent. The absence of an annulus is an apomorphic feature of Caprimulgus, apodiform, psittaciform, gruiform, and passerine sperm, homoplastic in at least some of these. In contrast to passerines, in Caprimulgus the cytoplasmic microtubules in the spermatid are restricted to a transient longitudinal manchette. The structure of the spermatid and spermatozoon is consistent with placement of the Caprimulgidae near the Psittacidae, but is less supportive of close proximity to the Apodidae, from DNA-DNA hybridization and some other analyses.

Chordoid meningioma (CM) is a meningioma containing regions that are histologically similar to chordoma, with trabeculae of eosinophilic, vacuolated cells in a myxoid background and corresponds to ...

The hibernating golden hamster (Mesocricetus auratus) is becoming a useful rodent model to study the neurophysiological role of some neuromediators on vital behaviors such as sleep and thermoregulation. Recent works have shown that the histamine neuroreceptor subtypes (H-sub(1-3)R) are able to modulate such behaviors. Here, specific subtype(s) and cerebral nuclei that were actively operating on feeding behaviors in pubertal and adult hamsters were identified. Of the subtypes assessed, H-sub-3R antagonist (thioperamide) provoked significant (p < .001) changes in behavior (very low total food and water intake) in adults, whereas it did not significantly modify these behaviors in pubertals. The H-sub-3R antagonist's role seemed to be related to elevated amounts of stress-induced damaged neurons displaying, mainly, shrunken crenated cell membranes and altered synaptic processes in limbic areas such as amygdala, cortex, and hippocampus. At the transcription level, an evident expression pattern of H-sub-3R messenger RNA appeared in pubertals, especially in neurons of the cortex and hippocampus, whereas the same trend was featured in amygdalar areas of hibernating adult hamsters, suggesting early H-sub-3R regulatory activities, at least in limbic sites of this rodent model.

The spermatozoon of Apus apus is typical of non-passerines in many respects. Features shared with palaeognaths and the Galloanserae are the conical acrosome, shorter than the nucleus; the presence of a proximal as well as distal centriole; the elongate midpiece with mitochondria grouped around an elongate distal centriole; and the presence of a fibrous or amorphous sheath around the principal piece of the axoneme. The perforatorium and endonuclear canal are lost in A. apus as in some other non-passerines. All non-passerines differ from palaeognaths in that the latter have a transversely ribbed fibrous sheath whereas in non-passerines it is amorphous, as in Apus, or absent. The absence of an annulus is an apomorphic but homoplastic feature of swift, psittaciform, gruiform and passerine spermatozoa. The long distal centriole, penetrating the entire midpiece, is a remarkably plesiomorphic feature of the swift spermatozoa, known elsewhere only in palaeognaths. The long centriole of Apus, if not a reversal, would be inconsistent with the former placement of the Apodiformes above the Psittaciformes from DNA-DNA hybridization. In contrast to passerines, in A. apus the microtubules in the spermatid are restricted to a transient single row encircling the cell. The form of the spermatozoon fully justifies the exclusion of swifts from the passerine family Hirundinidae.

We examined, from a morphological and ultrastructural point of view, the gill epithelium of Triturus italicus, both in basal conditions and after acute exposure to low pH. Our analysis of gill morphology began with the aim of determining lethal pH levels; we found that the pH value at which 50% of mortality occurs (LC50) is 4.0. We then investigated the effects of the larvae's exposure to a critical value of pH (pH 4.5). No change was observed in the gill during the first 24 hr. After 48 hr, a cellular response was evident and the gills appeared covered with a dense mucous layer. Observations that were carried out by light microscopy (LM) and electron microscopy, both scanning (SEM) and transmission (TEM), showed considerable histological and ultrastructural changes. As regards the principal filament, the alterations resulted in the presence of an external keratinized layer. More changes affected the mitochondria-rich cells (MRCs) in both a qualitative and quantitative way; this cell type shows a wide surface and seems to protrude more than the near pavement cells that, in normal conditions, partially cover the MRCs, thus reducing their external surface. The microvilli were extremely lengthened and often anatomized each other. Changes in the secondary filament affected the thickness of the epithelium, which appeared considerably smaller in the gills of the newts exposed to acid stress. The ciliated cells appeared to be more numerous than in the control specimens and the MRCs showed a widening of the apical surface.

The appearance and the modification of the gill apparatus in Rana dalmatina tadpoles have been described in the different phases of larval development. The morphology and ultrastructure have been studied using light microscopy and both scanning and transmission electron microscopy. The organization of the gills during the initial phases of development (external gills or transient gills) brings to mind the characteristics of Urodela larvae in which the gills appear to consist of three tufts of filaments supported by the gill arches III, IV and V. The cellular composition of the transient gills appears to be extremely simple and the presence of specialized cells is not noted. Basal cells, pavement cells and ciliated cells form the thin mono- or bilayered epithelium. In the persistent gills (or internal gills) of the R. dalmatina tadpole (Orton’s larval type 4) the gill arches carry four rows of gill tufts branching out to the ventral region. Meanwhile, from the dorsal portion of the arch the gill filters present an axial portion from which there is much branching out, which confers a characteristic appearance on this part of the gills. The cellular composition of the gill tufts and of the filters is different: in the gill tufts basal cells, pavement cells, ciliated cells, cubic cells and mitochondria-rich cells (MRCs) have been recognized, while in the gill filters the last cellular type does not appear. The MRC has highly variable forms and dimensions and is characterized by the presence of numerous mitochondria in the cytoplasm. Often the MRCs manifest themselves grouped together, in groups of three or more. The pavement cells and the cubic cells demonstrate identical ultrastructural characteristics and have an external surface area characterized by the presence of short superficial microridges and numerous vacuoles in the apical cytoplasm.

We sampled 154 sites of Triturus italicus from central and southern Italy to infer conservation guidelines. In most sites, the species lives in small populations (tens of individuals) in natural (ponds, pools, streams) and artificial (ditches, wells, tanks, troughs) bodies of water. Its biological cycle regarding in-water movements and reproductive patterns is very variable. The species seems in decline, with the exception of Latium and Calabria, and 22 populations have become extinct. The main causes of the species disappearance are likely due to loss of habitats caused by trasformation or destruction of bodies of water by modern farming techniques, pollution, and introduction of allochtonous species (such as fishes and pond turtles). A status of “endangered” is suggested for the species, or “vulnerable” for the populations of Latium and Calabria. The species deserves a conservation plan which includes measures such as habitat protection, scientific monitoring aimed at abundance and genetic analysis, and...

Information is given on the distribution and ecology of amphibians in the Pollino National Park, based on a summary of the data contained in the Herpetology Laboratory database at the Ecology Department (University of Calabria) and updated with information obtained during recent sampling. A total of 412 stations was monitored revealing the presence of 11 amphibian species. Two of these (Bombina pachypus, Salamandrina terdigitata) are included in annex II and six (Rana italica, R. dalmatina, Hyla intermedia, Bufo viridis, Triturus italicus, T. carnifex) in annex IV of the ‘Habitat’ directive. Some species represent interesting Italian (T. carnifex, H. intermedia), Apennine (B. pachypus, R. italica, S. terdigitata) and southern Italian (T. italicus) endemisms, which further valorize the heritage of the Park's fauna. For each species the state of conservation was checked and evaluated in order to propose suitable strategies for protection.

Labial and maxillary palps in 22 species of ground beetle larvae revealed the presence of four different types of sensilla: sensilla digitiformia, sensilla campaniformia, sensilla basiconica and sensilla chaetica. Using transmission electron microscopy techniques, the role of most of them was defined: sensilla campaniformia and sensilla chaetica are mechanoreceptors; while sensilla basiconica are chemoreceptors. Sensilla digitiformia can be both mechanoreceptors and chemoreceptors, though hygroreception may also be postulated on the basis of their abundance on the mouthparts of some hygrophilous species that depend on damp or wet patchy biotopes/seasons. The number and location of the sensory structures has been found to vary among the investigated taxa. The differences are related to the larval behavioural types, and are an adaptive response to different lifestyles, feeding habits and use of space.