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.

Is trace element concentration correlated to parasite abundance? A case study in a population of the green frog Pelophylax synkl. hispanicus from the Neto River (Calabria, southern Italy).

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.

Effects of Two Sublethal Concentrations of Mercury Chloride on the Morphology and Metallothionein Activity in the Liver of Zebrafish (Danio rerio).

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.

Endosulfan effects on Rana dalmatina tadpoles: quantitative developmental and behavioural analysis.

Endosulfan is an organochlorine pesticide that was recently labeled as a persistent organic pollutant, but it is still widely employed, particularly in developing countries. The goal of this study is to evaluate the acute (LC(50)) and chronic effects (developmental and behavioural traits) of this insecticide on Rana dalmatina tadpoles after exposure to ecologically relevant concentrations (0.005, 0.01, and 0.05 mg/L) by applying video-tracking techniques to evaluate the quantitative effect of endosulfan on amphibian behavioural patterns. The 96 h LC(50) value was 0.074 mg endosulfan/L. Tadpoles chronically exposed to 0.01 and 0.05 mg endosulfan/L underwent high mortality rate, decreased larval growth, delayed development, and increased incidence of malformations, and they did not reach metamorphosis by the end of the experiment. Moreover, tadpoles exposed to these concentrations exhibited several abnormalities in swimming patterns, such as shorter distance moved, swirling, resting, and unusual use of space. The exposure to 0.005 mg endosulfan/L did not cause any significant effects on behaviour, larval growth, or development, but we observed a significant decrease in both survival and time to metamorphosis. We showed that developmental abnormalities are dose-dependent and that the pesticide effects could differ depending on the endosulfan concentration and the species tested. We also validated the hypothesis that behavioural analysis, along with the use of new analytical methods, could be a useful tool in amphibian ecotoxicological studies.

Survived the Glaciations, Will They Survive the Fish? Allochthonous Ichthyofauna and Alpine Endemic Newts: A Road Map for a Conservation Strategy.

The Calabrian Alpine newt (Ichthyosaura alpestris inexpectata) is a glacial relict with small and extremely localised populations in the Catena Costiera (Calabria, Southern Italy) and is considered to be "Endangered" by the Italian IUCN assessment. Climate-induced habitat loss and recent fish introductions in three lakes of the Special Area of Conservation (SAC) Laghi di Fagnano threaten the subspecies' survival in the core of its restricted range. Considering these challenges, understanding the distribution and abundance of this newt is crucial. We surveyed the spatially clustered wetlands in the SAC and neighbouring areas. First, we provide the updated distribution of this subspecies, highlighting fish-invaded and fishless sites historically known to host Calabrian Alpine newt populations and two new breeding sites that have been recently colonised. Then, we provide a rough estimate of the abundance, body size and body condition of breeding adults and habitat characteristics in fish-invaded and fishless ponds. We did not detect Calabrian Alpine newts at two historically known sites now invaded by fish. Our results indicate a reduction in occupied sites and small-size populations. These observations highlight the need for future strategies, such as fish removal, the creation of alternative breeding habitats and captive breeding, to preserve this endemic taxon.

Parasitic Load, Hematological Parameters, and Trace Elements Accumulation in the Lesser Spotted Dogfish Scyliorhinus canicula from the Central Tyrrhenian Sea.

Parasitological, hematological, and ecotoxicological analyses were carried out on a population of lesser spotted dogfish Scyliorhinus canicula from the central Mediterranean Sea. Parasitological analyses highlighted a poor helminthic community, highly dominated by a single taxon represented by the cestode Nybelinia sp. No differences in the parasitic load between females and males were observed. Hematological analyses showed that the number of leukocytes was significantly lower in the sharks that resulted in parasitism, and this could be due to the ability of some trace elements, such as arsenic, weakening the immune system and exposing animals to a higher risk of parasite infection, although further hematological and parasitological analyses are required on a larger number of samples. Trace elements analyses in the vertebrae, skin, and liver highlighted that the most abundant and potentially toxic elements were lead (Pb), arsenic (As), and cadmium (Cd). Other trace elements were also abundant, such as manganese (Mn), zinc (Zn), nickel (Ni), copper (Cu), and iron (Fe). Pb, As, and Mn showed the highest concentrations in vertebrae, while Cd, Cu, and Zn were the highest in the liver, probably due to their concentration in the prey items of the sharks; Fe and Ni showed the highest concentrations in the skin, due to their presence in the water column, especially along the coast where animals were collected. The concentration of some trace elements analyzed in the vertebrae decreased with the growth of the sharks. 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.

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.

Ultrastructure of popliteal vein aneurysm.

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.

Toxicity of chlorpyrifos to larval Rana dalmatina: acute and chronic effects on survival, development, growth and gill apparatus.

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.

Human sperm anatomy: ultrastructural localization of 1alpha,25-dihydroxyvitamin D receptor and its possible role in the human male gamete.

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.

Endosulfan acute toxicity in Bufo bufo gills: ultrastructural changes and nitric oxide synthase localization.

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.

Effects of a sublethal concentration of sodium lauryl sulphate on the morphology and Na+/K+ ATPase activity in the gill of the ornate wrasse (Thalassoma pavo).

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.

Large cell variant ovarian small cell carcinoma: case report.

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.

Chronic exposures to fungicide pyrimethanil: multi-organ effects on Italian tree frog (Hyla intermedia).

Amphibian habitats are easily contaminated by several pollutants, and in agricultural landscapes the likely exposure scenario is represented by pesticides. Many of these substances are known or suspected to act as endocrine disrupting chemicals (EDCs). The goal of the present study was to assess the effects of pyrimethanil, a common-used but also overlooked fungicide, on liver, kidney and gonadal differentiation of Hyla intermedia. Through a multi-organ evaluation, we demonstrated that a long term exposure to two environmentally relevant concentrations of pyrimethanil (5 and 50 µg/L) elicits a range of toxic responses. First we showed that pyrimethanil induces underdevelopment of ovaries and interferes with normal sexual differentiation, thus revealing the endocrine disruption potential of this fungicide. Moreover we revealed that all considered organs are seriously affected by this fungicide and both necrosis and apoptosis contribute to the histological response. This is the first report on the effects of pyrimethanil on gonads, liver and kidney histology of a non-model species and it demonstrates that the hazardous properties of this fungicide can result from several pathological processes affecting different key compartments of amphibian.

Feeding differences in pubertal and aged golden hamsters (Mesocricetus auratus) are related to specific cerebral expression pattern of histamine subtype 3 receptor.

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.

Ultrastructure of the spermatid of Caprimulgus europaeus Linnaeus 1758, the European nightjar (Aves; Caprimulgidae), with phylogenetic implications.

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.

Transmission electron microscopy study of the effects produced by wide-band low-power millimeter waves on MCF-7 human breast cancer cells in culture.

Our previous work showed that low-power wide-band millimeter waves (MMW) inhibit the growth of the MCF-7 human breast carcinoma cell line, also causing a marked reduction of the density of microvilli at the apical membrane of the MCF-7 cells, as revealed by scanning electron microscopy. The aim of the present work was to investigate the ultrastructural changes induced by such electromagnetic radiations on this cell line. A transmission electron microscopy study was performed on MCF-7 cells irradiated under the same experimental conditions previously adopted. Transmission electron microscopy analysis revealed several ultrastructural features of the MMW-irradiated cells pertinent to cells subjected to sublethal injury. The antiproliferative effect of the millimeter radiation was confirmed. MMW, in the 52-78 GHz frequency range, act as stress factor on the cells that survive in a non-steady low-mitogenetic metabolic state.

Antiproliferative effect of linalool on RPMI 7932 human melanoma cell line: ultrastructural studies.

Linalool, a small monoterpene molecule, is used widely for its flavoring and fragrant properties in many cosmetic products. In this work, we investigated the antiproliferative effect of two different linalool solutions on RPMI 7932 human melanoma and NCTC 2544 normal keratinocites cell lines using the trypan blue method. Morphological changes in cells were investigated by scanning electron microscopy (SEM) and transmission electron microscopy (TEM). In addition, apoptosis was evaluated using caspase 3-antibody. Linalool showed a selective inhibitory effect on the growth of melanoma cells in a concentrationdependent manner, inducing several morphological changes, as revealed by SEM and TEM analysis. Moreover, the labelling for caspase-3 is abundant in the melanoma cells and almost absent in the normal keratinocites cells. The results suggest that linalool could be used as drug and/or as model drug for developing potential therapeutic agents for melanoma.

The ultrastructure of the book lungs of the Italian trap-door spider Cteniza sp. (Araneae, Mygalomorphae, Ctenizidae).

The fine structure of book lungs is not homogeneous across Arachnids and is considered phylogenetically informative, however few reports on the ultrastructural features of this organ have been published. In this study, we examined the general morphology and ultrastructure of adult spiders of the genus Cteniza. The respiratory system of Cteniza sp. consists of two pairs of well-developed book lungs, which is considered indicative of primitive spiders. The general organization of the book lungs is similar to that described for other arachnids and consists of leaves of alternating air and hemolymph channels. The air channels are lined with cuticle and open to an atrium that leads to a slit-like spiracle. The air channels are held open by cuticular trabeculae. The space holders in the hemolymph channels are pillar trabeculae formed by two cells from the opposed walls. The pillar cells have a complex ultrastructure that includes an interdigitating connection, gap junctions, microtubules and hemidesmosomes. These features apparently help strengthen the pillar cells and their interconnections with each other and the underlying cuticle. The cytoskeleton resembles that of arthropod tendon cells where substantial structural support is needed.

Selective inhibition of tumoral cells growth by low power millimeter waves.

The effects of low power millimetric wave (MMW) radiation on the growth of tumor and healthy cells were studied. A wide-band frequency range between 53.57-78.33 GHz with a radiation density power of 27 x 10(-17) watt/Hz were used. The radiating energy was low enough not to increase the temperature of the cellular samples (cold irradiation). One hour of radiation treatment given every other day to three tumoral human stable cell lines, produced a noticeable inhibition of the cellular growth. The analogous treatment given to two healthy cell lines gave a weak growth stimulation. A scanning electron microscopy study of MCF-7-and K562-irradiated cells revealed that MMW irradiation induced profound morphological changes of the membrane. Finally, we also provided a mechanistic indication, based on millimeter wave spectroscopy of the cells: water is the primary absorber of these electromagnetic waves. Our work provides interesting evidence that wide band low power MMW irradiation, in the appropriate frequency range, could be used in the future as a cold means to cause selective inhibition of tumor cell growth.