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

Abstract 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

Updating Distribution, Ecology, and Hotspots for Three Amphibian Species to Set Conservation Priorities in a European Glacial Refugium

The Calabrian Peninsula (Southern Italy) has acted as a glacial refugium and is now considered a hotspot for the genetic diversity of several species. Even if it hosts the highest diversity of many Italian endemic amphibian species, the distribution of some of these needs an update to address conservation measures. We took advantage of a vast dataset for three Italian species (Bombina pachypus, Salamandrina terdigitata, Triturus carnifex), two of which are endemic, deriving from a 40-year field surveys dataset (1982–2022), to update their distribution and basic ecological requirements. We evaluated changes in their distribution, projecting them on a broader spatial scale through a kernel density estimation, inferring statistically-significant hotspots using Corine Land Cover patches, and assessing the protected areas’ coverage. We confirmed that Pollino, Catena Costiera, Sila and Aspromonte massifs are the main statistically-significant hotspots. Kernel densities showed a diversified pattern of gains/losses, sometimes overlapping, depending on the species. The whole outcomes obtained allow us to pinpoint specific areas where effective conservation measures need to be applied. Ousr findings reveal that local-scale monitoring and management should be planned, especially within the existing nationally-designated protected areas, which have been shown to protect far less with respect to the Natura 2000 sites

Tossicità ed alterazioni dell’apparato branchiale di Bufo bufo (Amphibia, Anura) dopo esposizione acuta e cronica ad endosulfan

Dottorato di Ricerca in Biologia Animale,XX Ciclo,a.a. 2004-2007Università della Calabri

Alterazioni morfo-funzionali indotte da contaminanti ambientali in due specie di Teleostei

Scuola di Dottorato "Life Sciences", XXVII Ciclo, SSD a.a. 2014Università della Calabri

Gill alterations as biomarkers of chronic exposure to endosulfan in Bufo bufo tadpoles

Endosulfan sprayed on agricultural fields accumulates in temporary pools due to surface runoff or sediment transport and may result in high water concentrations in spring and summer, coinciding with breeding and crucial stages of amphibian larval development. In the present study, Bufo bufo tadpoles were exposed to three different concentrations of endosulfan (0.01, 0.05 and 0.1 mg/L) until they reached complete metamorphosis. The aim of the study was to investigate the effects of endosulfan, at environmentally relevant concentrations on gill morphology and ultrastructure. Modifications in ultrastructure and cell composition were observed at all concentrations after 96 h. The main gill effects recorded in treated animals were: mucous secretion, the appearance of tubular vesicles cells (TVC) and a degeneration phenomenon. Comparing these results with our previous findings in which we used growth, developmental rate and behaviour as endpoints, we also demonstrated that the first effect of endosulfan on Bufo bufo was gill alteration, thus supporting the role of a morphological approach in toxicological studies. This study provides additional information on the role of morphological studies in demonstrating the effects of exposure to environmental pollutants. In this context, the use of amphibian gills, as effective biomarkers, is a valuable approach in evaluating exposure to agrochemicals

Effetti dell’endosulfan sugli Anfibi: uno studio morfologico e comportamentale

Dottorato di Ricerca in Biologia Animale, XIV Ciclo a.a. 2010-2011The declining amphibian population is a global phenomenon in conservation biology that has attracted the attention of many scientists (Russell et al., 1995; Stallard, 2001; Sparling e Fellers, 2009). As for the majority of recent reductions in Earth biodiversity, the amphibian decline can also be attributed to direct human impact on the environment such as the use of pesticides and other chemical pollutants. Therefore, understanding the impacts of agrochemicals on amphibians is a challenging proposition. Amphibians are sensitive to the action of pollutants and they are considered excellent bioindicators of the environmental quality due to their biological, ecological and anatomical characteristics. For these reasons, amphibians are broadly used as typical targets in evaluating the effects of chemicals on aquatic and agricultural ecosystems (Schuytema e Nebeker, 1996; Pollet e Bendell-Young, 2000; Venturino et al., 2003; Bernabò et al., 2008). Decreased species richness, reduced populations, and high deformity incidences have been reported in agroecosystems, which might be linked to the extensive use of pesticides (Berrill et al., 1994, 1997; Bonin et al., 1997; Knutson et al., 2002; Davidson, 2004; Bridges et al., 2004; Relyea, 2005). As outlined by Boone et al. (2005) there is a lack of data for many commonly used pesticides with regard to their effects on amphibians. We chose endosulfan, an organochlorine globally used on a wide variety of crops and other sectors. The environmental persistence and toxicity of endosulfan forced several national authorities to ban it and to propose its inclusion as a persistent organic pollutant in the Stockholm convention (Kelly et al., 2007; EPA, 2010). Endosulfan reaches aquatic systems through direct application, as well as spray drift and runoff from agricultural areas (Leonard et al., 1999, 2000, 2001; Broomhall, 2002; Jergentz et al., 2004) and levels in nature pose a risk to aquatic ecosystems. The last EPA (2010) report on endosulfan, highlights the need for further work to consider amphibians as model species. Previous studies on the effects of endosulfan on amphibians showed detrimental effects on survival, growth, development and metamorphosis, and even caused deformity, severe changes in gill morphology and behavioural alterations (Bernabò et al., 2008; Brunelli et al., 2009, 2010; Jones et al., 2009; Shenoy et al., 2009). Therefore, we proposed to assess the effects of chronic exposure to ecologically relevant concentrations of endosulfan on two anuran species during larval development. In particular, we focused attention on the morphological endpoint evaluating the effects on Bufo bufo skin, to validate the hypothesis of our research group using a morphological approach in a toxicological study. Recently we demonstrated that environmentally relevant endosulfan concentrations (0.1, 0.05 and 0.01 mg/L) may affect Bufo bufo gills morphology and ultrastructure (Brunelli et al., 2010). On this basis we proposed completing the study on endosulfan toxicity in Bufo bufo tadpoles evaluating the effects of the same sublethal concentrations on skin. In fact, the thin and permeable skin of amphibians directly interacts with all environmental constituents, including toxicants. Moreover there is a gap in literature on endosulfan effects on this target organ. We also analyzed the effects of endosulfan on Rana dalmatina tadpoles. Firstly, we examined the short-term toxicity of endosulfan determining the LC50 value and after this preliminary analysis we conducted a classical toxicological study evaluating the effects of sublethal concentrations of endosulfan (0.005, 0.01 and 0.05 mg/L) on survival, growth, development, metamorphosis, deformities presence in Rana dalmatina tadpoles and, in collaboration with the University of Liege we applied new tools for evaluating behavioural patterns. For the first time we used video-tracking analyses to test the quantitative effect of endosulfan on the locomotion. We exposed Bufo bufo tadpoles from Gosner stage 25 until stage 42 to a chronic static renewal system according to the experimental design and conditions of our previous studies (according to Brunelli et al., 2010). and the epidermis was removed after 96 h, 14 and 20 days and analyzed by Trasmission Electron Microscopy (TEM). Exposure to endosulfan caused a general defensive response by mucous secretion and the appearance of large secretory vescicles in Bufo bufo larval epidermis. The exposure to all concentrations, already after 96 h, caused an increase of intercellular spaces, alteration of the cell-cells interactions, cellular degeneration, in particular of the typical larval bufonidae cells (Riesenzellens). The epidermal-dermal junctions and the figures of Eberth in the basal layer, were completely lost at the end of exposure. Our results were successful in showing that environmentally relevant concentrations of endosulfan negatively affected the epidermis of Bufo bufo tadpoles after long-term exposure, resulting in ultrastructure modification in the treated animals compared to controls and that epidermal damage was strongly correlated with exposure time. These results are consistent with our previous findings (Bernabò et al., 2008; Brunelli et al., 2010) concerning the morphological alteration pattern of the gill epithelium during long term exposure. We confirmed that the morphological approach is an useful tool in evaluating the effects of environmental contamination at sublethal levels in sensitive organisms like amphibians, and we supported the role of amphibian skin as a good biomarker following pesticide exposure. Rana dalmatina tadpoles were exposed to several nominal concentrations of endosulfan to assess the sensitivity of the species by estimating the LC50 value at 96 h in a static exposure system. We found that the nominal 96 h LC50 value was 0.074 mg/L. After this preliminary analysis, Rana dalmatina tadpoles were exposed to sublethal concentrations of endosulfan (0.005, 0.01, 0.05 mg/L) in a chronic static renewal system from Gosner stage 25 for the entire course of larval development. Mortality, larval growth (mass), body length, development, time to metamorphosis and deformities presence were monitored regularly over the entire course of larval development. The behaviour (swimming activity) was recorded after 7, 14, 21 and 28 days of exposure and analyzed by a video-tracking software Ehovision 7 XT (Noldus Information Technology, The Netherlands). We analyzed behavioural traits such as: distance moved, velocity, immobility and space use. Our results showed that long-term exposure to environmentally relavant concentrations of endosulfan impaired survival, larval growth, development, metamorphosis, swimming activity and caused severe morphological alteration in tadpoles exposed to 0.01 and 0.05 mg endosulfan/L (medium and high concentrations). With regards to the inhibition of metamorphosis in the highest concentrations, this has a profound implication in the light of amphibian decline. Instead, the lowest concentration of endosulfan (0.005 mg/L) did not cause any significant effects on growth, development and on swimming activity, although we have observed a slightly significant reduction in survival without decreasing the success of metamorphosis. In natural environments, increased incidences of deformities, a reduced larval growth could increase susceptibility to predation (Rohr et al., 2003), delay metamorphosis or result in metamorphosis at a smaller size having negative effects on future fitness, growth and overwinter survival (Brodie and Formanowicz, 1983; Semlitsch et al., 1988; Smith, 1987; Boone and Semlitsch, 2002; Altwegg and Reyer, 2003; Boone and James, 2003). We used video-tracking analyses for the first time to test the quantitative effects of a pesticide on the behaviour of amphibians. And we observed that behavioural effects occurred from 7 days of exposure and tadpoles exposed to the medium and high concentrations of endosulfan exhibited several anomalies in swimming activity such as: shorter distance moved, slower speed, swirling, resting and different space use. Our findings are in agreement with the previous studies of our research group on endosulfan effects on Bufo bufo (Brunelli et al., 2009) and, with the use of video-tracking analysis, we had advantages over standard visual methods. In fact we precisely quantified swimming activity anomalies. We demonstrated that quantitative ecotoxicology is thus a valuable tool to assess conservation concerns when other techniques cannot detect detrimental effects. Our study thus confirms and extends the results of previous studies on the toxicity of this organochlorine pesticide in showing its possible role in amphibian decline.Università della Calabri

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

Artificial Neural Network to Predict Varicocele Impact on Male Fertility through Testicular Endocannabinoid Gene Expression Profiles

The relationship between varicocele and fertility has always been a matter of debate because of the absence of predictive clinical indicators or molecular markers able to define the severity of this disease. Even though accumulated evidence demonstrated that the endocannabinoid system (ECS) plays a central role in male reproductive biology, particularly in the testicular compartment, to date no data point to a role for ECS in the etiopathogenesis of varicocele. Therefore, the present research has been designed to investigate the relationship between testicular ECS gene expression and fertility, using a validated animal model of experimental varicocele (VAR), taking advantage of traditional statistical approaches and artificial neural network (ANN). Experimental induction of VAR led to a clear reduction of spermatogenesis in left testes ranging from a mild (Johnsen score 7: 21%) to a severe (Johnsen score 4: 58%) damage of the germinal epithelium. However, the mean number of new-borns recorded after two sequential matings was quite variable and independent of the Johnsen score. While the gene expression of biosynthetic and degrading enzymes of AEA (NAPE-PLD and FAAH, respectively) and of 2-AG (DAGLα and MAGL, respectively), as well as their binding cannabinoid receptors (CB1 and CB2), did not change between testes and among groups, a significant downregulation of vanilloid (TRPV1) expression was recorded in left testes of VAR rats and positively correlated with animal fertility. Interestingly, an ANN trained by inserting the left and right testicular ECS gene expression profiles (inputs) was able to predict varicocele impact on male fertility in terms of mean number of new-borns delivered (outputs), with a very high accuracy (average prediction error of 1%). The present study provides unprecedented information on testicular ECS gene expression patterns during varicocele, by developing a freely available predictive ANN model that may open new perspectives in the diagnosis of varicocele-associated infertility