Dipartimento di Biologia, Ecologia e Scienze della Terra - Tesi di dottorato

Permanent URI for this collectionhttp://localhost:4000/handle/10955/34

Questa collezione raccoglie le Tesi di Dottorato afferenti al Dipartimento Dipartimento di Biologia, Ecologia e Scienze della Terra dell'Università della Calabria.

Browse

Subject: search.filters.subject.Biologia

Search Results

Now showing 1 - 6 of 6
  • No Thumbnail Available
    Item
  • No Thumbnail Available
    Item
    Effetti dell’endosulfan sugli Anfibi: uno studio morfologico e comportamentale
    (2011) Lavorato, Manuela; Cerra, ,Maria Carmela; Brunelli, Elvira; Denoel, Mathieu; Bernabò, Ilaria
    The 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.
  • No Thumbnail Available
    Item
    Gene expression patterns and stress response in the copepod Calanus helgolandicus
    (2011) Lauritano, Chiara; Cerra, Maria Carmela; Branno, Margherita; Ianora, Adrianna; Procaccini, Gabriele
    Diatoms and dinoflagellates are dominant photosynthetic organisms in the world‟s oceans and are considered essential in the transfer of energy through marine food chains. However, these unicellular organisms produce secondary metabolites such as products deriving from the oxidation of fatty acids collectively termed oxylipins (including polyunsaturated aldehydes or PUAs; by diatoms) or potent neurotoxins (brevetoxins; by dinoflagellates). It is often assumed that harmful algae toxins are grazing deterrents to discourage zooplankton grazers from eating these algae. Some laboratory studies have suggested that some toxic algae are either not eaten by various grazers or that grazers ingesting toxic algae suffer adverse effects such as reduced feeding rates, diminished reproductive success, behavioral modification or increased mortality (Cohen et al., 2007, Kubanek et al., 2007; Prince et al., 2006). The aim of this thesis was to study in the copepod Calanus helgolandicus the effects of toxic diets at the molecular level. Expression level analyses by the sensible technique reverse transcription-quantitative polymerase chain reaction (RT-qPCR) allowed the study of specific genes of interest (GOI) which are known to have a primary role in generic stress responses, defense systems (e.g. aldehyde, free fatty acid and free radical detoxification) or apoptosis regulation in other organisms, from humans to marine organisms (Bouraoui, et al., 2009; Einsporn, et al., 2009; Hasselberg, et al., 2004; Kim, et al., 2008; Olsvik, et al., 2009; Salazar-Medina, et al., 2010; Snyder, 2000; Vasiliou, et al., 2004; Wan, et al., 2011). The GOI analyzed were two heat shock proteins (HSP70 and HSP40), six Aldehyde dehydrogenases (ALDH2, ALDH3, ALDH6, ALDH7, ALDH8, ALDH9), Cytochrome P450-4 (CYP4), Catalase (CAT), Superoxide Dismutase (SOD), Glutathione S-Transferase (GST), Glutathione Synthase (GSH-S), Inhibitor of Apoptosis Protein (IAP), Cell Cycle and Apoptosis Regulatory 1Protein (CARP), Cellular Apoptosis Susceptibility Protein (CAS), actin (ACT) and Alpha and Beta tubulins (ATUB and BTUB). These GOI were analyzed in various experimental conditions: copepods exposed to algae which produce or do not produce toxic metabolites, including dinoflagellates (Prorocentrum minimum, Rhodomonas baltica or Karenia brevis) and diatoms (Chaetoceros socialis and Skeletonema marinoi), during field or laboratory experiments. In addition, the effect of the oxylipin producing diatom Skeletonema marinoi has been tested on two different C. helgolandicus populations: the Mediterranean population collected in the Adriatic Sea and the Atlantic population collected in the English Channel. According to the results obtained, expression levels of the specific GOI changed depending on the tested algae, times of exposure, copepod population analyzed and field/laboratory experiments. Gene expression level patterns in the different experimental conditions tested may help to understand the copepod response to stressful conditions. The identification of new genes, for example using cDNA libraries or new generation sequencing, and the application of new tools, such as functional proteomic approaches, may allow for a more comprehensive overview of how copepods respond to specific stressors in the laboratory, but also to predict the response under natural environmental conditions and the effects of these responses on higher trophic levels.
  • No Thumbnail Available
    Item
    Ecologia e biologia riproduttiva in una popolazione alloctona di Trachemys scripta elegans in Calabria
    (2012-12-14) Crescente, Antonio; Cerra, Maria Carmela; Tripepi, Sandro
    This research was carried out from 2009 to 2011 in the Lake of Angitola on the reproductive biology of the slider turtle Trachemys scripta elegans, an alien species that in this site has been naturalized since the 1980’s. According to the International Union for Conservation of Nature (IUCN), invasive species are one of the most important causes of biodiversity decline on a global scale. As a consequence, the impact of introduced species on local ecosystems is important to evaluate. Pet voluntary introductions are done by private people and usually concern only the release of a few individuals. However, the number of releases is as numerous as the number of pet owners that don't want to keep their pet anymore and consequently decide to "release" it. Although only a small percentage of these alien species will become invasive, the environmental impact would be insidious and often irreversible. In fact on a global scale alien species may be as damaging to native species and ecosystems as the loss and degradation of habitats. We can describe 3 steps in the biological invasions processes, comprising the initial dispersal, when an organism moves long distances to areas outside its native range, for example through human assisted dispersal; establishment of self-sustaining populations within the non-native range; and invasion of the new range. However, when species are invasive, they have strongly positive demographic trends and are often numerous, therefore their management can be extremely difficult and expensive. Our experimental model was Trachemys scripta elegans, the most popular freshwater turtle in the pet trade in the world. It is a medium to large-sized turtle as females reach a carapace length of up to 24 cm and males up to 20 cm. This reptile lives in a wide variety of freshwater habitats and it is a habitat generalist. It prefers larg pools and ponds with soft bottoms, and with many aquatic plants and suitable basking sites for thermoregulation. Trachemys scripta elegans is an opportunistic omnivore subsisting on a wide variety of plant and animal foods. The food preferences change with age. Juveniles are highly carnivorous. Instead the adults, mainly eat vegetable food. In the late 1970s a several turtle farms were established in southern USA, where the hatchlings are packed, in hundreds in small boxes, and are shipped abroad. At least 80% of them die during the first year in captivity. If released terrapins survive in nature, they pose a threat to local wildlife. Only in 1996 the total exports of Trachemys scripta elegans from USA were 8 million individuals, of which 3 million individuals were imported by Europe. With effect from 1997 the EU implemented a ban on the import of this species, in fact already in 1975 the U.S. Food and Drug Administration banned the sale of turtles under 10 cm carapace length in the United States and Canada because they transmitted human salmonellosis. Trachemys scripta elegans distribution includes eastern USA, in particular the Mississipi valley from Illinois to the Gulf of Mexico. It now has a far greater distribution area that include a lot of countries in America, Europe and Asia, where it has naturalized in some cases. Trachemys scripta elegans released in seminatural conditions were first recorded in Campobasso in the early 1970s, but sightings only becoming more common in the mid-80s. It is presently known in all of Italy except for Valle d’Aosta, Sardinia and Campania. Reproduction is common in captivity, but little is known about its reproduction in nature. Some authors suggest that naturalization wouldn’t be a serious problem, but it is known that this species is certainly able to reproduce in Friuli Venezia Giulia, Latium, Emilia Romagna and in Calabria. This research was aimed to investigate reproductive biology of Trachemys scripta elegans in order: • to evaluate if reproduction is an effective phenomenon that could interest the study area; • to estimate the reproductive success in natural condition; • to determine preferences of Trachemys scripta elegans in the nest site selection; • to define the step of the invasion process of slider turtle in the Lake of Angitola, in order to suggest conservation measures. The research was conducted at Lake of Angitola, a 196 ha artificial lake located about 5 km from Pizzo Calabro. The lake became a WWF oasis in 1975, and ten years later was declared an International Ramsar area due to the large presence of migratory birds. Today the lake is included in the Regional Park of Serre and in 2005 the area was proposed as a Site of Community Importance (SIC) by ministerial decree. The climate is typically Mediterranean; the vegetation is very varied: the shrub layer is formed by aromatic herbs; the arboreal layer includes a forests of genus Quercus . The animal communities are very interesting, especially the bird population; in fact we can see more than 150 different species per year. The study was conducted from three years, and included 3 monitors per month, in particular from April to October, when the turtles were more active. The lake’s shores were divided into eight transects using environmental criteria. Data collection were sampling focused on visual census observations, on the nesting sites research and on trapping. Also to analyze the nest site selection we divided the whole lake perimeter by differing vegetative covers and differing soil types to study the use of these selected environments by nesting females. Vegetative cover was classified by five categories by a buffer of 50 m, and including shrub land, grass, fruit growing and reforestation; mixed category was used in cases where no one coverage was present in more than 75% of the subdivisions. Soil type was classified by using the methodology of Casanova (1991), yielding three main categories of dirt, sand and clay substrates and the possible combination of them. Collected variables used in this research were tested with InStat 3.0 software; in particular we used Chi-square goodness of fit, Kruskal-Wallis and Spearman Rank Correlation tests. The results of the visual census showed that Cavalcavia was the preferred basking area for Trachemys thermoregulation. In fact, in this area was observed the highest percentage of turtle presence that performed basking on the emerged deadwood. The heterogeneity χ² test, that was used to compare number of observation and the areas, was statistically significant (N=2015; χ²=893,03; d.f.=5; P‹0,0001). Monthly monitoring activities of basking showed an increasing of turtle presence from April to July, and it is possible to observe a subsequent decreasing of basking activities from August to October. During the research we found 229 nests. The greatest distance of a nest from the shoreline was 100 m, but the greatest number of nests was found between 0 and 20 m from the shoreline. Kruskal-Wallis non-parametric test association between number of eggs and months of the year was statistically significant (KW=14,318; P=0,0137). “Movrella” and “Ceramida” were the main nesting areas; in these transects there are a lot of emerged spaces that female turtles can use for nesting activities. The correlation between the number of eggs and the depth of the nest was statistically extremely significant (N=229; r=0,2105; P<0,0001). More than 85% of nests were found in mixed soil type habitats, particurarly in dirt-sand and dirt-clay soil types. Chi squared test between number of nests and soil type categories was statistically significant (N=229; χ²=116,22; d.f.=3; P<0,0001). More than 65% of nests were found in environments with mixed vegetative cover (Grass+Shrubland). Chi squared test associations between number of nests and the 4 vegetative cover types were statistically significant (N=229; χ²=116,22; d.f.=3; P‹0,0001). We collected by trapping, and marked 26 specimens and in particular 6 juveniles; the more effective trapping method was represented by “basking trap”, that we used on the water surface. In Europe reproduction under natural conditions has been reported only for Spain and France. As we said before, little is known about Trachemys scripta elegans reproduction out of captivity in Italy, but in 2002 Ficetola recorded the first reproduction case of Trachemys scripta elegans on the Po Delta on sympatric condition with an Emys orbicularis population. The population of Angitola represents one of the largest reproductive population of Europe and the most important for our country. In Italy Trachemys scripta elegans deposition in seminatural conditions takes place once a year at the end of June. We recorded for the first time that in our study area deposition takes place twice a year, as reported for the American populations: we think that the first event in April-May, with hatching in June and July, and the second event in July- August, with hatching in September-October. Some tropical Trachemys species tend to nest in open areas that receive direct sunlight for at least part of each day. Analysis of the use of vegetative cover in our study area revealed that Trachemys scripta elegans nests were not randomly distributed within the general area used for nesting, and it suggesting that females were actively selecting nesting sites. In fact the largest number of nests around the lake was found in areas with mixed vegetative cover, as reported for Trachemys callirostris in Colombia. Mixed vegetative cover might reduce thermal stress to the nesting females, as well as lower the detectability of nests to natural predators and human hunters. It has been reported that Trachemys nesting females prefer only dirt soil, but in our study area the largest number of nests in our study area were found in habitat with mixed soil type categories, these zones were characterized by heterogeneous granulometry that increase the ventilation of the nest and the humidity degree for hatching success. We investigated experimentally the occupation of basking sites thus providing a better knowledge on this behavioral pattern. During thermoregulation turtles of lake Angitola mainly bask along the emergent deadwood. Floating (thermoregulation on the water surface) is showed especially during the summer season. This population performs basking from April to October at different times of the day: for example during the cooler months the time of sunlight exposure increases and the basking behavior occurs during the hottest hours of the day. These results are also important for further evaluations on competition for occupation of basking places between this allocthonous specie and Emys orbicularis, our endangered European pond turtle. In fact as reported in literature that Trachemys scripta elegans requires less exposure time for thermoregulation than Emys orbicularis, and for this reason American slider turtle is the winning species in competition for basking sites in sympatric conditions. The invasion process of the Trachemys population inhabiting the lake of Angitola is at the second step, in fact we are in front of a self-sustaining and growing population. Moreover the local population of Emys orbicularis, historically present in the lake until the early 2000's, nowadays is probably extinct. In conclusion it is very important to complete echo-ethological investigations for better understanding the biology and ecology of this invader, and to provide a monitoring and controlling plans, and to define translocation and eradication plans, but this operations could be extremely difficult and more expansive. The results of this research were objects of a scientific paper and of same participation for congresses. Two more papers are in preparation.
  • No Thumbnail Available
    Item
    Angiotensin II and mechanisms of oxidative damage in HUVECs
    (2011-12-14) Carino, Annarita; Cerra, Maria Carmela; Martino, Guglielmo
    The endothelium is essential for the maintenance of vascular homeostasis. Central to this role is the production of endothelium – derived nitric oxide (EDNO), synthesized by endothelial isoform of nitric oxide synthase (eNOS). Endothelial dysfunction, manifested as impaired EDNO bioactivity, is an important early event in the development of various vascular disease, including hypertension, diabetes, genesis of tumors and atherosclerosis. Endothelial dysfunction is an early feature of atherosclerosis vascular disease, characterized by a decrease in nitric oxide (NO) bioavailability and a concomitant increase in vascular superoxide (O2 . -) formation. Loss of NO bioavailability precedes the development of overt atherosclerosis and is an independent predictor of adverse cardiovascular events. Indeed, decreased NO and enhanced production of reactive oxygen species (ROS) have been recognized as major determinants of age-associated endothelial dysfunction. The degree of impairment of EDNO bioactivity is a determinant of future vascular complications. Accordingly, growing interest exists in the pathologic mechanism involved. However it is clear that immunologic mechanisms operating in the context of common cardiovascular risk factors lead to impaired endothelial function, mainly as a consequence of decreased NO bioavailability and excessive oxidative stress. The work submitted in this thesis describes on one side studies aimed to investigate cellular mechanisms underlying endothelial dysfunction and vascular damages driven by oxidative stress in the context of aging, hypertension and atherosclerosis using in vitro models. In addition, we desired to evaluate the efficacy of reducing agents such as flavonoid to monitor whether they actually have an action to recover from the cellular oxidative damage by these natural compounds and how real is their action at the level of microcirculation in vitro models. On the other side, we present studies focused on the pathophysiology of microcirculation as far as functional aspects are concerned in the context to better understand the functioning of the Renin- Angiotensin-System in particular if the Angiotensin IV is involved in mechanisms of oxidative stress and in Calcium intracellular levels.
  • No Thumbnail Available
    Item