Pollination ecology and reproductive success in isolated populations of flowering plants: Primula apennina Widmer, Dictamnus albus L. and Convolvulus lineatus L.

This research focuses on reproductive biology and pollination ecology of entomophilous angiosperms, with particular concern to reproductive success in small and isolated populations of species that occur at their distribution limits or are endemic. I considered three perennial herbs as model species: Primula apennina Widmer, Dictamnus albus L. and Convolvulus lineatus L. I carried out field work on natural populations and performed laboratory analyses on specific critical aspects (resource allocation, pollen viability, stigmatic receptivity, physiological self-incompatibility, seed viability), through which I analysed different aspects related to plant fitness, such as production of viable seed, demographic structure of populations, type and efficiency of plant-pollinator system, and limiting factors.

Functional Genomics and Cell Biology of the Dolphin (Tursiops runcatus): Establishment of Novel Molecular Tools to Study Marine Mammals in Changing Environments

The dolphin (Tursiops truncatus) is a mammal that is adapted to life in a totally aquatic environment. Despite the popularity and even iconic status of the dolphin, our knowledge of its physiology, its unique adaptations and the effects on it of environmental stressors are limited. One approach to improve this limited understanding is the implementation of established cellular and molecular methods to provide sensitive and insightful information for dolphin biology. We initiated our studies with the analysis of wild dolphin peripheral blood leukocytes, which have the potential to be informative of the animal’s global immune status. Transcriptomic profiles from almost 200 individual samples were analyzed using a newly developed species-specific microarray to assess its value as a prognostic and diagnostic tool. Functional genomics analyses were informative of stress-induced gene expression profiles and also of geographical location specific transcriptomic signatures, determined by the interaction of genetic, disease and environmental factors. We have developed quantitative metrics to unambiguously characterize the phenotypic properties of dolphin cells in culture. These quantitative metrics can provide identifiable characteristics and baseline data which will enable identification of changes in the cells due to time in culture. We have also developed a novel protocol to isolate primary cultures from cryopreserved tissue of stranded marine mammals, establishing a tissue (and cell) biorepository, a new approach that can provide a solution to the limited availability of samples. The work presented represents the development and application of tools for the study of the biology, health and physiology of the dolphin, and establishes their relevance for future studies of the impact on the dolphin of environmental infection and stress.

Food lipids: their effects on quality and oxidative stability of animal tissues and emulsions

Lipolysis and oxidation of lipids in foods are the major biochemical and chemical processes that cause food quality deterioration, leading to the characteristic, unpalatable odour and flavour called rancidity. In addition to unpalatability, rancidity may give rise to toxic levels of certain compounds like aldehydes, hydroperoxides, epoxides and cholesterol oxidation products. In this PhD study chromatographic and spectroscopic techniques were employed to determine the degree of lipid oxidation in different animal products and its relationship with technological parameters like feeding fat sources, packaging, processing and storage conditions. To achieve this goal capillary gas chromatography (CGC) was employed not only to determine the fatty acids profile but also, after solid phase extraction, the amount of sterols (cholesterol and phytosterols) and cholesterol oxidation products (COPs). To determine hydroperoxides, primary products of oxidation and quantify secondary products UV/VIS absorbance spectroscopy was applied. Beef and pork meat in this study were analysed. In actual fact, lipid oxidation is a major deterioration reaction in meat, meat products and results in adverse changes in the colour, flavour, texture of meat and develops different compounds which should be a risk to human health as oxysterols. On beef and pork meat, a study of lipid fraction during storage was carried out to evaluate its shelf-life and some nutritional features life saturated/unsaturated fatty acids ratio and sterols content, in according to the interest that has been growing around functional food in the last years. The last part of this research was focused on the study of lipid oxidation in emulsions. In oil-in-water emulsions antioxidant activity of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) was evaluated. The rates of lipid oxidation of 1.0% stripped soybean oil-in-water emulsions with DOPC were followed by monitoring lipid hydroperoxide and hexanal as indicators of primary and secondary oxidation products and the droplet surface charge or zeta potential (ζ) of the emulsions with varying concentrations of DOPC were tested. This manuscript reports the main results obtained in the three activities briefly summarized as follows: 1. study on effects of feeding composition on the photoxidative stability of lipids from beef meat, evaluated during storage under commercial retail conditions; 2. evaluation of effects of diets and storage conditions on the oxidative stability of pork meat lipids; 3. study on oxidative behavior of DOPC in stripped soybean oil-in-water emulsions stabilized by nonionic surfactant.

Taxonomy, phylogeny and reproductive ecology of Gentiana lutea L.

This research focuses on taxonomy, phylogeny and reproductive ecology of Gentiana lutea. L.. Taxonomic analysis is a critical step in botanical studies, as it is necessary to recognize taxonomical unit. Herbarium specimens were observed to assess the reliability of several subspecies-diagnostic characters. The analysis of G. lutea genetic variability and the comparison with that of the other species of sect. Gentiana were performed to elucidate phylogenetic relationships among G. lutea subspecies and to propose a phylogenetic hypothesis for the evolution and the colonization dynamics of the section. Appropriate scientific information is critical for the assessment of species conservation status and for effective management plans. I carried out field work on five natural populations and performed laboratory analyses on specific critical aspects, with special regard to G. lutea breeding system and type and efficiency of plant-pollinator system. Bracts length is a reliable character to identify subsp. vardjanii, however it is not exclusive, hence to clearly identify subsp. vardjanii, other traits have to be considered. The phylogenetic hypotheses obtained from nuclear and chloroplast data are not congruent. Nuclear markers show a monophyly of sect. Gentiana, a strongly species identity of G. lutea and clear genetic identity of subsp. vardjanii. The little information emerging from plastid markers indicate a weak signal of hybridization and incomplete sorting of ancestral lineages. G. lutea shows a striking variation in intra-floral dichogamy probably evolved to reduce pollen-stigma interference. Although the species is partially self-compatible, pollen vectors are necessary for a successful reproduction, and moreover it shows a strong inbreeding depression. G. lutea is a generalist species: within its spectrum of visitors is possible to recognize "nectar thieves" and pollinators with sedentary or dynamic behaviour. Pollen limitation is frequent and it could be mainly explained by poor pollen quality.

Phenotypic and genotypic characterisation of antimicrobial resistance in Escherichia coli indicator of animal, food and human origin.

This thesis presents AMR phenotypic evaluation and whole genome sequencing analysis of 288 Escherichia coli strains isolated from different sources (livestock, companion animal, wildlife, food and human) in Italy. Our data reflects general resistance trends in Europe, reporting tetracycline, ampicillin, sulfisoxazole and aminoglycosides resistance as the most common phenotypic AMR profile among livestock, pets, wildlife and humans. Identification of human and animal (livestock and companion animal) AMR profiles in niches with a rare (fishery, mollusc) or absent (vegetable, wild animal, wild boar) direct exposure to antimicrobials, suggests widespread environmental pollution with ARGs conferring resistance to these antimicrobials. Phenotypic resistance to highest priority critically important antimicrobials was mainly observed in food-producing animals and related food such as rabbit, poultry, beef and swine. Discrepancies between AMR phenotypic pattern and genetic profile were observed. In particular, phenotypic aminoglycoside, cephalosporin, meropenem, colistin resistance and ESBL profile did not have a genetic explanation in different cases. This data could suggest the diffusion of new genetic variants of ARGs, associated to these antimicrobial classes. Generally, our collection shows a virulence profile typical of extraintestinal pathogenic Escherichia coli (ExPEC) pathotype. Different pandemic and emerging ExPEC lineages were identified, in particular in poultry meat (ST10; ST23; ST69, ST117; ST131). Rabbit was suggested as a source of ST20-ST40 potential hybrid pathogens. Wildlife carried a high average number (10) of VAGs (mostly associated to ExPEC pathotype) and different predominant ExPEC lineages (ST23, ST117, ST648), suggesting its possible involvement in maintenance and diffusion of virulence determinants. In conclusion, our study provides important knowledge related to the phenotypic/genetic AMR and virulence profiles circulating in E. coli in Italy. The role of different niches in AMR dynamics has been discussed. In particular, food-producing animals are worthy of continued investigation as a source of potential zoonotic pathogens, meanwhile wildlife might contribute to VAGs spread.

Reproductive and developmental toxicity study using Sprague-Dawley rats exposed under various calendars to the weedkiller Glyphosate and commercial formulations Glyphosate-based

Glyphosate-based herbicides (GBHs) are the most globally used herbicides raising the risk of environmental exposition. Carcinogenic effects are only one component of the multiple adverse health effects of Glyphosate and GBHs that have been reported. Questions related to hazards and corresponding risks identified in relation to endocrine disrupting effects are rising. The present study investigated the possible reproductive/developmental toxicity of GBHs administered to male and female Sprague-Dawley rats under various calendar of treatment. Assessments included maternal and reproductive outcome of F0 and F1 dams exposed to GBHs throughout pregnancy and lactation and developmental landmarks and sexual characteristics of offspring. The study was designed in two stages. In the first stage Glyphosate, or its commercial formulation Roundup Bioflow, was administered to rats at the dose of 1.75 mg/kg bw/day (Glyphosate US Acceptable Daily Intake) from the prenatal period until adulthood. In the second stage, multiple toxicological parameters were simultaneously assessed, including multigeneration reproductive/developmental toxicity of Glyphosate and two GBHs (Roundup Bioflow and Ranger Pro). Man-equivalent doses, beginning from 0.5 mg/kg bw/day (ADI Europe) up to 50 mg/kg bw/day (NOAEL Glyphosate), were administered to male and female rats, covering specific windows of biological susceptibility. The results of stage 1 and preliminary data from stage 2 experiments characterize GBHs as probable endocrine disruptors as suggested by: 1) androgen-like effects of Roundup Bioflow, including a significant increase of anogenital distances in both males and females, delay of first estrous and increased testosterone in females; 2) slight puberty onset anticipation in the high dose of Ranger Pro group, observed in the F1 generation treated from in utero life until adulthood; 3) a delayed balano-preputial separation achievement in the high dose of Ranger Pro-treated males exposed only during the peri-pubertal period, indicating a direct and specific effect of GBHs depending on the timing of exposure.

Reproduction of Mediterranean zooxanthellate and azooxanthellate scleractinian corals along environmental gradients

Despite extensive studies focus mainly on sexual reproductive characteristics in tropical scleractinian species, there is limited knowledge on temperate regions. The Mediterranean is a biodiversity hotspot under intense pressure from anthropogenic impacts. Climatic models further predict that the Mediterranean basin will be one of the most impacted regions by the ongoing warming trend. This makes it a potential model of more global patterns to occur in the world’s marine biota, and a natural focus of interest for research on climate. The present research contributed to increase data on reproductive modes and sexuality of temperate scleractinian corals, highlighting their developmental plasticity, showing different forms of propagation and different responses to environmental change. For the first time, sexuality and reproductive mode in Caryophyllia inornata were determined. An unusual embryogenesis without a clear seasonal pattern was observed, suggesting the possibility of an asexual origin. Sexual reproduction of Astroides calycularis was governed by annual changes in seawater temperature, as observed for other Mediterranean dendrophylliids. Defining the reproductive biology of these species is the starting point for studying their potential response to variations of environmental parameters, on a global climate change context. The results on the influence of temperature on reproductive output of the zooxanthellate (symbiosis with unicellular algae) Balanophyllia europaea and the non-zooxanthellate Leptopsammia pruvoti suggest that the latter may be quite tolerant to temperature increase, since the zooxanthellate species resulted less efficient at warm temperatures. A possible explanation could be related to their different trophic system. In B. europaea thermal tolerance is primarily governed by the symbiotic algae, making it more sensitive to temperature changes. On the contrary, the absence of symbionts in L. pruvoti might make it more resistant to temperature. In a progressively warming Mediterranean, the efficiency on scleractinian reproduction could be influenced in different ways, reflecting their extraordinary adaptability.