Genetic and morphological variation and differentiation of Raja clavata populations in the European seas: marker development and preliminary data

In this study the population structure and connectivity of the Mediterranean and Atlantic Raja clavata (L., 1758) were investigated by analyzing the genetic variation of six population samples (N = 144) at seven nuclear microsatellite loci. The genetic dataset was generated by selecting population samples available in the tissue databases of the GenoDREAM laboratory (University of Bologna) and of the Department of Life Sciences and Environment (University of Cagliari), all collected during past scientific surveys (MEDITS, GRUND) from different geographical locations in the Mediterranean basin and North-east Atlantic sea, as North Sea, Sardinian coasts, Tuscany coasts and Cyprus Island. This thesis deals with to estimate the genetic diversity and differentiation among 6 geographical samples, in particular, to assess the presence of any barrier (geographic, hydrogeological or biological) to gene flow evaluating both the genetic diversity (nucleotide diversity, observed and expected heterozygosity, Hardy- Weinberg equilibrium analysis) and population differentiation (Fst estimates, population structure analysis). In addition to molecular analysis, quantitative representation and statistical analysis of morphological individuals shape are performed using geometric morphometrics methods and statistical tests. Geometric coordinates call landmarks are fixed in 158 individuals belonging to two population samples of Raja clavata and in population samples of closely related species, Raja straeleni (cryptic sibling) and Raja asterias, to assess significant morphological differences at multiple taxonomic levels. The results obtained from the analysis of the microsatellite dataset suggested a geographic and genetic separation between populations from Central-Western and Eastern Mediterranean basins. Furthermore, the analysis also showed that there was no separation between geographic samples from North Atlantic Ocean and central-Western Mediterranean, grouping them to a panmictic population. The Landmark-based geometric morphometry method results showed significant differences of body shape able to discriminate taxa at tested levels (from species to populations).

Morphological-molecular taxonomy and population genetic structure of common and Egyptian soles of the Adriatic Sea

A total of 352 specimens were analyzed to achieve the different aims of this thesis. 255 central-northern Adriatic specimens of S. solea and S. aegyptiaca were molecularly analysed using microsatellite locus Sos(AC)40 and 205 also morphologically due to evaluate the abundance and the distribution of the cryptic species S. aegyptiaca and to confirm morphologic analyses. Morphological and molecular analyses comparated show a correspondence of 96%. A combined morphologic approach could be proposed to apply multiple criteria on the analyzed external morphological keys. The Adriatic Egyptian soles may lives in shallow waters (up 30 m) and in brackish lagoon. 127 samples of Adriatic common sole added to 326 samples of previous studies showed, using mitochondrial marker (CytB), that the Adriatic Sea as contact zone between Tyrrhenian and Aegean Sea, the divergence within the Adriatic Sea is low but significant between central-north and south, with a longitudinal strong gene flow in central-northern side. It’s also showed as in the Adriatic Sea two near-panmictic populations of common sole exist.

Genetic variability and differentiation in the Mediterranean endemic starry ray Raja asterias (Delaroche, 1809)

The present study deal with the population structure and connectivity of the Mediterranean endemic starry ray Raja asterias (Delaroche, 1809) in the Western and Eastern Mediterranean basin. A panel of eight microsatellite loci which cross-amplify in Rajidae (El Nagar, 2010) was used to assess population connectivity and structure. Those aims were investigated by analyzing the genetic variation of 9 population sample for a total of 185 individuals collected during past scientific surveys (MEDITS, GRUND), commercial trawling and also directly at fish markets. The purpose of this thesis is to estimate the genetic divergence occurring between the Mediterranean populations and, in particular, to assess the presence of any barrier (geographic, hydrogeological and biological) to gene flow for this species. Different statistical approaches were performed to reach this aim evaluating both the genetic diversity (nucleotide diversity, allelic richness, observed and expected heterozygosity and Hardy-Weinberg equilibrium test) and the population differentiation patterns (pairwise Fst estimated and population structure analysis). The results obtained from the analysis of the microsatellite dataset suggest a geographic and genetic separation between the starry ray populations of the Mediterranean basin into three or four distinct groups: Western and Eastern Mediterranean basins and Sicilian coast always clustering as an independent group and Algeria which could be or not considered another separate group. The data were discussed from both an evolutionary and a conservation point of view and in relation to previous results obtained by the analysis of mitochondrial marker. A comparison with other Mediterranean demersal skate species was performed in order to better contextualise our results. Finally, our results could offer useful information to protect vulnerable species as R. asterias and developing effective conservation plans in the Mediterranean.

Reproduction and artificial restocking of Acipenser naccarii (Bonaparte, 1836). Genetic and physiological characterization through microsatellite markers and radioimmunoassay of sexual steroids

The Adriatic sturgeon, Acipenser naccarii (Bonaparte, 1836), is a highly threatened species due to human activities, particularly overfishing and habitat destruction. Its peculiar ecology and biology (restricted areal and anadromy) makes this species particularly vulnerable. In March 2010 the IUCN has identified the Adriatic sturgeon as a critically endangered species according to the Red List of Threatened Species. Due to its rapid decline, starting from the 80s, at present there is no evidence of natural reproduction in wild environment, which makes the Adriatic sturgeon dependenton captive breeding programs that need to be improved in order to be effective for the survival of the species. For this purpose this study aims to characterize artificial restocking population of Adriatic sturgeon, with both genetic and physiological analysis in order to establish an efficient restocking program for future reproductions. The research is structured on two levels: First genetically, by analyzing 9 microsatellite loci. This gives information relatively about parent allocation and kinship between individuals that were sampled for this study. Hence to predict which reproduction events are the most optimal in terms of incrementing genetic diversity, by the estimation of multilocus pairwise band sharing coefficients. Second step, physiological analysis: testosterone (T) concentration levels in each individual were measured for sexing, without sacrificing the lives of the animals with the use of an invasive examination of the gonads. The combination of interdisciplinary analysis is important to obtain an overall picture in order to indicate the main broodstock participating in reproduction events and future optimal potential participants, in order to ensure a valid management for restocking program and their monitoring.

A novel marker for heart failure: Cross sectional area assessment in a large vein by conductance catheter.

The present study investigates the feasibility of a new application able to check the heart failure status in a patient through the estimation of the venous distension. In this way it would be possible to follow up patients, avoiding invasive or expensive exams such as cardiac catheterization and echocardiography. Moreover, the devices would also be able to diagnose the decline of the disease, in order to allow a new adaptation to therapy, and vice versa to check the improvement in the patient’s conditions after the CRT device implant. This thesis is essentially divided into three parts: an analytical model was used to obtain an estimation of the error committed for the calculation of the CSA and to understand how the accuracy and sensitivity depend on the different configurations of the electrodes and the catheter position inside the vein; secondly, an in-vitro experiment was carried out in order to verify the practical feasibility for these kinds of measurements, in a very simplified model; in the end, several animal experiments were done to test the in-vivo practicability of the proposed method. The obtained results showed the feasibility of this approach. In fact, the error committed in the estimation of CSA, during the animal experiments, can be considered acceptable (CSAerror_max ≈ -14%). Moreover, it has been demonstrated that the conductance catheter allows assessing, not only the vein CSA, but also the breathing of the animal.