Abitudini alimentari di Merluccius merluccius in Alto-Medio Adriatico

Lo studio delle relazioni trofiche del nasello tramite analisi del contenuto stomacale è particolarmente importante per la gestione della pesca sia di questa specie che delle specie appartenenti alla sua alimentazione. Lo scopo della tesi è stato quello di esaminare l’alimentazione di questa specie utilizzando 200 campioni di nasello provenienti dalla Campagna Medits 2014, e confrontare la metodologia morfologica utilizzata con quella molecolare, determinando i vantaggi di ognuna ed i possibili limiti. La campagna di strascico è stata sviluppata secondo uno schema random stratificato in relazione alla profondità e all’interno di ciascun strato. Lo sbarcato è stato preventivamente sottoposto ad analisi biometriche registrando lunghezza totale, peso, sesso e maturità sessuale di ogni singolo individuo; successivamente sono stati conservati in etanolo gli stomaci prelevati dai 200 naselli scelti per classe di taglia. Sono state riconosciute le prede dei campioni di nasello sia analizzandole qualitativamente (al livello tassonomico più basso possibile), che quantitativamente, considerando alcuni degli indici maggiormente utilizzati. È stato poi calcolato l’indice trofico del nasello e sono stati infine confrontati i risultati con quelli delle analisi molecolari evidenziando le prede non riconosciute e gli errori di identificazione. La metodologia morfologica ha sottolineato che l’alimentazione del nasello dipende fortemente dalla taglia del predatore e dal loro grado di maturità sessuale, ma non varia significativamente in base al sesso. Ha infatti evidenziato una taglia del nasello, corrispondente alla taglia di prima maturità sessuale, in cui la specie cambia gradualmente la sua alimentazione, passando da piccoli crostacei a pesci di grandezza sempre maggiore. Risulta essere un forte predatore specializzato, ma che al tempo stesso risulta essere una specie opportunista, scegliendo il proprio cibo da ciò che ha a disposizione. Il confronto con l’analisi molecolare è stato utile per descrivere i pro e i contro di ciascuna metodologia, dimostrando che è necessario sfruttarle entrambe per avere risultati efficaci e completi dell’alimentazione di una specie.

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.

Struttura di età di nasello (Merluccius merluccius, Linnaeus, 1758) in alto-medio Adriatico mediante analisi degli otoliti e frequenza di taglia

The aim of present study is to define the general framework of Merluccius merluccius population structure, to estimate the growth rate and to assess the recruitment dynamics of juveniles from Northern and Central Adriatic, through otoliths analysis. The otoliths of hake specimens collected during the MedITS trawl survey in the 2012 in GSA 17, were cleaned and 102 otoliths out of 506 were embedded, sectioned, grindined and polished to obtain frontal and sagittal sections. The whole sample were analysed under stereomicroscope and optical microscope, with camera and connected to PC provided of an image analyses program. The frequency analysis of size classes and age revealed that the species is dominated by hake with >200mm TL and > one year old. The fish average size of M. merluccius at the end of the first year of life is about 199 mm TL. Allometrics analyses between fish TL and Feret (major axis), MiniFeret (minor axis), Area, Perimeter, showed a direct proportionality among lengths. Among the 88 otoliths sections analysed, the number of daily increments read ranged from 86 to 206, within 55 and 175mm TL range. The age estimate ranged from about 2-3 to 9 months and the growth rate from 20.99 to 27.15mm TL. The hatch-date distribution, obtained by back calculation, showed that the hatching occurs in November-March. In conclusion, strong preventive measures are needed for hake adults because the success of this species seems to be linked to deep water ecosystem protection where big spawners dwell.

Molecular phylogenesis of Mediterranean Octocorals

In order to support the conservation of the Mediterranean octocorals improvements on information regarding their taxonomic units and phylogenetic relationships are strongly needed. In the present thesis work, phylogenetic analyses based on the mitochondrial mtMSH and 16S genes were performed including 15 Mediterranean octocorals species on the 56 recognized to date. Moreover, an extended datasets with Atlanto/Pacific congeners Octocorallia species was implemented to clarify their phylogenetic relationships and estimate the divergence times of the Mediterranean species. Results indicated that: 1) there are similarity and differences among molecular and morphological traits depending on the taxonomical level considered; 2) the molecular phylogeny of the Mediterranean octocorals retrace the previous relationships based on wide octocorals analyses; and 3) the divergence time among Mediterranean and Atlanto/Pacific species varies depending on analysed taxa. At higher taxonomic level, the Mediterranean trees supported the division of the Mediterranean Octocorallia into one major clade (Alcyoniina-Holaxonia) plus two unresolved branch including the single species available of Scleraxonia and Stolonifera respectively. This topology was better supported including the Atlanto/Pacific congeners species. The molecular evidence suggested that Alcyonium palmatum and Corallium rubrum species are the youngest with a divergence time estimated around 4 MYA. Particularly, C. rubrum results were in agreement with the hypothesis that recent orogenesis process of the Mediterranean Sea promoted the allopatric speciation of this specie. Increasing the sample design and implementing the emerging next-generation genomic-sequencing technologies, further studies would be able to improve the understanding of the Mediterranean octocorals phylogenetic relationships and evolution.

Feeding entrainment of locomotor activity and clock gene expression in Senegalese sole, Solea senegalensis

The present study was conducted to investigate the influence of restricted food access on Solea senegalensis behaviour and daily expression of clock genes in central (diencephalon and optic tectum) and pheripheral (liver) tissues. The Senegalese sole is a marine teleost fish belonging to the Class of Actinopterygii, Order Pleuronectiformes and Family Soleidae. Its geographical distribution in the Mediterranean sea is fairly broad, covering the south and east of the Iberian Peninsula, the North of Africa and Middle East until the coast of Turkey. From a commercial perspective Solea senegalensis has acquired in recent years, a key role in aquacolture industry of the Iberian Peninsula. The Senegalese sole is also acquiring an important relevance in chronobiological studies as the number of published works focused on the sole circadian system has increased in the last few years. The molecular mechanisms underlying sole circadian rhythms has also been explored recently, both in adults and developing sole. Moreover, the consideration of the Pleuronectiformes Order as one of the most evolved teleost groups make the Senegalese sole a species of high interest under a comparative and phylogenetic point of view. All these facts have reinforced the election of Senegalese sole as model species for the present study. The animals were kept under 12L:12D photoperiod conditions and divided into three experimental groups depending on the feeding time: fed at midlight (ML), middark (MD) or random (RND) times. Throughout the experiment, the existence of a daily activity rhythm and it synchronization to the light-dark and feeding cycles was checked. To this end locomotor activity was registred by means of two infrared photocells placed in pvc tube 10 cm below the water surface (upper photocell) and the other one was located 10 cm above the bottom of the tank (bottom photocell). The photocell were connected to a computer so that every time a fish interrupted the infrared light beam, it produced an output signal that was recorded. The number of light beam interruptions was stored every 10 minutes by specialized software for data acquisition.

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).

Rhythms of anorexigenic neuropeptides in senegalese sole: Effects of light, feeding regimes and developmental stage.

Increasing knowledge on the endocrine mechanisms that regulate feeding and growth in cultured fish can contribute to make improvement in fish holding conditions and feeding strategies, supporting the development of new techniques that could ameliorate feeding, food conversion efficiency and growth in aquaculture practice. The main objective of this study was to investigate how daily mRNA expression of three specific anorexigenic hormones, i.e. the corticotropin-releasing hormone (CRH) and the paralogues α- and β- proopiomelanocortin (POMC), is modulated by different photoperiods, light spectra and feeding regimes, in both adult and larvae of Solea senegalensis. In addition, as Senegalese sole exhibits a shift from diurnal to nocturnal in locomotor activity and feeding habits during metamorphic process, we tried to elucidate if this shift is accompanied by relevant daily variations in the expression of these anorexigenic hormones before, during and after the completion of metamorphosis. In order to reach this main objective, three main experiments were developed. In a first experiment, adults were reared under LD (12 h light: 12h dark) cycle and fed at mid-light (ML), mid-dark (MD) and at random (RND). In a second experiment, adult specimens were reared in constant darkness (DD) and fed at subjective mid-light (sML) or at RND. Larvae of Senegalese sole were reared under LD cycle with white, blue or red light for 40 days. Our results show an independence of crh mRNA expression from the feeding time and suggest an endogenous control of crh expression in sole. Both pomc paralogues showed significant daily rhythms under LD conditions. The rhythms were maintained or were even more robust under DD conditions for pomc_a, but were completely abolished for pomc_b. Our results indicate an endogenous control of pomc_a expression by the molecular clock in telencephalon and diencephalon, but not in the pituitary gland. Our findings confirm for the first time the significant influence that ambient lighting has on larval growth and development in Senegalese sole, revealing an important effect of light spectra upon functional elements of this species. Our results also emphasize the importance of maintaining cycling light-dark conditions of the adequate wavelengths in aquaculture practices during early development of sole.

Inference of Raja miraletus population structure using mitochondrial and nuclear DNA: comparing the resolution power of molecular markers in exploring species and population boundaries

This study focused on the role of oceanographic discontinuities and the presence of transitional areas in shaping the population structure and the phylogeography of the Raja miraletus species complex, coupled with the test of the effective occurrence of past speciation events. The comparisons between the Atlantic African and the North-Eastern Atlantic-Mediterranean geographic populations were unravelled using both Cytochrome Oxidase I and eight microsatellite loci. This approach guaranteed a robust dataset for the identification of a speciation event between the Atlantic African clade, corresponding to the ex Raja ocellifera nominal species, and the NE Atlantic-Mediterranean R. miraletus clade. As a matter of fact, the origin of the Atlantic Africa and the NE Atlantic-Mediterranean deep split dated about 11.74MYA and was likely due to the synergic influence currents and two upwelling areas crossing the Western African Waters. Within the Mediterranean Sea, particular attention was also paid to the transitional area represented by Adventura and Maltese Bank, that might have contributed in sustaining the connectivity of the Western and the Eastern Mediterranean geographical populations. Furthermore, the geology of the easternmost part of Sicily and the geo-morphological depression of the Calabrian Arc could have driven the differentiation of the Eastern Mediterranean Sea. Although bathymetric and oceanographic discontinuity could represent barriers to dispersal and migration between Eastern and Western Mediterranean samples, a clear and complete genetic separation among them was not detected. Results produced by this work identified a speciation event defining Raja ocellifera and R. miraletus as two different species, and describing the R. miraletus species complex as the most ancient cryptic speciation event in the family Rajidae, representing another example of how strictly connected the environment, the behavioural habits and the evolutionary and ecologic drivers are.