Aspectos biológicos y ecológicos del tiburón ángel "Squatina squatina" (Linnaeus 1758) en la isla de Gran Canaria

Esta tesis contiene los cambios sugeridos por el tribunal

Genetic variability and structuring of deep Corallium rubrum (Linnaeus, 1758) populations

ABSTRACT Given the decline of shallow-water red coral populations resulting from over-exploitation and mass mortality events, deeper populations below 50 metres depth (mesophotic populations) are currently the most harvested; unfortunately, very little is known about their biology and ecology. The persistence of these populations is tightly linked to their adult density, reproductive success, larval dispersal and recruitment. Moreover, for their conservation, it is paramount understand processes such as connectivity within and among populations. Here, for the first time, genetic variability and structuring of Corallium rubrum populations collected in the Tyrrhenian Sea ranging from 58 to 118 metres were analyzed using ten microsatellite loci and two mitochondrial markers (mtMSH and MtC). The aims of the work were 1) to examine patterns of genetic diversity within each geographic area (Elba, Ischia and Praiano) and 2) to define population structuring at different spatial scales (from tens of metres to hundreds of kilometres). Based on microsatellite data set, significant deviations from Hardy-Weinberg equilibrium due to elevated heterozygote deficiencies were detected in all samples, probably related to the presence of null alleles and/or inbreeding, as was previously observed in shallow-water populations. Moreover, significant levels of genetic differentiation were observed at all spatial scale, suggesting a recent isolation of populations. Biological factors which act at small spatial scale and/or abiotic factors at larger scale (e.g. summer gyres or absence of suitable substrata for settlement) could determine this genetic isolation. Using mitochondrial markers, significant differences were found only at wider scale (between Tuscany and Campania regions). These results could be related to the different mutation rate of the molecular makers or to the occurrence of some historical links within regions. A significant isolation by distance pattern was then observed using both data sets, confirming the restricted larval dispersal capability of the species. Therefore, the hypothesis that deeper populations may act as a source of larvae helping recovery of threatened shallow-water populations is not proved. Conservation strategies have to take into account these results, and management plans of deep and currently harvested populations have to be defined at a regional or sub regional level, similarly to shallow-water populations. Nevertheless, further investigations should be needed to understand better the genetic structuring of this species in the mesophotic zone, e.g. extending studies to other Mediterranean deep-water populations.

Identification of stock units of Mullus barbatus (Linnaeus, 1758) in the Italian seas: development of genomic 2b-RAD markers and biological variation analysis.

This thesis is settled within the STOCKMAPPING project, which represents one of the studies that were developed in the framework of RITMARE Flagship project. The main goals of STOCKMAPPING were the creation of a genomic mapping for stocks of demersal target species and the assembling of a database of population genomic, in order to identify stocks and stocks boundaries. The thesis focuses on three main objectives representing the core for the initial assessment of the methodologies and structure that would be applied to the entire STOCKMAPPING project: individuation of an analytical design to identify and locate stocks and stocks boundaries of Mullus barbatus, application of a multidisciplinary approach to validate biological methods and an initial assessment and improvement for the genotyping by sequencing technique utilized (2b-RAD). The first step is the individuation of an analytical design that has to take in to account the biological characteristics of red mullet and being representative for STOCKMAPPING commitments. In this framework a reduction and selection steps was needed due to budget reduction. Sampling areas were ranked according the individuation of four priorities. To guarantee a multidisciplinary approach the biological data associated to the collected samples were used to investigate differences between sampling areas and GSAs. Genomic techniques were applied to red mullet for the first time so an initial assessment of molecular protocols for DNA extraction and 2b-RAD processing were needed. At the end 192 good quality DNAs have been extracted and eight samples have been processed with 2b-RAD. Utilizing the software Stacks for sequences analyses a great number of SNPs markers among the eight samples have been identified. Several tests have been performed changing the main parameter of the Stacks pipeline in order to identify the most explicative and functional sets of parameters.

Studio della presenza di capodogli (Physeter macrocephalus, Linnaeus 1758) in due aree del Mar Balear mediante metodi acustici passivi

I capodogli (Physeter macrocephalus) sono mammiferi marini con distribuzione cosmopolita; sono presenti anche nel Mar Mediterraneo, soprattutto nella zona occidentale del bacino. Per individuare la presenza di questi animali possono essere utilizzate differenti modalità. Questo studio è stato condotto tramite l’uso di metodi acustici passivi, in quanto i capodogli (P. macrocephalus) sono animali che emettono suoni. L’uso di metodi acustici ha alcuni vantaggi, infatti permette di individuare la presenza di un animale anche se si trova al di sotto della superficie dell’acqua, purché stia emettendo suoni. Le registrazioni possono essere effettuate indipendentemente dalle condizioni meteorologiche e per lunghi periodi di tempo, permettendo la continuità nella raccolta dei dati. Il lavoro di tesi è stato svolto nell’ambito di un Progetto di Ricerca de L’Oceanogràfic di Valencia. Le registrazioni sono state effettuate tramite lo strumento EAR (Ecological Acoustic Recorder) in due aree del Mar Balear, il Mar di Bamba e il Parco Nazionale di Cabrera, in differenti periodi, profondità e con diverse frequenze di campionamento (50 kHz e 5 kHz). I dati ottenuti sono stati analizzati tramite i programmi X-BAT (Extensible Bioacoustic Tool) e Adobe Audition 3.0 al fine di rilevare i pulsi emessi dai capodogli. Per entrambe le aree di studio i dati sono stati elaborati in modo da ottenere modelli giornalieri e stagionali delle ore positive e del numero di pulsi emessi. La presenza di P. macrocephalus è stata confermata in entrambe le aree di studio, e dal confronto delle analisi spettrali dei pulsi si può affermare che, nonostante le differenze, i pulsi registrati in entrambe le aree sono effettivamente emessi da capodogli. Il lavoro di Tesi permette di suggerire che per futuri studi sia applicata la frequenza di registrazione di 20 kHz utilizzando lo strumento SAMARUC, alla cui progettazione ha partecipato L’Oceanogràfic di Valencia.

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.

Influenze ontogenetiche e ambientali sui fattori di condizione di Merluccius merluccius Linnaeus, 1758 (Pisces: Gadiformes) e Galeus melastomus Rafinesque, 1810 (Pisces: Carcharhiniformes): Implicazioni sulla gestione delle risorse

In ecologia della pesca, i fattori o indicatori della “condizione” di un organismo forniscono molte informazioni sulle caratteristiche di adattamento dei pesci all’ambiente e sul loro ruolo nell’ecosistema. La “condizione” include molte caratteristiche strutturali ed energetiche che possono variare in funzione dell’ontogenesi, del ciclo riproduttivo, ma anche in funzione delle caratteristiche dell’ambiente, incluso il grado di stress al quale è sottoposta una specie (es. la pressione di pesca). L’obiettivo del presente studio sperimentale è stato valutare eventuali differenze nell’abbondanza, nei parametri di popolazione, nella struttura demografica e negli indicatori di “condizione” di due specie, Merluccius merluccius (Pisces: Gadiformes) e Galeus melastomus (Pisces: Carcharhiniformes), in due diverse aree: Toscana settentrionale e meridionale, differenti per caratteristiche ambientali e pressione di pesca. Nella prima parte dell’analisi, sono stati confrontati gli indici di densità e biomassa, la struttura di taglia delle due popolazioni, su dati estratti dagli archivi storici delle campagne di pesca sperimentale MEDITS dal 1994 al 2013. Nella seconda parte dell’analisi invece, sono stati analizzati 1000 individui provenienti dalla campagna MEDITS 2014, integrati con campioni provenienti dallo sbarcato commerciale per il biennio 2014-2015. Gli individui di M. merluccius sono stati ripartiti in due classi di taglia (I = individui ≤ 18 cm LT; II = individui > 18 cm LT), quelli di G. melastomus in tre classi di taglia (I = individui ≤ 20 cm LT; II = individui 20 cm< x ≤ 35 cm LT; III= individui > 35 cm LT), suddivisi rispettivamente in 50 maschi e 50 femmine, per ogni classe. E’ stato condotto lo studio della crescita relativa attraverso l’analisi della relazione taglia/peso e lo studio della condizione tramite i seguenti indicatori: il fattore K di Fulton, l’indice epatosomatico (HSI) e l’indice gonadosomatico (GSI). I risultati di questa tesi hanno evidenziato differenze nei popolamenti, riconducibili alle diverse condizioni ambientali e alla pressione di pesca, tra le due aree indagate. L’area sud, interessata da un più intenso sforzo di pesca esercitato sui fondali della piattaforma e della scarpata continentale e da una morfologia del fondale differente, mostra una diversità in termini di crescita relativa e stato della “condizione”, che risulta più elevata in entrambe le specie, rispetto all’area settentrionale, caratterizzata invece da uno sforzo di pesca meno intenso, incentrato sull’ampia piattaforma continentale.

Gene expression responses to acute and chronic heat stress in the common reef-building coral Pocillopora verrucosa

Global climate change is impacting coral reefs worldwide, with approximately 19% of reefs being permanently degraded, 15% showing symptoms of imminent collapse, and 20% at risk of becoming critically affected in the next few decades. This alarming level of reef degradation is mainly due to an increase in frequency and intensity of natural and anthropogenic disturbances. Recent evidence has called into question whether corals have the capacity to acclimatize or adapt to climate changes and some groups of corals showed inherent physiological tolerance to environmental stressors. The aim of the present study was to evaluate mRNA expression patterns underlying differences in thermal tolerance in specimen of the common reef-building coral Pocillopora verrucosa collected at different locations in Bangka Island waters (North Sulawesi, Indonesia). Part of the experimental work was carried out at the CoralEye Reef Research Outpost (Bangka Island). This includes sampling of corals at selected sites and at different depths (3 and 12 m) as well as their experimental exposure to an increased water temperature under controlled conditions for 3 and 7 days. Levels of mRNAs encoding ATP synthase (ATPs) NADH dehydrogenase (NDH) and a 70kDa Heat Shock Protein (HSP70) were evaluated by quantitative real time PCR. Transcriptional profiles evaluated under field conditions suggested an adaptation to peculiar local environmental conditions in corals collected at different sites and at the low depth. Nevertheless, high–depth collected corals showed a less pronounced site-to-site separation suggesting more homogenous environmental conditions. Exposure to an elevated temperature under controlled conditions pointed out that corals adapted to the high depth are more sensitive to the effects of thermal stress, so that reacted to thermal challenge by significantly over-expressing the selected gene products. Being continuously exposed to fluctuating environmental conditions, low-depth adapted corals are more resilient to the stress stimulus, and indeed showed unaffected or down-regulated mRNA expression profiles. Overall these results highlight that transcriptional profiles of selected genes involved in cellular stress response are modulated by natural seasonal temperature changes in P. verrucosa. Moreover, specimens living in more variable habitats (low-depth) exhibit higher basal HSP70 mRNA levels, possibly enhancing physiological tolerance to environmental stressors.

Sclerocronologia di Chamelea gallina (Linnaeus, 1758) in Adriatico

La conoscenza dell’età e della crescita dei bivalvi è un requisito fondamentale per uno studio di dinamica di popolazione e per una gestione ottimale della risorsa. Lo scopo di questa tesi è quello di individuare una metodologia valida per riconoscere la precisa natura dell’origine dei microincrementi di crescita nel guscio di Chamelea gallina, riconoscendo le discontinuità annuali all’interno del guscio. Sono stati prelevati gusci di individui di C. gallina provenienti da un precedente campionamento nella baia della Vallugola il 06/09/2013. Le valve sono state incluse in resina epossidica e sezionate dall’umbone al margine centrale e levigate così da poter osservare le fettine ottenute allo stereomicroscopio a luce trasmessa. Il vantaggio riscontrato di questa metodologia è rappresentato dalla inaccessibilità delle strutture di deposizione alle perturbazioni ambientali anche se risulta impossibile annullare i possibili effetti dovuti all’attività di pesca o di altri eventi stressanti. Sono stati individuati solamente organismi con uno o due anni di età. I picchi di rallentamento della crescita sincronizzati in ogni classe di taglia potrebbero indicare tassi di accrescimento costanti entro le classi di taglia. Si può notare inoltre una certa ciclicità con cui si presentano i picchi di rallentamento della crescita. Essi si presentano circa ogni 10-12 crestine corrispondenti ipoteticamente ad una annualità. Un limite di questa metodica è dato dal fatto che si riesce a leggere l’informazione contenuta nel guscio ma, senza una validazione non è possibile associarla ad un preciso periodo di vita dell’organismo. A causa delle tempistiche limitate, non è stato possibile condurre esperimenti di crescita in campo per validare la periodicità delle micro linee di accrescimento ma sarebbe auspicabile in futuro riuscire a svolgere almeno una delle diverse metodiche di validazione.

Suitability of real-time quantitative PCR to estimate the relative telomere length in European Hake (Merluccius merluccius Linnaeus, 1758)

Telomere length measurement has been proposed as a promising tool to estimate the age of individuals in natural populations. We used real-time quantitative PCR (qPCR) to measure relative telomere length in four tissues (brain, kidney, liver and muscle) of European hake (Merluccius merluccius) in different groups based upon body length an otolith age estimate. We observed a high level of inter-individual differences in the measurements of relative telomere length in hakes of similar age and body length groups. The results of qPCR analysis showed a great variability in all measures and a lack of repeatability and reproducibility with significant statistical differences in the results of the different assays. The paper discusses the technical reasons for the variability in qPCR obtained in this work and by other authors.

Metabolic rates and tissue composition of the coral Pocillopora verrucosa over 12 latitudes in the Red Sea characterized by strong temperature and nutrient gradient

Global warming was reported to cause growth reductions in tropical shallow water corals in both, cooler and warmer, regions of the coral species range. This suggests regional adaptation with less heat-tolerant populations in cooler and more thermo-tolerant populations in warmer regions. Here, we investigated seasonal changes in the in situ metabolic performance of the widely distributed hermatypic coral Pocillopora verrucosa along 12 degrees latitudes featuring a steep temperature gradient between the northern (28.5 degrees N, 21-27 degrees C) and southern (16.5 degrees N, 28-33 degrees C) reaches of the Red Sea. Surprisingly, we found little indication for regional adaptation, but strong indications for high phenotypic plasticity: Calcification rates in two seasons (winter, summer) were found to be highest at 28-29 degrees C throughout all populations independent of their geographic location. Mucus release increased with temperature and nutrient supply, both being highest in the south. Genetic characterization of the coral host revealed low inter-regional variation and differences in the Symbiodinium clade composition only at the most northern and most southern region. This suggests variable acclimatization potential to ocean warming of coral populations across the Red Sea: high acclimatization potential in northern populations, but limited ability to cope with ocean warming in southern populations already existing at the upper thermal margin for corals