R-spondin 1/Dickkopf-1/beta-catenin machinery is involved in testicular embryonic angiogenesis

Testicular vasculogenesis is one of the key processes regulating male gonad morphogenesis. The knowledge of the molecular cues underlining this phenomenon is one of today's most challenging issues and could represent a major contribution toward a better understanding of the onset of testicular morphogenetic disorders. R-spondin 1 has been clearly established as a candidate for mammalian ovary determination. Conversely, very little information is available on the expression and role of R-spondin 1 during testicular morphogenesis. This study aims to clarify the distribution pattern of R-spondin 1 and other partners of its machinery during the entire period of testicular morphogenesis and to indicate the role of this system in testicular development. Our whole mount immunofluorescence results clearly demonstrate that R-spondin 1 is always detectable in the testicular coelomic partition, where testicular vasculature is organized, while Dickkopf-1 is never detectable in this area. Moreover, organ culture experiments of embryonic male UGRs demonstrated that Dickkopf-1 acted as an inhibitor of testis vasculature formation. Consistent with this observation, real-time PCR analyses demonstrated that DKK1 is able to slightly but significantly decrease the expression level of the endothelial marker Pecam1. The latter experiments allowed us to observe that DKK1 administration also perturbs the expression level of the Pdgf-b chain, which is consistent with some authors' observations relating this factor with prenatal testicular patterning and angiogenesis. Interestingly, the DKK1 induced inhibition of testicular angiogenesis was rescued by the co-administration of R-spondin 1. In addition, R-spondin 1 alone was sufficient to enhance, in culture, testicular angiogenesis

Distribution of the Mediterranean ribbed limpet Patella ferruginea Gmelin, 1791 along the Ligurian coast and implications for conservation actions

Patella ferruginea is a limpet endemic to the Western Mediterranean Sea. It is presently considered the most threatened marine macroinvertebrate in the region and has been included in several international conservation directives. Its populations were widespread throughout the Western Mediterranean in the late Pleistocene period, and remained broadly distributed until the 19th century. Presently this species is confined into small populations in a few restricted areas due to human harvesting for food and baits, construction of coastal infrastructures and the effects of seawater pollution. In particular, the species is reported as presently disappeared from the whole of the Italian continental coast and measures are in progress to reintroduce the species through translocation and reproduction in controlled conditions along the Ligurian coasts of the Northwestern Mediterranean.Recent surveys implemented in the framework of the present work along the Ligurian coasts, to assess the most suitable sites for reintroduction, resulted in the discovery of 32 specimens of this endemic limpet, which previously was thought to have vanished from the area. These findings shed new light on the ability of species to naturally disperse, the relevance of the measures set in place to restore presently rarefied populations and may provide information to aid in the selection and management of sites within the Natura 2000 Ecological network

Implementation of controlled reproduction techniques on marine invertebrates: gastropod molluscs of Patella genus Sperimentazione di tecniche di riproduzione controllata di invertebrati marini: Molluschi gasteropodi, Genere Patella

Il progetto di dottorato \ue8 stato principalmente focalizzato sull\u2019implementazione delle tecniche di riproduzione controllata nel genere Patella. Le specie oggetto di studio sono state P. caerulea e pi\uf9 in particolare P. ferruginea, specie protetta e in via di estinzione, il cui ripopolamento \ue8 oggetto del progetto europeo ReLife (LIFE15 NAT/IT/000771). Essendo P. ferruginea una specie protetta, inizialmente abbiamo preferito testare i trattamenti di induzione non letale su una specie cogenerica, P. caerulea, comune e abbondante lungo le coste italiane. I vari trattamenti testati hanno dato ottimi risultati, e il trattamento del \u201cbubbling\u201d \ue8 risultato il migliore, sia in termini di tempo di risposta alla stimolazione degli esemplari, sia perch\ue9 si tratta di un metodo poco costoso e facilmente ripetibile. \uc8 stato possibile stilare un protocollo di spawning non letale, ed effettuare una fecondazione artificiale con successivo sviluppo larvale e insediamento dei giovanili (Ferranti et al., 2018). Inoltre, sono stati condotti dei monitoraggi lungo la costa ligure, che hanno permesso di riportare la presenza di esemplari di P. ferruginea, considerata estinta lungo queste coste (Espinosa et al., 2014). La distribuzione di P. ferruginea, lungo le coste liguri, \ue8 estremamente scarsa e frammentata, infatti la specie \ue8 stata rinvenuta come esemplari isolati o piccoli gruppi, come nell'AMP delle Cinque Terre. Questi ritrovamenti hanno per\uf2 permesso di ampliare la mappatura della presenza di P. ferruginea lungo le coste del Mediterraneo (Ferranti et al., 2019). Inoltre, per comprendere meglio il ciclo riproduttivo e i tempi di rilascio dei gameti in P. ferruginea alle nostre latitudini, abbiamo posizionato degli esemplari in mare per la maturazione in condizioni naturali, fino all\u2019inizio del periodo riproduttivo. Gli esemplari successivamente sono stati sottoposti a biopsia della gonade sia prima di essere messi in mare per determinare il sesso (Guallart et al., 2013a), che all'inizio del periodo riproduttivo previsto per comprendere lo stadio di maturazione. L\u2019osservazione del materiale ottenuto tramite biopsia ha permesso di determinare che gli esemplari avevano raggiunto la maturazione gonadica e che uno di loro aveva effettuato il cambio di sesso, evento noto in letteratura, ma osservato qui per la prima volta alla latitudine del Mediterraneo nord-occidentale (Ferranti et al., 2021). Tali risultati implicano che la specie pu\uf2 completare il suo ciclo riproduttivo lungo le coste liguri. Sempre al fine di comprendere meglio i tempi di maturazione delle gonadi, abbiamo applicato le tecniche della Risonanza Magnetica su esemplari di patelle. La Risonanza Magnetica, attraverso un approccio non invasivo, ha permesso di osservare lo spessore della gonade e anche di capire se un esemplare avesse rilasciato o meno gameti, al fine di applicare eventualmente tecniche di stimolazione al rilascio dei gameti solo al momento giusto. I risultati indicano che la risonanza magnetica pu\uf2 essere molto utile come strumento aggiuntivo, sia per valutare la maturit\ue0 sessuale di P. ferruginea, che per ridurre al minimo il numero di prove di induzione per ogni esemplare, e di conseguenza lo stress (Guallart et al., 2020). Ancora pi\uf9 rilevante \ue8 l'aver fornito l\u2019evidenza, per la prima volta, che l\u2019emissione delle uova pu\uf2 essere indotta in P. ferruginea e dimostrare che \ue8 possibile ottenere giovanili, in condizioni controllate, attraverso metodi non invasivi. Questi risultati hanno permesso di elaborare un protocollo preliminare sull'induzione all\u2019emissione spontanea dei gameti da parte di P. ferruginea, senza sacrificare i riproduttori di una specie protetta. Inoltre, questo ci ha permesso di effettuare una fecondazione artificiale che ha portato allo sviluppo, insediamento, metamorfosi e crescita larvale di un numero piuttosto elevato di individui giovanili di P. ferruginea (Ferranti et al., in prep.). Di conseguenza, sapendo che la specie \ue8 presente lungo la costa ligure, che riesce a raggiungere la maturit\ue0 anche a queste latitudini e che \ue8 possibile riprodurre P. ferruginea in condizioni controllate fino allo stadio giovanile, possiamo affermare che esiste la possibilit\ue0 di ripopolamento e reintroduzione in ambiente naturale di esemplari di P. ferruginea, recuperando questa specie nell\u2019area in oggetto, come previsto dal progetto ReLife. Inoltre, la reintroduzione realizzata da esemplari ottenuti mediante tecniche di acquacoltura, senza influenzare sostanzialmente le popolazioni donatrici, \ue8 anche in accordo con la strategia spagnola per la conservazione della specie (MMAMRM, 2008), e in generale, le azioni di ripopolamento attraverso la riproduzione controllata, potrebbero consentire in futuro di ripopolare altre aree costiere, dove le popolazioni di P. ferruginea sono scomparse e/o in regressione, per un recupero della specie a livello globale.The PhD project was mainly focused on the implementation of controlled reproduction techniques in the Patella genus. The species considered were P. caerulea and more particularly P. ferruginea, a protected and endangered species, whose repopulation is the subject of the European ReLife project (LIFE15 NAT/IT/000771). Being P. ferruginea a protected species, we initially preferred to test the non-lethal induction treatments on a cogeneric species, P. caerulea, common and abundant along the Italian coasts. The various treatments tested have provided excellent results, and the \u201cbubbling\u201d treatment was the best, both in terms of time of response to stimulation of the specimens and of replicability and ease of use. The results obtained made it possible to draw up a non-lethal spawning protocol, and to carry out artificial fertilization with subsequent larval development and settlement of the juveniles (Ferranti et al., 2018). In addition, monitoring was carried out along the Ligurian coast, which allowed reporting the presence of P. ferruginea, considered extinct along these coasts (Espinosa et al., 2014). P. ferruginea distribution, along the Ligurian coasts, is extremely scarce and fragmented; in fact the species has been found as isolated specimens or small groups, such as in the Cinque Terre MPA. However, these findings permitted to expand the mapping of the presence of P. ferruginea along the Mediterranean coasts (Ferranti et al., 2019). Furthermore, to better understand the reproductive cycle and the timing of P. ferruginea natural spawning at our latitudes, we placed specimens at sea for maturation in natural conditions, up to the beginning of the expected reproductive period. Specimens were later subjected to a biopsy of the gonad, both before being placed in the sea to determine sex (Guallart et al., 2013a), and at the beginning of the expected reproductive period to understand the stage of maturation. The observation of the material obtained through biopsy made it possible to determine that the specimens had reached gonadal maturation and that one of them had performed sex change, an event known in the literature, but observed here for the first time at the north-western Mediterranean latitude (Ferranti et al., 2021). This implies that the species can complete its reproductive cycle along the Ligurian coasts. Always in view of a better understanding of the timing of gonad maturation, we applied the Magnetic Resonance Imaging (MRI) techniques on limpets. MRI, through a non-invasive approach, allowed to observe the thickness of the gonad and also to understand whether or not a specimen had released gametes, in order to eventually apply stimulation techniques to spawning only at the right time, reducing stress on specimens. The results indicated that MRI can be very useful as an additional tool, both in evaluating the sexual maturity of P. ferruginea and in minimizing the number of induction trials for each specimen, and consequently the stress (Guallart et al., 2020). Even more relevant is having provided evidence, for the first time, that spawning can be induced in P. ferruginea and demonstrated the feasibility of obtaining juveniles, under controlled conditions through low-invasive methods. These results allowed drawing up a preliminary protocol on the induction of spawning of P. ferruginea, which allows for the spontaneous release of gametes, without sacrificing the broodstock of a protected species. Furthermore, this allowed us to carry out an artificial fertilization that led to larval development, settlement, metamorphosis and growth of a pretty large number of P. ferruginea juveniles (Ferranti et al., in prep.). Consequently, knowing that the species is present along the Ligurian coast, that it manages to reach maturity even at these latitudes, and that it is possible to reproduce P. ferruginea under controlled conditions until it reaches the juvenile stage, we provide evidence of the feasibility of P. ferruginea restocking and reintroduction in the natural environment, recovering this species in this area, as expected from the ReLife project. Moreover, the reintroduction made from specimens obtained through aquaculture techniques, without substantially affecting donor populations, is also in accordance with the Spanish strategy for the conservation of the species (MMAMRM, 2008), and generally, repopulation actions through controlled reproduction, could allow in the future to repopulate other coastal areas, where populations of P. ferruginea are disappeared and/or in regression, for the recovery of the species at a global level

Artificial reproduction protocol, from spawning to metamorphosis, through noninvasive methods in Patella caerulea Linnaeus, 1758

Controlled reproduction is a requirement for developing effective mollusc cultivation for commercial or restoration purposes. In this study, a protocol for spawning induc- tion using noninvasive methods in limpets was developed, using the common Mediterranean species, Patella caerulea Linnaeus, 1758. Six nonlethal spawning induction treatments were tested: three chemical (two concentrations of H2O2 and KCl) and three physical (bubbling, warm and cold thermal shock). All treatments, except thermal shocks, induced the spawning of fertile gametes. Bubbling resulted the best treatment in providing spawning response, being the easiest and least inva- sive method tested. After eggs fertilization, larval development was followed until metamorphosis, testing fed and unfed conditions. Settlement took place after 7 days. The developed protocol represents a benchmark for further application to other limpets, for aquaculture or repopulation

Effect of temperature and duration of immersion on the stability of prepared feeds in echinoculture

The aim of this study was to provide new insights into the stability in water of the prepared feeds for echinoculture. The stability tests of four prepared feeds were carried out in laboratory in two different trials corresponding to different temperature levels (24°C and 19°C) and four different times of immersion (up to 72 h). Temperature and duration of immersion significantly affected the stability of feeds. Feed 1 was the most stable whereas Feed 3 was the least stable feed, as it started to dissolve in a few hours. Feed 2 and Feed 4 remained stable at 19°C all along the trial, whereas at 24°C remained intact for only 30 h. To optimize the echinoculture practice at temperature >19°C, we recommend a feeding regime at least 3 times a week, whereas at lower temperature we suggest an intermittent feeding regime

Mathematical simulation of the suspended solids diffusion during dredging operations on the continental shelf off the coast of Lazio (Central Tyrrhenian Sea, Italy)

Artificial nourishmentisapreferredstrategytoremedycoastalerosionalongasandycoast.Toconducta pilot studyontheenvironmentalimpactoftheuseofsubmergedsandydepositstoartificially nourish beaches, oceanographicandsedimentologicalstudieshavebeenperformedinpotentialdredgingsites