Research and development of hatchery techniques to optimise juvenile production of the edible Sea Urchin, Paracentrotus lividus

Research and development in aquaculture has supported the knowledge-based development of the sector over the last decades. In particular, species diversification is playing an important role to ensure sustainability of the industry and helping to reduce pressure on wild stocks of those aquatic species for which farming technology is still at the early stages. Due to the increasing pressures on more traditional carnivorous marine finfish species (aquafeed reliance on fishmeal and fish oil, environmental impact, market price) low trophic organisms are receiving more attention to provide sustainable alternatives and integrate production activities with the aim of reducing environmental impacts and to provide secondary high value crops. Integrated Multi-Trophic Aquaculture (IMTA) systems are therefore at the forefront of innovation in the industry. Several invertebrate species have been investigated and tested as integral part of IMTA (mussels, oysters, abalone and macroalgae) and echinoderms have also been considered as good candidates for the future development of this technology. In order to allow for a more widespread uptake of integrated aquaculture, several technical and biological challenges need to be overcome, including a reliable supply of juveniles. In recent years, this has prompted investigation on Echiniculture as a whole and on hatchery technologies in particular. This PhD investigated key constraints in edible sea urchin (Paracentrotus lividus) juvenile production with the aim to improve commercial sea urchin hatchery outputs. The research firstly focused on larval nutrition (Chapter 3 and 4) and specifically tested the hypothesis that larvae required higher dietary inputs of long chain fatty acids than those provided by Dunaliella tertiolecta, a microalgae species widely used in echinoderm larval rearing. Fatty acid composition of P. lividus eggs, investigated in Chapter 3, supported this hypothesis, which was further confirmed by the results obtained in Chapter 4 where microalgae (Cricosphaera elongata, Pleurochrisis carterae and Tetraselmis suecica) with a more balanced fatty acid profile, in particular richer in long chain fatty acids, were employed. This resulted in a significantly improved larval development and survival. Results also indicated that these alternative microalgae species could be successfully grown without modification of the microalgae production protocols in the hatchery where the experimentation had taken place. The third experimental chapter compared static and flow through systems which provides more stable water quality through constant water exchange and reduces larval handling and associated stress. Results indicated that larval survival was significantly improved by the flow-through system and the need for tank cleaning was reduced (three versus seven times per larval cycle when using flow-through and static rearing systems respectively). However, water quality, based on the parameters assessed (NH4, PO4-3, NO2 and NO3), did not show any significant differences between systems. Reduced handling could have therefore played the most important role in promoting larval survival. Both these trials resulted in a significant 5 to 20 % increased survival. A follow-up study, combining flow-through with more suitable microalgae, should be carried out and could result in even further enhanced survival. Then, chapters 6 and 7 focused on broodstock nutrition and subsequent improvement of gamete quantity and quality. These two trials aimed to explore and describe the biological effects that some important nutrients, such as proteins, lipids, fatty acids and carotenoids, have on urchins’ somatic and gonadal growth, gonad biochemical composition during gametogenesis, fecundity and maternal provisioning to developing embryos. Results from the experiment described in Chapter 6 indicated that higher protein content can improve somatic growth in P. lividus adults and that more expensive, protein-, lipid- and energy-rich diets do not significantly enhance fecundity or offspring performance. Results, moreover, highlighted the need for a specifically formulated broodstock diet and gave some insights into what its composition should be, especially in relation to carotenoids. In Chapter 7, fatty acid profiles of P. lividus gonads throughout gametogenesis were studied for the first time. It was observed that, among Long Chain Polyunsaturated Fatty Acids (LC-PUFAs), Eicosapentaenoic acid (EPA) and Docosahexaenoic acid (DHA) are primarily accumulated during gametogenesis, whilst Arachidonic acid (ARA) appears to be independent of dietary input. In addition, it was clearly shown that ARA is the only LC-PUFA accumulated in the eggs along with Non Methylene Interrupted Fatty Acids (NMI FAs). As well as looking at the biological effects of different diets on fatty acid profiles of gonadal and larval tissues, the work also expanded on a more fundamental level to explore the metabolic pathway through which precursors could be used by sea urchins for the endogenous production of long chain fatty acids (Chapter 8). Three Expressed Sequence Tags (ESTs) for putative fatty acyl desaturases, one of which was closely related to Octopus vulgaris ∆5-like fatty acyl desaturase, were identified. The newly cloned putative desaturase of P. lividus possessed all typical features of other fatty acyl desaturases. However, because of time constraints, functional characterisation, originally planned, of the new protein could not be performed and further research effort is needed to investigate this important aspect of sea urchin physiology. Overall, the aim of this research project has been achieved as it provided a set of exploitable results and protocols to improve hatchery practices for the production of P. lividus juvenile. However, more research is required to investigate some of the underlying mechanisms behind the observed biological effects such as delay in larval development when T. suecica was used as larval feed, increased broodstock fecundity, improved larval survival in the flow-through system and higher gonadal concentration of some fatty acids (mainly DHA) than provided in the feed

Combinatorial and supramolecular saproaches to monodentate phosphorus-ligands for transition metal catalyzed asymmetric reactions.

In recent years, monodentate phosphorus ligands (e.g. phosphites, phosphonites, phosphoramidites and phosphinamines) have held the stage in asymmetric catalysis.1 In addition to their outstanding activity and selectivity, comparable or even superior to those of bidentate ligands, the convenient, fast and practical preparation from commercially available materials underlines their potential for industrial applications. Furthermore, the modular nature of all these ligands allows the synthesis of a variety of representatives, thereby making a combinatorial approach possible. My PhD research project deals with the synthesis of libraries of new chiral monodentate P-ligands and their screening in asymmetric transition metal-catalyzed reactions through two innovative approaches: \uf05f\uf020combination of binaphthol-derived phosphites and C1-symmetric phosphinamines for the selective generation of heteroleptic catalysts in Rh- and Pd-mediated reactions (Ligand Combination Approach, Research line 1); \uf05f\uf020supramolecular ligand-ligand interactions for highly selective transition metal catalysis (Supramolecular Approach, Research line 2). Research line 1 In 2002-3, the groups of Reetz and Feringa independently used a binary mixture of chiral monodentate Pligands in several asymmetric rhodium-catalyzed reactions. By mixing two different ligands (La and Lb) in the presence of a transition metal [M], three species can be formed: [M]LaLa, [M]LbLb (homocomplexes) and [M]LaLb (heterocomplex). The heterocomplex is often more reactive and more (regio-, diastereo-, enantio-) selective than either of the two homocomplexes. Moreover, under thermodynamic control the heterocomplex : homocomplexes ratios usually exceed the statistical value (2 :1 :1). The ideal case would constitute an equilibrium completely in favor of the heterocomplex [M]LaLb, because a single well-defined catalyst would then exist in the reaction mixture and the undesired competition of the less selective homocomplexes would be avoided. We were intrigued by the remarkable selectivities reported for the mixtures of chiral ligands (binolderived phosphites, phosphonites and phosphoramidites) with achiral phosphines.4 In particular, the 1:1 mixture of a chiral phosphite with an achiral phosphine was reported to induce reversal of the enantioselectivity in the Rh-catalysed hydrogenation of N-acetamido acrylate (compared to the chiral phosphite alone). The only possible explanation for this peculiar behaviour is the selective formation of the phosphite-phosphine Rh-heterocomplex, favoured by electronically matching one \uf073-donor ligand (phosphine) and one \uf070-acceptor ligand (phosphite). As enantiomerically pure chiral phosphines are not easy to synthesize, we were wondering whether the phosphine ligands could be substituted by other \uf073- donor phosphorus ligands still retaining the thermodynamic preference for the formation of the heterocomplex. For this reason, we turned our attention to chiral phosphinamines, which are easy to prepare enantiomerically pure, and have electronic properties similar to those of phosphines.5 DFT calculations showed that the phosphite-phosphinamine rhodium heterocomplex is more stable than the two homocomplexes by 11.29 kcal/mol We synthesized a library of monodentate chiral phosphites 1a-d by reacting enantiopure BINOL-PCl with different chiral and achiral alcohols. Monodentate chiral phosphinamines 2a-e were prepared by reaction of Ph2PCl with a number of C2-symmetric secondary amines and C1-symmetric secondary and primary amines. Complexation studies were performed by means of 31P-NMR, using Rh(acac)(C2H4)2 as the rhodium source. When C1-symmetric phosphinamine ligands were employed, the cis-heterocomplexes were formed with selectivity ranging from moderate (70%) to excellent (_ 99%). The homo- and heterocombinations of phosphites and C1-symmetric phosphinamines were then screened in the rhodium-catalyzed hydrogenation of methyl 2-acetamidoacrylate. Remarkably, the 1:1 combination of a BINOL-derived phosphite and a phosphinamine induced reversal of the enantioselectivity, compared both to the phosphite and the phosphinamine alone. This heterocombination induced a peculiar stereochemical outcome also in the palladium-catalyzed asymmetric allylic substitution of rac-1,3- The heterocomplexes formed in this way are expected to have reduced degrees of freedom7 compared to the complexes of normal monodentate ligands, and thus supramolecular ligands somehow resemble traditional bidentate ligands. According to this analogy, they are often referred to as supramolecular bidentate ligands or self-assembled ligands, thanks to their ability to spontaneously form bidentate systems in solution. These terms also apply to those supramolecular ligands that are only capable of noncomplementary interactions: indeed these systems are still capable of forming rigid and conformationally restricted complexes, although they cannot selectively form heterocomplexes when used in a mixture, which quite reduces their "combinatorial appeal". Prompted by the studies of Prof. Reek and co-workers on the synthesis of supramolecular bidentate heterocomplexes through complementary interactions (e.g. coordinative bondings)8 and by the efficient resolution strategy of racemic N-benzyl _-amino acids (N-Bn-AA) by liquid-liquid extraction using a chiral salen\u2013cobalt(III) complex as enantioselective receptor, accomplished in our laboratories, we decided to use the salen-cobalt(III)-N-benzyl-L-serine complex as a chiral platform for the preparation of new families of supramolecular mono- and bi-dentate P-ligands (Scheme 4 A and B). The salen-cobalt(III)-N-Bn-AA complexes, in fact, possess a rigid framework with the salen ligand in a cis-_-folded arrangement around the octahedral cobalt ion. The remaining two cis coordination sites are occupied by the N-benzyl _-amino acid, which is thus accommodated in the \u201cbinding pocket\u201d of the chiral cobalt complex. 9b Ligands 3 were synthesized in good yields starting from the symmetrical tetra-t-butyl-salen which was transformed into the corresponding cobalt(III) acetate complex, and the acetate ion was exchanged with Nbenzyl- L-serine. In the case of symmetric salen backbone, the complex was obtained pure in high yield and the phosphite moieties were then introduced by reaction with different diol-derived chlorophosphite. In the case of the supramolecular bidentate ligands 4, the unsymmetrical hydroxymethyl-containing salen afforded the corresponding cobalt(III) acetate complex as a mixture of two inseparable diastereoisomers. Complexation of monodentate ligands 3 to rhodium(I) was studied by 31P-NMR spectroscopy, which showed the formation of the desired Rh-complexes. The reactivity of supramolecular monodentate Pligands was investigated in two Pd-catalyzed-allylic alkylation on (E)-1,3-diphenylallyl acetate and Pdcatalized desymmetrization of meso-cyclopenten-2-ene-1,4-diol biscarbamate. We are now exploring new synthetic pathways to obtain the unsymmetrical (S,S)-salen-ligand and the corresponding unsymmetrical (S,S)-salen-cobalt(III)-N-Bn-L-serine bidentate P-ligand in a pure diastereoisomeric fashion. We have also investigated the design and synthesis of a novel class of chiral monodentate phosphite ligands, named PhthalaPhos, which contain a phthalic acid primary diamide moiety. Such phthalamidic group displays both donor and acceptor hydrogen bonding properties that, in principle, can give rise to supramolecular interactions both between the ligands and with the reaction substrate The pre-catalytic Rh complex of one of these ligands was fully characterized and studied by NMR, IR and mass spectroscopy, which confirmed the presence of hydrogen bonds between the coordinated ligands, and the formation of a supramolecular bidentate ligand. The catalytic properties of these new ligands were assessed in the rhodium-catalyzed hydrogenation of benchmark olefins (e.g. methyl 2-acetoamidoacrylate and N-(1-phenylvinyl)acetamide), taking the known phosphite 9 as a touchstone. Four ligands gave e.e. values higher than 97% with methyl 2-acetoamidoacrylate as substrate, and six reached the same level of performance with N-(1-phenylvinyl)acetamide. Remarkably, the reference ligand 9, featuring the same BINOL phosphite moiety, gave only 84% and 90% e.e. respectively for the same substrates, thus suggesting that the phthalimide residue significantly influences the catalytic properties of these ligands. Evaluation of the catalytic properties of the Phthalaphos ligands were also extended to the Rh-catalyzed hydrogenation of more challenging substrates of potential industrial interest, such as N-(3,4-dihydronaphthalen-1-yl)acetamide and (E)-methyl 2-(acetamidomethyl)-3-phenylacrylate. The results of this screening were quite variegated both in terms of activity and enantioselectivity, but the employment of a Phthalaphos ligand gave the highest e.e. value ever obtained on N-(3,4-dihydronaphthalen-1-yl)acetamide with phosphite ligands and the highest e.e. value ever obtained with (E)-methyl 2-(acetamidomethyl)-3-phenylacrylate

The new advanced Cities From the Green and Digital to the Smart Cities

The city has always brought within itself the characteristics of the historical period in which it existed and of the population that inhabited it. In the last years new kind of cities were born, some of them are called green, some others digital and then some are the Smart Cities. The latter has the same features of the first two cities, but them are also committed into satisfying the expectations of its population and caring about citizens’ welfare first. The Smart Cities are the result of technological progress and the search for environmental sustainability. The city was born as a consequence, almost obvious, of the improvement of human skills: many nearby houses represented a possibility of bringing together resources and abilities in an almost uninhabited land. Small villages have become towns, cities and metropolitan cities in which all the technological discoveries were applied and made concrete in the services that these cities could then offer to their inhabitants. The cities, and its various existing types, up to the green cities and digital cities and finally the Smart Cities will be described, as the apex of the discoveries of the digital and sustainable people, who respects the environment in which they live, indeed, which regenerates the environment from past pollution. In these cities, people live within a virtual environment too. These cities are in continuous evolution, extremely rapid, in a competition to each other, made up of skills and technological discoveries, which contain within their borders innovative services for all of its inhabitants, and all its stakeholders. This paper discovers the way of the first inhabited centers towards Smart Cities

Itv as a community-to-community collaborative system

Web-forums and Instant Messengers provide a unique opportunity for the users to create and support spontaneous communities. However, such systems tend to focus on the one-to-one and one-to-many approaches. Community-to- Community interaction, described as two groups of people which communicate by means of a network, is still missing. This work describes early implementation of an interactive television (iTV) chat system, which dynamically creates chat rooms where users can discuss, share their TV experience and access web resources.337-33

Stereoscopic Estimation of Volcanic Ash Cloud-Top Height from Two Geostationary Satellites

The characterization of volcanic ash clouds released into the atmosphere during explosive eruptions includes cloud height as a fundamental physical parameter. A novel application is proposed of a method based on parallax data acquired from two geostationary instruments for estimating ash cloud-top height (ACTH). An improved version of the method with a detailed discussion of height retrieval accuracy was applied to estimate ACTH from two datasets acquired by two satellites in favorable positions to fully exploit the parallax effect. A combination of MSG SEVIRI (HRV band; 1000 m nadir spatial resolution, 5 min temporal resolution) and Meteosat-7 MVIRI (VIS band, 2500 m nadir spatial resolution, 30 min temporal resolution) was implemented. Since MVIRI does not acquire data at exactly the same time as SEVIRI, a correction procedure enables compensation for wind advection in the atmosphere. The method was applied to the Mt. Etna, Sicily, Italy, eruption of 23 November 2013. The height of the volcanic cloud was tracked with a top height of ~8.5 km. The ash cloud estimate was applied to the visible channels to show the potential accuracy that will soon be achievable also in the infrared range using the next generation of multispectral imagers. The new constellation of geostationary meteorological satellites will enable full exploitation of this technique for continuous global ACTH monitoring

The use of biorefinery by-products and natural detritus as feed sources for Oysters (Crassostrea gigas) juveniles

New research is currently underway to explore the potential of macroalgae for the production of biofuels. Marine biofuels in general and macroalgae in particular, offer a number of advantages over terrestrial biofuels including reduced competition for freshwater resources and for land use. Sugars can be extracted from macroalgae and processed into biofuels by anaerobic digestion and fermentation. This process generates significant waste biomass, which, if used, could improve the economic sustainability of the biorefinery sector. Bivalves’ aquaculture relies heavily on the production of unicellular algae to feed juvenile individuals and this can represent a bottleneck for the bivalve industry especially in locations where sunlight is limited. Previous research explored the use of macroalgae derived digestate as alternative or integrative feed for juvenile bivalves, exploiting the notion that organic particulate matter (detritus) is an integral part of this animal class natural diet. The prospect of using waste products from the emerging biorefinery industry to solve a bottleneck for aquaculture businesses and, by so doing, improving profitability of both, is an exciting one. In this paper we describe the main nutritional profiles (Protein, Lipid, Carbohydrates and Fatty acids) of the tested diets and investigate the potential for the use of a biorefinery a by-product as replacement option for bivalves’ production, by benchmarking it against aquaculture industry standards (live microalgae and commercially available algae paste) and natural detritus constituted by farmed sea urchin digesta. Both the digestate and the natural detritus supported the survival and growth of bivalve spat, especially when used at 50% inclusion rate, over the course of 4-week preliminary trials. Data suggest that a synergistic effect of the nutritional profiles of the diets employed may underpin the observed results

Reproductive performance and offspring quality of non-ablated Pacific white shrimp (Litopenaeus vannamei) under intensive commercial scale conditions

This study evaluated reproductive performance of non-ablated Litopenaeus vannamei and the quality of their offspring under commercial conditions. Five tanks were stocked with non-ablated female and other five with ablated individuals as control. Two different larval rearing trials (Larviculture I and II) have been conducted. Six larviculture tanks (n = 3) were used on the first trial (LI) and ten for the second one (LII) (n = 3). Postlarvae from LII were used for nursery and grow-out. Spawning event and hatching rate per day were similar between both treatments. Mating success, mortality of female and number of eggs and nauplii per tank per day of non-ablated group were significantly lower than ablated female. Non-ablated female fecundity (number of eggs and nauplii per spawned female per day) was significantly higher than control. There was no significant difference between daily larval stage index of larvae in LI and LII. The response to the salinity stress test, and final survival and weight in LI was similar. However in LII, postlarvae derived from non-ablated had significantly higher survival to salinity stress test. Identical survival, final weight, weekly growth, feed conversion rate and yield were observed in nursery. The same was observed in grow-out, including weight gain and specific growth rate. Overall this study demonstrates that non-ablated females can have comparable level of productivity to ablated females in intensive commercial hatchery conditions. Their offspring perform comparably in all culture stages with evidence of enhanced resistance to stress in larvae derived from non-ablated female broodstock

Effects of larval diet and metamorphosis cue on survival and growth of sea urchin post-larvae (Paracentrotus lividus; Lamarck, 1816)

In this study, we present the results of two experiments; in the first one we evaluated the effects of four larval dietary treatments on the survival and growth of the sea urchin Paracentrotus lividus, larvae and post-larvae. In the second experiment we have measured the effects of two different settlement substrates, combined with the presence of conspecifics, on metamorphosis, survival and growth of post-larvae. The microalgae dietary treatments consisted in: Dunaliella tertiolecta (Duna); 50% mixture of Isochrysis galbana and D. tertiolecta (ID); 50% mixture of Chaetoceros gracilis and D. tertiolecta (CD); 33% mixture of I. galbana, C. gracilis and D. tertiolecta (ICD). Although all dietary treatments resulted in a good survival at competence, significant difference in post-larval survival was observed between treatments, and indeed, only larvae fed Duna and CD survived to 180 days post settlement (DPS).  In the second experiment, the settlement substrates consisted in a film of cultured Ulvella lens or a naturally developing biofilm of diatoms, and the employed rearing water was either natural seawater or seawater previously exposed to P. lividus adults. At 10 DPS, larger (p<0.05) post-larvae were observed in the natural biofilm treatment, whilst the presence of conspecifics significantly increased larval settlement in both substrates (p<0.01).  These results indicate that it is important to consider the survival of post-larvae and juveniles to establish the efficiency of the dietary treatment on the hatchery production of P. lividus. Furthermore, it suggests that improved settlement protocols, such as the use of conspecifics, could contribute to increase hatchery outputs. Finally, it confirms the suitability of U. lens as settlement cue but also highlights that further research is required to establish its effectiveness for post-larvae first feedin

New diagnostic SNP molecular markers for the Mytilus species complex

The development of diagnostic markers has been a long-standing interest of population geneticists as it allows clarification of taxonomic uncertainties. Historically, there has been much debate on the taxonomic status of species belonging to the Mytilus species complex (M. edulis, M. galloprovincialis and M. trossulus), and whether they are discrete species. We analysed reference pure specimens of M. edulis, M. galloprovincialis and M. trossulus, using Restriction site associated DNA (RAD) sequencing and identified over 6,000 SNP markers separating the three species unambiguously. We developed a panel of diagnostic SNP markers for the genotyping of Mytilus species complex as well as the identification of hybrids and interspecies introgression events in Mytilus species. We validated a panel of twelve diagnostic SNP markers which can be used for species genotyping. Being able to accurately identify species and hybrids within the Mytilus species complex is important for the selective mussel stock management, the exclusion of invasive species, basic physiology and bio-diversity studies

A Retrospective Cohort Study of Traumatic Root Fractures in Primary Dentition: Can Splinting Type Improve Therapeutic Outcomes?

(1) Introduction: Deciduous traumatic dental injuries pose a serious global health concern. Root fractures show an incidence rate of approximately 2%; however, the literature is limited regarding the appropriate treatment and prognosis of affected teeth. This retrospective study aims to analyze the long-term outcomes of orthodontic splinting using brackets compared with composite resin-only splinting in two homogeneous samples affected by root fractures. The study also examines the onset and patterns of root resorption. (2) Methods: The first group included 25 patients with 27 deciduous upper incisors affected by root fracture; Group 2 consisted of 35 patients with 38 root fractures of maxillary deciduous teeth. The categorical data for both groups were analyzed using the chi-squared homogeneity test. Age groups were compared using the Mann–Whitney U test; p < 0.005 was considered statistically significant. (3) Results: In both groups, the male gender predominated similarly. In Group 1, early decidual loss occurred in 16% of cases, whereas in Group 2 it occurred in 51% of cases. Pulp canal obliteration was observed in 68% of deciduous teeth in Group 1, compared with 30% in Group 2. No complications affecting permanent teeth were noted in Group 1; in Group 2, 29% developed enamel dysplasia and 26% experienced delayed eruption, with statistically significant differences in these proportions. Apical fragment resorption was observed in 92% of cases in Group 1 and 30% in Group 2. (4) Conclusions: The treatment of deciduous tooth root fractures using orthodontic splints can yield significant benefits, such as reduction of early tooth loss. Furthermore, the study confirms that early resorption of the distal fragment of fractured roots is a common physiological phenomenon in primary teeth and typically occurs within a year following trauma