Effects of habitat characteristics on cryptic fish assemblages

Habitat structure is known to influence the abundance of fishes on temperate reefs. Biotic interactions play a major role in determining the distribution and abundance of species. The significance of these forces in affecting the abundance of fishes may hinge on the presence of organisms that either create or alter habitat. On temperate reefs, for example, macroalgae are considered autogenic ecosystem engineers because they control resource availability to other species through their physical structure and provide much of the structure used by fish. On both coral and temperate reefs, small cryptic reef fishes may comprise up to half of the fish numbers and constitute a diverse community containing many specialized species. Small cryptic fishes (<100 mm total length) may be responsible for the passage of 57% of the energy flow and constitute ca. 35% of the overall reef fish biomass on coral reefs. These benthic fish exploit restricted habitats where food and shelter are obtained in, or in relation to, conditions of substrate complexity and/or restricted living space. A range of mechanisms has been proposed to account for the diversity and the abundance of small fishes: (1) lifehistory strategies that promote short generation times, (2) habitat associations and behaviour that reduce predation and (3) resource partitioning that allows small species to coexist with larger competitors. Despite their abundance and potential importance within reef systems, little is known of the community ecology of cryptic fishes. Specifically on habitat associations many theories suggested a not clear direction on this subject. My research contributes to the development of marine fish ecology by addressing the effects of habitat characteristics upon distribution of cryptobenthic fish assemblages. My focus was on the important shallow, coastal ecosystems that often serve as nursery habitat for many fish and where different type of habitat is likely to both play important roles in organism distribution and survival. My research included three related studies: (1) identification of structuring forces on cryptic fish assemblages, such as physical and biological forcing; (2) macroalgae as potential tools for cryptic fish and identification of different habitat feature that could explain cryptic fish assemblages distribution; (3) canopy formers loss: consequences on cryptic fish and relationship with benthos modifications. I found that: (1) cryptic fish assemblages differ between landward and seaward sides of coastal breakwaters in Adriatic Sea. These differences are explained by 50% of the habitat characteristics on two sides, mainly due to presence of the Codium fragile, sand and oyster assemblages. Microhabitat structure influence cryptic fish assemblages. (2) Different habitat support different cryptic fish assemblages. High heterogeneity on benthic assemblages reflect different fish assemblages. Biogenic components that explain different and diverse cryptic fish assemblages are: anemonia bed, mussel bed, macroalgal stands and Cystoseira barbata, as canopy formers. (3) Canopy forming loss is not relevant in structuring directly cryptic fish assemblages. A removal of canopy forming algae did not affect the structure of cryptic fish assemblages. Canopy formers algae on Conero cliff, does not seem to act as structuring force, probably due to its regressive status. In conclusion, cryptic fish have been shown to have species-specific associations with habitat features relating to the biological and non biological components afforded by fish. Canopy formers algae do not explain cryptic fish assemblages distribution and the results of this study and information from the literature (both from the Mediterranean Sea and elsewhere) show that there are no univocal responses of fish assemblages. Further exanimations on an non regressive status of Cystoseira canopy habitat are needed to define and evaluate the relationship between canopy formers and fish on Mediterranean sea.

Fine tuning of on-growing diets for farmed marine fish species as a key tool to sustain the aquaculture development

The aim of this thesis was to investigate some important key factors able to promote the prospected growth of the aquaculture sector. The limited availability of fishmeal and fish oil led the attention of the aquafeed industry to reduce the dependency on marine raw materials in favor of vegetable ingredients. In Chapter 2, we reported the effects of fishmeal replacement by a mixture of plant proteins in turbot (Psetta maxima L.) juveniles. At the end of the trial, it was found that over the 15% plant protein inclusion can cause stress and exert negative effects on growth performance and welfare. Climate change aroused the attention of the aquafeed industry toward the production of specific diets capable to counteract high temperatures. In Chapter 3, we investigated the most suitable dietary lipid level for gilthead seabream (Sparus aurata L.) reared at Mediterranean summer temperature. In this trial, it was highlighted that 18% dietary lipid allows a protein sparing effect, thus making the farming of this species economically and environmentally more sustainable. The introduction of new farmed fish species makes necessary the development of new species-specific diets. In Chapter 4, we assessed growth response and feed utilization of common sole (Solea solea L.) juveniles fed graded dietary lipid levels. At the end of the trial, it was found that increasing dietary lipids over 8% led to a substantial decline in growth performance and feed utilization indices. In Chapter 5, we investigated the suitability of mussel meal as alternative ingredient in diets for common sole juveniles. Mussel meal proved to be a very effective alternative ingredient for enhancing growth performance, feed palatability and feed utilization in sole irrespectively to the tested inclusion levels. This thesis highlighted the importance of formulating more specific diets in order to support the aquaculture growth in a sustainable way.

Effects of allelopathic compounds produced by native and invasive marine macroalgae on the associated communities

In this study, the production of bioactive secondary metabolites called "allelochemicals" by algae has been investigated, specifically focusing on polyunsaturated aldehydes (PUAs). PUAs are known to have adverse effects on planktonic grazers and on phytoplankton; however, their effect on benthic communities has been poorly studied. Macroalgae are ecosystem engineers that play an important role in the structure of the habitat and associated communities, presenting a great variability in their morphology and structural complexity, which is a primary factor in the structuring of associated communities. In recent decades, it has been seen how the introduction of invasive species can modify the benthic habitat structure, causing cascading effects on the trophic chain. The thesis includes several field and laboratory studies. Field studies examined aldehyde production by native and invasive macroalgal species (Sargassum muticum, in the Adriatic Sea, and Rugulopterix okamurae in the Strait of Gibraltar), their structural complexity, together with their associated phyto and meiobenthos. Two laboratory studies were conducted. The first one, based on microcosms experiments, evaluated the effect of PUA (produced by the diatom Skeletonema marinoi, or as decadienal analytical standard) on meiofauna. The second one evaluated the inhibitory effect of dilkamural, an allelopathic compound isolated from R. okamurae, on unicellular phototrophs. Our results showed that PUAs produced by macroalgae were species-specific and had a significant impact on the benthic community. The morphology of macroalgae was an important factor in shaping associated communities, particularly for microphytobenthos. Invasive species, such as S. muticum and R. okamurae, could reduce the biodiversity of native benthic communities and simplify the habitat. Dilkamural was hypothesized to be an allelochemical defense, and laboratory toxicity tests confirmed this hypothesis. Overall, this thesis sheds light on the importance of allelochemicals and macroalgal structural complexity in the benthic environment and highlights the potential impact of invasive species.

Functional Genomics and Cell Biology of the Dolphin (Tursiops runcatus): Establishment of Novel Molecular Tools to Study Marine Mammals in Changing Environments

The dolphin (Tursiops truncatus) is a mammal that is adapted to life in a totally aquatic environment. Despite the popularity and even iconic status of the dolphin, our knowledge of its physiology, its unique adaptations and the effects on it of environmental stressors are limited. One approach to improve this limited understanding is the implementation of established cellular and molecular methods to provide sensitive and insightful information for dolphin biology. We initiated our studies with the analysis of wild dolphin peripheral blood leukocytes, which have the potential to be informative of the animal’s global immune status. Transcriptomic profiles from almost 200 individual samples were analyzed using a newly developed species-specific microarray to assess its value as a prognostic and diagnostic tool. Functional genomics analyses were informative of stress-induced gene expression profiles and also of geographical location specific transcriptomic signatures, determined by the interaction of genetic, disease and environmental factors. We have developed quantitative metrics to unambiguously characterize the phenotypic properties of dolphin cells in culture. These quantitative metrics can provide identifiable characteristics and baseline data which will enable identification of changes in the cells due to time in culture. We have also developed a novel protocol to isolate primary cultures from cryopreserved tissue of stranded marine mammals, establishing a tissue (and cell) biorepository, a new approach that can provide a solution to the limited availability of samples. The work presented represents the development and application of tools for the study of the biology, health and physiology of the dolphin, and establishes their relevance for future studies of the impact on the dolphin of environmental infection and stress.

Urine proteome in animals of veterinary interest: species comparison and new biomarkers of nephropathy

Urine is considered an ideal source of biomarkers, however in veterinary medicine a complete study on the urine proteome is still lacking. The present work aimed to apply proteomic techniques to the separation of the urine proteome in dogs, cats, horses, cows and some non-conventional species. High resolution electrophoresis (HRE) was also validated for the quantification of albuminuria in dogs and cats. In healthy cats, applying SDS-PAGE and 2DE coupled to mass spectrometry (MS), was produced a reference map of the urine proteome. Moreover, 13 differentially represented urine proteins were linked with CKD, suggesting uromodulin, cauxin, CFAD, Apo-H, RBP and CYSM as candidate biomarkers to be investigated further. In dogs, applying SDS-PAGE coupled to MS, was highlighted a specific pattern in healthy animals showing important differences in patients affected by leishmaniasis. In particular, uromodulin could be a putative biomarker of tubular damage while arginine esterase and low MW proteins needs to be investigated further. In cows, applying SDS-PAGE, were highlighted different patterns between heifers and cows showing some interesting changes during pregnancy. In particular, putative alpha-fetoprotein and b-PAP needs to be further investigated. In horses, applying SDS-PAGE, was produced a reference profile characterized by 13±4 protein bands and the most represented one was the putative uromodulin. Proteinuric horses showed the decrease of the putative uromodulin band and the appearance of 2 to 4 protein bands at higher MW and a greater variability in the range of MW between 49 and 17 kDa. In felids and giraffes was quantified proteinuria reporting the first data for UTP and UPC. Moreover, by means of SDS-PAGE, were highlighted species-specific electrophoretic patterns in big felids and giraffes.

Controlled reproduction in Anguilla anguilla (L.): advanced studies on broodstock management, spawning techniques and system design for artificial seed production.

The thesis aims to expose the advances achieved in the practices of captive breeding of the European eel (Anguilla anguilla). Aspects investigated concern both approaches livestock (breeding selection, response to hormonal stimulation, reproductive performance, incubation of eggs) and physiological aspects (endocrine plasma profiles of players), as well as engineering aspects. Studies conducted on various populations of wild eel have shown that the main determining factor in the selection of wild females destined to captive breeding must be the Silver Index which may determine the stage of pubertal development. The hormonal induction protocol adopted, with increasing doses of carp pituitary extract, it has proven useful to ovarian development, with a synchronization effect that is positively reflected on egg production. The studies on the effects of photoperiod show how the condition of total darkness can positively influence practices of reproductions in captivity. The effects of photoperiod were also investigated at the physiological level, observing the plasma levels of steroids ( E2, T) and thyroid hormones (T3 and T4) and the expression in the liver of vitellogenin (vtg1 and vtg2) and estradiol membrane receptor (ESR1). From the comparison between spontaneous deposition and insemination techniques through the stripping is inferred as the first ports to a better qualitative and quantitative yield in the production of eggs capable of being fertilized, also the presence of a percentage of oocytes completely transparent can be used to obtain eggs at a good rate of fertility. Finally, the design and implementation of a system for recirculating aquaculture suited to meet the needs of species-specific eel showed how to improve the reproductive results, it would be preferable to adopt low-flow and low density incubation.

Genomic and functional insights into the interactions between vaginal Lactobacillus strains and pathogens

The vaginal microbiota of healthy pre-menopausal women is typically dominated by one Lactobacillus species among L. crispatus, L. gasseri, L. jensenii and L. iners. Thanks to a series of antimicrobial activities, strains belonging to these species represent the first barrier against infections and maintain niche homeostasis. On the other hands, the increase abundance in pathogen species is associated with the onset of numerous diseases, leading also to an increase risk of other infections acquisition. The deciphering of factors which influence Lactobacillus survival, as well as the interactions between lactobacilli-pathogens and pathogens-pathogens represent an important topic of study for improving woman health and investigating effective probiotic strategies. Here, we investigated environmental factors and genetic traits that lead to the dominance of either L. crispatus or L. gasseri in the vaginal niche and the possible applications of liposomes loaded with L. gasseri biosurfactants for the treatment and prevention of Staphylococcus aureus biofilm infections. Furthermore, considering the increasing relevance acquired by bacterial extracellular vesicles (EVs) we analysed the role of EVs derived from vaginal lactobacilli and pathogens on both bacterial growth and HIV-1 infections. As a result, we reported for the first time i) common and species-specific genotypic and phenotypic features of L. crispatus and L. gasseri ii) significant antibiofilm activity of liposomes loading vaginal Lactobacillus biosurfactants against multi-drug resistant S. aureus strains iii) absence of growth regulation mediated by EVs derived from lactobacilli on pathogen cultures and vice versa iv) anti-HIV-1 activity of protein derived from L. gasseri EVs and unexpected antiviral effect of pathogen-derived EVs on HIV-1 infections in vitro. In conclusion, this PhD thesis explored characteristics and possible applications of vaginal lactobacilli for the human health, as well as promising antiviral effects of both lactobacilli and pathogen derived EVs.

Modelling spillover effects in spatial stochastic frontier analysis

In the last two decades, authors have begun to expand classical stochastic frontier (SF) models in order to include also some spatial components. Indeed, firms tend to concentrate in clusters, taking advantage of positive agglomeration externalities due to cooperation, shared ideas and emulation, resulting in increased productivity levels. Until now scholars have introduced spatial dependence into SF models following two different paths: evaluating global and local spatial spillover effects related to the frontier or considering spatial cross-sectional correlation in the inefficiency and/or in the error term. In this thesis, we extend the current literature on spatial SF models introducing two novel specifications for panel data. First, besides considering productivity and input spillovers, we introduce the possibility to evaluate the specific spatial effects arising from each inefficiency determinant through their spatial lags aiming to capture also knowledge spillovers. Second, we develop a very comprehensive spatial SF model that includes both frontier and error-based spillovers in order to consider four different sources of spatial dependence (i.e. productivity and input spillovers related to the frontier function and behavioural and environmental correlation associated with the two error terms). Finally, we test the finite sample properties of the two proposed spatial SF models through simulations, and we provide two empirical applications to the Italian accommodation and agricultural sectors. From a practical perspective, policymakers, based on results from these models, can rely on precise, detailed and distinct insights on the spillover effects affecting the productive performance of neighbouring spatial units obtaining interesting and relevant suggestions for policy decisions.

Characterisation of probiotic strains for the control and prevention of enteropathogens in the food chain

60 strains (belonging to the genera Lactobacillus, Bifidobacterium, Leuconostoc and Enterococcus) were tested for their capacity to inhibit the growth of 3 strains of Campylobacter jejuni: Lactobacilli and bifidobacteria were left to grow in MRS or TPY broth at 37°C overnight in anaerobic conditions; Campylobacter jejuni was inoculated in blood agar plates at 37°C for 24-48 hours in microaerophilic conditions. The inhibition experiments were carried out in vitro using ”Spot agar test” and “Well diffusion assay” techniques testing both cellular activity and that of the surnatant. 11 strains proved to inhibit the growth of Campylobacter jejuni. These strains were subsequently analised analised in order to evaluate the resistance to particular situations of stress which are found in the gastrointestinal tract and during the industrial transformation processes (Starvation stress, osmotic stress, heat stress, resistance to pH and to bile salts). Resistance to starvation stress: all strains seemed to resist the stress (except one strain). Resistance to osmotic stress: all strains were relatively resistant to the concentrations of 6% w/v of NaCl (except one strain). Resistance to heat stress: only one strain showed little resistance to the 55°C temperature. Resistance to pH: In the presence of a low pH (2.5), many strains rapidly lost their viability after approximately 1 hour. Resistance to bile salts: Except for one strain, all strains seemed to be relatively resistant to the 2% w/v concentration of bile salts. Afterward, strains were identified by using phenotipic and molecular techniques. Phenotipic identification was carried out by using API 50 CHL (bioMérieux) and API 20 STREP identification system (bioMérieux); molecular identification with species-specific PCR: the molecular techniques confirmed the results by phenotipic identification. For testing the antibiotic resistance profile, bacterial strains were subcultured in MRS or TPY broth and incubated for 18 h at 37°C under anaerobic conditions. Antibiotics tested (Tetracycline, Trimethoprim, Cefuroxime, Kanamycin, Chloramphenicol, Vancomycin, Ampycillin, Sterptomycin, Erythromycin) were diluted to the final concentrations of: 2,4,8,16,32,64,128,256 mg/ml. Then, 20 μl fresh bacterial culture (final concentration in the plates approximately 106 cfu/ml) were added to 160 μl MRS or TPY broth and 20 μl antibiotic solution. As positive control the bacterial culture (20 ul) was added to broth (160 ul) and water (20 ul). Test was performed on plates P96, that after the inoculum were incubated for 24 h at 37oC, then the antibiotic resistance was determined by measuring the Optical Density (OD) at 620 nm with Multiscan EX. All strains showed a similar behaviour: resistance to all antibiotic tested. Further studies are needed.

Autenticazione d’origine, stima della Freshness quality e studio del profilo nutrizionale come elementi fondanti per la valorizzazione dell’alimento ittico

Questa tesi è dedicata alla qualità dell'alimento ittico in tre delle sue possibili accezioni. Dopo aver spiegato il complicato rapporto del consumatore con gli alimenti ittici e come l'Unione Europea abbia cercato di fare chiarezza al riguardo, gli argomenti di discussione saranno: Autenticazione d'origine La polpa di 160 esemplari di spigola (Dicentrachus labrax), suddivisi tra selvatici, allevati intensivamente e allevati estensivamente, provenienti dall'Italia e dall'estero per un totale di 18 fonti indagate, è stati analizzata individualmente per caratterizzarne la componente lipidica, isotopica e minerale e verificare le potenzialità di queste informazioni ai fini della autenticazione di origine in senso lato. Stima della Freshness Quality Numerosi lotti di seppia (Sepia officinalis), nasello (Merluccius merluccius) e triglia di fango (Mullus barbatus) sono stati sottoposti a due possibili modalità di stoccaggio sotto ghiaccio fondente, per indagare come, nell’arco della loro vita commerciale, ne evolvessero importanti connotati chimici (cataboliti dell’ATP e loro rapporti), fisici (proprietà dielettriche dei tessuti) e sensoriali (Quality Index Methods specie-specifici. Studio del profilo nutrizionale La componente lipidica di numerosi lotti di mazzancolla (Penaeus kerathurus), canocchia (Squilla mantis) e seppia (Sepia officinalis) è stata caratterizzata allo stato crudo e dopo cottura secondo tecniche “dedicate” per stabilire il contributo di queste matrici come fonte di acidi grassi polinsaturi della serie omega 3 e per pervenire alla determinazione dei loro coefficienti di ritenzione vera.

Biomineralization in calcifying marine organisms

The objective of this theses is to contribute to the wide discussion about the biological control level on the biomineralization operated by calcifying organisms. In particular the intra-crystalline organic matrix associated with different coral species was studied and its role in the process was investigated. The main goals obtained from the research on corals included: (i) the discovery of the species specific role of the intra-crystalline organic matrix molecules in the precipitation of calcium carbonate; (ii) the definition of the role of magnesium ions in the control of the macromolecules assembly/aggregation and in the consequent calcium carbonate polymorphic selectivity; (iii) the discovery that in corals the biomineralization process is not affected by the sea water acidity, as consequence corals are able to construct their skeletons independently from the environmental conditions as far they survive. At the same time, investigations on different kind of vaterite, biogenic and synthetic, were also carried out and confirm the importance of the organism control on the biomineralization process and in particular on the co-existence of different crystalline structures of vaterite for enabling optimization of specific functions, through the employment of OM and acidic macromolecules.

Insights on epidemiology and control of Marek’s disease through traditional and novel molecular tools

Marek's disease (MD) is a contagious, lymphoproliferative and neuropathic disease of poultry caused by a ubiquitous lymphotropic and oncogenic virus, Gallid alphaherpesvirus 2 (GaHV-2). MD has been reported in all poultry-rearing countries and is among the viral diseases with the highest economic impact in the poultry industry worldwide, including Italy. MD has been also recognized as one of the leading causes of mortality in backyard poultry. The present doctoral thesis aimed at exploring Marek's disease virus molecular epidemiology in Italian commercial and backyard chicken flocks and, for the first time, in commercial turkeys affected by clinical MD. Molecular biology techniques targeting the full-length meq gene, the major GaHV-2 oncogene, were used to detect and characterize the circulating GaHV-2 strains searching for genetic markers of virulence. A final study focused on the development of rapid, sensitive, and species-specific loop-mediated isothermal amplification assays coupled with a lateral flow device readout for the detection of conventional and recombinant HVT-based vaccines is included in the thesis. HVT vaccines, currently used to protect chickens from MD, are referred to as "leaky", as they do not impede the infection, replication, and shedding of field GaHV-2: vaccinal and field viruses can coexist in the vaccinated host and molecular tests able to discriminate between GaHV-2 and HVT are required. These new simple, fast, and accurate tests for the monitoring of MD vaccination success in the field could be greatly beneficial for field veterinarians, small laboratories, and more broadly for resource-limited settings.

Small Area Estimation of Economic Security

The objective of this thesis is the small area estimation of an economic security indicator. Economic security is a complex concept that carries a variety of meanings. In the literature there is no a formal unambiguous definition for economic security and in this work we refer to the definition recently provided for its opposite, economic insecurity, as the “anxiety produced by the possible exposure to adverse economic events and by the anticipation of the difficulty to recover from them” (Bossert and D’Ambrosio, 2013). In the last decade interest for economic insecurity/security has grown constantly, especially since the financial crisis of 2008, but even more in the last year after the economic consequences due to the Covid-19 pandemic. In this research, economic security is measures through a longitudinal indicator that takes into account the income levels of Italian households, from 2014 to 2016. The target areas are groups of Italian provinces, for which the indicator is estimated using longitudinal data taken from EU-SILC survey. We notice that the sample size is too low to obtain reliable estimates for our target areas. Therefore we resort to some Small Area Estimation strategies to improve the reliability of the results. In particular we consider small area models specified at area level. Besides the basic Fay-Herriot area-level model, we propose to consider some longitudinal extensions, including time-specific random effects following an autoregressive processes of order 1 (AR1) and a moving average of order 1 (MA1). We found that all the small area models used show a significant efficiency gain, especially MA1 model.

Multidisciplinary approach to horse welfare in different management systems

From its domestication until nowadays, the horse has assumed multiple roles in human society. Over time, this condition and the lack of specific regulation have led to the development of different kinds of management systems for this species. This Ph.D. research project aims to investigate horses' welfare in different management practices and housing systems, considering a multidisciplinary approach, taking into account biological function, naturalness, and affective dimension. The results are presented in five articles that evidence risk factors that can mine horse welfare, and examine tools and parameters that can be employed for its assessment. Our research shows the importance of considering the evolutionary history and the species-specific and behavioural needs of horses in their management and housing. Sociality, free movement, diet composition and foraging routine, and the workload that these animals undergo are important factors that should be taken into account. Furthermore, this research has evidenced the importance of employing different parameters (e.g., behaviour, endocrinological parameters, and immune activity) in welfare assessment and proposes the use of horsehair DHEA (dehydroepiandrosterone) as a possible useful additional non-invasive measure for the investigation of long-term stress conditions. Finally, our results underline the importance of considering the affective dimension in welfare research. Recently, Judgement Bias Tests (JBT), which are based on the influence of affective states on the decision-making process, have been widely employed in animal welfare research. However, our studies show that the use of spatial JBT in horses can have some limitations. Still today several management systems do not fulfill species-specific needs of horses, thus the implementation of specific regulations could ameliorate horse welfare. A multidisciplinary approach to welfare assessment is fundamental, but it should be always remembered the individual and its own characteristics, which can influence not only physiological, immunological, and behavioural responses but also emotional and cognitive dimensions.