ROLE OF BEHAVIOUR IN MARINE ORGANISMS: POTENTIAL EFFECTS UNDER FUTURE OCEAN CONDITIONS.

Over the last 250 years, the intensive burning of fossil fuels along with industrial processes and land uses (e.g. clearing forests and agriculture) has contributed to an increase in atmospheric CO2 from approximately 280 to 410 ppm, with a further increase (from 730 to 1020 ppm) projected by the end of this century. About 30% of the anthropogenic CO2 has been absorbed by the ocean, with a consequent decrease of the ocean’s surface pH causing a phenomenon better known as Ocean Acidification (OA). The average pH of the surface ocean has declined from 8.2 by 0.1 units since pre-industrial times as a result of CO2 emissions and a further reduction of 0.3–0.5 pH units is expected to occur by the 2100. This increased concentration of atmospheric CO2 has driven an increase in atmospheric and oceanic temperatures enhanced at a rate of ~ 0.2˚C per decade in the past 30 years. These rapid changing ocean conditions in pCO2 and temperature are considered two of the major threats to marine biodiversity, leading to changes in the distribution, physiology and behaviour of marine organisms, with potential consequences in community and ecosystem functioning and structure. Despite the increasing interest and amount of literature on this topic, the effects of OA and ocean warming (OW) on marine fauna is difficult to predict, especially because a wide range of impacts have been found across different life stages-and species suggesting that tolerance thresholds to such stressors can vary among life stages experienced by an organism or even between species. In this regard, an increased number of studies has been conducted to better understand the mechanisms by which species can cope with these rapid environmental changes. The first response of animals to a changing environment is predominantly through modification of their behaviour. To date, only a few climate change biology studies have considered behavioural plasticity as a way that animals can adjust their performance under rapid climate change, especially for marine ectotherms. The general objective of this thesis was to evaluate the effects of ocean warming and acidification on different aspects of behaviour in marine ectotherms. To achieve this aim I investigated the behavioural responses of two marine fish and one invertebrate, through field-based and laboratory experiments

Effects of Human Pressures on Ecosystem Structure, Functioning and Services in Salt Marshes

Salt marsh ecosystems are recognized as highly productive, diverse systems which provide key services to society, but are facing an increasing number of multiple human pressures resulting in global losses of these ecosystems. I aimed to assess the effects of human pressures on salt marsh status and functioning, focusing on the perennial Spartina spp. grasses across the Northern Adriatic Sea, using a combination of field observational studies, manipulation of pressures on salt marshes, and wave mesocosm experiments to measure erosion resistance. Major findings include: 1. Macrobenthos diversity and abundance in Spartina spp. patches were influenced by sediment and nutrient variables, and indirectly influenced by Spartina spp. which modified the sdiments via organic enrichment; 2. Spartina spp. below-ground biomass decreased resistance to erosion, with a more pronounced effect in sandy compared to silty sediments; 3. A heat wave that occurred in 2015 synergistically interacted with high nutrient loads to change the vegetation composition in experimentally fertilized plots, with a rapid shift from Spartina spp. grasses to Salicornia spp. succulents, and the heat wave triggered changes in sediment properties that reduced the erosion resistance of salt marsh sediments; 4. After exposure to different inundation levels through a salt marsh transplantation experiment, plant survival (indicated by % live shoot biomass) and % silt were the most significant predictors of erosion resistance. Overall, biomass and sediment properties were the main factors influencing lateral resistance to erosion in our experiments. The research in this thesis fills important knowledge gaps regarding the status of salt marshes and their functioning across lagoons of the Northern Adriatic Sea. These results help to consolidate the evidence of the important role of salt marsh vegetation in coastal protection, and demonstrate the conditions under which erosion resistance in salt marshes is most effective

Effects of local and global stressors on the status and future persistence of intertidal canopy-forming algae

Marine canopy-forming seaweeds are among the most important habitat-forming species along temperate and polar rocky coasts. They form diverse, productive and valuable "forest" habitats that play a key role in coastal primary production, nutrient cycling and disturbance regulation, and facilitate abundant algae and animal communities. They are also some of the most heavily impacted coastal habitats, facing increasing pressures from urban sprawl, pollution, overfishing and climatic instabilities. Identifying the type and strength of interactions between multiple anthropogenic and natural stressors can help setting achievable management targets for degraded ecosystems and support ecological resilience through local actions. My research focuses primarily on understanding the effects of multiple local and global stressors on canopy-forming seaweeds of the genus Cystoseira, with and emphasis on field investigation and experimentation. I approached my research integrating different approaches: 1) I investigated which are the factors driving the loss of canopy-forming seaweeds at global level. The results allowed to detect important synergistic interactions between nutrient enrichment, caused mainly by human activities, and different other stressors such as heavy metals, the presence of competitors, low light and increasing temperature. This suggested that local management of nutrient levels would provide the greatest opportunity for preventing the shift from canopy to mat-forming algae; 2) as second step, I analysed the status of the intertidal Cystoseira populations around the Italian coast, and explored which factors are most likely to influence it. The results reported severe depletion of intertidal populations of Cystoseira, and identified urbanization as one of the main factors related to these poor conditions, confirming the need for urgent management actions to reduce human pressures on these valuable habitat forming species; 3) then, I experimentally investigated the effects of extreme events caused by climate change. I focused my attention on the effects of abrupt increases of air temperature caused by heat-wave events on the intertidal C. compressa around the Italian coast. The results indicated that extreme heat-wave negatively affect the photosynthetic activity of C. compressa, and that local biodiversity and thermal history of the alga seem to play a role reducing or increasing respectively the impact of such extreme events. 4) I also explored the possible overlooked role of the epiphytic bacteria growing on C. compressa. Bacteria can interact with seaweeds in symbiotic, pathological and opportunistic ways, modulating the health, performance and resilience of their hosts and could, therefore play a critical role on the responses of Cystoseira spp. to stress factors. I characterised for the first time the epiphytic bacteria associated to the surface of C. compressa using Illumina Miseq sequences of V1-V3 hypervariable regions of 16S rRNA gene, and investigated their seasonal variations and their relationships with the bacterial populations in the surrounding seawater. I found that bacterial populations associated to C. compressa were clearly distinct from those in the surrounding media, and identified a clear successional pattern, interestingly characterized by an increase in abundance of potential microbial pathogens associated to older thalli of C. compressa; 5) the previous quantitative descriptive work represented an important base-knowledge to further explore experimentally whether surface bacteria could influence the responses of their hosting Cystoseira populations to stressors. I analysed experimentally in the field the interacting effects of nutrient enrichment and heat-wave events on C. compressa population, and explored whether any resulting changes in the photosynthetic activity of C. compressa were associated to changes in the epiphytic bacterial communities. The heat wave caused marked declines of the photosynthetic activity of the intertidal C. compressa. These effects persisted for at least 3 hours, while recovery generally occurred after 24 hours. The heat-wave altered the structure of the epiphytic bacteria of C. compressa. Thalli exposed to the heat-wave presented an increase of OTUs previously shown to be associated with the natural degradation of the thalli of C. compressa, or implied in causing disease or damage to macroalgae. As observed for the photosynthetic activity, these differences decreased over time, suggesting that the microbial community has the ability to recover. Differently from previous work, this experiment did not detect significant effects related to nutrient enrichment, suggesting that the effects of nutrients could be context dependent. These results open new questions concerning the mechanisms by which the epibacterial community could influence the responses and future persistence of these important canopy-forming seaweeds

Use of bioassays and biomarkers in Daphnia magna to assess the effect of pharmaceutical residuals in freshwater ecosystems

Widespread occurrence of pharmaceuticals residues has been reported in aquatic ecosystems. However, their toxic effects on aquatic biota remain unclear. Generally, the acute toxicity has been assessed in laboratory experiments, while chronic toxicity studies have rarely been performed. Of importance appears also the assessment of mixture effects, since pharmaceuticals never occur in waters alone. The aim of the present work is to evaluate acute and chronic toxic response in the crustacean Daphnia magna exposed to single pharmaceuticals and mixtures. We tested fluoxetine, a SSRI widely prescribed as antidepressant, and propranolol, a non selective β-adrenergic receptor-blocking agent used to treat hypertension. Acute immobilization and chronic reproduction tests were performed according to OECD guidelines 202 and 211, respectively. Single chemicals were first tested separately. Toxicity of binary mixtures was then assessed using a fixed ratio experimental design with concentrations based on Toxic Units. The conceptual model of Concentration Addition was adopted in this study, as we assumed that the mixture effect mirrors the sum of the single substances for compounds having similar mode of action. The MixTox statistical method was applied to analyze the experimental results. Results showed a significant deviation from CA model that indicated antagonism between chemicals in both the acute and the chronic mixture tests. The study was integrated assessing the effects of fluoxetine on a battery of biomarkers. We wanted to evaluate the organism biological vulnerability caused by low concentrations of pharmaceutical occurring in the aquatic environment. We assessed the acetylcholinesterase and glutathione s-transferase enzymatic activities and the malondialdehyde production. No treatment induced significant alteration of biomarkers with respect to the control. Biological assays and the MixTox model application proved to be useful tools for pharmaceutical risk assessment. Although promising, the application of biomarkers in Daphnia magna needs further elucidation

Application of Sequence Based Typing (SBT) Technique to Typing Strains of Legionella spp.: Development of an Environmental Risk Map

The main purpose of my PhD is the assessment of the environmental prevalence of Legionella spp. in artificial aquatic environments, considered a habitat at major risk of proliferation, in order to elaborate a risk map. The water distribution system for its characteristics: material of pipelines, aging of network, and absence of adequate maintenance procedures, become an habitat where the microorganism can survive protect by biofilm. During the course of 3 years I had the opportunity to study and evaluate the different habitats correlated to Legionella growth and dissemination; in particular, I have studied the ability of Legionella to colonize these aquatic environments in which, as reported by the WHO, there are suitable conditions for their survival and multiplication, creating a risk to public health. The focus of my research is the hot water network of an hospital, and at the same time I could assess the presence of the microorganism in other conditions as the water lines circuit of the dental unit and a cooling towers, recently correlated to single case and epidemic events. The techniques used for detection and enumeration of Legionella are recognized and approved by the national and international scientific community: they are the traditional culture method (considered the gold standard) and molecular techniques (in particular PCR and sequencing multilocus), a main topic of my PhD activity. As suggested by Italian Guidelines for the Control and Prevention of Legionellosis published in May 2015, molecular techniques have been applied alongside the culture technique, according to the official or experimental protocols

Reproduction and Population Structure in Temperate and Tropical Corals in Relation to Environmental Parameters

In the marine realm, two of the main stressors causing significant changes are ocean warming and acidification. Of particular concern are organisms reliant on the generation of calcium carbonate, such as corals. Reproduction and population structure are strongly related to environmental parameters and may be indicators for stability or decline of coral populations. Part of my research focused on the reproductive cycle of the Mediterranean non-zooxanthellate coral Caryophyllia inornata in relation to seasonal variations of seawater temperature and photoperiod. Moreover, an unusual embryogenesis was found in females, males and inactive individuals during the entire year, indicating a possible agamic origin of embryos. This was a pilot study for a broader investigation along a wide latitudinal gradient of temperature and solar radiation. Reproductive traits of this coral do not vary along the gradient as observed for another non-zooxanthellate species, Leptopsammia pruvoti, indicating that these species could be quite tolerant to environmental changes, probably due to the lack of symbiosis with the zooxanthellae. A study on the spermatogenesis of the zooxanthellate coral Balanophyllia europaea living along a natural pCO2 gradient showed no significant variations with low pH. We hypothesized that high CO2 levels probably enhance zooxanthellae photosynthesis, leading to an increase of the available energy for gonadal development. The ability to reproduce in particular conditions is related to population structure, providing information on coral responses to the environment. I investigated the population structure of the tropical coral Montastraea cavernosa along a depth gradient in Bermuda. Deeper populations were characterized by smaller but more numerous colonies compared to shallow ones, with no variation in the percent cover among depths. Thus, mesophotic populations of M. cavernosa in Bermuda seem quite stable, indicating that these reefs may serve as a source of propagules to maintain shallower reefs and help guide future management and conservation strategies

Studio sui calamari Loligo vulgaris (Lamarck, 1798) e Loligo forbesii (Steenstrup, 1856) delle acque della Sardegna

This thesis aims to deepening knowledge on the biology and ecology of the two squids Loligo vulgaris (Lamarck, 1798) and Loligo forbesii (Steenstrup, 1856) from Sardinian waters. Considering their socio- economic importance in the Sardinian fisheries, the annual fluctuations in abundance highlighted the possibility of set management measures for their rational exploitation on the base of the knowledge of their life cycle Even if their different preferential bathymetric range, the study of the reproductive process through macroscopic histological and gravimetric analysis of the reproductive systems showed similarities between the two squids both in morphology of the gonads and in the pattern of reproduction. They are similar because they show an extended period of sexual maturity; they have an intermittent spawning with intermediate characteristics between coastal and deep spawning. In particular specific results on the reproductive output of L.vulgaris, showed a potential fecundity of about 4000 eggs (size 0.30-3 mm) and 436 spermatophores (size 7.25-17.37 mm) according to the few studies so far carried out on the species. The reading of the age from the eye lens, tested here for the first time on cephalopods decapods, has allowed to estimate for both species a life cycle of about 18-20 months. For both squids were also performed morphometric analysis of the beaks and calculated specific correlations with the animal size. Considering the taxonomic value of the beak such information may be helpful in studies on diets of predators of these species

Genetic Background, Range Shifts and Associated Microbial Responses of Canopy Algae under Changing Environment

Canopy-forming macroalgae are experiencing a general global decline, mostly driven by human pressures on coastal ecosystems and global changes. In contrast to their high ecological relevance, little is known about their resilience and vulnerability to changing environmental conditions. This thesis aimed to understand genetic diversity, dispersal and connectivity of canopy-forming algae and on how these forest formers can be affected by global changes with consequences for population resilience. I focused on intertidal brown macroalgae of the genus Cystoseira, one of the leading canopy-forming seaweed genera along European coasts, coupling molecular and ecological approaches. First I investigated the projected ditribution of of the closely related C. amentacea, C. tamariscifolia, and C. compressa under predicted climatic sccenario and the interplay of distribution shifts with genetic strcture and connectivity. We found that considerable diversity could be lost in the future, particularly whithin Mediterranean area. I then modeled the effect of habitat fragmentation on species dispersal and connectivity by simulating stepping-stone dispersal dynamics for the speceis that showed to be more vulerable to climate change. Results helped describing and predicting the potential impacts of habitat fragmentation and loss. Finally, because the potential role of associated bacteria in macroalgal adaptation and resilience, I explored the association and functional role of the microbial community with Cystoseira spp. I showed that speceis-specific bacterial communitiy could be found and that the structure of Cystoseira associated bacterial communities followed a geographical isolation-by-distance pattern, clearly distinguishable among basins (Atlantic, Mediterranean and Adriatic) possibly driven by population genetic structure of the host. Also, a consistent set of shared bacteria formed the core community of C. compressa across its distribution and was characterized by key ecological functions for the holobiont system. Overall the knowledge adquired in this doctoral thesis can contribute improving the conservation and management of Cystoseira populations