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.

The role of biological processes in affecting the dynamics and fate of microplastics in coastal marine systems

Microplastics (MP) are omnipresent contaminants in the marine environment. Ingestion of MP has been reported for a wide range of marine biota, but to what extent the uptake by organisms affects the dynamics and fate of MP in the marine system has received little attention. My thesis explored this topic by integrating laboratory tests and experiments, field quantitative surveys of MP distribution and dynamics, and the use of specialised analytical techniques such as Attenuated-Total-Reflectance- (ATR) and imaging- Fourier Transformed Infrared Spectroscopy (FTIR). I compared different methodologies to extract MP from wild invertebrate specimens, and selected the use of potassium hydroxide (KOH) as the most cost-effective approach. I used this approach to analyse the MP contamination in various invertebrate species with different ecological traits from European salt marshes. I found that 96% of the analysed specimens (330) did not contain any MP. As preliminary environmental analyses showed high levels of environmental MP contamination, I hypothesised that most MP do not accumulate into organisms but are rather fast egested. I subsequently used laboratory multi-trophic experiments and a long-term field experiment using the filter-feeding mussel Mytilus galloprovincialis and the detritus feeding polychaete Hediste diversicolor to test the aforementioned hypothesis. Overall, results showed that MP are ingested but rapidly egested by marine invertebrates, which may limit MP transfer via predator-prey interactions but at the same time enhance their transfer via detrital pathways in the sediments. These processes seem to be extremely variable over time, with potential unexplored environmental consequences. This rapid dynamics also limits the conclusions that can be derived from static observations of MP contents in marine organisms, not fully capturing the real levels of potential contaminations by marine species. This emphasises the need to consider such dynamics in future work to measure the uptake rates by organisms in natural systems.

Effects of stochastic hypoxia events on the benthic communities of transitional waters

Hypoxia is one of the most important and faster spreading threats to marine life and its occurrence has significantly increased in the last century. The effects of hypoxia on marine organisms and communities has mostly been studied in light of the intensity of the disturbance but not a lot of attention has been given to its interaction with other stressors and the timing of its appearance. In this thesis I started to explore these topics through laboratory and manipulative field experiments. I studied the interactive effects of thermal stress and hypoxia on a European native bivalve species (Cerastoderma edule; Linnaeus, 1758 ) and a non native one (Ruditapes philippinarum; Adams & Reeve, 1850) through a laboratory experiment performed in the Netherlands. The non native species displayed a greater tolerance to oxygen depletion than the native one. The first field experiment was performed in an Italian brackish coastal lagoon (Pialassa Baiona) and tested the effects of different timing regimes of hypoxia on the benthic community. It emerged that the main factor affecting the community is the duration of the hypoxia. The ability of the communities to recover after repeated hypoxic periods was explored in the second manipulative field experiment. We imposed three different timing regimes of hypoxia on sediment patches in Pialassa Baiona and we monitored the changes of both the benthic and the microbial communities after the disturbances. The preliminary analyses of the data from this last work suggest that the experimental manipulations caused limited detrimental effects on the communities. Overall this thesis work suggests that the duration of hypoxic events, their repetitive nature and the associated thermal stress are key factors in determining their effects on the communities and that management measures should point towards a reduction of the duration of the single hypoxic periods more than their frequency.

Methylmercury and polycyclic aromatic hydrocarbons in Mediterranean seafood: a molecular anthropological perspective

Seafood carries several contaminants, among which mercury and polycyclic aromatic hydrocarbons are those that cause major concern. Evidence exists that human populations are exposed to these environmental chemicals since ancient times, which may have driven the positive selection of specific genetic polymorphisms related to chemicals toxicokinetic. Both mercury and polycyclic aromatic hydrocarbons are able to cause DNA methylation changes in humans. Some Mediterranean populations may be particularly exposed to these contaminants, being the Mediterranean Sea at a high-risk for contamination by toxic compounds, and because of their traditionally high consumption of locally caught seafood. Starting from these premises the present thesis aims to contribute to the understanding of the molecular impact of seafood consumption on the biology of the Mediterranean population. To this end the work has been divided into four main parts: (1) the development and meta-analysis of a georeferenced database on polycyclic aromatic hydrocarbons in Mediterranean seafood aimed at identifying geographical patterns of contamination and trends that could be related to the biology of the marine organisms, to the physico-chemical properties of each hydrocarbon and to the oceanographic characteristic of the Mediterranean; (2) the development and validation of a food frequency questionnaire to estimate the intake of mercury through seafood consumption among a population living in a geographic area that is usually considered a contamination hotspot; (3) the creation of a biobank made of biological samples from members of several Italian communities together with information on their dietary habits, lifestyle and general health; (4) a review of the literature on the genetic component of individual susceptibility to methylmercury and polycyclic aromatic hydrocarbons exposure in humans, to the effects that these pollutants have on human DNA methylation, and to the evidence that Mediterranean coastal communities represent an informative case study to investigate the potential molecular impact of these chemicals.

Relationship between phenotype and environment in marine calcifying organisms: exploring growth and shell properties of different mollusks species in past and modern scenario

Coastal ecosystems represent an inestimable source of biodiversity, being among the most productive areas on the planet. Despite the great ecological and economic value of those environments, many threats endanger the species living in this ecosystem, like the rapid warming and the sea acidification, among many other. Benthic calcifying organisms (e.g. mollusks, corals and echinoderms) in particular, are among the most exposed to those hazards. These organisms use calcium carbonate as a structural and protective material through the biomineralization process, biologically controlled by the organism, but nevertheless, strongly influenced by the environmental surroundings. Evaluating how a changing environment can influence the process of biomineralization is critical to understand how those species of great ecological and economic importance will face the ongoing climate change. This thesis investigates the mechanism of biomineralization in different mollusks’ species of the Adriatic Sea, providing detailed descriptions of shells skeletal, biometric and growth parameters. Applying a multidisciplinary and multi-scale research approach, the influence of external environmental factors on the process of shell formation has been investigated. To achieve this purpose analysis were conducted both on current populations and on fossil remain, which allows to investigate ecological responses to past climate transitions. Mollusks’ shells in fact are one of the best tools to understand climate change in the past, present and future, since they record the environmental conditions prevailed during their life, reflected on the geochemical properties, microstructure and growth of the shell. This approach allowed to overcome the time scale limit imposed by field and laboratory survey, and better understand species long term adaptive response to changing environment, a crucial issue to define proper conservation and management strategies. Furthermore, the investigation of fossil record of mollusks assemblages offered the opportunity to evaluate the long-term biotic response to anthropogenic stressors in the north Adriatic Sea.

Natural patterns and anthropogenic disturbance in north Adriatic marine benthic assemblages: descriptive and methodological studies

Marine soft bottom systems show a high variability across multiple spatial and temporal scales. Both natural and anthropogenic sources of disturbance act together in affecting benthic sedimentary characteristics and species distribution. The description of such spatial variability is required to understand the ecological processes behind them. However, in order to have a better estimate of spatial patterns, methods that take into account the complexity of the sedimentary system are required. This PhD thesis aims to give a significant contribution both in improving the methodological approaches to the study of biological variability in soft bottom habitats and in increasing the knowledge of the effect that different process (both natural and anthropogenic) could have on the benthic communities of a large area in the North Adriatic Sea. Beta diversity is a measure of the variability in species composition, and Whittaker’s index has become the most widely used measure of beta-diversity. However, application of the Whittaker index to soft bottom assemblages of the Adriatic Sea highlighted its sensitivity to rare species (species recorded in a single sample). This over-weighting of rare species induces biased estimates of the heterogeneity, thus it becomes difficult to compare assemblages containing a high proportion of rare species. In benthic communities, the unusual large number of rare species is frequently attributed to a combination of sampling errors and insufficient sampling effort. In order to reduce the influence of rare species on the measure of beta diversity, I have developed an alternative index based on simple probabilistic considerations. It turns out that this probability index is an ordinary Michaelis-Menten transformation of Whittaker's index but behaves more favourably when species heterogeneity increases. The suggested index therefore seems appropriate when comparing patterns of complexity in marine benthic assemblages. Although the new index makes an important contribution to the study of biodiversity in sedimentary environment, it remains to be seen which processes, and at what scales, influence benthic patterns. The ability to predict the effects of ecological phenomena on benthic fauna highly depends on both spatial and temporal scales of variation. Once defined, implicitly or explicitly, these scales influence the questions asked, the methodological approaches and the interpretation of results. Problem often arise when representative samples are not taken and results are over-generalized, as can happen when results from small-scale experiments are used for resource planning and management. Such issues, although globally recognized, are far from been resolved in the North Adriatic Sea. This area is potentially affected by both natural (e.g. river inflow, eutrophication) and anthropogenic (e.g. gas extraction, fish-trawling) sources of disturbance. Although few studies in this area aimed at understanding which of these processes mainly affect macrobenthos, these have been conducted at a small spatial scale, as they were designated to examine local changes in benthic communities or particular species. However, in order to better describe all the putative processes occurring in the entire area, a high sampling effort performed at a large spatial scale is required. The sedimentary environment of the western part of the Adriatic Sea was extensively studied in this thesis. I have described, in detail, spatial patterns both in terms of sedimentary characteristics and macrobenthic organisms and have suggested putative processes (natural or of human origin) that might affect the benthic environment of the entire area. In particular I have examined the effect of off shore gas platforms on benthic diversity and tested their effect over a background of natural spatial variability. The results obtained suggest that natural processes in the North Adriatic such as river outflow and euthrophication show an inter-annual variability that might have important consequences on benthic assemblages, affecting for example their spatial pattern moving away from the coast and along a North to South gradient. Depth-related factors, such as food supply, light, temperature and salinity play an important role in explaining large scale benthic spatial variability (i.e., affecting both the abundance patterns and beta diversity). Nonetheless, more locally, effects probably related to an organic enrichment or pollution from Po river input has been observed. All these processes, together with few human-induced sources of variability (e.g. fishing disturbance), have a higher effect on macrofauna distribution than any effect related to the presence of gas platforms. The main effect of gas platforms is restricted mainly to small spatial scales and related to a change in habitat complexity due to a natural dislodgement or structure cleaning of mussels that colonize their legs. The accumulation of mussels on the sediment reasonably affects benthic infauna composition. All the components of the study presented in this thesis highlight the need to carefully consider methodological aspects related to the study of sedimentary habitats. With particular regards to the North Adriatic Sea, a multi-scale analysis along natural and anthopogenic gradients was useful for detecting the influence of all the processes affecting the sedimentary environment. In the future, applying a similar approach may lead to an unambiguous assessment of the state of the benthic community in the North Adriatic Sea. Such assessment may be useful in understanding if any anthropogenic source of disturbance has a negative effect on the marine environment, and if so, planning sustainable strategies for a proper management of the affected area.

Impact of anthropogenic stressors on the microbial communities in marine holobionts and ecosystems

Free-living or host-associated marine microbiomes play a determinant role in supporting the functioning and biodiversity of marine ecosystems, providing essential ecological services, and promoting the health of the entire biosphere. Currently, the fast and restless increase of World’s human population strongly impacts life on Earth in the forms of ocean pollution, coastal zone destruction, overexploitation of marine resources, and climate change. Thanks to their phylogenetic, metabolic, and functional diversity, marine microbiomes represent the Earth’s biggest reservoir of solutions against the major threats that are now impacting marine ecosystems, possibly providing valuable insights for biotechnological applications to preserve the health of the ocean ecosystems. Microbial-based mitigation strategies heavily rely on the available knowledge on the specific role and composition of holobionts associated microbial communities, thus highlighting the importance of pioneer studies on microbial-mediated adaptive mechanisms in the marine habitats. In this context, we propose different models representing ecologically important, widely distributed, and habitat-forming organisms, to further investigate the ability of marine holobionts to dynamically adapt to natural environmental variations, as well as to anthropogenic stress factors. In this PhD thesis, we were able to supply the characterization of the microbial community associated with the model anthozoan cnidaria Corynactis viridis throughout a seasonal gradient, to provide critical insights into microbiome-host interactions in a biomonitoring perspective. We also dissected in details the microbial-derived mitigation strategies implemented by the benthonic anthozoan Anemonia viridis and the gastropod Patella caerulea as models of adaptation to anthropogenic stressors, in the context of bioremediation of human-impacted habitats and for the monitoring and preservation of coastal marine ecosystems, respectively. Finally, we provided a functional model of adaptation to future ocean acidification conditions by characterizing the microbial community associated with the temperate coral Balanophyllia europaea naturally living at low pH conditions, to implement microbial based actions to mitigate climate change.

Multiscale comparative analysis of marine biominerals and their ecological implications

Marine biomineralizing organisms provide a fundamental link between biology and environment. Calcified structure are important archives that can provide us main means of understanding organism adaptation, habits, environmental characteristics, and to look back in time and explore the past climate and their evolutionary history. In fact, biomineralized structures retain an unparalleled record of current and past ocean conditions through the investigation of their microchemistry and isotopes. This thesis considers aspects of two different biomineralization systems: fish otolith and coral skeletons at macro-, micro- and nanoscale, with the aim to understand how their morphology, structural characteristics and compositions can provide information of their functionality, and the environmental, behavioural, and evolutionary context in which organisms are framed. To this end, I applied a multidisciplinary approach in the scope to investigate calcified structures as “information recorders” and as models to study the phenotypic plasticity.

Monitoring of Mediterranean marine benthic biodiversity through novel, standardized, and integrated approaches

Marine healthy ecosystems support life on Earth and human well-being thanks to their biodiversity, which is proven to decline mainly due to anthropogenic stressors. Monitoring how marine biodiversity changes trough space and time is needed to properly define and enroll effective actions towards habitat conservation and preservation. This is particularly needed in those areas that are very rich in species compared to their low surface extension and are characterized by strong anthropic pressures, such as the Mediterranean Sea. Subtidal rocky benthic Mediterranean habitats have a complex structural architecture, hosting a panoply of tiny organisms (cryptofauna) that inhabit crevices and caves, but that are still unknown. Different artificial standardized sampling structures (SSS) and methods have been developed and employed to characterize the cryptofauna, allowing for data replicability and comparability across regions. Organisms growing on these artificial structures can be identified coupling morphological taxonomy and DNA barcoding and metabarcoding. The metabarcoding allows for the identification of organisms in a bulk sample without morphological analysis, and it is based on comparing the genetic similarities of the assessed organisms with barcoding sequences present in online barcoding repositories. Nevertheless, barcoded species nowadays represent only a small portion of known species, and barcoding reference databases are not always curated and updated on a regular basis. In this Thesis I used an integrative approach to characterize benthic marine biodiversity, specifically coupling morphological and molecular techniques with the employment of SSS. Moreover, I upgraded the actual status of COI (cytochrome c oxidase subunit I) barcoding of marine metazoans, and I built a customized COI barcoding reference database for metabarcoding studies on temperate biogenic reefs. This work implemented the knowledge about diversity of Mediterranean marine communities, laying the groundworks for monitoring marine and environmental changes that will occur in the next future as consequences of anthropic and climate threats.