Molecular biomineralization of octocoral skeletons: calcite versus aragonite

Aragonite and calcite represent the two most common polymorphs of calcium carbonate (CaCO3) formed biogenically by organisms. The mechanisms that allow animals to selectively deposit aragonite and/or calcite has been extensively studied in molluscs, but information on corals (class Anthozoa, phylum Cnidaria) is lacking. Contrary to scleractinian corals, exclusively producing aragonite skeletons, members of the coral subclass Octocorallia exhibit both calcitic and aragonitic skeletal structures. They thus represent an interesting target to study biological and environmental control over CaCO3 polymorphs in corals. In this project we selected different octocoral species - characterized by aragonite or calcite skeletons - to investigate the evolution and mechanisms underlying aragonite and calcite biomineralization in corals. Main objectives of this study were 1) the characterization of the molecular machinery employed to deposit the two different CaCO3 polymorphs, and 2) study the effects of seawater chemistry on skeleton mineralogy and gene expression. In the introductory section (Chapter 1) relevant concepts, terminology and background information is provided. Chapter 2 and 3 aimed at filling the gap in terms of availability of -omic resources for octocorals compared to scleractinians. New resources generated as part of the project include reference transcriptomes and skeletal proteomes for four octocoral species with different biomineralization strategies. The transcriptomic analysis presented in Chapter 2 provides a taxonomically comprehensive presence map for homologs of coral calcification genes across early-branching metazoans. By sensibly increasing taxonomic sampling, we expanded the distribution for several genes and reported homologs presence in previously unsurveyed groups. Homologs datasets were used for phylogenetic inferences, which provided insight into the evolution of acidic proteins and allowed to propose an alternative evolutionary scenario for the scleractinian protein galaxin senso stricto. In Chapter 3 several new proteins with putative functions in octocoral biomineralization are described. A comparative characterization of skeleton proteomes in Octocorallia and Scleractinia is also provided. This analysis highlighted an extremely low overlap in terms of proteins presence between aragonite and calcite-forming species, while at the same time identifying a small set of proteins that constitute the core proteome of octocoral sclerites. Instances of similarity between scleractinians and octocorals are also listed, and include galaxin-related proteins, carbonic anhydrases and multicopper oxidases. Finally, as in scleractinians, some octocoral skeletogenic proteins appear to have acquired their role in calcification as the result of secondary co-option and following the enrichment - within the sequence - of acidic residues. Chapter 4 and 5 focused on the interaction between environmental conditions and calcification in octocorals and scleractinians. Chapter 4 revolves around the effect of the magnesium-calcium molar ratio (mMg:mCa) and its effects on the skeleton polymorph. Exposure to calcite-inducing mMg:mCa did not cause a polymorph switch in H. coerulea, while calcite was incorporated in the skeleton of M. digitata. We did not observe changes in expression for skeletogenic proteins, with the exception of one gene coding for the uncharacterized skeleton organic matrix protein 5 (in M. digitata) and endothelin converting enzyme 1 (in H. coerulea). However, carbonic anhydrases and different calcium transporters and channels were affected, suggesting a potential response to changes in mMg:mCa centered around ions transport, rather than a direct involvement of the organic matrix. In Chapter 5, we exposed the octocoral Pinnigrogia flava to sublethal seawater temperature and lower pH (~7.3). We showed how the calcification process in this octocoral is decoupled from the response to stress. Increasing water temperature triggered a stress response but did not affect calcification, while acidification downregulated the expression of several calcification-related genes without causing stress. This represents a mechanistic explanation for the higher tolerance to anthropic stressors exhibited by octocorals. Finally in Chapter 6, an optimized protocol for 16S sequencing in bacteria, using the Illumina MiniSeq available at the Chair for Geobiology & Paleontology of the Department of Earth- and Environmental Sciences at Ludwig-Maximilians-Universität München in Munich (Germany), is presented. This protocol allowed to characterize bacterial communities from different sources, including aquarium seawater, and could thus represent a valuable tool to perform microbiome characterizations from marine organisms in the future. This dissertation contributes to our understanding of the mechanisms underlying the formation of aragonite and calcite skeletons in corals. It includes the first characterizations of octocoral skeleton proteomes, and led to the identification of several - previously unknown - genes with putative calcification-related functions. These novel targets represent a valuable groundwork for further studies, including functional investigations aiming at elucidating the exact mechanisms behind coral biomineralization. It also shed new light on the calcification responses triggered by predicted past and future environmental conditions, providing a better understanding on how corals reacted to changes during their evolutionary history, and their ability to cope with future ones

Molecular biomineralization of octocoral skeletons: calcite versus aragonite

Aragonite and calcite represent the two most common polymorphs of calcium carbonate (CaCO3) formed biogenically by organisms. The mechanisms that allow animals to selectively deposit aragonite and/or calcite has been extensively studied in molluscs, but information on corals (class Anthozoa, phylum Cnidaria) is lacking. Contrary to scleractinian corals, exclusively producing aragonite skeletons, members of the coral subclass Octocorallia exhibit both calcitic and aragonitic skeletal structures. They thus represent an interesting target to study biological and environmental control over CaCO3 polymorphs in corals. In this project we selected different octocoral species - characterized by aragonite or calcite skeletons - to investigate the evolution and mechanisms underlying aragonite and calcite biomineralization in corals. Main objectives of this study were 1) the characterization of the molecular machinery employed to deposit the two different CaCO3 polymorphs, and 2) study the effects of seawater chemistry on skeleton mineralogy and gene expression. In the introductory section (Chapter 1) relevant concepts, terminology and background information is provided. Chapter 2 and 3 aimed at filling the gap in terms of availability of -omic resources for octocorals compared to scleractinians. New resources generated as part of the project include reference transcriptomes and skeletal proteomes for four octocoral species with different biomineralization strategies. The transcriptomic analysis presented in Chapter 2 provides a taxonomically comprehensive presence map for homologs of coral calcification genes across early-branching metazoans. By sensibly increasing taxonomic sampling, we expanded the distribution for several genes and reported homologs presence in previously unsurveyed groups. Homologs datasets were used for phylogenetic inferences, which provided insight into the evolution of acidic proteins and allowed to propose an alternative evolutionary scenario for the scleractinian protein galaxin senso stricto. In Chapter 3 several new proteins with putative functions in octocoral biomineralization are described. A comparative characterization of skeleton proteomes in Octocorallia and Scleractinia is also provided. This analysis highlighted an extremely low overlap in terms of proteins presence between aragonite and calcite-forming species, while at the same time identifying a small set of proteins that constitute the core proteome of octocoral sclerites. Instances of similarity between scleractinians and octocorals are also listed, and include galaxin-related proteins, carbonic anhydrases and multicopper oxidases. Finally, as in scleractinians, some octocoral skeletogenic proteins appear to have acquired their role in calcification as the result of secondary co-option and following the enrichment - within the sequence - of acidic residues. Chapter 4 and 5 focused on the interaction between environmental conditions and calcification in octocorals and scleractinians. Chapter 4 revolves around the effect of the magnesium-calcium molar ratio (mMg:mCa) and its effects on the skeleton polymorph. Exposure to calcite-inducing mMg:mCa did not cause a polymorph switch in H. coerulea, while calcite was incorporated in the skeleton of M. digitata. We did not observe changes in expression for skeletogenic proteins, with the exception of one gene coding for the uncharacterized skeleton organic matrix protein 5 (in M. digitata) and endothelin converting enzyme 1 (in H. coerulea). However, carbonic anhydrases and different calcium transporters and channels were affected, suggesting a potential response to changes in mMg:mCa centered around ions transport, rather than a direct involvement of the organic matrix. In Chapter 5, we exposed the octocoral Pinnigrogia flava to sublethal seawater temperature and lower pH (~7.3). We showed how the calcification process in this octocoral is decoupled from the response to stress. Increasing water temperature triggered a stress response but did not affect calcification, while acidification downregulated the expression of several calcification-related genes without causing stress. This represents a mechanistic explanation for the higher tolerance to anthropic stressors exhibited by octocorals. Finally in Chapter 6, an optimized protocol for 16S sequencing in bacteria, using the Illumina MiniSeq available at the Chair for Geobiology & Paleontology of the Department of Earth- and Environmental Sciences at Ludwig-Maximilians-Universität München in Munich (Germany), is presented. This protocol allowed to characterize bacterial communities from different sources, including aquarium seawater, and could thus represent a valuable tool to perform microbiome characterizations from marine organisms in the future. This dissertation contributes to our understanding of the mechanisms underlying the formation of aragonite and calcite skeletons in corals. It includes the first characterizations of octocoral skeleton proteomes, and led to the identification of several - previously unknown - genes with putative calcification-related functions. These novel targets represent a valuable groundwork for further studies, including functional investigations aiming at elucidating the exact mechanisms behind coral biomineralization. It also shed new light on the calcification responses triggered by predicted past and future environmental conditions, providing a better understanding on how corals reacted to changes during their evolutionary history, and their ability to cope with future ones

The Biology and Evolution of Calcite and Aragonite Mineralization in Octocorallia

Octocorallia (class Anthozoa, phylum Cnidaria) is a group of calcifying corals displaying a wide diversity of mineral skeletons. This includes skeletal structures composed of different calcium carbonate polymorphs (aragonite and calcite). This represents a unique feature among anthozoans, as scleractinian corals (subclass Hexacorallia), main reef builders and focus of biomineralization research, are all characterized by an aragonite exoskeleton. From an evolutionary perspective, the presence of aragonitic skeletons in Octocorallia is puzzling as it is observed in very few species and has apparently originated during a Calcite sea (i.e., time interval characterized by calcite-inducing seawater conditions). Despite this, octocorals have been systematically overlooked in biomineralization studies. Here we review what is known about octocoral biomineralization, focusing on the evolutionary and biological processes that underlie calcite and aragonite formation. Although differences in research focus between octocorals and scleractinians are often mentioned, we highlight how strong variability also exists between different octocoral groups. Different main aspects of octocoral biomineralization have been in fact studied in a small set of species, including the (calcitic) gorgonian Leptogorgia virgulata and/or the precious coral Corallium rubrum. These include descriptions of calcifying cells (scleroblasts), calcium transport and chemistry of the calcification fluids. With the exception of few histological observations, no information on these features is available for aragonitic octocorals. Availability of sequencing data is also heterogeneous between groups, with no transcriptome or genome available, for instance, for the clade Calcaxonia. Although calcite represents by far the most common polymorph deposited by octocorals, we argue that studying aragonite-forming could provide insight on octocoral, and more generally anthozoan, biomineralization. First and foremost it would allow to compare calcification processes between octocoral groups, highlighting homologies and differences. Secondly, similarities (exoskeleton) between Heliopora and scleractinian skeletons, would provide further insight on which biomineralization features are driven by skeleton characteristics (shared by scleractinians and aragonitic octocorals) and those driven by taxonomy (shared by octocorals regardless of skeleton polymorph). Including the diversity of anthozoan mineralization strategies into biomineralization studies remains thus essential to comprehensively study how skeletons form and evolved within this ecologically important group of marine animals

A Unified Simulation Framework for Visual and Behavioral Fidelity in Crowd Analysis

Simulation is a powerful tool to easily generate annotated data, and a highly desirable feature, especially in those domains where learning models need large training datasets. Machine learning and deep learning solutions, have proven to be extremely data-hungry and sometimes, the available real-world data are not sufficient to effectively model the given task. Despite the initial skepticism of a portion of the scientific community, the potential of simulation has been largely confirmed in many application areas, and the recent developments in terms of rendering and virtualization engines, have shown a good ability also in representing complex scenes. This includes environmental factors, such as weather conditions and surface reflectance, as well as human-related events, like human actions and behaviors. We present a human crowd simulator, called UniCrowd, and its associated validation pipeline. We show how the simulator can generate annotated data, suitable for computer vision tasks, in particular for detection and segmentation, as well as the related applications, as crowd counting, human pose estimation, trajectory analysis and prediction, and anomaly detection

Introducing Neuroberry, a platform for pervasive EEG signaling in the IoT domain

The emergence of inexpensive off-the-shelf wireless EEG devices led researchers to explore novel paradigms in the field of Human Computer Interaction. In fact, the compliance of these devices with the IoT principles towards pervasive EEG signaling in smart home environments enables new models of interaction and a different perspective from traditional affective computing. In this paper, the implementation of wireless EEG (Emotiv EPOC and Mindawave) IoT connectivity of real time raw signals, through IoT hardware devices and through the Raspberry Pi 2, is presented