Abstract

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.