First paleoproteome study of fossil fish otoliths and the pristine preservation of the biomineral crystal host.

Otoliths are calcium carbonate components of the stato-acoustical organ responsible for hearing and maintenance of the body balance in teleost fish. During their formation, control over, e.g., morphology and carbonate polymorph is influenced by complex insoluble collagen-like protein and soluble non-collagenous protein assemblages; many of these proteins are incorporated into their aragonite crystal structure. However, in the fossil record these proteins are considered lost through diagenetic processes, hampering studies of past biomineralization mechanisms. Here we report the presence of 11 fish-specific proteins (and several isoforms) in Miocene (ca. 14.8-14.6 Ma) phycid hake otoliths. These fossil otoliths were preserved in water-impermeable clays and exhibit microscopic and crystallographic features indistinguishable from modern representatives, consistent with an exceptionally pristine state of preservation. Indeed, these fossil otoliths retain ca. 10% of the proteins sequenced from modern counterparts, including proteins specific to inner ear development, such as otolin-1-like proteins involved in the arrangement of the otoliths into the sensory epithelium and otogelin/otogelin-like proteins that are located in the acellular membranes of the inner ear in modern fish. The specificity of these proteins excludes the possibility of external contamination. Identification of a fraction of identical proteins in modern and fossil phycid hake otoliths implies a highly conserved inner ear biomineralization process through time.

Back to the Future: Reintegrating Biology to Understand How Past Eco-evolutionary Change Can Predict Future Outcomes.

During the last few decades, biologists have made remarkable progress in understanding the fundamental processes that shape life. But despite the unprecedented level of knowledge now available, large gaps still remain in our understanding of the complex interplay of eco-evolutionary mechanisms across scales of life. Rapidly changing environments on Earth provide a pressing need to understand the potential implications of eco-evolutionary dynamics, which can be achieved by improving existing eco-evolutionary models and fostering convergence among the sub-fields of biology. We propose a new, data-driven approach that harnesses our knowledge of the functioning of biological systems to expand current conceptual frameworks and develop corresponding models that can more accurately represent and predict future eco-evolutionary outcomes. We suggest a roadmap toward achieving this goal. This long-term vision will move biology in a direction that can wield these predictive models for scientific applications that benefit humanity and increase the resilience of natural biological systems. We identify short, medium, and long-term key objectives to connect our current state of knowledge to this long-term vision, iteratively progressing across three stages: (1) utilizing knowledge of biological systems to better inform eco-evolutionary models, (2) generating models with more accurate predictions, and (3) applying predictive models to benefit the biosphere. Within each stage, we outline avenues of investigation and scientific applications related to the timescales over which evolution occurs, the parameter space of eco-evolutionary processes, and the dynamic interactions between these mechanisms. The ability to accurately model, monitor, and anticipate eco-evolutionary changes would be transformational to humanity's interaction with the global environment, providing novel tools to benefit human health, protect the natural world, and manage our planet's biosphere.

Sclerites of the soft coral Ovabunda macrospiculata (Xeniidae) are predominantly the metastable CaCO3 polymorph vaterite.

Soft corals (Cnidaria, Anthozoa, Octocorallia, Alcyonacea) produce internal sclerites of calcium carbonate previously shown to be composed of calcite, the most stable calcium carbonate polymorph. Here we apply multiple imaging and physical chemistry analyses to extracted and in-vivo sclerites of the abundant Red Sea soft coral, Ovabunda macrospiculata, to detail their mineralogy. We show that this species' sclerites are comprised predominantly of the less stable calcium carbonate polymorph vaterite (> 95%), with much smaller components of aragonite and calcite. Use of this mineral, which is typically considered to be metastable, by these soft corals has implications for how it is formed as well as how it will persist during the anticipated anthropogenic climate change in the coming decades. This first documentation of vaterite dominating the mineral composition of O. macrospiculata sclerites is likely just the beginning of establishing its presence in other soft corals. STATEMENT OF SIGNIFICANCE: Vaterite is typically considered to be a metastable polymorph of calcium carbonate. While calcium carbonate structures formed within the tissues of octocorals (phylum Cnidaria), have previously been reported to be composed of the more stable polymorphs aragonite and calcite, we observed that vaterite dominates the mineralogy of sclerites of Ovabunda macrospiculata from the Red Sea. Based on electron microscopy, Raman spectroscopy, and X-ray diffraction analysis, vaterite appears to be the dominant polymorph in sclerites both in the tissue and after extraction and preservation. Although this is the first documentation of vaterite in soft coral sclerites, it likely will be found in sclerites of other related taxa as well.

Genetic basis of stony coral biomineralization: History, trends and future prospects.

Despite their simple body plan, stony corals (order Scleractinia, phylum Cnidaria) can produce massive and complex exoskeletal structures in shallow, tropical and subtropical regions of Earth's oceans. The species-specific macromorphologies of their aragonite skeletons suggest a highly coordinated biomineralization process that is rooted in their genomes, and which has persisted across major climatic shifts over the past 400 + million years. The mechanisms by which stony corals produce their skeletons has been the subject of interest for at least the last 160 years, and the pace of understanding the process has increased dramatically in the past decade since the sequencing of the first coral genome in 2011. In this review, we detail what is known to date about the genetic basis of the stony coral biomineralization process, with a focus on advances in the last several years as well as ways that physical and chemical tools can be combined with genetics, and then propose next steps forward for the coming decade.

The spatial network of skeletal proteins in a stony coral.

Coral skeletons are materials composed of inorganic aragonitic fibres and organic molecules including proteins, sugars and lipids that are highly organized to form a solid biomaterial upon which the animals live. The skeleton contains tens of proteins, all of which are encoded in the animal genome and secreted during the biomineralization process. While recent advances are revealing the functions and evolutionary history of some of these proteins, how they are spatially arranged in the skeleton is unknown. Using a combination of chemical cross-linking and high-resolution tandem mass spectrometry, we identify, for the first time, the spatial interactions of the proteins embedded within the skeleton of the stony coral Stylophora pistillata. Our subsequent network analysis revealed that several coral acid-rich proteins are invariably associated with carbonic anhydrase(s), alpha-collagen, cadherins and other calcium-binding proteins. These spatial arrangements clearly show that protein-protein interactions in coral skeletons are highly coordinated and are key to understanding the formation and persistence of coral skeletons through time.

Evolution of Protein-Mediated Biomineralization in Scleractinian Corals.

While recent strides have been made in understanding the biological process by which stony corals calcify, much remains to be revealed, including the ubiquity across taxa of specific biomolecules involved. Several proteins associated with this process have been identified through proteomic profiling of the skeletal organic matrix (SOM) extracted from three scleractinian species. However, the evolutionary history of this putative "biomineralization toolkit," including the appearance of these proteins' throughout metazoan evolution, remains to be resolved. Here we used a phylogenetic approach to examine the evolution of the known scleractinians' SOM proteins across the Metazoa. Our analysis reveals an evolutionary process dominated by the co-option of genes that originated before the cnidarian diversification. Each one of the three species appears to express a unique set of the more ancient genes, representing the independent co-option of SOM proteins, as well as a substantial proportion of proteins that evolved independently. In addition, in some instances, the different species expressed multiple orthologous proteins sharing the same evolutionary history. Furthermore, the non-random clustering of multiple SOM proteins within scleractinian-specific branches suggests the conservation of protein function between distinct species for what we posit is part of the scleractinian "core biomineralization toolkit." This "core set" contains proteins that are likely fundamental to the scleractinian biomineralization mechanism. From this analysis, we infer that the scleractinians' ability to calcify was achieved primarily through multiple lineage-specific protein expansions, which resulted in a new functional role that was not present in the parent gene.

First sequencing of ancient coral skeletal proteins.

Here we report the first recovery, sequencing, and identification of fossil biomineral proteins from a Pleistocene fossil invertebrate, the stony coral Orbicella annularis. This fossil retains total hydrolysable amino acids of a roughly similar composition to extracts from modern O. annularis skeletons, with the amino acid data rich in Asx (Asp + Asn) and Glx (Glu + Gln) typical of invertebrate skeletal proteins. It also retains several proteins, including a highly acidic protein, also known from modern coral skeletal proteomes that we sequenced by LC-MS/MS over multiple trials in the best-preserved fossil coral specimen. A combination of degradation or amino acid racemization inhibition of trypsin digestion appears to limit greater recovery. Nevertheless, our workflow determines optimal samples for effective sequencing of fossil coral proteins, allowing comparison of modern and fossil invertebrate protein sequences, and will likely lead to further improvements of the methods. Sequencing of endogenous organic molecules in fossil invertebrate biominerals provides an ancient record of composition, potentially clarifying evolutionary changes and biotic responses to paleoenvironments.

Optimization of skeletal protein preparation for LC-MS/MS sequencing yields additional coral skeletal proteins in Stylophora pistillata.

Stony corals generate their calcium carbonate exoskeleton in a highly controlled biomineralization process mediated by a variety of macromolecules including proteins. Fully identifying and classifying these proteins is crucial to understanding their role in exoskeleton formation, yet no optimal method to purify and characterize the full suite of extracted coral skeletal proteins has been established and hence their complete composition remains obscure. Here, we tested four skeletal protein purification protocols using acetone precipitation and ultrafiltration dialysis filters to present a comprehensive scleractinian coral skeletal proteome. We identified a total of 60 proteins in the coral skeleton, 44 of which were not present in previously published stony coral skeletal proteomes. Extracted protein purification protocols carried out in this study revealed that no one method captures all proteins and each protocol revealed a unique set of method-exclusive proteins. To better understand the general mechanism of skeletal protein transportation, we further examined the proteins' gene ontology, transmembrane domains, and signal peptides. We found that transmembrane domain proteins and signal peptide secretion pathways, by themselves, could not explain the transportation of proteins to the skeleton. We therefore propose that some proteins are transported to the skeleton via non-traditional secretion pathways.

How corals made rocks through the ages.

Hard, or stony, corals make rocks that can, on geological time scales, lead to the formation of massive reefs in shallow tropical and subtropical seas. In both historical and contemporary oceans, reef-building corals retain information about the marine environment in their skeletons, which is an organic-inorganic composite material. The elemental and isotopic composition of their skeletons is frequently used to reconstruct the environmental history of Earth's oceans over time, including temperature, pH, and salinity. Interpretation of this information requires knowledge of how the organisms formed their skeletons. The basic mechanism of formation of calcium carbonate skeleton in stony corals has been studied for decades. While some researchers consider coral skeletons as mainly passive recorders of ocean conditions, it has become increasingly clear that biological processes play key roles in the biomineralization mechanism. Understanding the role of the animal in living stony coral biomineralization and how it evolved has profound implications for interpreting environmental signatures in fossil corals to understand past ocean conditions. Here we review historical hypotheses and discuss the present understanding of how corals evolved and how their skeletons changed over geological time. We specifically explain how biological processes, particularly those occurring at the subcellular level, critically control the formation of calcium carbonate structures. We examine the different models that address the current debate including the tissue-skeleton interface, skeletal organic matrix, and biomineralization pathways. Finally, we consider how understanding the biological control of coral biomineralization is critical to informing future models of coral vulnerability to inevitable global change, particularly increasing ocean acidification.

Nanoscale Visualization of Biomineral Formation in Coral Proto-Polyps.

Calcium carbonate platforms produced by reef-building stony corals over geologic time are pervasive features around the world [1]; however, the mechanism by which these organisms produce the mineral is poorly understood (see review by [2]). It is generally assumed that stony corals precipitate calcium carbonate extracellularly as aragonite in a calcifying medium between the calicoblastic ectoderm and pre-existing skeleton, separated from the overlying seawater [2]. The calicoblastic ectoderm produces extracellular matrix (ECM) proteins, secreted to the calcifying medium [3-6], which appear to provide the nucleation, alteration, elongation, and inhibition mechanisms of the biomineral [7] and remain occluded and preserved in the skeleton [8-10]. Here we show in cell cultures of the stony coral Stylophora pistillata that calcium is concentrated in intracellular pockets that are subsequently exported from the cell where a nucleation process leads to the formation of extracellular aragonite crystals. Analysis of the growing crystals by lattice light-sheet microscopy suggests that the crystals elongate from the cells' surfaces outward.

Comparative genomics explains the evolutionary success of reef-forming corals.

Transcriptome and genome data from twenty stony coral species and a selection of reference bilaterians were studied to elucidate coral evolutionary history. We identified genes that encode the proteins responsible for the precipitation and aggregation of the aragonite skeleton on which the organisms live, and revealed a network of environmental sensors that coordinate responses of the host animals to temperature, light, and pH. Furthermore, we describe a variety of stress-related pathways, including apoptotic pathways that allow the host animals to detoxify reactive oxygen and nitrogen species that are generated by their intracellular photosynthetic symbionts, and determine the fate of corals under environmental stress. Some of these genes arose through horizontal gene transfer and comprise at least 0.2% of the animal gene inventory. Our analysis elucidates the evolutionary strategies that have allowed symbiotic corals to adapt and thrive for hundreds of millions of years.

Temporal and spatial expression patterns of biomineralization proteins during early development in the stony coral Pocillopora damicornis.

Reef-building corals begin as non-calcifying larvae that, upon settling, rapidly begin to accrete skeleton and a protein-rich skeletal organic matrix that attach them to the reef. Here, we characterized the temporal and spatial expression pattern of a suite of biomineralization genes during three stages of larval development in the reef-building coral Pocillopora damicornis: stage I, newly released; stage II, oral-aborally compressed and stage III, settled and calcifying spat. Transcriptome analysis revealed 3882 differentially expressed genes that clustered into four distinctly different patterns of expression change across the three developmental stages. Immunolocalization analysis further reveals the spatial arrangement of coral acid-rich proteins (CARPs) in the overall architecture of the emerging skeleton. These results provide the first analysis of the timing of the biomineralization 'toolkit' in the early life history of a stony coral.

Immunolocalization of skeletal matrix proteins in tissue and mineral of the coral Stylophora pistillata.

The precipitation and assembly of calcium carbonate skeletons by stony corals is a precisely controlled process regulated by the secretion of an ECM. Recently, it has been reported that the proteome of the skeletal organic matrix (SOM) contains a group of coral acid-rich proteins as well as an assemblage of adhesion and structural proteins, which together, create a framework for the precipitation of aragonite. To date, we are aware of no report that has investigated the localization of individual SOM proteins in the skeleton. In particular, no data are available on the ultrastructural mapping of these proteins in the calcification site or the skeleton. This information is crucial to assessing the role of these proteins in biomineralization. Immunological techniques represent a valuable approach to localize a single component within a calcified skeleton. By using immunogold labeling and immunohistochemical assays, here we show the spatial arrangement of key matrix proteins in tissue and skeleton of the common zooxanthellate coral, Stylophora pistillata. To our knowledge, our results reveal for the first time that, at the nanoscale, skeletal proteins are embedded within the aragonite crystals in a highly ordered arrangement consistent with a diel calcification pattern. In the tissue, these proteins are not restricted to the calcifying epithelium, suggesting that they also play other roles in the coral's metabolic pathways.

Cloning and characterization of four novel coral acid-rich proteins that precipitate carbonates in vitro.

Biomineralization is a widely dispersed and highly regulated but poorly understood process by which organisms precipitate minerals from a wide variety of elements [1]. For many years, it has been hypothesized that the biological precipitation of carbonates is catalyzed by and organized on an extracellular organic matrix containing a suite of proteins, lipids, and polysaccharides [2, 3]. The structures of these molecules, their evolutionary history, and the biophysical mechanisms responsible for calcification remain enigmatic. Despite the recognition that mineralized tissues contain proteins that are unusually rich in aspartic and glutamic acids [4-6], the role of these proteins in biomineralization remains elusive [5, 6]. Here we report, for the first time, the identification, cloning, amino acid sequence, and characterization of four highly acidic proteins, derived from expression of genes obtained from the common stony coral, Stylophora pistillata. Each of these four proteins can spontaneously catalyze the precipitation of calcium carbonate in vitro. Our results demonstrate that coral acid-rich proteins (CARPs) not only bind Ca(2+) stoichiometrically but also precipitate aragonite in vitro in seawater at pH 8.2 and 7.6, via an electrostatic interaction with protons on bicarbonate anions. Phylogenetic analysis suggests that at least one of the CARPs arose from a gene fusion. Similar, highly acidic proteins appear to have evolved several times independently in metazoans through convergence. Based purely on thermodynamic grounds, the predicted change in surface ocean pH in the next decades would appear to have minimal effect on the capacity of these acid-rich proteins to precipitate carbonates.

Proteomic analysis of skeletal organic matrix from the stony coral Stylophora pistillata.

It has long been recognized that a suite of proteins exists in coral skeletons that is critical for the oriented precipitation of calcium carbonate crystals, yet these proteins remain poorly characterized. Using liquid chromatography-tandem mass spectrometry analysis of proteins extracted from the cell-free skeleton of the hermatypic coral, Stylophora pistillata, combined with a draft genome assembly from the cnidarian host cells of the same species, we identified 36 coral skeletal organic matrix proteins. The proteome of the coral skeleton contains an assemblage of adhesion and structural proteins as well as two highly acidic proteins that may constitute a unique coral skeletal organic matrix protein subfamily. We compared the 36 skeletal organic matrix protein sequences to genome and transcriptome data from three other corals, three additional invertebrates, one vertebrate, and three single-celled organisms. This work represents a unique extensive proteomic analysis of biomineralization-related proteins in corals from which we identify a biomineralization "toolkit," an organic scaffold upon which aragonite crystals can be deposited in specific orientations to form a phenotypically identifiable structure.

Aragonite precipitation by "proto-polyps" in coral cell cultures.

The mechanisms of coral calcification at the molecular, cellular and tissue levels are poorly understood. In this study, we examine calcium carbonate precipitation using novel coral tissue cultures that aggregate to form "proto-polyps". Our goal is to establish an experimental system in which calcification is facilitated at the cellular level, while simultaneously allowing in vitro manipulations of the calcifying fluid. This novel coral culturing technique enables us to study the mechanisms of biomineralization and their implications for geochemical proxies. Viable cell cultures of the hermatypic, zooxanthellate coral, Stylophora pistillata, have been maintained for 6 to 8 weeks. Using an enriched seawater medium with aragonite saturation state similar to open ocean surface waters (Ω(arag)~4), the primary cell cultures assemble into "proto-polyps" which form an extracellular organic matrix (ECM) and precipitate aragonite crystals. These extracellular aragonite crystals, about 10 µm in length, are formed on the external face of the proto-polyps and are identified by their distinctive elongated crystallography and X-ray diffraction pattern. The precipitation of aragonite is independent of photosynthesis by the zooxanthellae, and does not occur in control experiments lacking coral cells or when the coral cells are poisoned with sodium azide. Our results demonstrate that proto-polyps, aggregated from primary coral tissue culture, function (from a biomineralization perspective) similarly to whole corals. This approach provides a novel tool for investigating the biophysical mechanism of calcification in these organisms.

Brevetoxin persistence in sediments and seagrass epiphytes of east Florida coastal waters.

A bloom of Karenia brevis Davis developed in September 2007 near Jacksonville, Florida and subsequently progressed south through east Florida coastal waters and the Atlantic Intracoastal Waterway (ICW). Maximum cell abundances exceeded 106 cells L-1 through October in the northern ICW between Jacksonville and the Indian River Lagoon. The bloom progressed further south during November, and terminated in December 2007 at densities of 104 cells L-1 in the ICW south of Jupiter Inlet, Florida. Brevetoxins were subsequently sampled in sediments and seagrass epiphytes in July and August 2008 in the ICW. Sediment brevetoxins occurred at concentrations of 11-15 ng PbTx-3 equivalents (g dry wt sediment)-1 in three of five basins in the northern ICW during summer 2008. Seagrass beds occur south of the Mosquito Lagoon in the ICW. Brevetoxins were detected in six of the nine seagrass beds sampled between the Mosquito Lagoon and Jupiter Inlet at concentrations of 6-18 ng (g dry wt epiphytes)-1. The highest brevetoxins concentrations were found in sediments near Patrick Air Force Base at 89 ng (g dry wt sediment)-1. In general, brevetoxins occurred in either seagrass epiphytes or sediments. Blades of the resident seagrass species have a maximum life span of less than six months, so it is postulated that brevetoxins could be transferred between epibenthic communities of individual blades in seagrass beds. The occurrence of brevetoxins in east Florida coast sediments and seagrass epiphytes up to eight months after bloom termination supports observations from the Florida west coast that brevetoxins can persist in marine ecosystems in the absence of sustained blooms. Furthermore, our observations show that brevetoxins can persist in sediments where seagrass communities are absent.