Ocean pH fluctuations affect mussel larvae at key developmental transitions.

Coastal marine ecosystems experience dynamic fluctuations in seawater carbonate chemistry. The importance of this variation in the context of ocean acidification requires knowing what aspect of variability biological processes respond to. We conducted four experiments (ranging from 3 to 22 days) with different variability regimes (pHT 7.4-8.1) assessing the impact of diel fluctuations in carbonate chemistry on the early development of the mussel Mytilus galloprovincialis. Larval shell growth was consistently correlated to mean exposures, regardless of variability regimes, indicating that calcification responds instantaneously to seawater chemistry. Larval development was impacted by timing of exposure, revealing sensitivity of two developmental processes: development of the shell field, and transition from the first to the second larval shell. Fluorescent staining revealed developmental delay of the shell field at low pH, and abnormal development thereof was correlated with hinge defects in D-veligers. This study shows, for the first time, that ocean acidification affects larval soft-tissue development, independent from calcification. Multiple developmental processes additively underpin the teratogenic effect of ocean acidification on bivalve larvae. These results explain why trochophores are the most sensitive life-history stage in marine bivalves and suggest that short-term variability in carbonate chemistry can impact early larval development.

Coral calcifying fluid pH is modulated by seawater carbonate chemistry not solely seawater pH.

Reef coral calcification depends on regulation of pH in the internal calcifying fluid (CF) in which the coral skeleton forms. However, little is known about calcifying fluid pH (pHCF) regulation, despite its importance in determining the response of corals to ocean acidification. Here, we investigate pHCF in the coral Stylophora pistillata in seawater maintained at constant pH with manipulated carbonate chemistry to alter dissolved inorganic carbon (DIC) concentration, and therefore total alkalinity (AT). We also investigate the intracellular pH of calcifying cells, photosynthesis, respiration and calcification rates under the same conditions. Our results show that despite constant pH in the surrounding seawater, pHCF is sensitive to shifts in carbonate chemistry associated with changes in [DIC] and [AT], revealing that seawater pH is not the sole driver of pHCF Notably, when we synthesize our results with published data, we identify linear relationships of pHCF with the seawater [DIC]/[H+] ratio, [AT]/ [H+] ratio and [[Formula: see text]]. Our findings contribute new insights into the mechanisms determining the sensitivity of coral calcification to changes in seawater carbonate chemistry, which are needed for predicting effects of environmental change on coral reefs and for robust interpretations of isotopic palaeoenvironmental records in coral skeletons.

Imaging intracellular pH in a reef coral and symbiotic anemone.

The challenges corals and symbiotic cnidarians face from global environmental change brings new urgency to understanding fundamental elements of their physiology. Intracellular pH (pHi) influences almost all aspects of cellular physiology but has never been described in anthozoans or symbiotic cnidarians, despite its pivotal role in carbon concentration for photosynthesis and calcification. Using confocal microscopy and the pH sensitive probe carboxy SNARF-1, we mapped pHi in short-term light and dark-incubated cells of the reef coral Stylophora pistillata and the symbiotic anemone Anemonia viridis. In all cells isolated from both species, pHi was markedly lower than the surrounding seawater pH of 8.1. In cells that contained symbiotic algae, mean values of pHi were significantly higher in light treated cells than dark treated cells (7.41 +/- 0.22 versus 7.13 +/- 0.24 for S. pistillata; and 7.29 +/- 0.15 versus 7.01 +/- 0.27 for A. viridis). In contrast, there was no significant difference in pHi in light and dark treated cells without algal symbionts. Close inspection of the interface between host cytoplasm and algal symbionts revealed a distinct area of lower pH adjacent to the symbionts in both light and dark treated cells, possibly associated with the symbiosome membrane complex. These findings are significant developments for the elucidation of models of inorganic carbon transport for photosynthesis and calcification and also provide a cell imaging procedure for future investigations into how pHi and other fundamental intracellular parameters in corals respond to changes in the external environment such as reductions in seawater pH.

Effects of light and darkness on pH regulation in three coral species exposed to seawater acidification.

The resilience of corals to ocean acidification has been proposed to rely on regulation of extracellular calcifying medium pH (pHECM), but few studies have compared the capacity of coral species to control this parameter at elevated pCO2. Furthermore, exposure to light and darkness influences both pH regulation and calcification in corals, but little is known about its effect under conditions of seawater acidification. Here we investigated the effect of acidification in light and darkness on pHECM, calcifying cell intracellular pH (pHI), calcification, photosynthesis and respiration in three coral species: Stylophora pistillata, Pocillopora damicornis and Acropora hyacinthus. We show that S. pistillata was able to maintain pHECM under acidification in light and darkness, but pHECM decreased in P. damicornis and A. hyacinthus to a much greater extent in darkness than in the light. Acidification depressed calcifying cell pHI in all three species, but we identified an unexpected positive effect of light on pHI. Calcification rate and pHECM decreased together under acidification, but there are inconsistencies in their relationship indicating that other physiological parameters are likely to shape how coral calcification responds to acidification. Overall our study reveals interspecies differences in coral regulation of pHECM and pHI when exposed to acidification, influenced by exposure to light and darkness.

Ion transporter gene expression is linked to the thermal sensitivity of calcification in the reef coral Stylophora pistillata.

Coral calcification underpins biodiverse reef ecosystems, but the physiology underlying the thermal sensitivity of corals to changing seawater temperatures remains unclear. Furthermore, light is also a key factor in modulating calcification rates, but a mechanistic understanding of how light interacts with temperature to affect coral calcification is lacking. Here, we characterized the thermal performance curve (TPC) of calcification of the wide-spread, model coral species Stylophora pistillata, and used gene expression analysis to investigate the role of ion transport mechanisms in thermally-driven declines in day and nighttime calcification. Focusing on genes linked to transport of dissolved inorganic carbon (DIC), calcium and H+, our study reveals a high degree of coherence between physiological responses (e.g. calcification and respiration) with distinct gene expression patterns to the different temperatures in day and night conditions. At low temperatures, calcification and gene expression linked to DIC transport processes were downregulated, but showed little response to light. By contrast, at elevated temperature, light had a positive effect on calcification and stimulated a more functionally diverse gene expression response of ion transporters. Overall, our findings highlight the role of mechanisms linked to DIC, calcium and H+ transport in the thermal sensitivity of coral calcification and how this sensitivity is influenced by light.

In vivo pH measurement at the site of calcification in an octocoral.

Calcareous octocorals are ecologically important calcifiers, but little is known about their biomineralization physiology, relative to scleractinian corals. Many marine calcifiers promote calcification by up-regulating pH at calcification sites against the surrounding seawater. Here, we investigated pH in the red octocoral Corallium rubrum which forms sclerites and an axial skeleton. To achieve this, we cultured microcolonies on coverslips facilitating microscopy of calcification sites of sclerites and axial skeleton. Initially we conducted extensive characterisation of the structural arrangement of biominerals and calcifying cells in context with other tissues, and then measured pH by live tissue imaging. Our results reveal that developing sclerites are enveloped by two scleroblasts and an extracellular calcifying medium of pH 7.97 ± 0.15. Similarly, axial skeleton crystals are surrounded by cells and a calcifying medium of pH 7.89 ± 0.09. In both cases, calcifying media are more alkaline compared to calcifying cells and fluids in gastrovascular canals, but importantly they are not pH up-regulated with respect to the surrounding seawater, contrary to what is observed in scleractinians. This points to a potential vulnerability of this species to decrease in seawater pH and is consistent with reports that red coral calcification is sensitive to ocean acidification.

Soluble organic matrix of two Scleractinian corals: partial and comparative analysis.

This study is a biochemical and molecular analysis of the soluble organic matrix (SOM) of two Scleractinian corals differing in their morphological characteristics: Stylophora pistillata, a branched robust coral and Pavona cactus, a leafy complex coral. Soluble organic matrix of both coral species were shown to contain high amounts of potentially acidic amino acids and glycine. However, proportions of glycosaminoglycans and SDS-PAGE analyses of soluble organic matrix proteins were very different. Three proteins of S. pistillata and at least five proteins of P. cactus were detected by silver staining, some of them being able to bind calcium. Internal peptide sequences of two matrix proteins (one from each species) were obtained. One sequence of S. pistillata is unusual because it contains a long poly-aspartate domain, as described in proteins belonging to the calsequestrin family and in proteins from molluscan species. This domain suggests an essential role for this protein in the control of mineralization.

Morphological plasticity of the coral skeleton under CO2-driven seawater acidification.

Ocean acidification causes corals to calcify at reduced rates, but current understanding of the underlying processes is limited. Here, we conduct a mechanistic study into how seawater acidification alters skeletal growth of the coral Stylophora pistillata. Reductions in colony calcification rates are manifested as increases in skeletal porosity at lower pH, while linear extension of skeletons remains unchanged. Inspection of the microstructure of skeletons and measurements of pH at the site of calcification indicate that dissolution is not responsible for changes in skeletal porosity. Instead, changes occur by enlargement of corallite-calyxes and thinning of associated skeletal elements, constituting a modification in skeleton architecture. We also detect increases in the organic matrix protein content of skeletons formed under lower pH. Overall, our study reveals that seawater acidification not only causes decreases in calcification, but can also cause morphological change of the coral skeleton to a more porous and potentially fragile phenotype.

Cloning of a calcium channel alpha1 subunit from the reef-building coral, Stylophora pistillata.

While the mechanisms of cellular Ca2+ entry associated with cell activation are well characterized, the pathway of continuous uptake of the large amount of Ca2+ needed in the biomineralization process remains largely unknown. Scleractinian corals are one of the major calcifying groups of organisms. Recent studies have suggested that a voltage-dependent Ca2+ channel is involved in the transepithelial transport of Ca2+ used for coral calcification. We report here the cloning and sequencing of a cDNA coding a coral alpha1 subunit Ca2+ channel. This channel is closely related to the L-type family found in vertebrates and invertebrates. Immunohistochemical analysis shows that this channel is present within the calicoblastic ectoderm, the site involved in calcium carbonate precipitation. These data and previous results provide molecular evidence that voltage-dependent Ca2+ channels are involved in calcification. Cnidarians are the most primitive organisms in which a Ca2+ channel has been cloned up to now; evolutionary perspectives on Ca2+ channel diversity are discussed.

Organic matrix synthesis in the scleractinian coral stylophora pistillata: role in biomineralization and potential target of the organotin tributyltin

The kinetics of organic matrix biosynthesis and incorporation into scleractinian coral skeleton was studied using microcolonies of Stylophora pistillata. [14C]Aspartic acid was used to label the organic matrix since this acidic amino acid can represent up to 50 mol % of organic matrix proteins. External aspartate was rapidly incorporated into tissue protein without any detectable lag phase, suggesting either a small intracellular pool of aspartic acid or a pool with a fast turn-over rate. The incorporation of 14C-labelled macromolecules into the skeleton was linear over time, after an initial delay of 20 min. Rates of calcification, measured by the incorporation of 45Ca into the skeleton, and of organic matrix biosynthesis and incorporation into the skeleton were constant. Inhibition of calcification by the Ca2+ channel inhibitor verapamil reduced the incorporation of organic matrix proteins into the skeleton. Similarly, organic matrix incorporation into the skeleton, but not protein synthesis for incorporation into the tissue compartment, was dependent on the state of polymerization of both actin and tubulin, as shown by the sensitivity of this process to cytochalasin B and colchicin. These drugs may inhibit exocytosis of organic matrix proteins into the subcalicoblastic space. Finally, inhibition of protein synthesis by emetin or cycloheximide and inhibition of N-glycosylation by tunicamycin reduced both the incorporation of macromolecules into the skeleton and the rate of calcification. This suggests that organic matrix biosynthesis and its migration towards the site of calcification may be a prerequisite step in the calcification process. On the basis of these results, we investigated the effects of tributyltin (TBT), a component of antifouling painting known to interfere with biomineralization processes. Our results have shown that this xenobiotic significantly inhibits protein synthesis and the subsequent incorporation of protein into coral skeleton. This effect was correlated with a reduction in the rate of calcification. Protein synthesis was shown to be the parameter most sensitive to TBT (IC50=0.2 micromol l-1), followed by aspartic acid uptake by coral tissue (IC50=0.6 micromol l-1), skeletogenesis (IC50=3 micromol l-1) and Ca2+ uptake by coral tissue (IC50=20 micromol l-1). These results suggest that the mode of action of TBT on calcification may be the inhibition of organic matrix biosynthesis.

Coral calcifying fluid pH dictates response to ocean acidification.

Ocean acidification driven by rising levels of CO2 impairs calcification, threatening coral reef growth. Predicting how corals respond to CO2 requires a better understanding of how calcification is controlled. Here we show how spatial variations in the pH of the internal calcifying fluid (pHcf) in coral (Stylophora pistillata) colonies correlates with differential sensitivity of calcification to acidification. Coral apexes had the highest pHcf and experienced the smallest changes in pHcf in response to acidification. Lateral growth was associated with lower pHcf and greater changes with acidification. Calcification showed a pattern similar to pHcf, with lateral growth being more strongly affected by acidification than apical. Regulation of pHcf is therefore spatially variable within a coral and critical to determining the sensitivity of calcification to ocean acidification.

Comparative analysis of the soluble organic matrix of axial skeleton and sclerites of Corallium rubrum: insights for biomineralization.

We analysed the soluble organic matrix (SOM) of two biominerals formed by the same organism but differing by their morphological characteristics: the axial skeleton and the sclerites of Corallium rubrum. The results of 1D SDS-PAGE electrophoresis show for the two biominerals that SOM proteins bands have similar apparent molecular weight but differ in quantity. Further analysis by 2D electrophoresis reveals each protein band as a line of spots with different isoelectric points. Our results suggest that each SOM protein band consists of a mix of proteins and/or one unique protein with post-translational modifications. By immunohistochemistry, we show that antibodies raised against the SOM of axial skeleton and sclerites label the SOM of the two biominerals but also label the insoluble organic matrix suggesting the presence of common epitopes between the two biominerals and the two organic fractions.

Evidence of low molecular weight components in the organic matrix of the reef building coral, Stylophora pistillata.

Biominerals contain both inorganic and organic components. Organic components are collectively termed the organic matrix, and this matrix has been reported to play a crucial role in mineralization. Several matrix proteins have been characterized in vertebrates, but only a few in invertebrates, primarily in Molluscs and Echinoderms. Methods classically used to extract organic matrix proteins eliminate potential low molecular weight matrix components, since cut-offs ranging from 3.5 to 10 kDa are used to desalt matrix extracts. Consequently, the presence of such components remains unknown and these are never subjected to further analyses. In the present study, we have used microcolonies from the Scleractinian coral Stylophora pistillata to study newly synthesized matrix components by labelling them with 14C-labelled amino acids. Radioactive matrix components were investigated by a method in which both total organic matrix and fractions of matrix below and above 5 kDa were analyzed. Using this method and SDS-PAGE analyses, we were able to detect the presence of low molecular mass matrix components (<3.5 kDa), but no free amino acids in the skeletal organic matrix. Since more than 98% of the 14C-labelled amino acids were incorporated into low molecular weight molecules, these probably form the bulk of newly synthesized organic matrix components. Our results suggest that these low molecular weight components may be peptides, which can be involved in the regulation of coral skeleton mineralization.

Daily variations of endolymph composition: relationship with the otolith calcification process in trout.

Ionic and organic parameters of the otolith calcification process in the trout Oncorhynchus mykiss were analysed in plasma and endolymph over the day:night cycle. Plasma pH remained constant and total CO(2) concentration was significantly lower (by 21%) during the day than at night. Calcifying parameters (total CO(2), total calcium concentration) were measured in the proximal and distal endolymphs and were unchanged in the latter during the day:night cycle, but fluctuated in the former. Non-collagenous protein and collagen concentrations in endolymph were higher (1.5- and 10-fold, respectively) during the day than at night. As there was no change in total calcium concentration, we propose that Ca(2+) increases during the dark period and was maximal by the end of the night when the total CO(2) concentration has also increased (by 14%). Measurements of endolymph pH in situ revealed significant differences between samples from proximal and distal endolymph (7.38 and 7.87, respectively), but no variation between values obtained during the day and at night. Thus, the saturation state of aragonite (Sa) in the proximal endolymph should fluctuate around unity during the day:night cycle, and CaCO(3) precipitation should occur when supersaturation is reached. The electrophoretic pattern of proximal endolymph showed variations in both major and minor components. Immunoblotting of endolymph, using a rabbit antiserum raised against the otolith soluble organic matrix revealed an increase in the expression of two proteins (65 kDa and 75 kDa) during the day period. We propose that organic matrix and calcium carbonate deposition on the otolith vary antiphasically: organic matrix deposition begins by the end of the day period, when the concentration of organic precursors is maximal in the endolymph, whereas CaCO(3) precipitation starts once the solubility of CaCO(3) is exceeded.