Denis Allemand

Coral Calcification, Cells to Reefs

In spite of more than one century and half of studies, mechanisms of coral biomineralization, leading to coral growth and reef formation, still remain poorly known, although major global threats to coral reefs, such as ocean acidification, primarily affect this process. Coral skeletons are used as environmental archives but the vital processes that govern incorporation of trace elements and stable isotope are still unknown. Our knowledge on coral physiology is restricted to the organismal level due to the lack of appropriate cell model, however the advent of new approaches, such as coral genomic, is changing drastically our knowledge on these animals even if only a few data are available concerning the field of biomineralization. This chapter reviews our present knowledge and discusses the different theories on coral calcification, from the molecular to the reef level. Conclusion is presented in a list of key issues to be resolved in order to understand the intimate mechanisms of calcification of corals, essential to determine the origin of the sensitivity of corals to ocean acidification, to improve paleoceanographic reconstructions or coral reef management, or “just” to understand how genes of a soft organism control the formation of an extracellular 3D-skeleton.

Impact of seawater acidification on pH at the tissue–skeleton interface and calcification in reef corals

Insight into the response of reef corals and other major marine calcifiers to ocean acidification is limited by a lack of knowledge about how seawater pH and carbonate chemistry impact the physiological processes that drive biomineralization. Ocean acidification is proposed to reduce calcification rates in corals by causing declines in internal pH at the calcifying tissue–skeleton interface where biomineralization takes place. Here, we performed an in vivo study on how partial-pressure CO2-driven seawater acidification impacts intracellular pH in coral calcifying cells and extracellular pH in the fluid at the tissue–skeleton interface [subcalicoblastic medium (SCM)] in the coral Stylophora pistillata. We also measured calcification in corals grown under the same conditions of seawater acidification by measuring lateral growth of colonies and growth of aragonite crystals under the calcifying tissue. Our findings confirm that seawater acidification decreases pH of the SCM, but this decrease is gradual relative to the surrounding seawater, leading to an increasing pH gradient between the SCM and seawater. Reductions in calcification rate, both at the level of crystals and whole colonies, were only observed in our lowest pH treatment when pH was significantly depressed in the calcifying cells in addition to the SCM. Overall, our findings suggest that reef corals may mitigate the effects of seawater acidification by regulating pH in the SCM, but they also highlight the role of calcifying cell pH homeostasis in determining the response of reef corals to changes in external seawater pH and carbonate chemistry.

Live Tissue Imaging Shows Reef Corals Elevate pH under Their Calcifying Tissue Relative to Seawater

The threat posed to coral reefs by changes in seawater pH and carbonate chemistry (ocean acidification) raises the need for a better mechanistic understanding of physiological processes linked to coral calcification. Current models of coral calcification argue that corals elevate extracellular pH under their calcifying tissue relative to seawater to promote skeleton formation, but pH measurements taken from the calcifying tissue of living, intact corals have not been achieved to date. We performed live tissue imaging of the reef coral Stylophora pistillata to determine extracellular pH under the calcifying tissue and intracellular pH in calicoblastic cells. We worked with actively calcifying corals under flowing seawater and show that extracellular pH (pHe) under the calicoblastic epithelium is elevated by ∼0.5 and ∼0.2 pH units relative to the surrounding seawater in light and dark conditions respectively. By contrast, the intracellular pH (pHi) of the calicoblastic epithelium remains stable in the light and dark. Estimates of aragonite saturation states derived from our data indicate the elevation in subcalicoblastic pHe favour calcification and may thus be a critical step in the calcification process. However, the observed close association of the calicoblastic epithelium with the underlying crystals suggests that the calicoblastic cells influence the growth of the coral skeleton by other processes in addition to pHe modification. The procedure used in the current study provides a novel, tangible approach for future investigations into these processes and the impact of environmental change on the cellular mechanisms underpinning coral calcification.

Carbonic anhydrase in the scleractinian coral stylophora pistillata: characterization, localization, and role in biomineralization

Carbonic anhydrases (CA) play an important role in biomineralization from invertebrates to vertebrates. Previous experiments have investigated the role of CA in coral calcification, mainly by pharmacological approaches. This study reports the molecular cloning, sequencing, and immunolocalization of a CA isolated from the scleractinian coral Stylophora pistillata, named STPCA. Results show that STPCA is a secreted form of α-CA, which possesses a CA catalytic function, similar to the secreted human CAVI. We localized this enzyme at the calicoblastic ectoderm level, which is responsible for the precipitation of the skeleton. This localization supports the role of STPCA in the calcification process. In symbiotic scleractinian corals, calcification is stimulated by light, a phenomenon called “light-enhanced calcification” (LEC). The mechanism by which symbiont photosynthesis stimulates calcification is still enigmatic. We tested the hypothesis that coral genes are differentially expressed under light and dark conditions. By real-time PCR, we investigated the differential expression of STPCA to determine its role in the LEC phenomenon. Results show that the STPCA gene is expressed 2-fold more during the dark than the light. We suggest that in the dark, up-regulation of the STPCA gene represents a mechanism to cope with night acidosis.

Symbiosis-induced adaptation to oxidative stress.

SUMMARY Cnidarians in symbiosis with photosynthetic protists must withstand daily hyperoxic/anoxic transitions within their host cells. Comparative studies between symbiotic (Anemonia viridis) and non-symbiotic (Actinia schmidti) sea anemones show striking differences in their response to oxidative stress. First, the basal expression of SOD is very different. Symbiotic animal cells have a higher isoform diversity (number and classes) and a higher activity than the non-symbiotic cells. Second, the symbiotic animal cells of A. viridis also maintain unaltered basal values for cellular damage when exposed to experimental hyperoxia (100% O2) or to experimental thermal stress (elevated temperature +7°C above ambient). Under such conditions, A. schmidti modifies its SOD activity significantly. Electrophoretic patterns diversify, global activities diminish and cell damage biomarkers increase. These data suggest symbiotic cells adapt to stress while non-symbiotic cells remain acutely sensitive. In addition to being toxic, high O2 partial pressure (PO2) may also constitute a preconditioning step for symbiotic animal cells, leading to an adaptation to the hyperoxic condition and, thus, to oxidative stress. Furthermore, in aposymbiotic animal cells of A. viridis, repression of some animal SOD isoforms is observed. Meanwhile, in cultured symbionts, new activity bands are induced, suggesting that the host might protect its zooxanthellae in hospite. Similar results have been observed in other symbiotic organisms, such as the sea anemone Aiptasia pulchella and the scleractinian coral Stylophora pistillata. Molecular or physical interactions between the two symbiotic partners may explain such variations in SOD activity and might confer oxidative stress tolerance to the animal host.

Inorganic carbon uptake for photosynthesis by the symbiotic coral/dinoflagellate association : I. Photosynthetic performances of symbionts and dependence on sea water bicarbonate

The aim of this and the accompanying paper is to investigate the mechanisms of dissolved inorganic carbon (DIC) uptake by the scleractinian coral Galaxea fascicularis, and its delivery to the endosymbiotic photosynthetic dinoflagellates (zooxanthellae). For this purpose, a comparison was made between the photosynthetic performance of zooxanthellae in intact symbiosis within microcolonies of Galaxea fascicularis, freshly isolated zooxanthellae (FIZ) and cultured zooxanthellae (CZ) under different conditions. Discrimination between CO2 or HCO3− uptake was achieved by several means including changes in DIC concentration, pH variations, pharmacology or modifications of ion concentration in seawater. In this paper, the photosynthesis/irradiance curves of G. fascicularis microcolonies, FIZ and CZ are presented. It is shown that zooxanthellae inside their host have lower photosynthetic performance than isolated zooxanthellae. Light saturation (Ik) occurred at higher irradiance in the intact association than in isolated symbionts. Light utilization efficiency (α) was minimum in the intact association and increased in FIZ and CZ. G. fascicularis microcolonies, FIZ and CZ were tested for their ability to utilize HCO3− as a source of DIC for photosynthesis. Two main approaches were used, the first consisting of changing the bicarbonate concentration by adding HCC3− to bicarbonate-free artificial seawater at constant pH, and the second of modifying the pH of the seawater in a closed or open system. At saturating light intensity, the DIC concentration saturating for photosynthesis is no more than that of normal sea water. At pH 8.2, a half-maximal rate of photosynthetic O2 evolution is achieved at 408, 71 and 178 μM HCO3− for coral, FIZ and CZ respectively. The photosynthetic O2 production with constant inorganic carbon but varying pH reached an optimum at pH 8 to 9 suggesting that HCO3− is the main species taken up initially. FIZ and CZ possess the ability to utilize both CO2 and HCO3− as substrates for transport. The rate of non-enzymatic dehydration of HCO3− exceeds the rate of photosynthesis in coral and FIZ, but not in CZ. The results presented in this paper suggest that G. fascicularis microcolonies are able to take up bicarbonate to supply symbiont photosynthesis, although zooxanthellae in hospite seems DIC-limited. FIZ seem to absorb CO2 and HCO3− indiscriminately while CZ use HCO3−.

Interactions between zooplankton feeding, photosynthesis and skeletal growth in the scleractinian coral Stylophora pistillata

SUMMARY We investigated the effect of zooplankton feeding on tissue and skeletal growth of the scleractinian coral Stylophora pistillata. Microcolonies were divided into two groups: starved corals (SC), which were not fed during the experiment, and fed corals (FC), which were abundantly fed with Artemia salina nauplii and freshly collected zooplankton. Changes in tissue growth, photosynthesis and calcification rates were measured after 3 and 8 weeks of incubation. Calcification is the deposition of both an organic matrix and a calcium carbonate layer, so we measured the effect of feeding on both these parameters, using incorporation of 14C-aspartic acid and 45Ca, respectively. Aspartic acid is one of the major components of the organic matrix in scleractinian corals. For both sampling times, protein concentrations were twice as high in FC than in SC (0.73 vs 0.42 mg P–1 cm–2 skeleton) and chlorophyll c2 concentrations were 3–4 times higher in fed corals (2.1±0.3 μg cm–2). Cell specific density (CSD), which corresponds to the number of algal cells inside a host cell, was also significantly higher in FC (1.416±0.028) than in SC (1.316±0.015). Fed corals therefore displayed a higher rate of photosynthesis per unit area (Pgmax= 570±60 nmol O2 cm–2 h–1 and Ik=403±27 μmol photons m–2 s–1). After 8 weeks, both light and dark calcification rates were twofold greater in FC (3323±508 and 416±58 nmol Ca2+ 2 h–1 g–1 dry skeletal mass) compared to SC (1560±217 and 225±35 nmol Ca2+ 2 h–1 g–1 dry skeletal mass, respectively, under light and dark conditions). Aspartic acid incorporation rates were also significantly higher in FC (10.44±0.69 and 1.36± 0.26%RAV 2 h–1 g–1 dry skeletal mass, where RAV is total radioactivity initially present in the external medium) than in SC (6.51±0.45 and 0.44±0.02%RAV 2 h–1 g–1 dry skeletal mass under dark and light conditions, respectively). Rates of dark aspartic acid incorporation were lower than the rates measured in the light. Our results suggest that the increase in the rates of calcification in fed corals might be induced by a feeding-stimulation of organic matrix synthesis.

Coral Calcification Under Ocean Acidification and Global Change

Coral reefs are unique marine ecosystems that form huge morphological structures (frameworks) in today’s oceans. These include coral islands (atolls), barrier reefs, and fringing reefs that form the most impressive products of CaCO3 biomineralization. The framework builders are mainly hermatypic corals, calcareous algae, foraminifera, and mollusks that together are responsible for almost 50% of the net annual CaCO3 precipitation in the oceans. The reef ecosystem acts as a huge filtration system that extracts plankton from the vast fluxes of ocean water that flow through the framework. The existence of these wave resistant structures in spite of chemical, biological, and physical erosion depends on their exceedingly high rates of calcification. Coral mortality due to bleaching (caused by global warming) and ocean acidification caused by atmospheric CO2 increase are now the major threats to the existence of these unique ecosystems. When the rates of dissolution and erosion become higher than the rates of precipitation, the entire coral ecosystem starts to collapse and will eventually be reduced to piles of rubble while its magnificent and high diversity fauna will vanish. The loss to nature and to humanity would be unprecedented and it may occur within the next 50 years. In this chapter, we discuss the issue of ocean acidification and its major effects of corals from the cell level to the reef communities. Based on the recently published literature, it can be generalized that calcification in corals is strongly reduced when seawater become slightly acidified. Ocean acidification lowers both the pH and the CO 3 2− ion concentration in the surface ocean, but calcification at the organism level responds mainly to CO 3 2− and not to pH. Most reports show that the symbiotic algae are not sensitive to changes in the carbonate chemistry. The potential mechanisms responsible for coral sensitivity to acidification are either direct input of seawater to the biomineralization site or high sensitivity of the enzymes involved in calcification to pH and/or CO2 concentrations. Increase in pH at the biomineralization site is most probably the most energy demanding process that is influenced by ocean acidification. While hermatypic corals and other calcifiers reduce their rates of calcification, chemical and biological dissolution increase and hence net calcification of the entire coral reef is decreasing dramatically. Community metabolism in several sites and in field enclosures show in some cases net dissolution. Using the relations between aragonite saturation (Ωarag) and community calcification, it is possible to predict that coral reefs globally may start to dissolve when atmospheric CO2 doubles.

Calcein labelling and electrophysiology: insights on coral tissue permeability and calcification

The mechanisms behind the transfer of molecules from the surrounding sea water to the site of coral calcification are not well understood, but are critical for understanding how coral reefs are formed. We conducted experiments with the fluorescent dye calcein, which binds to calcium and is incorporated into growing calcium carbonate crystals, to determine the permeability properties of coral cells and tissues to this molecule, and to determine how it is incorporated into the coral skeleton. We also compared rates of calcein incorporation with rates of calcification measured by the alkalinity anomaly technique. Finally, by an electrophysiological approach, we investigated the electrical resistance of coral tissues in order to better understand the role of tissues in ionic permeability. Our results show that (i) calcein passes through coral tissues by a paracellular pathway, (ii) intercellular junctions control and restrict the diffusion of molecules, (iii) intercellular junctions should have pores of a size higher than 13 Å and lower than 20 nm, and (iv) the resistance of the tissues owing to paracellular junctions has a value of 477 ± 21 Ohm cm2. We discuss the implication of our results for the transport of calcium involved in the calcification process.

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.