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 CO 2 -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.he impacts of ocean acidification on marine calcifying organisms are predicted to be varied and in many cases deleterious (1-3). Though several studies on marine calcifiers have investigated how rates of calcification respond to ocean acidification scenarios (4), comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. A mechanistic understanding of calcification responses to shifts in external seawater pH and carbonate chemistry is critical to predicting how corals and other major marine calcifiers respond and potentially acclimate to ocean acidification (5).The calcium carbonate (aragonite) skeletons of scleractinian corals make up a large component of shallow and deepwater reefs at both tropical and temperate latitudes. Corals form their skeletons by producing aragonite crystals in a fluid-filled medium called the subcalicoblastic medium (SCM) underlying the calcifying tissue [calicoblastic epithelium (CE)] (Fig. S1). The calicoblastic epithelium promotes calcification by exerting biological control over the SCM in a number of ways (reviewed in refs. 6 and 7). One important process is that the CE elevates pH in the SCM (pH SCM ) relative to the exterior seawater pH (8), possibly by the removal of protons via a by a Ca 2+ ATPase (9-12). This increase in pH SCM favors the conversion of bicarbonate to carbonate, elevating the saturation state ...

SUMMARYThe regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of reef-building corals and symbiotic cnidarians. Here, we investigated the hypothesis that dynamic changes in the pHi of coral host cells are controlled by the photosynthetic activity of the coralʼs dinoflagellate symbionts. Using live cell imaging and the pHsensitive dye SNARF-1, we tracked pH in symbiont-containing and symbiont-free cells isolated from the reef coral Stylophora pistillata. We characterised the response of coral pHi in the presence of a photosynthetic inhibitor, the dynamics of coral pHi during light exposure and how pHi values vary on exposure to a range of irradiance levels lying within the coralʼs photosynthesis-irradiance response curve. Our results demonstrate that increases in coral pHi are dependent on photosynthetic activity of intracellular symbionts and that pHi recovers under darkness to values that match those of symbiont-free cells. Furthermore, we show that the timing of the pHi response is governed by irradiance level and that pHi increases to irradiancespecific steady-state values. Minimum steady-state pHi values of 7.05±0.05 were obtained under darkness and maximum values of 7.46±0.07 were obtained under saturating irradiance. As changes in pHi often affect organism homeostasis, there is a need for continued research into acid/base regulation in symbiotic corals. More generally, these results represent the first characterization of photosynthesis-driven pHi changes in animal cells. Supplementary material available online at

The regulation of intracellular pH (pHi) is a fundamental aspect of cell physiology that has received little attention in studies of the phylum Cnidaria, which includes ecologically important sea anemones and reef-building corals. Like all organisms, cnidarians must maintain pH homeostasis to counterbalance reductions in pHi, which can arise because of changes in either intrinsic or extrinsic parameters. Corals and sea anemones face natural daily changes in internal fluids, where the extracellular pH can range from 8.9 during the day to 7.4 at night. Furthermore, cnidarians are likely to experience future CO 2 -driven declines in seawater pH, a process known as ocean acidification. Here, we carried out the first mechanistic investigation to determine how cnidarian pHi regulation responds to decreases in extracellular and intracellular pH. Using the anemone Anemonia viridis, we employed confocal live cell imaging and a pH-sensitive dye to track the dynamics of pHi after intracellular acidosis induced by acute exposure to decreases in seawater pH and NH 4 Cl prepulses. The investigation was conducted on cells that contained intracellular symbiotic algae (Symbiodinium sp.) and on symbiont-free endoderm cells. Experiments using inhibitors and Na + -free seawater indicate a potential role of Na + /H + plasma membrane exchangers (NHEs) in mediating pHi recovery following intracellular acidosis in both cell types. We also measured the buffering capacity of cells, and obtained values between 20.8 and 43.8 mM per pH unit, which are comparable to those in other invertebrates. Our findings provide the first steps towards a better understanding of acid-base regulation in these basal metazoans, for which information on cell physiology is extremely limited.

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