An aposymbiotic primary coral polyp counteracts acidification by active pH regulation

Corals build their skeletons using extracellular calcifying fluid located in the tissue–skeleton interface. However, the mechanism by which corals control the transport of calcium and other ions from seawater and the mechanism of constant alkalization of calcifying fluid are largely unknown. To address these questions, we performed direct pH imaging at calcification sites (subcalicoblastic medium, SCM) to visualize active pH upregulation in live aposymbiotic primary coral polyps treated with HCl-acidified seawater. Active alkalization was observed in all individuals using vital staining method while the movement of HPTS and Alexa Fluor to SCM suggests that certain ions such as H+ could diffuse via a paracellular pathway to SCM. Among them, we discovered acid-induced oscillations in the pH of SCM (pHSCM), observed in 24% of polyps examined. In addition, we discovered acid-induced pH up-regulation waves in 21% of polyps examined, which propagated among SCMs after exposure to acidified seawater. Our results showed that corals can regulate pHSCM more dynamically than was previously believed. These observations will have important implications for determining how corals regulate pHSCM during calcification. We propose that corals can sense ambient seawater pH via their innate pH-sensitive systems and regulate pHSCM using several unknown pH-regulating ion transporters that coordinate with multicellular signaling occurring in coral tissue.

calcifying fluid is higher than that of seawater and systematically decreases with seawater pH (0.3-0.6 units) 12 . These studies suggested that corals could remove protons (upregulate pH) in the SCM in acidified seawater, given that the decrease in pH at calcification sites was not steep relative to that of seawater.
The use of boron isotopes has provided details about the pH conditions for calcification, but this usage relies on an indirect geochemical technique using a boron isotope pH proxy and cannot reveal the mechanisms of pH upregulation by corals at calcification sites 12,13 . By contrast, it is worth noting that an in vivo pH imaging method using live coral tissues permits direct pH measurements at calcification sites, providing detailed information of pH dynamics in coral tissue 1,6 . Applying this advanced physiological technique will facilitate deeper understanding both of the physiological characteristics of the SCM and the manner in which the pH SCM during coral calcification responds to ambient seawater conditions, such as changes seawater pH 6 .
To study the regulation of pH SCM , we performed a new time-lapse pH imaging method monitoring change in pH of the calcifying fluid during seawater acidification [7][8][9][10] . Acidified seawater was prepared by using HCl in this experiment because we aimed to monitor acid-induced pH upregulation in coral. The physiological responses of marine calcifiers (e.g., coccolithophores 14 , sea urchin and bivalve 15 ) in HCl-acidified seawater are not strictly similar to that in the situation of CO 2 -driven ocean acidification from the aspect of marine CO 2 chemistry. However, we recognize that our experimental results would help us to understand the physiological responses of corals to acid stress such as daily changes in seawater pH and near-future ocean acidification. We discovered novel pH upregulation, acid-induced oscillations of pH SCM , and occasional acid-induced pH upregulation waves (velocity: 1-6 μ m/s), which propagated among the SCMs induced by surrounding seawater acidification. In the present study, we have shown that corals can regulate pH SCM more dynamically than was previously believed, which would be regulated by several types of pH-regulating ion transporters that coordinate with multicellular signaling in coral tissue.

Results and Discussion
Visualization of skeletal formation of aposymbiotic primary coral polyps on a glass-based dish. We used aposymbiotic primary polyps of Acropora digitifera 16 , which can be easily prepared with the metamorphosis-inducing peptide Hym-248 17 in a glass-based dish (Fig. 1a, black arrows). Primary polyps without symbiotic algae (Fig. 1b) began calcification shortly after larval settlement with skeletal formation of CaCO 3 structures (Fig. 1c,c: black arrow). Because the skeletal growth of aposymbiotic primary polyps of A. digitifera is very sensitive to exposure to acidified seawater 18 , this system is convenient for observing changes in pH SCM after exposure to acidified seawater. Calcification occurs at the interface between the tissue and the glass substrate 1,6 . Our system allowed the visualization of the coral primary polyp SCM ( Fig. 1d: red dotted line) which could estimate pH SCM without the effects of photosynthesis and respiration by symbiotic algae 18,19 . Continuous pH imaging during coral calcification. To investigate whether coral primary polyps upregulate pH at calcification sites, we applied time-lapse pH imaging (1 min time-interval and 12-h observation) of SCM during calcification. We used 8-hydroxypyrene-1,3,6-trisulfonic acid (HPTS) 20 (Fig. 1a), a highly water-soluble compound with low toxicity that cannot penetrate the cell membrane 21 , as a pH-sensitive fluorescent indicator (Fig. S1). High concentrations of HPTS (1 mM) were diluted continuously in the seawater to enhance the fluorescent signals from the extracellular space (the SCM) and to offset the autofluorescence of primary polyp tissues.
Coral primary polyps without symbiotic algae of A. digitifera had the ability to alkalize their SCMs (Fig. 1e,f: white arrows, pseudocolor in orange), relative to the pH of ambient seawater (pH 8.1, pseudocolor in yellow). Alkalization of the SCMs is expected to correlate with increases in carbonate ion (CO 3 2− ) concentration and to promote the precipitation of CaCO 3 1,3,6 . Initial crystals emerged ( Fig. 1f: yellow arrows) and gradually developed at the location of the SCMs (Fig. 1f: yellow dotted lines) during continuous pH imaging. However, 26 h after incubation, most SCMs disappeared and SCM areas were replaced with crystals ( Fig. 1f; see also Supporting Movie 1). Thus, long-term incubation combining HPTS pH measurements is applicable to monitoring pH SCM dynamics without major effects on normal developmental processes in aposymbiotic coral primary polyps.
Spatial variations of pH SCM and calcification sensitivity to acidified seawater were observed in adult Stylophora pistillata 22 . In our study, the different pH SCM values in some defined regions of interest (ROIs, Fig. 1e: white frames) and periodic fluctuations in pH SCM were detected even within primary polyps of A. digitifera examined (Fig. 1g). The observed range of alkalization relative to the seawater (average ± S.D. measured for 241 points) for ROI 2, 3, and 4 were 8.67 ± 0.21, 8.47 ± 0.12, and 8.60 ± 0.15, respectively. At the start of the experiment, the pH SCM values were different between ROIs ( Fig. 1g: ROI 2-4) and they were gradually alkalized 15 h after metamorphosis. Surprisingly, sudden drops in pH were observed (0.5-0.8 pH units; Fig. 1g: ROI 2 and 4, red arrows), after which the pH increased within 30 min. We cannot explain this phenomenon, but we speculate that primary polyps temporarily stopped the alkalization of pH SCM or discharged acidic compounds 3,23-25 (such as coral acid-rich proteins) into the SCMs.
Active pH upregulation of SCM induced by acidified seawater. The mechanisms behind active pH SCM upregulation in corals are unknown. To visualize the dynamics of pH SCM with changes in ambient seawater pH, we induced pH SCM changes by adding HCl-acidified seawater (n = 19). Acidified HPTS containing seawater was labeled using Alexa Fluor ® 568 dye to simultaneously monitor the putative diffusing H + into SCM via the paracellular pathway. Alexa Fluor ® 568 dye is the red fluorescent anionic compounds (10,000 MW in size) which cannot pass through the cell membrane and have no overlapping fluorescence with HPTS for pH measurements in our experimental system (Fig. S1g,h).
Four minutes after the start of the experiment, acidified seawater was added and red fluorescence from Alexa Fluor ® 568 was detected in ambient seawater 1 min later, but no red fluorescence was observed in the SCMs  (Fig. 2c), suggesting that H + could diffuse via a paracellular pathway [7][8][9][10] . However, the decrease in pH SCM stopped within 5 min of addition of acidified seawater, and the pH SCM was then maintained at a higher value than that of the ambient seawater (Fig. 2d). Approximately 1 pH unit upregulation was detected in all of the primary polyps after exposed to acidified seawater (n = 19).
Acid-induced pH oscillations and pH wave in an aposymbiotic primary polyp. Interestingly, acid-induced oscillations in pH SCM (hereafter called pH oscillations) were detected approximately 90 min after the start of the experiment in this individual polyp ( Fig. 2d: ROI 2). The oscillation amplitudes were 0.3-0.6 pH units at approximately 10-min intervals, but clear pH oscillations, like those observed for ROI 2, were not detected in the other ROIs. Although slight pH fluctuations (< 0.3 pH units) were detected for ROI 3-5 after adding acidified seawater, they were too small to be evaluated. The pH oscillations were detected in five of the 19 polyps, but no significant change in pH SCM was detected except under acidified seawater conditions (n = 3; Fig. S1g). Thus, we concluded that the acid-induced pH oscillations described above were a biological response of corals exposed to acidified seawater. This finding suggests the existence of an unknown pH regulation mechanism induced by the continuous diffusing H + into the SCMs (Fig. 2c). Such an acid-induced biological response (< 2 h) has not yet been reported in corals, although cellular mechanisms in response to alterations in external pH have been examined in many other organisms. For example, a sour taste is detected immediately by taste receptor cells that respond to acids in human beings 26 . Even bacteria such as Escherichia coli can recover their cytoplasmic pH within 2 min after exposure by acid stress 27 . In order to understand the detailed mechanism of acid-induced biological responses in corals, ion transporters mediating pH regulation in calicoblastic cells should be investigated in the near future 28,29 .
We also discovered an acid-induced pH regulation wave (hereafter, a pH wave) in four of the 19 coral primary polyps. To characterize the behavior of the pH regulation waves, we evaluated a representative case of polyp tissue in which a pH wave crossed from the upper left to the lower right ( Fig. 3a: white arrow) at 3-min intervals ( Fig. 3a; see also Supporting Movie 4). We found that the velocity of the pH wave was approximately 1-6 μ m/s in this primary polyp. Four ROIs (2)(3)(4)(5) in SCMs and ROI 1 in ambient seawater were defined in the direction of wave propagation (Fig. 3b: white arrow), and a pH wave appeared to increase the pH SCM as it moved in this direction, as shown by the white arrow ( Fig. 3b; also see area encircled in black dashed lines in Fig. 3c).
We assume that the pH wave propagation and pH oscillation observed in corals might be mediated by intercellular calcium signaling, given that cytosolic calcium signaling is a general phenomenon of signaling in excitable and non-excitable cells [30][31][32] , including corals 3,28,29,33 . The velocity of the pH wave was similar to fast calcium waves 33-35 mediated by Ca 2+ channels, Ca 2+ pumps, and GAP junctions, that are major components of the human ion trafficking system and have also been identified in the coral genomic data 34,36 . Furthermore, a study using the hippocampal neurons showed that electrical stimulation induced intracellular pH and Ca 2+ oscillations simultaneously 37 . Thus, the general framework of pH regulation in SCMs (pH SCM alkalization and pH SCM response to ambient seawater) is presumably coordinated by multicellular calcium signaling throughout the calicoblastic cells. These multicellular cell-to-cell communication may play a significant role in balancing the pH homeostasis of entire calcifying tissue during the skeletal formation and growth. The pH SCM alkalization can be partly explained by the activation of ATP-driven Ca 2+ /H + antiporters (i.e., Ca 2+ -ATPase) located at the plasma membrane 3,38 . However, it is unknown whether the regulation of pH SCM (pH sensing, pH oscillation, and the pH wave observed in this study) can be achieved only via activation of Ca 2+ -ATPase.
Despite the ambient seawater pH decrease to approximately 7.4, more extreme pH SCM decrease (0.1-0.3 pH units from the ambient seawater pH) were observed 15 min after the start of the experiment (in five of the 19 polyps; Fig. 3b,c). In addition, the pH SCM in ROI 2 considerably increased (1.4 pH unit) within a 2-min interval (17-19 min after the start of the experiment) and suddenly stopped alkalization at 20 min (Fig. 3c: ROI 2), while continuous H + diffusion into SCMs. These dynamic pH SCM upregulations are noteworthy. To further understand the mechanism underlying pH SCM upregulation in corals, we must focus on pH-sensitive ion channels 39 , given that the presence in the A. digitifera genome of well-known pH-sensitive ion channels (e.g., transient receptor potential channels, acid-sensing channels, and chloride channels) has been reported 34,36 . Other ion transporters such as bicarbonate ion transporters 40 or carbonic anhydrase 41 might be indispensable for the alkalization and supply process of dissolved inorganic carbon at calcification sites 4,42 . Further experiments should be needed for understanding the pH regulation mechanism in corals.
In summary, we found that corals can regulate pH SCM more dynamicaly than they were previously believed, suggesting that pH-sensing and -regulating mechanisms underlie coral calcification. We propose that several types of pH-sensitive ion transporters regulate alkalization of SCM in a coordinated manner. Understanding the pH upregulation of corals observed in this study will be essential for predicting how they regulate pH at the calcification site and how they respond to ongoing ocean acidification. In addition, our observations suggest not only that pH SCM dynamics were regulated by surrounding calicoblastic epithelium cells but also that the pH among SCMs was coordinated at the tissue level via long-range multicellular signaling beneath the primary polyp. These findings pave the way for future studies of the multicellular regulation mechanisms involved in intercellular calcium signaling during skeletal formation. However, we could not clearly observe intracellular calcium signals because of the difficulty of monitoring these signals in the presence of autofluorescence from coral tissue or staining of calicoblastic epithelium cells with fluorescent calcium indicators 31,32 . In future, it is crucial to investigate the physiological mechanisms proposed above to analyze the physiological adaptation and cellular response of corals to environmental disturbances such as ocean warming, ocean acidification, and eutrophication.

Methods
Sample preparation. The scleractinian coral A. digitifera, which is one of the most common species in the Ryukyu Islands of Japan 43 , was used. Gravid colonies of A. digitifera were collected from a fringing reef at Sesoko Island, Motobu-cho, Okinawa, Japan. The colonies were kept in a running seawater tank under natural light conditions at Sesoko Station, Tropical Biosphere Research Center, University of the Ryukyus, Okinawa, Japan. Coral spawning occurred at night around the time of the full moon in the spring and summer seasons of 2015. Gametes were collected after spawning, as described by Inoue et al. 19 Primary polyps were prepared by induction of the settlement of planula larvae (3-30 days old) using the coral metamorphosis inducer peptide Hym-248 17 . Hym-248 induces the synchronous metamorphosis and settlement of Acropora planulae and is a useful tool for studies of Acropora larval metamorphosis. Approximately 10-20 larvae were placed in a glass-based dish (No. 1S, thickness: 0.15-0.18 mm; IWAKI Glass, Tokyo, Japan) with 40 μ L of filtered seawater (FSW: pore size 0.22 μ m). Next, a 10-μ L aliquot of 2 × 10 −4 M Hym-248 in FSW was added to the dish and the larvae were incubated for 2 h to induce metamorphosis. Larvae that settled at the seawater surface and on the side of the dish were removed. The pH of the seawater was measured with a portable pH meter (D-71, Horiba Ltd., Kyoto, Japan) against a total hydrogen ion concentration scale 44,45 . A stock solution of 10-mM HPTS (Sigma-Aldrich, St. Louis, MO, USA) was prepared and the pH was adjusted to 8.1 (Total scale: pH T ) using sodium hydroxide (NaOH). This solution was then diluted with FSW and buffered to pH 8.1 with NaOH to obtain a final concentration of 1 mM (FSW-HPTS: salinity of approximately 35). After a 2-h incubation period with Hym-248, the solution was made to a volume of 2000 μ L with FSW-HPTS. The specimens were incubated with FSW-HPTS in an incubator (VERSOS, Hiroshima, Japan) in the dark at 27.0 ± 0.1 °C prior to pH imaging. Bright-field, dark-field, and polarization images were acquired with a Keyence VHX-2000 digital microscope (Osaka, Japan). pH imaging. We used an inverted confocal imaging system (A1+ confocal microscope system; Nikon Instruments Inc, Tokyo, Japan) equipped with a high-resolution galvano scanner and operated with NIS Elements software (Nikon Instruments Inc. Tokyo, Japan). A CFI Plan Apo × 10 objective lens (NA 0.45, Nikon) was used to capture the whole image of the bottom of the coral tissue. The screen display resolution for viewing confocal microscope images was 1024 × 1024 pixels (0.86 μ m = 1 pixel). Primary polyps at 14-18 h after metamorphosis were placed on a glass-based dish at room temperature (27 ± 0.2 °C). An acid-induced biological response in pH SCM was induced with acidified HPTS solution containing Alexa Fluor ® 568 (Thermo Fisher Scientific, Waltham, MA, USA). Before the start of the experiment, 1000 μ L of FSW-HPTS was gently extracted. During pH imaging, a mixed solution of 1000 μ L of acidified HPTS (1 mM, pH 7.0-7.2) and Alexa Fluor ® 568 (20 μ M) was added 4 min after the start of recording to label the adding acidified FSW-HPTS. The dual-excitation ratiometric indicator HPTS was excited at wavelengths of 405 and 440 nm, and fluorescence was detected at 510-530 nm. Alexa Fluor ® 568 was excited at 561 nm and fluorescence was detected at 610-640 nm 1 s after HPTS fluorescence was detected. Intervals were 1 min apart. pH calibration was performed by determining the ratio of HPTS fluorescence in FSW containing 1-mM HPTS adjusted with HCl and NaOH to the range of pH 6-9 at 25.0 ± 0.1 °C (Fig. S1). We could not find spectral shift of HPTS between 25 °C and 27 °C using spectro-photometer (Jasco Corporation, Tokyo, Japan). Ratio images that fitted the pH values were created using NIS Elements software. ImageJ (Rasband, WS. ImageJ. US National Institutes of Health, Bethesda, Maryland, USA. http://imagej.nih.gov/ij/; 1997-2014) was used to measure the velocity of pH waves.