Oxygen and Heterotrophy Affect Calcification of the Scleractinian Coral Galaxea fascicularis

Heterotrophy is known to stimulate calcification of scleractinian corals, possibly through enhanced organic matrix synthesis and photosynthesis, and increased supply of metabolic DIC. In contrast to the positive long-term effects of heterotrophy, inhibition of calcification has been observed during feeding, which may be explained by a temporal oxygen limitation in coral tissue. To test this hypothesis, we measured the short-term effects of zooplankton feeding on light and dark calcification rates of the scleractinian coral Galaxea fascicularis (n = 4) at oxygen saturation levels ranging from 13 to 280%. Significant main and interactive effects of oxygen, heterotrophy and light on calcification rates were found (three-way factorial repeated measures ANOVA, p<0.05). Light and dark calcification rates of unfed corals were severely affected by hypoxia and hyperoxia, with optimal rates at 110% saturation. Light calcification rates of fed corals exhibited a similar trend, with highest rates at 150% saturation. In contrast, dark calcification rates of fed corals were close to zero under all oxygen saturations. We conclude that oxygen exerts a strong control over light and dark calcification rates of corals, and propose that in situ calcification rates are highly dynamic. Nevertheless, the inhibitory effect of heterotrophy on dark calcification appears to be oxygen-independent. We hypothesize that dark calcification is impaired during zooplankton feeding by a temporal decrease of the pH and aragonite saturation state of the calcifying medium, caused by increased respiration rates. This may invoke a transient reallocation of metabolic energy to soft tissue growth and organic matrix synthesis. These insights enhance our understanding of how oxygen and heterotrophy affect coral calcification, both in situ as well as in aquaculture.     

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 alpha-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.     

Comprehensive characterization of skeletal tissue growth anomalies of the finger coral Porites compressa

The scleractinian finger coral Porites compressa has been documented to develop raised growth anomalies of unknown origin, commonly referred to as “tumors”. These skeletal tissue anomalies (STAs) are circumscribed nodule-like areas of enlarged skeleton and tissue with fewer polyps and zooxanthellae than adjacent tissue. A field survey of the STA prevalence in Oahu, Kaneohe Bay, Hawaii, was complemented by laboratory analysis to reveal biochemical, histological and skeletal differences between anomalous and reference tissue. MutY, Hsp90a1, GRP75 and metallothionein, proteins known to be up-regulated in hyperplastic tissues, were over expressed in the STAs compared to adjacent normal-appearing and reference tissues. Histological analysis was further accompanied by elemental and micro-structural analyses of skeleton. Anomalous skeleton was of similar aragonite composition to adjacent skeleton but more porous as evidenced by an increased rate of vertical extension without thickening. Polyp structure was retained throughout the lesion, but abnormal polyps were hypertrophied, with increased mass of aboral tissue lining the skeleton, and thickened areas of skeletogenic calicoblastic epithelium along the basal floor. The latter were highly metabolically active and infiltrated with chromophore cells. These observations qualify the STAs as hyperplasia and are the first report in poritid corals of chromophore infiltration processes in active calicoblastic epithelium areas.     

Calcification process dynamics in coral primary polyps as observed using a calcein incubation method

Calcification processes are largely unknown in scleractinian corals. In this study, live confocal imaging was used to elucidate the spatiotemporal dynamics of the calcification process in aposymbiotic primary polyps of the coral species Acropora digitifera. The fluorophore calcein was used as a calcium deposition marker and a visible indicator of extracellular fluid distribution at the tissue-skeleton interface (subcalicoblastic medium, SCM) in primary polyp tissues. Under continuous incubation in calcein-containing seawater, initial crystallization and skeletal growth were visualized among the calicoblastic cells in live primary polyp tissues. Additionally, the distribution of calcein-stained SCM and contraction movements of the pockets of SCM were captured at intervals of a few minutes. Our experimental system provided several new insights into coral calcification, particularly as a first step in monitoring the relationship between cellular dynamics and calcification in vivo. Our study suggests that coral calcification initiates at intercellular spaces, a finding that may contribute to the general understanding of coral calcification processes.     

Effects of variations in carbonate chemistry on the calcification rates of Madracis auretenra (= Madracis mirabilis sensu Wells, 1973): bicarbonate concentrations best predict calcification rates

Physiological data and models of coral calcification indicate that corals utilize a combination of seawater bicarbonate and (mainly) respiratory CO₂ for calcification, not seawater carbonate. However, a number of investigators are attributing observed negative effects of experimental seawater acidification by CO₂ or hydrochloric acid additions to a reduction in seawater carbonate ion concentration and thus aragonite saturation state. Thus, there is a discrepancy between the physiological and geochemical views of coral biomineralization. Furthermore, not all calcifying organisms respond negatively to decreased pH or saturation state. Together, these discrepancies suggest that other physiological mechanisms, such as a direct effect of reduced pH on calcium or bicarbonate ion transport and/or variable ability to regulate internal pH, are responsible for the variability in reported experimental effects of acidification on calcification. To distinguish the effects of pH, carbonate concentration and bicarbonate concentration on coral calcification, incubations were performed with the coral Madracis auretenra (= Madracis mirabilis sensu Wells, 1973) in modified seawater chemistries. Carbonate parameters were manipulated to isolate the effects of each parameter more effectively than in previous studies, with a total of six different chemistries. Among treatment differences were highly significant. The corals responded strongly to variation in bicarbonate concentration, but not consistently to carbonate concentration, aragonite saturation state or pH. Corals calcified at normal or elevated rates under low pH (7.6–7.8) when the seawater bicarbonate concentrations were above 1800 μm . Conversely, corals incubated at normal pH had low calcification rates if the bicarbonate concentration was lowered. These results demonstrate that coral responses to ocean acidification are more diverse than currently thought, and question the reliability of using carbonate concentration or aragonite saturation state as the sole predictor of the effects of ocean acidification on coral calcification.     

Similar controls on calcification under ocean acidification across unrelated coral reef taxa

Ocean acidification (OA) is a major threat to marine ecosystems, particularly coral reefs which are heavily reliant on calcareous species. OA decreases seawater pH and calcium carbonate saturation state (Ω), and increases the concentration of dissolved inorganic carbon (DIC). Intense scientific effort has attempted to determine the mechanisms via which ocean acidification (OA) influences calcification, led by early hypotheses that calcium carbonate saturation state (Ω) is the main driver. We grew corals and coralline algae for 8–21 weeks, under treatments where the seawater parameters Ω, pH, and DIC were manipulated to examine their differential effects on calcification rates and calcifying fluid chemistry (Ω_cf, pH_cf, and DIC_cf). Here, using long duration experiments, we provide geochemical evidence that differing physiological controls on carbonate chemistry at the site of calcification, rather than seawater Ω, are the main determinants of calcification. We found that changes in seawater pH and DIC rather than Ω had the greatest effects on calcification and calcifying fluid chemistry, though the effects of seawater carbonate chemistry were limited. Our results demonstrate the capacity of organisms from taxa with vastly different calcification mechanisms to regulate their internal chemistry under extreme chemical conditions. These findings provide an explanation for the resistance of some species to OA, while also demonstrating how changes in seawater DIC and pH under OA influence calcification of key coral reef taxa.     

Light-enhanced calcification and dark decalcification in isolates of the soft coral Cladiella sp. during tissue recovery

Light-enhanced calcification is a general characteristic of zooxanthellate corals, suggesting a link between calcification by the coral and photosynthesis by the zooxanthellae, but the relationship between zooxanthellae and coral hosts during this process has not been elucidated. We hypothesized that the effects of tissue injury on the coral fragments used in experiments studying calcification might obscure that link. To detect the effects of tissue injury on light-enhanced calcification, we measured calcification rates (sclerite formation) in the soft coral Cladiella sp. by the alkalinity anomaly method during a 36-day experiment following injury associated with coral fragmentation. In the 2 weeks after colony fragmentation, the calcification response did not show a relation with light intensity. The typical light-enhanced calcification pattern was not noticed until day 15 of tissue recovery. The calcification rate of this soft coral increased with light intensity and time of tissue recovery and was comparable to that of hard corals exposed to similar experimental conditions. However, Cladiella sp. decalcified in the dark. The diurnal calcification-decalcification cycles probably control sclerite size and shape.     

Wound repair in Montipora capitata

We documented the microscopic morphology of tissue healing in Montipora capitata. Fragments from two healthy coral colonies were traumatized by scraping tissue and skeleton and monitored in flow-through seawater tables every 2-4 days for 40 days for gross and cellular changes. Grossly, corals appeared healed and repigmented by Day 40. Histologically, traumatized issues were undistinguishable from intact untraumatized tissues by Day 12. We suspect that the calicoblastic epidermis of basal body wall is pluripotential and can develop into surface epidermis when needed.     

Dark calcification and the daily rhythm of calcification in the scleractinian coral, <Emphasis Type="Italic">Galaxea fascicularis</Emphasis>

The rate of calcification in the scleractinian coral Galaxea fascicularis was followed during the daytime using ⁴⁵Ca tracer. The coral began the day with a low calcification rate, which increased over time to a maximum in the afternoon. Since the experiments were carried out under a fixed light intensity, these results suggest that an intrinsic rhythm exists in the coral such that the calcification rate is regulated during the daytime. When corals were incubated for an extended period in the dark, the calcification rate was constant for the first 4 h of incubation and then declined, until after one day of dark incubation, calcification ceased, possibly as a result of the depletion of coral energy reserves. The addition of glucose and Artemia reduced the dark calcification rate for the short duration of the experiment, indicating an expenditure of oxygen in respiration. Artificial hypoxia reduced the rate of dark calcification to about 25% compared to aerated coral samples. It is suggested that G. fascicularis obtains its oxygen needs from the surrounding seawater during the nighttime, whereas during the day time the coral exports oxygen to the seawater.     

Effects of increased pCO2 on zinc uptake and calcification in the tropical coral Stylophora pistillata

Zinc (Zn) is an essential element for corals. We investigated the effects of ocean acidification on zinc incorporation, photosynthesis, and gross calcification in the scleractinian coral Stylophora pistillata. Colonies were maintained at normal pH_T (8.1) and at two low-pH conditions (7.8 and 7.5) for 5 weeks. Corals were exposed to ⁶⁵Zn dissolved in seawater to assess uptake rates. After 5 weeks, corals raised at pH_T (8.1) exhibited higher ⁶⁵Zn activity in the coral tissue and skeleton, compared with corals raised at a lower pH. Photosynthesis, photosynthetic efficiency, and gross calcification, measured by ⁴⁵Ca incorporation, were however unchanged even at the lowest pH.     

Low temperature FESEM of the calcifying interface of a scleractinian coral

The ultrastructural nature of the calcifying interface in the scleractinian coral Galaxea fascicularis has been investigated using high-resolution, low temperature field emission scanning electron microscopy (FESEM). This technique permitted structural analyses of soft tissue and skeleton in G. fascicularis in a frozen-hydrated state, without the need for chemical fixation or decalcification. Structural comparisons are made between frozen-hydrated polyps and polyps that have undergone conventional fixation and decalcification. Vesicles expelled by the calicoblastic ectodermal cells into sub-skeletal spaces and previously suggested to play a role in calcification were commonly observed in fixed samples but were distinctly absent in frozen-hydrated preparations. We propose that these vesicles are fixation artefacts. Two distinct types of vesicles (380 and 70 nm in diameter, respectively), were predominant throughout the calicoblastic ectodermal cells of frozen-hydrated preparations, but these were never seen to be entering, or to be contained within, sub-skeletal spaces, nor did they contain any crystalline material. In frozen-hydrated preparations, membranous sheets were seen to surround and isolate portions of aboral mesogloea and to form junctional complexes with calicoblastic cells. The calicoblastic ectoderm was closely associated with the underlying skeleton, with sub-skeletal spaces significantly smaller (P<0.0001) in frozen-hydrated polyps compared to fixed polyps. A network of organic filaments (26 nm in diameter) extended from the apical membranes of calicoblastic cells into these small sub-skeletal cavities. A thin sheath was also frequently observed adjacent to the apical membrane of calicoblastic cells.     

The calcifying interface in a stony coral primary polyp: An interplay between seawater and an extracellular calcifying space

Stony coral exoskeletons build the foundation for the most biologically diverse marine ecosystems on Earth, coral reefs, which face major threats due to many anthropogenic–related stressors. Therefore, understanding coral biomineralization mechanisms is crucial for coral reef management in the coming decades and for using coral skeletons in geochemical studies. This study combines in–vivo imaging with cryo-electron microscopy and cryo–elemental mapping to gain novel insights into the biological microenvironment and the ion pathways that facilitate biomineralization in primary polyps of the stony coral Stylophora pistillata. We document increased tissue permeability in the primary polyp and a highly dispersed cell packing in the tissue directly responsible for producing the coral skeleton. This tissue arrangement may facilitate the intimate involvement of seawater at the mineralization site, also documented here. We further observe an extensive filopodial network containing carbon-rich vesicles extruding from some of the calicoblastic cells. Single-cell RNA-Sequencing data interrogation supports these morphological observations by showing higher expression of genes involved in filopodia and vesicle structure and function in the calicoblastic cells. These observations provide a new conceptual framework for resolving the ion pathway from the external seawater to the tissue-mineral interface in stony coral biomineralization processes.     

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.     

A novel mechanism for iron incorporation into coral skeletons

Intertidal corals living in seawater with high concentrations of iron incorporate the metal into their skeletons. Cross-sections of the coral skeleton reveal orange-stained banding patterns reflecting periods of high availability of iron. The mechanism of metal incorporation involves deposition of iron compounds on to skeletal spines that are exposed as a result of temporary tissue retraction during periods of extreme stress. Subsequent tissue recovery and calcification trap the iron compounds which provide a visible environmental signature in the coral skeleton. This previously unrecognised mechanism has significant implications for the reconstruction of past environments from chemical analysis of annually-banded massive coral skeletons.     

A coral polyp model of photosynthesis, respiration and calcification incorporating a transcellular ion transport mechanism

A numerical simulation model of coral polyp photosynthesis, respiration and calcification was developed. The model is constructed with three components (ambient seawater, coelenteron and calcifying fluid), and incorporates photosynthesis, respiration and calcification processes with transcellular ion transport by Ca-ATPase activity and passive transmembrane CO₂ transport and diffusion. The model calculates dissolved inorganic carbon and total alkalinity in the ambient seawater, coelenteron and calcifying fluid, dissolved oxygen (DO) in the seawater and coelenteron and stored organic carbon (CH₂O). To reconstruct the drastic variation between light and dark respiration, respiration rate dependency on DO in the coelenteron is incorporated. The calcification rate depends on the aragonite saturation state in the calcifying fluid (Ωa _cal). Our simulation result was a good approximation of “light-enhanced calcification.” In our model, the mechanism is expressed as follows: (1) DO in the coelenteron is increased by photosynthesis, (2) respiration is stimulated by increased DO in the light (or respiration is limited by DO depletion in the dark), then (3) calcification increases due to Ca-ATPase, which is driven by the energy generated by respiration. The model simulation results were effective in reproducing the basic responses of the internal CO₂ system and DO. The daily calcification rate, the gross photosynthetic rate and the respiration rate under a high-flow condition increased compared to those under the zero-flow condition, but the net photosynthetic rate decreased. The calculated calcification rate responses to variations in the ambient aragonite saturation state (Ωa _amb) were nonlinear, and the responses agreed with experimental results of previous studies. Our model predicted that in response to ocean acidification (1) coral calcification will decrease, but will remain at a higher value until Ωa _amb decreases to 1, by maintaining a higher Ωa _cal due to the transcellular ion transport mechanism and (2) the net photosynthetic rate will increase.     

Antibodies against the organic matrix in scleractinians: a new tool to study coral biomineralization

Soluble organic matrix (SOM) synthesis and secretion were investigated in two scleractinian corals using antibodies raised against this organic matrix. Results demonstrate that even if other cell types, including zooxanthellae, can supply precursors for SOM synthesis, only calicoblastic cells facing the skeleton are directly responsible for the synthesis and secretion of the SOM components. Results also indicate that, as is the case for other biominerals, skeleton formation is biologically controlled and not chemically dominated as originally believed. In addition to advancing the understanding of mechanisms of coral biomineralization, these antibodies could have numerous applications: for example as markers of skeletogenesis, as tools for cell culture, and in comparative studies among calcifying organisms.     

Sponge erosion under acidification and warming scenarios: differential impacts on living and dead coral

Ocean acidification will disproportionately impact the growth of calcifying organisms in coral reef ecosystems. Simultaneously, sponge bioerosion rates have been shown to increase as seawater pH decreases. We conducted a 20‐week experiment that included a 4‐week acclimation period with a high number of replicate tanks and a fully orthogonal design with two levels of temperature (ambient and +1 °C), three levels of pH (8.1, 7.8, and 7.6), and two levels of boring sponge (Cliona varians, present and absent) to account for differences in sponge attachment and carbonate change for both living and dead coral substrate (Porites furcata). Net coral calcification, net dissolution/bioerosion, coral and sponge survival, sponge attachment, and sponge symbiont health were evaluated. Additionally, we used the empirical data from the experiment to develop a stochastic simulation of carbonate change for small coral clusters (i.e., simulated reefs). Our findings suggest differential impacts of temperature, pH and sponge presence for living and dead corals. Net coral calcification (mg CaCO₃ cm^?2 day^?1) was significantly reduced in treatments with increased temperature (+1 °C) and when sponges were present; acidification had no significant effect on coral calcification. Net dissolution of dead coral was primarily driven by pH, regardless of sponge presence or seawater temperature. A reevaluation of the current paradigm of coral carbonate change under future acidification and warming scenarios should include ecologically relevant timescales, species interactions, and community organization to more accurately predict ecosystem‐level response to future conditions.     

The nature of the skeleton and skeletogenic tissues in the Cnidaria

Calcification of zooxanthellate and non-zooxanthellate corals from 2 classes and 3 orders of Cnidaria was investigated using scanning and transmission electron microscopy and light microscopy. The ultrastructure of the skeleton and skeletogenic tissues (the calicoblastic ectoderm) from areas of active and non-active skeleton deposition were investigated. The results show that the fundamental cellular mechanism of calcification is similar in all 3 orders, and that the role of endosymbiotic zooxanthellae may be one that is concerned with the removal of waste products of the calcification process. The results are discussed with respect to the concepts of calcification and its evolution in the Cnidaria.     

Microsensor study of photosynthesis and calcification in the scleractinian coral, Galaxea fascicularis: active internal carbon cycle

Sources of inorganic carbon (Ci) for photosynthesis and calcification and the mechanisms involved in their uptake in scleractinian corals were investigated in microcolonies of Galaxea fascicularis. Direct measurements of Ca²⁺, pH and O₂ on the surface and inside the polyp's coelenteron were made with microsensors. Gross photosynthesis (Pg) and net photosynthesis (Pn) were measured on the surface. Light respiration (LR) was calculated from Pg and Pn. The effect of light/dark and dark/light switches on Ca²⁺ and pH dynamics on the surface and inside the coelenteron were followed. To evaluate the different sources of Ci for photosynthesis and calcification, Ci-free seawater and 6-Ethoxyzolamide and Acetazolamide, inhibitors for carbonic anhydrase (CA) were used.In normal seawater, Pg was about seven times higher than Pn, the LR was ca. 80-90% of the Pg. Thus, most of the O₂ produced in Pg are immediately consumed in respiration, indicating the presence of a highly active internal C-cycle. As the internal C-cycle is highly active, a large part of the Ci for calcification will have passed through the metabolism of the symbiont. The high LR provides ATP for energy requiring processes in light.Ci for photosynthesis and calcification can come from seawater in the form of free Ci, respiration of photosynthates (internal C-cycle) or respiration of the ingested plankton. These sources form a common carbon pool (C-pool) that is used for the different processes.In Ci-free seawater, Pg decreased by about 12.5%, indicating that most of the photosynthetically fixed Ci can temporarily be supplied from internal sources. The initial decalcification, observed directly upon the switch to Ci-free seawater, showed that the Ca-pools in the coral are exchangeable. Part of the Pg in Ci-free seawater may depend on this decalcification for its Ci supply.Three localities of CA were defined. One on the surface facing seawater and one on endodermal cells facing the coelenteron, while the third is intracellular. The inhibition of CA decreased Pg by about 30%, while it increased the concentration of Ca²⁺ as a result of a decrease in its precipitation. The reduction of photosynthesis and calcification by CA inhibition demonstrated that both processes need the enzyme for the supply of Ci. The pH on the surface and inside the coelenteron decreased upon 6-Ethoxyzolamide addition indicating a role of CA in pH control.     

Confocal laser scanning light microscopy of the extra-thecal epithelia of undecalcified scleractinian corals

The extra-thecal epithelia of cryofixed undecalcified, freeze-substituted polyps of the scleractinian corals Galaxea fascicularis and Tubastrea faulkneri and axial and basal polyps of Acropora formosa have been examined, in anhydrously prepared thick slices, by confocal laser scanning light microscopy. The avoidance of chemical fixation and decalcification makes it possible to determine whether previously seen structures are real or artefactual products of swelling, shrinkage and distortion. All of the epithelia of all the corals examined are characterised by well defined intercellular spaces. Mucocytes are present in all cell layers in Galaxea and Tubastrea but are not present in any cell layers in the axial polyp of Acropora although they are abundant in the oral ectoderm of the basal polyps in this coral. Zooxanthellae are absent in Tubastrea, the epithelia of the exert septa of Galaxea and the axial polyp of Acropora. The calicoblastic ectoderm is generally composed of thin squamous cells with large intercellular spaces. At rapidly calcifying regions such as the tips of the exert septa of Galaxea, the calicoblastic cells are elongated with extensive arborisation of the basal regions of the cells. They are separated by large intercellular spaces and contain numerous fluorescent granules. The apical regions of these cells appear to be closely applied to the surface of the skeleton. There is no evidence of a space between the apical region of the calicoblastic cells and the skeleton.