Myriad Mapping of nanoscale minerals reveals calcium carbonate hemihydrate in forming nacre and coral biominerals.

Calcium carbonate (CaCO3) is abundant on Earth, is a major component of marine biominerals and thus of sedimentary and metamorphic rocks and it plays a major role in the global carbon cycle by storing atmospheric CO2 into solid biominerals. Six crystalline polymorphs of CaCO3 are known-3 anhydrous: calcite, aragonite, vaterite, and 3 hydrated: ikaite (CaCO3·6H2O), monohydrocalcite (CaCO3·1H2O, MHC), and calcium carbonate hemihydrate (CaCO3·½H2O, CCHH). CCHH was recently discovered and characterized, but exclusively as a synthetic material, not as a naturally occurring mineral. Here, analyzing 200 million spectra with Myriad Mapping (MM) of nanoscale mineral phases, we find CCHH and MHC, along with amorphous precursors, on freshly deposited coral skeleton and nacre surfaces, but not on sea urchin spines. Thus, biomineralization pathways are more complex and diverse than previously understood, opening new questions on isotopes and climate. Crystalline precursors are more accessible than amorphous ones to other spectroscopies and diffraction, in natural and bio-inspired materials.

Spatial variability of and effect of light on the cœlenteron pH of a reef coral.

Coral reefs, the largest bioconstruction on Earth, are formed by calcium carbonate skeletons of corals. Coral skeleton formation commonly referred to as calcification occurs in a specific compartment, the extracellular calcifying medium (ECM), located between the aboral ectoderm and the skeleton. Calcification models often assume a direct link between the surrounding seawater and the ECM. However, the ECM is separated from the seawater by several tissue layers and the cœlenteron, which contains the cœlenteric fluid found in both polyps and cœnosarc (tissue connecting the polyps). Symbiotic dinoflagellate-containing cells line the cœlenteron and their photosynthetic activity contributes to changes in the chemistry of the cœlenteric fluid, particularly with respect to pH. The aim of our study is to compare cœlenteron pH between the cœnosarc and polyps and to compare areas of high or low dinoflagellate density based on tissue coloration. To achieve this, we use liquid ion exchange (LIX) pH microsensors to profile pH in the cœlenteron of polyps and the cœnosarc in different regions of the coral colony in light and darkness. We interpret our results in terms of what light and dark exposure means for proton gradients between the ECM and the coelenteron, and how this could affect calcification.

An Increase of Seawater Temperature Upregulates the Expression of Vibrio parahaemolyticus Virulence Factors Implicated in Adhesion and Biofilm Formation.

Climate change driven seawater temperature (SWT) increases results in greater abundance and geographical expansion of marine pathogens, among which Vibrio parahaemolyticus (Vp) causes serious economic and health issues. In addition, plastic pollution in the ocean constitutes a vector for harmful pathogens dissemination. We investigate the effect of elevated SWT on the expression of genes implicated in adhesion and biofilm formation on abiotic surfaces in the clinical Vp strain RIMD2210633, which expresses hemolysins. Among the genes studied, the multivalent adhesion molecule-7 and the GlcNAc-binding protein A were involved in the adhesion of Vp to abiotic and biotic surfaces, whereas the type IV pili, the mannose-sensitive hemagglutinin, and the chitin-regulated pilins facilitate attachment and biofilm formation. Data presented here show that at 21°C, Vp is still viable but does not either proliferate or express the virulence factors studied. Interestingly, at 27°C and as early as 1 h of incubation, all factors are transiently expressed in free-living bacteria only and even more upregulated at 31°C. These results clearly show that increased SWT has an important impact on the adhesion properties of free-living Vp to plastic support and thus emphasize the role of climate change in the spread of this pathogenic bacteria.

Faster Crystallization during Coral Skeleton Formation Correlates with Resilience to Ocean Acidification.

The mature skeletons of hard corals, termed stony or scleractinian corals, are made of aragonite (CaCO3). During their formation, particles attaching to the skeleton's growing surface are calcium carbonate, transiently amorphous. Here we show that amorphous particles are observed frequently and reproducibly just outside the skeleton, where a calicoblastic cell layer envelops and deposits the forming skeleton. The observation of particles in these locations, therefore, is consistent with nucleation and growth of particles in intracellular vesicles. The observed extraskeletal particles range in size between 0.2 and 1.0 µm and contain more of the amorphous precursor phases than the skeleton surface or bulk, where they gradually crystallize to aragonite. This observation was repeated in three diverse genera of corals, Acropora sp., Stylophora pistillata─differently sensitive to ocean acidification (OA)─and Turbinaria peltata, demonstrating that intracellular particles are a major source of material during the additive manufacturing of coral skeletons. Thus, particles are formed away from seawater, in a presumed intracellular calcifying fluid (ICF) in closed vesicles and not, as previously assumed, in the extracellular calcifying fluid (ECF), which, unlike ICF, is partly open to seawater. After particle attachment, the growing skeleton surface remains exposed to ECF, and, remarkably, its crystallization rate varies significantly across genera. The skeleton surface layers containing amorphous pixels vary in thickness across genera: ∼2.1 µm in Acropora, 1.1 µm in Stylophora, and 0.9 µm in Turbinaria. Thus, the slow-crystallizing Acropora skeleton surface remains amorphous and soluble longer, including overnight, when the pH in the ECF drops. Increased skeleton surface solubility is consistent with Acropora's vulnerability to OA, whereas the Stylophora skeleton surface layer crystallizes faster, consistent with Stylophora's resilience to OA. Turbinaria, whose response to OA has not yet been tested, is expected to be even more resilient than Stylophora, based on the present data.

Telomere dysfunction is associated with dark-induced bleaching in the reef coral Stylophora pistillata.

Telomere DNA length is a complex trait controlled by both multiple loci and environmental factors. A growing number of studies are focusing on the impact of stress and stress accumulation on telomere length and the link with survival and fitness in ecological contexts. Here, we investigated the telomere changes occurring in a symbiotic coral, Stylophora pistillata, that has experienced continuous darkness over 6 months. This stress condition led to the loss of its symbionts in a similar manner to that observed during large-scale bleaching events due to climate changes and anthropogenic activities, threatening reef ecosystems worldwide. We found that continuous darkness was associated with telomere length shortening. This result, together with a phylogenetic analysis of the telomere coral proteins and a transcriptome survey of the continuous darkness condition, paves the way for future studies on the role of telomeres in the coral stress response and the importance of environmentally induced telomere shortening in endangered coral species.

Effects of Ocean Acidification on Resident and Active Microbial Communities of Stylophora pistillata.

Ocean warming and ocean acidification (OA) are direct consequences of climate change and affect coral reefs worldwide. While the effect of ocean warming manifests itself in increased frequency and severity of coral bleaching, the effects of ocean acidification on corals are less clear. In particular, long-term effects of OA on the bacterial communities associated with corals are largely unknown. In this study, we investigated the effects of ocean acidification on the resident and active microbiome of long-term aquaria-maintained Stylophora pistillata colonies by assessing 16S rRNA gene diversity on the DNA (resident community) and RNA level (active community). Coral colony fragments of S. pistillata were kept in aquaria for 2 years at four different pCO2 levels ranging from current pH conditions to increased acidification scenarios (i.e., pH 7.2, 7.4, 7.8, and 8). We identified 154 bacterial families encompassing 2,047 taxa (OTUs) in the resident and 89 bacterial families including 1,659 OTUs in the active communities. Resident communities were dominated by members of Alteromonadaceae, Flavobacteriaceae, and Colwelliaceae, while active communities were dominated by families Cyclobacteriacea and Amoebophilaceae. Besides the overall differences between resident and active community composition, significant differences were seen between the control (pH 8) and the two lower pH treatments (7.2 and 7.4) in the active community, but only between pH 8 and 7.2 in the resident community. Our analyses revealed profound differences between the resident and active microbial communities, and we found that OA exerted stronger effects on the active community. Further, our results suggest that rDNA- and rRNA-based sequencing should be considered complementary tools to investigate the effects of environmental change on microbial assemblage structure and activity.

New Insights From Transcriptomic Data Reveal Differential Effects of CO2 Acidification Stress on Photosynthesis of an Endosymbiotic Dinoflagellate in hospite.

Ocean acidification (OA) has both detrimental as well as beneficial effects on marine life; it negatively affects calcifiers while enhancing the productivity of photosynthetic organisms. To date, many studies have focused on the impacts of OA on calcification in reef-building corals, a process particularly susceptible to acidification. However, little is known about the effects of OA on their photosynthetic algal partners, with some studies suggesting potential benefits for symbiont productivity. Here, we investigated the transcriptomic response of the endosymbiont Symbiodinium microadriaticum (CCMP2467) in the Red Sea coral Stylophora pistillata subjected to different long-term (2 years) OA treatments (pH 8.0, 7.8, 7.4, 7.2). Transcriptomic analyses revealed that symbionts from corals under lower pH treatments responded to acidification by increasing the expression of genes related to photosynthesis and carbon-concentrating mechanisms. These processes were mostly up-regulated and associated metabolic pathways were significantly enriched, suggesting an overall positive effect of OA on the expression of photosynthesis-related genes. To test this conclusion on a physiological level, we analyzed the symbiont's photochemical performance across treatments. However, in contrast to the beneficial effects suggested by the observed gene expression changes, we found significant impairment of photosynthesis with increasing pCO2. Collectively, our data suggest that over-expression of photosynthesis-related genes is not a beneficial effect of OA but rather an acclimation response of the holobiont to different water chemistries. Our study highlights the complex effects of ocean acidification on these symbiotic organisms and the role of the host in determining symbiont productivity and performance.

A Cystine-Cysteine Intercellular Shuttle Prevents Ferroptosis in xCTKO Pancreatic Ductal Adenocarcinoma Cells.

In our previous study, we showed that a cystine transporter (xCT) plays a pivotal role in ferroptosis of pancreatic ductal adenocarcinoma (PDAC) cells in vitro. However, in vivo xCTKO cells grew normally indicating that a mechanism exists to drastically suppress the ferroptotic phenotype. We hypothesized that plasma and neighboring cells within the tumor mass provide a source of cysteine to confer full ferroptosis resistance to xCTKO PDAC cells. To evaluate this hypothesis, we (co-) cultured xCTKO PDAC cells with different xCT-proficient cells or with their conditioned media. Our data unequivocally showed that the presence of a cysteine/cystine shuttle between neighboring cells is the mechanism that provides redox and nutrient balance, and thus ferroptotic resistance in xCTKO cells. Interestingly, although a glutathione shuttle between cells represents a good alternative hypothesis as a "rescue-mechanism", our data clearly demonstrated that the xCTKO phenotype is suppressed even with conditioned media from cells lacking the glutathione biosynthesis enzyme. Furthermore, we demonstrated that prevention of lipid hydroperoxide accumulation in vivo is mediated by import of cysteine into xCTKO cells via several genetically and pharmacologically identified transporters (ASCT1, ASCT2, LAT1, SNATs). Collectively, these data highlight the importance of the tumor environment in the ferroptosis sensitivity of cancer cells.

The Evolution of Calcification in Reef-Building Corals.

Corals build the structural foundation of coral reefs, one of the most diverse and productive ecosystems on our planet. Although the process of coral calcification that allows corals to build these immense structures has been extensively investigated, we still know little about the evolutionary processes that allowed the soft-bodied ancestor of corals to become the ecosystem builders they are today. Using a combination of phylogenomics, proteomics, and immunohistochemistry, we show that scleractinian corals likely acquired the ability to calcify sometime between ∼308 and ∼265 Ma through a combination of lineage-specific gene duplications and the co-option of existing genes to the calcification process. Our results suggest that coral calcification did not require extensive evolutionary changes, but rather few coral-specific gene duplications and a series of small, gradual optimizations of ancestral proteins and their co-option to the calcification process.

A role for primary cilia in coral calcification?

Cilia are evolutionarily conserved organelles that extend from the surface of cells and are found in diverse organisms from protozoans to multicellular organisms. Motile cilia play various biological functions by their beating motion, including mixing fluids and transporting food particles. Non-motile cilia act as sensors that signal cells about their microenvironment. In corals, cilia have been described in some of the cell layers but never in the calcifying epithelium, which is responsible for skeleton formation. In the present study, we used scanning electron microscopy and immunolabelling to investigate the cellular ciliature of the different tissue layers of the coral Stylophora pistillata, with a focus on the calcifying calicoblastic ectoderm. We show that the cilium of the calcifying cells is different from the cilium of the other cell layers. It is much shorter, and more importantly, its base is structurally distinct from the base observed in cilia of the other tissue layers. Based on these structural observations, we conclude that the cilium of the calcifying cells is a primary cilium. From what is known in other organisms, primary cilia are sensors that signal cells about their microenvironment. We discuss the implications of the presence of a primary cilium in the calcifying epithelium for our understanding of the cellular physiology driving coral calcification and its environmental sensitivity.

Paracellular transport to the coral calcifying medium: effects of environmental parameters.

Coral calcification relies on the transport of ions and molecules to the extracellular calcifying medium (ECM). Little is known about paracellular transport (via intercellular junctions) in corals and other marine calcifiers. Here, we investigated whether the permeability of the paracellular pathway varied in different environmental conditions in the coral Stylophora pistillata Using the fluorescent dye calcein, we characterised the dynamics of calcein influx from seawater to the ECM and showed that increases in paracellular permeability (leakiness) induced by hyperosmotic treatment could be detected by changes in calcein influx rates. We then used the calcein-imaging approach to investigate the effects of two environmental stressors on paracellular permeability: seawater acidification and temperature change. Under conditions of seawater acidification (pH 7.2) known to depress pH in the ECM and the calcifying cells of S. pistillata, we observed a decrease in half-times of calcein influx, indicating increased paracellular permeability. By contrast, high temperature (31°C) had no effect, whereas low temperature (20°C) caused decreases in paracellular permeability. Overall, our study establishes an approach to conduct further in vivo investigation of paracellular transport and suggests that changes in paracellular permeability could form an uncharacterised aspect of the physiological response of S. pistillata to seawater acidification.

Ubiquitous macropinocytosis in anthozoans.

Transport of fluids, molecules, nutrients or nanoparticles through coral tissues are poorly documented. Here, we followed the flow of various tracers from the external seawater to within the cells of all tissues in living animals. After entering the general coelenteric cavity, we show that nanoparticles disperse throughout the tissues via the paracellular pathway. Then, the ubiquitous entry gate to within the cells' cytoplasm is macropinocytosis. Most cells form large vesicles of 350-600 nm in diameter at their apical side, continuously internalizing their surrounding medium. Macropinocytosis was confirmed using specific inhibitors of PI3K and actin polymerization. Nanoparticle internalization dynamics is size dependent and differs between tissues. Furthermore, we reveal that macropinocytosis is likely a major endocytic pathway in other anthozoan species. The fact that nearly all cells of an animal are continuously soaking in the environment challenges many aspects of the classical physiology viewpoints acquired from the study of bilaterians.

Effect of salinity and temperature on the expression of genes involved in branchial ion transport processes in European sea bass.

The responses of European sea bass to temperature increase and salinity decrease were investigated measuring mRNA expression levels of main genes involved in ion transport. Juvenile fish were pre-acclimated to seawater (SW) at 18 °C (temperate) or 24 °C (warm) for two weeks and then transferred for two weeks to either fresh water (FW) or SW at the respective temperature. Unlike temperate conditions, there is no change in Na+/K+-ATPase α1a (nka α1a) and Na+/H+ exchanger 3 (nhe3) mRNA expression following FW transfer in warm conditions. This is linked to the high expression of these genes in warm SW compared to temperate SW. Na+/Cl--cotransporter (ncc2a) expression however is increased following FW transfer in temperate and warm conditions. Main transporters involved in ion excretion (Na+/K+/2Cl--1 cotransporter, nkcc1 and cystic fibrosis transmembrane conductance regulator, cftr) as well as nitrogen excretion (Rh-glycoproteins, rhcg1 and rhbg) and acid-base regulation (V-H+-ATPase, vha-a and b) are highly expressed in SW warm conditions vs FW warm. Overall, our results suggest a higher activation of ion transport processes in warm conditions and more strikingly in SW. This is linked to a strong interplay between diverse ion transporters in order to coordinate physiological responses at the gill level.

Genetic Ablation of the Cystine Transporter xCT in PDAC Cells Inhibits mTORC1, Growth, Survival, and Tumor Formation via Nutrient and Oxidative Stresses.

Although chemoresistance remains a primary challenge in the treatment of pancreatic ductal adenocarcinoma (PDAC), exploiting oxidative stress might offer novel therapeutic clues. Here we explored the potential of targeting cystine/glutamate exchanger (SLC7A11/xCT), which contributes to the maintenance of intracellular glutathione (GSH). Genomic disruption of xCT via CRISPR-Cas9 was achieved in two PDAC cell lines, MiaPaCa-2 and Capan-2, and xCT-KO clones were cultivated in the presence of N-acetylcysteine. Although several cystine/cysteine transporters have been identified, our findings demonstrate that, in vitro, xCT plays the major role in intracellular cysteine balance and GSH biosynthesis. As a consequence, both xCT-KO cell lines exhibited amino acid stress with activation of GCN2 and subsequent induction of ATF4, inhibition of mTORC1, proliferation arrest, and cell death. Tumor xenograft growth was delayed but not suppressed in xCT-KO cells, which indicated both the key role of xCT and also the presence of additional mechanisms for cysteine homeostasis in vivo. Moreover, rapid depletion of intracellular GSH in xCT-KO cells led to accumulation of lipid peroxides and cell swelling. These two hallmarks of ferroptotic cell death were prevented by vitamin E or iron chelation. Finally, in vitro pharmacologic inhibition of xCT by low concentrations of erastin phenocopied xCT-KO and potentiated the cytotoxic effects of both gemcitabine and cisplatin in PDAC cell lines. In conclusion, our findings strongly support that inhibition of xCT, by its dual induction of nutritional and oxidative cellular stresses, has great potential as an anticancer strategy. SIGNIFICANCE: The cystine/glutamate exchanger xCT is essential for amino acid and redox homeostasis and its inhibition has potential for anticancer therapy by inducing ferroptosis.

Inhibition of the amino-acid transporter LAT1 demonstrates anti-neoplastic activity in medulloblastoma.

Most cases of medulloblastoma (MB) occur in young children. While the overall survival rate can be relatively high, current treatments combining surgery, chemo- and radiotherapy are very destructive for patient development and quality of life. Moreover, aggressive forms and recurrences of MB cannot be controlled by classical therapies. Therefore, new therapeutic approaches yielding good efficacy and low toxicity for healthy tissues are required to improve patient outcome. Cancer cells sustain their proliferation by optimizing their nutrient uptake capacities. The L-type amino acid transporter 1 (LAT1) is an essential amino acid carrier overexpressed in aggressive human cancers that was described as a potential therapeutic target. In this study, we investigated the therapeutic potential of JPH203, a LAT1-specific pharmacological inhibitor, on two independent MB cell lines belonging to subgroups 3 (HD-MB03) and Shh (DAOY). We show that while displaying low toxicity towards normal cerebral cells, JPH203 disrupts AA homeostasis, mTORC1 activity, proliferation and survival in MB cells. Moreover, we demonstrate that a long-term treatment with JPH203 does not lead to resistance in MB cells. Therefore, this study suggests that targeting LAT1 with JPH203 is a promising therapeutic approach for MB treatment.

Full in vivo characterization of carbonate chemistry at the site of calcification in corals.

Reef-building corals form their calcium carbonate skeletons within an extracellular calcifying medium (ECM). Despite the critical role of the ECM in coral calcification, ECM carbonate chemistry is poorly constrained in vivo, and full ECM carbonate chemistry has never been characterized based solely on direct in vivo measurements. Here, we measure pHECM in the growing edge of Stylophora pistillata by simultaneously using microsensors and the fluorescent dye SNARF-1, showing that, when measured at the same time and place, the results agree. We then conduct microscope-guided microsensor measurements of pH, [Ca2+], and [CO3 2-] in the ECM and, from this, determine [DIC]ECM and aragonite saturation state (Ωarag), showing that all parameters are elevated with respect to the surrounding seawater. Our study provides the most complete in vivo characterization of ECM carbonate chemistry parameters in a coral species to date, pointing to the key role of calcium- and carbon-concentrating mechanisms in coral calcification.

Epigenome-associated phenotypic acclimatization to ocean acidification in a reef-building coral.

There are increasing concerns that the current rate of climate change might outpace the ability of reef-building corals to adapt to future conditions. Work on model systems has shown that environmentally induced alterations in DNA methylation can lead to phenotypic acclimatization. While DNA methylation has been reported in corals and is thought to associate with phenotypic plasticity, potential mechanisms linked to changes in whole-genome methylation have yet to be elucidated. We show that DNA methylation significantly reduces spurious transcription in the coral Stylophora pistillata. Furthermore, we find that DNA methylation also reduces transcriptional noise by fine-tuning the expression of highly expressed genes. Analysis of DNA methylation patterns of corals subjected to long-term pH stress showed widespread changes in pathways regulating cell cycle and body size. Correspondingly, we found significant increases in cell and polyp sizes that resulted in more porous skeletons, supporting the hypothesis that linear extension rates are maintained under conditions of reduced calcification. These findings suggest an epigenetic component in phenotypic acclimatization that provides corals with an additional mechanism to cope with environmental change.

The glutamine transporter ASCT2 (SLC1A5) promotes tumor growth independently of the amino acid transporter LAT1 (SLC7A5).

The transporters for glutamine and essential amino acids, ASCT2 (solute carrier family 1 member 5, SLC1A5) and LAT1 (solute carrier family 7 member 5, SLC7A5), respectively, are overexpressed in aggressive cancers and have been identified as cancer-promoting targets. Moreover, previous work has suggested that glutamine influx via ASCT2 triggers essential amino acids entry via the LAT1 exchanger, thus activating mechanistic target of rapamycin complex 1 (mTORC1) and stimulating growth. Here, to further investigate whether these two transporters are functionally coupled, we compared the respective knockout (KO) of either LAT1 or ASCT2 in colon (LS174T) and lung (A549) adenocarcinoma cell lines. Although ASCT2KO significantly reduced glutamine import (>60% reduction), no impact on leucine uptake was observed in both cell lines. Although an in vitro growth-reduction phenotype was observed in A549-ASCT2KO cells only, we found that genetic disruption of ASCT2 strongly decreased tumor growth in both cell lines. However, in sharp contrast to LAT1KO cells, ASCT2KO cells displayed no amino acid (AA) stress response (GCN2/EIF2a/ATF4) or altered mTORC1 activity (S6K1/S6). We therefore conclude that ASCT2KO reduces tumor growth by limiting AA import, but that this effect is independent of LAT1 activity. These data were further supported by in vitro cell proliferation experiments performed in the absence of glutamine. Together these results confirm and extend ASCT2's pro-tumoral role and indicate that the proposed functional coupling model of ASCT2 and LAT1 is not universal across different cancer types.

Computing the carbonate chemistry of the coral calcifying medium and its response to ocean acidification.

Critical to determining vulnerability or resilience of reef corals to Ocean Acidification (OA) is a clearer understanding of the extent to which corals can control carbonate chemistry in their Extracellular Calcifying Medium (ECM) where the CaCO3 skeleton is produced. Here, we employ a mathematical framework to calculate ECM aragonite saturation state (Ωarag.(ECM)) and carbonate system ion concentration using measurements of calcification rate, seawater characteristics (temperature, salinity and pH) and ECM pH (pH(ECM)). Our calculations of ECM carbonate chemistry at current-day seawater pH, indicate that Ωarag.(ECM) ranges from ∼10 to 38 (mean 20.41), i.e. about 5 to 6-fold higher than seawater. Accordingly, Dissolved Inorganic Carbon (DIC) and Total Alkalinity (TA) were calculated to be around 3 times higher in the ECM than in seawater. We also assessed the effects of acidification on ECM chemical properties of the coral Stylophora pistillata. At reduced seawater pH our calculations indicate that Ωarag.(ECM) remains almost constant. DIC(ECM) and TA(ECM) gradually increase as seawater pH declines, reaching values about 5 to 6-fold higher than in seawater, respectively for DIC and TA. We propose that these ECM characteristics buffer the effect of acidification and explain why certain corals continue to produce CaCO3 even when seawater chemistry is less favourable.

Coral Carbonic Anhydrases: Regulation by Ocean Acidification.

Global change is a major threat to the oceans, as it implies temperature increase and acidification. Ocean acidification (OA) involving decreasing pH and changes in seawater carbonate chemistry challenges the capacity of corals to form their skeletons. Despite the large number of studies that have investigated how rates of calcification respond to ocean acidification scenarios, comparatively few studies tackle how ocean acidification impacts the physiological mechanisms that drive calcification itself. The aim of our paper was to determine how the carbonic anhydrases, which play a major role in calcification, are potentially regulated by ocean acidification. For this we measured the effect of pH on enzyme activity of two carbonic anhydrase isoforms that have been previously characterized in the scleractinian coral Stylophora pistillata. In addition we looked at gene expression of these enzymes in vivo. For both isoforms, our results show (1) a change in gene expression under OA (2) an effect of OA and temperature on carbonic anhydrase activity. We suggest that temperature increase could counterbalance the effect of OA on enzyme activity. Finally we point out that caution must, thus, be taken when interpreting transcriptomic data on carbonic anhydrases in ocean acidification and temperature stress experiments, as the effect of these stressors on the physiological function of CA will depend both on gene expression and enzyme activity.