An improved approach investigating epithelial ion transport in scleractinian corals

Coral epithelia control ion fluxes to the calcification site influencing biomineralization and proxy incorporation. However, data on in vivo characteristics of coral tissue such as permeability, selectivity, and active ion transport are scarce but important for calcification and proxy modeling. To investigate ion permeability and ion fluxes across coral tissues in vivo, we developed an electrophysiological approach for the assessment of active and passive epithelial transport properties. Growing Stylophora pistillata corals in a thin layer over permeable filters allowed ion exchange at the site of skeleton formation for reproducible measurements of electrophysiological properties of coral tissues in a modified Ussing chamber. Compared to former applications, electrical measurements on these coral filter units were dominated by tissue characteristics with minimal influence of skeleton or physical stress. Coral tissues were cation selective. Their overall high electrical resistance characterized them as tight epithelia indicating low paracellular permeability for passive ion diffusion. This includes ions relevant for calcification. A small short-circuit current indicates active charge transport across the entire coral tissue. The present approach is applicable to corals laterally overgrowing substrates. It allows the electrophysiological characterization of coral tissue in vivo in response to environmental conditions. This will improve our knowledge on transepithelial transport relevant for biomineralization in corals

Genes Related to Ion-Transport and Energy Production Are Upregulated in Response to CO₂-Driven pH Decrease in Corals: New Insights from Transcriptome Analysis

Since the preindustrial era, the average surface ocean pH has declined by 0.1 pH units and is predicted to decline by an additional 0.3 units by the year 2100. Although subtle, this decreasing pH has profound effects on the seawater saturation state of carbonate minerals and is thus predicted to impact on calcifying organisms. Among these are the scleractinian corals, which are the main builders of tropical coral reefs. Several recent studies have evaluated the physiological impact of low pH, particularly in relation to coral growth and calcification. However, very few studies have focused on the impact of low pH at the global molecular level. In this context we investigated global transcriptomic modifications in a scleractinian coral (Pocillopora damicornis) exposed to pH 7.4 compared to pH 8.1 during a 3-week period. The RNAseq approach shows that 16% of our transcriptome was affected by the treatment with 6% of upregulations and 10% of downregulations. A more detailed analysis suggests that the downregulations are less coordinated than the upregulations and allowed the identification of several biological functions of interest. In order to better understand the links between these functions and the pH, transcript abundance of 48 candidate genes was quantified by q-RT-PCR (corals exposed at pH 7.2 and 7.8 for 3 weeks). The combined results of these two approaches suggest that pH ≥ 7.4 induces an upregulation of genes coding for proteins involved in calcium and carbonate transport, conversion of CO₂ into HCO₃⁻ and organic matrix that may sustain calcification. Concomitantly, genes coding for heterotrophic and autotrophic related proteins are upregulated. This can reflect that low pH may increase the coral energy requirements, leading to an increase of energetic metabolism with the mobilization of energy reserves. In addition, the uncoordinated downregulations measured can reflect a general trade-off mechanism that may enable energy reallocation

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

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.This publication is based on work supported by the KAUST Office of Sponsored Research under award no. FCC/1/1973- 22-01. Part of this study was conducted as part of the Centre Scientifique de Monaco Research Program, which is supported by the Government of the Principality of Monaco

Proton gradients across the coral calcifying cell layer: Effects of light, ocean acidification and carbonate chemistry

International audienceIn corals, pH regulation of the extracellular calcifying medium (ECM) by the calcifying cell layer is a crucial step in the calcification process and is potentially important to influencing how corals respond to ocean acidification. Here, we analyzed the growing edge of the reef coral Stylophora pistillata to make the first characterization of the proton gradient across the coral calcifying epithelium. At seawater pH 8 we found that while the calcifying epithelium elevates pH in the ECM on its apical side above that of seawater, pH on its basal side in the mesoglea is markedly lower, highlighting that the calcifying cells are exposed to a microenvironment distinct from the external environment. Coral symbiont photosynthesis elevates pH in the mesoglea, but experimental ocean acidification and decreased seawater inorganic carbon concentration lead to large declines in mesoglea pH relative to the ECM, which is maintained relatively stable. Together, our results indicate that the coral calcifying epithelium is functionally polarized and that environmental variation impacts pH ECM regulation through its effects on the basal side of the calcifying cells

Proton gradients across the coral calcifying cell layer: effects of light, ocean acidification and carbonate chemistry

Internal pH measurements made in the extracellular calcifying medium (ECM), calcifying (calicoblastic) epithelium and mesoglea of the coral Stylophora pistillata using the fluorescent dye SNARF-1 and confocal microscopy. The measurements were made in light and darkness three experiments. Experiment 1 involved using coral samples maintained at pH 8 seawater. Experiment 2 involved placing samples in 4 seawater acidification conditions: pH 8, 7.8, 7.4 and 7.2 for 1 week. Experiment 3 involved placing samples in 4 levels of dissolved inorganic carbon concentration: elevated; ambient, low and very low for one week. The research was carried out at the Centre Scientifique de Monaco between 2014-2017. The aim of the experiment was to determine the pH gradient across the calcifying cell layer and determine how it responded to the three experiments

Seawater carbonate chemistry and proton gradients across the coral calcifying cell layer

In corals, pH regulation of the extracellular calcifying medium (ECM) by the calcifying cell layer is a crucial step in the calcification process and is potentially important to influencing how corals respond to ocean acidification. Here, we analyzed the growing edge of the reef coral Stylophora pistillata to make the first characterization of the proton gradient across the coral calcifying epithelium. At seawater pH 8 we found that while the calcifying epithelium elevates pH in the ECM on its apical side above that of seawater, pH on its basal side in the mesoglea is markedly lower, highlighting that the calcifying cells are exposed to a microenvironment distinct from the external environment. Coral symbiont photosynthesis elevates pH in the mesoglea, but experimental ocean acidification and decreased seawater inorganic carbon concentration lead to large declines in mesoglea pH relative to the ECM, which is maintained relatively stable. Together, our results indicate that the coral calcifying epithelium is functionally polarized and that environmental variation impacts pHECM regulation through its effects on the basal side of the calcifying cells

Half-times of calcein influx to the coral calcifying medium under conditions of seawater acidification and temperature variation

Half-time of calcein influx was measured in the coral Stylophora pistillata as a method to investigate paracellular transport. pH was also measured in the extracellular calcifying medium (pHECM) and in the calcifying cells (pHi). The measurements were made at the Centre Scientifique de Monaco between 2016 and 2019. Measurements were made using fluorescent dyes calcein and SNARF-1 using a confocal microscope

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

Abstract 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

Time lapse of increasing calcein fluorescence in the extracellular calcifying medium of the coral Stylophora pistillata following addition of calcein to seawater

Half-time of calcein influx was measured in the coral Stylophora pistillata as a method to investigate paracellular transport. To calculate half-life of calcein influx, time lapses of calcein influx into the extracellular calcifying medium was captured using confocal microscopy. The time lapse data set associated with figure 2 was recorded at 25 degrees in a seawater pH of 8. The study was carried on microcolonies of S. pistillata on glass coverslips. The study was conducted the Centre Scientifque de Monaco between 2016 and 2019