Molecular assessment of the effect of light and heterotrophy in the scleractinian coral Stylophora pistillata

Abstract

Corals acquire nutrients via the transfer of photosynthates by their endosymbionts (autotrophy), or via zooplankton predation by the animal (heterotrophy). During stress events, corals lose their endosymbionts, and undergo starvation, unless they increase their heterotrophic capacities. Molecular mechanisms by which heterotrophy sustains metabolism in stressed corals remain elusive. Here for the first time, to the best of our knowledge, we identified specific genes expressed in heterotrophically fed and unfed colonies of the scleractinian coral Stylophora pistillata, maintained under normal and light-stress conditions. Physiological parameters and gene expression profiling demonstrated that fed corals better resisted stress than unfed ones by exhibiting less oxidative damage and protein degradation. Processes affected in light-stressed unfed corals (HLU), were related to energy and metabolite supply, carbohydrate biosynthesis, ion and nutrient transport, oxidative stress, Ca²⁺ homeostasis, metabolism and calcification (carbonic anhydrases, calcium-transporting ATPase, bone morphogenetic proteins). Two genes (cp2u1 and cp1a2), which belong to the cytochrome P450 superfamily, were also upregulated 249 and 10 times, respectively, in HLU corals. In contrast, few of these processes were affected in light-stressed fed corals (HLF) because feeding supplied antioxidants and energetic molecules, which help repair oxidative damage. Altogether, these results show that heterotrophy helps prevent the cascade of metabolic problems downstream of oxidative stress.

1. Introduction

Reefs are degrading rapidly worldwide owing to increased frequency and intensity of anthropogenic disturbances [1]. Under stress events, many coral species bleach, i.e. lose their symbiotic dinoflagellates of the genus Symbiodinium [2]. Because endosymbionts sustain most of the host metabolism through the transfer of photosynthetic products, their loss induces host starvation and can lead to the death of the coral colony [3]. Mass bleaching events are expected to increase in frequency in the future [4], so it is urgent to understand the physiological changes induced by the loss of endosymbionts and how corals can survive without this nutritional source.

Whether bleaching leads to mortality depends on several factors: the duration and intensity of the stress event, the tissue thickness, the endosymbiont contribution to the coral metabolism and the heterotrophic capacities of the coral host [5–8]. Heterotrophy—the capture of planktonic prey and the uptake of dissolved organic matter—is one of the main mechanisms by which some species can survive bleaching. Species able to increase their heterotrophic input were shown to be more resilient to bleaching and to recover faster [3,9], because a particulate food supply compensates for the loss of autotrophic products. It also allows for the maintenance of higher symbiont and pigment concentrations within the tissue, and thus facilitates the conservation of energy reserves [10,11]. Nutritional status will thus not only determine the resistance and resilience of corals to stress, but also their capacity to grow and reproduce. Although studies have started to assess some of the mechanisms by which heterotrophy sustains coral metabolism, others remain to be understood. In particular, the underlying mechanisms and molecular pathways involved in the physiological changes are unknown. To date, the changes in gene expression in response to feeding under normal or stressful conditions have not been investigated.

The aim of this study was to identify genes differentially expressed both in fed and unfed coral colonies maintained under normal and light-stress conditions. Solar bleaching is widespread throughout the tropics, especially on shallow reef flats [12], causing colourless lesions on coral surfaces directly facing the sun. Here we seek to find changes in gene expression which will target specific biological functions that are enhanced or repressed by irradiance levels and feeding behaviour. To complement this, we have combined an array of physiological tools, such as calcification, photosynthesis and tissue analyses. No study to date has explored the association between transcriptome expression response and different feeding and irradiance levels, although heterotrophy is important for coral health, and gene expression has been used to understand the coral metabolic response to environmental changes [13,14]. Such analysis is timely owing to the impact of environmental changes on the future of coral reefs.

2. Material and methods

(a). Experimental set-up

Four colonies of the scleractinian coral Stylophora pistillata, collected in the Red Sea under CITES permit no. DCI/89/32, were used. They were divided into 64 nubbins (16 nubbins per colony), and equally dispatched (four nubbins per colony) into four aquaria (30 l). All colonies were in symbiosis with Symbiodinium clade A1, identified using large, partial chloroplast subunit (23S)-rDNA sequences [15]. Aquaria were kept at 26°C ± 0.5°C with 500 W heaters (Rena) connected to temperature controllers (ElliWell PC 902/T), and were provided with light (photosynthetically active radiation, PAR) from overhead metal halide lamps (OSRAM H-QI 400 W) at an intensity of 200 µmoles photons m⁻² s⁻¹. Seawater was renewed at a rate of 2 l h⁻¹, mixed with a Rena pump, and no food was supplied, in order to keep the corals under autotrophy. The physico-chemical conditions in the aquaria were closely regulated as shown in electronic supplementary material, figure S1. Nubbins were maintained for seven weeks to monitor their rates of calcification and photosynthesis under these control conditions. For each measurement, one nubbin per colony and one per aquarium (n = 16) were selected. Nubbins were then frozen for determination of protein, chlorophyll (Chla) and symbiont density, as described below.

After these first measurements, two tanks remained at 200 µmoles photons m⁻² s⁻¹, whereas PAR was gradually increased over a period of 5 days in the two other aquaria to 500 µmoles photons m⁻² s⁻¹ to induce a light stress. Under each of the two light conditions, nubbins in one aquarium were kept unfed, whereas the others were fed every day for a period of 3 h with Artemia salina nauplii (ca 2000 nauplii l⁻¹). Four conditions were achieved: low light unfed (LLU), low light fed (LLF), high light unfed (HLU) and high light fed (HLF). The experiment was performed for 15 days, until a significant decrease in maximal photosynthetic efficiency (F_v/F_m) could be detected by pulse amplitude modulated (PAM) fluorometry [16]. Four nubbins per treatment (one per colony, total of n = 16) were snap frozen for gene expression analysis. The remaining two nubbins per colony and treatment (n = 32) were used to assess rates of calcification, photosynthesis, respiration and tissue biomass (Chla, endosymbiont density and protein).

(b). Measurements

Calcification rates were determined using the buoyant weight method [17]. The same nubbins were then used for photosynthesis and respiration measurements. Rates of respiration (R) in the dark, and net or gross photosynthesis (P_n and P_g) at 200 and 500 µmol photons m⁻² s⁻¹ were assessed for each nubbin using the respirometry [18]. The maximum quantum yield of photosystem II (PSII) fluorescence (F_v/F_m) was also assessed using a diving-PAM fluorometer (Walz GmbH, Germany), as described in electronic supplementary material. Samples were then frozen for determination of total chlorophyll (Chla + c₂), protein and symbiont concentrations, according to Godinot et al. [19]. Data were normalized to the skeletal surface area of each fragment (µmol O₂ cm⁻² h^–1) measured using the wax-dipping technique [20]. Data were checked for normality using the Kolmogorov–Smirnov test with the Lilliefors correction, and for variance homoscedasticity using the Levene test. The effect of treatment was tested using a factorial analysis of variance (ANOVA) with two factors (light and feeding). When there were significant interactions, the analyses were followed by the a posteriori Tukey's test.

(c). Gene expression analysis

Total RNA was extracted with TRIzol^® reagent (Invitrogen). DNAse treatment of samples was applied to avoid genomic DNA contamination (DNAse I, Invitrogen). RNA concentrations were measured using a NanoDrop spectrophotometer (ND-1000), and sample quality was checked using a bioanalyser (Agilent). Microarray experiments were performed using 200 ng of labelled RNA samples (Agilent low-input quick Amp labelling kit) hybridized against the microarray in a custom Agilent two-colour gene expression platform with 8 × 15 K probes per slide. Oligonucleotide probes (60 mers) were designed based on approximately 12 000 genes predicted to encode proteins retrieved from a de novo assembly of 454-sequenced EST libraries of S. pistillata [21]. The intensity of the emitted fluorescence from each target spot on the array was detected using an Agilent G2565BA microarray scanner. Raw and processed data were deposited under the Gene Expression Omnibus, with accession number GSE53661 (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=wjudmswwtxmvxqr&acc=GSE53661). Stylophora pistillata expressed sequencing tag (EST) data are also stored in the Cnidarian Database of Centre Scientifique de Monaco: http://data.centrescientifique.mc/CSMdata-home.html.

(d). Microarray data analysis

Measurements from all arrays were subjected to correction and LOESS within-array normalization using Agilent Feature Extraction software (Agilent Technologies, Santa Clara, CA). The remaining analyses were performed using Partek^® Genomics Suite software (v. 6.6, Copyright 2012, Partek Inc., St Louis, MO). Data from the four biological replicates and two technical replicates were used to perform two-way ANOVA. Genes with significantly up- or downregulated expression (false discovery rate (FDR) p-value < 0.05) were identified, with a cut-off of at least a 1.5-fold change. We then focused on expressed genes or pathways enriched as indicated by DAVID enrichment analysis program [22]. The gene ontology (GO) classification analysis was retrieved from the DAVID database and compared according to the different treatments. The DAVID GO fold change is defined as the ratio between the proportion of the submitted list to the proportion of the background one.

(e). Validation of microarray data

Reverse transcription was performed using RTIII superscript (Invitrogen) on 2 µg of DNAse-treated RNA using random hexamer primers. Validations were performed using real-time quantitative PCR (qPCR) as described in Moya et al. [23] (electronic supplementary material).

3. Results

(a). Coral holobiont physiology

Under low light, food provision did not significantly change the physiology of S. pistillata, except that symbiont density was higher in fed corals (figure 1). After exposing corals to high light for 15 days, several parameters were affected, especially in the unfed (HLU) treatment (electronic supplementary material, table S1). Symbiont density and total chlorophyll content of HLU corals were 80% lower compared with LLU nubbins (figure 1a,b), although the chlorophyll content per symbiont cell remained unchanged (ca 2.3 × 10⁻⁶ µg total chl symbiont⁻¹; data not shown). The maximal photosynthetic efficiency changed from 0.650 ± 0.010 in LLU corals to 0.438 ± 0.019 in HLU corals (Tukey's test, p = 0.001). In addition, rates of net photosynthesis, and respiration per surface area (figure 1c,d), as well as skeletal growth (% increase per day, figure 1e) were 80%, 42% and 45% lower in HLU compared with LLU corals. Only protein content remained unchanged (figure 1f). Conversely to HLU corals, HLF corals decreased their symbiont density and chlorophyll content by only 26% (figure 1a,b), and their rates of net photosynthesis by 45% (figure 1c) compared with LLF corals. The maximal photosynthetic efficiency of the symbionts was slightly reduced from 0.680 ± 0.013 in LLF to 0.571 ± 0.031 (Tukey's test, p = 0.02) in HLF corals. Thus, chlorophyll per symbiont cell (ca 1.6 × 10⁻⁶ µg total chl symbiont⁻¹; results not shown), respiration rates (figure 1d), skeletal growth (figure 1e) and protein content (figure 1f) remained unchanged between LLF and HLF corals. Therefore, HLF corals maintained an equivalent metabolism compared with LLU corals, except for their total chlorophyll content (figure 1b) and their rates of photosynthesis (figure 1c), which were slightly lower.

Figure 1.

Changes in physiological parameters after 15 days under the different culture conditions (LL, low light; HL, high light; F, fed; U, starved/unfed). Significant differences between treatments are shown with different letters. For P_g, differences were tested at 500 µmol photons m⁻² s⁻¹. All data are represented.

(b). Gene expression

The principal component analysis captured 37.3% of the total variability for the different treatments (electronic supplementary material, figure S2). Replicate samples were grouped in three clusters. The first cluster included all samples maintained under low light (LLF and LLU). HLF samples were grouped in a second cluster, whereas HLU samples constituted a third cluster. There was no significant difference except for two genes with less than twofold in gene expression between LLF and LLU (ANOVA FDR, p-value > 0.05), in agreement with the physiological observations. Therefore, we have decided that each treatment sample will be normalized to the control LLF condition, which represents the best growth condition for the corals, and presented in our Venn diagram (figure 2a), and in the heat map (figure 2b,c). The Venn diagram (figure 2a) of all differentially expressed genes (having an FDR p-value < 0.05) highlighted 770 genes that commonly changed in HLU and HLF versus the control condition. The HLU versus LLF comparison showed a much higher number of differentially expressed genes (total of 3482) than the HLF versus LLF comparison (total of 956 genes; ANOVA FDR, p-value < 0.05, figure 2c). While changing the control condition to HLF, 1360 genes were differentially expressed in the HLU (ANOVA FDR, p-value > 0.05) from which about 87% were already expressed in the LLF control condition expressed lists (figure 2a). These findings suggest that high light induced more cellular changes in starved than in fed corals. Finally, 1616 genes were changed between HLU versus LLU.

Figure 2.

(a) Venn diagram shows the gene expression profiles under the two conditions (HLU and HLF versus LLF), where LLF is used as a control for comparative expression. (b) Hierarchical clustering shows heat map of the expression prolife analysed based on the Venn diagram results with biological repeats. (c) The heat map of expression after averaging the biological repeats.

Variations in global gene expression were further explored through GO analysis (figure 3 and electronic supplementary material, table S2). Six enriched GO categories were changed both in HLU and HLF treatments compared with the control condition, with more than a sixfold change in processes such as electron transport chain, cellular respiration (with genes related to succinate and NADH dehydrogenases (cytochromes b and c)), and carbohydrate biosynthesis (with genes related to glycosyl transferase, glycogen synthase and glucose phosphate isomerase). Other processes affected were oxidoreduction (threefold change, with genes of thioredoxine, NADH, phosphogluconate and succinate dehydrogenases/reductases (cytochromes c and P450)), ketone metabolism and other metabolic processes (1.5-fold change). The above changes in GO terms were linked to the decrease in rates of photosynthesis observed in both coral groups (HLU and HLF; figure 1). The 2712 genes that significantly changed only in HLU corals compared with the control condition were grouped in seven other GO categories. The highest fold change was observed in the processes of ion transmembrane transport (8.36-fold change), in ATP-metabolic processes (5.20-fold change), protein-folding (4.37-fold change), and in metabolites and energy (3.05-fold change in aco1, aconitase 1 (uptake and sequestration of iron), aifm3 (apoptosis-inducing factor) and ptgs1 (regulation of cell proliferation). Finally, cellular and metabolic processes included 260 and 217 genes, which represented a 1.5-fold change. Most of these processes were linked to the supply of energy by the symbionts, whose density had decreased by more than 80% in the HLU corals, inducing an equivalent decrease in the photosynthesis rate.

Figure 3.

Variations in global gene expression between treatments explored through GO analysis terms. A total of 2712 genes that significantly changed only in HLU corals compared with control conditions were grouped in seven GO categories, and 770 genes were commonly changed under HLU and HLF in comparison to the control conditions.

To gain further insights into the cellular processes occurring during light stress, we investigated the genes that were the most up- or downregulated (cut-off more than twofold change). The analysis pointed to a group of upregulated genes involved in stress response and DNA- or protein-repair processes (figure 4 and table 1): redox regulation (thioredoxin (txnl), peroxidoxin (prdx5), superoxide dismutase (sodc), methionine sulfoxide (mrsa), quinone oxidoreductase (qor) and hydroxyacylglutathione hydrolase (glo2)), molecular chaperones (heat shock protein 40, 60, 70 (dnaj, hsp60, hsp70)), protein degradation (serine protease: prs23), DNA damage (topoisomerase 2-beta: top2b) and metabolism (enolase (eno2), phosphoglucomutase (pgm1) and Ca²⁺ transporting ATPase (atp2b1)). HLU corals exhibited the highest number and fold change of these regulated genes (total of 17) compared with the HLF corals (total of 7). The largest modification in expression level was observed in HLU corals, with an upregulation of two genes (cp2u1 and cp1a2) that belong to the cytochrome P450 superfamily. The fold change increase was 249 for cp2u1 and 10 for cp1a2 in the HLU versus LLF condition, respectively. Conversely, lower values were observed under the HLF versus LLF treatment (1.9 for cp2u1 and a non-significant −1.3 for cp1a2).

Figure 4.

Fold-change values of selected upregulated and downregulated genes. The colour-scale legend indicating the relative fold change is shown on the right. This figure was prepared using Expander software.

Table 1.

List of genes significantly up- or downregulated in HLF and HLU corals (fold change >2) compared with those in the control condition.

clone ID	annotation	putative function
FKBP14	peptidyl-prolyl cis–trans isomerase	accelerates protein folding
CAH6, 13, 14	carbonic anhydrases	reversible hydration of carbon dioxide
CAHA	carbaryl hydrolase	hydrolase, carbon-nitrogen ligase
CAH1, 2	carbonic anhydrases 1, 2	reversible hydration of carbon dioxide
CTR4	copper transporter complex	copper ion transmembrane transporter
LRP1	lipoprotein receptor	lipid homeostasis, clearance of apoptotic cells
SLC23A2	solute carrier family	ascorbic acid transporter
SLC6A1	solute carrier family 6 member 1
HPCL1	hyppocalcin-like 1	calcium-binding protein
CAH12	carbonic anhydrase 12	reversible hydration of carbon dioxide
BMPR2	bone morphogenic protein receptor 2	bone formation
CA2D2	α2δ calcium channel subunit	mediates entry of calcium ion into cells
PTPRC	protein tyrosine phosphatase receptor	regulation of cellular processes
PF2R	prostaglandin F receptor	receptor for prostaglandin
CALM	calmodulin 1	calcium-binding protein
S22aF	solute carrier family 22	organic-ion transporter
TXNL1	thioredoxin-like 1	oxidoreductase activity
PRDX5	peroxidoxin	antioxidant activity
SOD	superoxide dismutase	antioxidant activity
MsrA	methionine sulfoxide reductase	antioxidant activity
SelB	selenocysteinyl-tRNA-specific	insertion of selenocystein into proteins
QOR	quinone oxydoreductase	quinone reductase activity
GLO2	hydroxyacyl glutathione hydrolase	Tiolesterase
TOP2B	popoisomerase 2	DNA repair
HSP60, 70	heat shock protein 60, 70	chaperones
DnaJ	heat shock protein 40	chaperones
PRS29	putative serine protease 29	enzyme-cutting peptide bonds
Enog	enolase 2	magnesium ion binding
PGM1	phosphoglucomutase 1	breakdown and synthesis of glucose
CP2U1, CP1A2	cytochrome P450	steroid metabolism, lipid biosynthesis

In addition, 20 significantly differently expressed genes linked to calcification (calm, fkb14, ca2d2, atp2b1, lrp1, hpcl1, pf2r), calcium pumps (bmpr2, ca2d2, cah1, cah2, cah6, cah12, cah13, cah14, caha) and solute carrier family (s23a2, sc6a1, ctr4, s22af) were downregulated under HLU versus LLF conditions, whereas a slight decrease (cah14, cah13) or increase (sc6a1) of these genes was observed under LLF and HLF conditions, respectively (post hoc tests, p-value < 0.05). Three candidate genes that were more downregulated under HLU than under HLF conditions were evaluated using qPCR assays for the validation of the microarray results (electronic supplementary material, figure S3). The expression of calm, s22af and sc6a1 (compared with the housekeeping gene L40A) was significantly higher (ANOVA, p-value < 0.05) in the HLF compared with the HLU.

4. Discussion

This study was designed to determine and evaluate the physiological and molecular responses of a scleractinian coral, maintained under autotrophy or both auto- and heterotrophy, to an increase in light intensity affecting its nutritional status. Light-stress-driven changes in gene expression were well correlated with physiological changes, and documented a much higher response in unfed than in fed colonies of S. pistillata, as summarized in figure 5. Comparison of the two feeding conditions clearly showed that unfed colonies were affected by oxidative stress owing to a decrease in metabolic and energy processes. This condition prevented DNA repair and degradation of unfolded protein. Unfed colonies thus experienced a disruption in intracellular Ca²⁺ homeostasis, leading to a significant decrease in calcification. Conversely, well-fed heterotrophic corals better resisted the stress, because feeding protected them from oxidative damage by supplying antioxidants and energy-rich molecules that contributed to protein/DNA repair.

Figure 5.

A schematic conceptual model summarizing the molecular and biological changes induced by high light in unfed and fed corals. Green represents mild stress, whereas red represents severe stress. ER, endoplasmic reticulum; M, mitochondrion; ROS, reactive oxygen species; zoox, zooxanthellae (symbionts).

Bleaching was the physiological response of unfed coral colonies to light stress. These colonies rapidly lost ca 80% of their symbiont density and chlorophyll concentrations, as previously observed in the field [12], reducing photosynthesis (and autotrophic carbon acquisition) to very low levels. Photosystem II was the principal target for damage in these corals, with chronic photoinhibition [24]. Respiration rates were also lower under the HLU condition, and resulted either from a decline in lipid or protein levels, or from an acclimation response to compensate for reduced primary production [11,25]. On the contrary, fed corals lost only 15% of their photosynthetic pigments and slightly decreased their photosynthetic efficiency under high light. Therefore, they responded to irradiance increase with slight bleaching or adaptation to a new light environment [16].

Analysis of the gene expression changes identified molecular pathways involved in the coral responses to light stress. Overall, the fold change in gene expression, and the number of genes differentially expressed, were much lower in HLF than in HLU corals, confirming that feeding allowed coral colonies to avoid the cascade of molecular, and then phenotypic, changes in response to light stress. Genes linked to lipids or carbohydrates were consistently changed in HLU corals: diglyceride acetyltransferase 1 (Dgat1), glyceraldehyde-3-phosphate dehydrogenases (gapdhs) and enolase (enol2). These changes suggest that glycolysis might have been impacted by light stress in HLU corals, which relied on their storage lipids during stress to produce energy and compensate for a decrease in carbon acquisition by the symbionts. Conversely, the provision of external food to HLF corals may have enhanced the nocturnal recovery of the symbiont photosynthetic machinery, allowing the maintenance of high photosynthetic rates and energy supply to the coral host [10,26].

Other results from the gene expression of HLU corals also point to a cellular stress response, which was similar to that monitored in heat-stressed S. pistillata [27] or other coral species and coral larvae [14,27–30]. The processes enriched in light-stressed, unfed corals were involved in oxidative stress, DNA repair, redox regulation and molecular chaperones. The observed changes suggest that high light led to reactive oxygen species formation in the electron transport chain of the symbionts of HLU corals. In addition, in HLU corals, we observed the upregulation of two genes (cp2u1 and cp1a2) belonging to the cytochrome P450 superfamily, involved in cell detoxification and protection from oxidative stress [31]. cp2u1 (or family 2, subfamily U, polypeptide 1) encodes for an enzyme that catalyses the hydroxylation of arachidonic acid (AA) and other long-chain fatty acids, which protect from oxidative stress by activating peroxisome receptor gamma [32]. The other upregulated cyp gene (family 1, subfamily A, polypeptide 2) or cp1a2 also oxidizes steroids, fatty acids and xenobiotics, and might play the same role as cp2u1. The fact that cp2u1 was upregulated more than 200 times in HLU corals versus two times in HLF ones is consistent with the oxidative stress observed under high light. Because HLU corals were highly stressed by light, genes responsible for sensing and repairing DNA damage were finally activated. Therefore, while oxidative damage began to accumulate in the cells of HLU corals, thioredoxin and peroxiredoxin expression patterns increased to repair proteins and to regulate cellular redox balance. Superoxide dismutase (SOD) and molecular chaperone expressions were also upregulated probably to limit oxidative stress and to assist in protein folding.

Overall, unfed corals experienced a stress response to high light that was similar to the coral response to thermal or acidification stress [27,33,34], suggesting that the same cell-protection mechanisms are conserved among coral species. The decrease in the expression of genes related to energy metabolism and the large decrease in the photosynthetic rates of HLU corals suggest that they were not able to afford the energetic demand of cellular processes such as protein degradation, refolding and DNA repair. Fed corals presented fewer differentially expressed genes (figure 4), and no important bleaching or PSII photoinhibition was observed, suggesting that they were more resistant to high light. The food supply might have brought antioxidant products to corals. A. salina is, indeed, well known for being rich in vitamin E, which is present in unsaturated fatty acids and other antioxidant molecules [35]. This is also true of many other natural plankton species [36]. The involvement of vitamins in the protection against antioxidant stress in fed versus unfed corals is further supported by the downregulation of the solute carrier s23a2 by twofold under the HLU versus HLF corals. s23a2 encodes for a sodium/ascorbate co-transporter, which accounts for the specific uptake of vitamin C [37]. In addition to oxidative stress, feeding might have supplied energetic molecules to maintain coral metabolism and to enhance protein repair, which is a process incurring high metabolic costs and energy expenditure [38,39]. Altogether, these genotypic and phenotypic patterns suggest that HLF corals avoided severe bleaching by decreasing oxidative stress and by having more energy to repair photo-oxidative damage.

In HLU colonies, oxidative stress also induced a disruption in intracellular Ca²⁺ homeostasis. This disruption is marked by a sustained increase in intracellular Ca²⁺, which led to cytoskeletal rearrangement, cell adhesion changes, decreased calcification and the initiation of cell death. This was evidenced by the downregulation in HLU corals of several genes related to calcium pathways: calmodulin (calm) and fkb14 (peptidyl-prolyl cis–trans isomerase). Because calm interacts with members of all families of Ca²⁺ channels at the plasma membrane, endoplasmic reticulum and mitochondria, many other calcium-related genes were also downregulated in HLU corals: at2b1 (a plasma membrane calcium pump that regulates the calcium level in the cells [40]); pf2r (a receptor whose activity is mediated by a phosphatidylinositol–calcium messenger system, and that is involved in the regulation of apoptotic processes [41]); and lrp1 (which encodes a receptor involved in intracellular signalling and clearance of apoptotic cells [42]). A similar disruption in Ca²⁺ homeostasis was observed in corals experiencing heat-stress [14,43,44], again suggesting that this process is consistently affected among coral species during stress events.

This disruption in Ca²⁺ homeostasis and the almost complete loss of endosymbionts in HLU corals were linked to the severely depressed calcification. We believe that the loss of endosymbionts, indeed, induced a loss of nutrients used to build the organic matrix [45], whereas the disruption in Ca²⁺ homeostasis affected the calcium pumps used to pump calcium from the external medium to the site of calcification. Two other significant groups of genes involved in calcification were downregulated in HLU colonies. These genes are related to carbonic anhydrases (CAs), which catalyse the reversible hydration of carbon dioxide into bicarbonate [46], and those related to bone morphogenetic proteins (BMPs). BMPs are members of the transforming growth factor beta superfamily, and play a significant role in coral biomineralization [47]. As far as we know, this is the first report of the downregulation of CAs owing to light stress, although a similar pattern was observed in corals subjected to thermal-induced bleaching [38,48] or acidification stress [27]. Conversely, Vidal-Dupiol et al. [49] observed the upregulation of several CAs under acidification stresses at pH 7.8 and 7.4, but downregulation at a lower pH (7.2).

Finally, in HLU corals, several genes related to the solute carrier proteins (SLC) were affected by high light stress and downregulated: SLC6A1 is an amino acid transporter, [50]; SLC26-A6, which functions as a bicarbonate/chloride exchanger in vertebrates [51], was also downregulated in corals experiencing heat stress [43]; SLC7A4 or CTR4 is a cationic amino acid transporter. Its downregulation can impact protein or membrane formation. Finally, PTPRC encodes for a protein that belongs to the tyrosine phosphatase (PTP) family. PTPs are known to be signalling molecules that regulate a variety of cellular processes, including cell growth, differentiation, mitosis and oncogenic transformation [52].

Overall, our results represent the first transcriptomic study comparing the stress response of heterotrophically fed and unfed colonies of the scleractinian coral Stylophora pistillata. Before gene expression can serve as a universal biomarker, we must understand its limitations, particularly with respect to physiological differences in stress tolerance. This case study highlights a different stress response for corals relying on heterotrophy, because food provision protected them against antioxidant stress and bleaching, while maintaining symbiont density and photosynthetic performance at a high level during stress (figure 5). Plankton concentration and particulate matter can be high in coral reefs, allowing corals to increase their heterotrophic input when needed [48,53]. Therefore, we hypothesize that nutrition is an important player in the resilience of coral reefs, and heterotrophic capacity will serve as a selective positive force in future environmental changes.

Data accessibility

The EST sequences reported in this paper were deposited with GenBank under accession numbers GSE53661 and are available through a website that we established for these data: http://data.centrescientifique.mc/.

Authors' contributions

All authors carried out the laboratory work, participated in data analysis and drafted the manuscript.

Competing interests

We declare we have no competing interests.

Funding

This study was supported by the Scientific Center of Monaco, and was partially funded by the Israeli Science Foundation (grant no. 243/10) to O. Levy, as well as by the Groupement de Recherche International Coral Reefs.