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Integral Light-Harvesting Complex Expression In Symbiodinium Within The Coral Acropora aspera Under Thermal Stress

Coral reef success is largely dependent on the symbiosis between coral hosts and dinoflagellate symbionts belonging to the genus Symbiodinium. Elevated temperatures can result in the expulsion of Symbiodinium or loss of their photosynthetic pigments and is known as coral bleaching. It has been postulated that the expression of light-harvesting protein complexes (LHCs), which bind chlorophylls (chl) and carotenoids, are important in photobleaching. This study explored the effect a sixteen-day thermal stress (increasing daily from 25–34 °C) on integral LHC (chlorophyll a-chlorophyll c2-peridinin protein complex (acpPC)) gene expression in Symbiodinium within the coral Acropora aspera. Thermal stress leads to a decrease in Symbiodinium photosynthetic efficiency by day eight, while symbiont density was significantly lower on day sixteen. Over this time period, the gene expression of five Symbiodinium acpPC genes was quantified. Three acpPC genes exhibited up-regulated expression when corals were exposed to temperatures above 31.5 °C (acpPCSym_1:1, day sixteen; acpPCSym_15, day twelve; and acpPCSym_18, day ten and day sixteen). In contrast, the expression of acpPCSym_5:1 and acpPCSym_10:1 was unchanged throughout the experiment. Interestingly, the three acpPC genes with increased expression cluster together in a phylogenetic analysis of light-harvesting complexes.

Photosynthetic eukaryotic dinoflagellates belonging to the genus Symbiodinium form symbiotic relationships with a variety of marine taxa. Endosymbiotic associations observed between these photosynthetic dinoflagellates and corals are generally classed as mutualistic, as both the host and symbiont benefit from the relationship 1,2 . The photosynthetic symbiont acquires nutrients such as inorganic carbon, nitrogen and phosphate from host cells 3 , in turn symbionts then provide up to 90% of the energy required by corals to grow and reproduce. These relationships between scleractinian corals and Symbiodinium are critical for the proliferation of reefs supporting diverse marine ecosystems. Coral bleaching, the loss of photosynthetic pigments or endosymbionts from host cells occurs under stress conditions such as elevated sea temperatures of only a few degrees above long-term maxima 4,5 . While ocean temperature fluctuations occur on a daily basis, the mean sea surface temperature is predicted to rise by approximately 1-2 °C over the next century and is expected to lead to more mass bleaching events 6,7 .
Experimentation on Symbiodinium and the coral holobiont has focussed on many environmental factors implicated in the onset of coral bleaching including elevated seawater temperatures, eutrophication and disease. The effect of high sea-surface temperatures have been a key focus due to mass coral bleaching events (~42% GBR reefs bleached in 1998 and ~54% reefs bleached in 2002 8 ), attributed to global climate change 7 with the 1998 bleaching event coinciding with an El Niño Southern Oscillation event 4,9 . Differential thermal stress sensitivity is observed across the diverse Symbiodinium species complex, with both heat tolerant and heat sensitive species observed within the same clade 10 . Differences in photoinhibition sensitivity have either been acquired independently by thermally tolerant types or have been acquired in the common ancestor of all Symbiodinium types and since lost in thermally sensitive species 10 . Elucidation of sites of thermal sensitivity within Symbiodinium has focussed on potential points where damage results in a decrease in photosynthetic efficiency 11,12 . These potential points include damage to the D1 protein of photosystem II (PSII) 12 , Scientific RepoRts | 6:25081 | DOI: 10.1038/srep25081 inhibition of the de novo synthesis of the D1 protein 13 , the enzyme Rubisco 14 , thylakoid membrane integrity 10 , the carbon concentrating mechanism 15 and LHCs 16 . However, none of these sites have conclusively been demonstrated as the initial site of thermal damage.
LHCs, also called antenna proteins, are found in photoautotrophic organisms and are an array of protein, chl and accessory pigment molecules with roles in light-harvesting and photoprotection 17,18 . In Symbiodinium the light-harvesting system can be divided into two associated complexes, the highly conserved core LHCs, and variable periphery LHCs 19 . Studies of green plant LHCs have elucidated structural information, which has further improved the understanding of light capture and transfer, and the arrangement of peripheral LHCs in photosynthetic eukaryotes 18,20 . Peripheral LHCs may be categorized within the large gene super-family based on associated pigments into three related groups of pigment binding proteins 21,22 . The first group binds chl a and b, the second binds chl a and c and the third group binds chl a and phycobilins. The chl a/c lineage LHCs are additionally divided into the fucoxanthin-chl a/c and peridinin-chl a/c (PCP) complexes, which are found in dinoflagellates [21][22][23] .
In Symbiodinium two types of peripheral LHCs are found, PCP and acpPC 19 . Dinoflagellate PCPs share no sequence similarity with other known LHCs and are water soluble complexes found on the luminal periphery of thylakoid membranes 24 . In contrast, dinoflagellate acpPCs are integral thylakoid membrane complexes that share sequence similarity with the chl a/c subfamily of LHCs 24 . Further, characterisation of Symbiodinium C3 acpPCs and Symbiodinium A1.1 LHCs cluster sequences with three clades within the chl a/c binding LHC family, indicating high diversity of these proteins within species 21 . Dinoflagellate and Symbiodinium PCPs are well studied due to their unique features [25][26][27][28] . In Symbiodinium the LHCs have been shown to decrease energy transfer and dissociate from the photosystem reaction centres following photoinhibition in order to protect cells during stress events 12,[29][30][31][32] . Decreasing the number of peripheral LHCs available to absorb and transfer energy is a proposed photoprotection mechanism, as this reduces the amount of light reaching the reaction centres and limits the risk of possible photodamage to the D1 reaction centre proteins 30 . Further, kinetic studies have revealed that LHC proteins disassociate and reattach to thylakoid membranes under increased light levels to ameliorate stress 32 . However, few studies have examined the effect of thermal stress on Symbiodinium acpPC expression 16,32 .
Targeted studies of Symbiodinium transcript levels have shown that changes in gene expression occur on a relatively small scale. Quantitative-PCR has been used to determine changes in a variety of genes of interest related to stress response [33][34][35][36] . The validation of housekeeping genes for use in Symbiodinium has allowed for a reference to be established in order to determine differential gene expression under various conditions 36,37 . Although significant changes have been observed in Symbiodinium physiology and large fold changes in host gene expression have been recorded in cells exposed to stress, changes in Symbiodinium gene expression occur at a far smaller scale (± <5-fold) [33][34][35] .
This study focussed on the response of acpPC genes in Symbiodinium cells during a prolonged thermal stress event (sixteen days) in Acropora aspera. We find that the response of five acpPC genes in Symbiodinium under stress varies across a LHC phylogeny. This is the first study to investigate the effect of thermal stress on gene expression patterns in the LHC superfamily within a coral host.

Results
Symbiodinium density. Over a period of sixteen days, branches of A. aspera were exposed to temperature increasing from ambient levels (~25 °C) to a bleaching temperature of ~34 °C (Fig. 1). As has previously been found, this temperature increase led to a significant decrease in Symbiodinium cell densities (p < 0.001) over the course of the experiment, with average densities of 6.0 × 10 5 cells cm −2 in the treatment corals on day sixteen, compared to 1.6 × 10 6 cells cm −2 in the control corals on the same day (Fig. 2a).
Chl pigment content. Chl a content per Symbiodinium cell increased over the experimental period in the heated treatments from day eight of the experimental period (Fig. 2b). Analysis of the chl a content found that there were significant differences between day (p < 0.001, df = 5) and treatment (p < 0.05, df = 1) but not in the interaction treatment × day (p > 0.05, df = 5) (Fig. 2b). Similarly chl c content per Symbiodinium cell increased from day ten onwards in the experiment period (Fig. 2c). Analysis of the chl c content found that there were significant differences between day (p < 0.01, df = 5) and treatment (p < 0.01, df = 1) but not in the interaction treatment × day (p > 0.05, df = 5) (Fig. 2c). The ratio of chl c to chl a was unchanged between control and treatment conditions throughout the experiment (Fig. 2d).

Chl fluorescence and photosynthetic efficiency. Maximum quantum yield of photosynthesis (F v /F m )
was measured following sunset during the experiment. For corals maintained at control temperatures, F v /F m was between 0.602 and 0.692 (average 0.657) (Fig. 3a). Analysis of F v /F m found that there was a significant effect between day and treatment (p < 0.001, df = 5) and differences between day (p < 0.001, df = 5) and treatment (p < 0.001, df = 1) (Fig. 3a). A sequential Bonferroni post hoc analysis found that F v /F m decreased in the heated treatment on days eight, ten, twelve and sixteen of the experiment compared to controls from the same days (p < 0.01) (Fig. 3a).
Non-photochemical quenching (NPQ) was also measured over the course of the experiment. As with F v /F m , there were significant interaction effects between treatment × day (p < 0.001, df = 5), and between day (p < 0.001, df = 5) and treatment (p < 0.05, df = 1) (Fig. 3b). Post hoc analysis demonstrated NPQ was significantly increased by heating on days ten, twelve and fourteen before declining to zero on the final day of the experiment (Fig. 3b).
Gene expression under thermal stress. The expression of five acpPC genes (acpPCSym_1:1, acp-PCSym_5:1, acpPCSym_10:1, acpPCSym_15 and acpPCSym_18) from three distinct LHC clades ( Fig. 4) was determined. Over the course of the experiment three acpPC genes were found to have significant increases in gene expression, acpPCSym_1:1 on day sixteen (1.74-fold, p = 0.001), acpPCSym_15 on day twelve (1.33-fold, p = 0.014) and acpPCSym_18 on days ten (2.44-fold, p = 0.012) and sixteen (2.08-fold, p = 0.020) (Fig. 5a,d,e). These three genes belong to two distinct LHC clades (Fig. 4), both acpPCSym_15 and acpPCSym_18 belong to Clade 1, while acpPCSym_1:1 belongs to Clade 2. The largest fold change seen in these genes was a 2.44-fold increase in acpPCSym_18 compared to control. For the remaining two acpPCs (acpPCSym_5:1 and acp-PCSym_10:1) which both belong to Clade 3b (Fig. 4) no significant changes in gene expression were detected Error bars represent ± s.e.m., n = 9-12, some error bars obscured by data point markers. The statistical difference (post hoc sequential Bonferroni analysis) between treatment and control is indicated as *p < 0.05 or **p < 0.01. (Fig. 5b,c). In addition to the five acpPCs the expression of the psbA gene was also determined. During the course of the experiment no significant differences in psbA expression between control and treatment conditions were found (Fig. 5f).

Discussion
This study investigated the effect of increased temperatures on the expression of five acpPC genes in Symbiodinium under prolonged thermal stress of A. aspera and is the first to examine the expression of acpPC genes from different LHC Clades. The sixteen-day thermal regime was selected to enable sampling at temperatures leading up to, and inclusive of, a bleaching event and is the first experiment to investigate differential expression of integral antenna proteins in Symbiodinium within a coral host under thermal stress. Quantitative PCR was used to quantify the expression of five acpPC genes that are dispersed though out three clades of the chl a/c lineage of the LHC phylogeny (Fig. 4).
Over the course of the experiment temperature significantly effected Symbiodinium density and physiology. Symbiont cells decreased to approximately half the density in thermally stressed corals compared to control corals (Fig. 2a) as has been found in variety of other studies 4,35,38 . In addition chl a and chl c levels were elevated over the course of the experiment (Fig. 2b,c) in a manner seen before in this species (Gierz and Leggat, unpublished data) 35 . However, a statistical difference was only observed on day sixteen in chl c (Fig. 2c), this is consistent with other studies where an increase in chl pigments were observed 35 . In corals, heat-related increases in chl a have previously been recorded at low symbiont densities 39,40 , though in other experiments Symbiodinium pigmentation may be unchanged or decreased 41,42 . Increases in chl pigments have been attributed to repackaging of chls in the chloroplast membrane, with evidence that specific pigment-protein complexes may absorb more light at specific wavelengths 43 . In phytoplankton, chl a-specific absorption of different pigment-protein complexes from the same organism can be highly variable 43 . Therefore, it is possible that the increases in Symbiodinium pigments observed in heated corals may be attributed to alterations in the type of pigment-protein complexes expressed under thermal stress.
Imaging-pulse amplitude modulated (PAM) fluorometry analysis demonstrated that Symbiodinium cells exposed to elevated temperatures exhibited decreased photosynthetic efficiency (Fig. 3a), this is consistent with previous studies demonstrating the response of cells to elevated temperatures. Decreases in dark-adapted yield occurred throughout the experiment despite small changes in symbiont density, F v /F m levels of ~0.00 were recorded on day sixteen despite cell density being approximately five hundred thousand per cm 2 , indicating cells were incapable of photosynthesis at the end of the stress period. Increased NPQ response in cells at days eight, ten and twelve (Fig. 3b) illustrates that the cells were dissipating excess light energy. However, this NPQ response was not present on day sixteen of thermal stress indicating that the symbionts had passed a threshold where photosynthetic processes were no longer functioning. Together, the photosynthetic efficiency results, Symbiodinium densities and changes to pigment levels, demonstrate that in this experiment Symbiodinium were subjected to the full range of temperatures that are seen in a bleaching event, with responses from initial thermal stress through to Symbiodinium expulsion. As such it is reasonable to conclude that acpPC expression patterns are representative of what would be seen in a natural bleaching event.
Expression of acpPC genes was found to vary in Symbiodinium cells throughout the experiment. Functionally little is known about the diversity of LHCs, for example whether complexes only associate with specific photosystems, are some more efficient at light capture or energy transfer, are others favoured for photoprotection or do some display increased stability under high temperatures. Characterisation of Symbiodinium acpPCs has shown that there is large diversity within the gene super-family 21 . Analysis of the Symbiodinium genome has provided more of an insight into the diversification of the LHC family 23 , reinforcing theories on gene duplication and A. aspera nubbins exposed to control conditions (solid line) and heated treatment (dashed line). Values represent average obtained from twelve biological replicates across four replicate tanks. Error bars represent ± s.e.m., n = 12, some error bars obscured by data point markers. The statistical difference (post hoc sequential Bonferroni analysis) between treatment and control is indicated as *p < 0.05 or **p < 0.01. deletion events leading to the current structure of the Symbiodinium genome. The complexity observed in the integral LHC family has been attributed to multiple rounds of intra-and inter-genic gene duplication events 20,23 . A large gene super-family encodes integral LHCs, and a significant level of sequence similarity has been detected between the protein complexes 21,23 . Phylogenetic analysis of LHCs and LHC-like protein super-families indicate that the ancestor is most likely a central group of two-helix stress-enhanced proteins that had previously evolved from a gene-duplication event of the high-light induced proteins of cyanobacteria 44,45 . However, based upon sequence divergence, it is reasonable to assume that different clades of acpPC may have different functions. As such, the differences in expression between those acpPC (Fig. 5a-e) from Clade 1 and 2 versus Clade 3b (Fig. 4), may be indicative of functional roles, with Clade 1 and 2 possibly being involved in stress response while those of Clade 3b are constitutively expressed under the conditions used here.
Some ways in which acpPC may functionally vary is in the binding of varied pigment ratios, specificity for association to photosystems and response to stress events. For example it has been found that a variety of acpPC transcripts are missing key chl and pigment binging residues 21 . In addition it is not clear to which photosystems different acpPCs bind. In green plants, ten highly conserved genes encoding chl a/b binding proteins have been identified, associated with photosystem I (PSI) are four pigment -protein complexes (encoded by genes Lhca1, Lhca2, Lhca3 and Lhca4), and associated with PSII are six pigment -protein complexes (encoded by genes Lhcb1, Lhcb2, Lhcb3, Lhcb4, Lhcb5, Lhcb6) 20 . This can be contrasted to Symbiodinium where there is high sequence diversity coupled with high copy number, and as yet it is not clear which proteins bind to PSI or PSII 21,23 . It has been suggested that this sequence diversity allows for functional diversity such as, stress response 21 , attachment/dissociation 32 and enhanced photoprotection 46 . As such, it will only be with the linkage of more transcriptome and genome studies, and the analysis of chl and accessory pigments binding residues, linked to functional studies, that we will be able to elucidate the reason for the expansion of this gene family in dinoflagellates.
Core photosystem genes, psaA and psbA have previously been investigated in Symbiodinium under thermal stress. Decreases of psaA and psbA are hypothesised to significantly impair the mechanisms associated with coping with thermal stress 47 . In this study, the expression of the psbA gene, which encodes the core PSII D1 protein, was also quantified (Fig. 5f). Over the course of the experiment psbA expression increased on days eight and ten (Fig. 5f) although expression in treatment samples was not statistically significant. However, on day sixteen, psbA expression decreased (Fig. 5f), potentially to reduce light absorption to limit the amount of energy captured under stress conditions as a photoprotective mechanism.
As in previous studies, investigating transcript abundance in Symbiodinium very small changes in gene expression were observed in this study. In the five acpPC genes quantified, the largest observed change was a 2.44 fold increase (acpPCSym_18) on day ten of the thermal stress experiment. In Symbiodinium in hospite, these small changes in transcripts have been observed previously [33][34][35][36] and it is postulated that regulation is most likely post-translational and not at the transcriptional level 33,47,48 .
This study exploited a bleaching experiment to investigate the effect of thermal stress on photosynthetic genes. Quantitative PCR was used to determine the expression of five integral LHC genes. Three LHC genes (acpPCSym_1:1, acpPCSym_15 and acpPCSym_18) were found to have increased expression over the duration of the experiment and interestingly, grouped in Clade 1 and Clade 2 of the LHC phylogeny. Additionally two LHC genes (acpPCSym_5:1 and acpPCSym_10:1) grouped with Clade 3b did not exhibit differences in expression. Though transcriptional changes were detected, expression changes observed were less than 2.5 fold throughout the experiment. This is consistent with previous studies where small-scale changes in gene expression were also observed. Given that we currently do not know how the diverse range of LHCs are associated with the photosystems, both PSII and PSI, their specific functional roles, (e.g., light harvesting efficiency versus photoprotection), or the importance of Symbiodinium photosynthesis to the survival of corals, it is imperative that future research focuses on the specific roles of LHCs.

Methods
Thermal stress experimental design. Coral fragments (n = 312) were collected (under Great Barrier Reef Marine Park Authority permit G13/36402.1) from four different colonies of Acropora aspera (tan morph, approximately 78 from each colony) on the reef flat of Heron Island at low tide in May 2013. Heron Island colonies of A. aspera have been demonstrated to associate only with Symbiodinium clade C3 35,49,50 . Coral fragments were taken to the Heron Island Research Station and placed in a holding tub supplied with filtered water from the reef flat. Fragments were randomly selected and placed upright in racks. Nubbin racks were then transferred to eight 65 L replicate tanks and allowed to acclimate for five-days, at the end of this period tissue regrowth was observed on the cut section of the nubbins. Each of the replicate tanks were supplied with a flow of sand-filtered water pumped from the reef flat into two sump tanks, each sump tank then supplied water to four experimental tanks forming a semi-closed system.
The eight tanks were assigned to one of two treatments, control conditions (ambient temperature) and thermal-stress treatment conditions. The control conditions remained at the ambient seawater temperature (~24 °C) for the duration of the experiment (Fig. 1). Fluctuations observed in control conditions are natural daily temperature fluctuations (Fig. 1). The thermal-stress treatment temperature was increased by 0.7 °C for eleven days (25-32.3 °C), and then held at 33 °C for three days and then at 34 °C for a further three days (Fig. 1) to simulate a bleaching event. To achieve the required temperatures for the thermal treatment a three hundred Watt Eheim Jager (Eheim, Deisizou, Germany) heater was used in the heated sump as well as four 25 W Aqua One glass heaters. To ensure diurnal temperature variation in thermal treatment tanks the Eheim heater was turned off overnight to reflect natural fluctuations. Temperatures in each tank were recorded every 10 minutes with HOBO ® temperature/alarm pendant data loggers (Onset, Massachusetts, USA). Light levels were monitored over the course of the experiment, every 10 minutes with Odyssey Photosynthetic Active Radiation (PAR) recorders (Dataflow Systems Limited, New Zealand).
Imaging-PAM fluorometry. Imaging-PAM fluorometry (MAXI Imaging-PAM, Waltz, Effletrich, Germany) was used to measure photosynthetic efficiency of Symbiodinium within A. aspera. Imaging-PAM analysis was performed on the first day and every day from the third day following sunset. Nubbins were dark-adapted for twenty minutes prior to imaging-PAM analysis. Three replicate nubbins from each of the eight replicate tanks were designated for Imaging-PAM analysis and used throughout the experiment. Corals were measured in the same order at each imaging-PAM analysis. Dark-adapted yield and maximal fluorescence were determined using a weak pulse of light, followed by a saturating pulse of 2,700 μ mol quanta m −2 s −1 of photosynthetically active radiation (PAR) for 800 ms. Induction recovery curves were used to examine the photosynthetic efficiency and ability of symbionts to recover from light stress throughout the experiment. The induction recovery analysis utilised fifteen pulses of saturating light (2,700 μ mol quanta m −2 s −1) with a constant actinic light (111 μ mol quanta m −2 s −1 ) over a 5-minute period, followed by a dark recovery period of 14 min. Data from the induction recovery curve was used to determine photo-kinetic parameters, such as F v /F m and NPQ. Following analysis, nubbins were returned to experimental tanks.
Pigment quantification and Symbiodinium cell count. At 2 pm on days zero, eight, ten, twelve and sixteen, three replicate nubbins were taken from each replicate tank (n = 9-12) and were stripped of tissue using a Waterpik ™ dental irrigator using seawater. On day zero the blastate was centrifuged at 3,076 g for 5 min immediately following tissue removal. On days eight, ten, twelve and sixteen the blastate was homogenised with an immersion blender for 5 s and centrifuged at 3,076 g for 3 min to pellet algal cells. Total pelleted cells were resuspended in 50 mL of seawater. Aliquots (1 mL) were taken for cell density approximation. The remaining cells were centrifuged at 3,076 g for 3 min to pellet cells and stored at − 80 °C for chl a and c quantification. Chl was extracted in 90% acetone for 20 h in the dark at 4 °C and quantified using the equations of Jeffrey and Humphrey (1975) 51 . Cell number was determined using a Neubauer haemocytometer, with replicate cell counts performed (n = 5). Surface area of waterpiked nubbins was determined using the wax dipping method 52 .
Gene expression analysis. At 2 pm on days five, eight, ten, twelve and sixteen, three replicate nubbins were taken from each replicate tank (total twelve nubbins per treatment) and snap-frozen in liquid nitrogen and stored at − 80 °C for later mRNA isolation. Coral branches that had been snap-frozen in liquid nitrogen were crushed with a hydraulic press before transfer to a mortar chilled with liquid nitrogen and ground finely with a chilled pestle. The powder was then divided into cryotubes and stored at − 80 °C. Messenger RNA was isolated from cells using the Dynabeads ® mRNA DIRECT ™ kit as per the protocol outlined in Leggat et al. 33 . Extracted mRNA was quantified spectrophotometrically using the Nucleic acid: RNA-40 setting on a NanoDrop-1000 (NanoDrop Technologies, Wilmington USA). DNase treatment of 0.1 μ g of mRNA per reaction was carried out using RQ1 RNase-Free DNase (Promega) in a total volume of 9 μ l. cDNA was reverse transcribed from DNase treated mRNA isolated from Symbiodinium cells for quantitative real-time-PCR (qRT-PCR). Reverse transcription was performed using the SuperScript ™ III First-Strand Synthesis SuperMix for qRT-PCR (Invitrogen). The 2x RT Reaction Mix contains the following components oligo (dT) 20 (2.5 μ M), random hexamers (2.5 ng.μ l −1 ), 10 mM MgCl 2 , and dNTPs. Template cDNA dilution series were prepared to optimize quantification accuracy. For analysis, cDNA was diluted 1:40 prior to use as a template in the qRT-PCR analysis. Quantitative RT-PCR was performed using a Rotor-Gene ™ 6000 (Corbett Life Science, Australia). The qRT-PCR was performed in a final volume of 15 μ l, containing 7.5 μ l of GoTaq ® qPCR Master Mix (Promega, USA), 4 μ l of diluted template and gene specific primers (266 nM). qRT-PCR conditions were as follows: 95 °C for 2 min; 40 cycles of 95 °C for 15 s and 61 °C for 60 s. All qRT-PCR were followed by a melt curve analysis from 55 to 95 °C to ensure single product amplification ( Supplementary Fig. S1). Each Rotor-Disc ™ -100 included three technical replicates for ten biological samples with three genes. Non-template controls for each primer set were performed in triplicate in each run.
Housekeeping genes used in our analysis were selected from previously established reference genes, which included Proliferating Cell Nuclear Antigen (PCNA) 36 , cyclophin (Cyc) 37 , glyceraldehyde 3-phosphate dehydrogenase (GAPDH) 37 , S4 ribosomal protein (Rp-S4) 37 and S-adenosyl-L-methionine synthetase (SAM) 37 . Five acpPC (acpPCSym_1:1, acpPCSym_5:1, acpPCSym_10:1, acpPCSym_15 and acpPCSym_18) Symbiodinium C3 primers were designed against acpPC sequences from an EST library 53 obtained from the NCBI Genbank database (www.ncbi.nlm.nih.gov). Primers were designed for acpPC genes in Symbiodinium using the software DNASTAR Primer Select (Lasergene 11) ( Table 1). The psbA primers were previously established for use in Symbiodinium 47 . Relative expression analysis was performed using qBASE plus 2.5 software (Biogazelle; http://www.biogazelle. com/products/qbasePLUS) 54,55 . Validation of housekeeping genes (expression stability and the optimum number of genes) in this experiment was performed using geNorm (qBASE plus) 54,55 . Data analyses. Statistics software package (SPSS Statistics v 22.0, IBM, USA) was used for all statistical analyses. A generalized linear model with 'day' and 'treatment' as main effects and 'day × treatment' as an interaction was used for pairwise comparisons of cell density, chl a and c, imaging-PAM and gene expression data. The sequential Bonferroni post hoc test was used to adjust for the false discovery rate (or type I error).