Nutrient gradients simulate different adjustments of coral-algal symbiosis


 Background: Eutrophication is one of the major causes of coral reef degradation but the effect of eutrophication on coral and its symbiont algae remains unclear, particularly for the larval stage of coral. In the present study, the physiological and transcriptomic responses of the larvae of an ecologically important scleractinian coral Pocillopora damicornis were analyzed after a 5-day exposure to elevated nitrate in order to assess the survival and adaptation of coral-algal symbiosis under elevated nutrients. Results: The results showed that multiple larval transcripts were significantly correlated with Symbiodiniaceae transcripts. The major differentially expressed transcripts in coral/Symbiodiniaceae included those responsible for energy synthesis/comsumption, nitrogen metabolism and stressor response. Slightly elevated nitrate concentration could in fact promote the health of coral meta-organism. With increase in nitrate concentrations, coral larvae showed significant stress response to maintain the coral-algal symbiosis and coral-algal symbiosis was impaired, while Symbiodiniaceae switched photosynthetic states for ATP synthesis, material transport and nitrogen metabolism for symbiosis maintenance under the control of the coral hosts.Conclusions: Our results suggest that adjustment of coral-algal symbiosis via coral control and a shift in Symbiodiniaceae photosynthetic states serves as the basis of coral meta-organism adaptation under eutrophication stresses. The larvae of P. damicornis and Symbiodiniaceae displayed different transcriptomic responses to nitrate enrichment. Coral larva meta-organism can adapt to moderately elevated nutrient concentration while extreme eutrophication can impair coral-algal symbiosis and affect coral larvae survival ultimately.

widely acceptable theory is that high nutrients can destabilize coral-algal symbiosis, making coral vulnerable under thermal stress [24]. Symbiodiniaceae can keep more photosynthates under high nitrogen concentrations [25] which may bring in limitation of CO 2 and cause coral bleaching eventually. Besides, high nitrogen concentration followed by elevated nitrogen fixation in coral meta-organisms led to a phosphate starvation and raised cell division rate although coral control Symbiodiniaceae with a nitrogen limited internal environment [26], affecting the susceptibility of Symbiodiniaceae to thermal stress and thus reforming the coral-algal symbiosis [24]. Furthermore, nitrogen concentration can affect the photosynthesis efficiency as well as status in algae, which may finally affect coral-algal symbiosis [4].
Dispersal and recruitment of coral larvae play critical roles in establishing coral reefs [27], but the impact of eutrophication on coral larvae remains largely unknown. It was reported that coral larvae retained high energy requirement and less physiology capacity, leading to high susceptibility to environmental changes [28,29]. For example, larvae of Pocillopora damicornis reduced oxygen consumption under high partial pressure of CO 2 (pCO 2 ) but elevated pCO 2 did not affect adults significantly [30,31]. P. damicornis is a hermaphroditic brooder and is one of the most widespread corals in the world [27,32]. In the present study, we exposed P. damicornis larvae to four different nitrate concentrations along with ambient seawater as the control and then examined the photosynthetic physiology and the transcriptome changes of coral meta-organisms, with an aim to explore the potential mechanism of coral adaptation under eutrophication.

De novo assembly of reference transcriptome for coral and Symbiodiniaceae
The reference transcriptome with a total of 122 GB clean reads was generated from all the samples from all the treatments and control to estimate differential gene expression under enriched nutrient conditions. In total, 677,207 transcripts were assembled with alignments of all the samples over 98%. Statistics based on all the transcripts and the longest isoform per gene including N50, median and average contig length were listed in  [33,34]. This is because coral larvae were used for RNA extraction, not Symbiodiniaceae pure culture and some Symbiodiniaceae transcripts were discarded if they can be found in coral database. The numbers of transcripts in the present study was much bigger than those of the predicted genes and transcripts in a previous study [27]. Because one gene may have different transcripts and we didn't exclude high similar transcripts to avoid missing any information. Only limited number of bacterial transcripts were detected, maybe due to sequencing depth; and these bacteria included cyanobacteria and some other nitrogen cycling related bacteria, suggesting that bacteria also played certain roles in coral adaptation under nutrient enrichment (Supplementary Table S4, Additional File 1) but further analysis was not conducted in the present study.

Different expressed transcripts in coral and Symbiodiniaceae
For coral transcripts, the samples in 5, 10, 20, and 40 treatment groups had 24, 32, 37, and 48 up-regulated genes and 20, 26, 26, and 48 down-regulated genes in comparison to those in the control, respectively (FDR < 0.05, |logFC| > 1). Referring to Symbiodiniaceae transcripts, the samples in 5, 10, 20, and 40 treatment groups showed 1, 58, 2, and 16 upregulated genes and 1, 300, 4, and 17 down-regulated genes in comparison to those in the control, respectively (FDR<0.05, |logFC| > 1). The transcript differentially expressed in at least one treatment was regarded as differential expressed transcript. In total, 176 coral transcripts and 378 Symbiodiniaceae transcripts were retained as the differentially expressed ones for following analysis. Figure 1 shows that nMDS of coral differentially expressed transcripts in different samples were well separated by nitrate concentrations.
The samples from the highest nitrate concentration treatment showed large distances with the samples in the control. The samples in 5, 10, and 20 groups lay between 40 group and the control. There was no significant difference among samples in the same group.
However, Symbiodiniaceae differentially expressed transcripts did not show a clear gradient through nitrate concentrations. The samples from 10 group were well separated from other samples. Among 5, 20, and 40 and the control groups, 40 and the control still had the longest distance, while the samples from 5 and 20 groups cluster together.
Heatmap of coral and Symbiodiniaceae transcripts showed similar pattern with nMDS ( Supplementary Fig. S2, Additional File 1), even though many transcripts displayed different expression levels across the samples within the same group.

Function partitioning and diversity of coral and Symbiodiniaceae from different treatments
Most differentially expressed transcripts in corals were related to energy consumption, membrane transform and stressor response. In Symbiodiniaceae, however, the highly differentially expressed transcripts were related to photosynthesis, nitrogen cycling and stressor response (Supplementary Table S5 When mapping the differentially expressed transcripts to KEGG pathways, both coral and Symbiodiniaceae differentially expressed transcripts had large numbers assigned to purine/thiamine metabolism and antibiotics biosynthesis pathways. Symbiodiniaceae had more differentially expressed transcripts assigned to nitrogen compound metabolism pathway ( Table 2). These indicated that nutrient enrichment can affect energy metabolism of coral meta-organism, coral meta-organism establish defense mechanism for potential pathogens.
According to GSEA results of coral transcripts, 54, 147, 126, and 88 gene sets were identified as up-regulated and 45, 228, 36, and 99 gene sets as down-regulated in 5, 10, 20 and 40 groups, respectively. For Symbiodiniaceae transcripts, 337, 408, 324, and 398 gene sets were up-regulated and 17, 156, 127, and 161 gene sets were down-regulated in 5, 10, 20 and 50 groups, respectively. The dissimilarity through nitrate gradients of coral and Symbiodiniaceae transcripts depicted by GSEA was different from that by pairwise analysis because GSEA took more genes into consideration, forming a more complete picture of functional differences.
The main ATP generation related transcripts were more increased in Symbiodiniaceae than those in corals in all treatments, especially 5 and 40 group (Fig. 3a), indicating the increasing energy demand of coral meta-organism under eutrophic conditions might more rely on Symbiodiniaceae. Coral and Symbiodiniaceae enriched different sets of stress response genes in the treatments (Fig. 3c) with similar functions of stress response transcripts found in their total transcriptome. Coral genes related to immune processes were down-regulated in 10 group but up-regulated in 5 and 40 groups. Coral genes related to defense response to virus were down-regulated in 10 and 40 groups whereas genes related to defense response to bacteria were up-regulated in these two groups, indicating that coral becomes vulnerable and may suffer from attacks from potential pathogens and virus due to increased nutrients. Most Symbiodiniaceae stressor-related transcripts were activated through nitrate concentrations yet some transcripts potentially related to stressors were depressed in some groups. Coral stress response genes were mostly related to bacteria/virus defense and inflammatory response, while Symbiodiniaceae stress response genes were mainly related to detoxification, chemical stimulus and oxidative stress, implying they potentially worked together to undertake various stressors.
The sets of functional genes that differentially expressed in at least two pairs were shown in differential expression heatmap of gene sets, exhibiting more details of coral meta- and 40 groups but deactivated in 20 group. Catabolic process of organonitrogen compound was highly expressed in 5 and 10 group but decreased in 20 and 40 groups when compared to the control, suggesting that Symbiodiniaceae can help with nitrogen metabolism and transport for coral meta-organism adaptation under nutrient stress.

Coral-algal symbiosis under different nitrate concentrations
The average Symbiodiniaceae density in P. damicornis larvae increased in all treatments compared with the control but there were no significant differences among treatment groups (p = 0.05) (Fig. 4a). The P G was increased when larvae were exposed to nitrate enrichment in general and was the highest in 5 group, slightly increased in 10 and 20 groups, and moderately increased in 40 group (Fig. 4b). In 5 group, the average Symbiodiniaceae density slightly increased but the P G and P N were the highest, suggesting the photosynthesis efficiency of Symbiodiniaceae in 5 group was substantially enhanced when nitrate concentration was high. However, further increase in nitrate concentration led to Symbiodiniaceae overgrowth but did not increased its photosynthesis efficiency ( Fig. 4b). These were consistent with transcriptome changes that Symbiodiniaceae worked best in 5 group. The correlation between coral transcripts and Symbiodiniaceae photobiological parameters differed in different nitrate concentrations (Fig. 4c)  biosynthetic process was more overexpressed in 5, 10 and 40 together with higher overexpressed PSII in these three groups, suggesting Symbiodiniaceae in 5, 10 and 40 groups appeared to be at different photosynthetic states from 20 group and this was consistent with variation trend of PSII expression level. Meanwhile, the elevated expression levels of stressor response genes increased over nitrate concentration gradient, may also suggesting enhanced damage repairing occurred. Thus, increased PSII expression of Symbiodiniaceae in the present study could not only contribute to damage repairing but also balance ATP generation via switching photosynthetic states.
Furthermore, most of differentially expressed Symbiodiniaceae transcripts were related to energy generation/consumption and antibiotic generation. To conclude, Symbiodiniaceae regulates photosystem states and active stressor response pathways for adaptation under nutrient stress.

Coral-algal symbiosis works for coral meta-organism adaptation under eutrophication
Coral-algal symbiosis is fundamental to extend the capacity of coral meta-organism to face various environmental stressors [4]. Energy related transcripts of coral and Symbiodiniaceae were closely correlated and accompanied with adjustment of Symbiodiniaceae photosystem, indicating that Symbiodiniaceae adaptation is critical for coral hosts' high energy requirement during nitrogen assimilation under nutrient stress.
No nitrogen metabolism related genes of coral larvae were enriched in all the treatments; and the differentially expressed coral nitrogen metabolism related transcripts were significantly correlated with Symbiodiniaceae transcripts. Furthermore, several nitrogen metabolism related genes of Symbiodiniaceae were enriched in the treatments. These results indicate that coral larvae rely on Symbiodiniaceae to metabolize nitrogen compounds, which is consistent with previous studies [12,47].
Previous studies have revealed that coral get can control Symbiodiniaceae in three ways: 1) coral stimulates Symbiodiniaceae to release their photosynthates [48,49]; 2) coral digests/degrades Symbiodiniaceae to control Symbiodiniaceae density [50]; and 3) coral limits the nutrients uptake of Symbiodiniaceae [26,51]. In the present study, Symbiodiniaceae density did not increase significantly in 5 group, indicating that coral larvae might have regulated the Symbiodiniaceae density by digesting algae or limiting the nitrogen availability to avoid overgrowth of Symbiodiniaceae and to enhance the photosynthetic rate for high energy requirement. In 5 group, most coral larval transcripts for protein regulation did not enrich and coral larval development transcripts did not decrease significantly, together with the highest photosynthetic efficiency of Symbiodiniaceae in this group, suggesting an optimal growth status of coral larva metaorganism under this nitrate concentration. When nitrate concentration was raised to 10 µM, Symbiodiniaceae density increased but photosynthetic rate decreased in comparison to the number in 5 group. Symbiodiniaceae density in 20 group was the highest among all treatments, while the photosynthetic rate was lower than that in 5 group. Besides, Symbiodiniaceae in 20 group might at a different state of photosynthesis from other groups and ATP biosynthesis process transcripts were at the lowest expression level compared with among all the treatments. Many coral protein regulation transcripts were extremely over expressed and many stressor response transcripts were absent in 20 group, suggesting that the nitrogen concentration might be beyond the control limitation of coral larvae. Under this nitrogen concentration, the stabilization of coral-algal symbiosis might have been broken, leading to Symbiodiniaceae overgrowth, coral physiology altered, and ATP generation declined. When nitrate concentration increased to 40 µM, the density of Symbiodiniaceae was lower than that in 20 group, together with tremendous over expression of many stressor response transcripts in Symbiodiniaceae, suggesting that the exceed nitrogen may be harmful to Symbiodiniaceae.
Based on previous genomic research on coral and Symbiodiniaceae [52,53], we identified 15 kinds of gene sets, maintaining coral-algal symbiosis potentially, in the total coral/Symbiodiniaceae transcriptome, including material transporting and stress response, and propose a conceptual model to decipher coral meta-organism adaptation under eutrophication condition (Fig. 6a). More transporting genes showed enriched in Symbiodiniaceae, especially no negative transporting response were found in 5 group, inferring an optimal survival condition of coral larva meta-organism under slightly increasing nitrate concentration and coral larva meta-organism mostly relied on Symbiodiniaceae for adaptation under eutrophication, consistent with results from other analysis (Fig. 6b). Metal ion transport, contributing to Symbiodiniaceae growth and coralalgal symbiosis maintenance, decreased in 10, 20 and 40 groups suggesting coral-algal symbiosis impairment under eutrophication. Combined with the results from GSEA and correlation analysis, these indicated that coral-algal symbiosis was affected under eutrophication, and coral larvae regulated the coral-algal symbiosis with Symbiodiniaceae adjusting photosystems and transport for maintaining the symbiosis. Thus, the model assumes that control/protection from coral hosts and adjustment of Symbiodiniaceae photosystem/transport are key strategies for coral-algal symbiosis to adapt to nutrient stress. Coral-algal symbiosis serves as the basis to balance the energy and nitrogen cycling under eutrophication. When nitrate concentration excesses control limits of coral larvae, coral-algal symbiosis becomes unstable and coral meta-organism becomes susceptible to environmental stressors.

Conclusion
The present study showed that the larvae of P. damicornis and Symbiodiniaceae displayed different transcriptomic responses to nitrate enrichment. We proposed that coral larvae can adapt to moderately elevated nutrient concentration via coral-algal symbiosis adjustment, especially photosystem/transporting adjustment and potential photosynthetic state transition of Symbiodiniaceae for maintaining coral-algal symbiosis under nutrient stress. Under eutrophication, coral protects Symbiodiniaceae from extreme nutrient stress and Symbiodiniaceae regulate the photosystems for efficient energy synthesis and nitrogen metabolism. These results provide knowledged-base for costal management and coral reef conservation and suggest that management of nutrient enrichment is significant for coral recruitment.

Study area and sample collection
Luhuitou fringing reef, located in southeast Sanya Bay of Hainan Island in the South China Sea (Supplementary Fig S1, Additional File 1). It is about 3 km long and 250-500 m wide, and receives sewage discharges and runoffs from both the Sanya Bay and Sanya River and thus, it is largely influenced by anthropogenic activities. The nutrient level in Luhuitou fringing reef has been enriched and the nitrate concentration is much higher than those in typically oligotrophic waters of coral reefs [54]. The live coral coverage has been lost since the 1960s due to anthropogenic impacts and global climate change [2].
Ten healthy colonies of the adult P. damicornis were collected from the Luhuitou fringing reef and immediately transferred to the nearby CAS-HKUST Sanya Joint Laboratory of Marine Science Research. Colonies were placed in individual larval collection apparatus with running sand-filtered seawater until larvae were released before new moon. Planula larvae from different colonies were pooled and randomly assigned to experimental treatments.

Experimental setup
The nitrate enrichment experiment was conducted in small aquaria (350 ml) equipped with recirculating pump. There were five treatments with each containing four replicates. The control aquarium received only 0.5 µm filtered natural seawater while for four nutrientenriched treatments, aquaria were supplied with potassium nitrate at the target concertation of 5 µM, 10 µM, 20 µM and 40 µM. The nitrate concertation in the control is 2.5 µM, reflecting the reef environment from which corals were collected. The nitrate of the seawater in the aquaria was determined by nutrient autoanalyser (Seal Analytical AA3, Germany) during the experiment. The actual nitrate concertation during the experiment is given in Table S1 in the Additional File 1. Aquaria were illuminated with T5 fluorescent bulbs (Giesemann, German) on a 12 hr/12 hr light/dark cycle, which provided a mean irradiance of 300 µmol photons m -2 s -1 . The seawater temperature of the system was kept constant at 29°C using temperature controllers (WEIPRO, China). The experiment was maintained under above conditions for 5 days.
At the end of the experiment, 20 larvae for each replicate were randomly selected for physiological measurements as described below. The remaining larvae were aliquoted and preserved at −80°C for transcriptomic analyses (with about 20 larvae per aliquot).

Photosynthesis and respiration
Rates of net photosynthesis (P N ) and dark respiration (R D ) were assessed per treatment in 2 ml glass chambers. Chambers were equipped with magnetic stir bars and a temperaturecompensated oxygen minisensor (Ocean optics, USA) connected to the 4-Channel Microsensor Oxygen Meter (Presens, Germany). P N was measured during the midday, whereas R D was measured in darkness. Chambers with seawater as blank controls for each treatment. P N and R D were then calculated by regressing oxygen production/consumption against incubation time and expressed as nmol O 2 larva − 1 min − 1 .
Gross photosynthesis (P G ) was expressed by adding R D to P N . After measurements, larvae were persevered at −80°C before symbiont densities and pigment concentrations were measured.

Symbiont density and pigment
Algal symbiont density and pigment were determined following the protocol described in

Transcriptome sequencing
Total RNA was extracted from the aliquots of 20 P. damicornis larvae in each replicate,

Statistical analysis
Each sample was mapped and calculated counts against P. damicornis and Symbiodiniaceae final transcriptomes, respectively, by RSEM to estimate the response of coral meta-organism to the nutrient stress [59]. The pairwise differential expression analysis of each treatment against the control was established using edgeR with logFC more than 1 or less than -1 as well as FDR of 0.05 to identify a differentially expressed coral/Symbiodiniaceae transcript. The log base 2 of counts per million reads (CPM) for each transcript, which was differentially expressed in at least one pairwise analysis, was Distance-based redundancy analysis (dbRDA) was conducted in Primer-e among coral differentially expressed genes, pigment, density and net photosynthesis of Symbiodiniaceae. Co-expression network was generated with significant correlations between coral and Symbiodiniaceae (p ≤ 0.1), and analyzed in Cytoscape to visualize the correlations between coral and Symbiodiniaceae. The coral-Symbiodiniaceae transcripts with betweenness centrality higher than 0.005 were regarded as core transcripts and were used to conduct combined GO graphs to visualize the function annotations of these transcripts.

Availability of data and material
All raw sequence data has been submitted to NCBI under SRA accession number

Competing interests
The authors declare no competing interests.        Coral-algal symbiosis related pathways and concept model for coral adaptation under eutrophic conditions. Representing the primary potential coral-algal symbiosis related pathways in coral meta-organisms (a) and enrichments of related gene sets in different treatments (b). Red, blue and light blue represent