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 Table S2 of the Additional File 1. After filtration, 110,546 transcripts of coral and 35,044 transcripts of Symbiodiniaceae were kept for downstream analysis, displayed a BUSCO transcriptome completeness of 89% and 37%, respectively (Supplementary Table S3, Additional File 1). The completeness of coral transcriptome was similar with previous research but not the Symbiodiniaceae transcriptome [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 up-regulated 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, Additional File 1). Veen diagram exhibited the core differentially expressed transcripts in coral and Symbioniaceae under nitrate enrichment (Fig. 2). Fifteen coral transcripts were found to differentially expressed in four treatments. Most of them were related to development and reproduction, suggesting development of coral larvae were affected under nutrient stress (Supplementary Fig. S3a, Additional File 1). Nineteen Symbiodiniaceae transcripts were found to differentially express in at least two comparisons. Transcripts for transport are majority of these Symbiodiniaceae transcripts (Supplementary Fig. S3b, Additional File 1), indicating coral-algal symbiosis were affected under eutrophication and Symbiodiaceae played critical role in coral larval adaptation. Nitrogen compound metabolic transcripts were found in both 15 coral and 19 Symbiodiniaceae transcripts.
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-organism functions in different treatments (Supplementary Fig. S4, Additional File 1). Among the coral transcripts, carbohydrate and carbohydrate derivative transport gene sets were up-regulated in 10 and 20 groups, but showed no significant difference in 5 and 40 groups, suggesting coral tends to require more energy under high nitrate concentrations. No nitrogen metabolic related functional gene sets in coral were enriched in those treatments.
For Symbiodiniaceae transcripts, transcripts belonging to different photosystems differentially expressed in response to nitrate enrichment, suggesting Symbiodiniaceae were at different metabolic states of photosynthesis under different nitrate conditions with different proportion of increased photosystem I/II (PSI/PSII) related transcripts: in 5, 10 and 40 groups, PSII related transcripts were more overexpressed than the transcripts related to PSI, while in 20 group, PSI transcripts were more overexpressed (Fig. 3b, Supplementary Fig. S4, Additional File 1). And PSII in 5 group had the highest proportion in increased PSI/PSII transcripts. These demonstrating photosystem adjustments of Symbiodiniaceae under different nitrate concentrations. However, light harvesting related transcripts decreased in 10, 20 and 40 groups and only highly expressed in 5 group, suggesting Symbiodiniaceae worked better under slightly increased nitrate concentration while impaired under eutrophic conditions. Consistent with results of photosystem related transcripts, carbon fixation and carbohydrate biosynthetic processes were especially highly overexpressed in 5 group, decreased in 10 group, and either overexpressed or showed no differences in other groups. Carbohydrate derivative biosynthetic process increased in all groups compared with the control. Carbohydrate derivative catabolic process was activated in 5 and 10 groups but depressed in 20 and 40 groups. These results suggest Symbiodiniaceae physiological functions were affected under high nitrate concentrations. Nitrogen compound transport and metabolic process were induced in 5, 10 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 PG 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 PG and PN 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). Coral transcripts from different group distributed along dbRDA axis 1 over nitrate concentration gradient. Axis 1 was positively correlated with Chl c and negatively correlated with Symbiodiniaceae density, implying that with increasing nitrate, Chl c and Symbiodiniaceae density were two main factors contributing to coral transcriptome variation.
Co-expression network of coral-Symbiodiniaceae differentially expressed transcripts showed a compact organized network, driven by coral transcripts (Fig. 5). In total, 15,179 out of 66,704 significant correlations (p ≤ 0.1) were detected in the network. All the top 30 highest betweenness centrality of the network were coral transcripts and betweenness centrality of Symbiodiniaceae also exhibited lower than corals’ considering the quantity effects, indicating coral transcripts played more important roles in the network. GO map of core transcripts in coral showed that the coral transcripts that were highly correlated with Symbiodiniaceae transcripts were related to stressors response (GO0051716, GO0050896 etc.), nitrogen compound metabolic process (GO0034641, GO0006807 etc.) and ATP/carbohydrate binding (GO0005524, GO0097367 etc.) (Supplementary Fig. S5a-c, Additional File 1). Symbiodiniaceae core transcripts exhibited nitrogen compound metabolism/transport (GO0006807, GO0015696 etc.) and ATP binding (GO0005524) (Supplementary Fig. S5d-f, Additional File 1), suggesting that coral-algal symbiosis works for coral nitrogen metabolism and adaptation under high nitrate concentrations. Based on KEGG pathway analysis, the core coral transcripts were assigned to purine and thiamine metabolism pathways responsible for energy metabolism (Table 2).