Coral collection, maintenance, and sampling

Corals were collected under the Israel Nature and National Parks Protection Authority permit number 2014/40227 issued to Y.R. and O.L. Ten mature colonies of Acropora eurystoma (measuring > 50 cm in diameter) were collected from 4–5-m depth from the coral nursery of the Inter-University Institute (IUI) at the Gulf of Aqaba/Eilat Red Sea (28.6929°N, 34.7299°E) in February 2014. This location was chosen since it has no light contamination during nighttime. Corals were divided in two groups and placed in flow-through aquarium systems in IUI in Eilat. The aquarium systems are part of the Red Sea Simulator (RSS), an outdoor system that is exposed to full spectrum sunlight (1800 µmol m−2 s−1 PAR at midday on a cloudless day) with a 15 h photo-period on the longest day and 11 h on the shortest day76. Seawater temperature and pH (Polilyte Plus H Arc 120, Hamilton) were monitored and tested every hour through automated monitoring (sensor-carrying robot) to ensure all corals were under the same conditions (Table 1 and Figs. 2–3). Each experimental group, consisting of five coral colonies, was under a specific light regime; the first was natural light cycles and moon phases (AMB corals). The second group had artificial light contamination (ELP corals) from small white LED light strips 6000–6500 K (4.8 mol quanta m−2 s−1 at the top of the aquarium and 1.5–2 µmol quanta m−2 s−1 at coral height) that were placed behind the glass walls and were turned on every day at sunset. Light intensity in the ELP treatment was carefully adjusted to mimic the same levels of light during the nighttime that coral reefs in Eilat are exposed to based on Tamir et al.16. Light was measured using a LI-COR underwater quantum sensor LI-193, spectrum measurements were made using Ocean optics JAZ spectrometer (supplementary Fig. 1). In all aquarium systems sunlight was reduced with a neutral density filter (LEE Filters no. 210) based on Levy et al.77, and was equal to the light intensity that penetrates the water at 5-m depth at the northern part of the Gulf of Aquaba/Eilat in the Red Sea16. A black plastic sheet was placed between the two aquarium systems to prevent light contamination between treatments. Corals were kept under experimental conditions for 4 months before sampling started at the full moon day in June 2014. Second sampling day was during the new moon day of June 2014. At each sampling day there were four sampling times: sunrise, noon, sunset, and high moon. In total there were two sampling days, 13 June 2014 (full moon) and 25 June 2014 (new moon) and eight sampling times, four for each sampling day.

Prior to each collection, a container was filled with liquid nitrogen and brought to the sampling area. A small branch from the top of each colony, including the axial polyp, measuring an average of 5 cm in length, was sampled from each coral using pliers. The sampled branch was placed in a small piece of aluminum foil with a tag containing the sample ID, time and colony, snap frozen in liquid nitrogen and transferred to a −80 freezer. During the entire experiment corals were evaluated daily and no signs of bleaching or excess algal cover were noticed.

Physiology

Coral fragments from the last sampling day (n = 5 for each group) were tested for protein concentration, zooxanthellae density, and Chlorophyll concentration to assess the health of the corals. Tissue was removed from frozen coral fragments using an airbrush and ice-cold filtered (0.22 uM) seawater. Skeletons were retained for surface area determination using the wax dip technique78. Tissue samples were homogenized for 30 s using an electrical tissue homogenizer (Kinematica Polytron™ PT2100 Benchtop Homogenizer). A sub-sample (100 µL) of the supernatant was taken to determine host protein concentration by a colorimetric method79 using a multi-scan spectrum spectrophotometer (595 nm, 450 nm, Biotek HT Synergy plate reader) and bovine serum albumin as a standard (Quick Start Bradford Protein Assay, BIO-RAD). Protein concentration was used as a biomass and normalization index for the coral fragments. Samples were centrifuged to separate host and symbiont tissues. Further centrifugation and washing with filtered seawater was performed to isolate symbiont cells for cell counts (hemocytometer) and photosynthetic pigment extraction. Pigments were extracted for 24 h in 90% acetone at 4 °C in the dark and Chlorophyll (Chl) a and c2 concentrations were measured spectrometrically at 630, 664, and 750 nm with a multiskan spectrum microplate spectrophotometer (Thermo Fisher Scientific, USA). Chlorophyll concentration was determined as previously described80 and normalized to zooxanthellae cells and surface area. Zooxanthellae were counted with a hemocytometer under a microscope and normalized to coral surface area. Each result is an average of five fragments per treatment. Physiology assay results of both treatments were compared with each other in each parameter using the R package unpaired two-samples t-test to represent statistical relevance.

RNA extraction

Total RNA was extracted from all 80 fragments using TRIzol reagent (Invitrogen) and a modified version of the manufacturer’s protocol that included an additional chloroform extraction and magnesium chloride precipitation overnight. A small branch was cut off and placed in pre-cooled aluminum foil. The branch was crushed into a fine powder with a hammer, while the foil packet was occasionally dipped in liquid nitrogen to keep it frozen. The aluminum foil content was transferred to a 15 ml flacon pre-filled with 2 μL of TRIzol and left at room temperature for 5 min. The tubes were then centrifuged at 7500 × g for 5 min at 4 °C to remove the skeleton powder. In total, 1500 μL of the supernatant from each sample was transferred to a 2 mL tube with 300 μL of chloroform, shaken vigorously and kept at room temperature for 10 min, followed by centrifuging at 12,000 × g for 15 min at 4 °C. After this centrifugation, there were two visible phases. Eight hundred microliters of the aquatic phase was transferred to a 2 mL tube containing 600 μL of chilled isopropanol. Following a 10-min incubation at room temperature, the tubes were centrifuged at 12,000 × g for 10 min at 4 °C. The remaining supernatant was removed and the visible pellet was washed with 1 mL of 75% ethanol and then centrifuged at 7500 × g for 5 min at 4 °C. The final step of the ethanol wash was performed a second time and was followed by removal of the ethanol and drying of the tubes in a clean chemical hood. The dry pellets were covered in 500 µL RNAse free water and incubated at 57 °C for about 5 min until the pellet dissolved. Five hundred microliters of 5 M lithium chloride was added to the tube, gently mixed and stored in a −20 freezer overnight. The following morning, samples were defrosted on ice and centrifuged at 15,000 × g for 30 min at 4 °C. The supernatant was removed without touching the pellet and 1 mL of 75% ethanol was added. Samples were centrifuged at 7500 × g for 5 min at 4 °C and the supernatant was removed. The final step was again repeated, for a total of three ethanol washes. After the last wash, the ethanol was removed and the tubes were dried in a clean chemical hood. The dry pellets were covered in 40 μl RNase free water and incubated at 57 °C for about 5 min until the pellet dissolved. Purified RNA samples were analyzed using a NanoDrop 1000 spectrophotometer (ThermoScientific) to assess RNA quantity and a 2100 Bioanalyzer (Agilent) to assess RNA quality (RIN > 8.5).

Next-generation sequencing

From each sampling point we had three replicates sent for sequencing from each treatment. In total, 1.5-μg RNA from each of the 48 samples was sent for sequencing. RNA samples were prepared using the Illumina TruSeq RNA Library Preparation Kit v2, according to manufacturer’s protocol. Libraries from each sampling point were run on lanes of an Illumina HiSeq2000 machine using the multiplexing strategy of the TruSeq protocol. Protocol starts with poly A selection that results in RNA selection only. Paired-end reads, 100 bases long, were obtained for each sample. All sequencing libraries were trimmed using TrimeGalore version 0.4.0 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove adapters, primers, and low quality bases. All the libraries were merged and de novo assembly was performed using Trinity (version 2.2.0) with default parameters yielding 553,905 contigs (transcripts) with an N50 of 1406 and a median contig length of 415. The run_RSEM_align_n_estimate.pl script included with the Trinity software was used to map the reads for each library back to the assembled transcriptome and calculate sample-specific abundance for each transcript. In order to reduce the number of erroneous isoforms, all transcripts with an isoform percentage value (IsoPct%) < 1 were excluded from further analysis, yielding 545,469 contigs. These contigs were then further filtered for rRNA and other possible contaminations. BLASTP searches against the SILVA database yielded 416 rRNA matches. BLASTN searches against the NT database (100% identity, 90% query coverage) returned 174 bacteria, 76 Symbiodinium, and other non-Acropora matches. 553,114 contigs remained after filtering the assembly.

Putative coding regions were extracted from the transcriptome assemblies using TransDecoder software (www.transdecoder.sourceforge.net), with minimum length of 50 amino acids, providing all the CoDing Sequences and proteins from the assembly. The library contained 278,796 open reading frame (ORF) encoding contigs. Bowtie (v2.1.0) was used to map reads from each sample against these coding sequences. We performed a Symbiodinium specific search against a custom Symbiodinium database (symbs.reefgenomics.org) that revealed 21,992 transcripts that match Symbiodinium transcriptomes (80% query coverage and 90% identity and an e-value of 1e−10). In total, 66,589 coral transcripts were a match to the A. digitifera transcriptome. The remaining assembled contigs from coral and dinoflagellates were separated with Psytrans (https://github.com/sylvainforet/psytrans) using the Acropora digitifera v0.981 and Symbiodinium goreaui (Clade C, type C1)82 (downloaded from http://symbs.reefgenomics.org/download/) predicted coding sequences as references.

Psytrans classified 101,801 transcripts as coral and 88,418 Symb transcripts. We compared the GC-content of the adi-matched, symb-matched, coral-classified, and symb-classified contigs using a kernel-density plot. The GC-content of the coral-classified and symb-classified transcripts matched their corresponding transcriptome (supplementary fig. 4). In total, we identified 168,390 A. eurystoma transcripts. Transcriptome completeness was assessed by comparing the contigs to the Benchmarking Universal Single-Copy Orthologue v. 2 (BUSCO)83 with the metazoan orthologue set using the gVolante server84. The A. eurystoma transcriptome was found to be comprehensive, accounting for 94% of the core genes, and 97% if we include partial coverage. Note that the average number of orthologs per core genes is 1.8, meaning there are several transcripts that may represent one gene.

Transcriptome annotation

All sequencing libraries were trimmed and merged, and a de novo transcriptome was assembled for the Acropora eurystoma coral. Annotations were assigned by blasting the newly identified transcriptome against Acropora digitifera, Homo sapiens, Swissprot and UniProt50 database using BLASTP (NCBI). We filtered the BLASTP results in order to increase the certainty of obtaining true homologs. Annotation was assigned using the best matches obtained with an e-value threshold of 5 × 10−5, > = 30% alignment identity, and > = 70% query coverage. In total, we were able to assign annotation to 119,625 (42%) contigs.

Differential expression

RNA-Seq results were analyzed using the R package GOSEQ (v1.18.0) to detect statistically significantly over-represented genes that cluster into functional groups, requiring a Benjamini–Hochberg-corrected P-value of ≤ 0.05 when comparing between samples from both experimental groups. Using the normalized data from the DESeq comparisons, we performed hierarchical clustering and generated heat maps of all the significantly expressed genes using the heatmap.2 function from the R BIOCONDUCTOR package GPLOTS (v2.17.0). Heatmap.2 was used with the default clustering method and scaling the data by rows. Genes with an adjusted P-value (Benjamini–Hochberg) of ≤0.05 and a minimum 1.5-fold change were considered differentially expressed. We began by comparing the expression profile of genes within the different treatments (ELP and AMB), to find gene groups that are differentially expressed during each moon condition and time of day, regardless of light condition. We continued by comparing the two experimental groups looking for different genes, for example, comparing between ELP and AMB samples during each moon condition, regardless of the time of day, to find differentially expressed genes responding to the moon phase; comparing between ELP and AMB samples during the day time sampling periods, regardless the moon phase, to find differentially expressed genes responding to the duration of the day, comparing all ELP samples to all AMB samples in order to find specific pathways that are different between the two light regimes.

Pathway analysis

Pathway analysis of annotated sequences was performed using the IPA software (http://www.ingenuity.com). Only differentially expressed genes resulting from the DESEQ analysis were used as input for the software. The dynamic canonical pathways contained in IPA are well characterized metabolic and cell-signaling pathways that are compiled from the literature and the Kyoto encyclopedia of genes and genomes (KEGG). The IPA canonical pathways display the genes/proteins involved, their interactions, and the cellular and metabolic reactions in which the pathway is involved. Expression values were z-score normalized and a comparison was conducted in order to identify enriched pathways with a significant differential expression between the two experimental groups. All IPA results were filtered with a P < 0.05.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.