SHORT REPORT

Signaling cascades and the importance of

moonlight in coral broadcast mass
spawning
Paulina Kaniewska1,2†, Shahar Alon3†‡, Sarit Karako-Lampert4,
Ove Hoegh-Guldberg5*, Oren Levy4*
1
Australian Institute of Marine Science, Queensland, Australia; 2School of Biological
Sciences, The University of Queensland, St Lucia, Australia; 3George S Wise Faculty
of Life Sciences, Department of Neurobiology, Tel Aviv University, Tel Aviv, Israel;
4
Mina and Everard Goodman Faculty of Life Sciences, Bar-Ilan University, Ramat
Gan, Israel; 5Global Change Institute and ARC Centre of Excellence for Coral Reef
Studies, The University of Queensland, St Lucia, Australia

Abstract Many reef-building corals participate in a mass-spawning event that occurs yearly on
the Great Barrier Reef. This coral reproductive event is one of earth’s most prominent examples of
synchronised behavior, and coral reproductive success is vital to the persistence of coral reef
ecosystems. Although several environmental cues have been implicated in the timing of mass
*For correspondence: oveh@uq. spawning, the specific sensory cues that function together with endogenous clock mechanisms to
edu.au (OHG); [email protected] ensure accurate timing of gamete release are largely unknown. Here, we show that moonlight is an
(OL) important external stimulus for mass spawning synchrony and describe the potential mechanisms
†
underlying the ability of corals to detect environmental triggers for the signaling cascades that
These authors contributed
ultimately result in gamete release. Our study increases the understanding of reproductive
equally to this work
chronobiology in corals and strongly supports the hypothesis that coral gamete release is achieved
Present address: ‡Media Lab, by a complex array of potential neurohormones and light-sensing molecules.
Massachusetts Institute of DOI:10.7554/eLife.09991.001
Technology, Cambridge, United
States

Competing interests: The

authors declare that no Introduction
competing interests exist. Adaptation to environmental cycles including daily, tidal, lunar, or annual cycles over millions of
years, has enabled marine organisms to synchronize many aspects of their biology, thereby creating
Funding: See page 12
important biological rhythms for coping with a variable world (Tessmar-Raible et al., 2011). One of
Received: 09 July 2015 nature’s greatest examples of synchronized behavior is the coral spawning that occurs on the Great
Accepted: 04 November 2015 Barrier Reef (GBR). The ‘mass-spawning events’ on the GBR is the Earth’s largest reproduction event.
Published: 15 December 2015 During these annual events (Babcock et al., 1986; Harrison et al., 2011) changes in the intensity of
Reviewing editor: Sonja Pyott, moonlight trigger the spawning of more than 130 species of scleractinian corals as well as hundreds
University Medical Center of other invertebrates over a couple of nights (Babcock et al., 1986; Harrison et al., 2011). Broad-
Groningen, Netherlands cast spawning requires coral colonies to carefully synchronize the release of egg and sperm into the
water column in order to optimize fertilization success (Harrison et al., 2011; Levitan, 2005). Envi-
Copyright Kaniewska et al.
ronmental factors, such as sea surface temperature, lunar phase and the daily light cycle, influence
This article is distributed under
reproductive timing in coral and induce spawning (Babcock et al., 1986; Babcock et al., 1994;
the terms of the Creative
Commons Attribution License, Harrison et al., 1984). However, the mechanism by which corals fine-tune and coordinate spawning
which permits unrestricted use remains unclear.
and redistribution provided that Seasonal patterns are crucial for inducing gametogenesis and spawning in broadcast spawning
the original author and source are species (Mendes and Woodley, 2002), where rising sea temperature is the most likely environmen-
credited. tal driver behind the maturation of eggs and sperm (Babcock et al., 1986; Harrison et al., 1984;

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eLife digest Sexual reproduction in corals is possibly the most important process for
replenishing degraded coral reefs. Most corals are “broadcast spawners” that reproduce by
releasing their egg cells and sperm cells into the sea water surface. To maximize their chances of
reproductive success, most coral in the Great Barrier Reef – over 130 species – spawn on the same
night, during a time window that is approximately 30-60 minutes long. This is the largest-scale mass
spawning event of coral in the world, and is triggered by changes in sea water temperature, tides,
sunrise and sunset and by the intensity of the moonlight.
How corals tune their spawning behavior with the phases of the moonlight was an unanswered
question for decades. Now, Kaniewska, Alon et al. have exposed the coral Acropora millepora –
which makes up part of the Great Barrier Reef – to different light treatments and sampled the corals
before, during and after their spawning periods. This revealed that light causes changes to gene
expression and signaling processes inside cells. These changes are specifically related to the release
of egg and sperm cells, and occur only on the night of spawning.
Furthermore, by exposing corals to light conditions that mimic artificial urban “light pollution”,
Kaniewska, Alon et al. caused a mismatch in certain cellular signaling processes that prevented the
corals from spawning. Reducing the exposure of corals to artificial lighting could therefore help to
protect and regenerate coral reefs.
Future work will involve comparing these results with information about a coral species from
another part of the world to investigate whether there is a universal mechanism used by corals to
control when they spawn.
DOI:10.7554/eLife.09991.002

Oliver et al., 1988). The phase of the moon, on the other hand, coordinates the timing of mass
spawning, selecting neap tides that reduce gamete dilution in the water column (Babcock et al.,
1986; Babcock et al., 1994; Mendes and Woodley, 2002; Willis et al., 1985). Spawning occurs a
few days after the full moon and at a precise time after sunset, although the time of day and night(s)
on which spawning and gamete release occurs is highly species-specific. In addition to being influ-
enced by moonlight (Babcock et al., 1986; Harrison et al., 1990; Glynn et al., 1991), the timing of
spawning is thought to be regulated by the onset of darkness (Babcock et al., 1986), the duration
of regionally calm weather (van Woesik, 2010), food availability (Fadlallah, 1981), twilight chroma-
ticity (Sweeney et al., 2011), and salinity (Jokiel, 1985). How these exogenous factors function
together with endogenous mechanisms in corals to achieve mass spawning harmonization is
unknown.

Results and discussion

To explore the interaction of exogenous and endogenous factors, we characterized the transcrip-
tome from 16 Acropora millepora colonies over 8 days leading up to the spawning night, as well as
during and after spawning (Figure 1). These results were compared with transcriptomes of four A.
millepora colonies sampled 3 months prior to the month of spawning, during full and new moon
days at midday and night (12:00, 18:00 and 24:00). A comparison of new and full moon samples,
when spawning did not occur, revealed changes in gene expression according to the day within the
month, and relative to levels of moonlight (Figure 1—figure supplement 1, Supplementary file
1,2), thereby demonstrating a response by RNA transcription to the lunar cycle. Notably, many of
the genes showing higher expression levels at midnight on full moon days compared with midnight
on new moon days, were linked to rhythmic processes (circadian clock related genes) and light sig-
nalling, including cryptochrome 1, cryptochrome 2, and thyrotroph embryonic factor.
To elucidate the role of natural moonlight phases and the effect of ‘light pollution’ on mass
spawning synchronization, 16 large A. millepora colonies were collected from the Heron Island reef
flat (GBR, Australia) and transferred to large outdoor aquaria, which were exposed to natural sun-
light, moonlight and flow-through seawater from the reef flat. Coral colonies were exposed to one
of the following treatments: ambient (maintained under ambient conditions of daylight and moon-
light, N=6), light (illuminated using artificial light, low intensity ~ 5 mmole quanta m-2 s-1 daily from

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Figure 1. Changes in gene expression during mass spawning of the coral, Acropora millepora. (A) A. millepora colony releasing gametes. (B) Schematic
representing the sampling regime of coral branches from A. millepora colonies (colonies were sampled for 8 days leading up to the spawning night,
during the spawning night and 1 day following the spawning event), blue arrows indicate timing of experiment start, full moon and observed spawning.
Additionally, colonies were sampled prior to the November spawning month (the colonies were sampled at 12:00, 18:00 and 24:00 in August on new
moon and full moon days). (C) Hierarchical clustering of A. millepora gene expression data for the 184 coral transcripts that were only variable during
the spawning day using sampling points in August and November. ‘New’ and ‘Full’ denote new moon and full moon conditions, respectively.
DOI: 10.7554/eLife.09991.003
The following figure supplements are available for Figure 1:
Figure supplement 1. Hierarchical clustering of Acropora millepora gene expression during full moon versus new moon days at 12:00, 18:00 and 00:00.
DOI: 10.7554/eLife.09991.004
Figure supplement 2. Hierarchical clustering of the expression patterns of genes that are both expressed and non-constant (Materials and methods),
indicates that a sizable number of genes are highly variable only on the spawning day.
DOI: 10.7554/eLife.09991.005

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sunset to midnight, then kept in the dark until sunrise, N=5), or dark (covered with a black shade
from sunset to sunrise to block moonlight, N=5) (Figure 2A) beginning 8 days prior to the spawning
night. We sampled corals from all treatments during the days leading up to the spawning event at
midday, at moonrise and at hourly intervals during the spawning night after sunset; this included
sampling of released gametes (Figure 1B). During the spawning night, colonies exposed to ambient
conditions spawned in a similar manner to those on the reef (gametes were released at approxi-
mately 21:30–22:30); however, no sign of spawning behavior occurred in either the light or dark
treatments. For ambient corals, we identified 184 transcripts that were only variable during the
spawning day and showed a clear change in expression (mostly induction) just prior to and at the
time of gamete release (Figure 1C); however, such changes were not observed in corals that were
kept in the dark (Figure 2B). Colonies exposed to the light treatment showed a mismatch of gene
expression profiles, and the expression of the genes that were highly variable during spawning
occurred prematurely (around sunset at 18:15 and 19:30, Figure 2B). Moreover, these colonies did
not release gametes, implying that ‘light pollution’ disrupted spawning synchronization. Gene
enrichment analysis revealed that coral transcripts, which were only variable during spawning, were
enriched in Gene Ontology (GO) processes of the cell cycle, GTPase activity and signal transduction
(Figure 3A). An analysis of transcripts that were highly up-regulated or down-regulated during
spawning showed a larger proportion of transcripts (177) that increased their expression levels dur-
ing spawning (Figure 2—figure supplement 1A), a substantially smaller portion of 29 transcripts
that were down-regulated (Figure 2—figure supplement 1B) and no correlation with gene expres-
sion profiles from the released gametes. Fifty four out of the 177 up-regulated genes, and none of
the 29 down-regulated genes, were also found to be variable during the spawning day
(Supplementary file 1). The up-regulated coral genes were enriched in G protein-coupled pro-
cesses, signal transduction and respiratory processes (Figure 3B). Down-regulated genes were
enriched in processes which generate and maintain rhythms in the physiology of organisms (rhythmic
processes) such as circadian clock related genes (Figure 3C) and included some additional genes,
which were not clustered. A qPCR validation (Supplementary file 3) of both up-regulated and
down-regulated genes showed strong correlation between the qPCR and RNA-seq data based on
log2 fold change measured on the spawning night in ambient conditions at 19:30 versus 22:00
(r2=0.92, p<0.0001) (Figure 2—figure supplement 2).
Since corals do not have specialized visual structures, light detection is likely mediated through
photosensitive molecules such as opsins or cryptochromes (Reitzel et al., 2013) that help the corals
to adapt and to be synchronized with the external irradiance levels (Panda, 2002). Furthermore, spe-
cific classes of G-proteins can be activated by opsins in response to light in coral larvae
(Mason et al., 2012). Neuropeptides are another diverse class of signalling molecules that act
through G-protein coupled receptors. These peptides have been implicated in an array of biological
processes such as reproduction, metabolism, feeding, circadian rhythms, adaptive behaviors and
cognition (Grimmelikhuijzen and Hauser, 2012). The importance of neuropeptides in life phase
transitions has been recorded across a range of taxa such as annelids, cnidarians, insects and mam-
mals (Schoofs and Beets, 2013). Our results indicate that gamete release from a broadcast spawn-
ing coral is potentially controlled by a series of G-protein-coupled signalling pathways and that the
trigger for release might be a non-visual ocular photoreceptor such as the melanopsin-like coral
homolog and/or a range of neuropeptide candidates (Figure 4, Supplementary file 1). Genes
induced during spawning included two melanopsin (opn4b)-like homologs, a battery of G-protein-
coupled receptors (GPCRs), and a homolog of synaptotagmin 7 (Figure 3, Figure 4,
Supplementary file 1). Melanopsin (Opn4) has been shown to be required for light-induced circa-
dian phase shifting in mammals (Panda, 2002). Given the broad changes in expression patterns in
GPCRs and their potential triggers, it seems that neuropeptide signalling appears to be central in
controlling gamete release in broadcast coral spawning, where such a simple nervous system likely
evolved in an ancestor common to all cnidarians (Grimmelikhuijzen and Hauser, 2012). Our data
are consistent with the activation of GPCR receptors and consequent GPCR signalling cascades fol-
lowed by changes in cell migration (eg, RAP2B, UNC5D, IF2B1), immunity/cell death (eg. TLR2,
TLR6, TRAF5, DAB2P, FAK1, NLRC5) respiration (NU4M, NU5M, COX1, NDUA4, CYB) and cytoskel-
etal organization (eg. PAXI, INF2, NUP43) which together lead to synchronized gamete release
(Figure 4).

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Figure 2. Disrupting synchronized mass spawning in the coral, Acropora millepora. (A) Beginning 8 days prior to the spawning night, A. millepora
colonies were exposed to one of the following treatments: ambient (A), in which colonies were exposed to natural day and night cycles with full
exposure to moon light; light (L), in which colonies were exposed to natural daylight during the day and artificial photosynthetically active radiation
(PAR) light (~5 mmol quanta m-2 s-1) at sunset every day for ~6 hrs (between 18:15 and 24:00) and then left in the dark until sunrise; or dark (D), in which
colonies were exposed to natural daylight during the day and left in the dark from 18:15 to sunrise. (B) Hierarchical clustering of A. millepora gene
expression data for the 184 coral transcripts that were only variable during the spawning night, depicting gene expression changes between treatments
A, L and D denote ambient, light and dark treatments, respectively.
DOI: 10.7554/eLife.09991.006
The following figure supplements are available for Figure 2:
Figure supplement 1. Hierarchical clustering of Acropora millepora gene expression data for (A) the 177 coral transcripts that were up-regulated and
(B) the 29 coral transcripts that were down-regulated during the spawning night.
DOI: 10.7554/eLife.09991.007
Figure 2. continued on next page

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Figure 2. Continued
Figure supplement 2. Correlation of gene expression Log2 fold change between values obtained from RNA-seq analysis and expression values
obtained using quantitative PCR (qPCR).
DOI: 10.7554/eLife.09991.008
Figure supplement 3. The effect of light quantity and quality on the timing of broadcast spawning in the coral Acropora millepora at Heron Island.
DOI: 10.7554/eLife.09991.009

Figure 3. Gene ontology enrichment analysis for Acropora millepora gene variability during mass spawning. Gene enrichments (false discovery
rate<0.1) across GO categories are shown. GOseq was used to test for enriched GO categories of genes that were (A) variable during spawning, (B) up-
regulated during spawning or (C) down-regulated during spawning. The left and right identifiers represent the gene names and UniProt accession
numbers, respectively.
DOI: 10.7554/eLife.09991.010

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Figure 4. Proposed model for signalling events during spawning and gamete release in Acropora millepora. The release of gametes occurs in the
presence of moonlight or another signal (such as an olfactory signal) that stimulates melanopsin-like homologs and/or other neuropeptides and
commences GPCR signalling cascades upon receptor activation. This effect results in cell migration, follicle rupture, changes in immunity/cell death,
respiration and cytoskeletal organization, and the combination of which results in a synchronized gamete release. Blue labels indicate genes being up-
regulated; orange labels indicate genes down-regulated.
DOI: 10.7554/eLife.09991.011

Our study does not provide direct evidence as to whether the melanopsin-like homologs that are
up-regulated during spawning are true photopigments, as they have been shown to be in mammals
(Peirson et al., 2009). The sequential expression pattern of these melanopsin-like homologs, how-
ever, suggests that they are important for signalling events during mass spawning in corals regard-
less of whether they convey a light, pheromone or other signal. The discovery of potential
neuropeptide/GPCR-coupled signalling mechanisms are consistent with neurohormones playing a
role in synchronized spawning events in tropical abalone (York et al., 2012) and in the settlement
behavior of coral larvae (Grasso et al., 2011). Additionally, an increase in a tachykinin-like peptide
receptor suggests that a pheromonal trigger may be involved (Winther et al., 2006). Given that the

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transient receptor potential protein superfamily represents calcium permeable channels which have
important roles in phototransduction in Drosophila and in mammalian vision (Panda, 2002;
Pan et al., 2011), our results showing an up-regulation of Trp-related protein 4 fits with the pro-
posed hypothesis that cytoplasmic calcium may act as a secondary messenger for coral photorecep-
tors (Hilton et al., 2012).
Although the cues for the synchronization of gamete release in broadcast spawning corals are
complex, our results indicate that nocturnal illumination is an important factor and that changes in
the light regime surrounding corals can desynchronize the timing of gamete release. In this respect,
only colonies exposed to ambient light conditions at night spawned. We further tested the concept
of light regime changes causing a mismatch in the timing of gamete release through another experi-
ment in which A. millepora colonies were exposed to treatments where the length of the day was
extended artificially (between sunset and midnight), through exposure to a suite of light quantity
and quality regimes. We found that colonies exposed to ambient conditions and treatments with
light sources in the red light region (620–700 nm) of the spectrum spawned at 21:30, which was syn-
chronous with A. millepora colonies spawning in the field. However, colonies exposed to light in the
green (500–620 nm) and blue (400–500 nm) regions of the spectrum and to PAR (400–700 nm) at
low-dim, medium and high light intensities phase shifted their spawning time by a 6–8 hr delay or
spawned 2 nights later as compared with colonies under ambient conditions (Figure 2—figure sup-
plement 3). Our results suggest that detection of light in the blue and green parts of the spectrum
is important for gamete release, which is congruent with suggestions that blue light detection is a
key element for coral spawning behavior (Gorbunov and Falkowski, 2002) and evidence that A. mil-
lepora larvae settle more readily under blue and green light as compared with red light
(Strader et al., 2015). Furthermore, our results from both experiments (Figure 2, Figure 2—figure
supplement 3) on the effect of ‘light pollution’ on coral spawning behavior suggest that disruption
or phase shift delay in spawning time can occur rapidly, that is, within 7 days of exposure to changes
in nocturnal light regimes. These findings differ from previous studies which suggested that entrain-
ment to moonlight rhythms occurs over several months (Willis et al., 1985).
Our results on the effects of light on the timing of spawning are crucial because sexual reproduc-
tion is one of the most important processes for the persistence of reefs. The interplay between
endogenous clocks and external cues in an era of industrialization and global change (when artificial
lights compete with moonlight to affect reproductive timing and fertilization success in broadcasting
species) should be considered in plans to protect coral reefs and marine ecosystems.

Materials and methods

Coral collection and experimental design
Twenty whole colonies of A. millepora were collected on November 9, 2011 from the Heron Island
reef flat (23 33’S, 151 54’E), GBR, Australia. Small branches were cut from the central portion of
each colony prior to specimen collection to confirm pink-coloured eggs, which are present when a
colony is reproductively mature (Harrison et al., 1984). Four of the colonies were transported close
to the shore but were left in the field on the reef flat (treatment F). The remaining 16 colonies were
transferred to large, outdoor flow-through aquaria and were exposed to natural sunlight, moonlight
and flow-through seawater from the reef flat. Colonies in the outside aquaria were divided into the
following three treatments: ambient (A), in which the colonies (N=6) were exposed to natural day
and night cycles and full exposure to moonlight; light (L), in which the colonies (N=5) were exposed
to natural daylight during the day and to artificial PAR light (~5 mmol quanta m-2 s-1) post sunset
every day for ~6 hrs between 18:15 and 24:00 and then left in the dark until sunrise; and dark (D), in
which the colonies (N=5) were exposed to natural daylight during the day and left in the dark
between 18:15 and sunrise. The experiment was conducted at Heron Island Research Station in an
area that was maintained in darkness at night to avoid artificial light contamination from non-experi-
mental sources. Branches from colonies were collected from each of the three treatments on 4 d
(November 10, 12, 14, and 15, 2011) leading up to the spawning night (November 16, 2011)
(Figure 2A). The corals were sampled on these days at noon and during moonrise, which was
between 21:00 and 23:00 depending on the day. On November 16 (the spawning night), we sam-
pled the corals at noon, 18:15, 19:30, 21:00, 22:00, 22:30 and 00:20. The release of gametes

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occurred between 21:30 and 22:30, and the colonies in the Ambient treatment began to show signs
of setting at 19:30. We also sampled released gametes from colonies in the Ambient treatment.
Sampled coral branches were snap-frozen in liquid nitrogen and stored at -80˚C until processing for
total RNA extraction. Additional branches were sampled (N=4) during August from the reef flat at a
2-m depth during new moon and full moon days at 12:00, 18:00, and 24:00.

Total RNA isolation

Total RNA from the coral branches was isolated by homogenizing 100 mg coral tissue in 1 ml TRIzol
(Invitrogen) according to the manufacturer’s instructions. The RNA was then extracted once with 1
volume chloroform and precipitated in ½ volume isopropanol, washed in 1 volume of 75% ethanol
and subsequently dissolved in RNase-free water. These samples were then processed through a 5 M
LiCl precipitation overnight at 20˚C, washed three times with 75% ethanol and subsequently dis-
solved in RNase-free water. The integrity and quality of the total RNA was assessed using a Bioana-
lyzer (Agilent Technology). Only the samples showing intact RNA (RNA integrity number >8) were
used for the RNA-seq analysis.

RNA-Seq analysis
The Illumina TruSeq protocol was used to prepare libraries from the RNA samples. Overall, 12 librar-
ies of full moon and new moon samples were run on one lane and 24 libraries of samples from the
spawning experiment were run on two additional lanes in the Illumina HiSeq2000 machine using the
multiplexing strategy of the TruSeq protocol. On average, ~10 million single-end reads were
obtained for each sample of the full moon and new moon conditions, and ~15 million paired-end
reads were obtained for each sample in the spawning experiment. The sequencing data reported in
this study was deposited to the Sequence Read Archive (SRA), under accession SRP055723. The
reads were 100 bases long. TopHat (Trapnell et al., 2009) was used to align the reads against the
A. millepora genome, keeping only uniquely aligned reads with up to two mismatches per read.
Only reads that were aligned to the protein coding regions of the A. millepora genes (Moya, 2012)
were used. A custom script written in Perl was used to parse the output of TopHat (given in the
Sequence Alignment/Map (SAM) format [http://samtools.sourceforge.net/]) and to convert it into
the raw number of reads aligned to each A. millepora gene (available via Dryad data repository:
doi:10.5061/dryad.541g6). The A. millepora gene information was downloaded from the NCBI data-
base, we retained only genes that were found to be significant similar (Blastx E-value<1e-6) to the
Swiss-Prot proteins data set. On average, ~40% of the reads in the full moon and new moon condi-
tions and ~30% of the reads of the spawning conditions passed all filters and were mapped to the
coding regions of the 12,384 A. millepora genes with Swiss-Prot homology. These numbers are com-
parable to the ones obtained by a similar analysis in a better annotated animal, the zebrafish, in
which between 10% to 56% of the reads mapped to the coding regions of known genes (Ben-
Moshe at al., 2014; Tovin et al., 2012).

Comparison of transcriptome under full versus new moon

Statistically significant differences between the number of reads aligned to each A. millepora gene
(the expression profile) in the tested conditions were identified as described (Alon et al., 2011).
Briefly, the expression profiles were normalized using a variation of the TMM normalization method
(Robinson and Oshlack, 2010). Subsequently, we searched for expression differences between sam-
ples associated with the full versus new moon conditions that could not be explained by the
expected Poisson noise with a p-value <0.05 and using a Bonferroni correction for multiple testing
(Alon et al., 2011). Each one of the full moon and new moon samples was sequenced twice (techni-
cal duplicates), and as the correlation between the gene’s expression levels in these duplicates was
very high (average Pearson’s correlation >0.999) the average expression level is presented in the
figures.

Clustering analysis
The data from the full moon and new moon experiments and the spawning experiment were com-
bined and normalized as described above. We then performed a hierarchical clustering (MATLAB,
MathWorks) of all of the genes that (1) had expression levels above the median of all of the gene

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expression levels in at least one sample and (2) had a variance in the expression level that was
above the median variance of all of the genes. The gene’s variance was normalized by the gene’s
mean expression level (the expected variance assuming Poisson noise). Overall, 4756 genes out of
the original list of 12,384 genes passed these two filters and were therefore considered to be both
expressed and to have an expression profile that was not constant. Clustering of the expression pat-
terns of these genes indicates that a sizable number of genes are highly variable only on the spawn-
ing day (Figure 1—figure supplement 2). These genes are characterized and identified below.

Spawning analysis
The genes that were highly variable during the spawning day in ambient light conditions (regular
light-dark cycles), which ultimately led to the spawning event, could be responding to at least two
factors: (1) moonlight (either directly via light or indirectly through the time of the month) or (2) the
time of day. Alternatively, these genes can also be variable on other days, such as the days prior to
or after the spawning day. Finally, these highly variable genes could also be part of the molecular
mechanism of spawning. To identify the latter group, we selected all of the genes that were highly
variable during the spawning day in ambient conditions but significantly less variable in the full
moon and new moon conditions at different times of the day (without spawning events) and the
days prior to the spawning events (all under ambient conditions).
Variability was estimated using a gene’s variance divided by its expected variance assuming Pois-
son noise in the gene expression levels, which is equal to the gene’s mean expression level. All of
the gene variability measures were sorted, and we selected genes that were in the top 10% on the
spawning day (i.e., highly variable) but not in the top quartile (i.e., not as variable) in the previous
days and in the full moon and new moon experiments. We noted that changing the cut-offs pro-
duced similar results, for example by selecting genes that were in the top 5–25% on the spawning
day but not in the top 25–50% in the previous days and in the full moon and new moon experiments.
Additionally, we pursued high top expression levels (more than 100 reads) of these genes in any one
of the mentioned conditions (only one quarter of the genes showed this expression level or higher).
We also identified genes with higher or lower expressions on the spawning day compared with
the previous days and those of the full moon and new moon experiments. We selected for at least a
twofold difference between the mean expression level on the spawning day and (A) the mean
expression on the previous days, as well as (B) the mean expression in the full moon and new moon
experiments. Thus, the difference in expression level had to be at least twofold in respect to both
(A) and (B). Additionally, we filtered for greater than 100 reads in the top expression level in the
higher condition.
The genes detected were analyzed using the software GOseq (Young et al., 2010) to find statisti-
cally significant over-represented functional groups with a Benjamini-Hochberg false discovery rate
equal to or less than 0.1. We compared the gene’s expression pattern in the described conditions
with the expression pattern of the released gametes that were sequenced, and found no significant
correlation between the gene’s expression patterns (average Pearson’s correlation of 0.22 between
the released gametes and all the other described conditions). Specifically, for the genes detected
above as having higher or lower expression on the spawning day, the correlation with the released
gametes sample was even lower (average Pearson’s correlation of 0.05 between the released gam-
etes and all the spawning day conditions).

QPCR validation
The expression patterns of the RNA-seq data for selected genes with variable expressions during
spawning (Supplementary file 1) were validated via quantitative PCR (qPCR). Primers were
designed from RNA-seq data using the Primer Express Software v3.0 (Applied Biosystems, USA).
Total RNA (1000 ng) was reverse transcribed with a SuperScript VILO cDNA Synthesis Kit (Invitrogen)
following the manufacturer’s instructions. Transcript levels were determined via qPCR assays using
an Eppendorf 5075 (Applied Biosystems, USA) robot to dispense SYBR Green PCR master mix
(Applied Biosystems, UK) into 384-well plates, and assays were run in a 7900HT Fast Real-time PCR
System (Applied Biosystems, USA). The PCR conditions included an initial denaturation for 10 min at
95˚C, followed by 45 cycles of 95˚C for 15 s and 60˚C for 1 min. Finally, a dissociation step included
95˚C for 2 min, 60˚C for 15 s and 95˚C for 15 s. The final reaction volume was 10 ml and included

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 Short report Ecology

300 nM of primers. All reactions were carried out with two technical replicates (which showed no dif-
ference in expression levels). For each candidate gene sample (from five replicates), the ambient col-
onies at 19:30 were tested against the ambient colonies at 22:00 on the spawning night. A no-
template control and a no-reverse transcription control were performed for each gene and treat-
ment to ensure that the cDNA samples and PCR reagents did not undergo DNA contamination.
Additionally, to ensure the specificity of the primers for the coral cDNA, they were tested on cDNA
and genomic DNA from Symbiodinium sp. as a template in a PCR. This procedure avoided any
amplification of non-coral DNA. The comparative delta CT method (Walker, 2002) and a maximal
PCR efficiency for each gene (E=2) were used to determine the relative quantities of mRNA tran-
scripts from each sample. Each value was normalized to two reference genes, adenosylhomocystei-
nase (AdoHcyase) and ribosomal protein L7 (Rpl7). The selection of reference genes for this
experiment was performed using a pool of reference genes (Supplementary file 3) and analysing
their expression stability via the geNorm software (Vandesompele et al., 2002). For this study, the
most stable expression was found for AdoHcyase and Rpl7 (M value=0.225), and a minimum of two
reference genes was recommended (V2/3=0.102). The real-time dissociation curve was used to
check for the presence of a unique PCR product. Relative expression values for each gene were cal-
culated by showing a ratio of the relative expression at 22:00 over the relative expression at 19:30.
The results of the qPCR and RNA-seq analyses are presented on a log2 scale. A Welch t-test was
used to test for significant differences in the expression levels (obtained via quantitative real-time
PCR) of each gene at 19:30 compared with that at 22:00 (Supplementary file 3).

Effect of light quantity and quality on mass spawning synchronization

Ninety healthy colonies of A. millepora (which were checked for maturity as described above) were
transferred from the Heron Island reef flat and placed in the outdoor flow-through aquaria which
was continuously flushed with seawater obtained from the Heron Island reef flat. Five colonies were
placed into each aquarium, which were shaded during the day to simulate PAR levels at a 3-m
depth. The coral colonies were acclimated to ambient seawater temperatures for 48 hrs prior to the
experiment. At night, the shading was removed after sunset to expose the corals to natural moon-
light. We simulated later ‘sunsets’ to determine if light intensity and light spectra can phase shift-
spawning time. The coral colonies were divided between aquaria exposed to the light intensity treat-
ments (n=15) and light spectra treatments (n=10); the coral colonies were divided into three aquaria
for each of the seven treatments. The coral colonies were irradiated with artificial light every day for
6 hrs between 18:00 and 24:00 (midnight). These colonies also received the ambient light/dark cycle
during the day. We tested three light intensities (100 [‘high’], 50 [‘medium’] and 0.75–1 [‘low-dim’]
mmole quanta m-2 s-1 [using T8 fluorescent lamps]) and three light spectra (blue [400–500 nm, Lee fil-
ter model, deep blue 120 40% transmission], green [500–620 nm, Lee filter model, dark green 124
60% transmission] and red [620–700 nm, Lee filter model, bright red 026, 80% transmission]). The
light intensity was adjusted to 1 mmol quanta m-2 s-1 for all three light spectra using a neutral density
filter (Lee filter model, neutral density 210 (0.6) 20% transmission) when needed. The light was mea-
sured using a LI-COR (LI-192S) light meter. As a control, we maintained another set of corals that
were exposed to ambient light/dark cycles. The corals were divided into four large tables; the first
table included the control tanks, the second the ‘high’ light intensity treatment, the third the
‘medium’ light and the fourth table the ‘low’ light treatment corals. At the ends of each table, we
added another four tanks into which were projected the blue, green and red light spectra. To avoid
any light ‘leaks’, black plastic screens were placed between the treatments. The experiment was con-
ducted on a roof deck at the Heron Island Research Station to avoid artificial light ‘contamination’
and increase exposure to moonlight when the corals were not artificially irradiated. The experiment
began on October 31, 2006 and ended on November 15, 2006. The corals were monitored at night
each day after the full moon (November 5, 2006); moonrise began at 17:53, and moonset was at
04:22 (November 6). Each night, the corals were monitored between 18:00 and 03:00 or until spawn-
ing ceased. The times when the gamete bundles appeared in the polyp mouths (‘setting’) were
recorded, as well as the times at which the first and last gamete bundles were released into the
water column. The data were analyzed based on the timing of gamete release. Spawning in the field
for the same species was observed via snorkeling or scuba diving. The major spawning on the reef
and in our control aquaria occurred on the night of November 13, 2006, which was 8 nights after the
full moon night. After the spawning event on November 13, the corals were held in the aquaria and

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 Short report Ecology

monitored for later spawning in the ambient light/dark cycles with no artificial light until November
15. At the end of the experiment, the coral colonies were returned to the Heron Island reef flat.

Acknowledgements
The authors thank A Tarrant for comments on drafts of the manuscript and Heron Island Research
Station staff for their help during experiments. This work was supported by the Australian Research
Council (PK and OHG). The sequencing data reported in this study has been deposited to the
Sequence Read Archive (SRA), under accession SRP055723. The authors also thank Prof. P Harrison
for his supportive review and the two additional anonymous reviewers in improving this manuscript.

Additional information
Funding
Funder Author
Australian Research Council O Hoegh-Guldberg

The funders had no role in study design, data collection and interpretation, or the
decision to submit the work for publication.

Author contributions
PK, Conception and designs, Acquisition of data, Analysis and interpretation of data, Drafting or
revising the article, Contributed unpublished essential data or reagents; SA, Acquisition of data,
Analysis and interpretation of data, Drafting or revising the article; SKL, Analysis and interpretation
of data; OHG, Drafting or revising the article, Contributed unpublished essential data or reagents;
OL, Conception and design, Acquistion of data, Analysis and interpretation of data, Drafting or
revising the article, Contributed unpublished essential data or reagents

Additional files
Supplementary files
. Supplementary file 1. Acropora millepora genes up-regulated during broadcast spawning, genes

also detected to be variable during the spawning day are highlighted. The time of tides and moon-
rise during November 2011 spawning event.
DOI: 10.7554/eLife.09991.012
.Supplementary file 2. Gene ontology enrichment analysis for Acropora millepora gene variability
between new moon and full moon days. Gene enrichments (false discovery rate<0.1) across GO cat-
egories are shown. GOseq was used to test for enriched GO categories.
DOI: 10.7554/eLife.09991.013
.Supplementary file 3. List of candidate genes used in qPCR expression analysis.
DOI: 10.7554/eLife.09991.014

Major datasets
The following datasets were generated:
Database, license,
Dataset ID and accessibility in-
Author(s) Year Dataset title and/or URL formation
Kaniewska P, Alon 2015 Data from: Unwinding the mystery http://dx.doi.org/10. Available at Dryad
S, Karako-Lampert of coral broadcast mass spawning, 5061/dryad.541g6 Digital Repository un-
S, Hoegh-Guldberg signaling cascades and the impor- der a CC0 Public Do-
O, Levy O tance of moonlight main Dedication
Kaniewska P, Alon 2015 Acropora Raw RNA Sequencing http://www.ncbi.nlm.nih. Publicly available at
S, Karako-Lampert Data gov/sra/?term= the NCBI Short Read
S, Hoegh-Guldberg SRP055723 Archive (Accession
O, Levy O no: SRP055723).

Kaniewska et al. eLife 2015;4:e09991. DOI: 10.7554/eLife.09991 12 of 14

 Short report Ecology

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