Symbiont transmission and reproductive mode influence responses of three Hawaiian coral larvae to elevated temperature and nutrients

Abstract

Elevated temperatures and nutrients are degrading coral reef ecosystems, but the understanding of how early life stages of reef corals respond to these stressors remains limited. Here, we test the impact of temperature (mean ~ 27 °C vs. ~ 29 °C) and nitrate and phosphate enrichment (ambient, + 5 µM nitrate, + 1 µM phosphate and combined + 5 µM nitrate with 1 µM phosphate) on coral larvae using three Hawaiian coral species with different modes of symbiont transmission and reproduction: Lobactis scutaria (horizontal, gonochoric broadcast spawner), Pocillopora acuta (vertical, hermaphroditic brooder) and Montipora capitata (vertical, hermaphroditic broadcast spawner). Temperature and nutrient effects were species specific and appear antagonistic for L. scutaria and M. capitata, but not for P. acuta. Larvae survivorship in all species was lowest under nitrate enrichment at 27 °C. M. capitata and L. scutaria survivorship increased at 29 °C. However, positive effects of warming on survivorship were lost under high nitrate, but phosphate attenuated nitrate effects when N/P ratios were balanced. P. acuta larvae exhibited high survivorship (> 91%) in all treatments and showed little change in larval size, but lower respiration rates at 29 °C. Elevated nutrients (+N+P) led to the greatest loss in larvae size for aposymbiotic L. scutaria, while positive growth in symbiotic M. capitata larvae was reduced under warming and highest in +N+P treatments. Overall, we report a greater sensitivity of broadcast spawners to warming and nutrient changes compared to a brooding coral species. These results suggest variability in biological responses to warming and nutrient enrichment is influenced by life-history traits, including the presence of symbionts (vertical transmission), in addition to nutrient type and nutrient stoichiometry.

Full text

REPORT
Symbiont transmission and reproductive mode influence
responses of three Hawaiian coral larvae to elevated temperature
and nutrients
Rebecca M. Kitchen
1
•Madeline Piscetta
2
•Mariana Rocha de Souza
3
•
Elizabeth A. Lenz
3
•Daniel W. H. Schar
3
•Ruth D. Gates
3
•Christopher B. Wall
3,4
Received: 20 December 2019 / Accepted: 10 February 2020
ÓSpringer-Verlag GmbH Germany, part of Springer Nature 2020
Abstract Elevated temperatures and nutrients are degrad-
ing coral reef ecosystems, but the understanding of how
early life stages of reef corals respond to these stressors
remains limited. Here, we test the impact of temperature
(mean *27 °C vs. *29 °C) and nitrate and phosphate
enrichment (ambient, ?5lM nitrate, ?1lM phosphate
and combined ?5lM nitrate with 1 lM phosphate) on
coral larvae using three Hawaiian coral species with dif-
ferent modes of symbiont transmission and reproduction:
Lobactis scutaria (horizontal, gonochoric broadcast spaw-
ner), Pocillopora acuta (vertical, hermaphroditic brooder)
and Montipora capitata (vertical, hermaphroditic broadcast
spawner). Temperature and nutrient effects were species
specific and appear antagonistic for L. scutaria and M.
capitata, but not for P. acuta. Larvae survivorship in all
species was lowest under nitrate enrichment at 27 °C. M.
capitata and L. scutaria survivorship increased at 29 °C.
However, positive effects of warming on survivorship were
lost under high nitrate, but phosphate attenuated nitrate
effects when N/P ratios were balanced. P. acuta larvae
exhibited high survivorship ([91%) in all treatments and
showed little change in larval size, but lower respiration
rates at 29 °C. Elevated nutrients (?N?P) led to the
greatest loss in larvae size for aposymbiotic L. scutaria,
while positive growth in symbiotic M. capitata larvae was
reduced under warming and highest in ?N?P treatments.
Overall, we report a greater sensitivity of broadcast
spawners to warming and nutrient changes compared to a
brooding coral species. These results suggest variability in
biological responses to warming and nutrient enrichment is
influenced by life-history traits, including the presence of
symbionts (vertical transmission), in addition to nutrient
type and nutrient stoichiometry.
Keywords Coral reefs Larvae Nitrogen Phosphorus 
Ka¯ne‘ohe bay Lobactis scutaria Pocillopora acuta 
Montipora capitata
Introduction
Coral reef ecosystems are threatened by global and local
stressors generated by anthropogenic activities [e.g., ocean
warming and acidification (Hughes et al. 2017; Smale et al.
2019), sedimentation (Rogers 1990) and nutrient enrich-
ment (D’Angelo and Wiedenmann 2014)]. In particular,
ocean warming from anthropogenic climate change is
rapidly contributing to the decline of coral reefs through
global episodes of coral bleaching (Hughes et al. 2017).
Bleaching is the disruption of the mutualistic interaction of
the coral and its endosymbiotic algae [family Symbio-
diniaceae (LaJeunesse et al. 2018)] and can cause high
coral mortality with cascading negative effects on the
Topic Editor Anastazia Banaszak
Electronic supplementary material The online version of this
article (https://doi.org/10.1007/s00338-020-01905-x) contains sup-
plementary material, which is available to authorized users.
&Christopher B. Wall
cbwall@hawaii.edu
1
Marine Science Center, Northeastern University, 430 Nahant
Road, Nahant, MA 01908, USA
2
Rosenstiel School of Marine and Atmospheric Science,
University of Miami, 4600 Rickenbacker Causeway, Miami,
FL 33149, USA
3
Hawai‘i Institute of Marine Biology, University of Hawai‘i at
Ma¯ noa, 46-007 Lilipuna Road, Kaneohe, HI 96744, USA
4
Pacific Biosciences Research Center, University of Hawai‘i
at Ma¯ noa, 1933 East-West Rd., Honolulu, HI 96816, USA
123
Coral Reefs
https://doi.org/10.1007/s00338-020-01905-x

function and services provided by reef ecosystems (Baker
et al. 2008; Figueiredo et al. 2014; Fisch et al. 2019).
Rising sea surface temperatures are also linked to
increasing frequencies of severe storm events worldwide
(Hoyos et al. 2006) with the potential to increase coastal
runoff and nutrient pollution on coral reefs. Freshwater
input and terrestrial runoff from storm events can cause
spikes in nitrogen and phosphorus levels in normally
oligotrophic coral reefs (Drupp et al. 2011). As reef-
building corals are adapted to nutrient-poor conditions,
nutrient enrichment from coastal runoff, agriculture and
sewage in the form of nitrate and phosphate can cause
localized reef degradation (Szmant 2002), decreased cal-
cification rates (Silbiger et al. 2018), disruptions in repro-
ductive and early life stages (Harrison and Ward 2001; Cox
and Ward 2002; Lam et al. 2015; Serrano et al. 2018) and
increased levels of coral disease and bleaching (Vega
Thurber et al. 2014). Shifts in seawater nutrient concen-
tration and/or their stoichiometric balance (i.e., moles of
nitrogen/moles of phosphate) can have different effects on
reef corals. For instance, elevated nitrate levels under low
phosphate (i.e., high N/P ratios) interfere with symbiont
function, causing phosphate starvation, lipid depletion and
photosynthetic malfunction, thereby lowering bleaching
thresholds in response to light and temperature (Wieden-
mann et al. 2013; Ezzat et al. 2016). However, mitigating
dissolved inorganic nutrient pollution and improving water
quality on coral reefs can reduce the sensitivity of corals to
thermal stress (Wooldridge and Done 2009). Therefore,
coastal nutrient concentrations may be central to under-
mining or supporting the physiological resistance of corals
to ocean warming and climate change.
Early developmental stages are vulnerable points in
coral life cycles, and environmental stressors may impact
coral larvae differently or to a greater degree compared to
adult colonies (Putnam et al. 2010). Corals sexually
reproduce by broadcast spawning (i.e., external fertiliza-
tion) or brooding (i.e., internal fertilization), and eggs or
larvae released can inherit symbionts either from their
parent or from the environment (i.e., vertical and horizontal
transmission, respectively). Larvae produced through
broadcast spawning and brooding receive significant par-
ental investment in the form of lipid energy reserves pro-
visioned in the biomass of spawned eggs (85% dry weight)
and brooded larvae (40–60% dry weight) (Harii et al.
2007,2010), and these reserves support the buoyancy,
dispersal and metabolism of larvae during planktonic
stages (Harii et al. 2007; Figueiredo et al. 2012). Consid-
ering the symbiosis between corals and Symbiodiniaceae is
nascent or yet to be established in early life stages, coral
larvae may be wholly (aposymbiotic) or partially (symbi-
otic) dependent on energy reserves for respiratory needs
during planktonic stages, early metamorphosis and
settlement. Early onset of symbiosis through vertical
transmission or rapid symbiont uptake by aposymbiotic
larvae confers the benefits of autotrophic nutrition (Harii
et al. 2007) and benefits post-settlement survival (Suzuki
et al. 2013). However, damage to symbiont photomachin-
ery within larvae by environmental stress (i.e., elevated
light, temperature, nutrient enrichment) creates reactive
oxidative stress and can reduce larvae survivorship (Baird
et al. 2006; Yakovleva et al. 2009). Thus, environmental
conditions, including nutrient enrichment, with the poten-
tial to disrupt the function or establishment of the coral–
Symbiodiniaceae symbiosis or influence coral larvae res-
piration (Edmunds et al. 2001), photosynthesis and energy
reserves catabolism (Rivest et al. 2017) can create energy
shortages with consequences for larval performance (e.g.,
growth, pelagic duration, settlement and survivorship)
(Nakamura et al. 2011).
Although many studies have tested the effects of tem-
perature and/or nutrient enrichment (i.e., nitrogen [nitrate,
ammonium, urea] or phosphate) on adult corals (as
reviewed by Fabricius 2005; Shantz and Burkepile 2014),
studies on the responses of early life stages of coral to
elevated temperature and/or nutrients are sparse and results
often inconclusive. For instance, in the broadcast-spawned
aposymbiotic larvae of Orbicella faveolata and Diploria
strigosa thermal stress increased larvae mortality (Bassim
and Sammarco 2003; Serrano et al. 2018) and increased O.
faveolata respiration (Serrano et al. 2018). However, in
brooded symbiotic larvae the respiration of Porites
astreoides both increased (Olsen et al. 2013) and remained
unchanged (Ross et al. 2012; Serrano et al. 2018)in
response to elevated temperatures. Similar variability has
been noted for Pocillopora damicornis, with elevated
temperatures increasing (Rivest and Hofmann 2014) and
decreasing (Putnam et al. 2013) respiration rates, possibly
due to a combination of environmental history effects on
parents (Rivest et al. 2017), parental energy investments
and maternal bet hedging (Edmunds et al. 2001; Cumbo
et al. 2013). In the few studies examining nutrient effects
on early life stages of corals, nitrate enrichment was shown
to increase respiration and settlement of brooded P.
astreoides larvae (Serrano et al. 2018). Elevated ammo-
nium also increased D. strigosa mortality and decreased
larval settlement, and these effects were additive with
elevated temperatures (Bassim and Sammarco 2003). Ele-
vated ammonium and phosphate impaired fertilization and
development of embryos in broadcast-spawning corals
Acropora longicyathus and Goniastrea aspera (Harrison
and Ward 2001), but nutrients (nitrate, ammonium and
phosphate) had no effect on the fertilization in A. millepora
(Humphrey et al. 2008). In part, uncertainty in nutrient
effects can originate in nutrient identity (i.e., ammonium,
nitrate, urea), their ecological sources (i.e., fish-derived
Coral Reefs
123

[ammonium], fertilizer runoff [nitrate]) (Shantz and Bur-
kepile 2014) and whether nitrogen enrichment is con-
comitant with phosphate limitations (Ezzat et al. 2016).
Considering nutrient enrichment interferes with the sym-
bionts’ ability to uptake nutrients (Ezzat et al. 2016),
maintain photochemical function (Wiedenmann et al.
2013) and remain within host tissues (Rosset et al.
2017a,b), it is reasonable that coral larvae responses to
nutrient enrichment may depend on the presence or
absence of Symbiodiniaceae. Therefore, modes of sym-
biont transmission (i.e., inherited in egg/larvae, acquired
from environment) and size or energy constraints imposed
on larvae from parental investments (i.e., broadcast
spawned, brooded) may be important factors in disentan-
gling previously inconsistent responses of coral larvae to
dissolve nutrients.
Here, we test the effects of thermal stress and nutrient
enrichment on the larvae of three Hawaiian coral species—
Lobactis scutaria (formerly Fungia scutaria [see Gitten-
berger et al. 2011]), Pocillopora acuta and Montipora
capitata—with different reproductive strategies and sym-
biont transmission modes. Lobactis scutaria and M. capi-
tata are broadcast spawners, while P. acuta is a brooder; L.
scutaria relies on horizontal transmission, while M. capi-
tata and P. acuta acquire symbionts through vertical
transmission (Fig. 1; Table 1). Coral reefs in Ka¯ne‘ohe
Bay have a history of anthropogenic disturbance (i.e.,
sewage pollution, urbanization) coupled with strong natural
stressors (i.e., history of thermal anomalies, high pCO
2
variance) (Bahr et al. 2015) which have created a model
system for studying resilience in corals. Based on the
current understanding of temperature and nutrient stress on
adults of these coral species and larval responses in other
studies, we tested the following hypotheses: (1) the com-
bined effects of temperature and nutrients will decrease
larval survivorship (Edmunds et al. 2001; Schnitzler et al.
2012; Graham et al. 2015; Serrano et al. 2018), respiration
rates (Putnam et al. 2013) and reduce larvae growth (Ed-
munds et al. 2005) and symbiont densities as is observed in
bleached corals; (2) elevated nutrients (nitrate and phos-
phate) will increase the density of DIN-limited endosym-
bionts in symbiotic larvae, but greater symbiont densities
will exacerbate effects of thermal stress (Fabricius 2005;
Cunning and Baker 2013; Shantz et al. 2016). We also
hypothesize that symbiotic larvae will be more sensitive to
nutrient (especially nitrate) enrichment than aposymbiotic
larvae, as endosymbiont populations will utilize nutrients
for growth (Falkowski et al. 1993; Ezzat et al. 2015). In
addition, a greater size and parental investment in brooded
larvae will result in less sensitivity to temperature stress
compared to smaller broadcast-spawned larvae (Baird et al.
2009).
Materials and methods
Gamete collection and larval rearing
Adult corals of L. scutaria (Lamarck 1801), P. acuta
(Lamarck 1816) and M. capitata (Dana 1846) originated
from patch reefs within Ka¯ne‘ohe Bay, Hawai‘i (HIMB
Special Activities Permit 2018, Division of Aquatic
Resources, Hawaii). Gametes or planulae were collected
from parent colonies, and all larvae were reared at the
Hawai‘i Institute of Marine Biology (HIMB, see Supple-
mental Material).
Gametes were collected from L. scutaria adults
(n= 153) on the night of 28 July 2018 and placed into
ambient-temperature, sand-filtered seawater in 19-L buck-
ets. Pooled eggs and sperm were concentrated in the
buckets and allowed to fertilize for 1 h. Remaining sperm
were removed by siphoning the bottom of the buckets, and
eggs were rinsed by replenishing with 1-lm filtered sea-
water (FSW). Embryos were left to develop in buckets
overnight. The following morning, ciliated larvae were
randomly selected and placed into treatment tubes (detailed
below). Adult P. acuta colonies (n= 30) were isolated on
July 30, 2019, into containers with ambient-temperature,
sand-filtered seawater that would flow into vessels with
153-lm mesh. Larvae released from 15 different parent
colonies were collected the following morning, evenly
mixed and placed into treatment tubes on the day of col-
lection. For M. capitata, positively buoyant egg–sperm
bundles released from adult colonies were collected in situ
from Ka¯ne‘ohe Bay on the night of August 10, 2018, using
a 123-lm mesh sieve. Gamete bundles were rinsed with
1-lm FSW in 9.5-L buckets, with one layer of bundles over
the surface of 2 L of FSW. Fertilization occurred in the
buckets for 1 h. Fertilized eggs were gently poured into 5-L
flow-through conical tanks filled with 1-lm ambient-tem-
perature FSW overnight under gentle water motion. M.
capitata larvae were placed into treatment tubes on the
following day once the embryos had reached the prawn
chip stage.
Experimental treatments and sampling
Ecologically relevant temperature treatments were selected
based on average sea surface temperatures during the
summer months in Ka¯ne‘ohe Bay (*27 °C) (NOAA
2019) and mean temperatures recorded during local
bleaching events (*29 °C) (Coles et al. 2018; Wall et al.
2019). Ambient nitrate and phosphate concentrations in
Ka¯ne‘ohe Bay during summer months are low, being ca.
0.5 and 0.1 lM, respectively (Drupp et al. 2011; Wall et al.
2019). Elevated nutrient concentrations (5.0 lM nitrate,
Coral Reefs
123

1.0 lM phosphate) were chosen based on peak annual
values measured in Ka¯ne‘ohe Bay, which often pulse and
subside during rainy season months (September–February)
(Drupp et al. 2011).
Larvae were exposed to two temperatures (mean of
*27 and 29 °C, AT and HT) and four nutrient concen-
trations (ambient, ?5lM nitrate [NO
3
-
], ?1lM phos-
phate [PO
3
4-
] and combined ?5lM nitrate with ?1lM
phosphate), resulting in eight fully crossed treatments:
ambient-temperature ambient-nutrients (AT/A), high-tem-
perature ambient-nutrients (HT/A), ambient-temperature
high-nitrate (AT/N), high-temperature high-nitrate (HT/N),
ambient-temperature high-phosphate (AT/P), high-tem-
perature high-phosphate (HT/P), ambient-temperature
high-nitrate and phosphate (AT/NP) and high-temperature
high-nitrate and phosphate (HT/NP). For each species, five
a
c
e
0.5 mm
0.1 mm
1 mm
1 mm
b
d
f
g
Fig. 1 Adult and larvae of the
Hawaiian corals (a,b)Lobactis
scutaria and (c,d)Pocillopora
acuta, with Montipora capitata
(e) adult colonies, (f) eggs and
(g) planulae larvae. Settling P.
acuta larvae (d, top right).
Lobactis scutaria and M.
capitata are broadcast
spawners; P. acuta is a brooder.
Lobactis scutaria larvae are
initially aposymbiotic, while P.
acuta and M. capitata inherit
Symbiodiniaceae through
vertical transmission. (PC: all
authors, except [d: R. Ritson-
Williams])
Coral Reefs
123

tube replicates (50-mL Falcon tubes) were used for each
treatment with an initial number of 40 larvae in each tube
replicate for L. scutaria and M. capitata (n= 1600) and 20
larvae in each tube replicate for the larger P. acuta larvae
(n= 800). Larvae of each species were exposed to these
treatments for 5 d, and water changes were conducted
daily. In order to expose all species to treatments for the
same duration, the length of the experiment was based on
approximately the length of time L. scutaria could be kept
alive without the uptake of algal symbionts (Schwarz et al.
1999), which was 4–5 d.
Temperature treatments were set up at HIMB in two
outdoor, shallow water tables with a shade cloth overhead.
Water tables received a steady flow of ambient Ka¯ne‘ohe
Bay seawater (ca. 26–27 °C) with the heated water
table having a 300-W submersible heater to raise temper-
atures 1–2 °C above ambient. Temperature in each tank
was recorded at 15-min intervals with HOBO Water
Temperature Pro v2 Data Loggers (Onset Computer, USA)
throughout the experiment with seawater table tempera-
tures monitored throughout each day using a certified
digital thermometer (5-077-8, accuracy = 0.05 °C, Control
Company, USA). The treatment tubes containing the larvae
were placed upside-down in racks within each water
treatment bath to minimize shading.
Dissolved nutrients analyses
Conical centrifuge tubes containing 45 mL of 0.2-lm FSW
were spiked with nutrient stock solutions (Milli-Q water
with sodium nitrate [NaNO
3
] to 0.2 mM NO
3
-
, and
sodium phosphate dibasic anhydrous [Na
2
HPO
4
] to 1.0 lM
PO
4
3-
) to obtain nutrient enrichment treatments (see
Supplemental Material). Measurements showed that nutri-
ent spikes did not alter salinity and pH values beyond
normal ranges for Ka¯ne‘ohe Bay (NOAA 2019) (Table S1).
Treatment seawater was changed daily, and outflow water
at the end of the first and final days of the experiment (Day
4 or 5) was saved from all tubes (n= 5 tubes species
-1
)to
confirm the spiked nutrient treatments remained above
ambient treatment concentrations for the 24-h incubations
between water changes (Table S2). Collected water sam-
ples were kept in a dark cooler on ice, filtered (0.7-lm GF/
F), frozen (-20 °C) and analyzed for nitrate ?nitrite
(NO
3
-
?NO
2
-
or N ?N) and phosphate concentrations
(Strickland and Parsons 1972; Parsons et al. 1984), with
[NO
3
-
] values reported for each treatment.
Biological response variables
Survivorship (i.e., percentage of larvae alive) in each
replicate tube was measured daily during water changes;
swimming larvae or settled spat were counted with the aid
of a dissecting microscope. Larval dark respiration was
measured on the last day of the experiment (L. scutaria
[Day 4], P. acuta and M. capitata [Day 5]) using PreSens
Measurement Studio software (PreSens Precision Sensing
GmbH, Germany) and noninvasive fluorescent oxygen
sensors (PreSens Sensor Spots) affixed on 2-mL glass vials
(n= 3–5 vials treatment
-1
species
-1
). Due to low sur-
vivorship, replicate tubes in each treatment were pooled for
L. scutaria and M. capitata, with ten larvae treatment
-1
in
each respiration vial. P. acuta larvae exhibited high sur-
vivorship and were not pooled; instead, larvae (n= 10)
were randomly sampled from each of the five replicate
treatment tubes. Temperature treatments were maintained
using heat blocks, and temperature probes in separate
seawater–blank vials in each heat block were used to
monitor temperature during respiration measurements. P.
acuta and M. capitata larvae were dark adapted for
15–30 min, and respiration was measured in darkness, with
oxygen concentrations measured over 30–120-min inter-
vals to acquire a constant slope. Aposymbiotic L. scutaria
larvae were not dark adapted and had respiration measured
under indirect incandescent light. Final respiration rates
were corrected against seawater-only negative controls
Table 1 Details of the biological traits and reproductive strategies of the three species of Hawaiian corals studied (Lobactis scutaria, Pocil-
lopora acuta and Montipora capitata)
Species Growth form and sexuality Reproductive strategy Symbiont acquisition mode Initial larval size
a
(lm
2
mean ±SE)
L. scutaria Solitary, gonochoric Broadcast spawner Horizontal transmission: aposymbiotic larvae
acquire free-living symbionts 3–5 d after
spawning event (Schwarz et al. 1999)
47.42 ±0.02
P. acuta Colonial, hermaphroditic Brooder Vertical transmission: symbiotic, fully formed
larvae released from adults (Cumbo et al. 2013)
843.91 ±0.22
M. capitata Colonial, hermaphroditic Broadcast spawner Vertical transmission: eggs equipped with symbionts
that develop into symbiotic larvae once externally
fertilized (Padilla-Gamin
˜o et al. 2012)
219.00 ±0.07
a
Larval size at the beginning of the experiment
Coral Reefs
123

(n= 10), normalized by the number of larvae within each
vial and expressed as nmol O
2
larva
-1
min
-1
.
Symbiont cell density for P. acuta and M. capitata was
measured by sonicating preserved larvae, vortexing and
resuspending concentrated algal cells and counting cells
using a hemocytometer (n= 6–8 replicate counts) and an
Olympus BX51 compound microscope (see Supplemental
Materials). Symbiont cell densities were normalized to the
number of larvae within each tube and are presented as
mean symbiont cells larva
-1
.
To calculate the change in size, larvae were preserved in
diluted zinc formaldehyde fixative (1:4 Z-fix, Sigma-
Aldrich Inc., to 0.2-lm filtered seawater FSW) at the start
and the end of the experiment and stored at 4 °C. For each
species, we used two replicate sets of larvae (n= 10 larvae
set
-1
) at the start of the experiment and 3–5 sets of larvae
treatment
-1
at the end of the experiment. Larvae size at
each sampling point was quantified using MagnaFire
software and an Olympus SZX7 dissecting microscope
equipped with an Olympus America KIH036577 Micro-
scope Camera. Only larvae that were still intact within the
z-fix solution were included in the size analysis. Planar
area was calculated using ImageJ, version 1.51j8 (Schnei-
der et al. 2012).
Statistical analyses
Due to fundamental differences between the three species
studied, biological responses for each species were ana-
lyzed separately. Survivorship data were analyzed using a
Cox proportional hazards model using the survminer
package in R (Kassambara et al. 2019) with nutrient and
temperature treatments as fixed effects and replicate tube as
a random effect. The inclusion of random effects did not
alter model outputs and was dropped from the model.
Model assumptions were checked by testing and visualiz-
ing the Schoenfeld and Martingale residuals. Due to
complete mortality in one P. acuta HT/P tube, this tube
was removed from the analysis. Post hoc testing was
completed using the survival package (Therneau 2015).
Dark respiration and symbiont cell densities were ana-
lyzed with a linear model with nutrient and temperature
treatments as fixed effects; model assumptions were con-
firmed with Shapiro–Wilk and Levene’s tests. One outlier
was removed from both P. acuta and M. capitata respira-
tion rates data. L. scutaria respiration rates were not ana-
lyzed because the oxygen consumption rates for these
small larvae (n= 4–10 vial
-1
) were low and indistin-
guishable from background controls. Post hoc testing was
completed using the emmeans package (Lenth 2019).
Larval size for each species was analyzed using Kruskal–
Wallis nonparametric tests with nutrient and temperature
treatments as fixed effects. Model assumptions were
checked through visualization of residuals, and post hoc
testing was completed using the Dunn test. All analyses
were completed in R, version 3.6.1 (R Core Team 2019).
Data and scripts are publicly available on GitHub https://
github.com/cbwall/Coral-larvae-temp-and-nutrients (Wall
2020).
Results
Nutrient analyses
Mean seawater temperatures followed natural oscillations
throughout the day and averaged from 26.97 to 27.37 °Cin
the AT treatment and 28.72 to 29.17 °C in the HT treat-
ment (Table S1; Fig. 2). On average, daily maximum
temperatures were 28 °C and 31 °C in the AT and HT
treatments, respectively. Nutrients in ambient seawater had
mean (±SE, n= 8) [NO
3
-
] and [PO
4
3-
] of 0.47 and
0.10 lmol L
-1
, with nitrite contributing an additional
0.22 lmol L
-1
. Nitrite concentrations remained low and
constant across all treatments (\0.5 lmol L
-1
). Nutrient
treatments remained elevated during the 24-h incubations
between daily nutrient spikes, with similar nutrient
26
28
30
32
a
26
28
30
32
Temperature (°C)
b
26
28
30
32
012345
Day
cM. capitata
L. scutaria Ambient (27°C)
High (29°C)
P. acuta
Fig. 2 Seawater temperatures for three corals (a)Lobactis scutaria,
(b)Pocillopora acuta and (c)Montipora capitata exposed to
orthogonal ambient (27 °C) and high (29 °C) temperatures and four
orthogonal nutrient concentrations (Table S2). Dashed lines indicate
mean temperature for each treatment
Coral Reefs
123

concentrations in both temperature treatments (Table S2).
Instantaneous light levels measured across the seawater
table at midday averaged 70 ±9lmol photons m
-2
s
-1
(mean ±SE, n= 26) across experimental days.
Survivorship
Larval survivorship in M. capitata was the most sensitive
to treatment conditions, while P. acuta was the most tol-
erant. L. scutaria survivorship was low overall, with ca.
25–50% survivorship in all treatments within 48 h and
18–29% survivorship by day 4. Overall, L. scutaria sur-
vivorship was negatively affected by nutrient additions
(p\0.001), although nutrient effects were temperature
dependent (p= 0.003) and more pronounced at cooler
temperatures (Table 2, Fig. 3a; Fig. S3). Nitrate alone
reduced survivorship at both temperature treatments;
however, the combined ?N?P treatment increased sur-
vivorship at HT relative to the ambient nutrient control. For
P. acuta larvae, survivorship was impacted by both tem-
perature (p= 0.037) and nutrients (p= 0.003) (Table 2,
S3; Fig. 3b); although, these effects were negligible and
survivorship was [91% in all treatments. Similar to L.
scutaria,P. acuta larvae had the lowest survivorship (91%)
in the AT/N treatment and highest survivorship (98%) in
the HT/NP treatment.
Montipora capitata larval survivorship was the most
sensitive to treatment effects and was affected by temper-
ature, nutrients and their interaction (p\0.001) (Table 2,
S3; Fig. 3c). Overall, survivorship was significantly higher
at HT relative to AT (60% versus 44%) and was reduced by
20% in nitrate enriched treatments compared to all others.
Highest survivorship was observed under ambient nutrients
at HT (71%) and lowest under elevated nitrate at AT
(22%). Relative to nutrient controls, larval survivorship at
27 °C declined with elevated nitrate but increased under
elevated phosphate. Moreover, the negative effects of
nitrate at AT were ameliorated when nitrate and phosphate
were both elevated in the ?N?P treatment (55%). This
pattern contrasts with observations at HT, where M. capi-
tata survivorship declined in all nutrient-enriched treat-
ments, being lowest in treatments where nitrate was
elevated (i.e., HT/N, HT/NP).
Respiration
Dark respiration rates were only assessed for P. acuta and
M. capitata due to low signal in L. scutaria larvae (see
Materials and Methods). Treatment effects were only seen
in P. acuta larvae which showed ca. 39% less oxygen
consumption when exposed to elevated temperature
(p\0.001) (Table 2, S4; Fig. 4a). Dark respiration rates
of M. capitata larvae were lower compared to those of P.
acuta, but were not affected by treatments (pC0.209)
(Fig. 4b).
Symbiont cell densities
Symbiodiniaceae cell densities normalized per larvae of P.
acuta were not affected by treatments (p= 0.262)
(Tables 2, S4; Fig. 3c). M. capitata symbiont densities,
however, were affected by nutrient treatments (p= 0.008)
(Table 2; Fig. 3d) and ranged between 2000 and 2400
symbiont cells larva
-1
across all treatments. Nitrate- and
phosphate-enriched treatments had the highest densities
(23% and 21% above controls, respectively) with inter-
mediate densities (6% above controls) in the ?N?P
treatment (Fig. 4d).
Larval size
Changes in L. scutaria larvae size were influenced by
nutrients (p\0.001) and its interaction with temperature
(p\0.001). Mean larval size decreased in all treatments
Table 2 Summary of
significant statistics across all
biological metrics for the three
coral species studied
Species Effect Biological response
Survivorship Respiration Symbiont density Larval size
L. scutaria Temp 0.208 – – 0.687
Nutrients < 0.001 –– < 0.001
Temp 9nutrients 0.003 –– < 0.001
P. acuta Temp 0.037 < 0.001 0.285 0.117
Nutrients 0.003 0.798 0.640 0.507
Temp 9nutrients 0.205 0.174 0.262 0.191
M. capitata Temp < 0.001 0.839 0.922 0.011
Nutrients < 0.001 0.902 0.008 0.002
Temp 9nutrients < 0.001 0.209 0.068 < 0.001
For complete statistical analyses output, see Supplemental Material
Dashes represent responses that were not measured
Coral Reefs
123

29°C +Nitrate
0.00
0.25
0.50
0.75
1.00
024487296
Survivorship
024487296120
Hours
b
024487296120
c
L. scutaria
aP. acuta M. capitata
27°C +Phosphate +N+P
Control
*‡
§
§
Fig. 3 Survivorship for larvae of three coral species exposed to
ambient (solid lines) and high (dashed lines) seawater temperatures
and four nutrient concentrations of ambient (control) and elevated
nitrate (?N) and phosphate (?P) (Table 2). Symbols represent
significant effects (p\0.05) of temperature (asterisk), nutrients
(double tagger), or their interaction (section sign). Values are
mean ±SE (n= 35–200)
0
5000
10000
cd
Nutrient Treatment
‡
symbiont cells larva-1
0.0
0.1
0.2 ab
Ambient (27°C)
High (29°C)
P. acuta M. capitata
Respiration
(nmol O2 consumed larva-1 min-1)
Control
+Nitrate
+Phosphate
+N+P
Control
+Nitrate
+Phosphate
+N+P
abb ab
*
Fig. 4 Coral larva (a,b) dark
respiration rates and (c,
d) symbiont cell densities for
Pocillopora acuta and
Montipora capitata in response
to orthogonal temperature and
nutrient treatments. Lobactis
scutaria respiration rates were
poorly resolved and larvae are
aposymbiotic (see Materials and
Methods). Symbols represent
significant effects (p\0.05) of
temperature (asterisk) or
nutrients (double tagger).
Values are mean ±SE
(n= 38–50)
Coral Reefs
123

by 57–72%, but the largest change in size significantly
different from ambient nutrient controls was observed in
both ?N?P treatments and the HT/P treatments (Fig. 5a).
In contrast, treatments had no significant effect on P. acuta
larval size (Tables 2, S5; Fig. 5b). For M. capitata, larvae
increased in size by 25–57% and this was influenced by
temperature (p= 0.011), nutrients (p= 0.002) and their
interaction (p\0.001). Change in M. capitata larval size
was on average 10% less in HT relative to AT and 7–20%
lower in nitrate or phosphate treatments compared to both
ambient nutrient controls and the ?N?P treatments, which
saw the greatest positive change in larvae size. The AT/A
treatment and the AT/NP treatments grew the most (ca.
56%), while the HT/N treatment grew the least (25%).
Growth rates of all other treatments were between these
two size classes (average percent changes ranging from 30
to 41%) and were not significantly different from one
another (Tables 2, S5, Fig. 5c).
Discussion
We investigated the performance of three Hawaiian corals
with different symbiont transmission and reproductive
strategies to temperature and nutrient effects. Counter to
our initial hypotheses, a 2 °C elevation in temperature
alone or in combination with elevated nutrients did not
cause overly adverse effects in symbiotic species. In fact,
the naturally cycling elevated temperature regime used
here appeared to ameliorate some of the negative effects of
nutrients on the survivorship of the broadcast-spawned
larvae. Nitrate adversely affected both symbiotic and
aposymbiotic larvae. Phosphate exhibited a stronger neg-
ative effect on the aposymbiotic L. scutaria, and the
combination of ?N?P mitigated the harmful effects of
nitrate in most situations. We also show that the brooded P.
acuta larvae were more tolerant to treatment conditions,
displaying little change in survivorship and no discernible
change in symbiont cell density or larval size, but having
lower respiration rates at high temperature. These results
highlight the responsiveness of coral larvae to changes in
temperature and dissolved nutrients and suggest species-
specific responses may in part originate from fundamental
differences in life-history strategies and the presence of
Symbiodiniaceae in early life stages in relation to modes of
symbiont inheritance.
Thermal stress weakens coral–endosymbiont interac-
tions and reduces larval survivorship (Edmunds et al. 2001;
Randall and Szmant 2009; Schnitzler et al. 2012; Graham
et al. 2017; Serrano et al. 2018); however, the warm (and
variable) temperature treatments (*29 °C) in our study
positively affected larval survivorship and did not produce
lower symbiont densities relative to 27 °C. It should be
noted, however, that many previous studies used higher-
aab b
−100
−50
0
50
100
Control
+Nitrate
+Phosphate
+N+P
% change in larval area
a
Nutrient Treatment
b
b
a
b
bab
b
ab
a
c
P. acuta M. capitata
L. scutaria
Ambient (27°C)
High (29°C)
Control
+Nitrate
+Phosphate
+N+P
Control
+Nitrate
+Phosphate
+N+P
ab
aa
bb
§
§
Fig. 5 Percent change in larvae size in three coral species in response to orthogonal temperature and nutrient treatments. Symbols (section sign)
represent significant treatment interactions (p\0.05), while letters indicate post hoc contrasts. Values are mean ±SE (n= 15–49)
Coral Reefs
123

temperature elevations than used in the current study and
revealed negative affects of warming on larvae. For
instance, in studies where temperature was [29 °C, L.
scutaria larvae were unable to establish symbiosis with
Symbiodiniaceae and mortality increased (31 °C) (Schnit-
zler et al. 2012). While P. damicornis larval survivorship
was unaffected by temperatures (27, 30, 32 °C), moderate
bleaching was observed at temperatures above 27 °C
(Haryanti et al. 2015). Here, we observed survivorship in
P. acuta to be resistant to temperature effects, while
aposymbiotic L. scutaria and symbiotic M. capitata larvae
had higher survivorship in the high-temperature treatment
(*29 °C). The absence of clear negative effects of ele-
vated temperature on larval survivorship in the three coral
species used in our study, however, should not be inter-
preted as evidence for negligible effects of rising sea sur-
face temperatures on corals and their offspring. Instead, the
positive effects of elevated temperature on larvae sur-
vivorship (primarily in M. capitata) may be attributed to
the variable thermal regimes mimicking diel temperature
cycles compared to stable treatments, or alternatively,
to the level of thermotolerance in corals from Ka¯ne‘ohe
Bay (Coles et al. 2018). For instance, adult and juvenile
corals exhibit different physiological responses to variable
conditions of temperature (Putnam et al. 2010; Mayfield
et al. 2012), and adult L. scutaria, P. damicornis and M.
capitata corals in Ka¯ne‘ohe Bay have become less sensi-
tive to elevated temperatures over the last 30 yr (Coles
et al. 2018). While our treatments reached daily tempera-
ture maximums of 30–32 °C, these early-stage larvae
appear well equipped to tolerate these high and variable
temperatures, although latent effects of elevated tempera-
ture may manifest post-settlement. Therefore, under eco-
logically relevant and oscillating temperature regimes,
larvae from broadcast-spawning coral species benefited
from small increases in temperature; however, this effect
was most pronounced if larvae had already formed sym-
biosis with Symbiodiniaceae.
Although responses to temperature were moderate,
nutrient enrichment had more pronounced effects. Nutri-
ent-enrichment effects were most negative for survivorship
of M. capitata larvae. Principally, nitrate enrichment re-
duced larval survivorship, whereas the effects of elevated
phosphate were more benign. This is consistent with the
findings of Rosset et al. (2017a,b) that stated high-nitrate/
low-phosphate (i.e., phosphate starvation) conditions are
more detrimental to adult coral survival than high-phos-
phate/low-nitrate conditions. Interestingly in the current
study, elevated nitrate and phosphate together did not
reduce larval survivorship, indicating that negative nutrient
enrichment effects are in part linked to nutrient stoi-
chiometry and the nitrate/phosphate (N/P) ratio. This fur-
ther suggests that phosphate starvation is a significant
driver of negative nutrient impacts on larval survivorship.
This may be especially true in symbiotic larvae, where
greater nitrate availability may weaken host regulation of
symbiont nutrient availability and the integrity of the
symbiosis (Falkowski et al. 1993; Ezzat et al. 2015). In
addition, Ezzat et al. (2016) displayed that adult coral
phosphate uptake increases during thermal stress while
nitrogen acquisition rates decline, suggesting a pivotal role
of phosphate in endosymbiont function. Our results, how-
ever, suggest the capacity for nutrient concentrations to
destabilize coral symbioses and increase thermal sensitivity
may require threshold concentrations or chronic exposures
to elevated nutrients beyond those applied in the current
study.
One of the few studies comparing temperature and
nutrient effects on broadcast-spawned and brooded coral
larvae hypothesized the presence of symbionts in brooded
larvae of P. astreoides increased the sensitivity of this
species to elevated nitrate compared to the aposymbiotic O.
faveolata larvae (Serrano et al. 2018). Elevated nitrate
effects on Symbiodiniaceae can adversely affect a coral’s
thermal tolerance by lowering thresholds to light- and
temperature-induced bleaching (Wiedenmann et al. 2013;
Ezzat et al. 2016); however, temperature and nutrient
treatments in the present study did not reduce symbiont
densities. In fact, M. capitata symbiont densities were
marginally higher in nutrient-enriched treatments com-
pared to controls. In this context, nutrient concentrations
appear to have produced positive outcomes for Symbio-
diniaceae by increasing symbiont cell densities, with the
possibility that corals received more autotrophic nutrition.
However, these effects did not correlate with host fitness,
as the species most affected by temperature and nutrient
treatments (M. capitata) had lowest survivorship under
high nitrate, further highlighting the importance of nutrient
stoichiometry (Wiedenmann et al. 2013). These results
agree with Serrano et al. (2018) that vertical symbiont
transmission relates to nutrient sensitivity, but show these
effects are most significant in broadcast-spawning corals,
possibly due to the small larval size and lower stocks of
inherited energy reserves and symbiont cells compared to
brooders.
Treatment effects on larval respiration rates were lim-
ited to the brooded larvae of P. acuta, which showed lower
respiration at higher temperatures. These rates were similar
to those measured in other pocilloporid brooded larvae
(Edmunds et al. 2011; Cumbo et al. 2013; Putnam et al.
2013) and adults (Courtial et al. 2018) and agree with the
general pattern of elevated temperature reducing photo-
synthesis/respiration (P/R) ratios in corals (Coles and
Jokiel 1977; Edmunds et al. 2005). Larval respiration may
show a hyperbolic relationship with temperature (Edmunds
et al. 2011), increasing to a threshold and decreasing
Coral Reefs
123

thereafter once thermal stress damages proteins and dis-
rupts cellular processes (Hochachka and Somero 2002). In
symbiotic larvae, temperature effects on metabolism may
also be influenced by reactive oxygen species generated by
Symbiodiniaceae leading to symbiont expulsion (Weis
2008), as well as larval death (Yakovleva et al. 2009). The
expression of symbiont proteins central in photosynthesis
(i.e., Rubisco) can also decline at elevated temperatures,
leading to energy deficits that may also influence metabolic
costs and respiration rates (Putnam et al. 2013). In our
study, considering the lack of temperature effects on
symbiont densities, lower P. acuta respiration rates at
elevated temperatures may be attributed to host responses
to temperature; however, temperature and nutrient effects
on coral energy usage and acquisition should be further
explored. Finally, the null effect of treatments on M. cap-
itata respiration indicates cellular metabolism in this spe-
cies is less sensitive to 2 °C temperature changes. It should
be noted, however, respiration rates in the present study
(nmol O
2
min
-1
larva
-1
) are not normalized to units of
tissue biomass, and the temperature dependency coeffi-
cients (i.e., Q
10
) for biomass-normalized respiration rates
(lgO
2
h
-1
mg DW
-1
) may not differ among coral species
or life stages at sub-stressful temperature ranges
(25–30 °C) despite differences in overall respiration rates
(Haryanti and Hidaka 2015). Therefore, the metabolism
and respiration rates of adult and juvenile corals may be
equally sensitive to changes in temperature below thermal
thresholds, but the ability for P. damicornis and P. acuta
(this study) larvae to lower respiration rates at high tem-
peratures may benefit larvae survivorship as a metabolic
cost-saving strategy (Haryanti and Hidaka 2015).
Although, the capacity for larvae to mitigate negative
effects of thermal stress may depend on environmental
history and biological attributes such as brood quality (i.e.,
energy reserves, size, competency) and maternal invest-
ments (Putnam et al. 2010; Cumbo et al. 2013).
Changes in larvae size were influenced by the interac-
tion of temperature and nutrients and these effects differed
substantially among species. Larval size in reef corals is
not a good indicator of survivorship (Nozawa and Okubo
2011); however, larger brooded larvae have the potential
for extended pelagic durations (100 d; Richmond 1987),
and we observed minimal changes in larvae size of brooded
P. acuta larvae compared to other species. Larvae size
declined by[50% in all treatments in L. scutatia, possibly
due to the lack of algal symbionts and complete reliance on
energy reserves to meet metabolic costs. Conversely, M.
capitata larval size increased over the 5-d experiment
(*20–50%). The positive change in larvae size may relate
to the lifecycle ontogeny in M. capitata, but could also be
an effect of these larvae inheriting symbionts from their
parents and benefitting from symbiont-derived autotrophy.
Indeed, algal symbionts can transfer 13–27% of photo-
synthates to the host larvae (Richmond 1981), and sym-
biont photosynthesis provides coral larvae with the
capacity to use their lipid energy reserves at lower rates
(Harii et al. 2010), potentially extending larval
survivorship.
Understanding the effect of predicted environmental
changes on coral larvae is essential to predict the success of
coral reefs worldwide. Here, we show the effects of
warming and nutrients are species specific, and were more
pronounced in symbiotic broadcast-spawning species,
affecting respiration, symbiont density and larval sur-
vivorship. Other long-lasting effects of these stressors
could manifest post-settlement, thereby compromising
coral recruitment (Randall and Szmant 2009; Ross et al.
2012; Humanes et al. 2017) and creating profound, long-
lasting effects on the health of coral reefs. Future studies
should include different coral species and test the response
of corals in different life stages to the combined effect of
multiple stressors predicted for future oceans such as
warming, nutrient enrichment and acidification.
Acknowledgements This study was funded by the Paul G. Allen
Family Foundation, Colonel Willys E. Lord and Sandina L. Lord
Endowed Scholarship, an NSF graduate research fellowship to
E.A.L., a Coordenac¸a
˜o de Aperfeic¸oamento de Pessoal de Nı
´vel
Superior—Brasil (CAPES) fellowship to M.R.S. and an Environ-
mental Protection Agency STAR Fellowship Assistance Agreement
(FP-91779401-1) to C.B.W. The views expressed in this publication
have not been reviewed or endorsed by the EPA and are solely those
of the authors. We thank the SOEST Laboratory for Analytical Bio-
geochemistry (SLAB) at the University of Hawai‘i at Ma¯noa for
assistance with the nutrient analysis. We would also like to thank K.
Hughes, J. Davidson, C. Drury, A. Huffmyer, C. Harris and D. Chee
for their support. This is HIMB contribution 1786 and SOEST con-
tribution number 10905. We dedicate this manuscript to the life and
legacy of our dear friend and mentor Dr. Ruth Gates.
Compliance with ethical standards
Conflict of interest The authors declare that they have no conflicts of
interest.
References
Baird AH, Gilmour JP, Kamiki TM, Nonaka M, Pratchett MS,
Yamamoto HH, Yamasaki H (2006) Temperature tolerance of
symbiotic and non-symbiotic coral larvae. In: Proceedings of the
10th international coral reef symposium, pp 38–42
Baird AH, Guest JR, Willis BL (2009) Systematic and biogeograph-
ical patterns in the reproductive biology of Scleractinian corals.
Annu Rev Ecol Evol Syst 40(1):551–571
Bahr KD, Jokiel PL, Toonen RJ (2015) The unnatural history of
Ka¯ ne‘ohe Bay: coral reef resilience in the face of centuries of
anthropogenic impacts. PeerJ 3:e950
Baker AC, Glynn PW, Riegl B (2008) Climate change and coral reef
bleaching: an ecological assessment of long-term impacts,
Coral Reefs
123

recovery trends and future outlook. Estuar Coast Shelf Sci
80(4):435–471
Bassim K, Sammarco P (2003) Effects of temperature and ammonium
on larval development and survivorship in a scleractinian coral
(Diploria strigosa). Mar Biol 142(24):241–252
Coles SL, Bahr KD, Ku’ulei SR, May SL, McGowan AE, Tsang A,
Bumgarner J, Han JH (2018) Evidence of acclimatization or
adaptation in Hawaiian corals to higher ocean temperatures.
PeerJ 6:e5347
Coles S, Jokiel PL (1977) Effects of temperature on photosynthesis
and respiration in hermatypic corals. Mar Biol 43:209–216
Courtial L, Bielsa VP, Houlbre
`que F, Farrier-Page
`s C (2018) Effects
of ultraviolet radiation and nutrient level on the physiological
response and organic matter release of the scleractinian coral
Pocillopora damicornis following thermal stress. PLoS ONE
13(10):e0205261
Cox EF, Ward S (2002) Impact of elevated ammonium on reproduc-
tion in two Hawaiian scleractinian corals with different life
history patterns. Mar Pollut Bull 44:1230–1235
Cumbo VR, Fan TY, Edmunds PJ (2013) Effects of exposure duration
on the response of Pocillopora damicornis larvae to elevated
temperature and high pCO
2
. J Exp Mar Bio Ecol 439:100–107
Cunning R, Baker AC (2013) Excess algal symbionts increase the
susceptibility of reef corals to bleaching. Nat Clim Change
3:259–262
D’Angelo C, Wiedenmann J (2014) Impacts of nutrient enrichment on
coral reefs: new perspectives and implications for coastal
management and reef survival. Curr Opin Environ Sustain
7:82–93
Drupp P, De Carlo EH, Mackenzie FT, Bienfang P, Sabine CL (2011)
Nutrient inputs, phytoplankton response, and CO
2
variations in a
semi-enclosed subtropical embayment, Ka¯ ne‘ohe Bay, Hawai‘i.
Aquat Geochem 17:473–498
Edmunds P, Gates R, Gleason D (2001) The biology of larvae from
the reef coral Porites astreoides, and their response to temper-
ature disturbances. Mar Biol 139(5):981–989
Edmunds PJ, Gates RD, Leggat W, Hoegh-Guldberg O, Allen-Requa
L (2005) The effect of temperature on the size and population
density of dinoflagellates in larvae of the reef coral Porites
astreoides. Invertebr Biol 124(3):185–193
Edmunds PJ, Cumbo V, Fan TY (2011) Effects of temperature on the
respiration of brooded larvae from tropical reef corals. J Exp
Biol 214:2783–2790
Ezzat L, Maguer JF, Grover R, Ferrier-Page
`s C (2015) New insights
into carbon acquisition and exchanges within the coral–dinoflag-
ellate symbiosis under NH
4
?
and NO
3
-
supply. Proc Biol Sci
282(1812):20150610
Ezzat L, Maguer JF, Grover R, Ferrier-Page
`s C (2016) Limited
phosphorus availability is the Achilles heel of tropical reef corals
in a warming ocean. Sci Rep 6(1):31768
Fabricius KE (2005) Effects of terrestrial runoff on the ecology of
corals and coral reefs: review and synthesis. Mar Pollut Bull
50(2):125–146
Falkowski PG, Dubinsky Z, Muscatine L, McCloskey L (1993)
Population control in symbiotic corals. Bioscience
43(9):606–611
Figueiredo J, Baird AH, Cohen MF, Flot JF, Kamiki T, Meziane T,
Tsuchiya M, Yamasaki H (2012) Ontogenetic change in the lipid
and fatty acid composition of scleractinian coral larvae. Coral
Reefs 31:613–619
Figueiredo J, Baird AH, Harii S, Connolly SR (2014) Increased local
retention of reef coral larvae as a result of ocean warming. Nat
Clim Change 4:498–502
Fisch J, Drury C, Towle EK, Winter RN, Miller MW (2019)
Physiological and reproductive repercussions of consecutive
summer bleaching events of the threatened Caribbean coral
Orbicella faveolata. Coral Reefs 38:863–876
Gittenberger A, Reijnen BT, Hoeksema BW (2011) A molecularly
based phylogeny reconstruction of mushroom corals (Sclerac-
tinia:Fungiidae) with taxonomic consequences and evolutionary
implications for life history traits. Contrib Zool 80(2):107–132
Graham NAJ, Jennings S, Macneil MA, Mouillot D, Wilson SK
(2015) Predicting climate-driven regime shifts versus rebound
potential in coral reefs. Nature 518:94–97
Graham EM, Baird AH, Connolly SR, Sewell MA, Willis BL (2017)
Uncoupling temperature-dependent mortality from lipid deple-
tion for scleractinian coral larvae. Coral Reefs 36(1):97–104
Harii S, Nadaoka K, Yamamoto M, Iwao K (2007) Temporal changes
in settlement, lipid content and lipid composition of larvae of the
spawning hermatypic coral Acropora tenuis. Mar Ecol Prog Ser
346:89–96
Harii S, Yamamoto M, Hoegh-Guldberg O (2010) The relative
contribution of dinoflagellate photosynthesis and stored lipids to
the survivorship of symbiotic larvae of the reef-building corals.
Mar Biol 157:1215–1224
Harrison P, Ward S (2001) Elevated levels of nitrogen and
phosphorus reduce fertilisation success of gametes from scler-
actinian reef corals. Mar Biol 139(6):1057–1068
Haryanti D, Hidaka M (2015) Temperature dependence of respiration
in larvae and adult colonies of the corals Acropora tenuis and
Pocillopora damicornis. J Mar Sci Eng 3(3):509–519
Haryanti D, Yasuda N, Harii S, Hidaka M (2015) High tolerance of
symbiotic larvae of Pocillopora damicornis to thermal stress.
Zool Stud 54:52
Hochachka PW, Somero GN (2002) Biochemical adaptation: mech-
anism and process in physiological evolution. Oxford University
Press, New York
Hoyos CD, Agudelo PA, Webster PJ, Curry JA (2006) Deconvolution
of the factors contributing to the increase in global hurricane
intensity. Science 312(5770):94–97
Hughes TP, Kerry JT, Alvarez-Noriega M, Alvarez-Romero JG,
Anderson KD, Baird AH, Babcock RC, Beger M, Bellwood DR,
Berkelmans R, Bridge TC (2017) Global warming and recurrent
mass bleaching of corals. Nature 543(7645):373
Humanes A, Ricardo GF, Willis BL, Fabricius KE, Negri AP (2017)
Cumulative effects of suspended sediments, organic nutrients
and temperature stress on early life history stages of the coral
Acropora tenuis. Sci Rep 7(1):44101
Humphrey C, Weber M, Lott C, Cooper T, Fabricius K (2008) Effects
of suspended sediments, dissolved inorganic nutrients and
salinity on fertilisation and embryo development in the coral
Acropora millepora (Ehrenberg, 1834). Coral Reefs
27(4):837–850
Kassambara A, Kosinski M and Biecek P (2019) Survminer: drawing
survival curves using ‘ggplot2’. R package version 0.4.6. https://
CRAN.R-project.org/package=survminer
LaJeunesse TC, Parkinson JE, Gabrielson PW, Jeong HJ, Reimer JD,
Voolstra CR, Santos SR (2018) Systematic revision of Symbio-
diniaceae highlights the antiquity and diversity of coral
endosymbionts. Curr Biol 28(16):2570–2580
Lam EK, Ap Chui, Kwok CK, Ip AHA, Chn SW, Leung HN, Yeung
LC, Ang PO (2015) High levels of inorganic nutrients affect
fertilization kinetics, early development and settlement of the
scleractinian coral Platygyra acuta. Coral Reefs 34(3):837–848
Lenth R (2019) Emmeans: estimated marginal means, aka least-
squares means. R package version 1.3.5. https://CRAN.R-
project.org/package=emmeans
Mayfield AB, Chan PH, Putnam HM, Chen CS, Fan TY (2012) The
effects of a variable temperature regime on the physiology of the
reef-building coral Seriatopora hystrix: results from a labora-
tory-based reciprocal transplant. J Exp Biol 215:4183–4195
Coral Reefs
123

Nakamura M, Ohki S, Suzuki A, Sakai K (2011) Coral larvae under
ocean acidification: survival, metabolism, and metamorphosis.
PLoS ONE 6(1):e14521
NOAA (2019) National Data Buoy Center, Station MOKH1-1612480.
Mokuoloe, HI
Nozawa Y, Okubo N (2011) Survival dynamics of reef coral larvae
with special consideration of larval size and the genus Acropora.
Biol Bull 220:15–22
Olsen K, Ritson-Williams R, Ochrietor JD, Paul VJ, Ross C (2013)
Detecting hyperthermal stress in larvae of the hermatypic coral
Porites astreoides: the suitability of using biomarkers of
oxidative stress versus heat-shock protein transcriptional expres-
sion. Mar Biol 160:2609–2618
Padilla-Gamin
˜o JL, Pochon X, Bird C, Concepcion GT, Gates RD
(2012) From parent to gamete: vertical transmission of symbio-
dinium (Dinophyceae) ITS2 sequence assemblages in the reef
building coral montipora capitata. PLoS One 7:e38440
Parsons TR, Malta Y, Lalli M (1984) A manual of chemical and
biological methods for seawater analyses. Pergamon Press, New
York
Putnam HM, Edmunds PJ, Fan T-Y (2010) Effect of a fluctuating
thermal regime on adult and larval reef corals. Invertebr Biol
129:199–209
Putnam HM, Mayfield AB, Fan TY, Chen CS, Gates RD (2013) The
physiological and molecular responses of larvae from the reef-
building coral Pocillopora damicornis exposed to near-future
increases in temperature and pCO
2
. Mar Biol 160(8):2157–2173
R Core Team (2019) R: A language and environment for statistical
computing. R foundation for statistical computing, Vienna,
Austria
Rosset S, Wiedenmann J, Reed AJ, D’Angelo C (2017a) Phosphate
deficiency promotes coral bleaching and is reflected by the
ultrastructure of symbiotic dinoflagellates. Mar Pollut Bull
118(1):180–187
Randall CJ, Szmant AM (2009) Elevated temperature reduces
survivorship and settlement of the larvae of the Caribbean
scleractinian coral, Favia fragum (Esper). Coral Reefs
28(2):537–545
Richmond RH (1981) Energetic considerations in the dispersal of
Pocillopora damicornis (Linnaeus) planulae. In: Proceedings of
the 4th international coral reef symposium, vol 2. pp 153–156
Richmond RH (1987) Energetics, competency, and long-distance
dispersal of planula larvae of the coral Pocillopora damicornis.
Mar Biol 93(4):527–533
Rivest EB, Hofmann GE (2014) Responses of the metabolism of the
larvae of Pocillopora damicornis to ocean acidification and
warming. PLoS ONE. https://doi.org/10.1371/journal.pone.
0096172
Rivest EB, Chen C-S, Fan T-Y, Li H-H, Hofmann GE (2017) Lipid
consumption in coral larvae differs among sites: a consideration
of environmental history in a global ocean change scenario. Proc
R Soc B: Biol Sci 284:20162825
Rogers CS (1990) Responses of coral reefs and reef organisms to
sedimentation. Mar Ecol Prog Ser 62(1):185–202
Ross C, Ritson-Williams R, Olsen K, Paul VJ (2012) Short-term and
latent post-settlement effects associated with elevated tempera-
ture and oxidative stress on larvae from the coral Porites
astreoides. Coral Reefs 32(1):71–79
Rosset S, Wiedenmann J, Reed AJ, D’Angelo C (2017b) Phosphate
deficiency promotes coral bleaching and is reflected by the
ultrastructure of symbiotic dinoflagellates. Mar Pollut Bull
118(1–2):180–187
Schneider CA, Rasband WS, Eliceiri KW (2012) NIH image to
ImageJ: 25 years of image analysis. Nat Methods 9(7):671–675
Schnitzler CE, Hollingsworh LL, Krupp DA, Weis VM (2012)
Elevated temperature impairs onset of symbiosis and reduces
survivorship in larvae of the Hawaiian coral, Fungia scutaria.
Mar Biol 159(3):633–642
Schwarz JA, Krupp DA, Weis VM (1999) Late larval development
and onset of symbiosis in the scleractinian coral Fungia scutaria.
Biol Bull 196(1):70–79
Serrano XM, Miller MW, Hendee JC, Jensen BA, Gapayao JZ,
Pasparakis C, Grosell M, Baker AC (2018) Effects of thermal
stress and nitrate enrichment on the larval performance of two
Caribbean reef corals. Coral Reefs 37(1):173–182
Shantz AA, Burkepile DE (2014) Context-dependent effects of
nutrient loading on the coral-algal mutualism. Ecology
95:1995–2005
Shantz AA, Lemoine NP, Burkepile DE (2016) Nutrient loading alters
the performance of key nutrient exchange mutualisms. Ecol Lett
19(1):20–28
Silbiger NJ, Nelson CE, Remple K, Sevilla JK, Quinlan ZA, Putnam
HM, Fox MD, Donahue MJ (2018) Nutrient pollution disrupts
key ecosystem functions on coral reefs. Proc Biol Sci
285(1880):20172718
Smale DA, Wernberg T, Oliver EC, Thomsen M, Harvey BP, Straub
SC, Burrows MT, Alexander LV, Benthuysen JA, Donat MG,
Feng M (2019) Marine heatwaves threaten global biodiversity
and the provision of ecosystem services. Nat Clim Change 4:1
Strickland JDH, Parsons TR (1972) A practical handbook of seawater
analysis, 2nd edn. In: Bulletin-Fisheries Research Board of
Canada, vol 167
Suzuki G, Yamashita H, Kai S, Hayashibara T, Suzuki K, Iehisa Y,
Okada W, Ando W, Komori T (2013) Early uptake of specific
symbionts enhances the post-settlement survival of Acropora
corals. Mar Ecol Prog Ser 494:149–158
Szmant AM (2002) Nutrient enrichment on coral reefs: Is it a major
cause of coral reef decline? Estuaries 25(4):743–766
Therneau T (2015) A package for survival analysis in S. version 2.38.
https://CRAN.R-project.org/package=survival
Vega Thurber RL, Burkepile DE, Fuchs C, Shantz AA, McMinds R,
Zaneveld JR (2014) Chronic nutrient enrichment increases
prevalence and severity of coral disease and bleaching. Glob
Chang Biol 20(2):544–554
Wall C (2020) cbwall/Coral-larvae-temp-and-nutrients: temperature
and nutrient effects on coral larvae (Version pub.ver). Zenodo.
http://doi.org/10.5281/zenodo.3674230
Wall CB, Ritson-Williams R, Popp BN, Gates RD (2019) Spatial
variation in the biochemical and isotopic composition of corals
during bleaching and recovery. Limnol Oceanogr 64:2011–2028
Weis VM (2008) Cellular mechanisms of Cnidarian bleaching: stress
causes the collapse of symbiosis. J Exp Biol 211(19):3059–3066
Wiedenmann J, D’Angelo C, Smith EG, Hunt AN, Legiret FE, Postle
AD, Achterberg EP (2013) Nutrient enrichment can increase the
susceptibility of reef corals to bleaching. Nat Clim Change
3:160–164
Wooldridge SA, Done TJ (2009) Improved water quality can
ameliorate effects of climate change on corals. Ecol Appl
19(6):1492–1499
Yakovleva IM, Baird AH, Yamamoto HH, Bhagooli R, Nonaka M,
Hidaka M (2009) Algal symbionts increase oxidative damage
and death in coral larvae at high temperatures. Mar Ecol Prog
Ser 378:105–112
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