Warm temperature alters the chemical cue preference of Acropora tenuis and Heliopora coerulea larvae

Marine Pollution Bulletin 161 (2020) 111755

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Warm temperature alters the chemical cue preference of Acropora tenuis and Heliopora coerulea larvae Jeric P. Da-Anoy, Patrick C. Cabaitan, Cecilia Conaco * Marine Science Institute, University of the Philippines, Diliman, Quezon City 1101, Philippines

A R T I C L E I N F O

A B S T R A C T

Keywords: Allelopathy Chemical cues Chemosensation Thermal stress

Larvae released into the water column rely on chemical cues from the benthos for successful settlement. How­ ever, larval preference for substrates may be affected by rising seawater temperature brought about by global climate change. In this study, we examined the effect of elevated temperature on chemical cue preference by larvae of the scleractinian coral, Acropora tenuis, and the octocoral, Heliopora coerulea, collected from north­ western Philippines. At ambient temperature (28 ◦ C), both H. coerulea and A. tenuis larvae showed preference for substrates containing either crustose coralline algae or crude ethanolic extracts from conspecific or congeneric corals. In contrast, at higher temperature (30 ◦ C), greater preference was shown for substrates containing the crude extract from conspecific or congeneric corals. These results demonstrate that elevated temperature can change larval substrate preference, which will have downstream impacts on crucial biological processes, such as larval settlement and recruitment.

1. Introduction Settlement is a critical stage in the life history of corals, as this is the time when larvae select a suitable microhabitat that is crucial for their survival (Mundy and Babcock, 2000; Harrington et al., 2004). At this stage, coral larvae rely on sensory systems to detect signals from the substratum (Tran and Hadfield, 2013). The ability to process external cues is important for communication and habitat selection, as well as for discrimination of conspecifics and avoidance of competitors (Kuffner et al., 2006; Lecchini and Nakamura, 2013; Dixson et al., 2014). For coral larvae, the detection of appropriate cues leads to successful set­ tlement and recruitment, ensuring the continuous turnover and replenishment of coral populations (Hughes and Tanner, 2000; Dor­ opoulos et al., 2015). However, environmental perturbations such as ocean warming can disrupt these processes and result in recruitment failure, which can have profound impacts on reef community structure, population dynamics, and connectivity of coral populations (Babcock and Mundy, 1996; Raimondi and Morse, 2000; Hughes et al., 2018). The larval sensory system likely consists of chemoreceptors distrib­ uted in the aboral region of the planula or along the sides of the body in the aboral portion (Tran and Hadfield, 2013). Chemoreceptors, such as G protein-coupled receptors (GPCRs) and their associated signaling pathways, are potentially involved in the reception of settlement cues

and the mediation of metamorphosis in various marine invertebrate larvae, including sponges (Ueda et al., 2016), echinoderms (AmadorCano et al., 2006), hydrozoans (Freeman and Ridgway, 1990; Leitz and Klingmann, 1990), and scyphozoans (Hofmann and Brand, 1987). In coral larvae, increased expression of sensory and signal transduction genes, including GPCRs, ion channels, and neuropeptide signaling components, is observed during development (Strader et al., 2018). Alternative mechanisms for the reception of settlement and meta­ morphosis cues unrelated to GPCRs may also be involved, as cesium or potassium ions and protein kinase C activators can induce meta­ morphosis in some coral larvae (Tran and Hadfield, 2012). Coral larvae respond to a variety of external cues, including light (Mundy and Babcock, 1998), sound (Vermeij et al., 2006), and reef topography (Whalan et al., 2015) to guide habitat selection. However, the most effective inducers of larval settlement and metamorphosis are chemical cues from crustose coralline algae (CCA) (Harrington et al., 2004; Ritson-Williams et al., 2010; Tebben et al., 2015) and microbial biofilms (Heyward and Negri, 1999; Negri et al., 2001; Webster et al., 2004). High molecular weight sulfated galactans and monoacetylated glycerolipids in CCA provide strong signals for settlement and meta­ morphosis (Tebben et al., 2015). On the other hand, tetrabromopyrrole (TBP), which is produced by CCA-associated bacteria, can promote metamorphosis of coral larvae without substrate attachment (Tebben

* Corresponding author. E-mail address: [email protected] (C. Conaco). https://doi.org/10.1016/j.marpolbul.2020.111755 Received 27 June 2020; Received in revised form 1 October 2020; Accepted 7 October 2020 Available online 22 October 2020 0025-326X/© 2020 Elsevier Ltd. All rights reserved.

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et al., 2011). Coral larvae also exhibit different settlement preferences, with some preferring to settle near certain types of CCA or CCA extracts, while others prefer biofilm-covered substrate or naturally occurring reef biofilms (Webster et al., 2004; Golbuu and Richmond, 2007). Larvae can even distinguish chemical signals associated with healthy coral reef substrates versus those associated with degraded, seaweed-dominated substrates (Dixson et al., 2014). Adult corals can influence settlement of larvae through the produc­ tion of allelopathic chemicals that are harmless to conspecifics but may be toxic to heterospecific larvae (Chadwick and Morrow, 2011). Alle­ lopathy is used by benthic organisms for defense and spatial competition in the marine environment. The production of toxic chemicals has been demonstrated for many scleractinian species (Gunthorpe and Cameron, 1990). For example, extracts of Tubastraea faulkneri were toxic to 11 species of coral larvae but not to larvae from conspecifics (Koh et al., 2000). Heterospecific coral extracts also reduced survival and settle­ ment of Pocillopora damicornis larvae (Da-Anoy et al., 2017). Moreover, allelopathy has been experimentally demonstrated in many octocorals (Sammarco et al., 1983). Some octocoral compounds act as antipredatory agents (Pawlik et al., 1987; Fenical and Pawlik, 1991; Har­ vell et al., 1993; Epifanio et al., 1999; Heyward and Negri, 1999; Maia et al., 1999; Koh et al., 2000), while others inhibit larval settlement, increase resistance to fungal pathogens, or inhibit overgrowth by other sessile organisms (Standing et al., 1984; Rodríguez, 1995; Kim et al., 2000). The hermatypic octocoral, Heliopora coerulea, has been hypoth­ esized to produce allelopathic compounds that can inhibit settlement of scleractinian coral larvae (Atrigenio et al., 2017), although this remains to be empirically tested. Conditions in the external environment influence early larval development, settlement, and recruitment of corals. Rising seawater temperature has been shown to cause a higher incidence of develop­ mental aberrations and lower survival of coral larvae (Edmunds et al., 2001; Keshavmurthy et al., 2014). Elevated temperature also results in reduced competency periods and lower dispersal potential (Edmunds et al., 2001; O’Connor et al., 2007; Heyward and Negri, 2010; Figueir­ edo et al., 2014), as well as low settlement success and post-settlement survival (Krupp et al., 2006; Randall and Szmant, 2009). However, larvae of different coral species vary in terms of their response to elevated temperature. For example, raising temperature to 32 ◦ C significantly reduced gamete fertilization, larval survivorship, and larval settlement of Acropora tenuis (Humanes et al., 2016). On the other hand, larvae of H. coerulea were able to survive up to 9 days at 33 ◦ C, although with significant reduction in survival and settlement rates (Conaco and Cabaitan, 2020). Larvae of A. solitaryensis and Favites chinensis, in turn, showed accelerated acquisition of competency and increased settlement rates but experienced greater post-settlement mortality at higher tem­ perature (Nozawa and Harrison, 2007). Notably, exposure to microalgal allelochemicals can exacerbate the oxidative stress induced by elevated temperatures, resulting in lower larval survival, as demonstrated in Porites astreoides (Ritson-Williams et al., 2016). Environmental stressors may also affect sensory pathways crucial for habitat selection by inducing changes in the physiology of the organism or by altering pro­ duction of the cues (Rivest et al., 2019). In fact, changes in preference for settlement substrate under variable temperature conditions have been demonstrated for both Stylophora pistillata and A. millepora larvae (Put­ nam et al., 2008; Winkler et al., 2015). However, the effect of temper­ ature on the ability of larvae from different coral species to sense or distinguish settlement cues and allelochemicals produced by other corals remains to be determined. This study thus aims to understand how ocean warming affects the settlement preferences of larvae from the scleractinian coral, A. tenuis, and the octocoral, H. coerulea. A. tenuis is a broadcast spawning coral with larvae that are expected to disperse widely, whereas H. coerulea produces brooded planula that can settle close to the parent colony soon after release (Harii and Kayanne, 2003; Harii et al., 2007). Both species are abundant in Bolinao, northwestern Philippines, a region that

˜ aflor regularly experiences periods of high sea surface temperature (Pen et al., 2009). Yet, H. coerulea has rapidly become the dominant species on these reefs (Vergara et al., 2010; Atrigenio et al., 2017; Quimpo et al., 2020). Assessing how habitat selection is affected by environmental disturbances is an important step towards understanding the capacity of different types of corals to cope with a changing ocean environment and to gauge the potential impacts of these disturbances on the dynamics of coral reef populations. 2. Materials and methods 2.1. Coral collection Colonies of the scleractinian corals, Acropora tenuis, Acropora dig­ itifera, Pocillopora acuta, Porites cylindrica, Seriatopora caliendrum, and Stylophora pistillata, and of the octocoral, Heliopora coerulea, were collected from the fore reef at about 5–8 m in depth within the BolinaoAnda Reef Complex, Pangasinan, northwestern Philippines, and trans­ ported to the Bolinao Marine Laboratory. Coral identities were verified based on the World List of Scleractinia (Hoeksema and Cairns, 2019). Prior to collection, small fragments were chipped from selected colonies to check for the presence or absence of mature oocytes (Harrison et al., 1984; Baird et al., 2002; Guest et al., 2005). In the Bolinao Reef Com­ plex, coral spawning typically occurs during the summer from April to May (Villanueva, 2016; Jamodiong et al., 2018). Colony fragments (about 17–20 cm in diameter) from six non-gravid colonies of each species were collected for extract preparation. Extracts were derived only from non-gravid corals to exclude potential chemical cues that may be associated with developing oocytes. Colonies with mature oocytes were tagged for larval collection. Crustose coralline algae (CCA) was scraped from coral rubble collected from the reef. Coral sample collec­ tion and processing were conducted with permission of the Philippines Department of Agriculture Bureau of Fisheries and Aquatic Resources (DA-BFAR Gratuitous Permit No. 0102-15 and 0169-19). 2.2. Crude extract preparation Crude ethanolic extracts were prepared by crushing and immediately soaking non-gravid coral colony fragments in absolute ethanol, followed by incubation at 4 ◦ C for 72 h (Gunthorpe and Cameron, 1990). The extracts were filtered twice through a double thickness of Whatman number 1 filter paper (8 μm pore size), concentrated in vacuo, and freeze-dried to powder form. Crude ethanol extracts from the corals were incorporated into agarose gel plugs following a modification of the method described by Thacker et al. (1998). Briefly, 2000 μg of powdered ethanolic extracts were placed into 50 ml plastic tubes. 100 μl of dichloromethane (DCM) was added to facilitate dispersal and resus­ pension of the powdered extracts. Tubes were air dried to remove re­ sidual DCM, leaving the extract as a fine layer coating the bottom of the tubes. 2 ml of 1% agarose in filtered seawater (FSW) was then added to resuspend the extract and obtain a gel mixture at 1000 μg/ml concen­ tration. To make CCA plugs, 2000 μg of fine CCA scrapings were resuspended in 2 ml of 1% agarose in FSW to obtain a gel mixture at 1000 μg/ml. The gel mixtures were allowed to solidify and then cut into circular plugs with a volume of about 150 μl (6 mm in diameter, 5 mm in height). 2.3. Coral larvae collection Larvae were obtained from six gravid colonies each of H. coerulea and A. tenuis that were collected in April 2017. We used larvae from A. tenuis in our experiments because this species spawns around the same time when H. coerulea releases larvae (Villanueva, 2016; dela Cruz and Harrison, 2017). Coral spawning and larval rearing were conducted following previously described procedures (Guest et al., 2010). Each H. coerulea colony was placed into a separate plastic bucket (25 2

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Marine Pollution Bulletin 161 (2020) 111755

cm in diameter and 25 cm in depth) supplied with aerated flow-through sand-filtered seawater. Seawater flow was stopped in the evening (18:00 h) to avoid loss of larvae that are released from the corals be­ tween 01:00 to 09:00 h (Villanueva, 2016). All six colonies of H. coerulea released a total of around 3500 larvae from 12 to 15 April 2017. Released larvae were collected by filtering through a nylon screen (215 μm mesh) the following morning (09:00 h). Larvae released from the 6 colonies during the first day of planulation, which ranged from ~250 to 400 (295 ± 21, average ± SE) planulae per colony, were pooled and used in the succeeding experiments. Larvae were kept in a glass container with aerated 0.45 μm-filtered seawater. Gravid A. tenuis colonies were maintained in a tank with flowing seawater until ready to spawn. Colonies were then transferred into a tank with static, filtered seawater at 18:00 h. Coral colonies were sup­ ported with concrete blocks to maintain an upright position for spawning. All colonies spawned between 18:30 to 19:00 h on 10 April 2017. All released gamete bundles were pooled into one plastic bucket (25 cm in diameter and 25 cm in depth) for fertilization, following established protocols (dela Cruz and Harrison, 2017). Egg-sperm bun­ dles from different colonies were gently mixed to induce separation of the gametes. Fertilization was allowed to proceed for 30 min. Fertilized eggs were collected and washed to remove excess sperm. Developing embryos were reared in a polyethylene tank (2 × 0.8 × 0.3 m in size) containing static, 1 μm-filtered seawater with gentle aeration. Seawater in the rearing tanks was changed daily to maintain the health of the larvae. Larvae for experiments were obtained at 1–2 days post-release for H. coerulea and at 4–5 days post-fertilization for A. tenuis, when majority are capable of settlement (Fig. S1) (Conaco and Cabaitan, 2020; dela Cruz and Harrison, 2017). After spawning and planulation, all adult colonies were alive and were returned to the reef. Average sea surface temperature on the reef during the spawning period ranged from about 27 ◦ C to 29 ◦ C (Guzman et al., 2019), which is similar to the seawater temperature in the rearing tanks.

2017; Conaco and Cabaitan, 2020). Fifty actively swimming larvae in 20 ml FSW (2.5 larvae/ml) were placed into scintillation vials that were capped then submerged into duplicate tanks maintained at the desired temperatures. Four replicate vials were included for each temperature treatment per species of larvae (n = 4, two vials per tank). Submersible temperature loggers (Onset HOBO) were placed in each tank to record water temperature every 10 min during the course of the experiment (Fig. S2). After 24 h, the number of surviving larvae in each vial was counted using a light microscope.

2.4. Effect of conspecific and heterospecific coral extracts on larval survival

2.7. Effect of elevated temperature on larval preference for coral extracts

2.6. Larval preference for coral-derived crude extracts To determine which extracts were most preferred by each species of larvae, settlement experiments were conducted in circular polystyrene plates (10 cm diameter) with 60 ml FSW. Each test plate was marked into eight equal segments. A gel plug containing 1000 μg/ml of crude extract from each of 6 coral species or the controls was placed in the center of each segment (Fig. S3). This concentration was used to elicit the most robust effect from the coral larvae. A gel plug containing CCA scrapings was used as positive control, while a gel plug with FSW served as negative control. Sixty actively swimming larvae of A. tenuis or H. coerulea were introduced into the center of each plate (1 larva/ml). Plates were incubated undisturbed in a room at 26–28 ◦ C temperature with illumination maintained at ca. 600–700 lx using 20 W LED daylight lamps (Firefly) following a 12L:12D photoperiod. After 24 h of incuba­ tion, larvae that had moved towards a gel plug were enumerated. Only larvae found on the plug or that were within at least 3 mm around each of the gel plugs were counted as settled larvae. Here, settled larvae refers to individuals that moved towards a preferred gel plug but did not necessarily show signs of metamorphosis. Larvae that remained at the center of the test plate or that were outside of the designated areas around the gel plugs were not included in the analyses. The experiment was conducted with 5 replicate plates for each species of coral larvae (n = 5). Gel plugs were randomly arranged in each replicate plate.

To determine the effect of temperature on larval settlement prefer­ ence, settlement experiments were conducted in circular polystyrene plates (6 cm diameter) containing 20 ml of FSW. Each test plate was marked into four equal segments. A gel plug containing 1000 μg/ml of crude extract from A. digitifera, H. coerulea, CCA scrapings (positive control), or FSW (negative control) was placed in each of the four seg­ ments (Fig. S3). Sixty actively swimming larvae of A. tenuis or H. coerulea were introduced into the center of each plate (3 larvae/ml). Test plates were incubated in tanks containing static seawater maintained at either 28.01 ± 0.02 ◦ C (control) or 30.58 ± 0.01 ◦ C (treatment) using sub­ mersible heaters. Only the two coral extracts most preferred by coral larvae (as identified in Section 2.6) and temperatures that did not cause larval mortality (as identified in Section 2.5) were tested in this exper­ iment. The same experimental conditions described in Section 2.5 and the same methods for counting coral larvae described in Section 2.6 were used in this experiment. The experiment was conducted with 4 replicate plates for each species of coral larvae per temperature treat­ ment (n = 4).

To determine the extract concentration range within larval tolerance limits, 10 actively swimming A. tenuis or H. coerulea larvae were intro­ duced into 10 ml of FSW (1 larva/ml) containing different concentra­ tions of coral extract (1, 10, 100, 500, 1000 μg/ml) in a 50 ml polystyrene tube. FSW alone was used as the negative control. Three replicate tubes were included for each extract concentration per species of larvae (n = 3). Tubes were incubated at room temperature (26–28 ◦ C). Surviving and dead larvae were counted after 24 h. Mortality was defined by movement arrest with leaking gastrovascular material and marked tissue degeneration (Goh, 1991; Epstein et al., 2000). The me­ dian lethal concentration (LC50) values or the concentration causing 50% mortality of larvae were calculated for each crude coral extract using probit analysis (Finney, 1971). 2.5. Effect of elevated temperature on larval survival To determine the temperature tolerance of the coral larvae, tem­ perature manipulation experiments were conducted in 40 l poly­ propylene incubation tanks (n = 2) with constantly aerated 10 μmfiltered seawater in a flow-through system (2 l/min flow rate). Tem­ perature was regulated using 300 W submersible heaters (Eheim Jaeger) and illumination was maintained at ca. 600–700 lx using 20 W LED daylight lamps (Firefly) following a 12L:12D photoperiod. For A. tenuis larvae, treatments included 28.01 ± 0.02 ◦ C (control, mean ± SE, n = 2), 30.58 ± 0.01 ◦ C, and 32.42 ± 0.02 ◦ C, while for H. coerulea larvae, treatments included 28.01 ± 0.02 ◦ C (control), 30.58 ± 0.01 ◦ C, and 34.07 ± 0.03 ◦ C. The highest temperature treatment for each species was based on thermal thresholds reported in other studies (Humanes et al.,

2.8. Statistical analyses Analyses were conducted separately for each species of coral larvae to distinguish intra-species differences in response to the treatments. All data were checked for homogeneity and normality using Levene’s and Kolmogorov-Smirnov tests, respectively. Differences in larval survival when exposed to varying concentrations of different coral extracts were examined using Two-way analysis of variance (ANOVA), whereas dif­ ferences in larval survival exposed to different temperature treatments were examined using One-way ANOVA. To determine the preference of 3

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each species of coral larvae for different coral-derived crude extracts, the Ivlev preference index was computed using the formula:

Table 1 Summary of statistical analyses for larval survival upon exposure to different coral-derived extracts (A) or different temperatures (B), and percent larval set­ tlement (C and E) and larval settlement preference (D and F) in response to different coral extracts (C and D) or different coral extracts at different tem­ peratures (E and F).

Ei = ri − ni/ri + ni where ri is the proportion of all settled larvae on the ith substrate (i.e., gel plug containing crude extract from different coral species or the controls), and ni is the proportional abundance of substrate (Ivlev, 1961; Roff et al., 2015). General Linear Model (GLM) was used to examine the differences in larval settlement and larval preference for various coral extracts at different temperatures. Significant results in ANOVA and GLM were further tested with Tukey’s HSD post hoc test to compare pairwise differences in larval survival, differences in percent larval settlement and settlement preference between coral extracts or between temperature treatments. All statistical analyses were conducted using Statistica (version 8.0).

Factors

Acropora tenuis

Heliopora coerulea

F

F

p

p

A) Effect of different coral-derived extracts on larval survival (Two-way ANOVA, n = 3) Extract 1019.38 < 0.001 32.30 < 0.001 Concentration 1938.60 < 0.001 25.30 < 0.001 Extract*Concentration 194.88 < 0.001 19.30 < 0.001 B) Effect of temperature on larval survival (One-way ANOVA, n = 4) Temperature 468.30 < 0.001 5.77 0.03 C) Percent larval settlement on different coral-derived crude extracts (GLM, n = 5) Extract 49.97 < 0.001 164.16 < 0.001

3. Results

D) Larval settlement preference (preference index) on different coral-derived crude extracts (GLM, n = 5) Extract 25.82 < 0.001 16.39 < 0.001

3.1. Effect of conspecific and heterospecific coral extracts on larval survival

E) Effect of temperature on percent larval settlement on different coral-derived crude extracts (GLM, n = 4) Extract 62.65 < 0.001 22.61 < 0.001 Temperature 1.26 0.274 11.82 < 0.001 Extract*Temperature 6.76 < 0.01 3.22 < 0.01

Different concentrations of coral-derived crude extracts showed varying toxicity against A. tenuis and H. coerulea larvae (Fig. 1; Table 1A; Table S1). No mortality was observed for A. tenuis larvae in the presence of varying concentrations of the congeneric extract from A. digitifera (Fig. 1A). On the other hand, most heterospecific coral extracts caused a significant reduction in the survival of A. tenuis larvae, particularly at concentrations of 100 μg/ml or greater, except for the S. caliendrum extract, which only reduced larval survival to 83.33 ± 3.33% (mean ± standard error) at 1000 μg/ml (Two-way ANOVA, Extract*Concentra­ tion: F = 194.88, p < 0.001). The LC50 of the crude extracts on A. tenuis larvae was 31.37 μg/ml for H. coerulea, 71.43 μg/ml for P. acuta, 95.64

F) Effect of temperature on larval settlement preference (preference index) on different coral-derived crude extracts (GLM, n = 4) Extract 98.29 < 0.001 24.00 < 0.001 Temperature 17.57 < 0.001 0.74 0.397 Extract*Temperature 19.20 < 0.001 4.89 < 0.001

Fig. 1. Survival of larvae exposed to coral-derived crude extracts. Mean percent survival of Acropora tenuis (A) and Heliopora coerulea (B) larvae after 24 h of exposure to different crude extract concentrations is shown (n = 3). Overlapping data points have been staggered for visualization purposes. Error bars represent standard error. Asterisks indicate significant differences compared to the FSW control (Tukey’s HSD tests, p < 0.05). 4

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μg/ml for P. cylindrica, and 102.40 μg/ml for S. pistillata.

extract was significantly greater than for any heterospecific extract (Fig. 3A; Tukey’s post hoc tests: p < 0.05), including H. coerulea (6.00 ± 1.13%), P. cylindrica (5.33 ± 0.97%), P. acuta (5.67 ± 1.55%), S. caliendrum (8.67 ± 0.62%), S. pistillata (7.00 ± 0.62%), or the FSW controls (1.67 ± 0.91%). On the other hand, the settlement of H. coerulea larvae on gel plugs with the conspecific H. coerulea extract was not significantly different from its settlement on gel plugs with P. cylindrica (6.67 ± 0.53%) or S. pistillata extracts (6.00 ± 0.85%) (Tukey’s post hoc test: p > 0.05), but was significantly higher than its settlement on gel plugs with extracts from P. acuta (4.33 ± 0.85%), S. caliendrum (4. 67 ± 0.82%), and A. digitifera (4.67 ± 0.33%), or the FSW controls (1.33 ± 1.33%) (Fig. 3B; Tukey’s post hoc test: p < 0.05). Both A. tenuis and H. coerulea larvae had a clear preference for CCA and congeneric or conspecific extracts compared to extracts from other coral species (Fig. S4; Table 1D; Table S4). No mortality was observed for either A. tenuis or H. coerulea larvae after 24 h of exposure to gel plugs con­ taining the coral-derived crude extracts. This suggests that the amount of extract that had diffused out of the gel plugs probably did not exceed concentrations that were lethal to the larvae.

Similarly, conspecific extracts did not affect the survival of H. coerulea larvae. However, in contrast to what was observed for A. tenuis, H. coerulea larvae showed high percent survival upon exposure to varying concentrations of heterospecific coral extracts (Fig. 1B). At the highest concentration that was applied, the A. digitifera extract only reduced H. coerulea larval survival to 96.67 ± 3.33% while the S. caliendrum extract reduced survival to 73.33 ± 3.33% (Two-way ANOVA, Extract*Concentration: F = 19.30, p < 0.001). The LC50 of the crude extracts on H. coerulea larvae was 1023.912 μg/ml for S. caliendrum, and 1812.52 μg/ml for A. digitifera.

3.2. Effect of elevated temperature on coral larval survival Exposure to seawater temperature of 28 ◦ C or 30 ◦ C for 24 h did not result in mortality of either A. tenuis or H. coerulea larvae (Fig. 2; Table 1B; Table S2). However, raising the temperature to 32 ◦ C reduced survival of A. tenuis larvae to 28.50 ± 3.30%, which was significantly lower than the controls at 28 ◦ C (Tukey’s post hoc test: p < 0.05). In contrast, the survival of H. coerulea larvae remained at 95.00 ± 2.08% even at a temperature of 34 ◦ C (Tukey’s post hoc test: p > 0.05).

3.4. Effect of elevated temperature on larval substrate preference

3.3. Larval preference for coral-derived crude extracts

Temperature affected the settlement preferences of both A. tenuis and H. coerulea larvae (Fig. 4; Table 1E; Table S5). At ambient temperature (28 ◦ C), A. tenuis larvae showed high settlement on gel plugs containing either CCA or the congeneric A. digitifera extract, with 51.35 ± 3.15% and 57.50 ± 8.54% settlement, respectively (Fig. 4A). These values were significantly higher than larval settlement on gel plugs with the H. coerulea extract and FSW (Tukey’s post hoc test: p < 0.05). However, at a temperature of 30 ◦ C (ambient +2 ◦ C), A. tenuis larvae showed significantly greater settlement on gel plugs with the congeneric A. digitifera extract (60.00 ± 5.40%) compared to CCA (32.50 ± 3.23%), H. coerulea (17.50 ± 3.23%) extract, or the FSW control (6.25 ± 2.39%)

Settlement of A. tenuis and H. corulea larvae varied significantly for different coral-derived crude extracts (Fig. 3; Table 1C; Table S3). The gel plugs containing CCA induced the highest settlement for larvae of both A. tenuis (34.67 ± 2.95%,) and H. coerulea (18.00 ± 1.70%). This settlement rate was similar to that elicited by conspecific and congeneric extracts (Tukey’s post hoc tests: p > 0.05), with 26.67 ± 2.64% of A. tenuis larvae and 13.00 ± 1.43% of H. coerulea larvae settling near the A. digitifera or H. coerulea extracts, respectively. The settlement of A. tenuis larvae on gel plugs with CCA or with the congeneric A. digitifera

Fig. 2. Effect of elevated temperature on survival of larvae. Mean percent of survival of Acropora tenuis (A) and Heliopora coerulea (B) larvae after 24 h of exposure to different temperatures is shown (n = 4). Error bars represent standard error. Asterisks indicate significant differences compared to the control (28 ◦ C) temperature (Tukey’s HSD tests, p < 0.05). 5

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Fig. 3. Settlement preference of larvae for different coralderived crude extracts. Boxplots show percent settlement of larvae from Acropora tenuis (A) or Heliopora coerulea (B) on gel plugs containing each extract (n = 5). Treatments include filtered seawater (FSW), crustose coralline algae (CCA), and extracts from A. digitifera (Adig), H. coerulea (Hcoe), P. cylindrica (Pcyl), P. acuta (Pacu), S. caliendrum (Scal), and S. pistillata (Spis). Letters indicate treatments with significant differences based on Tukey’s HSD tests, which were conducted separately for each of the two species of coral larvae.

Fig. 4. Effect of different temperature re­ gimes on the settlement preference of larvae. Boxplots show percent settlement of larvae from Acropora tenuis (A-B) or Heliopora coerulea (C-D) on gel plugs containing different extracts when incubated at 28 ◦ C (A and C) or 30 ◦ C (B and D) (n = 4). Treatments include filtered seawater (FSW), crustose coralline algae (CCA), A. digitifera extract (Adig), and H. coerulea extract (Hcoe). Letters indicate treatments with significant differences based on Tukey’s HSD tests, which were conducted separately for each of the two species of coral larvae.

(Fig. 4B; Tukey’s post hoc test: p < 0.05). A. tenuis larvae had a clear settlement preference for congeneric extracts compared to extracts from other coral species or CCA when exposed to 30 ◦ C (Fig. S5A-B; Table 1F; Table S6). A similar pattern was observed for H. coerulea larvae (Fig. 4, Table 1E; Table S5). The larvae showed greater settlement on gel plugs

with CCA (27.92 ± 3.62%) and the conspecific H. coerulea extract (27.50 ± 3.08%) at ambient temperature (Fig. 4C). These values were significantly higher than settlement on gel plugs with A. digitifera extract (15.83 ± 2.40%) or FSW (14.17 ± 1.44%) (Tukey’s post hoc test: p < 0.05). When temperature was increased to 30 ◦ C (ambient +2 ◦ C), H. coerulea larvae exhibited greater settlement on gel plugs with the 6

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conspecific H. coerulea extract (25.83 ± 1.44%) compared to gel plugs with CCA (14.58 ± 2.08%), A. digitifera extract (14.17 ± 0.48%), or FSW (6.25 ± 1.84%) (Fig. 4D; Tukey’s post hoc test: p < 0.05). H. coerulea larvae had a clear preference for the conspecific extract compared to extracts from A. digitifera or CCA when exposed to 30 ◦ C (Fig. S5C-D, Table 1F; Table S6). No larval mortality was observed after 24 h of exposure, suggesting that the amount of extract that had diffused out of the gel plugs even in the higher temperature treatment likely did not exceed concentrations that were lethal to the larvae.

that the population on the reef is maintained through local retention of larvae (Figueiredo et al., 2014). It is important to note, however, that our experiments were conducted in setups with small volume, limited water flow, and variable larval density, which could result in declining water quality over time and that may have affected the observed re­ sponses of the larvae. In addition, the concentrations of the cues that were used may not necessarily mimic concentrations encountered on the reef. Thus, further studies are needed to examine the influence of other factors on chemosensation in coral larvae. The similar settlement preference behavior exhibited by both types of larvae under warmer conditions suggests that similar mechanisms may be induced by temperature fluctuations encountered during larval settlement. Increased preference for conspecific cues could be due to a temperature-mediated change in the larvae, such as a shift in the expression of cell surface receptors that sense chemical cues (Meyer et al., 2011; Polato et al., 2013). Alternatively, modified cue discrimi­ nation could be due to a change in the quality of the chemical cues themselves (Roggatz et al., 2016), as elevated temperature may cause degradation of heat-labile compounds or disrupt the CCA-associated bacteria that produce the compounds (Webster et al., 2011). Temperature-induced changes in chemosensation have been reported in other organisms. For example, high temperature reduced the ability of female rock lizards to detect scent marks from males, which was likely due to rapid evaporation and degradation of chemical stimuli (Martín and L´ opez, 2013). Spiny lobsters exposed to high temperatures, high salinity, and low pH also lost their ability to sense conspecific cues that indicate suitable shelters and cues from diseased conspecifics and predators that indicate shelters to be avoided (Ross and Behringer, 2019). On the other hand, low water temperature reduced both loco­ motor activity and freshwater preference in glass eels (Edeline et al., 2006). The exact mechanisms underlying temperature-induced changes in the detection and response to chemical cues in coral larvae, as well as in other organisms, remain to be explored. Taken together, our findings shed more light on the factors that may have contributed to the present-day dominance of H. coerulea over A. tenuis and other scleractinian corals in the Bolinao-Anda Reef Com­ plex. First, H. coerulea larvae are better able to tolerate warm temper­ ature conditions and heterospecific compounds potentially secreted by scleractinian corals. Second, adult colonies of H. coerulea may produce allelochemicals that are toxic to the larvae of some scleractinian corals, such as A. tenuis, and that may inhibit their settlement. Third, warmer temperature induces a shift in settlement preference from CCA to conspecific and congeneric cues, which may further enhance local retention of larvae on natal reefs. These results suggest that, apart from its significant impacts on the physiology and health of marine organ­ isms, ocean warming may also indirectly affect coral distribution pat­ terns by altering the settlement preferences of coral larvae.

4. Discussion In the present study, we demonstrated that ethanolic extracts derived from heterospecific corals, but not the conspecific or congeneric ex­ tracts, significantly decreased the survival of coral larvae (Fig. 1). The effects were concentration-dependent, suggesting that allelopathic ef­ fects potentially increase with proximity to the source. However, larvae from different coral species had varying tolerance for the extracts. Whereas A. tenuis larvae were susceptible to most heterospecific ex­ tracts, H. coerulea larvae were robust, suggesting that they could tolerate high concentrations of allelochemicals exuded by competing scler­ actinian corals. These findings indicate that H. coerulea larvae may be able to settle near other corals as their survival at this stage is unaffected by heterospecific compounds. Furthermore, these findings suggest that the octocoral, H. coerulea, can produce compounds that could impact the settlement of other coral larvae. This supports a previous report that larvae from scleractinian corals, such as Acropora, Pocillopora, and Por­ ites, did not settle on tiles placed near H. coerulea colonies (Atrigenio et al., 2017). Coral larvae have varying tolerance to temperature (Fig. 2). A. tenuis larvae were more sensitive to elevated temperature compared to H. coerulea. Other studies reported similar observations on the thermo­ sensitivity of acroporid larvae. For example, increased temperature significantly reduced gamete fertilization, larval survivorship, and set­ tlement success in A. tenuis (Humanes et al., 2016; Humanes et al., 2017). The larvae of A. millepora but not those of the merulinids, Favites chinensis and Mycedium elephantotus, also showed significantly reduced fertilization and higher frequency of embryonic abnormalities at tem­ peratures 4 ◦ C above ambient (Negri et al., 2007). In contrast, H. coerulea larvae were able to tolerate higher temperatures. In fact, H. coerulea larvae have been shown to survive temperatures of up to 6 ◦ C above ambient for up to 9 days, albeit with a marked reduction in sur­ vival and settlement (Conaco and Cabaitan, 2020). This level of thermal tolerance exceeds that of most scleractinian coral larvae (Cumbo et al., 2013; Pitts et al., 2020). Adult colonies of H. coerulea are similarly able to tolerate warmer temperatures, as observed on Shiraho Reef in 1998, where anomalously high seawater temperatures resulted in widespread bleaching of scleractinians but not H. coerulea (Kayanne et al., 2002). This suggests that H. coerulea may be able to withstand predicted future ocean temperatures that could be higher by 1.2–3.2 ◦ C compared to present day conditions (IPCC, 2014). Both A. tenuis and H. coerulea coral larvae showed greater preference for CCA and their conspecifics or congenerics under ambient water temperature conditions (Figs. 3 and 4). Larval preference for hetero­ specific extracts was low, even for extracts that were not lethal at the higher concentrations. Notably, exposure to elevated temperature caused a decrease in larval preference for CCA while retaining higher levels of affinity for the conspecific extracts. This suggests that, at warmer temperature, larvae may prefer to settle near parental or conspecific colonies. A temperature-induced shift in larval settlement preference has also been observed in S. pistillata and A. millepora larvae, both of which showed enhanced settlement on CCA compared to bare substrates at warmer temperatures (Putnam et al., 2008; Winkler et al., 2015). A shift in preference for conspecific cues under warmer ocean conditions may provide coral larvae with the advantage of being able to settle quickly in a habitat where conditions are favorable and ensures

CRediT authorship contribution statement JPD, PCC, and CC conceived and designed the research. All authors performed the experiments. PCC and CC provided supervision, mate­ rials, and funds. JPD, PCC, and CC prepared the first draft of the manuscript. All authors analyzed the data, edited the manuscript, and gave approval for publication. Declaration of competing interest The authors declare that they have no conflict of interest. Acknowledgements The authors extend their gratitude to the staff of the Bolinao Marine Laboratory, Jake Ivan Baquiran, and Michael Angelou Nada for assis­ tance with coral collection and experiments, and Dr. Aaron Joseph L. Villaraza of the University of the Philippines Institute of Chemistry and 7

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Thatcher Pancho for assistance with extract preparation. This study was funded by the Department of Science and Technology Philippine Council for Agriculture, Aquatic and Natural Resources Research and Develop­ ment Coral Genomics Program to CC and Ocean Acidification Program (QMSR-MRRD-MEC-295-1449) to CC and PCC.

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