Optimizing Lighting Regimes for Rearing Orbicella Faveolata and Acropora Cervicornis Recruits Paul D

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7-23-2019 Optimizing lighting regimes for rearing Orbicella faveolata and Acropora cervicornis recruits Paul D. Kreh Nova Southeastern University, [email protected]

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NSUWorks Citation Paul D. Kreh. 2019. Optimizing lighting regimes for rearing Orbicella faveolata and Acropora cervicornis recruits. Master's thesis. Nova Southeastern University. Retrieved from NSUWorks, . (518) https://nsuworks.nova.edu/occ_stuetd/518.

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Submitted in Partial Fulfillment of the Requirements for the Degree of

Master of Science M.S. Marine Biology

Nova Southeastern University Halmos College of Natural Sciences and Oceanography

July 2019

Approved: Thesis Committee

Major Professor: Joana Figueiredo Ph.D.

Committee Member: Brian Walker Ph.D.

Committee Member: Bernhard Riegl Ph.D.

This thesis is available at NSUWorks: https://nsuworks.nova.edu/occ_stuetd/518

NOVA SOUTHEASTERN UNIVERSITY

HALMOS COLLEGE OF NATURAL SCIENCES AND OCEANOGRAPHY

Optimizing lighting regimes for rearing Orbicella faveolata and

Acropora cervicornis recruits

By:

Paul Kreh

Submitted to the Faculty of

Halmos College of Natural Sciences and Oceanography

in partial fulfillment of the requirements for

the degree of Master of Science with a specialty in:

Marine Science

Nova Southeastern University

July 2019

TABLE OF CONTENTS

ABSTRACT…………………………………………………………………………………….....3

INTRODUCTION………………………………………………………………………………...4

METHODS………………………………………………………………………………………..7

1. Study Species……………………………………………………………………….....7 2. Coral Collection…………………………………………………………………….....7 3. Coral Spawning and Fertilization……………………………………………………..8 4. Larval Rearing and Settlement Stages……………………………………………...... 8 5. Experimental System …………………………………………………………………9 6. Experimental Design………………………………………………………………....10

DATA ANALYSIS ……………………………………………………………………………..11

RESULTS ……………………………………………………….………………………………11

DISCUSSION …………………………………………………………………………………...20

ACKNOWLEGEMENTS……………………………………………………………………….25

LITERATURE CITED ………………………………………………………………………….26

Abstract: Coral reef decline worldwide has led to the need for coral reef restoration. The use of sexual reproduction in restoration efforts is required to increase genetic diversity; however, the procedures for rearing newly-settled coral recruits ex situ still need to be optimized. Recruits initially require low light irradiance, but it is unclear when higher irradiances are required to enhance growth and survival. Here we determined the optimal light regime for Orbicella faveolata and Acropora cervicornis recruits. Newly settled recruits were reared under treatments with varied rates of increasing irradiance (after reaching 5 weeks of age), and their survival, growth, and coloration was assessed weekly until they were 16 weeks old. Orbicella faveolata and Acropora cervicornis growth and survival were significantly affected by light irradiance regimes. Coloration also varied between treatments with a general trend of darkening pigmentation over the sixteen weeks. We found that low irradiances (< 40 µmol photons m-2s-1) were optimal for new recruits up to 8-10 weeks of age, which is possibly related to the full establishment of symbiosis and/or the ability to feed and digest food. Aposymbiotic recruits were able to survive for a longer period under low irradiances but experienced high mortality when exposed to higher irradiance, regardless of their age, possibly due to low levels or the lack of mycosporine like amino acids and other antioxidants produced by the Symbiodiniaceae that protect against high irradiances and reactive oxygen species. After Weeks 8-10, high irradiance levels similar to the ones that are optimal for adults (> 120 µmol photons m-2s-1) were required by zooxanthellate coral to survive and to boost their growth. This further suggests that the acquisition of symbionts from the family Symbiodiniaceae is at least one key component in the shift toward tolerating higher irradiances.

Keywords: Coral, Irradiance, Recruits, Juveniles, Light

Introduction: Coral reefs are one of the world’s most ecologically important ecosystems, providing both ecosystem services and economic revenue. Scleractinian corals, the main reef builders, provide a three-dimensional structure that serves as habitat for a highly diverse assemblage of plants and animals (Moberg and Folke 1999; Spalding et al. 2001). In fact, coral reefs cover less than one percent of the ocean floor, yet they support twenty-five percent of all marine fish species (Spalding et al. 2001). Through the reduction of wave action, coral reefs create sedimentary environments promoting coastal habitats such as mangrove forests and seagrass beds (Moberg and Folke 1999), which are important nurseries for many economically important reef dwelling fish (Nagelkerken et al. 2002). In addition to their ecological importance, reefs can also generate approximately US$ 30 billion per year globally and are a main source of revenue for many island nations (Moberg and Folke 1999; Cesar et al. 2003; Burke et al. 2011; Spalding et al. 2017). South Florida reef related tourism contributes approximately US$ 1.16 billion per year (Spalding et al. 2017). Despite their economic and ecological significance, the combination of global and local stressors has led coral reefs to suffer massive declines worldwide over the last several decades (Burke et al. 2011). From the 1970’s to the early 2010’s mean coral cover has decreased globally as much as 50% (Gardener et al. 2003; Bruno and Selig 2007; De’ath et al. 2012; GCRMN 2014). Two of the greatest global threats to corals are ocean warming and acidification (Hoegh-Guldberg et al. 2007, 2017; Veron et al. 2009; Hughes et al. 2018), which are caused by the release of greenhouse gasses, i.e. CO2 into the atmosphere from human activities (Crowley 2000; Knowlton 2001; Loya et al. 2001; Hoegh-Guldberg et al. 2007, 2017; IPCC 2014). The resulting increase in sea surface temperature causes coral to expel their photosynthetic algal symbionts from family Symbiodiniaceae in a process referred to as “coral bleaching” (Glynn 1993; Spalding et al. 2001). Coral bleaching can lead to lower growth, lower reproductive success, and increased susceptibility to disease, or death is symbiosis is not restored (Burke et al. 2011). Local stressors include increases in sedimentation, eutrophication, pollution, and unsustainable fishing practices (Burke et al. 2011). These stressors can cause turbidity on reefs to rise, reducing light levels and clogging corals’ feeding structures (Erftemeijer et al. 2012), increased competition with macroalgae (Lapointe et al. 2005; Box and Mumby 2007; Teichberg et al. 2010), and remove herbivorous fish from the reef that reduce macroalgal overgrowth

(Scheffer et al. 2001; Bellwood et al. 2004; Mumby et al. 2006). Several studies suggest that local stressors may compound global stressors further exacerbating coral reef degradation (Carilli et al. 2009; Ban et al. 2013; Fourney and Figueiredo 2017). In light of coral decline, there has been an effort to protect (Hughes et al. 2003; Mora et al. 2006; Mora 2008) and, in the most affected regions, restore degraded reefs (Young et al. 2012). Current coral restoration efforts primarily utilize techniques based on the fragmentation of adult colonies, an asexual form of reproduction. The predominant technique for coral reef restoration “coral gardening” has been successful in the restoration of branching corals such as the Acropora species (Levy et al. 2010; Lirman et al. 2010l; Miller et al. 2016). It consists of fragmenting donor colonies and subsequent growth of these fragments in coral nurseries on PVC “coral trees” (Nedimyer et al. 2011) or in “ropes”, followed by outplanting the fragments onto the reef (Young et al. 2012). Recently, a technique has been developed for the restoration of mounding, plating, and boulder corals, in which a coral is cut into pieces 1-3cm in diameter (i.e. microfragmentation), epoxied onto a tile, where they grow and are able to fuse together due to their identical genetic makeup (i.e. microfusion) (Forsman et al. 2015). This is advantageous, because corals with only a few polyps have a larger perimeter to surface area ratio, allowing them to grow faster than larger corals, be outplanted sooner, and become sexually mature sooner (Forsman et al. 2015). While these methods may be helpful in regenerating coral cover, they can at best maintain genetic diversity by duplicating existing genotypes and thus, can lead to genetic bottlenecking by preventing genetic recombination (Baums 2008). Additionally, for gonochoric species, fragmentation of few individuals for outplanting may lead to a single-gendered population or one with a skewed sex ratio (Shearer et al. 2009). To increase the genetic diversity in declining populations, restoration efforts should include sexual reproduction. While sexually producing corals is a relatively new field, the methodology is fairly well established. For broadcast spawning corals, gametes can be collected by tenting wild colonies during a spawning event, by collecting adult colonies before spawning for collection of gametes ex situ (Barton et al. 2015), or by inducing captive adult colonies to spawn in aquaria by mimicking the natural cycles of temperature, sun, and moonlight (Craggs et al. 2017). After collection eggs and sperm are mixed at a concentration between 104-106 sperm cells/mL in order to maximize fertilization success and reduce chances of polyspermy (Oliver and Babcock 1992). Embryos/larvae can then be reared in stagnant tanks or recirculating tanks with a very small

water flow until acquisition of competency, after which settlement tiles (typically preconditioned in situ for the acquisition of natural biofilms and crustose coralline algae) are provided to induce larval settlement and metamorphosis (Heyward and Negri 1999; Webster et al. 2004). Although spawning, larval rearing, and settlement procedures are relatively well developed, there is little information in the literature on optimal conditions for rearing coral recruits. Often it is assumed that the ideal conditions for coral recruits mimic what adult corals experience on their reefs of origin, however adults and newly settled coral recruits occupy different niches, particularly in terms of light irradiance. While the ideal light irradiance to rear coral recruits has yet to be determined, several recent studies have shown that the optimal levels for adults are deleterious to recruits. Average irradiance levels at the surface can reach intensities of over 1200 µmol photons m-2s-1 but due to the attenuation of solar irradiances, at a depth of 10m where corals typically occur, irradiances on sunny days reach as much as 400 µmol photons m-2s-1 on sunny days (Lesser et al. 2000; Sinniger et al. 2019). However, many studies suggest coral larvae preferentially settle in cracks and crevices, areas of lower irradiance. Field and lab studies frequently report that, under light irradiance levels typical of shallow areas, coral larvae settle preferentially on the undersides of settlement tiles in high irradiance (Babcock and Mundy 1996), but under low irradiance (e.g. deeper waters) larvae settle on the upside of tiles (Bak and Engel 1979; Birkland et al. 1981; Babcock and Mundy 1996). Additionally, a recent study found that recruits of the species Porites astreoides showed higher survival rates when covered by coarse sediments, which provide cryptic covering from excessive light while still allowing water circulation for access to oxygen and food, than when exposed to direct light (Fourney and Figueiredo 2017). Newly settled recruits of Acropora tenuis and Acropora millepora reared under lower light (180 µmol photons m-2s-1) also displayed higher survival and darker pigmentation (full symbiont establishment) than those reared at high irradiances (390 µmol photons m-2s-1) (Abrego et al. 2012). These results suggest that the light levels preferred by adult corals with a full assemblage of Symbiodiniaceae are detrimental to the health of new coral recruits. The reasons for this light sensitivity are not well studied but it is possible that exposure to high irradiance levels before symbiosis is fully established and/or feeding structures are well developed, can lead to oxidative stress similar to what is seen in high temperatures. A proper understanding of the effects of light

on recruits, as well as when they develop full symbiosis and light tolerances of adults, is crucial in the reduction of mortality rates when culturing coral for outplanting. To ensure higher genetic diversity, coral restoration practices should utilize sexual reproduction of corals in combination with existing fragmentation (coral gardening) practices. Additionally, procedures for growing sexually-produced corals must be optimized in order to maintain a large number of colonies for outplanting. Reducing the cost of propagation per individual and increasing survival rates will be key to achieving this goal; in particular, proper light irradiance levels must be established. This study aimed to optimize light irradiance levels during the growout of Orbicella faveolata and Acropora cervicornis, two of the most important reef building species on the Florida Reef Tract, in land-based coral nurseries.

Methods: 1. Study Species: The staghorn coral, Acropora cervicornis, and the mountainous star coral, Orbicella faveolata, were selected for this study due to their threatened status on the IUCN Red List of Threatened Species (Aronson et al. 2008a, 2008b). Both species are hermaphroditic broadcast spawners, meaning that colonies are simultaneously male and female and release gamete bundles into the water column for fertilization (Szmant 1986, Vargas-Angel et al. 2006). Acropora cervicornis spawns in August (Szmant 1986; Vargas-Angel et al. 2006), while O. faveolata spawns during August and September (Szmant 1986; Levitan et al. 2004).

2. Coral Collection: On July 29, 2018 and August 2, 2018 fragments from adult A. cervicornis colonies were collected via SCUBA from the Coral Restoration Foundation Nursery and brought to the Keys Marine Laboratory for spawning. They were brought up to the boat carefully bubble wrapped and placed in coolers with water changes performed every 20 minutes to maintain temperature and water quality. Fragments were maintained and monitored for spawning in flow through systems.

3. Coral Spawning and Fertilization The adult colonies of Acropora cervicornis were monitored every fifteen minutes from 8pm until midnight for signs of gamete release. Corals spawned on August 2-4, 2018; upon release, the gamete bundles were carefully collected by skimming bundles off the surface with a polystyrene cup and placed into a cooler in similar proportions from each genotype that released, where they were allowed to break down into eggs and sperm. Once gamete bundles were broken down, they were diluted with fresh sterile seawater to a sperm concentration of approximately 106 cells/mL to ensure maximum fertilization and sperm and allowed to fertilize for 1hr. and 30min. The eggs were then washed to rid them of sperm with fresh sterile seawater using a fat separator. A. cervicornis embryos/larvae were reared in upwelling conical tanks initially with a small aerator and after two days with a low but constant water change was allowed (100 µm filters prevented the larvae from escaping the tanks). Larvae of Orbicella faveolata were donated by Dana Williams (NOAA). These larvae were obtained from gametes spawned on August 2nd, 2018 at 11:30 pm by 14 colonies at Horseshoe Reef, in the upper Florida Keys.

4. Larval Rearing and Settlement One day before larvae started acquiring competence (i.e. day 4 for Acropora cervicornis and day 2 for Orbicella faveolata), the larvae were moved to polystyrene containers filled with water that had semi-stacked ceramic settlement tiles layering the bottom. The settlement tiles for this experiment had been preconditioned at the NSU Layer Cakes Nursery (26.12453, -80.09703) for 3 weeks to accumulate CCA and its associated bacteria biofilm. The polystyrene containers were maintained at ambient laboratory light levels (<10 µmol photons m-2s-1) with daily water changes (1µm filtered seawater) to maintain water quality. Polystyrene containers were placed into a water bath with heaters to maintain temperature at 26 °C, and pumps to achieve homogenous temperature throughout the water bath; the water level in the bath matched that of the polystyrene container to ensure that no temperature gradients were established. After 24-48 hours, tiles were censused for settlement and metamorphosis. Tiles with newly settled recruits were photographed using an Olympus LC20 digital camera attached to an Olympus SZ61 dissecting stereoscope. Recruits in each photo were labeled. The software CellSens® was used to measure the initial surface area of each coral polyp. If the container still had swimming

larvae, new tiles were added so that remaining larvae could settle; this process continued daily for 3-4 days. All tiles with newly settled recruits were initially kept at 10 µmol photons m-2s-1 (found to promote higher survival and growth in newly settled Porites astreoides, McMahon, 2018), and then randomly distributed between the experimental treatments.

5. Experimental System: Experiments were conducted indoors in a 1500L recirculating system with two raceways (250 x 60 x 30cm) (Figure 1). Raceways had flow rate of 350L/h and shared a sump. The sump system contained bioballs for biological filtration, a protein skimmer, UV sterilizer, and phosphate and calcium carbonate reactors. Submersible heaters and a chiller connected to temperature controllers were used to maintain the system at 28±1°C mimicking historical Figure 1: This figure shows the experimental system and design. The direction of the arrows represents the direction of water flow through the temperature on the system. “A” represents the adult colonies that were included to act as symbiont donors and the numbers “1-5” represent the treatment locations reef after within the system. The black dotted lines represent black plastic sheets used spawning. to separate the treatments and prevent light scattering.

Temperature was monitored daily using a YSI Pro 20 temperature probe. Reverse osmosis water was added to the sump as needed in order to maintain a salinity of 35ppt. Weekly water quality tests were performed to assess ammonia, nitrate, nitrite, and phosphate levels; if necessary partial water changes were executed. The use of a shared sump ensured that all water conditions were the same throughout all five treatments with the exception of light irradiance.

6. Experimental Design: To determine the optimal lighting regime for the two species, each raceway was divided into six sections. The first section of each raceway housed adult corals of the species Acropora cervicornis and Montastraea cavernosa to act as symbiont donors. To produce 5 different irradiances, sections 2-6 were equipped with AI Hydra 26 HD lights and divided by plastic sheets to block light scattering between treatments. Within each raceway, treatments were randomly assigned to sections 2-6. All treatments were replicated twice within the system (once per raceway). Five week old recruits of both species were randomly assigned to one of five treatments (minimum of 14 individuals per treatment per species): corals in treatments 1 and 2 were placed at 60 µmol photons m-2s-1, corals in treatments 3 and 4 were paced at 40 µmol photons m-2s-1, and corals in treatment 5 were set at 20 µmol photons m-2s-1. After one week (and then every week until recruits were 16 weeks old), tiles were censused under the Olympus microscope to assess coral recruit survival (0 = Alive and 1 = Dead) and pigmentation (scale 1 – 6, 1 = pale, 6 = dark, based on the “Coral Color Reference Card” Siebak et al. 2006. Figure 2). Pictures were also taken and the CellSens® program was utilized to measure growth (surface area). In the following weeks, due to the limited number of recruits we had available, the irradiance for all treatments was increased adaptively based off of recruit health, as determined by mortality, coloration, and growth in order to prevent high mortality rates. Specifically, when the treatments under higher light irradiance had low mortality, good coloration, and good growth during the previous week the other treatments, at lower light levels, would be bumped up to match it while the one at higher light would be increased even further. Treatments with high mortality or low/no growth in a given week were maintained at the same irradiance for an additional week.

Data Analysis: To evaluate the effect of the light irradiance regimes on the survival of coral recruits, a survival analysis was performed (event of interest = mortality). To assess how survival varied between treatments a Kaplan- Meier estimator was used to estimate the survival curves for each treatment and then a Mantel-Haenszel test was run to compare treatments. Figure 2: Coral Color Reference To evaluate the effect of light irradiance on the Card Siebak et al. 2006. growth of corals a generalized additive model was utilized with time as the continuous predictor and treatment as a factor. All analyses were performed using R Studio software version 3.4.3.

Results: Orbicella faveolata: The lighting regime treatments (Figure 3) significantly affected survival of Orbicella faveolata recruits (p= 1.71x10-5). The treatment where the light irradiance was kept low in the first 9 weeks (£ 40 µmol photons m-2s-1), but then increased more rapidly (Treatment 3) had the highest survival at 50%, while treatment 1 reached 0% survival by the final week of data collection (Figure 4). When recruits were exposed to higher irradiance earlier on (Treatment 1) and/or maintained under low irradiance past 9 weeks (Treatments 1 and 5), survival was significantly reduced (Figures 3 and 4). The large mortality in Treatment 5 at Week 7 corresponds with the increase of irradiance from 20 to 40 µmol photons m-2s-1.

O. fav Time (Weeks) Treatment 1-4 5 6 7 8 9 10 11 12 13 14 15 16 1 10 60 60 60 60 60 80 100 120 140 140 180 180 2 10 60 80 80 80 80 100 120 140 160 160 180 180 3 10 40 40 40 40 60 80 120 140 180 180 180 180 4 10 40 40 60 60 60 100 120 160 160 160 180 180 5 10 20 20 40 40 40 60 80 120 140 140 180 180 Figure 3: Lighting irradiance regime for O. faveolata treatments. The different line colors represent the different treatments.

Figure 4: Kaplan-Meier survival curves for the newly settled Orbicella faveolata recruits. The curves show the probability of survival for each treatment over time while the different colors of the lines represent the different lighting regime treatments. Letters represent treatments that are not significantly different.

The coloration of the Orbicella faveolata recruits varied slightly between treatments, but all showed a darkening trend as time progressed, with a slight paling in color if maintained under very high irradiance (³ 180 µmol photons m-2s-1) (Figure 5). Treatment 5 remained pale longer than the others because it was kept under lower light (£ 80 µmol photon m-2s-1) up to Week 11; as light intensity was increased, the death of recruits that had not established symbiosis caused higher mortalities in that treatment.

Orbicella faveolata coloration Treatment 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5 Treatment 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5

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Figure 5: Coloration for Orbicella faveolata recruits over time. Pie charts show the percentage of each treatment at each color intensity for each week.

The size of the recruits (Figure ^6) increased over time and was significantly different between treatments (all p < 0.05). Light regime explained 57.8% of the variation seen in size over the 16-week study period. From Weeks 0-5 when irradiances in all treatments were low (£60 µmol photons m-2s-1), there was little growth; however, after Week 6 higher irradiances lead to better growth. The treatment in which corals were exposed to higher light irradiance earlier on (Treatment 2) had the highest growth throughout the study. It should be noted that despite having the highest growth, Treatment 2 did have significantly lower survival than Treatment 3. When kept under brighter irradiances initially (Weeks 6-7), but increased very slowly thereafter (Treatment 1) recruits displayed the lowest growth and survival. After Week 11 Treatment 1 lagged behind Treatments 2-4, in terms of intensity (light irradiance), all of which had better survival and growth.

The optimal lighting irradiance regime for O. faveolata was as follows: Weeks 0-4: 10 µmol photons m-2s-1, Weeks 5-7: 40 µmol photons m-2s-1, Weeks 8- 10: 80 µmol photons m- 2s-1, Week 11: 120 µmol photons m-2s-1, Week 12: 140 µmol photons m-2s-1, Weeks 13-14: 160 µmol photons m-2s- 1, and Weeks 15-16: 180 µmol photons m-2s-1.

Figure 6: Growth curves for O. faveolata treatments generated from the Generalized Additive Model.

Acropora cervicornis

Lighting irradiance treatment (Figure 7) significantly affected recruit survival (p=7.79x10-3). Recruits displayed highest survival when kept under low light irradiance (£40 µmol photons m- 2s-1) for the first 9 weeks and then moved to higher irradiance levels more rapidly (Survival of treatments 1-4 respectively were 25.0%, 5.9%, 27.8%, and 20.0% at Week 16). When the recruits were maintained under low irradiance for a longer period of time (Treatment 5), recruits showed higher mortality when irradiance was eventually increased (Week 7) and did not survive past Week 11 (Figures 7 & 8). A large portion (79.8%) of the A. cervicornis recruits used for this study never reached a coloration 3d or higher on the coral color reference card (Figure 2), and therefore never established adequate levels of symbiosis, causing survival across all treatments to be relatively low.

Acer Time (Weeks) Treatment 1-4 5 6 7 8 9 10 11 12 13 14 15 16 1 10 60 60 60 80 80 100 100 120 140 140 160 180 2 10 60 60 60 60 80 120 120 120 140 160 180 180 3 10 40 40 40 80 100 120 120 140 160 160 180 180 4 10 40 40 40 60 80 100 120 140 140 140 180 180 5 10 20 20 40 60 60 80 100 100 100 100 100 100 Figure 7: Lighting irradiance regime for A. cervicornis treatments. The different line colors represent the different treatments.

Figure 8: Kaplan-Meier survival curves for the newly settled Acropora cervicornis recruits. The curves show the probability of survival for each treatment over time while the different colors of the lines represent the different lighting regime treatments. Letters represent treatments that are not significantly different.

Similarly, to the Orbicella faveolata recruits, the color of the Acropora cervicornis recruits showed a darkening trend as time progressed with a slight paling if maintained under very high irradiance (³ 180 µmol photons m-2s-1) (Figure 9). Exposure to low light levels (Treatment 5) did not allow corals to ever darken.

Acropora cervicornis coloration Treatment 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5 Treatment 1 Treatment 2 Treatment 3 Treatment 4 Treatment 5

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Figure 9: Coloration for Acropora cervicornis recruits over time. Pie charts show the percentage of each treatment at each color intensity for each week.

Recruit size increased significantly over time, with recruits in Treatments 1 & 3 having significantly faster growth (p<0.05). The lighting regime explained 32.3% of the variation in recruit growth over the 16-week study period (Figure 10). No treatments show substantial growth for the first 5 weeks of the study during which all were under low irradiances. From Weeks 6-8, recruits did not grow. The overall decreases in mean size during this period is likely due to mortality of some of the bigger individuals that had not acquired symbionts yet and died during this period. The sudden increase of growth at Week 8 for treatments 1, 3, 4, and 5 coincides with the period when light irradiance reaches a values ³ 60 µmol photons m-2s-1. The recruits in Treatment 2 remained at 60 µmol photons m-2s-1 for longer than the other treatments and then were moved to 120 µmol photons m-2s-1 over the next 2 weeks where they remained for another 6 weeks (Figure 7). It is possible that this fast increase, as opposed to a more gradual one, is responsible for the lower growth of Treatment 2 (Figure 10). It should also be noted that while the curve for Treatment 5 continues to Week 16, after Week 11 it is an estimate, as all individuals had died by Week 11.

For A. cervicornis the optimal regime was as follows; Weeks 0-4: 10 µmol photons m- 2s-1, Weeks 5-7: 40 µmol photons m-2s-1, Week 8: 80 µmol photons m-2s-1, Week 9: 100 µmol photons m-2s-1, Weeks10-11: 120 µmol photons m-2s-1, Week 12: 140 µmol photons m-2s-1, Weeks 13-14: 160 µmol photons m-2s-1, and Weeks 15-16: 180 µmol photons m-2s-1. Figure 10: Growth curves for A. cervicornis treatments generated from the Generalized Additive Model.

Discussion:

This study found that the survival of Orbicella faveolata and Acropora cervicornis recruits was significantly increased if the corals are maintained under low light irradiances for the first 7 weeks (Weeks 0-4: 10 µmol photons m-2s-1, Weeks 5-7: 40 µmol photons m-2s-1). After this initial period, recruits survived better and grew significantly faster when irradiance was quickly increased to higher light irradiance similar to the levels preferred by adults (O. faveolata: Weeks 8-10: 80 µmol photons m-2s-1 , Week 11: 120 µmol photons m-2s-1, Week 12: 140 µmol photons m-2s-1, Weeks 13-14: 160 µmol photons m-2s-1, and Weeks 15-16: 180 µmol photons m-2s-1; A. cervicornis: Week 8: 80 µmol photons m-2s-1 , Week 9: 100 µmol photons m- 2s-1, Weeks 10-11: 120 µmol photons m-2s-1, Week 12: 140 µmol photons m-2s-1, Weeks 13-14: 160 µmol photons m-2s-1, and Weeks 15-16: 180 µmol photons m-2s-1). The results of this study do not allow us to explain the reasons for this switch but it appears to be at least partly related to the full establishment of symbiosis (during Weeks 8-10) and potentially with the ability to feed (development of tentacles) and/or digest food. Azooxanthellate recruits displayed high mortality when exposed to higher light irradiance levels, regardless of their age; likely because they lack the mycosporine like amino acids (MAAs) that the Symbiodiniaceae provide which protect them from high irradiance and UV. At the time of settlement, the recruits of both study species were aposymbiotic, thus they had to acquire their algal symbionts from the water column around them. Symbiodiniaceae provide the coral with as much as 90% of their carbon needs (Falkowski et al. 1984) as well as compounds that act as sunscreens and antioxidants for the coral host. MAAs are produced by the algal symbionts and transferred to the coral host where they are able to act as sunscreens against high irradiance and UV radiation as well as antioxidants to help mitigate the effects of oxidative stress (Yakovleva et al. 2004; Furla et al. 2005). MAAs produced by the symbionts are concentrated into the host ectodermal tissue, particularly in areas with exposure to high irradiances such as the oral disk and tentacles, protecting both the symbionts and the host from photo damage and oxidative stress (Shick et al. 1995; Furla et al. 2005). The mechanisms of production of MAAs are not present in the coral host (Shick and Dunlap 2002); therefore, before the development of symbiosis newly settled coral recruits are ill equipped to handle higher irradiance levels. Recruits of both species that were kept under low light irradiances (20 µmol photons m-2s-1) for a

longer period of time (Treatment 5) were able to survive with low levels symbiodinium in these conditions. However, once irradiance was increased (after Week 7) recruits with light pigmentation (and likely lower concentrations of MMAs) were presumably not as protected from the increased irradiance and could not benefit from photosynthetic feeding and consequently did not survive as well as those with darker pigmentation (and potentially higher MMA concentrations). Sensitivity to high light irradiance persists even when the corals already have Symbiodiniaceae, albeit at lower densities. The survival curves for O. faveolata show that the 5- 7 week old recruits were sensitive to higher irradiances, indicated by significantly greater mortality in the 60 µmol photons m-2s-1 treatments (1 and 2) than in the 40 µmol photons m-2s-1 (3 and 4), with A. cervicornis following a similar trend. The ultimate cause of this apparent sensitivity to high irradiances is unclear, but there are some indicators in the literature as to why this may be the case. Research has shown that early on in the coral lifecycle symbionts can be detrimental to their hosts. For instance, newly settled Porites astreoides and Agaricia acaricites corals, despite having zooxanthellae, display faster growth under lower lighrt irradiances during the first weeks after their settlement (McMahon 2018). Additionally, under high temperatures and high solar irradiance coral larvae already infected with algal symbionts show higher instances of cellular damage due to reactive oxygen species (ROS) and have higher levels of antioxidants than aposymbiotic larvae (Yakovleva et al. 2009; Nesa et al. 2012; Chamberland et al. 2017). It is possible that this holds true for newly settled recruits so that higher light irradiance increases ROS and antioxidants causing higher mortality in our treatments initially under higher irradiances. Eight to ten weeks after settlement, O. faveolata and A. cervicornis recruits switched preferences from low to higher light irradiance, likely due in part to the full establishment of symbiosis. From Weeks 8-14, mortality was greatly reduced and growth in most treatments increased as irradiance was increased. These results are consistent with previous studies who found light to be essential for adult corals in the production of the photosynthates that they utilize for growth and calcification (Falkowski et al. 1984; Wijgerde et al. 2012). Increasing the quantity of light (i.e. irradiance) can also positively affect both growth and calcification through increased photosynthetic rates (Houlbrèque et al. 2004; Moya et al. 2006). For instance, coral growth decreases with depth, which is generally attributed to the attenuation of light as depth

increases, thus corals under lower light grow slower (Baker and Weber 1975; Huston 1985; Miller 1995). The alignment of the acquisition of full coloration (d4-d6) around Weeks 8-10 along with the increased survival and growth of the treatments points to the full establishment of algal symbiosis as the probable cause for the recruits’ sudden tolerance and necessity of higher light irradiances. It is unclear as to what aspect of symbiosis contributes most to the switch in light tolerance; it could be higher MMA concentrations, enhanced photosynthetic food production, or more likely a combination of the two. The lighting regime with low light for the first few weeks followed by a rapid increase that we found to be optimal for O. faveolata and A. cervicornis recruits is similar to what has been found for other species, including species with zooxanthellate larvae, offering an alternate explanation for the switch in preference from low to high light irradiance. A study similar to the current one sought to optimize the light irradiance regimes of two common Caribbean corals, P. astreoides and A. agaricites (McMahon 2018). When the optimized regimes for those two species are compared to those with the highest survivals in this study, striking resemblances become clear (Figure 11). In both studies, corals were sensitive to higher irradiances at the beginning of the study and then switched to requiring higher irradiance for both growth and survival. The timing of these events is slightly different between species, likely because P. astreoides and A. agaricites parent colonies provide their larvae with symbionts before release, and differences in specie-specific lighting requirements of adults. The reduced growth of newly settled P. astreoides and A. agaricites corals under high light irradiance despite the presence of zooxanthellae demonstrates that the absence of symbionts cannot be the only explanation for the sensitivity of newly settled recruits to high irradiance.

Acer Time (Weeks) Treatment 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 Aagar 10 10 20 20 40 40 80 80 120 120 150 150 150 150 150 150 Acer 10 10 10 10 40 40 40 80 100 120 120 140 160 160 180 180 Ofav 10 10 10 10 40 40 40 40 60 80 120 140 180 180 180 180 Past 10 10 10 20 20 40 40 80 80 120 120 140 140 150 150 150 Figure 11: Compares the optimal irradiances of Agaricia agaricites (Aagar), Acropora cervicornis (Acer), Orbicella faveolata (Ofav), and Porites astreoides (Past).

Another possible explanation for light sensitivity of recruits during the first 8 weeks after settlement is that symbionts could act as an energy drain on the recruits. While corals rely heavily on the photosynthetic products of their algal symbionts, they must also provide essential nutrients in return (Muscatine and Porter, 1977; Lesser, 2004) and heterotrophy is an essential source of such nutrients (Lesser 2004; Houlbrèque and Ferrier‐Pagès 2009). It is possible that under higher initial irradiances (Treatments 1 and 2) recruits that established higher levels of symbiosis early on were not able to obtain enough nutrients heterotrophically to ‘feed’ the zooxanthellae, and thus had to ‘feed’ them with their energy reserves, diverting energy from maintenance and growth. While corals appear to have fully developed tentacles as soon as 3 weeks post settlement, it is possible that due to their small size, they may not be able to feed at a rate adequate for keeping up with the nutritional needs of their symbionts. Alternatively, corals may be able to catch food using their tentacles but could lack the necessary enzymes or internal structures to fully digest their prey. Studies have also shown that heterotrophic feeding can increase photosynthetic rates and in turn growth (Houlbrèque et al. 2004). Thus, the switch

undergone by recruits around Weeks 8-9 could be facilitated by the full development of feeding structures and digestive enzymes. Future studies may wish to analyze symbiont density and cell counts, MMA concentrations, and the development of tentacles and digestive enzymes in order to more clearly determine the causes of light sensitivity immediately following settlement and the cause of the shift in light requirements seen around 8 weeks post settlement.

Figure 12: a.) Three-week-old O. faveolata; b.) Six-week-old O. faveolata; c.) eight-week-old O. faveolata; d.) Sixteen-week-old O. faveolata; e.) Three-week-old A. cervicornis; f.) Six-week-old A. cervicornis; g.) Eight-week-old A. cervicornis; h.) Sixteen-week-old A. cervicornis. **Individuals in this figure are not the same in all pictures and pictures are at different magnifications**

Optimization techniques for rearing coral recruits and understanding what environmental conditions are limiting is essential as restoration continues to rely more heavily on propagating sexually produced corals. As such, our recommendations for the optimal lighting regimes under which to rear recruits of A. cervicornis and O. faveolata and may serve as a suitable starting point for studying rearing conditions for other closely related species. Successfully increasing growth rate and production of more genetically diverse corals will reduce the grow-out period by allowing sooner micro-fragmentation, reaching colony outplant size sooner, and ultimately allow higher numbers of genetically diverse corals to be outplanted for the restoration of degraded reefs.

Acknowledgements: This study was funded by the Mote Marine Laboratory’s Protect Our Reefs Grant (POR- 2016-23) and NSU President’s Faculty Research and Development Grant (2017).

I would like to than Dana Williams with NOAA for the donation of the Orbicella faveolata larvae used in this study. To my lab mates present and past I thank you for all of the input, edits, and moral support over the last year. Specifically, I would like to acknowledge Morgan Stephenson, Samantha King, and Allan Anderson for collecting the Acropora cervicornis gametes used for this study Nicholas McMahon for setting up the raceway system for this study. Thank you to my committee members, Dr. Walker and Dr. Riegl, for their input and guidance in the thesis writing process. I would like to thank my friends and family for their unwavering support and encouragement while pursuing my master’s degree. Lastly, I would like to acknowledge my advisor Joana Figueiredo for trusting me with this project and for advising, teaching, and guiding me throughout my time at NSU.

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