CALIFORNIA STATE UNIVERSITY, NORTHRIDGE

THE EFFECTS OF OCEAN ACIDIFICATION ON THE PHYSIOLOGY OF CORAL

RECRUITS

A thesis submitted in partial fulfillment

of the requirements for the degree of

Master of Science in Biology

By

Aaron Matthew Dufault

December 2012

The thesis of Aaron Matthew Dufault is approved:

Dr. Peter Edmunds, Chair Date

Dr. Robert Carpenter Date

Dr. Steve Dudgeon Date

California State University, Northridge

ii Acknowledgments

I’d like to thank my advisor Dr. Pete Edmunds for his dedicated guidance throughout my MS research. I appreciate Dr. Edmunds’ commitment to fostering independent and critically minded students, which has helped me grow as a scientist while under his mentoring.

I would also like to thank my committee members, Dr. Robert Carpenter and Dr. Steve Dudgeon for their constructive criticism, statistical advice and for bringing their own expertise to my project ideas.

I am grateful to Dr. Vivian Cumbo for her advice, patience, and all around uplifting attitude while in the field. Your encouragement has helped me focus on the novelty of my findings, which I greatly appreciate.

I would like to thank the other polyp lab members including Lianne, Jacobson, Sylvia Zamudio, Chris Wall and Darren Brown for their constructive input, field/lab assistance and their vital role in fostering my progress as a scientist during my time at CSUN. I would also like to thank fellow CSUN marine biology graduate students Anya Brown, Jenny Gowan, Jesse Tootell and Brenton Spies for their constructive input.

I’d like to also acknowledge the crucial role of the lab of Dr. Tony Fan at the National Museum of Marine Biology and Aquarium (NMMBA) in Taiwan. Your assistance in the lab and the field were incredibly helpful in ensuring our experiments ran smoothly. Specifically I’d like to thank Yao-Hung Chen, Okay Chan, Neo Zong-yu Wu, John Chen Wei-ta, and Tony Yang. I am also very grateful for the assistance Hollie Putnam, Anderson Mayfield and Steve Doo provided while in the field.

I would like to thank my fiancé and future wife Molly Kleinman for her support throughout this time. I appreciate your support and encouragement through my extended field trips and putting up with me working long hours even while home.

This work would not have been performed if not for the financial support from the National Science Foundation (OCE 08-44785). Partial funding was also provided by CSUN Graduate Research and Internation programs, Associated Students, and University Corporation.

iii TABLE OF CONTENTS

Signature Page ii

Acknowledgements iii

Abstract v

Chapter 1 General Introduction 1

Chapter 2 The effects of diurnally oscillating pCO2 on the calcification and survival of coral recruits

Introduction 9 Methods 11 Results 18 Discussion 20 Tables 26 Figures 27

Chapter 3 The importance of light in mediating the effects of ocean acidification on coral recruits

Introduction 31 Methods 35 Results 42 Discussion 44 Tables 51 Figures 53

Chapter 4 The response of carbonic anhydrase activity to ocean acidification: implications for coral calcification

Introduction 57 Methods 59 Results 65 Discussion 66 Tables 69 Figures 71

Chapter 5 Conclusion 72

References 76

iv Abstract

The Effects of Ocean Acidification on the Physiology of Coral Recruits

By

Aaron M. Dufault

Master of Science in Biology

Ocean acidification (OA), caused by the dissolution of anthropogenic CO2 into the surface waters of the ocean, threatens the fate of calcifying marine organisms. The effects of OA on adult coral calcification have been well-studied over the past decade and generally results in decreased calcification rates with increasing pCO2, although the effects of OA on early life history stages are less well-studied. This thesis addresses the effects of OA on coral recruit physiology with an emphasis on filling key gaps in the ecological relevance of previous manipulative OA coral studies. Chapter I: In March and

June 2010, two experiments were conducted exposing newly settled Seriatopora caliendrum recruits to low (440, 456 µatm), high (663, 837 µatm; March,June respectively) and diurnally oscillating pCO2 which mimicked the conditions at Hobihu reef, Taiwan where adult corals were collected. Calcification and survival of coral recruits was elevated in diurnally oscillating pCO2 relative to static ambient and high pCO2, hypothesized to be the result of increased DIC stored in coral tissues at night.

Chapter II: In March 2011, newly settled Pocillopora damicornis recruits were exposed to low (493 µatm) and high pCO2 (878µatm) in varying light intensities (226, 122, 70, 41,

31 µmol photons m-2 s-1) to test the effects of light and OA on coral recruit physiology.

Coral recruit calcification and survival in both pCO2 treatments was light-dependent, with

v large differences in calcification at intermediate light intensities (41, 70 µmol photons m-2 s-1) though calcification at high and low light intensities did not differ (226, 31 µmol photons m-2 s-1). Survivorship was not correlated with size and was highest in both

-2 -1 ambient and high pCO2 at 122 µmol photons m s . Chapter III: Finally, the activity of carbonic anhydrase in S. caliendrum juveniles (< 3 cm ) exposed to ambient, high and diurnally oscillating pCO2 was measured to elucidate the mechanistic basis for increased calcification in diurnally oscillating pCO2. CA activity was decreased in both high and diurnally oscillating pCO2 during the day, which is consistent with the DIC buildup hypothesis proposed in Chapter I. Together these findings provide novel insight into the physiology of corals exposed to OA under ecologically relevant seawater chemistry and light conditions. Coral recruits are biologically quite different than their adult counterparts therefore further work is needed to determine the extent to which these results apply to adult corals.

vi Chapter 1

Introduction

Ocean acidification and Corals

Current atmospheric CO2 concentrations are unprecedented over the last 800,000 y (Tripati et al. 2009), and present levels of ~ 391 ppm are predicted to rise to > 800 ppm by 2100 due to the continued burning of hydrocarbon-based fuels (A2 scenario, IPCC

2007). Rising atmospheric CO2 has two primary implications for marine organisms: 1) increased atmospheric CO2 causes an enhanced greenhouse effect leading to warmer ocean temperatures, and 2) atmospheric CO2 dissolves into the surface waters of the ocean, leading to a process called ocean acidification. The enhanced greenhouse effect is predicted to raise global mean temperatures by ~ 4°C by the year 2100 (Sokolov et al.

2009) which stands to effect marine organisms around the earth (Doney et al. 2009).

Ocean acidification is the coined term describing the following series of reactions that take place as anthropogenic CO2 dissolves into the surface waters of the ocean:

Equation 1:

CO2 g ↔CO!(aq),

CO2 aq +H2O l ↔H2CO3 aq , + - H2CO3 aq ↔H aq +HCO3(aq), + 2- HCO3 aq ↔H aq +CO3 (aq)

The results of anthropogenic CO2 dissolving into ocean waters are the net

2- - decrease in pH and CO3 and increased pCO2, HCO3 , and total dissolved inorganic carbon (DIC). By the year 2100, ocean pH is predicted to decrease by as much as 0.5 units from current levels (Caldeira and Wickett 2003). For many calcifying marine organisms, OA decreases the rate of biogenic CaCO3 deposition used to create their

1 skeletons (Doney et al. 2009). The cause of this decline in calcification often is attributed

- 2+ to a decrease in the availability of CO3 that can combine with Ca to form CaCO3.

- However the source of inorganic carbon used in the calcification process, whether HCO3

2- - or CO3 is a debated topic (Jury et al. 2010). Biological evidence points towards HCO3 being the primary external source of carbon for calcification due to the identification of

- HCO3 transporters in a wide range of invertebrate and vertebrate taxa including corals

2- (Furla et al. 2000). No CO3 transporter has been identified in corals, although a strong

2- positive correlation between [CO3 ] and coral calcification rates remains for most corals

(Schneider and Erez 2006, Erez et al. 2011, Allemand et al. 2011, Pandolfi et al. 2011).

Recent evidence reveals calcification of a few corals remaining unaffected or stimulated

2- by increased pCO2 under certain conditions (i.e., decreased CO3 , Anthony et al. 2008,

Jury et al. 2010, De Putron et al. 2011). Despite the few examples of negligible or increased calcification by corals in OA conditions, these studies highlight the complexities of the biogenic calcification process and suggest responses by corals to OA may be more diverse than previously thought (Jury et al. 2010). Decreased calcification instead may be the result of multiple interactions between increased pCO2, decreased pH, and the change in the concentrations of DIC species resulting from OA, although it has not been tested thoroughly (except Schneider and Erez 2006, Jury et al. 2010).

OA and early coral life history stages

In a recent review on the effects of OA on corals, ~25 scleractinian coral species throughout the past decade have been empirically tested in OA conditions (Erez et al.

2011), a mere 3% of the total 794 species scleractinian order (Veron 2000). The majority

2 of these studies have tested the response of adult corals to OA, and only recently have early life history stages (larvae/recruits/juveniles) been explored (Albright et al. 2008,

Cohen et al. 2009, Albright 2011). There is reason to believe early life history stages are biologically quite different from their adult counterparts (Hamdoun and Epel 2007), as this life history stage marks a crucial transition from a solitary pelagic larva to a colonial benthic sedentary existence. For coral recruits, their size often determines their ability to withstand environmental perturbations, and it influences their competitive ability with other corals, invertebrates and macroalgae, which all are competing for similar benthic substrata (Babcock 1991, Dunston et al. 1998). This results in strong selection for rapid calcification by newly settled coral recruits and is of fundamental importance in reaching large sizes quickly. For coral recruits, calcification decreases with increasing pCO2, as has been reported for adult corals (Albright 2008, Cohen et al. 2009, Albright 2010,

Anlauf et al. 2010, DePutron et al. 2010, Suwa et al. 2010). Examples of reduced calcification of coral recruits in OA conditions include a 16-35% reduction in calcification measured by the cross sectional area of Porites astreoides exposed to 560 and 800 µatm pCO2 (Albright et al. 2011). Calcification of P. astreioides and Favia fragum also were found to decrease calcification rapidly, but only after pCO2 reached

>800-850 µatm (De Putron et al. 2011). Despite a robust trend of decreased calcification in response to OA for juvenile and adult corals, exceptions to the decreased calcification trend exist. Anlauf et al. (2011) reported temperature-dependent effects of OA on coral calcification, with the calcification of F. fragum recruits in 1000 µatm pCO2 and 29.9 °C unaffected, although it was reduced ~30% at 28.9°C. There is evidence that coral recruits may be better able to withstand the negative effects of OA on coral calcification, for

3 example with F. fragum continuing to calcify in aragonite subsaturating conditions

(Cohen et al. 2009, De Putron et al. 2011). Calcification in aragonite subsaturating conditions has not been shown in adult corals and suggests the effects of OA on the calcification of coral recruits may be more variable than previously shown for adults.

The negative effects of ocean acidification for early life history stages of coral are not limited to calcification (Albright 2011). Other impacts of OA on early life history stages of corals include fertilization success (Albright et al. 2010, 2011), metabolism

(Albright et al. 2011) and settlement success (Suwa et al. 2010, Albright et al. 2011,

Anlauf et al. 2011,). However the negative impacts of OA do not appear to be lethal for

Acropora digitifera and Acropora tenuis larvae, for larvae survival of these species was unaffected by >2000 µatm pCO2 (Suwa et al. 2010, Nakamura et al. 2011). Similarly, survival of Porites panamensis recruits also was unaffected by OA treatments up to 1000

µatm pCO2, even when accompanied by a significant decrease in calcification (Anlauf et al. 2011). Despite the negative impacts of OA on coral larvae prior to settlement, successful survival of coral recruits post-settlement occurs irrespective of calcification rates.

Diurnal Oscillations of pCO2 on Shallow Reefs

Despite the rapidly growing literature documenting the responses of corals to OA for adult and juvenile corals, to date, all previous studies have attempted to employ constant OA treatments (Erez et al. 2011) that represent conditions similar to open ocean environments. This approach of employing constant OA treatments has proved useful in

4 establishing baseline responses of corals to future OA conditions (Erez et al. 2011), but it has overlooked aspects of the natural variation in pCO2 that has long been known to occur in many nearshore environments (Smith 1973). Shallow coral reefs (<10 m) undergo diurnal oscillations in pCO2 and pH (Kayanne et al 1995, Gattuso et al. 1997,

Ohde and Van Woesik 1999) driven by the metabolism of the benthic community

(Andersson et al. 2011). Throughout the day, photosynthesizing organisms take up

! dissolved inorganic carbon (DIC) in the form of dissolved CO2 and HCO! from the seawater, decreasing the available DIC in the seawater and increasing pH. This uptake of

CO2 by photosynthesis ceases at night, while respiration by the benthic community continues to produce CO2, resulting in increased DIC and reduced pH at night.

Combined, photosynthesis and respiration cause a daily cyclical change in DIC that can vary up to ~ 750 µatm pCO2 and 0.7 pH on some shallow reefs (Rukan Sho, Japan, Ohde and Van Woesik 1999). The magnitude of diurnal oscillation is influenced primarily by the metabolism of the benthic community, but is also affected by reef bathymetry and water residence time, which determines the spatial extent of shallow habitats and the flux of seawater across them (Ohde and Van Woesik 1999, Kayanne et al. 2008).

The effects of diurnally oscillating pCO2 on coral physiology are unknown, however evidence from multiple phyla in aquatic and marine habitats suggests the response by organisms to oscillatory abiotic conditions differ from static or steady-state effects (Cox and Rutherford 2000, Měráková and Gvoždík 2009, Putnam and Edmunds

2010, Oliver and Palumbi 2011). For corals, exposure to diurnally oscillating thermal regimes is more representative of the conditions experienced by corals on many reefs and

5 may enhance the response of corals to subsequent thermal stresses (Coles 1975, Warner et al. 1996, Castillo and Helmuth 2005, Oliver and Palumbi 2011). Warner et al. (1996) found the photochemical efficiency of PS II of corals commonly found in thermally variable back-reef environments was less affected by heat stress than corals in more thermally stable fore-reef habitats, while Pocillopora damicornis larvae exposed to diurnally varying temperatures increased dark-adapted quantum yield of PSII more than when they were exposed to steady temperature perturbations (Putnam et al. 2010).

Similarly, Acropora hyacinthus from lagoons with high thermal variability had the highest survivorship and the lowest decline in photochemical efficiency of photosystem

II when exposed to stressful temperatures (Oliver and Palumbi 2011). Because of the correlation between coral calcification and pCO2 levels, and the differing response by corals to oscillating environment conditions, there is reason to believe calcification by corals exposed to diurnally oscillating OA conditions may differ from those in static conditions.

Light and Ocean Acidification

Throughout the past decade of work addressing the response by corals to ocean acidification, advancements have been made in the experimental methodology used in

OA studies. Examples include the means used to create OA conditions, which have progressed from acid additions commonly used in early studies to gas mixing systems delivering a constant flow of premixed gas to treatment aquaria. Despite the advancements made to deliver ecologically relevant and precise OA conditions to corals, one aspect of experimental design crucial to corals has been neglected, light. Light drives

6 photosynthesis by the symbiotic algae Symbiodinium spp., which has helped hermatypic corals to maintain high rates of calcification during the day, termed light-enhanced calcification (Kawagati and Sakumoto 1948, Goreau 1959, Goreau and Goreau 1959,

Pearse and Muscatine 1971, Vandermuelen et al. 1972). Furthermore, calcification is influenced by light intensity, resembling a hyperbolic tangent function between coral symbiont photosynthesis and light intensity (Chalker and Taylor 1978, Chalker 1981). In the early work by Chalker (1981), calcification by 2 hermatypic coral species (Acropora cervicornis, Acropora formosa) reached maximum rates at light intensities similar to those required for maximum for photosynthesis (~400 µmol photons m-2 s-1 A. cervicornis, 100 µmol photons m-2 s-1 A. formosa).

The interaction of light and OA has been scarcely studied for corals, despite both of their roles in determining calcification rates in corals (Gattuso et al. 1999, Erez et al.

2011). Light intensities used in studies of the effects of OA on coral calcification range from 10 µmol photons m-2 s-1 (Albright et al. 2008) to 1600 µmol photons m-2 s-1

(Marshall and Clode 2002), thereby spanning a range of light intensities from those that do not saturate photosynthesis, to light intensities causing photoinhibition and potentially photodamage. Despite the effects of light intensity and OA on coral calcification, the combined effects have only been addressed explicitly by a single study (Marubini et al.

2001). Porities compressa exposed to 80, 170, and 800 µmol photons m-2 s-1 and OA conditions (186, 440 µatm pCO2) decreased calcification with increasing pCO2, with the effect intensified at higher light intensities (Marubini et al. 2001). However, Marubini et al. (2001) manipulated DIC seawater chemistry through the addition of acid, and as a

7 result total alkalinity also decreased, potentially confounding their results. Despite the influence of light intensity on coral calcification, any interactive effects of OA and light remain unknown.

Purpose and Questions

The primary research focus of this thesis is an evaluation of the effects of OA on the early life history stages of brooded corals. Research questions were designed to address key gaps in the ecological relevance of current coral OA literature, with a focus on elucidating the physiological mechanism underlying the observed processes. The research questions asked in each chapter are: 1) What are the effects of diurnally- oscillating pCO2 on the growth and survival of coral recruits, 2) How does light and increased pCO2 affect the physiology of coral recruits, and 3) How does activity of the enzyme carbonic anhydrase contribute to the calcification response of coral recruits in diurnally oscillating pCO2.

8 Chapter 2

Effects of diurnally-oscillating pCO2 on the calcification and survival of coral

recruits

Introduction

Ocean acidification (OA) arising from the dissolution of atmospheric CO2 in seawater is one of the most serious threats facing marine ecosystems (Orr et al. 2005,

Pelejero et al. 2005, Kiessling and Simpson 2011), because most calcifying organisms deposit less CaCO3 at high pCO2 (Kleypas and Langdon 2006, Doney et al. 2009, Ries et al. 2009). Some of the most striking examples of these effects are exhibited by scleractinian corals (Pandolfi et al. 2011), yet despite the negative implications of these trends for coral reef ecosystems (Erez et al. 2011), progress has been slow in elucidating the mechanisms underlying the response of corals to high pCO2 (Cohen and Holcomb

2009, Jury et al. 2010, Allemand et al. 2011).

Most evidence describing the effects of OA on corals has come from the exposure of adult colonies to seawater DIC chemistries simulating the consequences of high pCO2

(Erez et al. 2011). Most recently, attention has turned to early life stages of corals

(Albright 2011), because these may be more susceptible than adults to environmental challenges (Kurihara 2008), and play important roles in recruitment and population growth (Hughes and Jackson 1985). Early results from these efforts show that larval and newly recruiting corals can also be affected negatively by OA (Cohen et al. 2009, Suwa et al. 2010, Albright and Langdon 2011, de Putron et al. 2011, Nakamura et al. 2011).

9 An important advantage of studying early life stages is that they allow the reaction of environmentally naive organisms to novel conditions to be evaluated, thereby allowing the response to immediate conditions to be tested without the complexity of time- integrated effects that apply to studies of adults. The experimental benefits of studying early life stages of corals are strong for calcification, for which a contrast of pelagic larvae and benthic recruits supports a contrast of uncalcified and calcified tissue.

To date, all experimental studies of the response of corals to OA have come through manipulations of pH or DIC chemistry to create steady levels of perturbed conditions

(Kleypas and Langdon 2006). While such studies have advanced understanding of the responses of corals to OA, they have overlooked natural variation in pCO2 that occurs as seawater flows over shallow reefs (Andersson and Mackenzie 2011). The best-known example of these effects involve diurnal oscillations in pCO2 and pH (Gattuso et al. 1997,

Ohde and Van Woesik 1999, Bates et al. 2010), which can involve changes between day and night of >600 µatm pCO2 and 0.5 pH (Ohde and Van Woesik 1999). The magnitude of these oscillations is influenced first, by the benthic community, which drives DIC chemistry through metabolism, and second, by water residence time, which affects the flux of gases into the seawater (Ohde and Van Woesik 1999).

Oscillatory conditions result in biological outcomes that can differ from those arising under stable conditions (Coles 1975, Oliver and Palumbi 2011), and are more than simply the sum of exposure times to each extreme. For instance, diurnal temperature oscillations promote survival and enhanced thermal tolerance in nymphs of the mayfly

10 Deleatidium aumnale (Cox and Rutherford 2000), and favor rapid swimming in larvae of the newt Triturus alpestris (Měráková and Gvoždík 2009), in both cases relative to steady conditions. Likewise, exposure of the corals Pocillopora damicornis and Porites rus to diurnally varying temperatures reduces their Symbiodinium content and dark-adapted quantum yield of PSII more than exposure to steady temperature perturbations similar in magnitude to those at the extremes of the diurnal treatment (Putnam et al. 2011).

Similarly, exposure of Acropora hyacinthus to diurnally-varying temperature in tidal pools increases their resistance to elevated thermal stress compared to corals in more homogeneous conditions (Oliver and Palumbi 2011). Together, these results suggest that corals might respond to ecologically relevant natural oscillations in pCO2 in ways differing from those recorded under steady-state conditions. To test the effects of diurnally-oscillating pCO2 on coral recruit calcification and survival, I subjected newly settled Seriatopora caliendrum recruits to ecologically relevant oscillations in pCO2. A second experiment testing both diurnally-oscillating pCO2 of an increased magnitude and oscillating pCO2 on a reverse phase was used to provide insight into the mechanistic basis of the response of S. caliendrum recruits to diurnally-oscillating pCO2.

Methods

Larvae were obtained from the brooding coral Seriatopora caliendrum, which is a common branching coral on reefs along the southern coast of Taiwan and throughout the

Indo-Pacific (Veron 2000, Dai and Horng 2009). Colonies of S. caliendrum (~20-cm diameter) were collected from 5 to 7 m depth on Hobihu Reef, Nanwan Bay, in March and June of 2010, and returned to the National Museum of Marine Biology and

11 Aquarium (NMMBA) where they were placed into individual flow-through seawater tanks and exposed to sunlight at an intensity of 131 ± 13 µmol photons m-2 s-1

-2 -1 (Experiment I) or 78 ± 3 µmol photons m s (Experiment II; mean ± SEM). Overflow water from each tank passed through mesh-lined (110 µm) cups to capture larvae, which were released during the night (Fan et al. 2006). Larvae were collected at 08:00 hrs, pooled across colonies and retained in a 1L beaker until processed for the experiment.

Following collection, ~500 larvae were placed into plastic containers (12 x 24 x12 cm) at ~ 11:00 hrs, each of which was fitted with mesh windows (110 µm mesh size) to allow the passage of seawater, and floated in the same tanks containing the adult colonies.

Seriatopora caliendrum larvae were settled onto clean, pre-weighed glass microscope coverslips (22 x 22 mm, ~150 µm thick). The coverslips allowed the size of the settled coral recruits to be assessed gravimetrically (± 0.0001 g, Mettler-Toledo AX205). Six coverslips were placed in each container and the larvae left to settle for 24 h under a natural light/dark cycle. The following day, > 90% of larvae had settled on the coverslips, with up to 40 on each coverslip. The remaining swimming larvae were discarded, and coverslips with coral recruits (n = 18 Experiment I, n = 36 Experiment II) were assigned randomly to the pCO2 treatments.

In Experiment I, treatments consisted of steady ambient pCO2, steady high pCO2, and diurnally-oscillating pCO2 on a natural phase (Table 1); this design was augmented in Experiment II by including a diurnally-oscillating pCO2 on a reverse phase. The oscillatory treatments were created by moving the coverslips (and attached recruits)

12 between high and ambient pCO2 with the transfers accomplished at dawn and dusk

(07:00 hrs and 19:00 hrs under the laboratory conditions) without exposing the recruits to air. In the diurnally-oscillating pCO2 on a natural phase, high pCO2 was administered at night, and low pCO2 during the day, but in the diurnally oscillating pCO2 on a reverse phase, this sequence was reversed. The diurnally oscillating pCO2 on a natural phase mimicked the cycle of pCO2 in seawater passing over shallow reefs (Bates et al. 2010), and the diurnally oscillating pCO2 on a reverse phase was used to test coral calcification under novel conditions.

The pCO2 treatments were created in six, 30L aquaria, with two maintained at ambient pCO2, two at high pCO2, and a pair of tanks retained at ambient and high pCO2 and used to create the oscillatory pCO2 treatment. All tanks were maintained at 25.4 ±

0.4 °C in March (Exp. I n=128) and 25.1 ± 0.2 °C in June (mean ± SEM, Exp. II n =136), using independent heaters and chillers. Ambient seawater temperature was 25.5 ± 0.2°C in March, and although it was warmer in June (27.8 ± 0.2 °C) the same incubation temperature was employed to facilitate a direct contrast between the two experiments.

The tanks were filled with filtered seawater (1 µm), and the seawater was changed partially (10 to 15% volume) each night. All tanks were illuminated from 07:00 hrs to

19:00 hrs with lamps fitted with a metal halide bulb (Phillips 150 W 10,000k) and two

39-W fluorescent bulbs (Phillips T5 460 nm) that provided light at an intensity of 179 ± 2

µmol photons m-2 s-1 (mean ± SEM, n = 64) in Experiment I, and 305 ± 20 µmol photons in Experiment II (n=120) (measured with a LI-192 sensor, LI-COR Biosciences Lincoln,

Nebraska).

13 pCO2 Manipulation

The dissolved inorganic carbon (DIC) content of seawater was manipulated by bubbling premixed gas of a known pCO2, or by bubbling unmodified air for the ambient treatment. Due to bubbling ambient air from inside a closed building, the ambient pCO2 treatments were consistently higher than the 390ppm global atmospheric average (IPCC

2007). To mix the gas for the high pCO2 treatments, a system employing a variable timed solenoid valve was used, which controlled the flow of air and CO2 into a mixing chamber to reach the target pCO2 of 800 µatm. This target value was selected to provide a conservative estimate for the atmospheric pCO2 by 2100 following the business as usual emission scenario A1 (IPCC 2007). The solenoid valve was connected to an infrared gas analyzer (S151, Qubit Systems, Ontario Canada), which monitored the output gas and provided dynamic control of the duty cycle of the solenoid, thereby providing a consistent concentration of mixed gas to the treatment tanks.

Seawater Chemistry

Seawater DIC chemistry was measured every 2 d during each experiment. Total alkalinity (TA) and pH were measured following standard operating procedures (SOP 3b and 6b respectively of Dickson et al. [2007]). TA was measured using an open cell automatic titrator (Mettler-Toledo, Model DS50) filled with certified HCl titrant supplied by Dr. Andrew Dickson, and TA was calculated using an Excel™ (Microsoft 2008) spreadsheet (Fangue et al. 2010). The accuracy and precision of the TA measurements were tested against certified reference materials (CRM, Dr. Andrew Dickson) and maintained within <5% of certified values in March, and <1% of certified values in June.

14 pH was measured on the total scale (± 0.001 pH) using the dye m-cresol purple and a spectrophotometer assay (Metertech SP8001). Salinity was measured with a conductivity meter (± 0.1 accuracy, WTW 340i), and temperature was measured with a certified digital thermometer (± 0.05°C accuracy, Fisher Scientific Traceable Digital

! Thermometer). TA and pH were used to calculate the DIC parameters (pCO2, HCO! ,

!! CO! , and aragonite saturation state (ΩA)) using CO2SYS (Fangue et al. 2010).

Field Seawater Sampling

To assess the diurnal change in DIC seawater chemistry in Nanwan Bay, seawater was sampled over Hobihu Reef, where corals used in this study were collected. Seawater was sampled every two hours over July 8–9 and 13–14, 2010, at Hobihu Reef, using 300 ml borosilicate bottles filled 0.5 m above the substratum at 3 m and 6 m depth following

SOP 1 (Dickson et al. 2007). Loggers (Onset Computer Corporation®, Model: HOBO

Pro v2, ± 0.2 °C) were placed in duplicate at each depth to quantify temperature through the sampling period, which was used to calculate seawater chemistry parameters.

Duplicate loggers differed by ± ~ 0.1 °C, and were averaged to describe seawater temperature at each depth.

Calcification

Upon completion of the experiments, coverslips with coral recruits were placed in bleach (6% NaOCl) for 8 hrs to dissolve the tissue on the small corals and leave behind the CaCO3 skeleton. Coverslips then were rinsed with deionized water to remove the bleach, and air-dried for 24 h at ~ 27 °C. Calcification was measured using the summed

15 weight of the CaCO3 deposited by recruits on each coverslip, and also as the planar area of the basal plate of each recruit. Coverslips without recruits but subjected to identical treatments served as procedural controls, and these did not change in weight in either experiment (Paired t-test, Experiment I, t = 2.80, df = 11, I P = 0.99; Experiment II, t =

0.48, df = 3, P = 0.64). In Experiment I, the change in weight of each coverslip was divided by the number of corallites to provide a mean weight that was used as a statistical replicate. As some (≈ 5 %) recruits died during the experiment, this technique slightly underestimated calcification. To remove this bias in Experiment II, only recruits alive at the end of the experiment were used for growth measurements.

To measure the area of the recruits corallites were photographed (Nikon Coolpix

4500, 4.0 megapixel resolution) through a compound microscope (Zeiss Axiolab e, 40x magnification). Images were analyzed using Image J software (Wayne Rasband, Version

1.42q), with 50 corallites in each treatment measured for Experiment I, and 25 in each treatment for Experiment II. Only corallites that were not touching neighboring corallites were selected for analysis, and each corallite was treated as a statistical replicate.

Survivorship

To evaluate survivorship during Experiment II, recruits were photographed

(Canon 40d, 10 megapixel resolution) every 2 d throughout the 6 d experiment. Images were used to score the recruits as alive or dead based on the presence of tissue, which is easily discernable from photographs.

16 Statistical analysis

The weight and area of corallites were analyzed with a nested ANOVA in which pCO2 treatment was the independent variable and replicate tanks were nested within treatments. To satisfy the normality assumptions of ANOVA, calcification normalized to weight in Experiment I was 4th power transformed (Zar 1999), to correct for positive skewing, which was created by a small number of unusually heavy corallites. All other data met the homogeneity of variances (Levene’s test) and normality assumptions of

ANOVA (Shapiro-Wilks test). When tank effects were not significant (P > 0.25;

Underwood 1997), tank was removed from the statistical model and the each coverslip treated as a replicate. Posthoc analyses of calcification data were completed using a

Tukey test. To test for differences between natural and reverse phase diurnal pCO2, a

Bonferroni adjusted one-tailed t-test (α = 0.05) was performed to test the hypotheses that any stimulatory effect of natural phase diurnal pCO2 on calcification for both weight and area was diminished in the reverse phase.

To analyze survivorship in Experiment II, a Kaplan-Meier product-limit (KM) analysis was used (Machin et al. 2006). Nonparametric survival analyses such as the KM have the advantage over parametric analyses of accommodating non-normal distributions, which are common in survival data. For this analysis, the probability of individual recruits surviving is assumed to be independent of all other recruits, and because KM analyses cannot accommodate nested experimental designs, replicate corallites were pooled within each treatment. Survival was analyzed using the statistical program JMP®

17 (Version 9.0.2, 2010 SAS Institute Inc.) and a log-rank test was used to test for differential survival among treatments.

Results

Treatment conditions

Experimental pCO2 conditions in Experiment I (420-597 µatm pCO2, Table 1) mimicked the conditions found at 6-m depth on Hobihu reef (365-515 µatm, Fig. 1), where the parent colonies originally were collected. In Experiment II, the pCO2 range was expanded (448-845 µatm pCO2) to more closely match the experimental conditions to those occurring on other shallow reefs (Ohde and Van Woesik 1999).

Calcification

Seriatopora caliendrum larvae rapidly settled onto microscope coverslips at mean densities of 3.4 ± 1.5 recruits cm-2 (± SEM, n = 18) in Experiment I, and at 6.3 ± 2.2 recruits cm-2 (± SEM, n = 36) in Experiment II. Recruits began calcifying shortly after settlement, with septa and a basal plate visible within 24 h. In Experiment I, single corallites weighed 60 - 130 µg with areas 1.1 - 2.8 mm2 after 3 d; in Experiment II, corallites weighed 110 – 210 µg with areas 1.0 – 3.0 mm2 after 6 d. When standardized by time, the mean growth of recruits across all treatments was 26% slower in Experiment

II compared to Experiment I (Fig. 1, t = 5.86, df = 25, P < 0.001).

In Experiment I, the growth of Seriatopora caliendrum recruits was affected by treatments on both weight (F2,17 = 5.155 P = 0.020) and area (F2,199 = 6.265 P = 0.003)

18 scales, with the highest growth under diurnally-oscillating pCO2 on a natural-phase (Fig.

2). Increased growth under diurnally-oscillating pCO2 on a natural-phase was more pronounced for weight than area, and amounted to a 17-19% and 10-6% growth increase relative to recruits in the steady 456 µatm and 624 µatm pCO2 conditions; growth on weight and area scales did not vary between steady 456 µatm and 624 µatm pCO2 (Fig.

2). In Experiment II, the weight of corallites was affected by the treatments (F2,30 = 3.899,

P = 0.032, Fig. 2), and relative to steady ambient pCO2, was elevated 16% under both diurnally oscillating pCO2 on a natural-phase and high pCO2; the area of the corallites was unaffected (F2,99 = 1.786, P = 0.173).

In Experiment II, recruits exposed to the diurnally-oscillating pCO2 on a reverse- phase differed in area from those grown under diurnally-oscillating pCO2 on a natural- phase (t = 2.16, df = 60, P = 0.018), and their mean area was reduced 11% relative to recruits in the diurnally oscillating pCO2 on a the natural-phase. The weight of recruits did not differ between pCO2 regimes oscillating on either a reverse- or natural- phase (t =

0.49, df = 17, P = 0.315) (Fig. 3).

Survivorship

At the start of Experiment II during which survivorship was measured, there were

331 recruits in the ambient pCO2 treatment, 179 in the high pCO2 treatment, 354 in the natural-phase diurnal pCO2 treatment, and 354 in the reverse-phase diurnal pCO2 treatment. These recruits were scattered among a varying number of coverslips in each treatment, with a mean of 30.1 ± 1.7 (± SEM, n = 42) recruits coverslip-1. At the end of

19 the 6 d experiment, there were 256 (72%) survivors in ambient pCO2, 101 (67%) in the high pCO2, 305 (85%) in the natural-phase diurnal pCO2, and 225 (55%) in the reverse-

2 phase diurnal pCO2 (Fig. 4). Survivorship differed significantly among treatments (χ =

107, df = 3, P < 0.001) with the highest survival in the natural-phase diurnal pCO2 treatment.

Discussion

My experiments show for the first time that diurnally-oscillating pCO2 can increase calcification and survival in coral recruits that are ≤ 6 d old. Despite the growing literature addressing the response of corals to OA (Albright 2011, Erez et al.

2011, Pandolfi et al. 2011), to my knowledge, only one previous study has employed a measure of fitness as a response variable for corals exposed to high pCO2 (Anlauf et al.

2011), and it demonstrated the survivorship of Porites panamensis larvae was unaffected by 861-950 µatm pCO2. Additionally, the impact on corals of diurnally-oscillating pCO2 with a natural phase relationship - as occurs routinely on shallow reefs (Gattuso et al.

1997, Ohde and Van Woesik 1999, Bates et al. 2010, Andersson and Mackenzie 2011) - has not previously been considered. Instead, studies of the effects of OA on corals have employed steady DIC regimes mimicking those expected in open oceans under increased atmospheric pCO2 (Erez et al. 2011), and only recently have the limitations of these scenarios for the conditions in near-shore habitats been recognized (Andersson and

Mackenzie 2011). In the present study, I show that newly-settled coral recruits grown for

3-6 d in diurnally oscillating seawater DIC chemistry with a natural phase relationship

20 survive better than their counterparts in static treatments of either ambient or elevated pCO2.

Most scleractinian corals that have been studied respond to elevated pCO2 with decreased calcification (Erez et al. 2011), although a few exceptions to this trend have been found (Anthony et al. 2008, Jury et al. 2010, Edmunds 2011). The present results contribute to these exceptions, with calcification of newly-recruited Seriatopora caliendrum elevated 16-19% by weight (relative to ambient pCO2) in both diurnally oscillating pCO2 on a the natural-phase, and high pCO2 treatments, and these effects were more pronounced in our first experiment. The decreased calcification in my second experiment probably reflects my selection of a 25 °C experimental temperature, which was chosen to facilitate a contrast with my earlier experiment, even though the ambient temperature on the reef (where the parental colonies were collected) was 3 °C warmer during Experiment II compared to Experiment I. Stimulatory effects on calcification of pCO2 as high as 845 µatm have not been observed in coral recruits, and instead the calcification of coral recruits (< 50 d old and based on 7 species) has been reported to decrease up to 84% when exposed to pCO2 ranging from 560-3585 µatm (Albright 2011).

Recruits of Porites astreoides, Favia fragum and P. panamensis departed somewhat from this trend, with calcification in P. astreoides and F. fragum remaining constant until pCO2 was > ~800-850 µatm (calculated from de Putron et al. 2011), and calcification in

P. panamensis declined only 3% at a pCO2 of 926 µatm (Anlauf et al. 2011). It is important to note that corallite size determined from planar area or skeletal mass (as used in the present study and several others (Albright and Langdon 2008, de Putron et al. 2011,

21 Anlauf et al. 2011)) do not reflect the presence of biomass, which exerts a strong biological control on the process of mineralization (Hofmann et al. 2010) that can be altered to beneficial effect under OA conditions (Edmunds 2011). Thus, most contemporary studies of the effects of OA on coral recruits are limited in their capacity to ascribe process to the experimental outcomes and, likewise, here I was unable to determine the extent to which biomass contributed to the increased calcification in diurnally-oscillating pCO2 on a natural-phase, and high pCO2.

Null or stimulatory effects of high pCO2 on coral calcification, similar to those reported here, have been reported for at least three species of corals studied as adult colonies. Anthony et al. (2008) reported that calcification of adult Porites lobata increased ~23% under 520-700 ppm pCO2 at 28-29 °C, Jury et al. (2010) reported that calcification in Madracis auretenra remained unaffected or increasing up to 21% when exposed to 876, 1406, 1480 ppm pCO2 at 27-28 °C, and Edmunds (2011) reported that area-normalized calcification in massive Porites spp. was unaffected by 815 µatm pCO2.

Clearly, the outcome of experiments exposing reef corals to conditions simulating the effects of OA are likely to be dependent on the pCO2 regime applied, the length of the experiment, seawater temperature, and light regime (as well as many other physical and chemical conditions), but nevertheless the aforementioned studies, as well as the present analyses, demonstrates that there is intrinsic variability in the response of corals to OA

(Pandolfi et al. 2011).

22 While studies of the effects of OA on coral calcification are beginning to be published in large numbers, little has been done to determine the effects on fitness traits such as survivorship, fecundity, and fertilization success. There is evidence, however, that fertilization efficiency in broadcast spawning corals declines with increased pCO2, especially at low sperm concentrations (Albright et al. 2010, Albright and Langdon 2011), although survivorship of coral recruits has been found to be unresponsive to high pCO2

(Anlauf et al. 2011). In the present study, there were striking differences in the survival of Seriatopora caliendrum recruits, with the highest 6-d survivorship (85%) in diurnally- oscillating pCO2 on a natural-phase. Survivorship decreased to 67-72% in static 464 and

830 µatm pCO2, and was only 55% in the diurnally-oscillating pCO2 on a reverse phase.

For very young corals (i.e., colonies <2 cm), in situ survival is size-dependent, with strong selective value ascribed to rapid growth (Babcock 1991, Babcock and Mundy

1996, Dunstan and Johnson 1998). However, this was not the case in the present study for recruits in high pCO2 and diurnally-oscillating pCO2 on a natural-phase in Experiment

II, where recruits of similar sizes and equivalent growth rates exhibited considerably different survivorship. This result implies that the survival of coral recruits in this experiment was independent of size and growth rate.

While it was beyond the scope of this study to identify the mechanism underlying the stimulatory effect on calcification and survival of diurnally oscillating pCO2 on a natural-phase, my results are consistent with a testable hypotheses evoking night-time build up of DIC in the coral tissue. According to this hypothesis, nocturnal increases in

! !! seawater pCO2 favors the accumulation of DIC (HCO! , CO! , CO2) in the coral tissue at

23 night through increased flux of DIC (Furla et al. 2000b), leading to the creation of an intracellular DIC pool that could support calcification during the early part of the following day. In the present study, coral recruits in diurnally oscillating pCO2 on a

! natural-phase were exposed to elevated DIC mostly in the form of HCO! at night, which is thought to be used as the primarily DIC source for calcification and photosynthesis in corals during the day (Marubini and Thake 1999, Schneider and Erez 2006, Herfort et al.

! 2008, Jury et al. 2010); during the night, HCO! might then be sequestered in the coral tissue.

In the coral Stylophora pistillata, the DIC pool equilibrated with surrounding seawater after ~3 h of calcification (Allemand et al. 2011 adapted from Furla et al.

2000b), indicating active DIC storage is possible. Short-term storage of DIC would benefit corals if calcification becomes DIC-limited during periods of light-enhanced calcification (Marubini and Thake 1999, Herfort et al. 2008, Jury et al. 2010), and potentially could sustain calcification until it became depleted. One means by which corals could store DIC is through storage in the cytosol that utilized the enzyme carbonic anhydrase (CA) in the oral ectoderm to transport DIC from seawater into coral tissues

! (Bertucci et al. 2011). This could be achieved by converting HCO! to CO2 for passive

! transport across cell membranes, and then converting it back to HCO! in the ectoderm to be later transferred throughout the coral for photosynthesis or calcification (Bertucci et al.

2011). The hypothesized role of night-time DIC storage to explain stimulation of calcification in diurnally oscillating pCO2 on a natural phase also has the potential to account for the erosion of this effect in diurnally oscillating pCO2 on a reverse phase.

24 Corals in this reverse-phase treatment calcified less than those in the natural-phase treatment, and this support the “DIC storage hypothesis” as these corals were not subjected to the increased DIC at night, and instead were subjected to low DIC. The DIC storage hypothesis could be tested experimentally by exposing corals to labeled DIC and evaluating the incorporation into host tissues under ambient, high and natural-phased diurnally oscillating pCO2 at night and into the onset of light-enhanced calcification.

More information is needed to determine the range of environmental conditions under which natural-phase diurnal pCO2 can affect scleractinian corals, and also to evaluate the types of coral life stages that are affected by these chemical conditions. It is not possible to know the extent to which results from recruits <6 d old apply to older corals, and indeed, there is reason to suspect that spat biologically are quite different from adults (Hamdoun and Epel 2007). However, identifying the extent to which newly recruited, juvenile, and adult corals are able to benefit from diurnally oscillating pCO2 will play an important role in understand the complexities of coral responses to ocean acidification and climate change.

25

26

Figure 1. pCO2 and pH at Hobihu reef over two 24 hour periods. Samples were taken at 3 m (filled circles) and 6 m (open squares) depth every 2 hours. 02:00 and 04:00 hrs samples on 7/13-7/14/2010 were not collected due to inclement weather.

27

Figure 2. Calcification of Seriatopora caliendrum recruits in ambient, high, and diurnally oscillating pCO2 treatments during Experiments I and II. Calcification was measured by weight (a, c) and area (b, d). Mean ± S.E.M. displayed (weight n = 6-12, area n = 30-79).

28

Figure 3. Calcification of Seriatopora caliendrum recruits in Experiment II under natural-phase diurnal (grey bars) and reverse-phase diurnal pCO2 (white bars) treatments using weight (left ordinate) and area (right ordinate) scales. Significant paired t-tests between pCO2 treatments (p<0.05) denoted by “*”. Mean ± S.E.M. displayed (weight n = 32-36, area n = 11-12).

29

Figure 4. Survivorship of Seriatopora caliendrum recruits during Experiment II as calculated by Kaplan-Meier product-limit analysis for ambient pCO2, high pCO2, natural- phased diurnal pCO2, and reverse-diurnal pCO2 treatments. The numbers of recruits at the start of the experiment in each treatment are shown in the legend.

30 Chapter 3

The importance of light in mediating the effects of ocean acidification on coral

recruits.

Introduction

Light is of fundamental importance to reef-building (hermatypic) corals, and through photosynthesis by their symbiotic Symbiodinium, has helped corals dominate many shallow tropical seas (Dubinsky and Falkowski 2011). Corals provide habitat for fish and invertebrates, thereby creating one of the most diverse ecosystems on the planet.

For corals, light plays direct and indirect roles in a range of behaviors and processes including photosynthesis (Goreau 1959), calcification (Pearse and Muscatine 1971), larval settlement/metamorphosis (Babcock and Mundy 1996), spawning/larval release

(Babcock et al. 1986), feeding (Houlbreque and Ferrier-Pages 2009), growth morphology

(Graus and Macintyre 1982, Muko et al. 2000), gene expression (Levy et al. 2011), and contributes to bleaching stress (Fitt et al. 2001). Of these, the effects of light on photosynthesis and calcification are among the most thoroughly studied for corals.

The influence of light on coral calcification is relatively well-understood, and was a dominant theme in coral biology in the 1930’s – 1980’s (Yonge and Nicholls 1931,

Kawaguti and Sakumoto 1948, Goreau 1959, Goreau and Goreau 1959, Pearse and

Muscatine 1971, Vandermuelen et al. 1972). In the presence of light, symbiotic corals exhibit increased calcification compared to the dark, termed light-enhanced calcification

(Kawagati and Sakumoto 1948, Goreau 1959). In the last 30 y, little empirical work on

31 the role of light on the calcification rates of corals has been done to explore the mechanistic basis of the effects of varying light intensities on coral calcification (except

Coles and Jokiel 1978, Chalker and Taylor 1978, Chalker 1981). One of the most prominent studies (i.e., Chalker 1981) showed a correlation between calcification and light intensity in corals, and found calcification of 3 hermatypic corals to be maximized at irradiances similar to those promoting maximum photosynthesis in the same corals (eg.,

~400 µmol photons m-2 s-1 Acropora cervicornis). The shape of the relationship between calcification and light intensity closely resembled a hyperbolic tangent function that has been used to describe the light-saturation curves of coral photosynthesis (Chalker and

Taylor 1978, Chalker 1981). Since the work of Chalker (1981), the role of light and photosynthesis on calcification has been discussed in reviews by Barnes and Chalker

(1990), Gattuso (1999), and Allemand et al. (2011) although little new substantive work has contributed to elucidating a mechanistic relationship between light and calcification.

Calcification again has become a central topic in current coral literature, with the majority of recent studies exploring the effects of ocean acidification (OA) on calcification. Concurrent with increasing global temperatures resulting from climate change, OA is expected to reduce the pH of the ocean from 0.3-0.5 by 2100 (IPCC 2007), which is a 40-60% increase in acidity (concentration of H+ ions) over preindustrial levels

(IPCC 2007). When subjected to conditions simulating the effects of OA in the form of increased pCO2 (800-1000 µatm), decreased pH (7.80-7.75), increased DIC and decreased carbonate ions, corals generally exhibit decreased calcification rates (Erez et al.

2011). The underlying mechanism(s) responsible for the decrease in calcification is

32 debated widely among coral biologists (Erez et al. 2011), with the discussion centered on

2- - the form of DIC (CO3 or HCO3 ) used in the calcification process (Allemand et al. 2011).

This debate highlights the lack of knowledge of the mechanism underlying the effects of

OA on the biogenic calcification process, which limits the understanding of whether if corals can acclimatize or adapt to climate change and ocean acidification (Baird and

Maynard 2008). Recent work has revealed that some corals can increase calcification and/or are unaffected by increased pCO2, which contrasts the general trend of decreased calcification by corals subjected increased pCO2 (Anthony 2008, Jury 2010, Edmunds

2011, Thesis Ch. 2). These exceptions to the decreasing calcification trend allude to the complexity of the responses of corals to OA.

Initial investigations of the effect of OA on corals has focused on adult corals, and has overlooked the possible effects on early life history processes such as settlement and post-settlement survival, which are crucial in structuring adult coral populations (Erez et al. 2011). There is a growing interest in exploring the response of early coral life history stages to OA (reviewed in Albright 2011) due to their potentially increased vulnerability to environmental challenges (Edmunds 2004, Maier et al. 2009). Calcification by newly- settled corals generally is impeded by pCO2 of ~800 µatm (Albright et al. 2008, Albright et al. 2010, Albright 2011, Albright and Langdon 2011, DePutron et al. 2011) which is hypothesized to lead to decreased fitness in situ as a result of the strong selective pressure for rapid growth in early life history stages of corals (Babcock 1991, Dustan and Johnson

1998). OA is well documented to reduce the rate at which corals deposit CaCO3 (mass), although it is unclear how reduced deposition translates to decreased size (area) of coral

33 recruits. Despite reduced rates of calcification in Porites panamensis survival does not appear to be negatively impacted by pCO2 conditions (~540-1006 µatm, Anlauf et al.

2011). Seriatopora caliendrum recruits of similar sizes displayed significantly different survivorship to OA conditions (464, 830 µatm pCO2, Chapter 1) confirming the previous results of Anlauf et al. (2011) that survivorship under OA conditions is independent of size, and instead, likely is a physiological response to the OA conditions. However the effect of OA on the survival of coral recruit is still relatively unknown and warrants further investigation.

Current coral literature addressing the effects of OA on corals highlight the range of methods used to create OA conditions in tanks (Erez et al. 2011), which is likely to contribute to the variability in coral responses to OA. Light is crucial for symbiotic corals to maintain a high rate of calcification (Goreau 1959), however light has not been controlled rigorously in OA studies. Light intensities in OA studies have ranged from 10

µmol photons m-2 s-1 (Albright et al. 2008) to 1600 µmol photons m-2 s-1 (Marshall and

Clode 2002) and typically lack ecological relevance to the locations from which the corals were collected. Calcification by symbiotic corals is correlated with light intensity, although to date only one study has investigated the interactive effects of light (80, 170,

800 µmol photons m-2 s-1) and OA on coral calcification (Marubini et al. 2001).

Marubini et al. (2001) reported decreased calcification of Porites compressa in increased pCO2, with the effect intensifying with light intensity. However because DIC seawater chemistry was manipulated through the addition of acid (HCl), total alkalinity also decreased (Kleypas and Langdon 2006) as a result of the acid addition and this may have

34 confounded their results. Furthermore, the pCO2 levels created through this acid addition

(186, 440 µatm) fall short of the expected levels of pCO2 by the end of the century to which most studies are compared (IPCC 2007). To test for light-dependent effects on coral calcification and survivorship in OA conditions, I exposed Pocillopora damicornis recruits to varying light intensities combined with increased pCO2.

Methods

I used an orthogonal design in which light intensity and pCO2 were manipulated to explore the individual and combined effects of pCO2 and light on Pocillopra damicornis recruits. P. damicornis is a cosmopolitan species throughout the Indo-Pacific

(Veron 2000), including on the coast of southern Taiwan, and is used commonly in laboratory studies. Adult P. damicornis colonies (~20 cm diameter) were collected from

5 to 7 m depth on Hobihu Reef, Nanwan Bay, Taiwan in early March 2011 and returned to the National Museum of Marine Biology and Aquarium (NMMBA). Colonies were placed into individual 15L flow-through seawater tanks and exposed to sunlight at an intensity of 103 ± 2 µmol photons m-2 s-1 (Mean ± SEM, measured with a LI-192 sensor,

LI-COR Biosciences Lincoln, Nebraska). Overflow seawater from each tank passed through mesh-lined (110 µm) cups to capture larvae, which were released during the night starting at 22:00 h (Fan et al. 2006). Larvae were collected at 08:00 hrs, pooled across colonies and retained in a 1L beaker for 4 h until ready to be used for settlement.

Following collection, ~ 2000 Pocillopora damicornis larvae were placed at

~11:00 hrs into each of 2 plastic containers (24 x 24 x12 cm) that were used to settle the

35 larvae. The containers were fitted with mesh windows (110 µm) to allow the circulation of seawater, and floated in the same tanks containing the adult colonies. Glazed porcelain tiles (2 cm x 2 cm x 8mm), which had accumulated natural biofilms after ~1 month incubation in aquaria, were placed into the floating plastic container with the glazed surface facing up. Larvae were allowed to settle for 24h onto the glazed surface of the tiles and, thereafter, unsettled larvae were discarded. Recruits that settled onto the glass surface were easily removed, which allowed for gravimetric analysis of individual coral recruits. Groups of 3 tiles with attached recruits were haphazardly assigned to each of 20 plastic containers (15 x 8 x 8cm), which had two 24-cm2 holes (6 x 4cm) cut in the side to allow for the exchange of seawater.

Each of the 20 containers was fitted with a lid composed of one or more sheets of neutral density (ND) gel filter (Lee Filters, Hampshire, England) to create 5 different light treatments. ND filters have an advantage over other shading techniques (e.g., shadecloth), by blocking out a constant proportion of all wavelengths that are used by photosynthesizing organisms (400-700 nm) and can be stacked to create multiple light treatments. However, ultraviolet (UV) wavelengths <300 nm are filtered out completely by the ND filter (Fig. 1). A single 0.3 ND filter blocked ~50% of the visible light and by stacking multiple filters five light treatments were created (100%, 50% 25%, 12.5%, and

6.25% of total light ~250 µmol photons m-2 s-1). Light treatments were crossed with ambient (~400 µatm) and high (~800µatm) pCO2 to test the effect of varying light intensities and pCO2 on coral recruit growth and survival. To create the 100% light treatment, a layer of clear plastic film (Glad Products Company ©) was placed over the

36 top of containers as a procedural control. The transmission characteristics of the ND filter and clear plastic film were assessed using a spectrophotometer (Spectronic Genesys 5,

Milton Roy, Fig. 1)

Containers fitted with lids representing 5 light treatments were placed into 120 L aquaria for 5 days (March 16-21), with two maintained at ambient pCO2 and two at high pCO2. All tanks were maintained at 24 °C using independent heaters and chillers controlled by an aquarium controller (±0.1°C Aquacontroller Apex Neptune Systems©,

San Jose, CA). The experimental temperature mimicked ambient seawater temperature at

Hobihu reef a week prior to the experiment (23.6 ± 0.6°C mean ± SD, TY Fan unpublished data) where the adult colonies providing larvae were collected. The tanks were filled with filtered seawater (1 µm), and the seawater was changed partially (10-

15% volume) everyday to correct any changes in salinity caused by evaporation. All tanks were illuminated from 07:00 hrs to 19:00 hrs with lamps fitted with a metal halide bulb (Phillips 150 W 10,000k) and two 39 W fluorescent bulbs (Phillips T5 460 nm) to create average light intensities of 265 ± 18 µmol photons m-2 s-1 (mean ± SD, n= 140, 4π sensor LI-COR Biosciences).

Light inside each container was measured twice daily using the photosynthetically active radiation (PAR) cosine sensor on a diving PAM (Heinz Walz GmbH,

Effeltrich Germany). The small size (1 mm diameter) of the sensor allows for light measurements ~1 cm over the coral recruits while inside the plastic container. The PAR sensor on the diving PAM was calibrated using a separate light meter (LI-1400

37 Datalogger, LI-192 quanta sensor, LI-COR Biosciences Lincoln, Nebraska). To place our experimental light conditions in an ecological context, the intensity of light in the field at Hobihu reef was measured using an underwater light intensity recorder (MDS-

MkV/L, Alec Electronics USA, Leslie Santee, CA) that was secured upright at ~5 m depth from March 5 – March 10, 2011 recording light every 10 min. The average light intensity from 0600 hrs to 1800 hrs at Hobihu reef where corals were collected was 392 ±

283 µmol photons m-2 s-1 (Mean ± SD, n=72) and reached daily maximum intensities of

~1151±503 µmol photons m-2 s-1 (n=6).

Seawater Chemistry

The dissolved inorganic carbon (DIC) content of seawater was manipulated in the aquaria by bubbling premixed gas of a known pCO2, or by bubbling air for the ambient treatment. To mix the gas for the high pCO2 treatment, a system employing a variable timed solenoid valve was used, which controlled the flow of air and CO2 into a mixing chamber to reach the target pCO2 of 900 µatm. This target value was selected to provide a moderate-to-high treatment pCO2 following the business as usual emission scenario A1 for atmospheric CO2 concentrations by the year 2100 (IPCC 2007). The solenoid valve was connected to an infrared gas analyzer (S151, Qubit Systems, Ontario Canada), which monitored the output gas and provided dynamic control of the duty cycle of the solenoid, thereby providing a consistent concentration of mixed gas to the treatment tanks.

Seawater DIC chemistry was measured daily through the experiment. Total alkalinity (TA) and pH were measured following standard operating procedures (SOP) 3b

38 and 6b respectively of Dickson et al. (2007). TA was measured using an open cell automatic titrator (Mettler-Toledo, Model DS50) filled with certified HCl titrant supplied by Dr. Andrew Dickson, and TA was calculated using an Excel™ (Microsoft 2008) spreadsheet (Fangue et al. 2010). The accuracy and precision of the TA measurements were tested against certified reference materials (CRM, Dr. Andrew Dickson) and maintained within 0.7% of certified values. pH was measured on the total scale (± 0.001 pH) using the dye m-cresol purple and a spectrophotometric assay (SOP 6b). Salinity was measured with a conductivity meter (± 0.1 accuracy, WTW 340i), and temperature was measured with a certified digital thermometer (± 0.05°C accuracy, Fisher Scientific

Traceable Digital Thermometer). TA and pH were used to calculate the remaining DIC

! !! parameters including pCO2, HCO! , CO! and aragonite saturation state (ΩA) using

CO2SYS (Pierrot et al. 2006).

Measured Variables

Upon completion of the experiment, to ascertain the effects of light and pCO2 on

Pocillopora damicornis physiology, recruits were sampled for calcification, survivorship,

Symbiodinium concentration, and total protein. To measure the skeletal deposition of coral recruits, individual recruit corallites were assessed gravimetrically. Tiles with remaining coral recruits after Symbiodinium and protein sampling (discussed below), were placed in bleach (6% NaOCl) for 8 hrs to dissolve the tissue from the skeletons leaving the CaCO3 skeleton behind. Tiles then were rinsed with deionized water to remove the bleach, and dried in a fume hood for 72 h at ~ 27 °C. Dried corallites were

39 removed from the glass surface of the tile using a razor blade and weighed individually on a microbalance (± 100 ng Mettler-Toledo UMT2).

Survival of coral recruits was assessed daily for the duration of the experiment.

Due to high densities of recruitment on some tiles (up to 55 recruits tile-1, average 19.2

±0.2, mean ± SE), assessing survival by enumerating under a microscope was logistically difficult. Instead, individual tiles were photographed using a macro lens (Canon 60mm f2.8) fitted to a digital SLR camera (Canon 40d, 10 megapixel resolution). The images were used to score the recruits as alive or dead based on the presence of tissue, which is easily discernable from images.

Symbiodinium density in coral recruits was measured due to the role played by the symbionts in providing photosynthetically derived carbon to the coral host (Dubinksy and

Falkowski 2011). Corals in low light intensities often have higher symbiont densities and/or chlorophyll concentrations to maintain sufficient photosynthesis in low light (Fitt et al. 2000, Stambler 2011). To quantify the density of Symbiodinium in P. damicornis recruits in this study, Symbiodinium were counted in samples of 4 recruits fixed in 10% formalin and homogenized using a microcentrifuge tube pestle. Cells were counted using a hemocytometer (5 replicate counts were performed per sample) and Symbiodinium density was standardized to individual recruit (cells recruit-1).

Currently the effects of OA on coral physiology, with the exception of calcification, are not well understood (Erez et al. 2011) although recent evidence suggests

40 coral biomass may increase in response to OA (Edmunds 2011). Total protein of coral tissue provides one means to measure indirectly the biomass of P. damicornis recruits and is used to standardize calcification rates to coral biomass. Three recruits were harvested for each total protein sample. Total protein content was analyzed using the

Bio-Rad protein assay (Bio-Rad Laboratories) in a microtiter plate, which utilizes a 96 well plate spectrophotometer (Biotek Synergy H4 Hybrid Reader (Biotek, VT USA)). To prepare samples for the assay, 400 µl of deionized (DI) water and 40 µl of 1M NaOH was added to the frozen recruits and the pH measured to ensure it was >10, which is needed to solubilize protein. The resulting mixture was then homogenized using a sonicator for 15 secs at 10% amplitude. Homogenized samples were heated for 5 h at 50 °C and neutralized with 1M HCl to pH 7.0-7.5 (verified using pH paper) and processed in triplicate. Total protein content was compared to a standard curve composed of bovine serum albumin (BSA) standards.

Statistical Analysis

Calcification rate, Symbiodinium density and protein concentration were analyzed using two-way nested ANOVA’s with tank as a random factor nested in light and pCO2 treatments using the statistical program JMP® (Version 9.0.2, 2010 SAS Institute Inc.).

To test for the normality and homogeneity of variance assumptions of ANOVA, normality was assessed using a Shapiro-Wilks test and homogeneity of variances were assessed graphically by plotting ANOVA residuals against the predicted residuals. When the nested tank factor was not significant (p > 0.25; Underwood 1997), it was removed from the statistical model and replicates were pooled across tanks within treatments.

41

To analyze survivorship, a Kaplan-Meier product-limit (KM) analysis was used

(Machin et al. 2006). For this analysis, the probability of individual recruits surviving is assumed to be independent of all other recruits, and because KM analysis cannot accommodate nested experimental designs, replicate corallites were pooled within treatments. KM analysis was performed using the statistical software JMP, and log-rank tests (Machin et al. 2006) were used to test for differential survival among light and pCO2 treatments. To test the multiple treatments in this study that a log rank test cannot accommodate, log-rank tests were used to compare individual light treatments between ambient and high pCO2.

Results

The neutral density (ND) filters created 5 light treatments (100%, 50%, 25%,

12.5%, 6.25%) characterized by mean light intensities of 226 ± 9.4, 122 ± 3.7, 70 ± 2.6,

41 ± 2.1 and 31 ± 1.7 µmol photons m-2 s-1 respectively (±S.E., n=10). For the duration of the experiment, pCO2 treatments were maintained at 493±27 µatm (ambient pCO2) and

878±26 µatm (high pCO2) (±S.E., n=5). Other seawater chemistry parameters including

- 2- HCO3 , CO3 , Ω and other physical tank parameters are summarized in Table 1.

Pocillopora damicornis larvae settled onto tiles at densities of 19.2±1.6 corals tile-1 (±S.E. n=61). Over the course of the experiment individual corals grew rapidly,

-1 depositing CaCO3 ranging in weight from 104 to 558 µg recruit . Protein content of P. damicornis recruits did not differ significantly across light or pCO2 treatments (data not

-1 shown, ANOVA F9,34=0.816, p=0.607) and averaged 0.057 ± 0.001 mg recruit (± SE

42 n=35). Calcification of coral recruits standardized to the average total protein content

-1 -1 ranged from 1.12 – 1.57 mg CaCO3 mg protein day , and was significantly affected by pCO2 (F1,130=9.01 p=0.003) and the interactive effect of light and pCO2 (F4,130=3.57 p=0.009, Fig. 2). Calcification in the ambient pCO2 treatment resembled a parabolic function against light intensity, with calcification maximized at 70 µmol photons m-2 s-1 and decreasing slightly thereafter. In contrast, calcification in the high pCO2 treatment was affected by light in a relationship that was the inverse of that observed in the ambient pCO2 conditions. The effects of light and pCO2 on coral calcification were therefore most pronounced at intermediate light intensities (122, 70 and 41 µmol photons m-2 s-1).

The greatest discrepancy between ambient and high pCO2 growth occurred at 70 µmol

-2 -1 photons m s , with corals in the high pCO2 treatment calcifying 29% slower than corals

-2 -1 in ambient pCO2. At both the minimum (31 µmol photons m s ) and maximum (226

-2 -1 µmol photons m s ) light intensity, calcification was similar between pCO2 treatments.

Survivorship of Pocillopora damicornis recruits calculated from the K-M analysis varied significantly across light and pCO2 treatments (Fig. 2 and 3, Table 2). Recruit survival remained high (>90%) in all treatments until day 4, when differential survival among treatments became apparent. Despite calcification being similar at both the lowest

(31 µmol photons m-2 s-1) and highest (222 µmol photons m-2 s-1) light treatments,

-2 -1 survival was 34% higher (p=0.012, Table 2) in ambient pCO2 at 31 µmol photons m s ,

-2 -1 while recruits in 222 µmol photons m s and high pCO2 survived 38% better

-2 -1 (p=0.0001) than those in ambient pCO2. At 70 µmol photons m s survival of recruits was 34%, the lowest among light treatments in ambient pCO2 but was elevated 38%

43 (p=0.000) at high pCO2 coinciding with a 29% decrease in growth. Survival of recruits

-2 -1 was maximized at 122 µmol photons m s in both ambient and high pCO2.

Symbiodinium concentrations in Pocillopora damicornis recruits largely were unaffected by both light and pCO2 treatments (Fig. 4). Substantial variability in the number of Symbiodinium recruit-1 was found, both within and among treatments, although due to mortality across all treatments there were insufficient replicates to support statistical analysis. Mean Symbiodinium concentrations were 23,365 ± 7240 cells recruits-1 (±SD n=33) across all treatments, with the highest concentrations in the two

-2 -1 lowest light treatments (31 and 41 µmol photons m s ) in ambient pCO2. Recruits in

-2 -1 high pCO2 also had the highest symbiont concentrations at 31µmol photons m s and

-2 -1 decreased drastically at 41 µmol photons m s in contrast to ambient pCO2. At the highest light intensity (226 µmol photons m-2 s-1) Symbiodinium concentrations were relatively equal between pCO2 treatments.

Discussion

The calcification and survival of Pocillopora damicornis was influenced heavily by pCO2 and light. Calcification differed between pCO2 treatments at intermediate light intensities, although it remained similar at the lowest and highest light levels. Previously only a single study (Marubini et al. 2001) addressed the combined effect of pCO2 and light on coral calcification, and it found coral calcification in elevated pCO2 (641µatm) resembled a hyperbolic tangent function of light intensity. In contrast, in the present study the hyperbolic tangent relationship in high pCO2 was ambiguous and corals in

44 ambient pCO2 showed responses similar to photoinhibition at 122 and 226 µmol photons m-2 s-1 (Ralph et al. 2002). The results of the present experiment suggest the decreased calcification in some other studies as a result of OA conditions (Anlauf et al. 2010,

Albright 2011, de Putron et al. 2011) may be light dependent. Furthermore, the calcification by corals in high pCO2 suggests the negative effects of OA on coral calcification may be counteracted at higher light intensities.

The sensitivity of calcification by adult corals in OA conditions varies among studies (Langdon and Atkinson 2005), however calcification generally decreases with increasing pCO2 (Erez et al. 2011). The calcification of coral recruits in this study was similar at both high and low light intensities in both pCO2 treatments, with the greatest difference at growth in intermediate light intensities. Previous investigations of the effects of OA on the calcification of coral recruits have largely disregarded the role of light in contributing to the observed calcification response (Suwa et al. 2010, Albright et al. 2011, de Putron et al. 2011). Experiments addressing calcification in response to OA for coral recruits have used light intensities varying between ~10 µmol photons m-2 s-1

(Albright et al. 2008) and 61 µmol photons m-2 s-1 (Cohen et al. 2009, de Putron et al.

2011), although it has been unreported in others (Suwa et al. 2010, Albright et al. 2010,

Albright et al. 2011). Anlauf et al. (2011) reported ambient light levels of 135 ± 79 µmol photons m-2 s-1 (± SD) recorded over 3 years prior to their experiment in the same location although no light levels were measured during their experiment.

45 Measurements of in situ light intensities where coral colonies/nubbins or juveniles are collected prior to experimental manipulations rarely are made but can be useful in gauging the ecological relevance of the experimental light intensities. Light levels in the present experiment ranged from ~30-225 µmol photons m-2 s-1 which likely are ecologically relevant light intensities for coral recruits settling in cracks or crevices on the reef (Nozawa 2008). This preference for spatial refuges in the reef substrata is hypothesized to enhance their post-settlement survival by protection from grazing predators. Preliminary settlement experiments performed before the present study, revealed P. damicornis larvae seek out cracks or crevices when exposed to light intensities > ~150µmol photons m-2 s-1 during settlement, although they readily settle on exposed flat surfaces in low light intensities (~50-100 µmol photons m-2 s-1). Light- dependent settlement patterns of coral larvae were also shown by Babcock and Mundy

(1996), where Platygyra sinensis and Oxypora lacera coral larvae preferentially settled at specific depths mediated by light intensity. Depth dependent settlement patterns may also indicate a preference for low light during settlement.

The limited data available that describes the relationship between coral calcification and irradiance shows calcification and photosynthesis of Acropora formosa and Acropora cervicornis exposed to 0-1200 µmol photons m-2 s-1 saturating at similar irradiances (~500µmol photons m-2 s-1 A. cervicornis; 100µmol photons m-2 s-1 A. formosa [Chalker 1981]). For the single study that has explored the interactive roles of irradiance and OA on coral calcification (Marubini et al. 2001), calcification rates of

Porites compressa saturated at ~ 195 µmol m-2 s-1 (Marubini et al. 2001). In a second

46 experiment, P. compressa in 80, 150, and 700 µmol photons m-2 s-1 were exposed to 186 and 440 µatm pCO2 and exhibited a positive calcification relationship with increasing irradiance (Marubini et al. 2001). However, the pCO2 treatments used were created by

- 2- acid additions that do not accurately reflect the changes in DIC (pCO2, HCO3 , CO3 ) that are created by gas dissolution as occurs naturally. For early life history stages, no empirical work has addressed the effects of OA and light irradiance on coral calcification.

Comparing light intensities of previous OA studies with coral recruits might help determine to what extent the calcification trends at varying light intensities in the present study may influence the calcification of corals in previous OA studies. Porites astreoides recruits exposed to a mean light intensity of < 10 µmol photons m-2 s-1 and incubated in seawater acidified by the addition of HCl for one month, exhibited a 45-84% reduction in cross sectional area (a correlate of CaCO3 deposition) with increasing pCO2 (decreasing

Ω, Albright et al. 2008). When the experiment was repeated using ‘natural’ light (no quantitative light data reported) and seawater DIC was manipulated by CO2 bubbling, the effect of the OA treatment on the area of P. astreiodes decreased to a 16-35% reduction in area; a ~60% reduced effect relative to the first experiment (Albright et al. 2011).

Disparities in the DIC conditions resulting from acid vs. gas bubbling methods between

Albright et al. (2008) and Albright et al. (2011) limits assigning a cause to the reduced

OA treatment effect on calcification reported by Albright et al. (2011). However the decreased effect of OA on calcification in Albright et al. (2011) is consistent with our results showing increasing light can offset the negative effects of OA on coral calcification. In another study, P. astreoides and Favia fragum recruits were incubated at a light intensity of 61 µmol photons m-2 s-1 and their calcification decreased 22-37%

47 below 2.5-2.8 Ω, although they continued to calcify in aragonite subsaturating conditions

(Ω < 1) (de Putron et al. 2011). For Pocillopora damicornis recruits in the present study, the negative effects of high pCO2 on calcification were most pronounced at ~70µmol

-2 -1 photons m s . This calcification disparity between pCO2 treatments at 70 µmol photons m-2 s-1 suggests the reduced calcification reported for P. astreoides and F. fragum recruits to high pCO2 might have been less severe at higher light intensities (de Putron et al.

2011).

Survival of Pocillopora damicornis recruits in the present study was not

2 2 correlated with calcification rates (ambient pCO2 r =0.03, high pCO2 r =0.18) that have been shown previously for size differences (area) in situ (Babcock 1991). To the contrary, in the present study the survival of P. damicornis recruits in ambient pCO2 was lowest when calcification was high, alluding to a possible energetic tradeoff between high calcification rates and tissue growth and quality (Anthony et al. 2002). This is the first evidence that reduced coral calcification under high pCO2 may result in a survival advantage for the coral. The protein content of P. damicornis recruits and Symbiodinium density did not differ between pCO2 or light treatments, indicating recruits in each treatment had similar protein biomass and symbiont densities. Recruits survived ~34%

-2 -1 better at 31 and 41 µmol photons m s in ambient pCO2, but was 9-38% lower than

-2 -1 high pCO2 for the 3 highest light treatments (70, 122, 226 µmol photons m s ). The energetic demands of increased calcification at this early life history stage likely come at a cost to somatic tissue growth and maintenance, yet any added energetic costs of increased calcification alone do not explain the survival trends of P. damicornis recruits

48 across all pCO2 and light treatments. For example recruits in high pCO2 at 41 µmol photons m-2 s-1 had the lowest survival of all the light treatments and this was accompanied by moderate calcification rates. However, survival of P. damicornis

-2 -1 recruits at 122 µmol photons m s was highest in both pCO2 treatments, suggesting that an optimal irradiance exists that maximizes coral survival which may differ from irradiances that maximize coral calcification.

The results of this study indicate that coral calcification and survival in OA conditions are light dependent. The calcification in light and pCO2 treatments may be explained by competition between the coral and the symbiont for DIC, affecting the photosynthetic rate of the symbiont at the various light intensities. During light-enhanced calcification, corals and their Symbiodinium can become DIC limited (Marubini and

- Thake 2001, Herfort et al. 2008) yet in high pCO2, DIC is increased in the form of HCO3 and pCO2, which can increase the productivity of the Symbiodinium (Crawley et al. 2010).

Photosynthesis in low light treatments was likely similar between pCO2 treatments because of a low demand for DIC due to light limitation, resulting in similar calcification rates. As light and photosynthesis increase in this study, photosynthesis remains light limited in both treatments however the negative effects of OA (Erez et al. 2011) on calcification become apparent in high pCO2 until 70 µmol photons m-2 s-1. From 70 –

220 µmol photons m-2 s-1, photoinhibition may be occurring leading to photodamage, which the results in decreased calcification rates. The increased calcification and photosynthesis in high pCO2 might then overcome the negative effects of OA on coral calcification. Accompanying photosynthesis-irradiance curves along with the current

49 study could determine how photosynthesis may have contributed to the interaction between light and pCO2.

The light-dependent calcification and survival findings presented here have implications for calcification of both adult and juvenile corals in response to OA. Light levels in this study, although lower than what is found in situ on Hobihu reef, may elicit similar light-dependent effects on calcification in coral recruits exposed to OA that may be observed at higher light intensities in adults. Similar calcification rates to this study may be observed at higher light intensities for adult corals caused by the photoacclimation to higher light levels than is present for coral recruits. The response of corals to OA appears to be more complex than previously thought (Erez et al 2011) and warrants further investigation to identify the conditions under which light-dependent effects of OA are manifested.

50

Table 1. Physical and seawater DIC chemistry parameters over the 5d duration of the experiment. Values are means ± S.E. (n=5). Temperature and salinity were measured twice daily (n=10).

- 2- pCO2 TA (µmol pCO2 HCO3 (µmol CO3 (mmol -1 -1 -1 Treatment Temp.(°C) Salinity kg SW ) pHTotal (µatm) kg SW ) kg SW ) Ambient 24.01±0.14 33.9±0.1 2276±13 7.97±0.02 493±27 1846±30 175±3 High 23.98±0.10 33.9±0.1 2274±10 7.75±0.01 877±26 1992±20 115±2

51

Table 2. Results from log rank tests on the survivorship of Pocillopora damicornis recruits calculated by the Kaplan Meier analysis. Comparisons between all light treatments in ambient and high pCO2 treatments as well as individual tests of light treatments between pCO2 treatments are shown.

pCO2 Treatment Comparisons Chi Square DF p-value Ambient χ2=60.5 4 p<0.0001 High χ2=209.9 4 p<0.0001 Light Treatment Comparisons (PAR) 100% (226) χ2=22.8 1 p<0.0001 50% (122) χ2=4.9 1 p<0.0267 25% (70) χ2=34.0 1 p<0.0001 12.5% (41) χ2=22.5 1 p<0.0001 6.25% (31) χ2=6.3 1 p<0.0123

52

Figure 1. Percent transmittance of light through a single neutral density filter (circles) used to create the 4 reduced light treatments (122, 70, 41, 31 µmol photons m-2 s-1) and the plastic film (squares) used to create the 100% (226 µmol photons m-2 s-1) light treatment.

53

Figure 2. Calcification of Pocillopora damicornis recruits standardized to protein (0.057 mg recruit-1) in the five light treatments (6.25%, 12.5%, 25%, 50%, 100%). Corals were incubated for 5 days in ambient (~493µatm) or high (~878µatm) pCO2. Values are mean ± SE (Calcification n~6-20, photosynthetically active radiation n=10). Percent of recruits alive (white) and dead (black-ambient pCO2, gray-high pCO2) calculated by the K-M analysis at the end of experiment for each treatment indicated by the pie chart next to each datapoint (n=43-169).

54

55

Figure 4. Symbiodinium cell content per Pocillopora damicornis recruit incubated in the 5 light treatments under ambient (white bars: pCO2 ~ 493µatm) or high (grey bars: pCO2 ~ 878µatm) pCO2. Values are means ± S.D. The number of replicates in each sample (n) is shown above each bar. Note the break on the y-axis.

56 Chapter 4

The response of carbonic anhydrase activity to ocean acidification: Implications for

coral calcification

Introduction

The effects of ocean acidification (OA) on coral growth are well established, with corals frequently calcifying slower as pCO2 increases (Erez et al. 2011). Despite a rapidly growing literature documenting the effects of OA on coral growth, the mechanism(s) responsible for the decrease in calcification has remained largely speculative (Erez et al.

2011). Recent studies investigating the capacity of corals to calcify in seawater differing in aragonite saturation states demonstrates that some corals exhibit non-linear calcification rates in response to ocean acidification and alludes to the complexity of coral responses to OA (Atkinson and Langdon 2005, DePutron et al. 2011). However, the mechanism(s) responsible for the variability in coral calcification have not been thoroughly tested and may require a more reductionist approach than has been used previously.

Carbonic anhydrases (CA) are group of enzymes used by a wide range of invertebrates and vertebrates for a suite of physiological processes including pH

- regulation, dissolved CO2 and HCO3 transport and exchange, CO2 fixation, electrolyte secretion in animal organs and calcification (Supuran 2008, Xu et al. 2008). Carbonic anhydrases have been identified in a diverse range of cnidarians including multiple species of corals and anemones (Weis et al. 1989). For corals, CA not only helps to

57 - maintain pH balance, but also converts HCO3 (which makes up the majority of DIC in seawater) to CO2 making carbon available for photosynthesis by the Symbiodinium (Weis et al. 1989, Weis et al. 1991). More recently, a specific type of α - CA was hypothesized to play a role in calcification of corals because it was localized only in the calicoblastic ectoderm where calcification occurs (Moya et al. 2008, Bertucci et al. 2011). CA

- localized in the calicoblastic ectoderm may convert CO2 to HCO3 (or vice versa) into a preferred species for calcification (Tambutte et al. 2007, Bertucci et al. 2011) facilitating the availability of DIC to the carbon-intensive process or by removing carbonic acid from the site of skeletogenesis (Moya et al. 2008), raising the aragonite saturation state (Ω) in the calcifying medium leading to more rapid aragonite deposition. Furthermore, CA in the oral body wall may trap DIC in coral tissues by converting CO2 that passively

- diffuses across membranes into HCO3 , which likely requires active transport to cross cell

! membranes. This conversion of CO2 to HCO! by CA could trap DIC in host tissues so that it may used later to fuel photosynthesis or calcification.

In a previous experiment in Chapter 2, we found increased calcification in recruits of the coral Seriatopora hystrix exposed to diurnally oscillating pCO2 on a natural phase, similar to the diurnal swings in pCO2 present on many shallow reefs (Andersson et al.

2011). I hypothesized that increased calcification upon exposure to diurnally oscillating pCO2 may have been supported by a buildup of DIC in host tissues at night (Chapter 2).

This DIC buildup may have helped fuel calcification in the early part of the day without the negative impacts of high pCO2 on light-enhanced calcification. In the present study I tested the hypothesis of increased DIC supporting increased calcification by subjecting

58 juvenile Seriatopora hystrix corals (< 3 cm diameter) to ambient (471 µatm), high (958

µatm), and diurnally oscillating pCO2 (471-958 µatm) seawater and measuring the resulting carbonic anhydrase activity in the coral. I hypothesized that CA activity at night would be similar in both diurnally-oscillating on a natural phase and steady high pCO2 treatments compared to ambient pCO2. This similarity in CA activity is between the natural phase diurnally oscillating and high pCO2 treatments may be due to the availability to increased DIC in the seawater present in high and diurnally oscillating pCO2, which is thought to compose ~25% of the DIC needed for calcification (Furla et al.

2000b).

Methods

Juvenile Seriatopora caliendrum colonies (<3 cm diameter) were collected on

July 4, 2011 from Hobihu Reef, Nanwan Bay, Taiwan. Juvenile colonies were brought to the National Museum of Marine Biology and Aquarium and allowed to acclimate to ambient treatment conditions for 5 days. This period was sufficient to stabilize the corals photophysiological performance indicating acclimation to the treatment conditions

(Abrego and Ulstrup 2008, Chris Wall unpublished data). At the start of the experiment,

8 juvenile S. caliendrum corals were placed into 1050 L (240x74x60cm) aquaria maintained at ambient or high pCO2 and temperature controlled using separate chillers

(± 0.1 °C Aquatec, Aquasystems). An additional 8 corals were exposed to diurnally oscillating pCO2, which was created by moving the colonies between ambient pCO2 during the day (6:00 hrs) into high pCO2 at night (18:00 hrs). Sand-filtered seawater was supplied to each tank at 6 L min-1 which replenished the entire tank volume ~8 times in a

59 24 h period. Two water pumps (Rio 1100 1454 L h-1, TAAM Inc.) provided additional flow in each tank. Photosynthetically active radiation (PAR) in each tank was 148 ± 3

µmol photons m-2 s-1 (mean ± SEM, n=168) and was measured using a 4π light sensor

(LI-192 sensor, LI-COR Biosciences Lincoln, Nebraska). Corals were incubated in treatment aquaria for 6-9 days.

Carbonic Anhydrase Assay

Two corals from each treatment were sampled over two consecutive days at 7:00 hrs, 1 h after lights were turned on (July 15,16) and at 24:00 hrs (July 17,18) ~ 6 h after lights were turned off. Sampling times were chosen in order to best address the hypothesis of DIC buildup which postulates corals in high and diurnally oscillating pCO2 maintain a DIC pool during the early morning hours (1-3 h after sunrise). CA activity in the coral is hypothesized to be elevated during the day due to CA providing CO2 to the

- Symbiodinium through the conversion of HCO3 to CO2, which can be taken up by the symbionts for photosynthesis. Nighttime sampling (24:00 hrs) was used to determine CA activity when photosynthesis was not occurring which is known to increase CA activity

(Weis et al. 1989). Sample preparation and the carbonic anhydrase (CA) assay were modified from Weis et al. (1989) and summarized below. Coral tissue was removed from the skeleton using an airbrush filled with 1µm filtered seawater, and the resulting tissue slurry centrifuged at ~900 x g for 2 minutes to pellet the Symbiodinium in the sample. To ensure the majority of Symbiodinium were removed from the animal supernatant through centrifugation, Symbiodinium counts were performed using a hemocytometer on both centrifuged and non-centrifuged samples. The procedure revealed centrifugation

60 removed > 99% of Symbiodinium cells from the animal homogenate. After centrifugation, the animal tissue homogenate (supernatant) was decanted (~8 mL) and placed on ice. 1 mL of animal homogenate was removed for total protein analysis and immediately placed in liquid N2 to stop protein degradation. The protein sample was transferred to a -80 °C freezer for later analysis. The animal homogenate then was diluted

1:1 with cold barbital buffer (25 mM Sodium barbital (5,5-diethylpyrimidine-

2,4,6(1H,3H,5H)-trione), 5mM EDTA, 5 mM Dithiothreitol (DTT), and 10 mM MgSO4

[Weis et al. 1989]) and kept on ice at 2 °C until the assay was performed ~2 h later. Half of each animal homogenate sample (~4 mL) was separated and boiled for 10 min to denature all proteins and serve as a negative control for CA activity. Crystallized

-1 mammalian CA was prepared to 0.55 µg mL in cold (2 °C) distilled H2O and used as a positive control (Sigma-Aldrich, C3934, ≥2500 W-A units mg-1 protein).

CA activity was measured indirectly through a decrease in pH of the animal

- homogenate caused by the hydration of CO2 to HCO3 , which was catalyzed by CA.

300ml of CO2 saturated Milli-Q (© Millipore Corporation) water bubbled with CO2 gas was used as the CO2 substrate for the assay. To ensure the water remained saturated with

CO2, CO2 gas was bubbled into the water between each assay (~20 min) for 10 min and the bottle stoppered to avoid gas exchange. 1 mL of 2°C animal homogenate or CA positive control, and an additional 1ml of cold 50 mM barbital buffer were added to a test tube on ice and mixed with a small (1.5 x 8 mm) magnetic stir bar. A pH probe (Fisher scientific Orion 3 portable pH Meter, ±0.01 pH) was placed into the mixing homogenate and allowed to equilibrate to the temperature of the homogenate for 3 minutes. An

61 additional 1ml of saturated CO2 water was then added rapidly to the solution and the pH recorded every 10 s for 3 min. To control for pH change not associated with CA activity, the boiled coral tissue homogenate (denatured control) was measured in the same manner.

CA activity was calculated as follows (Weis et al. 1989):

Equation 1:

ΔpH of animal homogenate - ΔpH denatured control min−1 ( ) mg soluble protein pH in the CA assay declined linearly at a mean rate of ~1.2 pH units min-1 until ~1 min and thereafter CA activity reduced as the CO2 substrate was depleted. Thus CA activity calculations were based on pH changes recorded during the first minute of the assay.

Diamox (acetazolamide - (N-(5-sulfamoyl-1,3,4-thiadiazol-2-yl)acetamide)) a specific inhibitor of CA activity was used to test for CA inhibition and confirm the pH change was caused by CA activity. Diamox was mixed with the 50 mM barbital buffer to a concentration of 10-5 M and added to the coral tissue homogenate in place of the normal

50mM barbital buffer used in the assay. Percent inhibition of CA activity was calculated from the following equation:

Equation 2:

"(CA activity with Diamox) % 100 −$ •100' # CA Activity &

Protein

Tissue samples of homogenized coral were analyzed for total protein following a modified BCA (bicinchoninic acid) protein assay. To lyse any remaining animal cells in

62 the protein sample after the initial tissue removal using the airbrush, RIPA buffer (Radio-

Immunoprecipitation Assay) composed of TRIS buffer 50mM, NP-40 1% (NonidetP40),

Na-deoxycholate 0.25%, NaCl 150 mM and a protease inhibitor (0.04% total volume) were added to each protein sample in a 1:1.5 protein sample to buffer ratio while on ice.

Samples were sonicated in an ice bath for 5 min then left on ice for an additional 10-20 min. To remove any remaining debris, samples were centrifuged for 5 min at 1500 x g at

4 °C. 25 µl of protein extract was added to 200 µl of bovine serum albumin (BSA) and the absorbances were measured at 595 nm using a Biotek Synergy H4 Hybrid Reader

(Biotek, VT USA). The standard curve was created using deionized water and BSA to concentrations of 0, 0.4, 0.8, 1.2, 1.6 and 2.0 mg/mL BSA.

Seawater Chemistry

The dissolved inorganic carbon (DIC) content of seawater in the high pCO2 tank was manipulated by bubbling 100% CO2 gas. The flow of CO2 gas was controlled by an aquarium controller fitted with a pH probe (Aquacontroller apex module, Neptune

Sytems Inc., San Jose, CA), which monitored pH (±0.01pH) of the tank and controlled a solenoid valve (ASCO Valves 120v) that regulated the flow of CO2 to the tank. The pH probe was calibrated at the start of the experiment using pH 4,7,10 buffers (Mettler

Toledo). The pH set point programmed into the aquarium controller used to regulate the flow of CO2 was calculated from the results using CO2SYS (Pierrot et al. 2006) from the targeted seawater pCO2 level of 900 µatm, the total alkalinity (TA), salinity and temperature of the tank. The 900 µatm target value in the high pCO2 treatment represented the high estimate for the business-as-usual emission scenario A1 for atmospheric CO2 concentrations by the year 2100 (IPCC 2007). The ambient pCO2 tank

63 was bubbled constantly with ambient air. Seawater DIC chemistry was measured daily through the experiment. Total alkalinity (TA) and pH were measured following standard operating procedures (SOP 3b and 6b respectively of Dickson et al. [2007]). TA was measured using an open cell automatic titrator (Mettler-Toledo, Model DS50) filled with certified HCl titrant supplied by Dr. Andrew Dickson, and TA was calculated using an

Excel™ (Microsoft 2008) spreadsheet (Fangue et al. 2010). The accuracy and precision of the TA measurements were tested against certified reference materials (CRM, Dr.

Andrew Dickson) and maintained within <1% of certified values. pH was measured on the total scale (± 0.001 pH) using the dye m-cresol purple and a spectrophotometer assay

(SOP 6b). Salinity was measured with a conductivity meter (± 0.1 accuracy, WTW 340i), and temperature was measured with a certified digital thermometer (± 0.05°C accuracy,

Fisher Scientific Traceable Digital Thermometer). TA and pH were used to calculate the

! 2- DIC parameters (pCO2, HCO! and CO3 , and aragonite saturation state [ΩA]) using

CO2SYS.

Statistical Analysis

Carbonic anhydrase activity of the corals in ambient, high and natural-phased diurnally oscillating pCO2 treatments was analyzed using a two-way ANOVA in which pCO2 treatment and sampling time (day vs. night) were treated as fixed factors. To meet the assumptions of the ANOVA, normality was tested using a Shapiro-Wilks goodness of fit test, and homogeneity of variances was assessed graphically. To test the hypothesis that CA activity would be similar in high and diurnally oscillating pCO2 treatments driven by the increased DIC at night in the surrounding seawater, an a-priori contrast

64 comparing ambient versus the natural phased diurnally-variable and high pCO2 treatments was performed for both early morning and night sampling times.

Results

The activity of carbonic anhydrase, which is the rate at which CO2 is hydrated to

- HCO3 resulting in a decrease in pH, was inhibited 96.5 ± 2.2% (± SEM n=4) by the CA specific inhibitor diamox, and CA activity was similar to that in the denatured control

(Table 1). The mammalian CA used as the positive control exhibited increased activity compared to CA activity of Seriatopora caliendrum however, when standardized to total protein of the mammalian CA, activity was ~680% higher in the positive control than in

Seriatopora caliendrum. Together the denatured control, the inhibition of CA activity by

Diamox, and the mammalian CA activity results demonstrate that the pH change recorded in the assay was caused by the activity of CA.

Carbonic anhydrase activity in juvenile Seriatopora caliendrum corals exposed to ambient (471 µatm), high (958 µatm), and natural-phased diurnally oscillating pCO2

(471-958 µatm, Table 2) was elevated 21% during the early morning relative to the night across all treatments (ANOVA F1,22=14.93 p=0.001, Fig. 1, Table 1). CA activity in ambient pCO2 during the morning was ~19% higher than both high and natural-phased diurnally-oscillating pCO2 though the contrast between ambient and the high and diurnally-oscillating pCO2, treatments was not significant (a-priori contrast F1,17=3.67 p=0.072). Comparing ambient to high and diurnally oscillating pCO2 treatments at night revealed no clear trend in CA activity (a priori contrast F1,17=1.32 p=0.267) and no

65 interaction between the sampling time and pCO2 treatment (ANOVA F2,22=0.187 p=0.778).

Discussion

Carbonic anhydrase activity has been hypothesized to play a role in the calcification of corals however before the present study, CA activity in response to ocean acidification conditions, which generally causes decreased calcification rates had not been explored for corals. Dufault et al. (2011) hypothesized the increased calcification observed in natural-phased diurnally oscillating pCO2 in Seriatopora caliendrum recruits was the result of increased access to DIC caused by the forming of an intracellular DIC pool that contributed to increased calcification rates during the early morning. I hypothesize the decreased trend of CA activity of S. caliendrum juveniles in high and natural-phased diurnally oscillating pCO2 compared to ambient pCO2 in the present study are indicative of increased DIC and supports the proposed hypothesis. A power analysis of the a-priori contrasts revealed low statistical power, which contributes to the non- significant findings of the contrast (Cohen 1988). At the time of the morning sampling

(~1 h after lights on), corals in diurnally oscillating pCO2 had been exposed to ambient pCO2 for one hour but displayed similar CA activity to corals in high pCO2. CA activity in vertebrates is elevated by hypoxic conditions (Loncaster et al. 2001) though for corals, increased CA activity occurs during the day when conditions in coral tissues are likely hyperoxic due to the oxygen production by photosynthesis of the Symbiodinium (Weis et al. 1989, Levy et al. 2011). I hypothesize decreased CA activity is indicative of sufficient carbon being available in coral tissues to meet the demands for both photosynthesis and

66 calcification, resulting in carbonic anhydrase down-regulation. This increased DIC could facilitate elevated rates of calcification observed in Dufault et al. (2011) as well as increased photosynthesis.

CA activity of Seriatopora caliendrum juveniles was similar to that of other symbiotic cnidarians, although it was slightly elevated compared to 6 other scleractinians

(Weis et al. 1989). The increased CA activity by S. caliendrum juveniles in this study may be a result of their juvenile life history stage and small size. Adult branching corals often show spatial distributions of Symbiodinium, which often decrease in density near the branch tips. The branch tip is also the site of the most rapid calcification in the coral

(Pearse and Muscatine 1971) and CA activity in corals is reported to be higher in corals, which harbor Symbiodinium (Weis et al. 1989). For juvenile corals the distance between the site of greatest calcification and Symbiodinium densities likely overlaps due to the small size of the corals used (< 3cm). This could lead to a greater demand of DIC per unit protein and thus greater CA activity to support those carbon demands that is alleviated in high and natural-phased diurnally oscillating pCO2 due to the hypothesized

DIC buildup during the night. Futhermore, CA activity not only is affected by the density of Symbiodinium but also by their photosynthetic rate (Weis et al. 1989). Corals from low light environments at 30 m depth exhibited 33% decreased CA activity compared to those in a high light environment at 3 m, hypothesized to be the result of decreased photosynthetic demand for DIC in corals at 30 m depth (Weis et al. 1989).

However, in the present study light intensity was similar between treatment tanks (145±5,

152±5 µmol photons m-2 s-1) and likely did not contribute to CA activity.

67

Recent work on the role of CA in corals has focused on identifying specific isozymes of CA, and this effort has led to the identification of novel CA types performing specific roles for the coral, some of which are linked to coral calcification (Moya et al.

2008). Genes coding for a specific CA in Stylophora pistillata (STPCA) localized in calicoblastic cells display 2 fold up-regulation during the night to facilitate the production

- of HCO3 from CO2 in the subcalicoblastic environment and also help in the removal of carbonic acid from the subcalicoblastic space which would keep calcification thermodynamically favorable (Moya et al. 2008, Bertucci et al. 2011). A similar nighttime increase of CA activity in calicoblastic cells may be occurring in Seriatopora caliendrum juveniles, however the methods pioneered by Weis et al (1989) and used in this study, do not distinguish between different isozymes of CA in the coral. Findings of previous studies suggest CA localized in the calicoblastic cells may make up only a minor fraction of the total CA present in the coral and likely to not contribute the CA trends in this study (Weis et al. 1989, Weis 1991).

CA activity clearly plays a fundamental role in contributing to light-enhanced calcification (Moya et al. 2008, Bertucci et al. 2011), though CA activity in response to the surrounding pCO2 environment has not been studied. As new studies progress towards finding the mechanistic basis for decreased calcification as a result of ocean acidification (increased pCO2), coupling manipulative experiments testing the effects of

OA on coral calcification with CA enzymatic assays may help elucidate the processes contributing to the decline in coral calcification at low pH.

68

Table 1. Summarized activity of coral tissue homogenates used in the experiment.

CA Mixture Δ pH units min.-1 Δ pH units min.-1 mg protein-1 Avg. Day 1.18 5.58 Avg. Night 1.21 4.60 Denatured Control 0.35 1.58 CA control 1.98 3827.6 10-5 Diamox 0.33 1.27

69

70

Figure 1. Activity of carbonic anhydrase of juvenile Seriatopora caliendrum in ambient, high, and diurnally-oscillating DIC seawater chemistry sampled during the early morning (7:00 h) and night (24:00). Values are means ± SEM (n=4, except ambient 24:00 n=3)

71 Chapter 5

Conclusion

The effect of OA on both adult and juvenile coral calcification generally is negative (Albright 2011, Pandolfi et al. 2011,). For Seriatopora caliendrum and

Pocillopora damicornis recruits used in Chapters II and III, the ubiquitous decline in coral calcification to OA conditions was not observed. These results differ from the majority of previous findings on the effects of OA on coral calcification for both adult and juvenile corals. The cause of this discrepancy may be a species-specific response of the corals used in these experiments, which are all brooding corals. However, as the number of coral species studied under OA conditions has grown over the last decade, which span a range of life history strategies and growth morphologies (Kleypas and

Langdon 2006), exceptions to the commonplace decreased coral calcification findings are becoming more prevalent (Jury et al. 2010, de Putron et al. 2011, Edmunds 2011). The diversity of responses by corals to OA underpins the current knowledge on the biogenic calcification process and suggests some corals may be able to maintain calcification rates in seawater that is thermodynamically unfavorable for aragonite precipitation (Allemand et al. 2011). Identifying the corals and life history stages that are unaffected or even benefit from OA may inform how coral community structure may change in the future.

A central focus of this thesis was to identify keep gaps in the ecological relevance of current OA studies, the first being the discrepancies between the static OA treatments used in manipulative experiments and the actual conditions present on coral reefs. The

DIC seawater conditions on natural reefs are rarely static, and instead often fluctuate on

72 daily to seasonal temporal scales (Andersson and Mackenzie 2011). In Chapter II,

Seriatopora caliendrum recruits were exposed to natural-phase diurnally-oscillating pCO2, similar to what is observed in the field. Corals in natural-phase diurnally- oscillating pCO2 increased calcification rates and survived better than recruits exposed to static pCO2. The idea that varying abiotic conditions produce differing physiological outcomes compared to static conditions has been shown for a broad range of taxa including corals to varying thermal regimes (Coles et al. 1975, Oliver and Palumbi 2011).

It is still unclear as to what extent corals may benefit from diurnally oscillating pCO2.

However the combined calcification and fitness advantage in coral recruits exposed to oscillating pCO2 conditions warrants further investigation and may prove useful in identifying locally acclimatized or adapted corals that can withstand the negative effects of OA.

Chapter III addressed the role of varying light intensities contributing to the effects of OA on coral calcification. Despite the well-founded effect of light intensity on coral calcification (Chalker 1981, Gattuso et al. 1999), light largely has been disregarded as a contributing factor in the calcification response by corals in OA studies (Erez et al.

2011). Pocillopora damicornis recruits exposed to varying light intensities showed light- dependent effects of OA on coral calcification. Survival was also light-dependent but was not correlated with recruit size, as has been described for other juvenile corals

(Babcock and Mundy 1996). A common conclusion from reduced coral calcification rates due to OA, is the inference that lower calcification rates are indicative of a less fit coral, although survival of calcifying corals has been observed in only a single study

73 (Anlauf et al. 2011). Interestingly, recruits in this experiment that achieved the highest calcification rate in ambient pCO2 had the lowest survival. The light-dependent effects on calcification in OA conditions found in this experiment stresses the importance of measuring all contributing factors that contribute to calcification trends in OA coral experiments.

In Chapter II, the increased calcification in diurnally-oscillating pCO2 was hypothesized to be the result of DIC storage in host tissues. Carbonic anhydrase in corals

! acts as a carbon concentration mechanism converting HCO! to CO2 so that it may be used for photosynthesis, although it also is hypothesized to play a role calcification (Weis et al.

1989, Moya et al. 2008, Bertucci et al. 2011). To elucidate the physiological mechanism driving the increased calcification, in Chapter IV S. caliendrum juveniles were exposed to similar treatment conditions used in Chapter II and the carbonic anhydrase activity was measured. Carbonic anhydrase activity during the day was reduced in both high and diurnally oscillating pCO2 relative to ambient pCO2. The decrease in CA activity suggests similar DIC concentrations in coral tissues even though corals were exposed to different DIC treatments at the time of sampling indicating DIC storage in coral tissues.

This result adds merit to the hypothesis proposed in Chapter II accounting for the increased calcification in natural-phase diurnally-oscillating pCO2 though tissue DIC concentrations must be explicitly measured to confirm this hypothesis.

Despite the findings of increased calcification and survival of recruits in natural- phase diurnally oscillating pCO2, light-dependent effects of calcification and survival in

74 OA conditions and decreased CA activity in diurnally oscillating and high pCO2 presented in this thesis, it remains unclear to what extent the trends observed here in coral recruits can be extrapolated to adults. Early life history stages of coral are biologically quite different from adults (Hamdoun and Epel 2007) however for S. caliendrum and P. damicornis recruits, OA does not appear to negatively affect coral recruit calcification or survival and may even promote them certain instances given ecologically relevant treatment conditions.

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