Differential effects of copper on three species of scleractinian corals and their algal symbionts (Symbiodinium spp.)

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Contents lists available at ScienceDirect

Aquatic Toxicology

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ifferential effects of copper on three species of scleractinian corals and theirlgal symbionts (Symbiodinium spp.)

.K. Bielmyera,∗, M. Grosellb, R. Bhagooli c, A.C. Bakerb, C. Langdonb, P. Gilletteb, T.R. Capob

Department of Biology, Valdosta State University, 1500 N. Patterson St, Valdosta, GA 31698, United StatesDivision of Marine Biology and Fisheries, Rosenstiel School of Marine and Atmospheric Science, University of Miami, Miami, FL, United StatesDepartment of Biosciences, Faculty of Science, University of Mauritius, Reduit, Mauritius

r t i c l e i n f o

rticle history:eceived 19 August 2009eceived in revised form7 December 2009ccepted 22 December 2009

eywords:etal

oxicityoral

a b s t r a c t

Land-based sources of pollution have been identified as significant stressors linked to the widespreaddeclines of coral cover in coastal reef ecosystems over the last 30 years. Metal contaminants, althoughnoted as a concern, have not been closely monitored in these sensitive ecosystems, nor have their potentialimpacts on coral-algal symbioses been characterized. In this study, three species of laboratory-reared scle-ractinian corals, Acropora cervicornis, Pocillopora damicornis, and Montastraea faveolata each containingdifferent algal symbionts (Symbiodinium A3, C1 and D1a, respectively) were exposed to copper (rang-ing from 2 to 20 �g/L) for 5 weeks. At the end of the exposure period, copper had accumulated in theendosymbiotic dinoflagellate (“zooxanthellae”) and animal tissue of A. cervicornis and the animal tissueof M. faveolata; however, no copper accumulation was detected in the zooxanthellae or animal tissue ofP. damicornis. The three coral species exhibited significantly different sensitivities to copper, with effectsoccurring in A. cervicornis and P. damicornis at copper concentrations as low as 4 �g/L. Copper exposureaffected zooxanthellae photosynthesis in A. cervicornis and P. damicornis, and carbonic anhydrase was sig-

nificantly decreased in A. cervicornis and M. faveolata. Likewise, significant decreases in skeletal growthwere observed in A. cervicornis and P. damicornis after copper exposure. Based on preliminary results, nochanges in Symbiodinium communities were apparent in response to increasing copper concentration.These results indicate that the relationships between physiological/toxicological endpoints and copperaccumulation between coral species differ, suggesting different mechanisms of toxicity and/or suscepti-bility. This may be driven, in part, by differences in the algal symbiont communities of the coral species in question.

. Introduction

Coral reefs are among the most biologically productive andiverse ecosystems in the world and are among the world’sost fragile and endangered ecosystems. Built by mutualistic

artnerships between scleractinian (stony) corals and endosym-iotic dinoflagellates in the genus Symbiodinium (often referredo as “zooxanthellae”), reef structures represent essential habi-at for a wide variety of marine animals. Some feed directly onorals while others feed on organisms that establish themselvesmong or on the coral (Edmunds and Carpenter, 2001). These

Please cite this article in press as: Bielmyer, G.K., et al., Differential effecsymbionts (Symbiodinium spp.). Aquat. Toxicol. (2010), doi:10.1016/j.aqua

ragile ecosystems face severe threats from climate change andisease, overfishing, habitat destruction and poor water qualityHughes et al., 2003). In the Caribbean, coral reefs have experi-nced an estimated 80% reduction in coral cover in the last three

∗ Corresponding author. Tel.: +1 229 333 5766.E-mail address: [email protected] (G.K. Bielmyer).

166-445X/$ – see front matter. Published by Elsevier B.V.oi:10.1016/j.aquatox.2009.12.021

Published by Elsevier B.V.

decades (Gardner et al., 2003), and similar trends are present inthe Indo-Pacific (Bruno and Selig, 2007). Most corals thrive in anarrow range of water quality and temperature regimes and thusserve as bioindicators of our oceans’ health. Reefs in near shoreenvironments close to heavily populated areas with substantialanthropogenic inputs are particularly threatened from the com-bined effects of multiple interacting stressors. However, data on thepotential effects of waterborne pollutants, such as heavy metals,is lacking, despite the common occurrence of metal contamina-tion in marine environments (Esquivel, 1983; Howard and Brown,1984; Brown and Howard, 1985; Peters et al., 1997; Esslemont,2000; Reichelt-Brushett and Harrison, 2000, 2004). Countries inthe Caribbean, like other developing countries, have establishedmany new industries over the last several decades such as food pro-

ts of copper on three species of scleractinian corals and their algaltox.2009.12.021

cessing; metal mining and smelting; metal plating and finishing;battery, cement, fertilizer, cosmetics and pharmaceutical manufac-turing; and weaving and dying, all of which potentially contributeto heavy metal contamination (Ross and DeLorenzo, 1997; Guzmanand Jimenez, 1992). In addition, marine disposal of municipal solid

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aste (Knap et al., 1991) and sacrificial anodes on boats (McConchiend Harriott, 1992) also increases metal concentrations in marinenvironments. Due to recent restrictions on the use of organotin-ased antifoulants, copper oxide paints, often complemented withrganic “antifouling boosting” compounds, are more commonlysed and thus have been found in surface waters off the coast ofouth Florida (Evans et al., 2000; Voulvoulis et al., 2000; Gardinalit al., 2004).

The continued degradation of the Florida Keys coral reef tract iselieved to be, due, at least in part, to the contamination of coastalaters. Limited studies have reported the presence of heavy met-

ls in coral tissue, water, and sediment samples taken from theoast of Florida and Hawaii (Glynn et al., 1984, 1989; Hanna anduir, 1990; Gardinali et al., 2002; Owen et al., 2002) as well as Aus-

ralia (Denton and Burdon-Jones, 1986a,b,c; Esslemont, 1999, 2000;aynes and Johnson, 2000). Several studies have demonstrated

hat metals substantially accumulate in the zooxanthellae and ani-al tissue fractions of branching corals (Reichelt-Brushett andcOrist, 2003; Anu et al., 2007; Mitchelmore et al., 2007). Further,

eichelt-Brushett and McOrist (2003) demonstrated that copperccumulation in the zooxanthellae and animal tissue was prefere-ential to the skeleton. No significant differences were reportedetween skeletal copper accumulation in pristine and metal-ontaminated coral reefs (Bastidas and Garcia, 1999). Howard andrown (1984) suggested that metals may accumulate in the skeletal

ractions after long-term exposure; however, they are not likely toffect living tissue. Additional laboratory-based studies are neededo better understand the extent of metal accumulation and the

echanisms of metal toxicity by manipulating external factorsnd minimizing variability. Moreover, laboratory research in thisrea may also suggest potential biomarkers for field monitoringituations. Replicate coral fragments taken from individual coralolonies are ideally suited for use in toxicological laboratory studiesecause they are genetically identical, displaying minimal variancend typically have ≥90% survival using approved protocols (Shafirt al., 2002, 2003). Since clonal replicates have by definition theame genetic composition, if the clone is unusual, all results will benusual and generalizations are limited.

When coral-algal symbioses are stressed by high temperatures,igh irradiance (both in the visual and ultraviolet regions of theolar spectrum) or other environmental changes, such as man-ade pollutants, they experience coral “bleaching” (loss of algal

ymbionts, or a reduction in their per-cell pigment concentra-ions). This results in the visible paling of coral colonies, which turnhite, as the coral’s calcium carbonate skeleton becomes visible

hrough the translucent tissue (Brown and Howard, 1985; Downst al., 2000). The loss of zooxanthellae in response to metal expo-ure has also been reported in several studies (Harland and Brown,989; Peters et al., 1997; Reichelt-Brushett and McOrist, 2003) andhis results in generally reduced metal concentrations in the coral.epending on the intensity and duration of the stress, corals may

ecover from bleaching, but if the algal symbiont communities areot restored relatively quickly (typically 2–4 weeks), corals mayie. Mass mortality from coral bleaching has been severe in somelaces, such as the 1982–1983 El Nino event in the eastern Pacific,he 1997–1998 El Nino event in the western Indian Ocean, the002 event on the Great Barrier Reef, and the 2005 event in theE Caribbean (including the Florida Keys), although recovery in

ome locations has been surprisingly rapid (Baker et al., 2008). Theffects of heavy metal pollutants on the algal symbionts of coralsan potentially be measured before they leave or are expelled from

Please cite this article in press as: Bielmyer, G.K., et al., Differential effecsymbionts (Symbiodinium spp.). Aquat. Toxicol. (2010), doi:10.1016/j.aqua

he coral. Chlorophyll fluorescence can be used to measure a vari-ty of photosynthetic parameters, and instruments such as a Pulsemplitude Modulated (PAM) fluorometer can be used to distin-uish between photon energy captured by a chlorophyll-a pigmentolecule used to drive photosynthesis versus that emitted as flu-

PRESScology xxx (2010) xxx–xxx

orescence or converted to heat in many species of zooxanthellae(Warner et al., 1996; Hoegh-Guldberg and Jones, 1999; Ralph et al.,1999).

Another potentially important biomarker of coral stress is theactivity of the enzyme carbonic anhydrase (CA). In fish, bivalvemollusks, and more recently in anemones and corals, researchershave demonstrated that metals directly inhibit CA (Bundy, 1977;Henry, 1996; Morgan et al., 2004; Gilbert and Guzman, 2001). CAcatalyzes the interconversion between CO2 and HCO3

− and is there-fore important in respiration and metabolism (Bundy, 1977; Henry,1996) and potentially for generating CO3

2− which is used for calci-fication. Coral growth has been shown to be an effective indicator ofthe overall health of a coral reef ecosystem and reduced growth canreflect impaired photosynthetic output of the zooxanthellae and/orchanges in enzyme activity (Moya et al., 2008).

The objectives of the present study were to assess copperaccumulation and the responses of three species of coral: the west-ern Atlantic species Acropora cervicornis (a threatened species ofstaghorn coral) and Montastraea faveolata (Star coral) and the Indo-Pacific species Pocillopora damicornis (cauliflower coral), followingchronic waterborne copper exposure. A. cervicornis, a fast grow-ing species, and M. annularis, a moderately fast growing coral, areboth important reef builders found in the Caribbean (Gladfelter etal., 1978). P. damicornis has a much larger geographic range. Theoverall goals of this study were to determine sensitivity and phys-iological response of coral to copper exposure and to elucidate themechanism(s) of copper toxicity in these species.

2. Methods

2.1. Test organisms

A. cervicornis was collected from Biscayne National Park inFebruary, 2005; P. damicornis was purchased from The Coral Nurs-ery in October, 2003; and M. faveolata was collected from Key WestHarbor in December, 2003. Since then, all coral species have beenmaintained and cultured at the at the Coral Resource Facility at theUniversity of Miami’s Rosenstiel School of Marine and AtmosphericSciences (RSMAS) in an isolated re-circulating seawater system. Foreach species, coral fragments were cut from a single colony andthen mounted on tiles to facilitate handling and the measurementsof non-terminal endpoints (described below). Coral fragments wereallowed to recover from handling and observed for normal growthand performance such that only healthy coral fragments were usedfor experimentation. Coral fragments were then acclimated to thetest conditions for 3 weeks prior to the start of the experiment. Eachtile contained three fragments of one coral species.

2.2. Test conditions

A. cervicornis, M. faveolata, and P. damicornis were exposed tocopper, as CuCl2 for 5 weeks in a flow-through system. Concen-trated copper solutions and gravity fed natural filtered seawaterwere continually mixed and then distributed into testing cham-bers (5 gallon aquaria) at a flow rate of 13.4 ± 5.05 mL/min.Measured copper concentrations (average ± standard error) wereas follows: 2.38 ± 0.23 (control; n = 16), 4.04 ± 0.24 �g/L (n = 20),10.63 ± 0.46 �g/L (n = 17), and 20.31 ± 1.55 �g/L (n = 18). Therewere 4 replicate tanks per treatment, each with two tiles (6 coralfragments) of each species per tank (total n = 144 samples per

ts of copper on three species of scleractinian corals and their algaltox.2009.12.021

species). Water samples were collected twice weekly and acidifiedwith concentrated trace metal grade nitric acid (1%; Fisher Scien-tific, Pittsburgh, PA) for later copper analysis. Temperature, anddissolved oxygen (DO) were measured with a YSI (model 55), salin-ity with a refractometer (Reichert TC), and light was measured with

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light meter (Biospherical Instruments Inc., QSL-2101) twice pereek throughout the experiment. Water quality conditions in the

xperimental chambers were as follows (average ± standard devi-tion): 26.5 ± 0.93 ◦C, 6.62 ± 0.34 mg/L DO, 36.1 ± 0.73 g/L salinity,nd 172.4 ± 15.72 �E m−2 s−1 light. At the end of the exposure,hotosynthetic parameters of algal symbionts and growth ratesf the coral hosts were measured in all coral fragments. Sur-ival was verified by polyp extension and if necessary microscopicxamination with a dissecting microscope. Additionally, tissuesere preserved (−80◦ C) for later analysis of Cu distribution and

ccumulation, CA activity and Symbiodinium genetic identifica-ion.

.3. Coral fractionation

A preliminary 2-week experiment demonstrated that copperccumulation occurred predominantly in the coral soft tissue andlgal symbiont tissue fractions in all three coral species. As such,he coral’s CaCO3 skeletal fractions were not analyzed for copperccumulation in this experiment. Coral fragments were weighednd then separated into coral soft tissue, algal symbiont, and coralkeletal fractions. The fragments were first rinsed and then blastedith filtered (0.22 �m) seawater using an airbrush to separate the

oral soft tissue and algal symbionts from the exoskeleton. The vol-me of seawater used was normalized in all samples. This “blastate”f combined soft tissue fractions was then mixed and homogenizedsing a Dounce-type glass homogenizer and the algal symbiontsere then isolated from the coral soft tissue via centrifugation formin at 50,000 × g.

.4. Copper analysis

Three coral fragments from each of the four replicate tanks (12ragments) per treatment were used for copper analysis. Soft tis-ue fractions (coral and zooxanthellae) were individually dried inre-weighed 15 mL polypropylene centrifuge tubes at 80 ◦F for 24 ho determine dry weight. Each fraction was then digested with 1Nitric acid. Digested soft tissue fractions were then measured foropper using graphite furnace atomic absorption spectrophotom-try (GFAAS; Varian 220FS Mulgrave, Victoria, Australia). Wateramples were measured for low copper concentrations (<15 �g/L)y first co-precipitating with iron (Weisel et al., 1984), followed byFAAS (detection limit = 0.5 �g/L copper). For higher copper con-entrations, solvent extraction using chelating agents was usedKinrade and Van Loon, 1974) followed by AAS with flame detec-ion.

.5. Carbonic anhydrase

Three coral fragments from each of the four replicate tanks12 fragments) per treatment were used for CA measurement.ntact coral fragments were analyzed for total CA activity usinghe method of Henry (1991). Tissue samples for the assay wererepared according to the methods of Gilbert and Guzman (2001).

n brief, 1–2 mL of reaction buffer (10 mM TRIS base, 225 mMannitol, 75 mM sucrose) was added to each coral fragment. The

ragments were then sonicated (Kontes rod sonicator, Pittsburg,A, USA) on ice for approximately 1 min. The activity of CA washen determined by the Delta pH Assay, which uses a sensitive

Please cite this article in press as: Bielmyer, G.K., et al., Differential effecsymbionts (Symbiodinium spp.). Aquat. Toxicol. (2010), doi:10.1016/j.aqua

ecording pH meter to directly measure the rate of change in pHn a buffer containing CA upon the addition of substrate (e.g. CO2or hydration; Henry, 1991). Total protein of the sonicated sam-les was determined by the Bradford Assay, using bovine serumlbumin as standards. CA activity is expressed as �mol/min/mgrotein.

PRESScology xxx (2010) xxx–xxx 3

2.6. Algal photosynthesis measurements

PAM fluorometry was used on all of the coral fragments todetermine the following parameters (Genty et al., 1989; Ralph etal., 1999): effective quantum yield of photosystem II (�PSII), andusing the rapid light curves (RLCs) the relative electron transportrate (rETR), non-photochemical quenching (NPQ) of photosystemII, the efficiency of the system (˛), and the minimum amount of lightnecessary to saturate the system (Ek) can be derived. The �PSII pro-vides an indication of the amount of energy used in photochemistry(photosynthesis). The rETR is closely related to the photosyntheticactivity and is an approximation of the rate of electrons pumpedthrough the photosynthetic chain. NPQ refers to the amount of heatdissipated at PSII. The fluorometer uses a range of flashing pulsedlights to directly measure photosynthesis and rETR, ˛ and Ek arederived parameters from the RLCs. If Ek is low and ˛ is low thenless light is needed to drive photosynthesis and the efficiency isreduced, indicating a less capable system and possible release ofthe symbiont. However, a situation where Ek is high and ˛ is lowmay be indicative of toxicity to the symbiont.

2.7. Identification of algal symbionts (Symbiodinium spp.)

After 5 weeks exposure to copper, DNA was extracted andpurified from the algal tissue fraction of A. cervicornis (n = 4), M.faveolata (n = 3) and P. damicornis (n = 4) using standard organic pro-tocols (Baker et al., 1997). One sample was analyzed from each ofthe 4 copper treatments, except for M. faveolata which was notanalyzed from the 10 �g/L copper treatment. The Internal Tran-scribed Spacer-2 (ITS-2) region of Symbiodinium ribosomal DNA(rDNA) was then amplified using the primers “ITSintfor2” and“ITS2revclamp” (LaJeunesse, 2001) using the following conditions:an initial denaturing step of 94 ◦C for 3 min followed by 35 cycles of1 min at 94 ◦C, 1 min at 58 ◦C and 1 min at 74 ◦C, followed by a singlecycle of 7 min at 74 ◦C. Amplified DNA was then analyzed by dena-turing gradient gel electrophoresis (DGGE; CBScientific, Del Mar,CA, USA) using a denaturant gradient of 35–75%. Prominent bandscharacterizing different profiles (as described by LaJeunesse, 2002)were then excised, bead beaten and re-amplified without the GCclamp using the same profile described above. Sequencing was per-formed using an Applied Biosystems 3730xl DNA Analyzer (FosterCity, CA, USA). Sequences were identified using BLAST searches ofGenBank, and exact matches were reported using the nomenclatureestablished by LaJeunesse (2001).

2.8. Coral growth

Linear extension rate of each coral species was measured usingan optical micrometer (Keyence LS-7000) consisting of a highintensity green LED light source and a linear CCD receiver. Theinstrument quantifies the dimensions of the shadow cast by thecoral fragment and provides a digital display to precisely mea-sure linear extension. Using this method, it is possible to determinechanges in growth rate in a matter of days. At the start and end ofthe exposure period, tiles containing coral fragments were removedfrom the test chambers and measured for growth.

2.9. Data analysis

All data are presented as the mean ± standard error of the

ts of copper on three species of scleractinian corals and their algaltox.2009.12.021

mean. Data were analyzed to determine significant differencesbetween treatments using a one way repeated measures ANOVAfollowed by a Student–Newman–Keuls multiple comparison pro-cedure (˛ = 0.05) with the commercial statistical packages SigmaPlot® 8.0 and Sigma Stat® 3.0.

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Fig. 1. Copper accumulation in (A) zooxanthellae and (B) coral soft tissue of Acrop-ora cervicornis (circles), Montastraea faveolata (triangles), and Pocillopora damicornis(iF

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Fig. 2. Denaturing gradient gel electrophoresis of Symbiodinium ITS-2 rDNA from

squares), after waterborne copper exposure for 5 weeks. Asterisks indicate a signif-cant difference from control values (˛ = 0.05). Data are presented as mean ± SEM.or each treatment n = 4 (12 fragments).

. Results

.1. Copper accumulation

Copper accumulated concentration-dependently in A. cervicor-is exposed to copper for 5 weeks (Fig. 1A and B). Significant

ncreases in copper were measured in the zooxanthellae of A. cervi-ornis after exposure to 10 and 20 �g/L Cu (Fig. 1A); whereas copperccumulation in the animal soft tissue of A. cervicornis was signifi-antly elevated at all copper exposure concentrations, as comparedo control values (Fig. 1B). Increased copper accumulation waseither detected in the zooxanthellae nor animal soft tissue of. damicornis after exposure to copper concentrations as high as0 �g/L for 5 weeks (Fig. 1A and B). Increased copper accumula-ion was not detected in the zooxanthellae of M. faveolata (Fig. 1A);owever, a concentration-dependent increase was observed in thenimal soft tissue after exposure to the highest two copper treat-

Please cite this article in press as: Bielmyer, G.K., et al., Differential effecsymbionts (Symbiodinium spp.). Aquat. Toxicol. (2010), doi:10.1016/j.aqua

ents, as compared to controls (Fig. 1B). Copper concentrations inontrol algal symbiont and animal soft tissue of A. cervicornis and. damicornis were similar; however, copper concentrations were–10 times higher in the algae and 2–3 times higher in the animaloft tissue of control M. faveolata (Fig. 1A and B).

Acropora cervicornis (lanes 1–4), Montastraea faveolata (lanes 5–7), and Pocilloporadamicornis (lanes 8–11) exposed to various copper concentrations.

3.2. Symbiodinium identification

DGGE analysis indicated the presence of different Symbiodiniumin each coral species (Fig. 2). A. cervicornis contained SymbiodiniumA3 (match to Genbank EU074857), P. damicornis contained Sym-biodinium C1 (Genbank EU074884) and M. faveolata containedSymbiodinium D1a (Genbank EU074901, EU074903 and EU074906).No changes in Symbiodinium communities were detected over thecourse of the experiment.

3.3. Algal photosynthesis

In agreement with increasing copper accumulation in the algalsymbionts of A. cervicornis, the �PSII significantly decreased andNPQ significantly increased in a concentration-dependent man-ner (Fig. 3A and B). Likewise, rETR, Ek, and ˛ decreased withincreasing copper exposure (Fig. 3C–E). Additionally, visual obser-vations of bleaching were noted in the 10 and 20 �g/L coppertreatments. The algal symbionts of P. damicornis exhibited a signif-icantly decreased �PSII in all copper treatments; however, ˛ wassignificantly lower and Ek significantly higher only at the highestcopper treatment (Fig. 4A, D and E). No significant differences inrETR or NPQ were observed (Fig. 4B and C). Visual bleaching wasobserved in the highest two treatments of P. damicornis. No signif-icant decreases in photosynthetic parameters were detected in thezooxanthellae of M. faveolata (data not shown) in agreement withthe lack of copper accumulation. Likewise, no visual bleaching wasobserved.

ts of copper on three species of scleractinian corals and their algaltox.2009.12.021

3.4. CA activity

CA activity decreased with increasing exposure concentrationsof copper. Significant effects were observed in A. cervicornis exposedto 10 and 20 �g/L copper and in M. faveolata exposed to 20 �g/L

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ig. 3. (A) Effective quantum yield of photosystem II (�PSII); (B) the relative elecfficiency of the system (˛), and (E) the minimum amount of light necessary to satuifference from control values (˛ = 0.05). Data are presented as mean ± SEM. For ea

opper (Fig. 5). These findings are consistent with the increasingopper accumulation observed in the animal tissue of these corals.lthough CA activity appeared lower in P. damicornis exposed toopper, no significant effects of copper were detected, as comparedo controls (Fig. 5).

.5. Growth

Concurrent with physiological impairment, a decrease inrowth was observed in A. cervicornis exposed to copper, withignificant effects observed after exposure to 20 �g/L (Fig. 6A). P.amicornis exposed to copper concentrations as low as 4 �g/L Cu

Please cite this article in press as: Bielmyer, G.K., et al., Differential effecsymbionts (Symbiodinium spp.). Aquat. Toxicol. (2010), doi:10.1016/j.aqua

xhibited a significant decrease in growth (Fig. 6B). The growthate in P. damicornis was about 5 times greater than in A. cervicornisFig. 6A and B). Growth effects due to copper exposure in M. fave-lata were not detected possibly because control growth was notignificant over the 5-week exposure duration.

ansport rate; (C) non-photochemical quenching (NPQ) of photosystem II; (D) thee system (Ek) in Acropora cervicornis zooxanthellae. Asterisks indicate a significanttment n = 4 (24 coral fragments).

4. Discussion

A. cervicornis exposed to copper concentrations as low as 4 �g/Lfor 5 weeks in the laboratory accumulated significant tissue copper.As the copper concentration increased, photosynthetic impairmentwas observed, demonstrating that more energy was converted intoheat and less energy was being used for photosynthesis in A. cervi-cornis zooxanthellae. These data suggest that the system was lesscapable, because less light was needed to drive photosynthesisand the efficiency of the zooxanthellae was reduced; potentiallydue to changes in the photosynthetic activity and/or a loss of thesymbiont. Likewise, bleaching in A. cervicornis was also observed,

ts of copper on three species of scleractinian corals and their algaltox.2009.12.021

similar to that reported by Jones (1997) in the coral Acropora for-mosa after increasing copper exposure. Exposure of A. formosa to20 and 40 �g/L copper resulted in a significant decrease in zoox-anthellae density after 48 h and 24 h, respectively, and 80% of thecorals died after 48-h exposure to 40 �g/L copper (Jones, 1997).

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ig. 4. (A) Effective quantum yield of photosystem II (�PSII); (B) the relative elecfficiency of the system (˛), and (E) the minimum amount of light necessary to saturifference from control values (˛ = 0.05). Data are presented as mean ± SEM. For ea

eters et al. (1997) suggested that the expulsion of zooxanthellaeay be a mechanism of metal detoxification, since zooxanthellae

ave been found to accumulate heavy metals to a larger extentnd thus be more tolerant than their symbiotic hosts. However, weound significant copper accumulation in coral tissue in this speciesf Acropora, indicating this mechanism may not apply equally to alloral and/or algal symbiont taxa.

Increased copper exposure and accumulation in A. cervicornislso resulted in a reduced CA activity, which may have affected theymbiont by reducing the CO2 available for photosynthesis. Weis et

Please cite this article in press as: Bielmyer, G.K., et al., Differential effecsymbionts (Symbiodinium spp.). Aquat. Toxicol. (2010), doi:10.1016/j.aqua

l. (1989) suggested that CA may have a role in the photosyntheticetabolism of algal/cnidarian symbiosis by enabling zooxanthel-

ae to maintain high photosynthesis rates by supplying the CO2rom bicarbonate (Weis et al., 1989). Relatively high CA activityas reported in both the zooxanthellae and coral fractions of Acro-

ransport rate; (C) non-photochemical quenching (NPQ) of photosystem II; (D) thesystem (Ek) in Pocillopora damicornis zooxanthellae. Asterisks indicate a significanttment n = 4 (24 coral fragments).

pora hebes (Isa and Yamazato, 1984) and P. damicornis (Grahamand Smillie, 1976). Weis (1991) suggested that CA activity mightbe increased by the presence of symbiotic algae in the anemoneAiptasia pulchella, and that the algae might aid in the detoxificationprocess upon exposure to metals.

Not only does CA control respiration and the interchangebetween HCO3

− and CO2, but the enzyme also facilitates the for-mation of CO3

2−, via conversion of CO2 to HCO3− which acts as a

substrate for CaCO3 formation. Thus, suppression of CA activity, aswas observed in A. cervicornis, could have decreased the substrate

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available for use in calcification and skeletal growth of the animalhost. The decreased growth in A. cervicornis could have also been aresult of increased energy expenditure on copper detoxification andexpulsion of zooxanthellae, as well as reduced nutrient availabilitybecause of toxicity to, or absence of, the symbiont. A. cervicornis

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Fig. 5. Carbonic anhydrase (CA) activity in (A) Acropora cervicornis (circles), (B) Mon-tastraea faveolata (triangles), and (C) Pocillopora damicornis (open squares), afterwaterborne copper exposure for 5 weeks. Asterisks indicate a significant differencefrom control values (˛ = 0.05). Data are presented as mean ± SEM. For each treatmentn = 4 (12 coral fragments).

Fig. 6. Coral growth rate (�m/day) in (A) Acropora cervicornis and (B) Pocilloporadamicornis after waterborne copper exposure for 5 weeks. Asterisks indicate astatistically significant difference from controls (˛ = 0.05). Data are presented asmean ± SEM. For each treatment n = 4 (24 coral fragments).

PRESScology xxx (2010) xxx–xxx 7

has historically been one of the most important Caribbean coralsin terms of its contribution to fish habitat and reef growth, withbranches increasing in length by 10–20 cm per year. A strong cor-relation was observed between CA activity and growth, as well asrelative electron transport rate and growth in A. cervicornis, and itseems likely that impairment of both physiological parameters ledto the observed decrease in growth.

When P. damicornis was exposed to copper under the sameconditions as A. cervicornis, tissue copper accumulation was notobserved, however, effects on both coral and zooxanthellae weredetected. Mitchelmore et al. (2007) demonstrated copper accumu-lation in both the algal and animal fractions of P. damicornis aftera 14-d exposure to copper concentrations as low as 5 �g/L in thelaboratory. We conducted a preliminary 14-d experiment and alsomeasured significant tissue copper accumulation in P. damicornisexposed to 20 �g/L copper (data not shown). It is possible thatafter 5 weeks of copper exposure in the present study, the coralhad mobilized homeostatic control systems to excrete copper, pre-venting detection of elevated levels, but the toxic effects (reductionof algal quantum yields) were still present. It is also possible thatcopper had accumulated in the exoskeleton of P. damicornis, whichwas not analyzed in this experiment. Esslemont (2000) reportedelevated copper levels in the skeletal fractions of P. damicorniscollected from a polluted site, as compared to a pristine site. Inthis study, zooxanthellae were present in P. damicornis; however,reduced photosynthetic efficiency was observed after exposure to20 �g/L copper. Several studies have reported decreased zooxan-thellae primary production rate (Alutoin et al., 2001) and growthrate (Goh and Chou, 1997), as a consequence of copper exposure.The mechanisms of toxicity and detoxification are still unclearin P. damicornis; however, zooxanthellae expulsion may also beinvolved. P. damicornis and Montipora verrucosa exposed to 10 �g/Lcopper for 48 h exhibited symptoms of severe stress, such as with-drawn polyps and bleaching, and by the sixth day of exposure allof the corals had died (Evans, 1977).

CA activity did not appear to be a sensitive endpoint in P.damicornis; however, another possibility is that CA activity wasrecovering from initial impairment, similar to the tissue copperin copper-exposed P. damicornis. Further investigation of coppertoxicity in P. damicornis over time is needed to better elucidatethe mechanism. Despite the unclear physiological effects after 5weeks of copper exposure, growth of P. damicornis was significantlydecreased in all copper treatments tested demonstrating that thisspecies is relatively sensitive to copper nonetheless.

Unlike both A. cervicornis and P. damicornis, M. faveolata exhib-ited copper accumulation in the animal tissue only. Concurrentwith the lack of significant copper accumulation in the zooxanthel-lae, no negative effects on zooxanthellae electron transport ratewere observed. Jones (2004) demonstrated that 60 �g/L copperinduced rapid bleaching in coral Seriatopora hystrix, without affect-ing the quantum yield of the algae. Tests with isolated S. hystrixindicated that copper concentrations as high as 300 �g/L copperwere required to affect photosynthesis (Jones, 2004). In this study,the elevated background (control) tissue copper concentrations inSymbiodinium D1a in M. faveolata may indicate that these sym-bionts have a different copper requirement and potentially moremetal binding proteins than either the Symbiodinium A3 in A. cervi-cornis or the Symbiodinium C1 in P. damicornis. Symbiodinium D1amay therefore have a more efficient copper homeostasis mecha-nism. Despite the slightly elevated background tissue copper in M.faveolata, as compared to A. cervicornis and P. damicornis, M. faveo-

ts of copper on three species of scleractinian corals and their algaltox.2009.12.021

lata still exhibited significant animal tissue copper accumulationwith increasing copper exposure, similar to the other two coralspecies tested. This increasing tissue copper burden paralleled thedecrease in CA activity in M. faveolata. Gilbert and Guzman (2001)reported a significant decrease in CA activity in the anemones

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ondylactis gigantea and Stichodactyla helianthus exposed to ele-ated copper, nickel, lead, and vanadium in the laboratory, as wells in the coral Montastraea cavernosa obtained from metal-pollutedites compared to those obtained from pristine sites. Additionally,A activity was affected before any observation of algae expul-ion, as was observed in this study. Hence, CA activity may be anmportant biomonitoring tool for assessing coral health prior torreversible damage.

M. faveolata and A. cervicornis occupy many of the same habitats,et these coral species exhibited significantly different sensitivitieso copper and it is likely that they also have different sensitivitieso other environmental parameters. However, these coral specieslso typically host different algal symbionts, and the colonies usedn this experiment were no exception in this regard. Symbiodinium1a is typically viewed as relatively heat tolerant, and therefore

esistant to bleaching. The results reported here indicate it may beroadly tolerant of a variety of environmental stressors, includingetals. In 2006, A. cervicornis corals, which exhibit the fastest linear

rowth rates of all western Atlantic corals, were listed as “threat-ned” by NOAA-Fisheries under the US Endangered Species Act of973, as amended, due to the recent drastic declines in these coralopulations.

The diversity of host–symbiont combinations in corals is inher-ntly complex, and reflects significant potential flexibility in manyperhaps all) scleractinian coral species (Baker, 2003). Conse-uently, the responses of these diverse combinations of hosts andymbionts (different “holobionts”) to metal exposure are also likelyo vary, as illustrated in this study. Each coral species containedifferent Symbiodinium and each exhibited different sensitivi-ies to copper. However, despite the fact that clear differencesere observed in the tolerances of different types of Symbio-

inium to copper, there was no apparent change in Symbiodiniumommunities in this experiment in response to different copperoncentrations. This might be more conclusively explored usingner resolution molecular markers, such as quantitative PCR (qPCR)o detect changes in the relative abundances of “minor” symbiontsver time and with an increase in sample size.

Results of this study demonstrated copper toxicity to sclerac-inian corals at concentrations near the acute U.S. Ambient Wateruality Criterion (WQC) of 3 �g/L (U.S. EPA, 2003). The criteria used

o protect tropical reef ecosystems were derived mainly using tox-city data from temperate organisms, which although relativelybundant, may not be appropriate for other organisms (Bielmyernd Grosell, in press). In recent years, several different marinerganisms have been reported to be sensitive to copper exposuret levels near or below current WQC (Beiras and Albentosa, 2004;ielmyer et al., 2005; Bielmyer et al., 2006; Main et al., 2010). Also,

t is important to note that many reefs around the world are locatedn areas with far less stringent water quality standards than in the.S., and therefore may be at an even higher risk. Peters et al. (1997)

eported copper concentrations in polluted coral reef ecosystemsanging from 10 to 1800 �g/L Cu in the water column and 7.5 to8 �g Cu/g dry weight in coral soft tissue. The highest copper con-entration used in this study (20 �g/L) caused toxic effects in allf the corals tested and is well within the range of concentrationseasured in metal-polluted environments.

. Conclusions

Increased copper exposure in marine environments may impair

Please cite this article in press as: Bielmyer, G.K., et al., Differential effecsymbionts (Symbiodinium spp.). Aquat. Toxicol. (2010), doi:10.1016/j.aqua

oral physiology, possibly through CA activity (Gilbert and Guzman,001), and cause declines in photosynthetic efficiency of algal sym-ionts. The combined effects are likely to impact growth rates offfected corals. In an era of climate change and ocean acidifica-ion, where factors impacting growth and resilience factors are

PRESScology xxx (2010) xxx–xxx

becoming important, understanding the biological effects of metalexposure to these keystone tropical organisms may be critical.

Differences in copper sensitivity may vary with organism physi-ology (Grosell et al., 2007); therefore it is imperative to understandmechanisms of copper toxicity in ecologically important species,such as corals. The mechanisms of copper toxicity appear to be dif-ferent among the three species tested, nevertheless, biomarkerssuch as CA activity and electron transport rate, were sufficientlysensitive to detect deleterious effects, and may be used in con-junction with other endpoints to assess the health of coral atmetal-impacted sites.

Acknowledgements

The authors would like to thank Paul Jones, Rebecca Albright,Alyssa Anderson, and Alain Pierre-Louis from the University ofMiami for help in data collection and Margaret Miller and CherylWoodley from NOAA for helpful scientific dialogue. AB and CL werefunded by NSF OCE-0547169.

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