Evaluation of thermal acclimation capacity in corals with different thermal histories based on catalase concentrations and antioxidant potentials

Comparative Biochemistry and Physiology, Part A 144 (2006) 155 – 162 www.elsevier.com/locate/cbpa

Evaluation of thermal acclimation capacity in corals with different thermal histories based on catalase concentrations and antioxidant potentials Sean P. Griffin a,⁎, Ranjeet Bhagooli b,c , Ernesto Weil a a

c

University of Puerto Rico, Dept. of Marine Science, P.O. Box 908, Lajas, PR 00667, Puerto Rico b Biodiversity and Environment Institute, Reduit, Mauritius University of the Ryukyus, Department of Chemistry, Biology and Marine Sciences, Senbaru 1, Nishihara, Okinawa 903-0213, Japan Received 1 October 2005; received in revised form 8 February 2006; accepted 15 February 2006 Available online 6 March 2006

Abstract Colonies of Pocillopora damicornis from Kaneohe Bay and colonies of Pocillopora meandrina from a thermal outfall site and a control site at Kahe were exposed to three different temperatures (29, 32 and 33 °C) in outdoor aquaria on running water tables for five days. Samples (n = 3) were taken from each treatment at 0800, 1200 and 1600 h. ELISAs using catalase antibodies and ferric reducing/antioxidant potential (FRAP) assays were run on the samples to determine how antioxidant levels changed throughout the experiment. Light levels during the experiment were highest in the morning (≈ 1000–1500 μmol quanta m− 2 s− 1) and decreased to 25–60 μmol quanta m− 2 s− 1 by 1100 h and remained low until sunset. Antioxidant concentrations were highest in the morning for P. damicornis from Kaneohe and P. meandrina outfall samples. There was no significant change through the day for P. meandrina samples from the control site. The difference in response between the outfall samples and the control samples suggests that P. meandrina has acclimated to elevated temperatures found at the outfall site. © 2006 Elsevier Inc. All rights reserved. Keywords: Acclimation capacity; Antioxidants; Catalase; Coral; ELISA; FRAP; Light; Temperature

1. Introduction Potentially harmful reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH•− ) and superoxide radical (O2•−) are generated during normal aerobic cellular metabolism in the coral host and by photosynthesis in the endosymbiotic algae present within the coral tissue. As temperatures increase, more ROS are produced (Dykens and Shick, 1982; Dykens et al., 1992; Lesser, 1996, 1997; Nii and Muscatine, 1997; Downs et al., 2000, 2002). The cells react to this increase in ROS by boosting up the concentrations of antioxidants such as catalase, glutathione, α-tocopherol, ascorbic acid, superoxide dismutase (SOD), peroxidase etc. to reduce ROS and prevent potential harm to the cell (Beckman and Ames, 1998; Halliwell and Gutteridge, 1999). Photosynthesis in the zooxanthellae increases with light and results in a cascading effect which causes an increase in O2 in ⁎ Corresponding author. Tel./fax: +1787 890 0374. E-mail address: [email protected] (S.P. Griffin). 1095-6433/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.cbpa.2006.02.017

the cell, which in turn cause an increase in ROS (Dykens and Shick, 1982). High levels of light can also cause photodamage and photoinhibition to the zooxanthellae (Jones et al., 1998; Brown et al., 1999; Hoegh-Guldberg and Jones, 1999; Bhagooli and Hidaka, 2003). It has also been experimentally demonstrated that elevated temperature lowers the threshold photoinhibitory light levels in in hospite zooxanthellae (Bhagooli and Hidaka, 2004). Pigments such as fluorescent proteins (Salih et al., 2000) in the coral, UV-absorbing compounds (mycosporine-like amino acids, MAAs) and xanthophylls protect the symbiont from UV and/or solar damage that can occur during increased irradiance through dissipation of excess absorbed excitation energy in the zooxanthellae and enzymes with antioxidant functions. The concentration of pigments is the result of photoacclimation to a certain range of light intensity that the coral has been acclimated to (Lewis, 1995; Jokiel et al., 1997; Dunlap and Shick, 1998) while xanthophylls and enzymes such as superoxide dismutase (SOD) and catalase react to short term fluctuations in light and temperature over a period of hours (Ambarsari et al., 1997;

156

S.P. Griffin et al. / Comparative Biochemistry and Physiology, Part A 144 (2006) 155–162

•− Brown et al., 1999). SOD reduces O2 to H2O2 and CAT converts H2O2 to water. Accumulation of O2•− and H2O2 can be toxic to cells if not scavenged by SOD and catalase (Beckman and Ames, 1998). Griffin and Bhagooli (2004) showed that FRAP could be a useful tool in determining antioxidant potential in corals. The corals in those experiments were only exposed to elevated temperatures for 3 h. In the present study, FRAP was used to determine the diurnal change in antioxidant potentials in corals exposed to elevated temperatures over the course of 5 days. ELISAs using catalase antibodies were run to collaborate the FRAP results. This research compared the physiological responses between two coral species: Pocillopora damicornis and Pocillopora meandrina. To investigate the thermal acclimation capacities of corals with different thermal histories, comparisons were made between 2 populations of P. meandrina from sites with different temperature anomalies: an area influenced by the effluent from the Hawaiian Electric Company's Kahe Generating Station's thermal outfall and a control site. Results from this study are important because they will give insight into whether corals will acclimate to global increases in sea surface temperatures.

2. Materials and methods 2.1. Coral sampling Six colonies of P. damicornis were collected from 1 m depth in Kaneohe Bay from the reefs surrounding the Hawaii Institute of Marine Biology's marine lab on Coconut Island. Colonies of P. meandrina were collected from 3–5 m depth from 2 sites at Kahe on the southwest side of Oahu Island. Six colonies were collected from in front of and from the edges of the 2 thermal outfall pipes leaving the Hawaiian Electric Company's Kahe Generating Station. These colonies are frequently exposed to thermally charged seawater leaving the Outfall reaching up to 5 °C higher than the surrounding ambient sea surface temperatures. These colonies are referred to as the Outfall samples. Six P. meandrina colonies were also taken from reefs in Kahe where there was minimal, if any, influence from the thermal outfall. These colonies are referred to as the Kahe samples. The corals were brought to the running seawater table on Coconut Island and made into nubbins by breaking off individual branches from the colonies. They were then left for 4 days in aquaria with running sea water and screens (∼30% of surface irradiance) to filter direct sunlight to prevent stress from high UV levels.

quanta m− 2 s− 1 from 1100 h until sunset. Nubbins from colonies of P. damicornis, and P meandrina from Kahe and Outfall were placed in each tank (29, 32 and 33 °C) at 0700 h on the first day of experiments. Three nubbins were removed for each species, site and treatment at 0800, 1200 and 1600 h each day for 3 days and at 0800 h on days 4 and 5. Samples were not removed at 1200 and 1600 h on days 4 and 5 because there were not enough nubbins remaining. 2.3. Protein extraction for FRAP FRAP requires reaction of antioxidants and chemical reductants to reduce a ferric tripyridyltriazine (FeIII-TPTZ) complex and produce a colored ferrous tripyridyltriazine (FeIITPTZ) form. ELISAs analyze denatured proteins which would not react in the FRAP assay. Therefore, it was necessary to follow different protocols for extracting proteins from the samples for the FRAP and ELISA assays. Samples of frozen coral were grounded in a mortar and pestle and suspended in 1 mL of 10 mM Tris–HCl buffer (pH 7.5). The samples were then centrifuged for 10 min at 10,000 rpm to separate out any skeleton and other cellular debris. The supernatant was then removed and placed in 1.5 mL Eppendorf micro centrifuge tubes. A protease inhibitor cocktail (Sigma cat. #P8340) was then added to the remaining supernatant (ratio of 1:100) to prevent the destruction of any of the proteins in the sample. These samples were then stored at − 80 °C until further analysis. Protein concentration was determined following the method of Ghosh et al. (1988). 2.4. Protein extraction for ELISAs Fifteen to 20 mg of ground frozen coral tissue was added to a buffer solution of 50 mM Tris pH 7.8, 10 mM Di-Sodium EDTA pH 8.0, 3% polyvinylpolypyrriolidine (PVPP), 2% SDS, and 20 mM Dithiolthrietol (DTT). The samples were then vortexed, heated at 80 °C for 3 min, aerated, mixed again and reheated for another 3 min. The samples were allowed to cool at room temperature for 5–10 min, vortexed and then centrifuged for 10 min at 14,000 rpm. The supernatant was removed, carefully avoiding the mucus on the top layer and placed in another Eppendorf tube with 100 uL of buffer. They were then mixed and recentrifuged for another 10 min and the supernatant was removed avoiding the top mucus layer. Pierce's Protease Inhibitor Cocktail was then added to the final sample (100 × dilution) and stored at − 80 °C until further analysis. Protein concentration was determined using the method of Ghosh et al. (1988).

2.2. Temperature and light experiments 2.5. FRAP analysis Aquaria with temperatures of 32 and 33 °C were set up on running water tables outside. Aquarium heaters were used to maintain relatively constant temperatures throughout the experiment. Another aquarium with ambient temperatures (29 °C) was also set up as a control. Light levels were monitored throughout the day, and light intensity fluctuated from 1000– 1500 μmol quanta m− 2 s− 1 in the morning to 25–60 μmol

This method was modified from Benzie and Strain's protocol (1996, 1999) as described earlier in Chapter 3 and by Griffin and Bhagooli (2004). The working FRAP reagent was produced by mixing 300 mM Acetate buffer (pH 3.6), 10 mM TPTZ solution and 20 mM FeCl3·6H2O in a 10:1:1 ratio just before use and heated to 37 °C. The 300 mM Acetate buffer was

S.P. Griffin et al. / Comparative Biochemistry and Physiology, Part A 144 (2006) 155–162

prepared by mixing 3.1 g of sodium acetate trihydrate (C2H3NaO2·3H2O) with 16 mL glacial acetic acid and brought to 1 L with distilled water. The TPTZ solution was prepared by making a solution of 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) in 40 mM HCl. 150 μL of working FRAP reagent was added to each well in a 96 well (300 μL) micro titer plate. A blank reading was then taken at 600 nm using an ELISA plate reader (Packard SpectraCount, Packard Biosciences). Twenty μL of sample was then added to each well. Each sample was run in triplicate. After addition of sample to the FRAP reagent, a second reading at 600 nm was performed after 8 min. The initial blank reading for each well with just FRAP reagent was then subtracted from the final reading of FRAP reagent with sample to determine the FRAP value for each sample. The change in absorbance after 8 min from the initial blank reading was then compared to that of a standard that was run simultaneously. Standards of known Fe (II) concentrations (FeSO4·7H2O) were run in triplicate using several concentrations between 25–1000 μM (25, 50, 75, 100, 150, 200, 500, and 1000 μM). A calibration curve was then prepared by plotting the average FRAP value for each standard versus its concentration.

157

ciences (RPN2106) and exposed to Biomax Scientific Imaging Film from Kodak (#191 7012) in a dark room. The films were then developed using a developer and fixer from Kodak. Concentrations for each sample were estimated by comparing them with the diluted reference standard from each assay. A standard curve was set up by determining the density of each reference dilution using Scion Image. Scion Image was then used to determine the density of each sample, and the relative % concentration was calculated using the standard curve from the diluted reference standard on each ELISA. 2.7. Statistical analysis A Three-way ANOVA was used to compare FRAP results looking at species/location, temperature, and time of day. A Three-way ANOVA was also used to compare the catalase ELISA results using the same 3 factors. An All Pairwise Multiple Comparison Procedures (Tukey Test) was used to test for significant differences between each treatment. Sigma Stat was used and did not give p values when p was greater than 0.05. Significance was determined using a p ≤ 0.05. 3. Results

2.6. ELISA analysis A catalase primary antibody specific to the coral host from Envirtue Biotechnologies was used during this experiment. After separation of proteins in the samples by SDS-PAGE electrophoresis, the proteins were then transferred from the gel to a PVDF membrane. Western blots were performed to test the antibodies for nonspecific binding and appropriate band size. Bands were at 70–72 kD and there was no nonspecific binding. ELISAs were performed using a 96 well Dot Blot and polyvinylidene fluoride (PVDF) membranes. Samples were diluted with 5 mM DTT in Tris Buffered Saline (TBS) solution to 0.05 μg/μL and 100 μL were added to each well for a total of 5 μg/well. Each sample was run in triplicate and a reference standard was run in columns 1, 5 and 9 with dilutions of 5, 2.5, 1, 0.5, 0.25, 0.1 and 0.05 μg/well. The same reference standard was used in every ELISA to allow comparisons between each assay. A Kahe sample from the 33 °C treatment was chosen as a reference standard for this experiment because it expressed high concentrations of catalase proteins. After the samples were pulled through the membrane by the vacuum, the membrane was incubated in blocking solution (5% powder non-fat milk in TBS) for 1 h. The membrane was then incubated with a catalase primary antibody from Envirtue Biotechnologies Inc (2 μL antibody in 30 mL blocking solution). After one hour, the membrane was removed and washed 4 times in TBS for 10 min each wash. It was then incubated with a secondary antibody (Peroxidase-conjugated Affini Pure Donkey Anti-Rabbit IgG #711-036-152 from Jackson) diluted in blocking solution (2 μL antibody/40 mL solution). After incubating for an hour in the secondary antibody, the membranes were washed 4 times in TBS as before. The membranes were then saturated with a chemiluminescent developing solution from Amersham Bios-

Mean FRAP values for P. meandrina Outfall samples (Fig. 1A) from the control treatment decreased from 3.76 ± 0.24 μM/ μg in the morning to 3.45 ± 0.28 μM/μg at noon and to 2.66 ± 0.27 μM/μg in the afternoon. There was a significant difference (Tukey Test; p b 0.05) between FRAP values in the morning and in the afternoon. Mean FRAP values from the 32 °C treatments decreased throughout the day but there was no significant difference (Tukey Test; p N 0.05) between the different times. Mean FRAP values from the 33 °C treatment decreased significantly (Tukey Test; p b 0.05) from 5.31 ± 0.46 μM/μg in the morning to 2.73 ± 0.36 μM/μg at noon and 2.56 ± 0.33 μM/ μg in the afternoon. The results from each temperature treatment were combined for each collection time to compare daily patterns between collection sites. When FRAP values at each time (0800, 1200 and 1600 h) were combined from each treatment, there was a significant decrease (Tukey Test; p b 0.05) from 3.83 ± 0.19 μM/ μg in the morning to 2.77 ± 0.18 μM/μg at noon and 2.41 ± 0.17 μM/μg in the afternoon. Higher FRAP levels in the morning would suggest that there was a higher antioxidant potential which correlates to higher concentrations of oxidants in the coral cytosol. Mean catalase concentrations (%) for P. meandrina Outfall samples (Fig. 1B) from the Control treatment appeared to decrease from 4.41 ± 1.11% in the morning and 5.13 ± 1.40% at noon to 3.00 ± 1.00% in the afternoon, but there was no significant difference (Tukey Test; p N 0.05) between time of day. Catalase concentrations for the 32 °C treatment were at 4.47 ± 0.80% in the morning, 2.59 ± 0.81% at noon and 2.70 ± 0.65% in the afternoon, but there was no significant difference (Tukey Test; p N 0.05) between these values. Catalase concentrations in the samples from the 33 °C treatment showed a significant decrease (Tukey Test; p b 0.05) from 23.00 ± 9.07%

158

S.P. Griffin et al. / Comparative Biochemistry and Physiology, Part A 144 (2006) 155–162 6

Mean FRAP Value (uM/ug)

A (FRAP)

A

5 A

A AB

4 B B

B B

3

A A A B

2 1 0 All Temp AM Noon PM

29 C

32 C

33 C

Temperature (˚C)

FRAP values and catalase concentrations for the Outfall and Kahe samples from the Control and 32 °C samples fluctuated throughout the experiment (Fig. 3). There were no significant differences between the 2 treatments within either site (Outfall or Kahe) for P. meandrina, but there was a significant effect (ANOVA; p b 0.001) for site depending on the temperature present. There was a significant difference between how the Kahe and Outfall samples reacted at 32 and 33 °C but not within the Control. FRAP values rose after 1 h (Day 1 AM) in the 33 °C Outfall samples and then decreased until the colonies were dead (afternoon of Day 2) while there was no significant difference in FRAP values for the 33 °C Kahe samples over time (Fig. 4A). 6

Mean Catalase Concentration (%)

35

B (Catalase)

A

30 25 20 15

A

10

A A A B B

5

A A A

Mean FRAP Value (uM/ug)

A (FRAP)

B B

5 4 3 2 1 0 All Temps

All Temp AM Noon PM

29 C

32 C

33 C

Temperature (˚C)

29 C

32 C

33 C

Temperature (˚C)

AM Noon PM

0

Fig. 1. (A) Mean FRAP value (uM/ug) and standard errors of the mean are given for P. meandrina Outfall samples for all temperatures combined (All Temp) and each temperature (29, 32 and 33 °C) at different times of the day (AM, Noon and PM). (B) Mean catalase concentrations (%) and standard errors of the mean are given for P. meandrina Outfall samples for all temperatures combined (All Temp) and each temperature (29, 32 and 33 °C) at different times of the day (AM, Noon and PM). Control samples were in aquaria with running sea water at ambient sea surface temperatures (29 °C). Tukey groups are given to show significant differences (p b 0.05) between times of day within each temperature treatment.

in the morning to 3.38 ± 0.90% at noon and to 1.54 ± 0.45% in the afternoon. When catalase concentrations from each time (0800, 1200 and 1600 h) were combined from each treatment, there was a significant decrease (Tukey Test; p b 0.05) from 10.63 ± 0.99% in the morning to 3.70 ± 0.93% at noon and 2.41 ± 0.88 in the afternoon. Higher levels of catalase antibodies in the morning would indicate higher concentrations of H2O2 in the coral cytosol. In contrast, the results for P. meandrina Kahe samples showed there were no significant differences (Tukey Test; p N 0.05) in mean FRAP values (Fig. 2A) or mean Catalase concentrations (Fig. 2B) between time of day (0800, 1200 and 1600 h) for any of the treatments (Control, 32 and 33 °C) or when the treatments were combined.

Mean Catalase Concentrations (%)

35

B (Catalase) 30 25 20 15 10 5 0 All Temps AM Kahe Noon Kahe PM Kahe

29 C

32 C

33 C

Temperature

Fig. 2. (A) Mean FRAP value (uM/ug) and standard errors of the mean are given for P. meandrina Kahe samples for all temperatures combined (All Temp) and each temperature (29, 32 and 33 °C) at different times of the day (AM, Noon and PM). (B) Mean catalase concentrations (%) and standard errors of the mean are given for P. meandrina Kahe samples for all temperatures combined (All Temp) and each temperature (29, 32 and 33 °C) at different times of the day (AM, Noon and PM). Control samples were in aquaria with running sea water at ambient sea surface temperatures (29 °C). There were no significant differences (p N 0.05) between times of day within any of the temperature treatments for the Kahe samples.

S.P. Griffin et al. / Comparative Biochemistry and Physiology, Part A 144 (2006) 155–162

159

morning to 6.01 ± 1.01% at noon and to 3.49 ± 0.51% in the afternoon. There was a significant difference (Tukey Test; p b 0.05) between the morning and afternoon concentrations. At 33 °C, concentrations in the morning (3.35 ± 1.72%) and at noon (3.70 ± 0.75%) decreased significantly (Tukey Test; p b 0.05) in the afternoon to 0.28 ± 0.02%. When the catalase concentrations from each time (0800, 1200 and 1600 h) were combined from each treatment, there was a decrease from 6.02 ± 0.70% in the morning to 4.45 ± 0.85% at noon and to 2.78 ± 0.88% in the afternoon. There was a significant difference (Tukey Test; p b 0.05) between the morning and afternoon concentrations. Significantly higher catalase concentrations in the morning compared to the afternoon suggest that there were higher concentrations of H2O2 in the coral cytosol in the morning.

Catalase concentrations also rose after 1 h (day 1, AM) in the 33 °C Outfall samples and then decreased until the colonies were dead (Fig. 4B). Kahe samples had a less pronounced increase in catalase concentrations that did not appear until day 1 at noon. FRAP values for the P. damicornis samples are not available. Due to the small size of the P. damicornis samples, there was not enough tissue to run the FRAP assay. The mean catalase concentrations for P. damicornis are shown in Fig. 5. Concentrations decreased in the Control treatment from 5.24 ± 1.02% in the morning to 3.63 ± 0.79 at noon and 4.59 ± 1.03% in the afternoon but there was no significant difference (Tukey Test; p N 0.05) between these values. In the 32 °C treatment, catalase concentrations decreased from 9.48 ± 1.88% in the

7

B (Catalase) 30

4 3 2

Concentrations (%)

5

Mean Catalase

Mean FRAP Value (uM/ug)

A (FRAP) 6

20

10

1 0

0 0

20

40

60

80

100

0

20

Hours of Exposure

40

60

80

100

80

100

Hours of Exposure

Outfall 29 C Outfall 29 C Outfall 32 C

Outfall 32 C

7

D (Catalase)

30

Mean Catalase

5 4 3 2 1 0

Concentrations (%)

Mean FRAP Value (uM/ug)

6

20

10

C (FRAP) 0

20

40

60

80

100

0 0

20

Hours of Exposure

40

60

Hours of Exposure

Kahe 29 C

Kahe 29 C

Kahe 32 C

Kahe 32 C

Fig. 3. (A) Mean FRAP values (uM/ug) and standard errors of the mean are given for 29 and 32 °C P. meandrina Outfall samples from 0 h of exposure until 100 h. (B) Mean catalase concentrations (%) and standard errors of the means are given for 29 and 32 °C P. meandrina Outfall samples from 0 h of exposure until 100 h. (C) Mean FRAP values (uM/ug) and standard errors of the mean are given for 29 and 32 °C P. meandrina Kahe samples from 0 h of exposure until 100 h. (D) Mean catalase concentrations (%) and standard errors of the means are given for 29 and 32 °C P. meandrina Kahe samples from 0 h of exposure until 100 h. Control samples were in aquaria with running sea water at ambient sea surface temperatures (29 °C).

160

S.P. Griffin et al. / Comparative Biochemistry and Physiology, Part A 144 (2006) 155–162

A

5 4

3 2 1

Mean Catalase Concentrations (%)

Mean FRAP Value (uM/ug)

6

A

10

8 A A A

6

0

Day 1 AM

Kahe 33 C

Day 1 Noon Day 1 PM

A A B

B

2 B

All temp

29 C

32 C

33 C

Day 2 Noon Day 2 PM

AM Noon PM

Time (Days/ Hour)

Outfall 33 C

35

Mean Catalase Concentration (%)

AB

4

0

0

AB

A

30

B

25 20

Temperature (˚C)

Fig. 5. Mean catalase concentrations (%) and standard errors of the mean are given for P. damicornis samples for all temperatures combined (All Temp) and each temperature (29, 32 and 33 °C) at different times of the day (AM, Noon and PM). Control samples were in aquaria with running sea water at ambient sea surface temperatures (29 °C). Tukey groups are given to show significant differences (p b 0.05) between time of day within each temperature treatment.

15 10 5 0

0

Day 1 AM

Kahe 33 C

Day 1 Noon Day 1 PM

Day 2 Noon Day 2 PM

Time (Days/ Hour)

Outfall 33 C

Fig. 4. (A) Mean FRAP Values and standard errors of the mean are given for P. meandrina Outfall and Kahe samples from the 33 °C temperature treatment from Time 0 through Day 2. (B) Mean catalase concentrations (%) and standard error of the means are given for 33 °C Kahe and Outfall samples from initial Time 0 through Day 2. In the 33 °C treatment, all the P. meandrina colonies from both Kahe and Outfall sites were dead on Day 3.

Catalase concentrations for 33 °C P. damicornis samples fluctuated during the first day and peaked on the morning of Day 2. Catalase concentrations then decreased until all colonies were dead on Day 3 at noon (Fig. 6A). Catalase concentrations for P. damicornis from the Control and 32 °C samples fluctuated throughout the experiment and there were no significant differences between the 2 treatments (Fig. 6B). A temperature of 33 °C proved to be a lethal temperature for P. damicornis and both populations of P. meandrina. This resulted in an initial increase in catalase concentrations and antioxidant potential which was followed by a subsequent decrease until the corals perished. The timing of the increase varied from one hour after exposure for the Outfall samples (Fig. 4B), 5 h for the Kahe samples (Fig. 4B) and 24 h for P. damicornis (Fig. 6A). Visual observations of bleaching (Table 1) showed that P. meandrina samples from Kahe bleached faster than the Outfall

samples at 33 °C. 4% of the Kahe samples bleached on the first day when exposed to 33 °C while none of the Outfall or P. damicornis samples had bleached yet. On day 2, more than 90% of the Kahe samples were bleached or had tissue sloughing while about 55% of the Outfall samples were bleaching or losing tissue. In the morning of day 3, 100% of the Kahe colonies were dead or bleached completely white and around 85% of the Outfall samples were bleached or paling. By noon of day 3, however, all of the P. meandrina colonies from both sites were dead. On day 5, 100% of the P. meandrina samples from Kahe and the P. damicornis samples were bleached in the 32 °C treatment while none of the Outfall samples in the 32 °C tank were bleached on day 5 (Table 1). No bleaching occurred on any of the samples Table 1 Bleaching and mortality observations for P. meandrina samples from the Outfall site and the control site (Kahe) and P. damicornis Temp. Sample Day 1 (n = 39)

Day 2 (n = 30)

Day 3 (n = 21)

29 °C Outfall Kahe P. dam 30 °C Outfall Kahe P. dam 31 °C Outfall Kahe P. dam 32 °C Outfall Kahe P. dam 33 °C Outfall Kahe P. dam

0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 55% 90% 18%

0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 85% 100% 80%

0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 4% 0%

Day 4 (n = 12)

Day 5 (n = 3)

0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 0%

0% 0% 0% 0% 0% 0% 0% 0% 0% 0% 100% 100%

100%

S.P. Griffin et al. / Comparative Biochemistry and Physiology, Part A 144 (2006) 155–162

Mean Catalase Concentration (%)

35

A

30 25 20 15 10 5 0

0

Day 1 AM Day 1 Noon Day 1 PM Day 2 AM Day 2 Noon Day 2 PM Day 3 AM

Mean Catalase Concentrations (%)

Time (Day/ Hour)

B

30

20

10

0 0

20

40

60

80

100

Hours of Exposure 29 C 32 C

Fig. 6. (A) Mean catalase concentrations and standard errors of the mean are given for 33 °C P. damicornis samples from Time 0 until Day 3 AM. All of the P. damicornis colonies were dead by Noon on Day 3. (B) Mean catalase concentrations and standard errors of the mean are given for P. damicornis Control and 32 °C Samples from 0 h of exposure until 100 h. Control samples were in aquaria with running sea water at ambient sea surface temperatures (29 °C).

in the control treatment suggesting that elevated temperatures were the cause for coral bleaching and subsequent mortality in the 32 and 33 °C treatments. 4. Discussion In general, the results for both assays (FRAP and catalase ELISAs) showed similar patterns to temperature and time of day for P. meandrina from the Outfall site. Both FRAP and catalase data had higher levels in the morning than at noon and in the afternoon. FRAP values and catalase concentrations peaked when irradiance was the highest in the morning and decreased after light levels fell by noon. Neither test showed a significant difference in time of day for P. meandrina samples from Kahe. The Outfall samples displayed similar physiological behavior to the P. damicornis samples with higher concentrations of catalase and antioxidant potential in the morning when light levels were highest. P. damicornis is usually found in shallower waters than P.

161

meandrina and therefore experiences more exposure to light and higher fluctuations in temperature. P. meandrina are also regarded as one of the more thermosensitive species on Hawaiian reefs (Coles, 1975). The similarity between the Outfall samples and P. damicornis suggests that the Outfall samples have acclimated to different environmental conditions and now behave differently than their conspecifics from a nearby population with more static environmental parameters and lower temperatures. Ambient sea surfaces temperatures around Hawaii during June of 2003 were 29 °C which resulted in control temperatures of 29 °C during the experiment. This is seasonally high for Oahu, Hawaii which has a normal summer ambient temperature of 26 °C (Jokiel and Coles, 1977). This could explain why there were no statistically significant differences between the Control and 32 °C for either of the species over the course of the 5 day experiment (Figs. 2 and 4C). Control samples were already facing higher than normal temperatures; therefore, no significant differences were found for the catalase concentrations and FRAP potential between the Control and 32 °C. But on day 5, 100% of the Kahe and P. damicornis samples were bleached in the 32 °C treatment. None of the Outfall samples in the 32 °C tank were bleached on day 5 nor were any of the control samples bleached. If the experiment had been run longer, bleaching tolerance levels could have been tested for the Outfall samples. How long could the Outfall samples tolerate 32 °C while the other samples at that temperature were visibly suffering? The similarities between the results for the catalase ELISAs and FRAP assay show that either test would be suitable for determining changes in antioxidants levels in corals. ELISAs are more sensitive to specific changes depending on the antibodies chosen, while the FRAP assay provides a general inexpensive and reproducible method for determining antioxidant potential. Both of these assays when used along with other tests of cellular parameters such as heat shock proteins, metallothionein, ubiquitin and other proteins can be very useful in thoroughly determining the status of cells exposed to different environmental conditions. More information would be able to be gathered if an assay was available that provided continuous data over the course of time. For example in the 33 °C treatment catalase concentrations appear to peak for P. meandrina Outfall samples after 1 h while the peak comes after 5 h for the Kahe samples (Fig. 4B). This peak could have been earlier or later in either case, but the limitations of taking samples at specific times instead of having continuous data available do not allow for this. The results of this research show that P. meandrina from the Outfall site has acclimated to the environmental conditions at that site as a result of the heated effluent from the electric plant. This population now exhibits different physiological response compared with conspecifics from a nearby control population. Their reactions are similar to P. damicornis which is a species adapted to shallower environments with greater fluctuations in temperature and irradiance. The Kahe generating station began in 1971 and resulted in high mortality to corals in the area (Jokiel and Coles, 1974). In December of 1976, operation of the 2 large offshore outfall pipes began (AECOS, 2002). Acclimation to these conditions has occurred in less than 30 years. This implicates the possibility for some coral species to acclimate to

162

S.P. Griffin et al. / Comparative Biochemistry and Physiology, Part A 144 (2006) 155–162

the present trends in global warming although only after initial episodes of mass mortality which could already be happening on reefs around the world. 5. Conclusions – P. meandrina from the Outfall site can tolerate elevated temperatures for longer periods of time than conspecifics from the control population at Kahe and P. damicornis. – Temperatures of 33 °C for over 3 days are lethal for P. damicornis and both populations of P. meandrina. – Catalase ELISA's and FRAP assays displayed similar behavioral patterns in the samples. ELISA's can very useful for determining specific antioxidant concentrations such as catalase while the FRAP assay can be used for a general indicator of antioxidant potential. – The difference in behavior between the outfall samples and the control samples suggests that P. meandrina has acclimated to elevated temperatures found at the outfall site. Acknowledgements The authors would like to thank the Hawaii Institute of Marine Biology (HIMB) where this research was conducted, and Drs. Joanne Leong, Teresa Lewis, and Fenny Cox who were extremely helpful in giving advice and letting us use their laboratory and materials. We would also like to thank Craig Downs and Envirtue Biotechnologies Inc for supplying the antibodies used in this research, Dr. Cheryl Woodley for lending us the Dot Blot for the ELISAs, the Pauley Foundation for the opportunity to conduct research at Hawaii Institute of Marine Biology (HIMB), and the University of Puerto Rico Sea Grant College Program for partial funding. Craig Downs and Dr. Cheryl Woodley were also helpful in giving us advice and answering our many questions. S.P.G. thanks Alliance for Graduate Education and the Professoriate (AGEP) of the National Science Foundation (NSF) and the University of Puerto Rico for their financial support. R.B. is thankful to the Ministry of Education, Culture, Sports, Science and Technology, Japan for fellowship. Corals were collected under the scientific collecting permit #2003-06. References AECOS Inc, 2002. Annual Report. Kahe Generating Station. NPDES Monitoring Program. Ambarsari, I., Brown, B.E., Barlow, R.G., Britton, G., Cummings, D.G., 1997. Fluctuations in algal chlorophylls and carotenoid pigments during solar bleaching in the coral Goniastrea aspera at Phuket, Thailand. Mar. Ecol. Prog. Ser. 159, 303–307. Beckman, K.B., Ames, B.N., 1998. The free radical theory of aging matures. Physiol. Rev. 78 (2), 548–571. Benzie, I.F.F., Strain, J.J., 1996. The ferric reducing ability of plasma (FRAP) as a measure of “antioxidant power”: the FRAP assay. Anal. Biochem. 239, 70–76. Benzie, I.F.F., Strain, J.J., 1999. Ferric reducing/antioxidant power assay: direct measure of total antioxidant activity of biological fluids and modified

version for simultaneous measurement of total antioxidant power and ascorbic acid concentration. Methods Enzymol. 299, 15–27. Bhagooli, R., Hidaka, M., 2003. Comparison of stress susceptibility of in hospite and isolated zooxanthellae among five coral species. J. Exp. Mar. Biol. Ecol. 291, 181–197. Bhagooli, R., Hidaka, M., 2004. Photoinhibition, bleaching susceptibility and mortality in two scleractinian corals, Platygyra ryukyuensis and Stylophora pistillata, in response to thermal and light stresses. Comp. Biochem. Physiol. A 137, 547–555. Brown, B.E., Ambarsari, I., Warner, M.E., Fitt, W.K., Dunne, R.P., Gibb, S.W., Cummings, D.G., 1999. Diurnal changes in photochemical efficiency and xanthophylls concentrations in shallow water reef corals: evidence for photoinhibition and photoprotection. Coral Reefs 18, 99–105. Coles, S.L., 1975. A comparison of effects of elevated temperature versus temperature fluctuations on reef corals at Kahe Point, Oahu. Pac. Sci. 29, 15–18. Downs, C.A., Mueller, E., Phillips, S., Fauth, J.E., Woodley, C., 2000. A molecular biomarker system for assessing the health of coral (Montastraea faveolata) during heat stress. Mar. Biotechnol. 2, 1–12. Downs, C.A., Fauth, J.E., Halas, J.C., Dustan, P., Bemiss, J., Woodley, C.M., 2002. Oxidative stress and seasonal coral bleaching. Free Radic. Biol. Med. 33, 533–543. Dunlap, W.C., Shick, J.M., 1998. Ultraviolet radiation-absorbing myrcosporinelike amino acids in coral reef organisms: a biochemical and environmental perspective. J. Phycol. 34, 418–430. Dykens, J.A., Shick, M., 1982. Oxygen production by endosymbiotic algae controls superoxide dismutase activity in their animal host. Nature 297, 579–580. Dykens, J.A., Shick, J.M., Benoit, C., Buettner, G.R., Winston, G.W., 1992. Oxygen radical production in the sea anemone Anthopleura elegantissima and its endosymbiotic algae. J. Exp. Biol. 168, 219–241. Ghosh, S., Gepstein, S., Heikkila, J.J., Dumbroff, B.G., 1988. Use of a scanning densitometer or an ELISA plate reader for measurement of nanogram amounts of protein in crude extracts from biological tissue. Anal. Biochem. 169, 227–233. Griffin, S.P., Bhagooli, R., 2004. Measuring antioxidant potential in corals using the FRAP assay. J. Exp. Mar. Biol. Ecol. 302, 201–211. Halliwell, B., Gutteridge, J.M.C., 1999. Free Radicals in Biology and Medicine, 3rd edition. Oxford University Press. Hoegh-Guldberg, O., Jones, R., 1999. Photoinhibition and photoprotection in the symbiotic dinoflaggelates from reef-building corals. Mar. Ecol. Prog. Ser. 183, 73–86. Jokiel, P.L., Coles, S.L., 1974. Effects of heated effluent on hermatypic corals at Kahe Point, Oahu. Pac. Sci. 28, 1–18. Jokiel, P.L., Coles, S.L., 1977. Effects of temperatura on the mortality and growth of Hawaiian reef corals. Mar. Biol. 43, 201–208. Jokiel, P.L., Lesser, M.P., Ondrusek, M.E., 1997. UV-absorbing compounds in the coral Pocillopora damicornis: interactive effects of UV radiation, photosynthetically active radiation, and water flow. Limnol. Oceanogr. 42, 1468–1473. Jones, R.J., Hoegh-Guldberg, O., Larkum, W.D., Schreiber, U., 1998. Temperature-induced bleaching of corals begins with impairment of the carbon dioxide fixation mechanism in zooxanthellae. Plant Cell Environ. 21, 1219–1230. Lesser, M.P., 1996. Elevated temperatures and ultraviolet radiation causes oxidative stress and inhibit photosynthesis in symbiotic dinoflagellates. Limnol. Oceanogr. 41, 271–283. Lesser, M.P., 1997. Oxidative stress causes coral bleaching during exposure to elevated temperatures. Coral Reefs 16, 187–192. Lewis, S., 1995. Response of a Pacific stony coral to short-term exposure of ultraviolet and visible light. Ultraviolet Radiation and Coral ReefsHIMB Tech. Report, vol. 41, pp. 89–106. Nii, C.M., Muscatine, L., 1997. Oxidative stress in the symbiotic sea anemone Aiptasia pulchella (Carlgren, 1943): contribution of the animal to superoxide ion production at elevated temperature. Biol. Bull. 192, 444–456. Salih, A., Larkum, A., Cox, G., Kuhl, M., Hoegh-Guldberg, O., 2000. Fluorescent pigments in corals are photoprotective. Nature. 408, 850–853.