By Maria Bynagle and Emily Peterson under the advisement of Suzanne Strom
Shannon Point Marine Center, Western Washington University, Bellingham, WA 98225-9181

INTRODUCTION
Coral bleaching is an increasingly widespread event and a serious environmental and economic problem for many tropical countries. A major world-wide bleaching event was observed during the 1997-1998 ENSO. In general, coral bleaching is strongly associated with elevated seawater temperatures and increased exposure to UV radiation. Our goal in this project is to understand the pigmentation changes associated with temperature and UV stress in the coral Montastraea faveolata.

Specifically,

1.  Did bleaching occur? Was bleaching induced by temperature stress, UV stress, or both?
2. Are observed stress induced changes in coral pigmentation caused by expulsion of zooxanthellae or by changes in the pigment content of zooxanthellae retained in the host?
3. Does the pigment content of expelled zooxanthellae differ from the pigment content of zooxanthellae retained in the host?
4. Is there a change in zooxanthellar pigment composition as the algae respond to temperature and UV stress?

METHODS

• Plugs (2.1 cm dia.) of the boulder coral Montastraea faveolata were collected from three North Perry Reef colonies (Lee Stocking Island, Bahamas) at a depth of 15+1 m.

• Two experiments were conducted, one at ambient temperature and one at elevated temperature. The temperature was kept constant using a temperature-controlled water circulation system.

• We used natural density screen and mylar to simulate three different light environments.

• Three coral plugs from each treatment were sampled every 24 hours. The water from each holding dish was collected for the expelled zooxanthellae. The tissue was  removed from the skeleton of the coral using an Interplaq water pik for the zooxanthellae retained in the host.

• After repeated centrifugation, the final resuspension of coral zooxanthellae was filtered through 30 mm Nitex mesh. The zooxanthellar suspension was vacuum filtered onto GF/C filters, wrapped in foil and placed in liquid nitrogen for later analysis by HPLC.

• Filters were ground using 100% acetone. An internal standard, canthaxanthin, was added to the samples after they were ground and centrifuged.

• HPLC pigment separation was by a modification of the method of Mantoura and Llewellyn (1983). A methanol-acetone gradient was used on a C-18 reverse phase column with 5 mm particle size, flow rate 1 mL/min.

• The pigments present in the eluent were detected and quantified using fluorescence and absorbance detection (Mantoura and Repeta, 1997).

• Bleaching was evident at both temperatures in enhanced light treatments.
• No bleaching occurred in response to temperature stress alone (as shown by ambient temp exp).
• Temperature and UV stress combined to cause a more severe bleaching response (as shown by differing reef + UV response in the two temp conditions).

Ambient Temperature

• Bleaching, which occurred in the enhanced light treatment, was a result of zooxanthellar expulsion.

1. Pigment/cm² decreased (Figure 1)
2. Pigment/cell remained constant (Figure 2)
3. Cell density decreased (Figure 3)

• Enhanced light treatment showed bleaching as a result of both zooxanthellar expulsion and loss of pigment within retained zooxanthellae.

1. Pigment/cm² decreased (Figure 1)
2. Pigment/cell decreased (Figure 2)
3. Cell density decreased (Figure 3)

 
• Reef + UV treatment exhibited bleaching due only to loss of pigment in retained zooxanthellae.

 
1. Pigment/cm² decreased (Figure 1)
2. Pigment/cell decreased (Figure 2)
3. Cell density remained constant (Figure 3)

• In all cases, expelled zooxanthellae have far more unknown xanthophyll than retained zooxanthellae (Figures 4 and 5).
• Without temperature stress, expelled and retained zooxanthellae had similar chlorophyll a content (Figure 6 ).
• With temperature stress, expelled zooxanthellae had less chlorophyll a than retained zooxanthellae (Figure 7).

• Stress caused an increase in photoprotective pigments relative to the total pigment content (Figures 8 and 9 and Figures 10 and 11).
• Expelled zooxanthellae showed a much larger increase in the photoprotective/total pigment ratio over time compared to the retained zooxanthellae (Figures 10 and 11).
• The continuous increase in the pigment ratio for expelled zooxanthellae shows that they were experiencing stress within the animal prior to their expulsion (Figures 10 and 11).

Conclusions

• Exposure to enhanced light intensities caused bleaching; temperature stress led to bleaching at lower light intensities.
• Different environmental stresses (e.g. UV, temperature) seem to cause bleaching through different pigment loss mechanisms.
• "Unknown xanthophylls" appeared to be produced in response to stress, especially in expelled zooxanthellae.