Abstract

The majority of global monitoring programs report that coral reefs are declining and health factors such as coral diseases and bleaching are recognized as key threats. Understanding the relationships between histologic alterations in tissue structure and physiologic responses of an organism to stress is important in defining the health status of corals. Ongoing efforts to standardize the histologic methods used with scleractinian corals have resulted in a developing literature correlating gross observations of coral diseases with histopathologic evidence of disease. However, coral physiology is still poorly understood, both on the level of the colony and individual polyp. Proteomics is a method to investigate the physiology of an organism through the study of proteins, including the way they function and interact with each other inside cells.

This study was undertaken to develop complimentary histologic and proteomic methods for individual animals isolated from apparently healthy scleractinian coral colonies. Surgical and histologic procedures were developed that facilitated the evaluation of individual coral polyps with a fixation method and embedding matrix that allowed concurrent proteomic analysis. The key factors in tissue preparation involved frozen processing of the tissue following alcohol fixation and rapid decalcification.

Histologic evaluation of an individual coral polyp from Goniastrea retiformis revealed good tissue preservation that facilitated standard H&E staining as well as special stains such as alcian blue-periodic acid-Schiff (AB-PAS). Figure 1 is an image of an individual coral polyp stained with H&E and showing good preservation of the central mesenteries of the body cavity. Figure 2 is a higher magnification of the oral disk area stained with H&E and showing good preservation of the apical epidermis (arrow 1), gastrodermis (arrow 2) and nematocyst aggregates (arrow 3) within a tentacle. Figure 3 is a higher magnification of the apical surface showing the basal distribution of epidermal nuclei (arrow 1), an intact mucin layer composed mostly of acid mucopolysaccharides (arrow 2) and prominent zooxanthellae in the gastrodermis (arrow 3).

Click on the image to see a larger view

	Figure 1. Individual coral polyp. H&E stain. Original magnification=2x
	Figure 2. Oral disk area of individual coral polyp. H&E stain. Original magnification=63x
	Figure 3. Epidermis of individual coral polyp AB-PAS stain. Original magnification=400x

Two-dimensional SDS-polyacrylamide gel analysis was used to establish the protein profile of individual polyps from frozen histologic sections of apparently healthy coral. Gels were stained using a mass spectroscopy-compatible silver stain. Digital images of silver-stained gels (Figure 4) were captured using a gel documentation system and calibrated against commercial isoelectric point and molecular weight standards to determine relative position, isoelectric point, molecular weight, optical density and total area of individual protein spots. MALDI-TOF mass spectroscopy of peptide fragments is currently being used to identify proteins of interest.

Click on the image to see a larger view

Figure 4. Representative image of characteristic protein profile from histologic section of an individual coral polyp. Proteins in the upper right hand corner have isoelectric points (pI) > 7 and molecular weights > 100, 000 daltons.

The combination of histologic and proteomic analyses outlined above should provide the most detailed evaluation of coral health to date, while minimizing the sampling-induced damage to endangered coral populations. The parametric and non-parametric data obtained using these methods will support the complex systematic covariate analyses necessary to more accurately define the health status of coral.