Coral reefs are often referred to as the rain forests of the sea, not only due to their amazing diversity and importance to ocean ecology, but also because of their economic impact. They play an essential role in the protection of coastlines, act as nurseries for many fish and invertebrate species, and attract fishermen and tourists. Successful tourism depends on maintaining vivacious coral reefs, and if the ecology of the reef is dramatically altered, the tropical paradise may lose its appeal. There are many stressors to coral reef ecosystems; such as warming oceans, overfishing, sedimentation due to coastal development, physical damage caused by boat anchors, and pollution due to discharge, oil or chemical spills.
Since the early 1970s, there has been an effort to characterize marine diseases, including the application of techniques to identify coral pathogens and understand coral resistance to disease. Most of the causative agents of diseases, the factors contributing to their spread, and the impacts on coral populations remain unknown.
Histological techniques can provide a wealth of information about coral disease and functioning. At George Mason University in the Peters Lab, we study what causes coral health decline, as well as unknown coral pathogens and bacteria through histology and other novel techniques like fluorescence in situ hybridization (FISH) and laser capture microdissection (LCM).
Figure 1: Staghorn corals (Acropora cervicornis) collected from the Florida Keys displaying bleaching and tissue loss.
Figure 2: White band disease on elkhorn coral (Acropora palmata).
In addition to stony corals (Figure 1), soft corals (gorgonians) such as sea fans are also studied (Figure 2). Many types of diseases affect corals and are named based on their appearance on the coral tissue: white band disease (Figures 3 and 4), black band, red band, etc. A few diseases are named based upon their etiologies such as Aspergillosis sydowii, a fungus causes the disease Aspergillus in gorgonians. The bacteria, Serratia marcescens, has been identified as the cause of “white pox disease” or Serratiosis in acroporid corals. Other diseases can affect corals through rapid tissue necrosis, dark spots and skeletal anomalies.
Figure 3. Gram-negative rod-shaped bacterial aggregates in the skeleton producing epidermis lining the gastrovascular canals in acroporid corals infected with white band disease were reported by Dr. Esther Peters. The role of the aggregates remains unknown in the disease process, as they can also be found in apparently healthy corals.
Fixed coral samples are shipped to our lab after having been collected from coral reefs nation-wide. Coral tissue collection is very different compared to clinical or even veterinary collection because most coral samples are retrieved by snorkeling at shallow depths, and SCUBA diving at deeper depths. The collection of coral tissue requires specific training and preventing contamination underwater can be challenging. Usually additional data is collected and sent to us about the sample and its environment such as species, location, health or condition, water quality and adjacent organisms. To detect the causal agent, timing of sampling is critical because the presence of the pathogen can be missed as other bacteria move in onto degraded tissue. Using histological techniques on corals allows us to see pathological cell and tissue changes such as degeneration, atrophy, infectious agents and parasites.
Corals are unique because coral polyps excrete a calcium carbonate exoskeleton. Colonial coral polyps are connected by a thin living tissue called the coenosarc, though others can be solitary polyps. Working with coral in histology can be difficult at times due this unique morphology and thin tissue layer.
For example, when working with undulating brain coral it can take many sections to see the same area of multiple polyps. After arrival at the lab, coral samples are photographed and trimmed. Our histotechnique procedures are similar to others; however, we often enrobe coral samples in agarose or HistoGel to preserve a margin where atrophy or tissue loss is occurring, prior to decalcification. For coral samples, the calcium carbonate exoskeleton must be removed to facilitate sectioning and examination of the tissues. Animal samples that contain bone need to be decalcified, but in corals once the crystalline calcium is decalcified there is nothing left to retain the underlying support structure, hence the need for enrobing.
Next, trimmed coral samples are placed into labeled cassettes and into a jar or beaker of EDTA solution. The jar or beaker is then placed on the shaker table (100 rpm) and checked every 12-48 hours. A dissecting needle is used to gently touch and probe the tissue to check for decalcification of hard material which may take days to weeks to decalcify depending on skeletal density and size. Decalcified samples are then rinsed with tap water and placed into 70% undenatured ethanol to begin the dehydration process of tissue prior to processing. Our coral samples are trimmed once again depending on morphological characteristics and goal of the study, processed (dehydrated and processed in paraffin), embedded in Paraplast, and ready to section using the microtome. For coral tissue, we cut sections at 6 micrometers or thinner. Once slides are made and are dry, we stain our slides of coral tissue. Most often, we use Harris’s Hematoxylin and Eosin (H&E) stain but we also apply special stains to identify microorganisms such as bacteria, fungi, protozoans and other parasites. Our lab employs a Giemsa stain to illustrate the presence of Rickettsiales-like organisms (RLOs) and other microorganisms found in coral samples. These are hypothesized to be presumed pathogens for coral disease and have been observed by Dr. Esther Peters. Other special stains such as Cason’s trichrome, Movat’s Pentachrome, and modified Heidenhain’s Azocarmine-Aniline Blue are used to identify various connective tissues within the sample.
Figure 4. A healthy section of sea fan (Gorgonia ventalina) tissue stained using H&E.
Using histotechniques and biomedical tools to understand coral disease and resistance to disease will have major management implications on coral reef ecosystems worldwide. The rapid degradation of coral reefs and the stressors responsible for facilitating these changes are increasing in severity. Research on coral disease requires an interdisciplinary approach to identify and characterize diseases, their consequences, and their relationships with anthropogenic stressors. Reducing stressors on coral reefs such as overfishing, pollution, sedimentation, temperature, and even harmful chemicals from sunscreen (i.e., oxybenzone and octinoxate) can enable coral recovery at a local scale. By reducing these stressors, corals can more easily fight off the infections and bacteria causing mass mortality.
If you are interested in learning more about coral histology, please check out the new e-book written by our very own Dr. Esther Peters and Kathy Price called “Histological Techniques for Corals.” It is a laboratory manual containing coral tissue sample processing and histoslide preparation methods. The link for the e-book can be found here: books2read.com/u/boZP19.