TOPICAL SCIENCE
BY TIMOTHY HOVANEC

Bacterial and Coral Bleaching

TERRY SIEGEL
A picture of a Diploria sp. coral in Terry Siegel’s 135-gallon reef tank. Several Diploria species were among the 17 species of corals found to be infected with a fast-growing disease that emerged in the Florida Reef Tract in 1995.
Coral bleaching is a topic that has been discussed in this column several times because it is a constant worry to hobbyists who are concerned that their corals may bleach, why this might happen, and what they can I do to prevent it. There are several theories about what causes bleaching and how to prevent it — subjects for endless discussion.

In this month’s column I will introduce two papers that were just published, and that establish a relationship between coral bleaching and bacteria. The first paper discusses what organism was responsible for the large coral disease outbreak on Florida reefs in 1995. The second paper investigates how the virulence of a specific bacterium, which has been shown to cause coral bleaching, can increase and what biochemical mechanism is responsible for this increased virulence.

The first paper is a “scientific correspondence” (Richardson, L. L., W. M. Goldberg, K. G. Kuta, R. B. Aronson, G. W. Smith, K. B. Ritchie, J. C. Halas, J. S. Feingold and S. L. Miller. 1998. Florida’s mystery coral-killer identified. Nature 392:557-559), and as such it is short and does not provide many experimental details that would normally accompany a paper. It is basically a kind of late-breaking finding in the form of a letter. A full scientific paper, which will provide many more details, will surely be published in the future by these same authors in some journal.

In 1995, a fast-spreading coral disease emerged in Florida. It started in the Florida Reef Tract and within four months of being first discovered had spread 120 miles in the Florida Keys. The disease was characterized by starting at the colony base and spreading rapidly. By studying the distribution of infected corals the authors showed that the incidence of the disease was greatest at 14 meters, but no possible reasons are given for this (such as water temperature).

Further, the incidence is related to coral density, which indicates that the disease may be contagious. The disease was found in 17 corals (and one hydrocoral). The first coral infected was Dichocoenia stokesi. Other coral species infected included Agaricia agaricites, A. lamarcki, Diploria labyrinthiformis, D. strigosa, Montastrea anularis, Siderastrea siderea and Solenastrea bournoni. The disease occurred in other areas of Florida in 1996 and 1997, from June to October. Thus, there is a seasonality to the disease, although the authors do not discuss any possible reasons, such as water temperature, for this.

To find possible causes of the disease, the authors used transmission electron microscopy (TEM). They did not find any evidence of bacteria in the bleached tissue, but along the edge of the disease (where it was attacking fresh coral tissue) they found relatively large numbers of bacteria compared with non-diseased areas. They isolated a bacterium and characterized it using the polymerase chain reaction (PCR) to amplify 16S ribosomal RNA genes. This allowed them to compare the bacterium to other organisms. They also characterized the bacterium by physiology using an instrument called a Biolog. With this instrument the bacterium is exposed to a wide variety of carbon sources. By determining which types of carbon the bacterium can (and cannot) grow on, the metabolism of the bacterium can be inferred.

The combination of these two methods placed the bacterium in the alpha subdivision of the class Proteobacteria. Thus, the bacterium is gram-negative. Their genetic data suggest that the bacterium is a member of the genus Sphingomonas. The bacterial origin of the disease is also borne out by the facts that they were able to infect healthy colonies of D. stokesi with isolates of the bacterium, and when they put diseased colonies in aquaria with healthy colonies, the healthy colonies became diseased.

There is no discussion about cures or antibiotics. Perhaps when a more complete paper is published this research will be included. At this time, what the article shows is that the disease is a bacterium and that it is capable of spreading rapidly and is infectious.

This leads to the second paper, which looks at how the virulence of a bacterium is increased with increasing water temperatures (Toren, A., L. Landau, A. Kushmaro, Y. Loya, and E. Rosenberg. 1998. Effect of temperature on adhesion of Vibrio strain AK-1 to Oculina patagonica and on coral bleaching. Appl Environ Microbiol 64:1379-1384). These researchers found that a bacterium, which they named Vibrio AK-1, can cause coral bleaching in Oculina patagonica, but the bleaching is correlated with increased water temperature (see Kushmaro, A., Y. Loya, M. Fine and E. Rosenberg. 1996. Bacterial infection and coral bleaching. Nature 380:396, and Kushmaro, A., E. Rosenberg, M. Fine, and Y. Loya. 1997. Bleaching of the coral Oculina patagonica by Vibrio AK-1. Mar Ecol Prog Ser 147:159-165).

What this new paper discloses is how the virulence of the bacterium is increased with rising water temperatures. It is well established, but not universally accepted, that increased water temperatures are related to coral bleaching incidents. But questions remain about exactly how the increased temperature causes the bleaching. Does the higher water temperature cause some physiological problem for the coral or the zooxanthellae, or is there something else going on?

This paper investigated what effects water temperature had on the physiology of the bacteria, which allowed them to infect corals. Previous research had demonstrated that at 16 degrees Celsius (61 degrees Fahrenheit), the Vibrio AK-1 could not infect the corals. At 25 degrees Celsius (77 degrees Fahrenheit) some infection occurred, while at 29 degrees Celsius (84 degrees Fahrenheit) the infection rate greatly increased.

The research question is: What are the temperature-related mechanisms that allow greater infection at higher temperatures? To infect a coral, the bacteria must first adhere to the coral tissue. This is not as easy as it seems. Adhesion can be quite specific because it involves numerous biochemical reactions and organic compounds, such as proteins and polysaccharides. To adhere to the coral, specific recognition sites must be present on the coral. Furthermore, the function of proteins can be dependent on a specific temperature or range of temperatures. These are some of the factors this paper investigated.

To do the work, they first isolated the bacterium. They determined that it appeared to be a new species of Vibrio by using various biochemical, physiological and molecular tests (they called it Vibrio AK-1). They then grew up large numbers of the bacterium. At the same time, they put colonies of the coral Oculina patagonica in seawater of three different temperatures (16, 23 and 29 degrees Celsius) so they could acclimatize to the water temperatures. The researchers then inoculated the corals with four different concentrations of the bacterium. Thus, they had a test matrix of temperature and bacterial concentration.

What the results showed was that at 29 degrees Celsius, even a small dose of the bacterial cells caused infection and bleaching, while at 16 degrees, even the largest dosage of cells failed to cause bleaching. Thus, water temperature was more important than bacterial numbers for successful infection of the coral colonies.

Next, they looked at adhesion of the bacterial cells to the coral. They did this by adding a certain number of cells to a volume of seawater that contained a coral colony (for the control, no colony added). They then took water samples after various durations of incubation and counted the bacterial cells still in the water. Because some cells did adhere to the glass sides of the culture vessel, a correction factor was determined. With this method in place, they could then look at how water temperature was related to adhesion. Further, they could examine how various chemicals prevented the bacterial adhesion to the coral.

The results of several experiments showed that the water temperature the bacteria was grown at was a critical factor, not the temperature the corals were in. Normally, no infection would occur at 16 degrees Celsius, but if corals held in 16-degree water were inoculated with bacterial cells grown at 25 degrees, there was a significant amount of beaching. This result suggested that the critical factor is the temperature the bacterium is grown at that determines the virulence of the bacterial cells. The thinking here is that at higher water temperatures, the bacteria become more virulent rather then the coral becoming more susceptible to infection.

In another experiment, they found that a chemical called beta-D-galactopyranoside was a receptor for the Vibrio. They suggest that residues of this chemical on the surface of the coral act as a receptor for the Vibrio AK-1. In the inhibition experiments, they determined that a chemical called methyl-beta-D-galactopyranoside could prevent the adhesion of the Vibrio, although the results of this test were confounded by the fact that there was poor recovery of the bacterial cells. They knew how many cells they added to the water and they could determine how many cells adhered to the coral. So, when they added the chemical to cause de-adhesion, they should have been able to recover close to the original number of cells, but they were able to recover only 40 to 50 percent of the original number of cells.

They do not speculate about whether this chemical would be a “cure” for coral bleaching. There are many potential problems with it working. For instance, the adhesion and de-adhesion can be host specific, which means it may only work with certain species pairs of coral and bacteria. It would take much laboratory work to determine how widely effective this, or any such inhibitor of adhesion, would be.

These two papers show that coral bleaching is complex and many factors must be taken into account. However, it seems that the causative agent is bacterial and there are more than one species of bacteria that can cause bleaching. Much work remains to be done, especially in applying these results to the benefit of reefkeepers, because none of these studies were focused on cure and prevention with captive corals when they were being conducted. These papers can serve as a start for the applied research that is needed to cure bleaching in home aquaria. For the time being, they point out the importance of controlling water temperature.

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