DISCUSSION
Yellow band disease appears to affect zooxanthellae rather than host tissue in Montastraea spp. This hypothesis is based on the fact that corals exposed to 20°C for 96 h all survived while still showing signs of YBD. Corals exposed to 31°C showed an 80% mortality rate, and the 20% that survived had YB lesions covering the entire surface (host tissue remained alive). When the four Vibrio spp. were inoculated together, the YBD signs developed more rapidly and closely matched the disease progression and cell impairment features found in field samples of YBD (8). This suggests that these four species may act as a consortium. Field specimens of corals with YBD remain alive for years, depending upon the stress conditions placed upon the coral. As the lesion advances, the tissue remains alive, albeit without its symbionts. Over time, if stress conditions remain and the lesion advances quickly, the tissue starts to die and the skeleton is exposed. The symbionts are either degenerate, vacuolated, or absent. YBD has an appearance similar to that of thermal coral bleaching and can sometimes be confused with it. It is important to take careful photographs and analyze samples using microbiological probes to confirm the differences between YBD and thermal bleaching.
While Koch's postulates have ostensibly been fulfilled for each of the four Vibrio sp. isolates tested during this study, the postulates lack the flexibility to be applied directly to disease symptoms that may be induced by more than one bacterium. The poorly understood and complex nature of the microbial community associated with the coral makes it very difficult to ascertain the stress conditions needed to initiate the disease. The isolates may be acting as destabilizing agents within the microbial community, allowing some normally innocuous member of the coral-associated microbial community to opportunistically cause infection. It is also possible that each of the isolates directly induces the disease symptoms, either by some common pathogenic mechanism or by disparate means. Variations in the proportions of different bacteria in the consortium could affect the color, width, and seasonal progress rate of the lesions. These questions merit further research, as the answers could determine whether YBD continues to be treated as a single disease or as a collection of symptoms common to several diseases.
The temperatures that induce symbiotic algal stress are between 26 and 30°C, depending on the geographic region. In YBD-infected corals, neither a complete expulsion of dead and damaged algae nor a release of mucus from the coral gastrodermis occurs, as in bleaching. The symbiotic zooxanthellae appear to be degraded inside the gastroderm instead of being expelled during YBD. The corals that are in a weakened state due to higher temperatures are likely to be more susceptible to disease. Past studies have shown that many Vibrio spp., including Vibrio cholerae, are present on the surfaces of marine species but are not toxic until triggered by various stress factors (11).
Temperature may be causing the YB Vibrio spp. to reach their toxic state, but direct demonstration of toxin production remains to be established. Future work investigating the specific virulence factors, including biochemical characterization of the isolates, is warranted (53). Due to the close sequence homology between the Vibrio spp. isolated in this study and V. alginolyticus and Vibrio parahaemolyticus, such studies should include the examination of possible roles of known toxins produced by these species (i.e., various hemolysins) and determining if reference strains of V. parahaemolyticus and V. alginolyticus are capable of inducing disease signs in Montastraea spp.
The symbiotic zooxanthellae appear to be lysed. Similar cell wall damage is evident in harmful algal-bloom species when free-floating dinoflagellates are attacked by Pseudoalteromonas (45). If the mechanism of Pseudoalteromonas can be identified, it may provide insights into how a similar phenomenon occurs in YBD.
Our data suggested that the Vibrio spp. had a stronger effect on the symbiotic dinoflagellate when the temperature was increased. Histological examinations of whole tissues with symbiotic zooxanthellae harbored in the gastrodermal tissue and subjected to various temperatures, namely, 24.5, 29, and 33°C, showed the effects of temperature and pathogens together. These changes are not present when a coral undergoes thermal bleaching without pathogens, as they are expelled through the gastroderm as wholly intact cells. When pathogens are present, they do not trigger the coral to expel zooxanthellae, or to bleach; the increased sea surface temperatures cause the pathogens to be more virulent, resulting in zooxanthella cells dying in situ (Fig. 4).
The degeneration of YB-infected zooxanthella cells is consistent with a decrease in Chl a and c2 pigments, thereby possibly disrupting biochemical reactions normally taking place on the thylakoid membranes during photosynthesis, as seen in thermal bleaching (40). Symbiotic zooxanthellae, chlorophyll a and c2, and densities begin to substantially decrease from the border of the healthy tissue toward the yellow band lesion. The loss of photosynthetic capabilities of the reef builder Montastraea slowly diminishes due to YB exposure (Fig. 8).
According to electron micrographs, disfigured chloroplasts are evident in the zooxanthellae. Redistribution and degeneration of algal organelles accompanied by vacuolization, fragmentation, swelling, and displacement of cytological features are evident (Fig. 6). These symbiotic zooxanthellae are broken down during bacterially induced stress, hindering normal growth of the coral. We further clarified the characteristics of this breakdown due to the decrease in cell density and mitotic indices of YBD-exposed corals. The measurement of zooxanthella loss as a bioassay for assessing stress in coral has been used before (39, 46, 63, 68) (Fig. 7). Future research focusing on longer exposure times of the pathogenic bacteria and its mechanism of action are needed.
This research provides evidence for the correlation between increased temperature and the infectious natures of four marine Vibrio spp. in a major reef-building coral in the Caribbean. YBD can therefore be best interpreted as a primary disease of symbiotic algae that only secondarily affects host coral tissues of the reef builder Montastraea spp. Links of global warming and temperature stress to coral bleaching have been hypothesized (19, 23, 26, 27). High temperatures associated with global warming may exacerbate YBD (8) and other coral diseases (29, 31, 33).
Previous documentation indicated that V. shilonii (44) and V. coralliilyticus (3) caused coral-bleaching-like effects with increased temperature (43). We provide new evidence that indicates that YBD is a symbiont infection that results in in situ zooxanthella degradation rather than expulsion from host tissue on Montastraea spp,. as in thermal bleaching. When Chl a and c2 pigment concentrations decrease due to stress, photosynthesis becomes impaired, decreasing energy and slowing growth (15, 40, 66). It has been found that under thermal-bleaching conditions, Montastrea spp. and other corals have lower zooxanthella chlorophyll a and c2 concentrations than normal pigmented colonies (14, 40, 42). Any future research should make use of various molecular techniques, such as denaturing gradient gel electrophoresis, which could provide further insight into the dynamics of the total microbial community during the disease process. Further experiments using fluorescence in situ hybridization probes of the four bacterial species, exposing the symbiotic algae to YBD-associated Vibrio spp., and comparing the results to normal thermal bleaching would be interesting.
ACKNOWLEDGMENTS We thank the Leslie Jones Foundation and the Global Coral Reef Alliance (GCRA) for partially funding this research.
We also thank the Mote Marine Laboratory (Summerland Key facility) and Patricia Sobecky at the Georgia Institute of Technology for laboratory space needed to carry out important laboratory experiments during this project. We thank Derek Kelly of the New York City Fire Department for field assistance. For scientific discussions and feedback, we thank Robert Trench, Forest Rohwer, Les Kaufman, John Willams, Andrew Baker, Norm Wainwright, and Esther Peters. Special thanks for editorial comments are due to Kathryn Winiarski of the New York Academy of Medicine, Jeff Houdret of the GCRA, Joseph Claro of the science research program at St. Francis Prep Science Research High School in New York City, and an anonymous reviewer who improved the clarity of the manuscript.
FOOTNOTES
* Corresponding author. Present address: Department of Biological Sciences, Pace University, 1 Pace Plaza, New York, NY 10038. Phone: (917) 620-5287. E-mail: cnidaria@earthlink.net
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