Cytotoxic activity of Black Band Disease (BBD) extracts against the symbiotic dinoflagellate Symbiodinium sp. Jorge FRIAS-LOPEZ*, James S. KLAUS & Bruce W. FOUKE Department of Geology, University of Illinois, 1301 W. Green Street, Urbana, IL 61801, USA. *Corresponding author. Jorge Frias-Lopez, Present address: MIT, Department of Civil and Environmental Engineering, 15 Vassar St. 48-336b e-mail:jfrias@mit.edu Abstract. Black band disease (BBD) is an infectious bacterial disease caused by the migration of a black mat of microorganisms across the surface of coral colonies, consuming healthy coral tissue and leaving dead skeleton behind. Although a complex community of bacteria composes the infectious bacterial mat, the most abundant organism present in the mat is a large filamentous, non-heterocyst cyanobacterium, identified as Phormidium corallyticum. In spite of our knowledge on the composition of the BBD mat, the mechanisms by which this disease destroys coral tissue remains unclear. The results presented in this paper show that an extract from the BBD mat has toxic effects on the growth and viability of the symbiotic dinoflagellate Symbiodinium sp. Nonetheless, neither the pellet recovered from the extraction nor extracts from other bacteria have such an effect, suggesting that neither LPS nor membrane proteins are involved in BBD toxicity. Moreover, this compound has a molecular weight lower than 10,000 Da and is heat stable. All these results indicate that there is a toxic compound possibly involved in the mechanism of pathogenesis of BBD in corals. Further studies will involve the isolation and characterization of such compound. Keywords coral, black band disease, toxin, cyanobacteria. Introduction Coral diseases have recently been recognized as key elements in the health status of coral reefs. Reports describing new coral diseases and their incidence have been increasingly appearing in the literature. However, despite the new awareness of their importance, our knowledge on the etiology and origin of these diseases is still limited. Black band disease (BBD) is one of the most widespread and well-studied coral diseases. It is well established that BBD is an infectious disease caused by the activity of a complex bacterial mat. This bacterial mat forms a ring-shaped black band that moves from top-to-bottom across coral colonies. BBD is able to infect a large number of different species of corals. In the Caribbean, BBD has frequently been detected infecting colonies of Diploria strigosa , D. labyrinthoformis , Montastrea annularis , and M. cavernosa (Antonius 1981; Edmunds 1991; Rützler and Santavy 1983). These are among the most ecologically important scleractinian corals in those reefs and therefore coral mortality caused by BBD is an important force in the restructuring of these coral reef communities (Edmunds 1991; Kuta and Richardson 1997). Moreover, once the coral is infected by BBD the bacterial mat migrates at a high rate, destroying the coral colony in a short period of time. Although the bacterial mat causing BBD has been characterized, the cause, origin and mechanisms of infection of the disease remain unknown. By far, the most abundant organism in the BBD microbial mat is a large filamentous cyanobacterium, identified optically as Phormidium corallyticum (Richardson 1998; Rützler and Santavy 1983). Nonetheless, the BBD mat is composed of highly complex bacterial communities, with a number of different bacterial species that are not found on healthy coral or surrounding seawater (Cooney et al. 2002; Frias-Lopez et al. 2002). The role of specific BBD microorganisms in the progression of the disease remains unclear. In this study, we present results indicating that the production of a small toxic compound that has cytotoxic activity against Symbiodinium sp., the symbiotic dinoflagellate living in corals. These results may explain in part the role of Phormidium corallyticum in the disease as well as the mechanism of tissue destruction. Material and Methods BBD Sample Collection Sampling was conducted using standard SCUBA techniques on the coral reefs of Curaçao, Netherlands Antilles. Colonies of Diploria strigosa exhibiting the distinct BBD ring were sampled at water depths of approximately 3m near the Curaçao Water Plant. Portions of active BBD mats were physically peeled off the infected coral surfaces using forceps and placed in 15ml Falcon tubes filled with RNAlater (Ambion, Austin, TX) and samples were then immediately frozen at –20 o C.