Black band disease (BBD) is a cyanobacterial dominated pathogenic consortium that affects corals worldwide. Recently two cyanobacteria (Oscillatoria strains 101-1 and 100-1) were isolated into culture from BBD. The aim of this study was to characterize the strains and assess their role in BBD pathogenesis. Light, scanning electron and transmission electron microscopy, coupled with 16S rRNA gene sequencing, were used for taxonomic characterization. Cyanotoxin production was assessed by enzyme-linked immunosorbent assay. Toxin identification was performed by high performance liquid chromatography. The ability of the strains to initiate BBD was tested on host coral fragments of Siderastrea siderea and Diploria strigosa under controlled laboratory conditions. Results showed that both Oscillatoria sp. strains caused infection that resulted in complete lysis of coral tissue. Both strains produced a cyanotoxin, identified as microcystin-LR, with production affected by different environmental factors. This study provides evidence that BBD Oscillatoria is a key component in BBD pathogenicity.

Coral diseases were unknown in the scientific community fifty years ago. Since the discovery of a coral disease in 1965, there has been an exponential increase in the number of known coral diseases, as the abundance, prevalence, distribution, and number of host species affected has also significantly increased. Coral diseases are recognized as contributing significantly to the dramatic losses of coral cover on a global basis, particularly in the Caribbean. The apparent sudden emergence of coral diseases suggests that they may be a symptom of an overall trend associated with changing environmental conditions. However, not much evidence has been gathered to address this question. The following studies were designed to build a comprehensive argument to support this hypothesis for one important coral disease – black band disease (BBD). A meta-analysis of clone libraries identifying the microbial communities associated with BBD reveal important information including that a single cyanobacterial operational taxonomic unit (OTU) was by far the most prevalent OTU in diseased samples, and that the alphaproteobacteria, which include some of the most common bacteria in marine waters, were the most diversely represented. The analysis also showed that samples exhibited regional similarities. An fine and ultrastructural characterization of the disease revealed that the cyanobacteria are prolific borers through the coral skeleton, and that the cyanobacteria penetrate coral tissue, leading to their presence ahead of the main migrating disease band. It was further found that apparently healthy corals exposed to toxins found in BBD, exhibited similar tissue degradation to those infected with BBD. Comparing the disease progression to biofilm formation, it was determined that scouting cyanobacteria may contribute to the migration of the disease through progressive biofilm development over intact coral tissue. Together, these studies provide significant evidence for the hypothesis that BBD is an opportunistic disease, caused by common environmental bacteria, facilitated by the changing environmental conditions associated with climate change.

The black band disease (BBD) microbial consortium often causes mortality of reef-building corals. Microbial chemical interactions (i.e., quorum sensing (QS) and antimicrobial production) may be involved in the BBD disease process. Culture filtrates (CFs) from over 150 bacterial isolates from BBD and the surface mucopolysaccharide layer (SML) of healthy and diseased corals were screened for acyl homoserine lactone (AHL) and Autoinducer-2 (AI-2) QS signals using bacterial reporter strains. AHLs were detected in all BBD mat samples and nine CFs. More than half of the CFs (~55%) tested positive for AI-2. Approximately 27% of growth challenges conducted among 19 isolates showed significant growth inhibition. These findings demonstrate that QS is actively occurring within the BBD microbial mat and that culturable bacteria from BBD and the coral SML are able to produce QS signals and antimicrobial compounds. This is the first study to identify AHL production in association with active coral disease.

On the reefs of Bermuda, the coral Diploria labyrinthiformis is rarely infected with black band disease (BBD) while BBD-infected colonies of D. strigosa are common. This study investigated several possible explanations for the documented difference in BBD prevalence including: 1) temperature and light conditions differentially affect the progression of the disease among these two species; 2) the bacterial communities associated with each species are different and 3) the bacterial composition of BBD in Bermuda is unique from that of other geographical regions where D. labyrinthiformis has been reported with BBD. Results suggest that light and temperature are important variables in determining BBD progression for both species; reef location, rather than coral species dictates the coral associated bacterial diversity; and the BBD bacterial mat in Bermuda is unique compared to other regions of the Caribbean. Many factors are likely responsible for the low occurrence of BBD among D. labyrinthiformis in Bermuda.

Coral reefs are the proverbial rainforests of the ocean, but these spectacular structures are under threat from globally rising sea surface temperatures and ocean acidification. The Red Sea and the Persian/Arabian Gulf (PAG) display unusually high sea surface temperatures, and therefore, provide a model for studying environmental change. Corals are so-called holobionts consisting of the coral host, photosynthetic algae (Symbiodiniaceae), along with other microorganisms, such as bacteria, archaea, fungi, and viruses. While the importance of bacteria to coral holobiont functioning is acknowledged, little is known about changes in the microbial communities under natural environmental stressors in the Red Sea and the PAG. Accordingly, I investigated microbial community and mucus differences in bleached, healthy, and diseased corals. Analysis of the composition of mucus-associated microbial communities of bleached and healthy Porites lobata colonies from the Red Sea and the PAG were stable, although some regional differences were present. In a distinct study investigating coral disease, a broad range of corals in the Red Sea were shown to be infected with black band disease (BBD). Investigating the microbial community associated with BBD revealed the presence of the three main indicators for BBD (cyanobacteria, sulfate-reducing bacteria (SRB), and sulfide-oxidizing bacteria (SOB). Last, I investigated the chemical composition (carbohydrates) of the surface mucus layer of a range of Red Sea corals. Given that coral mucus represents a first line of defense, I was interested to examine whether mucus carbohydrate composition would point to a role of adaptation to the extreme environment of the Red Sea. This analysis showed that mucus consists of conserved sugars that are globally conserved. In summary, this thesis characterizes the microbial communities associated with a range of coral species in different health states (bleached, healthy, and diseased). The microbial community patterns I characterized support the notion that bacteria contribute to coral holobiont health and possibly adaptation to extreme environmental conditions in the Red Sea and PAG.

Planktothricoides raciborskii and Roseofilum reptotaenium are physiologically similar, yet ecologically distinct organisms found in a hot spring outflow and coral black band disease (BBD), respectively. The aim of this study was to elucidate the relationship between R. reptotaenium and sulfide in BBD, to compare microcystin (MC) production in response to environmental factors, and to determine chemotactic responses to MC and sulfide by the two organisms. Results showed that the pathogenicity of R. reptotaenium in BBD is dependent on sulfate-reducing bacteria as secondary pathogens. Roseofilum reptotaenium produced significantly more MC than P. raciborskii, as measured using ELISA. Roseofilum reptotaenium was negatively chemotactic to sulfide, determined using horizontal and vertical gradients in agar, while P. raciborskii was not affected. Neither cyanobacterium was chemotactic to MC in the agar assays. The ecophysiology of P. raciborskii and R. reptotaenium in relation to MC production and response to sulfide reflected their pathogenic versus non-pathogenic status.

Black band disease (BBD) of corals is a temperature dependent, highly virulent, polymicrobial disease affecting reef-building corals globally. The microbial consortium of BBD is primarily comprised of functional physiological groups that include photosynthetic cyanobacteria, sulfate reducers, sulfide oxidizers and a vast repertoire of heterotrophic bacteria. Quorum sensing (QS), the cell-density dependent communication phenomenon in bacteria, is known to induce expression of genes for a variety of virulence factors in diseases worldwide. Microbes capable of QS release signals such as acyl homoserine lactones (AHLs) and autoinducer-2 (AI-2), which coordinate microbial interaction. The focus of the present study was to investigate the presence and potential role of QS in BBD pathogenicity, utilizing culture dependent and independent methodologies. Isolates across coral health states including BBD, were screened for production of QS signals, and AHL and AI-2 production capabilities were analyzed via LC-MS/MS. The effect of temperature on AHLs was also examined. Additionally, antimicrobial production capabilities of isolates were tested. BBD metagenomes were utilized to screen for sequences related to QS, antimicrobial synthesis, and antimicrobial resistance genes. BBD isolates represented a significantly higher proportion of isolates capable of producing QS signals in comparison to healthy coral isolates. Several AHLs produced by coral derived bacterial cultures were identified, and three AHLs, specifically 3OHC4, 3OHC5 and 3OHC6, showed a significant increase in production at an elevated temperature of 30 °C, which correlates with increased BBD incidence on reefs with increasing water temperature. Most of the BBD cultured isolates were identified as vibrios. Several sequences related to QS, antimicrobial synthesis and resistance genes were detected in the BBD metagenomes. Based on the findings of this study, a model for potential microbial interactions amongst BBD heterotrophs, centered around QS, is proposed. Taken together, the findings from this study provide a clearer understanding of the potential role of QS in BBD, and serve as the basis for further studies aimed at elucidating the pathogenesis of an intricate coral disease.