Coral disease pathogens of the Indo-Pacific Ocean

Since the identification of coral diseases in the Caribbean in the early 1970's, the number of reported coral disease syndromes, their prevalence and spread worldwide have rapidly increased. Despite increasing reports of coral epizootics resulting in mortalities, little is known about the direct causes of coral diseases. Currently, the study of coral pathogens, their natural reservoirs and possible vectors are still in their early infancy with only five causative agents identified and confirmed by fulfilling Henle-Koch's postulates. Uncertainty regarding the causes of disease has sparked a sharp debate about, whether coral diseases occurring in complex aquatic environments are only caused by primary pathogens, or by secondary pathogens in combinations with other factors, such as ocean warming or anthropogenic stress. The aim of this study is to identify coral pathogens that are directly associated with the following Indo-Pacific scleractinian coral diseases: black band disease (BBD), red bands and white syndromes (WS's), and to clarify their role in disease onset. Filamentous cyanobacteria forming red and black bands on three scleractinian corals from Palau were isolated, cultured and identified based on 16S rRNA gene identity as belonging to a single ribotype. Following trials of a range of specialized media and culture conditions, two media, Grund and ASN III, were identified as the best for successful isolation and culturing. Cultured cyanobacteria were examined under a light microscope to establish purity, color and morphological appearance. DNA extraction and partial sequencing of the 16S rRNA gene of both red and black cyanobacterial isolates demonstrated 100% sequence identity. These isolated strains were also found to have 99% sequence identity with an uncultured cyanobacterial strain previously identified by molecular techniques as belonging to a cyanobacterial ribotype associated with BBD infected corals in the Caribbean. Based on these findings, it is concluded that the classification of these two syndromes as separate coral diseases be postponed until further evidence is collected. Coral pathogens from white syndrome (WS) epizootics in the Indo-Pacific were also investigated. Bacterial isolates were obtained from corals displaying disease signs at three WS outbreak sites: Nikko Bay in the Republic of Palau, Nelly Bay in the central Great Barrier Reef (GBR) and Majuro Atoll in the Republic of the Marshall Islands, and used in laboratory-based infection trials involving exposure of healthy corals to putative bacterial pathogens in order to satisfy three separate criteria for establishing causality: Henle-Koch’s postulates, Evan’s rules and Hill’s criteria. Phylogenetic 16S rRNA gene analysis demonstrated that all six pathogens identified in this study were members of the γ-Proteobacteria family Vibrionacae, each with greater than 98% sequence identity with the previously characterized coral bleaching pathogen Vibrio coralliilyticus. Tests to determine the ability of putative coral pathogens to adhere to corals demonstrated that only the pathogenic strains could transit from aquaria seawater to coral mucus in less than 12 hours. Screening for proteolytic activity of more than 150 coral derived bacterial isolates by a biochemical assay and specific primers for a Vibrio family zinc-metalloprotease demonstrated a significant association between the presence of isolates capable of proteolytic activity and observed disease signs. A Vibrio zinc-metalloprotease, derived from the supernatants of six identified WS pathogens, demonstrated rapid photoinactivation of susceptible Symbiodinium endosymbionts followed by lesions in coral tissue. Symbiodinium photosystem II inactivation was diagnosed by an imaging pulse amplitude modulation fluorometer in two bioassays, performed by exposing Symbiodinium cells and coral juveniles to noninhibited and EDTA-inhibited supernatants. Sequencing of protein bands (using nano LC/MS/MS) retrieved from pathogen supernatants (via protein electrophoresis) identified the Vibrio zinc-metalloprotease as a member of the thermolysin family and a potential virulence factor in the infection process. This virulence factor, which has been previously identified from numerous Vibrio pathogens of fish, mollusks and humans, showed highest activity when pathogen cultures were grown at 27oC and inoculated into susceptible Symbiodinium cultures that were acclimatized to the same temperature. This is the first study to identify coral pathogens on the GBR by fulfilling Henle-Koch's postulates, and the first study to investigate the phylogeny of cyanobacterial strains isolated from corals displaying both red band and black band disease signs. This study also presents novel findings on the aetiology of Indo-Pacific coral diseases, in particular the role of a bacterial virulence factor in causing WS disease signs and the potential effects of host and environmental conditions on its performance. Findings from this study will enable better monitoring of Indo-Pacific coral diseases and their spread in the future, including better understanding of coral pathogen virulence mechanisms and coral disease aetiologies.