Viruses: contributors to and mitigators of black band disease in corals

Coral reefs worldwide have seen a considerable increase in coral disease prevalence and disease-related mortality over the last few decades. Black band disease (BBD) is a common and widespread disease of reef-building corals that causes tissue loss at a rate of up to two cm/day. The polymicrobial disease is characterised by a dense, dark mat comprising numerous bacterial taxa including filamentous cyanobacteria, the most prevalent bacterium in terms of biomass. BBD-associated cyanobacteria contribute to the disease by providing a framework and structure to the other bacteria and by fuelling the disease progression with their photosynthesis products. The cyanobacterium Roseofilum reptotaenium of the genus Oscillatoria is ubiquitously present in BBD. Despite years of bacterium-centred research on BBD, key questions, such as the direct cause of the initial disease onset and disease mitigation, remain unclear. Viruses are known agents of a number of diseases in the marine environment, but little is known about their roles in coral disease, including BBD. In this thesis, I examine whether viruses that infect bacteria (bacteriophages) are involved in the virulence of the main BBD-associated cyanobacteria and progression of BBD. This includes: (1) the characterisation of the bacteriophage community that targets BBD-associated cyanobacteria, (2) the establishment of methodologies to cultivate the main BBD cyanobacterium, R. reptotaenium, and to isolate lytic bacteriophages that target R. reptotaenium, (3) an assessment of the role of lytic bacteriophages as potential mitigators of the disease by lysing R. reptotaenium and (4) an examination of lysogenic bacteriophages as potential contributors to the virulence of their hosts by introducing virulence genes into BBD cyanobacteria genomes. (1) Using amplicon next generation sequencing and bioinformatic analyses, I described the BBD bacteriophage community and showed that bacteriophages are part of the BBD microbial consortium. The bacteriophage community of tissue samples of BBD and healthy corals showed a generally higher variability than the communities prevalent in seawater samples. Nevertheless, several BBD-associated bacteriophage taxa were exclusively abundant in BBD samples and I hypothesised these infect one of the main BBD cyanobacteria; other bacteriophage taxa were more prevalent in healthy coral tissues. (2) To further clarify the role of BBD-associated bacteriophages, I optimised existing methodologies for the isolation and cultivation of one of the main BBD cyanobacteria, R. reptotaenium. The newly established protocols serve as a basis for the isolation of lytic and lysogenic bacteriophages that target R. reptotaenium. (3) Lysis of R. reptotaenium cultures was successfully achieved using protocols commonly applied for lytic bacteriophage isolation. Three bacteriophages that potentially target the R. reptotaenium were isolated from the culture medium, their genomes sequenced, and used in a phage therapy trial on BBD-affected corals. The isolated bacteriophages are closely related to Cellulophaga phages and therefore not known to infect cyanobacteria and not suitable for BBD phage therapy. Currently, phage therapy of BBD might be possible, but requires extensive optimisation in order to successfully mitigate the disease. (4) To investigate whether lysogenic bacteriophages are present in the R. reptotaenium genome and whether they may increase cyanobacterial virulence, I sequenced and assembled the first draft genome of R. reptotaenium and in conjunction analysed a previously published genome of the BBD cyanobacterium Geitlerinema sp. BBD_1991. Both cyanobacteria were equipped with adaptive, heritable defence systems that help to prevent bacteriophage infections (clustered regularly interspaced short palindromic repeats – CRISPR-Cas systems). Bacteriophage taxa that previously infected the cyanobacteria were reconstructed by analysing the target sequences (spacers) of the CRISPR-Cas defence systems, and were found to include taxa of the BBD bacteriophage community (Chapter 2). In addition, potential prophages were identified in three regions of the R. reptotaenium AO1 genome and in five regions of the Geitlerinema sp. BBD_1991 genome. These genomic regions contained putative virulence genes relevant of BBD, such as an NAD-dependent epimerase/dehydratase (a gene with homologue function to the third and fourth most expressed gene in BBD), lysozyme/metalloendopeptidases and other genes for lipopolysaccharide modification. My findings suggest that the BBD disease mat is a hot-spot for phage infections. The presence of CRISPR-Cas defence systems in the cyanobacterial host genomes provides evidence of a constant arms race between BBD-associated cyanobacteria and bacteriophages. Maintaining such a defence system likely reduces the number of successful bacteriophage infections and mortality in the cyanobacteria, supporting the progress of BBD. The disease related genes in potential prophage regions suggest a role of bacteriophages as contributors to the virulence of BBD cyanobacteria. In addition, bacteriophages that successfully lyse cyanobacteria within the mat might be redistributing organic matter to the BBD microbial consortium as part of a viral shunt and also possibly contribute to the progression of the disease. Since the application of antibiotics is not feasible to control BBD due to the complexity of coralmicrobial interactions, the use of a lytic bacteriophage in a phage therapy is a promising approach and should be developed further. This study is one of the first to investigate the multi-faceted role that bacteriophages have in coral disease and reveals bacteriophages as potential contributors to and mitigators of BBD. Applying the concepts and approaches of this study to other coral diseases is likely to show bacteriophages as hidden drivers, e.g. in the coral disease white syndrome and potentially even coral bleaching. Future studies should focus on characterising the associated virus community of healthy and diseased coral colonies with viral metagenomic approaches to obtain information on baseline communities and investigate individual virus-host interactions with quantitative PCR, classic virology methods and targeted amplicon sequencing. Viruses in coral diseases may hold the key to important answers regarding the virulence and pathogenicity of pathogens, as well as innovative mitigation strategies to improve coral health with phage therapy approaches