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.

Coral reefs are in decline worldwide and coral disease is a significant contributing factor. However, etiologies of coral diseases are still not well understood. In contrast with the Caribbean, extremely little is known about coral diseases in the Philippines. In 2005, off Southeast Negros Island, Philippines, I investigated relationships between environmental parameters and prevalence of the two most common coral diseases, ulcerative white spot (UWS) and massive Porites growth anomalies (MPGAs). Samples were collected along a disease prevalence gradient 40.5 km long. Principal component analyses showed prevalence of MPGAs was positively correlated with water column nitrogen, organic carbon of surface sediments, and colony density. UWS was positively correlated with water column phosphorus. This is the first quantitative evidence linking anthropogenically-impacted water and sediment to a higher prevalence of these diseases. Histological and cytological alterations were investigated by comparing tissues from two distinct types of MPGA lesions (types 1 and 2) and healthy coral using light and electron microscopy. Skeletal abnormalities and sloughing, swelling, thinning, and loss of tissues in MPGAs resembled tissues exposed to bacterial or fungal toxins. Both lesion types had decreases in symbiotic zooxanthellae, which supply nutrients to corals. Notable alterations included migrations of chromophore cells (amoebocytes) (1) nocturnally to outer epithelia to perform wound-healing, including plugging gaps and secreting melanin in degraded tissues, and (2) diurnally to the interior of the tissue possibly to prevent shading zooxanthellae in order to maximize photosynthate production. Depletion of melanin (active in wound healing) in type 2 lesions suggested type 2 tissues were overtaxed and less stable. MPGAs contained an abundance of endolithic fungi and virus-like particles, which may result from higher nutrient levels and play roles in disease development. Swollen cells and mucus frequently blocked gastrovascular canals (GVCs) in MPGAs. Type 1 lesions appeared to compensate for impeded flow of wastes and nutrients through these canals with proliferation of new GVCs, which were responsible for the observed thickened tissues. In contrast, type 2 tissues were thin and more degraded. Dysplasia and putative neoplasia were also observed in MPGAs which may result from the tissue regeneration capacity being overwhelmed.

Coral reef ecosystems are in rapid decline due to global and local anthropogenic factors. Being among the most diverse ecosystems on Earth, a loss will decrease species diversity, and remove food source for people along the coast. The coral together with its symbionts (i.e. Symbiodinium, bacteria, and other microorganisms) is called the ‘coral holobiont’. The coral host offers its associated symbionts suitable habitats and nutrients, while Symbiodinium and coral-associated bacteria provide the host with photosynthates and vital nutrients. Association of corals with certain types of Symbiodinium and bacteria confer coral stress tolerance, and lack or loss of these symbionts coincides with diseased or bleached corals. However, a detailed understanding of the coral holobiont diversity and structure in regard to diseases and health states or across global scales is missing. This dissertation addressed coral-associated symbiont diversity, specifically of Symbiodinium and bacteria, in various coral species from different geographic locations and different health states. The main aims were (1) to expand the scope of existing technologies, (2) to establish a standardized framework to facilitate comparison of symbiont assemblages over coral species and sites, (3) to assess Symbiodinium diversity in the Arabian Seas, and (4) to elucidate whether coral health states have conserved bacterial footprints. In summary, a next generation sequencing pipeline for Symbiodinium diversity typing of the ITS2 marker is developed and applied to describe Symbiodinium diversity in corals around the Arabian Peninsula. The data show that corals in the Arabian Seas are dominated by a single Symbiodinium type, but harbor a rich variety of types in low abundant. Further, association with different Symbiodinium types is structured according to geographic locations. In addition, the application of 16S rRNA gene microarrays to investigate how differences in microbiome structure relate to differences in health and disease demonstrate that coral species share common microbial footprints in phenotypically similar diseases that are conserved between regional seas. Moreover, corals harbor bacteria that are species-specific and distinct from the diseased microbial footprints. The existence of conserved coral disease microbiomes allows for cataloging diseases based on bacterial assemblage over coral species boundaries and will greatly facilitate future comparative analyses.

Caribbean reef ecosystems have undergone major ecological changes in the last 30 – 40 years, with the result that ecological systems once dominated by structurally complex Acropora cervicornis and Montastraea annularis corals now consist mainly of flattened carbonate substrates with macroalgal overgrowth. A need for greater understanding of coral reef ecosystems is imperative if we are to attempt to conserve them. The threespot damselfish, Stegastes planifrons, is herbivorous damselfish species ubiquitous to Caribbean reefs, where it has been termed a keystone species. Aggressive in nature, S. planifrons defends territories of around 70 cm in diameter from other roving herbivorous fish and urchins, in apparent effort to maintain the algal resources therein for its own use. The predilection of Stegastes planifrons for basing its territories on the now Critically Endangered staghorn coral, Acropora cervicornis, and the Endangered boulder coral Montastraea annularis is well known, however the likely ecological implications of this fact have not been investigated. Using a combination of experimental and observational methodologies we examine the ecological implications of coral microhabitat choice and use by S. planifrons. We also assess the magnitude of the direct and indirect effects of S. planifrons’ territorial behaviour on macroalgal dynamics both within and outside of territory confines, at the reef-wide level. We find that coral microhabitat is a more important determinant of algal community structure than damselfish presence, and that this can be explained by a previously unrecognised effect of coral microhabitat on the grazing behaviour of roving herbivorous fishes - on which S. planifrons’ territorial behaviour has little effect. In a modification of the space availability hypothesis of Williams et al (2001) we suggest that Acropora cervicornis acts as a grazing fish „exclusion zone‟, and we further hypothesise that the existence of large stands of this coral prior to the Caribbean „phase shift‟ may have acted to concentrate the grazing pressure of excluded roving fish onto the remaining areas of the reef. We further hypothesise that the loss of such „exclusion zones‟ and accompanied effective dilution of grazing pressure may have been on a scale large enough to have been a significant underlying factor in the proliferation of macroalgae seen on modern day Caribbean reefs. In the absence of demonstrable direct or indirect effects on benthic algal communities we question the continued keystone status of S. planifrons, particularly since the status 6 was originally based on interference behaviour involving the important grazing urchin Diadema antillarum, which is now functionally absent from Caribbean reefs. Implications of the context-dependant nature of keystone status are also discussed. We find that the effect of S. planifrons on coral community may be more important than its effects on benthic algal community. In examining the factors involved in habitat coral choice we establish a significant preference for 100% live coral substrate over substrates with a supply of algal food. Territory selection was followed by a high rate of coral biting – a behaviour which has previously been shown to result in coral tissue death and the fast establishment of algal turf communities on which S. planifrons likes to feed (Kaufman 1977). We also demonstrate a novel and significant association between S. planifrons presence and disease incidence its primary habitat coral, the Critically Endangered staghorn coral Acropora cervicornis, and a significant correlation between areas of fish biting and the later onset of disease. Changes to the overall role of damselfish on today's Caribbean reefs are discussed in light of these insights.

The iconic pillar coral, Dendrogyra cylindrus, is one of five Caribbean species listed in 2014 under the US Federal Endangered Species Act because of its extreme low abundance and continued decline in US waters. Until recently, little was known about the demographics or genetic diversity of Florida’s D. cylindrus population. This study represents the first time two holobiont partners (coral animal and associated photosynthetic algal endosymbionts) have been closely examined, spatially and temporally, in this little-studied species. The aim was to explore the influences of coral animal genotypes, mutualistic photosynthetic algal strains, and hyperthermal stress on bleaching and disease processes, resistance, and recovery through two consecutive hyperthermal events on the Florida Reef Tract (FRT) in 2014 and 2015. Through geographically stratified, triannual assessments and tissue sampling of D. cylindrus colonies across three regions of the FRT from April 2014 to April 2016, I compared genotypic identities of the coral animal to bleaching and disease status and recovery. Additionally, I characterized the algal endosymbionts (Symbiodiniaceae family) in D. cylindrus between regions of the FRT using Illumina amplicon sequencing of the partial chloroplast 23S rDNA Domain V gene and correlated them to differential responses during bleaching and recovery. Finally, I examined the effects of hyperthermal stress on disease prevalence and changes in disease susceptibility in D. cylindrus throughout two consecutive hyperthermal events in 2014 and 2015. Genotypic differences in D. cylindrus were associated with full or partial bleaching and/or disease resistance associated with some genets. Additionally, this study characterized unexpected diversity in the Symbiodiniaceae community within D. cylindrus and a site-specific, species-level switch in endosymbionts associated with acquired bleaching resistance during the 2015 hyperthermal event. Finally, this study demonstrated that two consecutive hyperthermal events were associated with an increase in prevalence of white plague in D. cylindrus and contributed to its susceptibility to black band disease, documented for the first time on the FRT. Through understanding the response of the D. cylindrus holobiont partners to biotic and abiotic stressors, such as hyperthermal bleaching and associated diseases, we gained valuable insights into how this threatened species may respond to a changing climate.

Climate change is now widely regarded as the single greatest threat to coral reefs. Climate change poses a range of different threats and reef ecosystems are widely reported as being amongst the first ecosystems to be severely affected by increases in global average temperatures. Coral reef managers responding effectively to the climate change threat will require research and monitoring, communication, impact mitigation and informed planning. Aspects of this thesis cover all of those working areas.

Les coraux vivent en association symbiotique avec des algues dinoflagellées du genre Symbiodinium, distinguées en différents clades (A à I). Un même hôte peut abriter seul ou plusieurs de ces clades, a priori dépendant d’un contexte environnemental, suggérant qu’ils possèdent des propriétés physiologiques distinctes. Ainsi, les capacités de flexibilité et d’acquisition de clades pourraient être une issue pour la survie de l’holobionte face à divers stress. Dans le cadre de cette thèse, ces capacités ont été explorées chez plusieurs espèces coralliennes, décrites avec différentes flexibilités et sensibilités aux conditions environnementales. La diversité et la dynamique des communautés de Symbiodinium associées aux coraux ont été estimées à l’aide de la technique de PCR en temps réel et évaluées dans différents contextes environnementaux: naturels, lors d’un suivi spatio-temporel de 18 mois à Moorea, ou contrôlés, lors d'une incubation à différentes pCO2. Les coraux ont montré la possibilité d’héberger jusqu’à 3 ou 4 clades distincts, reconsidérant la notion de spécialiste (i.e. symbionte unique). Néanmoins, propre à l’hôte corallien, il a été mis en évidence la régulation de ‘signatures symbiotiques’ caractérisées par des profils particuliers d’association de clades, stables et durables. Des modifications sporadiques de ces profils, par switching et shuffling ont put être observées, indépendamment d’un contexte environnemental donné, suggérant la dynamique comme un mécanisme constitutif chez les coraux. Néanmoins, en cas de d’apparition d’épizooties de Vibrio spp. et d’une maladie corallienne il est suggéré que le clade D participe activement au ‘fitness’ de leur hôte. / Corals live in symbiosis with dinoflagellate algae of the genus Symbiodinium, divided into 9 clades (A to I). The same host can harbour one or more of these clades depending on the environmental context. This lead to the suggestion that each clade has its unique physiological property. Therefore the capacity of flexibility and acquisition of clades may be an issue for the survival of the holobiont against various stresses. In this thesis, these capacities have been explored in several coral species, described with various flexibilities and sensitivities facing different environmental conditions. The diversity and dynamics of Symbiodinium communities associated to corals were estimated by using the real-time PCR technique, and evaluated in different environmental contexts : natural, in an 18-month spatio-temporal survey around Moorea, or controlled, upon incubation with different pCO2. Some corals showed the ability to host up to 3 or 4 distinct clades, leading to a reconsideration of the specialist-concept (i.e. host associated with one symbiont type). Nevertheless, depending on the coral host, ‘symbiotic signature’ regulations have been revealed. Latter are characterized by specific clade combination profils, which are stable and sustainable. In addition, sporadic changes by switching and shuffling, were observed in a given profil, regardless of a given environmental context, suggesting the dynamics as an constitutive mechanism in corals. However, records of coral disease together with Vibrio spp outbreaks, suggested an active participation of clade D for the ‘fitness’ of the coral host.

The high latitude thickets of Acropora cervicornis off Broward County flourish despite the presence of natural and anthropogenic impacts. These populations provided a unique study area in contrast to disease-stricken populations of the Florida Keys. This study used time-sequenced photographs to examine how A. cervicornis was affected by tissue loss attributed to white-band disease during 2007–2008. Variables monitored included healthy colony skeletal extension rates, diseased colony skeletal extension rates, and tissue loss. The transmissibility of the three white-band syndromes found in the Scooter and Oakland thickets was examined through tissue grafting experiments. Skeletal extension rates of healthy and diseased colonies were generally not significantly different. Mean skeletal extension for A. cervicornis colonies in Broward County was observed to be 9.6 cm/y (SD=3.95, Range: 1.02–19.9). Mean linear tissue loss from disease signs was 2.6 mm/d (SD=4.3, Range: 0.023–16.8). Although the majority of active disease lesions caused severe tissue loss upon contact with healthy branches, in 25% of the cases there was no tissue loss. Disease signs were also observed in 10% of the control grafting trials. A. cervicornis thickets in Broward County were growing at rates similar to those observed in this species elsewhere in Florida, but faster than other areas of the Western Atlantic. Tissue loss rate from disease lesions was lower than reported elsewhere. White-band disease and/or other tissue loss syndromes are always present in Broward County, but the low prevalence of affected colonies, inconsistent transmission of a presumptive agent that causes the disease signs, and optimum branch skeletal extension seems to limit effects on the thickets. Results of this research are significant as the current protected status of acroporid corals no longer allows manipulative research such as coral grafting for transmissibility of potential disease pathogens.

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.