Sponge-derived matter is assimilated by coral holobionts.

Coral reef biodiversity is maintained by a complex network of nutrient recycling among organisms. Sponges assimilate nutrients produced by other organisms like coral and algae, releasing them as particulate and dissolved matter, but to date, only a single trophic link between sponge-derived dissolved matter and a macroalgae has been identified. We sought to determine if sponge-coral nutrient exchange is reciprocal using a stable isotope 'pulse-chase' experiment to trace the uptake of 13C and 15N sponge-derived matter by the coral holobiont for three coral species (Acropora cervicornis, Orbicella faveolata, and Eunicea flexuosa). Coral holobionts incorporated 2.3-26.8x more 15N than 13C from sponge-derived matter and A. cervicornis incorporated more of both C and N than the other corals. Differential isotopic incorporation among coral species aligns with their ecophysiological characteristics (e.g., morphology, Symbiodiniaceae density). Our results elucidate a recycling pathway on coral reefs that has implications for improving coral aquaculture and management approaches.

Limited Metabolomic Overlap between Commensal Bacteria and Marine Sponge Holobionts Revealed by Large Scale Culturing and Mass Spectrometry-Based Metabolomics: An Undergraduate Laboratory Pedagogical Effort at Georgia Tech.

Sponges are the richest source of bioactive organic small molecules, referred to as natural products, in the marine environment. It is well established that laboratory culturing-resistant symbiotic bacteria residing within the eukaryotic sponge host matrix often synthesize the natural products that are detected in the sponge tissue extracts. However, the contributions of the culturing-amenable commensal bacteria that are also associated with the sponge host to the overall metabolome of the sponge holobiont are not well defined. In this study, we cultured a large library of bacteria from three marine sponges commonly found in the Florida Keys. Metabolomes of isolated bacterial strains and that of the sponge holobiont were compared using mass spectrometry to reveal minimal metabolomic overlap between commensal bacteria and the sponge hosts. We also find that the phylogenetic overlap between cultured commensal bacteria and that of the sponge microbiome is minimal. Despite these observations, the commensal bacteria were found to be a rich resource for novel natural product discovery. Mass spectrometry-based metabolomics provided structural insights into these cryptic natural products. Pedagogic innovation in the form of laboratory curricula development is described which provided undergraduate students with hands-on instruction in microbiology and natural product discovery using metabolomic data mining strategies.

Transmission studies and the composition of prokaryotic communities associated with healthy and diseased Aplysina cauliformis sponges suggest that Aplysina Red Band Syndrome is a prokaryotic polymicrobial disease.

Aplysina cauliformis, the Caribbean purple rope sponge, is commonly affected by Aplysina Red Band Syndrome (ARBS). This transmissible disease manifests as circular lesions with red margins and results in bare spongin fibers. Leptolyngbya spp. appear to be responsible for the characteristic red coloration but transmission studies with a sponge-derived isolate failed to establish disease, leaving the etiology of ARBS unknown. To investigate the cause of ARBS, contact transmission experiments were performed between healthy and diseased sponges separated by filters with varying pore sizes. Transmission occurred when sponges were separated by filters with pore sizes ≥ 2.5 µm, suggesting a prokaryotic pathogen(s) but not completely eliminating eukaryotic pathogen(s). Using 16S rRNA gene sequencing methods, 38 prokaryotic taxa were significantly enriched in diseased sponges, including Leptolyngbya, whereas seven taxa were only found in some, but not all, of the ARBS-affected sponges. These results do not implicate a single taxon, but rather a suite of taxa that changed in relative abundance with disease, suggesting a polymicrobial etiology as well as dysbiosis. As a better understanding of dysbiosis is gained, changes in the composition of associated prokaryotic communities may have increasing importance for evaluating and maintaining the health of individuals and imperiled coral reef ecosystems.

Microbiome Analyses Demonstrate Specific Communities Within Five Shark Species.

Profiles of symbiotic microbial communities ("microbiomes") can provide insight into the natural history and ecology of their hosts. Using high throughput DNA sequencing of the 16S rRNA V4 region, microbiomes of five shark species in South Florida (nurse, lemon, sandbar, Caribbean reef, and tiger) have been characterized for the first time. The microbiomes show species specific microbiome composition, distinct from surrounding seawater. Shark anatomical location (gills, teeth, skin, cloaca) affected the diversity of microbiomes. An in-depth analysis of teeth communities revealed species specific microbial communities. For example, the genus Haemophilus, explained 7.0% of the differences of the teeth microbiomes of lemon and Caribbean reef sharks. Lemon shark teeth communities (n = 11) contained a high abundance of both Vibrio (10.8 ± 26.0%) and Corynebacterium (1.6 ± 5.1%), genera that can include human pathogenic taxa. The Vibrio (2.8 ± 6.34%) and Kordia (3.1 ± 6.0%) genera and Salmonella enterica (2.6 ± 6.4%) were the most abundant members of nurse shark teeth microbial communities. The Vibrio genus was highly represented in the sandbar shark (54.0 ± 46.0%) and tiger shark (5.8 ± 12.3%) teeth microbiomes. The prevalence of genera containing potential human pathogens could be informative in shark bite treatment protocols and future research to confirm or deny human pathogenicity. We conclude that South Florida sharks host species specific microbiomes that are distinct from their surrounding environment and vary due to differences in microbial community composition among shark species and diversity and composition among anatomical locations. Additionally, when considering the confounding effects of both species and location, microbial community diversity and composition varies.

Microbial community shift under exposure of dredged sediments from a eutrophic bay.

Microbial communities occur in almost every habitat. To evaluate the homeostasis disruption of in situ microbiomes, dredged sediments from Guanabara Bay-Brazil (GB) were mixed with sediments from outside of the bay (D) in three different proportions (25%, 50%, and 75%) which we called GBD25, GBD50, and GBD75. Grain size, TOC, and metals-as indicators of complex contamination-dehydrogenase (DHA) and esterase enzymes (EST)-as indicators of microbial community availability-were determined. Microbial community composition was addressed by amplifying the 16S rRNA gene for DGGE analysis and sequencing using MiSeq platform (Illumina).We applied the quality ratio index (QR) to the GB, D, and every GBD mixture to integrate geochemical parameters with our microbiome data. QR indicated high environmental risk for GB and every GBD mixture, and low risk for D. The community shifted from aerobic to anaerobic profile, consistent with the characteristics of GB. Sample D was dominated by JTB255 marine benthic group, related to low impacted areas. Milano-WF1B-44 was the most representative of GB, often found in anaerobic and sulfur enriched environments. In GBD, the denitrifying sulfur-oxidizing bacteria, Sulfurovum, was the most representative, typically found in suboxic or anoxic niches. The canonical correspondence analysis was able to explain 60% of the community composition variation and exhibit the decrease of environmental quality as the contamination increases. Physiological and taxonomic shifts of the microbial assemblage in sediments were inferred by QR, which was suitable to determine sediment risk. The study produced sufficient information to improve the dredging plan and management.

Host population genetics and biogeography structure the microbiome of the sponge Cliona delitrix.

Sponges occur across diverse marine biomes and host internal microbial communities that can provide critical ecological functions. While strong patterns of host specificity have been observed consistently in sponge microbiomes, the precise ecological relationships between hosts and their symbiotic microbial communities remain to be fully delineated. In the current study, we investigate the relative roles of host population genetics and biogeography in structuring the microbial communities hosted by the excavating sponge Cliona delitrix. A total of 53 samples, previously used to demarcate the population genetic structure of C. delitrix, were selected from two locations in the Caribbean Sea and from eight locations across the reefs of Florida and the Bahamas. Microbial community diversity and composition were measured using Illumina-based high-throughput sequencing of the 16S rRNA V4 region and related to host population structure and geographic distribution. Most operational taxonomic units (OTUs) specific to Cliona delitrix microbiomes were rare, while other OTUs were shared with congeneric hosts. Across a large regional scale (>1,000 km), geographic distance was associated with considerable variability of the sponge microbiome, suggesting a distance-decay relationship, but little impact over smaller spatial scales (<300 km) was observed. Host population structure had a moderate effect on the structure of these microbial communities, regardless of geographic distance. These results support the interplay between geographic, environmental, and host factors as forces determining the community structure of microbiomes associated with C. delitrix. Moreover, these data suggest that the mechanisms of host regulation can be observed at the population genetic scale, prior to the onset of speciation.

Microbial symbionts and ecological divergence of Caribbean sponges: A new perspective on an ancient association.

Marine sponges host diverse communities of microbial symbionts that expand the metabolic capabilities of their host, but the abundance and structure of these communities is highly variable across sponge species. Specificity in these interactions may fuel host niche partitioning on crowded coral reefs by allowing individual sponge species to exploit unique sources of carbon and nitrogen, but this hypothesis is yet to be tested. Given the presence of high sponge biomass and the coexistence of diverse sponge species, the Caribbean Sea provides a unique system in which to investigate this hypothesis. To test for ecological divergence among sympatric Caribbean sponges and investigate whether these trends are mediated by microbial symbionts, we measured stable isotope (δ13C and δ15N) ratios and characterized the microbial community structure of sponge species at sites within four regions spanning a 1700 km latitudinal gradient. There was a low (median of 8.2 %) overlap in the isotopic niches of sympatric species; in addition, host identity accounted for over 75% of the dissimilarity in both δ13C and δ15N values and microbiome community structure among individual samples within a site. There was also a strong phylogenetic signal in both δ15N values and microbial community diversity across host phylogeny, as well as a correlation between microbial community structure and variation in δ13C and δ15N values across samples. Together, this evidence supports a hypothesis of strong evolutionary selection for ecological divergence across sponge lineages and suggests that this divergence is at least partially mediated by associations with microbial symbionts.

Diverse deep-sea anglerfishes share a genetically reduced luminous symbiont that is acquired from the environment.

Deep-sea anglerfishes are relatively abundant and diverse, but their luminescent bacterial symbionts remain enigmatic. The genomes of two symbiont species have qualities common to vertically transmitted, host-dependent bacteria. However, a number of traits suggest that these symbionts may be environmentally acquired. To determine how anglerfish symbionts are transmitted, we analyzed bacteria-host codivergence across six diverse anglerfish genera. Most of the anglerfish species surveyed shared a common species of symbiont. Only one other symbiont species was found, which had a specific relationship with one anglerfish species, Cryptopsaras couesii. Host and symbiont phylogenies lacked congruence, and there was no statistical support for codivergence broadly. We also recovered symbiont-specific gene sequences from water collected near hosts, suggesting environmental persistence of symbionts. Based on these results we conclude that diverse anglerfishes share symbionts that are acquired from the environment, and that these bacteria have undergone extreme genome reduction although they are not vertically transmitted.

Gut microbiome diversity is associated with sleep physiology in humans.

The human gut microbiome can influence health through the brain-gut-microbiome axis. Growing evidence suggests that the gut microbiome can influence sleep quality. Previous studies that have examined sleep deprivation and the human gut microbiome have yielded conflicting results. A recent study found that sleep deprivation leads to changes in gut microbiome composition while a different study found that sleep deprivation does not lead to changes in gut microbiome. Accordingly, the relationship between sleep physiology and the gut microbiome remains unclear. To address this uncertainty, we used actigraphy to quantify sleep measures coupled with gut microbiome sampling to determine how the gut microbiome correlates with various measures of sleep physiology. We measured immune system biomarkers and carried out a neurobehavioral assessment as these variables might modify the relationship between sleep and gut microbiome composition. We found that total microbiome diversity was positively correlated with increased sleep efficiency and total sleep time, and was negatively correlated with wake after sleep onset. We found positive correlations between total microbiome diversity and interleukin-6, a cytokine previously noted for its effects on sleep. Analysis of microbiome composition revealed that within phyla richness of Bacteroidetes and Firmicutes were positively correlated with sleep efficiency, interleukin-6 concentrations and abstract thinking. Finally, we found that several taxa (Lachnospiraceae, Corynebacterium, and Blautia) were negatively correlated with sleep measures. Our findings initiate linkages between gut microbiome composition, sleep physiology, the immune system and cognition. They may lead to mechanisms to improve sleep through the manipulation of the gut microbiome.

Characterization of the microbiome and bioluminescent symbionts across life stages of Ceratioid Anglerfishes of the Gulf of Mexico.

The interdependence of diverse organisms through symbiosis reaches even the deepest parts of the oceans. As part of the DEEPEND project (deependconsortium.org) research on deep Gulf of Mexico biodiversity, we profiled the bacterial communities ('microbiomes') and luminous symbionts of 36 specimens of adult and larval deep-sea anglerfishes of the suborder Ceratioidei using 16S rDNA. Transmission electron microscopy was used to characterize the location of symbionts in adult light organs (esca). Whole larval microbiomes, and adult skin and gut microbiomes, were dominated by bacteria in the genera Moritella and Pseudoalteromonas. 16S rDNA sequencing results from adult fishes corroborate the previously published identity of ceratioid bioluminescent symbionts and support the findings that these symbionts do not consistently exhibit host specificity at the host family level. Bioluminescent symbiont amplicon sequence variants were absent from larval ceratioid samples, but were found at all depths in the seawater, with a highest abundance found at mesopelagic depths. As adults spend the majority of their lives in the meso- and bathypelagic zones, the trend in symbiont abundance is consistent with their life history. These findings support the hypothesis that bioluminescent symbionts are not present throughout host development, and that ceratioids acquire their bioluminescent symbionts from the environment.

Culture-independent Characterization of the Microbiome of Healthy Pulp.

INTRODUCTION: Advances in culture-independent molecular biotechnologies have driven a greater appreciation for the function of mutualistic microorganisms in the maintenance of states of health in humans. The purpose of this study was to test the long-held hypothesis that healthy pulp lack bacteria. METHODS: Strict inclusion criteria were used to identify 10 pristine teeth from 10 healthy patients that were scheduled to be electively extracted in compliance with an orthodontic treatment plan. Using a rigorous disinfection protocol to isolate the operating field, the pulp space was accessed, and pulp tissue was collected in vivo from each tooth using a barbed broach. Genomic DNA was extracted from each pulp sample and analyzed for the presence of bacterial DNA using universal 16S ribosomal RNA polymerase chain reaction primers and MiSeq sequencing (Illumina, San Diego, CA) of community amplicons. RESULTS: One hundred percent (10/10) of the tested pulp tissues demonstrated the presence of bacterial DNA, with a mean of 343 operational taxonomic units per sample (range, 191-479). These were derived from 12 genera in which Ralstonia, Actinetobacter, and Staphylococcus were predominant (43%-78% of total community). None of the negative-field controls and none of the instruments used in the study tested positive for the presence of contaminating DNA. CONCLUSIONS: This study presents evidence to support the conclusion that the pulp spaces of pristine healthy teeth contain detectable bacterial DNA.

Depth-Dependent Environmental Drivers of Microbial Plankton Community Structure in the Northern Gulf of Mexico.

The Gulf of Mexico (GoM) is a dynamic marine ecosystem influenced by multiple natural and anthropogenic processes and inputs, such as the intrusion of warm oligotrophic water via the Loop Current, freshwater and nutrient input by the Mississippi River, and hydrocarbon inputs via natural seeps and industrial spills. Microbial plankton communities are important to pelagic food webs including in the GoM but understanding the drivers of the natural dynamics of these passively distributed microorganisms can be challenging in such a large and heterogeneous system. As part of the DEEPEND consortium, we applied high throughput 16S rRNA sequencing to investigate the spatial and temporal dynamics of pelagic microbial plankton related to several environmental conditions during two offshore cruises in 2015. Our results show dramatic community shifts across depths, especially between photic and aphotic zones. Though we only have two time points within a single year, observed temporal shifts in microbial plankton communities were restricted to the seasonally influenced epipelagic zone (0-200 m), and appear mainly driven by changes in temperature. Environmental selection in microbial plankton communities was depth-specific, with variables such as turbidity, salinity, and abundance of photosynthetic taxa strongly correlating with community structure in the epipelagic zone, while variables such as oxygen and specific nutrient concentrations were correlated with community structure at deeper depths.

The sponge microbiome project.

Marine sponges (phylum Porifera) are a diverse, phylogenetically deep-branching clade known for forming intimate partnerships with complex communities of microorganisms. To date, 16S rRNA gene sequencing studies have largely utilised different extraction and amplification methodologies to target the microbial communities of a limited number of sponge species, severely limiting comparative analyses of sponge microbial diversity and structure. Here, we provide an extensive and standardised dataset that will facilitate sponge microbiome comparisons across large spatial, temporal, and environmental scales. Samples from marine sponges (n = 3569 specimens), seawater (n = 370), marine sediments (n = 65) and other environments (n = 29) were collected from different locations across the globe. This dataset incorporates at least 268 different sponge species, including several yet unidentified taxa. The V4 region of the 16S rRNA gene was amplified and sequenced from extracted DNA using standardised procedures. Raw sequences (total of 1.1 billion sequences) were processed and clustered with (i) a standard protocol using QIIME closed-reference picking resulting in 39 543 operational taxonomic units (OTU) at 97% sequence identity, (ii) a de novo clustering using Mothur resulting in 518 246 OTUs, and (iii) a new high-resolution Deblur protocol resulting in 83 908 unique bacterial sequences. Abundance tables, representative sequences, taxonomic classifications, and metadata are provided. This dataset represents a comprehensive resource of sponge-associated microbial communities based on 16S rRNA gene sequences that can be used to address overarching hypotheses regarding host-associated prokaryotes, including host specificity, convergent evolution, environmental drivers of microbiome structure, and the sponge-associated rare biosphere.

Diversity, structure and convergent evolution of the global sponge microbiome.

Sponges (phylum Porifera) are early-diverging metazoa renowned for establishing complex microbial symbioses. Here we present a global Porifera microbiome survey, set out to establish the ecological and evolutionary drivers of these host-microbe interactions. We show that sponges are a reservoir of exceptional microbial diversity and major contributors to the total microbial diversity of the world's oceans. Little commonality in species composition or structure is evident across the phylum, although symbiont communities are characterized by specialists and generalists rather than opportunists. Core sponge microbiomes are stable and characterized by generalist symbionts exhibiting amensal and/or commensal interactions. Symbionts that are phylogenetically unique to sponges do not disproportionally contribute to the core microbiome, and host phylogeny impacts complexity rather than composition of the symbiont community. Our findings support a model of independent assembly and evolution in symbiont communities across the entire host phylum, with convergent forces resulting in analogous community organization and interactions.

Sponge distribution and the presence of photosymbionts in Moorea, French Polynesia.

Photosymbionts play an important role in the ecology and evolution of diverse host species within the marine environment. Although sponge-photosymbiont interactions have been well described from geographically disparate sites worldwide, our understanding of these interactions from shallow water systems within French Polynesia is limited. We surveyed diverse habitats around the north coast of Moorea, French Polynesia and screened sponges for the presence of photosymbionts. Overall sponge abundance and diversity were low, with <1% cover and only eight putative species identified by 28S barcoding from surveys at 21 sites. Of these eight species, seven were found predominately in shaded or semi-cryptic habitats under overhangs or within caverns. Lendenfeldia chondrodes was the only species that supported a high abundance of photosymbionts and was also the only species found in exposed, illuminated habitats. Interestingly, L. chondrodes was found at three distinct sites, with a massive, fan-shaped growth form at two of the lagoon sites and a thin, encrusting growth form within a bay site. These two growth forms differed in their photosymbiont abundance, with massive individuals of L. chondrodes having higher photosymbiont abundance than encrusting individuals from the bay. We present evidence that some sponges from French Polynesia support abundant photosymbiont communities and provide initial support for the role of these communities in host ecology.

Variation in species diversity and functional traits of sponge communities near human populations in Bocas del Toro, Panama.

Recent studies have renewed interest in sponge ecology by emphasizing the functional importance of sponges in a broad array of ecosystem services. Many critically important habitats occupied by sponges face chronic stressors that might lead to alterations in their diversity, relatedness, and functional attributes. We addressed whether proximity to human activity might be a significant factor in structuring sponge community composition, as well as potential functional roles, by monitoring sponge diversity and abundance at two structurally similar sites that vary in distance to areas of high coastal development in Bocas Del Toro, Panama. We surveyed sponge communities at each site using belt transects and differences between two sites were compared using the following variables: (1) sponge species richness, Shannon diversity, and inverse Simpson's diversity; (2) phylogenetic diversity; (3) taxonomic and phylogenetic beta diversity; (4) trait diversity and dissimilarity; and (5) phylogenetic and trait patterns in community structure. We observed significantly higher sponge diversity at Punta Caracol, the site most distant from human development (∼5 km). Although phylogenetic diversity was lower at Saigon Bay, the site adjacent to a large village including many houses, businesses, and an airport, the sites did not exhibit significantly different patterns of phylogenetic relatedness in species composition. However, each site had a distinct taxonomic and phylogenetic composition (beta diversity). In addition, the sponge community at Saigon included a higher relative abundance of sponges with high microbial abundance and high chlorophyll a concentration, whereas the community at Punta Caracol had a more even distribution of these traits, yielding a significant difference in functional trait diversity between sites. These results suggest that lower diversity and potentially altered community function might be associated with proximity to human populations. This study highlights the importance of evaluating functional traits and phylogenetic diversity in addition to common diversity metrics when assessing potential environmental impacts on benthic communities.

Metabolic diversity and niche structure in sponges from the Miskito Cays, Honduras.

Hosting symbionts provides many eukaryotes with access to the products of microbial metabolism that are crucial for host performance. On tropical coral reefs, many (High Microbial Abundance [HMA]) but not all (Low Microbial Abundance [LMA]) marine sponges host abundant symbiont communities. Although recent research has revealed substantial variation in these sponge-microbe associations (termed holobionts), little is known about the ecological implications of this diversity. We investigated the expansion of diverse sponge species across isotopic niche space by calculating niche size (as standard ellipse area [SEA c ]) and assessing the relative placement of common sponge species in bivariate (δ (13)C and δ (15)N) plots. Sponges for this study were collected from the relatively isolated reefs within the Miskito Cays of Honduras. These reefs support diverse communities of HMA and LMA species that together span a gradient of photosymbiont abundance, as revealed by chlorophyll a analysis. HMA sponges occupied unique niche space compared to LMA species, but the placement of some HMA sponges was driven by photosymbiont abundance. In addition, photosymbiont abundance explained a significant portion of the variation in isotope values, suggesting that access to autotrophic metabolism provided by photosymbionts is an important predictor in the location of species within isotopic space. Host identity accounted for over 70% of the variation in isotope values within the Miskito Cays and there was substantial variation in the placement of individual species within isotopic niche space, suggesting that holobiont metabolic diversity may allow taxonomically diverse sponge species to utilize unique sources of nutrients within a reef system. This study provides initial evidence that microbial symbionts allow sponges to expand into novel physiochemical niche space. This expansion may reduce competitive interactions within coral reefs and promote diversification of these communities.

Phylogenetic signal in the community structure of host-specific microbiomes of tropical marine sponges.

Sponges (Porifera) can host diverse and abundant communities of microbial symbionts that make crucial contributions to host metabolism. Although these communities are often host-specific and hypothesized to co-evolve with their hosts, correlations between host phylogeny and microbiome community structure are rarely tested. As part of the Earth Microbiome Project (EMP), we surveyed the microbiomes associated with 20 species of tropical marine sponges collected over a narrow geographic range. We tested whether (1) univariate metrics of microbiome diversity displayed significant phylogenetic signal across the host phylogeny; (2) host identity and host phylogeny were significant factors in multivariate analyses of taxonomic and phylogenetic dissimilarity; and (3) different minimum read thresholds impacted these results. We observed significant differences in univariate metrics of diversity among host species for all read thresholds, with strong phylogenetic signal in the inverse Simpson's index of diversity (D). We observed a surprisingly wide range of variability in community dissimilarity within host species (4-73%); this variability was not related to microbial abundance within a host species. Taxonomic and phylogenetic dissimilarity were significantly impacted by host identity and host phylogeny when these factors were considered individually; when tested together, the effect of host phylogeny was reduced, but remained significant. In our dataset, this outcome is largely due to closely related host sponges harboring distinct microbial taxa. Host identity maintained a strong statistical signal at all minimum read thresholds. Although the identity of specific microbial taxa varied substantially among host sponges, closely related hosts tended to harbor microbial communities with similar patterns of relative abundance. We hypothesize that microbiomes with low D might be structured by regulation of the microbial community by the host or by the presence of competitively dominant symbionts that are themselves under selection for host specificity.

Exploring individual- to population-level impacts of disease on coral reef sponges: using spatial analysis to assess the fate, dynamics, and transmission of Aplysina Red Band Syndrome (ARBS).

BACKGROUND: Marine diseases are of increasing concern for coral reef ecosystems, but often their causes, dynamics and impacts are unknown. The current study investigated the epidemiology of Aplysina Red Band Syndrome (ARBS), a disease affecting the Caribbean sponge Aplysina cauliformis, at both the individual and population levels. The fates of marked healthy and ARBS-infected sponges were examined over the course of a year. Population-level impacts and transmission mechanisms of ARBS were investigated by monitoring two populations of A. cauliformis over a three year period using digital photography and diver-collected data, and analyzing these data with GIS techniques of spatial analysis. In this study, three commonly used spatial statistics (Ripley's K, Getis-Ord General G, and Moran's Index) were compared to each other and with direct measurements of individual interactions using join-counts, to determine the ideal method for investigating disease dynamics and transmission mechanisms in this system. During the study period, Hurricane Irene directly impacted these populations, providing an opportunity to assess potential storm effects on A. cauliformis and ARBS. RESULTS: Infection with ARBS caused increased loss of healthy sponge tissue over time and a higher likelihood of individual mortality. Hurricane Irene had a dramatic effect on A. cauliformis populations by greatly reducing sponge biomass on the reef, especially among diseased individuals. Spatial analysis showed that direct contact between A. cauliformis individuals was the likely transmission mechanism for ARBS within a population, evidenced by a significantly higher number of contact-joins between diseased sponges compared to random. Of the spatial statistics compared, the Moran's Index best represented true connections between diseased sponges in the survey area. This study showed that spatial analysis can be a powerful tool for investigating disease dynamics and transmission in a coral reef ecosystem.