Severe hurricanes increase recruitment and gene flow in the clonal sponge Aplysina cauliformis.

Upright branching sponges, such as Aplysina cauliformis, provide critical three-dimensional habitat for other organisms and assist in stabilizing coral reef substrata, but are highly susceptible to breakage during storms. Breakage can increase sponge fragmentation, contributing to population clonality and inbreeding. Conversely, storms could provide opportunities for new genotypes to enter populations via larval recruitment, resulting in greater genetic diversity in locations with frequent storms. The unprecedented occurrence of two Category 5 hurricanes in close succession during 2017 in the U.S. Virgin Islands (USVI) provided a unique opportunity to evaluate whether recolonization of newly available substrata on coral reefs was due to local (e.g. re-growth of remnants, fragmentation, larval recruitment) or remote (e.g. larval transport and immigration) sponge genotypes. We sampled A. cauliformis adults and juveniles from four reefs around St. Thomas and two in St. Croix (USVI). Using a 2bRAD protocol, all samples were genotyped for single-nucleotide polymorphisms (SNPs). Results showed that these major storm events favoured sponge larval recruitment but did not increase the genetic diversity of A. cauliformis populations. Recolonization of substratum post-storms via clonality was lower (15%) than expected and instead was mainly due to sexual reproduction (85%) via local larval recruitment. Storms did enhance gene flow among and within reef sites located south of St. Thomas and north of St. Croix. Therefore, populations of clonal marine species with low pelagic dispersion, such as A. cauliformis, may benefit from increased frequency and magnitude of hurricanes for the maintenance of genetic diversity and to combat inbreeding, enhancing the resilience of Caribbean sponge communities to extreme storm events.

Cophylogeny and convergence shape holobiont evolution in sponge-microbe symbioses.

Symbiotic microbial communities of sponges serve critical functions that have shaped the evolution of reef ecosystems since their origins. Symbiont abundance varies tremendously among sponges, with many species classified as either low microbial abundance (LMA) or high microbial abundance (HMA), but the evolutionary dynamics of these symbiotic states remain unknown. This study examines the LMA/HMA dichotomy across an exhaustive sampling of Caribbean sponge biodiversity and predicts that the LMA symbiotic state is the ancestral state among sponges. Conversely, HMA symbioses, consisting of more specialized microorganisms, have evolved multiple times by recruiting similar assemblages, mostly since the rise of scleractinian-dominated reefs. Additionally, HMA symbioses show stronger signals of phylosymbiosis and cophylogeny, consistent with stronger co-evolutionary interaction in these complex holobionts. These results indicate that HMA holobionts are characterized by increased endemism, metabolic dependence and chemical defences. The selective forces driving these patterns may include the concurrent increase in dissolved organic matter in reef ecosystems or the diversification of spongivorous fishes.

Microbiome diversity and metabolic capacity determines the trophic ecology of the holobiont in Caribbean sponges.

Sponges are increasingly recognized as an ecologically important taxon on coral reefs, representing significant biomass and biodiversity where sponges have replaced scleractinian corals. Most sponge species can be divided into two symbiotic states based on symbiont community structure and abundance (i.e., the microbiome), and are characterized as high microbial abundance (HMA) or low microbial abundance (LMA) sponges. Across the Caribbean, sponge species of the HMA or LMA symbiotic states differ in metabolic capacity, as well as their trophic ecology. A metagenetic analysis of symbiont 16 S rRNA and metagenomes showed that HMA sponge microbiomes are more functionally diverse than LMA microbiomes, offer greater metabolic functional capacity and redundancy, and encode for the biosynthesis of secondary metabolites. Stable isotope analyses showed that HMA and LMA sponges primarily consume dissolved organic matter (DOM) derived from external autotrophic sources, or live particulate organic matter (POM) in the form of bacterioplankton, respectively, resulting in a low degree of resource competition between these symbiont states. As many coral reefs have undergone phase shifts from coral- to macroalgal-dominated reefs, the role of DOM, and the potential for future declines in POM due to decreased picoplankton productivity, may result in an increased abundance of chemically defended HMA sponges on tropical coral reefs.

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.

Combined and independent effects of hypoxia and tributyltin on mRNA expression and physiology of the Eastern oyster (Crassostrea virginica).

Oyster reefs are vital to estuarine health, but they experience multiple stressors and globally declining populations. This study examined effects of hypoxia and tributyltin (TBT) on adult Eastern oysters (Crassostrea virginica) exposed either in the laboratory or the field following a natural hypoxic event. In the laboratory, oysters were exposed to either hypoxia followed by a recovery period, or to hypoxia combined with TBT. mRNA expression of HIF1-α and Tß-4 along with hemocyte counts, biomarkers of hypoxic stress and immune health, respectively, were measured. In field-deployed oysters, HIF1-α and Tß-4 expression increased, while no effect on hemocytes was observed. In contrast, after 6 and 8 days of laboratory-based hypoxia exposure, both Tß-4 expression and hemocyte counts declined. After 8 days of exposure to hypoxia + TBT, oysters substantially up-regulated HIF1-α and down-regulated Tß-4, although hemocyte counts were unaffected. Results suggest that hypoxic exposure induces immunosuppression which could increase vulnerability to pathogens.

Proteomic profiling of healthy and diseased hybrid soft corals Sinularia maxima × S. polydactyla.

Emerging diseases of marine invertebrates have been implicated as one of the major causes of the continuing decline in coral reefs worldwide. To date, most of the focus on marine diseases has been aimed at hard (scleractinian) corals, which are the main reef builders worldwide. However, soft (alcyonacean) corals are also essential components of tropical reefs, representing food, habitat and the 'glue' that consolidates reefs, and they are subject to the same stressors as hard corals. Sinularia maxima and S. polydactyla are the dominant soft corals on the shallow reefs of Guam, where they hybridize. In addition to both parent species, the hybrid soft coral population in Guam is particularly affected by Sinularia tissue loss disease. Using label-free shotgun proteomics, we identified differences in protein expression between healthy and diseased colonies of the hybrid S. maxima × S. polydactyla. This study provided qualitative and quantitative data on specific proteins that were differentially expressed under the stress of disease. In particular, metabolic proteins were down-regulated, whereas proteins related to stress and to symbiont photosynthesis were up-regulated in the diseased soft corals. These results indicate that soft corals are responding to pathogenesis at the level of the proteome, and that this label-free approach can be used to identify and quantify protein biomarkers of sub-lethal stress in studies of marine disease.

Molecular community profiling reveals impacts of time, space, and disease status on the bacterial community associated with the Caribbean sponge Aplysina cauliformis.

Reports of marine sponge diseases have increased in recent years, but few etiologic agents have been identified. Aplysina red band syndrome (ARBS), a condition observed in the Caribbean sponge Aplysina cauliformis, is characterized by a rust-colored leading margin. Culture-independent methods (terminal restriction fragment length polymorphism and clone library analyses) were used to assess bacterial communities associated with healthy and ARBS-affected sponges from two locations over 2 years. Although the bacterial communities associated with healthy and ARBS-affected sponges were significantly different, the sponges maintained a core bacterial community across space, time, and health status. Ten terminal restriction fragments were shown to change significantly between sponge health conditions, with six increasing in abundance with disease and four decreasing. The prevalence of the photosymbiont Synechococcus spongiarum decreased with ARBS infection, suggesting a functional consequence of disease. After cultivating a red-pigmented Leptolyngbya strain from ARBS lesions, transmission studies were conducted to determine whether this organism was the ARBS pathogen. Despite significantly increased abundance of Leptolyngbya spp. in diseased sponges, signs of ARBS were not observed in healthy sponges following 24 days of contact with the cultured strain. Additional work with this model system is needed to increase our understanding of the dynamics of marine diseases.

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.

Marine proteomics: a critical assessment of an emerging technology.

The application of proteomics to marine sciences has increased in recent years because the proteome represents the interface between genotypic and phenotypic variability and, thus, corresponds to the broadest possible biomarker for eco-physiological responses and adaptations. Likewise, proteomics can provide important functional information regarding biosynthetic pathways, as well as insights into mechanism of action, of novel marine natural products. The goal of this review is to (1) explore the application of proteomics methodologies to marine systems, (2) assess the technical approaches that have been used, and (3) evaluate the pros and cons of this proteomic research, with the intent of providing a critical analysis of its future roles in marine sciences. To date, proteomics techniques have been utilized to investigate marine microbe, plant, invertebrate, and vertebrate physiology, developmental biology, seafood safety, susceptibility to disease, and responses to environmental change. However, marine proteomics studies often suffer from poor experimental design, sample processing/optimization difficulties, and data analysis/interpretation issues. Moreover, a major limitation is the lack of available annotated genomes and proteomes for most marine organisms, including several "model species". Even with these challenges in mind, there is no doubt that marine proteomics is a rapidly expanding and powerful integrative molecular research tool from which our knowledge of the marine environment, and the natural products from this resource, will be significantly expanded.

Spatial variability in secondary metabolites of the indo-pacific sponge Stylissa massa.

Chemical diversity represents a measure of selective pressures acting on genotypic variability. In order to understand patterns of chemical ecology and biodiversity in the environment, it is necessary to enhance our knowledge of chemical diversity within and among species. Many sponges produce variable levels of secondary metabolites in response to diverse biotic and abiotic environmental factors. This study evaluated intra-specific variability in secondary metabolites in the common Indo-Pacific sponge Stylissa massa over various geographic scales, from local to ocean basin. Several major metabolites were quantified in extracts from sponges collected in American Samoa, Pohnpei, Saipan, and at several sites and depths in Guam. Concentrations of several of these metabolites varied geographically across the Pacific basin, with American Samoa and Pohnpei exhibiting the greatest differences, and Guam and Saipan more similar to each other. There were also significant differences in concentrations among different sites and depths within Guam. The crude extract of S. massa exhibited feeding deterrence against the omnivorous pufferfish Canthigaster solandri at natural concentrations, however, none of the isolated compounds was deterrent at the maximum natural concentrations observed, nor were mixtures of these compounds, thus emphasizing the need for bioassay-guided isolation to characterize specific chemical defenses. Antibacterial activity against a panel of ecologically relevant pathogens was minimal. Depth transplants, predator exclusion, and UV protection experiments were performed, but although temporal variability in compound concentrations was observed, there was no evidence that secondary metabolite concentration in S. massa was induced by any of these factors. Although the reasons behind the variability observed in the chemical constituents of S. massa are still in question, all sponges are not created equal from a chemical standpoint, and these studies provide further insights into patterns of chemical diversity within S. massa.

Trade-offs in defensive metabolite production but not ecological function in healthy and diseased sponges.

Diseases of marine organisms, and sponges in particular, are increasingly reported worldwide. Prior research indicates that the survival of sponges on reefs is due largely to their production of biologically active secondary metabolites that provide protection from a diversity of stressors. Aplysina Red Band Syndrome (ARBS) is an emerging disease affecting Caribbean rope sponges (Aplysina spp.), but it is not known whether secondary metabolites play a role in disease susceptibility and resistance. To investigate whether differences in secondary metabolites may explain variability in susceptibility to ARBS in Aplysina cauliformis, we used high performance liquid chromatography (HPLC) to generate chemical profiles from healthy tissue in both healthy and diseased sponges, and quantified peak areas for 15 metabolites. Analyses of healthy and diseased sponges revealed qualitative and quantitative differences in their chemical profiles. Aplysamine-1 and fistularin-3 were produced in significantly higher concentrations by healthy sponges, whereas aerothionin and 11-oxoaerothionin were found only in diseased sponges. At natural concentrations, extracts from both healthy and diseased sponges deterred feeding by an omnivorous reef fish. Fistularin-3 deterred feeding at concentrations found in healthy sponges, but not at concentrations found in diseased sponges. Aerothionin deterred feeding at concentrations found in diseased sponges, and may at least partially replace the loss of fistularin-3 as a feeding deterrent compound following pathogenesis, suggesting a trade-off in the production of feeding deterrent compounds. Extracts from healthy and diseased sponges inhibited bacterial growth, and both aplysamine-1 and fistularin-3 displayed selective antibacterial activity. Despite differences in secondary metabolite production between healthy and diseased sponges, the stress associated with ARBS does not appear to compromise the ability of A. cauliformis to maintain defenses against some of its natural enemies.

Chemical constituents of the deep reef caribbean sponges Plakortis angulospiculatus and Plakortis halichondrioides and their anti-inflammatory activities.

Chemical investigations of two collections of the deep reef Caribbean sponge Plakortis angulospiculatus resulted in the isolation of a new compound (1) along with the known compound spiculoic acid B (2) belonging to the spiculoic acid class and four other new compounds (3-6) belonging to the zyggomphic acid class. Three new aromatic compounds (7-9) were isolated from the Caribbean sponge Plakortis halichondrioides. The structural determination of the compounds was based on extensive NMR and mass spectroscopic analysis. The isolated compounds 1-7 were tested for their anti-inflammatory activity using in vitro assays for inhibition of inducible nitric oxide synthase and nuclear factor kappa B (NFκB) activity, as well as inhibition of intracellular reactive oxygen species generation as a result of oxidative stress. The cytotoxicity of these compounds was also evaluated to determine the selectivity index of their bioactivity with respect to cytotoxicity. Compounds 1 and 4 were more potent than the positive control in inhibiting NFκB activity and had IC(50) values of 0.47 and 2.28 µM, respectively.

Colony versus population variation in susceptibility and resistance to dark spot syndrome in the Caribbean coral Siderastrea siderea.

Scleractinian corals appear to be increasingly susceptible to pathogenic diseases, yet it is poorly understood why certain individuals, populations or species are more susceptible to diseases than others. Clearly an understanding of mechanisms of disease resistance in corals is essential to our understanding of patterns of disease incidence and virulence; this work must begin by examining the colony and population levels of organization. The Caribbean coral Siderastrea siderea exhibits variability in susceptibility to dark spot syndrome (DSS), a disease of unknown origin that can result in tissue necrosis. On the reef scale, variability in DSS prevalence in S. siderea occurred through time, but was not correlated with site, seawater temperature or depth. We monitored colonies of S. siderea affected by DSS, as well as their nearest neighbor controls, for 2 years in the Bahamas and found a marked decline in extent of DSS infection in October of both years. A preliminary survey of antimicrobial activity in S. siderea indicated selective activity against certain ecologically relevant bacteria. To assess whether changes in chemical defenses were responsible for the observed temporal variability in DSS prevalence, we sampled S. siderea for qualitative and quantitative analysis of chemical variability between resistant and susceptible colonies of S. siderea. These data suggest that phenotypic plasticity in antimicrobial activity may impact microbial settlement and/or survival.