Peter J. Schupp

Assessing the complex sponge microbiota: core, variable and species-specific bacterial communities in marine sponges.

Marine sponges are well known for their associations with highly diverse, yet very specific and often highly similar microbiota. The aim of this study was to identify potential bacterial sub-populations in relation to sponge phylogeny and sampling sites and to define the core bacterial community. 16S ribosomal RNA gene amplicon pyrosequencing was applied to 32 sponge species from eight locations around the worlds oceans, thereby generating 2567 operational taxonomic units (OTUs at the 97% sequence similarity level) in total and up to 364 different OTUs per sponge species. The taxonomic richness detected in this study comprised 25 bacterial phyla with Proteobacteria, Chloroflexi and Poribacteria being most diverse in sponges. Among these phyla were nine candidate phyla, six of them found for the first time in sponges. Similarity comparison of bacterial communities revealed no correlation with host phylogeny but a tropical sub-population in that tropical sponges have more similar bacterial communities to each other than to subtropical sponges. A minimal core bacterial community consisting of very few OTUs (97%, 95% and 90%) was found. These microbes have a global distribution and are probably acquired via environmental transmission. In contrast, a large species-specific bacterial community was detected, which is represented by OTUs present in only a single sponge species. The species-specific bacterial community is probably mainly vertically transmitted. It is proposed that different sponges contain different bacterial species, however, these bacteria are still closely related to each other explaining the observed similarity of bacterial communities in sponges in this and previous studies. This global analysis represents the most comprehensive study of bacterial symbionts in sponges to date and provides novel insights into the complex structure of these unique associations.

Marine Biofilm Bacteria Evade Eukaryotic Predation by Targeted Chemical Defense

Many plants and animals are defended from predation or herbivory by inhibitory secondary metabolites, which in the marine environment are very common among sessile organisms. Among bacteria, where there is the greatest metabolic potential, little is known about chemical defenses against bacterivorous consumers. An emerging hypothesis is that sessile bacterial communities organized as biofilms serve as bacterial refuge from predation. By testing growth and survival of two common bacterivorous nanoflagellates, we find evidence that chemically mediated resistance against protozoan predators is common among biofilm populations in a diverse set of marine bacteria. Using bioassay-guided chemical and genetic analysis, we identified one of the most effective antiprotozoal compounds as violacein, an alkaloid that we demonstrate is produced predominately within biofilm cells. Nanomolar concentrations of violacein inhibit protozoan feeding by inducing a conserved eukaryotic cell death program. Such biofilm-specific chemical defenses could contribute to the successful persistence of biofilm bacteria in various environments and provide the ecological and evolutionary context for a number of eukaryote-targeting bacterial metabolites.

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 worlds 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.

Chitin-based scaffolds are an integral part of the skeleton of the marine demosponge Ianthella basta

The skeletons of demosponges, such as Ianthella basta, are known to be a composite material containing organic constituents. Here, we show that a filigree chitin-based scaffold is an integral component of the I. basta skeleton. These chitin-based scaffolds can be isolated from the sponge skeletons using an isolation and purification technique based on treatment with alkaline solutions. Solid-state (13)C NMR, Raman, and FT-IR spectroscopies, as well as chitinase digestion, reveal that the isolated material indeed consists of chitin. The morphology of the scaffolds has been determined by light and electron microscopy. It consists of cross-linked chitin fibers approximately 40-100 nm in diameter forming a micro-structured network. The overall shape of this network closely resembles the shape of the integer sponge skeleton. Solid-state (13)C NMR spectroscopy was used to characterize the sponge skeleton on a molecular level. The (13)C NMR signals of the chitin-based scaffolds are relatively broad, indicating a high amount of disordered chitin, possibly in the form of surface-exposed molecules. X-ray diffraction confirms that the scaffolds isolated from I. basta consist of partially disordered and loosely packed chitin with large surfaces. The spectroscopic signature of these chitin-based scaffolds is closer to that of alpha-chitin than beta-chitin.

Three-dimensional chitin-based scaffolds from Verongida sponges (Demospongiae: Porifera). Part I. Isolation and identification of chitin.

Marine invertebrate organisms including sponges (Porifera) not only provide an abundant source of biologically active secondary metabolites but also inspire investigations to develop biomimetic composites, scaffolds and templates for practical use in materials science, biomedicine and tissue engineering. Here, we presented a detailed study of the structural and physico-chemical properties of three-dimensional skeletal scaffolds of the marine sponges Aiolochroia crassa, Aplysina aerophoba, A. cauliformis, A. cavernicola, and A. fulva (Verongida: Demospongiae). We show that these fibrous scaffolds have a multilayered design and are made of chitin. (13)C solid-state NMR spectroscopy, NEXAFS, and IR spectroscopy as well as chitinase digestion and test were applied in order to unequivocally prove the existence of alpha-chitin in all investigated species.

Calcium carbonate and secondary metabolites in tropical seaweeds : variable effects on herbivorous fishes

On coral reefs where herbivorous fishes are both numerous and diverse, many seaweeds combine mineral and chemical defenses. We hypothesized that combined defenses affect a greater number of herbivore species than either single defense, and in this study we experimentally tested this hypothesis. We first incorporated calcium carbonate (aragonite form) alone at a range of concentrations into an artificial diet composed of freeze—dried samples of the noncalcified, green alga Enteromorpha calathrata, carragenan, cornstarch, and water. In field experiments where diets were made available to a natural fish community, aragonite had variable effects: in some trials aragonite deterred feeding, in others it had no significant effect, and in one trial it even simulated feeding. Parrotfishes (Scaridae) and the surgeonfish Ctenochaetus striatus (Acanthuridae) showed a significant preference for food containing aragonite in field observations, while the surgeonfish Naso vlaminigii and three damselfishes (Pomacentridae) were observed to feed preferentially on food without added aragonite. In the laboratory, the surgeonfishes Acanthurus nigrofuscus, A. triostegus, A. xanthopterus, and Naso unicornis and the rabbitfish Siganus spinus (Siganidae) significantly avoided food with added aragonite. However, the parrotfishes Scarus sordidus and S. schlegeli consumed significantly more food containing aragonite the plain food. Ctenochaetus stiatus avoided aragonite only at the highest aragonite concentration and showed no preference at lower aragonite concentrations. Artificial diets containing natural concentrations of aragonite (80% dry mass), dieter—penoid secondary metabolites from Halimeda macroloba (1% dry mass), and a combination of both defenses (80% aragonite and 1% aragonite—free dry mass of diterpenes) were tested in the laboratory against four fish species. Scarus sordidus and Ctenochaetus striatus avoided secondary metabolites but not aragonite, and Naso lituratus and Signaus spinus were deterred by aragonite but not by secondary metabolites. All fishes were deterred by one defense or the other, and all fishes but Scarus sordidus avoided food containing the combined defenses. Combinations of aragonite and secondary metabolites increased the number of fish species that were effectively deterred from grazing relative to either single defense.

Evidence for Acyl Homoserine Lactone Signal Production in Bacteria Associated with Marine Sponges

ABSTRACT We report for the first time the production of acyl homoserine lactones (AHLs) by bacteria associated with marine sponges. Given the involvement of AHLs in bacterial colonization of many higher organisms, we speculate that such quorum sensing signals could play a part in interactions between sponges and the dense bacterial communities living within them.

Three-dimensional chitin-based scaffolds from Verongida sponges (Demospongiae: Porifera). Part II: Biomimetic potential and applications

In order to evaluate the biomedical potential of three-dimensional chitinous scaffolds of poriferan origin, chondrocyte culturing experiments were performed. It was shown for the first time that freshly isolated chondrocytes attached well to the chitin scaffold and synthesized an extracellular matrix similar to that found in other cartilage tissue engineering constructs. Chitin scaffolds also supported deposition of a proteoglycan-rich extracellular matrix of chondrocytes seeded bioconstructs in an in vivo environment. We suggest that chitin sponge scaffolds, apart from the demonstrated biomedical applications, are highly optimized structures for use as filtering systems, templates for biomineralization as well as metallization in order to produce catalysts.

Apratoxin E, a Cytotoxic Peptolide from a Guamanian Collection of the Marine Cyanobacterium Lyngbya bouillonii

A collection of the marine cyanobacterium Lyngbya bouillonii from Guam afforded apratoxin E (1), a new peptide-polyketide hybrid of the apratoxin class of cytotoxins. The planar structure of 1 was elucidated by NMR spectroscopic analysis and mass spectrometry. Configurational assignments of stereocenters in the peptide portion were made by chiral HPLC analysis of the acid hydrolysate. The relative configuration in the polyketide moiety was assigned by comparison of NMR data including proton-proton coupling constants with those of the known analogues. Apratoxin E (1) displayed strong cytotoxicity against several cancer cell lines derived from colon, cervix, and bone, ranging from 21 to 72 nM, suggesting that the alpha,beta-unsaturation of the modified cysteine residue is not essential for apratoxin activity. The 5- to 15-fold reduced activity compared with apratoxin A (2) is attributed to the dehydration in the long-chain polyketide unit, which could affect the conformation of the molecule.

The HMA-LMA Dichotomy Revisited: an Electron Microscopical Survey of 56 Sponge Species

The dichotomy between high microbial abundance (HMA) and low microbial abundance (LMA) sponges has been long recognized. In the present study, 56 sponge species from three geographic regions (greater Caribbean, Mediterranean, Red Sea) were investigated by transmission electron microscopy for the presence of microorganisms in the mesohyl matrix. Additionally, bacterial enumeration by DAPI-counting was performed on a subset of samples. Of the 56 species investigated, 28 were identified as belonging to the HMA and 28 to the LMA category. The sponge orders Agelasida and Verongida consisted exclusively of HMA species, and the Poecilosclerida were composed only of LMA sponges. Other taxa contained both types of microbial associations (e.g., marine Haplosclerida, Homoscleromorpha, Dictyoceratida), and a clear phylogenetic pattern could not be identified. For a few sponge species, an intermediate microbial load was determined, and the microscopy data did not suffice to reliably determine HMA or LMA status. To experimentally determine the HMA or LMA status of a sponge species, we therefore recommend a combination of transmission electron microscopy and 16S rRNA gene sequence data. This study significantly expands previous reports on microbial abundances in sponge tissues and contributes to a better understanding of the HMA-LMA dichotomy in sponge-microbe symbioses.