Microbially mediated nutrient cycles in marine sponges.

Efficient nutrient cycles mediated by symbiotic microorganisms with their hosts are vital to support the high productivity of coral reef ecosystems. In these ecosystems, marine sponges are important habitat-forming organisms in the benthic community and harbor abundant microbial symbionts. However, few studies have reviewed the critical microbially mediated nutrient cycling processes in marine sponges. To bridge this gap, in this review article, we summarize existing knowledge and recent advances in understanding microbially mediated carbon (C), nitrogen (N), phosphorus (P) and sulfur (S) cycles in sponges, propose a conceptual model that describes potential interactions and constraints in the major nutrient cycles, and suggest that shifting redox state induced by animal behavior like sponge pumping can exert great influence on the activities of symbiotic microbial communities. Constraints include the lack of knowledge on spatial and temporal variations and host behavior; more studies are needed in these areas. Sponge microbiomes may have a significant impact on the nutrient cycles in the world's coral reef ecosystems.

In situ determination of Si, N, and P utilization by the demosponge Tethya citrina: A benthic-chamber approach.

Sponges consume dissolved silicon (DSi) to build their skeletons. Few studies have attempted to quantify DSi utilization by these organisms and all available determinations come from laboratory measurements. Here we measured DSi consumption rates of the sponge Tethya citrina in its natural habitat, conducting 24h incubations in benthic chambers. Sponges consumed DSi at an average rate of 0.046 ± 0.018 µmol h-1 mL-1 when DSi availability in its habitat was 8.3 ± 1.8 µM. Such DSi consumption rates significantly matched the values predicted by a kinetic model elsewhere developed previously for this species through laboratory incubations. These results support the use of laboratory incubations as a suitable approach to learn about DSi consumption. During the field incubations, utilization of other dissolved inorganic nutrients by this low-microbial-abundance (LMA) sponge was also measured. The sponges were net sources of ammonium (-0.043 ± 0.031 µmol h-1 mL-1), nitrate (-0.063 ± 0.031 µmol h-1 mL-1), nitrite (-0.007 ± 0.003 µmol h-1 mL-1), and phosphate (-0.004 ± 0.005 µmol h-1 mL-1), in agreement with the general pattern in other LMA species. The detected effluxes were among the lowest reported for sponges, which agreed with the low respiration rates characterizing this species (0.35 ± 0.11 µmol-O2 h-1 mL-1). Despite relatively low flux, the dense population of T. citrina modifies the availability of dissolved inorganic nutrients in the demersal water of its habitat, contributing up to 14% of nitrate and nitrite stocks. Through these effects, the bottom layer contacting the benthic communities where siliceous LMA sponges abound can be partially depleted in DSi, but can benefit from inputs of N and P dissolved inorganic nutrients that are critical to primary producers.

Scrobiculosides A and B from the deep-sea sponge Pachastrella scrobiculosa.

Two new steroidal saponins, scrobiculosides A and B, were isolated from the deep-sea sponge Pachastrella scrobiculosa, collected at a depth of 200 m off Miura Peninsula, Japan. The aglycones of scrobiculosides A and B feature a vinylic cyclopropane and a ∆24,25 exomethylene on the side chains, respectively. Both saponins have a common sugar moiety composed of ß-D-galactopyranosyl-(1 → 2)-6-acetyl-ß-D-glucopyranoside, with the exception of an acetyl group on C6″ in scrobiculoside A. Scrobiculoside A exhibited cytotoxicity against HL-60 and P388 cells, with IC50 values of 52 and 61 µM, respectively.

Manzamine A Exerts Anticancer Activity against Human Colorectal Cancer Cells.

Marine sponges are known to produce numerous bioactive secondary metabolites as defense strategies to avoid predation. Manzamine A is a sponge-derived ß-carboline-fused pentacyclic alkaloid with various bioactivities, including recently reported anticancer activity on pancreatic cancer. However, its cytotoxicity and mode of action against other tumors remain unclear. In this study, we exhibit that manzamine A reduced cell proliferation in several colorectal cancer (CRC) cell lines. To further investigate the manzamine A triggered molecular regulation, we analyzed the gene expression with microarray and revealed that pathways including cell cycle, DNA repair, mRNA metabolism, and apoptosis were dysregulated. We verified that manzamine A induced cell cycle arrest at G0/G1 phase via inhibition of cyclin-dependent kinases by p53/p21/p27 and triggered a caspase-dependent apoptotic cell death through mitochondrial membrane potential depletion. Additionally, we performed bioinformatics analysis and demonstrated that manzamine A abolished epithelial⁻mesenchymal transition process. Several mesenchymal transcriptional factors, such as Snail, Slug, and Twist were suppressed and epithelial marker E-cadherin was induced simultaneously in HCT116 cells by manzamine A, leading to the epithelial-like phenotype and suppression of migration. These findings suggest that manzamine A may serve as a starting point for the development of an anticancer drug for the treatment of metastatic CRC.

The sponge holobiont in a changing ocean: from microbes to ecosystems.

The recognition that all macroorganisms live in symbiotic association with microbial communities has opened up a new field in biology. Animals, plants, and algae are now considered holobionts, complex ecosystems consisting of the host, the microbiota, and the interactions among them. Accordingly, ecological concepts can be applied to understand the host-derived and microbial processes that govern the dynamics of the interactive networks within the holobiont. In marine systems, holobionts are further integrated into larger and more complex communities and ecosystems, a concept referred to as "nested ecosystems." In this review, we discuss the concept of holobionts as dynamic ecosystems that interact at multiple scales and respond to environmental change. We focus on the symbiosis of sponges with their microbial communities-a symbiosis that has resulted in one of the most diverse and complex holobionts in the marine environment. In recent years, the field of sponge microbiology has remarkably advanced in terms of curated databases, standardized protocols, and information on the functions of the microbiota. Like a Russian doll, these microbial processes are translated into sponge holobiont functions that impact the surrounding ecosystem. For example, the sponge-associated microbial metabolisms, fueled by the high filtering capacity of the sponge host, substantially affect the biogeochemical cycling of key nutrients like carbon, nitrogen, and phosphorous. Since sponge holobionts are increasingly threatened by anthropogenic stressors that jeopardize the stability of the holobiont ecosystem, we discuss the link between environmental perturbations, dysbiosis, and sponge diseases. Experimental studies suggest that the microbial community composition is tightly linked to holobiont health, but whether dysbiosis is a cause or a consequence of holobiont collapse remains unresolved. Moreover, the potential role of the microbiome in mediating the capacity for holobionts to acclimate and adapt to environmental change is unknown. Future studies should aim to identify the mechanisms underlying holobiont dynamics at multiple scales, from the microbiome to the ecosystem, and develop management strategies to preserve the key functions provided by the sponge holobiont in our present and future oceans.

Aquaporin in Chondrosia reniformis Nardo, 1847 and Its Possible Role in the Interaction Between Cells and Engulfed Siliceous Particles.

The sponge Chondrosia reniformis selectively engulfs siliceous particles that, when in crystalline form, become quickly dissolved in its ectosome. The molecular mechanism, identity, and physiological significance of the cells involved in this process are not completely understood. In the present study, we applied light and electronic microscopic techniques to show how the quartz particles in C. reniformis are enveloped through collagen fibers and host cells near the surface of these organisms. As various aquaporins from bacteria, animals, and plants bidirectionally conduct metalloids-including silicon ions--through the cell membrane, the presence and potential involvement of aquaporins in quartz dissolution in C. reniformis have been investigated. An aquaporin-like transcript (CrAQP) was isolated according to the transcriptome sequencing results in C. reniformis The full-length CrAQP cDNA is 907 nucleotides long, with a 795-base pair (bp), open reading frame encoding a protein of 265 amino acids, a 29-bp, 5'-non-coding region, and a 83-bp, 3'-untranslated region. The Bayesian phylogenetic inference suggests that CrAqp is closely related to the Aqp8L grade, which is also implicated in H2O2 transport. Quantification of CrAQP mRNA through qPCR indicated that the transcript level was higher in the ectosome than in the choanosome. Immunofluorescence of a mammalian AQP8 in C. reniformis showed positivity in some cells near the quartz particles, a finding that may support the initial hypothesis of the potential involvement of CrAQP in quartz erosion. However, the features of the primary structure of this protein offer a new viewpoint about the functional role of these molecules in this process: that CrAQP may be involved in the permeation of H2O2 released during silica erosion.

Molecular Cloning, Characterization, and Expression Analysis of a Prolyl 4-Hydroxylase from the Marine Sponge Chondrosia reniformis.

Prolyl 4-hydroxylase (P4H) catalyzes the hydroxylation of proline residues in collagen. P4H has two functional subunits, α and ß. Here, we report the cDNA cloning, characterization, and expression analysis of the α and ß subunits of the P4H derived from the marine sponge Chondrosia reniformis. The amino acid sequence of the α subunit is 533 residues long with an M r of 59.14 kDa, while the ß subunit counts 526 residues with an M r of 58.75 kDa. Phylogenetic analyses showed that αP4H and ßP4H are more related to the mammalian sequences than to known invertebrate P4Hs. Western blot analysis of sponge lysate protein cross-linking revealed a band of 240 kDa corresponding to an α2ß2 tetramer structure. This result suggests that P4H from marine sponges shares the same quaternary structure with vertebrate homologous enzymes. Gene expression analyses showed that αP4H transcript is higher in the choanosome than in the ectosome, while the study of factors affecting its expression in sponge fragmorphs revealed that soluble silicates had no effect on the αP4H levels, whereas ascorbic acid strongly upregulated the αP4H mRNA. Finally, treatment with two different tumor necrosis factor (TNF)-alpha inhibitors determined a significant downregulation of αP4H gene expression in fragmorphs demonstrating, for the first time in Porifera, a positive involvement of TNF in sponge matrix biosynthesis. The molecular characterization of P4H genes involved in collagen hydroxylation, including the mechanisms that regulate their expression, is a key step for future recombinant sponge collagen production and may be pivotal to understand pathological mechanisms related to extracellular matrix deposition in higher organisms.

Sponge derived bromotyrosines: structural diversity through natural combinatorial chemistry.

Sponge derived bromotyrosines are a multifaceted class of marine bioactive compounds that are important for the chemical defense of sponges but also for drug discovery programs as well as for technical applications in the field of antifouling constituents. These compounds, which are mainly accumulated by Verongid sponges, exhibit a diverse range of bioactivities including antibiotic, cytotoxic and antifouling effects. In spite of the simple biogenetic building blocks, which consist only of brominated tyrosine and tyramine units, an impressive diversity of different compounds is obtained through different linkages between these precursors and through structural modifications of the side chains and/or aromatic rings resembling strategies that are known from combinatorial chemistry. As examples for bioactive, structurally divergent bromotyrosines psammaplin A, Aplysina alkaloids featuring aerothionin, aeroplysinin-1 and the dienone, and the bastadins, including the synthetically derived hemibastadin congeners, have been selected for this review. Whereas all of these natural products are believed to be involved in the chemical defense of sponges, some of them may also be of particular relevance to drug discovery due to their interaction with specific molecular targets in eukaryotic cells. These targets involve important enzymes and receptors, such as histone deacetylases (HDAC) and DNA methyltransferases (DNMT), which are inhibited by psammaplin A, as well as ryanodine receptors that are targeted by bastadine type compounds. The hemibastadins such as the synthetically derived dibromohemibastadin are of particular interest due to their antifouling activity. For the latter, a phenoloxidase which catalyzes the bioglue formation needed for firm attachment of fouling organisms to a given substrate was identified as a molecular target. The Aplysina alkaloids finally provide a vivid example for dynamic wound induced bioconversions of natural products that generate highly efficient chemical weapons precisely when and where needed.

Sponges as sentinels: Metal accumulation using transplanted sponges across a metal gradient.

To be effective sentinels, organisms must be able to be readily translocated to contamination hotspots. The authors sought to assess metal accumulation in genetically identical explants of a relatively common estuarine sponge, Suberites cf. diversicolor. Explants were transplanted to 7 locations across a metal contamination gradient in a large coastal estuary in southeastern Australia to establish, first, that explants of this species could be successfully translocated; second, that explants accumulated metals (cadmium, copper, lead, selenium, and zinc) sufficiently rapidly to be effective sentinels; third, that rates of metal accumulation in explants were in agreement with metal concentrations within sediments (<63-µm fraction) at each of the transplant locations; and finally, that changes in explant biomass correlated with overall metal load. Suberites were readily transplanted, with no mortality observed for the 2 mo of transplantation. Metal accumulation for lead, cadmium, and zinc was in close agreement with sediment metal concentrations, and explants showed dramatic increases in these metals in the heavily contaminated northern sections of the estuarine lake. No striking patterns were apparent for copper and selenium. Finally, growth was negatively correlated with total metal load and standardized total metal load in our explants. Taken together, these outcomes confirm that explants of this sponge are amenable to translocation and show considerable promise as biomonitors.

Scalarane sesterterpenes from the Paracel Islands marine sponge Hyrtios sp.

A new scalarane sesterterpene, sesterstamide (1), together with four known sesterterpenes (2-5), were isolated from the Paracel Islands marine sponge Hyrtios sp. The chemical structures were established on the basis of spectroscopic analysis and comparison with known compounds. The cytotoxic and antileishmanial activities of the isolated compounds were also evaluated.

Antifungal activity of metabolites from the marine sponges Amphimedon sp. and Monanchora arbuscula against Aspergillus flavus strains isolated from peanuts (Arachis hypogaea).

Contamination of preharvest and stored peanuts (Arachis hypogaea L.) by aflatoxigenic strains of Aspergillus flavus is an important economical and food safety problem in many tropical and subtropical areas of the world. The present investigation reports the antifungal activity of a halitoxins/amphitoxins enriched extract obtained from the sponge Amphimedon sp. (HAEEAsp), and of batzelladine L isolated from the sponge Monanchora arbuscula on Aspergillus flavus isolated from stored peanuts. A PCR system directed against the ITS region and aflatoxin biosynthetic pathway genes of A. flavus was applied for identification of aflatoxin producing strains. The HAEEAsp extract and batzelladine L showed minimal inhibitory concentration (MIC) in the range between 1.9 to 15.6 microg/mL and between 1.9 to 7.8 microg/mL, respectively. The minimal fungicide concentration (MFC) of HAEEAsp extract and batzelladine L was in the range between 3.9 to 31.3 microg/mL and 3.9 to 15.6 microg/mL, respectively. These results indicate that these marine alkaloids may be further explored for the development of potential lead compounds active against aflatoxigenic fungi.

5alpha,8alpha-epidioxysterols from a Formosan sponge, Axinyssa sp.

One new 5alpha,8alpha-epidioxysterol, 3-acetylaxinysterol (1), along with one known sterol, axinysterol (2), were isolated from a Formosan sponge, Axinyssa sp.. The structures of the compounds were determined by spectroscopic methods and the absolute configuration of 2 was further confirmed by single-crystal X-ray diffraction analysis for the first time. Compound 2 exhibited significant cytotoxicity against K562 and Molt 4 cancer cell lines.

N-containing metabolites from the marine sponge Agelas clathrodes.

A new bisuracil analogue, 3,3-bis(uracil-1-yl)-propan-1-aminium (1), together with four known N-containing metabolites (2-5), were isolated from the South China Sea sponge Agelas clathrodes. Their chemical structures were established on the basis of spectroscopic and spectrometric analysis and comparison with known compounds. Compound 1 is an unusual naturally occurring bisuracil analogue, and compound 2 was isolated from a natural source for the first time. Compounds 2 and 4 exhibit moderate cytotoxicity against cancer cell line SGC7901.

Base-mediated transformations of 3,5-dibromoverongiaquinol from the sponge Aplysina sp. to cavernicolins-1, -2 and a subereatensin analogue.

Treatment of 3,5-dibromoverongiaquinol (1) with NaHCO3-MeOH at room temperature gave cavernicolin-1 (2), cavernicolin-2 (3), the 4,7-dimethoxy analogue of subereatensin (4) and dimethyl ketal (5). These transformations may clarify the origin of the cavernicolins and subereatensin isolated from the extracts of some verongid sponges.

Common genetic denominators for Ca++-based skeleton in Metazoa: role of osteoclast-stimulating factor and of carbonic anhydrase in a calcareous sponge.

Calcium-based matrices serve predominantly as inorganic, hard skeletal systems in Metazoa from calcareous sponges [phylum Porifera; class Calcarea] to proto- and deuterostomian multicellular animals. The calcareous sponges form their skeletal elements, the spicules, from amorphous calcium carbonate (ACC). Treatment of spicules from Sycon raphanus with sodium hypochlorite (NaOCl) results in the disintegration of the ACC in those skeletal elements. Until now a distinct protein/enzyme involved in ACC metabolism could not been identified in those animals. We applied the technique of phage display combinatorial libraries to identify oligopeptides that bind to NaOCl-treated spicules: those oligopeptides allowed us to detect proteins that bind to those spicules. Two molecules have been identified, the (putative) enzyme carbonic anhydrase and the (putative) osteoclast-stimulating factor (OSTF), that are involved in the catabolism of ACC. The complete cDNAs were isolated and the recombinant proteins were prepared to raise antibodies. In turn, immunofluorescence staining of tissue slices and qPCR analyses have been performed. The data show that sponges, cultivated under standard condition (10 mM CaCl(2)) show low levels of transcripts/proteins for carbonic anhydrase or OSTF, compared to those animals that had been cultivated under Ca(2+)-depletion condition (1 mM CaCl(2)). Our data identify with the carbonic anhydrase and the OSTF the first two molecules which remain conserved in cells, potentially involved in Ca-based skeletal dissolution, from sponges (sclerocytes) to human (osteoclast).

Diacarperoxide S, new norterpene cyclic peroxide from the sponge Diacarnus megaspinorhabdosa.

Re-investigation of the methanolic extract of the sponge Diacarnus megaspinorhabdosa afforded one new norsesterpene cyclic peroxide diacarperoxide S (2), in addition to two known compounds sigmosceptrellin B (1) and nebularine (3). The structures of the isolated compounds were established on the basis of one and two dimensional NMR spectroscopic studies (1H, 13C, DEPT, COSY, HMQC, HMBC, and ROESY) as well as on mass spectral analysis. The isolated compounds were evaluated for their cytotoxic as well as antimicrobial activities.

Bioactive compounds from marine sponges and their symbiotic microbes: a potential source of nutraceuticals.

Sponges are considered as the chemical factory in marine environment because of its immense production of chemically diverse compounds. Other than the chemical diversity, these compounds possess remarkable bioactivities. This great potential has aroused applications of sponge-derived compounds as therapeutics and at present, a number of promising compounds are in clinical and preclinical trials. Recently, nutraceuticals have received considerable interest among the health conscious community because of its multiple therapeutic effects. Natural health-promoting substances gain continuous popularity as nutraceuticals due to its reduced risk of side effects. This overview discusses the potentials of marine sponge-derived bioactivities as natural health-promoting compounds.

Recently confirmed apoptosis-inducing lead compounds isolated from marine sponge of potential relevance in cancer treatment.

Despite intense efforts to develop non-cytotoxic anticancer treatments, effective agents are still not available. Therefore, novel apoptosis-inducing drug leads that may be developed into effective targeted cancer therapies are of interest to the cancer research community. Targeted cancer therapies affect specific aberrant apoptotic pathways that characterize different cancer types and, for this reason, it is a more desirable type of therapy than chemotherapy or radiotherapy, as it is less harmful to normal cells. In this regard, marine sponge derived metabolites that induce apoptosis continue to be a promising source of new drug leads for cancer treatments. A PubMed query from 01/01/2005 to 31/01/2011 combined with hand-curation of the retrieved articles allowed for the identification of 39 recently confirmed apoptosis-inducing anticancer lead compounds isolated from the marine sponge that are selectively discussed in this review.

Biosilica-based strategies for treatment of osteoporosis and other bone diseases.

Osteoporosis is a common disease in later life, which has become a growing public health problem. This degenerative bone disease primarily affects postmenopausal women, but also men may suffer from reduced bone mineral density. The development of prophylactic treatments and medications of osteoporosis has become an urgent issue due to the increasing proportion of the elderly in the population. Apart from medical/hormonal treatments, current strategies for prophylaxis of osteoporosis are primarily based on calcium supplementation as a main constituent of bone hydroxyapatite mineral. Despite previous reports suggesting an essential role in skeletal growth and development, the significance of the trace element silicon in human bone formation has attracted major scientific interest only rather recently. The interest in silicon has been further increased by the latest discoveries in the field of biosilicification, the formation of the inorganic silica skeleton of the oldest still extant animals on Earth, the sponges, which revealed new insights in the biological function of this element. Sponges make use of silicon to build up their inorganic skeleton which consists of biogenously formed polymeric silica (biosilica). The formation of biosilica is mediated by specific enzymes, silicateins, which have been isolated, characterized, and expressed in a recombinant way. Epidemiological studies revealed that dietary silicon reduces the risk of osteoporosis and other bone diseases. Recent results allowed for the first time to understand the molecular mechanism underlying the protective effect of silicic acid/biosilica against osteoporosis. Biosilica was shown to modulate the ratio of expression of two cytokines involved in bone formation-RANKL and osteoprotegerin. Hence, biosilica has been proposed to have a potential in prophylaxis and therapy of osteoporosis and related bone diseases.

Terpenoid metabolites from Spongia spp. and their effects on nucleic acid biosynthesis in sea urchin eggs.

19-Norspongia-13(16),14-diene-3-one (1) was isolated for the first time from a natural source, along with a series of known spongiane diterpenoids (2-11) and sesquiterpene (12) from two unidentified species belonging to the genus Spongia. The effects of 1, 4, 5, 8-12 on biosynthesis of nucleic acids and embryonic development of the sea urchin Strongylocentrotus intermedius have been studied. All the compounds inhibit sea urchin embryo development at concentration of 20 microg/mL and above and DNA biosynthesis at the dose of 10 microg/mL. The inhibitory effect of diterpenoids at least partly may be explained by the inhibition of thymidine kinase activity.