Whole genome sequence of the deep-sea sponge Geodia barretti (Metazoa, Porifera, Demospongiae).

Sponges are among the earliest branching extant animals. As such, genetic data from this group are valuable for understanding the evolution of various traits and processes in other animals. However, like many marine organisms, they are notoriously difficult to sequence, and hence, genomic data are scarce. Here, we present the draft genome assembly for the North Atlantic deep-sea high microbial abundance species Geodia barretti Bowerbank 1858, from a single individual collected on the West Coast of Sweden. The nuclear genome assembly has 4,535 scaffolds, an N50 of 48,447 bp and a total length of 144 Mb; the mitochondrial genome is 17,996 bp long. BUSCO completeness was 71.5%. The genome was annotated using a combination of ab initio and evidence-based methods finding 31,884 protein-coding genes.

First continuous marine sponge cell line established.

The potential of sponge-derived chemicals for pharmaceutical applications remains largely unexploited due to limited available biomass. Although many have attempted to culture marine sponge cells in vitro to create a scalable production platform for such biopharmaceuticals, these efforts have been mostly unsuccessful. We recently showed that Geodia barretti sponge cells could divide rapidly in M1 medium. In this study we established the first continuous marine sponge cell line, originating from G. barretti. G. barretti cells cultured in OpM1 medium, a modification of M1, grew more rapidly and to a higher density than in M1. Cells in OpM1 reached 1.74 population doublings after 30 min, more than twofold higher than the already rapid growth rate of 0.74 population doublings in 30 min in M1. The maximum number of population doublings increased from 5 doublings in M1 to at least 98 doublings in OpM1. Subcultured cells could be cryopreserved and used to inoculate new cultures. With these results, we have overcome a major obstacle that has blocked the path to producing biopharmaceuticals with sponge cells at industrial scale for decades.

Geobarrettin D, a Rare Herbipoline-Containing 6-Bromoindole Alkaloid from Geodia barretti.

Geobarrettin D (1), a new bromoindole alkaloid, was isolated from the marine sponge Geodia barretti collected from Icelandic waters. Its structure was elucidated by 1D, and 2D NMR (including 1H-15N HSQC, 1H-15N HMBC spectra), as well as HRESIMS data. Geobarrettin D (1) is a new 6-bromoindole featuring an unusual purinium herbipoline moiety. Geobarrettin D (1) decreased secretion of the pro-inflammatory cytokine IL-12p40 by human monocyte derived dendritic cells, without affecting secretion of the anti-inflammatory cytokine IL-10. Thus, compound 1 shows anti-inflammatory activity.

Is the North Atlantic Geodia barretti (Porifera, Tetractinellida, Geodiidae) present on the Southwest Indian Ridge?

There are currently 163 species of Geodia Lamarck, 1815 described worldwide, many of which are found in deep waters, but none of which have been recorded from the Southwest Indian Ridge (SWIR). Spicule morphology and barcodes (Folmer COI, 28S (C2D2), partial 18S) suggest that a specimen of Geodia collected on the SWIR at a depth of 2236 m is closely comparable to Geodia barretti Bowerbank, 1858. Geodia barretti is the most studied and thus well-known deep-sea Geodia species, due to its wide North Atlantic distribution and key role in boreal sponge grounds. This unexpected and markedly disjunct record would extend the distribution range of this species considerably, consequently challenging our knowledge about interoceanic deep-sea sponges.

Evaluating the secondary bioactive metabolites in Geodia corticostylifera.

Ophiactis savignyi could be discovered all over the world in tropical marine environments. People could have aided in the spread of O. savignyi, particularly in the western and eastern populations of Panama's Isthmus. The brittle star Ophiactis savignyi, often known as savigny's brittle star, coexists alongside the sponge Geodia corticostylifera. The focus of this research has been to assess the functional relevance of G. corticostylifera secondary metabolites as antifoulant against mussels, protection against generalist fish, and chemical cues to affiliated brittle stars. Both in flow-through and static seawater laboratory studies, O. savignyi which has previously been connected with sponges, was given both treated and control mimics at the same time. The sponge extract was also tested for its ability to protect fish against predators and fouling. Deterrence test using chemicals indicated that the normal level of the sponge extract may also suppress generalist fish predation in the field as well as the mussel Perna perna's normal attachment in clinical contexts. According to the findings, G. corticostylifera crude extract has many roles in the aquatic environments, apparently being accountable for this sponge's tighter relationship with O. savignyi, which protects the ophiuroid and inhibits epibionts on itself.

Barrettides: A Peptide Family Specifically Produced by the Deep-Sea Sponge Geodia barretti.

Natural product discovery by isolation and structure elucidation is a laborious task often requiring ample quantities of biological starting material and frequently resulting in the rediscovery of previously known compounds. However, peptides are a compound class amenable to an alternative genomic, transcriptomic, and in silico discovery route by similarity searches of known peptide sequences against sequencing data. Based on the sequences of barrettides A and B, we identified five new barrettide sequences (barrettides C-G) predicted from the North Atlantic deep-sea demosponge Geodia barretti (Geodiidae). We synthesized, folded, and investigated one of the newly described barrettides, barrettide C (NVVPCFCVEDETSGAKTCIPDNCDASRGTNP, disulfide connectivity I-IV, II-III). Co-elution experiments of synthetic and sponge-derived barrettide C confirmed its native conformation. NMR spectroscopy and the anti-biofouling activity on larval settlement of the bay barnacle Amphibalanus improvisus (IC50 0.64 µM) show that barrettide C is highly similar to barrettides A and B in both structure and function. Several lines of evidence suggest that barrettides are produced by the sponge itself and not one of its microbial symbionts.

Taxonomy of Geodia and Rhabdastrella from the Brazilian coast: a new species, new synonyms and redescription of Geodia tylastra (Demospongiae: Astrophorina: Geodiidae and Ancorinidae).

In this contribution we revise the type specimens of Geodia tylastra, Rhabdastrella fibrosa and Geodia corticostylifera, adding new photographs of preserved specimens, skeleton sections, and scanning electron micrographs of the spicules. We demonstrate that the three species are synonyms, with priority to the older name G. tylastra Boury-Esnault, 1973. We also show that Rhabdastrella fibrosa sensu Van Soest (2017) differs from the holotype in spicule composition and is here given a new name, Rhabdastrella vansoesti sp. nov. In addition, we describe a new species of Geodia, G. polytriaena sp. nov., and new records of G. glariosa and G. tylastra from Sergipe State, in the Northeast Brazilian coast. With the description of Geodia polytriaena sp. nov. and the synonymyzation of G. corticostylifera, 20 species of Geodia are now known in the Tropical Western Atlantic Ocean.

Bacterial precursors and unsaturated long-chain fatty acids are biomarkers of North-Atlantic deep-sea demosponges.

Sponges produce distinct fatty acids (FAs) that (potentially) can be used as chemotaxonomic and ecological biomarkers to study endosymbiont-host interactions and the functional ecology of sponges. Here, we present FA profiles of five common habitat-building deep-sea sponges (class Demospongiae, order Tetractinellida), which are classified as high microbial abundance (HMA) species. Geodia hentscheli, G. parva, G. atlantica, G. barretti, and Stelletta rhaphidiophora were collected from boreal and Arctic sponge grounds in the North-Atlantic Ocean. Bacterial FAs dominated in all five species and particularly isomeric mixtures of mid-chain branched FAs (MBFAs, 8- and 9-Me-C16:0 and 10- and 11-Me-C18:0) were found in high abundance (together ≥ 20% of total FAs) aside more common bacterial markers. In addition, the sponges produced long-chain linear, mid- and a(i)-branched unsaturated FAs (LCFAs) with a chain length of 24‒28 C atoms and had predominantly the typical Δ5,9 unsaturation, although the Δ9,19 and (yet undescribed) Δ11,21 unsaturations were also identified. G. parva and S. rhaphidiophora each produced distinct LCFAs, while G. atlantica, G. barretti, and G. hentscheli produced similar LCFAs, but in different ratios. The different bacterial precursors varied in carbon isotopic composition (δ13C), with MBFAs being more enriched compared to other bacterial (linear and a(i)-branched) FAs. We propose biosynthetic pathways for different LCFAs from their bacterial precursors, that are consistent with small isotopic differences found in LCFAs. Indeed, FA profiles of deep-sea sponges can serve as chemotaxonomic markers and support the concept that sponges acquire building blocks from their endosymbiotic bacteria.

The Molecular Machinery of Gametogenesis in Geodia Demosponges (Porifera): Evolutionary Origins of a Conserved Toolkit across Animals.

All animals are capable of undergoing gametogenesis. The ability of forming haploid cells from diploid cells through meiosis and recombination appeared early in eukaryotes, whereas further gamete differentiation is mostly a metazoan signature. Morphologically, the gametogenic process presents many similarities across animal taxa, but little is known about its conservation at the molecular level. Porifera are the earliest divergent animals and therefore are an ideal phylum to understand evolution of the gametogenic toolkits. Although sponge gametogenesis is well known at the histological level, the molecular toolkits for gamete production are largely unknown. Our goal was to identify the genes and their expression levels which regulate oogenesis and spermatogenesis in five gonochoristic and oviparous species of the genus Geodia, using both RNAseq and proteomic analyses. In the early stages of both female and male gametogenesis, genes involved in germ cell fate and cell-renewal were upregulated. Then, molecular signals involved in retinoic acid pathway could trigger the meiotic processes. During later stages of oogenesis, female sponges expressed genes involved in cell growth, vitellogenesis, and extracellular matrix reassembly, which are conserved elements of oocyte maturation in Metazoa. Likewise, in spermatogenesis, genes regulating the whole meiotic cycle, chromatin compaction, and flagellum axoneme formation, that are common across Metazoa were overexpressed in the sponges. Finally, molecular signals possibly related to sperm capacitation were identified during late stages of spermatogenesis for the first time in Porifera. In conclusion, the activated molecular toolkit during gametogenesis in sponges was remarkably similar to that deployed during gametogenesis in vertebrates.

6-Bromoindole Derivatives from the Icelandic Marine Sponge Geodia barretti: Isolation and Anti-Inflammatory Activity.

An UPLC-qTOF-MS-based dereplication study led to the targeted isolation of seven bromoindole alkaloids from the sub-Arctic sponge Geodia barretti. This includes three new metabolites, namely geobarrettin A⁻C (1⁻3) and four known compounds, barettin (4), 8,9-dihydrobarettin (5), 6-bromoconicamin (6), and l-6-bromohypaphorine (7). The chemical structures of compounds 1⁻7 were elucidated by extensive analysis of the NMR and HRESIMS data. The absolute stereochemistry of geobarrettin A (1) was assigned by ECD analysis and Marfey's method employing the new reagent l-Nα-(1-fluoro-2,4-dinitrophenyl)tryptophanamide (l-FDTA). The isolated compounds were screened for anti-inflammatory activity using human dendritic cells (DCs). Both 2 and 3 reduced DC secretion of IL-12p40, but 3 concomitantly increased IL-10 production. Maturing DCs treated with 2 or 3 before co-culturing with allogeneic CD4⁺ T cells decreased T cell secretion of IFN-γ, indicating a reduction in Th1 differentiation. Although barettin (4) reduced DC secretion of IL-12p40 and IL-10 (IC50 values 11.8 and 21.0 µM for IL-10 and IL-12p40, respectively), maturing DCs in the presence of 4 did not affect the ability of T cells to secrete IFN-γ or IL-17, but reduced their secretion of IL-10. These results indicate that 2 and 3 may be useful for the treatment of inflammation, mainly of the Th1 type.

Evaluating the Effects of an Organic Extract from the Mediterranean Sponge Geodia cydonium on Human Breast Cancer Cell Lines.

Marine sponges are an excellent source of bioactive secondary metabolites for pharmacological applications. In the present study, we evaluated the chemistry, cytotoxicity and metabolomics of an organic extract from the Mediterranean marine sponge Geodia cydonium, collected in coastal waters of the Gulf of Naples. We identified an active fraction able to block proliferation of breast cancer cell lines MCF-7, MDA-MB231, and MDA-MB468 and to induce cellular apoptosis, whereas it was inactive on normal breast cells (MCF-10A). Metabolomic studies showed that this active fraction was able to interfere with amino acid metabolism, as well as to modulate glycolysis and glycosphingolipid metabolic pathways. In addition, the evaluation of the cytokinome profile on the polar fractions of three treated breast cancer cell lines (compared to untreated cells) demonstrated that this fraction induced a slight anti-inflammatory effect. Finally, the chemical entities present in this fraction were analyzed by liquid chromatography high resolution mass spectrometry combined with molecular networking.

Microbiome analysis of a disease affecting the deep-sea sponge Geodia barretti.

Reports of sponge disease are becoming increasingly frequent, although almost all instances involve shallow-water, tropical species. Here, we describe the first disease affecting the deep-water sponge, Geodia barretti. The disease is characterised by brown/black discolouration of the sponge tissue, extensive levels of tissue disintegration and increased levels of fouling. Disease prevalence was quantified using video survey transects conducted between 100 and 220 m in Korsfjorden, Norway, and the microbial communities of healthy and diseased sponges were compared using 16S rRNA gene sequencing. Highly divergent community profiles were evident between the different health states, with distinct community shifts involving higher relative abundances of Bacteroidetes, Firmicutes and Deltaproteobacteria in diseased individuals. In addition, three operational taxonomic units were exclusively present in diseased individuals and were shared between the disease lesions and the apparently healthy tissue of diseased individuals, suggesting a non-localised infection or dysbiosis. Genomic analysis of the G. barretti microbiome combined with experimental work to assess the mechanisms of infection will further elucidate the role of microorganisms in the disease.

Metabolomic Profiling Reveals the N-Acyl-Taurine Geodiataurine in Extracts from the Marine Sponge Geodia macandrewii (Bowerbank).

A metabolomic approach was used to identify known and new natural products from the marine sponges Geodia baretti and G. macandrewii. G. baretti is known to produce bioactive natural products such as barettin (1), 8,9-dihydrobarettin (2), and bromobenzisoxazolone barettin (3), while secondary metabolites from G. macandrewii are not reported in the literature. Specimens of the two sponges were collected from different sites along the coast of Norway, and their extracts were analyzed using UHPLC-HR-MS. Metabolomic analyses revealed that extracts from both species contained barettin (1) and 8,9-dihydrobarettin (2), and all samples of G. baretti contained higher amounts of both compounds compared to G. macandrewii. The analysis of the MS data also revealed that samples of G. macandrewii contained a compound that was not present in any of the G. baretti samples. This new compound was isolated and identified as the N-acyl-taurine geodiataurine (4), and it was tested for antioxidant, anticancer, and antibacterial properties.

Sub-lethal effects of water-based drilling muds on the deep-water sponge Geodia barretti.

Offshore oil and gas activities can result in the discharge of large amounts of drilling muds. While these materials have generally been regarded as non-toxic to marine organisms, recent studies have demonstrated negative impacts to suspension feeding organisms. We exposed the arctic-boreal sponge Geodia barretti to the primary particulate components of two water-based drilling muds; barite and bentonite. Sponges were exposed to barite, bentonite and a natural reference sediment at a range of total suspended solid concentrations (TSS = 0, 10, 50 or 100 mg/L) for 12 h after which we measured a suite of biomarker responses (lysosomal membrane stability, lipid peroxidation and glutathione). In addition, we compared biomarker responses, organic energy content and metal accumulation in sponges, which had been continuously or intermittently exposed to suspended barite and natural sediment for 14 d at relevant concentrations (10 and 30 mg TSS/L). Lysosomal membrane stability was reduced in the sponges exposed to barite at 50 and 100 mg TSS/L after just 12 h and at 30 mg TSS/L for both continuous and intermittent exposures over 14 d. Evidence of compromised cellular viability was accompanied by barite analysis revealing concentrations of Cu and Pb well above reference sediments and Norwegian sediment quality guidelines. Metal bioaccumulation in sponge tissues was low and the total organic energy content (determined by the elemental composition of organic tissue) was not affected. Intermittent exposures to barite resulted in less toxicity than continuous exposure to barite. Short term exposures to bentonite did not alter any biomarker responses. This is the first time that these biomarkers have been used to indicate contaminant exposure in an arctic-boreal sponge. Our results illustrate the potential toxicity of barite and the importance of assessments that reflect the ways in which these contaminants are delivered under environmentally realistic conditions.

Marine AChE inhibitors isolated from Geodia barretti: natural compounds and their synthetic analogs.

Barettin, 8,9-dihydrobarettin, bromoconicamin and a novel brominated marine indole were isolated from the boreal sponge Geodia barretti collected off the Norwegian coast. The compounds were evaluated as inhibitors of electric eel acetylcholinesterase. Barettin and 8,9-dihydrobarettin displayed significant inhibition of the enzyme, with inhibition constants (Ki) of 29 and 19 µM respectively towards acetylcholinesterase via a reversible noncompetitive mechanism. These activities are comparable to those of several other natural acetylcholinesterase inhibitors of marine origin. Bromoconicamin was less potent against acetylcholinesterase, and the novel compound was inactive. Based on the inhibitory activity, a library of 22 simplified synthetic analogs was designed and prepared to probe the role of the brominated indole, common to all the isolated compounds. From the structure-activity investigation it was shown that the brominated indole motif is not sufficient to generate a high acetylcholinesterase inhibitory activity, even when combined with natural cationic ligands for the acetylcholinesterase active site. The four natural compounds were also analysed for their butyrylcholinesterase inhibitory activity in addition and shown to display comparable activities. The study illustrates how both barettin and 8,9-dihydrobarettin display additional bioactivities which may help to explain their biological role in the producing organism. The findings also provide new insights into the structure-activity relationship of both natural and synthetic acetylcholinesterase inhibitors.

Anti-Inflammatory Effects of a Methanol Extract from the Marine Sponge Geodia cydonium on the Human Breast Cancer MCF-7 Cell Line.

Many research groups are working to find new possible anti-inflammatory molecules, and marine sponges represent a rich source of biologically active compounds with pharmacological applications. In the present study, we tested different concentrations of the methanol extract from the marine sponge, Geodia cydonium, on normal human breast epithelial cells (MCF-10A) and human breast cancer cells (MCF-7). Our results show that this extract has no cytotoxic effects on both cell lines whereas it induces a decrease in levels of VEGF and five proinflammatory cytokines (CCL2, CXCL8, CXCL10, IFN-γ, and TNF-α) only in MCF-7 cells in a dose-dependent manner, thereby indicating an anti-inflammatory effect. Moreover, interactomic analysis suggests that all six cytokines are involved in a network and are connected with some HUB nodes such as NF-kB subunits and ESR1 (estrogen receptor 1). We also report a decrease in the expression of two NFKB1 and c-Rel subunits by RT-qPCR experiments only in MCF-7 cells after extract treatment, confirming NF-kB inactivation. These data highlight the potential of G. cydonium for future drug discovery against major diseases, such as breast cancer.

Isolation, Characterization, and Synthesis of the Barrettides: Disulfide-Containing Peptides from the Marine Sponge Geodia barretti.

Two disulfide-containing peptides, barrettides A (1) and B (2), from the cold-water marine sponge Geodia barretti are described. Those 31 amino acid residue long peptides were sequenced using mass spectrometry methods and structurally characterized using NMR spectroscopy. The structure of 1 was confirmed by total synthesis using the solid-phase peptide synthesis approach that was developed. The two peptides were found to differ only at a single position in their sequence. The three-dimensional structure of 1 revealed that these peptides possess a unique fold consisting of a long ß-hairpin structure that is cross-braced by two disulfide bonds in a ladder-like arrangement. The peptides are amphipathic in nature with the hydrophobic and charged residues clustered on separate faces of the molecule. The barrettides were found not to inhibit the growth of either Escherichia coli or Staphylococcus aureus but displayed antifouling activity against barnacle larvae (Balanus improvisus) without lethal effects in the concentrations tested.

Modeling the distribution of Geodia sponges and sponge grounds in the Northwest Atlantic.

Deep-sea sponge grounds provide structurally complex habitat for fish and invertebrates and enhance local biodiversity. They are also vulnerable to bottom-contact fisheries and prime candidates for Vulnerable Marine Ecosystem designation and related conservation action. This study uses species distribution modeling, based on presence and absence observations of Geodia spp. and sponge grounds derived from research trawl catches, as well as spatially continuous data on the physical and biological ocean environment derived from satellite data and oceanographic models, to model the distribution of Geodia sponges and sponge grounds in the Northwest Atlantic. Most models produce excellent fits with validation data although fits are reduced when models are extrapolated to new areas, especially when oceanographic regimes differ between areas. Depth and minimum bottom salinity were important predictors in most models, and a Geodia spp. minimum bottom salinity tolerance threshold in the 34.3-34.8 psu range was hypothesized on the basis of model structure. The models indicated two currently unsampled regions within the study area, the deeper parts of Baffin Bay and the Newfoundland and Labrador slopes, where future sponge grounds are most likely to be found.

Antioxidant and anti-inflammatory activities of barettin.

In this paper, we present novel bioactivity for barettin isolated from the marine sponge Geodia barretti. We found that barettin showed strong antioxidant activity in biochemical assays as well as in a lipid peroxidation cell assay. A de-brominated synthetic analogue of barettin did not show the same activity in the antioxidant cell assay, indicating that bromine is important for cellular activity. Barettin was also able to inhibit the secretion of the inflammatory cytokines IL-1ß and TNFα from LPS-stimulated THP-1 cells. This combination of anti-inflammatory and antioxidant activities could indicate that barettin has an atheroprotective effect and may therefore be an interesting product to prevent development of atherosclerosis.

A novel interdomain interface in crystallins: structural characterization of the ßγ-crystallin from Geodia cydonium at 0.99 Å resolution.

The ßγ-crystallin superfamily includes highly diverse proteins belonging to all of the kingdoms of life. Based on structural topology, these proteins are considered to be evolutionarily related to the long-lived ßγ-crystallins that constitute the vertebrate eye lens. This study reports the crystallographic structure at 0.99 Å resolution of the two-domain ßγ-crystallin (geodin) from the sponge Geodia cydonium. This is the most ancient member of the ßγ-crystallin superfamily in metazoans. The X-ray structure shows that the geodin domains adopt the typical ßγ-crystallin fold with a paired Greek-key motif, thus confirming the hypothesis that the crystallin-type scaffold used in the evolution of bacteria and moulds was recruited very early in metazoans. As a significant new structural feature, the sponge protein possesses a unique interdomain interface made up by pairing between the second motif of the first domain and the first motif of the second domain. The atomic resolution also allowed a detailed analysis of the calcium-binding site of the protein.