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A new species of ‘lithistid’ (rock sponge) Sollasipelta subterranea sp. nov. is described from near-shore submarine caves of Okinawa and Shimoji Islands, the Ryukyu Islands, southwestern Japan. This new species is characterized by the smooth dentate ectosomal pseudophyllotriaenes, slender choanosomal oxea/style/subtylostyles and two types of amphiasters, one of which is in two size classes. Sollasipelta subterranea sp. nov. is morphologically closest to Sollasipelta sollasi (Lévi & Lévi, 1989), known from the Philippines deep sea, and our examination of the type specimen of S. sollasi revealed that the pseudophyllotriaenes and desmas are substantially different between the two species, and both species possess two types of amphiasters, one of which has two size classes. Sollasipelta cavernicola (Vacelet & Vasseur, 1965) and S. punctata (Lévi & Lévi, 1983) are transferred to the genus Daedalopelta Sollas, 1888, based on possession of one type of amphiastes as microscleres. Sollasipelta mixta (Vacelet, Vasseur and Lévi, 1976) is formally transferred to the genus Neopelta Schmidt, 1880, based on the morphological characters. A key to species of the genus Sollasipelta Van Soest & Hooper, 2020 is also provided. Sollasipelta subterranea sp. nov. represents the first ‘lithistid’ sponge from submarine caves of the Western Pacific and the first species of sponge inhabiting anchialine cave environments in the Indo-West Pacific. This is also a new record of the family Neopeltidae from Japanese waters.

A simple methylenedioxy dibromoindole alkaloid, amakusamine (1), was isolated from a marine sponge of the genus Psammocinia, and its structure was determined from spectroscopic data, time-dependent density-functional theory calculations, and synthesis. Compound 1 inhibited the receptor activator of nuclear factor-κB ligand (RANKL)-induced formation of multinuclear osteoclasts with an IC50 value of 10.5 μM in RAW264 cells. The structure-activity relationship of 1 was also investigated with synthetic derivatives.

A new rearranged nitrogenous bisabolone-type sesquiterpene, halichonic acid B (1), was isolated from a marine sponge Axinyssa sp. together with halichonic acid (2) and (6R,7S)-7-amino-7,8-dihydro-α-bisabolene (3). The structure of 1 was determined by extensive NMR and MS analyses, revealing an unprecedented carbon framework, and its absolute configuration was elucidated by time-dependent density-functional theory (TDDFT)-based electronic circular dichroism (ECD) spectrum calculation. We propose that 1 and 2 may be biosynthesized in the same pathway, involving the reaction between farnesyl pyrophosphate and glycine, followed by cyclization.

A previously unreported heterodetic cyclic peptide, homophymamide A (1), was isolated from a Homophymia sp. marine sponge. The structure of homophymamide A was determined to be a lower homologue of anabaenopeptins by spectroscopic analysis, chemical degradation, and chemical synthesis. Analysis of the acidic hydrolysate showed that the racemization of Lys took place, leading us to pose a cautionary note on the configurational assignment of peptides that contain a ureido bond.

Oshimalides A (1) and B (2) were isolated from a Luffariella sp. marine sponge. The absolute configurations of the stereogenic centers in the cyclohexenone ring were determined by the modified Mosher's analysis of the reduction product. The absolute configuration of the stereogenic center in the dihydropyran ring was assigned by analysis of the 1H NMR data of the vicinal diols which were prepared by AD-mix reagents stereoselectively.

Myrindole A, a bis-indole alkaloid, was isolated from the deep-sea sponge Myrmekioderma sp. The high degree of unsaturation of the molecule complicated the assignment of its structure by standard 2D-NMR experiments but was ultimately achieved by a combination of 1H-15N-HMBC and 1,n-ADEQUATE experiments as well as the comparison of measured and calculated CD spectra. Myrindole A showed antimicrobial activity against Gram-positive and Gram-negative bacteria.

The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), 1 of the oldest metazoans. However, most bacterial members of sponge microbiomes have not been cultivated or sequenced, and therefore, remain unrecognized. Unequivocally linking metabolic functions to a cellular source in sponge microbiomes is, therefore, a challenge. Here, we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge (sponge that hosts a rich bacterial community) Theonella swinhoei, previously shown to contain ‘Entotheonella’ symbionts that produce most of the bioactive substances isolated from the sponge. As an exception, the antifungal aurantosides had remained unassigned to a source. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, ‘Candidatus Poriflexus aureus’ from a new Chloroflexi lineage as the aurantoside producer. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter., PNAS Nexus, 1 (1), ISSN:2752-6542

Among over 20,000 species of Annelida, only two branching species with a highly modified body-pattern are known until now: the Syllidae Syllis ramosa McIntosh, 1879, and Ramisyllis multicaudata Glasby et al. (Zoological Journal of the Linnean Society, 164, 481–497, 2012). Both have unusual ramified bodies with one head and multiple branches and live inside the canals of host sponges. Using an integrative approach (combining morphology, internal anatomy, ecology, phylogeny, genetic divergence, and the complete mitochondrial genome), we describe a new branching species from Japan, Ramisyllis kingghidorahi n. sp., inhabiting an undescribed species of Petrosia (Porifera: Demospongiae) from shallow waters. We compare the new species with its closest relative, R. multicaudata; emend the diagnosis of Ramisyllis; and discuss previous reports of S. ramosa. This study suggests a much higher diversity of branching syllids than currently known. Finally, we discuss possible explanations for the feeding behaviour in the new species in relation to its highly ciliated wall of the digestive tubes (especially at the distal branches and anus), and provide a hypothesis for the evolution of branching body patterns as the result of an adaptation to the host sponge labyrinthic canal system.

Hexactinellid sponges are important members of deep-sea benthic ecosystems because they provide available hard substrate habitats for filter-feeding invertebrates. However, symbioses between hexactinellid sponges and their symbionts are poorly known. Zoantharians associated with hexactinellid sponges have been reported widely from deep-sea marine ecosystems, either on the bodies or stalks of hexactinellid sponges. Despite these records, there has been a lack of research on their diversity and phylogenetic relationships. In this study, 20 specimens associated with amphidiscophoran and hexasterophoran sponges were collected from the waters of Australia and Japan in the Pacific, and from Curaçao in the southern Caribbean, and these were examined in addition to museum specimens. Based on molecular phylogenetic analyses and morphological observations, we formally describe two new genera and three new species of Zoantharia and report several previously described species. The results suggest at least two independent origins for the symbioses between hexactinellid sponges and zoantharians. Our results demonstrate that the diversity of hexactinellid sponge-associated zoantharians is much higher than has been previously thought. The new taxa described in this work further reconfirm that the deep-sea harbours high levels of undescribed zoantharian diversity.

The production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. An example is defensive symbiosis that might account for much of the chemical richness of marine invertebrates including sponges (Porifera), one of the oldest metazoans. However, as most complex microbiomes remain largely uncultivated and lack reference genomes, unequivocally linking metabolic functions to a cellular source is a challenge. Here we report an analysis pipeline of microfluidic encapsulation, Raman microscopy, and integrated digital genomics (MERMAID) for an efficient identification of uncultivated producers. We applied this method to the chemically rich bacteriosponge Theonella swinhoei, previously shown to contain ‘Entotheonella’ symbionts providing most of its bioactive substances except for the antifungal aurantosides that lacked biosynthetic gene candidates in the metagenome. Raman-guided single-bacterial analysis and sequencing revealed a cryptic, distinct multiproducer, ‘Candidatus Poriflexus aureus’ from a new Chloroflexi lineage. Its exceptionally large genome contains numerous biosynthetic loci and suggested an even higher chemical richness of this sponge than previously appreciated. This study highlights the importance of complementary technologies to uncover microbiome functions, reveals remarkable parallels between distantly related symbionts of the same host, and adds functional support for diverse chemically prolific lineages being present in microbial dark matter.Significance StatementThe production of bioactive metabolites is increasingly recognized as an important function of host-associated bacteria. However, the acquisition of integrated genomic and metabolic data from uncultivated environmental bacteria is still challenging. In this work, we explored the combination of Raman microscopy and single-cell sequencing to localize chemical features to a specific bacterium in an uncultivated microbiome, and we specified the bacteria in the uncultured lineage as a producer of aurantoside, an antifungal natural product, from a chemically and microbially complex sponge. This study offers a new methodology as well as insights into chemical functions of uncultivated life.

A new rearranged nitrogenous bisabolone-type sesquiterpene, halichonic acid B (1), was isolated from a marine sponge Axinyssa sp. together with halichonic acid (2) and (6R,7S)-7-amino-7,8-dihydro-α-bisabolene (3). The structure of 1 was determined by extensive NMR and MS analyses, revealing an unprecedented carbon framework, and its absolute configuration was elucidated by time-dependent density-functional theory (TDDFT)-based electronic circular dichroism (ECD) spectrum calculation. We propose that 1 and 2 may be biosynthesized in the same pathway, involving the reaction between farnesyl pyrophosphate and glycine, followed by cyclization.

N 6-Isopentenyladenosine (i6A) was isolated from a marine sponge Oceanapia sp. as the major cytotoxic constituent along with N6-isopentenyladenosine 5ʹ-monophosphate (i6AP) which was inactive. The structures of i6A and i6AP were assigned by a combination of the analysis of NMR spectroscopy and mass spectrometry. This is the first isolation of i6A and i6AP from a marine sponge.

We screened 868 marine extracts in search of hematopoietic molecules resulted in findings of several extracts that proliferated Ba/F3-HuMpl cells but not the cells expressed with other hematopoietic cytokine receptors, EPO and G-CSF. Separation of the most potent extract of a Micronesian sponge Corticium sp., guided by the cell proliferation assay using Ba/F3-HuMpl cells resulted in an isolation of thrombocorticin (ThC), a novel 14 kDa protein as an active principal. ThC displayed concentration-dependent proliferation of Ba/F3-HuMpl cells, and had a stronger activity than that of eltrombopag, a small molecule drug used to treat thrombocytopenia. ThC induced phosphorylation of STAT5, suggesting that it activates Jak/STAT pathway as in the case of TPO. These results together indicated that ThC is a specific agonist for c-Mpl, although the size and shape differs largely from TPO. Here we present isolation, characterization and biological activity of ThC.

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 β-d-galactopyranosyl-(1 → 2)-6-acetyl-β-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.

The geochemistry of calcifying marine organisms is an excellent proxy for reconstructing paleoceanographic history, but studies of hypercalcified demosponges (sclerosponges) are considerably fewer than those of corals, foraminifers, and bivalves. For this study, we first generated near-annual resolved stable carbon and oxygen isotope (δ13C and δ18O) and element/Ca ratios (Sr/Ca, Ba/Ca, Pb/Ca, U/Ca) time series for 1880–2015 from sclerosponge samples (Acanthochaetetes wellsi) collected at Miyako Island and Okinawa Island in the Ryukyu Islands of southwestern Japan. The δ13C records exhibited a typical variation of anthropogenically derived Suess effects, demonstrating that the rates of decrease of –0.0043‰/year before 1960 and – 0.024‰/year after 1960 in the northwestern subtropical Pacific were respectively similar to and about 1.4 times higher than those of the Caribbean Sea in the tropical Atlantic. Spectral analysis of the δ18O time series revealed significant periodicity of approximately 2, 3, 6.5, 7–10, and 20–30 year/cycle, indicating that sea surface conditions in the southern Ryukyu Islands had been dominated by interannual and decadal variations in temperature and seawater δ18O since the late nineteenth century. The Sr/Ca and U/Ca ratios for the species A. wellsi (high-Mg calcite) might not be a robust proxy for seawater temperatures, unlike Astrosclera willeyana and Ceratoporella nicholsoni sclerosponges (aragonite). An evident increasing Pb/Ca trend after 1950 found in the samples is probably attributable to Pb emissions from industrial activities and atmospheric aerosols in eastern Asian countries. The Ba/Ca variations differ greatly among sampling sites, which might be attributable to the respective local environments. This evidence demonstrates that more high-resolution age determinations and geochemical profilings enable delineation of secular variations in ocean environments on annual and interannual timescales. Results of our study suggest that if sclerosponges living in deeper ocean environments are collected, spatial and vertical oceanographic variations for the last several centuries will be reconstructed along with coral proxy records.

A previously unreported heterodetic cyclic peptide, homophymamide A (

Marine sponges are an excellent source of biologically active secondary metabolites. We focus on deep-sea sponges for our discovery study. A marine sponge Cladocroce sp. exhibited cytotoxic activity in the bioactivity screening. From this sponge a previously unreported cytotoxic glycosphingolipid, calyxoside B, was isolated and the structure of this compound was elucidated by analyses of MS and NMR spectra and chemical derivatization. We converted the ketone in the middle of a long aliphatic chain into an oxime to which was applied Beckmann rearrangement to afford two positional isomers of amides. The products were subjected to acidic hydrolysis followed by LC-MS analysis, permitting us to assign unequivocally the position of the ketone. Calyxoside B shows cytotoxicity against HeLa cells with an IC50 value of 31 µM and also weakly stimulated the production of cytokines in mice.

Myrindole A, a bis-indole alkaloid, was isolated from the deep-sea sponge

Natural products are important sources for drug development. Discovery of natural products that inhibit cell cycle progression significantly contributes to the progress of cancer biology and the development of new antitumor agents. In this study, cell cycle inhibitory activity was evaluated with our extract library of natural resources, including marine invertebrates, fungi, and bacteria, using HeLa/Fucci2 cells which allow classification of the cell cycle phases of living cells. Screening of the extract library revealed that the extract of the marine sponge Dactylospongia metachromia inhibited cell cycle progression at S/G2/M phases. Bioassay-guided fractionation afforded a new sesquiterpene quinone, neoisosmenospongine (1), and four known compounds, nakijiquinone I, N, and Q (2-4) and (-)-dictyoceratin-C (5). The chemical structure of 1 was elucidated by interpretating the NMR and mass spectroscopic data, and the absolute configuration was determined by comparison of the experimental and calculated ECD spectra. Fluorescent imaging of HeLa/Fucci2 cells revealed that 1-4 inhibited the cell cycle progression at S/G2/M phases. This study demonstrated that fluorescent image-based high-content screening using HeLa/Fucci2 cells is an effective approach for isolating cell cycle inhibitors from natural resources.

The geochemistry of the calcium carbonates of marine organisms is an excellent proxy for reconstruction of the paleoceanographic history. However, previous studies of hypercalcified demosponges (sclerosponges) are considerably fewer than those of corals, foraminifers, and bivalves. Here, we investigated stable oxygen (δ18O) and carbon (δ13C) isotopes and minor and trace element (Mg, Sr, Ba, Pb, and U) to Ca ratios of 36 living sclerosponges (Astrosclera willeyana) collected from Kume Island in the Ryukyu Islands, southwestern Japan, to evaluate the utility of geochemistry as a paleoenvironmental proxy. The δ18O, δ13C, and Sr/Ca deviations of the coevally precipitated skeleton were extremely small and almost constant at all skeletal portions, strongly suggesting that within-skeletal variations in the chemical components are negligibly small for non-symbiotic sclerosponges. Mean δ18O, δ13C, and Sr/Ca values (N = 36), falling within a quite narrow range, showed no significant evidence for intraspecific (inter-specimen) variations in the sclerosponges. The sclerosponges δ18O and δ13C were consistent with those of the aragonites precipitated in isotopic equilibrium with seawater at the growth site. The sclerosponge Sr/Ca was close to that of inorganically precipitated aragonite, and the estimated partition coefficient of 1.1 (almost unity) is identical to previously reported values for different species (Ceratoporella nicholsoni). Consequently, these results suggest that A. willeyana sclerosponge, having little vital effects on the geochemistry, is a robust indicator of paleocean environments (seawater δ18O, temperature, and dissolved inorganic carbon δ13C). Further, our evaluation study documents that sclerosponges living in deeper ocean environments can support the reconstruction of spatial and vertical paleoceanographic changes in conjunction with coral proxy records. The sclerosponge U/Ca showed little within-skeletal and intraspecific variations, but the heterogeneity and individual difference of the Mg/Ca, Ba/Ca, and Pb/Ca were relatively large, the reasons of which still remain unresolved.

Stellatolide H (1) was isolated from a deep-sea sponge Discodermia sp. as the cytotoxic constituent. The planar structure of 1 was elucidated on the basis of the NMR spectroscopic and mass spectrometric data. The absolute configurations of the constituent amino acid residues were determined by the Marfey’s method. Stellatolide H (1) is a peptide lactone of the callipeltin class with its N-terminus blocked by 3-hydroxy-6,8-dimethyldeca-(4Z,6E)-dienoic acid (Hdda).

Poecillastrin H (1), a chondropsin-type macrolide with a conjugated pentaene moiety, was isolated from the Characella sp. marine sponge. The planar structure of 1 was elucidated by analysis of spectroscopic data. The absolute configuration of the β-hydroxyaspartic acid residue (β-OHAsp) was determined to be d-threo by Marfey’s analysis, and the mode of lactone ring formation through the OHAsp residue was determined by chemical degradation. Poecillastrin H was extremely sensitive toward light and showed potent cytotoxic activity against 3Y1 cells with an IC50 value of 4.1 nM.

Morphology-guided cell-based screening of the extract of a Mycale sp. marine sponge led to the isolation of two trisoxazole macrolides, miuramides A (1) and B (2), which induced characteristic morphological changes in 3Y1 cells. The structure of 1 including absolute configuration was elucidated by a combination of the analysis of spectroscopic data, derivatization, and degradation. Both compounds exhibit potent cytotoxicity against 3Y1 cells.

A new species in a new genus of sea anemone, Tempuractis rinkai gen. et sp. nov., was discovered at several localities along the temperate rocky shores of Japan. The new species is approximately 4 mm in length and has been assigned to family Edwardsiidae, because it has eight macrocnemes, lacks sphincter and basal muscles, and possesses rounded aboral end. The sea anemone, however, also has a peculiar body shape unlike that of any other known taxa. This new species resembles some genera, especially Drillactis and Nematostella, in smooth column surface without nemathybomes or tenaculi, but is distinguishable from them by several morphological features: the presence of holotrichs and absence of nematosomes. Furthermore, this edwardsiid species exhibits a peculiar symbiotic ecology with sponges. Therefore, a new genus, Tempuractis, is proposed for this species. In the field, T. rinkai sp. nov. was always found living inside homosclerophorid sponge of the genus Oscarella, which suggests a possible obligate symbiosis between Porifera and Actiniaria. The benefit of this symbiosis is discussed on the basis of observations of live specimens, both in the aquarium and field. This is the first report of symbiosis between a sea anemone and a homoscleromorph sponge.

Poecillastrin E (1), F (2), and G (3) were isolated from a marine sponge Poecillastra sp. as the cytotoxic constituents. Their planar structures were determined by analyzing the MS and NMR spectra. They are closely related to the known poecillastrin C (4). The absolute configuration of the β-hydroxyaspartic acid (OHAsp) residue was determined to be D-threo by Marfey's analysis of the hydrolysate. The mode of lactone ring formation of OHAsp residue in 1–3 was determined by selective reduction of the ester linkage followed by acid hydrolysis.

A new compound, halichonic acid (1), was isolated from a marine sponge Halichondria sp., together with (6R,7S)-7-amino-7,8-dihydro-α-bisabolene (2). The structure of 1 was elucidated by spectroscopic analysis and ECD spectrum calculation to be a rearranged bisabolene-type sesquiterpene having a 3-azabicyclo[3.3.1]nonene moiety. Compound 2 was cytotoxic against HeLa cells with an IC50 value of 50 μM, whereas 1 did not show cytotoxicity even at 50 μM. It is possible that 1 is biosynthesized from farnesyl pyrophosphate and glycine, with rearrangement.

Natural products that inhibit cell cycle progression show promise as anticancer agents and chemical probes. In our research on biologically active natural products that affect cell cycle progression of HeLa/fluorescent ubiquitination-based cell cycle indicator (Fucci)2 cells, the extract of the marine sponge Neopetrosia chaliniformis was revealed to inhibit cell proliferation. Purification of the extract afforded four new pyridine alkaloids, neopetrosidines A–D (1–4). Their structures were elucidated by the interpretation of spectroscopic data and chemical degradation. Compounds 1–4 were found to inhibit cell proliferation of HeLa/Fucci2 cells, and time-lapse imaging showed that 1 exerts its effect by increasing the duration of the cell cycle. Furthermore, we show that 1 perturbs bioenergetics to exhibit a cytostatic effect by reducing the mitochondrial membrane potential.

New sesquiterpene quinones, metachromins X (1) and Y (2), together with the known metachromins C (3), J (4), and T (5), were isolated as inhibitors of cell cycle progression in the HeLa/Fucci2 cells. The structure of 1 was assigned by spectroscopic data and confirmed by a total synthesis. The planar structure of 2 was determined by interpretation of spectroscopic data, whereas its absolute configuration was analyzed by a combination of chiral HPLC and CD spectroscopy. Metachromins X (1) and C (3) arrested the cell cycle progression of HeLa/Fucci2 cells at S/G2/M phase.

Two new species of thinly encrusting sponge Hamacantha (Demospongiae, Merliida, Hamacanthidae) are described from Japan. Hamacantha (Vomerula) mamoi sp. nov. was collected from Sagami Bay, and Hamacantha (Vomerula) umisachii sp. nov. from off Hachijo Island. This is the first record of Hamacantha and the Hamacanthidae from Japan. H. (V.) mamoi sp. nov. is characterized by having styles, two types of diancistras and one of sigmas. Only two known species have the same spicule composition: H. (V.) acerata Lévi, 1993 and H. (V.) esperioides Ridley & Dendy, 1886, described from New Caledonia, and south-west Africa and south-east South America, respectively. H. (V.) acerata has larger diancistras and much smaller sigmas compared with those of H. (V.) mamoi sp. nov. H. (V.) esperioides can be separated by having larger styles and smaller sigmas than those of H. (V.) mamoi sp. nov. H. (V.) umisachii sp. nov is characterized by having styles, diancistras, cyrtancistra-like diancistras and sigmas. Hamacantha (V.) carteri Topsent, 1904 seems to have similar spicule composition, however the size of all spicule types is different.

Two new triterpene galactosides, melophluosides A and B, were isolated from a marine sponge Melophlus sarasinorum collected in Indonesia. Their structures were determined by the analysis of spectroscopic data and chemical reactions. The absolute configuration of cyclohexenone moiety was determined by the calculated ECD spectrum of a simplified model. Melophluosides A and B exhibited moderate cytotoxicity against HeLa cells (IC50, 11.6 and 9.7 µM, respectively).

From a marine sponge Pachastrella sp., new congeners of the microsclerodermins, microsclerodermins N and O, have been isolated. Their structures were elucidated by analysis of NMR spectroscopic data, chemical derivatization, and degradation studies. Microsclerodermins N and O are unique by possessing a p-ethoxyphenyl moiety instead of the frequently observed p-methoxyphenyl moiety at the terminus of the ω-phenyl-polyhydroxylated β-amino acid residue. The absolute configurations of all the stereogenic centers were shown to be identical with those of microsclerodermin B. Microsclerodermins N and O exhibit cytotoxic activity against HeLa cells.

Bioassay-guided fractionation of the extract of the rare deep-sea marine sponge Pleroma sp. afforded seven new linear acetylenes, yakushynols A–F (1–6) and neopetroformyne E (7). The structures of 1–7 were determined by a combination of the analysis of spectroscopic data and chemical derivatization. Compounds 1–6 are the first examples of the sponge-derived acetylenes of the size of duryne with oxidation at the sixth carbon from the terminus. Compounds 1–5 and 7 exhibited moderate cytotoxic activity. A biosynthetic route of neopetroformyne A was inferred from the structural transition among sponge-derived linear acetylenes.

The purpose of this study was to develop a novel electrical retrieval method (ER method) for living sponge-associated microorganisms from marine sponges frozen at −80 °C. A −0.3-V vs. Ag/AgCl constant potential applied for 2 h at 9 °C induced the attachment of the sponge-associated microorganisms to an indium tin oxide/glass (ITO) or a gallium-doped zinc oxide/glass (GZO) working electrode. The electrically attached microorganisms from homogenized Spirastrella insignis tissues had intact cell membranes and showed intracellular dehydrogenase activity. Dead microorganisms were not attracted to the electrode when the homogenized tissues were autoclaved for 15 min at 121 °C before use. The electrically attached microorganisms included cultivable microorganisms retrieved after detachment from the electrode by application of a 9-MHz sine-wave potential. Using the ER method, we obtained 32 phyla and 72 classes of bacteria and 3 archaea of Crenarchaeota thermoprotei, Marine Group I, and Thaumarchaeota incertae sedis from marine sponges S. insignis and Callyspongia confoederata. Employment of the ER method for extraction and purification of the living microorganisms holds potential of single-cell cultivation for genome, transcriptome, proteome, and metabolome analyses of bioactive compounds producing sponge-associated microorganisms.