Comparison of Cryopreservation Techniques for Cells of the Marine Sponge Dysidea etheria.

BACKGROUND: Cryopreservation is a commonly used method for the long-term storage of cell lines and provides a stable source of cells for experiments, allowing researchers to study species that are not geographically nearby, and useful to progress studies on sponge cell biotechnology. OBJECTIVE: The marine sponge Dysidea etheria was chosen as our model organism to evaluate the impact and effectiveness of two commonly used cryoprotectants, dimethyl sulfoxide (DMSO) and glycerol. MATERIALS AND METHODS: By testing a range of concentrations (3-10% DMSO, 10-50% glycerol), we determined the optimal cryoprotectant for D. etheria based on its ability to preserve viable cells and optimize recovery after cryopreservation. RESULTS: Cells cryopreserved in DMSO had significantly higher viability after cryopreservation than those cryopreserved in glycerol. Cells cryopreserved in glycerol had irregular morphology as well as lower recovery of viable cells than those from DMSO treatments. CONCLUSION: Our results demonstrate that the optimal cryoprotectant for sponge cells, without a significant loss of viability, is 5-8% DMSO. This approach can be used to optimize cryopreservation methods for cells of other marine invertebrate species.

Phylogeny and systematics of demospongiae in light of new small-subunit ribosomal DNA (18S) sequences.

The most diverse and species-rich class of the phylum Porifera is Demospongiae. In recent years, the systematics of this clade, which contains more than 7000 species, has developed rapidly in light of new studies combining molecular and morphological observations. We add more than 500 new, nearly complete 18S sequences (an increase of more than 200%) in an attempt to further enhance understanding of the phylogeny of Demospongiae. Our study specifically targets representation of type species and genera that have never been sampled for any molecular data in an effort to accelerate progress in classifying this diverse lineage. Our analyses recover four highly supported subclasses of Demospongiae: Keratosa, Myxospongiae, Haploscleromorpha, and Heteroscleromorpha. Within Keratosa, neither Dendroceratida, nor its two families, Darwinellidae and Dictyodendrillidae, are monophyletic and Dictyoceratida is divided into two lineages, one predominantly composed of Dysideidae and the second containing the remaining families (Irciniidae, Spongiidae, Thorectidae, and Verticillitidae). Within Myxospongiae, we find Chondrosida to be paraphyletic with respect to the Verongida. We amend the latter to include species of the genus Chondrosia and erect a new order Chondrillida to contain remaining taxa from Chondrosida, which we now discard. Even with increased taxon sampling of Haploscleromorpha, our analyses are consistent with previous studies; however, Haliclona species are interspersed in even more clades. Haploscleromorpha contains five highly supported clades, each more diverse than previously recognized, and current families are mostly polyphyletic. In addition, we reassign Janulum spinispiculum to Haploscleromorpha and resurrect Reniera filholi as Janulum filholi comb. nov. Within the large clade Heteroscleromorpha, we confirmed 12 recently identified clades based on alternative data, as well as a sister-group relationship between the freshwater Spongillida and the family Vetulinidae. We transfer Stylissa flabelliformis to the genus Scopalina within the family Scopalinidae, which is of uncertain position. Our analyses uncover a large, strongly supported clade containing all heteroscleromorphs other than Spongillida, Vetulinidae, and Scopalinidae. Within this clade, there is a major division separating Axinellidae, Biemnida, Tetractinellida, Bubaridae, Stelligeridae, Raspailiidae, and some species of Petromica, Topsentia, and Axinyssa from Agelasida, Polymastiidae, Placospongiidae, Clionaidae, Spirastrellidae, Tethyidae, Poecilosclerida, Halichondriidae, Suberitidae, and Trachycladus. Among numerous results: (1) Spirophorina and its family Tetillidae are paraphyletic with respect to a strongly supported Astrophorina within Tetractinellida; (2) Agelasida is the earliest diverging lineage within the second clade listed above; and (3) Merlia and Desmacella appear to be the earliest diverging lineages of Poecilosclerida.

Use of real-time qPCR to quantify members of the unculturable heterotrophic bacterial community in a deep sea marine sponge, Vetulina sp.

In this report, real-time quantitative PCR (TaqMan qPCR) of the small subunit (SSU) 16S-like rRNA molecule, a universal phylogenetic marker, was used to quantify the relative abundance of individual bacterial members of a diverse, yet mostly unculturable, microbial community from a marine sponge. Molecular phylogenetic analyses of bacterial communities derived from Caribbean Lithistid sponges have shown a wide diversity of microbes that included at least six major subdivisions; however, very little overlap was observed between the culturable and unculturable microbial communities. Based on sequence data of three culture-independent Lithistid-derived representative bacteria, we designed probe/primer sets for TaqMan qPCR to quantitatively characterize selected microbial residents in a Lithistid sponge, Vetulina, metagenome. TaqMan assays included specificity testing, DNA limit of detection analysis, and quantification of specific microbial rRNA sequences such as Nitrospira-like microbes and Actinobacteria up to 172 million copies per microgram per Lithistid sponge metagenome. By contrast, qPCR amplification with probes designed for common previously cultured sponge-associated bacteria in the genera Rheinheimera and Marinomonas and a representative of the CFB group resulted in only minimal detection of the Rheiheimera in total DNA extracted from the sponge. These data verify that a large portion of the microbial community within Lithistid sponges may consist of currently unculturable microorganisms.

Oceans and human health: Emerging public health risks in the marine environment.

There has been an increasing recognition of the inter-relationship between human health and the oceans. Traditionally, the focus of research and concern has been on the impact of human activities on the oceans, particularly through anthropogenic pollution and the exploitation of marine resources. More recently, there has been recognition of the potential direct impact of the oceans on human health, both detrimental and beneficial. Areas identified include: global change, harmful algal blooms (HABs), microbial and chemical contamination of marine waters and seafood, and marine models and natural products from the seas. It is hoped that through the recognition of the inter-dependence of the health of both humans and the oceans, efforts will be made to restore and preserve the oceans.

Characterization of genetic markers for in vitro cell line identification of the marine sponge Axinella corrugata.

The marine sponge Axinella corrugata is being developed as a model organism for in vitro marine invertebrate research. Molecular genetics methods such as DNA fingerprinting [amplified fragment length polymorphism (AFLP) and single-stranded conformation polymorphism (SSCP)] and single-locus DNA sequence analyses were applied to this model to meet the primary objective of identifying positive A. corrugata-specific molecular markers that will aid in verifying cell identity in vitro and distinguish sponge cells from potential microbial contaminants. The extent of intra- and interspecific variation in these markers from geographically distinct samples of A. corrugata and closely related sponge taxa was also assessed. Two novel nuclear loci along with intervening transcribed spacer (ITS) regions of nuclear rRNA were characterized, although the latter appeared to better meet primary marker criteria, such as taxonomic specificity and high frequency of detection (via polymerase chain reaction [PCR]) from different individuals (n > 40) and cell cultures. Phylogenetic and phylogeographic analyses of ITS DNA sequences helped clarify taxonomies and also suggested species boundaries between and among western Atlantic and eastern Atlantic/Indian Ocean A. corrugata and Axinellidae samples. Patterns of genetic variation have important implications for the systematics, evolution, and chemical ecology of A. corrugata and related axinellids and are discussed.

Three new peroxides from the sponge Plakinastrella species.

Two new five-membered-ring peroxide acids, plakinic acid F (3) and epiplakinic acid F (4), and a new peroxide-lactone, plakortolide F (5), were isolated from a sponge of the genus Plakinastrella collected from Felicite Island, Seychelles. The structures were elucidated through spectral analysis. The free acids 3 and 4 exhibit moderate antifungal activity against Candida albicans with minimum inhibitory concentrations of 25 micrograms/mL (SDB) and 3.1 micrograms/mL (RPMI) for 3, and 25 micrograms/mL (SDB) and 6.25 micrograms/mL (RPMI) for 4, respectively. Both also showed moderate in vitro inhibition of Aspergillus fumigatus with IC90's of 25 micrograms/mL.

Quantitative assessment of marine sponge cells in vitro: development of improved growth medium.

As sources of natural products with potential human therapeutic value, marine sponges are important subjects for cell culture studies. A critical component of any cell culture system is its growth medium. Proceeding from the hypotheses that the thawed, cryopreserved, primary cells would display detectable differential responses and that those responses could be comparatively quantified, this study has established that multiwell screening assays are useful tools for improving medium formulations in cell cultures of the marine sponge, Teichaxinella morchella. Fluorescent probe signals were correlated with known cell densities and viabilities in a 96-well format. Analysis of variance and post-test methods were applied to judge the significance of signal differences seen in a variety of medium formulations. Results from a series of experiments suggested that reducing glutamine and selenium concentrations in the standard medium would result in greater DNA, protein, and esterase activity signals. This was confirmed by the direct comparison of the standard and improved medium formulations. Significantly higher protein content and esterase activity were associated with the improved medium. DNA content was also higher, though not significantly. The result is a new medium formulation that may be more able to support cell growth and division, providing an improved cell culture system for marine sponge cell studies. The assays can be used in additional studies to further improve the in vitro conditions for marine sponge cell culture.

Eryloside F, a novel penasterol disaccharide possessing potent thrombin receptor antagonist activity.

We report the discovery of Eryloside F, a novel disaccharide of the steroidal carboxylic acid penasterol, isolated from an extract of the marine sponge Erylus formosus. The compound is a potent thrombin receptor antagonist, and furthermore inhibits human platelet aggregation in vitro.

Sterols of the marine sponge Petrosia weinbergi: implications for the absolute configurations of the antiviral orthoesterols and weinbersterols.

The marine sponge Petrosia weinbergi was found to contain isofucosterol and clionasterol as its major sterols. The rare cyclopropyl sterol (24S,28S)-24,28-methylenestigmast-5-en-3beta-ol, previously detected as only 0.07% of the total sterols of a pelagophytic alga Pulvinaria sp., made up 6.6% of the total sterols. These sterols are believed to be the biosynthetic precursors of the antiviral orthoesterols and weinbersterols found in the same sponge. Based on the side chains of the isolated sterols, the absolute configurations of the antiviral steroid side chains are assigned to be (24R,28S)- for orthoesterol B, (24R)- for orthoesterol C, and (24S,28S)- for weinbersterols A and B.

Secobatzellines A and B, two new enzyme inhibitors from a deep-water Caribbean sponge of the genus Batzella.

Secobatzelline A (1), a new batzelline natural analogue, and secobatzelline B (2), a likely artifact formed during the isolation procedure, have been isolated from a deep-water marine sponge of the genus Batzella. Secobatzellines A and B inhibited the phosphatase activity of calcineurin, and secobatzelline A inhibited the peptidase activity of CPP32. Both compounds showed in vitro cytotoxicity against P-388 and A-549 cell lines. The isolation and structure elucidation of secobatzellines A (1) and B (2) are described.

Discorhabdin P, a new enzyme inhibitor from a deep-water Caribbean sponge of the genus Batzella.

Discorhabdin P (1), a new discorhabdin analogue, has been isolated from a deep-water marine sponge of the genus Batzella. Discorhabdin P (1) inhibited the phosphatase activity of calcineurin and the peptidase activity of CPP32. It also showed in vitro cytotoxicity against P-388 and A-549 cell lines. The isolation and structure elucidation of discorhabdin P (1) are described.

Isolation and characterization of niphatevirin, a human-immunodeficiency-virus-inhibitory glycoprotein from the marine sponge Niphates erecta.

Anti-human immunodeficiency virus (HIV)-bioassay-guided fractionation of aqueous extracts of the Caribbean sponge Niphates erecta led to isolation of a novel anti-HIV protein, named niphatevirin. The protein was purified to homogeneity by ethanol precipitation, ammonium sulfate precipitation, gel-permeation chromatography and concanavalin-A-Sepharose affinity chromatography. Niphatevirin potently inhibited the cytopathic effects of HIV-1 infection in cultured human lymphoblastoid (CEM-SS) cells; the effective concentration of drug that results in 50% protection of the cells through inhibition of cell lethality, cell-cell fusion and syncytium formation was approximately 10 nM. Delay of addition of niphatevirin to infected cultures by two hours markedly decreased (approximately 50%) cytoprotection; delay of addition by eight hours resulted in no antiviral activity. Niphatevirin bound to CD4 in a manner that prevented the binding of gp120, but did not directly bind gp120. Niphatevirin (6.5 microM) was inactive in both hemagglutination and hemolysis assays. Niphatevirin had a molecular mass of about 19 kDa by matrix-assisted laser-desorption ionization-time of flight (MALDI-TOF) mass spectrometry, and a native molecular mass of approximately 18 kDa by gel-filtration chromatography. The protein had an acidic isoelectric point of 4.2-4.6, and was shown by periodate acid Schiff's staining to be glycosylated.

Characterization of an interleukin 6 cytokine family antagonist protein from a marine sponge, Callyspongia sp.

An inhibitor of IL-6 binding to the human hepatoma line HepG2 and myeloma cell line U266 was identified in a saline extract of the marine sponge, Callyspongia sp. Functional activity, measured through the increase in haptoglobin production by HepG2 cells stimulated with IL-6, could be strongly inhibited by the extract. Similarly, IL-6-induced production of IgM by the B cell line SKW6.4 was substantially reduced. In neither cell line was there evidence of toxicity produced by the extract. Other sponges of the Callyspongia species were found to contain analogous activity. The activity was destroyed by trypsin treatment or boiling of the extract, suggesting that the inhibition is due to a protein. When the binding of IL-6 to its receptor complex was dissected in vitro, inhibition of binding of IL-6 to soluble receptor by the extract was not detected, but binding of the IL-6-sIL-6R complex to soluble gp130 was inhibited in a dose-dependent fashion. This was borne out in cellular assays since the extract inhibited activation of HepG2 cells stimulated with oncostatin M or leukemia inhibitory factor, cytokines which also use gp130 for signal transduction. These results suggest that the Callyspongia extract contains a protein which blocks the interaction of the IL-6 family of cytokines with their signal transduction moiety, gp130. Elucidation of the structure and mode of action of such a protein would be helpful in designing gp130 antagonists to inhibit the functions of this cytokine family, overproduction of which has been associated with cancer and pathologies of autoimmune disease and AIDS.

Aplysillin A, a thrombin receptor antagonist from the marine sponge Aplysina fistularis fulva.

Aplysillin A [1], an unusual disulfate ester of a 1,4-diphenyl-1,3-butadiene, was isolated from a deep water specimen of a sponge of the genus Aplysina. Its structure was determined through spectroscopic methods. The compound weakly inhibited binding to the thrombin receptor with an IC50 value of 20 microM.

Phylogeny and classification of lithistid sponges (porifera: Demospongiae): a preliminary assessment using ribosomal DNA sequence comparisons.

Traditionally, lithistid sponges have been combined within one order linked by the common possession of an interlocking siliceous desma skeleton. However, molecular data, morphology, and paleontology confirm recent hypothesis of polyphyly for this enigmatic group of sponges. Parsimony and neighbor-joining analysis of partial 18S rDNA sequences derived from amplified genomic rDNA of lithistids and other demosponges suggest that tetracladinid and dicranocladinid lithistids are monophyletic and closely related to choristid demosponges. Rhizomorinid families, in contrast, are indicated as being polyphyletic, with a diversity of points of origin within the tetractinomorph and the ceractinomorph poriferan subclasses. These arrangements are discussed in light of several hypotheses of demosponge evolution.

Verongamine, a novel bromotyrosine-derived histamine H3-antagonist from the marine sponge Verongula gigantea.

The novel bromotyrosine derivative verongamine [1] has been isolated from the marine sponge Verongula gigantea. The isolation of verongamine was facilitated by employing histamine-H3 bioassay-guided fractionation. Verongamine is a specific histamine-H3 antagonist at concentrations as low as 1 microgram/ml.

Discobahamins A and B, new peptides from the Bahamian deep water marine sponge Discodermia sp.

Discobahamin A [1] and discobahamin B [2] are two bioactive peptides isolated from a new species of the Bahamian deep water marine sponge Discodermia. The discobahamins are inhibitors of the growth of Candida albicans, and the isolation and structure elucidation of 1 and 2 by nmr and chemical methods is described.

Isolation and structure elucidation of 34-sulfatobastadin 13, an inhibitor of the endothelin A receptor, from a marine sponge of the genus Ianthella.

34-Sulfatobastadin 13 [1] was isolated from the sponge Ianthella sp. Its structure was elucidated by nmr techniques and chemical transformation to bastadin 13 [2]. Compound 1 weakly inhibited binding to the endothelin A receptor (ETA), while compound 2 inhibited growth of Bacillus subtilis.

Sesquiterpene-derived metabolites from the deep water marine sponge Poecillastra sollasi.

Six sesquiterpene-derived compounds, 1-6, which we call sollasins a-f, have been isolated from a deep water specimen of the sponge Poecillastra sollasi. The structures were elucidated by comparison of spectral data to related metabolites and confirmed using spectroscopic methods. The compounds inhibit the growth of the pathogenic fungi Candida albicans and Cryptococcus neoformans and the P-388 and A-549 tumor cell lines. Compounds 3 and 4 show weak inhibition of binding of [125I] angiotensin II to rat aorta smooth muscle cell membranes.