Metabolomic fingerprinting of Brazilian marine sponges: a case study of Plakinidae species from Fernando de Noronha Archipelago.

Marine sponges from the Plakinidae family are well known for hosting cytotoxic secondary metabolites and the Brazilian Atlantic coast and its oceanic islands have been considered as a hotspot for the discovery of new Plakinidae species. Herein, we report the chemical profile among cytotoxic extracts obtained from four species of Plakinidae, collected in Fernando de Noronha Archipelago (PE, Northeastern Brazil). Crude organic extracts of Plakinastrella microspiculifera, Plakortis angulospiculatus, Plakortis insularis, and Plakortis petrupaulensis showed strong antiproliferative effects against two different cancer cell lines (HCT-116: 86.7-100%; MCF-7: 74.9-89.5%) at 50 µg/mL, by the MTT assay. However, at a lower concentration (5 µg/mL), high variability in inhibition of cell growth was observed (HCT-116: 17.3-68.7%; MCF-7: 0.00-55.5%), even within two samples of Plakortis insularis which were collected in the west and east sides of the Archipelago. To discriminate the chemical profile, the samples were investigated by UHPLC-HRMS under positive ionization mode. The produced data was uploaded to the Global Natural Products Social Molecular Networking and organized based on spectral similarities for purposes of comparison and annotation. Compounds such as dipeptides, nucleosides and derivatives, polyketides, and thiazine alkaloids were annotated and metabolomic differences were perceived among the species. To the best of our knowledge, this is the first assessment for cytotoxic activity and chemical profiling for Plakinastrella microspiculifera, Plakortis insularis and Plakortis petrupaulensis, revealing other biotechnologically relevant members of the Plakinidae family.

Bacillus Strains Associated to Homoscleromorpha Sponges are Highly Active Against Multidrug Resistant Bacteria.

The search for new, powerful antimicrobials is essential to respond to the current worldwide spread of antibiotic-resistant pathogens. Sponge-associated bacteria have great potential for production of antimicrobials against resistant and multidrug resistant (MDR) pathogenic bacteria, but only few species of the Class Homoscleromorpha have been screened for these activities so far. The aim of this study was to isolate and identify sponge-associated bacteria active against antibiotic-resistant pathogens from sponges of classes Homoscleromorpha and Demospongiae. By employing five different growth conditions, a total of 239 colony-forming units were isolated and remained viable. Among these, 17 (7.1%) isolates presented antimicrobial activity against pathogenic and (multi)drug resistant bacteria including vancomycin-resistant Enterococcus faecalis, Escherichia coli, Citrobacter freundii, Klebsiella penumoniae, Staphylococcus spp. and Streptococcus spp. Bioactive bacteria belonging to genera Bacillus and Vibrio were identified at species level and the DNA fingerprint patterns showed that strains of the same genus were not clonally related. The most active strains belong to genus Bacillus and were isolated from Oscarella sp., Plakina cyanorosea and Chondrilla caribensis. Our results show for the first time that sponge-associated strains of Bacillus pumilus and Bacillus muralis have high anti-MDR activity, and that the Homoscleromorpha may be a better source of such anti-MDR active bacteria than the Demospongiae. These results suggest that marine bacteria associated to homoscleromorph sponges may be an interesting source of new antimicrobial substances with biotechnological potential to treat infections caused by antibiotic-resistant bacteria.

Paraclostridium is the Main Genus of Anaerobic Bacteria Isolated from New Species of the Marine Sponge Plakina in the Brazilian Southeast Coast.

Despite the broad assessment of sponge bacterial diversity through cultivation-independent and dependent strategies, the knowledge focusing on cultivable anaerobes from this holobiont is still incipient. Plakina is a genus with the highest number of described species from the smallest of poriferan classes, Homoscleromorpha. The Brazilian Atlantic coast has been presenting itself as a hotspot for the discovery of new plakinidae species, with initial surveys just now concerning to characterize their microbiome. The current study aimed to isolate and identify strict anaerobes from recently described species of Plakina collected at the coast of Cabo Frio, RJ. Samples of four sympatric morphotypes of Plakina cyanorosea and Plakina cabofriense were collected on the coast of Cabo Frio, RJ. Using five different culture media, a total of 93 bacterial isolates were recovered, among which 60 were strict anaerobes and, ultimately, 34 remaining viable. A total of 76.5% from these strains were mostly identified as Clostridium bifermentans by mass spectrometry and 82.4% identified by 16S rRNA sequencing, almost all of them affiliated to the genus Paraclostridium, and with one isolate identified as Clostridium butyricum by both techniques. None of the anaerobic bacteria exhibited antimicrobial activity by the adopted screening test. The present work highlights not only the need for cultivation and characterization of the anaerobic microbiota from marine sponges but also adds the existing scarce knowledge of culturable bacterial communities from Homoscleromorph sponges from Brazilian coast.

Culturable bacterial communities associated to Brazilian Oscarella species (Porifera: Homoscleromorpha) and their antagonistic interactions.

Sponges offer an excellent model to investigate invertebrate-microorganism interactions. Furthermore, bacteria associated with marine sponges represent a rich source of bioactive metabolites. The aim of this study was to characterize the bacteria inhabiting a genus of sponges, Oscarella, and their potentiality for antimicrobial production. Bacterial isolates were recovered from different Oscarella specimens, among which 337 were phylogenetically identified. The culturable community was dominated by Proteobacteria and Firmicutes, and Vibrio was the most frequently isolated genus, followed by Shewanella. When tested for antimicrobial production, bacteria of the 12 genera isolated were capable of producing antimicrobial substances. The majority of strains were involved in antagonistic interactions and inhibitory activities were also observed against bacteria of medical importance. It was more pronounced in some isolated genera (Acinetobacter, Bacillus, Photobacterium, Shewanella and Vibrio). These findings suggest that chemical antagonism could play a significant role in shaping bacterial communities within Oscarella, a genus classified as low-microbial abundance sponge. Moreover, the identified strains may contribute to the search for new sources of antimicrobial substances, an important strategy for developing therapies to treat infections caused by multidrug-resistant bacteria. This study was the first to investigate the diversity and antagonistic activity of bacteria isolated from Oscarella spp. It highlights the biotechnological potential of sponge-associated bacteria.

An example of the importance of labels and fieldbooks in scientific collections: A freshwater sponge misunderstood for a marine new genus and species.

The demosponge genus Crelloxea Hechtel, 1983 was created to allocate a single species, Crelloxea spinosa Hechtel, 1983, described based on specimens collected by Jacques Laborel in northeastern Brazil in 1964 and deposited at the Porifera Collection of the Yale Peabody Museum. The genus Crelloxea was originally defined as "Crellidae with dermal and interstitial acanthoxeas and acanthostrongyles, with skeletal oxea and without microscleres or echinators" (Hechtel, 1983). Crelloxea was allocated in the marine sponge family Crellidae (Order Poecilosclerida), which is characterized by a tangential crust of spined ectosomal spicules (oxeas, anisoxeas or styles), a choanosomal plumose skeleton of smooth tornotes, sometimes a basal skeleton of acanthostyles erect on the substrate, microscleres usually arcuate chelae or absent, and surface with areolated pore fields (van Soest, 2002). Nowadays, Crelloxea is considered a junior synonym of Crella (Grayella) Carter, 1869 (van Soest, 2002; van Soest et al., 2015).

Mercury and methylmercury detoxification potential by sponge-associated bacteria.

Ionic and organic forms of mercury (Hg) are powerful cytotoxic and neurotoxic agents in both humans and wild life. The aim of this study was to analyze the resistance profile and potential detoxification of inorganic and organic forms of Hg of bacteria isolated from marine sponges on the coast of Rio de Janeiro, Brazil. Out of the 1,236 colony forming units associated with eleven species of marine sponges, 100 morphologically different bacterial strains were analyzed in this study. Of these, 21 strains were resistant to Hg, 14 of which were classified as highly resistant because they grew despite exposure to 100 µM HgCl2. Fifteen resistant strains reduced Hg and presented merA in their genomes. The remaining six strains produced biosurfactants, suggesting that they may tolerate Hg by sequestration. Eleven strains grew in the presence of methylmercury. Our results suggest a potential for mercury detoxification by marine sponge-associated resistant bacteria, either through reduction or sequestration, as well as the possibility of bioremediation of toxic waste containing mercury.

Potential application in mercury bioremediation of a marine sponge-isolated Bacillus cereus strain Pj1.

Sponges are sessile marine invertebrates that can live for many years in the same location, and therefore, they have the capability to accumulate anthropogenic pollutants such as metals over a long period. Almost all marine sponges harbor a large number of microorganisms within their tissues. The Bacillus cereus strain Pj1 was isolated from a marine sponge, Polymastia janeirensis, and was found to be resistant to 100 µM HgCl(2) and to 10 µM methylmercury (MeHg). Pj1 was also highly resistant to other metals, including CdCl(2) and Pb(NO(3))(2), alone or in combination. The mer operon was located on the bacterial chromosome, and the volatilization test indicated that the B. cereus Pj1 was able to reduce Hg(2+)-Hg(0). Cold vapor atomic absorption spectrometry demonstrated that Pj1 volatilized 80 % of the total MeHg that it was exposed to and produced elemental Hg when incubated with 1.5 µM MeHg. Pj1 also demonstrated sensitivity to all antibiotics tested. In addition, Pj1 demonstrated a potential for biosurfactant production, presenting an emulsification activity better than synthetic surfactants. The results of this study indicate that B. cereus Pj1 is a strain that can potentially be applied in the bioremediation of HgCl(2) and MeHg contamination in aquatic environments.

Taxonomy of Placospongia Gray, 1867 from Northern and Northeastern Brazil, with description of two new species (Porifera: Demospongiae: Clionaida).

The demosponge genus Placospongia includes 13 valid species, of which six occur in the Brazilian Exclusive Economic Zone. In the present study, we describe two new species of Placospongia from Northern (off the Amazon River mouth) and Northeastern Brazilian continental shelf and oceanic islands (i.e., Fernando de Noronha Archipelago), viz., Placospongia beatrizae sp. nov. and Placospongia amphinolastra sp. nov., and redescribe the holotype of Placospongia cristata Boury-Esnault, 1973, a poorly known species. In addition, we revise the Brazilian records of the allegedly cosmopolitan species P. carinata, P. melobesioides and P. intermedia, and provide new records of P. colombiensis and P. ruetzleri from Brazil. We demonstrate that P. carinata sensu Coelho & Mello-Leito and P. intermedia sensu Muricy & Moraes and Jimenez et al. belong in fact to P. ruetzleri, and that P. melobesioides sensu Mothes et al. and Gonzlez-Faras is a new species, described here as Placospongia beatrizae sp. nov. With the description of the two new species and synonymyzation of previous records, six species of Placospongia are currently known in Brazilian waters, two of which are endemic. A key to Placospongia species is also provided.

Four new species of Plakinidae (Porifera: Homoscleromorpha) from Brazil.

Seven species of Plakinidae are currently known from Brazil, belonging to genera Plakina (P. trilopha), Plakinastrella (P. microspiculfera, P. onkodes), and Plakortis (P. angulospiculatus, P. halichondrioides, P. insularis, and P microrhabdifera). In this study we describe four new species of the genera Plakinastrella and Plakortis, increasing the number of plakinid species in Brazil to eleven. Plakortis petrupaulensis sp. nov. is encrusting, light brown; the skeleton is disorganized with few traces of reticulation; lacunae are absent; the spicules are diods and tuberculate microrhabds; spheres are common and triods are absent. Plakortis spinalis sp. nov. is encrusting, gray, cartilaginous, has a reticulated ectosomal skeleton, and is distinguished by the spines in some of the diods and triods; microrhabds are also tuberculate and smooth spheres are common. Plakortis potiguarensis sp. nov. is encrusting, brown with gray tinges, and cartilaginous; the skeleton is disorganized and scarce, composed of thin, irregular diods, triods and relatively large microrhabds. Plakinastrella globularis sp. nov. has a globular shape and dark blue color in vivo; the ectosomal skeleton has a double tangential reticulation and the choanosomal skeleton is reticulated. The spicules are diods, triods, calthrops and microrhabds; spheres are common. Two of the new species have characters previously unknown in their respective genera: the spined diods and triods of Plakortis spinalis sp. nov. and the blue color and microrhabds of Plakinastrella globularis sp. nov. The presence of microrhabds in Plakinastrella suggests a close relationship of this genus with Plakortis.

Antibacterial activity and cytotoxicity analysis of halistanol trisulphate from marine sponge Petromica citrina.

OBJECTIVES: An aqueous extract and fraction from the marine sponge Petromica citrina have antibacterial activity. We performed a chemical and biological characterization of the antibiotic substance from P. citrina and investigated its mode of action on Staphylococcus aureus cells. METHODS: The inhibitory activity of the aqueous extract of P. citrina was determined against 14 bacteria belonging to type strains and clinical antibiotic-resistant strains. The aqueous extract was fractionated under bioassay guidance and the bioactive substance was identified by its (1)H-NMR, (13)C-NMR and mass spectra. The MIC and the MBC of this substance were determined. This substance was also subjected to cytotoxic bioassays. The mode of action on S. aureus cells was investigated by light and transmission electron microscopy analysis. RESULTS: P. citrina showed a large spectrum of activity against type strains and resistant-bacteria such as S. aureus, Staphylococcus epidermidis, Enterococcus faecalis, Mycobacterium fortuitum and Neisseria gonorrhoeae. The aqueous extract was fractionated and halistanol trisulphate (24ε,25-dimethylcholestane-2ß,3α,6α-triol trisodium sulphate) was isolated for the first time from P. citrina. Halistanol trisulphate had a bactericidal effect on exponentially growing S. aureus cells at the MIC (512 mg/L). Cytotoxicity biossays showed moderate toxicity against cancer cell line L929 (fibrosarcoma). This substance apparently acts by damaging the cell membrane, with subsequent cell lysis. CONCLUSIONS: Halistanol trisulphate is a broad-spectrum antibiotic isolated from P. citrina with a mode of action involving disruption of the cytoplasmic membrane. It is a new candidate for research on antibacterial substances.

Manned submersible dives reveal a singular assemblage of Hexactinellida (Porifera) off the Amazon River mouth, Northern Brazil.

The outer shelf and upper slope region under the influence of the Amazon River mouth harbors an extensive reef system with a rich sponge fauna, but no hexactinellids were recorded in the area so far. In this work, manned submersibles were used to describe for the first time three species of Hexactinellida off the Amazon River mouth: Hyalonema (Cyliconemaoida) alucia sp. nov., Dactylocalyx pumiceus and Claviscopulia facunda. We found two morphotypes of Dactylocalyx pumiceus, suggesting that it may be a complex of cryptic species. The populations of the three species off Par State were sparse, but a large aggregation of D. pumiceus was found off Maranho State from 145 to 230 m depth. These findings indicate that hexactinellid grounds may be more common along the Northern Brazilian continental outer shelf and slope than previously thought.

Oroidin inhibits the activity of the multidrug resistance target Pdr5p from yeast plasma membranes.

Oroidin was isolated from the marine sponge Agelassventres and inhibited the activity and function of Pdr5p, an enzyme responsible for the multidrug resistance phenotype in Saccharomyces cerevisiae. This compound may help in the development of new drugs that reverse this dangerous phenotype of pathogenic yeast and fungi.

Evaluation of Marine Brown Algae and Sponges from Brazil as Anticoagulant and Antiplatelet Products.

The ischemic disorders, in which platelet aggregation and blood coagulation are involved, represent a major cause of disability and death worldwide. The antithrombotic therapy has unsatisfactory performance and may produce side effects. So, there is a need to seek molecules with antithrombotic properties. Marine organisms produce substances with different well defined ecological functions. Moreover, some of these molecules also exhibit pharmacological properties such as antiviral, anticancer, antiophidic and anticoagulant properties. The aim of this study was to evaluate, through in vitro tests, the effect of two extracts of brown algae and ten marine sponges from Brazil on platelet aggregation and blood coagulation. Our results revealed that most of the extracts were capable of inhibiting platelet aggregation and clotting measured by plasma recalcification tests, prothrombin time, activated partial thromboplastin time, and fibrinogenolytic activity. On the other hand, five of ten species of sponges induced platelet aggregation. Thus, the marine organisms studied here may have molecules with antithrombotic properties, presenting biotechnological potential to antithrombotic therapy. Further chemical investigation should be conducted on the active species to discover useful molecules for the development of new drugs to treat clotting disorders.

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.

Taxonomy of some Indian and Pacific oceans Corticium, with the description of three new species from Western Australia (Porifera: Homosclerophorida).

The sponge genus Corticium has important evolutionary significance and great pharmacological potential, but its diversity remains poorly known. In this contribution we redescribe two poorly-known Indian and Pacific oceans species, viz., Corticium simplex and C. bargibanti, and describe three new species of Corticium from Western Australia. We observed for the first time the presence of rare calthrops in C. simplex, which has a wide distribution and may be a species complex. The three new species are mainly characterized by their spiculation: Corticium furcatum sp. nov. has large calthrops and exclusive candelabra with bifurcated rays in the apical actine; C. monolophum sp. nov. has large monolophose calthrops, smooth calthrops and candelabra; and C. verticillatum sp. nov. has acanthocalthrops with verticillated spines, which are exclusive to this species within the genus. The diversity of Corticium is raised to 11 species worldwide, but it still remains highly underestimated. A key to all valid species is here provided.

Genomic and in silico protein structural analyses provide insights into marine polysaccharide-degrading enzymes in the sponge-derived Pseudoalteromonas sp. PA2MD11.

Active heterotrophic metabolism is a critical metabolic role performed by sponge-associated microorganisms, but little is known about their capacity to metabolize marine polysaccharides (MPs). Here, we investigated the genome of the sponge-derived Pseudoalteromonas sp. strain PA2MD11 focusing on its macroalgal carbohydrate-degrading potential. Carbohydrate-active enzymes (CAZymes) for the depolymerization of agar and alginate were found in PA2MD11's genome, including glycoside hydrolases (GHs) and polysaccharide lyases (PLs) belonging to families GH16, GH50 and GH117, and PL6 and PL17, respectively. A gene potentially encoding a sulfatase was also identified, which may play a role in the strain's ability to consume carrageenans. The complete metabolism of agar and alginate by PA2MD11 could also be predicted and was consistent with the results obtained in physiological assays. The polysaccharide utilization locus (PUL) potentially involved in the metabolism of agarose contained mobile genetic elements from other marine Gammaproteobacteria and its unusual larger size might be due to gene duplication events. Homology modelling and structural protein analyses of the agarases, alginate lyases and sulfatase depicted clear conservation of catalytic machinery and protein folding together with suitable industrially-relevant features. Pseudoalteromonas sp. PA2MD11 is therefore a source of potential MP-degrading biocatalysts for biorefinery applications and in the preparation of pharmacologically-active oligosaccharides.

High reduction of staphylococcal biofilm by aqueous extract from marine sponge-isolated Enterobacter sp.

Staphylococcus aureus and Staphylococcus epidermidis are among the most important bacterial species responsible for biofilm formation on indwelling medical devices, including orthopaedic implants. The increasing resistance to antimicrobials, partly attributed to the ability to form biofilms, is a challenge for the development of new antimicrobial agents. In this study, the cell-free supernatant obtained from sponge-associated Enterobacter strain 84.3 culture inhibited biofilm formation (>65%) and dissociated mature biofilm (>85%) formed by S. aureus and S. epidermidis strains. The culture supernatant was subjected to solvent partitioning and the aqueous extract presented a concentration-dependent antibiofilm activity for each strain with a minimum biofilm eradication concentration (MBEC) ranging from 16 to 256 µg/mL. The effect of the aqueous extract on mature S. aureus biofilm was analyzed by confocal scanning laser microscopy, showing a significant reduction of the biofilm layer as well as diminished interactions among the cells. This extract is not toxic for mammalian cells (L929 cell line). Studies targeting substances with antibiofilm activity gained significant attention in recent years due to difficult-to-treat biofilm infections. Here, sponge-associated Enterobacter 84.3 proved to be a source of substances capable of eradicating staphylococcal biofilm, with potential medical use in the future.

Peeling the Layers Away: The Genomic Characterization of Bacillus pumilus 64-1, an Isolate With Antimicrobial Activity From the Marine Sponge Plakina cyanorosea (Porifera, Homoscleromorpha).

Bacillus pumilus 64-1, a bacterial strain isolated from the marine sponge Plakina cyanorosea, which exhibits antimicrobial activity against both pathogenic and drug-resistant Gram-positive and Gram-negative bacteria. This study aimed to conduct an in-depth genomic analysis of this bioactive sponge-derived strain. The nearly complete genome of strain 64-1 consists of 3.6 Mbp (41.5% GC), which includes 3,705 coding sequences (CDS). An open pangenome was observed when limiting to the type strains of the B. pumilus group and aquatic-derived B. pumilus representatives. The genome appears to encode for at least 12 potential biosynthetic gene clusters (BGCs), including both types I and III polyketide synthases (PKS), non-ribosomal peptide synthetases (NRPS), and one NRPS-T1PKS hybrid, among others. In particular, bacilysin and other bacteriocin-coding genes were found and may be associated with the detected antimicrobial activity. Strain 64-1 also appears to possess a broad repertoire of genes encoding for plant cell wall-degrading carbohydrate-active enzymes (CAZymes). A myriad of genes which may be involved in various process required by the strain in its marine habitat, such as those encoding for osmoprotectory transport systems and the biosynthesis of compatible solutes were also present. Several heavy metal tolerance genes are also present, together with various mobile elements including a region encoding for a type III-B Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) region, four prophage segments and transposase elements. This is the first report on the genomic characterization of a cultivable bacterial member of the Plakina cyanorosea holobiont.

Not That Close to Mommy: Horizontal Transmission Seeds the Microbiome Associated with the Marine Sponge Plakina cyanorosea.

Marine sponges are excellent examples of invertebrate-microbe symbioses. In this holobiont, the partnership has elegantly evolved by either transmitting key microbial associates through the host germline and/or capturing microorganisms from the surrounding seawater. We report here on the prokaryotic microbiota during different developmental stages of Plakina cyanorosea and their surrounding environmental samples by a 16S rRNA metabarcoding approach. In comparison with their source adults, larvae housed slightly richer and more diverse microbial communities, which are structurally more related to the environmental microbiota. In addition to the thaumarchaeal Nitrosopumilus, parental sponges were broadly dominated by Alpha- and Gamma-proteobacteria, while the offspring were particularly enriched in the Vibrionales, Alteromonodales, Enterobacterales orders and the Clostridia and Bacteroidia classes. An enterobacterial operational taxonomic unit (OTU) was the dominant member of the strict core microbiota. The most abundant and unique OTUs were not significantly enriched amongst the microbiomes from host specimens included in the sponge microbiome project. In a wider context, Oscarella and Plakina are the sponge genera with higher divergence in their associated microbiota compared to their Homoscleromorpha counterparts. Our results indicate that P. cyanorosea is a low microbial abundance sponge (LMA), which appears to heavily depend on the horizontal transmission of its microbial partners that likely help the sponge host in the adaptation to its habitat.

Marine Pseudomonas putida: a potential source of antimicrobial substances against antibiotic-resistant bacteria.

Bacteria isolated from marine sponges found off the coast of Rio de Janeiro, Brazil, were screened for the production of antimicrobial substances. We report a new Pseudomonas putida strain (designated P. putida Mm3) isolated from the sponge Mycale microsigmatosa that produces a powerful antimicrobial substance active against multidrug-resistant bacteria. P. putida Mm3 was identified on the basis of 16S rRNA gene sequencing and phenotypic tests. Molecular typing for Mm3 was performed by RAPD-PCR and comparison of the results to other Pseudomonas strains. Our results contribute to the search for new antimicrobial agents, an important strategy for developing alternative therapies to treat infections caused by multidrug-resistant bacteria.