Filamentous Marine Cyanobacteria Research Articles

The Kavaratamides: Discovery of Linear Lipodepsipeptides from the Marine Cyanobacterium Moorena bouillonii Using a Comparative Chemogeographic Analysis.

Kavaratamide A (1), a new linear lipodepsipeptide possessing an unusual isopropyl-O-methylpyrrolinone moiety, was discovered from the tropical marine filamentous cyanobacterium Moorena bouillonii collected from Kavaratti, India. A comparative chemogeographic analysis of M. bouillonii collected from six different geographical regions led to the prioritized isolation of this metabolite from India as distinctive among our data sets. AI-based structure annotation tools, including SMART 2.1 and DeepSAT, accelerated the structure elucidation by providing useful structural clues, and the full planar structure was elucidated based on comprehensive HRMS, MS/MS fragmentation, and NMR data interpretation. Subsequently, the absolute configuration of 1 was determined using advanced Marfey's analysis, modified Mosher's ester derivatization, and chiral-phase HPLC. The structures of kavaratamides B (2) and C (3) are proposed based on a detailed analysis of their MS/MS fragmentations. The biological activity of kavaratamide A was also investigated and found to show moderate cytotoxicity to the D283-medullablastoma cell line.

Barbamide Displays Affinity for Membrane-Bound Receptors and Impacts Store-Operated Calcium Entry in Mouse Sensory Neurons.

Marine cyanobacteria are a rich source of bio-active metabolites that have been utilized as leads for drug discovery and pharmacological tools for basic science research. Here, we describe the re-isolation of a well-known metabolite, barbamide, from Curaçao on three different occasions and the characterization of barbamide's biological interactions with targets of the mammalian nervous system. Barbamide was originally discovered as a molluscicidal agent from a filamentous marine cyanobacterium. In our hands, we found little evidence of toxicity against mammalian cell cultures. However, barbamide showed several affinities when screened for binding affinity for a panel of 45 receptors and transporters known to be involved in nociception and sensory neuron activity. We found high levels of binding affinity for the dopamine transporter, the kappa opioid receptor, and the sigma receptors (sigma-1 and sigma-2 also known as transmembrane protein 97; TMEM97). We tested barbamide in vitro in isolated sensory neurons from female mice to explore its functional impact on calcium flux in these cells. Barbamide by itself had no observable impact on calcium flux. However, barbamide enhanced the effect of the TRPV1 agonist capsaicin and enhanced store-operated calcium entry (SOCE) responses after depletion of intracellular calcium. Overall, these results demonstrate the biological potential of barbamide at sensory neurons with implications for future drug development projects surrounding this molecule.

Quantitative Analysis of the Trade-Offs of Colony Formation for Trichodesmium.

There is considerable debate about the benefits and trade-offs for colony formation in a major marine nitrogen fixer, Trichodesmium. To quantitatively analyze the trade-offs, we developed a metabolic model based on carbon fluxes to compare the performance of Trichodesmium colonies and free trichomes under different scenarios. Despite reported reductions in carbon fixation and nitrogen fixation rates for colonies relative to free trichomes, we found that model colonies can outperform individual cells in several cases. The formation of colonies can be advantageous when respiration rates account for a high proportion of the carbon fixation rate. Negative external influence on vital rates, such as mortality due to predation or micronutrient limitations, can also create a net benefit for colony formation relative to individual cells. In contrast, free trichomes also outcompete colonies in many scenarios, such as when respiration rates are equal for both colonies and individual cells or when there is a net positive external influence on rate processes (i.e., optimal environmental conditions regarding light and temperature or high nutrient availability). For both colonies and free trichomes, an increase in carbon fixation relative to nitrogen fixation rates would increase their relative competitiveness. These findings suggest that the formation of colonies in Trichodesmium might be linked to specific environmental and ecological circumstances. Our results provide a road map for empirical studies and models to evaluate the conditions under which colony formation in marine phytoplankton can be sustained in the natural environment. IMPORTANCE Trichodesmium is a marine filamentous cyanobacterium that fixes nitrogen and is an important contributor to the global nitrogen cycle. In the natural environment, Trichodesmium can exist as individual cells (trichomes) or as colonies (puffs and tufts). In this paper, we try to answer a longstanding question in marine microbial ecology: how does colony formation benefit the survival of Trichodesmium? To answer this question, we developed a carbon flux model that utilizes existing published rates to evaluate whether and when colony formation can be sustained. Enhanced respiration rates, influential external factors such as environmental conditions and ecological interactions, and variable carbon and nitrogen fixation rates can all create scenarios for colony formation to be a viable strategy. Our results show that colony formation is an ecologically beneficial strategy under specific conditions, enabling Trichodesmium to be a globally significant organism.

Heterologous Expression in Anabaena of the Columbamide Pathway from the Cyanobacterium Moorena bouillonii and Production of New Analogs.

Columbamides are chlorinated acyl amide natural products, several of which exhibit cannabinomimetic activity. These compounds were originally discovered from a culture of the filamentous marine cyanobacterium Moorena bouillonii PNG5-198 collected from the coastal waters of Papua New Guinea. The columbamide biosynthetic gene cluster (BGC) had been identified using bioinformatics, but not confirmed by experimental evidence. Here, we report the heterologous expression in Anabaena (Nostoc) PCC 7120 of the 28.5 kb BGC that encodes for columbamide biosynthesis. The production of columbamides in Anabaena is investigated under several different culture conditions, and several new columbamide analogs are identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and nuclear magnetic resonance (NMR). In addition to previously characterized columbamides A, B, and C, new columbamides I-M are produced in these experiments, and the structure of the most abundant monochlorinated analog, columbamide K (11), is fully characterized. The other new columbamide analogs are produced in only small quantities, and structures are proposed based on high-resolution-MS, MS/MS, and 1H NMR data. Overexpression of the pathway's predicted halogenases resulted in increased productions of di- and trichlorinated compounds. The most significant change in production of columbamides in Anabaena is correlated with the concentration of NaCl in the medium.

Micrococcin P1 and P2 from Epibiotic Bacteria Associated with Isolates of Moorea producens from Kenya.

Epibiotic bacteria associated with the filamentous marine cyanobacterium Moorea producens were explored as a novel source of antibiotics and to establish whether they can produce cyclodepsipeptides on their own. Here, we report the isolation of micrococcin P1 (1) (C48H49N13O9S6; obs. m/z 1144.21930/572.60381) and micrococcin P2 (2) (C48H47N13O9S6; obs. m/z 1142.20446/571.60370) from a strain of Bacillus marisflavi isolated from M. producens’ filaments. Interestingly, most bacteria isolated from M. producens’ filaments were found to be human pathogens. Stalked diatoms on the filaments suggested a possible terrestrial origin of some epibionts. CuSO4·5H2O assisted differential genomic DNA isolation and phylogenetic analysis showed that a Kenyan strain of M. producens differed from L. majuscula strain CCAP 1446/4 and L. majuscula clones. Organic extracts of the epibiotic bacteria Pseudoalteromonas carrageenovora and Ochrobactrum anthropi did not produce cyclodepsipeptides. Further characterization of 24 Firmicutes strains from M. producens identified extracts of B. marisflavi as most active. Our results showed that the genetic basis for synthesizing micrococcin P1 (1), discovered in Bacillus cereus ATCC 14579, is species/strain-dependent and this reinforces the need for molecular identification of M. producens species worldwide and their epibionts. These findings indicate that M. producens-associated bacteria are an overlooked source of antimicrobial compounds.

Targeted Discovery of Amantamide B, an Ion Channel Modulating Nonapeptide from a South China Sea Oscillatoria Cyanobacterium.

Amantamide B (1) is a new linear nonapeptide analogue of the cyanobacterial natural product amantamide A (2), and both have methyl ester and butanamide termini. These compounds were discovered in this study from the organic extract of a tropical marine filamentous cyanobacterium, Oscillatoria sp., collected around the Paracel Islands in the South China Sea. The use of LC-MS/MS molecular networking for sample prioritization and as an analytical dereplication tool facilitated the targeted isolation of 1 and 2. These molecules were characterized by spectroscopy and spectrometry, and configurational assignments were determined using chemical degradation and chiral-phase HPLC analysis. Compounds 1 and 2 modulated spontaneous calcium oscillations without notable cytotoxicity at 10 μM in short duration in vitro testing on primary cultured neocortical neurons, a model system that evaluates neuronal excitability and/or the potential activity on Ca2+ signaling. Both molecules were also found to be moderately cytotoxic in longer duration bioassays, with in vitro IC50 values of 1-10 μM against CCRF-CEM human T lymphoblastoid cells and U937 human histiocytic lymphoma cells. These formerly undiscovered bioactivities of known compound 2 expand upon its previously reported function as a selective CXCR7 agonist among 168 GPCR targets.

Metabolomic Characterization of a cf. Neolyngbya Cyanobacterium from the South China Sea Reveals Wenchangamide A, a Lipopeptide with In Vitro Apoptotic Potential in Colon Cancer Cells.

Metabolomics can be used to study complex mixtures of natural products, or secondary metabolites, for many different purposes. One productive application of metabolomics that has emerged in recent years is the guiding direction for isolating molecules with structural novelty through analysis of untargeted LC-MS/MS data. The metabolomics-driven investigation and bioassay-guided fractionation of a biomass assemblage from the South China Sea dominated by a marine filamentous cyanobacteria, cf. Neolyngbya sp., has led to the discovery of a natural product in this study, wenchangamide A (1). Wenchangamide A was found to concentration-dependently cause fast-onset apoptosis in HCT116 human colon cancer cells in vitro (24 h IC50 = 38 μM). Untargeted metabolomics, by way of MS/MS molecular networking, was used further to generate a structural proposal for a new natural product analogue of 1, here coined wenchangamide B, which was present in the organic extract and bioactive sub-fractions of the biomass examined. The wenchangamides are of interest for anticancer drug discovery, and the characterization of these molecules will facilitate the future discovery of related natural products and development of synthetic analogues.

Trichome Lengths of the Heterocystous N2-Fixing Cyanobacteria in the Tropical Marginal Seas of the Western North Pacific

Calothrix rhizosoleniae and Richelia intracellularis are heterocystous cyanobacteria found in the tropical oceans. C. rhizosoleniae commonly live epiphytically on diatom genera Chaetoceros (C-C) and Bacteriastrum (B-C) while R. intracellularis live endosymbiotically within Rhizosolenia (R-R), Guinardia (G-R), and Hemiaulus (H-R); although, they occasionally live freely (FL-C and FL-R). Both species have much shorter trichomes than the other marine filamentous cyanobacteria such as Trichodesmium spp. and Anabaena gerdii. We investigated the trichome lengths of C. rhizosoleniae and R. intracellularis in the South China Sea (SCS) and the Philippine Sea (PS) between 2006 and 2014. On average, H-R had the shortest trichome lengths (3.5 cells/trichome), followed by B-C and C-C (4.9–5.2 cells/trichome) and FL-C (5.9 cells/trichome), and R-R, G-R, and FL-R had the longest trichome lengths (7.4–8.3 cells/trichome). Field results showed the trichome lengths of C-C and B-C did not vary seasonally or regionally. However, FL-C and H-R from the SCS and during the cool season had longer trichomes, where/when the ambient nutrient concentrations were higher. R-R, G-R, and FL-R also showed regional and seasonal variations in trichome length. Ultrastructural analysis found no gas vesicles within the C. rhizosoleniae cells to assist in buoyancy regulation. Results suggest that the trichome lengths of C. rhizosoleniae and R. intracellularis might be regulated by their diatom hosts’ symbiotic styles and by ambient nutrients. Short trichome length might help C. rhizosoleniae and R. intracellularis to stay in the euphotic zone regardless as to whether they are free-living or symbiotic.

Marine Cyanobacteria: A Source of Lead Compounds and their Clinically-Relevant Molecular Targets.

The prokaryotic filamentous marine cyanobacteria are photosynthetic microbes that are found in diverse marine habitats, ranging from epiphytic to endolithic communities. Their successful colonization in nature is largely attributed to genetic diversity as well as the production of ecologically important natural products. These cyanobacterial natural products are also a source of potential drug leads for the development of therapeutic agents used in the treatment of diseases, such as cancer, parasitic infections and inflammation. Major sources of these biomedically important natural compounds are found predominately from marine cyanobacterial orders Oscillatoriales, Nostocales, Chroococcales and Synechococcales. Moreover, technological advances in genomic and metabolomics approaches, such as mass spectrometry and NMR spectroscopy, revealed that marine cyanobacteria are a treasure trove of structurally unique natural products. The high potency of a number of natural products are due to their specific interference with validated drug targets, such as proteasomes, proteases, histone deacetylases, microtubules, actin filaments and membrane receptors/channels. In this review, the chemistry and biology of selected potent cyanobacterial compounds as well as their synthetic analogues are presented based on their molecular targets. These molecules are discussed to reflect current research trends in drug discovery from marine cyanobacterial natural products.

Elevated CO2 impact on growth and lipid of marine cyanobacterium Phormidium valderianum BDU 20041– towards microalgal carbon sequestration

Abstract The growth of marine cyanobacteria at elevated CO2 levels is one of the feasible means to bioremediate the greenhouse gases. The present paper describes a marine filamentous cyanobacterium Phormidium valderianum BDU 20041 that can grow at elevated CO2 levels (15%), simulated and actual flue gas. Compared to ambient air and other tested CO2 concentrations, 3% CO2 showed higher biomass productivity, growth rate and carbon fixation rate. The organism was able to tolerate 15% CO2 and the simulated flue gas conditions making it a strong candidate for microalgal carbon capture. The lipid content of P. valderianum BDU 20041 at 15% CO2, simulated and actual flue gas conditions was higher than the ambient air. The conditions tested, i.e. 15% CO2, simulated and real flue gas show that this organism could be a potential entity in curbing carbon emission and a plausible biodiesel feedstock through hydrothermal liquefaction.

A Convolutional Neural Network-Based Approach for the Rapid Annotation of Molecularly Diverse Natural Products.

This report describes the first application of the novel NMR-based machine learning tool "Small Molecule Accurate Recognition Technology" (SMART 2.0) for mixture analysis and subsequent accelerated discovery and characterization of new natural products. The concept was applied to the extract of a filamentous marine cyanobacterium known to be a prolific producer of cytotoxic natural products. This environmental Symploca extract was roughly fractionated, and then prioritized and guided by cancer cell cytotoxicity, NMR-based SMART 2.0, and MS2-based molecular networking. This led to the isolation and rapid identification of a new chimeric swinholide-like macrolide, symplocolide A, as well as the annotation of swinholide A, samholides A-I, and several new derivatives. The planar structure of symplocolide A was confirmed to be a structural hybrid between swinholide A and luminaolide B by 1D/2D NMR and LC-MS2 analysis. A second example applies SMART 2.0 to the characterization of structurally novel cyclic peptides, and compares this approach to the recently appearing "atomic sort" method. This study exemplifies the revolutionary potential of combined traditional and deep learning-assisted analytical approaches to overcome longstanding challenges in natural products drug discovery.

The differential carbon-fixing and nitrogen-assimilating enzyme activities of Oscillatorian marine cyanobacterium Phormidium valderianum BDU 20041

We have identified an efficient filamentous marine cyanobacterium, Phormidium valderianum BDU 20041, which was adaptive to different concentrations of CO2. In our study, P. valderianum BDU 20041 was grown under ambient (0.04) and elevated (2, 3, 4, 5, 10, and 15%) CO2 concentration. Its optimal growth occurred at 3% CO2 supply. P. valderianum displayed similar carbon fixation activities through Rubisco, phosphoenolpyruvate carboxylase (PEPC), carbonic anhydrase, and malate dehydrogenase. The activity of Rubisco, the primary carbon-fixing enzyme, was found to be approximately 80% higher in P. valderianum grown under 4% CO2 than under ambient air. This was substantiated by Western blot. The nitrogen-assimilating enzymes shared a similar trend at higher CO2 concentrations. Our study is one of the few reports that examined the activities of CO2-fixing and nitrogen-assimilating enzymes together. Due to the active PEPC, this cyanobacterium was found to have active C3 and C4 enzymes that promoted higher CO2 fixation and cellular growth. Thus, it may be an efficient organism for biotechnological exploitation.

Biotechnological potential of Neolyngbya (Cyanobacteria), a new marine benthic filamentous genus from Brazil

Marine filamentous cyanobacteria are sources of biologically active secondary metabolites with potential biotechnological application. Despite the extension and environmental variety of the Brazilian coast, floristic surveys of filamentous cyanobacteria are scarce. Studies on their biotechnological potential are even more limited. In this work, we sampled Neolyngbya (Oscillatoriaceae) populations along the Brazilian coast and evaluated the antimicrobial, antioxidant, and cytotoxic activities of their organic extracts. We also assessed the strains' genetic potential for production of cyanotoxins. The strains were cultivated in SWBG-11 medium. Extraction of biomass was carried out with methanol, ethanol, and acetic acid. Antimicrobial (against seven bacterial strains and the yeast Candida albicans), antioxidant (DPPH and ABTS radicals), and cytotoxic (brine shrimp) potential were evaluated. PCR reactions were performed to analyze the genetic potential for the production of microcystin, saxitoxin, and cylindrospermopsin. Brazilian marine filamentous cyanobacteria presented relevant antimicrobial capacity. Seven extracts from four Neolyngbya strains inhibited growth of almost all bacteria, while nine extracts from four strains inhibited C. albicans, with a minimum inhibitory concentration (MIC) range from 2.5 to 10.0 mg·mL−1. Methanolic extracts presented the greatest antioxidant potential for both DPPH and ABTS radicals, ranging from 50.0 to 98.3% at a concentration of 10 mg·mL−1. Cytotoxicity assays showed a LC50 ranging from 0.8 to 6.6 mg·mL−1. We detected toxic genotypes in eight of the analyzed strains, in which the most frequent gene clusters were mcyG (microcystin) and cyrJ (cylindrospermopsin). All Neolyngbya strains screened in this study have potential for biotechnological application, which highlights the economical and pharmacological importance of studying this underestimated biodiversity in tropical areas.

Screening cyanobacteria from marine coastal waters of Thailand for biohydrogen production

Cyanobacteria are prokaryotic organisms capable of oxygenic photosynthesis. H2 can be produced by cyanobacterial bidirectional hydrogenase, but mainly during anaerobic dark fermentation. Here, we screened H2 producing cyanobacteria isolated from marine environments in Thailand and optimized physiological conditions for maximizing H2 production of the selected isolate. Most of the 54 cyanobacterial strains isolated and purified from samples of seawater, stones, sand, and shells in the Gulf of Thailand and the Andaman Sea, southern part of Thailand produced H2 when cells were incubated in nitrogen-deprived medium under dark/anaerobic condition. The filamentous non-heterocystous cyanobacterium Geitlerinema sp. RMK-SH10 gave the highest H2 yield with highest H2 production rate found in 7-day grown cells. Geitlerinema sp. RMK-SH10 showed maximum H2 production rate of 0.271 ± 0.013 μmol H2 mg−1 dry weight h−1 when incubated in NaNO3-free ASN III medium containing 0.2 M NaCl, 18.9 mmol C-atom of glucose L−1, and 0.1 μM Ni2+. These results suggest that the marine filamentous cyanobacterium Geitlerinema sp. RMK-SH10 has high potential as a H2 producer amenable for further improvement by genetic manipulation.

Morphological and genetic characterization of marine filamentous cyanobacterium Geitlerinema isolated from Thailand

Morphological and genetic characterization of marine filamentous cyanobacterium Geitlerinema isolated from Thailand

Collection, Culturing, and Genome Analyses of Tropical Marine Filamentous Benthic Cyanobacteria.

Decreasing sequencing costs has sparked widespread investigation of the use of microbial genomics to accelerate the discovery and development of natural products for therapeutic uses. Tropical marine filamentous cyanobacteria have historically produced many structurally novel natural products, and therefore present an excellent opportunity for the systematic discovery of new metabolites via the information derived from genomics and molecular genetics. Adequate knowledge transfer and institutional know-how are important to maintain the capability for studying filamentous cyanobacteria due to their unusual microbial morphology and characteristics. Here, we describe workflows, procedures, and commentary on sample collection, cultivation, genomic DNA generation, bioinformatics tools, and biosynthetic pathway analysis concerning filamentous cyanobacteria.

Identification of a new-to-science cyanobacterium, Toxifilum mysidocida gen. nov. & sp. nov. (Cyanobacteria, Cyanophyceae).

Cyanobacteria occupy many niches within terrestrial, planktonic, and benthic habitats. The diversity of habitats colonized, similarity of morphology, and phenotypic plasticity all contribute to the difficulty of cyanobacterial identification. An unknown marine filamentous cyanobacterium was isolated from an aquatic animal rearing facility having mysid mortality events. The cyanobacterium originated from Corpus Christi Bay, TX. Filaments are rarely solitary, benthic mat forming, unbranched, and narrowing at the ends. Cells are 2.1×3.1μm (width×length). Thylakoids are peripherally arranged on the outer third of the cell; cyanophycin granules and polyphosphate bodies are present. Molecular phylogenetic analysis in addition to morphology (transmission electron microscopy and scanning electron microscopy) and chemical composition all confirm it as a new genus and species we name Toxifilum mysidocida. At least one identified Leptolyngbya appears (based on genetic evidence and TEM) to belong to this new genus.

On-site concurrent carbon dioxide sequestration from flue gas and calcite formation in ossein effluent by a marine cyanobacterium Phormidium valderianum BDU 20041

In the recent past, there has been an upsurge in the use of microalgae to capture CO2. Microalgae are the most productive biological systems for generating biomass; they produce much more biomass than terrestrial organisms. In the present study, a cyanobacterium was exploited for combined CO2 capture and ossein effluent treatment. Phormidium valderianum BDU 20041, a non-heterocystous filamentous marine cyanobacterium was grown in an open tank (∼550L) in ossein effluent using flue gas (coal burnt) after cooling. After 10days with unscrubbed flue gas, the organism’s biomass productivity, specific growth rate, CO2 fixation rate, and lipid content were 30mgL−1d−1, 0.10μd−1, 56.4mgCL−1d−1, and 12.74%, respectively. The predominance of C14 and C18 fatty acids in the total lipid content revealed its suitability as a potential biofuel. Results of this study show that flue gas from a coke oven can be used for mass cultivation of P. valderianum BDU 20041, which efficiently captures CO2 and remediates ossein wastewater. Calcite formation by the organism in effluent was confirmed by scanning electron microscopy and energy dispersion spectroscopy studies. Biomass production employing flue gas coupled with the biomineralization of CO2 in ossein effluent at a point source offers a novel self-sustaining strategy to mitigate CO2 through outdoor cultivation and the subsequent generation of a biodiesel feedstock.

The Phormidolide Biosynthetic Gene Cluster: A trans-AT PKS Pathway Encoding a Toxic Macrocyclic Polyketide.

Phormidolide is a polyketide produced by a cultured filamentous marine cyanobacterium and incorporates a 16-membered macrolactone. Its complex structure is recognizably derived from a polyketide synthase pathway, but possesses unique and intriguing structural features that prompted interest in investigating its biosynthetic origin. Stable isotope incorporation experiments confirmed the polyketide nature of this compound. We further characterized the phormidolide gene cluster (phm) through genome sequencing followed by bioinformatic analysis. Two discrete trans-type acyltransferase (trans-AT) ORFs along with KS-AT adaptor regions (ATd) within the polyketide synthase (PKS) megasynthases, suggest that the phormidolide gene cluster is a trans-AT PKS. Insights gained from analysis of the mode of acetate incorporation and ensuing keto reduction prompted our reevaluation of the stereochemistry of phormidolide hydroxy groups located along the linear polyketide chain.

Genetic Diversity of Filamentous Cyanobacteria from Shore Regions of Okinawa

Benthic marine cyanobacteria are ubiquitous oxygenic photosynthetic bacteria and they form major components of various communities, including epiphytic, epilithic and microbial mats, in coral reef environments. Filamentous groups may form almost monospecific macroscopic colonies, and sometimes make potentially harmful algal blooms (HABs). HABs bring serious problems for marine tourism but marine filamentous cyanobacteria are good resource for drug discovery. However, cultivation and identification of such filamentous cyanobacteria are difficult and there are taxonomic confusions. In this study, we investigate 17 samples from coral reef area of 10 localities in Okinawa. Macroscopic mats or cushions of filamentous cyanobacteria were collected. The samples were divided into three parts: (1) for morphological observation, (2) for molecular work, and (3) cultivation for further study. Dominant cyanobacteira of each sample identified morphologically as Anabaena, Hydrocoleum, Lyngbya, Oscillatoria, Phormidium, Schizothrix and Symploca. Molecular results of partial 16S rDNA sequences revealed six groups that composed of heterocystous group, Leptolyngbya group, Symploca group, Moorea group, Oscillatoria group and unknown group, respectively. Some samples corroborated between morphology and molecular results but some were not. In addition, four out of six groups closely related with natural product producers. These results suggest that there are high biodiversity of marine benthic filamentous cyanobacteria in Okinawa that may become seeds of HABs as well as providing promising resources for drug discovery.