Intracellular Immunohistochemical Detection of Tetrodotoxin in Pleurobranchaea maculata (Gastropoda) and Stylochoplana sp. (Turbellaria)

Tetrodotoxin (TTX), is a potent neurotoxin targeting sodium channels that has been identified in multiple marine and terrestrial organisms. It was recently detected in the Opisthobranch Pleurobranchaea maculata and a Platyhelminthes Stylochoplana sp. from New Zealand. Knowledge on the distribution of TTX within these organisms is important to assist in elucidating the origin and ecological role of this toxin. Intracellular micro-distribution of TTX was investigated using a monoclonal antibody-based immunoenzymatic technique. Tetrodotoxin was strongly localized in neutral mucin cells and the basement membrane of the mantle, the oocytes and follicles of the gonad tissue, and in the digestive tissue of P. maculata. The ova and pharynx were the only two structures to contain TTX in Stylochoplana sp. Using liquid chromatography-mass spectrometry, TTX was identified in the larvae and eggs, but not the gelatinous egg cases of P. maculata. Tetrodotoxin was present in egg masses of Stylochoplana sp. These data suggest that TTX has a defensive function in adult P. maculata, who then invest this in their progeny for protection. Localization in the digestive tissue of P. maculata potentially indicates a dietary source of TTX. Stylochoplana sp. may use TTX in prey capture and for the protection of offspring.     

Investigating Diet as the Source ofTetrodotoxin in Pleurobranchaea maculata

The origin of tetrodotoxin (TTX) is highly debated; researchers have postulated either an endogenous or exogenous source with the host accumulating TTX symbiotically or via food chain transmission. The aim of this study was to determine whether the grey side-gilled sea slug (Pleurobranchaea maculata) could obtain TTX from a dietary source, and to attempt to identify this source through environmental surveys. Eighteen non-toxic P. maculata were maintained in aquariums and twelve were fed a TTX-containing diet. Three P. maculata were harvested after 1 h, 24 h, 17 days and 39 days and TTX concentrations in their stomach, gonad, mantle and remaining tissue/fluids determined using liquid chromatography-mass spectrometry. Tetrodotoxin was detected in all organs/tissue after 1 h with an average uptake of 32%. This decreased throughout the experiment (21%, 15% and 9%, respectively). Benthic surveys at sites with dense populations of toxic P. maculata detected very low or no TTX in other organisms. This study demonstrates that P. maculata can accumulate TTX through their diet. However, based on the absence of an identifiable TTX source in the environment, in concert with the extremely high TTX concentrations and short life spans of P. maculata, it is unlikely to be the sole TTX source for this species.     

Tetrodotoxin concentrations in Pleurobranchaea maculata: temporal, spatial and individual variability from New Zealand populations

Tetrodotoxin (TTX) is a potent neurotoxin that has been identified in a range of phylogenetically unrelated marine and terrestrial organisms. Tetrodotoxin was recently detected in New Zealand in Pleurobranchaea maculata (the grey side-gilled sea slug). From June 2010 to June 2011 wild specimens were collected from 10 locations around New Zealand. At one site (Narrow Neck Beach, Auckland) up to 10 individuals were collected monthly for 6 months. Attempts were also made to rear P. maculata in captivity. Tetrodotoxin was detected in samples from eight of the ten sites. The highest average (368.7 mg kg⁻¹) and maximum (1414.0 mg kg⁻¹) concentrations were measured in samples from Illiomama Rock (Auckland). Of the toxic populations tested there was significant variability in TTX concentrations among individuals, with the highest difference (62 fold) measured at Illiomama Rock. Tetrodotoxin concentrations in samples from Narrow Neck Beach varied temporally, ranging from an average of 184 mg kg⁻¹ in June 2010 to 17.5 mg kg⁻¹ by December 2010. There was no correlation between TTX levels and mass. The highest levels correspond with the egg laying season (June–August) and this, in concert with the detection of high levels of TTX in eggs and early larval stages, suggests that TTX may have a defensive function in P. maculata. Only one larva was successfully reared to full maturation and no TTX was detected.     

Bioprospecting from Marine Sediments of New Brunswick,Canada: Exploring the Relationship between Total Bacterial Diversity and Actinobacteria Diversity

Actinomycetes are an important resource for the discovery of natural products with therapeutic properties. Bioprospecting for actinomycetes typically proceeds without a priori knowledge of the bacterial diversity present in sampled habitats. In this study, we endeavored to determine if overall bacterial diversity in marine sediments, as determined by 16S rDNA amplicon pyrosequencing, could be correlated with culturable actinomycete diversity, and thus serve as a powerful tool in guiding future bioprospecting efforts. Overall bacterial diversity was investigated in eight marine sediments from four sites in New Brunswick, Canada, resulting in over 44,000 high quality sequences (x = 5610 per sample). Analysis revealed all sites exhibited significant diversity (H’ = 5.4 to 6.7). Furthermore, statistical analysis of species level bacterial communities (D = 0.03) indicated community composition varied according to site and was strongly influenced by sediment physiochemical composition. In contrast, cultured actinomycetes (n = 466, 98.3% Streptomyces) were ubiquitously distributed among all sites and distribution was not influenced by sediment composition, suggesting that the biogeography of culturable actinomycetes does not correlate with overall bacterial diversity in the samples examined. These actinomycetes provide a resource for future secondary metabolite discovery, as exemplified by the antimicrobial activity observed from preliminary investigation.     

New Insights on the Terpenome of the Red Seaweed Laurencia dendroidea (Florideophyceae,Rhodophyta)

The red seaweeds belonging to the genus Laurencia are well known as halogenated secondary metabolites producers, mainly terpenoids and acetogennins. Several of these chemicals exhibit important ecological roles and biotechnological applications. However, knowledge regarding the genes involved in the biosynthesis of these compounds is still very limited. We detected 20 different genes involved in the biosynthesis of terpenoid precursors, and 21 different genes coding for terpene synthases that are responsible for the chemical modifications of the terpenoid precursors, resulting in a high diversity of carbon chemical skeletons. In addition, we demonstrate through molecular and cytochemical approaches the occurrence of the mevalonate pathway involved in the biosynthesis of terpenes in L. dendroidea. This is the first report on terpene synthase genes in seaweeds, enabling further studies on possible heterologous biosynthesis of terpenes from L. dendroidea exhibiting ecological or biotechnological interest.     

The Bacterial (Vibrio alginolyticus) Production of Tetrodotoxin in the Ribbon Worm Lineus longissimus—Just a False Positive?

We test previous claims that the bacteria Vibrio alginolyticus produces tetrodotoxin (TTX) when living in symbiosis with the nemertean Lineus longissimus by a setup with bacteria cultivation for TTX production. Toxicity experiments on the shore crab, Carcinus maenas, demonstrated the presence of a paralytic toxin, but evidence from LC-MS and electrophysiological measurements of voltage-gated sodium channel–dependent nerve conductance in male Wistar rat tissue showed conclusively that this effect did not originate from TTX. However, a compound of similar molecular weight was found, albeit apparently non-toxic, and with different LC retention time and MS/MS fragmentation pattern than those of TTX. We conclude that C. maenas paralysis and death likely emanate from a compound <5 kDa, and via a different mechanism of action than that of TTX. The similarity in mass between TTX and the Vibrio-produced low-molecular-weight, non-toxic compound invokes that thorough analysis is required when assessing TTX production. Based on our findings, we suggest that re-examination of some published claims of TTX production may be warranted.     

Metabolomic Profiling and Genomic Study of a Marine Sponge-Associated Streptomyces sp

Metabolomics and genomics are two complementary platforms for analyzing an organism as they provide information on the phenotype and genotype, respectively. These two techniques were applied in the dereplication and identification of bioactive compounds from a Streptomyces sp. (SM8) isolated from the sponge Haliclona simulans from Irish waters. Streptomyces strain SM8 extracts showed antibacterial and antifungal activity. NMR analysis of the active fractions proved that hydroxylated saturated fatty acids were the major components present in the antibacterial fractions. Antimycin compounds were initially putatively identified in the antifungal fractions using LC-Orbitrap. Their presence was later confirmed by comparison to a standard. Genomic analysis of Streptomyces sp. SM8 revealed the presence of multiple secondary metabolism gene clusters, including a gene cluster for the biosynthesis of the antifungal antimycin family of compounds. The antimycin gene cluster of Streptomyces sp. SM8 was inactivated by disruption of the antimycin biosynthesis gene antC. Extracts from this mutant strain showed loss of antimycin production and significantly less antifungal activity than the wild-type strain. Three butenolides, 4,10-dihydroxy-10-methyl-dodec-2-en-1,4-olide (1), 4,11-dihydroxy-10-methyl-dodec-2-en-1,4-olide (2), and 4-hydroxy-10-methyl-11-oxo-dodec-2-en-1,4-olide (3) that had previously been reported from marine Streptomyces species were also isolated from SM8. Comparison of the extracts of Streptomyces strain SM8 and its host sponge, H. simulans, using LC-Orbitrap revealed the presence of metabolites common to both extracts, providing direct evidence linking sponge metabolites to a specific microbial symbiont.     

Insecticidal Activity and Insect Repellency of Four Species of Sea Lily (Comatulida: Comatulidae) From Taiwan

The methanol and ethyl acetate (EA) extracts of four species of sea lily (Himerometra magnipinna, Comaster multifidus, Comanthina sp., and Comatella maculata) were evaluated for their insecticidal activity against Yellow-fever mosquito larvae (Aedes aegypti) and their repellency against adult Asian Tiger mosquitoes (Aedes albopictus). The 24-hr minimum inhibition concentration (MIC) data revealed that the extracts from H. magnipinna and the C. maculata were the most active, killing mosquito larvae at 12.5 ppm. The toxicity of the extracts from these four sea lilies in descending order was H. magnipinna (12.5 ppm), C. maculata (12.5 ppm), C. multifidus (100 ppm), and Comanthina sp. (200 ppm). Furthermore, no significant difference in toxicity was found using either EA or methanol as the extraction solvent. The MIC at 12.5 ppm is promising as an insecticide lead. The repellency study results show that EA is a better solvent for one species (H. magnipinna), but the methanol is a better solvent overall. The repellency of these sea lily extracts in descending order was Comanthina sp. MeOH (ED₅₀ at 0.32%), followed by H. magnipinna EA (ED₅₀ at 0.38%), C. multifidus MeOH (ED₅₀ at 0.57%), C. maculata MeOH (ED₅₀ at 0.76%), C. multifidus EA (ED₅₀ at 1.25%), and H. magnipinna MeOH (ED₅₀ at 1.67%). A compound with ED₅₀ <0.5% is considered to be a promising repellant. Among the studied sea lilies, both Comanthina sp. and H. magnipinna have potential to be further developed as mosquito control agents due to their favorable toxicity and repellency.     

Sensitivity of Neurospora crassa to a Marine-Derived Aspergillus tubingensis Anhydride Exhibiting Antifungal Activity That Is Mediated by the MAS1 Protein

The fungus Aspergillus tubingensis (strain OY907) was isolated from the Mediterranean marine sponge Ircinia variabilis. Extracellular extracts produced by this strain were found to inhibit the growth of several fungi. Among the secreted extract components, a novel anhydride metabolite, tubingenoic anhydride A (1) as well as the known 2-carboxymethyl-3-hexylmaleic acid anhydride, asperic acid, and campyrone A and C were purified and their structure elucidated. Compound 1 and 2-carboxymethyl-3-hexylmaleic acid anhydride inhibited Neurospora crassa growth (MIC = 330 and 207 μM, respectively) and affected hyphal morphology. We produced a N. crassa mutant exhibiting tolerance to 1 and found that a yet-uncharacterized gene, designated mas-1, whose product is a cytosolic protein, confers sensitivity to this compound. The ∆mas-1 strain showed increased tolerance to sublethal concentrations of the chitin synthase inhibitor polyoxin D, when compared to the wild type. In addition, the expression of chitin synthase genes was highly elevated in the ∆mas-1 strain, suggesting the gene product is involved in cell wall biosynthesis and the novel anhydride interferes with its function.     

Cloning and expression analysis of kenaf (Hibiscus cannabinus L.) major lignin and cellulose biosynthesis gene sequences and polymer quantification during plant development

In order to apply state-of-the-art molecular breeding techniques in fibre crop it is necessary to have a good knowledge of major polymer biosynthesis gene sequences and their expression pattern. Polymerase chain reaction was employed to isolate sequences of the major genes for lignin and cellulose biosynthesis in a kenaf cultivar. CeSA, 4cl, c4h, cad, and ccr gene primers were designed according to their conservative regions; partial sequences of lignin and cellulose biosynthesis genes were obtained. One actin II gene sequence was also isolated from the kenaf genome as a housekeeping gene to be employed in qPCR analysis. Expression levels of genes c4h, cad and CeSA in bark and core from plants harvested at three different growth stages were evaluated. Using qPCR analyses it was found that the expression levels of the two biosynthesis lignin genes in bark tissues increased during plant growth, while a negative trend was recorded in core tissues. In both bark and core, the quantity of lignin was positively correlated to plant growth while cellulose content decreased.     

Bioactive compound synthetic capacity and ecological significance of marine bacterial genus pseudoalteromonas

The genus Pseudoalteromonas is a marine group of bacteria belonging to the class Gammaproteobacteria that has come to attention in the natural product and microbial ecology science fields in the last decade. Pigmented species of the genus have been shown to produce an array of low and high molecular weight compounds with antimicrobial, anti-fouling, algicidal and various pharmaceutically-relevant activities. Compounds formed include toxic proteins, polyanionic exopolymers, substituted phenolic and pyrolle-containing alkaloids, cyclic peptides and a range of bromine-substituted compounds. Ecologically, Pseudoalteromonas appears significant and to date has been shown to influence biofilm formation in various marine econiches; involved in predator-like interactions within the microbial loop; influence settlement, germination and metamorphosis of various invertebrate and algal species; and may also be adopted by marine flora and fauna as defensive agents. Studies have been so far limited to a relatively small subset of strains compared to the known diversity of the genus suggesting that many more discoveries of novel natural products as well as ecological connections these may have in the marine ecosystem remain to be made.     

Total Synthesis of Fellutamide B and Deoxy-Fellutamides B,C, and D

The total syntheses of the marine-derived lipopeptide natural product fellutamide B and deoxy-fellutamides B, C, and D are reported. These compounds were accessed through a novel solid-phase synthetic strategy using Weinreb amide-derived resin. As part of the synthesis, a new enantioselective route to (3R)-hydroxy lauric acid was developed utilizing a Brown allylation reaction followed by an oxidative cleavage-oxidation sequence as the key steps. The activity of these natural products, and natural product analogues was also assessed against Mycobacterium tuberculosis in vitro.     

Molecular and Chemical Analysis of the Lipopolysaccharide from Aeromonas hydrophila Strain AH-1 (Serotype O11)

A group of virulent Aeromonas hydrophila, A. sobria, and A. veronii biovar sobria strains isolated from humans and fish have been described; these strains classified to serotype O11 are serologically related by their lipopolysaccharide (LPS) O-antigen (O-polysaccharide), and the presence of an S-layer consisting of multiple copies of a crystalline surface array protein with a molecular weight of 52 kDa in the form of a crystalline surface array which lies peripheral to the cell wall. A. hydrophila strain AH-1 is one of them. We isolated the LPS from this strain and determined the structure of the O-polysaccharide, which was similar to that previously described for another strain of serotype O11. The genetics of the O11-antigen showed the genes (wb_O11 cluster) in two sections separated by genes involved in biosynthesis and assembly of the S-layer. The O11-antigen LPS is an example of an ABC-2-transporter-dependent pathway for O-antigen heteropolysaccharide (disaccharide) assembly. The genes involved in the biosynthesis of the LPS core (waa_O11 cluster) were also identified in three different chromosome regions being nearly identical to the ones described for A. hydrophila AH-3 (serotype O34). The genetic data and preliminary chemical analysis indicated that the LPS core for strain AH-1 is identical to the one for strain AH-3.     

Cytotoxicity of the ascidian Cystodytes dellechiajei against tumor cells and study of the involvement of associated microbiota in the production of cytotoxic compounds

Many cytotoxic compounds of therapeutic interest have been isolated from marine invertebrates, and some of them have been reported to be of microbial origin. Pyridoacridine alkaloids are the main compounds extracted from the ascidian Cystodytes dellechiajei. Here we describe the in vitro antiproliferative activity against different tumor cell lines of the ascidian extracts and provide some insights on the role of the microbial community associated with the tunicate in the production of these compounds. C. dellechiajei extracts showed remarkably high antiproliferative activity (IC₅₀ ≤5 μg/mL) in human lung carcinoma A-549, colon adenocarcinoma H-116, pancreatic adenocarcinoma PSN-1 and breast carcinoma SKBR3 cell lines. Moreover, we found that the maximum activity was located in the tunic tissue of the colony, which harbours a microbial community. In order to ascertain the involvement of this community in the synthesis of the bioactive compounds different approachs that included culture and culture independent methods were carried out. We undertook a screening for antiproliferative activities of the bacterial isolates from the ascidian, as well as a comprative analysis of the cytotoxic activities and the microbial communities from two color morphs of the ascidian, green and blue. In addition, the changes of the antiproliferative activities and the composition of the microbial communities were studied from ascidians kept in aquaria and treated with antibiotics for one month. Our data obtained from the different experiments did not point out to bacteria as the source of the cytotoxic compounds, suggesting thus an ascidian origin.     

A New Dibenz[b,e]oxepine Derivative, 1-Hydroxy-10-methoxy-dibenz[b,e]oxepin-6,11-dione,from a Marine-Derived Fungus,Beauveria bassiana TPU942

1-Hydroxy-10-methoxy-dibenz[b, e]oxepin-6,11-dione (1) was obtained from the culture broth of a marine-derived fungus, Beauveria bassiana TPU942, isolated from a marine sponge collected at Iriomote Island in Okinawa, together with two known compounds, chrysazin (2) and globosuxanthone A (3). The structure of 1 was elucidated on the basis of its spectroscopic data (HREIMS, 1D and 2D NMR experiments including ¹H–¹H COSY, HMQC and HMBC spectra). Dibenz[b, e]oxepines are rare in nature, and only six natural products have been reported. Therefore, compound 1 is the seventh natural product in this class. Compounds 2 and 3 showed an antifungal activity against Candida albicans, and 3 inhibited the cell growth against two human cancer cell lines, HCT-15 (colon) and Jurkat (T-cell lymphoma). Compound 1 did not show an apparent activity in the same bioassays.     

Gene Sequence Based Clustering Assists in Dereplication of Pseudoalteromonas luteoviolacea Strains with Identical Inhibitory Activity and Antibiotic Production

Some microbial species are chemically homogenous, and the same secondary metabolites are found in all strains. In contrast, we previously found that five strains of P. luteoviolacea were closely related by 16S rRNA gene sequence but produced two different antibiotic profiles. The purpose of the present study was to determine whether such bioactivity differences could be linked to genotypes allowing methods from phylogenetic analysis to aid in selection of strains for biodiscovery. Thirteen P. luteoviolacea strains divided into three chemotypes based on production of known antibiotics and four antibacterial profiles based on inhibition assays against Vibrio anguillarum and Staphylococcus aureus. To determine whether chemotype and inhibition profile are reflected by phylogenetic clustering we sequenced 16S rRNA, gyrB and recA genes. Clustering based on 16S rRNA gene sequences alone showed little correlation to chemotypes and inhibition profiles, while clustering based on concatenated 16S rRNA, gyrB, and recA gene sequences resulted in three clusters, two of which uniformly consisted of strains of identical chemotype and inhibition profile. A major time sink in natural products discovery is the effort spent rediscovering known compounds, and this study indicates that phylogeny clustering of bioactive species has the potential to be a useful dereplication tool in biodiscovery efforts.     

Assessment of the Antimicrobial Activity of Algae Extracts on Bacteria Responsible of External Otitis

External otitis is a diffuse inflammation around the external auditory canal and auricle, which is often occurred by microbial infection. This disease is generally treated using antibiotics, but the frequent occurrence of antibiotic resistance requires the development of new antibiotic agents. In this context, unexplored bioactive natural candidates could be a chance for the production of targeted drugs provided with antimicrobial activity. In this paper, microbial pathogens were isolated from patients with external otitis using ear swabs for over one year, and the antimicrobial activity of the two methanol extracts from selected marine (Dunaliella salina) and freshwater (Pseudokirchneriella subcapitata) microalgae was tested on the isolated pathogens. Totally, 114 bacterial and 11 fungal strains were isolated, of which Staphylococcus spp. (28.8%) and Pseudomonas aeruginosa (P. aeruginosa) (24.8%) were the major pathogens. Only three Staphylococcus aureus (S. aureus) strains and 11 coagulase-negative Staphylococci showed resistance to methicillin. The two algal extracts showed interesting antimicrobial properties, which mostly inhibited the growth of isolated S. aureus, P. aeruginosa, Escherichia coli, and Klebsiella spp. with MICs range of 1.4 × 10⁹ to 2.2 × 10¹⁰ cells/mL. These results suggest that the two algae have potential as resources for the development of antimicrobial agents.     

Comparative Analysis of Glycoside Hydrolases Activities from Phylogenetically Diverse Marine Bacteria of the Genus Arenibacter

A total of 16 marine strains belonging to the genus Arenibacter, recovered from diverse microbial communities associated with various marine habitats and collected from different locations, were evaluated in degradation of natural polysaccharides and chromogenic glycosides. Most strains were affiliated with five recognized species, and some presented three new species within the genus Arenibacter. No strains contained enzymes depolymerizing polysaccharides, but synthesized a wide spectrum of glycosidases. Highly active β-N-acetylglucosaminidases and α-N-acetylgalactosaminidases were the main glycosidases for all Arenibacter. The genes, encoding two new members of glycoside hydrolyses (GH) families, 20 and 109, were isolated and characterized from the genomes of Arenibacter latericius. Molecular genetic analysis using glycosidase-specific primers shows the absence of GH27 and GH36 genes. A sequence comparison with functionally-characterized GH20 and GH109 enzymes shows that both sequences are closest to the enzymes of chitinolytic bacteria Vibrio furnissii and Cellulomonas fimi of marine and terrestrial origin, as well as human pathogen Elisabethkingia meningoseptica and simbionts Akkermansia muciniphila, gut and non-gut Bacteroides, respectively. These results revealed that the genus Arenibacter is a highly taxonomic diverse group of microorganisms, which can participate in degradation of natural polymers in marine environments depending on their niche and habitat adaptations. They are new prospective candidates for biotechnological applications due to their production of unique glycosidases.     

Natural Products from Marine Fungi—Still an Underrepresented Resource

Marine fungi represent a huge potential for new natural products and an increased number of new metabolites have become known over the past years, while much of the hidden potential still needs to be uncovered. Representative examples of biodiversity studies of marine fungi and of natural products from a diverse selection of marine fungi from the author’s lab are highlighting important aspects of this research. If one considers the huge phylogenetic diversity of marine fungi and their almost ubiquitous distribution, and realizes that most of the published work on secondary metabolites of marine fungi has focused on just a few genera, strictly speaking Penicillium, Aspergillus and maybe also Fusarium and Cladosporium, the diversity of marine fungi is not adequately represented in investigations on their secondary metabolites and the less studied species deserve special attention. In addition to results on recently discovered new secondary metabolites of Penicillium species, the diversity of fungi in selected marine habitats is highlighted and examples of groups of secondary metabolites produced by representatives of a variety of different genera and their bioactivities are presented. Special focus is given to the production of groups of derivatives of metabolites by the fungi and to significant differences in biological activities due to small structural changes.