Metal pollution affects both native and non-indigenous biofouling recruitment in a subtropical island system.

Hull fouling has been a driving force behind the development of most modern marine antifouling coatings that mainly contain copper based biocides to inhibit growth of fouling organisms. Despite these efforts, several non-indigenous species continue to be transferred via hull-fouling worldwide. In this study we designed a disturbance gradient with three commercial antifouling paints applied to PVC settling plates with different concentrations of copper oxide and allowed colonization of fouling communities in four marinas located at the Madeira Archipelago (NE Atlantic). Overall, the antifouling treatments were effective in decreasing the diversity of fouling communities and spatial variability across marinas was observed. Increasing exposure to metal pollutants decreases both species cover and total diversity, independently of their native or NIS condition. However, evidences found suggest that long-term effects of copper based antifouling coatings can be modulated by metal-resistant species allowing a secondary substrate for the epibiosis of other species to establish.

HSP90 regulates larval settlement of the bryozoan Bugula neritina through the nitric oxide pathway.

The larvae of many sessile marine invertebrates go through a settlement process, during which planktonic larvae attach to a substrate and metamorphose into sessile juveniles. Larval attachment and metamorphosis (herein defined as 'settlement') are complex processes mediated by many signalling pathways. Nitric oxide (NO) signalling is one of the pathways that inhibits larval settlement in marine invertebrates across different phyla. NO is synthesized by NO synthase (NOS), which is a client of the molecular chaperone heat shock protein 90 (HSP90). In the present study, we provide evidence that NO, a gaseous messenger, regulates larval settlement of Bugula neritina By using pharmacological bioassays and western blotting, we demonstrated that NO inhibits larval settlement of B. neritina and that NO signals occur mainly in the sensory organ of swimming larvae. The settlement rate of B. neritina larvae decreased after heat shock treatment. Inhibition of HSP90 induced larval settlement, and attenuated the inhibition of NO donors during larval settlement. In addition, the expression level of both HSP90 and NOS declined upon settlement. These results demonstrate that HSP90 regulates the larval settlement of B. neritina by interacting with the NO pathway.

Brevianamides and Mycophenolic Acid Derivatives from the Deep-Sea-Derived Fungus Penicillium brevicompactum DFFSCS025.

Four new compounds (1-4), including two brevianamides and two mycochromenic acid derivatives along with six known compounds were isolated from the deep-sea-derived fungus Penicillium brevicompactum DFFSCS025. Their structures were elucidated by spectroscopic analysis. Moreover, the absolute configurations of 1 and 2 were determined by quantum chemical calculations of the electronic circular dichroism (ECD) spectra. Compound 9 showed moderate cytotoxicity against human colon cancer HCT116 cell line with IC50 value of 15.6 µM. In addition, 3 and 5 had significant antifouling activity against Bugula neritina larval settlement with EC50 values of 13.7 and 22.6 µM, respectively. The NMR data of 6, 8, and 9 were assigned for the first time.

Anti-HSV-1, antioxidant and antifouling phenolic compounds from the deep-sea-derived fungus Aspergillus versicolor SCSIO 41502.

Chemical investigation of the deep-sea-derived fungus Aspergillus versicolor SCSIO 41502 resulted in the isolation of three new anthraquinones, aspergilols G-I (1-3), one new diphenyl ether, 4-carbglyceryl-3,3'-dihydroxy-5,5'-dimethyldiphenyl ether (4), and one new benzaldehyde derivative, 2,4-dihydroxy-6-(4-methoxy-2-oxopentyl)-3-methylbenzaldehyde (5), along with 23 known phenolic compounds (6-28). The structures of new compounds were elucidated by extensive spectroscopic analysis. The absolute configuration of 3 was established by CD spectrum and the modified Mosher method. Compounds 2, 3 and 9 had evident antiviral activity towards HSV-1 with EC50 values of 4.68, 6.25, and 3.12µM, respectively. Compounds 15, 18, 20 and 22-24 showed more potent antioxidant activity than l-ascorbic acid with IC50 values of 18.92-52.27µM towards DPPH radicals. Comparison of the structures and antioxidant activities of 1-28 suggests that the number of phenolic hydroxyl group that can freely rotate can significantly affect the antioxidant activity of phenolic compounds. In addition, 4, 22-24 and 27 had significant antifouling activity against Bugula neritina larval settlement with EC50 values of 1.28, 2.61, 5.48, 1.59, and 3.40µg/ml, respectively.

Antifouling properties of zinc oxide nanorod coatings.

In laboratory experiments, the antifouling (AF) properties of zinc oxide (ZnO) nanorod coatings were investigated using the marine bacterium Acinetobacter sp. AZ4C, larvae of the bryozoan Bugula neritina and the microalga Tetraselmis sp. ZnO nanorod coatings were fabricated on microscope glass substrata by a simple hydrothermal technique using two different molar concentrations (5 and 10 mM) of zinc precursors. These coatings were tested for 5 h under artificial sunlight (1060 W m(-2) or 530 W m(-2)) and in the dark (no irradiation). In the presence of light, both the ZnO nanorod coatings significantly reduced the density of Acinetobacter sp. AZ4C and Tetraselmis sp. in comparison to the control (microscope glass substratum without a ZnO coating). High mortality and low settlement of B. neritina larvae was observed on ZnO nanorod coatings subjected to light irradiation. In darkness, neither mortality nor enhanced settlement of larvae was observed. Larvae of B. neritina were not affected by Zn(2+) ions. The AF effect of the ZnO nanorod coatings was thus attributed to the reactive oxygen species (ROS) produced by photocatalysis. It was concluded that ZnO nanorod coatings effectively prevented marine micro and macrofouling in static conditions.

Assessing the antifouling properties of cold-spray metal embedment using loading density gradients of metal particles.

Particles of copper, bronze and zinc were embedded into a polymer using cold-spray technology to produce loading density gradients of metal particles. The gradients were used to identify the species with the highest tolerance to the release of copper and zinc ions. The gradients also established the minimum effective release rates (MERRs) of copper and zinc ions needed to prevent the recruitment of fouling under field conditions. Watersipora sp. and Simplaria pseudomilitaris had the highest tolerances to the release of metal ions. Copper and bronze gradient tubes were similar in their MERRs of copper ions against Watersipora sp. (0.058 g m(-2) h(-1) and 0.054 g m(-2) h(-1), respectively) and against S. pseudomilitaris (0.030 g m(-2) h(-1) and 0.025 g m(-2) h(-1), respectively). Zinc was not an effective antifoulant, with failure within two weeks. In conclusion, cold-spray gradients were effective in determining MERRs and these outcomes provide the basis for the development of cold-spray surfaces with pre-determined life-spans using controlled MERRs.

Antifouling potentials of eight deep-sea-derived fungi from the South China Sea.

Marine-derived microbial secondary metabolites are promising potential sources of nontoxic antifouling agents. The search for environmentally friendly and low-toxic antifouling components guided us to investigate the antifouling potentials of eight novel fungal isolates from deep-sea sediments of the South China Sea. Sixteen crude ethyl acetate extracts of the eight fungal isolates showed distinct antibacterial activity against three marine bacteria (Loktanella hongkongensis UST950701-009, Micrococcus luteus UST950701-006 and Pseudoalteromonas piscida UST010620-005), or significant antilarval activity against larval settlement of bryozoan Bugula neritina. Furthermore, the extract of Aspergillus westerdijkiae DFFSCS013 displayed strong antifouling activity in a field trial lasting 4 months. By further bioassay-guided isolation, five antifouling alkaloids including brevianamide F, circumdatin F and L, notoamide C, and 5-chlorosclerotiamide were isolated from the extract of A. westerdijkiae DFFSCS013. This is the first report about the antifouling potentials of metabolites of the deep-sea-derived fungi from the South China Sea, and the first stage towards the development of non- or low-toxic antifouling agents from deep-sea-derived fungi.

Combining a photocatalyst with microtopography to develop effective antifouling materials.

Polydimethylsiloxane surfaces textured with a square-wave linear grating profile (0, 20, 200, 300 and 600 µm), and embedded with a range of photocatalytic titanium dioxide (TiO2) nanoparticle loadings (3.75, 7.5, 11.25 and 15 wt.%), were used to test the combined efficacy of these technologies as antifouling materials. Settlement of the fouling bryozoan species Bugula neritina was quantified in the laboratory under two intensities of UV light. The lowest settlement rates were observed on 20 µm surfaces. However, texture effects were not as critical to larval settlement as the presence of TiO2. In conjunction with UV light, TiO2 completely inhibited larval metamorphosis even at the lowest loading (3.75 wt.%) and the lowest intensity of UV light (24 W m(-2)). Recruitment of B. neritina was also quantified in field trials and showed similar results to laboratory assays. The lowest recruitment was observed on 20 and 200 µm surfaces, with recruitment being significantly lower on all surfaces containing TiO2. Therefore for B. neritina, although all TiO2 loadings were effective, 3.75 wt.% can be used as a minimum inhibitory concentration to deter larval settlement and the addition of a 20 µm texture further increases the deterrent effect.

Polyketides from a marine-derived fungus Xylariaceae sp.

Eighteen polyketides (1-18) including six citrinin derivatives, two phenol derivatives, one cyclopentenone, two naphthol derivatives, and seven tetralone derivatives were isolated from the culture broth of a marine-derived fungal strain Xylariaceae sp. SCSGAF0086. Five of these compounds (1, 2, 8, 9, and 10) were new, and their structures were determined by spectroscopic methods. Compounds 4, 6, 7, and 17 showed enzyme-inhibitory activities towards several tested enzymes, and 6 and 7 showed strong antifouling activity against Bugula neritina larvae settlement. This is the first time that the antifouling and enzyme-inhibitory activities of these compounds has been reported.

Pentacyclic hemiacetal sterol with antifouling and cytotoxic activities from the soft coral Nephthea sp.

A novel unusual pentacyclic hemiacetal sterol nephthoacetal (1), was isolated from soft coral Nephthea sp. The structure of this sterol was inferred from its two acetyl derivatives (2) and (3), by means of spectroscopic methods, and quantum chemical calculations. Anti-fouling activity of compounds 1-3 against Bugula neritina larvae was evaluated, sterol (1) exhibited significant inhibitory effect with EC(50) value of 2.5 µg/mL, while having low toxicity with LC(50)>25.0 µg/mL. The in vitro cytotoxic activity of compounds 1-3 against HeLa cells was also evaluated, all of them exhibited moderate cytotoxicity with IC(50) values of 12.3 (1), 10.1 (2), and 19.6 µg/mL (3), respectively.

Coumarins from the Herb Cnidium monnieri and chemically modified derivatives as antifoulants against Balanus albicostatus and Bugula neritina larvae.

In the search for new environmental friendly antifouling (AF) agents, four coumarins were isolated from the herbal plant Cnidium monnieri, known as osthole (1), imperatorin (2), isopimpinellin (3) and auraptenol (4). Furthermore, five coumarin derivatives, namely 8-epoxypentylcoumarin (5), meranzin hydrate (6), 2'-deoxymetranzin hydrate (7), 8-methylbutenalcoumarin (8), and micromarin-F (9) were synthesized from osthole. Compounds 1, 2, 4, 7 showed high inhibitory activities against larval settlement of Balanus albicostatus with EC(50) values of 4.64, 3.39, 3.38, 4.67 µg mL-1. Compound 8 could significantly inhibit larval settlement of Bugula neritina with an EC(50) value of 3.87 µg mL-1. The impact of functional groups on anti-larval settlement activities suggested that the groups on C-5' and C-2'/C-3' of isoamylene chian could affect the AF activities.

[Alkaloids from gorgonian-derived Streptomyces sp. SCSGAA0009 and their antibacterial and antilarval activities].

OBJECTIVE: To search for structurally novel and biologically active compounds from the secondary metabolites of gorgonian-derived actinomyces. METHODS: Strains of actinomyces with antimicrobial activities were screened by biological methods. Then, those active strains were cultured under different conditions to obtain crude extracts. Subsequently, the chemical diversities of the extracts were investigated by reverse phase high-performance liquid chromatography (RP-HPLC) and thin layer chromatography (TLC), while the bioactivities of the extracts were evaluated by antifouling and antibacterial assays. A target strain Streptomyces sp. SCSGAA0009 was selected to further study by large-scale fermentation on modified ISP2 medium. The compounds were isolated by column chromatography over silica gel, Sephadex LH-20, and semipreparative HPLC, and their structures were determinated by spectroscopic analysis. RESULTS: The target strain was identified as actinomyces, Streptomyces sp. SCSGAA0009. A new alkaloid N-(2-(1H-indol-3-yl)ethyl) propionamide (1) and a known compound phenazine-1-carboxylic acid (2) were purified from the culture broth. Compound 2 showed moderate antibacterial activity against Escherichia coli and Pseudoaltermonas piscida, and exhibited strong antilarval settlement activity towards Bugula neritina larva. CONCLUSION: Bioactive and new compounds can be achieved from the secondary metabolites of gorgonian-derived microorganisms from South China Sea.

Polyurethane seismic streamer skins: an application of cold spray metal embedment.

Cold spray metal embedment is an innovative antifouling (AF) technology that delivers metal particles with AF properties into many thermoplastic polymers. AF efficacy was quantified for low (22.1 ± 4.8 g m(-2)) and high (101.1 ± 10.8 g m(-2)) densities of copper particles embedded into polyurethane (PU) seismic streamer skins, which are used in geophysical exploration. Failure of each Cu-embedded treatment was defined as settlement of hard foulers. Low-density streamers failed after 42 days while high-density streamers failed after 210 days. Most importantly, the high-density streamers were completely free of hard foulers including the barnacle Amphibalanus reticulatus during this time period. In conclusion, cold-spray metal embedment is an effective AF technology for PU seismic streamer skins, under intense fouling conditions. Higher copper particle densities enhance AF longevity and the effect of density provides a tool to extend efficacy and enhance AF performance for specific polymers.

The mode of action of isocyanide in three aquatic organisms, Balanus amphitrite, Bugula neritina and Danio rerio.

Isocyanide is a potential antifouling compound in marine environments. In this study, we investigated its mode of action in three aquatic organisms. Two of them, the bryozoan Bugula neritina and the barnacle Balanus amphitrite, are major marine fouling invertebrates, and the other organism is the non-target species zebrafish Danio rerio. In the swimming larvae of B. neritina, isocyanide did not affect the total attachment rate (≤50 µg ml(-1)), but it did change the attachment site by increasing the percentage of attachment on the bottom of the container rather than on the wall or air-water inter-surface. Isocyanide binds several proteins in B. neritina as identified via SDS-PAGE-LC-MS/MS: 1) a 30 kD protein band containing two proteins similar to voltage dependent anion channels (VDAC), which control the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol and the release of apoptogenic factors from mitochondria of mammalian cells; and 2) an unknown 39 kD protein. In B. amphitrite cyprids, the isocyanide binding protein were 1) a protein similar to NADH-ubiquinone oxidoreductase, which is the "entry enzyme" of oxidative phosphorylation in mitochondria; and 2) cytochrome P450. In Danio rerio embryos, isocyanide caused "wavy" notochords, hydrocephalus, pericardial edema, poor blood circulation, and defects in pigmentation and hematopoiesis, which phenocopied copper deficiency. This is the first report on isocyanide binding proteins in fouling organisms, as well as the first description of its phenotype and potential toxicology in zebrafish.

Butenolide inhibits marine fouling by altering the primary metabolism of three target organisms.

Butenolide is a very promising antifouling compound that inhibits ship hull fouling by a variety of marine organisms, but its antifouling mechanism was previously unknown. Here we report the first study of butenolide's molecular targets in three representative fouling organisms. In the barnacle Balanus (=Amphibalanus) amphitrite, butenolide bound to acetyl-CoA acetyltransferase 1 (ACAT1), which is involved in ketone body metabolism. Both the substrate and the product of ACAT1 increased larval settlement under butenolide treatment, suggesting its functional involvement. In the bryozoan Bugula neritina, butenolide bound to very long chain acyl-CoA dehydrogenase (ACADVL), actin, and glutathione S-transferases (GSTs). ACADVL is the first enzyme in the very long chain fatty acid ß-oxidation pathway. The inhibition of this primary pathway for energy production in larvae by butenolide was supported by the finding that alternative energy sources (acetoacetate and pyruvate) increased larval attachment under butenolide treatment. In marine bacterium Vibrio sp. UST020129-010, butenolide bound to succinyl-CoA synthetase ß subunit (SCSß) and inhibited bacterial growth. ACAT1, ACADVL, and SCSß are all involved in primary metabolism for energy production. These findings suggest that butenolide inhibits fouling by influencing the primary metabolism of target organisms.

The effect of butenolide on behavioral and morphological changes in two marine fouling species, the barnacle Balanus amphitrite and the bryozoan Bugula neritina.

Butenolide [5-octylfuran-2(5H)-one] is a very promising antifouling compound. Here, the effects of butenolide on larval behavior and histology are compared in two major fouling organisms, viz. cypris larvae of Balanus amphitrite and swimming larvae of Bugula neritina. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC50=0.82 µg ml(-1)) and B. neritina (EC50=3 µg ml(-1)). Its effect on the attachment of cyprids of B. amphitrite was influenced by temperature, and butenolide increased attachment of larvae of B. neritina to the bottom of the experimental wells. At concentrations of 4 µg ml(-1) and 10 µg ml(-1), butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of secretory granules and altered the rough endoplasmic reticulum (RER) in the cement gland of B. amphitrite cyprids. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.

Heritable pollution tolerance in a marine invader.

The global spread of fouling invasive species is continuing despite the use of antifouling biocides. Furthermore, previous evidence suggests that non-indigenous species introduced via hull fouling may be capable of adapting to metal-polluted environments. Using a laboratory based toxicity assay, we investigated tolerance to copper in the non-indigenous bryozoan Watersipora subtorquata from four source populations. Individual colonies were collected from four sites within Port Hacking (Sydney, Australia) and their offspring exposed to a range of copper concentrations. This approach, using a full-sib, split-family design, tests for a genotype by environment (G×E) interaction. Settlement and complete metamorphosis (recruitment) were measured as ecologically relevant endpoints. Larval sizes were also measured for each colony. Successful recruitment was significantly reduced by the highest copper concentration of 80µgL(-1). While there was no difference in pollution tolerance between sites, there was a significant G×E interaction, with large variation in the response of colony offspring within sites. Larval size differed significantly both between sites and between colonies and was positively correlated with tolerance. The high level of variation in copper tolerance between colonies suggests that there is considerable potential within populations to adapt to elevated copper levels, as tolerance is a heritable trait. Also, colonies that produce large larvae are more tolerant to copper, suggesting that tolerance may be a direct consequence of larger size.

Changes in the proteome and phosphoproteome expression in the bryozoan Bugula neritina larvae in response to the antifouling agent butenolide.

Larval attachment and metamorphosis, commonly referred to as larval settlement, of marine sessile invertebrates can be triggered or blocked by chemical cues and affected by changes in overall protein expression pattern and phosphorylation dynamics. This study focuses on the effects of butenolide, an effective larval settlement inhibitor, on larval settlement at the proteome level in the bryozoan Bugula neritina. Liquid-phase IEF sample prefractionation combined with 2-DE and MALDI-TOF MS was used to identify the differentially expressed proteins. Substantial changes occurred both in protein abundance and in phosphorylation status during larval settlement and when settling larvae were challenged with butenolide. The proteins that responded to treatment were identified as structural proteins, molecular chaperones, mitochondrial peptidases and calcium-binding proteins. Compared with our earlier results, both genistein and butenolide inhibited larval settlement of B. neritina primarily by changes in protein abundance and the phosphorylation status of proteins but have different protein targets in the same species. Clearly, to design potent antifouling compounds and to understand the mode of action of compounds, more studies on the effects of different compounds on proteome and phosphoproteome of different larval species are required.

Does the survivorship of activated resting stages in toxic environments provide cues for ballast water treatment?

The toxic effects of three inorganic metals (Cu, Cr, Hg), three organic (phenol, formalin, ammonium) chemicals, ozone-enriched water and peroxides (H2O2) on embryonic development were tested in 8 species from the Porifera, Bryozoa and Crustacea. Toxicants with lower molecular weight showed stronger negative impacts on post-diapause embryos than chemicals with higher molecular weight if related to the toxicity of the chemicals to active adult stages. Only few embryos of the cladoceran Moina macrocopa and none of the cladoceran Wlassicsia pannonica treated with peroxides at concentration 0.3% developed further. Ozone-enriched water had no significant effect on post-diapause embryonic development in cladocerans. Ammonium (the product of NH4OH dissociation) in concentration 100 mg/l and higher killed all embryos of M. macrocopa inside protective membranes. Peroxides and ammonium are suggested for the purification of ship ballast waters as effective, non-expensive and non-persistent toxic chemicals. Resting stages of invertebrates including at least Crustaceans, Porifera and Bryozoa seem to allow not only dispersal among toxic industrial environments such as ship ballast compartments, but may also endure serious pollution events common in seaports and estuaries. Artemia cysts due to their strong protection against different toxic substances are recommended as a model for studies of toxic effects in diapausing stages in polluted estuaries and marine environments.

Antifouling sesterterpenes from the New Zealand marine sponge Semitaspongia bactriana.

Three antifouling (AF) sesterterpenes have been isolated from the New Zealand marine sponge Semitaspongia bactriana with toxicity against the diatom Nitzschia closterium and bryozoan Bugula neritina. The three metabolites have been characterised by spectroscopic techniques as 7E,12E,20Z-variabilin (1), cavernosolide (2) and lintenolide A (3) (also called spongianolide C) and have low micromolar activity against our two test species. The gamma-hydroxybutenolide containing sesterterpenes (2 and 3) show the most promise, with relative stability and suitable lipophilicity for incorporation of either these metabolites, or synthetic analogues, as biocides to produce paints or plastics with AF properties.