R. de Nys

Broad spectrum effects of secondary metabolites from the red alga delisea pulchra in antifouling assays

In this study the antifouling activity was investigated of a series of chemically related, halogenated furanones isolated from Delisea pulchra (Greville) Montagne, a red alga which is rarely fouled in the field. The metabolites were tested in laboratory assays against representatives of the three major groups of fouling organisms, the barnacle Balanus amphitrite amphitirite Darwin, the macroalga Ulva lactuca Linnaeus and a marine bacterium (strain SW 8). Settlement of barnacle cyprid larvae was strongly inhibited, with an EC50 of < 25 ng.ml‐1 (25 ppb) for some compounds. The settlement and growth of algal gametes was also strongly inhibited, in some cases at concentrations as low as 25 ngcm‐2. Growth of the marine bacterium SW8 was inhibited more strongly than by the common antibiotic gentamicin. Overall, activity of the D. pulchra metabolites was comparable to that of the heavy metals and biocides currently used in antifouling paints. However, no single compound was most active in all tests and some meta...

Inhibition of luminescence and virulence in the black tiger prawn (Penaeus monodon) pathogen Vibrio harveyi by intercellular signal antagonists.

ABSTRACT Expression of luminescence in the Penaeus monodonpathogen Vibrio harveyi is regulated by an intercellular quorum sensing mechanism involving the synthesis and detection of two signaling molecules, one of which is N-hydroxy butanoyl-l-homoserine lactone and the other of which is uncharacterized. Indirect evidence has suggested that virulence, associated with a toxic extracellular protein, and luminescence inV. harveyi are coregulated. In this study the effects of an acylated homoserine lactone antagonist produced by the marine algaDelisea pulchra on luminescence and toxin production in a virulent strain of V. harveyi were analyzed. Luminescence and toxin production were both inhibited by the signal antagonist at concentrations that had no impact on growth. Toxin production was found to be prematurely induced in V. harveyi cultures incubated in a 10% conditioned medium. Additionally, a significant reduction in the toxicity of concentrated supernatant extracts from V. harveyi cultures incubated in the presence of the signal antagonist, as measured by in vivo toxicity assays in mice and prawns, was observed. These results suggest that intercellular signaling antagonists have potential utility in the control of V. harveyi prawn infections.

Testing attachment point theory : diatom attachment on microtextured polyimide biomimics

Abstract This paper explores diatom attachment to a range of laser etched polyimide surfaces to directly test ‘attachment point theory’. Static bioassays were conducted on microtextured polyimide surfaces using four diatom species, Fallacia carpentariae, Nitzschia cf. paleacea, Amphora sp. and Navicula jeffreyi with cell sizes ranging from 1 – 14 μm. The microtextured polyimides were modelled from natural fouling resistant bivalve surfaces and had wavelengths above, below and at the same scale as the diatom cell sizes. Diatoms attached in significantly higher numbers to treatments where the numbers of attachment points was highest. The lowest diatom attachment occurred where cells were slightly larger than the microtexture wavelength, resulting in only two theoretical points of attachment. The results support attachment point theory and highlight the need to address larval/cell size in relation to the number of attachment points on a surface. Further studies examining a range of microtexture scales are needed to apply attachment point theory to a suite of fouling organisms and to develop structured surfaces to control the attachment and development of fouling communities.

The role of nano-roughness in antifouling

Nano-engineered superhydrophobic surfaces have been investigated for potential fouling resistance properties. Integrating hydrophobic materials with nanoscale roughness generates surfaces with superhydrophobicity that have water contact angles (θ) >150° and concomitant low hysteresis (<10°). Three superhydrophobic coatings (SHCs) differing in their chemical composition and architecture were tested against major fouling species (Amphora sp., Ulva rigida, Polysiphonia sphaerocarpa, Bugula neritina, Amphibalanus amphitrite) in settlement assays. The SHC which had nanoscale roughness alone (SHC 3) deterred the settlement of all the tested fouling organisms, compared to selective settlement on the SHCs with nano- and micro-scale architectures. The presence of air incursions or nanobubbles at the interface of the SHCs when immersed was characterized using small angle X-ray scattering, a technique sensitive to local changes in electron density contrast resulting from partial or complete wetting of a rough interface. The coating with broad spectrum antifouling properties (SHC 3) had a noticeably larger amount of unwetted interface when immersed, likely due to the comparatively high work of adhesion (60.77 mJ m−2 for SHC 3 compared to 5.78 mJ m−2 for the other two SHCs) required for creating solid/liquid interface from the solid/vapour interface. This is the first example of a non-toxic, fouling resistant surface against a broad spectrum of fouling organisms ranging from plant cells and non-motile spores, to complex invertebrate larvae with highly selective sensory mechanisms. The only physical property differentiating the immersed surfaces is the nano-architectured roughness which supports longer standing air incursions providing a novel non-toxic broad spectrum mechanism for the prevention of biofouling.

Attachment point theory revisited: the fouling response to a microtextured matrix

This paper examines attachment point theory in detail by testing the fouling attachment of several fouling groups to a microtextured matrix. Static bioassays were conducted on polycarbonate plates with nine equal regions, comprising eight scales of microtexture (4–512 µm) and one untextured region. The microtextures examined were continuous sinusoidal ridges and troughs of defined height and width. Attachment over the microtextured plates was examined for the diatom Amphora sp., the green alga Ulva rigida, the red alga Centroceras clavulatum, the serpulid tube worm Hydroides elegans and the bryozoan Bugula neritina. It was found that the size of the microtexture in relation to the size of the settling propagules/larvae was important in the selection of attachment sites. Attachment was generally lower when the microtexture wavelength was slightly smaller than the width of the settling propagules/larvae and increased when the wavelength was wider than their width. The effect of attachment points was weak for small motile microfoulers (Amphora sp. and U. rigida) (7 µm), strong for large macrofouling larvae (H. elegans and B. neritina) (129–321 µm) and non-existent for the non-motile algal spores (C. clavulatum) (37 µm). This study reinforces the potential of using attachment points to develop surfaces with increased fouling resistance or, alternatively, surfaces which promote the attachment of selected target sizes of motile propagules or larvae.

Biocrude yield and productivity from the hydrothermal liquefaction of marine and freshwater green macroalgae

Six species of marine and freshwater green macroalgae were cultivated in outdoor tanks and subsequently converted to biocrude through hydrothermal liquefaction (HTL) in a batch reactor. The influence of the biochemical composition of biomass on biocrude yield and composition was assessed. The freshwater macroalgae Oedogonium afforded the highest biocrude yield of all six species at 26.2%, dry weight (dw). Derbesia (19.7%dw) produced the highest biocrude yield for the marine species followed by Ulva (18.7%dw). In contrast to significantly different yields across species, the biocrudes elemental profiles were remarkably similar with higher heating values of 33-34MJkg(-1). Biocrude productivity was highest for marine Derbesia (2.4gm(-2)d(-1)) and Ulva (2.1gm(-2)d(-1)), and for freshwater Oedogonium (1.3gm(-2)d(-1)). These species were therefore identified as suitable feedstocks for scale-up and further HTL studies based on biocrude productivity, as a function of biomass productivity and the yield of biomass conversion to biocrude.

Chemical aspects of mass spawning in corals. I. Sperm-attractant molecules in the eggs of the scleractinian coral Montipora digitata

This paper provides the first evidence for sperm chemotaxis in the Scleractinia. Montipora digitata Dana, 1845 (Scleractinia: Coelenterata) is a hermaphroditic coral which reproduces bi-annually, releasing egg-sperm bundles during the mass spawning at Magnetic Island (19°10′S; 146°52′E) in late spring-early summer, and autumn each year. The buoyant egg-sperm bundles float to the surface where they break apart, releasing eggs and sperm into the ocean. Fertilisation occurs after ∼30 min. Unfertilized eggs were collected, washed free of sperm, and freeze-dried. The eggs were extracted with dichloromethane, fractionated by chromatography on silica gel, and the fractions assayed for their ability to attract M. digitata sperm. The active fraction was further fractionated by high-performance liquid chromatography, resulting in the isolation of three highly unsaturated fatty alcohols: (1) dodeca-2,4-diynol; (2) tetradec-13-ene-2,4-diynol; (3) (14Z)-heptadeca-14,16-diene-2,4-diynol. Of these three compounds, only Compound 1 attracted sperm of M. digitata. Synthetic Compound 1, produced from simple precursors by known reactions, possessed sperm-attracting activity comparable to the naturally derived attractant. Preliminary experiments suggest that the natural mixture of Compounds 1, 2 and 3 in the ratio 1:4:9 is more effective in attracting sperm from M. digitata than sperm from other Montipora species. Sperm attractants may act to reduce the incidence of hybridisation between different species of Montipora.

Chemically mediated interactions between the red algaPlocamium hamatum (Rhodophyta) and the octocoralSinularia cruciata (Alcyonacea)

Interactions between the red algaPlocamium hamatum J. Agardh (Rhodophyta) and other benthic organisms including the alcyonacean soft coralSinularia cruciata (Tixier-Durivault) were investigated on an inshore fringing reef environment in whichP. hamatum was the dominant large fleshy alga. Field observations of sessile reef organisms including octocorals and sponges living in close proximity toP. hamatum revealed that varying degrees of tissue necrosis were suffered by the invertebrates when in physical contact with the alga. In order to establish whether the chemical constituents of the alga, especially chloromertensene, played a role in this necrosis, manipulative field experiments were carried out in the Pelorus Channel, Palm Island group (18°34′S; 146°29′E), North Queensland, Australia, in November and December 1988. The first experiment involved the relocation of healthy plants and soft corals into contact and non-contact situations on a mesh grid. In all cases of contact betweenP. hamatum andS. cruciata, the soft coral suffered tissue necrosis (n=6,p=0.0022). The second experiment had the same design, but involved the use of artificial “plants” both uncoated and coated with natural levels of chloromertensene, in contact withS. cruciata. In all cases of contact with coated treatments, necrosis was observed inS. cruciata (n=4,p=0.025). In cases where uncoated artificial fronds were placed in contact with soft corals,S. cruciata showed minor abrasion effects, but no appreciable necrosis. Coated treatments were not fouled by epiphytes during the experiment and were not consumed by predators. Uncoated treatments were rapidly reduced in size by predation and any remaining material was biofouled. These experiments thus demonstrated that the deleterious effects observed in soft corals in the field were caused by contact with the algaP. hamatum, that these effects were indeed chemically mediated by chloromertensene, and that physical contact without chemical intervention caused no such deleterious effects. This is the first experimental evidence which conclusively demonstrates allelopathy between an alga and other marine organisms and identifies the compound responsible for the observed allelopathic effects.

Biomimetic characterisation of key surface parameters for the development of fouling resistant materials.

Material science provides a direct route to developing a new generation of non-toxic, surface effect-based antifouling technologies with applications ranging from biomedical science to marine transport. The surface topography of materials directly affects fouling resistance and fouling removal, the two key mechanisms for antifouling technologies. However, the field is hindered by the lack of quantified surface characteristics to guide the development of new antifouling materials. Using a biomimetic approach, key surface parameters are defined and quantified and correlated with fouling resistance and fouling removal from the shells of marine molluscs. Laser scanning confocal microscopy was used to acquire images for quantitative surface characterisation using three-dimensional surface parameters, and field assays correlated these with fouling resistance and fouling release. Principle component analysis produced a major component (explaining 54% of total variation between shell surfaces) that correlated with fouling resistance. The five surface parameters positively correlated to increased fouling resistance were, in order of importance, low fractal dimension, high skewness of both the roughness and waviness profiles, higher values of isotropy and lower values of mean surface roughness. The second component (accounting for 20% of variation between shells) positively correlated to fouling release, for which higher values of mean waviness almost exclusively dictated this relationship. This study provides quantified surface parameters to guide the development of new materials with surface properties that confer fouling resistance and release.

Chemical defense in a marine alga: heritability and the potential for selection by herbivores

Herbivores have major impacts on many marine algae and are assumed to have been important forces driving the evolution of algal chemical defenses. However, an evolutionary change in chemical defenses in response to herbivory requires that there is both heritable variation for that trait and a response by herbivores to such variation. The subtidal red alga Delisea pulchra produces four main secondary metabolites (furanones) that vary in concentration at a variety of spatial and temporal scales. Here we determined the heritability of furanone concentrations and then, with a series of feeding experiments, determined how feeding by herbivores associated with D. pulchra varied as a function of nonpolar extract and furanone concentration. Total furanone concentration showed signif- icant broad-sense heritability (h 0.234), but broad-sense heritability of the four individual 2 C furanones varied in magnitude, ranging from 0.058 to 0.321. Compound 3, which is often the most abundant furanone, was the only one of the four furanones to show significant genetic variation. However, all four furanones were strongly genetically correlated, indi- cating that selection acting on any one of them will cause a corresponding change in the others. With the exception of the mesograzer gastropodPhasianotrochus eximius,herbivores generally consumed D. pulchra at lower rates compared to other common macroalgae. Macrograzers (sea urchins and large gastropods) were mostly deterred by nonpolar extract that contained levels of furanones that spanned the range of furanone variation in the field. The amphipod Ampithoe ngana was not deterred at the lowest extract concentration, and P. eximius was not deterred by any naturally occurring extract concentration. Compound 3 was deterrent to macrograzers at 3 and 5 mg/g dry mass, but was not deterrent at 1 mg/ g dry mass. Compound 3 was also not deterrent to A. ngana at any of those concentrations. For the most part D. pulchra in the field contains concentrations of furanones that are strongly deterrent to most herbivores. However, at some places and times, furanones do occur at lower concentrations that are not deterrent to herbivores. Given the heritable variation for furanones, when this overlap occurs there is the potential for selection by herbivores for higher concentrations of furanones in D. pulchra.