Enhancing the settlement and attachment strength of pediveligers of Mytilus galloprovincialis bychanging surface wettability and microtopography

Surface wettability and microtopography can either enhance or deter larval settlement of many sessile marine organisms. This study quantifies the effect of these surface properties on the settlement of pediveligers of Mytilus galloprovincialis, using polymers spanning a range of wettability and microtextured polydimethylsiloxane (PDMS). Furthermore, the adhesion strength of settled pediveligers on microtextured PDMS surfaces was quantified using a flow chamber. Settlement was enhanced at the hydrophilic end of the wettability spectrum, where mean settlement on nylon reached 33.5 ± 13.1%. In contrast, mean settlement on the most hydrophobic polymer (PDMS) was 4.2 ± 3.2%. Microtopography had a much stronger effect compared to wettability, where 400 μm textured PDMS enhanced settlement above 90%. Settlement preferences were also positively correlated to adhesion strength at flow rates of 4 knots, with all initially settled pediveligers on smooth PDMS detaching, while 79.9 ± 5.7% of pediveligers remained on the 400 μm texture.     

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.     

Short-term testing of antifouling surfaces: the importance of colour

Abstract

Data from short-term biofouling assays are frequently used to evaluate the performance of antifouling (AF) coatings. There are a large number of factors, however, that may influence community development. One such factor is colour. The hypothesis was that differences in colour may impact the short-term development of a biofouling community and therefore bias the results. An experiment was designed to investigate the effect of black and white substrata on settlement of fouling organisms in the field. Both Ulva sp. and Spirorbis sp. had significantly higher settlement on black surfaces. This result emphasises the importance of considering colour and other factors when undertaking short-term testing of AF coatings.     

The Influence of Natural Surface Microtopographies on Fouling

Multiple antifouling strategies of marine organisms may consist of combinations of physical, chemical and mechanical mechanisms. In this study, the role of surface microtopography (?<?500?μm) of different marine organisms, such as Cancer pagurus, Mytilus edulis, Ophiura texturata and the eggcase of Scyliorhinus canicula, has been investigated as a possible component of their defence systems. High resolution resin replicates of these natural surface structures were exposed to natural fouling in field experiments. Abundances of recruits were determined and compared to those on untextured, but otherwise identical, control surfaces to quantify the influence of the different microtopographies on fouling rates. Antifouling effects of microtopographies varied with type of microtopography and coloniser species. The surface microtopography of C. pagurus significantly rejected macrofoulers. The surface structures of the eggcase and O. texturata had repellent effects on microfoulers. Barnacle settlement was temporarily reduced on surface microtopographies of M. edulis and the eggcase. These results emphasise the promising non-toxic antifouling properties of microtextured surfaces.     

Reduction of barnacle recruitment on micro‐textured surfaces: Analysis of effective topographic characteristics and evaluation of skin friction

This study investigates five designed micro‐textured surfaces and their effects on barnacle fouling and hydrodynamic drag. Three of the micro‐textures were developed in the present study and evaluated together with two commercial riblet films. All micro‐structures were arranged as longitudinal grooves with different profile depths, widths and angles of inclination. In field tests the recruitment of the barnacle Balanus improvisus on micro‐textured surfaces and smooth controls was evaluated. All micro‐textured surfaces reduced recruitment, and the most efficient texture reduced recruitment by 98%. For some micro‐textures the reduction of recruitment declined as settlement intensity increased. In a correlative analysis, the trigonometric inclination of the micro‐structures explained most of the recruitment reduction. The steepest angle of inclination caused a massive reduction in barnacle settlement. Surface micro‐structures may affect the boundary‐layer flow and the hydrodynamic drag (skin friction) of the surface. The skin friction was empirically measured in a flow channel using a sub‐set of the tested micro‐textures. The measurements of skin friction showed that the orientation of the microstructures is important, with a minimum friction when the grooves are parallel to the flow. For one of the micro‐textures the skin friction was ca 10% lower compared to a hydraulically smooth surface. It is concluded that, depending on the flow speed, micro‐textures will not significantly increase skin friction when arranged parallel to the flow, even at moderate protrusion through the viscous sub‐layer.     

The effect of vibration frequency and amplitude on biofouling deterrence

The use of vibration is proposed as a means of controlling the settlement of marine fouling organisms. In this study, panels with embedded lead zirconate titanate, known as PZT, were placed in the field over 3?months. The panels were vibrated at different velocity levels at frequencies between 70 and 445?Hz. It was found that barnacles (Amphibalanus variegatus Darwin and Elminius sp.) were the only fouling organisms affected by the applied vibration, and these organisms settled in significantly lower numbers when the plates were excited at specific frequencies and amplitudes. Panels vibrating at relatively higher frequencies, greater than 260?Hz, exhibited reduced barnacle settlement, whilst lower frequencies in the 70–100?Hz range had little or no effect. The settlement of other fouling organisms such as tubeworms, bryozoans, ascidians and algae did not appear to be affected by the applied excitation. The experimental results showed that increasing the velocity amplitude of vibration was a contributing factor in inhibiting barnacle settlement.     

Disruption-free imaging by Raman spectroscopy reveals a chemical sphere with antifouling metabolites around macroalgae

Investigations of the surface chemistry of marine organisms are essential to understand their chemically mediated interactions with fouling organisms. In this context, the concentration of natural products in the immediate vicinity of algal surfaces, as well as their biological activity, are of particular importance. However, due to lack of appropriate methods, the distribution of compounds within the chemical sphere around marine algae is unknown. This study demonstrates the suitability of confocal resonance Raman microspectroscopy for the determination of metabolites around algal surfaces with a micrometer resolution. The spatial distribution of carotenoids in the diffusion boundary layer of the brown alga Fucus vesiculosus and the green alga Ulva sp. was determined using the disruption-free optical method. A gradient of carotenoids was determined within 0 to 150 μm from the surface of thealgae, thereby demonstrating the release of the non-polar metabolites involved in antifouling processes. Thedifferences in the carotenoid composition of the brown and green algae were reflected in the spectra. Resonance Raman microspectroscopy also allowed visualization of the lateral distribution of fucoxanthin on the algal surface and localization of concentration maxima within a 50 × 50 μm² area. The results from this work show clearly that established dipping techniques suitable for the extraction of the diffusion boundary layer of macroalgae only provide an average of the local strongly variable concentrations of metabolites on algal surfaces.     

Settlement Behaviour of Marine Invertebrate Larvae Measured by EthoVision 3.0

Submerged marine surfaces are rapidly colonized by fouling organisms. Current research is aimed at finding new, non-toxic, or at least environmentally benign, solutions to this problem. Barnacles are a major target organism for such control as they constitute a key component of the hard fouling community. A range of standard settlement assays is available for screening test compounds against barnacle cypris larvae, but they generally provide little information on mechanism(s) of action. Towards this end, a quick and reliable video-tracking protocol has been developed to study the behaviour of the cypris larvae of the barnacle, Balanus amphitrite, at settlement. EthoVision 3.0 was used to track individual cyprids in 30-mm Petri dishes. Experiments were run to determine the optimal conditions vis-à-vis acclimation time, tracking duration, number of replicates, temperature and lighting. A protocol was arrived at involving a two Petri dish system with backlighting, and tracking over a 5-min period after first acclimating the cyprids to test conditions for 2 min. A minimum of twenty replicates was required to account for individual variability in cyprid behaviour from the same batch of larvae. This methodology should be widely applicable to both fundamental and applied studies of larval settlement and with further refinements, to that of smaller fouling organisms such as microalgae and bacteria.     

The effects of potential larval supply, settlement and post-settlement processes on the distribution of two species of filter-feeding caddisflies

1. Hydropsychid caddisfly larvae are often abundant in fast flow habitats and on highly textured substrata. Such patterns are usually inferred to result from habitat preference, but the roles of larval supply to the benthos and post‐settlement events are rarely examined for stream populations. We describe a manipulative field experiment that examined larval supply, habitat preference and post‐settlement events simultaneously for two co‐occurring species of hydropsychids. 2. Ten artificial channels were constructed in a natural riffle to create fast and slow flow habitats and within each channel we placed six artificial substrata whose surfaces had been modified to create three different texture treatments. We measured discharge through channels and monitored hydropsychid colonisation of substrata every 3–4 days. Half of the substrata had all hydropsychids and nets removed every 3–4 days (short‐term colonisation) whereas the remaining half were counted but nets and hydropsychids left attached (long‐term colonisation). Short‐term counts of nets and individuals were summed to allow comparison with long‐term colonisation substrata. 3. Smicrophylax sp. AV2 larvae were more abundant in fast flow channels than slow flow channels, but these differences were proportional to discharge through each of the channels, suggesting that supply of settlers can explain this pattern. Smicrophylax larvae were least abundant on smooth substrata, which suggests that this species selects habitats based on surface texture. Alternatively, Asmicridea sp. AV1 larvae were only found in fast flow channels and this most likely reflects an active habitat choice by this species, but there was no significant difference between different texture treatments. 4. There were no differences between summed, short‐ and long‐term counts of recruits for either species, but there were more nets than larvae, especially in slow flow channels, by the end of the experiment, suggesting that larval mortality or re‐dispersal after settlement could be considerable. 5. Our results indicate that supply, habitat selection at settlement and post‐settlement processes all contributed variously to the distribution of hydropsychid caddisfly larvae, but that each species was affected differentially by these factors. Larval supply and post‐settlement processes are rarely examined by stream researchers and our results demonstrate these factors deserve much more consideration. Calculating accurate larval supply rates to sites is challenging, but we suggest that such detailed information is necessary if we are to sort out what sets limits to distributions and the underlying population structure of stream invertebrate populations.     

Antifouling chromanols isolated from brown alga Sargassum horneri

Biofouling in aquatic environments have a wide range of detrimental effects on man-made structures and cause economic loss. Current antifouling compounds including Diuron, dichlorofluanid, and Irgarol are toxic and can accumulate in marine environments. Thus, effective and environmentally friendly antifoulants are needed. Six structurally similar compounds were isolated from the brown alga, Sargassum horneri, based on bioactivity-guided isolation by reversed-phased liquid flash chromatography and high-performance liquid chromatography. Six chemical constituents possessing antifouling activities were identified as chromanols consisting of polyprenyl chain by nuclear magnetic resonance and mass spectroscopy. Antifouling activities of these six compounds were determined against representative fouling organisms including a hard fouling organism the mussel Mytilus edulis, a soft fouling macroalga Ulva pertusa, the biofouling diatom Navicula annexa, and the biofouling bacteria Pseudomonas aeruginosa KNP-3 and Alteromonas sp. KNS-8. The compounds could inhibit larvae settlement of mussel M. edulis with an EC₅₀ of 0.11–3.34 μg mL^?1, spore settlement of U. pertusa zoospores (EC₅₀ of 0.01–0.43 μg mL^?1), and the diatom N. annexa (EC₅₀ of 0.008–0.19 μg mL^?1). The two biofouling bacteria were sensitive to the tested compounds (minimum inhibitory concentration of 1.68–36.8 and 1.02–30.4 μg mL^?1, respectively). From toxicity tests on juvenile Sebastes schlegelii fish, brine shrimp Artemia salina, and microalga Tetraselmis suecica, S3 had the lowest LC₅₀ values of 60.2, 108, and 6.7 μg mL^?1 and exhibited no observed effect concentration at 24.5, 41.6, and 3.1 μg mL^?1 for these three tested marine organisms, respectively.     

Mini review: Hydrodynamics of larval settlement into fouling communities

Abstract

Flowing water delivers planktonic larvae to surfaces, but also dislodges them. This paper reviews experiments in the field and in laboratory flumes, as well as mathematical models, which have revealed how the interaction of ambient water motion with a developing fouling community affects larval settlement. Although mean current velocities across fouling communities in harbours are low, instantaneous velocities can be much higher due to turbulence and to the velocity oscillations of wind chop and ship wakes. As a fouling community develops, its topography becomes more complex and the range of flow microhabitats on the spatial scale of larvae increases. In spite of the prevalence of waves in shallow coastal habitats, and in spite of the importance to settlement of the fine-scale instantaneous velocities encountered by larvae, most studies of flow effects on larval settlement have focused on unidirectional currents and on temporally- and spatially-averaged aspects of the flow.     

Antifouling activities expressed by marine surface associated Pseudoalteromonas species

Members of the marine bacterial genus Pseudoalteromonas have been found in association with living surfaces and are suggested to produce bioactive compounds against settlement of algal spores, invertebrate larvae, bacteria and fungi. To determine the extent by which these antifouling activities and the production of bioactive compounds are distributed amongst the members of the genus Pseudoalteromonas, 10 different Pseudoalteromonas species mostly derived from different host organisms were tested in a broad range of biofouling bioassays. These assays included the settlement of larvae of two ubiquitous invertebrates Hydroides elegans and Balanus amphitrite as well as the settlement of spores of the common fouling algae Ulva lactuca and Polysiphonia sp. The growth of bacteria and fungi, which are the initial fouling organisms on marine surfaces, was also assayed in the presence of each of the 10 Pseudoalteromonas species. It was found that most members of this genus produced a variety of bioactive compounds. The broadest range of inhibitory activities was expressed by Pseudoalteromonas tunicata which inhibited all target fouling organisms. Only two species, Pseudoalteromonas haloplanktis and Pseudoalteromonas nigrifaciens, displayed negligible activity in the bioassays. These were also the only two non-pigmented species tested in this study which indicates a correlation between production of bioactive compounds and expression of pigment. Three members, P. tunicata, Pseudoalteromonas citrea and Pseudoalteromonas rubra, were demonstrated to express autoinhibitory activity. It is suggested that most Pseudoalteromonas species are efficient producers of antifouling agents and that the production of inhibitory compounds by surface associated Pseudoalteromonas species may aid the host against colonisation of its surface.     

Charged hydrophilic polymer brushes and their relevance for understanding marine biofouling

The resistance of charged polymers to biofouling was investigated by subjecting cationic (PDMAEMA), anionic (PSPMA), neutral (PHEMA-co-PEG₁₀MA), and zwitterionic (PSBMA) brushes to assays testing protein adsorption; attachment of the marine bacterium Cobetia marina; settlement and adhesion strength of zoospores of the green alga Ulva linza; settlement of barnacle (Balanus amphitrite and B. improvisus) cypris larvae; and field immersion tests. Several results go beyond the expected dependence on direct electrostatic attraction; PSPMA showed good resistance towards attachment of C. marina, low settlement and adhesion of U. linza zoospores, and significantly lower biofouling than on PHEMA-co-PEG₁₀MA or PSBMA after a field test for one week. PDMAEMA showed potential as a contact-active anti-algal coating due to its capacity to damage attached spores. However, after field testing for eight weeks, there were no significant differences in biofouling coverage among the surfaces. While charged polymers are unsuitable as antifouling coatings in the natural environment, they provide valuable insights into fouling processes, and are relevant for studies due to charging of nominally neutral surfaces.     

Surface microtopographies of tropical sea stars: lack of an efficient physical defence mechanism against fouling

Abstract

The role of surface topography as a defence against fouling in tropical sea stars was investigated. The sea stars Linckia laevigata, Fromia indica, Cryptasterina pentagona and Archaster typicus are not fouled and have paxillae (modified ossicles with a median vertical pillar) on their aboral surfaces, which varied in diameter, height and distance depending on species and position on the aboral surface, providing unique and complex surface microtopographies for each species. The surfaces of the sea stars L. laevigata, F. indica and A. typicus were moderately wettable, with their mean seawater contact angles, calculated from captive bubble measurements, being 60.1°, 70.3° and 57.3°, respectively. The seawater contact angle of C. pentagona could not be measured. To evaluate the effectiveness of the surface microtopographies in deterring the settlement of fouling organisms, field experiments with resin replicas of the four sea star species were conducted at three sites around Townsville, Australia, for 8 weeks during the dry and wet seasons. The fouling community and total fouling cover did not differ significantly between replicas of L. laevigata, F. indica, C. pentagona, A. typicus and control surfaces at any site during the dry season. Significant differences between fouling communities on the replicas of the sea stars and control surfaces were detected at two sites during the wet season. However, these differences were transitory, and the total fouling cover did not differ significantly between replicas of sea stars and control surfaces at two of the three sites. In contrast to recent literature on the effects of biofouling control by natural surfaces in the marine environment, the surface microtopographies of tropical sea stars alone were not effective in deterring the settlement and growth of fouling organisms.     

Effects of adsorbed organic and primary fouling films on bryozoan settlement

Marine primary fouling films, which consist of molecular organic and microbial components, have been reported to facilitate colonization of immersed surfaces by marine fouling organisms. Larvae of the cosmopolitan fouling bryozoan Bugula neritina (Linnaeus) were offered various substrata for attachment and metamorphosis. The materials were offered (a) after detergent washing, (b) after sorption of dissolved organic molecular films, and (c) after formation of primary films consisting of both microbial and adsorbed organic material. Wettability of the substrata by sea water was determined by contact angle measurements for each substratum. On washed substrata, attachment was favored with contact angles greater than ≈45° (cos contact angle <0.7). Adsorbed surface films had no effect on the low settlement of larvae on glass and high settlement on plastics. Microbial primary films, however, made glass attractive and plastics unattractive. These settlement preference changes did not correlate with the changes in wettability observed on these substrata. Dispersion of larvae over the settlement surface was random except on wettable surfaces coated with bacterial films, where settlement was strongly clustered (contagious).     

Fouling-resistant surfaces of tropical sea stars

Abstract

Qualitative evidence suggests sea stars are free of fouling organisms; however the presence of fouling-resistant surfaces of sea stars has not previously been documented. Field surveys were conducted in northern Queensland, Australia, during the wet and dry seasons and several tropical sea star species were examined for surface-associated micro- and macro-organisms. Mean bacterial abundances on seven sea star species were approximately 10⁴ to 10⁵ cells cm^?2 during both seasons. There were no consistent trends in bacterial abundances with season, species and aboral positions on sea star arms. No common generalist fouling organisms, such as algae, barnacles, serpulid polychaetes, bryozoans and ascidians, were found on any specimens of 12 sea star species. However, low numbers of parasitic and commensal macro-organisms were found on six sea star species. The gastropods Parvioris fulvescens, Asterolamia hians, Thyca (Granulithyca) nardoafrianti and Thyca crystallina were found exclusively on the sea stars Archaster typicus, Astropecten indicus, Nardoa pauciforis and Linckia laevigata, respectively. The shrimp Periclimenes soror was only found on Acanthaster planci, and the polychaete Ophiodromus sp. on A. typicus. The copepods Stellicola illgi and Paramolgus sp. were only found on L. laevigata and Echinaster luzonicus, respectively. As no common generalist fouling organisms were discovered, sea stars offer an excellent model to investigate the mechanisms driving fouling-resistant surfaces and the selective settlement of specialist invertebrates.     

Larval Settlement: The Role of Surface Topography for Sessile Coral Reef Invertebrates

For sessile marine invertebrates with complex life cycles, habitat choice is directed by the larval phase. Defining which habitat-linked cues are implicated in sessile invertebrate larval settlement has largely concentrated on chemical cues which are thought to signal optimal habitat. There has been less effort establishing physical settlement cues, including the role of surface microtopography. This laboratory based study tested whether surface microtopography alone (without chemical cues) plays an important contributing role in the settlement of larvae of coral reef sessile invertebrates. We measured settlement to tiles, engineered with surface microtopography (holes) that closely matched the sizes (width) of larvae of a range of corals and sponges, in addition to surfaces with holes that were markedly larger than larvae. Larvae from two species of scleractinian corals (Acropora millepora and Ctenactis crassa) and three species of coral reef sponges (Luffariella variabilis, Carteriospongia foliascens and Ircinia sp.,) were used in experiments. L. variabilis, A. millepora and C. crassa showed markedly higher settlement to surface microtopography that closely matched their larval width. C. foliascens and Ircinia sp., showed no specificity to surface microtopography, settling just as often to microtopography as to flat surfaces. The findings of this study question the sole reliance on chemical based larval settlement cues, previously established for some coral and sponge species, and demonstrate that specific physical cues (surface complexity) can also play an important role in larval settlement of coral reef sessile invertebrates.