Using Virtual AChE Homology Screening to Identify Small Molecules With the Ability to Inhibit Marine Biofouling

The search for effective yet environmentally friendly strategies to prevent marine biofouling is hampered by the large taxonomic diversity amongst fouling organisms and a lack of well-defined conserved molecular targets. The acetylcholinesterase enzyme catalyses the breakdown of the neurotransmitter acetylcholine, and several natural antifouling allelochemicals have been reported to display acetylcholinesterase inhibitory activity. Our study is focussed on establishing if acetylcholinesterase can be used as a well-defined molecular target to accelerate discovery and development of novel antifoulants via sequential high-throughput in silico screening, in vitro enzymatic studies of identified compound libraries, and in vivo assessment of the most promising lead compounds. Using this approach, we identified potent cholinesterase inhibitors with inhibitory concentrations down to 3 μM from a 10,000 compound library. The most potent inhibitors were screened against five microfouling marine bacteria and marine microalgae and the macrofouling tunicate Ciona savignyi. No activity was seen against the microfoulers but a potent novel inhibitor of tunicate settlement and metamorphosis was discovered. Although only one of the identified active cholinesterase inhibitors displayed antifouling activity suggesting the link between cholinesterase inhibition and antifouling is limited to certain compound classes, the study highlights how in silico screening employed regularly for drug discovery can also facilitate discovery of antifouling leads.

Selective Metal Coordination in Antifouling Coatings

<p>Marine biofouling is the accumulation of biological material (e.g. microorganisms, soft- and hard-fouling organisms) on the surface of an object submerged in seawater, and it remains a worldwide problem for shipping industries. The fouling of ship hulls results in a reduction of speed and manoeuvrability due to frictional drag, as well as increased fuel consumption and accelerated corrosion, and the exorbitant expenses and losses of efficiency attributed to biofouling have prompted the development of antifouling coatings. Current antifouling paints use copper as a biocidal agent, but copper-based paints are increasingly being banned due to environmental concerns about the non-target effects of leached copper. This project aims to circumvent these concerns and tightening regulations via a revolutionary concept: the development of marine antifouling paints that incorporate Cu(II)-selective ligands to draw the biocidal ingredient (i.e. Cu(II)) from seawater. A multistage strategy emerged for the development of this technology. First, criteria were established for the project’s ideal ligand, and ligands were synthesised or selected based on these criteria. Second, the ligands were incorporated in coatings through covalent modification of the paint binder or additives. Third, methodology was developed and implemented to test each coating’s ability to coordinate and retain Cu(II), as well as its subsequent ability to prevent microfouling by marine bacteria. The suitability of two ligand classes was assessed: acylhydrazones and tetraaza macrocycles, specifically cyclen. Unlike the acylhydrazones, cyclen met the established criteria and was initially evaluated as a curing agent and/or surface-modifier in a two-pack epoxy system with resin Epikote™ 235. However, the Cu(II)-loading by these coatings was relatively low, being at most ~0.05% w/w, and the modification of silica, a common paint additive, with cyclen was explored as an alternative formulation route. The method for the functionalisation of silica with cyclen was optimised, and the maximum Cu(II)-loading achieved by the product was 2.60% w/w. The cyclen-functionalised silica was incorporated on the surface of an epoxy coating, and a bacterial adherence assay was developed to assess the cellular attachment of marine bacterium Vibrio harveyi to this coating, which was found to be undeterred. Yet, the development of the strategy and testing methodology by which the project’s goals may be achieved provides a solid foundation for future work.</p>

Selective Metal Coordination in Antifouling Coatings

<p>Marine biofouling is the accumulation of biological material (e.g. microorganisms, soft- and hard-fouling organisms) on the surface of an object submerged in seawater, and it remains a worldwide problem for shipping industries. The fouling of ship hulls results in a reduction of speed and manoeuvrability due to frictional drag, as well as increased fuel consumption and accelerated corrosion, and the exorbitant expenses and losses of efficiency attributed to biofouling have prompted the development of antifouling coatings. Current antifouling paints use copper as a biocidal agent, but copper-based paints are increasingly being banned due to environmental concerns about the non-target effects of leached copper. This project aims to circumvent these concerns and tightening regulations via a revolutionary concept: the development of marine antifouling paints that incorporate Cu(II)-selective ligands to draw the biocidal ingredient (i.e. Cu(II)) from seawater. A multistage strategy emerged for the development of this technology. First, criteria were established for the project’s ideal ligand, and ligands were synthesised or selected based on these criteria. Second, the ligands were incorporated in coatings through covalent modification of the paint binder or additives. Third, methodology was developed and implemented to test each coating’s ability to coordinate and retain Cu(II), as well as its subsequent ability to prevent microfouling by marine bacteria. The suitability of two ligand classes was assessed: acylhydrazones and tetraaza macrocycles, specifically cyclen. Unlike the acylhydrazones, cyclen met the established criteria and was initially evaluated as a curing agent and/or surface-modifier in a two-pack epoxy system with resin Epikote™ 235. However, the Cu(II)-loading by these coatings was relatively low, being at most ~0.05% w/w, and the modification of silica, a common paint additive, with cyclen was explored as an alternative formulation route. The method for the functionalisation of silica with cyclen was optimised, and the maximum Cu(II)-loading achieved by the product was 2.60% w/w. The cyclen-functionalised silica was incorporated on the surface of an epoxy coating, and a bacterial adherence assay was developed to assess the cellular attachment of marine bacterium Vibrio harveyi to this coating, which was found to be undeterred. Yet, the development of the strategy and testing methodology by which the project’s goals may be achieved provides a solid foundation for future work.</p>

FEEDING OF THE INVASIVE ROUND GOBY Neogobius melanostomus (Perciformes: Gobiidae) IN THE SOUTH-EASTERN BALTIC

The feeding of the Ponto-Caspian invasive round goby Neogobius melanostomus in the coastal zone of the Baltic Sea near the Curonian Spit, in the Vistula Lagoon and in the eastern part of the Gdansk Bay has been studied. The round goby in coastal waters prefers biotopes with various shelters - boulders, large pebbles, etc. In the diet of the round goby, there is a significant proportion of fouling organisms inhabiting these objects. If fouling organisms are absent for some reason, the goby switches to other types of prey: molluscs, free-living crustaceans, insect larvae and other groups of benthic and nektobenthic organisms. There is a connection between the food spectrum and the size of the round goby: large individuals consume larger prey. The round goby implements a food strategy, which consists in the fact that all organisms that are available territorially and in size are used for food. Such food plasticity is one of the reasons for the wide expansion of this species outside the native range.

Anti-fouling activity and toxicity of the Mediterranean alien sponge Paraleucilla magna Klautau, Monteiro & Borojevic, 2004 (Porifera, Calcarea)

Poriferans, as sessile organisms without rigid external covering, use secondary metabolites for protection from predators and fouling organisms. The present study tested the antifouling activity of ethanolic extract of the Mediterranean alien calcareous sponge Paraleucilla magna towards juvenile mussels Mytilus galloprovincialis. Furthermore, toxicity tests on nauplii of brine shrimp Artemia salina and two microalgae strains, Nannochloropsis sp. and Tetraselmis suecica, were also conducted. A total attachment inhibition of M. galloprovincialis was achieved at a concentration of 400 µg/mL of sponge extract. The 50% mortality of A. salina nauplii was recorded at a concentration of 500 µg/mL of ethanolic extract. The growth inhibitory effect on both marine microalgae strains has been registered at a concentration of 300 µg/mL. Our results suggest promising natural antifouling activity and low toxicity of the ethanolic extract of P. magna that could be used as antifouling compound.

The Antifouling and Drag-Reduction Performance of Alumina Reinforced Polydimethylsiloxane Coatings Containing Phenylmethylsilicone Oil

Fouling-release coatings reinforced with micro-alumina and nano-alumina were prepared based on polydimethylsiloxane (PDMS) containing phenylmethylsilicone oil. The surface properties, mechanical properties, leaching behavior of silicone oil, anti-fouling and drag-reduction performance of the coating were studied. The results show that the addition of alumina can significantly improve the tensile strength, elastic modulus and Shore’s hardness of the coating. The adhesion experiments of marine bacteria and Navicula Tenera show that the addition of alumina can reduce the antifouling performance of the coating, which is related to the stripping mode of fouling organisms. The fouling organisms leave the coating surface by shearing, and the energy required for shearing is proportional to the elastic modulus of the coating. At 800–1400 rpm, the addition of alumina will reduce the drag reduction performance of the coating, which is related to the drag reduction mechanism of PDMS. PDMS counteracts part of the resistance by surface deformation. The larger the elastic modulus is, the more difficult the surface deformation is. The experiment of silicone oil leaching shows that the increase of alumina addition amount and the decrease of particle size will inhibit the leaching of silicone oil.

Biocides in Antifouling Paint Formulations Currently Registered For Use

Antifouling paints incorporate biocides in their composition seeking to avoid or minimize the settlement and growing of undesirable fouling organisms. Therefore, biocides are released into the aquatic environments also affecting several non-target organisms and, thus, compromising ecosystems. Despite global efforts to investigate the environment occurrence and toxicity of biocides currently used in antifouling paints, the specific active ingredients that have been used in commercial products are poorly known. Thus, the present study assessed the frequencies of occurrence and relative concentrations of biocides in antifouling paint formulations registered for marketing worldwide. The main data were obtained from databases of governmental agencies, business associations and safety data sheets from paint manufacturers around the world. Results pointed out for 25 active ingredients currently used as biocides, where up to six biocides have been simultaneously used in the examined formulations. Cuprous oxide, copper pyrithione, zinc pyrithione, zineb, DCOIT and cuprous thiocyanate were the most frequently ones, with mean relative concentrations of 35.9±12.8 %, 2.9±1.6 %, 4.0±5.3 %, 5.4±2.0 %, 1.9±1.9 % and 18.1±8.0 % (w/w) of respective biocide present in the antifouling paint formulations. Surprisingly, antifouling paints containing TBT as active ingredient are still being registered for commercialization nowadays. These results can be applied as a proxy of biocides that are possibly being used by antifouling systems and, consequently, released into the aquatic environment, which can help to prioritize the active ingredients that should be addressed in future studies.

Composite Slow-Release Fouling Release Coating Inspired by Synergistic Anti-Fouling Effect of Scaly Fish

Inspired by the antifouling properties of scaly fish, the conventional silicone coating with phenylmethylsilicone oil (PSO/PDMS) composite coating was fabricated and modified with single layer polystyrene (PS) microsphere (PSO/PDMS-PS) arrays. The fish scale like micro-nano structures were fabricated on the surface of bio-inspired coating, which can reduce the contact area with the secreted protein membrane of fouling organisms effectively and prevent further adhesion between fouling organisms and bio-inspired coating. Meanwhile, PSO exuded to the coating surface has the similar function with mucus secreted by fish epidermis, which make the coating surface slithery and will be polished with the fouling organisms in turbulent waters. Compared to PSO/PDMS coating without any structure and conventional silicone coating, PSO/PDMS-PS showed better antiadhesion activity against both marine bacteria and benthic diatom (Navicula sp.). Additionally, the existence of PS microspheres can reduce the release rate of PSO greatly, which will extend the service life of coating. Compared to PSO/PDMS coating, the sustained release efficiency of PSO/PDMS-PS coating can reach 23.2%. This facile method for fabricating the bio-inspired composite slow-release antifouling coating shows a widely fabricating path for the development of synergistic anti-fouling coating.

Filtration and fertilisation effects of the bivalves Mytilus edulis and Magallana gigas on the kelp Saccharina latissima in tank culture

Biofouling by opportunistic epiphytes is a major concern in seaweed aquaculture. Colonisation of fouling organisms contributes to a reduction in algal performance as well as a lower quality crop. Further, epiphyte removal techniques often increase maintenance costs of cultivation systems. There have been a variety of methods to mitigate fouling in tank cultivations of seaweed, including the use of biological controls. Here, we present the use of filter feeding bivalves, the blue mussel (Mytilus edulis) and Pacific oyster (Magallana gigas), as a novel biofilter that also serves as a source of dissolved inorganic nitrogen in tank cultivations of the sugar kelp, Saccharina latissima. We observed significant reductions of fouling epiphytes on seaweed blades of around 50% by bivalve filtration, significant elevations of ammonium (NH4+) and phosphate (PO43−) by bivalves and alterations to kelp tissue quality when co-cultivated with bivalves rather than supplied with ambient seawater. Stable isotope ratios and seawater chlorophyll a concentrations provided evidence for bivalve biofiltration and the incorporation of their by-products into kelp tissue.

The Negative Relationship between Fouling Organisms and the Content of Eicosapentaenoic Acid and Docosahexaenoic Acid in Cultivated Pacific Oysters, Crassostrea gigas

Bivalves serve as an important aquaculture product, as they are the source of essential fatty acids, such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in our diet. However, their cultivation in the wild can be affected by fouling organisms that, in turn, affect their EPA and DHA content. The effects of fouling organisms on the EPA and DHA contents of cultivated bivalves have not been well documented. We examined the effects of fouling organisms on the EPA and DHA contents and condition index of cultured oysters, Crassostrea gigas, in an aquaculture system. We sampled two-year-old oysters from five sites in Shizugawa Bay, Japan, in August 2014. Most of the fouling organisms were sponges, macroalgae, and Mytilus galloprovincialis. A significant negative relationship existed between the DHA content in C. gigas and the presence of sponges and macroalgae. A lower C. gigas EPA content corresponded to a higher M. galloprovincialis fouling mass and a lower C. gigas condition index. This can be explained by dietary competition between C. gigas and M. galloprovincialis for diatoms, which were the main producer of EPA in our study sites. Our findings indicate that fouling organisms likely reduce the EPA and DHA content in cultivated oysters. Therefore, our results suggest that the current efforts to remove fouling organisms from oyster clusters is an effective strategy to enhance the content of EPA and DHA in oysters.