Mini-review: Molecular mechanisms of antifouling compounds

Abstract

Various antifouling (AF) coatings have been developed to protect submerged surfaces by deterring the settlement of the colonizing stages of fouling organisms. A review of the literature shows that effective AF compounds with specific targets are ones often considered non-toxic. Such compounds act variously on ion channels, quorum sensing systems, neurotransmitters, production/release of adhesive, and specific enzymes that regulate energy production or primary metabolism. In contrast, AF compounds with general targets may or may not act through toxic mechanisms. These compounds affect a variety of biological activities including algal photosynthesis, energy production, stress responses, genotoxic damage, immunosuppressed protein expression, oxidation, neurotransmission, surface chemistry, the formation of biofilms, and adhesive production/release. Among all the targets, adhesive production/release is the most common, possibly due to a more extensive research effort in this area. Overall, the specific molecular targets and the molecular mechanisms of most AF compounds have not been identified. Thus, the information available is insufficient to draw firm conclusions about the types of molecular targets to be used as sensitive biomarkers for future design and screening of compounds with AF potential. In this review, the relevant advantages and disadvantages of the molecular tools available for studying the molecular targets of AF compounds are highlighted briefly and the molecular mechanisms of the AF compounds, which are largely a source of speculation in the literature, are discussed.

Keywords:

• antifouling compounds
• mode of action
• molecular mechanisms
• molecular methodology
• molecular targets

Introduction

In the marine environment, the undesirable colonization of substrata by fouling organisms, with temporary or permanent adhesion to surfaces, is termed biofouling (Callow & Callow 2002). Biofouling is a serious global problem in marine systems, causing extensive material and economic costs worldwide (Yebra et al. 2004; Schultz et al. 2011). Biofouling not only creates technical and economic challenges for marine operations, it also generates environmental problems such as the spread of invasive species (Davidson et al. 2009).

Various antifouling (AF) technologies have been developed over a number of decades (Finnie & Williams 2010), including UV irradiation, ultrasound, electric fields, foul-release polymeric coatings, and AF paints such as self-polishing systems (Loschau & Kratke 2005; Finnie & Williams 2010; Guo et al. 2011, 2012; Martinelli et al. 2011). The most common practice to protect marine surfaces has been to coat them with AF paint-containing biocides. In the past, the most widely used and the best-performing AF compounds were based mainly on heavy metals such as copper, lead, mercury, arsenic, and cadmium (Omae 2003). Organotin was first introduced in the mid-1960s as a relatively environmentally friendly AF compound that was highly effective at deterring settlement. However, the high efficacy of organotin was due primarily to its acute toxicity to fouling organisms (Voulvoulis 2006).

In response to the restricted use/ban of tributyltin (TBT), marine coating industries have been actively developing alternative AF biocides. The most commonly used of the current AF coatings are based on copper (Cu) supplemented by booster biocides to control Cu-resistant fouling organisms (Voulvoulis 2006). The most commonly used booster biocides/co-biocides include Irgarol 1051 (2-methylthio-4-tertiary-butylamino-6-cyclopropylamino-s-triazine), diuron (1-(3,4-dichlorophenyl)-3,3-dimethylurea), zinc pyrithione (2-mercaptopyridine N-oxide zinc salt; ZnPT), copper pyrithione (2-mercaptopyridine N-oxide copper salt; CuPT), chlorothalonil (2,4,5,6-tetrachloro isophthalonitrile), and SeaNine 211 (4,5-dichloro-2-n-octyl-4-isothiazolin-3-one; DCOIT) (Konstantinou & Albanis 2004; Zhou et al. 2006). However, many of the booster biocides are also a threat to the marine environment (van Wezel & van Wlaardingen 2004); some can accumulate to high levels, despite claims for rapid degradation, and have a biocidal effect on non-target marine organisms (Konstantinou & Albanis 2004; Bellas 2006, 2007, 2008; Thomas & Brooks 2010). Some are reported to be more toxic than TBT. For instance, Bellas (2007) determined that Sea-Nine 211 (DCOIT) had a deleterious effect on the embryo-larval stage of the sea urchin, Paracentrotus lividus, despite its classification as a safe biocide (Braithwaite & Fletcher 2005). Booster biocides such as diuron and Irgarol 1051, which have been prevalent previously, have been banned by many EU countries in recent years (Cresswell et al. 2006) due to increasing evidence of environmental toxicity (Munoz et al. 2010).

The effects of TBT on the environment have also led to an increase in the regulations regarding the use of other AF biocides. The EU now requires a clear understanding of the mode of action of any biocidal product seeking approval for marketing purposes. This not only demands a thorough understanding of environmental risk, ecotoxicology, and the environmental fate of new biocides, but also calls for detailed studies on the molecular mechanisms/targets of AF compounds. This was not the case in the past when numerous AF compounds were first introduced into the marine environment. For instance, although there is substantial environmental data on some biocides, eg Irgarol 1051 and diuron, that facilitates the assessment of environmental safety and potential risk, there is little information on how these compounds act on target fouling organisms or on the molecular mechanisms. The same scenario applies to other compounds such as dichlofluanid (N′-Dimethyl-N-phenylsulfamide), SeaNine 211 (DCOIT), and zinc/copper pyrithione (ZnPT/CuPT) (Thomas & Brooks 2010 and references therein). As such, the long-term environmental impact and behavior of these biocides in marine ecosystems remain unknown. It is pertinent to ask whether the mistakes made with TBT-based coatings are being repeated by introducing so-called ‘environmentally friendly’ AF compounds into marine environments.

In short, the elucidation of molecular mechanisms has become an essential step in the development of ‘non-toxic’ antifoulants. A clear understanding of the mode of action of AF compounds will provide important insights into the identification of sensitive molecular targets or the signaling pathways responsible for the settlement of cells and larvae. Such information will assist in the establishment of targeted bioassays using sensitive biomarkers for the rapid screening of AF compounds. Research on molecular mechanisms will also provide essential information for the future discovery, synthesis, or optimization of potent antifoulants identifying moieties effective at targeting settlement. In this review, a brief overview of the relevant advantages and disadvantages of the tools commonly used to study the molecular targets of AF compounds is provided. The molecular mechanisms that are largely speculative in the literature are then highlighted.

Common methods for studying molecular targets

To identify molecular targets (genes, proteins, or pathways), the following methodologies are available.

Histology

Since the attachment of fouling organisms to favorable substrata relies on the exocytosis of adhesive proteins, efficacious antifoulants can inhibit the release of these adhesive proteins (Zhang, Wang et al. 2011). Light microscopy and transmission electron microscopy can be used to observe directly the detailed morphological changes in, for example, the cement gland of barnacle cyprids while confocal microscopy can be used to study the in situ secretory process of the cement gland of live cyprids (Ödling et al. 2006). Differential interference contrast optics support the in situ imaging of granule movement. If the AF compounds can be labeled with fluorescent probes, then the interactions between the compounds and fouling organisms can be visualized by fluorescence microscopy (Kitano et al. 2005). However, these techniques do not offer direct evidence regarding the genes, proteins or which pathways are being targeted by the compounds.

Behavioral responses

The repellent function of non-toxic antifoulants can be defined as a negative motor response, taxis or kinesis movement in response to stimuli that drive organisms away from the source of the stimulus (Railkin 2004) and thus, can be determined using locomotor activity, phototaxis, or settlement bioassays (Wu et al. 1997a, 1997b; Amsler et al. 2006; Faimali et al. 2006; Zhang, Wang et al. 2011). However, behavioral bioassays offer very little information about the molecular mechanisms of AF compounds since it is almost impossible to link the changes in behavior with changes in the genes, proteins, or pathways in the target organisms. Furthermore, the physiological conditions and the specific experimental setup are often substantive sources of variation that make experimental results difficult to interpret.

Gene expression

The level of expression of certain genes with known biological functions and sequences can be determined semi-quantitively by the quantitative real-time polymerase chain reaction (qRT-PCR) (Xu et al. 2009; Wang & Qian 2010) in response to AF compounds. For instance, we challenged barnacle cyprids with chemical cues and then extracted the RNA from both the treated and untreated larvae for the cDNA synthesis of target genes. The synthesized cDNA were then subjected to qRT-PCR reactions to determine the relative expression levels of particular genes (Xu et al. 2009; Wang & Qian 2010). This method is relatively simple and has often been used to confirm the results obtained by the methods discussed below. However, it is difficult to select target genes as information on the function of these genes in fouling organisms is limited. Furthermore, changes in the level of gene expression do not necessarily lead to changes in biological function. In addition, since this method does not provide any information on the effects of a compound on other genes, experimental results may not reveal the real gene target(s).

Protein expression

The global protein expression pattern (proteome) and protein post-translational phosphorylation dynamics (phosphoproteome) have been used to identify key proteins or phosphoproteins which show a response to chemical treatment (Thiyagarajan et al. 2009; Qian, Wong et al. 2010; Zhang et al. 2010).

Comparative proteomics

The comparative proteomic method has been used successfully to identify the proteins whose expression has been either up-regulated or down-regulated in response to AF treatments in fouling organisms (Thiyagarajan et al. 2009; Qian, Wong et al. 2010; Wang et al. 2010; Wong et al. 2010; Zhang et al. 2010). The findings from proteomic analysis were confirmed with western blotting using monoclonal antibodies (Zhang et al. 2010) or qRT-PCR to check expression at the mRNA level (Wong et al. 2010). A probe of the gene for a particular protein was designed and applied to a whole mount in situ hybridization to visualize the expression pattern of the selected proteins (Wang et al. 2010). Thus, functional analysis of the selected genes or proteins was conducted at both the protein and mRNA level using specific inhibitor assays (Zhang et al. 2012a). However, this approach neither provides direct evidence for the real target proteins of AF compounds nor identifies the low abundance proteins affected. It often requires complex and expensive technologies, and instrumentation for protein extraction, purification, separation, and identification as well as a large transcriptomic and/or genomic database of relevant organisms to identify the proteins. More importantly, it is difficult to confirm the findings of comparative proteomics, due to the technical limitations of western blotting, in situ hybridization and inhibitory bioassays.

Protein pull-down (affinity binding) technique

Affinity pull-down assays have been applied to identify the potential molecular targets of AF compounds by capturing the bound target proteins (Zhang et al. 2012b). Although this method is the most direct means of identifying direct binding targets, not all AF compounds are suitable for pull-down assays, given that the compounds may not be linked easily to the binding matrix without losing bioactivity.

Comparative transcriptomics

Comparative transcriptome analysis can be used to quantify the level of gene expression (Chen et al. 2011) in response to treatment with an AF compound. A normalized Expressed Sequence Tag library was constructed with the aid of 454-pyrosequencing technology for three developmental stages of Balanus amphitrite, viz nauplius, cyprid, and adult and several genes that might be involved in larval settlement were identified (De Gregoris et al. 2011). However, this approach does not provide direct evidence of the real targets of a compound because it only shows the up-regulated or down-regulated transcripts. Furthermore, the extraction of total RNA and mRNA can be technically challenging, while assembling and annotating the transcripts often requires a reference genome database for target organisms, which is often unavailable. More importantly, since there may be many up-regulated or down-regulated transcripts, it is impractical to confirm the expression level of every differentially expressed transcript using qRT-PCR, in situ hybridization or inhibitory bioassays. Furthermore, it is technically challenging and labor-intensive to design the probes, and analyze the expression profile of each transcript.

Microarrays

Microarray tests are considered a high throughput, cost-effective tool for studying the effects of chemical compounds on genes and gene networks (Yasokawa et al. 2010) because they can offer a high coverage of the genome (Schena et al. 1995). This allows for rapid prediction of the toxicological effects of AF compounds. The technique is also user friendly since it does not involve any radioactive or toxic substances. However, given that the genomic information for most fouling species is unavailable, gene information from other organisms has to be used to develop gene chips. This may not provide good coverage of the genes of the target organisms or may even yield false results. In addition, microarray analyses may not be sufficiently sensitive to detect rare transcripts. More importantly, understanding is often lacking concerning the implications of the variety of gene combinations and how they affect the behavior and treatment of chemical compounds.

Signal transduction pathways

Pharmacological assays using agonists and antagonists capable of activating or blocking specific signal transduction pathways provide fundamental knowledge of the molecular mechanisms associated with the settlement and metamorphosis of fouling organisms (Clare et al. 1995; He et al. 2012; Wong et al. 2012; Zhang, He et al. 2012). Given that information on the molecular mechanisms of AF activity is limited, the use of pharmacological agonists and antagonists to elucidate a signaling pathway can be a powerful tool for determining the signaling interactions upon treatment with a compound of interest.

In vitro assays

The extreme complexity of living organisms prevents the in vivo exploration of AF mechanisms, whereas in vitro assays eliminate factors that complicate the interpretation of in vivo assays and allow one cellular component or pathway to be studied. Okano et al. (1996, 1998) successfully isolated and cultured the cement gland and secretory cells of barnacle cyprids. A new AF assay based on inhibition of the activity of phenoloxidase purified from blue mussels has been used as a reliable candidate for rapid screening to search for sensitive, specific and efficient molecular biomarkers (Hellio et al. 2000). However, it is very challenging to extrapolate the in vitro results to intact organisms. Therefore, a combination of in vitro and in vivo studies should be used to incorporate their respective advantages.

AF compounds with proposed specific targets

AF compounds are known to demonstrate a fouling-deterrent effect in different ways and may have various modes of actions. AF compounds where specific molecular targets have been proposed, speculated about, or indicated in the literature can be grouped into five major categories (Table 1).

Ion channel inhibitors

The alkaloid-rich fraction from the Mediterranean sponge Crambe crambe inhibits the settlement of bryozoans (Becerro et al. 1997), probably due to the disruption of Ca²⁺ signaling by the channel blocker crambescidin 816 (Berlinck et al. 1993). The polymeric 3-alkylpyridinium salts (poly-APS) isolated from a Mediterranean sponge, Reniera sarai, can protect submerged surfaces from fouling by bacteria, fungi, microalgae, and barnacles (Faimali et al. 2003; Garaventa et al. 2003; Chelossi et al. 2006; Turk et al. 2007). It has been suggested that poly-APS acts as a surfactant, binding to and causing lesions in the lipid cell membrane and thus causing the lysis of the microorganism (Turk et al. 2007). Brominated metabolites, bastadins, extracted from the sponge Ianthella basta, inhibit the settlement of the cypris larvae of barnacles, probably by regulating the intracellular Ca²⁺ levels (Ortlepp et al. 2007). Isocyanide 1 (11-Isocyano-11-methyldodec-1-ene), which has a low EC₅₀ (0.046 μg ml⁻¹) and high safety ratio (LC₅₀/EC₅₀ > 652) against cypris larvae of B. amphitrite, is suspected to bind to voltage-dependent anion channels (VDAC) (Fusetani et al. 1996; Nogata & Kitano 2006). However, none of the proposed modes of action regarding ion channels has been examined fully and confirmed.

Table 1. Molecular mechanisms and bioactivity of AF compounds with proposed specific targets.

Proposed molecular mechanism and targets	Compounds	Activity	Category	Sources	Toxicity	References
Inhibitors of ion channel function
Blocks Ca^2+ channels	Crambescidins	AF (Bugula neritina)	Natural product	Mediterranean sponge Crambe crambe	Non-toxic	Berlinck et al. (1993); Becerro et al. (1997)
Surfactant; lysis of the cell membrane and microorganisms	poly-APS	Antibacterial, antialgal, antifungal. AF (barnacle Balanus amphitrite)	Natural product	Mediterranean sponge Reniera sarai	Non-toxic	Sepčić et al. (1998); Faimali et al. (2003); Garaventa et al. (2003), Chelossi et al. (2006); Turk et al. (2007)
Alteration of the intracellular Ca^2+ levels	Bastadins	AF (Balanus improvisus; blue mussel Mytilus edulis)	Natural product	Sponge Ianthella basta	Non-toxic	Ortlepp et al. (2007); Bayer et al. (2011)
Binds to voltage dependent anion channels	Isocyanide 1	AF (B. neritina)	Natural product	Sponge Acanthella cavernosa	Non-toxic	Nogata and Kitano (2006); Zhang, Zhang et al. (2012)
Inhibitors of quorum sensing
Inhibits quorum sensing	Manoalide	Antibacterial	Natural product	Sponge Luffariella variabilis	Non-toxic	Skindersoe et al. (2008)
Antagonist of AHL-dependent quorum sensing	Brominated alkaloids	Antibacterial	Natural product	Bryozoan Frusta foliacea	Non-toxic	Peters et al. (2003)
Antagonist of bacterial acylated homoserine lactone regulatory system	Halogenated Furanones	Antibacterial, AF (B. amphitrite, macroalga Ulva lactuca)	Natural product	Red alga Delisea pulchra	Non-toxic	de Nys et al. (1995); Manefield et al. (2002)
Inhibits N-octanoyl-DL-homoserine lactone dependent quorum sensing	Floridoside and isethionic acid mixture	Antibacterial	Natural product	Red alga Ahnfeltiopsis flabelliformis	Non-toxic	Liu et al. (2008)
Inhibits the responses of the LuxR-based reporter	Kojic acid	Antibacterial, antialgal	Natural product	Marine organisms	Non-toxic	Dobretsov et al. (2011)
Degrades AHL affecting quorum sensing and biofilm development	AHL acylase	Antibacterial	Shelf-stable	Acylase	Non-toxic	Xu (2004); Kristensen et al. (2008)
Blockers of neurotransmission
Inhibitor of serotonergic neurons	2,5,6-tribromo-1-methylgramine	AF (B. amphitrite)	Natural product	Bryozoan Zoobotryon pellucidum	Non-toxic	Kon-ya et al. (1994)
Inhibitor of AChE	poly-APS	Antibacterial, antialgal, antifungal, AF (B. amphitrite)	Natural product	Mediterranean sponge R. sarai	Non-toxic	Sepčić et al. (1998); Faimali et al. (2003); Garaventa et al. (2003); Chelossi et al. (2006); Turk et al. (2007)
Induces hyperactivity and activates octopamine receptors	Medetomidine and clonidine	AF (B. amphitrite, B. improvises, Hydroides elegans)	Shelf-stable	α 2-adrenoceptor agonist	Non-toxic	Dahlström et al. (2000); Lind et al. (2010)
Inhibitors of adhesive production/release
Strong inhibitors of blue mussel phenoloxidase	Bastadins	AF (B. improvisus, M. edulis)	Natural product	Sponge I.	Non-toxic	Ortlepp et al. (2007); Bayer et al. (2011)
Specifically inhibits the activity of phenoloxidase	Algal extract	Antibacterial, antifungal, AF (M. edulis)	Natural product	Enteromorpha intestinalis; Sargassum muticum; Polysiphonia lanosa	Non-toxic	Hellio et al. (2000)
Inhibits the activity of mussel phenoloxidase	Meroditerpe-noids	Antibacterial, antialgal, AF (macroalgae Sargassum muticum, Ulva intestinalis, blue mussel M. edulis)	Natural product	Brown alga Cystoseira baccata	Non-toxic	Mokrini et al. (2008)
Cleaves adhesives; removes proteinaceous cues	Neutral, cold-adapted protease	AF (B. amphitrite, B. neritina, Schizoporella sp.)	Natural product	Bacterium Pseudoalteromonas issachenkonii UST041101-043	Non-toxic	Dobretsov et al. (2007)
Influences the secretion of adhesive	Butenolide	Antibacterial, AF (B. amphitrite, H. elegans, B. neritina)	Natural product	Streptomyces albidoflavus strain UST040711-291	Non-toxic	Qian, Xu et al. (2010); Xu et al. (2010); Zhang et al. (2010); Zhang, Wong et al. (2011); Zhang, Kitano et al. (2012)
Cleaves the adhesive of spores	Trypsin	AF (Enteromorpha sp.)	Shelf-stable	Serine protease	Non-toxic	Callow et al. (2000)
Cleaves adhesives; removes proteinaceous cues	Papain and trypsin	AF (B. neritina)	Shelf-stable	Papain: cysteine protease; Trypsin: serine protease	Non-toxic	Dobretsov et al. (2007)
Reduces the strength of adhesion	Alcalase and Savinase	AF, antialgal, (B. amphitrite, U. linza)	Shelf-stable	Serine protease	Non-toxic	Pettitt et al. (2004)
Removes cyprid ‘footprint’ deposits and reduces the effectiveness of the adhesive	Alcalase (Subtilisin A)	AF (B. amphitrite)	Shelf-stable	Serine protease	Non-toxic	Aldred et al. (2008)
Reduces the strength of bacterial adhesion and degrades the matrix of the biofilm; reduces the strength of adhesion through proteolytic degradation of the adhesive	Subtilisin A	Antibacterial, antialgal, AF (B. amphitrite, green algae U.linza)	Shelf-stable	Serine protease	Non-toxic	Leroy et al. (2008); Tasso et al. (2009, 2012); Friedrichs et al. (2012)
Removes biofilm by degrading extracellular polysaccharide; oxidoreductases have a bactericidal acttion	Glucose oxidase/lactoperoxidase/ Pectinex Ultra	Antibacterial	Shelf-stable	Enzyme blend	Non-toxic	Johansen et al. (1997)
Hydrolyzes polysaccharide	Cellulase/alpha-amylase/protease	Antibacterial	Shelf-stable	Enzyme blend	Non-toxic	Wiatr (1990)
Inhibits the release of adhesive	SCH23390	AF (B. improvisus)	Shelf-stable	Dopamine receptor antagonist	Non-toxic	Martensson and Gunnarsson (2005)
Repellent and chemical antiadhesive	Benzoic acid	Antibacterial	Shelf-stable	Food preservative and antimicrobial agent	Non-toxic	Al-Juhni and Newby (2006)
Inhibits phenoloxidase activity in mussels	N-substituted imides	Antimicrobial (bacteria and fungi), AF (M. edulis)	Shelf-stable	Synthesized chemical	Non-toxic	Zentz et al. (2002)
Enzyme inhibitors
Chitinase inhibitor	Styloguanidine	AF (barnacle)	Natural product	Marine sponge Stylotella aurantium	Non-toxic	Kato et al. (1995)
Affects the expression of marker genes (Ran GTPase activating protein, ATP synthase)	12-Methyltetrade-canoid acid	AF (H. elegans)	Natural product	Streptomyces sp. UST040711-290; in deep sea sediment	Non-toxic	Xu et al. (2009)
Affects oxidative phosphorylation in mitochondria via binding to NADH-ubiquinone oxidoreductase	Isocyanide 1	AF (B. amphitrite)	Natural product	Sponge Acanthella cavernosa	Non-toxic	Nogata and Kitano (2006); Zhang, Zhang et al. (2012)
Binds to and influences protein expression and phosphorylation during energy metabolism	Butenolide	Antibacterial, AF (B. amphitrite, H. elegans, B. neritina)	Natural product	Streptomyces albidoflavus strain UST040711-291	Non-toxic	Qian, Xu et al. (2010); Xu et al. (2010); Zhang et al. (2010); Zhang, Wong et al. (2011); Zhang, Kitano et al. (2012)

Inhibitors of quorum sensing

Manoalide and its analogs isolated from the marine sponge Luffariella variabilis inhibit bacterial quorum sensing or cell–cell communication (Skindersoe et al. 2008). Brominated alkaloids isolated from the North Sea bryozoan Flustra foliacea demonstrate antagonistic effects on bacterial N-acyl homoserine lactones (AHL)-dependent quorum sensing (Peters et al. 2003). Halogenated furanones isolated from the red alga Delisea pulchra (de Nys et al. 1995; Rasmussen et al. 2000) and other furanones (Ren et al. 2001) accelerate the degradation of the transcriptional activator LuxR, inhibiting the AHL regulatory pathways of bacteria (Manefield et al. 2002). A mixture of floridoside and isethionic acid from the red alga Ahnfeltiopsis flabelliformis also inhibited the bacterial quorum sensing activity mediated by N-octanoyl-DL-homoserine lactone, resulting in an inhibition of biofilm formation (Liu et al. 2008). Kojic acid, a natural product, showed an inhibitory effect on the responses of the LuxR-based reporter after induction of N-3-oxo-hexanoyl-L-homoserine lactone, and also decreased the density of bacteria and diatom cells on glass slides in a controlled mesocosm experiment, thereby inhibiting the formation of microbial communities (Dobretsov et al. 2011). One shelf-stable AHL-acylase degraded the AHL involved in quorum sensing, thereby inhibiting biofilm formation (Xu 2004; Kristensen et al. 2008). The modes of action of these compounds on quorum sensing processes need to be studied in greater depth to confirm their molecular mechanisms.

Neurotransmission blockers

The 2,5,6-tribromo-1-methtyl gramine from the bryozoan Zoobotryon pellucidum prevented the settlement of barnacle cyprids by inhibiting serotonergic neurons (Kon-ya et al. 1994; Raveendran & Mol 2009). Serotonergic blockers can also inhibit the settlement of barnacle larvae (Yamamoto et al. 1996). It has also been suggested that poly-APS inhibits acetylcholinesterase (AChE) activity, thereby inhibiting the settlement of barnacles (Sepčić et al. 1998). Medetomidine and clonidine, classified as α₂-adrenoceptor agonists in vertebrates, can efficiently prevent the settlement of barnacle cyprids (Dahlström et al. 2000; Dahlström & Elwing 2006). Medetomidine is believed to inhibit the settlement of larvae through the hyperactive locomotor response and the activation of octopamine receptors (Lind et al. 2010). It also reduces pheromone-induced mate search behavior (Krang & Dahlström 2006).

Inhibitors of adhesive production/release

In addition to causing alteration in intracellular Ca²⁺ levels, bastadins can also inhibits mussel phenoloxidase, the enzyme responsible for the polymerization of the foot proteins, the process responsible for the firm attachment of mussels to substrata (Bayer et al. 2011). A crude extract of marine algae (Hellio et al. 2000) and meroditerpenoids isolated from the brown alga Cystoseira baccata (Mokrini et al. 2008) inhibited the activity of mussel phenoloxidase, thereby preventing mussels from attaching to substrata (Bayer et al. 2011). Following incorporation into water-based paints, neutral and cold-adapted proteases purified from the bacterium Pseudoalteromonas issachenkonii UST041101-043 inhibited the settlement of barnacles and bryozoans by either cleaving the adhesive substances or removing the proteinaceous settlement cues (Dobretsov et al. 2007).

Currently, the best-known study of the mode of action of an AF compound is butenolide. Butenolides with AF activity were first isolated from a marine strain of Streptomyces. A structure-function analysis revealed that the functional moiety of the butenolides responsible for AF activity lies within the 2-furanone ring, and that the lipophilicity of the compounds substantially affected their AF activity (Xu et al. 2010; Li et al. 2012). Based on these findings, Xu et al. (2010) and Zhang, Xiao et al. (2011) designed and chemically synthesized 5-octylfuran-2(5H)-one, a highly effective compound with low toxicity that outperformed SeaNine211 and other commercial AF compounds in both laboratory antilarval settlement bioassays and initial field AF panel trials. Treatment with butenolide inhibited the swelling of the secretory granules and altered the rough endoplasmic reticulum in the cement gland of cypris larvae of B. amphitrite. It also reduced the number of secretory granules in the pyriform-glandular complex (Zhang, Wang et al. 2011).

Different groups of commercial compounds such as enzymes, neurotransmitter agonists/antagonists, and antioxidants/oxidants have been screened for AF capabilities. Enzymes have been proposed repeatedly as alternatives to traditional hazardous AF paints because they can inhibit settlement and attachment through different mechanisms, such as degrading the proteinaceous components of adhesive polymers, degrading the biofilm matrix, interfering in bacterial intracellular communication, and catalyzing the production of repellent compounds (Kristensen et al. 2008). Enzymes can also remove the proteinaceous cues necessary for larval attachment (Dobretsov et al. 2007). For instance, proteolytic enzymes can cleave proteinaceous adhesives and reduce adhesion strength in a variety of fouling organisms including bacteria (Friedrichs et al. 2012), diatoms (Pettitt et al. 2004; Tasso et al. 2009), green algae (Callow et al. 2000; Pettitt et al. 2004; Tasso et al. 2009), bryozoans (Dobretsov et al. 2007), and barnacles (Pettitt et al. 2004; Aldred et al. 2008; Tasso et al. 2012). Several enzymes and enzyme blends can also cause oxidative damage through oxidoreductase-produced reactive oxygen species (eg hydrogen peroxide and hypohalogenic acids), which inhibit the formation of biofilms or degrade/remove biofilm matrices by hydrolyzing extracellular polymeric substances (Wiatr 1990; Johansen et al. 1997; Leroy et al. 2008; Friedrichs et al. 2012). The dopamine receptor antagonist SCH23390 inhibits barnacle settlement by blocking the release of adhesive (Martensson & Gunnarsson 2005). Benzoic acid, commonly used as an antimicrobial agent, inhibited bacterial attachment after being incorporated into coatings through repellency and chemical antiadhesive mechanisms (Al-Juhni & Newby 2006). N-substituted imides inhibited phenoloxidase activity involved in the attachment of mussels to the substratum (Zentz et al. 2002). However, the real molecular targets of these enzymes in fouling organisms have not yet been confirmed through carefully designed experiments.

Inhibitors of enzymes

Styloguanidines isolated from the marine sponge Stylotella aurantium control the molting cycle and the settlement of barnacle cyprids by inhibiting the activity of chitinase, a key enzyme involved in the ecdysis of crustaceans, through the hydrolysis of integumental chitin (Kato et al. 1995). The branched fatty acid 12-methyltetradecanoid down-regulates the Ran GTPase activating protein and up-regulates ATP synthase, which may contribute to its inhibitory effect on the settlement of polychaete larvae (Xu et al. 2009). However, the compound with the best-known mode of action is butenolide. Butenolide inhibits the settlement of barnacle cypris larvae by sustaining the expression level of stress-associated and metabolism-related proteins (vitellogenin), thus arresting development until a fully competent stage had been reached (Zhang et al. 2010). Butenolide also increases the expression and phosphorylation of the stress-induced protein HSP70 of swimming larvae of the bryozoan Bugula neritina, inducing substantial changes in the abundance and phosphorylation status of structural proteins, molecular chaperones, mitochondrial peptidases, and calcium-binding proteins (Qian, Wong et al. 2010). Furthermore, using affinity pull-down assays, Zhang, Zhang et al. (2012) showed that the molecular targets of butenolide were enzymes involved in the primary metabolism for energy production, particularly those related to lipid and fatty acid metabolism. For instance, in B. neritina, 5-octylfuran-2(5H)-one bound to the long-chain molecule acyl-coenzyme A dehydrogenase (ACADVL) (Zhang, Zhang et al. 2012), which catalyzes the first step of fatty acid β-oxidation (Zhang, Kitano et al. 2012). In H. elegans, an acyl-CoA dehydrogenase specific for long-chain lengths and an enoyl-CoA hydratase, which catalyzes the generation of acetyl-CoA (AC-CoA) from fatty acids, were up-regulated. In the cypris larvae of B. amphitrite, 5-octylfuran-2(5H)-one bound to AC-CoA acetyltransferase 1 (referred to as thiolase hereafter), which is involved in ketone body metabolism. Four aldehyde dehydrogenases (NAD+), long-chain fatty-acid CoA ligase, acyl-CoA dehydrogenase, Acss1, acyl-CoA synthetase, and acyl-CoA oxidase were also up-regulated in the cypris larvae (Zhang, Zhang et al. 2012). The structural model produced by autodock tools (AutoDock 4 program, http://autodock.scripps.edu) to predict the docking site of butenolide suggested that both the thiolase in B. amphitrite and the ACADVL in B. neritina contains a 5-octylfuran-2(5H)-one binding site in their substrate binding pocket. A series of bioassays were conducted to confirm whether the potential 5-octylfuran-2(5H)-one binding proteins were really involved in 5-octylfuran-2(5H)-one AF activity. In B. amphitrite, in the presence of butenolide, the addition of AC-CoA and acetoacetyl-CoA (ACAC-CoA), the substrate and product of ACAT1, respectively, significantly increased the settlement rate of cypris larvae. For cypris larvae of B. neritina, in the presence of butenolide, the addition of 2 of the substrates of ACADVL, ACAC, and pyruvate significantly increased the rate of settlement. Butenolide also bound to the succinyl-CoA synthetase β subunit, inhibiting growth in the marine bacterium Vibrio sp. UST020129-010 (Zhang, Zhang et al. 2012). These findings confirm that although 5-octylfuran-2(5H)-one targeted different enzymes in barnacles and bryozoans, both thiolase in B. amphitrite and ACADVL in B. neritina are involved in fatty acid β-oxidation for energy production, which provides the essential energy source in both species during their swimming larval stages. The scientific challenge is to determine whether 5-octylfuran-2(5H)-one targets enzymes along this metabolic (energy production) pathway in other major fouling organisms in addition to non-target organisms. If so, it can be concluded that 5-octylfuran-2(5H)-one impairs the lipid metabolisms of fouling organisms, limiting the availability of the energy source that is required by settling larvae to complete the energy intensive demands of attachment and metamorphosis. Such impairment and inhibition are reversible. Therefore, 5-octylfuran-2(5H)-one prevents these fouling organisms from settling through non-toxic inhibition mechanisms and suggests it could be developed into next-generation ‘ecofriendly’ marine coatings. Using the same pull-down assay, the following proteins were identified as isocyanide targets in B. neritina. Firstly, a 30 kD protein band containing 2 proteins similar to VDAC that control the direct coupling of the mitochondrial matrix to the energy maintenance of the cytosol and the release of apoptogenic factors from the mitochondria of mammalian cells. Secondly, an unknown 39 kD protein (Zhang, Kitano et al. 2012). In the cypris larvae of B. amphitrite, the isocyanide binds to NADH-ubiquinone oxidoreductase, which is the ‘entry enzyme’ for oxidative phosphorylation in mitochondria (Zhang, Kitano et al. 2012).

In summary, two protein families were affected by butenolide, isocyanide, polyether B (see below), and meleagrin (see below); a chitin binding protein and an ATPase appear to be mediated by these AF compounds. Therefore, it has been suggested that chitin-binding proteins mediating the molting process and ATPase mediating the energy process can be considered the common targets of action in all of the antifoulants examined. On the other hand, each AF compound up-regulated or down-regulated a specific set of proteins (compound-specific), clearly indicating a distinct and specific mode of action exhibited by each compound. Obviously, both common and specific targets identified for these 4 AF compounds are far from conclusive and a more in-depth study of molecular mechanisms of AF compounds is called for.

AF compounds with a proposed general mode of action

Many AF compounds act on target organisms through different/multiple pathways and their specific molecular targets have not yet been defined; these are considered as compounds with a general mode of action (Table 2).

Protein expression regulators

The incorporation of proteins into industrial coatings painted onto surfaces produced a stable oxygen-depleted layer consequent on microbial degradation of the protein component, and thereby efficiently reduced fouling by bryozoans and barnacles (Lindgren et al. 2009). Indeed, a number of compounds can inhibit the settlement of larvae of fouling organisms by altering their protein expression. For instance, polyether B, isolated from a sponge-associated bacterium Winogradskyella poriferorum (strain UST030701-295T), inhibited the formation of a marine biofilm and the settlement of barnacle and polychaete larvae (Dash et al. 2011). In addition, polyether B altered the expression levels of proteins related to energy metabolism, oxidative stress and molecular chaperones. These changes indicate that polyether B interfered with the redox-regulatory mechanisms governing the settlement of barnacle larvae (Dash et al. 2012). Genistein effectively inhibits the settlement of barnacles and bryozoans (Zhou et al. 2009), reducing the number of phosphoproteins in bryozoans, but substantially increasing them in barnacles (Thiyagarajan et al. 2009).

Table 2. Molecular mechanisms and bioactivity of AF compounds with proposed general targets.

Proposed molecular mechanism and targets	Compounds	Activity	Category	Sources	Toxicity	References
Regulators of protein expression
Influences the expression of proteins from cypris larvae associated with energy metabolism, phosphorylation, cytoskeleton and oxidative stress	Poly-ether B	Antibacterial, AF (Balanus amphitrite, Hydroides elegans)	Natural product	Winogradskyella poriferorum strain UST030701-295T, bacterium associated with a sponge	Non-toxic	Dash et al. (2011, 2012)
Affects the expression of protein involved in metabolic pathways, ECM-receptor interactions and regulation of the actin cytoskeleton	Meleagrin	AF (B. amphitrite)	Natural product	Fungus Penicillium meleagrinum	Non-toxic	He et al. (2012)
Forms an oxygen-depleted layer by microbial degradation of protein	Industrial protein paint	AF (barnacle; bryozoan)	Shelf-stable	Protein	Non-toxic	Lindgren et al. (2009)
Influences protein phosphorylation as an inhibitor of metamorphosis	Genistein	AF (B. amphitrite’ Bugula neritina)	Shelf-stable	Isoflavones	Non-toxic	Thiyagarajan et al. (2009); Zhou et al. (2009)
Oxidative stress inducers
Causes damage to the cell membrane and induces peroxidative processes	Unsaturated fatty acid	Antibacterial	Natural product	Brown algae Sargassum muticum and Desmarestia ligulata	Non-toxic	Rosell and Srivastava (1987); Desbois et al. (2009); Plouguerné et al. (2010)
Oxidative damage by active oxygen radicals	Hydrogen peroxide, hypohalogenic acid, and ozone	Antimacrofouling	Shelf-stable	Oxidants	Non-toxic	Nishimura et al. (1988); Bollyky and LePage (1991); Kristensen et al. (2008)
Blockers of neurotransmission
Stimulates the chemosensor network and induces metamorphosis into juvenile stage without cementation onto the surface	Noradrenaline	AF (B. amphitrite, B. improvisus, oysters (Crassostrea virginica, C. gigas)	Shelf-stable	Adrenergic receptor agonist	Non-toxic	Coon et al. (1985); Dahlström et al. (2000); Gohad et al. (2010, 2012)
Blocks neurotransmission for metamorphic transition	Idazoxan and phentolamine	AF (B. amphitrite, B. neritina, H. elegans)	Shelf-stable	α-Adrenergic antagonists	Non-toxic	Dahms et al. (2004)
Affects neuromuscular responses of the foot	L-3,4-DOPA	AF (zebra mussels Dreissena polymorpha)	Shelf-stable	Catecholamine transmitter	Non-toxic	Cope et al. (1997)
Affects nicotinic receptors; activates certain chemoreceptor neurons	Anabaseine, 2,3′-bipyridyl (2,3′-BP)	AF (B. amphitrite)	Shelf-stable	Nemertine pyridyl alkaloids	Non-toxic	Kem et al. (2003)
Surface modifiers
Blocks bacterial surface attachment sites	Zosteric acid	Antibacterial	Natural product	Zostera marina (eelgrass)	Non-toxic	Xu et al. (2005)
Blocks bacterial surface attachment sites	Capsaicin	Antibacterial, AF (D. polymorpha byssus)	Natural product	Capsicum frutescens L. (chilli peppers)	Non-toxic	Cope et al. (1997); Xu et al. (2005); Angarano et al. (2007); Peng et al. (2012)
Decreases cell surface hydrophobicity and inhibits the attachment to hydrophobic substrata	Pronase, trypsin, chymotrypsin	Antibacterial	Shelf-stable	Serine protease	Non-toxic	Paul and Jeffrey (1985)
Reduces the formation of biofilm by damaging the cell membrane and reducing surface hydrophobicity	Dichlorochalcone	Antibacterial and slimicidal activity	Shelf-stable	Chalcone	Non-toxic	Sivakumar et al. (2010)
Biofilm inhibitors and modifiers
Modifies the composition of the bacterial community within the biofilm and reduces the settlement of the larvae of fouling organisms	Kalihinol A	Antibacterial; AF (H. elegans)	Natural product	Sponge Acanthella cavernosa	Non-toxic	Yang et al. (2006)
Alters the cell density and species diversity of the bacterial community in the biofilm	Succinic acid	Antibacteria; AF (H. elegans)	Natural product	Fungus associated with sponge Acanthella cavernosa	Non-toxic	Yang, Lau et al. (2007)
Inhibits bacterial quorum sensing	Diketopiperazines	Antibacterial; AF (B. amphitrite)	Natural product	Streptomyces fungicidicus; fungus Letendraea helminthicola	Non-toxic	Holden et al. (1999); Li et al. (2006); Yang, Miao et al. (2007)
Inhibitors of adhesive production/release
Interferes with the oxidation reactions for byssal thread development; repels the attachment of marine life	Capsaicin	Antibacterial; AF (D. polymorpha byssus)	Natural product	Capsicum frutescens L. (chilli pepper)	Non-toxic	Cope et al. (1997); Xu et al. (2005); Angarano et al. (2007); Peng et al. (2012)
Interferes with the oxidation reaction for byssal thread production	Butylated hydroxyanisole, tert-butylhydroquinone, and tannic acid	AF (D. polymorpha)	Shelf-stable	Phenolic antioxidants	Non-toxic	Cope et al. (1997)
Lethal toxicity
Chronic intoxication or contact intoxication	Ivermectin	AF (B. improvisus)	Natural product	Streptomyces avermitilis	Toxic	Pinori et al. (2011)
Kills mussels by destroying the digestive system	Protein toxin	AF (D. polymorpha, D. bugensis)	Natural product	Bacterium Pseudomonas fluorescens CL0145A	Toxic	Molloy (2001)
Uncouples oxidative phosphorylation; immunosuppressor; endocrine disruptor; cytotoxic and genotoxic effects	Organotin	AF	Heavy metal	Organometallics	Toxic	Cooper et al. (1995); Bragadin and Marton (1997); Morcillo and Porte (2000); Patricolo et al. (2001); Pellerito et al. (2005)
Decreases synthesis of ATP; immunosuppressive effects; inhibits AChE activity	Copper, cadmium, lead, mercury, and zinc	AF	Heavy metal	Metallic ion	Toxic	Tedengren et al. (1999); Rao and Khan (2000); Tujula et al. (2001); Brown et al. (2004)
Inhibits photosynthesis and ATP synthesis; immunosuppressive effects	Irgarol 1051	Antialgal	Booster biocide	Herbicide	Toxic	Devilla et al. (2005), Arrhenius et al. (2006), Bragadin et al. (2006); Cima and Ballarin (2012)
Inhibits photosynthesis and ATP synthesis; immunosuppressive effects; induces synthesis of ascidian eicosanoids	Sea-Nine 211	AF	Booster biocide	Isothiazolone compound	Toxic	Knight et al. (1999); Bragadin et al. (2005), Devilla et al. (2005); Arrhenius et al. (2006); Cima et al. (2008)
Inhibits photosynthesis of marine algae by targeting photosystem II; cytotoxic and genotoxic; immunosuppressive effects	Diuron	Antialgal	Booster biocide	Herbicide	Toxic	Devilla et al. (2005); Menin et al. (2008); Akcha et al. (2012)
Inhibits photosynthesis of marine algae by targeting photosystem II	Copper pyrithione	Antialgal	Booster biocide	Fungicide	Toxic	Maraldo and Dahllöf (2004)
Inhibits photosynthesis and ATP synthesis	Zinc pyrithione	Antialgal	Booster biocide	Bactericide; fungicide; algicide	Toxic	Ermolayeva and Sanders (1995); Bragadin et al. (2003); Devilla et al. (2005)
Inhibits ATP synthesis; immunosuppressive effects	TCMS pyridine	AF	Booster biocide	Fungicide	Toxic	Bragadin et al. (2007); Menin et al. (2008)
Immunosuppressive effects	Chlorothalonil	AF	Booster biocide	Fungicide	Toxic	Cima et al. (2008)

Meleagrin, a natural alkaloid compound initially isolated from Penicillium meleagrinum (Kawai et al. 1984) inhibited the settlement of cyprids of B. amphitrite (He et al. 2012). Furthermore, it was found that 50 proteins of B. amphitrite were differentially expressed in response to meleagrin treatment: 26 proteins were associated with development/aging in the cypris larvae, and of those 24 were associated specifically with meleagrin treatment. Analysis of data from the Kyoto Encyclopedia of Genes and Genomes indicated that meleagrin affected several pathways, including metabolic pathways, ECM–receptor interactions, and the regulation of the actin cytoskeleton. Among the 24 proteins affected by meleagrin treatment, a vitellogenin-like protein was up-regulated, indicating endocrine disruption and prevention of the larval molting cycle. With the exception of mediation of the molting process involving chitin binding proteins and ATPase-mediated energy, most of the major proteins in this study were not found to be significant in butenolide and polyether B treated larvae, suggesting that meleagrin exhibits a distinct and specific mode of action.

Oxidative stress inducers

Unsaturated fatty acids isolated from the brown algae Sargassum muticum (Plouguerné et al. 2010) and Desmarestia ligulata (Rosell & Srivastava 1987) damaged the cell membranes and induced peroxidative stress in marine bacteria, thereby inhibiting bacterial growth and attachment (Desbois et al. 2009). Oxidants including hydrogen peroxide, hypohalogenic acid, and ozone prompt oxidative damage in fouling organisms through active oxygen radicals, thereby decreasing attachment (Nishimura et al. 1988; Bollyky & LePage 1991; Kristensen et al. 2008).

Blockers of neurotransmission

The adrenoceptor agonist noradrenaline can inhibit the settlement process and/or induce barnacle and oyster larvae to metamorphose into sessile juveniles without cementation onto the substratum (Coon et al. 1985, 1986; Dahlström et al. 2000; Gohad et al. 2010, 2012). Two α-adrenergic antagonists, idazoxan and phentolamine, inhibited the settlement of three marine invertebrate larvae (B. amphitrite, B. neritina, and H. elegans). The ability of these antagonists to accumulate on solid surfaces suggests they will be promising candidates for incorporation in controlled release AF paints (Dahms et al. 2004). The catecholamine transmitter L-3,4-DOPA may affect the neuromuscular response of the foot, which is necessary for the production of byssal threads in zebra mussels (Cope et al. 1997). Certain nemertine neurotoxins, such as anabaseine and 2,3′-bipyridyl, affect the nicotinic receptors in the central and peripheral nervous systems of crustaceans, activating the chemoreceptor neurons in walking legs, which leads to the inhibition of cyprid settlement in barnacles (Kem et al. 2003). However, detailed molecular mechanisms of how these compounds act have not been well documented in previous studies.

Surface modifiers

Zosteric acid extracted from the eelgrass Zostera marina is thought to block the surface attachment sites of bacteria thus preventing the formation of biofilms (Xu et al. 2005). Similarly, capsaicin isolated from the chilli pepper, Capsicum frutescens L., is believed to block attachment sites on bacterial surfaces also preventing the formation of biofilms (Xu et al. 2005). Serine proteases also decrease the hydrophobicity of bacterial cell surfaces and inhibit attachment to hydrophobic substrata (Paul & Jeffrey 1985). Marine paint containing dichlorochalcone damaged cell membranes, reduced surface hydrophobicity, and inhibited the formation of biofilms (Sivakumar et al. 2010). However, the molecular targets of these compounds in microfouling organisms have not been revealed.

Biofilm inhibitors

Kalihinol A, isolated from the marine sponge Acanthella cavernosa, changes the bacterial species composition of the biofilm, inhibiting the settlement of polychaete larvae (Yang et al. 2006). Succinic acid isolated from strains of fungi on the surface of the sponge A. cavernosa altered the bacterial density and the species diversity in the biofilm, and allowed fewer polychaete larvae to settle on the succinic acid-modulated biofilm (Yang, Lau et al. 2007). Diketopiperazines isolated from the deep sea bacterium Streptomyces fungicidicus (Li et al. 2006) and the sponge-associated fungus Letendraea helminthicola (Yang, Miao et al. 2007) inhibited the settlement of barnacle cypris larvae as well as the formation of a biofilm; the compounds have been confirmed to be bacterial quorum sensing agonists (Holden et al. 1999). However, the molecular targets of these compounds remain unknown.

Inhibitors of adhesive production/release

Capsaicin also inhibited the attachment of zebra mussels by byssal threads (Angarano et al. 2007; Peng et al. 2012). The effect was attributed to its antioxidant properties, ie inhibition of oxidation reactions during the production of byssal threads (Cope et al. 1997). Moreover, reattachment of zebra mussels can also be inhibited by several antioxidants (eg butylated hydroxyanisole, tert-butylhydroquinone, and tannic acid), which also inhibit the oxidation reactions occurring in the pathways of byssal thread development (Cope et al. 1997).

Lethal toxicity

Among the various avermectins produced by Streptomyces avermitilis, ivermectin enhances post-settlement mortality, but has no effect on the settlement of the cypris larvae of barnacles (Pinori et al. 2011). Protein toxins from the bacterium Pseudomonas fluorescens CL0145A are specifically lethal to mussels by destroying their digestive systems without causing non-target mortality (Molloy 2001). A substantial amount of information has been generated on the bioaccumulation, non-selective killing, toxicity, and molecular mechanism of organotin against marine organisms (Iwao 2003). For instance, organotin, known as an uncoupler of oxidative phosphorylation (Bragadin & Marton 1997), inhibits ATP synthesis in bacteria (Hunziker et al. 2002), depresses metabolic activity in mussels (Wang et al. 1992; Widdows & Page 1993; Huang & Wang 1995), and acts as a photosynthesis inhibitor in algae (Fent 1996). Organotin also acts as an immunosuppression agent in both vertebrates and invertebrates, inhibiting Ca²⁺-ATPase via direct interaction with calmodulin. This increases intracellular Ca²⁺ concentrations, disorganizes cytoskeletal components, decreases respiratory burst and phenoloxidase activity, induces cell apoptosis and inhibits phagocytosis in cultured hemocytes from ascidians (Raftos & Hutchinson 1997; Cima et al. 2002; Cima & Ballarin 1999, 2000, 2004) and bivalve molluscs (Fisher et al. 1990; Bouchard et al. 1999; Cima et al. 1999; Hagger et al. 2005). In addition, TBT acts as an endocrine disruptor that increases testosterone levels and decreases oestradiol levels in clams, suggesting a potential masculinization effect which has been linked to TBT exposure (Morcillo & Porte 2000). During the metamorphosis of ascidian larvae, TBT significantly decreases thyroid hormone metabolism and blocks the transformation of larvae (Patricolo et al. 2001). TBT treatment also alters the expression of AChE activity, which is considered to be a sensitive biomarker for environmental contamination (Mansueto et al. 2012). Cytotoxic and genotoxic effects of TBT, including the induction of stress response proteins and DNA damage, have also been observed in sea urchins (Pellerito et al. 2005), bivalve molluscs (Clayton et al. 2000; Mičić et al. 2001, 2002), and polychaetes (Hagger et al. 2002). In salmon, TBT exposure causes both the elevation and inhibition of P450aromA, P450aromB, and aromatase activity, in addition to elevating and inhibiting Erα and plasma Vtg while antagonizing the EE2-induced expression of those enzymes. In general, these findings suggest that the exposed salmon experience impaired steroidogenesis and modulation of receptor-mediated endocrine responses (Lyssimachou et al. 2006). Urushitani et al. (2011) summarized the possible mechanisms of TBT-induced imposex in marine gastropods: (1) TBT inhibits aromatase leading to an increase in androgen levels; (2) TBT inhibits acyl CoA-steroid acyltransferase and thus causes an increase in testosterone; (3) TBT inhibits the excretion of androgen sulfate conjugates, leading to an increase in androgen levels; (4) TBT interrupts the release of the penis morphogenetic/retrogressive factor; (5) TBT increases the level of an alanine–proline–glycinetryptophan amide neuropeptide; and (6) TBT activates retinoid X receptors (see relevant references cited in Urushitani et al. 2011). However, to confirm which, if any of these mechanisms is responsible requires a substantial amount of molecular research.

Many species of heavy metals are essential to the growth of marine organisms at low concentrations, but they become toxic at high concentrations. Heavy metals such as copper, cadmium, lead, mercury, and zinc are commonly used in AF coatings because of the acute toxicity they cause. As uncouplers of oxidative phosphorylation (Bragadin et al. 2001) or via opening pores in the mitochondrial membrane (Bragadin et al. 2003), metals can decrease the synthesis of ATP and alter the metabolic activity of various marine organisms such as barnacles and mussels (Rao et al. 1986; Redpath & Davenport 1988; Tedengren et al. 1999; Rao & Khan 2000). In addition, heavy metals released from marine paints can have immunosuppressive effects (Cheng & Sullivan 1984; Pipe et al. 1999; Tujula et al. 2001; Gómez-Mendikute & Cajaraville 2003; Cima & Ballarin 2012) as well as inducing stress responses (Tedengren et al. 1999; Radlowska & Pempkowiak 2002). Copper also inhibits the AChE activity in the hemocytes of limpets (Brown et al. 2004), alters the chemical profiles of biofilms and reduces the inductiveness of the biofilm for the settlement of polychaetes (Bao et al. 2010).

Organic booster biocides, combined with copper-based AF coatings, can control copper-resistant biofouling organisms and thus improve the efficacy of the formulations (Voulvoulis 2006), while concomitantly reducing the amount of copper in the marine coatings. As many suppliers claim, the majority of the booster biocides used currently in AF applications are supposed to degrade either biotically and/or abiotically. This favorable degradation profile prevents accumulation in the environment, so whilst they are lethal to the settling stages of fouling organisms, toxicity toward non-target organisms is low due to rapid degradation following the release from the AF paint film. However, the modes of action of most co-biocides have not been fully investigated. Based on the literature, several biocides including Irgarol 1051 (Hall et al. 1999; Jones & Kerswell 2003; Devilla et al. 2005; Arrhenius et al. 2006; Bragadin et al. 2006), Sea-Nine 211 (Bragadin et al. 2005; Devilla et al. 2005; Arrhenius et al. 2006), diuron (Jones & Kerswell 2003; Devilla et al. 2005; Ricart et al. 2009), copper pyrithione (Maraldo & Dahllöf 2004), and zinc pyrithione (Maraldo & Dahllöf 2004; Devilla et al. 2005) are suspected of inhibiting the photosynthesis of marine algae by targeting photosystem II. Irgarol 1051 exposure significantly increased the total and dinoflagellate multixenobiotic resistance, dinoflagellate Cu/Zn superoxide dismutase, dinoflagellate chloroplast small heat-shock proteins, and cnidarian protoporphyrinogen oxidase IX while decreasing cnidarian glutathione peroxidase, cnidarian ferrochelatase, cnidarian catalase, and cnidarian CYP 450-3 and -6 classes (Downs & Downs 2007). Both Irgarol 1051 and zinc pyrithione inhibit ATP synthesis by opening small pores in the mitochondrial membranes of rats (Bragadin et al. 2003, 2006), whereas Sea-Nine 211 and TCMS pyridine (2,3,5,6-tetrachloro-4-(methyl sulphonyl) pyridine) reduce succinic dehydrogenase activity in the respiratory chain (Bragadin et al. 2005, 2007). Zinc pyrithione can also interact with the polar head group of membrane phospholipids in bacteria, inhibiting membrane transport of substrates and decreasing intracellular ATP levels by inhibiting ATP synthesis (Ermolayeva & Sanders 1995; Dinning, AL-Adham, Austin et al. 1998; Dinning, AL-Adham, Eastwood et al. 1998). This can induce the expression of heat shock proteins, DNA fragmentation, and apoptosis in marine mussels (Marcheselli et al. 2011). ZnPT induces genotoxic effects in the marine mussel Mytilus galloprovincialis, as demonstrated by an amplified fragmentation of DNA and an increased frequency of apoptotic cells in the tissues of the exposed mussels (Marcheselli et al. 2011). In the yeast Sacharomyces cerevisiae, ZnPT strongly up-regulates genes related to ion transport and down-regulates those related to the biosynthesis of cytochrome (Yasokawa et al. 2010) causing severe iron starvation and thus limiting growth. Various biocides decrease metabolic activity as demonstrated by inhibited bioluminescence in bacteria (Zhou et al. 2006). Diuron also induces the expression of heat shock proteins, fragmentation of DNA, and apoptosis in oysters (Akcha et al. 2012). The in vitro treatment of ascidian hemocytes with Irgarol 1051 (Cima & Ballarin 2012), Sea-Nine 211 and chlorothalonil (Cima et al. 2008), diuron and TCMS pyridine (Menin et al. 2008) suppresses the cellular immune systems. These types of immunosuppressive effects can be caused by disturbances in the mitochondrial respiratory chains, sudden increases in intracellular Ca²⁺ concentrations, cell apoptosis due to severe oxidative stress, disorganized cytoskeletal components, and hindered phagocytic activity. Sea-Nine 211 may also exert its action through inducing the synthesis of ascidian eicosanoids involved in the settlement of invertebrate larvae (Knight et al. 1999).

Conclusions

AF compounds with specific molecular targets mainly act on ion channels, enzymes, adhesive glands, and adhesive production or serve as inhibitors of quorum sensing or blockers of neurotransmission (Table 1). In contrast, those without specific targets may act on neurotransmitters, proteins, processes of biofilm, production/release of adhesive, induction of oxidative stress, or they simply act as lethal toxins. It is clear that antifoulants with specific molecular targets probably function through non-toxic mechanisms whilst those with no clear molecular targets act as toxic compounds. This separation lends support to the approach of screening for specific molecular targets in the development of non-toxic AF compounds. Among the molecular targets identified, the most common are adhesive proteins, (adhesive production and release), which may reflect the extensive studies on adhesive materials or bioadhesion of different fouling organisms. The second most common or promising targets are enzymes related to energy production. Stress-related proteins may not be the direct targets of AF compounds, but they may represent an indirect physiological/biochemical response of organisms to exposure. Quorum sensing molecules can be considered as the best targets for inhibiting biofilm formation (Dobretsov et al. 2009).

Over the last few decades, many types of AF paint have been developed, marketed, but finally abandoned due to issues related to environmental toxicity. The tragedy of TBT could have been avoided if more had been learned about the molecular mechanism of action before its introduction into the marine environment. Unfortunately, booster biocides are often introduced into the market before a clear understanding of their mode of action has been gained. Indeed, the molecular mechanisms of booster biocides used currently in marine paints remain largely unknown as, until now, most studies have focused on their acute toxicity to marine organisms.

In contrast, although a large number of natural products with effective AF activities have been isolated and characterized, few of these compounds have been commercialized and introduced into the market due to the limited information available on their mode of action, possible environmental effects and environmental fate. A better understanding of the mechanisms of these effective, ‘environmentally friendly’ AF compounds will help the identification of the molecular biomarkers for the rapid screening of AF activity (Qian, Xu et al. 2010). However, given the lack of precise information about the genomic and proteomic background of major fouling organisms, identifying molecular targets and mapping the mechanisms associated with AF activity remains a challenge. In conclusion, it is essential to gain a better understanding of the molecular mechanisms associated with the settlement and metamorphosis of fouling organisms.

Acknowledgments

The authors would like to thank the three reviewers and the editor for their constructive comments on the MS. This study was supported by a grant from China Ocean Mineral Resources Research and Development (DY125-15-T-02), and an award (SA-C0040/UK-C0016) from the King Abdullah University of Science and Technology to P-Y Qian.