Article in press - uncorrected proof Botanica Marina 51 (2008): 191–201  2008 by Walter de Gruyter • Berlin • New York. DOI 10.1515/BOT.2008.027

Antifouling activity of natural products from Brazilian seaweedsa

Bernardo A.P. da Gama1,*, Ana G.V. Carvalho1, Kerstin Weidner2, Angelica R. Soares3, Ricardo Coutinho4, Beatriz G. Fleury5, Valeria L. Teixeira1 and Renato C. Pereira1 Programa de Po´s-Graduac¸a˜o em Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, Caixa Postal 100.644, CEP 24.001-970, Nitero´i, Rio de Janeiro, Brazil, e-mail: [email protected] 2 Interdisciplinary Ecology Center, Technical University Bergakademie Freiberg, Leipziger Straße 29, 09599 Freiberg, Germany 3 Nu´cleo em Ecologia e Desenvolvimento So´cioambiental de Macae´, Universidade Federal do Rio de Janeiro, Rua Rotary Club s/no., CEP 27901-000, Macae´, Rio de Janeiro, Brazil 4 Instituto de Estudos do Mar Almirante Paulo Moreira, Rua Kioto 253, CEP 28930-000, Arraial do Cabo, Rio de Janeiro, Brazil 5 Departamento de Ecologia, Universidade do Estado do Rio de Janeiro, Rua Sa˜o Francisco Xavier 524, PHLC Sala 220, CEP 20559-900, Rio de Janeiro, Brazil 1

* Corresponding author

Abstract Antifouling chemical defense is likely an evolutionary response to the ecological disadvantages of epibiosis, particularly for photosynthetic organisms. Seaweed natural products with antifouling activity can provide effective, environmentally friendly alternatives to currently used antifouling paint booster biocides. The aim of this work was to assess the antifouling potential of natural products from Brazilian littoral seaweeds. Crude organic extracts from 51 populations comprising 42 species of macroalgae from eight locations along the Brazilian coast were tested against a relevant fouling organism in laboratory bioassays, the brown mussel Perna perna. In five cases, antifouling activities of purified compounds were also tested. A total of nine macroalgae were also cultured and tested for the presence of inducible defenses against fouling. Ecologically relevant field tests were performed in 11 cases to confirm laboratory results. Despite the unbalanced number of macroalgae tested among different localities, there seems to be no latitudinal trend of increased antifouling activity towards lower latitudes, where fouling pressure is presumed to be higher. However, there was a clear phylogenetic pattern in antifouling activity, with red macroalgae having the highest proporThe outline of this paper was presented at the 13th International Congress on Marine Corrosion and Fouling, Rio de Janeiro, Brazil, 23–28 July 2006.

a

tion (55%) of active species (moderate or strong fouling inhibition), followed by brown macroalgae (14%). Green seaweeds never exhibited strong antifouling activity (G80% inhibition of byssal attachment relative to controls). Some degree of induced antifouling defense was observed in seven species (78%). These results appear to support known trends of secondary metabolite production among seaweeds and suggest that research efforts should be focused on tropical red macroalgae in the quest for new antifoulants. On the other hand, it seems clear that macroalgal groups, such as green algae, must have mechanisms of defenses against fouling that are not chemical. Keywords: antifoulants; biofouling; epibiosis; marine chemical ecology; secondary metabolites.

Introduction Biofouling is a conspicuous process in marine environments (Taylor and Wilson 2003, Railkin 2003). Overgrowth by fouling (epibiosis) may be detrimental to marine organisms serving as substrata, including seaweeds and marine invertebrates. Seaweeds should be particularly susceptible to fouling as they are sessile and restricted to the photic zone where conditions for fouling establishment and growth are optimal (de Nys et al. 1995). However, the consequences of epibiosis for host seaweeds are still inadequately known, but include reduced growth and reproduction (Orth and van Montfrans 1984, Brawley 1992, Williams and Seed 1992), increased drag and losses of algal tissue during storms (Dixon et al. 1981, Brawley 1992, Williams and Seed 1992), or losses to consumers that are attracted by palatable epibionts (Bernstein and Jung 1979, Pereira et al. 2003). Other effects caused by epibionts may include reduction of light for photosynthesis and nutrient depletion (Buschmann and Gome´z 1993). On the other hand, epibiosis can sometimes provide a defense or camouflage to seaweeds, such as bryozoans on kelps (Durante and Chia 1991), and epiphytes on other seaweeds (Karez et al. 2000). Nevertheless, epibiosis generally seems to be harmful to host seaweeds, either directly or by attracting consumers, in what is sometimes called a ‘‘shared doom’’ effect (Wahl and Hay 1995). Antifouling chemical defense has long been acknowledged (Davis et al. 1989, Pawlik 1992, Rittschof 2001, Steinberg et al. 2001, Fusetani 2004) and is probably an evolutionary response to the ecological disadvantages of epibiosis, particularly in photosynthetic organisms. Marine macroalgae as a whole are known to produce as many as 3600 secondary metabolites (from a total of ca. 15,000 marine natural products, according to Bhadury

2008/27

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and Wright 2004), most of which play ecological roles yet unknown. Commonly, defense against herbivores is presumed to be the main ecological role of seaweed secondary metabolites (Hay 1996), although a growing body of evidence points toward other/multiple roles, such as defense against epibiosis or antifouling (Pawlik 1992, Rittschof 2001, Steinberg et al. 2001, da Gama et al. 2002), which increase their adaptive value (Pereira and da Gama 2008). However, very few studies have systematically investigated the role of these compounds as antifoulants (but see Hellio et al. 2002). The fouling of ship hulls is one of the major problems currently facing marine technology (da Gama et al. 2003) and various types of antifouling paints have been applied in the past, including organotin-based paints, such as tributyltin (Willemsen and Ferrari 1993, Ponasik et al. 1998, Evans et al. 2000), until recently, when they were found to be toxic to many marine organisms. The use of these compounds is now either prohibited or controlled in many countries and was banned by the International Maritime Organization in January 2008 (Suzuki et al. 1992, Burridge et al. 1995, Horiguchi et al. 1995, Ruiz et al. 1995, Fent 1996, Grinwis et al. 1998, Hashimoto et al. 1998, Abarzua et al. 1999, Fischer et al. 1999, Hunter and Anderson 2000, Burgess et al. 2003, Omae 2003, Stupak et al. 2003, Yebra et al. 2004). At least 18 different booster biocides are used as alternative antifouling paints at present, but they may also pose a threat to the aquatic environment. Indeed, even the so-called ‘‘biocide-free’’ antifouling paints are toxic to marine organisms, as has been demonstrated recently (Karlsson and Eklund 2004, Lo¨schau and Kra¨tke 2005). Therefore, there is an urgent quest for new ‘‘environmentally friendly’’ antifoulants (Chambers et al. 2006). Many researchers try to employ chemical defense systems from marine life forms for this purpose (Rittschoff 2001, Fusetani 2004). Antifouling marine natural products have been recognized as a promising alternative to commercial antifoulants (Rittschof 2001, Bhadury and Wright 2004). Screening for seaweed natural products with antifouling activity can provide effective, environmentally friendly alternatives to currently used antifouling paint booster biocides (da Gama et al. 2002), while helping to understand the ecological functions and mechanisms of production of secondary metabolites. The aim of this work was to assess the antifouling potential of natural products from seaweeds of the wide Brazilian littoral, southwestern Atlantic Ocean, and is part of a broader, ongoing project dedicated to screening Brazilian marine organisms (algae and marine invertebrates) for their potential as producers of secondary metabolites with antifouling activity (da Gama and Pereira 1995, Pereira et al. 2002, da Gama et al. 2002, 2003, Clavico et al. 2006, Epifanio et al. 2006, Barbosa et al. 2007).

immediately transferred to the laboratory in isothermic boxes, where they were gently washed in seawater, sorted, cleaned from associated biota, and biovolume was determined (by water displacement in a graduated cylinder). Macroalgae were then identified and either freezedried or dried at room temperature to constant weight (dry weight, DW). Selection of seaweed species was mainly by biomass, which should be sufficient to allow laboratory and field testing and compound purification, where appropriate. Sampling sites were spread along 188 N (one site only) and 278 S (Table 1), most frequently at accessible places, such as in Rio de Janeiro, Sa˜o Paulo, and Espı´rito Santo states. Crude, whole-algal extracts and/or purified compounds from 51 seaweed assemblages comprising 42 species were prepared by immersing dried algae in pure dichloromethane (DCM, analytical grade; Merck, Darmstadt, Germany) or a mix (2:1) of dichloromethane and methanol (MeOH). The mixture was then filtered and solvents eliminated under reduced pressure in a rotatory evaporator at room temperature. Each species was extracted in triplicate. The remainder was then weighed (0.0001 g precision digital scale) and stored at -108C prior to use in bioassays. Further studies were, in some cases, performed with pure compounds obtained through bioassay-guided fractionation by usual phytochemical procedures (i.e., silica-gel column chromatography, preparative thin-layer chromatography) and identification through comparison of 13C- and 1H-NMR spectroscopic data with the relevant literature (data not shown). All the extracts/compounds were tested at natural concentrations, as detailed later. Authorities for the algal binomials are presented in Table 1. Induction treatments In nine cases (Lobophora variegata, Sargassum vulgare, Dictyota menstrualis, Stypopodium zonale, Osmundaria obtusiloba, Gracilaria cearensis, Pterocladiella capillacea, Chondrophycus flagellifera, and Codium decorticatum), seaweeds were submitted to stressful conditions (e.g., herbivore damage, HD) before the extraction process, for subsequent comparison with control macroalgae (nondamaged, ND) to test for defense induction (e.g., Karban et al. 1999, Weidner et al. 2004, Toth et al. 2005). Herbivore induction was performed exactly as described in a previous work (Weidner et al. 2004), i.e., stressed macroalgae were cultured in the laboratory for 7 days exposed to grazing by amphipods wElasmopus brasiliensis (Dana 1855), 30 individuals per experimental unit, ns5 per treatmentx prior to extraction. Control macroalgae were cultured under the same conditions except for absence of herbivores. Likewise, one red seaweed (Cryptonemia seminervis) was found simultaneously with and without epibionts, and both were tested to verify whether epibiont presence would elicit a defense response (with or without epibiosis in Figure 3).

Materials and methods Sampling of marine algae and extract preparation Seaweeds were collected by hand while SCUBA or free diving in the shallow subtidal zone. Seaweeds were

Antifouling assays with the mussel Perna perna Extracts and pure compounds (Figures 1–3) were tested in laboratory bioassays against the brown mussel Perna perna (Linnaeus, 1758). Mussels are relevant fouling

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Table 1 List of macroalgae tested by phylum, latitude and provenance. Division

Taxa assayed

Latitude

Provenance

Chlorophyta Phaeophyta Chlorophyta Phaeophyta Phaeophyta Rhodophyta Phaeophyta Rhodophyta Rhodophyta Rhodophyta

Penicillus sp. Stypopodium zonale (J.V. Lamouroux) Papenfuss Caulerpa cupressoides (West) C. Agardh Dictyota ciliolata Sonder ex Ku¨tzing Dictyota pfaffii Schnetter Laurencia sp. 1 Stypopodium zonale Laurencia aldingensis Saito et Womersley Laurencia scoparia C. Agardh Chondrophycus translucida (Fujii et Cordeiro-Marino) Garbary et Harper Anadyomene stellata (Wulfen) C. Agardh Chondria sp. Codium isthmocladum Vickers Codium intertextum F.S. Collins et Hervey Gracilaria domingensis (Ku¨tzing) Sonder ex Dickie Halimeda sp. Hydropuntia caudata (J. Agardh) Gurgel et Fredericq Ochtodes secundiramea (Montagne) M.A. Howe Zonaria tournefortii (J.V. Lamouroux) Montagne Ochtodes secundiramea Laurencia caduciramulosa Masuda et Kawaguchi Laurencia filiformis (C. Agardh) Montagne Codium decorticatum (Woodward) M.A. Howe Padina gymnospora (Ku¨tzing) Sonder Lobophora variegata (J.V. Lamouroux) Womersley ex Oliveira Peyssonnelia capensis Montagne Stypopodium zonale Condrophycus flagellifera (J. Agardh) K.W. Nam Cryptonemia seminervis (C. Agardh) J. Agardh Dictyota menstrualis (Hoyt) Schnetter: Ho¨rning et Weber-Peukert Gracilaria cearensis (Joly et Pinheiro) Joly et Pinheiro Laurencia sp. 2 Laurencia variegata Osmundaria obtusiloba (Mertens ex C. Agardh) R.E. Norris Pterocladiella capillacea (S.G. Gmelin) Santelices et Hommersand Sargassum vulgare C. Agardh Laurencia obtusa (Hudson) J.V. Lamouroux Laurencia obtusa Laurencia arbuscula Sonder Bryothamnion seaforthii (Turner) Ku¨tzing Dictyopteris delicatula J.V. Lamouroux Heterosiphonia gibbesii (Harvey) Falkenberg Jania rubens (L.) J.V. Lamouroux Laurencia filiformis Chaetomorpha sp. Hypnea sp. Laurencia sp. Ulva sp.

188 N 38 S 58 S 58 S 58 S 58 S 178 S 198 S 198 S 198 S

Jamaica PE: Fernando de Noronha Archipelago RN: Atol das Rocas Archipelago RN: Atol das Rocas Archipelago RN: Atol das Rocas Archipelago RN: Atol das Rocas Archipelago BA: Abrolhos Archipelago ES: Anchieta ES: Marataı´zes ES: Anchieta

208 208 208 208 208 208 208 208 208 208 228 228 228 228 238 238 238 238 238 238

S S S S S S S S S S S S S S S S S S S S

ES: Praia da Baleia ES: Praia da Baleia ES: Praia da Baleia ES: Praia da Baleia ES: Praia da Baleia ES: Praia da Baleia ES: Praia da Baleia ES: Praia da Baleia ES: Praia da Baleia ES: Praia de Camburi RJ: Angra dos Reis RJ: Angra dos Reis RJ: Praia de Itaipu, Nitero´i RJ: Praia de Itaipu, Nitero´i RJ: Praia do Forno, Bu´zios RJ: Praia do Forno, Bu´zios RJ: Praia do Forno, Bu´zios RJ: Praia Rasa, Bu´zios RJ: Praia Rasa, Bu´zios RJ: Praia Rasa, Bu´zios

238 238 238 238 238

S S S S S

RJ: RJ: RJ: RJ: RJ:

238 238 238 248 248 248 248 248 248 278 278 278 278

S S S S S S S S S S S S S

RJ: Praia Rasa, Bu´zios RJ: Prainha: Arraial do Cabo RJ: Cabo Frio Island, A. do Cabo SP: Ilha das Couves, Ubatuba SP: Praia Branca SP: Praia Branca SP: Praia Branca SP: Praia Branca SP: Praia Brava, Ubatuba SC: Praia do Poa´ SC: Praia do Poa´ SC: Praia do Poa´ SC: Praia do Poa´

Chlorophyta Rhodophyta Chlorophyta Chlorophyta Rhodophyta Chlorophyta Rhodophyta Rhodophyta Phaeophyta Rhodophyta Rhodophyta Rhodophyta Chlorophyta Phaeophyta Phaeophyta Rhodophyta Phaeophyta Rhodophyta Rhodophyta Phaeophyta Rhodophyta Rhodophyta Phaeophyta Rhodophyta Rhodophyta Phaeophyta Rhodophyta Rhodophyta Rhodophyta Rhodophyta Phaeophyta Rhodophyta Rhodophyta Rhodophyta Chlorophyta Rhodophyta Rhodophyta Chlorophyta

Praia Praia Praia Praia Praia

Rasa, Rasa, Rasa, Rasa, Rasa,

Bu´zios Bu´zios Bu´zios Bu´zios Bu´zios

Abbreviations for Brazilian states: PE, Pernambuco; RN, Rio Grande do Norte; BA, Bahia; ES, Espı´rito Santo; RJ, Rio de Janeiro; SP, Sa˜o Paulo; SC, Santa Catarina.

organisms in an ecological context, as they are usually found settled on seaweeds (Petersen 1984, Eyster and Pechenik 1988, Davis and Moreno 1995, Lasiak and Barnard 1995, Alfaro et al. 2004). Juvenile mussels were collected during low tide from the rocky coastal area of Itaipu (Nitero´i city, Rio de Janeiro, Brazil) and kept in a 400-l recirculating laboratory aquarium equipped with biological filtering, protein skimming and activated carbon at constant temperature (ca. 208C), salinity (ca. 35)

and aeration for 12 h. The mussels were then carefully disaggregated by cutting the byssal threads and those exhibiting substratum exploring behavior (actively exposing their foot and crawling) were selected for experiments. Antifouling activity was measured by the following procedure wdescribed in detail in da Gama et al. (2003)x modified from the method described by Ina et al. (1989) and Goto et al. (1992). Water-resistant filter paper was cut into 9-cm diameter circles and soaked in solvent

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Figure 1 Antifouling activity (% inhibition relative to mussel byssal attachment in controls) of algal extracts from the division Chlorophyta. In total, 10 replicates per treatment were carried out. Significant results are indicated whenever p-0.05. HD, algae submitted to herbivore damage; ND, non-damaged algae; RJ, Rio de Janeiro state.

Figure 2 Antifouling activity (% inhibition relative to mussel byssal attachment in controls) of algal extracts or pure compounds from the division Phaeophyta. In total, 10 replicates per treatment were carried out. Significant results are indicated whenever p-0.05. BA, Bahia; HD, algae submitted to herbivore damage; ND, non-damaged algae; PE, Pernambuco; RJ, Rio de Janeiro; RN, Rio Grande do Norte.

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Figure 3 Antifouling activity (% inhibition relative to mussel byssal attachment in controls) of algal extracts or pure compounds from the division Rhodophyta. In total, 10 replicates per treatment were carried out. Significant results are indicated whenever p-0.05. HD, algae submitted to herbivore damage; ND, non-damaged algae; w2xx, double natural concentration.

(control filter). Another 9-cm diameter set of filter papers (treatment filters) was cut in a chessboard pattern (1-cm squares) and soaked in a natural concentration of extracts, bioassay-guided fractions (data not shown) or purified compounds (determined as the extract equivalent to the DW of algasDW of filter paper). All filter paper circles were allowed to air dry. Entire filter circles were then placed in the bottom of sterile polystyrene Petri dishes, over which treated chessboard filters were placed. Dishes were filled with 80 ml of seawater and three mussels (1.5–2.5 cm length) added. In this way, mussels would have the same area of treated (superior, squared) and control (inferior, entire) filter paper on which to attach. A total of 10 replicates of each treatment were always used. Experimental dishes were kept in total darkness, as mussels have been shown to produce more byssal threads when held in the dark (Davis and Moreno 1995). Experiments were allowed to run for 12 h. Mussel attachment was recorded immediately after the start of the experiment and then after 12 h, when all records of attachment were checked, mussels were placed in plastic mesh bags tagged according to treatment, and suspended in a marine aquarium for 24 h to check for possible mortality due to exposure to the test substances (data not shown). After the trials, filter papers from some treatments were taken from dishes and allowed to air dry. The filter papers were then re-extracted, the solvents evaporated and the residue remaining applied to a thinlayer chromatography plate for comparison with the original crude extracts. This was necessary to ensure that

test extracts or compounds did not degrade during the experiments. Antifouling activity in the field In 11 cases (Table 2), antifouling activities of extracts/ purified compounds were also tested through more ecologically relevant field tests to confirm laboratory results. The method employed was modified from Henrikson and Pawlik (1995) by da Gama et al. (2002, 2003) and has some advantages over traditional field tests. Extracts or compounds are incorporated in a gel (at natural volumetric concentrations found within seaweeds) from where they can slowly diffuse into the water in a manner similar to that occurring in a living organism. Extracts are dispersed in through the gel (not only on the surface), which helps preserve the physical properties of the gel surface; hence, differences in larval settlement can only be attributed to chemical properties of the extracts. Finally, settlement of foulers on the experimental gel occurs under natural conditions of flow and diffusion, being exposed to a natural supply of larvae and spores of algae. Macroalgal extracts mixed with phytagel (Sigma-Aldrich, St Louis, USA) were poured into sterile polystyrene Petri dishes (treatments). Solvent alone was added to phytagel in the controls. Gel plates were prepared using a mixture of 1.52 g of phytagel and 35 ml of distilled water heated to boiling point. The mixture was vigorously stirred with a glass rod while 0.5 ml of methanol (or extract) was added, then poured into the circular

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Table 2 Algal extracts (or pure compound, within parentheses) tested for antifouling activity through field experiments, their provenance and synthesis of the main results obtained. Taxa Phaeophyta Canistrocarpus cervicornis (Ku¨tzing) De Paula et De Clerck Dictyota menstrualis (Hoyt) Schnetter, Ho¨rning et Weber-Peukert Dictyota pfaffii Schnetter Stypopodium zonale (J.V. Lamouroux) Papenfuss Rhodophyta Laurencia obtusa (Hudson) J.V. Lamouroux Laurencia obtusa (elatol) Laurencia obtusa Laurencia arbuscula Sonder Laurencia filiformis (C. Agardh) Montagne Laurencia sp. 1 Laurencia sp. 2

Provenance

Activity

RJ: Praia do Forno, Bu´zios RJ: Praia Rasa, Bu´zios

Biofilm stimulus Inactive

RN: Atol das Rocas RJ: Praia do Forno, Bu´zios

Active during 3rd week Biofilm stimulus

RJ: Ilha de Cabo Frio, Atol do Cabo RJ: Ilha de Cabo Frio, Atol do Cabo RJ: Prainha, Arraial do Cabo SP: Ilha das Couves, Ubatuba SP: Praia Brava, Ubatuba RN: Atol das Rocas RJ: Praia Rasa, Bu´zios

Strong macrofouling inhibition Strong macrofouling inhibition Strong macrofouling inhibition Inactive Inactive Inactive Inactive

Abbreviations for Brazilian states: PE, Pernambuco; RN, Rio Grande do Norte; BA, Bahia; ES, Espı´rito Santo; RJ, Rio de Janeiro; SP, Sa˜o Paulo; SC, Santa Catarina.

moulds (Petri dishes) for hardening. The extract/compound added to every 35 ml of gel was equivalent to an extract of 35 ml of fresh seaweed (endeavor to maintain the natural metabolite concentrations). Gels were kept in circular molds so as to allow extract diffusion through only one side of the experimental plates. This would theoretically reduce diffusion rates by one-half relative to the method of Henrikson and Pawlik (1995), as estimated in a previous study (da Gama et al. 2002). In each experiment, six 35-ml replicates were prepared for each extract/compound and for the control. One replicate of each treatment and one control replicate were randomly arranged and fastened to each one of six rectangular aluminum structures. These represented independent experimental units, avoiding problems of pseudoreplication (Hurlbert 1984). The structures were then submerged at a depth of 1 m, secured to a swivel (to ensure orientation parallel to water flow) to flotation rafts moored at Cabo Frio Island (Arraial do Cabo city, state of Rio de Janeiro, 238009040 S, 428009210 W). Settlement of foulers in the field was measured in situ weekly as percentage cover, using a dot-grid method (Foster et al. 1991). A high number of points (235) were used to avoid underestimating rare species and to reduce deviation among replicates (Dethier et al. 1993). To prevent death of fouling organisms, each structure was kept in a large aluminum tray containing seawater during measurements. The biofilm cover was measured by means of its macroscopic manifestation, i.e., the growth of microorganisms on gels created an easily estimated conspicuously thin, colored layer, covering an area of the plate. Biofilm samples taken to the laboratory always contained bacteria, benthic diatoms and ciliated protists, and sometimes small filamentous macroalgae, mainly Ectocarpaceae. In some cases, gels were cut into small pieces after the field assay, allowed to dry, and re-extracted with the solvents used previously. Thin-layer chromatography analysis was then performed to carry out a comparison with the original extracts, ensuring that experimental results were not confounded by degradation byproducts.

Statistical analysis One-way analyses of variance (ANOVA) were performed to compare mussel byssal thread attachment data among treatments. Whenever ANOVA detected significant results, Dunnett’s post hoc test was employed to compare treatments (extracts, pure compounds) with controls. When assumptions of normality and variance homogeneity were not met, even after data transformations, Kruskal-Wallis non-parametric tests were run. Differences were considered significant when p-0.05 (as5%).

Results A total of 42 seaweed species from eight provenances along the Brazilian coast were tested under different circumstances, comprising 68 extracts or pure compounds. From these, nine species were Chlorophyta (21%), nine were Phaeophyta (21%) and 24 were Rhodophyta (57%, Table 1). All results were expressed as mean percentage inhibition of mussel byssal thread attachment relative to the respective controls (i.e., antifouling activity). From the 11 test results with green macroalgae (Figure 1), three revealed significant antifouling activity (27%), although in all cases the intensity was moderate (i.e., fouling inhibition significant, but below 80% effect size, Figure 5). Of the 21 results from brown macroalgae (Figure 2), only three (14%) significantly inhibited fouling, but strongly so (Figure 5). On the other hand, two extracts and one purified compound from one of them had significant stimulatory activity (i.e., stimulated rather than inhibited mussel attachment). The largest group tested comprised red algae (36 tests, Figure 3), and the majority (20% or 55%) possessed antifouling activity, with more than 50% having strong fouling inhibition, including a pure compound (Figure 5). Despite the unequal number of macroalgae sampled by latitude (see Table 1), there was no correlation

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exhibited strong antifouling activity and five were inactive against fouling). Among the five pure compounds isolated and tested, namely the sesquiterpene elatol from the red seaweed Laurencia obtusa, a dolabellane diterpene from the brown alga Dictyota pfaffii, the diterpenes epitaondiol and atomaric acid, and the pigment fucoxanthin from the brown alga Stypopodium zonale, only two (elatol and the dolabellane diterpene) had significant antifouling activity (Figures 2 and 3), in both cases confirmed by further field testing (Table 2).

Discussion

Figure 4 Relationship between latitude of screened algal collection and antifouling activity (% inhibition relative to mussel byssal attachment in controls). Different symbols indicate macroalgal divisions. Negative latitude values indicate northern latitude, while positive values indicate southern latitude.

between latitude of origin and antifouling activity (Figure 4). Red algae had by far the highest proportion (55%) of active extracts (moderate or strong fouling inhibition), followed by brown algae (14%). Green seaweeds never exhibited strong antifouling activity (significant, G80% fouling inhibition relative to controls, Figure 5). From the nine seaweed species tested for induced antifouling defense, seven (78%) exhibited some degree of defense induction (Figures 1–3) after exposure to herbivore damage (HD) when compared to non-damaged (ND) control macroalgae (Figures 1–3). Significant defense inductions were recorded for Codium decorticatum (Chlorophyta) and Gracilaria cearensis (Rhodophyta). For Cryptonemia seminervis, tests of specimens collected with and without epibiosis showed that when epibiosis was present there was a significant fouling inhibition (Figure 3). Ecologically relevant field tests were performed in 11 cases to confirm laboratory results (Table 2, detailed data not shown). Three responses were from brown macroalgae (one inactive, one with antifouling activity and one stimulating fouling, in accordance with the laboratory results) and eight from red macroalgae (of which three

From the 68 extracts or pure compounds from Brazilian seaweeds tested against fouling in the present study, 26 (38%) showed some significant degree of fouling inhibition in the laboratory. Strong antifouling activity was observed for 16 macroalgal extracts (23.5%), the majority of which (12.75%) were from red seaweeds. On the other hand, a significant stimulation of mussel attachment was observed in three cases (all of them extracts/pure compound from the brown alga Stypopodium zonale). Field experiments performed to corroborate laboratory results showed fouling inhibition in four out of 11 cases (36%), of which three (75%) were rhodophyceans. These results suggest that, at least in the broad Brazilian littoral (ca. 8000 km), antifouling chemical defense is relatively widespread among the macroalgae. The absence of significant mortality of the test organisms points toward non-toxic inhibition of fouling (mortality data not shown). Despite the unbalanced number of macroalgae tested between different localities, there seems to be no latitudinal trend of increased antifouling activity towards lower latitudes, where fouling pressure is presumed to be higher (Railkin 2003). The concept that tropical seaweeds are better defended than their temperate counterparts has been widely suggested in the literature (Bolser and Hay 1996), especially in defense against herbivores, but this has also been disputed (e.g., Cetrulo and Hay 2000, Pereira and da Gama 2008). To the best of our knowledge, this is the first time that a latitudinal gradient in antifouling activity has been investigated. Despite the absence of latitudinal trends in antifouling defense, there was a clear phylogenetic pattern in antifouling activity, with red macroalgae showing the highest

Figure 5 Antifouling activity by algal phylum. Strong: significant, G80% inhibition relative to controls; moderate: significant, but -80% inhibition; inactive: any non-significant result (p)0.05).

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proportion (55%) of active species (moderate or strong fouling inhibition), followed by brown macroalgae (14%). Green seaweeds never exhibited strong antifouling activity (G80% inhibition of byssal attachment relative to controls). These results appear to support known trends of secondary metabolite production among seaweeds (see Harper et al. 2001, Blunt et al. 2005 for reviews) and appear to suggest that research efforts should be focused on the more prolific red macroalgae in the quest for new natural product antifoulants from seaweeds. On the other hand, it seems clear that algal groups, such as green macroalgae, must have mechanisms of defense other than chemical against fouling. Keats et al. (1994) and Littler and Littler (1999) described other antifouling mechanisms or strategies, such as epithallus sloughing and blade abandonment/proliferation, but those mechanisms have never been described for the majority of green algae. As pointed out by Steinberg and de Nys (2002), there is a number of largely unanswered fundamental questions about chemical inhibition of epibiota by seaweed secondary metabolites, e.g., (1) what effect do fouling organisms have on fitness of seaweeds, and therefore what is the selection pressure on seaweeds to evolve chemical defenses against fouling, and (2) how widespread is the deterrence of epibionts by seaweed secondary metabolites? The results presented here provide useful clues to both questions. First of all, if fouling pressure were such an intense selective pressure, antifouling chemical defenses would be expected to be evenly distributed across macroalgal phyla, which does not seem to be the case. In this study, defenses against fouling were clearly concentrated in the Rhodophyta, although our results should be interpreted with caution, as we used mainly laboratory assays employing only one fouling species. Secondly, antifouling defenses do not appear to follow latitudinal trends, which is in opposition to expectations, as fouling pressure is presumably higher in lower latitudes, where fouling development is faster and more intense (Railkin 2003). Similarly, some seaweeds appear to stimulate fouling, e.g., Stypopodium zonale (Littler et al. 1986, da Gama et al. 2002, 2003) and some of its metabolites. Marine macroalgae encompass lineages that diverged approximately one billion years ago. Recent results suggest that they feature natural immunity traits that are conserved, as well as others that appear to be phylum- or environment-specific (Potin et al. 2002). Within this context, antifouling chemical defense in seaweeds appears to be phylum-specific. On the other hand, from an analysis of the literature records of antifouling activity from seaweed natural products, such as the recent review by Bhadury and Wright (2004), it may be concluded that brown macroalgae are the major producers of antifouling defenses, in clear opposition to the results found here, where red macroalgae were by far more active against fouling. However, this could be a result of (1) a larger number of studies from temperate floras, in which brown macroalgae prevail, or (2) a large number of studies employing microbial assays in the laboratory as antifouling tests. The relationship between the inhibition of microorganisms and

inhibition of fouling in the field has long been controversial (Bakus et al. 1985, Steinberg and de Nys 2002), and thus the extrapolation of such results must be performed with caution. Another underappreciated aspect of antifouling chemical defense in seaweeds, besides latitudinal and phylogenetic variation, is variability in time or seasonality. Only two recent studies (Hellio et al. 2004, Marechal et al. 2004) have addressed this important question, which was neglected in the present work. Although there is a large body of evidence to show that algae are endowed with chemical defenses, the concept that many of these defenses are induced has emerged only recently. Therefore, Potin et al. (2002) suggest that the antifouling reactions of marine algae can no longer be based on the search for constitutive defense chemicals alone. Here, in addition to assessment of constitutive defenses, assays were performed to induce defense production (herbivore stress), and the majority of tested algae (88%) showed some degree of increased antifouling defense in comparison to control, undamaged algae; but temporal or seasonal variation in chemical defenses was not assessed. Weidner et al. (2004) found that of nine Brazilian algae tested for induced defense against herbivores, eight had at least a trend toward being less palatable after herbivore contact. The same trend seems to hold for antifouling chemical defenses, although more relevant studies should be performed, perhaps simulating the detrimental effects of epibiosis to properly assess antifouling responses. Many marine macroalgae, as well as benthic marine invertebrates, are relatively free of epibiosis (Paul 1992, Hellio et al. 2001, 2002, Steinberg et al. 2002, Kubanek et al. 2003) due to the production of biogenic compounds that possess antibacterial, antialgal, antifungal, antiprotozoan and anti-macrofouling properties. These agents are usually seaweed secondary metabolites (Abarzua and Jakubowski 1995, Abarzua et al. 1999, Etahiri et al. 2001, Hellio et al. 2001, 2002, Bhosale et al. 2002, da Gama et al. 2002, 2003, de Nys and Steinberg 2002). Therefore, the isolation and production of these natural products from marine macroalgae could be used effectively for the prevention of biofouling. On the other hand, Rittschoff (2001) highlighted a series of constraints for the use of natural products from marine organisms as commercial antifoulants, such as the difficulties in obtaining these compounds in large scale. We tested five pure compounds from marine algae as antifoulants, of which two exhibited significant antifouling activity in ecologically relevant field studies. Regardless of the limitations for the application of these compounds directly as commercial antifoulants, these molecules can serve as templates for the synthesis of new, environmentally friendly antifouling agents. In particular, smaller compounds (such as elatol) seem promising, although further studies are needed to elucidate its toxicology.

Acknowledgements The authors wish to thank many undergraduate and graduate students, as well as a number of collaborators, for their help in sampling algae or helping in experiments. B.A.P.G., R.C.P., V.L.T.

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and R.C. thank the Brazilian National Research Council, CNPq, for their Research Productivity fellowships. CNPq, CAPES and FAPERJ supported this research.

References

Abarzua, S. and S. Jakubowski. 1995. Biotechnological investigation for the prevention of biofouling. Biological and biochemical principles for the prevention of biofouling. Mar. Ecol. Prog. Ser. 123: 301–312. Abarzua, S., S. Jakubowski, S. Eckert and P. Fuchs. 1999. Biotechnological investigation for the prevention of marine biofouling II. Blue-green algae as potential producers of biogenic agents for the growth inhibition of microfouling organisms. Bot. Mar. 42: 459–465. Alfaro, A.C., A.G. Jeffs and R.G. Creese. 2004. Bottom-drifting algal/mussel spat associations along a sandy coastal region in northern New Zealand. Aquaculture 241: 269–290. Bakus, G.J., B. Schulte, S. Jhu, M. Wright, M. Green and P. Gome´z. 1990. Antibiosis and antifouling in marine sponges: laboratory versus field studies. In: (K. Ruetzler, ed.). New Perspectives in Sponge Biology. Proc. 3rd Int. Sponge Conf. WHOI, Woods Hole, MA. pp. 102–108. Barbosa, J.P., B.G. Fleury, B.A.P. da Gama, V.L. Teixeira and R.C. Pereira. 2007. Natural products as antifoulants in the Brazilian brown alga Dictyota pfaffii (Phaeophyta, Dictyotales). Biochem. Syst. Ecol. 35: 549–553. Bernstein, B.B. and N. Jung. 1979. Selective pressure and coevolution in a kelp canopy community in Southern California. Ecol. Monogr. 493: 335–355. Bhadury, P. and P.C. Wright. 2004. Exploitation of marine algae: biogenic compounds for potential antifouling applications. Planta 219: 561–578. Bhosale, S.H., V.L. Nagle and T.G. Jagtap. 2002. Antifouling potential of some marine organisms from India against species of Bacillus and Pseudomonas. Mar. Biotechnol. 4: 111–118. Blunt, J.W., B.R. Copp, M.H.G. Munro, P.T. Northcote and M.R. Prinsep. 2005. Marine natural products. Nat. Prod. Rep. 22: 15–61. Bolser, R.C. and M.E. Hay. 1996. Are tropical plants better defended? Palatability and defenses of temperate vs. tropical seaweeds. Ecology 77: 2269–2286. Brawley, S.H. 1992. Mesoherbivores. In: (D.H. John, S.J. Hawkins and J.H. Price, eds.) Plant-animal interactions in the marinebenthos. Systematics Association Special Volume 46. Clarendon, Oxford. pp. 235–263. Burgess, J.G., K.G. Boyd, E. Armstrong, Z. Jiang, L.M. Yan, M. Berggren, U. May, T. Pisacane, A. Granmo and D.R. Adams. 2003. The development of a marine natural product-based antifouling paint. Biofouling 19: 197–205. Burridge, T.R., T. Lavery and P.K.S. Lam. 1995. Effects of tributyltin and formaldehyde on the germination and growth of Phyllospora comosa (Labillardiere) C. Agardh (Phaeophyta: Fucales). Bull. Environ. Contam. Toxicol. 55: 525–532. Buschmann, A.H. and P. Gome´z. 1993. Interaction mechanisms between Gracilaria chilensis (Rhodophyta) and epiphytes. Hydrobiologia 261: 345–351. Cetrulo, G.L. and M.E. Hay. 2000. Activated chemical defenses in tropical versus temperate seaweeds. Mar. Ecol. Prog. Ser. 207: 243–253. Chambers, L.D., K.R. Stokes, F.C. Walsh and R.J.K. Wood. 2006. Modern approaches to marine antifouling coatings. Surf. Coat. Technol. 201: 3642–3652. Clavico, E.E.G., G. Muricy, B.A.P. da Gama, D. Batista, C.R.R. Ventura and R.C. Pereira. 2006. Ecological roles of natural products from the marine sponge Geodia corticostylifera. Mar. Biol. 148: 479–488.

da Gama, B.A.P. and R.C. Pereira. 1995. Produtos na˜o¸ ˜ o. Cieˆn. Hoje 19: 16–25. poluentes contra a bioincrustaca da Gama, B.A.P., R.C. Pereira, A.G.V. Carvalho, R. Coutinho and Y. Yoneshigue-Valentin. 2002. The effects of seaweed secondary metabolites on biofouling. Biofouling 18: 13–20. da Gama, B.A.P., R.C. Pereira, A.R. Soares, V.L. Teixeira and Y. Yoneshigue-Valentin. 2003. Is the mussel test a good indicator of antifouling activity? A comparison between laboratory and field assays. Biofouling 19: 161–169. Davis, A.R. and C.A. Moreno. 1995. Selection of substrata by juvenile Choromytilus chorus (Mytilidae): are chemical cues important? J. Exp. Mar. Biol. Ecol. 191: 167–180. Davis, A.R., N.M. Targett, O.J. McConnel and C.M. Young. 1989. Epibiosis of marine algae and benthic invertebrates: natural products chemistry and other mechanisms inhibiting settlement and overgrowth. Bioorg. Mar. Chem. 3: 85–114. de Nys, R. and P.D. Steinberg. 2002. Linking marine biology and biotechnology. Curr. Opin. Biotechnol. 13: 244–248. de Nys, R., P.D. Steinberg, P. Willemsen, S.A. Dworjanyn, C.L. Gabelish and R.J. King. 1995. Broad spectrum effects of secondary metabolites from the red alga Delisea pulchra in antifouling assays. Biofouling 8: 259–271. Dethier, M.N., E.S. Graham, L. Cohen and M. Tear. 1993. Visual versus random-point percent cover estimations: ‘objective’ is not always better. Mar. Ecol. Prog. Ser. 96: 93–100. Dixon, J., S.C. Schroeter and J. Kastendiek. 1981. Effects of the encrusting bryozoan, Membranipora membranacea, on the loss of blades and fronds by the giant kelp, Macrocystis pyrifera (Laminariales). J. Phycol. 17: 341–345. Durante, K.M. and F. Chia. 1991. Epiphytism on Agarum fimbriatum: can herbivore preferences explain distributions of epiphytic bryozoans? Mar. Ecol. Prog. Ser. 77: 279–287. Epifanio, R.A., B.A.P. da Gama and R.C. Pereira. 2006. Epoxypukalide as the antifouling agent from the Brazilian endemic sea fan Phyllogorgia dilatata Esper (Octocorallia, Gorgoniidae). Biochem. Syst. Ecol. 34: 446–448. Etahiri, S., V. Bultel-Ponce´, C. Caux and M. Guyot. 2001. New bromoditerpenes from the red alga Sphaerococcus coronopifolius. J. Nat. Prod. 64: 1024–1027. Evans, S.M., A.C. Birchenough and M.S. Brancato. 2000. The TBT ban: out of the frying pan into the fire? Mar. Pollut. Bull. 40: 204–211. Eyster, L.S. and J.A. Pechenik. 1988. Attachment of Mytilus edulis L. larvae on algal and byssal filaments is enhanced by water agitation. J. Exp. Mar. Biol. Ecol. 114: 99–110. Fent, K. 1996. Ecotoxicology of organotin compounds. Crit. Rev. Toxicol. 26: 1–117. Fischer, W.S., L.M. Oliver, W.W. Walker, C.S. Manning and T.F. Lytle. 1999. Decreased resistance of eastern oysters (Crassostrea virginica) to a protozoal pathogen (Perkinsus marinus) after sublethal exposure to tributyl tin oxide. Mar. Environ. Res. 47: 185–201. Foster, M.S., C. Harrold and D.D. Hardin. 1991. Point versus photo quadrat estimates of the cover of sessile marine organisms. J. Exp. Mar. Biol. Ecol. 146: 193–203. Fusetani, N. 2004. Biofouling and antifouling. Nat. Prod. Rep. 21: 94–104. Goto, R., R. Kado, K. Muramoto and H. Kamiya. 1992. Fatty acids as antifoulants in a marine sponge. Biofouling 6: 61–68. Grinwis, G.C.M., A. Boonstra, E.J. van den Brandhof, J.A.M.A. Dormans, M. Englesma, R.V. Kuiper, H. van Loveren, P.W. Wester, M.A. Vaal, A.D. Vethaak and J.G. Vos. 1998. Shortterm toxicity of bis (tri-n-butyl tin) oxide in flounder (Platichthys flesus): pathology and immune function. Aquat. Toxicol. 42: 15–36. Harper, M.K., T.S. Bugni, B.R. Copp, R.D. James, B.S. Lindsay, A.D. Richardson, P.C. Schnabel, D. Tasdemir, R.M. Van Wagoner, S.M. Verbitski and C.M. Ireland. 2001. Introduction to the chemical ecology of marine natural products. In: (J.B. McClintock and B.J. Baker, eds.) Marine chemical ecology. CRC, London. pp. 3–69.

Article in press - uncorrected proof 200 B.A.P. da Gama et al.: Brazilian seaweed antifoulants

Hashimoto, S., M. Watanabe, Y. Noda, T. Hayashi, Y. Kurita, Y. Takasu and A. Otsuki. 1998. Concentration and distribution of butyltin compounds in a heavy tanker route in the Strait of Malacca and in Tokyo Bay. Mar. Environ. Res. 45: 169–177. Hay, M.E. 1996. Marine chemical ecology: what’s known and what’s next? J. Exp. Mar. Biol. Ecol. 200: 103–134. Hellio, C., D. de La Broise, L. Dufosse´, Y. Le Gal and N. Bourgougnon. 2001. Inhibition of marine bacteria by extracts of macroalgae: potential use for environmentally friendly antifouling paints. Mar. Environ. Res. 52: 231–247. Hellio, C., J.P. Berge, J.P. Beaupoil, C. Le Gal and N. Bourgougnon. 2002. Screening of marine algal extracts for anti-settlement activities against microalgae and macroalgae. Biofouling 18: 205–215. Hellio, C., J.P. Marechal, B. Veron, G. Bremer, A.S. Clare and Y. Le Gal. 2004. Seasonal variation of antifouling activities of marine algae from the Brittany coast (France). Mar. Biotechnol. 6: 67–82. Henrikson, A.A. and J.R. Pawlik. 1995. A new antifouling assay method: results from field experiments using extracts of four marine organisms. J. Exp. Mar. Biol. Ecol. 194: 157–165. Horiguchi, T., H. Shiraishi, M. Shimizu, S. Yamazaki and M. Morita. 1995. Imposex in Japanese gastropods (Neogastropoda and Mesogastropoda): effects of tributyltin and triphenyltin from antifouling paints. Mar. Poll. Bull. 31: 402–405. Hunter, J.E. and C.D. Anderson. 2000. Antifouling paints and the environment. Technical paper. Antifouling Discussions IMO/ MEPC 44: 6–13. Hurlbert, S.H. 1984. Pseudoreplication and the design of ecological field experiments. Ecol. Monogr. 54: 187–211. Ina, K., R. Takasawa, A. Yagi, M. Yamashita, H. Etoh and K. Sakata. 1989. An improved bioassay method for antifouling substances using the blue mussel Mytilus edulis. Agric. Biol. Chem. 53: 3319–3321. Karban, R., A.A. Agrawal, J.S. Thaler and L.S. Adler. 1999. Induced plant responses and information content about risk of herbivory. Trends Ecol. Evol. 14: 443–447. Karez, R., S. Engelbert and U. Sommer. 2000. ‘Co-consumption’ and ‘protective coating’: two new proposed effects of epiphytes on their macroalgal hosts in mesograzer-epiphytehost interactions. Mar. Ecol. Prog. Ser. 205: 85–93. Karlsson, J. and B. Eklund. 2004. New biocide-free anti-fouling paints are toxic. Mar. Poll. Bull. 49: 456–464. Keats, D.W., P. Wilton and G. Maneveldt. 1994. Ecological significance of deep-layer sloughing in the eulittoral zone coralline alga, Spongites yendoi (Foslie) Chamberlain (Coralinaceae, Rhodophyta) in South Africa. J. Exp. Mar. Biol. Ecol. 175: 145–154. Kubanek, J., P.R. Jensen, P.A. Keifer, M.C. Sullards, D.O. Collins and W. Fenical. 2003. Seaweed resistance to microbial attack: a targeted chemical defense against marine fungi. Proc. Natl. Acad. Sci. USA 100: 6916–6921. Lasiak, T.A. and T.C.E. Barnard. 1995. Recruitment of the brown mussel Perna perna onto natural substrata – a refutation of the primary/secondary settlement hypothesis. Mar. Ecol. Prog. Ser. 120: 147–153. Littler, M.M. and D.S. Littler. 1999. Blade abandonment/proliferation: a novel mechanism for rapid epiphyte control in marine macrophytes. Ecology 80: 1736–1746. Littler, M.M., P.R. Taylor and D.S. Littler. 1986. Plant defense associations in the marine environment. Coral Reefs 5: 63–71. Lo¨schau, M. and R. Kra¨tke. 2005. Efficacy and toxicity of selfpolishing biocide-free antifouling paints. Environ. Poll. 138: 260–267. Marechal, J.P., G. Culioli, C. Hellio, H. Thomas-Guyon, M.E. Callow, A.S. Clare and A. Ortalo-Magne. 2004. Seasonal variation in antifouling activity of crude extracts of the brown alga Bifurcaria bifurcata (Cystoseiraceae) against cyprids of Balanus amphitrite and the marine bacteria Cobetia marina and

Pseudoalteromonas haloplanktis. J. Exp. Mar. Biol. Ecol. 313: 47–62. Omae, M. 2003. General aspects of tin-free antifouling paints. Chem. Rev. 103: 3431–3448. Orth, R.J. and J. van Montfrans. 1984. Epiphyte-seagrass relationships with an emphasis on the role of micrograzing: a review. Aquat. Bot. 18: 43–69. Paul, V.J. 1992. Ecological roles of marine natural products. Cornell University Press, New York. pp. 245. Pawlik, J.R. 1992. Chemical ecology of the settlement of benthic marine invertebrates. Oceanogr. Mar. Biol. 30: 273–335. Pereira, R.C. and B.A.P. da Gama. 2008. Macroalgal chemical defenses and their roles in structuring tropical marine communities. In: (C.D. Amsler, ed.) Algal chemical ecology. Springer-Verlag, Berlin. pp. 25–55. Pereira, R.C., A.G.V. Carvalho, B.A.P. da Gama and R. Coutinho. 2002. Field experimental evaluation of secondary metabolites from marine invertebrates as antifoulants. Braz. J. Biol. 62: 311–320. Pereira, R.C., B.A.P. da Gama, V.L. Teixeira and Y. YoneshigueValentin. 2003. Ecological roles of natural products of the Brazilian red seaweed Laurencia obtusa. Braz. J. Biol. 63: 665–672. Petersen, J.H. 1984. Larval settlement behavior in competing species – Mytilus californianus Conrad and Mytilus edulis L. J. Exp. Mar. Biol. Ecol. 82: 147–159. Ponasik, J.A., S. Conova, D. Kinghorn, W.A. Kinney, D. Rittschof and B. Ganem. 1998. Pseudoceratidine, a marine natural product with antifouling activity: synthetic and biological studies. Tetrahedron 54: 6977–6986. Potin, P., K. Bouarab, J.P. Salaun, G. Pohnert and B. Kloareg. 2002. Biotic interactions of marine algae. Curr. Opin. Plant Biol. 5: 308–317. Railkin, A.I. 2003. Marine biofouling: colonization processes and defenses. CRC Press, Boca Raton, FL. pp. 320. Rittschof, D. 2001. Natural products antifoulants and coatings development. In: (J.B. McClintock and B.J. Baker, eds.) Marine chemical ecology. Marine Science Series. CRC, London. pp. 543–566. Ruiz, J.M., G.W. Bryan and P.E. Gibbs. 1995. Effects of tributyltin (TBT) exposure on the veliger larvae development of the bivalve Scrobicularia plana (da Costa). J. Exp. Mar. Biol. Ecol. 186: 53–63. Steinberg, P.D. and R. de Nys. 2002. Chemical mediation of colonization of seaweed surfaces. J. Phycol. 38: 621–629. Steinberg, P.D., R. de Nys and S. Kjelleberg. 2001. Chemical mediation of surface colonization. In: (J.B. McClintock and B.J. Baker, eds.) Marine chemical ecology. CRC Press, Boca Raton, FL. pp. 355–387. Steinberg, P.D., R. de Nys and S. Kjelleberg. 2002. Chemical cues for surface colonization. J. Chem. Ecol. 28: 1935–1951. Stupak, M.E., M.T. Garcia and M.C. Perez. 2003. Non-toxic alternative compounds for marine antifouling paints. Int. Biodeterior. Biodegrad. 52: 49–52. Suzuki, T., R. Matsuda and Y. Saito. 1992. Molecular species of tri-n-butyltin compounds in marine products. J. Agric. Food Chem. 40: 1437–1443. Taylor, P.D. and M.A. Wilson. 2003. Paleoecology and evolution of marine hard substrate communities. Earth Sci. Rev. 62: 1–103. Toth, G.B., O. Langhamer and H. Pavia. 2005. Inducible and constitutive defenses of valuable seaweed tissues: consequences for herbivore fitness. Ecology 86: 612–618. Wahl, M. and M.E. Hay. 1995. Associational resistance and shared doom: effects of epibiosis on herbivory. Oecologia 102: 329–340. Weidner, K., B.G. Lages, B.A.P. da Gama, M. Molis, M. Wahl and R.C. Pereira. 2004. Effect of mesograzers and nutrient levels on induction of defenses in several Brazilian macroalgae. Mar. Ecol. Prog. Ser. 283: 113–125.

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Willemsen, P.R. and G.M. Ferrari. 1993. The use of anti-fouling compounds from sponges in anti-fouling paints. Surf. Coat. Int. 10: 423–427. Williams, G.A. and R. Seed. 1992. Interactions between macrofaunal epiphytes and their host algae. In: (D.H. John, S.J. Hawkins and J.H. Price, eds.) Plant-animal interactions in the marine benthos. Systematics Association Special Volume 46. Clarendon, Oxford. pp. 189–211.

Yebra, D.M., S. Kiil and K. Dam-Johansen. 2004. Antifouling technology – past, present and future steps towards efficient and environmentally friendly antifouling coatings. Prog. Org. Coat. 50: 75–104.

Received 24 April, 2007; accepted 20 March, 2008

Antifouling activity of natural products from Brazilian ...

currently facing marine technology (da Gama et al. .... states: PE, Pernambuco; RN, Rio Grande do Norte; BA, Bahia; ES, Espırito Santo; RJ, Rio de Janeiro; SP,.

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