Biofouling, 2002 Vol 18 (1), pp 13–20

The Effects of Seaweed Secondary Metabolites on Biofouling BERNARDO A P DA GAMAa,b, RENATO C PEREIRAa,*, ANA G V CARVALHOa, RICARDO COUTINHOc and YOCIE YONESHIGUE-VALENTINb a

Departamento de Biologia Marinha, Universidade Federal Fluminense (UFF)-CP 100.644 CEP 24001-970 Nitero´i,-RJ, Brazil; bPrograma de Po´sGraduc¸a˜o em Biotecnologia Vegetal, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de Janeiro (UFRJ)-Ilha do Funda˜o CEP 21949-900, Rio de Janeiro, RJ, Brazil; cInstituto de Estudos do Mar Almirante Paulo Moreira (IEAPM)-R. Kioto, 253 CEP 28930-000 Arraial do Cabo, RJ, Brazil

(Received 11 April 2001; in final form 20 August 2001)

Antifouling activity is one poorly investigated property of seaweed natural products. To determine, in the field, whether seaweeds contain chemicals able to influence the settlement of fouling organisms, crude organic extracts from Stypopodium zonale, Dictyota menstrualis (Phaeophyceae) and Laurencia obtusa (Rhodophyceae) were incorporated at natural volumetric concentrations, into hard stable gels that served as substrata for fouling in the experiments. Fouling organisms settled at a significantly higher rate on plates treated with S. zonale extracts than on control gels, while settlement was strongly inhibited on gels containing L. obtusa extracts. Fouling on gels treated with the D. menstrualis extract was not significantly different from the fouling found on control gels. The findings suggest that the broad antifouling properties of the crude extract of L. obtusa inhibit the settlement of fouling as well as hinder the development of settled fouling species, thereby reducing the richness of species. The results imply that L. obtusa possibly harbours powerful agents that can be explored for the development of antifouling technology. Keywords: antifouling; field assay; seaweeds; secondary metabolites

INTRODUCTION Seaweeds produce a wide range of secondary metabolites, many of which exhibit a broad spectrum of bioactivity (Paul, 1992). The ecological roles of secondary metabolites produced by seaweeds have only recently been investigated, in studies that chiefly emphasise chemical mediation as a defence against herbivores (Hay & Steinberg, 1992).

Preliminary studies of secondary metabolites from seaweeds have shown biological activity against bacteria (Sieburth & Conover, 1965; Al-Ogily & Knight-Jones, 1977), Mytilus edulis larvae (Katsuoka et al., 1990), epiphytes (Phillips & Towers, 1982; de Nys et al., 1991), larvae of the bryozoan Bugula neritina (Schmitt et al., 1995), and Balanus amphitrite larvae (Willemsen, 1994). Despite being acknowledged as producers of antifouling secondary metabolites, seaweeds have been overlooked in studies on chemical defences against fouling (see reviews by Clare, 1996; Rittschof, 2001). Research in this field has generally been laboratory-oriented (Bobzin & Faulkner, 1992) or focused on larval settlement (e.g. Rittschof et al., 1985; Davis & Wright, 1990), rather than directed to a more realistic field evaluation of the performance of secondary metabolites as antifoulants (da Gama, 1998). Although this type of knowledge is generally lacking (Clare, 1996), mechanisms by which marine organisms inhibit the settlement of fouling have been investigated at a variety of levels, from molecular to ecological approaches (Steinberg et al., 1998). In a molecular perspective, marine secondary metabolites appear to inhibit bacterial colonisation by interfering with bacterial AHL (acylated homoserine lactone) regulatory processes (Kjelleberg et al., 1997). On the other hand, there are few field or ecological studies investigating the effects of marine secondary metabolites on marine organisms (Willemsen & Ferrari, 1996). In this approach, settlement of fouling on gels containing extracts of marine organisms is measured as related to field conditions (Henrikson & Pawlik, 1995; 1998).

*Corresponding author; fax: þ55-27195934; e-mail: [email protected] ISSN 0892-7014 print/ISSN 1029-2454 online q 2002 Taylor & Francis Ltd DOI: 10.1080/08927010290017680


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Besides preventing the settlement of fouling on marine organisms, marine secondary metabolites may be the most desirable way of breaking the fouling chain and so, indirectly, of reducing the macrofouling settlement on man-made structures (Wilsanand et al., 1999). According to Clare (1996), in order to demonstrate that natural products really play an ecological role in antifouling, evidence must be provided 1) that the natural products are sequestered at, or released from the surface of the basibiont at a sufficient concentration to prevent biofouling, and 2) of an antifouling role against ecologically relevant epibionts, under realistic conditions. In the present study the antifouling properties of secondary metabolites from the seaweeds Laurencia obtusa (Hudson) Lamouroux, Dictyota menstrualis (Hoyt) Schnetter, Ho¨rnig & Weber-Peukert and Stypopodium zonale (Lamouroux) Papenfuss have been evaluated through field experiments.

MATERIALS AND METHODS Organisms Three species of seaweeds known to produce secondary metabolites (see Faulkner, 1999) were chosen for the experiments and collected from different areas on the Brazilian littoral: the brown seaweed Stypopodinum zonale from the Abrolhos Archipelago, Bahia State, Dictyota menstrualis from Praia Rasa, Bu´zios, Rio de Janeiro State, and the red alga Laurencia obtusa from Cabo Frio Island, Rio de Janeiro State. The seaweeds were washed in seawater in the laboratory (Marine Station of the IEAPM—Instituto de Estudos do Mar Almirante Paulo Moreira—Arraial do Cabo, RJ), to eliminate associated organisms. Volumes of the fresh seaweeds were obtained by water displacement into a graduated cylinder. Extraction Procedures After determination of the volume of fresh material (210 ml to prepare 6 replicates of 35 ml each), the algae were dried in the dark at room temperature (in order to avoid photolysis and thermal degradation of the metabolites) until a steady weight was obtained. Each alga was submitted to exhaustive and successive extraction in a combination of organic solvents (dichloromethane and methanol), in the proportion of 2:1, following standard procedures for natural products chemistry. To increase the effectiveness of extraction, the algae were submitted to ultrasound for 15 min (Branson model 3210). The solvent was eliminated in a rotary evaporator under reduced pressure and the remainder was weighed to

determine the natural volumetric concentration of extract for each species. In order to detect the presence of metabolites at the surface of the algae that exhibited antifouling activity (L. obtusa ), fresh specimens were extracted in hexane for 40 s, a time period insufficient to cause cell lysis (de Nys et al., 1998). The resulting extracts were compared by thin layer chromatography (TLC) with the extracts used in the experiments. Field Assays The method used here, developed by Henrikson and Pawlik (1995), affords the following advantages 1) the organic extracts can be included in the gel at the natural concentrations found in seaweeds, and the extracts can then slowly diffuse in the water in a manner similar to that occurring in a living organism; 2) the extracts are incorporated into the gel and not confined to the surface, and do not alter the physical properties of the gel surface, thus, the differences observed in larval settlement can only be attributed to the chemical properties of the extracts; 3) settlement of fouling on the experimental gel occurs under natural conditions of flow and diffusion, being exposed to a natural supply of larvae and spores of algae. Plastic Petri dishes containing phytagele (Sigma Chemical Co) were used. Extracts of the algae were added to some (treatments), and only solvent to others (controls). Control plates ðn ¼ 6Þ were prepared with a mixture of 1.52 g phytagel and 35 ml of distilled water, and then were heated to boiling point in a microwave oven. The mixture was then vigorously stirred with a glass rod, while adding 0.5 ml methanol, and poured into Petri dishes for hardening. In each treatment solution ðn ¼ 6Þ; an aliquot of the extract to be tested was mixed with the solution (diluted in 0.5 ml methanol) after cooling down to #608C. The extract added to every 35 ml of gel was equivalent to an extract of 35 ml of fresh material, in an attempt to maintain the natural concentrations of the metabolites. In the experiment, six replicates were prepared for each algal extract and for the control. One replicate of each treatment and one control replicate were randomly arranged and each fastened to one of six rectangular aluminium structures. These represented independent experimental units, eschewing problems of pseudo-replication (see Hulbert, 1984). The structures were then submerged to a depth of 1 m and secured to a swivel (to ensure orientation parallel to water flow) suspended from three flotation rafts moored at Cabo Frio Island (Arraial do Cabo, State of Rio de Janeiro). The settlement of fouling in the field was measured weekly as percentage cover, using a dotgrid method (Foster et al., 1991; Henrikson & Pawlik,



FIGURE 1 Total number of fouling species observed on the control ( –†– ) and treatments containing D. menstrualis ( –B–), S. zonale (– O– ), and L. obtusa (– P–) extracts over immersion time (weeks). Significant differences in species richness (ANOVA and Tukey HSD test) were observed during the 1st week (S. zonale – control, D. menstrualis and L. obtusa; p , 0:0001), the 3rd week (L. obtusa – control, D. menstrualis and S. zonale; p , 0:03) and the 6th week (L. obtusa – control, D. menstrualis and S. zonale; p , 0:02).

1995). A large number of points (235) was used to avoid underestimating rare species and to reduce deviation among replicates (Dethier et al., 1993; da Gama, 1998). To prevent the fouling organisms from dying, each structure was kept in a large aluminium 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 a conspicuous thin, brownish layer, covering an area of the plate that was estimated by the dot-grid method described. Samples of biofilm taken to the laboratory contained mainly bacteria, benthic diatoms and ciliated protists.

submitted to extraction. Fouling was removed from the gels (and the plate), and these were then cut into small pieces and extracted in organic solvents as described above. Two types of controls were also analysed, viz. 1) one control gel was extracted to determine whether the gel absorbed metabolites from the seawater: 2) the salts deposited in each extract were drawn out with solvent and weighed. The extracts were evaporated and the remaining weighed to obtain the amount of extract left in comparison to controls 1) and 2). The final amount of each treatment was compared to the initial amount (the extract aliquot mixed to the gel), to obtain the percentage retention.

Extract Degradation

Statistical Analysis

The subject of artefact generation is well known in the literature of natural products chemistry (e.g. Fenical, 1993; Cronin et al., 1995). To ensure that were no artefacts of degradation by heating in the crude extracts, TLC was performed on each extract, before and after heating to 80 –1008C.

Mono-factorial analysis of variance (ANOVA) and post hoc Tukey’s Honest Significant Difference (HSD) test were used to check for differences among percent cover of fouling among treatments. Each of the analyses considered only the data of that week.

RESULTS Diffusion of Extracts As extracts tend to diffuse in seawater, one important issue was to determine the amount of extract remaining on gels. After 5, 6 and 7 weeks, one replicate of each treatment was removed and

General Aspects The duration of the experiments was determined by two factors, viz. 1) retention of the extract in the experimental gels, verified as up to 7 weeks after


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FIGURE 2 Total cover, biofilm, multispecific turf of algae, and balanid cover (%) on the control ( –W– ) and treatments containing D. menstrualis ( –A–), S. zonale ( –K– ),and L. obtusa ( –L– ) extracts over time. Vertical bars ¼ SD: Significant differences were evaluated by ANOVA and the Tukey HSD test and are indicated (*) when p , 0:01:

immersion, and 2) a factor not foreseen originally, which determined the end of the experiment; after 6 weeks, i.e. predation by fish strongly affected the percentage cover of fouling on the treatments gel. Predation signals (marks of bites) were observed only after 6 weeks. On account of this, the time of immersion was restricted to 7 weeks between 9 July and 28 August 1997 (50 days) despite the fact that the results indicated that even after this period there was still extract retention in the gel. The surface extract of L. obtusa showed a TLC profile similar to that of the whole plant extract, confirming that the major compound was present at the surface of this alga.

Species Richness Field experiments revealed a total of 13 taxa, including turf-forming and crustose coralline algae (families Ectocarpaceae and Corallinaceae, respectively), tunicates (Ascidiacea), hydrozoans (Cnidaria), bryozoans (Ectoprocta), polychaetes (Annelida) and barnacles (Crustacea). It was not always possible to identify the organisms to the generic or specific level, owing to their small size and to the non-destructive sampling method used. Figure 1 shows the comparative richness of species among the treatments along the immersion period. An increase in species number was observed in all


FIGURE 3 Percentage retention of crude extracts of S. zonale (– †–), D. menstrualis ( –B–), and L. obtusa (– O–) after immersion for 5, 6 and 7 weeks.

the treatments and controls up to 4 weeks, after which it tended to stabilise (in treatments with extracts of L. obtusa and D. menstrualis ) or decrease (other treatments and the control). The treatment containing extract of L. obtusa was fouled by fewer species than the other treatments in the 3 and 6 week samplings ðp , 0:03Þ: In contrast, the treatment with the extract of S. zonale was fouled by more species ðp , 0:001Þ than the others after 1 week’s immersion.

Total Cover With respect to total cover of fouling through time (Figure 2), an increase was observed after 2 weeks’ immersion; this increase was significantly larger in the treatment bearing the S. zonale extract. After 3 weeks, a reduction in the total cover of all treatments and control was observed followed by an additional increase, although significantly smaller in the treatment with the L. obtusa extract. After 6 weeks, the same trend continued, with the L. obtusa extract presenting significantly higher antifouling activity, as compared to the control plates and to the other treatments. However, cover by fouling was reduced by fish predation, common in this particular area (Ferreira et al., 1998).

Specific Cover Figure 2 presents changes in the percentage cover of the main fouling organisms present on gels. Only


selected species with $10% cover in 1 week or significant differences in more than 1 week (ANOVA, and HSD Tukey test) have been used here. The groups meeting this criterion were biofilm, the multispecific turf of algae, and balanid barnacles. Other species were not present in all the treatments, and did not follow a recognisable distribution pattern. It is important to consider that the reduction in percentage cover after the 6th week was probably caused by fish grazing. It is hypothesised that selective predation on fouling organisms may have occurred in the treatments, as serpulid tube worms were observed to have been removed from gels leaving clear fish grazing scars in their place. A general pattern of community development was observed in all the treatments. After 2 weeks, biofilm, composed primarily of diatoms and bacteria, prevailed, followed by a multi-specific turf of algae (composed mainly of members of the Ectocarpaceae). The number of barnacles (Balanidae) increased steadily, reaching a maximum after 7 weeks (Figure 2). Significant differences were also observed in the cover of several species among treatments and control, which helped in the comprehension of the trends noticed in total cover among treatments. In chronological sequence, the following effects were noted. After 1 week, the treatment with the S. zonale extract had a significantly higher cover of biofilm ðp , 0:001Þ and Balanidae ðp , 0:01Þ than all the other treatments and the control. After 2 weeks, the treatment with the L. obtusa extract had a significantly smaller cover of biofilm ðp , 0:01Þ than all the other treatments and the control. After 3 weeks, gels treated with L. obtusa and D. menstrualis extracts had a lower cover of Ectocarpaceae ðp , 0:01Þ compared to the control and to the S. zonale treatment. In the 4th week, the L. obtusa treatment had significantly less turf ðp , 0:001Þ than the other treatments and the control, and the S. zonale treatment had significantly more ðp ¼ 0:01Þ than the other treatments. After 5 weeks, the gels treated with the L. obtusa extract had a turf cover that was significantly smaller ðp , 0:001Þ than the other treatments and the control, and a cover of balanids significantly smaller than the control ðp ¼ 0:05Þ: The later pattern was maintained for 6 –7 weeks, with the L. obtusa extract exhibiting a significantly lower cover of turf than the other treatments ðp , 0:001Þ: All the treatments, however, exhibited reduction in turf cover due to predation (data not shown). Diffusion of Extracts Figure 3 shows the final amount of crude extract of each alga remaining in the experimental gels after 5, 6 and 7 weeks, as compared to the controls and relative to the initial extract aliquots added before immersion. The percentage mean retention (all


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treatments pooled together) was 61:7% ^ 26:0 ðmean ^ SDÞ after 5 weeks, 43:1 ^ 16:6 after 6 weeks, and 25:4 ^ 12:7 after 7 weeks. The diffusion rate of extracts from the gels was probably neither constant over time nor the same for all extracts. TLC analysis comparing the extracts after 5 weeks showed that a number of substances were still on the gel after this period, despite the detectable quantitative loss. However, many spots probably containing more polar (water soluble) pigments or metabolites were lost to the seawater, although the colours of the remaining extracts were similar to those of the initial extracts. Gels treated with the S. zonale extract appeared to be the only exception, clearly retaining all the substances (visible or UVreactive) in the original extract. The L. obtusa extract was lost to the seawater more rapidly than either of the other extracts.

DISCUSSION Chemical defence may be species-specific for bacteria, microalgae, macroalgae or larvae, or may have a broad spectrum of bioactivity to fouling organisms. The present results show that the crude extract of L. obtusa was capable of inhibiting the settlement of a broad range of fouling organisms in field experiments. Laurencia species produce a diverse array of secondary metabolites (Faulkner, 1999 and previous reviews), exhibiting several kinds of biological activity, such as chemical defence against diverse marine herbivores (Hay et al., 1987; Paul et al., 1988) and other functions (e.g. Konig et al., 2000). For example, both the sesquiterpene compounds elatol and deschloroelatol from Laurencia rigida J. Agardh display very strong effects against invertebrate larvae and are highly toxic (de Nys et al., 1996). However, the selective activity and strong toxicity of both these compounds appears to rule out their possible application as commercial nontoxic antifoulants (de Nys et al., 1995; Rittschof, 2001). In addition, crude extract and two other sesquiterpene compounds, 5b-hydroxyaplysistatin and palisol, also extracted from L. rigida were found to reduce the establishment of algal spores and invertebrate larvae (Battista, 1995). The sesquiterpenoid palisadin A deterred feeding both by the herbivorous fish Zebraoma flavescens Bennett in laboratory assays and by herbivorous fish in the field (Paul et al., 1988). It was also observed that common fouling organisms are absent or rare on the L. obtusa studied here, which only carried free living organisms (Aplysia sp.) on its fronds. The combination of these field observations with the results of the present field assays, provides evidence that the secondary metabolites produced by L. obtusa can work effectively to prevent fouling. Furthermore, de Nys

et al., (1998) demonstrated that Australian L. obtusa, extracted during a time period insufficient to cause cell lysis, contained the metabolites aplysistatin and palisadin A, showing that these metabolites are present at the surface of the alga. The results of the present paper agree with these observations, as TLC analysis of the surface extract of Brazilian L. obtusa exhibited the major metabolite also present in the extract of the whole plant. Nevertheless, it was not possible to measure its concentration. Crude extract from the Brazilian D. menstrualis did not inhibit the settlement of fouling organisms. However, Dictyota species are rich sources of bioactive secondary metabolites known to deter feeding by herbivores such as fish, urchins, amphipods and crabs (Hay & Steinberg, 1992). Specimens of D. menstrualis from North Carolina were found to be unfouled (Schmitt et al., 1995), and the negative effects of secondary metabolites produced by this species (e.g. dictyol E, dictyol B acetate, pachydictyol A and dictyodial diterpenes) include larval mortality, abnormal development and reduced growth rate of invertebrate larvae (Schmitt et al., 1998). Therefore, these metabolites are effective chemical defences against fouling organisms and can select against the establishment of the kinds of larvae that avoid hosts producing these metabolites (Schmitt et al., 1998). Specimens of Brazilian D. menstrualis on the other hand have the diterpenes pachydictyol A and (6R)-6-hidroxydichotoma3,14-diene-1, 17-dial as two major secondary metabolites (Pereira et al., 2000b). Pachydictyol A is a compound known to be able to inhibit herbivory at the natural concentration found in the N. Carolina D. menstrualis (Hay & Steinberg, 1992), but it only inhibits herbivory in concentrations above those natural to the Brazilian D. menstrualis (previously identified as D. dichotoma (Hudson) Lamouroux, Pereira et al., 1994). Only the natural concentration of (6R)-6-hidroxydichotoma-3,14-diene-1, 17-dial inhibits herbivory by the amphipod Parhyale hawaiensis Dana (Pereira et al., 2000a; 2000b). Thus, the crude extract of D. menstrualis was not effective as an antifoulant in the field, probably because of the production of compounds other than the ones found in N. Carolina specimens. Colonisation by fouling agents on experimental plates containing the S. zonale extract occurred quickly, reaching 100% after one week of immersion in the sea. S. zonale is known to produce several secondary metabolites able to induce pronounced deleterious effects on fish in laboratory assays (Gerwick et al., 1979), or to inhibit herbivory in field assays (Hay et al., 1988). The major toxic metabolite found in the S. zonale from Belize is stypotriol, which is rapidly air oxidised to stypoldione (Gerwick & Fenical, 1981). However, in spite of its ability to be easily oxidised, stypotriol is a stable


compound in the crude extract (Gerwick et al., 1979). The Brazilian S. zonale collected at Fernando de Noronha Island has also been found to yield several similar secondary metabolites previously found in specimens from Belize (Soares, 2001). Besides stypotriol, several other compounds also contribute to the overall toxicity of S. zonale (Gerwick & Fenical, 1981) and stypoldione is a chemical deterrent to herbivory by fish (Hay et al., 1988). Hence, should there have been air oxidation of stypotriol to stypoldione in the crude extract studied here, this or other secondary metabolites produced by the Brazilian S. zonale could inhibit fouling. Nevertheless, the results obtained here, jointly with the observation that fronds of this species are generally colonised by bryozoans and polychaetes as well as by coralline algae in the field, provide strong evidence that secondary metabolites from the Brazilian S. zonale did not prevent fouling. Due to continuous predation observed after the 6th week of the field test, all the experiments were prematurely terminated. Analysis of the material extracted after this period revealed that 18.6% of the extracts were still retained in the gel, although the calculated amount of L. obtusa extract remaining on gels after 7 weeks was only 10.8%. The diffusion rate calculated for the data of Henrikson and Pawlik (1995) (12.7% week21) was smaller than that found here (18.2%). This is probably due to the fact that the plates used by the other authors were immersed in mesocosms of 1000 l; in the present study, the plates were immersed directly in the sea, therefore under more intense flow conditions. Also, in the present study, the diffusion rate inside the gel was deliberately reduced by allowing the extract to face the water on only one side. This procedure decreased the amount of biological material necessary to extract, allowing a less destructive collection of organisms. In addition, a reduction in the amount of extract also reduces the probability that intra-specific differences among plants may account for the differences in metabolite production. That is, it reduces the chances of specimens collected from different populations having very significant quantitative differences in metabolite production. To the authors’ knowledge, this is one of the first assignments of the ecological role of seaweed crude extract containing secondary metabolites as antifoulants, using an ecologically relevant bioassay. Although the substances that exhibit antifouling activity in L. obtusa are not yet known, the importance of this type of study for marine technology remains. Several marine organisms have defences probably developed in response to strong selective pressures, including fouling. By identifying the active agent, marine chemical ecology can provide potential alternatives for commercial highly toxic antifoulants.


Acknowledgements The National Brazilian Research Council (CNPq. Proc 62.0470/94.1 and 521914/96-5), who supported this research. RCP, YYV and RC thank CNPq for the Productivity Fellowships and BAPG thanks CAPES for the Sc D Fellowship. Comments by Cassiano Monteiro-Neto, Paul W Sammarco and two anonymous referees improved the manuscript.

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