J. Phycol. 44, ***–*** (2008)  2008 Phycological Society of America DOI: 10.1111/j.1529-8817.2008.00507.x

TRANSPORT AND DEFENSIVE ROLE OF ELATOL AT THE SURFACE OF THE RED SEAWEED LAURENCIA OBTUSA (CERAMIALES, RHODOPHYTA) 1 Daniela B. Sudatti Po´s-Graduac¸a˜o em Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, PO Box 100.644, CEP 24001-970, Nitero´i, Rio de Janeiro, Brazil

Silvana V. Rodrigues Departamento de Quı´mica Analı´tica, Universidade Federal Fluminense, Outeiro de Sa˜o Joa˜o Batista, s ⁄ n, Nitero´i, Rio de Janeiro, Brazil

Ricardo Coutinho Instituto de Estudos do Mar Almirante Paulo Moreira (IEAPM)-R. Kioto, 253 CEP 28930-000 Arraial do Cabo, Rio de Janeiro, Brazil

Bernardo A. P. da Gama Po´s-Graduac¸a˜o em Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, PO Box 100.644, CEP 24001-970, Nitero´i, Rio de Janeiro, Brazil

Leonardo T. Salgado Laborato´rio de Biomineralizac¸a˜o, Departamento de Histologia e Embriologia, Instituto de Cieˆncias Biome´dicas, Centro de Cieˆncias da Sau´de, Universidade Federal do Rio de Janeiro, Rio de Janeiro, Brazil

Gilberto M. Amado Filho Programa Zona Costeira, Instituto de Pesquisas Jardim Botaˆnico, Ministe´rio do Meio Ambiente, Rio de Janeiro, Brazil

and Renato C. Pereira2 Po´s-Graduac¸a˜o em Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense, PO Box 100.644, CEP 24001-970, Nitero´i, Rio de Janeiro, Brazil

Natural within-thallus concentrations of elatol produced by Laurencia obtusa (Huds.) J. V. Lamour. inhibit herbivory and prevent fouling. However, elatol occurs in larger amounts within the thallus compared with the quantities from the surface of this alga. We evaluated whether the surface elatol concentrations inhibit both herbivory and fouling and whether the content of corps en cerise can be transferred to the external cell walls. Surface elatol concentrations did not inhibit herbivory by sea urchins, settlement of barnacle larvae, or mussel attachment. Evidence of a connection between the corps en cerise, where elatol is probably stored, and the cell wall of L. obtusa was based on channel-like membranous connections that transport vesicles from the corps to the cell wall region. Therefore, L. obtusa presents a specific process of chemical transport between the cell storage structures and the plant surface. We hypothesized that if high amounts of elatol are capable of inhibiting herbivory and fouling, if the tested organisms are ecologically relevant, and if

elatol really occurs on the surface of L. obtusa and this seaweed can transport this compound to its surface, the low natural concentration of defensive chemicals on the surface of L. obtusa is probably not absolute but may be variable according to environmental conditions. We also hypothesized that herbivory and fouling would not exert the same selective force for the production of defensive chemicals on L. obtusa’s surface since the low concentrations of elatol were inefficient to inhibit either processes or distinguish selective pressures. Key index words: chemical defense; elatol; fouling; herbivory; Laurencia obtusa Abbreviation: Rf, retention factor

Secondary metabolites play an important role in mediating complex ecological interactions in the sea, including predator-prey and competitive interactions, settlement cues, and potential defenses against infection by microorganisms (see recent review by Paul et al. 2006). For seaweeds, most evidence for the chemical mediation of ecological

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Received 10 January 2007. Accepted 27 September 2007. Author for correspondence: e-mail [email protected].

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interactions comes from studies of chemical defense by a diverse array of secondary metabolites against herbivores, such as fishes, sea urchins, gastropods, and amphipods (Paul et al. 2001, Amsler and Fairhead 2006). In fact, herbivory is known to have a strong impact on seaweed populations and communities in a wide variety of marine habitats worldwide (John et al. 1992) and could potentially be a selective agent to chemical defense evolution in seaweeds (Vallim et al. 2005). Besides inhibiting herbivory, seaweed secondary metabolites are also known to inhibit fouling organisms—including bacteria (Sieburth and Conover 1965, Al-Ogily and Knight-Jones 1977), Mytilus edulis larvae (Katsuoka et al. 1990), epiphytes (Phillips and Towers 1982), competitors such as corals (de Nys et al. 1991), larvae of the bryozoan Bugula neritina (Schmitt et al. 1995), and larvae of the barnacle Balanus amphitrite (Willemsen 1994)—and to have a broad antifouling property against several fouling organisms, including turf-forming and crustose coralline algae, tunicates, hydrozoans, polychaetes, and barnacles (da Gama et al. 2002). However, unlike herbivory, the effects of fouling are less understood, but epibionts may diminish the light energy and nutrients reaching the basibiont plant, indirectly influencing abundance, distribution, and productivity as well as sexual and vegetative reproduction (Orth and Van Montfrans 1984). Additionally, epibionts may attract consumers that otherwise would not feed significantly on the host plant (Bernstein and Jung 1979, Karez et al. 2000, Pereira 2004). Thus, epibionts could also be potential agents to select for the production of defensive chemicals in seaweeds; yet this possibility has been little explored. One important reason for the lack of understanding of some chemically mediated interactions involving seaweeds is the paucity of knowledge on the presentation—the possible mechanisms that bring the compounds to the thallus surface and the localization of metabolites on ⁄ in seaweeds (Dworjanyn et al. 1999). For instance, variation in these chemicals between the surface and within the thalli of seaweeds has rarely been studied (e.g., de Nys et al. 1998). The exact quantification of secondary metabolites for interpreting ecological roles is essential for assessing studies in chemical ecology (de Nys et al. 1995, 1998, Schmitt et al. 1995). The determination of antifouling metabolites present near or at the surface of host organisms is important because antifouling function cannot be inferred without demonstrating the presence or the concentration of secondary metabolites in situ (de Nys et al. 1998). On the other hand, the quantification of these chemicals in the seaweed as a whole can be important to evaluate their defensive role against herbivores; many herbivores, such as fishes, echinoids and gastropods, consume parts or the entire thallus of seaweeds.

In order to guard against autotoxicity and provide storage for metabolites, the secondary metabolites in seaweeds can be localized in specific cellular structures (physodes, gland cells or corps en cerise). If these structures are at the surface of the thallus or at least connected to the surface of thallus in some way, then the compounds within thalli have the potential to be used as natural antifoulants (Steinberg and de Nys 2002). Some Laurencia species have been suggested to encapsulate secondary metabolites in their unusually refractile structures called corps en cerise (Young et al. 1980), but there is no current information on the role of these structures in chemical mediation on the surface of this seaweed. The Brazilian red seaweed L. obtusa produces the sesquiterpene elatol as its major secondary metabolite, whose natural concentrations (in whole thallus) inhibit the consumption of the alga by herbivores (Pereira et al. 2003) and the settlement of fouling organisms (da Gama et al. 2002). However, a recent study has demonstrated that the concentration of elatol in the Brazilian L. obtusa was the highest within the thallus (9.89 mg Æ g)1 of alga dry weight [dwt]), compared to the extremely low values found on the surface (0.0059 mg Æ g)1 of alga dwt or 0.5 to 10.0 ng Æ cm)2; Sudatti et al. 2006). In this study, we conducted laboratory bioassays and anatomical evaluations to understand the ecological roles or effectiveness of low concentrations of elatol on the surface of L. obtusa as a defense against herbivory and fouling. More specifically, we asked: (1) Is the low concentration of elatol found on the surface of L. obtusa capable of inhibiting both herbivory and fouling? (2) Would the metabolites found on the surface of this seaweed be ecologically irrelevant? Could the content of the corps en cerise from cortical cells of this species be transferred to external cell walls? MATERIALS AND METHODS

Seaweed. Laurencia obtusa is an intertidal red seaweed occurring along most of all the Brazilian littoral (Oliveira Filho 1977). Specimens of L. obtusa were collected in the midlittoral zone on the rocky coast of Cabo Frio Island (2259¢ S, 4259¢ W), Rio de Janeiro State, Brazil. In the present study, the population collected had a huge amount—about 9.89 mg Æ g)1 (Sudatti et al. 2006)—of the sesquiterpene elatol in the dwt of this seaweed, a known secondary metabolite with defensive properties against herbivores (Pereira et al. 2003) and fouling (da Gama et al. 2002). Isolation of elatol. Crude extract from L. obtusa (4.07% dwt) was obtained using an exhaustive and successive extraction in dichloromethane, following standard procedures for natural products. Elatol was isolated, based on the reference factor (Rf 0.66), through precoated TLC plates. The resulting extract, a transparent oil (about 0.45% dwt), was identified as the halogenated sesquiterpene elatol by using 1H NMR analysis and by matching the results found in the literature (Ko¨nig and Wright 1997). Surface levels were expressed as percentage of elatol mass (mg) per alga mass (g dwt) or elatol mass (ng) per alga surface (cm2), the values obtained varying from 5 · 10)6

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to 5 · 10)3% dwt and from 0.5 to 10 ng of elatol Æ cm)2, respectively. Based on these levels, deterrence bioassays were run to evaluate the ecological roles of surface elatol. Feeding deterrence: Lytechinus variegatus. The defensive property of elatol was tested by including the equivalent elatol surface concentrations, expressed as mass of elatol per surface area of alga, in an artificial food (method detailed in Hay et al. 1994). Surface concentrations tested were 0.5, 1.0, 5.0, and 10.0 ng of elatol Æ cm)2. This ranking was employed in the food choice test, in which the green seaweed Ulva sp. was incorporated into artificial food. For control food, a mass was prepared adding 0.45 g of agar to 10 mL of distilled water and heating in a microwave oven until the boiling point. This mixture was added to 6 mL of cold water containing 1 g of freeze-dried and ground Ulva. The treatment food was similarly prepared, but the elatol was first dissolved in hexane and added to 1 g of grounded Ulva, and the solvent was removed by evaporation. Control food also received solvent (without elatol) to assure that hexane presence was not an artifact confounding the results. Treatments and controls were hardened onto a screen and cut into small pieces (10 · 10 squares about 1.2 · 1.4 mm each), which were then simultaneously offered to the sea urchin Ly. variegatus. The defensive activity was estimated by comparing the number of consumed squares between treatment food and control food. Specimens of Ly. variegatus were kept in a recirculating laboratory aquarium at constant temperature (20C), salinity (35), and aeration. After an acclimation period, the bioassay was carried out separating each individual (n = 11–15 replicates) into a perforated plastic container (1 L seawater) placed in large tanks containing about 500 L of recirculating seawater. Fouling deterrence: Amphibalanus amphitrite. Adult A. amphitrite barnacles were induced to spawn (by increasing water temperature). Mass-spawned nauplii were collected, transferred to another sea aquarium, and fed on Skeletonema sp. Larvae reached the cyprid stage after 4 to 6 d. Equivalent elatol surface concentrations found in specimens of L. obtusa, expressed as mass of elatol per surface area of alga, were prepared in hexane solution and used to coat internal glass petri dishes. The treatment conditions were 0.5, 1.0, 5.0, and 10.0 ng of elatol Æ cm)2; control (glass petri dishes coated only with solvent—hexane); and blank control (glass petri dishes without coating). Ten cyprid larvae were pipetted into the dishes, which were filled with 20 mL of filtered natural seawater. Petri dishes were incubated for a week at room temperature (27C) under an entire dark cycle. During and after incubation periods, the number of settled larvae was counted using a stereomicroscope. Average settlement percentages were calculated using four replicates for each treatment. Fouling deterrence: Perna perna. This test was first proposed by Ina et al. (1989) and Goto et al. (1992) and was later modified by da Gama et al. (2003). Juvenile P. perna mussels were kept in a recirculating laboratory aquarium at constant temperature (20C), salinity (35), and aeration for 12 h. Individuals were carefully disaggregated and cleaned by cutting the byssus threads. They were sorted into size groups according to shell length. Two hundred and sixteen individuals, from the same group size (3–4 cm), exhibiting substrate-exploring behavior (actively exposing their foot and crawling) were selected for the experiments. Water-resistant filter paper (Darmstadt, Germany) was cut into 9 cm diameter circles and soaked in solvent, hexane (control filter). Another 9 cm set of filter papers (treatment filters) was cut in a chessboard pattern (1.5 cm squares) and soaked in elatol extract at a surface concentration solution (determined as the extract equivalent to the dwt of alga ) dwt of filter paper). All filter paper circles were allowed to air-dry. Entire filter paper circles were then placed at the bottom of

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petri dishes, over which treated chessboard filters were placed. Three mussel specimens were replaced to each petri dish filled with 70 mL of seawater. In this way, mussels would have the same area of treated (superior, squared) and control (inferior, entire) filter paper to attach. Twelve replicates of each treatment—blank control (filter paper), control (filter paper soaked into solvent), and treatment (filter paper soaked into elatol extract)—were used. The surface elatol concentrations were expressed as a percentage of alga dwt corresponding to 5 · 10)6, 5 · 10)5, 5 · 10)4, and 5 · 10)3 mg of elatol per gram of L. obtusa. The bioassay was allowed to run for 16 h. Mussel activity was recorded by considering the number of byssal threads attached to substratum (control or treated filter paper, shell of another mussel, or border of petri dish). Time-lapse optical microscopy. For corps en cerise observation, freshly collected specimens of L. obtusa were placed in an inverted Nikon Eclipse TE300 (Nikon, Tokyo, Japan) optical microscope mounted in a Newport (Newport RS 2000TM, Newport Co., Irvine, CA, USA) table stabilized against environment vibrations. The sample was observed in bright field with a Plan (low curvature of field) Apo (apochromatic), 100X, NA 1.4 objective (Nikon). The digitized images (10 frames per second) were acquired with a CCD camera connected to an Argus-20 system (Hammamatsu, Hammamatsu City, Japan) and an LG-3-16 PCI CCIR Scion frame grabber (Scion Co., Frederick, MD, USA). Using the ImageJ software (Abramoff et al. 2004), the images obtained were processed with a convolve low pass filter and with an enhanced contrast tool. Statistical analysis. One-tailed Wilcoxon matched pairs test, a nonparametric equivalent to the t-test for dependent samples, was used to evaluate the results from feeding assays. The data from bioassays with A. amphitrite were analyzed through the number of settled larvae (expressed as percentages in the graphs) by Kruskal–Wallis test due to severe violation of analysis of variance (ANOVA) assumptions. Data from P. perna bioassays (number of attached byssal threads) were analyzed pffiffiffi by oneway ANOVA after square root transformation (x¢ = x + 0.5) to meet assumptions (Zar 1999). For all analyses, the differences among treatments were considered significant whenever P < 0.05 (a = 5%). RESULTS

Feeding deterrence: Ly. variegatus. Elatol levels on the surface of L. obtusa did not deter feeding by the sea urchin Ly. variegatus (Fig. 1, P > 0.05, Wilcoxon

Fig. 1. Effects of elatol surface levels from Laurencia obtusa on feeding preference by the sea urchin Lytechinus variegatus at 0.5, 1.0, 5.0, and 10 ng of elatol Æ cm)2. Differences between means were considered significant when P < 0.05 (*, Wilcoxon matched pairs test). Vertical bars are standard errors; n = number of replicates.

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Fig. 2. Effects of elatol surface levels (5 · 10)6 to 5 · 10)3% dwt and 0.5–10 ng of elatol Æ cm)2) from Laurencia obtusa on (a) settlement larvae of Amphibalanus amphitrite barnacles (number of replicates = 4; number of larvae per dish = 10) and (b) byssal thread production of Perna perna mussels (number of replicates = 12; number of mussels per dish = 3). Differences between means were not significant (P > 0.05). Vertical bars are standard errors.

matched pairs tests), regardless of the concentration (ranging from 0.5 to 10 ng Æ cm)2). Fouling inhibition: A. amphitrite and P. perna. The settlement of barnacle cyprid larvae was equal in the treatments, solvent control (hexane), and blank control (Kruskal–Wallis: H5,24 = 1.455, P = 0.918; Fig. 2a), and similar results were obtained for the number of byssal threads produced by the mussel P. perna (Fig. 2b; ANOVA: F5,66 = 1.453, P = 0.217). Therefore, both bioassays demonstrated that elatol surface levels were not sufficient to avoid fouling by mussels or barnacles. Cellular observation. The presence of L. obtusa corps en cerise is restricted to cortical cells, and they were observed, in general, in a number ranging from 1 to 2 per cell (Fig. 3). Images of live samples obtained from a cortical cell in superficial view revealed the presence of membranous-like channel connections between the corps en cerise and the

Fig. 3. Differential interferential contrast (DIC) image of a transversal section of Laurencia obtusa. The algae were fixed in glutaraldehyde 5%, postfixed with 2% OsO4, embedded in Epon resin (Electron Microscopy Sciences, Hatfield, PA, USA), and the semithin sections obtained in a Reichert ultramicrotome (G. Reichert, Optische Werke A. G., Vienna, Austria). Corps en cerise (*) and many small and large vesicles (arrows) inside the cortical cells are seen. Scale bar, 6 lm.

cell periphery (Fig. S1, see the supplementary material). Temporal sequential images from live cells demonstrated the occurrence of vesicular transport from the corps en cerise to the cell wall region. The observed vesicular transport (see Video S1 in the supplementary material) occurs along a channel-like membranous connection (Fig. S1). DISCUSSION

The main conclusion from this study is that the low levels of elatol naturally occurring on the surface of L. obtusa were inefficient as chemical defenses against fouling (barnacle larvae settlement and juvenile mussel attachment) and herbivory by the sea urchin Ly. variegatus. To our knowledge, the application of surface concentrations of algal natural products to verify their ecological role as defensive chemicals against both consumers and fouling is a new initiative. Laboratory and field assays have demonstrated that secondary metabolites from seaweeds serve as defenses against consumers and fouling, but using only the whole plant extracts (Clare 1996, da Gama et al. 2002). There are several criteria needed to show that seaweeds use antifouling chemicals (Davis et al. 1989, Schmitt et al. 1995, Clare 1996, Hay 1996). First, the seaweed should be naturally devoid of fouling in the field; this is indeed true for the majority of the Brazilian specimens of L. obtusa studied. The second point is to verify whether the putative antifouling compound inhibits fouling at the concentration present at the seaweed surface. In this aspect, we concluded that elatol from L. obtusa

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at surface concentrations, as reproduced in experiments by coating petri dishes (barnacles) or soaking filter papers (mussels) in surface concentrations of elatol, in a manner hypothetically similar to surface exposition in nature, is ineffective as a chemical defense (0.5–10 ng Æ cm)2). Similarly, levels of elatol from Laurencia rigida also had no or minimal effects on Ulva lactuca settlement and germination and bacterial growth (Vibrio fisheri and Serratia sp.) at the highest concentrations (i.e., 1.0 ng Æ cm)2 and 10 ng Æ mL)1), respectively (de Nys et al. 1996). On the other hand, the settlement of A. amphitrite (as Balanus amphitrite) and Bugula neritina larvae was inhibited at 10 and 100 ng Æ cm)2, respectively (de Nys et al. 1996), in a presentation of compounds at test surfaces similar to the barnacle bioassays here described. Elatol activity on A. amphitrite from Australia and Brazil differs in that results presented herein suggest an intraspecific resistance possibly due to local conditions. Although the level of elatol at the surface of L. obtusa from Brazil is known to be very low (Sudatti et al. 2006), it seems to be realistic since using the same extraction method, comparable surface concentrations were observed for L. obtusa from Australia (de Nys et al. 1998). In the same way, polyphenols have been shown to inhibit the settlement of a range of fouling organisms (see review de Nys and Steinberg 1999), but when carefully measured in the field, the concentrations of these compounds near the surface of brown seaweeds are orders of magnitude lower than those needed to act as antifoulants (Jennings and Steinberg 1994, 1997). An alternative hypothesis to explain the absence of activity of surface concentrations would be that compounds other than elatol could play a role in antifouling defense of this alga, either additive or synergistic with that of elatol. This possibility deserves further study. The third important aspect is that the assays to verify antifouling properties need to use ecologically relevant organisms. Here, we used two important components of the fouling communities observed along the Brazilian littoral, the mussel P. perna and the barnacle A. amphitrite, to evaluate the antifouling properties of the sesquiterpene elatol. In this way, if both organisms were not inhibited by elatol, we could conclude that the concentration of this compound on the surface of the Brazilian L. obtusa is too low to inhibit fouling in situ. A recent study (Sudatti et al. 2006) revealed that the within-thalli concentration of elatol is higher (9.89 mg Æ g)1 of Brazilian L. obtusa [dwt]) than at the thalli surface (0.006 mg Æ g)1 of L. obtusa [dwt], or 0.5– 10.0 ng Æ cm)2). Barnacles are considered to be relevant fouling organisms but are rarely, if ever, found on algal thalli. However, barnacles are common model organisms in marine antifouling studies (e.g., Rittschof 2001). On the other hand, mussels frequently settle on seaweeds (Petersen 1984, Eyster and Pechenik 1988, Davis and Moreno 1995, Lasiak

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and Barnard 1995, Alfaro et al. 2004). Despite the absence of activity against barnacles and mussels, the possibility that natural surface concentrations of elatol may be active in inhibiting other epibiont species should not be completely excluded. Furanones from Delisea pulchra were the only other metabolites also quantified at the algal surface (de Nys et al. 1998, Dworjanyn et al. 1999) and studied as antifoulants (de Nys et al. 1995, Steinberg et al. 1998, Wright et al. 2000, Dworjanyn et al. 2006). No single furanone was effective against all organisms tested, but in some cases, concentrations lower than 25 ng Æ cm)2 were strongly inhibitory (de Nys et al. 1995). The surface extract and furanones inhibited the settlement of fouling algae when tested at concentrations ranging between 10 and 100 ng Æ cm)2 (Dworjanyn et al. 2006). Therefore, both elatol and furanones were present at low concentrations on the surface and showed different effects depending on the organism tested. The fourth important aspect is where the chemicals are encountered with reference to the seaweed (on its surface or in the surrounding water) and any interactions among the compounds produced by the seaweed. Elatol can be found on the surface of L. obtusa (da Gama et al. 2003, Sudatti et al. 2006), and other reasons besides the absolute quantity of elatol found at the time of L. obtusa collection may impact this chemically mediated process. For example, the dynamics of allocation of this chemical defense, which implies costs of production, transport, storage, and maintenance (Cronin 2001), may be important and need to be evaluated in future studies. For seaweeds, most of the investigations into costs of chemical defenses are restricted to polyphenols (Yates and Peckol 1993, Pavia et al. 1999), which not only have a defensive function (Schoenwaelder 2002a,b) but also reveal apparent trade-offs between growth and defense (Arnold and Targett 2003). The only evidence for the cost of production of a secondary metabolite was described for furanones in D. pulchra (Dworjanyn et al. 2006). On the other hand, variation in concentrations of elatol on the surface of L. obtusa during periods of the day (e.g., low-tide periods) may also be important to understand the interaction between fouling and this seaweed. Finally, the knowledge of the mechanism by which the compounds are translocated to the seaweed surface can help explain the chemically mediated process and confirm that chemicals measured on the surface of any seaweed are not a methodological artifact. Like other metabolites (Dworjanyn et al. 1999, Paul et al. 2006), halogenated compounds can be localized in subcellular vesicles known as corps en cerise in Laurencia species (Young et al. 1980) or in gland cells in D. pulchra (Dworjanyn et al. 1999, Paul et al. 2006). Although gland cells were recognized as a storage structure (Dworjanyn et al. 1999, Paul et al. 2006), the

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production and transport of halogenated metabolites remain unclear. Studies of Asparagopsis armata described a stalk-like structure as a possible mechanism to transfer material to the thalli surface (Paul et al. 2006). Similar to that observed for gland cells of A. armata, we have demonstrated the existence of connections between the corps en cerise and the cell walls of L. obtusa. Furthermore, we have shown the occurrence of channel-like membranous connections that transport vesicles from the corps en cerise to the cell wall region. These results indicate that L. obtusa presents a specific process of chemical transport between the cell storage structure and the plant surface, where defense compounds such as elatol can also be detected. These recent findings about transport from cell storage structure to the thallus surface, coupled with trade-offs and cost ⁄ benefit theories related to chemical defense production, lead us to a crucial question: is elatol accessible at the surface at all times? The range of elatol concentrations previously quantified (Sudatti et al. 2006) and tested in this work may not perfectly represent the actual concentration available in fouling interactions. If the releasing rates of elatol from storage cell structures change with time, surface concentrations would also vary under different stress situations. We believe that these features of storage cell structures can provide essential cues about surfacemediated chemical defenses and can change the approach of fouling experiments. Despite the doubts about extraction methods and the mechanism for elatol release, this work provides the first ecological approach to fouling and herbivory assays using surface concentrations. Low elatol levels have only been evaluated in broad-scale bioassay testing focused on comparison of antifouling activity of natural products to commercial biocides (de Nys et al. 1996). More generally, these data contribute to elucidating the multiple ecological functions of the natural products and the ecological interactions at the surface of the alga. Unlike the surface concentration, the whole plant extract from L. obtusa is known for its efficiency as a defense against the attachment of the mussel P. perna (da Gama et al. 2002) and the herbivore Ly. variegatus (Pereira et al. 2003), and against fouling under field conditions (da Gama et al. 2003), demonstrating a broad or multiple spectrum of action. Additionally, elatol at the intrathallus concentration has the same properties (B. A. P. da Gama, unpublished data), but the question whether surface and intrathallus concentrations had different effects remained. Although the herbivore Ly. variegatus was not inhibited by surface concentrations of elatol, sea urchins possibly respond to chemical cues from either prey or predators (Hagen et al. 2002). Ly. variegatus usually ‘‘handles’’ food items with pedicellariae prior to consumption and could possibly detect and respond to prey cues prior

to ingestion. The possibility that this type of herbivore could be inhibited by compounds at the algal surface could not be excluded without the experiments performed. Similarly, other herbivores, either small (such as amphipods and snails) or large (such as fishes), could equally detect and respond to surface compounds, although the former explanation seems more likely than the latter. On the other hand, sea urchins are predators that upon the first bite will penetrate the outer cells of the algal thallus, thus being exposed to within-thallus concentrations of metabolites. Preventing even one bite by surface concentrations would be beneficial in preventing a small wound that could be the portal of entry for pathogens (Hay and Steinberg 1992), but having internal concentrations of a defensive metabolite that do deter herbivores would generally be a good strategy, which has been demonstrated to occur in L. obtusa (Pereira et al. 2003). Herbivory is one of the key factors structuring algal communities, affecting species abundance and distribution (Carpenter 1986, Cyr and Pace 1993). Fouling organisms can decrease growth and reproduction of the host plant (D’Antonio 1985, Williams and Seed 1992), increase thalli weight contributing to dislodgment (Dixon et al. 1981), and increase host plant attractiveness to consumers (Bernstein and Jung 1979, Wahl and Hay 1995, Pereira et al. 2003). Some authors argue that the existence and abundance of marine natural products are maintained by natural selection driven by herbivores (Hay et al. 1987, Hay and Steinberg 1992, Wright et al. 2004). Although little is known about their relative importance to the selection of marine chemical compounds, herbivory and fouling can be distinct pressures guiding this process (Hay 1996). According to our results in an actual or ecological approach, we hypothesize that both processes would not exert the same selective force on chemical defense production, because the low concentrations of elatol were inefficient to inhibit both herbivory and fouling, or would not represent distinct selective pressures since both processes were not inhibited. However, further evidence is needed from a wide variety of seaweeds in order to reinforce or refute these notions. We are grateful to Conselho Nacional de Desenvolvimento Cientı´fico e Tecnolo´gico (CNPq) and Fundac¸a˜o de Amparo a` Pesquisa do Estado do Rio de Janeiro (FAPERJ) for financial support. R. C. P., B. A. P. G., R. C., and G. M. A. F. thank CNPq for their Research Productivity fellowships, and D. B. S. thanks CNPq for her PhD fellowship. The authors also wish to thank the staff of the Instituto de Estudos do Mar Alte, and Paulo Moreira (IEAPM – Brazilian Navy) for providing assistance during field work. Abramoff, M. D., Magelhaes, P. J. & Ram, S. J. 2004. Image processing with ImageJ. Biophotonics Int. 11:36–42. Alfaro, A. C., Jeffs, A. G. & Creese, R. G. 2004. Bottom-drifting algal ⁄ mussel spat associations along a sandy coastal region in northern New Zealand. Aquaculture 241:269–90.

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Supplementary Material The following supplementary material is available for this article: Fig S1. (a) Bright-field image of the superficial view of a live cortical cell from L. obtusa. Corp en cerise (*) and the membranous-like channel connections (arrowheads) are seen. Scale bar, 2 lm. (b–g) Selected frames from the temporal sequential images (bright-field optical microscopy; 10 frames per second) of live cells revealing the occurrence of vesicular transport from the corps en cerise. Video S1. A video file obtained by using the ‘‘time-lapse’’ video microscopy technique showing the vesicular transport of halogenated compounds along the membranous tubular connections from corps en cerise to cell periphery (mpeg format). This material is available as part of the online article from: http://www.blackwell-synergy.com/ doi/abs/10.1111/j.1529-8817.2008.00507.x. (This link will take you to the article abstract.) Please note: Blackwell Publishing is not responsible for the content or functionality of any supplementary materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.