The effect of epibionts on the susceptibility of the red ...

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Downloaded By: [da Gama, Bernardo A. P.] At: 13:34 31 March 2008

Biofouling Vol. 24, No. 3, May 2008, 209–218

The eﬀect of epibionts on the susceptibility of the red seaweed Cryptonemia seminervis to herbivory and fouling Bernardo A.P. da Gamaa*, Rodrigo P. de A. Santosb and Renato C. Pereiraa a Programa de Po´s-Graduac¸a˜o em Biologia Marinha, Universidade Federal Fluminense (UFF), Niteroi, Brasil; bPrograma de Po´s-Graduac¸a˜o em Cieˆncias Ambientais, Universidade Santa U´rsula (USU), Rio de Janeiro, Brasil

(Received 9 December 2007; ﬁnal version received 5 March 2008) Epibiosis or fouling on living organisms can have direct and indirect detrimental eﬀects, in particular on photosynthetic organisms such as seaweeds. It thus seems reasonable to hypothesize that macroalgae have been selected for the presence or induction of antifouling (AF) defences. The red seaweed Cryptonemia seminervis is usually found in nature with an elevated cover of epibionts. To assess the eﬀect of epibiosis on the susceptibility of this seaweed to herbivory and fouling, the abundance of fouling was evaluated and compared to herbivore consumption (by amphipods and sea urchins) of fouled (bryozoan and sponge) and non-fouled C. seminervis. Attachment of the mussel Perna perna to surfaces treated with extracts from seaweeds with and without epibionts was also assessed. Epibiosis corresponded to ca. 51% of the blade surface of C. seminervis, sometimes covering as much as 90% and up to 51% of the thallus weight, encompassing mainly the bryozoan Membranipora membranacea and an unidentiﬁed sponge. Algae colonized by M. membranacea were preferred compared to algae devoid of epibionts, a ‘shared doom’ eﬀect, either by the amphipod Elasmopus brasiliensis or by the urchin Lytechinus variegatus (p 5 0.01). Sponge epibiosis also increased consumption by both herbivores (p 5 0.001), suggesting that epibionts may act as lures to herbivores, attracting consumers that otherwise would not feed signiﬁcantly on the seaweed. Foods containing extracts from fouled C. seminervis were preferred by urchins over the alga devoid of epibionts. However, extracts from fouled alga inhibited mussel attachment when compared to epibiont-free alga. Diﬀerences might be a direct detrimental eﬀect of the presence of epibionts. On the other hand, epibiosis may induce the production of AF defences in C. seminervis. Keywords: epibiosis; Cryptonemia seminervis; Rhodophyta; epibiont-plant-herbivore interaction; bryozoans; sponges

Introduction Epibiosis or biofouling is a common process in the marine environment and is capable of creating a new interface between the basibiont and its environment (Laudien and Wahl 2004; Railkin 2004). However, epibiosis by many epiphytes and epizoans may be detrimental to the basibiont, including seaweeds and invertebrates, and is often thought to be facultative and interspeciﬁc (Wahl 1989; Wahl and Hay 1995). Epibiosis is known to alter predation on hermit crabs (Ross 1971), scallops (Pitcher and Butler 1987) and mussels (Wahl et al. 1997), aﬀect feeding by bivalves (Paine 1976), cause shell destruction in periwinkles (Warner 1997), and limit the buoyancy and swimming rate of copepods (McAllen and Scott 2000). Epibiosis also can aﬀect the ﬁtness of the basibionts by reducing growth rates, survivorship, gonad maturity and ability to produce fertile oﬀspring (Wahl 1996; Buschbaum and Saier 2001; Cruz-Rivera and Hay 2000; Chan and Chan 2005). In addition, epibiosis may provide a defence or camouﬂage for both

*Corresponding author. Email: [email protected] ISSN 0892-7014 print/ISSN 1029-2454 online Ó 2008 Taylor & Francis DOI: 10.1080/08927010802041253 http://www.informaworld.com

invertebrate or seaweed host organisms, such as bacteria on crustacean embryos (Gil-Turnes et al. 1989), bryozoans on whelks (Barkai and McQuaid 1988), algae on clams (Vance 1978), invertebrates and algae on mussels (Laudien and Wahl 2004), sponges on scallops (Bloom 1975; Forester 1979), and bryozoan (Durante and Chia 1991) and algal epibionts on seaweeds (Karez et al. 2000). Seaweeds are particularly susceptible to fouling by epibiota because they are sessile and restricted to the photic zone where conditions for the growth of fouling organisms are optimal (de Nys et al. 1995). The consequences of epibiosis for seaweeds, however, are poorly known, but include reduced growth and reproduction (Orth and van Montfrans 1984; Brawley 1992; Williams and Seed 1992), increased drag and consequently tissue loss during storms (Dixon et al. 1981; Brawley 1992; Williams and Seed 1992), or increased susceptibility to consumers that are attracted to seaweeds possessing fouling organisms (Bernstein and Jung 1979; Pereira et al. 2003).

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Field observations on the common Brazilian red seaweed Cryptonemia seminervis (C. Agardh) J. Agardh (Rhodophyta, Halymeniaceae) reveal that this alga is usually found with a high cover of epibionts. This leads to questions concerning the role of epibiosis on algae, such as does epibiosis directly or indirectly aﬀect seaweed consumption? Does epibiosis aﬀect epibiont attachment? To answer these questions, amphipod and sea urchin consumption of non-fouled C. seminervis and C. seminervis fouled by bryozoans or sponges were compared, as well as the eﬀects of epibiosis on chemical defences against herbivores and an ecologically relevant epibiont.

analysis. Samples of epibionts were then removed, labelled, preserved and sent to specialists for taxonomic identiﬁcation. Additional blades (n ¼ 10) of C. seminervis with a range of epibiont fouling were then selected from aquaria and used to evaluate the relationship between epibiont weight increase and epibiont cover. Thalli were photographed and blade and epibiont cover determined as described previously. Epibionts were then removed and weighed (Sartorius balance, Germany, 0.0001 g precision) and regression analysis performed with area as the independent and weight increase as the dependent variable.

Materials and methods Seaweed collection

Eﬀect of epibionts on feeding by herbivores Feeding trials

Cryptonemia seminervis (C. Agardh) J. Agardh (¼ Cryptonemia luxurians) is a red seaweed commonly found along the Brazilian littoral, especially in shallow (53 m depth) wave-washed areas with strong currents. Praia Rasa (Shallow Beach) is located on the southeastern Brazilian coast between Armac¸a˜o de Bu´zios and Cabo Frio municipalities (228450 1500 S, 418530 1500 W), and has a rocky shore with a gentle slope. This site is considered to have the most diverse algal ﬂora of Rio de Janeiro state (Yoneshigue-Braga 1985). Approximately 100 plants of this seaweed (with and without epibiosis, usually found intermingled) were haphazardly collected at Praia Rasa by free diving (2 divers) along a 10 m-long transect positioned at ca. 1 m depth parallel to the shoreline during low tide. All sampled thalli were at least 5–7 cm in leaﬂet length, and presumably adult plants capable of being fertile. Sampled thalli were placed in coolers, immediately returned to the laboratory, and held in 400 l aquaria in seawater (salinity 35%) at 208C, under an irradiance of 200 mmol photons m72 s71 (12:12 h light: dark, daylight spectrum ﬂuorescent lamps).

Experiments were conducted on C. seminervis blades with and without epibionts (whole plant assays) and on artiﬁcial foods (artiﬁcial diet assays) made with extracts from blades with and without epibionts to assess epibiont eﬀects on food choice in two invertebrate herbivores, viz. Lytechinus variegatus (Lamarck 1816) and Elasmopus brasiliensis (Dana, 1855).

Estimation of the degree of epibiosis on C. seminervis Single blades (5–7 cm in length) were selected from independent thalli for use in experiments. Both sides of each blade were photographed using a digital camera (Nikon Coolpix885, Nikon Corp., Japan) at the maximum resolution (3.21 megapixels), together with a ruler for scale. These images were then analysed using the software ImageJ (Abramoﬀ et al. 2004). The area corresponding to each epibiont species (colony or individual) was identiﬁed and measured together with the area corresponding to the whole blade. Thus, the contribution of each epibiont species was determined as well as the area of each seaweed thallus covered by epibiosis. These data were converted to percentages for

Whole plant assays Epibiont-covered algae were divided into two parts, one of which had all epibionts carefully removed, while the other retained the ﬁeld epibionts in order to isolate the eﬀects of epibiont cover from those attributable to pre-existent chemical diﬀerences between naturally fouled and clean thalli. However, chemical diﬀerences were not observed on thin layer chromatography proﬁles of thalli with and devoid of epibiosis (data not shown). Epibionts were removed by hand whenever possible, and by scrubbing the thallus surface with a very soft toothbrush. Samples were then observed under an optical microscope to assess surface damage, because this can inﬂuence the concentrations of defence compounds at the plant surface (eg de Nys et al. 1998). Damaged individuals were thus excluded and never employed in assays. Only the two most common epibiont species (the bryozoan Membranipora membranacea [Linnaeus 1767] and a sponge of the order Haplosclerida) were used in feeding experiments (eg Wahl and Hay 1995; Wahl et al. 1997) because those were the only epibionts abundant enough to allow appropriate replication. Pairs of C. seminervis thalli of equal area, with and without epibionts, were simultaneously oﬀered to the sea urchin Lytechinus variegatus for sponge (N ¼ 35 replicates) and bryozoan (N ¼ 50) experiments. Similar feeding trials were performed with the amphipod Elasmopus brasiliensis with sponge (N ¼ 30) and bryozoan (N ¼ 30)

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Biofouling epibionts. All seaweed pieces oﬀered to these herbivores were weighed before and after the experiments to verify weight loss (wet weight, ww, 0.0001 g precision) attributable to urchin and amphipod feeding. Similarly, all algal pieces were photographed prior and after the experiments and images used to estimate losses due to consumption, using the ImageJ software as previously described. Equal numbers of control pieces (N ¼ 30–50) were included to estimate autogenic changes and corrections made according to the procedures of Peterson and Renaud (1989) and Cronin and Hay (1996). Careful observations were made during experiments to verify whether herbivores fed exclusively or predominantly on the epibiont species or on the seaweed, including image analysis as described previously. This was necessary in order to ensure that weight diﬀerences in particular were the result of consumption of epibiont-covered algae, and not due only to the removal of epibionts by consumers. Experiments with sea urchins were allowed to run overnight (sea urchins are active feeders only at night) until 30–40% of biomass was eaten in either treatment, which generally took only 3–4 h. Amphipods are slower grazers, thus taking more time to attain measurable consumption (3–5 days). Artiﬁcial diet assays Defensive properties of the crude extracts from thalli covered by epibionts were assessed by feeding trials with the sea urchin L. variegatus on artiﬁcially prepared food. Natural concentrations of both extracts were embedded into an agar-based artiﬁcial food, prepared according to Hay et al. (1994) and Pereira et al. (2000). Fouled and non fouled specimens of C. seminervis were collected, and fouled thalli carefully cleaned from epibionts as described previously. Extracts from each set of algae (fouling-cleaned and unfouled algae) were individually (ie each plant yielded one extract) prepared. This was necessary to assess diﬀerences in chemical defences that could have led to the presence of epibionts (eg genetic diﬀerences among algae could have led to diﬀerential susceptibility to epibiosis) or resulted from the production of defences in fouled algae. Prior to assays, the algae were completely cleaned from epibionts, freeze-dried, weighed (dry weight, dw) and then extracted thrice with dichloromethane (DCM). Solvents were evaporated in vacuo to yield the crude extracts. Artiﬁcial food blocks were prepared by (1) adding 0.72 g of agar to 20.0 ml of distilled water and heating the mixture in a microwave oven to the boiling point; (2) adding natural concentrations of crude extracts (ie an aliquot of extract equivalent to 2.0 g of algal dw, diluted in DCM) to 2.0 g of powdered Ulva fasciata

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(Chlorophyta), a preferred food item, and eliminating the solvent in vacuo; (3) adding the Ulva powder to 16 ml of water at room temperature, and adding this to the heated mixture, and (4) pouring the mixture (containing extracts of algae with or without epibiosis) onto a screen mesh (plastic window screen mesh, 1.2 mm sided squares), allowing it to harden and cool and then cutting it into small pieces (7 6 10 squares), which were then simultaneously oﬀered to the sea urchin L. variegatus. The assays were carried out in perforated plastic containers (1 l) holding one individual of L. variegatus (N ¼ 22), and placed in large tanks containing ca. 1000 l of recirculating seawater. For all assays, the defensive activities of crude extracts were estimated by comparing the number of squares consumed between experimental foods (with and without epibiosis). Antifouling assays Crude extracts of algae originally collected with or without epibiosis were prepared as described previously, and their eﬀects on the attachment of the common fouling mussel Perna perna (Linnaeus 1758) were assessed as an indicator of epibiosis deterrence or antifouling (AF) activity (Da Gama et al. 2003). Juveniles of this species are commonly found to recruit on several species of algae. Young specimens of P. perna were collected during low tide from the rocky coastal area of Itaipu (22858´2600 S, 438020 4800 W, Nitero´i city, Rio de Janeiro, Brazil) and kept in a 500 l recirculating laboratory aquarium (equipped with biological ﬁltering, protein skimming and activated carbon) at a constant temperature (208C), salinity (ca. 35%) and aeration for 6 h. Individuals were disaggregated by carefully cutting the byssal threads and divided into size groups according to shell length, ranging from 0.5 to 2.5 cm in a plastic tray with seawater. Only individuals exhibiting substratum exploring behaviour (actively exposing their foot and crawling) were selected for experiments. AF activity was measured by the following procedure, fully described in Da Gama et al. (2003). Water-resistant ﬁlter paper was cut into 9 cm diameter circles and soaked in solvent (DCM, within-treatment control surface). Another 9 cm-diameter set of ﬁlter papers (extract with and without epibiosis) was cut in a chess board pattern (1 cm2) and soaked in a natural concentration of extracts (determined as the extract equivalent to the dw of alga ¼ dw of ﬁlter paper). All ﬁlter paper circles were allowed to air dry. Entire ﬁlter circles were then placed at the bottom of sterile polystyrene Petri dishes (9 cm diameter), over which treated (extracts of algae with or without epibiosis) chess board ﬁlters were placed. Dishes were ﬁlled with

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80 ml of seawater and three mussel specimens were added. In this way, mussels would have the same area of the Petri dish with (superior, squared) and without extract (inferior, entire) for attachment (Figure 2b). Ten replicates per treatment (C. seminervis with and without epibionts) were used. Experimental units were kept in total darkness, as mussels have been shown to produce more byssal threads when held in the dark (Davis and Moreno 1995) and allowed to run for 12 h. Mussel activities were recorded immediately after the start of the experiment, and then after 12 h. The activities recorded were substratum exploring behaviour (data not shown), and number of byssal threads attached to each substratum. After the end of the experiment, all records of attachment were checked, mussels were placed in plastic mesh bags tagged according to treatment, and suspended in a sea aquarium for 24 h to check for possible mortality due to exposure to the test substances. Data analysis Data from all feeding experiments were analysed through t-tests for dependent samples whenever the assumption of normality was met. When data distributions were not normal, the non-parametric Wilcoxon signed-rank test was employed (Zar 1999). Weight and area data (%) were arcsin-transformed prior to analyses. Controls for autogenic change in algal biomass did not show any signiﬁcant diﬀerence (comparing biomass and area from equal numbers of control plants before and after experiments through ttests for dependent samples, p 4 0.25) and thus were not considered for biomass consumption calculations. Attachment data from two independent experiments performed simultaneously were analysed through Mann–Whitney U-test since distributions were not normal even after attempted transformations (Zar 1999). Data were considered as statistically diﬀerent whenever p 5 0.05 (a ¼ 5%). Results Estimation of the degree of epibiosis on C. seminervis From the 86 thalli of C. seminervis examined in detail, epibiosis corresponded to a mean of 51%+24.5% (SD) of the thallus surface, sometimes reaching a cover as great as 90% (epibiosis cover varied from 0 to 90%). Encrusting bryozoans of the species Membranipora membranacea were by far the most abundant, comprising up to 90% of the epibiont cover on C. seminervis hosts compared to remaining components of epibiosis on this red seaweed. Unidentiﬁed sponges of the order Haplosclerida were less frequent, but still abundant on some blades (comprising 6.9% of the epibiont cover),

while calcareous tube worms from the family Serpulidae (Hydroides spp.) and crustose coralline red seaweeds (family Corallinaceae) were rarely found and comprised 1.9 and 1.2% of the epibiont cover, respectively. Eﬀects of epibiosis on the alga: weight increase Epibiosis caused up to 51% weight increase in C. seminervis thalli, and weight increased linearly with area overgrown (weight ¼ 11.248 þ 0.463*area). A high correlation between the area of the thallus covered by epibionts and the weight increase was observed (Pearson’s r ¼ 0.834, p 5 0.05). Eﬀect of epibiont species on consumption by two herbivore species Epibiosis by either bryozoans or sponges caused a signiﬁcant increase in consumption (as measured by the weight change of the sets of fouled algae in comparison to epibiont-free algae) by both herbivores tested, the amphipod E. brasiliensis and the sea urchin L. variegatus (Figure 1a and d, p 5 0.001 and p ¼ 0.01, respectively), although sponge epibionts (Figure 1a and 1b) seemed to be even more attractive to these herbivores than bryozoans (Figure 1c and 1d). In all experiments, both epibiont species and host seaweeds were equally consumed by sea urchins and amphipods. The controls for autogenic changes showed neither signiﬁcant growth rates nor loss of biomass during the time considered for the experiments (p 4 0.25, t-tests for dependent samples). Inﬂuence of epibionts on defensive chemistry Artiﬁcial food containing non-polar extracts of seaweeds previously overgrown by epibionts was signiﬁcantly more consumed than food containing extracts of seaweeds devoid of epibiosis (Figure 2a, p 5 0.001, Wilcoxon test). On the other hand, the extracts of epibiont-covered seaweeds were signiﬁcantly more active against fouling than extracts of seaweeds that never presented epibiont overgrowth (Figure 2b, p 5 0.05, Wilcoxon test), suggesting that some kind of AF metabolite could be produced as a response to epibiosis. There was no mortality of mussels due to exposure to extracts. Discussion Most of the C. seminervis individuals sampled had some degree of epibiosis, usually colonies of the encrusting calcareous bryozoan M. membranacea, which covered up to 90% of the thalli, and less

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frequently, unidentiﬁed sponges of the order Haplosclerida. This study has shown that both epibionts increase consumption by amphipods and sea urchins compared to clean thalli. Littler and Littler (2000) reported that C. seminervis from the Caribbean is also found partially or entirely encrusted by bryozoans. Along the Atlantic coast of Nova Scotia, M. membranacea is an invasive species, to which a disruption in sea urchin-kelp dynamics has been attributed (Scheibling et al. 1999). This bryozoan encrusts kelp fronds causing fragmentation and loss, repeatedly decimating kelp beds. By removing the dominant canopy species, M. membranacea has possibly facilitated further invasion by the Asian green alga, Codium fragile spp. tomentosoides (Scheibling et al. 1999). Although the bryozoan increase in C. seminervis fragmentation was not currently assessed, encrusted thalli of this seaweed were frequently found adrift along the studied site, Praia Rasa. Epibionts are known to either increase or decrease the susceptibility of basibionts to consumption, depending on epibiont identity (eg Karez et al. 2000, Enderlein et al. 2003). This investigation suggests that epibiosis by the bryozoan M. membranacea and an unidentiﬁed sponge of the order Haplosclerida can alter the susceptibility of C. seminervis to sea urchin and amphipod grazing. Epibiont cover on this seaweed may act as a lure to herbivores, ie epibionts may attract consumers that otherwise would not feed signiﬁcantly on the host plant. This could be due to an increased nutritional value of fouled compared to clean algae. Likewise, herbivores could be attracted to algal cues possibly resulting from fouling damage on seaweeds (Wahl and Hay 1995; Leonardi et al. 2006). There are intrinsic diﬃculties to separate and thus interpret the consumption of epibionts from the algal consumption, or from the unit composed by epibionts and basibionts. Regardless of the method limitation, however, the results seem to suggest that epibionts have a ‘shared doom’ eﬀect (sensu Wahl and Hay 1995), rather than provide a ‘protective coating’ (Karez et al. 2000) to C. seminervis.

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Figure 1. Consumption of the red seaweed C. seminervis with and without sponge (a and b) or bryozoan epibionts (c and d) by the sea urchin L. variegatus (b and d) or by the amphipod E. brasiliensis (a and c). Data shown are mean percentage of initial biomass (wet weight) removed (left axis) and mean percentage of initial area consumed (right axis) + SEs. N ¼ number of replicates. Signiﬁcant diﬀerences are indicated whenever p 5 0.05. Autogenic changes in equal numbers of controls not exposed to grazers accounted for a non-signiﬁcant growth of 51% in a and c (p 4 0.25).

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Figure 2. Inﬂuence of the presence of epibiosis on C. seminervis on the defensive activity of crude extracts against (a) herbivory and (b) fouling. AF activity (%) is expressed relative to mean number of mussel byssal threads attached to controls. Error bars are SEs over mean values. N ¼ number of replicates. Signiﬁcant diﬀerences are indicated whenever p 5 0.05.

Epibiosis is also known to have a considerable eﬀect on macroalgal production, and some papers have demonstrated experimentally that epibiont algae such as Ulva can reach weight loads of ca. 60% of total biomass in intertidal cultures of Gracilaria chilensis from southern Chile (eg Buschmann and Gomez 1993). Their study evaluated the relative importance of light reduction, addition of weight to the host thalli, and nutrient depletion as mechanisms determining the interactive eﬀects of epibionts on Gracilaria cultivation, and concluded that epibionts can signiﬁcantly depress algal biomass production. The addition of weight to the host algae and the consequent increase of dislodgement appear to be the main mechanisms involved in this interaction (Buschmann and Gomez 1993). In the present study, epibiont cover caused a weight increase as high as 50%, which could possibly incur a

considerable growth decrease in Cryptonemia. Theoretically, this detrimental eﬀect of epibiosis could, by itself, lead to some sort of response by the basibiont alga, such as defence induction. However, unlike defence induction against herbivores (eg Cronin 2001, Paul et al. 2001, Macaya et al. 2005, Amsler and Fairhead 2006, Diaz et al. 2006, Coleman et al. 2007, Long and Trussell 2007, Svensson et al. 2007, Toth et al. 2007), inducible defences against epibionts have never been demonstrated experimentally. According to herbivory data, the present results showed a clear relationship between the presence of epibiosis and increased consumption. This study supports previous ﬁndings, which have shown that host plants can be fouled by higher-preference epibionts, thus suﬀering not only the direct negative eﬀects of being fouled (Davis et al. 1989; Wahl 1989; Williams and Seed 1992), but also experiencing increased grazing rates (Wahl and Hay 1995; Karez et al. 2000). None of the common epibionts on Cryptonemia provided ‘associational resistance’ (sensu Wahl and Hay 1995) or a ‘protective coating’ to this alga (Karez et al. 2000). The eﬀect of epibiosis on susceptibility to consumers may vary with herbivore type. Studies of associational resistance in the marine environment evaluated mainly larger generalist herbivores, such as sea urchins and ﬁshes (Hay 1986; Littler et al. 1986; Pﬁster and Hay 1988). At ﬁrst, these studies with larger generalist herbivores might not be predictive for smaller mesograzers that live and feed on seaweeds (Hay 1992). For example, feeding preferences by amphipods can be a result of food value, habitat quality and epiphytes (Brawley 1992; Hay 1992; Duﬀy and Hay 1994). However, the data presented here veriﬁed that the amphipod E. brasiliensis and the sea urchin L. variegatus were aﬀected similarly by epibionts on seaweeds, although sea urchins were less aﬀected by bryozoans than amphipods. Amphipods, unlike sea urchins, being small and having small mouth pieces, would be expected to feed predominantly if not exclusively in a selective form, either preferring to consume the algae or the epibionts, depending on the species-speciﬁc nature of amphipod feeding preferences (eg Duﬀy 1990). However, this was not the case, since during the experiments, observations and image analyses have shown that both herbivore types, amphipods and sea urchins, fed equally on epibionts and basibiont algae, ie consumed the whole layer of epibiont–basibiont association in a given part of the thallus. Indeed, E. brasiliensis is abundantly found either on macroalgae or on sessile invertebrates such as hydroids and branching bryozoans, such as Bugula neritina and Zoobotryon verticillatum (Weidner et al. 2004).

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Biofouling According to an interpretation of preferences of herbivores related to the presence of epibiosis on host seaweeds, each partner would contribute to the chemical aura sensed by sea urchins (Wahl and Hay 1995). This may also be true in the present study, because the presence of M. membranacea or the less abundant Haplosclerida sponge on C. seminervis clearly alters herbivore preference either by the sea urchin L. variegatus or by the amphipod E. brasiliensis. However, is the observed preference in consumption between clean vs. fouled specimens of C. seminervis a direct eﬀect of the presence of epibionts? Is the presence of epibiosis in host plants caused by previous chemical diﬀerences among individuals or does it cause chemical diﬀerences to exist among individuals (ie epibionts trigger the production of chemical defences)? Several studies have clearly shown that seaweed natural products function as feeding deterrents toward herbivores but their eﬀects may vary depending on the type of herbivore (see Hay 1996; Paul et al. 2001 for reviews). Chemical defences are often active against large generalist herbivores such as ﬁshes and sea urchins, but not against smaller, less mobile mesograzers such as amphipods. Previous studies revealed that diﬀerences in chemistry between clean and fouled hosts were not responsible for the diﬀerential consumption by sea urchins (Wahl and Hay 1995). The complementary assays revealed that artiﬁcial food with crude extracts from fouled C. seminervis were preferentially consumed by herbivores compared to food prepared with extracts from epibiont-free specimens. At ﬁrst, the results revealed that chemical defences against herbivores would not have been triggered by the presence of epibiosis. This would have seemed logical, given the costs that could incur from the attractiveness created by epibionts. As far as is known, there is no information on secondary metabolites produced by Cryptonemia. Weidner et al. (2004) showed that C. seminervis from the same site in the Brazilian coast did not exhibit induced defences against amphipod grazers. However, a series of biologically active galactans was identiﬁed from other species of Cryptonemia (C. crenulata, Talarico et al. 2004; Zibetti et al. 2005), that could play some yet unknown ecological role. Although chemical defences against herbivores were not found, it is possible that defences have been allocated to the primary source of harm, ie epibionts, and not to herbivores. Extracts from naturally fouled C. seminervis specimens signiﬁcantly inhibited attachment of P. perna in laboratory assays, when compared to extracts of algae devoid of epibiosis in nature. Several studies have documented the induction of secondary metabolites, toughness, or resistance to herbivores in response to natural or simulated grazing

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(Lowell et al. 1991; Cronin and Hay 1996; Hammerstrom et al. 1998; Pavia and Toth 2000; Weidner et al. 2004; Macaya et al. 2005; Diaz et al. 2006; Coleman et al. 2007). However, there is no evidence to date of induction of defences against fouling in macroalgae, although it is now clear that fouling pressure surely exerts a strong selective pressure on basibionts that could have led to the development of AF defences in a number of organisms (eg Bakus et al. 1986; Bingham and Young 1991; Becker and Wahl 1996; Armstrong et al. 2001; Da Gama et al. 2002; Hellio et al. 2004; Bers et al. 2006; Guenther and de Nys, 2007; Guenther et al. 2007). On the other hand, there is no information about the identity of chemicals capable of inhibiting fouling in Cryptonemia species. Another red alga, Gracilaria conferta, responds to cues of microbial infestation (oligosaccharides) with oxidative bursts, a possible algal defence system against epibionts likely to be present in other red seaweeds (Weinberger et al. 1999; Weinberger and Friedlander 2000a, 2000b). Inhibition of fouling could be a case of induced defence in response to epibiosis on C. seminervis, perhaps to prevent further colonisation. The induction of defences against fouling after an organism is already colonised may seem useless at ﬁrst, but epibiosis, besides the direct eﬀects and the eﬀects on consumption, may alter friction forces and ﬂow patterns in a way, which can lead to higher settlement rates of propagules (Abelson et al. 1994; Abelson and Denny 1997; Koehl 2007), and consequently to more heavily fouled surfaces. In this case, some unknown level of epibiosis would be needed to trigger the production of defences that would then prevent further settlement or growth of foulers. However, further experimental evidence is needed to support this. Manipulations of nutrient and light availability revealed the crucial role of epibiota in mediating resource availability for the brown seaweed, Fucus vesiculosus (Jormalainen et al. 2003). Nutrient enhancement alone increased epibiota and decreased phlorotannins. Cleaning the thallus resulted in increased growth and together with nutrient enhancement, also in a trade-oﬀ with phlorotannins (Jormalainen et al. 2003). Diﬀerences in feeding preference might be a direct eﬀect of the presence of epibionts, a shared doom eﬀect of epibiosis. However, diﬀerences in chemistry between clean and fouled C. seminervis could be induced by epibionts, or a response to resource availability mediated by epibiosis. Although naturally produced chemicals are undoubtedly important in mediating interactions at the surfaces of living marine organisms such as seaweeds, the chemical ecology of surface interactions such as epibiosis has lagged behind that of seaweed/herbivore interactions, the area which has dominated the ﬁeld of

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macroalgal chemical ecology. This is for a number of reasons, including methodological issues such as the diﬃculty in identifying, quantifying, and testing chemical signals for colonisation in situ relative to analogous experiments and measurements for seaweed-herbivore studies (Steinberg and de Nys 2002). Additional studies are still needed to cast more light upon the relationship among macroalgae, epibiosis and consumers.

Acknowledgments FAPERJ, CNPq, and CAPES supported this research. BAPG and RCP thank CNPq for their Research Productivity Fellowships. The authors thank R. Villac¸a, A. Gu¨th, and S.M. Ribeiro for seaweed, amphipod and sponge identiﬁcation, respectively. J.H.S. Miyamoto, N.G. Silveira, M.A.O. Lacerda and L. Avellar helped either in ﬁeld work or in preliminary assays. Comments by Steven N. Murray and anonymous reviewers greatly improved this work. All experiments performed comply with Brazilian environmental protection laws.

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