Prompt induction of chemical defenses in the red seaweed Laurencia dendroidea: The role of herbivory and epibiosis

Journal of Sea Research 138 (2018) 48–55

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Prompt induction of chemical defenses in the red seaweed Laurencia dendroidea: The role of herbivory and epibiosis

T

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Daniela Bueno Sudattia, , Mutue Toyota Fujiib, Silvana Vianna Rodriguesc, Alexander Turrad, Renato Crespo Pereiraa,e a

Departamento de Biologia Marinha, Instituto de Biologia, Universidade Federal Fluminense (UFF), P.O. Box 100.644, Niterói, Rio de Janeiro CEP 24.001-970, Brazil Departamento de Ficologia, Instituto de Botânica, São Paulo, Brazil c Departamento de Química Analítica, Universidade Federal Fluminense, Outeiro de São João Batista, s/n, Niterói, Rio de Janeiro, Brazil d Departamento de Oceanografia Biológica, Instituto Oceanográfico, Universidade de São Paulo, São Paulo, SP 05508-900, Brazil e Instituto de Pesquisas Jardim Botânico do Rio de Janeiro, Rio de Janeiro, Brazil b

A R T I C LE I N FO

A B S T R A C T

Keywords: Inducible chemical defense Simulated herbivory Microalgal epibiosis Bacterial epibiosis Bacterial infestation Algal susceptibility Elatol Chemical defense

Induced chemical defense is still - uncertain process in seaweed, especially in tropical areas. Attention has been given to changes on phlorotannin contents due herbivore pressure, while knowledge considering - other secondary metabolites or other environmental factors as inducers is limited. We used laboratory experiments to assess induced chemical defense in the tropical red seaweed Laurencia dendroidea in response to simulated herbivory, presence of epibionts (microalga and bacterium), and bacterial infestation, addressing the following questions: 1) does inducible chemical defense by simulated herbivory, as well as microalgal and bacterial epibiosis, occur?; 2) are chemical defenses induced by bacterial infestation after simulated herbivory?; 3) is there a trade-off between inducible defense and seaweed growth?; and 4) how do variations in chemical defense affect seaweed susceptibility to specialist and generalist consumers? Inducible defense was measured as changes in sesquiterpene elatol levels, and growth was taken to infer whether induced chemical defense incurred metabolic costs. Specimens of L. dendroidea under simulated herbivory produced higher concentrations of elatol up to 2 days after treatment, followed by relaxation after 7 days, while epibiosis (by microalga and bacterium) and bacterial infestation did not induce a defense response. Simulated herbivory resulted in reduced L. dendroidea growth, reinforcing that induced chemical defense is a cost-saving strategy as stated by the Optimal Defense Theory (ODT), -in addition there was a tendency of epibiosis for biomass decreasing. The defensive effect varied according to elatol concentration and consumer identity, since aplysiids tolerated 10-fold natural concentrations (NC) whereas sea urchins were inhibited by NC. Our results highlight that inducible chemical defenses may also be a more rapid process than previously evidenced in seaweeds. Higher levels of elatol promoted decreased consumption, demonstrating that seaweed susceptibility is dose-dependent and induction is an important ecological strategy depending on environmental conditions.

1. Introduction In marine systems, grazing pressure by herbivores strongly influences distribution, abundance and community structure of seaweeds (Lubchenco and Gaines, 1981; Carpenter, 1986). Seaweeds are exposed to a broad variety of herbivorous pressure, from mobile fishes and sea urchins (Humphries et al., 2014) to more sedentary crustaceans (e.g., amphipods) (Butler and Mojica, 2012) and mollusks (Williams and Walker, 1999). Herbivory has been considered an important selective force favoring production and evolution of chemical defense in these organisms (Wright et al., 2004), which can respond to this pressure in

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Corresponding author. E-mail address: [email protected] (D.B. Sudatti).

https://doi.org/10.1016/j.seares.2018.04.007 Received 8 June 2017; Received in revised form 3 April 2018; Accepted 29 April 2018 Available online 01 May 2018 1385-1101/ © 2018 Elsevier B.V. All rights reserved.

two ways, i.e., through production of constitutive (Pereira and da Gama, 2008) and/or induced chemical defenses (Jormalainen and Honkanen, 2008). Constitutive chemical defenses represent those secondary metabolites that are continuously produced (and sometimes stored as inactive forms) in sufficient amounts to inhibit adverse agents, such as herbivores. Constitutive chemical defenses have been extensively studied in seaweeds over the last 20 years, involving studies of green, brown and red seaweed species from cold to tropical waters, mainly focused on compounds such as terpenes and phlorotannins (Amsler, 2008). Induced chemical defenses are produced by seaweeds in response to

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for their own defense, as aplysiids (Rogers et al., 1995, 2000), whereas L. variegatus avoids it (Pereira et al., 2003). Natural populations of L. dendroidea present wide variations in the levels of chemical defenses they produce (Sudatti et al., 2006; Oliveira et al., 2013), which probably determines the - magnitude of - defense response. Thus, we specifically addressed the following questions in this red seaweed: 1) does inducible chemical defense by simulated herbivory, as well as microalgal and bacterial epibiosis, occur?; 2) are chemical defenses induced by bacterial infestation after simulated herbivory?; 3) is there a tradeoff between inducible defense and seaweed growth?; and 4) how do variations in chemical defense affect seaweed susceptibility to specialist and generalist consumers?

herbivory, leading to the herbivore having a reduced preference for the seaweed or reduced performance, as well as increasing seaweed fitness and promoting phenotypic plasticity (Toth and Pavia, 2007; Jormalainen and Honkanen, 2008). While constitutive chemical defenses occur broadly, meta-analyses have revealed that induced chemical defenses are more common in brown compared to red and green seaweeds (Toth and Pavia, 2007; Jormalainen and Honkanen, 2008). In addition, induced chemical defenses are mainly reported for brown temperate seaweeds and involve changes in the production of a single chemical group, the phlorotannins (for reviews see Toth and Pavia, 2007 and Jormalainen and Honkanen, 2008; as well as Macaya and Thiel, 2008; Haavisto et al., 2010, 2014; Reynolds and Sotka, 2011; Flöthe et al. 2014 a,b,c). Induced defenses have commonly been described from vascular terrestrial plants, with herbivory resistance being theorized as a tradeoff between benefits and costs (Karban and Baldwin, 1997). When herbivores are present, a plant may increase its resource allocation to defense in a manner that will protect it from future attacks rather than, for example, investing those resources in growth or reproduction. This concept is the central basis of the Optimal Defense Theory (ODT), which predicts that organisms allocate defenses in a way that maximizes fitness, but that such defenses are costly in the absence of enemies (Rhoades, 1979). In fact, some authors have demonstrated that resource allocation to seaweed defense results in reduced growth, lower photosynthetic rates and reduced fecundity (e.g. Dworjanyn et al., 2006). Thus, induced defense is considered a cost-saving strategy to protect organisms when natural enemies are present, as continuous production of defensive chemicals infers biosynthetic, storage and transport investments (Dworjanyn et al., 2006; Paradas et al., 2010). Despite the considerable attention given to plant-herbivore interactions, other interactions in marine systems can also be understood as evolutionary forces on chemical defense production, such as space competition. Seaweeds are frequently used as a substrate for the settlement of epibionts, which negatively affect the basibionts by impeding growth and reproduction (Orth and van Montfrans, 1984; Brawley, 1992; Williams and Seed, 1992), facilitating drag and consequent tissue loss during storms (Dixon et al., 1981; Brawley, 1992; Williams and Seed, 1992), or increasing the susceptibility to consumers that are attracted to seaweeds possessing such fouling organisms (Bernstein and Jung, 1979; Pereira et al., 2003). Macro- and microepibionts are both relevant fouling organisms. For example, bacterial biofilms are known to interfere in successional processes (Wahl and Hay, 1995; Steinberg et al., 2002) and other biological interactions, such as chemical defenses (Da Gama et al., 2014). Bacterial adhesion is inversely related to levels of furanones in the red seaweed Delisea pulchra (Maximilien et al., 1998) and it promotes an increase in the transport of corps en cerise in Laurencia dendroidea (Salgado et al., 2008; Paradas et al., 2010). Epibionts can also act beyond the thallus surface, assuming a pathogenic role inside the thallus (Weinberger, 2007). Breach of the thallus barrier may also occur due to lesions caused by herbivory, which may facilitate the entry of microorganisms such as bacteria (Hay, 1996; Amsler, 2001). Herbivory and epibiosis can potentially exert a selective pressure on the production and evolution of chemical defenses in seaweeds. Therefore, we explored the dynamics of elatol levels in clones of Laurencia dendroidea (Ceramiales: Rhodomelaceae) to monitor inducible defense when this seaweed was subjected to herbivory and epibiotic stimuli. The genus Laurencia has been studied for decades due to its production of halogenated terpenoids (Harizani et al., 2016), but the ecological roles and production dynamics of these chemicals are still under investigation. The sesquiterpene elatol exhibits important constitutive chemical defense action against herbivory (Pereira et al., 2003) and fouling organisms (Da Gama et al., 2003), and it is the major defense compound in populations of Laurencia dendroidea along the Brazilian coast (Oliveira et al., 2013). Some herbivores are known to feed on species of Laurencia and to sequester halogenated compounds

2. Material and methods Laboratory bioassays were conducted to detect induced changes in elatol levels in cultivated clones of L. dendroidea (isolation and biomass propagation as Sudatti et al., 2011, voucher SP399789, Instituto de Botânica, São Paulo state, Brazil) under conditions of simulated herbivory, microalgal and bacterial epibiosis, and bacteria infestation following simulated herbivory. Associated bacterial biofilm was also cultured (see Paradas et al., 2010) and the microalga Phaeodactylum tricornutum - (CEPA UB7) was sourced from the Microalgae Laboratory, Elizabeth Aidar Collection, Universidade Federal Fluminense, Rio de Janeiro state, Brazil. General conditions for seaweed cultivation were: sterile seawater enriched with 50% von Stosch solution (VS/2), at 22 ± 2 °C, salinity 32 ± 1% and irradiance 60–80 μmol photons m−2 s−1, provided by cool-white fluorescent lamps with 14:10 h light:dark cycle, without aeration. Prior to each bioassay, clonal replicates of L. dendroidea (three thallus tips measuring c.a. 1 cm in length) were isolated in small glass flasks (50 ml) and allowed to acclimate for 7 days (d) under the same conditions described above. It was assumed as the experimental unit for the induction bioassays described -below. Different individuals were used on bioassays, gametophyte for herbivory and bacterial epibiosis, and sporophyte for microalgal epibiosis. 2.1. Herbivory induction Herbivory induction was simulated using a small pin to remove biomass along the thallus promoting little holes; thereby wounding L. dendroidea and reflecting consumption by mesoherbivores (an important group reported to feed on and induce chemical defense in seaweeds; Hay, 1992). Each wound was equidistant (2 mm). The treatment condition consisted of wounded seaweeds (T, n = 4), whereas the control condition (C, n = 4) was represented by seaweeds without wounds. The induced defense response was evaluated by measuring levels of elatol after ¼ d, 2 d and 6 d. At the same timepoints, growth was also estimated as: final biomass – initial biomass per time-point – wet weight (ww) after removing excess water with paper towel. 2.2. Microalgal induction We chose the diatom P. tricornutum for this bioassay due to its epibiotic nature (Hellio et al., 2002), its importance as a promoter of bacterial biofilms (Dugdale et al., 2006; Molino and Wetherbee, 2008), as well as its broad tolerance to different cultivation conditions that facilitated acclimation to our culture conditions for L. dendroidea. The only modification consisted in enriched VS/2 media with silicate (Na2SiO4.5H2O) in the same Conway media ratio (80 mg ml−1; Walne, 1966), in which this microalga was originally kept. The experimental conditions (n = 5) were: treatment (T, seaweed + microalgae in VS/ 2 + Na2SiO4.5H2O); control (C, seaweed in VS/2 + Na2SiO4.5H2O); null control (NC, seaweed in VS/2). Five milliliters of P. tricornutum stock culture (3.08 × 106 cells ml−1) were inoculated into the 49

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prepared these by adding 0.45 g agar to 10 ml of distilled water and heating it in a microwave until boiling point. This mixture was added to 6 ml of cold water containing 1 g of freeze-dried and ground Ulva. Treatment foods contained elatol or crude extract of L. dendroidea that had been incorporated into the ground Ulva, and the carrier solvent (CH2Cl2) was removed by evaporation. Control foods received the same proportions of the carrier solvent to ensure that its presence did not bias our results. Wafers were supported in a plastic net marked with 10 × 10 squares (Hay et al., 1994). Artificial foods were offered to consumers in separate small aquaria (2 l) with circulating seawater for 24 h and 8 h, respectively, for sea urchins (n = 15 per tested concentration) and mollusks (n = 5 per tested concentration). Bioassay durations were established according to species voracity, with aplysiid specimens feeding faster than sea urchins. At the end of bioassays, consumption of artificial foods was measured by counting the numbers of removed squares.

treatment with cultivated seaweed clones. The induced defense response was evaluated by measuring levels of elatol after 2 d and 7 d. At the same time-points, growth of L. dendroidea was estimated, as described above. After 2 d and 7 d, 5 ml samples of media culture were extracted from each experimental condition, i.e. treatments, and fixed with acetic lugol solution (1% v/v) to estimate microalgal biomass (cells ml−1) in Neubauer chambers under a conventional microscope (Guillard and Sieracki, 2005) as a measure of microalgal culture viability. 2.3. Bacterial induction and infestation after simulated herbivory Bacterial biofilms were isolated from the surfaces of L. dendroidea (as detailed in Paradas et al., 2010) and re-inoculated onto cultivated clones of this seaweed as inducers. For bacterial isolation, a sample of seaweed was transferred to Erlenmeyer flasks containing sterile seawater (50 ml) and stirred for 5 min to break down the bacterial biofilm (Epstein and Rossel, 1995). Released bacteria were cultivated in a medium of Bacto™Peptone (0.5 g, BD Biosciences), seawater (450 ml) and an aqueous soluble extract of L. dendroidea tissue (2 ml). This soluble extract corresponded to the supernatant fraction obtained by centrifugation (Eppendorf centrifuge 5810 R) of 20 g fresh weight blended seaweed (blender Philips RI2044/51). We used this soluble extract to represent the natural nutrients and chemical signals available to the community of bacteria living on L. dendroidea. Five milliliters of this medium containing 3.48 × 106 cells cm−3 cultured bacteria was inoculated into vials with clones of L. dendroidea. The induced defense response was assessed by levels of elatol found in L. dendroidea after 2 d and 7 d under the following conditions: treatment 1 (T1, unwounded seaweed with bacteria); control 1 (C1, only unwounded seaweed); treatment 2 (T2, wounded seaweed with bacteria); control 2 (C2, only wounded seaweed). For each experimental condition (n = 5), growth was estimated as described above. After 2 d and 7 d, 1 ml of media culture was extracted from all experimental conditions, fixed in 4% formol, and bacterial biomass was evaluated by epifluorescence microscopy (Kepner and Pratt, 1994) cell counts to determine bacterial culture viability.

2.5. Chemical analysis Crude extracts in 20 ml hexane were obtained from each individual clone of L. dendroidea at intervals of one week, repeated three times. Elatol quantification was performed by gas chromatography coupled to electron capture (GC-ECD) using a previously-described external standardization method (Sudatti et al., 2006), with a few modifications. The GC-ECD system (Chrompack, Netherlands) was fitted with a RTX-5 capillary column (30 m × 0.25 mm, 5% phenyl, 95% dimethylpolysiloxane; Restek, USA). The oven temperature program was as follows: 80 °C (1 min), 10 °C min−1 to 250 °C (16 min), then −15 °C min−1 to 80 °C. Nitrogen (99.999%, White Martins) was used as carrier (28 cm s−1), makeup (35 ml s−1) and purge (15 ml s−1). The detector temperature was set at 320 °C and the injection pressure was 120 kPa. On column injections were carried out (0.5 μl). Isolation of standard elatol was carried out using pre-coated TLC (thin layer chromatography) plates, and identified by TLC (Merck Al TLC 20 × 20 cm silica gel 60F254) and 1H NMR (nuclear magnetic resonance), and compared with the literature (König and Wright, 1997; Machado et al., 2011). 2.6. Statistical analysis

2.4. Feeding deterrence by elatol and crude extracts of L. dendroidea The effects of simulated herbivory, microalgal and bacterial inductions and time-period on elatol levels and growth (= biomass) in L. dendroidea were estimated using two-way analysis of variance (ANOVA) followed by Tukey's test where appropriate (α = 5%). We predicted that presence of these stimuli (simulated herbivory, bacterial and microalgal epibiosis and bacterial infestation after simulated herbivory) would promote an increase in elatol levels and a decrease in growth, according to the trade-off model outlined by ODT. We also used analysis of variance (one-way ANOVA with post hoc Tukey's test, α = 5%) to compare the consumption of artificial food wafers containing different levels of chemical defense in each bioassay (with sea urchin and mollusk, independently). We expected that consumption would be lower for artificial food wafers with higher elatol concentrations, indicating that susceptibility varied according to the induced defense response in individuals of this natural population. L. dendroidea populations are known to present huge intra- and interpopulational variability in chemical defense contents (Oliveira et al., 2013), which implies different levels of induced defense response.

To analyze how chemical defense variation may affect the susceptibility of L. dendroidea to herbivory, different concentrations of elatol or crude extract based on natural concentrations (NC) of the sampled population (10 mg elatol g−1 of alga dry weight – dw) were evaluated in a feeding preference bioassay using artificial food wafers, as described in Hay et al. (1994). Two consumers were tested; the specialist Aplysia brasiliana and the generalist sea urchin Lytechinus variegatus. Specimens of A. brasiliana and L. variegatus were collected from Forno Inlet, Armação de Búzios, RJ (22°46′4″S, 41°52′57″W) and Itaipu Beach, Niterói, RJ (22°97′43″S, 43°04′78″W), respectively. Both consumers were transported to our laboratory at University Federal Fluminense, kept in individual aquaria (2 l) with recycling seawater, and acclimated for one week prior to bioassays. We varied the range of tested concentrations according to each organism. For sea urchins, the levels of elatol tested were: -NC; 0.1 NC; 0.01 NC; 0.001 NC; 0.0001 NC; and control (palatable seaweed). For aplysiids, levels of crude extract tested were: NC; 0.1 NC; and 10 NC. There was no control condition for the aplysiid bioassay because this mollusk naturally eats L. dendroidea. To conduct the aplysiid bioassay, we applied crude extract instead of elatol due to a scarcity of the pure compound; the highest concentration we could use was 100 mg elatol g−1 of algal dw, but elatol is the major compound in the crude extract so any activity can be attributed to this metabolite. Concentrations of elatol or crude extract were incorporated into a ground palatable alga (Ulva sp.) to make artificial food wafers. We

3. Results 3.1. Herbivory induction Simulated herbivory promoted changes in elatol levels in L. dendroidea (Table 1). After 2 d, the elatol level in wounded clones was higher than in intact ones (Fig. 1A). When we compared treatment and control pairs, post hoc Tukey's test indicated a tendency for elatol 50

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Table 1 Two-way ANOVA evaluating the effect of time (days) and experimental condition on elatol levels and growth (=biomass) of L. dendroidea in induction bioassays: herbivory, and microalgal and bacterial epibiosis. Inducing factor Herbivory Elatol

Biomass

Microalgal epibiosis Elatol

Biomass

Bacterial epibiosis Elatol

Biomass

Variation source

df

F

P

Time (¼ d, 2 d, 6 d) Condition (wounded or unwounded) Time × condition Time (0 d, ¼ d, 2 d, 6 d) Condition (wounded or unwounded) Time × condition

2 1 2 3 1 3

2.14 16.68 1.82 9.72 10.21 1.18

0.146 < 0.001 0.190 < 0.001 0.002 0.243

Time (2 d, 7 d) Condition (diatoms or no diatoms) Time × condition Time (0 d, 2 d, 7 d) Condition (diatoms or no diatoms) Time × condition

1 2 1 2 2 4

0.25 0.55 0.12 26.50 2.08 0.54

0.621 0.582 0.885 < 0.001 0.139 0.768

Time (2 d, 7 d) Condition (unwounded and bacteria, unwounded and no bacteria, wounded and bacteria or wounded and no bacteria) Time × condition Time (0 d, 2 d, 7 d) Condition (unwounded and bacteria, unwounded and no bacteria, wounded and bacteria or wounded and no bacteria) Time × condition

1 3

65.44 13.22

< 0.001 < 0.001

3 2 3

10.99 20.57 12.51

< 0.001 < 0.001 < 0.001

6

8.55

< 0.001

Fig. 2. Effect of microalgal epibiosis on elatol levels (A) and growth (=biomass) (B) of L. dendroidea after 2 and 7 days of incubation. Treatment = seaweed with microalgae in VS/2 + Na2SiO4.5H2O; control = seaweed in VS/2 + Na2SiO4.5H2O; null control = seaweed in VS/2 (n = 5 per experimental condition). Symbols and statistical tests as in Fig. 1.

production to increase after ¼ d (T1/4d = C1/4d; P = 0.079), but there was a significant increase in treatment over control after 2 d (T2d > C2d; P = 0.037) and a relaxation or decrease in elatol content in - treatment after 6 d - (C6d = T6d; P = 0.960). Growth (= biomass) of L. dendroidea was equivalent in control and treatments pairs (C0d = T0d; C1/4d = T1/4d; C2d = T2d, C6d = T6d). However, there was evidence of a biomass increase in controls after 6 d (C0d < C6d) that was not observed in treatments (T0d = T1/4d = T2d = T6d; Fig. 1B). 3.2. Microalgal induction Microalgal epibiosis did not affect elatol levels (Table 1, Fig. 2A) or growth of L. dendroidea (Table 1, Fig. 2B). When compared within the same period, all algal biomasses were equivalent (T0d = C0d = NC0d; T2d = C2d = NC2d; T7d = C7d = NC7d), but clones maintained in the absence of epibionts grew faster than clones with epibionts (T0d = T2d = T7d; C0d = C2d < C7d; NC0d = NC2d < NC7d; Fig. 2B). After 2 d and 7 d of inoculation, microalgal densities (average ± SD) in treatment conditions were 4.5 × 105 ( ± 9.8 × 104) and 2.3 × 106 ( ± 4.19 × 105) cells ml−1, respectively. 3.3. Bacterial induction and infection We verified an interaction between elatol level and growth of L. dendroidea, as well as that elatol levels were influenced by treatment duration (Table 1). Higher levels of elatol were observed in wounded seaweed (C2) and wounded seaweed with bacteria (T2) compared to unwounded seaweed (C1) and unwounded seaweed with bacteria (T1) (T2 = C2 > T1 = C1, Fig. 3A). Additionally, elatol concentrations were higher in C2 than C1 (post hoc Tukey's test P < 0.001). After 7 d, elatol concentrations were equal in L. dendroidea clones maintained under all experimental conditions (Fig. 3A). After 2 d, L. dendroidea growth was equivalent in all experimental conditions (T1 = C1 = T2 = C2, Fig. 3B). However, after 7 days, L. dendroidea growth was higher in the absence

Fig. 1. Effect of herbivory (simulated wound damage) on elatol level (A) and growth (=biomass) (B) of L. dendroidea, after ¼, 2 and 6 days of incubation. Treatment = wounded seaweed; control = unwounded seaweed (n = 4 per experimental condition). Vertical bars represent mean + one standard deviation. Bars with the same pattern of horizontal lines are not significantly different. Differences between means were considered significant when P < 0.05, 2 way-ANOVA/Tukey's HSD test. 51

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Fig. 3. Effect of bacterial epibiosis on elatol levels (A) and growth (=biomass) (B) of L. dendroidea after 2 and 7 days of incubation. T1 = unwounded seaweed with bacteria; C1 = unwounded seaweed; T2 = wounded seaweed with bacteria; C2 = wounded seaweed (n = 5 per experimental condition). Symbols and statistical tests as in Fig. 1.

Fig. 4. Feeding deterrence of L. dendroidea according to chemical defense variability against - specialist consumer Aplysia brasiliana (crude extract, n = 5) and - generalist consumer Lytechinus variegatus (elatol, n = 15). Vertical bars represent mean + one standard error. Bars with the same pattern of horizontal lines are not significantly different. Differences between means were considered significant when P < 0.05, one-way ANOVA/Tukey's HSD test.

of bacteria (C1 = C2 > T1 = T2; Fig. 3B). After 2 d of inoculation, bacterial densities (average ± SD) in T1 and T2 were 1.55 × 108 ( ± 2.34 × 107) and 1.64 × 108 ( ± 1.14 × 107) cells cm3, respectively, and after 7 d they were 3.13 × 108 ( ± 2.16 × 106) and 3.22 × 108 ( ± 1.31 × 107) cells/cm3.

1996), direct evaluation of changes in metabolite levels is an effective way to consider quantitative variations in chemicals exhibiting important ecological roles. Despite controversy surrounding the reliability of simulating herbivory, we opted to use this procedure because the magnitude of damage could be controlled across all replicates (unlike the action of real herbivores) and we could minimize the possible introduction of pathogens from herbivores (Baldwin, 1990) (which would have confounded our bacterial induction and infestation bioassay). Additionally, encouraging results based on herbivore mimicry have been obtained for brown seaweeds (e.g. Van Alstyne, 1988; Renaud et al., 1990; Yates and Peckol, 1993; Peckol et al., 1996; Hammerstrom et al., 1998). Furthermore, manipulation of cultured clones allowed us to monitor the exact effects of the induction stimuli on elatol levels, eliminating variations due to individual responses and environmental factors (see Hammerstrom et al., 1998; Haavisto et al., 2017) that could have biased the results of our induction bioassays. We found that induction and relaxation of chemical defense in L. dendroidea are rapid processes, occurring within 2 and 6 days respectively; though defense began mobilizing after only 6 h (1/4 d). This timeframe for elatol induction would seem a more reasonable defensive strategy than the 11–20 day induction after damage or herbivory that has frequently been reported for seaweeds (Pavia and Toth, 2007). To be effective against herbivory, defense induction should be rapid and also quickly scaled back if no further attack is imminent (Hammerstrom et al., 1998). In other words, as a response to an ongoing or imminent attack (e.g. arising from chemical cues), induced chemical defense will be more effective if it is promptly applied. Relaxation of chemical defense also needs to be fast because, if the defense is prolonged, it will incur the costs of constitutive chemical defenses. Indeed, induced chemical defense in seaweeds may be more common at shorter timeframes and, for those studies in which no evidence of induction was found, it may have occurred but was relaxed before any measurements

3.4. Feeding deterrence by elatol and crude extract- of L. dendroidea The specialist herbivore A. brasiliana consumed artificial foods containing natural concentration, but artificial foods with the lowest level (0.1 NC) of elatol were consumed significantly more than those with the highest level (10 NC) (ANOVA: F(2; 12) = 5.28; P = 0.02; n = 5/Tukey test: P < 0.05; Fig. 4-), which indicates decreasing consumption as a function of increasing elatol concentration (y = −31.11x + 104.24; R2 = 0.98). For the sea urchin L. variegatus bioassay, only the artificial foods containing the lowest elatol level tested (0.0001 NC) was consumed to the same extent as the control. All other concentrations, ranging from 0.001 NC to 10 NC, inhibited sea urchin feeding (ANOVA: F(5, 84) = 11.62; P < 0.001; n = 15/Tukey test: P < 0.05; Fig. 4-). Natural concentration (10.0 mg elatol g−1 alga) elicited the lowest consumption by sea urchins. The sea urchin bioassay also revealed a decrease in consumption as a function of increasing elatol concentration (y = −13.82x + 75.412; R2 = 0.87). 4. Discussion We have demonstrated that simulated herbivory induces production of the sesquiterpene elatol, the major defensive secondary metabolite from the tropical red seaweed L. dendroidea. To our knowledge, no previous study has described an induced defense response based on mechanical damage in red seaweeds while monitoring a target secondary metabolite. As susceptibility to herbivores and other such threats can be dependent on metabolite concentration and the organisms involved in the interaction (Cronin and Hay, 1996; De Nys et al., 52

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clones was not significantly different from intact paired clones. By the end of the experiment (after 6 days), the biomass of unwounded clones had increased, which was not observed in wounded clones. Thus, we concluded that simulated herbivory prompted a response in elatol content after 2 days and growth seemed to be impeded for up to 6 days. Increased elatol content by simulated herbivory associated with impeded growth in L. dendroidea corroborates the Optimal Defense Theory (see Cronin, 2001), i.e., that induced chemical defense is a cost-saving strategy. However, there are no direct experimental evaluations of these costs or what is the ideal parameter to measure metabolic tradeoffs between primary and secondary metabolism in seaweeds. For example, a positive correlation between the levels of halogenated furanones and growth was observed in Delisea pulchra (Dworjanyn et al., 1999). However, specimens cultured under bromine deprivation grew faster, but lacked furanone production (Dworjanyn et al., 1999). Thus, the information relating to the costs of furanone production in D. pulchra is conflicting. Also, no evidence for costs linked to reduced growth in the production of defense in Fucus vesiculosus was reported by Rohde et al. (2004), but such evidence has been found for Alaria nana (Pfister, 1992), Ascophyllum nodosum (Pavia et al., 1999) and Bonnemaisonia hamifera (Nylund et al., 2013). Data revealed a trend for lower growth in clones of L. dendroidea hosting epibionts (bacterium and diatom) due to absence of significant biomass increase over time for the treatment cultures in our epibiosis bioassays which may be indicative of competition for nutrients between L. dendroidea and epibionts, since the densities of epibiont organisms increased throughout the experiments. Thus, even though epibionts did not influence elatol content, they could negatively affect growth of L. dendroidea in a longer time bioassay, impeding seaweed growth as observed for Odonthalia floccosa (Ruesink, 1998). The generalist sea urchin L. variegatus consumed less artificial foods containing elatol than the specialist herbivore A. brasiliana, which can tolerate elatol concentrations 10 times higher than those naturally found in L. dendroidea. This finding indicates that variations in elatol levels affect herbivores differently. Despite the difference in magnitude of effect, increasing elatol levels reduced seaweed consumption by both consumers. Elatol levels vary widely in L. dendroidea specimens at inter(Oliveira et al., 2013) and intra-population (Sudatti et al., 2006) scales, and this plasticity probably contributes to the magnitude of the induced defense response and individual fitness. The feeding behaviors of different species, and even within the same consumer species, must be considered in assessments of the susceptibility of seaweeds to herbivory, given that such behaviors are crucial components for the outcome evolutionary plant–herbivore interactions (Poore and Hill, 2006). Aplysiids are the only consumers that feed on large quantities of Laurencia seaweeds. These sea hares live in association with this seaweed and have evolved mechanisms for the detoxification of Laurencia chemicals (Rogers et al., 2000). Against a specialist herbivore, an induced defense response can be disadvantageous because the consumer can be attracted to plants with higher concentrations of defensive compounds (Agrawal and Karban, 1999). However, this does not seem to be the case for elatol in L. dendroidea, since increased levels of this compound inhibited A. brasiliana feeding behavior, despite this mollusk having a defensive compound sequestering mechanism (Rogers et al., 1995, 2000). For Laurencia and other chemically defended seaweeds in tropical habitats, other consumers, besides mesograzers, should be considered as inducers. Artificial foods with crude extracts of L. dendroidea were not completely rejected in our bioassays, but they were consumed less than our controls; a finding also reported when crabs and fishes were tested in laboratory assays (Pereira et al., 2003). Thus, the action mechanism of impact of different grazer types on chemically defended tropical seaweeds and the aplysiid associations - warrantmore in-depth investigation. Besides elatol's roles in constitutive chemical defense against fouling (Da Gama et al., 2002) and herbivores (Pereira et al., 2003), our results expand its actions to inducible defense. Haavisto et al. (2017) also

were made (Hammerstrom et al., 1998). Many seaweed extracts and natural products inhibit the settling or development of epibionts (e.g. Steinberg and de Nys, 2002), amongst them elatol (e.g. Da Gama et al., 2003). We did not find evidence of variations in the level of this metabolite in response to epibiosis by the diatom P. tricornutum. However, extracts of the red seaweed Cryptonemia seminervis under epibiotic pressure have been shown to inhibit fouling, presumably by inducing the action of an antifouling defense (Da Gama et al., 2008), and the green seaweed Codium decorticatum exhibited inducible antifouling defenses that were triggered by simulated epibiosis (Pereira et al., 2017). The presence of bacteria did not alter elatol content in our unwounded L. dendroidea treatments. In this bioassay, only wounding stimulated the production of elatol and there was no evidence that bacterial infestation after wounding exacerbated elatol production. However, the bacterial culture we used in the bioassay was isolated from the seaweed surface and, therefore, may have a beneficial association with the seaweed as opposed to being pathogenic. For example, the surface of the green seaweed Codium fragile is protected by an association with epiphytic bacteria that release repellent substances into the water column near the algal surface that prevent attachment of other organisms (Armstrong et al., 2001). Pathogen and fouling can negatively influence the development and reproduction of seaweeds, but in different ways since pathogens display an infectious or virulent action that is absent in epibiosis (Weinberger, 2007). However, the presence of bacteria commonly entails disadvantages for seaweeds related to diseases, reproductive impairment and growth decline (Lane and Kubanek, 2008), as observed here for L. dendroidea after 7 d, when bacteria densities achieved ca. 3 × 108 cells/cm3. Some additional aspects may explain the absence of defense induction in treatments with epibiosis and bacterial infestation. The first relates to the response reaction time. Perhaps sampling after 2 and 7 days was not an appropriate timeframe to detect changes in elatol contents relating to epibiosis and bacteria infestation since the response time of defense induction may be variable, depending on the species of seaweed and also the evaluated metabolite (Toth and Pavia, 2007). Second, epibiont attachment mechanisms interfere with the seaweed stem regeneration process (Ram et al., 2000). Epibionts on Gracilaria chilensis promote different kinds of lesions on the seaweed, resulting in varying degrees of damage to the host (Leonardi et al., 2006). Thus, the type of injury produced by epibionts can influence the defense process, similar to what occurs during herbivory induction when mechanical damage (Hammerstrom et al., 1998), water born cues and direct grazing (Macaya and Thiel, 2008) and/or components of salivate (Coleman et al., 2007) can elicit different degrees of defense. Third, also associated to injury type (i.e. to the seaweed medulla or cortex), some interactions with epibionts may involve regulatory mechanisms of metabolite availability only on the seaweed surface (Dworjanyn et al., 1999; Nylund et al., 2007). For example, bacteria are capable of inducing vesicular trafficking of corps en cerise (in which secondary metabolites are stored) to algal surfaces (Paradas et al., 2010), and variations in the amount of halogenated compounds on the plant surface is not always significantly and positively related to variation over the entire thallus (Dworjanyn et al., 1999). Thus, if epibiosis by diatom or bacterium only results in cortical damage whereas herbivory affects both the cortical and medullary regions, it may explain why we only found defense induction in wounded L. dendroidea because we only measured elatol concentrations within the thallus as a whole. In other words, epibiotic stimuli used here may only have influenced elatol levels at the surface of the seaweed, which were not subject of our study. Measurements of biomass change allow assessments of the effects of induction stimuli on the growth of seaweed and are an indirect way of addressing fitness parameters in terrestrial plants (Nykänen and Korivheva, 2004), but this approach has been little applied in studies of seaweeds (Toth et al., 2007). For L. dendroidea, growth of wounded 53

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highlighted that phlorotannins can act either as constitutive or inducible defense compounds according to herbivore pressure. Variation in levels of elatol, depending on stimuli, demonstrate that constitutive or inducible defenses are not obligatory constraints (Agrawal et al., 2010), since a single metabolite can have multiple functions. We suggest that constitutive and inducible defenses are not distinct defensive strategies, but are product of phenotypic plasticity in a same trait. Unpredictable elatol levels under stimuli (as described here) and under other circumstances, such as diel cycle variation (Sudatti et al., 2016), are probably an important strategy to deter herbivory at minimum cost (see Flöthe et al., 2014c), to decrease the predictability of food quality (Borell et al., 2004), to hamper physiological adaptations and coevolution of grazers (Gardner and Agrawal, 2002), and to deal with other natural threats (Hay, 2009). Based on qualitative and quantitative variation in secondary metabolites (Maschek and Baker, 2008) and induced chemical defenses in seaweeds (Toth and Pavia, 2007), it is plausible to argue that this character evolved by any other pressure besides herbivory, such as epibiosis. However, proving this statement will only be possible by reconstructing the evolutionary history of a particular character, but this information has not yet been verified for chemical defenses (Karban and Baldwin, 1997). Although we explored two types of epibiosis (presence of diatoms and bacteria), laboratory experiments imposed some limitations and we could not demonstrate a relationship between inducible defenses and fouling presence, in contrast to our observations for simulated herbivory. It is reasonable to postulate that herbivory currently modulates this character, but this may not have been true in the past. Additionally, the presence of antimicrobial activity and antifouling is undeniable (Lane and Kubanek, 2008), which increases the adaptive value assigned to secondary metabolites and indicates that other forces can operate the chemical defense system from seaweeds in natural ecosystems (Schmitt et al., 1995).

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