Detection of a chemical cue from the host seaweed Laurencia dendroidea by the associated mollusc Aplysia brasiliana

N. Nocchi; A. R. Soares; M. L. Souto; J. J. Fernández; M. N. Martin; R. C. Pereira

doi:10.1371/journal.pone.0187126

Abstract

Chemical cues from sessile hosts can attract mobile and associated organisms and they are also impotant to maintain associations and overall biodiversity, but the identity and molecular structures of these chemicals have been little explored in the marine environment. Secondary metabolites are recognized as possible chemical mediators in the association between species of Laurencia and Aplysia, but the identity of the compounds has not been established. Here, for the first time, we experimentally verify that the sesquiterpene (+)-elatol, a compound produced by the red seaweed Laurencia dendroidea, is a chemical cue attracting the associated sea hare Aplysia brasiliana. In addition to revealing the nature of the chemical mediation between these two species, we provide evidence of a chemical cue that allows young individuals of A. brasiliana to live in association with L. dendroidea. This study highlights the importance of chemical cues in Laurencia-Aplysia association.

Citation: Nocchi N, Soares AR, Souto ML, Fernández JJ, Martin MN, Pereira RC (2017) Detection of a chemical cue from the host seaweed Laurencia dendroidea by the associated mollusc Aplysia brasiliana. PLoS ONE 12(11): e0187126. https://doi.org/10.1371/journal.pone.0187126

Editor: Riccardo Mozzachiodi, Texas A&M University Corpus Christi, UNITED STATES

Received: July 7, 2017; Accepted: October 13, 2017; Published: November 2, 2017

Copyright: © 2017 Nocchi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: Financial Support was provided by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro and the Project of the Spain National Plan (CTQ2014-55888-C03-01). RCP thanks CNPq for Research Productivity fellowships. NN thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for a doctoral fellowship and CNPq for a sandwich doctoral fellowship.

Competing interests: The authors have declared that no competing interests exist.

Introduction

In the marine environment, chemical cues or waterborne infochemicals act as mediators in different intra- and inter-specific ecological interactions between organisms [1], such as chemical defense [2], settlement and recruitment of larvae [3], feeding stimuli [4], foraging behavior [5], selection and localization of prey [6], among others. Marine organisms produce a wide variety of molecules that mediate these ecological interactions [1, 7]. Despite their broad and crucial roles in population structure, community organization and ecosystem function in marine systems, the omnipresence of these chemical cues and their impacts are still inadequately recognized [1]. However, scientific efforts have been increasing to elucidate the mechanisms of chemical communication between organisms in marine environments [8].

Red seaweeds of the genus Laurencia are well established as rich producers of secondary metabolites [9]. Since investigations of this taxon began more than 60 years ago, more than 1,000 secondary metabolites such as terpenes and C₁₅ acetogenins (both halogenated or not) have been isolated from about 60 species [10]. These metabolites not only possess unique structural features, but they also exhibit significant ecological roles as antifoulants [11] and as a defense against herbivores [12]. Despite the strong defensive properties of these chemicals, Aplysia sea hares selectively graze on Laurencia species and have the ability to sequester and store secondary metabolites from these red seaweeds in a specialized digestive gland [13,14]. A huge number of molecules have been discovered from Aplysia species [15] and these sea hares possess secondary metabolites obtained either from their algal diets or by de novo direct synthesis [16], with the former being slightly modified. This feeding relationship makes Aplysia species promoters of chemical diversity, since most of the metabolites found in these molluscs are structurally similar, but distinct from those produced by Laurencia species [17].

The maintenance of the relationship between Laurencia and Aplysia species is probably due to the adaptative evolution of these herbivores to metabolism of the seaweed chemicals giving them the ability to use as a self-defense mechanism [18,19]. In addition, some photosynthetic proteins from Laurencia are converted to an effective chemical defense mechanism displayed by Aplysia, which releases them as a purple ink and opaline when attacked by predators [20]. In fact, these secretions contain vital metabolites for Aplysia species, which can act as feeding stimulants, feeding deterrents, and aversive chemicals [21].

In general, host seaweeds are preferred sites for metamorphosis of larvae in most species of sea hares [22]. For Aplysia species we observed that these organisms preferentially inhabit and grow better on red seaweeds, including Laurencia species [13,14]. Evidence has shown that chemical mediation is vital in interactions between species of Laurencia and Aplysia [17], but the nature of the compounds involved in this interaction has not been established [7]. Although Aplysia and Laurencia are rich sources of secondary metabolites, these compounds have been mainly studied in the context of bioprospecting for bioactive molecules [17,23].

Due to the close and chemically-mediated relationship between Laurencia algae and Aplysia sea hares, we hypothesized that secondary metabolites produced by Laurencia dendroidea can act as important chemical cues for the specialist predator Aplysia brasiliana. In this work, we addressed the following two questions, evaluated under laboratory conditions: (1) Which compounds in seawater are exudates from L. dendroidea? and (2) How does the mollusc A. brasiliana respond to L. dendroidea metabolites in bioassays? Although many studies assume its occurrence, to our knowledge this is the first study that tested and identified Laurencia chemical cues for Aplysia.

Materials and methods

Collection of organisms

Specimens of Laurencia dendroidea and young individuals (4.8 ± 1.5 SD cm in length; N = 194) of the sea hare Aplysia brasiliana found living in association with these algae were collected from the intertidal zone of Azeda Beach (Búzios, 22°44'33.6'' S, 41°52'55.6'' W, Rio de Janeiro State, Brazil) by free diving in September, 2014. After collection, all the organisms were transported to the laboratory in individual aquaria containing seawater with aeration and maintained under constant temperature (22°C), aeration, salinity (35), and a 12 h:12 h light:dark photoperiod. During the acclimatization period of 48 hours, the specimens of A. brasiliana were fed with fresh palatable green seaweed Ulva sp. before starting the assays.

Collection of organisms was supported by authorization SISBIO 34321–1 (Instituto Chico Mendes de Conservação da Biodiversidade).

Laurencia-conditioned seawater (LCW) and Laurencia-exuded metabolites in seawater extract (SEM)

For the laboratory detection of the L. dendroidea exuded metabolites in seawater, a proportion of 24 g of algae mass to 1 L of artificial seawater (“Sea salts” SIGMA, USA, in distilled water) was used for incubation in fifteen Erlenmeyer flasks of 3 liters each. They were kept for 12 hours in a BOD chamber at 20°C, under constant ilumination and constant aeration. Then, the algae were taken out from the flasks and the conditioned water was passed through a 0.45 μm filter Millipore system to yield the Laurencia-conditioned seawater (LCW). Part of this LCW was used immediately in Y-maze tests (20 L), and the remaining LCW (10 L) was used to identify the metabolites exuded by L. dendroidea. Artificial seawater (without the presence of L. dendroidea) was used as a control for chemical analyses and as a control stimulus in the Y-maze tests. The portion of the LCW separated for chemical analysis was passed through a glass column filled with DIAON HP20 resin (Sigma-Aldrich). Then the resin was washed with ultrapure water to eliminate salts and the metabolites retained in the resin were eluted with 600 mL of methanol (Tedia, HPLC), that was evaporated to yield the Laurencia-exuded metabolites in seawater extract (SEM). The same procedure was used to produce a representative control from artificial seawater. Both the SEM and the control extract from artificial seawater were analyzed by GC/MS.

Chemical extraction and identification of secondary metabolites

Whole fresh tissues of L. dendroidea (450 g) were extracted in EtOAc/MeOH (1:1 v/v). The resulting extract was filtered and the solvent was eliminated in a rotary evaporator under reduced pressure. The extract (1.4 g) was submited to size exclusion chromatography using Sephadex LH-20 (6 x 34 cm, Pharmacia Fine Chemicals^®) and eluted with n-hexane/CH₂Cl₂/MeOH (2:1:1). Seven fractions (FR_A to FR_G) were obtained and analyzed by Nuclear Magnetic Resonance (NMR) of ¹H. From the ¹H NMR analysis the fraction-enriched with sesquiterpenes (283 mg, FR_D) was subsequently processed by chromatography in medium-pressure Lobar LiChroprep Si-60 (40–63 μm, MERCK^®) using n-hexane/EtOAc (9:1) at 2 mL min^-1. Twelve fractions (FR₁ to FR₁₂) were obtained, which allowed isolation of compounds 1 (1.45 mg, FR₄) and 2 (120.49 mg, FR₅). Fractions FR₇ and FR₈ were purified using high-performance liquid chromatography (HPLC) with a μ-Porasil^™ silica column (125 Å; 1.9 x 15 cm) and eluted with n-hexane/EtOAc (9:1), which yielded compounds 3 (0.52 mg) and 4 (1.51 mg) from fraction FR₈ and compound 5 (2.7 mg) from fraction FR₇.

The chemical structures of the isolated compounds were established by NMR of ¹H and ¹³C, correlation spectroscopy (COSY), heteronuclear multiple-bond correlation (HMBC) and heteronuclear single quantum correlation (HSQC) in a Bruker^® Avance 600 MHz system using CDCl₃. Chemical shifts were reported in ppm referenced to solvent signals (CDCl₃: δ_H 7.26, δ_C 77.16). Mass spectrometric data were acquired using a VG-Autospec Fisons system in electrospray ionization mode. The specific rotations [α]_D were determined using a Polarimeter Perkin-Elmer^® (model 241), under conditions of 20°C, sodium D line λ = 589 nm and cell path length 1 dm. All data were compared with the available literature [24–29].

10-bromo-9-hydroxy-chamigra-2,7(14)-diene (1): oil; [α]_D -125 (c 0.008, CHCl₃), lit. [α]_D -110 (c 0.20, CHCl₃) [24]; ESI-HRMS (m/z): [M+Na]⁺ calcd. for C₁₅H₂₃⁷⁹BrONa, 321.0830 and C₁₅H₂₃⁸¹BrONa, 323.0810; found 321.0834 and 323.0807 (ratio 100:97), respectively. All other physical and spectroscopic data are in agreement with those previously reported [24].

(+)-elatol (2): oil; [α]_D +79 (c 0.06, CHCl₃), lit. [α]_D +75 (c 1.01, CHCl₃) [24,25]; ESI-HRMS (m/z): [M+Na]⁺ calcd. for C₁₅H₂₂O³⁵Cl⁷⁹BrNa, 355.0440 and C₁₅H₂₂O³⁵Cl⁸¹BrNa, 357.0420; found 355.0443 and 357.0435 (ratio 100:94), respectively. All other physical and spectral properties are in agreement with published data [25,26].

(Z)-10,15-dibromo-9-hydroxychamigra-1,3(15),7(14)-triene (3): colorless oil; [α]_D +10 (c 0.02, CHCl₃), lit. [α]_D +3 (c 0.002, CHCl₃) [26]; ESI-HRMS (m/z): [M+Na]⁺ calcd. for C₁₅H₂₀O⁷⁹Br₂Na, 396.9779; C₁₅H₂₀O⁷⁹Br⁸¹BrNa, 398.9758 and C₁₅H₂₀O⁸¹Br₂Na, 400.9738; found 396.9779, 398.9756, and 400.9749 (ratio 48:100:52), respectively. All other physical and spectroscopic data are comparable to those previously reported [27].

(E)-10,15-dibromo-9-hydroxychamigra-1,3(15),7(14)-triene (4): colorless oil; [α]_D -31 (c 0.016, CHCl₃), lit. [α]_D -40 (c 0.01, CHCl₃) [26]; ESI-HRMS (m/z): [M+Na]⁺ calcd. for C₁₅H₂₀O⁷⁹Br₂Na, 396.9779, C₁₅H₂₀O⁷⁹Br⁸¹BrNa, 398.9758 and C₁₅H₂₀O⁸¹Br₂Na, 400.9738; found 396.9784, 398.9761, and 400.9756 (ratio 48:100:52), respectively. All other physical and spectroscopic data are in agreement with those previously reported [27].

isoobtusol (5): white crystal; [α]_D +77 (c 0.03, CHCl₃), lit. [α]_D +25 (c 0.50, CHCl₃) [27–28]; ESI-HRMS (m/z): [M+Na]⁺ calcd. for C₁₅H₂₃⁷⁹Br⁸¹Br³⁵ClONa, 436.9681, C₁₅H₂₃⁷⁹Br₂³⁵ClONa, 436.9672 and C₁₅H₂₃⁸¹Br₂³⁵ClONa, 438.9661; 434.9732, 436.9694, and 438.9674 (ratio 48:100:73), respectively. All other physical and spectroscopic data are in agreement with those previously reported [27–29].

GC/MS analysis

Chemical profiles were obtained on a GC/MS Agilent 7890B, MS7000C (using an automatic injector CombiPAL with the control software MassHunter) in the electron impact mode (70 eV). Chromatographic separation was achieved with a HP-5MS UL capillary column (15 m x 0.25 mm x 0.25 μm). Helium was used as the carrier gas (flow rate of 1 mL min^-1). The oven temperature was 60°C for the first 2.5 min, then increased at a rate of 10°C.min^-1 to 280°C and held at the latter temperature for 6 min, with a total run time of 30.5 min. Injector and ion source temperatures were 300 and 240°C. Samples were injected in splitless mode. The detection was performed in the full scan mode, using a mass range of 60–400 m/z. All samples were solubilized in dichloromethane and filtered through a PTFE Syringe filter (pore width of 0.45μm; diameter of 13mm) before the injections.

The chemical profiles of all the extracts (whole algal extract, SEM and control) were obtained by GC/MS. The isolated compounds (1–5) and purified standards (6–15) (Fig 1) available in our laboratory were also analyzed by GC/MS to construct a L. dendroidea metabolites database. Compounds 6–15 were obtained as described by the literature [30–33]. Identification of metabolites exuded into seawater by L. dendroidea (SEM) was performed by comparing the peaks of the SEM chromatogram with its respective control (artificial seawater extract). A baseline alignment of each chromatogram was performed prior to comparison and the time-shift among peaks was corrected by the Correlation Warping Algorithm (COW), as described by Nielsen and collaborators (1998) [34], using the software COW (http://www2.biocentrum.dtu.dk/mycology/analysis/cow). Compounds present in the algal extract, and SEM extract were identified based on their mass spectra data, retention time, and comparison with pure compounds available in our laboratory.

Download:

Fig 1. Chemical structures of the metabolites found in L. dendroidea.

https://doi.org/10.1371/journal.pone.0187126.g001

The compound identified from the SEM extract (elatol) was quantified using external standardization by means of an analytical curve calculated from four different concentrations of purified elatol in dichloromethane (10 to 10⁻² μg mL^-1). Detection and quantification limits were determined according to United States Environmental Protection Agency provisions; we obtained an analytical curve in a concentration range almost 5 times the noise of the baseline. Then, the concentrations of three replicates of the standard were determined from this curve, and its standard deviation was calculated. The relative standard deviation (RSD) or coefficient of variation (CV) was 10%.

Y-maze experiments

Y-maze behavioural bioassays were performed to determine whether the specimens of A. brasiliana would be attracted by L. dendroidea compounds. The maze used in this study consisted of a Y-shaped plastic tube connected to two 10 L aquariums, with seawater flowing by gravity from the aquariums through each branch of the Y-maze (Fig 2). One of the aquariums contained artificial seawater with the tested stimuli (treatment), while the other was filled with pure artificial seawater, i.e. the control (see below). Water flowed from the two aquariums (treatment and control) through the Y-maze and exited at the base of the conjoined branch. Soluble coloring was used to verify the water plume and flow regulators were used to standardize the flux in the control and treatment aquaria before each experiment. The artificial seawater was used at 26°C and a salinity of 35.

Download:

Fig 2. The maze used in this study.

The control was in the left aquarium flowing pure seawater and the treatment was in the right aquarium flowing seawater plus a stimuli (e.g. Laurencia tallus or Laurencia-exuded metabolites in seawater extract (SEM).

https://doi.org/10.1371/journal.pone.0187126.g002

In the intervals between each assay, the entire Y-maze was washed with distilled water and rinsed with seawater and control and stimulus arms of the Y-maze were switched. The seawater flow in each branch of the Y-maze was controlled by flow controllers. Individuals of A. brasiliana were initially positioned at the base of the Y-maze at the start of each bioassay.

Six experiments were undertaken with individual A. brasiliana choosing between stimulus vs artificial seawater. Each experiment consisted to introduce one sea hare at the base of the Y-maze and its behavior in terms of choosing between the arms containing either the stimulus or the artificial seawater was observed for up to 15 min. Three responses were considered: (a) positive, when A. brasiliana swam to the end of the stimulus arm; (b) negative, when A. brasiliana swam to the end of the control arm; and (c) no response, when A. brasiliana did not choose either one of the arms within the 15 min time frame. The stimulus tested in each experiment was drive in the follow sequence: 1) Control stimulus (n = 21): both arms of the Y-maze tube were filled only with artificial seawater in order to verify the ability of A. brasiliana individuals to overcome the water flow in the branches of the Y-maze structure; 2) Fresh L. dendroidea (n = 32): one specimen of this seaweed was placed in the stimulus arm of the Y-maze, while a plastic L. dendroidea mimic with equivalent size and volume of this fresh seaweed was placed in the artificial seawater arm (control) to maintain an equivalent flow of water through the tube during the experiment; 3) Laurencia-conditioned seawater (LCW): the stimulus aquarium was filled with LCW while only artificial seawater was used in the control tank. A total of 43 replicates were used; 4) L. dendroidea extract: the effect of compounds obtained from fresh L. dendroidea on A. brasiliana was evaluated using the extract at its obtained concentration (the amount of extract per algae dry mass, 146 mg g^-1). A piece of filter paper was cut into squares 1.5 × 1.5 cm and each square was equivalently soaked into the extract previously dissolved in dichloromethane (concentration of extract determined as the extract equivalent to the dry weight of alga = dry weight of filter paper). For the control, an equivalent square piece of paper was soaked in dichloromethane without the extract. After evaporating the solvent, each piece of paper of control and treatment was positionated at its respective Y-maze arm. A total of 42 replicates was used; 5) Laurencia-exuded metabolites in seawater extract (SEM): To evaluate the effect of SEM extract (using the obtained concentration of extract per algal dry mass incubated, 5 mg g^-1) on the Aplysia specimens, the experiments were conducted in the same way as described for LCW above. A total of 30 replicates was used; and 6) Elatol-conditioned water (ECW): the effect of elatol (2), the compound identified in the SEM by GC/MS, on Aplysia specimens was investigated. The amount of treatment added was such that the final concentration was equal to the natural isolated concentration based on seawater volume used for extraction (0.02 mg L^-1). The compound was solubilized in 35μL DMSO and added to the artificial seawater in the stimulus arm. The same amount of 35μL DMSO was added to the artificial seawater in the control arm. A total of 26 replicates were used in this experiment. In the intervals between each assay, the entire Y-maze was washed and the control and stimulus arms of the Y-maze were switched in order to remove any remaining A. brasiliana scents or previous chemical cues. For each assay a new A. brasiliana specimen was used. In the end of the experiments all the animals were returned to field.

The significance of A. brasiliana behavioral choice was evaluated by a chi-squared test that compared the number of times that A. brasiliana moved to for the three response groups (positive response, negative resposnde and no response) against an equal random distribution. Differences between the groups were deemed statistically significant when p ≤ 0.05.

Results

GC/MS database of metabolites of Laurencia dendroidea

A total of 15 sesquiterpenes of L. dendroidea were analysed by gas chromatography/mass spectrometry (GC/MS) in order to obtain a GC/MS database of Laurencia metabolites (supp 1). Extract of L. dendroidea yielded compounds 1–5, which were purified using a series of chromatographic steps over Sephadex LH-20, silica gel and normal-phase high performance liquid chromatography (HPLC). The compounds were elucidated based on spectral data: optical rotation, infrared, electron ionization (EIMS) and/or high-resolution electron-impact mass spectroscopy (HREIMS), 1D and 2D NMR. The known halogenated chamigrane sesquiterpenes were identified by comparing their spectroscopic data with the literature as follows: 10-bromo-9-hydroxy-chamigra-2,7(14)-diene (1) [24], (+)-elatol (2) [25], (Z)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (3) [27], (E)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (4) [27] and isoobtusol (5) [28] (The NMR data and all the MS and NMR spectra of 1–5 are available in the supplemental material). Another 10 metabolites that were isolated previously by our research group [30–33] were used here to further populate the GC/MS Laurencia metabolite database: triquinane (6) [31,33]; debromoelatol (7) [30,31]; enone of desbromocartilagineol or laurencenone D (8) [35]; cartilagineol (9) [30,31]; obtusol (10) [31–32]; dendroidiol (11) [30,31]; nidificene (12) [31]; 3,10-dibromo-4-chloro-alpha-chamigrane (13) [31]; (+)-obtusane (14) [30] and rogiolol (15) [31].

Identification of metabolites in extracts of Laurencia dendroidea by GC/MS analysis

The L. dendroidea extract, Laurencia-exuded metabolites in seawater extract (SEM) and control extract (extract from artificial seawater) were analyzed by GC/MS. The retention times and mass spectroscopy patterns of the compounds in these extracts were compared to the L. dendroidea metabolites database (Table 1 and Fig 3). Compounds 1–8 were detected in the L. dendroidea extract (Fig 3A), whereas only compound 2 (elatol) was found in the exudate (SEM) (Fig 3B) at a concentration of 0.02 mg L^-1 (or 0.0007 mg g^-1 by algal wet weight; y = 1E+10x-3E+08; R² = 0.99). No metabolites were identified in the control extract (Fig 3C).

Download:

Fig 3. GC/MS chromatograms.

(A) L. dendroidea extract. (B) Laurencia-exuded metabolites in seawater extract (SEM). (C) Control extract (from artificial seawater). Compounds identified using the GC/MS Laurencia metabolite database: 10-bromo-9-hydroxy-chamigra-2,7(14)-diene (1), (+)-elatol (2), (Z)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (3), (E)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (4), isoobtusol (5), triquinane (6), debromoelatol (7) and enone of desbromocartilagineol or laurencenone D (8).

https://doi.org/10.1371/journal.pone.0187126.g003

Download:

Table 1. Retetion time (r_t); main ions (m/z) observed in the mass spectra (EIMS) and detection of sesquiterpenes in the L. dendroidea extract and Laurencia-exuded metabolites in seawater extract (SEM), as characterized by GC/MS analysis.

The presence of identified compounds in the samples was marked (x).

https://doi.org/10.1371/journal.pone.0187126.t001

Y-maze bioassays

Fig 4 shows that a significantly higher percentage of individuals of A. brasiliana (according to Chi-square tests) selected the arm of the Y-maze containing one type of chemical cue (Fig 4), represented by the presence of fresh L. dendroidea (p < 0.005; χ² = 25.187; DF = 2), LCW (p < 0.005; χ² = 16.791; DF = 2), L. dendroidea extract (p < 0.005; χ² = 18.143; DF = 2), SEM (p < 0.05; χ² = 15.20; DF = 2), or elatol-conditioned water (p < 0.005; χ² = 7.00; DF = 2). These results are reinforced by the absence of preferential displacement (no response) of 86% of the 21 individuals of A. brasiliana in our control tests (p < 0.005; χ² = 26.00; DF = 2).

Download:

Fig 4. Percent responses of A. brasiliana specimens to different treatments in Y-maze bioassays.

Control = artificial seawater without the presence of any L. dendroidea stimuli; LCW = L. dendroidea conditioned water; SEM = Laurencia-exuded metabolites in seawater extract; and ECW = elatol-conditioned water. Asterisks (*) denote significant differences between the response groups according to Chi-square tests (p ≤ 0.05).

https://doi.org/10.1371/journal.pone.0187126.g004

Discussion

Our results showed that the mollusc Aplysia brasiliana is able to detect and respond to chemicals produced and exuded by Laurencia dendroidea. This mollusc showed a positive chemotaxis by the presence of live seaweed, its water exudates in conditioned water (LCW) and also to its lipophilic extract (SEM).

Benthic marine invertebrates sense molecules from other organisms and use these compounds in different ecological contexts as communication, navigation and foraging behaviour [36]. These organisms produce and detect a wide range of polar and nonpolar compounds, but their identities are poorly understood [7]. Here, the lipophilic compounds exuded by L. dendroidea into seawater were detected by GC/MS analysis. Among them, the halogenated sesquiterpene (+)-elatol (2) was identified and revealed as one of the major compounds. In adition, the analysis of L. dendroidea extract revealed (+)-elatol as the major compound produced. These results indicated that (+)-elatol was exuded by alga into seawater.

The nonpolar (+)-elatol (2) is a known terpenoid isolated from Laurencia species that has multiple ecological functions, including anti-herbivory [12] and antifouling activities [37]. In our work, (+)-elatol attracted A. brasiliana in Y-maze experiments indicating its potencial as a chemical mediator in the interaction between Laurencia and Aplysia, its associated fauna.

Liposoluble terpenoids can mediate different alga-herbivore interactions. Halimedatetraacetate is a sesquiterpenoid produced by the green alga Halimeda incrassata identified as a foraging cue for the specialist gastropod Elysia tuca [38]. As one of the few herbivores that preferentially consume Halimeda species, Elysia may locate its prey by tracking Halimeda-derived chemical cues. This herbivore is frequently found associated to H. incrassata, which is able to sequester its chemical defenses to become unpalatable to its predators [39].

The oligophagous herbivorous molluscs belonging to the genus Aplysia feed primarily on red seaweeds and as well as the mentioned gastropod Elysa tuca, they are able to accumulate algal metabolites from these diets, which have been hypothesized to function as a defense against predators [13,16,18]. In fact, some of the halogenated sesquiterpenes isolated from L. dendroidea, such as the enantiomer of 10-bromo-9-hydroxy-chamigra-2,7(14)-diene (1), (+)-elatol (2), (Z)-10,15-dibromo-9-hydroxy-chamigra-1,3(15),7(14)-triene (3), (E)-10,15-dibromo-9-hydroxy-charmigra-1,3(15),7(14)-triene (4), isoobtusol (5), cartilagineol (9), obtusol (10) and rogiolol (15) have also been found in Aplysia species [16,18,26, 30, 40–43]. These findings reinforce the importance of these chemicals in the close relationship maintained between Laurencia and the sea hares belonging to the genus Aplysia.

Chemoreception can be the principal sensory system driving behavior for several marine organisms, including slowmoving molluscs belonging to the genus Aplysia [36]. Specific receptors involved in chemical signaling are able to detect and to bind to different compounds as water-soluble amino acids, organic acids or liposoluble terpenoids [44]. The sea hare Aplysia californica, for example, has a well-developed chemical sense with rhinophores and tentacles that help it to localize odors of its main food, i.e., seaweeds belonging to Plocamium and Laurencia genus [45]. A. brasiliana, which usually swims in order to find food [46], probably use similar sensors for localization. Our results provide evidence that secondary metabolites are important chemical cues mediating the interaction between A. brasiliana and L. dendroidea.

Identification of chemical communication and discrimination between evolved functions (signals) and unintentional releases (cues) are significant challenges in aquatic biology [8]. Chemical signals are intentionally released by the sender to convey information about presence of the signaler or the signaler’s environment changing the behavior of the receiver; this change of behavior must be profitable to sender and receiver. In general, signals must be honest and reliable. Already chemical cues are compounds unintentionally released by the sender that any other organism can use as a guide to display a particular behavior, benefiting the receiver exclusively [47] Distinguishing these two modes of information is essential in identifying, properly, a chemical substance that triggers a certain behavior in another individual, and only a closer look at the chemical compounds involved brings clarification about its ecological and evolutionary interactions [48]. Here, for the first time, the combination of experiments allowed to verify that the sea hare A. brasiliana is attracted by the sesquiterpene (+)-elatol as a chemical cue at different concentrations. In the experiments using conditioned water and pure elatol, the concentration of this compound was probably higher than that in natural conditions. On the other hand, in experiments using a unique piece of algal thallus, the exudated concentrations are expected to be similar to natural conditions, and the result obtained in this assay was similar that verified using conditioned water and pure elatol. In all cases, elatol present in the water was sufficient to attract A. brasiliana.

Additionaly, the diurnal production of elatol by L. dendroidea is dynamic and strongly correlated to the primary photosynthetic metabolism [49]. So, it is expected that its surface concentration is variable creating different scenarios for the chemical sinalization and interactions between the host seaweed and its associated fauna including the mollusc A. brasiliana.

Several species of Laurencia contain refractile membrane-bound vesicles (corps en cerise) in the outer cells or cortical layer, which act as locations for the biosynthesis and storage of halogenated secondary metabolites [50], including elatol. Through a transport system, these metabolites reach the surface and provide essential cues as surface-mediated chemicals [51]. A previous study revealed that elatol occurs in very low amounts on the surface of L. dendroidea (0.006 mg g^-1) when compared to the highest within-thallus quantities (9.89 mg g^-1) based on algal dry weight [52]. Low concentrations of elatol on the surface of L. dendroidea did not inhibit herbivory by sea urchins, settlement of barnacle larvae, or mussel attachment [51], but is probably enough to attract A. brasiliana.

Compared to adults, juvenile sea hares are known to be subject to high predation pressure [53]. In addition, juvenile sea hares have restricted diets, often eating only one or a few types of red seaweeds [54]. Several small sedentary herbivores (mesograzers such as amphipods, crabs and polychaetes) can significantly decrease their susceptibility to predation by living among and feeding on seaweeds that are avoided by predators [55]. In our bioassays, we only used juvenile A. brasiliana that were found associated with L. dendroidea in the field. Thus, young individuals of A. brasiliana are mesograzers that use L. dendroidea both food and as a chemical source that protects from predators.

In conclusion, our discovery of Aplysia foraging cues reveals that this mollusc uses prey secondary metabolites as foraging cues. The sesquterpene (+)-elatol can facilitate the encounter of A. brasiliana with L. dendroidea, but it is still not known if other compounds are involved in this process. Thus, elatol represents for this alga both an evolved function against natural enemies such as pressure by conusmers [12] and fouling [37], and also a cue that benefits the sea hare A. brasiliana.

Supporting information

S1 Table. NMR data and all the MS and NMR spectra of the isolated compounds 1–5 found in L. dendroidea.

https://doi.org/10.1371/journal.pone.0187126.s001

(DOC)

Acknowledgments

We are grateful to the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), the Fundação de Amparo a Pesquisa do Estado do Rio de Janeiro and the Project of the Spain National Plan (CTQ2014-55888-C03-01) for financial support. RCP thanks CNPq for Research Productivity fellowships. NN thanks Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for a doctoral fellowship and CNPq for a sandwich doctoral fellowship. We are also grateful to the peer reviewers for the substantial contributions in improving our manuscript.

References

1. Hay ME. Marine chemical ecology: chemical signals and cues structure marine populations, communities, and ecosystems. Ann Rev Mar Sci. 2009; 1: 193–212. pmid:21141035
- View Article
- PubMed/NCBI
- Google Scholar
2. Pereira RC, Da Gama BAP. Macroalgal chemical defenses and their roles in structuring tropical marine communities. In: Amsler CD, editor. Algal chemical ecology. Springer Berlin Heidelberg; 2008. pp. 25–55.
3. Tebben J, Motti CA, Nachshon Siboni, Tapiolas DM, Negri AP, Schupp PJ, et al. Chemical mediation of coral larval settlement by crustose coralline algae. Sci Rep. 2015; 5: 10803. pmid:26042834
- View Article
- PubMed/NCBI
- Google Scholar
4. Gillete R, Huang RC, Hatcher N, Moroz LL. Cost-benefit analysis potential in feeding behavior of a predatory snail by integration of hunger, taste, and pain. Proc Natl Acad Sci. 2000; 97: 3585–3590. pmid:10737805
- View Article
- PubMed/NCBI
- Google Scholar
5. Zimmer RK, Commins JE, Browne KA. Regulatory effects of environmental chemical signals on search behavior and foraging sucess. Ecology. 1999; 80: 1432–1446.
- View Article
- Google Scholar
6. Alino PM, Coll JC, Sammarco PW. Toxic prey discrimination in a highly specialized predator Chaetodon melannotus (Block et Schneider): visual vs. chemical cues. J Exp Mar Biol Ecol. 1992; 164: 209–220.
- View Article
- Google Scholar
7. Kamio M, Derby CD. Finding food: how marine invertebrates use chemical cues to track and select food. Nat Prod Rep. 2016; 34: 514–528. pmid:28217773
- View Article
- PubMed/NCBI
- Google Scholar
8. Núñez-Acuña G, Boltaña S, Gallardo-Escárate C. Pesticides drive stochastic changes in the chemoreception and neurotransmission system of marine ectoparasites. Int J Mol Sci. 2016; 17: 700. pmid:27258252
- View Article
- PubMed/NCBI
- Google Scholar
9. Wang B, Gloer JB, Ji N, Zhao J. Halogenated organic molecules of Rhodomelaceae origin: chemistry and biology. Chem Rev. 2013; 113: 3632–3685. pmid:23448097
- View Article
- PubMed/NCBI
- Google Scholar
10. Harizani M, Ioannou E, Roussis V. The Laurencia paradox: an endless source of chemodiversity. Prog Chem Org Nat Prod. 2016; 102: 91–252. pmid:27380407
- View Article
- PubMed/NCBI
- Google Scholar
11. Paradas WC, Tavares Salgado L, Pereira RC, Hellio C, Atella GC, de Lima Moreira D, et al. A novel antifouling defense strategy from red seaweed: exocytosis and deposition of fatty acid derivatives at the cell wall surface. Plant Cell Physiol. 2016; 57: 1008–1019. pmid:26936789
- View Article
- PubMed/NCBI
- Google Scholar
12. Pereira RC, Da Gama BAP, Teixeira VL, Yoneshigue-Valentin Y. Ecologial roles of natural products from the Brazilian red seaweed Laurencia obtusa. Braz J Biol. 2013; 63: 665–672.
- View Article
- Google Scholar
13. Pennings SC. Size-related shifts in herbivory: specialization in the sea hare Aplysia californica. J Exp Mar Biol Ecol. 1990; 142: 43–61.
- View Article
- Google Scholar
14. Rogers CN, Steinberger PD, De Nys R. Factors associated with oligophagy in two species of sea hares. J Exp Mar Biol Ecol. 1995; 192: 47–73.
- View Article
- Google Scholar
15. Kamiya H, Sakai R, Jimbo M. Bioactive molecules from sea hares. In: Cimino G. Gavagnin M, editors. Molluscs: from chemo-ecological study to biotechnological application. Springer Berlin Heidelberg; 2006. pp. 215–339.
16. Palaniveloo K, Vairappan CS. Chemical relationship between red algae genus Laurencia and sea hare Aplysia dactylomela Rang in the North Borneo Island. J Appl Phycol. 2014; 26: 1199–1205.
- View Article
- Google Scholar
17. Pereira RB, Andrade PB, Valentão P. Chemical diversity and biological properties of secondary metabolites from sea hares of Aplysia genus. Mar. Drugs. 2016; 14: 1–33. pmid:26907303
- View Article
- PubMed/NCBI
- Google Scholar
18. Ginsburg DW, Paul VJ. Chemical defenses in the sea hare Aplysia parvula: importance of diet and sequestration of algal secondary metabolites. Mar Ecol Prog Ser. 2001; 215: 261–274.
- View Article
- Google Scholar
19. Rogers CN, de Nys R, Steinberg PD. Effects of algal diet on the performance and susceptibility to predation of the sea hare Aplysia parvula. Mar Ecol Prog Ser. 2002; 236: 241–254.
- View Article
- Google Scholar
20. Kamio M, Grimes TV, Hutchins MH, van Dam R, Derby CD. The purple pigment aplysioviolin in sea hare ink deters predatory blue crabs through their chemical senses. Animal Behav. 2010; 80: 89–100.
- View Article
- Google Scholar
21. Derby CD. Escape by inking and secreting: marine molluscs avoid predators through a rich array of chemicals and mechanisms. Biol Bull. 2007; 213: 274–289. pmid:18083967
- View Article
- PubMed/NCBI
- Google Scholar
22. Switzer-Dunlap M. Settlement and metamorphosis of marine invertebrate larvae. In Chia FS, Rice ME, editors. Proc Symp Settl Metamorph Mar Invert Larvae. American Zoological Society Meeting, Toronto, Ontario, Canada; 1978. pp. 197–208.
23. Machado FLS, Kaiser CR, Costa SS, Gestinari LM, Soares AR. Atividade biológica de metabólitos secundários de algas marinhas do gênero Laurencia. Rev Bras Farmacogn. 2010; 20: 441–452.
- View Article
- Google Scholar
24. Vairappan CS, Suzuki M, Abe T, Masuda M. Halogenated metabolites with antibacterial activity from the Okinawan Laurencia species. Phytochemistry. 2001; 58: 517–523. pmid:11557086
- View Article
- PubMed/NCBI
- Google Scholar
25. Bansemir AJN, Michalik MLU, Lalk M. Extracts and sesquiterpene derivatives from the red alga Laurencia chondrioides with antibacterial activity against fish and human pathogenic bacteria. Chem. Biodiv. 2004; 1: 463–467. pmid:17191860
- View Article
- PubMed/NCBI
- Google Scholar
26. Brennan MR, Erickson KL, Minott DA, Pascoe K. O. Chamigrane metabolites from a Jamaican variety of Laurencia obtusa. Phytochemistry. 1987; 26: 1053–1057.
- View Article
- Google Scholar
27. Coll JC, Wright AD. Tropical marine algae. III. New sesquiterpenes from Laurencia majuscula (Rhodophyta, Rhodophyceae, Ceramiales, Rhodomelaceae). Aust J Chem. 1989; 42: 1591–1603.
- View Article
- Google Scholar
28. Wessels M, König GM, Wright AD. New natural product isolation and comparison of the secondary metabolite content of three distinct samples of the sea hare Aplysia dactylomela from Tenerife. J Nat Prod. 2000; 63: 920–928. pmid:10924166
- View Article
- PubMed/NCBI
- Google Scholar
29. Vairappan CS. Potent antibacterial activity of halogenated metabolites from Malaysian red algae, Laurencia majuscula (Rhodomelaceae, Ceramiales). Biomol Engin. 2003; 20: 255–259.
- View Article
- Google Scholar
30. da Silva Machado FL, Ventura TL, Gestinari LM, Cassano V, Resende JA, Kaiser CR, et al. Sesquiterpenes from the Brazilian red alga Laurencia dendroidea J. Agardh. Molecules. 2014; 19: 3181–3192. pmid:24642907
- View Article
- PubMed/NCBI
- Google Scholar
31. Machado FL, Duarte HM, Gestinari LM, Cassano V, Kaiser CR, Soares AR. Geographic distribution of natural products produced by red algae Laurencia dendroidea J. Agardh. Chem Biodivers. 2016; 13: 845–851. pmid:27224289
- View Article
- PubMed/NCBI
- Google Scholar
32. da Silva Machado FL, Lima WP, Duarte HM, Rossi-Bergmannc B, Gestinari LM, et al. Chemical diversity and antileishmanial activity of crude extracts of Laurencia complex (Ceramiales, Rhodophyta) from Brazil. Rev Bras Farmacogn. 2014; 24: 635–643.
- View Article
- Google Scholar
33. da Silva Machado FL, Pacienza-Lima W, Rossi-Bergmann B, de Souza Gestinari LM, Fujii MT, Campos de Paula J, et al. Antileishmanial sesquiterpenes from the Brazilian red alga Laurencia dendroidea. Planta Med. 2011; 77: 733–735. pmid:21058243
- View Article
- PubMed/NCBI
- Google Scholar
34. Nielsen NPV, Carstensen JM, Smedsgaard J. Aligning of single and multiple wavelength chromatographic profiles for chemometric data analysis using correlation optimized warping. J. Chromatogr. A. 1998; 805: 17–35.
- View Article
- Google Scholar
35. Kennedy DJ, Selby IA, Thomson RH. Chamigrane metabolites from Laurencia obtusa and L. scoparia. Phytochemistry. 1988; 27: 1761–1766.
- View Article
- Google Scholar
36. Weissburg J. Death from downstream: Chemosensory navigation and predator-prey processes. Brönmark C, Lars-Anders Hansson L-A, editors. Chemical ecology in aquatic systems. Oxford Scholarship Online; 2015.
37. Da Gama BAP, Pereira RC, Carvalho MGV, Coutinho R, Yoneshigue-Valentin Y. The effects of seaweed secondary metabolites on biofouling. Biofouling. 2002; 18: 13–20.
- View Article
- Google Scholar
38. Rasher DB, Stout EP, Engel S, Shearer TL, Kubanek J, Hay ME. Marine and terrestrial herbivores display convergent chemical ecology despite 400 million years of independent evolution. PNAS. 2015; 112: 12110–12115. pmid:26324909
- View Article
- PubMed/NCBI
- Google Scholar
39. Gavagnin M, Mollo E, Montanaro D, Ortea J, Cimino G. Chemical studies of Caribbean sacoglossans: dietary relationships with green algae and ecological implications. J Chem Ecol., 2000; 26: 1563–1578.
- View Article
- Google Scholar
40. Dias T, Brito I, Moujir L, Paiz N, Darias J, Cueto M. Cytotoxic sesquiterpenes from Aplysia dactylomela. J Nat Prod. 2005; 68: 1677–1679. pmid:16309323
- View Article
- PubMed/NCBI
- Google Scholar
41. Vairappan CS, Anangdan SP, Matsunaga S. Diet-derived halogenated metabolite from the sea hare Aplysia parvula. Malays J Sci. 2009; 28: 269–273.
- View Article
- Google Scholar
42. Jiménez-Romero C, Mayer AMS, Rodríguez AD. Dactyloditerpenol acetate, a new prenylbisabolane-type diterpene from Aplysia dactylomela with significant in vitro anti-neuroinflammatory activity. Bioorg Med Chem Lett. 2014; 24: 344–348. pmid:24279991
- View Article
- PubMed/NCBI
- Google Scholar
43. Díaz-Marrero AR, de la Rosa JM, Brito I, Darias J, Cueto M. Dactylomelatriol, a biogenetically intriguing omphalane-derived marine sesquiterpene. J Nat Prod. 2012; 75: 115–118. pmid:22220686
- View Article
- PubMed/NCBI
- Google Scholar
44. Mollo E, Fontana A, Roussis V, Polese G, Amodeo P, Ghiselin MT. Sensing marine biomolecules: smell, taste, and the evolutionary transition from aquatic to terrestrial life. Front Chem. 2014; 2: 1–6. pmid:25360437
- View Article
- PubMed/NCBI
- Google Scholar
45. Audesirk TE. Chemoreception in Aplysia californica. I. Behavioral localization of distance chemoreceptors used in food-finding. Behav Biol. 1975; 15: 45–55. pmid:1237288
- View Article
- PubMed/NCBI
- Google Scholar
46. Carefoot TH, Pennings SC. Influence of proximal stimuli on swimming in the sea hare Aplysia brasiliana. J Exp Mar Biol Ecol 2003; 288: 223–237.
- View Article
- Google Scholar
47. Steiger S, Schmitt T, Schaefer HM. The origin and dynamic evolution of chemical information transfer. Proc R Soc Lond B Biol Sci. 2010; rspb20102285. pmid:21177681
- View Article
- PubMed/NCBI
- Google Scholar
48. Saleh N, Scott AG, Bryning GP, Chittka L. Distinguishing signals and cues: bumblebees use general footprints to generate adaptive behaviour at flowers and nest. Arthropod-Plant Interactions. 2007; 1: 119–127.
- View Article
- Google Scholar
49. Sudatti DB, Fujii MT, Rodrigues SV, Turra A, Duarte HM, Soares AR, et al. Diel variation of sesquiterpene elatol production: a chemical defense mechanism of the red seaweed Laurencia dendroidea. Biochem Syst Ecol. 2016; 64: 131–135.
- View Article
- Google Scholar
50. Salgado LT, Viana NB, Andrade LR, Leal RN, da Gama BA, Attias M et al. Intra-cellular storage, transport and exocytosis of halogenated compounds in marine red alga Laurencia obtusa. J Struct Biol. 2008; 162: 345–355. pmid:18337120
- View Article
- PubMed/NCBI
- Google Scholar
51. Sudatti DB, Rodrigues SV, Coutinho R, Da Gama BA, Salgado LT, et al. Transport and defensive role of elatol at the surface of the red seaweed Laurencia obtusa (Ceramiales, Rhodophyta). J Phycol. 2008; 44: 584–591. pmid:27041418
- View Article
- PubMed/NCBI
- Google Scholar
52. Sudatti DB, Rodrigues SV, Pereira RC. Quantitative GC-ECD analysis of halogenated metabolites: determination of surface and within-thallus elatol of Laurencia obtusa. J Chem Ecol. 2006; 32: 835–843. pmid:16718572
- View Article
- PubMed/NCBI
- Google Scholar
53. Pennings SC, Nastisch S, Paul VJ. Vulnerability of sea hares to fish predators: importance of diet and fish species. Coral Reefs. 2001; 20: 320–324.
- View Article
- Google Scholar
54. Pennings SC. Size-related shifts in herbivory: specialization in the sea hare Aplysia californica Cooper. J Exp Mar Biol Ecol. 1990; 142: 43–61.
- View Article
- Google Scholar
55. Hay ME. Seaweed chemical defenses: their role in the evolution of feeding specialization and in mediating complex interactions. In Paul VJ, editor. Ecological roles of marine natural products. Comstock Publishing Associates; 1992. pp. 93–118.

[ref1] 1. Hay ME. Marine chemical ecology: chemical signals and cues structure marine populations, communities, and ecosystems. Ann Rev Mar Sci. 2009; 1: 193–212. pmid:21141035
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Pereira RC, Da Gama BAP. Macroalgal chemical defenses and their roles in structuring tropical marine communities. In: Amsler CD, editor. Algal chemical ecology. Springer Berlin Heidelberg; 2008. pp. 25–55.

[ref3] 3. Tebben J, Motti CA, Nachshon Siboni, Tapiolas DM, Negri AP, Schupp PJ, et al. Chemical mediation of coral larval settlement by crustose coralline algae. Sci Rep. 2015; 5: 10803. pmid:26042834
View Article
PubMed/NCBI
Google Scholar

[7] View Article

[8] PubMed/NCBI

[9] Google Scholar

[ref4] 4. Gillete R, Huang RC, Hatcher N, Moroz LL. Cost-benefit analysis potential in feeding behavior of a predatory snail by integration of hunger, taste, and pain. Proc Natl Acad Sci. 2000; 97: 3585–3590. pmid:10737805
View Article
PubMed/NCBI
Google Scholar

[11] View Article

[12] PubMed/NCBI

[13] Google Scholar

[ref5] 5. Zimmer RK, Commins JE, Browne KA. Regulatory effects of environmental chemical signals on search behavior and foraging sucess. Ecology. 1999; 80: 1432–1446.
View Article
Google Scholar

[15] View Article

[16] Google Scholar

[ref6] 6. Alino PM, Coll JC, Sammarco PW. Toxic prey discrimination in a highly specialized predator Chaetodon melannotus (Block et Schneider): visual vs. chemical cues. J Exp Mar Biol Ecol. 1992; 164: 209–220.
View Article
Google Scholar

[18] View Article

[19] Google Scholar

[ref7] 7. Kamio M, Derby CD. Finding food: how marine invertebrates use chemical cues to track and select food. Nat Prod Rep. 2016; 34: 514–528. pmid:28217773
View Article
PubMed/NCBI
Google Scholar

[21] View Article

[22] PubMed/NCBI

[23] Google Scholar

[ref8] 8. Núñez-Acuña G, Boltaña S, Gallardo-Escárate C. Pesticides drive stochastic changes in the chemoreception and neurotransmission system of marine ectoparasites. Int J Mol Sci. 2016; 17: 700. pmid:27258252
View Article
PubMed/NCBI
Google Scholar

[25] View Article

[26] PubMed/NCBI

[27] Google Scholar

[ref9] 9. Wang B, Gloer JB, Ji N, Zhao J. Halogenated organic molecules of Rhodomelaceae origin: chemistry and biology. Chem Rev. 2013; 113: 3632–3685. pmid:23448097
View Article
PubMed/NCBI
Google Scholar

[29] View Article

[30] PubMed/NCBI

[31] Google Scholar

[ref10] 10. Harizani M, Ioannou E, Roussis V. The Laurencia paradox: an endless source of chemodiversity. Prog Chem Org Nat Prod. 2016; 102: 91–252. pmid:27380407
View Article
PubMed/NCBI
Google Scholar

[33] View Article

[34] PubMed/NCBI

[35] Google Scholar

[ref11] 11. Paradas WC, Tavares Salgado L, Pereira RC, Hellio C, Atella GC, de Lima Moreira D, et al. A novel antifouling defense strategy from red seaweed: exocytosis and deposition of fatty acid derivatives at the cell wall surface. Plant Cell Physiol. 2016; 57: 1008–1019. pmid:26936789
View Article
PubMed/NCBI
Google Scholar

[37] View Article

[38] PubMed/NCBI

[39] Google Scholar

[ref12] 12. Pereira RC, Da Gama BAP, Teixeira VL, Yoneshigue-Valentin Y. Ecologial roles of natural products from the Brazilian red seaweed Laurencia obtusa. Braz J Biol. 2013; 63: 665–672.
View Article
Google Scholar

[41] View Article

[42] Google Scholar

[ref13] 13. Pennings SC. Size-related shifts in herbivory: specialization in the sea hare Aplysia californica. J Exp Mar Biol Ecol. 1990; 142: 43–61.
View Article
Google Scholar

[44] View Article

[45] Google Scholar

[ref14] 14. Rogers CN, Steinberger PD, De Nys R. Factors associated with oligophagy in two species of sea hares. J Exp Mar Biol Ecol. 1995; 192: 47–73.
View Article
Google Scholar

[47] View Article

[48] Google Scholar

[ref15] 15. Kamiya H, Sakai R, Jimbo M. Bioactive molecules from sea hares. In: Cimino G. Gavagnin M, editors. Molluscs: from chemo-ecological study to biotechnological application. Springer Berlin Heidelberg; 2006. pp. 215–339.

[ref16] 16. Palaniveloo K, Vairappan CS. Chemical relationship between red algae genus Laurencia and sea hare Aplysia dactylomela Rang in the North Borneo Island. J Appl Phycol. 2014; 26: 1199–1205.
View Article
Google Scholar

[51] View Article

[52] Google Scholar

[ref17] 17. Pereira RB, Andrade PB, Valentão P. Chemical diversity and biological properties of secondary metabolites from sea hares of Aplysia genus. Mar. Drugs. 2016; 14: 1–33. pmid:26907303
View Article
PubMed/NCBI
Google Scholar

[54] View Article

[55] PubMed/NCBI

[56] Google Scholar

[ref18] 18. Ginsburg DW, Paul VJ. Chemical defenses in the sea hare Aplysia parvula: importance of diet and sequestration of algal secondary metabolites. Mar Ecol Prog Ser. 2001; 215: 261–274.
View Article
Google Scholar

[58] View Article

[59] Google Scholar

[ref19] 19. Rogers CN, de Nys R, Steinberg PD. Effects of algal diet on the performance and susceptibility to predation of the sea hare Aplysia parvula. Mar Ecol Prog Ser. 2002; 236: 241–254.
View Article
Google Scholar

[61] View Article

[62] Google Scholar

[ref20] 20. Kamio M, Grimes TV, Hutchins MH, van Dam R, Derby CD. The purple pigment aplysioviolin in sea hare ink deters predatory blue crabs through their chemical senses. Animal Behav. 2010; 80: 89–100.
View Article
Google Scholar

[64] View Article

[65] Google Scholar

[ref21] 21. Derby CD. Escape by inking and secreting: marine molluscs avoid predators through a rich array of chemicals and mechanisms. Biol Bull. 2007; 213: 274–289. pmid:18083967
View Article
PubMed/NCBI
Google Scholar

[67] View Article

[68] PubMed/NCBI

[69] Google Scholar

[ref22] 22. Switzer-Dunlap M. Settlement and metamorphosis of marine invertebrate larvae. In Chia FS, Rice ME, editors. Proc Symp Settl Metamorph Mar Invert Larvae. American Zoological Society Meeting, Toronto, Ontario, Canada; 1978. pp. 197–208.

[ref23] 23. Machado FLS, Kaiser CR, Costa SS, Gestinari LM, Soares AR. Atividade biológica de metabólitos secundários de algas marinhas do gênero Laurencia. Rev Bras Farmacogn. 2010; 20: 441–452.
View Article
Google Scholar

[72] View Article

[73] Google Scholar

[ref24] 24. Vairappan CS, Suzuki M, Abe T, Masuda M. Halogenated metabolites with antibacterial activity from the Okinawan Laurencia species. Phytochemistry. 2001; 58: 517–523. pmid:11557086
View Article
PubMed/NCBI
Google Scholar

[75] View Article

[76] PubMed/NCBI

[77] Google Scholar

[ref25] 25. Bansemir AJN, Michalik MLU, Lalk M. Extracts and sesquiterpene derivatives from the red alga Laurencia chondrioides with antibacterial activity against fish and human pathogenic bacteria. Chem. Biodiv. 2004; 1: 463–467. pmid:17191860
View Article
PubMed/NCBI
Google Scholar

[79] View Article

[80] PubMed/NCBI

[81] Google Scholar

[ref26] 26. Brennan MR, Erickson KL, Minott DA, Pascoe K. O. Chamigrane metabolites from a Jamaican variety of Laurencia obtusa. Phytochemistry. 1987; 26: 1053–1057.
View Article
Google Scholar

[83] View Article

[84] Google Scholar

[ref27] 27. Coll JC, Wright AD. Tropical marine algae. III. New sesquiterpenes from Laurencia majuscula (Rhodophyta, Rhodophyceae, Ceramiales, Rhodomelaceae). Aust J Chem. 1989; 42: 1591–1603.
View Article
Google Scholar

[86] View Article

[87] Google Scholar

[ref28] 28. Wessels M, König GM, Wright AD. New natural product isolation and comparison of the secondary metabolite content of three distinct samples of the sea hare Aplysia dactylomela from Tenerife. J Nat Prod. 2000; 63: 920–928. pmid:10924166
View Article
PubMed/NCBI
Google Scholar

[89] View Article

[90] PubMed/NCBI

[91] Google Scholar

[ref29] 29. Vairappan CS. Potent antibacterial activity of halogenated metabolites from Malaysian red algae, Laurencia majuscula (Rhodomelaceae, Ceramiales). Biomol Engin. 2003; 20: 255–259.
View Article
Google Scholar

[93] View Article

[94] Google Scholar

[ref30] 30. da Silva Machado FL, Ventura TL, Gestinari LM, Cassano V, Resende JA, Kaiser CR, et al. Sesquiterpenes from the Brazilian red alga Laurencia dendroidea J. Agardh. Molecules. 2014; 19: 3181–3192. pmid:24642907
View Article
PubMed/NCBI
Google Scholar

[96] View Article

[97] PubMed/NCBI

[98] Google Scholar

[ref31] 31. Machado FL, Duarte HM, Gestinari LM, Cassano V, Kaiser CR, Soares AR. Geographic distribution of natural products produced by red algae Laurencia dendroidea J. Agardh. Chem Biodivers. 2016; 13: 845–851. pmid:27224289
View Article
PubMed/NCBI
Google Scholar

[100] View Article

[101] PubMed/NCBI

[102] Google Scholar

[ref32] 32. da Silva Machado FL, Lima WP, Duarte HM, Rossi-Bergmannc B, Gestinari LM, et al. Chemical diversity and antileishmanial activity of crude extracts of Laurencia complex (Ceramiales, Rhodophyta) from Brazil. Rev Bras Farmacogn. 2014; 24: 635–643.
View Article
Google Scholar

[104] View Article

[105] Google Scholar

[ref33] 33. da Silva Machado FL, Pacienza-Lima W, Rossi-Bergmann B, de Souza Gestinari LM, Fujii MT, Campos de Paula J, et al. Antileishmanial sesquiterpenes from the Brazilian red alga Laurencia dendroidea. Planta Med. 2011; 77: 733–735. pmid:21058243
View Article
PubMed/NCBI
Google Scholar

[107] View Article

[108] PubMed/NCBI

[109] Google Scholar

[ref34] 34. Nielsen NPV, Carstensen JM, Smedsgaard J. Aligning of single and multiple wavelength chromatographic profiles for chemometric data analysis using correlation optimized warping. J. Chromatogr. A. 1998; 805: 17–35.
View Article
Google Scholar

[111] View Article

[112] Google Scholar

[ref35] 35. Kennedy DJ, Selby IA, Thomson RH. Chamigrane metabolites from Laurencia obtusa and L. scoparia. Phytochemistry. 1988; 27: 1761–1766.
View Article
Google Scholar

[114] View Article

[115] Google Scholar

[ref36] 36. Weissburg J. Death from downstream: Chemosensory navigation and predator-prey processes. Brönmark C, Lars-Anders Hansson L-A, editors. Chemical ecology in aquatic systems. Oxford Scholarship Online; 2015.

[ref37] 37. Da Gama BAP, Pereira RC, Carvalho MGV, Coutinho R, Yoneshigue-Valentin Y. The effects of seaweed secondary metabolites on biofouling. Biofouling. 2002; 18: 13–20.
View Article
Google Scholar

[118] View Article

[119] Google Scholar

[ref38] 38. Rasher DB, Stout EP, Engel S, Shearer TL, Kubanek J, Hay ME. Marine and terrestrial herbivores display convergent chemical ecology despite 400 million years of independent evolution. PNAS. 2015; 112: 12110–12115. pmid:26324909
View Article
PubMed/NCBI
Google Scholar

[121] View Article

[122] PubMed/NCBI

[123] Google Scholar

[ref39] 39. Gavagnin M, Mollo E, Montanaro D, Ortea J, Cimino G. Chemical studies of Caribbean sacoglossans: dietary relationships with green algae and ecological implications. J Chem Ecol., 2000; 26: 1563–1578.
View Article
Google Scholar

[125] View Article

[126] Google Scholar

[ref40] 40. Dias T, Brito I, Moujir L, Paiz N, Darias J, Cueto M. Cytotoxic sesquiterpenes from Aplysia dactylomela. J Nat Prod. 2005; 68: 1677–1679. pmid:16309323
View Article
PubMed/NCBI
Google Scholar

[128] View Article

[129] PubMed/NCBI

[130] Google Scholar

[ref41] 41. Vairappan CS, Anangdan SP, Matsunaga S. Diet-derived halogenated metabolite from the sea hare Aplysia parvula. Malays J Sci. 2009; 28: 269–273.
View Article
Google Scholar

[132] View Article

[133] Google Scholar

[ref42] 42. Jiménez-Romero C, Mayer AMS, Rodríguez AD. Dactyloditerpenol acetate, a new prenylbisabolane-type diterpene from Aplysia dactylomela with significant in vitro anti-neuroinflammatory activity. Bioorg Med Chem Lett. 2014; 24: 344–348. pmid:24279991
View Article
PubMed/NCBI
Google Scholar

[135] View Article

[136] PubMed/NCBI

[137] Google Scholar

[ref43] 43. Díaz-Marrero AR, de la Rosa JM, Brito I, Darias J, Cueto M. Dactylomelatriol, a biogenetically intriguing omphalane-derived marine sesquiterpene. J Nat Prod. 2012; 75: 115–118. pmid:22220686
View Article
PubMed/NCBI
Google Scholar

[139] View Article

[140] PubMed/NCBI

[141] Google Scholar

[ref44] 44. Mollo E, Fontana A, Roussis V, Polese G, Amodeo P, Ghiselin MT. Sensing marine biomolecules: smell, taste, and the evolutionary transition from aquatic to terrestrial life. Front Chem. 2014; 2: 1–6. pmid:25360437
View Article
PubMed/NCBI
Google Scholar

[143] View Article

[144] PubMed/NCBI

[145] Google Scholar

[ref45] 45. Audesirk TE. Chemoreception in Aplysia californica. I. Behavioral localization of distance chemoreceptors used in food-finding. Behav Biol. 1975; 15: 45–55. pmid:1237288
View Article
PubMed/NCBI
Google Scholar

[147] View Article

[148] PubMed/NCBI

[149] Google Scholar

[ref46] 46. Carefoot TH, Pennings SC. Influence of proximal stimuli on swimming in the sea hare Aplysia brasiliana. J Exp Mar Biol Ecol 2003; 288: 223–237.
View Article
Google Scholar

[151] View Article

[152] Google Scholar

[ref47] 47. Steiger S, Schmitt T, Schaefer HM. The origin and dynamic evolution of chemical information transfer. Proc R Soc Lond B Biol Sci. 2010; rspb20102285. pmid:21177681
View Article
PubMed/NCBI
Google Scholar

[154] View Article

[155] PubMed/NCBI

[156] Google Scholar

[ref48] 48. Saleh N, Scott AG, Bryning GP, Chittka L. Distinguishing signals and cues: bumblebees use general footprints to generate adaptive behaviour at flowers and nest. Arthropod-Plant Interactions. 2007; 1: 119–127.
View Article
Google Scholar

[158] View Article

[159] Google Scholar

[ref49] 49. Sudatti DB, Fujii MT, Rodrigues SV, Turra A, Duarte HM, Soares AR, et al. Diel variation of sesquiterpene elatol production: a chemical defense mechanism of the red seaweed Laurencia dendroidea. Biochem Syst Ecol. 2016; 64: 131–135.
View Article
Google Scholar

[161] View Article

[162] Google Scholar

[ref50] 50. Salgado LT, Viana NB, Andrade LR, Leal RN, da Gama BA, Attias M et al. Intra-cellular storage, transport and exocytosis of halogenated compounds in marine red alga Laurencia obtusa. J Struct Biol. 2008; 162: 345–355. pmid:18337120
View Article
PubMed/NCBI
Google Scholar

[164] View Article

[165] PubMed/NCBI

[166] Google Scholar

[ref51] 51. Sudatti DB, Rodrigues SV, Coutinho R, Da Gama BA, Salgado LT, et al. Transport and defensive role of elatol at the surface of the red seaweed Laurencia obtusa (Ceramiales, Rhodophyta). J Phycol. 2008; 44: 584–591. pmid:27041418
View Article
PubMed/NCBI
Google Scholar

[168] View Article

[169] PubMed/NCBI

[170] Google Scholar

[ref52] 52. Sudatti DB, Rodrigues SV, Pereira RC. Quantitative GC-ECD analysis of halogenated metabolites: determination of surface and within-thallus elatol of Laurencia obtusa. J Chem Ecol. 2006; 32: 835–843. pmid:16718572
View Article
PubMed/NCBI
Google Scholar

[172] View Article

[173] PubMed/NCBI

[174] Google Scholar

[ref53] 53. Pennings SC, Nastisch S, Paul VJ. Vulnerability of sea hares to fish predators: importance of diet and fish species. Coral Reefs. 2001; 20: 320–324.
View Article
Google Scholar

[176] View Article

[177] Google Scholar

[ref54] 54. Pennings SC. Size-related shifts in herbivory: specialization in the sea hare Aplysia californica Cooper. J Exp Mar Biol Ecol. 1990; 142: 43–61.
View Article
Google Scholar

[179] View Article

[180] Google Scholar

[ref55] 55. Hay ME. Seaweed chemical defenses: their role in the evolution of feeding specialization and in mediating complex interactions. In Paul VJ, editor. Ecological roles of marine natural products. Comstock Publishing Associates; 1992. pp. 93–118.

Figures

Abstract

Introduction

Materials and methods

Collection of organisms

Laurencia-conditioned seawater (LCW) and Laurencia-exuded metabolites in seawater extract (SEM)

Chemical extraction and identification of secondary metabolites

GC/MS analysis

Y-maze experiments

Results

GC/MS database of metabolites of Laurencia dendroidea

Identification of metabolites in extracts of Laurencia dendroidea by GC/MS analysis

Y-maze bioassays

Discussion

Supporting information

S1 Table. NMR data and all the MS and NMR spectra of the isolated compounds 1–5 found in L. dendroidea.

Acknowledgments

References