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Author's personal copy J Chem Ecol (2013) 39:675–685 DOI 10.1007/s10886-013-0276-1
Lipophilic Defenses From Alcyonium Soft Corals of Antarctica Laura Núñez-Pons & Marianna Carbone & Jennifer Vázquez & Margherita Gavagnin & Conxita Avila
Received: 28 June 2012 / Revised: 7 February 2013 / Accepted: 25 February 2013 / Published online: 28 March 2013 # Springer Science+Business Media New York 2013
Abstract Alcyonacean soft corals lack physical or skeletal defenses and their nematocyst system is weak, leading to the conclusion that soft corals mainly rely on chemistry for protection from predators and microbes. Defensive chemicals of primary and secondary metabolic origin are exuded in the mucus surface layer, explaining the general lack of heavy fouling and predation in corals. In Antarctic ecosystems, where generalist predation is intense and mainly driven by invertebrate consumers, the genus Alcyonium is represented by eight species. Our goal was to investigate the understudied chemical ecology of Antarctic Alcyonium soft corals. We obtained six samples belonging to five species: A. antarcticum, A. grandis, A. haddoni, A. paucilobulatum, and A. roseum, and assessed the lipid-soluble fractions for the presence of defensive agents in these specimens. Ethyl ether extracts were tested in feeding bioassays with the sea star Odontaster validus and the amphipod Cheirimedon femoratus as putative sympatric predators. Repellent activities were observed towards both consumers in all but one of the samples assessed. Moreover, three of the extracts caused inhibition to a sympatric marine bacterium. The ether extracts afforded characteristic illudalane sesquiterpenoids in two of the samples, as well as particular wax esters subfractions in all the colonies analyzed. Both kinds of metabolites displayed significant deterrent activities demonstrating their likely defensive role. L. Núñez-Pons (*) : J. Vázquez : C. Avila Departament de Biologia Animal (Invertebrats), Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 643, 08028 Barcelona, Catalunya, Spain e-mail:
[email protected] M. Carbone : M. Gavagnin Istituto di Chimica Biomolecolare, CNR, Via Campi Flegrei 34, I 80078 Pozzuoli, Napoli, Italia
These results suggest that lipophilic chemicals are a first line protection strategy in Antarctic Alcyonium soft corals against predation and bacterial fouling. Keywords Chemical defense . Alcyopterosin sesquiterpenes . Wax esters . Deterrent metabolites . Sea star Odontaster validus . Amphipod Cheirimedon femoratus
Introduction The Southern Ocean is a fluctuant plankton depauperate system in which organisms tend to acquire opportunistic habits and accumulate lipid energy reserves in the form of wax esters and triglycerides (Arnaud, 1977; Sargent et al., 1977). Benthic communities are dominated by species with circumantarctic and eurybathic distributions, where predators such as voracious sea stars (Dayton et al., 1974; McClintock, 1994) and abundant amphipods (Bregazzi, 1972; De Broyer et al., 2007) usually share both shallow and deep habitats with sessile prey organisms (Dayton et al., 1974; Gutt et al., 2000). Until now, only Slattery and coworkers have investigated the ecology of Antarctic soft corals, and in these investigations they have demonstrated an extensive use of chemical defenses (reviewed in Slattery and McClintock, 1997). Anthozoans are the third dominant taxon in the benthos of the Weddell Sea, Antarctica (Arnaud, 1977). In these latitudes, the group includes eight soft coral species of the genus Alcyonium. Soft corals (order Alcyonacea) are a polyphyletic subgroup within the subclass Octocorallia (Alcyonaria), in which the colonies are composed of polyps connected by a fleshy tissue (coenenchyme). These corals lack calcium
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carbonate solid skeletons but contain an assortment of internal minute spiky sclerites that render shape and support (Brusca and Brusca, 2003). Mechanical defense achieved through sclerites is still under discussion, but these small devices are primarily considered structural (Harvell and Fenical, 1989; Sammarco and Coll, 1992; Van Alstyne et al., 1992; Slattery and McClintock, 1995; Kelman et al., 1999; O'Neal and Pawlik, 2002). Despite the flabby aspect and nutritious nature of soft corals (La Barre et al., 1986b), they are not attacked by generalist predators, and only specialist consumers (pycnogonids and opistobranchs) readily feed on them (Sammarco and Coll, 1992; Slattery et al., 1998; Avila et al., 1999). In shallow Antarctic waters for instance, no important predation is observed on Alcyonium corals, and only certain pycnogonid species have been recorded feeding on soft corals (Slattery and McClintock, 1997; personal observations). Octocorals, including soft corals and other coral groups, have weak nematocyst systems that lack stinging devices (i.e., mastigophores), and have a low density of a single type (rhabdoidic heteronemes) of cnidocysts (Schmidt, 1974; Brusca and Brusca, 2003). It is likely that soft corals are defended by repellent metabolites that may originate from primary or secondary metabolism (La Barre et al., 1986b; Wylie and Paul, 1989; Sammarco and Coll, 1992; Van Alstyne et al., 1992,1994; Kelman et al., 1999). Alcyonacea are rich in bioactive compounds that have a role in defense against predators, competition for space, antifouling, and reproduction enhancement (La Barre et al., 1986a; Coll et al., 1987; Pass et al., 1989; Wylie and Paul, 1989; Sammarco and Coll, 1992; Kelman et al., 1999; Wang et al., 2008). Most of these products are lipid-soluble, with terpenoids (di- and sesquiterpenes), and sterols predominating (Blunt et al., 2012), but the specific molecules responsible for reported bioactivities rarely have been identified (Wylie and Paul, 1989; Sammarco and Coll, 1992; Miyamoto et al., 1994; Slattery et al., 1997a, 1998, 2001; Fleury et al., 2008; Wang et al., 2008). A large proportion of soft corals are ichthyotoxic and deterrent, although these properties seem not to be correlated, nor to derive from the same compounds (La Barre et al., 1986b). Distastefulness rather than toxicity is important against predators (Paul, 1992). It is likely that protection is achieved through more than one metabolite or groups of metabolites, sometimes present in different fractions, and their bioactivities in the overall protection may be antagonistic or cooperative (Wylie and Paul, 1989; Van Alstyne et al., 1994; Kelman et al., 1998; Wang et al., 2008). The surface of living corals is covered with a complex muco-polysaccharide lipid material with anti-predatory and anti-fouling characteristics (Miyamoto et al., 1994; Slattery et al., 1997a; Kelman et al., 1999). This layer provides a matrix for bacterial colonization (Ducklow and Mitchell, 1979; Ritchie, 2006), but the layer presumably also contains inhibitors, since typically corals stay free from epibiosis and
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resist detrimental microbial invasion (Coll et al., 1987; Slattery et al., 1995; Kelman et al., 1998; Wang et al., 2008). Among the substances exuded within the mucus of soft corals are sterols, wax esters, terpenoid toxins, and unidentified UV-absorbing compounds (Coll et al., 1982; Miyamoto et al., 1994; Slattery et al., 1997a; Brown and Bythell, 2005; Wang et al., 2008). We hypothesized that Antarctic Alcyonium soft corals rely on defensive metabolites that prevent predation and fouling. The lipid-soluble fractions of our samples of Antarctic Alcyonium soft corals were selected for feeding bioassays to evaluate potential chemical defenses against two Antarctic predators, the sea star Odontaster validus and the amphipod Cheirimedon femoratus. Inhibitory activity against a sympatric marine bacterium also was tested. The same crude extracts were the source for several active terpenoids and wax ester fractions
Methods and Materials Sample Collection and Extraction Antarctic soft corals of the genus Alcyonium were collected in the Eastern Weddell Sea by trawling between 308 and 622 m depth during the ANT XXI/2 cruise (November 2003 – January 2004) on board the R/V Polarstern (AWI, Bremerhaven, Germany). Several specimens of the species A. haddoni were collected at 9 m depth by diving in Deception Island (South Shetland Archipelago, Antarctica) during the ACTIQUIM-1 campaign (December 2008 - January 2009). Colonial clumps of each species from a single collection site were grouped together as a single sample for further experimentation and analysis. Pictures of fresh animals were taken, and a voucher portion of each sample was conserved in 10 % formalin for taxonomy. The material was frozen at −20º C, and sent to the University of Barcelona. The literature was used to identify samples to the species level (Verseveldt and Van Ofwegen, 1992; Casas et al., 1997; Van Ofwegen et al., 2007), revealing that the collection included a total of 6 samples of 5 different species (Table 1). Each sample consisted of several colonies and was exhaustively extracted with acetone at room temperature while grinding the tissue with a mortar and pestle. After removal of the solvent in vacuo, residual water was partitioned three times against diethyl ether (Et2O) and once against butanol. The organic phases were combined to obtain an ether fraction (EE) and a butanol fraction (BE). The respective organic solvents were evaporated under reduced pressure, providing dry EE and BE fractions and an aqueous residue. The dry crude fractions were weighed for calculations of yield. Sample tissue concentrations for the ether fractions, hereafter referred to as “natural concentrations”, were calculated with respect to the total dry weight (DWT =dry
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Table 1 Alcyonium soft coral samples collected in the Southern Ocean (Antarctica). AGT: Agassiz Trawl, BT: Bottom Trawl, RD: Rauschert Dredge, SD: Scuba diving Species name and sample
Location
Latitude
Longitude
Gear
Depth (m)
Alcyonium Alcyonium Alcyonium Alcyonium Alcyonium Alcyonium
Weddell Sea Weddell Sea Deception Island Weddell Sea Weddell Sea Weddell Sea
70° 56′ S 72° 51.43′ S 62º 59.55′ S 72º 49.99′ S 71º 17.1′ S 71º 4′ S
10° 31′ W 19° 38.62′ W 60º 33.68′ W 19º 34.99′ W 12º 36′ W 11º 31.99′ W
BT BT SD RD AGT BT
337.2 597.6 9 622 416 308.8
antarcticum Wright & Studer, 1889 grandis Casas, Ramil and van Ofwegen, 1997 haddoni Wright & Studer, 1889 paucilobulatum Casas, Ramil and van Ofwegen, 1997 roseum 1 van Ofwegen et al., 2007 roseum 2 van Ofwegen et al., 2007
Purification Diethyl ether extracts were transferred to ICBCNR (Pozzuoli, Napoli, Italia), where they were processed
during several visits between 2007 and 2011. They were screened by TLC (light petroleum ether/Et2O in different ratio ranging from 100 % of light petroleum ether to 100 % of Et2O). The plates were developed with CeSO4 revealing a main spot at Rf 0.9 (light petroleum ether/Et2O, 9:1) in all Alcyonium samples, whereas a series of UV sensitive spots with Rf ranging from 0.25 to 0.75 (light petroleum ether/Et2O, 8:2) were observed only in the extracts of A. grandis and A. roseum sample 1. The Et2O extracts obtained from all species were fractionated on Sephadex LH-20 with a 1:1 mixture of chloroform/methanol. Fractions from A. grandis and A. roseum sample 1 extracts containing the UV sensitive metabolites were submitted to further purification. In particular, alcyopterosins 1–9 were isolated exclusively from A. grandis extract according to the previously reported procedure and identified by 1H NMR (Carbone et al., 2009). The fractionation of the A. roseum extract on Sephadex LH20 yielded a fraction containing two UV sensitive spots at Rf 0.75 and 0.42 (light petroleum ether/Et2O, 8:2), respectively. This fraction was further purified by preparative TLC chromatography (SiO2, light petroleum ether/Et2O, 8:2) to yield two previously unreported pure compounds, alcyopterosins 10 and 11, whose structures were elucidated by spectroscopic methods (see Chemical Analysis section in the Results). Additionally, fractionation of Alcyonium extracts on Sephadex LH20 led us to isolate a more lipophilic fraction
Table 2 Data of diethyl ether (Et2O) extract yields and of the fraction containing wax esters (12–13) and of the illudalane fractions of alcyopterosins (1–9) and (10–11) of the studied Antarctic Alcyonium soft coral samples. WW: wet weight of the sample, DW: total dry weight of the sample calculated as: DW = dry residue (DR) + dry diethyl ether extract
(EE) + dry butanolic extract (BE). [NEE]: Natural tissue concentration in mg of the dry Et2O extract (EE) per g of the total dry weight (DW) of the sample; [N(1–9)], [N(10–11)] and [N(12–13)]: Natural tissue concentrations in mg of the illudalane fraction of alcyopterosins (1–9) and (10–11), and the dry wax esters fractions (12–13) per g of the total dry weight (DW) of the sample
weight of the solid extracted residue + EE dry weight + BE dry weight). The weight of the material that partitioned into the aqueous fraction was not included in the dry weight calculation. The ether fractions were used for bioassays and chemical analysis, while the butanol fraction and aqueous residue were kept for future investigations (Table 2). Characterization 1H and 13C NMR spectra of samples dissolved in CDCl3 were recorded on DRX 600, Avance 400, and DPX 300 MHz Bruker spectrometers, with chemical shifts reported in ppm relative to CHCl3 (δ 7.26 for proton and δ 77.0 for carbon). Electro Spray Ionization Mass Spectroscopy (ESIMS) and High Resolution Electron Spray Ionization Mass Spectroscopy (HRESIMS) were measured on a Micromass Q-TOF Micro mass spectrometer coupled with a Waters Alliance 2695 HPLC. The instrument was calibrated with a polyethylene glycol (PEG) mixture representing molecular weights ranging from 200 to 1,000 g/mol. Silica gel chromatography was performed using precoated Merck F254 plates and Merck Kieselgel 60 powder. HPLC purification was carried out on a Shimadzu LC-10 AD liquid chromatograph equipped with a UV SPD-10A wavelength detector.
Species and sample
WW (g)
DW (g)
EE (mg)
[NEE] (mg g−1 DW)
A. antarcticum A. grandis A. haddoni A. paucilobulatum A. roseum 1 A. roseum 2
1.01 18.58 118.90 1.25 9.32 1.56
0.51 4.55 17.65 0.33 1.66 0.47
10.15 544.06 813.41 15.95 59.21 17.78
20.10 119.57 46.09 47.89 35.67 38.00
[N(1–9)] (mg g−1 DW)
[N(10–11)] (mg g−1 DW)
27.8 -
3.9 -
[N(12–13)] (mg g−1 DW) 3.12 20.58 2.88 8.06 1.34 4.49
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from all analyzed samples. The lipophilic fractions were submitted to silica gel column purification to give wax ester mixtures (Rf 0.9, light petroleum ether/Et2O, 9:1) from all Alcyonium samples. A preliminary LC-MS analysis (250 × 4.60 mm, Phenomenex Kromasil C18, 60 min gradient from 30 % to 100 % CH3OH in H2O) showed that all the wax mixtures comprised the same two main components 12 [m/z 529 (M + Na)+] and 13 [m/z 501 (M + Na)+]. With the aim of identifying their structures, the wax mixture from A. grandis was dissolved in anhydrous MeOH (1 ml) before adding an excess of Na2CO3. The reaction mixture was stirred at room temperature for 4 h, filtered, evaporated, and purified on a silica gel Pasteur-pipette column (light petroleum ether/Et2O gradient) to give fatty acid methyl ester and fatty alcohol fractions that were characterized by 1H NMR. The fatty alcohol mixture was dissolved in dry C5H5N (0.5 ml), treated with acetic anhydride (two drops) and mixed at room temperature for 8 h. After evaporation, the residue was filtered on a Pasteur pipette-SiO2 column (light petroleum ether/Et2O, 9:1) to give the corresponding acetyl derivatives, which were analyzed by both 1H NMR and mass spectrometry. In particular, the MS spectrum showed two main sodiated peaks at m/z 307 and 279, which were consistent with the presence of C16:0 and C14:0 alcohol methyl esters, respectively. In the same way, the fatty acid methyl ester mixture obtained from the methanolysis reaction was subjected to both 1H NMR and mass spectrometry analysis leading to the identification of a C18:1 fatty acid as the main component. These data indicated that the two main components of mixture from A. grandis and all others Alcyonium samples examined were C34:1 (12) and C32:1 (13) waxes. Feeding Deterrence Assays with Asteroids Sea stars belonging to the eurybathic, ubiquitous Antarctic species Odontaster validus, which has voracious omnivorous habits and circumpolar distribution (McClintock, 1994), were captured at Port Foster Bay in Deception Island, South Shetland Archipelago (62º 59.369′ S, 60º 33.424′ W). Collection of sea stars with diameters ranging from 7 to 10.5 cm was done during three campaigns: ECOQUIM-2 (January 2006), ACTIQUIM-1 (December 2008-January 2009), and ACTIQUIM-2 (January 2010), by scuba diving at 3–17 m depth (n>1500). Sea stars are extraoral feeders, extruding the cardiac stomach and bolting down whole shrimp food cubes (McClintock, 1994), so dry weight can be used to approximate the “defense per feeding cube”. Using dry weight eliminates the water content, which may produce remarkable deviations in marine samples, especially those with soft porous tissues that capture water. The detailed methodology of these feeding assays using asteroids as a
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model predator is described in previous papers (Avila et al., 2008). Briefly, the sea stars were maintained in large tanks with fresh seawater at the Spanish Base BAE “Gabriel de Castilla” (Deception Island) where all bioassays took place. Before initiating the assays, sea stars were starved for 5 d. Each test included treatment and control experiments that ran simultaneously, and each treatment was replicated 10 times in individual containers filled with 2.5 L of seawater and accommodating one sea star. The small shrimp food items (5×5×5 mm and 13.09±3.43 mg dry mass), comprised 12.4 % protein, 9.1 % carbohydrates, and 1.5 % lipids, and yielded 17.8 KJ g−1 dry wt (4.1 KJ g−1 wet wt) (Atwater and Benedict, 1902). The cubes were treated with solvent alone (Et2O), or with natural concentrations of lipophilic Et 2 O extracts or sub-fractions from Antarctic Alcyonium soft coral samples (Table 2). Crude extracts and sub-fractions were diluted in diethyl ether for addition to the feeding cubes, and solvent was removed under a flow hood. The illudalane mixture from A. grandis (1–9), as well as the wax ester fractions (12–13) were assayed at their corresponding natural concentrations. For mixture 1–9, the concentration used was 27.8 mg g−1 dry weight. Fractions containing the wax esters C34:1 (12) and C32:1 (13) were tested at concentrations (1–25 mg g−1 dry weight) corresponding to the range of concentrations found in our samples (Table 2). After 24 h, the number of shrimp cubes eaten in each test was recorded, and the remaining cubes were conserved. TLC was used to evaluate the test compounds in the uneaten food cubes at the end of each treatment period. Feeding repellence was statistically evaluated with Fisher’s Exact tests contrasting each treatment assay with the simultaneous control (Sokal and Rohlf, 1995). After the experiments, the asteroids were returned to the sea. Feeding Preference Assays with Amphipods Lyssianasid amphipods of the abundant, eurybathic Antarctic species Cheirimedon femoratus were used following methodologies described in previous papers (Núñez-Pons et al., 2012a, b). These amphipods are voracious omnivore-scavengers with a circumpolar distribution (Bregazzi, 1972; De Broyer et al., 2007). Hundreds of individuals were captured while scuba diving in Port Foster Bay (Deception Island, South Shetland Archipelago; 62º 59.369′ S, 60º 33.424′ W) with fishing nets, between 2 to 7 m depth. Baited traps using canned sardines also were deployed along the BAE’s coastline to trap amphipods during the campaigns ACTIQUIM-2 (January 2010) and ACTIQUIM-3 (January 2012). Artificial caviar-textured food pearls were prepared using a 10 mg ml−1 alginate aqueous solution along with 66.7 mg ml−1 of concentrated feeding stimulant (Phytoplan®; 19 KJ g−1 dry wt). The powdered dehydrated food was mixed into the cold alginate solution with a drop of green or red food coloring in order to obtain pearls that were easily distinguished by color. The colors for
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treatment or control pearls were randomly swapped throughout the experimentation period, and previous trials confirmed that color had no effect on feeding preferences (P>0.1.). The mixture was put into a syringe without needle and added dropwise to a solution of 0.09 M (1 %) CaCl2 where it polymerized, forming pearls that were 2.5 mm diam comprising 3.3 % protein, 1.4 % carbohydrates, and 1.3 % lipids, and yielding 18 KJ g−1 dry wt and 1.5 KJ g−1 wet wt (Atwater and Benedict, 1902). For extract-treated pearls, the dry Alcyonium Et2O extracts were dissolved in Et2O at the natural concentrations, applied to the dehydrated food (Phytoplan), and allowed to evaporate (Table 2). Control pearls were prepared with solvent alone. Wax ester fractions were tested at three concentrations within the range of the sample natural concentrations (2.5–10 mg g−1 dry weight; Table 2). The illudalane mixtures of alcyopterosins 1–9 and 10–11 from A. grandis and A. roseum sample 1, respectively, could not be tested in this assay because the available quantities were too small. Amphipods were maintained in 8 L aquariums and were starved for 1–2 d. Every assay consisted of 15 replicate containers filled with 500 ml of sea water and 15–20 amphipods, which were offered a simultaneous choice of 10 treatment and 10 control pearls. The assays ended when approximately half of either food types had been consumed, or 4 h after food presentation. The number of remaining pearls of each color (control or treatment) was recorded. Since our feeding trials were short in time, mass autogenic alterations were avoided, and there was no need to run controls in the absence of amphipods for changes unrelated to consumption (Peterson and Renaud, 1989). Each replicate was represented by a paired result: treatment and control. Since the assumptions of normality and homogeneity of variances were not met, data were compared by non-parametric procedures using R-command software and Exact Wilcoxon tests. Uneaten treatment pearls were assessed by TLC to check for possible alterations related to chemical degradation or loss of the test compounds. Once testing was over the amphipods were returned to the sea. Antibiotic Tests towards a Sympatric Marine Bacterium Antibiotic activities of the Et2O extracts and the purified wax ester fractions (12–13) were tested with an unidentified sympatric marine bacterium by an agar disc-diffusion method. Unfortunately, neither of the alcyopterosin-containing fractions (1–9 and 10–11) was available at the time this test was performed. The bacterium was obtained from a seawater sample collected at Crater 70, Deception Island (Antarctica), and a subsample was conserved at −20 °C in 7 % glycerol filtered-sterilized seawater and ultimately shipped to the University of Barcelona. A 1 ml aliquot of the seawater sample was added to Difco™ marine broth 2216 (Difco Laboratories), left for 24 h at 18–20 °C, and subsequently cultured on plates with Difco™ marine agar
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2216 (Difco Laboratories). The resulting bacterial colonies were isolated, and the strain exhibiting the fastest growth was chosen for the assays. A subsample of the selected strain was frozen at −20 °C and shipped to the University of Barcelona. Taxonomic identification of the selected strain by DNA amplification with real-time PCR (Polymerase Chain Reaction) techniques was unsuccessful. Rinse broth, a membrane rinsing fluid for bacterial culture, was inoculated with pure cultures of the selected strain and incubated at 18–20 °C until the turbidity corresponded to 0.5 on the McFarland scale (McFarland, 1907), which is equivalent to 1.5×10−8 colony forming units ml−1. A 0.1 ml suspension of this culture was evenly spread onto marine agar plates that were divided into six regions: three regions for testing an extract or wax ester fraction (12–13) in triplicate; another for the positive control, chloramphenicol; two regions for the negative controls, one with Et2O and one without solvent. Paper antimicrobial assay disks (BBL Microbiology Systems) 6 mm diam and carrying 20 μl of Et2O extracts, or wax esters fractions (12–13) diluted in Et2O, or chloramphenicol (30 μg), or solvent were placed in the middle of the appropriate testing region in the inoculated Petri dishes. Extract amounts added to the disks were equivalent to the natural concentrations as described above (Table 2). The plates were incubated for 1 d at 18–20 °C, and the diameters of the inhibition halos were recorded. When the diameter of the inhibition halo was larger than 7 mm, the test material was considered active. One active replicate indicated weak activity, two active replicates mild inhibition, and three active replicates strong activity (Mahon et al., 2003).
Results Soft Coral Organic Fractions The Alcyonium soft corals studied here belonged to five different species. Each coral species consisted of globular, massive, pale pinkish colonies with small white polyps, except for the sample of A. haddoni collected in a shallower location, which had brighter orange coloration and yellowish polyps. Colony shape, polyp arrangement, and sclerite morphology allowed us to identify the six samples as A. antarcticum, A. grandis, A. haddoni, A. paucilobulatum, and two samples of A. roseum from different collection sites, named sample 1 and sample 2 (Table 1) (Verseveldt and Van Ofwegen, 1992; Casas et al., 1997; Van Ofwegen et al., 2007). The six samples, each consisting of several colonies, yielded six diethyl ether extracts that were used for ecological and chemical analysis (Table 2). Chemical Analysis In the course of our chemical analyses of Antarctic soft corals, A. grandis and A. roseum sample 1 were found to contain a series of sesquiterpenoids belonging to the illudalane class (compounds 1–11). In particular,
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alcyopterosins 1–9 (Fig. 1) were isolated from A. grandis as described in our previous study of this species (Carbone et al., 2009), whereas two new compounds (10 and 11) were isolated exclusively from A. roseum sample 1 (Fig. 2). The conspecific A. roseum sample 2 did not possess any illudalane-related terpenoids. The structures of the new metabolites were established by NMR. Preliminary 1H NMR analysis of compounds 10 and 11 showed their close structural relationship, identifying for both metabolites the same illudalane aromatic carbon skeleton as that reported for alcyopterosins. Compound 10 exhibited the molecular formula C17H23O2Cl as deduced by HRESIMS from the molecular ion at 317.1296 (M + Na)+. The 1H NMR spectrum displayed five singlet signals at δ H 1.07 (3H), 1.13 (3H), 2.07 (3H), 2.23 (3H), and 2.34 (3H), which were attributed to two tertiary methyl groups (H3-14 and H3-15), an acetyl group, and two aromatic methyl groups (H3-12 and H3-13), respectively. Three methylene signals at δH 3.53 (2H, m, H2-10), 3.12 (2H, m, H2-5), 2.94 (1H, d, J=16 Hz, H-10a), 2.54 (1H, d, J=16 Hz, H-10b), an oxygenated methine at δH 5.96 (1H, s, H-1), and one aromatic methine at δH 6.92 (1H,s, H-8) completed the spectrum. These data were consistent with the alcyopterosin carbon skeleton containing a chlorine and an acetylated hydroxyl group on the five membered ring. The 13 C NMR spectrum displayed signals assigned to six aromatic carbons and nine sp3 carbons (four CH3, three CH2, and one quaternary C) along with signals at δ 170.7 (CO) and δ 21.0 (CH3) due to the carbons of the acetylated group. The location at C-1 of the oxygenated functionality was indicated clearly by diagnostic NOE observed between H312 and H-1, as well as between H3-13 and H-8, leading to structure 10. Characterization of compound 11 was achieved easily by comparison with 10. The molecular formula C17H21O3Cl of compound 11, established by HRESIMS on the sodiated molecular peak at 331.1073 (M + Na)+, indicated the presence of unsaturation as well as an additional oxygen with respect to compound 10. Analysis of
Fig. 1 Chemical structures of the nine illudalane compounds (alcyopterosins 1-9) purified from Alcyonium grandis
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Fig. 2 Chemical structures of the two new illudalane compounds (alcyopterosins 10-11) purified from Alcyonium roseum 1
NMR spectra revealed that 11 differed from 10 only in the absence of one aromatic methyl group (δH 2.23; δC 14.1), which was replaced by an aldehyde functionality (δH 10.35; δC 191.7). As for compound 10, NOE observed between H12 and H-1, and H3-13 and H-8 led us to locate the aldehyde and the acetylated hydroxyl groups as indicated in formula 11. Analysis of 2D-NMR experiments ( 1 H- 1H COSY, HSQC and HMBC) of both compounds 10 and 11 allowed the complete proton and carbon assignments as listed below. The absolute configuration of the unique chiral center C-1 in both compounds was unassigned. Compound 10: 1H NMR (CDCl3) δH 6.92 (1H, s, H-8), 5.96 (1H, s, H-1), 3.53 (2H, m, H2-4), 3.12 (2H, m, H25), 2.94 (1H, d, J=16 Hz, H-10a), 2.54 (1H, d, J= 16 Hz, H-10b), 2.34 (3H, s, H3-13), 2.23 (3H, s, H312), 2.07 (3H, s, -COCH3), 1.13 (3H, s, H3-15 or H314), 1.07 (3H, s, H3-14 or H3-15); 13C NMR (CDCl3) δC170.7 (−COCH3), 143.7 (C-2), 138.2 (C-9), 134.6 (C-6 and C-3), 133.2 (C-7), 124.8 (C-8), 83.2 (−1), 45. 8 (C-10), 43.8 (C-11), 42.2 (C-4), 33.1 (C-5), 27.6 (C14 or C-15), 22.4 (C-15 or C-14), 21.0 (−COCH3), 20.4 (C-13), 14.1 (C-12). HRESIMS (M + Na)+ m/z 317. 1296 (calculated for C17H23O2ClNa, 317.1284). Compound 11: [α]D +5.4 (c=0.07, CHCl3); 1H NMR (CDCl3) δH 10.35 (1H, s, H-12), 7.32 (1H, s, H-8), 6.31 (1H, s, H-1), 3.65 (1H, m, H2-4) 3.46 (2H, m, H2-5), 2. 95 (1H, d, J=16 Hz, H-10a), 2.58 (1H, d, J=16 Hz, H10b), 2.42 (3H, s, H3-13), 2.05 (3H, s, -COCH3), 1.25 (3H, s, H3-15 or H3-14), 1.17 (3H, s, H3-14 or H3-15); 13 C NMR (CDCl3) δC191.7 (C-12), 170.7 (−COCH3), 144.9 (C-2), 142.7 (C-9), 139.5 (C-7), 136.8 (C-6), 132. 6 (C-8), 131.6 (C-3), 81.4 (C-1), 44.8 (C-10), 44.1 (C11), 43.6 (C-4), 33.2 (C-5), 27.5 (C-14 or C-15), 22.4 (C-15 or C-14), 20.9 (C-13), 20.1 (−COCH 3 ). HRESIMS (M + Na)+ m/z 331.1073 (calculated for C17H21O3ClNa, 331.1077). Further chemical analysis carried out on the samples led to the isolation and identification of two common waxes, compounds 12 and 13. These metabolites were the main components of the wax mixture in each of the samples, with approximate concentrations ranging between 1.3 and 21 mg g−1 dry weight (Table 2). Both waxes consist of a C18-monounsaturated fatty acid (C18:1) esterified to an
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unsaturated alcohol. Compound 12 has a C16-saturated alcohol (16:0) while 13 has a C14-saturated alcohol (14:0), thus producing C34:1 and C32:1 wax esters, respectively (Fig. 3). The illudalane mixture of alcyopterosins 1–9 obtained from colonies of A. grandis was tested only in the sea star assay, whereas compounds 10–11 from A. roseum sample 1 were not assayed due to a limitation in the amount available. Waxes 12–13, which were common to all Alcyonium samples were used at natural sample concentrations in all assays. Feeding Deterrence Assays with Asteroids Control shrimp feeding cubes impregnated with solvent alone were 80– 100 % accepted, whereas treated food cubes were often rejected. The sea star Odontaster validus significantly rejected cubes treated with five out of the six soft coral lipophilic Et2O fractions, suggesting that all five Alcyonium species were chemically defended. However, the extract from A. roseum sample 2 was not significantly repellent (P>0.1) (Fig. 4). Shrimp food cubes treated with the fraction containing the illudalane mixture (1–9) were highly deterrent at the natural concentration. The wax ester fractions 12–13 at concentrations ranging between 2.5 and 25 mg g−1 dry weight were significantly rejected, while the lowest concentration, 1 mg g−1 dry weight, was not rejected (Fig. 4). Feeding Preference Assays with Amphipods In these experiments, only three species could be tested due to the lack of test material. All three extracts were unpalatable towards the amphipod Cheirimedon femoratus at their respective natural concentrations (Fig. 5). This amphipod is gregarious in its feeding habits, and was voracious with the control diet. When extracts were included in the alginate food pearls, consumption was almost completely inhibited. We could only test the wax ester fraction (12–13) at three mean concentrations. Food pearls containing wax esters at 5 or 10 mg g−1 total dry weight were significantly rejected with respect to the paired untreated control pearls, but concentrations of 2.5 mg g−1 total dry weight were accepted (Fig. 5). TLC, used to evaluate the uneaten shrimp feeding cubes and alginate pearls, showed that the extracts or sub-fractions containing mixtures of the isolated compounds remained in the food items after the bioassays. These compounds are
Fig. 4 Percentage acceptance in the feeding repellence bioassays with the sea star Odontaster validus using whole-colony lipophilic Et2O extracts from Alcyonium Antarctic soft corals, as well as sub-fractions of illudalane mixture (1-9) and wax esters WAX (12-13), the last ones at diverse concentrations. The paired results of control and extract treated shrimp cubes are shown for each test. *: significant differences (P10 %) within the tissues and mucus, making them unsuitable to most predators. Only few specialized consumers like crown-of-thorns starfishes (Acanthaster spp) possess a unique wax-digesting system allowing them to voraciously feed on living corals (Benson et al., 1975). Amphipods were deterred at higher wax concentrations (5 mg g−1 dry weight) compared to asteroids (2.5 mg g−1 dry weight). This could be because Antarctic amphipods make use of wax esters as energy reserve, while sea stars do not accumulate waxes (Sargent et al., 1977). Waxes such as 12–13 are common marine waxes, and also were obtained as part of more complex mixtures from several Antarctic gorgonians in our collections (unpublished results). Soft coral mucus is the medium into which allelochemicals are exuded as defense against predation, fouling and competition, keeping these activities near the coral’s surface (Coll et al., 1982; Miyamoto et al., 1994; Slattery et al., 1997a; Wang et al., 2008). We speculate that the bioactive illudalane terpenoids (alcyopterosins 1–11) and wax esters (12–13) are secreted into the mucus. Coral mucus secretion is variable in amount and composition, responding to disturbance or stimuli (Brown and Bythell, 2005). This may explain the diversity of concentrations found in the wax ester fractions (12–13) within the different samples. A latitudinal cline with a higher diversity of octocoral secondary metabolites in the tropics than in temperate regions
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has been proposed (Blunt et al., 2012). In polar waters, the research effort has been low, and therefore, it is not possible to draw many conclusions about patterns of metabolic diversity. Nonetheless, many Antarctic organisms, including cnidarians, have yielded a notable number of new natural products, many of them with interesting bioactivities (Avila et al., 2008). We believe that the ecological success of soft corals in Antarctic communities is probably related to the presence of noxious feeding repellents and antifouling compounds, derived from both primary and secondary metabolism. As far as we know, ours is one of the few studies in which ecologically relevant metabolites have been identified in Antarctic Alcyonium soft corals. Additional studies are needed on their biotic interactions and the defensive mechanisms of these and other Antarctic species. Acknowledgements We thank L. Ciavatta, F. Castelluccio, M. Rodríguez-Arias, M. Paone, S. Taboada, J. Cristobo, B. Figuerola, C. Angulo, and J. Moles for support and help in the lab. Thanks are due to S. Catazine for the artwork. Also we are grateful to W. Arntz and the crew of R/V Polarstern, UTM (CSIC), “Las Palmas” and BAE “Gabriel de Castilla” crews for logistic support. Funding was provided by the Ministry of Science and Innovation of Spain (CGL/2004-03356, ANT, CGL2007-65453/ANT and CGL2010-17415/ANT).
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