Marine benthic invertebrate samples were collected in the Eastern Weddell Sea (Antarctica) during the ANT XXI/2 cruise (November 2003–January 2004) on board the R/V “Polarstern” (AWI, Bremerhaven, Germany). Our samples were obtained from 7 stations at depths ranging from 230 to 600 m sampled with Agassiz and bottom trawls. During ACTIQUIM-I campaigns (2007–2008 and 2008–2009), algae and invertebrate samples from Deception, Livingston, and Snow Islands were sampled by SCUBA at 7 stations (ca. 0–15 m depth) (Table 1). Organisms were photographed alive and immediately frozen (−20°C) for chemical investigations. Organisms were selected according to potential interest based on ecological observations and/or previous ecological assays (Taboada et al., 2012). Voucher specimens or a portion of each sample was fixed in 10% formalin or 70% EtOH for taxonomical identification. These vouchers are deposited at the Dept. of Animal Biology (Invertebrates), University of Barcelona, Spain.

When necessary, several conspecific specimens from the same station were extracted together in order to obtain enough extract for the experiments. Frozen selected samples were weighted and immediately lyophilized for 24–48 h. Subsequently, dehydrated samples were re-weighted, pounded, and exhaustively extracted with absolute ethanol (1:10 w/v) helping with ultrasonic baths of 10 min. After filtering, the ethanol of the remaining extracts was evaporated in vacuo. The solid ethanol extracts were sequentially solubilized at different EtOH/H₂O concentrations: 125 μg/μL EtOH 100%, 5 μg/μL EtOH 4%, and 0.25 μg/μL EtOH 0.2% and freeze-dried immediately. Cell culture was performed only with ethanol extracts that remained soluble at EtOH 0.2%, as it is the highest ethanol concentration innocuous to cells. Ten species with soluble extracts at 0.2% EtOH were chosen to perform anti-inflammatory assays according to the solubility tests. We similarly extracted more individuals and/or the rest of the frozen samples selected in order to obtain enough extract for the anti-inflammatory assays. Infrared spectra of samples and replicates were performed for all the species in order to discard contamination due to extraction methods of the ten species. Wet, dry, and extract's weight of the species selected and those rejected are shown in Supplementary Tables S1, S2, respectively.

The mouse macrophage RAW 264.7 cell line (American Type Culture Collection, Manassas, VA, U.S.A.) was cultured in DMEM medium supplemented with 5% fetal bovine serum. Cells were incubated with marine organisms' extracts at concentrations of 50, 125, and 250 μg/mL (dissolved in 0.2% ethanol). The cells were stimulated with lipopolysaccharide (LPS; 1 μg/ml) and incubated for 24 h, in the presence or absence of extracts. Dexamethasone (1 μM) was used as a reference anti-inflammatory drug. Each test was performed in quadruplicate. Toxicity of the extracts at the concentrations tested was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5 diphenyltetrazolium bromide to colored formazan (MTT method). The production of inflammatory mediators was determined in cell supernatants by measuring: prostaglandin E₂ (PGE₂) and leukotriene B₄ (LTB₄) by radioimmunoassay and interleukin-1β (IL-1β) by ELISA. Statistical significance was established by ANOVA followed by Dunnett's test. Results indicate mean ± SE (at least n = 8 for each group). ^*P < 0.05, ^**P < 0.01 with respect to LPS control.

A total of 36 ethanol extracts from benthic algae and invertebrates were dissolved at decreasing ethanol concentrations, down to 0.2% EtOH, to perform the bioactivity assays at an innocuous ethanol concentration (Table 1). Ethanol extracts of ten species from five different phyla: Achrophyta (2), Porifera (4), Chordata (2), Echinodermata (1), and Hemichordata (1), resulted suitable, i.e., soluble at EtOH 0.2%, to cell culture (asterisks in Table 1).

EtOH extracts of the alga Desmarestia menziesii, the sponge Mycale (Oxymycale) acerata, and the seastar Odontaster validus exhibit cytotoxicity at the two higher concentrations (125 and 250 μg/mL; Figure 1). Certain cytotoxicity, only at the highest concentration, was found in the alga Desmarestia anceps, the sponge Isodictya toxophila, and the hemichordate Cephalodiscus sp. Finally, the sponges Isodictya erinacea and Homaxinella cf. balfourensis, the ascidiacean Synoicum adareanum, and the thaliacean Salpa cf. thompsoni were not cytotoxic at the concentrations tested.

Desmarestia menziesii extracts decreased the release of IL-1β and PGE₂ at cytotoxic concentrations (Supplementary Figure S1). Similarly, the extract of Odontaster validus reduced the release of IL-1β at the highest concentration, which had cytotoxic effects. The EtOH extract of the brown alga D. anceps inhibited PGE₂ release at non-cytotoxic concentrations (50 and 125 μg/mL), while showing no action on the other mediators. The extract of the salpid Salpa cf. thompsoni reduced significantly and dose-dependently the release of IL-1β and PGE₂. However, at the highest concentration (250 μg/mL) increased the release of LTB₄. The EtOH extract of the demosponge Homaxinella cf.balfourensis reduced significantly the release of IL-1β and PGE₂ only at the highest, but not cytotoxic, concentration (250 μg/mL). Finally, the extract of the colonial ascidiacean, Synoicum adareanum, increased the release of PGE₂ and LTB₄ at the highest concentration, having no effect at lower concentrations (Supplementary Figure S1).

The extract of Isodictya erinacea inhibited significantly and dose-dependently the release of IL-1β and PGE₂, although not affecting LTB₄ (Figure 1). The extracts of both the sponge Isodictya toxophila and the hemichordate Cephalodiscus sp. decreased the release of IL-1β and LTB₄ at two non-cytotoxic concentrations (50 and 125 μg/mL) and also diminished PGE₂ release at the intermediate concentration (125 μg/mL) (Figure 1). Mycale (Oxymycale) acerata's extract decreased the release of IL-1β, PGE₂, and LTB₄ at the lower concentration of 50 μg/mL, which was not cytotoxic (Figure 1).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.