The collection of wild organisms from the local environment was undertaken by staff of the Marine Biological Resource Centre according to the conditions of the scientific sampling permit accorded to the Roscoff Biological Station by the Brittany Prefecture (Permit 2013-6793 awarded 13/03/2013). The animal species used are not locally endangered and were collected in minimal quantities necessary to perform the study. The use of Sepia officinalis complied with the EU directive 2010/63/EU. No vertebrates were used in this study. Sponges [Pachymatisma johnstonia Bowerbank in Johnston, 1842, Polymastia sp. Bowerbank, 1863; C. celata Grant, 1826, Tethya aurantia (Pallas, 1766), Hymeniacidon sp. Bowerbank, 1858, Axinella sp. Schmidt, 1862 and Raspailia sp. Nardo, 1833], anemones and hydras [Actinia fragacea Tugwell, 1856, Eunicella verrucosa (Pallas, 1766), Alcyonium digitatum Linnaeus, 1758 and Alcyonium glomeratum Hassall, 1843], bryozoans [Flustra foliacea (Linnaeus, 1758)], echinoderms [Asteria rubens Linnaeus, 1758 and Paracentrotus lividus (Lamarck, 1816)] were collected in the vicinity of Roscoff (France, GPS coordinates 48.72, −3.98). S. officinalis Linnaeus, 1758 and Ciona intestinalis (Linnaeus, 1767) were cultivated in Roscoff Marine Station. After collection they were extensively washed with clean sea water to remove possible contaminations or epiphytes. Finally, they were frozen for at least 48 h at −80°C to be terminated, lyophilized and were finely ground using metallic balls and tissuelyser (Quiagen), at a frequency of 27 s⁻¹ until fine powder was obtained.

Alcohol insoluble residues (AIR) from samples were obtained as described in Hervé et al. (2015). Briefly, powdered materials were consecutively washed with an orbital shaker in 70% ethanol, 96% ethanol, chloroform: methanol (1:1, v/v), and acetone intercalated by spin down of materials (1,500 g, 15 min). Pellets and supernatants were kept and reserved at −80°C for further analysis. The final air-dried pellets were chemically fractionated using five sequential extractions: 2% CaCl₂ at 80°C, 0.2 M HCl, 3% Na₂CO₃, 50 mM diamino-cyclo-hexane-tetra-acetic acid (CDTA) and 4 M NaOH with 1% v/v NaBH₄ to avoid β elimination. The extractions were applied for 60 min and soft shaking (4 s⁻¹) with a tissuelyser. Each extractions were intercalated by a centrifugation step to separate pellet from supernatant (1,500 g, 15 min). The supernatants obtained from each extraction were frozen at –80°C and lyophilized. Each lyophilized sample was re-suspended in printing buffer (55.2% glycerol, 44% water, 0.8% Triton X-100) at 10 mg/300 μl and printed onto nitrocellulose membrane with a pore size of 0.45 μm (Whatman) at 22°C and 55% humidity) using an Arrayjet Sprint microarray printers and probed as described by Moller et al. (2007) with the following monoclonal antibodies (mAbs) or carbohydrate binding modules (CBM): anti-fucoidan (BAM1, BAM2, BAM3, and BAM4), anti-alginate (BAM7 and BAM10), anti-homogalacturonan (LM19), anti-extensin (LM1), anti-arabinogalactan proteins or AGP (JIM8), anti-xyloglucan (LM25) anti-xylan (LM10), and two CBMs against cellulose/xyloglucan epitopes (CBM30 and CBM3a) (see also Table 2). Probing procedure in brief: the printed arrays were blocked 1 h in PBS (140 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 1.7 mM KH₂PO₄, pH 7.5) with 5% w/v low fat milk powder (MPBS). Then, the arrays were incubated 2 h with the aforementioned mAbs and CBMs. Probes were diluted 1:10 and 10 μg/ml, respectively, in MPBS. Afterwards, the arrays were washed thoroughly in PBS and incubated for 2 h with secondary antibodies conjugated to alkaline phosphatase, anti-rat (Jackson Immuno Research) for the mAbs (BAM1, BAM2, BAM3 and BAM4, BAM7 and BMA10, LM19, LM1, JIM8, LM10 and LM25) and anti-His for the two CBMs (Sigma-Aldrich) diluted 1:5,000 (anti-rat) or 1/1,500 (anti-His tag) in MPBS. Once washed in PBS and distilled water, microarrays were developed in a solution containing 5-bromo-4-chloro-3-indolylphosphate and nitro blue tetrazolium (Sigma-Aldrich) in alkaline phosphatase buffer (100 mM NaCl, 5 mM MgCl₂, 100 mM diethanolamine, pH 9.5). The probed and developed arrays were scanned on a flatbed scanner and the heatmap was calculated as described by Hervé et al. (2015).

Piece of the tissue of ~1 cm³ size surrounding the ostium was excised from the specimen. The sample was fixed in 4% formaldehyde in PBS for 1 h, washed twice with PBS and dehydrated in a series of ethanol:water solutions (30%, 70% and twice with 100%, 30 min incubation time each step) followed by clearing with HistoChoice (Sigma-Aldrich) for 24 h. The HistoChoice solution was gradually replaced with melted paraffin (Sigma-Aldrich). The embedding was performed for 2 days at 60°C. The paraffin block was sectioned on the microtome (Thermo HM355) to generate 10 μm-thick sections which were adhered to SuperFrost slides. Slides were deparaffinised in pure xylene and rehydrated in ethanol:water series (100, 96, 70 50, 10%, pure water; 5 min incubation time each step). Haematoxylin and Eosin (H&E) staining was performed according to the manufacturer instructions (Fischer Scientific). For immunolocalisation sections were individualized using PapPen, blocked with 5% skimmed milk in PBS for 30 min, probed with primary antibodies diluted in 5% milk/PBS (1:10), incubated for 1 h washed twice with PBS and incubated for 1 h with anti-rat secondary antibody conjugated with Alexa Fluor 488 diluted in 1% BSA in PBS (1:500, Sigma). Sections were washed twice with PBS, stained with 1 μM propidium iodide in PBS, washed once and mounted in CitiFluor (Agar Scientific). Laser scanning confocal microscopy (LSCM) was done on Leica SP5 equipped with argon and argon/krypton lasers. At least 3 sections were observed in 2 independent experiment observed with the same confocal settings. The pictures were processed using GIMPII software.

For our analysis, we assembled a collection of 18 specimens of marine animals common for the Mediterranean and Northern Atlantic rim, collected from these marine environments or cultured at Station Biologique de Roscoff (Roscoff, France). The species were selected to represent a range of early emerging metazoans. Table 1 lists all analyzed species and their phylogenetic standing (according the Systema Naturae 2000). The specimens were frozen, lyophilized, homogenized and the resulting powders used to obtain alcohol insoluble residues AIR to remove non-polysaccharide components including proteins, lipids and metabolites. AIR was then fractionated by sequential extractions in the following order: (1) 2% CaCl₂ at 80°C, (2) 0.2M HCl, (3) 3% Na₂CO₃, (4) CDTA, and (5) 4 M NaOH with 1% NaBH₄.The rationale behind each extraction step was; the first two solvents (1, 2) were used to precipitate any negatively charged polysaccharides usually forming intermolecular bridges with calcium (e.g., alginates and pectins) and then to extract them with the a mild base (3). This would allow extracellular matrixes to become more accessible for further glycan removal by (4) which is a chelating agent and was used to complete the extraction of charged polysaccharides. The last solvent (5) is a well-established solvent for less soluble cell wall glycan components (for example hemicelluloses when applied to land plants) and can also partially dissolve cellulosic materials.

The extractions were printed as microarrays and probed with four anti-fucoidan mAbs (designated BAM1 to BAM4) recognizing distinct epitopes on fucoidan polysaccharides from the brown alga F. vesiculosus (Torode et al., 2015). In addition, we used a selection of probes directed against typical algal and plant cell wall components. These included: anti-alginates BAM7 and BAM10 mAbs (Torode et al., 2016); LM19 mAb recognizing homogalacturonan (HG) with low degree of esterification (Verhertbruggen et al., 2009); LM1 and JIM8 mAbs recognizing cell wall proteoglycans extensins and AGPs respectively (Pennell et al., 1991; Smallwood et al., 1995) and mAbs recognizing LM25 (xyloglucan; Pedersen et al., 2012), LM10 (xylan; McCartney et al., 2005). In addition to mAbs we used β-(1,4)-glucan (cellulose and xyloglucan)-specific carbohydrate binding modules CBM3a (Blake et al., 2006; Hernandez-Gomez et al., 2015) and CBM30 (Najmudin et al., 2006). We expected that these CBMs would bind to tunicin of C. intestinalis and hence serve as a positive control. Figure 1 shows simplified schematic of the whole CoMPP procedure and the Table 2 summarizes the probes used and the epitopes they recognized.

The quantification of the resulting developed arrays is presented as a heatmap where numbers are mean signal intensities resulting from antibody binding (Figure 2). We detected the presence of fucoidan epitopes in five organisms belonging to cephalopods, sponges, and tunicates. Signals were observed with BAM1 and BAM4 mAbs in six species whilst no binding was observed for BAM2 and BAM3 mAbs in any of the species (Figure 2). BAM4, which is specific for a sulfated epitope, bound to the S. officinalis (cephalopod) and C. intestinalis (tunicate) fractions, whereas BAM1 recognizing a non-sulfated epitope bound strongly to C. celata (demosponges). Interestingly, the effectiveness of different extraction buffers varied between the species. For instance the Na₂CO₃ extraction was the most efficient in extraction of BAM1 and BAM4 epitopes in several species, on the other hand only NaOH extraction revealed higher amounts of BAM4 epitopes in C. intestinalis (Figure 2). The differential extractability and binding support the notion of high structural diversity of these compounds in animals and possibly their intimate relationship (possibly covalent) to other matrix components. Interestingly, in our sample set up we could not detect fucoidans in echinoderms, which may be due to different structure not recognizable by BAM mAbs. Indeed Pereira et al. (1999), showed that fucoidans from sea urchin are more linear and repetitive than branched and heterogeneous as is the case in brown algae.

In the case of other plant-specific mAbs no significant binding was observed. Although low signal of two mAbs (mannan and xyloglucan) could be detected in the NaOH fractions of some species, binding signals were very low and fell below the 5% lower cut off used on the heat maps. On the other hand, as expected, both CBM3a and CBM30 bound strongly to NaOH fraction of C. intestinalis producing tunicin, demonstrating selectivity and sensitivity of the approach.

Profiling of animal samples for the presence of glycan structures in homogenized samples can be hampered by the contamination with plant matter ingested as food, or by the presence of algal or bacterial endosymbionts. To partially address this, we also undertook immunolocalisation analysis in C. celata. Sponges are considered to be the simplest form of animals (Gold et al., 2016) and the presence of some polysaccharides in the sponges' bodies could be an ancestral trait shared with other eukaryotic lineages. However, sponges are also known to host symbiotic microorganism (Hentschel et al., 2012) which increase the possibility of false positive hits. Sulfated polysaccharides have been reported in many species of sponges including C. celata, although not yet directly classified as fucoidans (Zierer and Mourão, 2000; Esteves et al., 2011). Glycan chains which contain fucose are also a part of the sponges' aggregation factors–a highly complex and diverse class of circular proteoglycans also called spongicans. These glycan structures of aggregation factors are responsible for mediating the cell-to-cell adhesion via calcium bridges (Misevic and Burger, 1993; Fernandez-Busquets and Burger, 2003; Bucior et al., 2004; Vilanova et al., 2009, 2016).

We therefore decided to further explore the presence of fucoidan epitopes in situ and to characterize their localization in C. celata using immunolocalisation. A piece of tissue surrounding the ostium (inhalant pore) was embedded in paraffin and the sections were probed with BAM1 and BAM4 and observed using laser scanning confocal microscope (LSCM) (Figure 3). The morphological structure and different cell types are shown in the Figure 3B. BAM1 labeled exclusively the visceral layer of the internal chambers (spongocoel; Figures 3C,D). Also BAM1 did not strongly label any other cell types like sclerocytes—the cells producing needle-like structures spicules (Figure 3E). BAM4 labeling corroborated the results from CoMPP and no specific labeling could be observed in any of the other tissues (Figure 3F). The close observation showed that the BAM1 labels exclusively the layer of the cells forming the internal chambers (spongocoel) and no signal could be detected between the cells (Figure 3G). This indicates that this particular epitope is most likely not a part of the aggregation factors. On the other hand, previously it has been shown that glycoprotein-rich mucus (glycocalyx) connects collars of choanocytes with function in filtering or trapping food particles (Leys and Hill, 2012). Hence, our results suggest that fucoidan epitopes in sponges are actually part of the components of this type of extracellular matrix in some sponges.

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.