Kang V et al. (2020), Molecular insights into the powerful mucus-base...

ECB-ART-49782

Open Biol 2020 Jun 01;106:200019. doi: 10.1098/rsob.200019.

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Molecular insights into the powerful mucus-based adhesion of limpets (Patella vulgata L.).

Kang V , Lengerer B , Wattiez R , Flammang P .

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Limpets (Patella vulgata L.) are renowned for their powerful attachments to rocks on wave-swept seashores. Unlike adult barnacles and mussels, limpets do not adhere permanently; instead, they repeatedly transition between long-term adhesion and locomotive adhesion depending on the tide. Recent studies on the adhesive secretions (bio-adhesives) of marine invertebrates have expanded our knowledge on the composition and function of temporary and permanent bio-adhesives. In comparison, our understanding of the limpets' transitory adhesion remains limited. In this study, we demonstrate that suction is not the primary attachment mechanism in P. vulgata; rather, they secrete specialized pedal mucus for glue-like adhesion. Through combined transcriptomics and proteomics, we identified 171 protein sequences from the pedal mucus. Several of these proteins contain conserved domains found in temporary bio-adhesives from sea stars, sea urchins, marine flatworms and sea anemones. Many of these proteins share homology with fibrous gel-forming glycoproteins, including fibrillin, hemolectin and SCO-spondin. Moreover, proteins with potential protein- and glycan-degrading domains could have an immune defence role or assist degrading adhesive mucus to facilitate the transition from stationary to locomotive states. We also discovered glycosylation patterns unique to the pedal mucus, indicating that specific sugars may be involved in transitory adhesion. Our findings elucidate the mechanisms underlying P. vulgata adhesion and provide opportunities for future studies on bio-adhesives that form strong attachments and resist degradation until necessary for locomotion.

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	Figure 1. Limpets (Patella vulgata) have evolved powerful attachments to withstand crashing tidal waves and predatory attacks. Here, one of the authors lifted a heavy rock by hooking onto a single limpet.
	Figure 2. Three types of pedal mucus were sampled from individual P. vulgata limpets. (a) Limpets were allowed to settle onto thin PVCA films; (b) Interfacial primary adhesive mucus (IPAM) was collected by peeling a thin PVCA film from a settled limpet and scraping the thin layer on the PVCA, while bulk primary adhesive mucus (BPAM) was sampled from the limpet pedal sole; (c) Secondary adhesive mucus (SAM) was sampled by gently detaching a limpet, wiping the pedal sole clean, leaving it upturned for at least 30 min, then gently collecting the SAM from the pedal sole.
	Figure 3. Representative in vivo sub-pedal pressure values from P. vulgata with schematics showing the different trial conditions. (a) Free locomotion: small pressure values (around −0.02 to +0.07 kPa) were observed when the limpet was undisturbed and locomoting over the sensor. (b) Simulated attack: when a ball bearing was used to simulate a predation event, the pressure was lower, at around −1.0 kPa. This negative peak decayed slowly, taking around 7 s to reach approximately 60% of the minimum pressure. (c) Normal pull off: when the limpet was allowed to settle over the sensor and then manually detached perpendicularly (arrow marks beginning of detachment), a sharp negative peak was recorded that reached −5.7 kPa, which returned to zero when the limpet detached (marked Ø). All tests were conducted under water.
	Figure 4. Overview of P. vulgata foot tissue and the chemistry of glandular secretions. (a–d) Alcian blue (pH 2.5) highlights glands in blue (carboxylate and sulfate moieties) and phloxine stains muscles in red. Regions used for higher-magnification images (b–d) are labelled as marginal groove (mg), anterior (at), middle (m) and posterior (pt). Scale bars: 100 µm in (b–d). Compass labels: A, anterior; P, posterior; D, dorsal; V, ventral. (e–k) Lectin stains highlight the different sugar residues present within specific glands. (e) LCA: stitched image showing the entire foot. Glands contain α-Man and/or α-Fuc linked to N-acetylchitobiose sugar residues. Side-wall glands are not stained. Dotted line marks the epithelium, and imaged regions are labelled as in (a). Scale bar 400 µm. (f) LCA (at): stained glands are found within the epithelium and up to approximately 300 µm into the foot. Scale bar 100 µm. Stained mucus and gland secretions are visible (inset, 150 µm box). (g) sWGA (at): secretions containing GlcNAc but not sialic acid are highlighted throughout the tissue. Scale bar 100 µm. Granules are secreted from long necks (inset, 200 µm box). (h) UEA I (at): specific glands contain α-Fuc. Scale bar 20 µm. (i) MAL II (at): infrequent glands deep in the tissue (approx. 100–150 µm) contain (α-2,3)-sialic acid. Scale bar 100 µm. Granules are secreted from long necks (inset, 370 µm box). (j) WGA (m): glands with GlcNAc are present throughout the foot tissue. Note the epithelium is strongly stained. Glands approximately 30 µm in size are full of granules (inset, 150 µm box). (k) RCA I (m): similar to MAL II but with GalNAc or galactose. Scale bar 50 µm. See text and table 1 for additional information on the lectins used and their ligands.
	Figure 5. Conserved protein domains present in a subset of limpet pedal mucus proteins. Amino acid lengths are shown after the N-terminal of each sequence. See legend for details.
	Figure 6. Comparison between P-vulgata_3 and SCO-spondin from Gallus gallus. P-vulgata_3 shares many conserved domains with the first one-third of SCO-spondin sequence.
	Figure 7. In situ hybridization (ISH) of five protein sequences confirms the presence and locality of the target mRNA within P. vulgata foot. (a) Alcian blue stain highlights the glands present in the foot and provides context for the ISH stains. Scale bar 100 µm. Probes for P-vulgata_1 (b), P-vulgata_3 (c) and P-vulgata_4 (d) localized to a specific band of glands approximately 30–110 µm away from the epithelium. Scale bars 100 µm, 20 µm, 20 µm, respectively. Note that in (b), the weak staining around 150 µm away from the epithelium and in the mucus is unspecific background staining (orange arrowheads) that is distinct from the specific expression sites at approximately 100 µm away from the epithelium (green arrowhead). (e) P-vulgata_7 stained the pedal sole epithelium. Scale bar 20 µm. (f) P-vulgata_11 localized at the side-wall epithelium. Scale bar 50 µm.

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