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

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.


Assembly settings
The full software setting for read alignment using bowtie2, v2.2.5 was as follows: Parameters: -q --phred64 --sensitive --dpad 0 --gbar 99999999 --mp 1,1 --np 1 --score-min L,0,-0.1 -I 1 -X 1000 --no-mixed --nodiscordant -p 1 -k 200. Supplementary Figure 2: Example of a limpet that managed to crawl up and adhere to plastic mesh. Figure 3: SDS-PAGE gels of protein extracts from P. vulgata pedal sole mucus stained for additional information. BPAM: bulk primary adhesive mucus; SAM: secondary adhesive mucus; IPAM: interfacial primary adhesive mucus. (a) Coomassie Blue stain identified at least 11 prominent protein bands, ranging from ~40 to greater than 250 kDa. Note the approximate protein band sizes to the right of the gel image. (b) Smeared purple bands from PAS staining confirms that the presence of glycosylation for specific proteins within IPAM and not in BPAM. The strong smearing at the top of the gel indicates large complexes that failed to properly migrate into the gel. Asialo-fetuin from bovine serum used to as a reference glycoprotein to illustrate smearing pattern. (c) Multi-coloured bands from Stains-All highlight several differences between BPAM and IPAM (blue for highly acidic proteins and Ca 2+ -binding proteins, purple for intact proteoglycans, and pink for weakly acidic proteins).  P-vulgata_14 *: Not applicable (NA), where a manuscript ID was not generated for a given Trinity-assigned protein ID as it was not included in downstream manual annotation and ISH was unsuccessful. Detailed protocol of in situ hybridization (ISH) RNA probe synthesis. Primers were designed with Primer3 (http://primer3.ut.ee, v4.1.0) and for antisense/sense probe production a T7/SP6 promoter region was added at the 5`end of the reverse/forward primers. cDNA was generated from isolated total RNA of the limpet tissue, (same limpet individual as the one sent for transcriptome sequencing) using Transcriptor First Strand cDNA Synthesis Kit (Roche). PCR reactions were performed with Q5® High-Fidelity DNA Polymerase (NEB) and their correct length verified on a 1 % agarose gel. The PCR products were purified with Wizard® SV Gel and PCR Clean-Up System (Promega). To synthesize single stranded digoxigenin-labelled RNA probes, T7/Sp6 polymerase (Promega) and DIG labelling mix (Roche) were used according to manufactures' protocols. DIG-labelled RNA probes were precipitated with isopropanol and bands length and intensity checked on a 1 % agarose gel. RNA probes were diluted in hybmix to an estimated concentration of 5ng/µl (based on gel images) and stored at -80°C until usage. Probes were used at a final concentration of approximately 0.2 ng/µl. We repeatedly observed unspecific background staining using sense probes, which should not be able to bind to any mRNA specifically (supplementary figure 4a). After extensive troubleshooting we realised that the precipitation with isopropanol was not sufficient to eliminate unbound DIG-labelled nucleotides, which were the source of this background staining. Please also see background staining in the in situ hybridization experiments addressing this issue.

List of primers used to synthesise ISH probes
Paraffin section in situ hybridization. For section in situ hybridization, Patella tissue was dissected and fixed in 4% PFA in PBS overnight. After several washes in PBS, the tissue was dehydrated in an ethanol series until 100% ethanol. Tissue pieces were treated with xylene for 8 and 5 min and immediately placed in 60°C preheated liquid paraffin wax. The tissue was moved to fresh preheated paraffin wax every hour for three times and left at 60°C overnight. Afterwards, the samples were moved to room temperature for the paraffin blocks to harden. The paraffin-embedded tissue was then cut into 14 µm thick sections using a Microm HM 340 E microtome. The sections were placed on Superfrost ultra plus® slides (Thermo scientific) and heated in an oven at 60 °C for 1 h and allowed to cool down for 15 min. After dewaxing in xylene and rehydration, sections were postfixed in 4% PFA in PBS for 20 min, washed several times in PBS and treated with 0.2 M HCl for 10 min. Following washes in PBS, sections were treated with Proteinase K (20 µg/ml) for 20 min. Sections were postfixed in 4 % PFA in PBS for 5 min, washed in PBS and placed in 0.1 M triethanolamine (pH 8) containing 0.5 % acetic anhydride for 20 min (exchanging the solution after 10 min). Afterwards sections were washed and gradually dehydrated with ethanol. To dry the sections, Chloroform was dropped on the sections and allowed to evaporate. Slides were placed in a humidity chamber and incubated with hybmix at 55 °C for 30 min. Sections were incubated with RNA probes diluted in hybmix (0.2 ng/µl) overnight at 55 °C. To avoid evaporation of the liquid, sections were covered with fresh parafilm pieces during this step. The following day the sections were incubated in 50 % formamide in 2x SSC at 62 °C (3 x 20 min). After washes with 1x SSC and PBS, sections were blocked in blocking reagent (Roche) for 1 h at 4 °C. Sections were incubated with Anti-digoxigenin-AP Fab fragments (Roche) 1:2000 in blocking reagent overnight at 4 °C. Following several washes, the signal was developed using the NBT/BCIP system (Roche) at 37 °C. Colour reaction was stopped with ethanol and sections washed several times with PBS. Sections were mounted in Mowiol® 4-88 (Roth, Germany), prepared according to the manufacturer's protocol and images were taken with a Zeiss Axioscope A1 microscope.
Background staining in the in situ hybridization experiments. For ISH negative control samples, we used DIG-labelled RNA sense probes that should not bind to any mRNA sequences. However, we repeatedly observed unspecific background staining in the negative controls in glands ~100-150 µm from the epithelium, in the epithelium and secreted mucus (supplementary figure 4a). No staining was observed when no ISH probes were added to the samples (not shown). For interpretation of ISH results in this study, we only considered probes that consistently stained differently to the background in two to three repeated ISH experiments. Using P-vulgata_3 as an example (supplementary figure 4c; modified version of Figure  3b), the positive stainings were distinct in intensity and locality compared to background stainings (supplementary figure 4a). Subsequent troubleshooting by B.L. in Mons, where all the ISH experiments were conducted, identified the probable cause as short DIG-labelled nucleotides that freely bound to unspecific regions of the tissue. When negative control probes were additionally purified through a Micro Bio-Spin® Chromatography Column (Biorad) and re-tested, the unspecific background staining issue was resolved (supplementary figure 4b). Unfortunately, data collection for the project had concluded several months prior to this clarification, and the lead author had already returned to Cambridge, UK, hence ISH could not be repeated again. Nevertheless, by carefully considering the differences between background and signal staining patterns and by upholding a high threshold for inclusion, we are confident that we have reported valid results for the five candidate probes (shown in Figure 3 of the main text).
Supplementary Figure 5: Unspecific background staining due to DIG-labelled nucleotides present in the ISH probes. (a) Unspecific background stains in the negative control samples were diffuse and localised to ~100-150 µm from the epithelium. Scale bar 20 µm. (b) When negative control probes were additionally purified through microspin columns, background stains were eradicated. Scale bar 20 µm. (c) P-vulgata_3 staining, showing the distinct staining pattern between the signal (green arrows) and the background (orange). Based on our prior experiences with ISH, the sharp intense stains indicated by the green arrows are indicative of positive stains. Additionally, the background and positive stains were segregated by location within the tissue. ISH probes that did not fit these criteria were not included in the analysis.
Additional information on PX26-30DV pressure sensor calibration: The pressure sensor was calibrated by connecting a syringe pump to an analogue pressure gauge (WIKA Instruments Ltd, UK), which in turn was connected to the sensor port. The syringe pump was used to increase the pressure (relative to ambient) from 0 to +40 kPa in 10 kPa bar steps, and the same to decrease the pressure from 0 to -40 kPa. Sensor output voltage was then plotted against pressure, and a linear regression (intercept set to 0) was fit to obtain the relationship Pressure (kPa) = 8.6979 * Volts (V), with a Pearson's correlation r 2 = 0.9995 (see below). This linear model was used to analyse the in vivo sub-pedal pressures from P. vulgata.
Additional information for manual vertical pull-off experiments: for the trial shown in Figure 3c, the limpet had been disturbed previously and had clamped down, corresponding to the slight negative pressure (around -1.4 kPa). The pull-off force was applied by gripping the limpet's shell with fingers and pulling upwards starting from ~20s until detachment at ~25s. Because of this method, we cannot state with confidence whether the applied detachment force was constant during the whole duration. For this particular trial, we observed an initial negative peak of around -2 kPa, followed by a second much larger peak of -5.7 kPa, and then the pressure returned to -2 kPa for ~3 s before full detachment. One possible explanation for this second dip is that as the limpet was being pulled, it attempted to clamp down, which would result in a decrease in sub-pedal pressure. Once it relaxed, the pressure returned to around -1.4 kPa, before being fully detached. We wrote in the Discussion that clamping can lead to a sub-pedal pressure reduction, although this still requires adhesion between the contact area and the surface, which we propose is provided by adhesive mucus.