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Research
Cite this article: Kang V, Lengerer B, Wattiez
R, Flammang P. 2020 Molecular insights into
the powerful mucus-based adhesion of limpets
(Patella vulgata L.). Open Biol. 10: 200019.
http://dx.doi.org/10.1098/rsob.200019
Received: 17 January 2020
Accepted: 14 May 2020
Subject Area:
molecular biology/genomics/cellular biology/
bioinformatics/genetics
Keywords:
bio-adhesion, adhesive proteins, glycosylation,
transitory adhesion, Patellogastropoda
Author for correspondence:
Victor Kang
e-mail: kwk22@cam.ac.uk
Electronic supplementary material is available
online at https://doi.org/10.6084/m9.figshare.
c.5004875.v1.
Molecular insights into the powerful
mucus-based adhesion of limpets
(Patella vulgata L.)
Victor Kang1, Birgit Lengerer2,3, Ruddy Wattiez4and Patrick Flammang2
1
Department of Zoology, University of Cambridge, Cambridge, UK
2
Biology of Marine Organisms and Biomimetics Unit, Research Institute for Biosciences,
University of Mons, Mons 7000, Belgium
3
Institute of Zoology, University of Innsbruck, 6020 Innsbruck, Austria
4
Laboratory of Proteomics and Microbiology, Research Institute for Biosciences, University of Mons,
Mons 7000, Belgium
VK, 0000-0003-0959-1364; BL, 0000-0002-5431-916X; PF, 0000-0001-9938-1154
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 lim-
pets’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 flat-
worms 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 oppor-
tunities for future studies on bio-adhesives that form strong attachments and
resist degradation until necessary for locomotion.
1. Introduction
Limpets (the Patellogastropoda) are an ancient and diverse group of marine
gastropods. The earliest fossil records date back to the Middle Ordovician
(approx. 450 million years ago), and extant species can be found on seashores
around the world [1]. The common limpet, Patella vulgata L. (Patellidae), is
widespread in Europe and is found in the upper intertidal zone, a challenging
habitat with strong forces from tidal waves and currents, as well as prolonged
exposure to air and predators [2–4]. Limpets have characteristic conical shells
and attach to the surface using their muscular pedal sole. The limpet’s powerful
attachment is well established, with recorded tenacity values (normal peak
attachment force divided by contact area) typically ranging between 0.1 and
0.2 MPa [4–7], reaching 0.7–1.1 MPa in some reports [6,8]. Such impressive
attachments help them resist strong tidal waves and thwart predatory attacks
[3,9] (figure 1). However, unlike adult mussels and barnacles that rely on
© 2020 The Authors. Published by the Royal Society under the terms of the Creative Commons Attribution
License http://creativecommons.org/licenses/by/4.0/, which permits unrestricted use, provided the original
author and source are credited.
filter-feeding and permanently adhere to surfaces in the inter-
tidal zone, limpets are active grazers of biofilm and detritus
[10]; hence, they can travel considerable distances while feed-
ing (up to 1.5 m [6]). They must therefore alternate between
powerful attachments during stationary periods at low tides
and locomotory adhesion at high tides [11]. We refer to this
sub-type of transitory adhesion as tidal transitory adhesion.
Despite over a century of research, the mechanisms respon-
sible for the patellid limpets’strong attachment remain
unresolved [4]. So far, proposed ideas include: suction
[5,11,12] (lowering of pressure beneath the attachment organ
by muscle contraction), clamping (muscles forcing shells
against the substrate to provide additional friction) [8,13], vis-
cous adhesion (resulting from the flow resistance of viscous
secretions beneath the pedal sole) [4] and glue-like adhesion
(via secretions that form chemical interactions to link the
pedal sole with the surface) [11,14,15]. Following a series of
studies ruling out suction in Patella [4,6,14], suction as a poten-
tial mechanism of attachment was re-introduced in a distantly
related family of limpets, Lottidae [5,12]. The authors found a
relationship between tenacity and ambient pressure, and
also measured pressures directly beneath the pedal sole.
Consequently, Smith proposed that limpets alternate between
actively creating suction for adhesive locomotion at high tide
and glue-like adhesion using adhesive mucus for powerful
long-term attachment at low tide [5,11,12]. Such relationships
have not been investigated in Patella. Furthermore, as Patellidae
separated from Lottidae around 191 million years ago [1], it is
unclear if this dual mechanism of suction and glue-like mucus
is also used by P. vulgata.
If limpets do secrete a specialized pedal mucus for
adhesion, a detailed biochemical characterization could offer
insights into how the molecular components interact to func-
tion as a bio-adhesive. Limpet pedal mucus, independent of
the species, is largely composed of water, around 90–95%
wet weight, with the rest being proteins, carbohydrates
and inorganic material [14–16]. Pedal mucus is highly resistant
to solubilization [14,15], although addition of protein and
sugar-degrading enzymes results in a complete breakdown
of the mucus [14]. Two different types of pedal mucus were
identified from Lottia limatula: a solid plaque (‘adhesive
mucus’) that was inconsistently left attached to the substrate
when stationary limpets were forcibly detached, and more vis-
cous pedal mucus from active limpets (‘non-adhesive mucus’)
[11,15]. The biochemistry of the adhesive and non-adhesive
mucus differed in two ways: first, there was around a twofold
increase in protein and carbohydrate content from non-
adhesive to adhesive pedal mucus [15]. Second, of the nine
protein bands isolated from L. limatula pedal mucus, one
protein (118 kDa) was present only in the adhesive samples,
while a 68 kDa protein was associated with the non-adhesive
mucus. The study concluded that L. limatula can control the
properties of the mucus and transition from a non-adhesive
to an adhesive type by modulating both the level and type of
proteins secreted.
Although studies have examined the physical and bio-
chemical properties of P. vulgata pedal mucus [14,17], none
identified the different types of mucus as seen in L. limatula.
Nine glands have been characterized from P. vulgata pedal
sole, five of which can secrete pedal mucus into the contact
zone (space between the pedal sole and the attachment surface)
[17]. Histochemical tests indicated that proteins and sugars are
stored within these glands, possibly as glycoproteins in some
of them [14]. Although putative locomotory or adhesive func-
tions were assigned to the glands, these designations were not
experimentally validated. Eight proteins ranging from 23 to
195 kDa were extracted from one type of P. vulgata pedal
mucus that is probably similar to ‘non-adhesive’mucus from
L. limatula based on sampling method [14]. This pedal mucus
is a viscoelastic material, exhibiting fluid and solid-like behav-
iour, and is probably a cross-linked gel [14,18]. It is not soluble
in water and requires strong reducing agents or harsh alkaline
conditions for solubilization, and proteolytic or glycosidic
enzymes for full degradation [14].
While these earlier efforts offer initial biochemical descrip-
tions of the limpet pedal mucus, our knowledge of its
molecular components and their function remains limited
compared with our understanding of other marine bio-
adhesive secretions. Advances in sequencing technology and
bioinformatics have allowed researchers to assemble and ana-
lyse transcriptomes and proteomes in order to characterize
the molecules and their interactions that govern bio-adhesive
systems. Consequently, our understanding of marine bio-
adhesives has drastically expanded over the last three decades
(example publications, among many others, include [19–22]).
However, the bulk of our knowledge stems from biological
systems that use either temporary adhesion (e.g. sea stars,
sea urchins, barnacle larvae and flatworms) or permanent
adhesion (e.g. mussels, adult barnacles and macro-algae)
[21]. Tidal transitory adhesion, as seen in limpets, requires
different functionalities; limpets need to attach weakly
during locomotion, but also form strong attachments for long
periods of time. Another crucial difference that makes limpet
adhesion special and worth investigating is that the long-
term attachment is reversible or degradable for locomotion.
At the same time, limpet adhesive mucus needs to withstand
unwanted microbial degradation during stationary periods
and potentially lower the risk of infection. A detailed molecular
characterization of limpet pedal mucus, therefore, can help us
understand how strong resistant biomaterials are synthesized
and function as bio-adhesives.
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.
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
2
In this study, we used a range of appropriate molecular
biology approaches to investigate tidal transitory adhesion in
Patella vulgata, including transcriptomics, proteomics, lectin-
based assays and in situ hybridization. We isolated and ident-
ified 171 candidate adhesive protein sequences from different
types of pedal mucus. We also localized specific sugar residues
to pedal glands and secretions. Fourteen candidate protein
sequences were individually annotated with conserved protein
domains, many of which are also present in published tempor-
ary adhesives from marine invertebrates. To elucidate the role
of active suction in P. vulgata and to complement our molecular
investigation, we performed sub-pedal pressure recordings
while limpets were freely locomoting, under stimulated
predatory attack and during manual detachment via normal
pull-offs. We conclude that P. vulgata limpets secrete pedal
mucus that has similar molecular constituents to temporary
bio-adhesives but with several clear distinctions. Furthermore,
we found no evidence of large reductions in sub-pedal
pressure, suggesting that suction is not the principal
mechanism underlying the limpet’s powerful attachment.
2. Material and methods
2.1. Animal collection and maintenance
Limpets (Patella vulgata) were collected on five occasions
from Sheringham, England. Individuals with shell widths of
around 20–35 mm were removed from exposed rock surfaces
during low tide using the following approach to minimize
damage to the pedal sole: a flat tool (e.g. flathead screw-
driver) was placed at a shallow angle to the horizontal and
slowly chiselled into the shell margin until the limpet was
cleanly dislodged. The pedal soles of all individuals were
visually inspected for damage before being brought to the lab-
oratory. Limpets were kept in a marine aquarium tank lined
with polyvinyl chloride acetate (PVCA) sheets for easy detach-
ments, and a pump was scheduled on a timer to simulate tidal
waves. Fit individuals (based on their resistance to dis-
lodgement and clamping response) were detached without
damaging their pedal soles for all the experiments.
2.2. In vivo pressure measurements
Pressure recordings were conducted in vivo to investigate the
range of pressure differences generated beneath limpet pedal
soles. Healthy individuals were placed on a custom-built
underwater set-up consisting of a horizontal platform made
from clear acrylic with four adjustable walls to constrain the
path of the moving limpet (see electronic supplementary
material, figure S1). A small circular hole (0.5 mm radius)
was laser-cut in the horizontal acrylic platform and connected
to a pressure sensor (PX26-30DV, Omega Engineering, UK; see
electronic supplementary material for details on calibration)
via flexible tubing. For recording pressures beneath the pedal
sole during locomotion, a limpet was first detached from the
main tank, its pedal sole gently cleaned with laboratory
tissue to remove existing mucus, then placed on the platform
so that it had to locomote anteriorly and over the pressure
sensor. In a second set of experiments, a predatory attack
was simulated using a method adapted from [8] to elicit a
clamping response: a stainless steel ball bearing (10 mm
radius; 32.3 g weight) was accelerated down a 6 cm path at
45° incline by gravity to fall on the shell of the limpet. Lastly,
limpets were allowed to locomote over the pressure sensor,
tapped on the shell to elicit clamping, then manually detached
with a vertical pull. The voltage output from the pressure trans-
ducer was recorded using a USB DAQ (NI-6001, National
Instruments, USA) and a MATLAB script (MATLAB version
R2017b, The MathWorks Inc., MA, USA). Analysis of the
recordings was conducted in MATLAB.
2.3. Adhesive mucus sampling for biochemical
characterization
Previous studies used different techniques to sample pedal
mucus: Grenon & Walker detached P. vulgata,wipedthesole
clean and left them upturned for 30 min before collecting the
secreted mucus [17]. The samples were pooled from multiple
individuals for biochemical characterization. Smith et al., on the
other hand, differentiated between adhesive (‘A’) and non-
adhesive (‘NA’) mucus: adhesive mucus was sampled by detach-
ing L. limatula that had settled onto glass walls of an aquarium
tank, whereupon roughly a third of the limpets left behind a
solid ‘glue’that remained firmly adhered on the glass, while
non-adhesive mucus was sampled by placing several individuals
in a plastic bag and making them move around without attaching
firmly for 4–8 h [15]. Mucus samples from multiple L. limatula
were then pooled for biochemical characterization.
For our experiments, we developed a specialized sampling
technique to collect three different types of P. vulgata pedal
mucus while minimizing contamination and damage to the
individual (figure 2). Limpets were allowed to settle onto
thin sheets of PVCA (around 200 µm thick) in an aquarium
tank with circulating artificial saltwater (ASW, made per man-
ufacturer instructions; Instant Ocean, Aquarium Systems, VA,
USA) at the Biology of Marine Organisms and Biomimetics
Unit, University of Mons, Belgium. Immediately before
sampling, the plastic sheets with limpets were removed from
the tank, cleaned of excess debris, and positions of each settled
limpet were marked using a permanent marker. The limpets
were then carefully detached from the thin PVCA sheet by
peeling (figure 2a)—this minimized both damage to the soft
pedal sole and possible contamination from other mucus
types. Since our collection method was similar to that used to
sample barnacle cement (‘primary cement’is secreted naturally
for attachment, while ‘secondary cement’is secreted only
during re-attachment [23]), we have adapted their terminology
to define our three types of pedal mucus. The thin mucus layer
found on the PVCA sheet, similar to the ‘adhesive’mucus from
Smith, was defined as ‘interfacial primary adhesive mucus’
(IPAM; figure 2b), while the mucus layer remaining on the
pedal sole was termed ‘bulk primary adhesive mucus’
(BPAM). IPAM was collected by scraping it off the plastic
sheets using sterilized glass squares with different extraction
buffers (see below for buffer descriptions). BPAM was collected
directly from the pedal soles of upturned limpets by making the
thin mucus film swell with a small volume of filtered ASW
(Instant Ocean, per manufacturer instructions), then removing
the swollen mucus using sterilized forceps. The third mucus
type was isolated using a previously described method [14]
with some modifications: after the BPAM was collected, the
pedal sole was thoroughly cleaned with ASW and laboratory
tissue paper, then the limpet was left upturned in a humid con-
tainer to stimulate fresh mucus production. After at least
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
3
30 min, a small volume of newly secreted mucus, called ‘sec-
ondary adhesive mucus’(SAM), was collected ( figure 2c).
It should be noted that as SAM was collected in air and its com-
position may vary if sampled from individuals left in an
aqueous environment. Samples from individual limpets were
kept separate and kept frozen at −20°C until further analysis.
2.4. Protein extraction and gel electrophoresis with
various staining methods
The three different mucus types (IPAM, BPAM, SAM) were
solubilized and extracted for gel electrophoresis using the pro-
tocol described previously with minor modifications [24]. The
extraction buffer, made with 1.5 M Tris-HCl buffer at pH 7.8, 5
M urea, 2% (weight/volume) SDS, and 0.5 M dithiothreitol
(DTT), was added to the samples and transferred to a glass
pestle tissue homogenizer and manually ground for up to
5 min to thoroughly disrupt the mucus. The homogenized
samples were then incubated at 60°C with agitation for 1 h, fol-
lowed by 5 min of cooling down to room temperature (RT).
Sulfhydryl groups were carbamidomethylated with iodoaceta-
mide used in a 2.5-fold excess (w/w) to DTT in the dark at RT
for 20 min. An equal quantity of β-mercaptoethanol was added
to stop the reaction, and the sample was centrifuged at 13 000
RPM for 15 min at 4°C. The supernatant containing solubilized
proteins was transferred to Eppendorf tubes and stored at
−20°C for future use.
For gel electrophoresis, Laemmli sample buffer (Bio-Rad)
was added to the protein extracts with 5% (v/v) β-mercaptoetha-
nol, heated for 2 min at 90°C, then centrifuged for 5 min at
16 000 g. 10% sodium dodecyl sulfate (SDS)–polyacrylamide
gels were used and subsequently stained with Coomassie Blue
(to visualize proteins), Periodic Acid Schiff (PAS; to visualize
carbohydrates) or Stains-All (colour depends on protein proper-
ties; blue for acidic proteins or Ca
2+
-binding proteins [25], purple
for proteoglycans, pink for less acidic proteins [26]).
2.5. Peptide sequencing using mass spectrometry
Mass spectrometry was performed as previously described [27]
with minor modifications. Proteins from whole extracts of
IPAM, BPAM and SAM from three individuals were precipi-
tated using cold acetone (repeated until DTT scent was not
detectable), then subjected to trypsin digest at 37°C overnight
(1 µg of trypsin per 50 µg of extracted protein; modified porcine
trypsin, sequencing grade from Promega). Tryptic peptides were
analysed by reverse-phase HPLC-ESI-MS/MS using an Eksi-
gent NanoLC 400 2D Ultra Plus HPLC system connected to a
TripleTOF 6000 quadrupole time-of-flight mass spectrometer
(AB Sciex, Concord, ON, Canada). After injection, peptide mix-
tures were transferred to a AB Sciex column (3C18-CL 75 µm ×
15 cm) and eluted at a flow rate of 300 nl min
−1
.MSdatawas
acquired using the TripleTOF 6000 mass spectrometer fitted
with a Nanospray III source (AB Sciex) using a pulled quartz
tip as the emitter (New Objectives, MA, USA).
2.6. RNA isolation and transcriptome generation
Total RNA was isolated from an individual P. vulgata limpet by
the following method: first, the pedal sole was carefully excised
from surrounding tissue on ice and divided into longitudinal
sections. Next, each section was immediately frozen in liquid
nitrogen, added to TRIzol (Life Technologies, Carlsbad, CA),
then homogenized using a hand-held mechanical tissue hom-
ogenizer. After going through the recommended protocol
using TRIzol reagent, quality of the isolated RNA was initially
checked using spectrophotometry, at which point a second
clean-up was conducted using RNeasy Mini-kit (Qiagen, CA,
USA) to digest genomic DNA and to further purify the RNA.
Subsequent sequencing, data processing and transcriptome
assembly were performed at the Beijing Genomic Institute,
China (BGI). Integrity of the isolated RNA was assessed by
gel electrophoresis and via Agilent Bioanalyser prior to
sequencing (RNA integrity number: 6). Illumina HiSeqXTen
platform was used to generate 150 bp paired-end reads, and
the raw reads were filtered to remove adaptors and low-quality
reads (see electronic supplementary material for more details).
Cleaned reads were used for de novo assembly of the transcrip-
tome with Trinity software v2.0.6 [28] and assembled into
Unigenes with Tgicl v2.0.6 [29]. Fragments per kilobase of
transcript per million mapped reads (FPKM) values were
calculated by first mapping clean reads to Unigenes with
interfacial primary
adhesive mucus
(IPAM)
bulk primary
adhesive mucus
(BPAM)
peel
flexible plastic
sheet
secondary
adhesive mucus
(SAM)
wait for >30 min
(b)(a)(c)
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.
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
4
bowtie2 [30] (v2.2.5, sensitive mode; see electronic supplemen-
tary material for full software settings), then RSEM [31]
(v1.2.12, default parameters) was used to quantify expression
levels. To assess transcriptome assembly and annotation
completeness, we conducted an analysis based on the Bench-
marking Universal Single-Copy Orthologs (BUSCO) using
BUSCO v3.0.2 [32] for metazoa_odb9 and eukaryote_odb9
datasets. Based on the metazoan dataset, the assembled tran-
scriptome was estimated to be 91.5% complete with 894
complete BUSCOs, 4.4% (43) fragmented BUSCOs and 4.1%
(41) missing BUSCOs from a total of 978 BUSCO groups
searched. Similar values were obtained with the eukaryota
dataset. Note that these BUSCO numbers are in line with
those from the Lottia gigantea reference genome [32]. Raw
sequencing reads and the assembled transcriptome has been
deposited to an NCBI BioProject database under accession
number PRJNA613775.
2.7. Searching for peptides against the transcriptome
The assembled transcriptome was translated into the six
open reading frames (ORF) on a Galaxy Project server
(https://usegalaxy.org [33]). MS/MS data were searched
for protein candidates against all ORFs using the software
ProteinPilot 5.0 (AB Sciex). Carbamidomethyl cysteine was
set as the fixed modification and trypsin as the digesting
enzyme. For all samples, candidates with false discovery
rates (FDR) above 0.01 were excluded from further analyses.
Only sequences that appeared in all three individuals were
compiled to form a single set of candidate proteins for IPAM,
BPAM and SAM. Additionally, these three lists were combined,
and duplicates removed to create a total candidate protein list
obtained from aligned transcriptome and proteome data.
Homology against known proteins was assessed using NCBI
Basic Local Alignment Search Tool for protein (blastp) against
UniProtKB/SwissProt databases (www.uniprot.org [34]) set
to the default parameters. Conserved protein domains were
searched on InterPro (v75.0) [35]. Clustal Omega (www.ebi.
ac.uk/Tools/msa/clustalo) [36] and Jalview Version 2
(v2.10.4b1) [37] were used for multiple sequence alignments
to check for conserved protein domains. The following
suite of prediction algorithms were used to characterize the
candidate proteins: DeepLoc 1.0 was used for localization pre-
dictions[38]; NetCGlyc 1.0, NetNGlyc and NetOGlyc 4.0 for C-,
N- and O-glycosylation predictions [39–41]; NetPhos 3.1 for
phosphorylation predictions [42]; and SulfoSite for predicting
tyrosine sulfation sites [43].
2.8. Expression analysis of candidates using in situ
hybridization
From the combined list of candidate proteins, a subset was
selected for further analysis using in situ hybridization (ISH)
based on the following criteria: first, we limited our selection
to proteins that were ranked highly by ProteinPilot to ensure
we were targeting proteins that were present in adhesive
mucus. Second, we included candidates with conserved
protein domains that were commonly associated with marine
bio-adhesives (e.g. vWFD, EGF, lectins). Finally, we sought to
sample proteins across the different types of mucus with the
goal of identifying candidates associated with specific types
of mucus (IPAM, BPAM and SAM). ISH probes were generated
based on a modified protocol from [44] and is described in
full in the electronic supplementary material. In summary,
cDNA was generated from isolated total RNA (same limpet
individual as the one sent for transcriptome sequencing)
using Transcriptor First Strand cDNA Synthesis Kit (Roche).
Gene-specific primers were designed with Primer 3 (http://
primer3.ut.ee, v4.1.0) and used to synthesize template DNA
for the production of digoxigenin-labelled (DIG) antisense
RNA probes (see electronic supplementary material for list of
primers used). Limpet tissues were fixed in 4% paraformalde-
hyde (PFA) in PBS, embedded in paraffin wax, then sectioned
into 14 µm sections using a Microm HM 340 E microtome.
Probes were added to tissue sections and developed using
NBT/BCIP system (Roche) at 37°C. Sections were mounted
and imaged with a Zeiss Axio Scope.A1 microscope.
2.9. Lectin staining to identify specific sugar residues
within limpet pedal sole
Lectin staining was used to provide additional insight into the
identity and locality of specific sugar residues within the
limpet adhesive organ (see [45] for additional information on
lectin-based staining method to investigate adhesive organs).
Nine biotinylated lectins (see table 1; Vector Laboratories,
USA) were applied to 5 µm paraffin sections of P. vulgata
pedal sole and visualized using Texas Red conjugated Strepta-
vidin. For the negative control, a section was prepared
alongside the rest, but no lectin stain was added. A few sections
were also stained with alcian blue at pH 2.5 and counterstained
with phloxine to facilitate the interpretation of the lectin stains
by providing an overview of the pedal sole morphology and
glands. Sample images were taken at the following regions to
aid in comparison between different staining patterns: mar-
ginal groove, anterior (immediately posterior to the marginal
groove), middle and posterior end of the foot. All images
were taken with a Zeiss Axio Scope.A1 microscope. Qualitative
assessment of lectin stain intensity was conducted based on
images taken with the same exposure settings. Images for
figures were post-processed in FIJI to enhance clarity [46].
3. Results
3.1. General observations on Patella vulgata attachment
and in vivo pressure recordings
During the course of this study, we recorded four key
observations about limpet attachments that provided novel
insights into their adhesion: first, when stationary limpets
were detached (by peeling from plastic sheets) and immedi-
ately placed onto a smooth flat surface (glass or plastic), their
adhesion was insufficient to hold their own weight when
turned upside-down (i.e. their re-attachment was not immedi-
ate). Once the limpets had time to locomote away from being
returned to a surface (around 1–2 min), they consistently left
behind a gel-like layer on the surface (probably similar to the
BPAM collected for proteomics; see electronic supplementary
material, video S1). Such samples resisted degradation in the
saltwater tank for several weeks. Second, stationary limpets
that were well attached remained so even when a length of
wire was pushed through from one margin of the pedal sole
to the other (electronic supplementary material, video S2).
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5
Third, limpets crawled up the vertical wall of a basket made up
of plastic fishing mesh placed in the aquarium and settled
slightly above the waterline with strong adhesion (electronic
supplementary material, figure S2). Lastly, limpets that died
while firmly adhered to plastic sheets remained well attached,
and the surface could be peeled away from them in a similar
way to living limpets. Each of these observations suggested a
mechanism of attachment that is not reliant on muscle-actuated
suction, which is typically dynamic (fast attachments and
detachments) [47], detaches from a disruption of the rim,
fails to seal on meshes or porous substrates and is often not
functional upon death of the animal.
To further investigate the contribution of reduced pressures
beneath limpet pedal soles, sub-pedal pressures were
measured while limpets attached to aclean smooth acrylic sur-
face. Plots from a representative experiment with three
different conditions (free locomotion, simulated attack and
normal pull-off) are shown in figure 3. When limpets were
freely locomoting, we recorded both positive and negative
pressures (relative to ambient pressure) beneath the pedal
sole, ranging from −0.79 to 1.0 kPa (n= 20 measurements
from 8 limpets). The average (± standard deviation, s.d.) mini-
mum and maximum recorded pressures per limpet were −0.42
± 0.23 kPa and 0.32 ± 0.33 kPa, respectively (n= 8). No discern-
ible difference in pressures was observed when limpets were
left to attach to the surface for 10 min or longer. When limpets
were disturbed with a simulated predatory attack, we saw a
distinct reduction in pressure (−2.3 ± 1.5 kPa, average ± s.d.;
n= 6 limpets). These pressure reductions decayed exponen-
tially over time, taking 3.7 ± 3.0 s (average ± s.d.; n=8
measurements from 5 limpets) to reach 60% of the minimum
pressure. The most negative pressure value (−5.7 kPa) was
recorded when an attached limpet was manually pulled off
perpendicularly from the surface (see electronic supplemen-
tary material for additional information). The average
pressure value for all manual detachments was −1.5 ± 1.9 kPa
(± s.d., n= 4 limpets).
3.2. Overview of the molecular components of Patella
vulgata pedal mucus
As a result of our revised mucus collection method (figure 2),
we successfully isolated three types of limpet pedal mucus
(IPAM, BPAM and SAM). We observed a number of qualitat-
ive differences between the types of mucus: first, IPAM was a
thin layer left on the surface when the limpet was detached
that sometimes felt like a raised solid patch. The thin layer
of IPAM became visible with crystal violet staining. BPAM,
on the other hand, was visible as an opaque swollen layer
on top of the pedal sole and could at times be removed as
an intact sheet of mucus. Lastly, the small quantities of
SAM produced on the pedal sole easily broke apart during
collection and did not form sheets like BPAM.
From the three types of pedal mucus collected from
P. vulgata, we used gel electrophoresis to visualize multiple
protein bands (electronic supplementary material, figure S3).
In total, at least 11 distinct protein bands were identified with
Coomassie Blue staining, with molecular weight estimates
ranging from 40 to 190 kDa, and a few protein bands larger
than 250 kDa. Unlike previous studies where mucus samples
were pooled from many individual limpets, sufficient amounts
of protein were collected to compare secreted proteins
between individuals, although no discernible differences
Table 1. List of lectin-based stains used to investigate sugar residues present in the limpet pedal sole.
lectin acronym target sugars
staining
intensity pedal sole side-wall
Lens culinaris agglutinin LCA α-Man, α-Fuc linked to
N-acetylchitobiose
+++ glands no
Ulex europaeus
agglutinin I
UEA I α-Fuc + epithelium,
glands
no
wheat germ agglutinin WGA GlcNAc (dimers or trimers
preferred), chitobiose
Sialic acid
++ epithelium,
glands (also throughout
the foot)
epithelium
succinylated wheat germ
agglutinin
sWGA GlcNAc (without sialic acid) +++ glands epithelium
Ricinus communis
agglutinin I
RCA I Gal, GalNAc + glands no
Maackia amurensis lectin II MAL II (α-2,3)-sialic acid + glands no
soy bean agglutinin SBA α-orβ-GalNAc, Gal + non-glandular structures
throughout the foot
no
Concanavalin A ConA α-Man +++ n.a.
a
n.a.
Jacalin Jacalin Galβ-(1–>3)-GalNAc −no no
none negative control n.a. −no no
a
n.a.: not applicable due to unspecific staining; —: no staining observed.
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
6
were observed. The most prominent protein bands across all
three adhesive mucus types were 45, 55, 130 and 160 kDa,
and a protein bandoutside the top range of the ladder (referred
to as ‘greater than 250 kDa’). While proteins from the three
mucus types were similar in size, one smeared band around
60 kDa was associated with the IPAM samples (electronic sup-
plementary material, figure S3a). Periodic acid–Schiff (PAS)
staining confirmed the presence of glycosylated proteins of
approximately 60 kDa in size (electronic supplementary
material, figure S3b). PAS staining also revealed large glycosy-
lated protein-based complexes that were not fully disrupted
and failed to migrate into the gel. Stains-all cationic dye pro-
vided further insights into the differences between the types
of mucus (electronic supplementary material, figure S3c). We
observed blue-stained protein bands (indicative of proteins
that are highly acidic, negatively charged, and/or bind Ca
2+
)
at around approximately 110 kDa in both BPAM and IPAM,
although the band stained more strongly in the latter. There
was also a strong blue staining at the top of the gel that over-
lapped with the positive PAS staining, supporting the
presence of large protein–sugar complexes that failed to enter
the gel. Meanwhile, purple-stained bands (proteoglycans) at
approximately 55 kDa and greater than 250 kDa and a bright
pink band (weakly acidic proteins) of approximately 160 kDa
were associated with BPAM only. Once again, there was a
smeared band approximately 60 kDa from all IPAM samples,
and this band stained pink to purple.
Lectin assays provided additional information on the
nature of the sugar residues present in limpet pedal mucus
(figure 4). Six of the nine tested lectins labelled specific
glands and secreted mucus; four specifically targeted glands
near the pedal sole and not the side-walls (table 1). We discov-
ered that Lens culinaris agglutinin (LCA), which recognizes
α-Mannose and/or α-Fucose linked to N-acetylchitobiose
sugar residues, specifically stained glands within the pedal
sole and not the side-wall. LCA revealed an anterior–posterior
gradient of stained glands, where the anterior of the foot
featured the highest density of staining, followed by the pos-
terior end of the foot, while the middle section was stained
least intensely (figure 4e). We observed oval glands within
the pedal sole epithelium, while flask-shaped subepithelial
glands were found in a zone of high glandular density extend-
ing from the epithelium to approximately 150 µm into the body
(dorsally). In contrast to the strong and dense staining from
LCA, the rest of the lectin stains localized to more specific
glands within the foot, the pedal sole or the side-wall. Maackia
amurensis lectin II (MAL II) stains highlighted distinct granular
contents within pedal sole glands, and succinylated wheat
germ agglutinin (sWGA) stained granular gland contents dis-
tributed throughout the foot tissue as well as in pedal sole
glands. Wheat germ agglutinin (WGA), unlike sWGA, did
not stain pedal sole glands. Both WGA and sWGA strongly
stained the epithelium. Notably, sWGA and LCA captured sev-
eral glands in the midst of secreting mucus to the outside. We
observed a dorsoventral gradient of glands with different
sugar residues: while LCA, sWGA and Ulex europaeus aggluti-
nin 1 (UEA I) stained glands both close to the epithelium and
deeper (dorsally) into the tissue, Ricinus communis agglutinin
I (RCA I) and MAL II localized to specific glands further
away from the epithelium. MAL II-stained glands particularly
deep in the limpet foot (approx. 60 to 150 µm away from the
epithelium), with granular contents secreted through long
necks to the outside.
3.3. Identification of putative limpet adhesive proteins
from transcriptomics and proteomics
Ade novo transcriptome was obtained from the Patella vulgata
pedal sole (electronic supplementary material, table S1).
Functional annotation was conducted using seven databases
(NR, NT, GO, KOG, KEGG, SwissProt and InterPro), which
yielded 37 261 annotations overall (see electronic supplemen-
tary material, table S2 for numbers from each database). This
F
t60% = 7 s
F
Ø
0.20
–0.20
–0.40
–0.60
–0.80
–1.00
0 5 10 15 20 25 30
time (s)
0 5 10 15 20 25 30
time (s)
0 5 10 15 20 25 30
time (s)
0
pressure (kPa)
0.20
–0.20
–0.40
–0.60
–0.80
–1.00
–6.0
–5.0
–4.0
–3.0
–2.0
–1.0
0
1.0
0
pressure (kPa)
pressure (kPa)
(b)
(a)
(c)
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.
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
7
transcriptome was used as a reference to map peptide
sequences of IPAM, BPAM and SAM protein extracts from
pedal mucus (n= 3 individuals). With this approach, 171
candidate sequences for limpet adhesive proteins were ident-
ified: 27% of them (46 sequences) were found in all three
mucus types (to classify as being present in a type of
mucus, a candidate sequence had to be found in all three
individuals), while 22% (37 sequences) were present only in
BPAM, 13% (23 sequences) in SAM and 9% (15 sequences)
only in IPAM. Note that, due to our selection criteria
(where a candidate protein had to be present in all three
limpet individuals in order to be attributed to BPAM, SAM
or IPAM), some proteins may not have been assigned to a
particular type of adhesive mucus. Nevertheless, this
at mpt
RCA I (m)
MAL II (at) WGA (m)
LCA (at) sWGA (at) UEA I (at)
AP
D
V
mg
*
at
m
pt
Alcian Blue
LCA
mg
*
at
m
pt
AP
D
V
(k)
(a)
(c)(d)
(b)
(e)
(f)(g)(h)
(i)(j)
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.
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
8
categorization helped to form initial ideas about the potential
functions of each isolated protein.
Fourteen sequences (P-vulgata_1 to 14; see figure 5) were
chosen for a more detailed analysis with manual annotation
of conserved protein domains and in situ hybridization. Six
of the 14 sequences were found in all three pedal mucus
types (P-vulgata_1 to 6); one was found in IPAM and BPAM
only (P-vulgata_7); one from IPAM and SAM (P-vulgata_11);
four from only IPAM (P-vulgata_8 to 10,P-vulgata_12), and
one from each of the SAM and BPAM samples (P-vulgata_13
and P-vulgata_14, respectively).
The following characteristics of the 14 sequences are sum-
marized in table 2: the type of mucus it was isolated from
(IPAM, BPAM, SAM), fragment per kilobase million (FPKM),
A2M TED
1604
830
392
157
219
2500
1854
1860
legend
1764
201
638
1320
669
vWFD
1113
ApeC
vWFD
P-vulgata_3
P-vulgata_4
P-vulgata_7
P-vulgata_9
P-vulgata_12
P-vulgata_2
P-vulgata_1
P-vulgata_6
P-vulgata_10
P-vulgata_11
P-vulgata_8
P-vulgata_5
P-vulgata_13
P-vulgata_14
Reprolysin
vWFD
vWFD
vWFD Gal-bd SF
Gal-bd
SF
C-type
lectin-like
SRCR
+ve charged
aa repeat
Peptidase_
M12A
ShKT
signal
peptide
A2M
CKCT
antistasin
A2M BRD
SUSHI
TIL
Glyco_
hydro_
family_22
missing start/
stop codon
EGF
Mucin_2
LDLrA
CUB
TSP-1
chitin
binding
type II
KSPI
WAP
MG2
Kringle
C8
SPTT
repeat
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.
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
9
Table 2. Summary of putative adhesive proteins and their characteristics. See figure 4 and text for details.
sequence ID
found in
which
mucus
types? FPKM
length
(amino
acids)
Start/
Stop
codon
cysteine
content (%
of length)
signal
peptide
predicted
localization
conserved protein
domains (InterPro)
homologous proteins of interest
(BLASTp)
predicted
glycosylation
predicted
phosphorylation
predicted
sulfation
P-vulgata_1 all 737 1854 N/Y 7.6% Y secreted vWFD x2, C-type lectin-like,
EGF x6, cystine-knot C-
terminal, KSPI x3
fibrillin, zonadhesin, alpha-tectorin,
IgGFc-binding protein
N, C, O numerous 1
P-vulgata_2 all 28 2500 Y/Y 7.8% Y cell membrane vWFD, EGF x11, KSPI x2,
TSP1 x1
fibrillin, zonadhesin, alpha-tectorin,
IgGFc-binding protein, adhesive
protein 1 [Minona ileanae]
N, C, O numerous 5
P-vulgata_3 all 2
a
1604 N/Y 8.6% N cell membrane C8 domain x2, vWFD x3, TIL
x2, LDL receptor class A
binding repeats x5
SCO-spondin, hemolectin/hemocytin,
mucin
N, O numerous 0
P-vulgata_4 all 17 830 Y/Y 5.7% N secreted vWFD zonadhesin, alpha-tectorin, IgGFc-binding
protein, Sfp-1 [Asterias rubens]
O numerous 0
P-vulgata_5 all 334 1320 Y/N 0.6% Y secreted M2-like, A2M BRD, A2M,
A2M TED
CD109-antigen-like, SIPC [Megabalanus
coccopoma]
N, O numerous 0
P-vulgata_6 all 1147 1860 Y/Y 5.9% N cell membrane TSP1 x4, cystine-knot C-
terminal
SCO-spondin, hemicentin, adhesion
protein 2 [Macrostomum lignano]
N, C, O numerous 2
P-vulgata_7 IPAM &
BPAM
21 122 392 Y/Y 5.9% Y secreted chitin binding type II BSMP14 N, O numerous 0
P-vulgata_8 IPAM 180 638 Y/N 3.9% Y secreted reprolysin ADAM family mig-17 N, O numerous 0
P-vulgata_9 IPAM 2607 157 Y/Y 5.7% Y secreted Glyco_hydro family 22 C-type lysozyme N, O numerous 0
P-vulgata_10 IPAM 9 1764 N/Y 5.2% N cell membrane SRCR x8, vWFD, galactose-
binding domain
superfamily
deleted in malignant brain tumours-1,
scavenger receptor cysteine-rich
protein type 12 precursor
N, C, O numerous 4
P-vulgata_11 IPAM &
SAM
4 201 N/Y 6.0% Y secreted Mucin x2 oikosin-like, mucin, cartilage intermediate
layer-like
O numerous 1
P-vulgata_12 IPAM 399 219 Y/Y 15.1% Y secreted WAP, antistasin-like n.a. N, O numerous 0
P-vulgata_13 SAM 162 669 N 9.7% N secreted galactose-binding domain
superfamily, EGF x5
n.a. N, O numerous 4
P-vulgata_14 BPAM 65 1113 N/Y 4.9% N secreted peptidase_M12A, ApeC, CUB,
Kringle, SUSHI x2, C-type
lectin-like, ShKT
n.a. N, C, O numerous 1
a
The sum of the FPKMs of two nearly identical contigs for P-vulgata_3 are shown.
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10
sequence length in amino acids, presence of start and
stop codons, cysteine content, signal peptide, predicted subcel-
lular localization, homologous proteins of interest based on
NCBI blastp, conserved protein domains based on InterPro,
predicted glycosylation (N-, C- or O-linked), predicted phos-
phorylation, and predicted sulfation. The cysteine content
ranged from 3.9% to 15.1%, with the exception of one protein
that had a low content of 0.6% (P-vulgata_5).The most
common protein domains included von Willebrand factor
type D (vWFD), epidermal growth factors (EGF), Kazal-type
serine protease inhibitors (KSPI) and scavenger receptor
cysteine-rich (SRCR). All proteins were predicted to have
post-translational modifications (PTMs), with at least one
type of glycosylation and numerous phosphorylation sites.
While half of the proteins were predicted to have sulfated tyro-
sine, this PTM was not detected in P. vulgata (neither on foot
sections nor on mucus trails) using an anti-sulfotyrosine
antibody [48].
BLAST analysis of the limpet proteins highlighted hom-
ology to a number of characterized proteins. Most notably,
three proteins (P-vulgata_1,2and 4) had the same group
of homologous proteins: fibrillin, zonadhesin, alpha-tectorin
and fragment crystallizable region of immunoglobulin G
(IgGFc)-binding protein. These proteins can participate in
ligand-binding, adhesion, oligomerization or fibril formation:
for example, zonadhesin is a multi-domain protein believed
to facilitate the binding of sperm to the egg zona pellucida
[49], while fibrillin is a large Ca
2+
-dependent glycoprotein
that forms microfibrils in the extracellular matrix [50]. More-
over, both P-vulgata_2 and 4had sequence similarities to
known adhesive proteins: adhesive protein 1 from Minona
ileanae (QEP99777.1) [51] and sea star footprint protein 1
(Sfp-1) from Asterias rubens (AHN92641.1) [24], respectively.
It is worth mentioning that P-vulgata_4 featured two positively
charged repeats at the C-terminus (amino acid sequence
RRSRRNRNKARRSRRNRN) that did not align with any of
the homologous proteins.
Two proteins—P-vulgata_3 and 6—were similar to SCO-
spondin, a large secreted glycoprotein from the thrombospon-
din family involved in neural development with binding sites
for sugars, proteins and lipids [52,53]. P-vulgata_3 in particular
was highly homologous to SCO-spondin, with homology to
NCBI reference sequences for SCO-spondin from multiple
unrelated species (e.g. 84% QC and 30.70% ID to SCO-spondin
from Gallus gallus, NP_001006351.2; see electronic supplemen-
tary material, figure S4a). Interestingly, P-vulgata_3 was highly
similar to a specific portion of SCO-spondin (figure 6). The
implications of this homology are discussed in the following
section. While P-vulgata_3 featured nearly all of the conserved
domains of SCO-spondin (vWFD, TIL, C8, LDLrA), P-vul-
gata_6 had just the repeating TSP-1-like domains in common.
As a result of these repeating domains, however, P-vulgata_6
aligned with a small portion of adhesion protein 2 from
Macrostomum lignano (QAX24810.1) [54].
P-vulgata_5 was homologous to the settlement-inducing
protein complex (SIPC) from barnacles (Megabalanus cocco-
poma; 91% QC, 27.70% ID; BAM28692.1), both of which
contain alpha-2 macroglobulin domains. This protein’s
homology to SIPC is discussed in the subsequent section.
P-vulgata_8 featured one type of domain, reprolysin,
which is a metallopeptidase (also called adamalysin M12B
peptidase). Clustal alignment with reprolysin consensus
sequence cd04267 from NCBI Conserved Domain Database
[55] highlighted similarities between the proteins but, impor-
tantly, the conserved catalytic HEXXH motif was absent in in
P-vulgata_8 (electronic supplementary material, figure S4b).
In addition, unlike other adamalysin peptidases, P-vulgata_8
featured nine tandem Ser-Pro-Thr-Thr repeats starting from
residue position 598. This was probably incomplete as the
stop codon was missing and the sequence terminated after
the first Serine of the next repeat (i.e. S/PTT). Enrichment
of Ser, Pro and Thr residues began upstream of the SPTT
repeat region (amino acid 550–638) and accounted for 73 of
the last 89 residues (82%) of the sequence.
P-vulgata_9 contained a predicted enzymatic domain, gly-
coside hydrolase family 22 (Glyco_hydro_family_22). This
sequence was similar to a C-type lysozyme from Haliotis
discus hannai, a species of abalone (92% QC, 48.99% ID;
ADR70995.1), as well as with an NCBI reference sequence for
lysozyme C from Canis lupus familiaris (88% QC, 38.03% ID;
NP_001300804.1). The conserved catalytic Glu residue found
in lysozymes was present in P-vulgata_9.
Due to the tandem SRCR repeats, P-vulgata_10 aligned
with an NCBI reference sequence for the deleted in malignant
brain tumours-1 protein isoform c precursor from Homo
sapiens (48% QC, 46.37% ID; NP_060049.2). Other notable
homologues included SRCR-rich proteins from sea stars
(Asterias rubens, QAA95957.1), sea urchins (Strongylocentrotus
purpuratus, NP_999762.1) and sea anemones (Exaiptasia
pallida, Aipgene2358 [56]).
No reliable homologues outside of hypothetical proteins
were identified for P-vulgata_12,13 and 14. However, it is
worth noting that the ShKT domain prediction in P-vulgata_14
was unexpected as it was first described as a potent ion channel
toxin in sea anemones [57]. Multi-sequence Clustal alignment of
P-vulgata_14 with four roundworm mucin proteins homolo-
gous to ShkT (Tc-MUC-1 to 4) [58] confirmed the presence of
the conserved cysteine residues (electronic supplementary
material, figure S4c). However, the catalytic dyad Lys-25/Tyr-
26 found in ShKT was absent in P_vulgata-14 and Tc-MUC-1
to 4.
3.4. Expression of putative limpet adhesive proteins
From the list of 171 candidate sequences isolated from the
three mucus types, 16 were selected for in situ hybridization
to determine their expression site within the limpet. These
16 sequences were selected based on their relative ranking,
SCO-spondin [Gallus gallus]5255
vWFD
vWFC
vWFD FA58
P-vulgata_3 1604
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.
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
11
domain similarity to published adhesive proteins and pres-
ence in a specific mucus type (electronic supplementary
material, table S3). Specific expression patterns were obtained
for P-vulgata_1,P-vulgata_3,P-vulgata_4,P-vulgata_7 and
P-vulgata_11 (figure 7). As some of our samples had unspeci-
fic background staining, we only analysed those that
produced distinct expression patterns (see figure 7a, which
shows the distinct specific and unspecific regions). A more
detailed account of the background staining is provided in
electronic supplementary material, figure S5. From the five
samples with distinct expression sites, P-vulgata_1,P-vul-
gata_3 and P-vulgata_4 stained a band of glands between
approximately 30 µm and approximately 110 µm away from
the epidermis, while P-vulgata_7 localized to glands within
the epidermis. These expression patterns confirm that the cor-
responding proteins were produced and secreted from glands
specific to the pedal sole. P-vulgata_11 specifically stained
glands in the side-wall, which might indicate a contamination
of the samples with side wall secretions.
4. Discussion
4.1. The role of sub-pedal pressure differences in limpet
attachments
Numerous studies have sought to understand the principles
behind the limpet’s powerful attachment by ascribing it to suc-
tion [5,11,12], clamping [13] or glue-like secretions [11,14,15].
Smith reported a significant reduction in sub-pedal pressures
when limpets (either Tectura scutum or Lottia gigantea) were
placed on an acrylic surface and slid across the pressure
gauge (approx. −20 kPa, relative to ambient), and an even
larger reduction when irritated to illicit a clamping response
(close to −50 kPa) [5]. Our sub-pedal pressure differential
measurements using Patella vulgata revealed much smaller
values, with the average minimum pressure during free
locomotion of −0.42 ± 0.23 kPa. Even with a simulated pre-
datory attack inducing a clamping response, our values
(−2.3 ± 1.5 kPa) were smaller than those reported by Smith.
Our locomotion pressures are in good agreement with pre-
vious sub-pedal pressure measurements from locomoting
P. vulgata (reported as 6 cm of water, which is around
0.6 kPa) [59]. When a predatory attack was simulated, the
limpet clamped its shell against the surface, resulting in a
rapid decrease in sub-pedal pressure, followed by a less nega-
tive pressure as the limpet relaxed slightly but did not quickly
return to ambient (taking around 3.7 s to reach 60% of the peak
minimum value). This response is similar to the clamping be-
haviour of Cellana tramoserica reported by Ellem et al., where
initial irritation (single tap to the limpet shell or the experimen-
tal set-up) caused a clamping force of 2–5 N that decayed in
1–2 s to pre-stressed levels, while continued irritation (continu-
ous tapping) resulted in a much higher force of 25 N that
decayed slowly over 5 min [13]. While we measured the
sub-pedal pressures and not the forces, we observed a similar
sustained decay in response to a simulated predatory attack.
Hence, when our sub-pedal pressure findings are considered
in combination with our observations on limpet attachment
(i.e. adhering strongly with a disrupted seal or climbing on a
mesh but not being able to re-attach quickly), it is likely that
P. vulgata does not actively generate suction for attachment.
Rather, it is likely that, at least in this limpet species, the most
critical element for adhesion is creating a strong yet reversible
connection between the pedal sole and the surface that is inde-
pendent of sub-pedal pressures. Consequently, when the
(a)(c)
(d)
(b)
(e)(f)
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.
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
12
limpet clamps down, the sole is able to sustain the reaction
force even when a seal is absent. If a continuous seal is present
or if influx is sufficiently low, then clamping will result in a
pressure reduction that may further contribute to the overall
attachment; however, ourpressure recordings and behavioural
observations indicate that low sub-pedal pressures are not
required for attachments in P. vulgata.
4.2. Generating adhesion through chemical interactions:
how limpet pedal mucus may function as a bio-
adhesive
Several studies have previously investigated the chemical con-
stituents of limpet pedal mucus using histochemistry and gel
electrophoresis [14,15,17,48]. Our results from lectin-binding
assays and transcriptome-assisted proteomics have revealed
novel insights into the biochemical properties of P. vulgata
pedal mucus.
Lectin staining of P. vulgata revealed a highly complex
glandular system within the foot. We found LCA to be a
good candidate for a comprehensive labelling of pedal sole
glands. This suggests that LCA’s target sugar residues
(α-Man and α-Fuc linked to N-acetylchitobiose) are specific
to the pedal sole mucus and not to secretions from other
parts of the body. Furthermore, we observed a dorsoventral
gradient of lectin staining, where the longest subepithelial
glands (whose cell bodiesare located approx. 60—150 µm dor-
sally from the pedal sole epithelium) are labelled with RCA I
and MAL II (N-acetylgalactosamine or sialic acid, respectively).
The density of these glands was also lower than that of the
shorter ones expressing other residues, such as mannose,
fucose, chitobiose, sialic acid or N-acetylglucosamine, which
were found closer to the epithelium. At the other end of the
spectrum, some glands were present within the epithelium,
containing fucose, N-acetylglucosamine, chitobiose or sialic
acid residues. A dorsoventral gradient may be important for
the timing of secretions: glands close to the surface would
require less time to secrete than those deep inside the foot,
where the secretion needs to be pushed out through long
thin necks. Spatial distribution of glands may also be important
for the digital mucus glands of tree frogs, where clusters of ven-
tral and dorsal glands within the attachment organ, the toe
pad, have distinct morphologies and locations that may be
related to their functions [60]. The tubular ventral glands,
which supplies mucus to the surface of the adhesive toe pad,
are situated deep within the organ and have long thin necks
to the ventral surface, similar to the glands found in limpets.
An alternative explanation for the dorsoventral gradient may
be related to the volume of the glands: one way to increase
the gland volume is to lengthen it with a long neck. Indeed,
tubular glands are significantly larger than dorsal glands in
tree frogs, and this is also likely to be the case for limpets.
Our findings, which highlight the usefulness of lectins in clar-
ifying limpet gland morphology and chemistry, also
emphasize the need for future work on understanding the
function of these glands and associated sugar chemistries.
From our transcriptome-assisted proteomics study of
limpet pedal mucus, 171 sequences were identified from the
limpet pedal mucus, of which 14 were selected and manually
characterized. To facilitate discussion about their putative
functions, these proteins have been assigned to three broad
functional categories based on their domain composition:
(i) proteins likely to be involved in oligomerization, ligand
binding (proteins, sugars, metals) and peptide stabilization
(disulfide bridges); (ii) enzymes or inhibitors; and (iii) proteins
with elements of both. It is worth mentioning that the tran-
scriptome was based on the sequencing data from the pedal
sole of a single limpet specimen. Since adhesive proteins are
often large and repetitive [24,54], they tend to be inadequately
assembled with short-read transcriptomics [51,61]. We sought
to increase mapped transcript lengths by reducing the com-
plexity of the input RNA and minimizing transcript variation
caused by pooling samples from multiple individuals.
Although our analysis showed that the transcriptome is of
good quality, we want to highlight that due to the limited
sample size, some transcripts and transcript variations may
not be represented in this dataset.
Proteins P-vulgata_4,6,7,10,11 and 13 fall into the first cat-
egory, sharing among them domains associated with multi-
protein complex formation (vWFD, EGF, SRCR and TSP-1)
and protein–carbohydrate binding (C-type lectin-like, galac-
tose binding-like domain superfamily and chitin-binding
type II). The ability to bind to other proteins and carbohydrates
(either free-existing or attached to glycosylated proteins) is an
important requirement for the cohesion of gastropod mucus
and underwater adhesives, which are often cross-linked and
are difficult to dissolve without potent denaturing agents
[14,62–66]. Furthermore, these proteins had elevated cysteine
residue contents of 5–9.7%, much higher than the average for
eukaryotes (1–2%), although this may decrease when the full
length of some of the proteins are sequenced. Nevertheless,
as all limpet mucus samples were challenging to dissolve
even in harsh extraction buffers and high heat, disulfide
bridges (either intra- or intermolecular bonds) are probably
present in limpet adhesive mucus. Besides cohesion, ligand
interactions are also important for adhesion to surfaces, and
the carbohydrate-binding domains may promote interactions
with surface-adsorbed or biofilm-based sugars [67].
Proteins P-vulgata_5,8,9,12 and 14 belong to the second
category, with each sequence having at least one enzymatic
(sugar-cleaving glycoside hydrolase and metallopeptidase)
or inhibitory domain (WAP, antistasin, macroglobulin and
ShkT). P-vulgata_8, while sharing some conserved residues
with other members of the adamalysin metallopeptidases,
lacks the canonical catalytic HEXXH motif. It is unclear, there-
fore, if P-vulgata_8 has any enzymatic function. This protein
warrants further investigation, however, as it was found only
in IPAM. Similarly, P-vulgata_9 was also found in IPAM and
is a secreted protein with a glycoside hydrolase domain
that cleaves sugar bonds within carbohydrates or linked to a
glycoprotein. Unlike P-vulgata_8, this protein did include the
catalytic residue, and may be involved in active degradation
of pedal mucus to transition from stationary to locomotive
states. Alternatively, since P-vulgata_9 is homologous to lyso-
zymes, it may have a defensive function, similar to cp-16 k, a
lysozyme-like protein found in barnacle cement [68]. Follow-
up studies on the activity of P-vulgata_8 and 9are necessary
to understand their respective roles in limpet IPAM.
P-vulgata_12 and 14, on the other hand, are more likely to
serve a defensive role. P-vulgata_12 contains a WAP domain,
which is often found in proteins with antiproteinase and
antimicrobial activities [69,70], as well as an antistasin-like
domain, a serine protease inhibitor (InterPro accession
number IPR004094). Both domains contain multiple intramole-
cular disulfide bridges, which may afford stability in harsh
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
13
physical conditions [71]. P-vulgata_12 therefore may be a
highly stable protease inhibitor present in IPAM to protect
the protein- and sugar-rich pedal mucus against foreign degra-
dation. P-vulgata_14, on the other hand, is notable as it contains
numerous domains for recognizing, binding and degrading
sugars and peptides. One of its domains, Peptidase_M12A, is
a zinc-dependent metallopeptidase, while Kringle, CUB and
SUSHI domains are involved in recognition processes and reg-
ulating proteolytic functions. Apextrin-like (ApeC) and C-type
lectin-like domains are both involved in innate immune
responses of invertebrates by binding to bacterial peptido-
glycans [72–74], although it should be noted that C-type
lectin-like domains can interact with other types of ligands,
such as proteins, lipids and inorganic compounds [74]. One
unexpected domain prediction is the ShKT, which is a potent
potassium ion channel inhibitor originally characterized from
the sea anemone Stichodactyla helianthus. Loukas et al. found
homologous regions (referred by its alternative name, six-
cysteine repeat, SXC) from four proteins (Tc-MUC-1 to 4)
encoding for secreted mucins in the parasitic nematode Toxo-
cara canis with no toxin-like function [58]. Our analysis of
P-vulgata_14, Tc-MUC-1 to 4, and ShKT confirmed sequence
homology to the ShKT domain; however, like Tc-MUC-1 to 4,
P-vulgata_14 lacks the functional dyad found in the ShKT
toxins and is unlikely to function as a potent ion channel inhibi-
tor. Instead, the domain could be involved in forming stable
disulfide bridges that occurs in the native structure of the
ShKT. The abundant target recognition and regulation-related
domains suggest that P-vulgata_14 acts as an antibacterial
agent within the pedal mucus. Interestingly, since this protein
was found only in the BPAM samples, possible roles of P-vul-
gata_14 are to: (i) prevent microbial degradation of the secreted
mucus, which has to remain functional over prolonged periods
of time (e.g. during high tide, when the limpet typically stops
foraging and remains stationary within the safety of its home
scar), and (ii) to minimize risk of infection. However, many
aspects of this protein need to be further investigated to
verify its purported function, such as its target specificity,
stability and how it interacts with the gel network.
P-vulgata_1,2and 3belong to the third category, with
numerous domains for diverse ligand-binding in combination
with protease inhibitor domains. These proteins represent
some of the longest, most well-represented and complex
sequences from the annotated set. Each protein contains at
least four different domains and can potentially interact with
proteins (through Ca
2+
-binding EGF, TSP-1, vWFD), glycans
(C-type lectin-like domain) or lipids (through lipoproteins
binding to LDLrA). These domains suggest that these proteins
can bind to diverse ligands that are soluble and/or adsorbed
to the surface, as well as undergoing homo- or hetero-
oligomerization (for example, vWFD, lectins and cystine
knot, C-terminal domains can self-oligomerize [74–76]). Such
functional domains could promote the formation of large net-
works with protein–protein and protein–glycan cross-links,
which is essential for cohesive strength and is a mechanism
to interact directly with surfaces or surface-adsorbed molecules
[67]. This hypothesis is supported by the type of homologous
proteins identified through blastp analysis: P-vulgata_1,2,
as well as 4, shares homology with fibrillin, zonadhesin,
alpha-tectorin and IgGFc-binding protein. Incidentally, the
similarities between these proteins have been reported pre-
viously in a study on the evolution of gel-forming mucin
proteins [77]. These proteins form microfibrils (fibrillin;
[50,78]), bind to glycoproteins (zonadhesin [79]), recruit col-
lagen fibrils to microvillar membrane surfaces (tectorin [80])
and form gels (IgGFc-binding protein [81]).
Similarly, P-vulgata_3 is highly homologous with the
consensus sequence for SCO-spondin, albeit only to roughly
one-third of the total length. SCO-spondin is a large secreted gly-
coprotein present in the central nervous system and can be found
either in a soluble state or aggregated into Reissner’s fibre [53,76].
The homologous segment contains vWFD, C8, TIL and LDLrA
domains, while the remainder features numerous copies of
TSP-1, SCO-spondin region repeats (SCOR), TIL and a CTCK
[76]. Interestingly, the TSP-1-rich segment from SCO-spondin
that is absent in P-vulgata_3 is specifically implicated in promot-
ing neuronal development [82,83], which appears to be a
superfluous function for adhesive proteins. Furthermore, in situ
hybridization localized P-vulgata_3 expression to the pedal sole,
ruling out contamination as a possible source of SCO-spondin-
like peptides. Hence, P-vulgata_3 may represent a re-purposing
of a highly conserved protein involved in neuronal development
to an adhesive one through the loss of the TSP-1 and SCOR
repeats. Alternatively, the TSP-1 motifs in SCO-spondin
sequences have been increasingly duplicated through evolution,
as evident in the lengthening observed in SCO-spondin-like pro-
teins from Echinodermata to Vertebrata [84]. Ancestral SCO-
spondin therefore may have served a conserved role in adhesion,
then gradually lengthened to facilitate an increasingly important
function in neuronal development. Indeed, the domains that
were conserved, including vWFD, C8, TIL and LDLrA, are
adhesive-like by themselves and can participate in protein–
protein interactions; for example, both C8 and TIL domains can
form interdimer disulfide bridges with vWFD [85].
Unlike the other annotated limpet adhesive proteins,
P-vulgata_3 is unique in having five highly conserved LDLrA
repeats that can bind to multiple targets other than lipopro-
teins, including glycoproteins like TSP-1 and reelin [53].
Lipids are believed to be an important component of barnacle
larvae and mussel permanent adhesives [86,87]. In barnacle
larvae, lipidaceous granules (probably in the form of lipopro-
teins or lipopolysaccharides) are secreted as a primer to
potentially displace water from the contact surface and to pro-
vide a protective and stabilizing environment for the
subsequent proteinaceous secretion [86]. Similarly, limpet
pedal mucus probably contains lipidic moieties (V. Kang
2020, unpublished data), which suggests an intriguing parallel
between permanent and tidal transitory adhesives that has yet
to be verified in temporary adhesive systems (but see [54,88]).
However, more work is needed to understand the role of
P-vulgata_3, including its relative abundance within the
different types of adhesive mucus. Although its transcript
expression level was low (table 2), it is difficult to draw con-
clusions about protein abundance based solely on the
transcript expression levels, especially when the FPKM is
derived from a single individual. Follow-up studies using tech-
niques like exponentially modified protein abundance index
(emPAI) can provide quantitative information on the abun-
dance of P-vulgata_3 and other adhesive proteins.
4.3. Comparing adhesive proteins from marine
invertebrates
Limpets exhibit tidal transitory adhesion, where they transition
from high-strength, semi-sessile attachment to locomotory
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
14
attachment. While it is currently not feasible to tease apart all
the nuances of what makes a protein suitable for temporary,
transitory or permanent adhesion, our results offer some
useful initial insights: (i) both transitory and temporary
adhesive proteins in general appear to be large multidomain
sequences, often with duplicated domains thought to facilitate
protein–ligand interactions; (ii) both transitory and temporary
bio-adhesives contain numerous glycosylated proteins;
(iii) both transitory and temporary adhesives probably do not
contain 3,4-dihydroxyphenyl-L-alanine (Dopa) (V. Kang
2020, unpublished data), which is more often associated with
permanent adhesives from mussels, sandcastle worms and
adult ascidians [89,90].
While none of the annotated limpet adhesive proteins
share homology with known permanent adhesive proteins,
P-vulgata_5 aligned well (QC 91%, ID 27.70%) with a glyco-
protein secreted by barnacle larvae for temporary adhesion,
called the barnacle settlement-inducing protein complex
(SIPC; NCBI Accession BAM28692.1). SIPC can adsorb to
surfaces and is part of larval footprints [91,92]. This further
supports the proposed adhesive function of P-vulgata_5 and
is another example of the similarity between limpet adhesi-
ves proteins and temporary adhesives. Indeed, proteins
P-vulgata_2,4and 6share homology with known temporary
adhesive proteins from flatworms (QEP99777.1 [51] and
QAX24810.1 [54]) and sea stars (AHN92641.1 [24]). All these
proteins also share similarities with the aforementioned
group of proteins (fibrillin, zonadhesin, alpha-tectorin,
IgGFc-binding protein). Moreover, a study examining the role
of sulfated biopolymers in marine bio-adhesives also high-
lighted the similarities between transitory and temporary
adhesive secretions, where such moieties may serve a cohesive
function in the adhesive material of limpets and sea stars but
not in tubeworms or sea cucumbers [48]. Moreover, a review
of adhesive secretions from marine invertebrates demonstrated
that, based on comparisons between the amino acid compo-
sitions of whole adhesives, there are similarities between
secretions from all species using non-permanent adhesion
(i.e. temporary and transitory) [93].
Another animal that uses a similar type of transitory
adhesion is the sea anemone (Actiniaria). Sea anemones com-
monly occupy a similar ecological niche as limpets, and they
can alternate between stationary and locomotive states [94].
One species, Epiactis prolifera, is capable of an average daily
movement of 0.18 pedal disc diameter [95]. While the exact
mechanism of pedal movement remains unclear, it seems
likely that a combination of retrograde pedal waves and
punctuated step-like movements is involved [95]. A recent
transcriptomic study of the glass anemone Exaiptasia pallida
has identified numerous upregulated genes in the pedal
disc that may be important in bio-adhesion [56]. Enriched
domains include protease inhibitors and metallopeptidases,
analogous to the limpet adhesive proteins that may serve a
role in defence or to transition the mucus from adhesive to
locomotive, and protein–ligand binding domains, similar to
both the limpet and temporary adhesives. Interestingly, the
most abundantly expressed sequence in the pedal disc, Aip-
gene2358, shared homology with P-vulgata_10, mainly from
the tandem repeats of scavenger receptor cysteine-rich
(SRCR) domains. Further studies are needed to ascertain
the functional role of SRCR repeats in adhesive secretions,
since SRCR participates in a wide range of activities, includ-
ing ligand binding to lipoproteins and selected polyanions
[96,97], immune responses in marine invertebrates [73], and
are also present in spider silk glands [98]. It should be
noted, however, that although both limpets and sea ane-
mones may participate in a similar type of adhesion and
share related protein domains, there are no striking analogies
that clearly differentiate transitory adhesive proteins from
temporary adhesive ones.
5. Conclusion
The common limpet, Patella vulgata, has intrigued researchers
for over a century with their impressive attachment strength.
While previous studies have proposed both suction and
glue-like attachment as mechanisms underlying limpet
adhesion, we found only slight pressure differences gener-
ated beneath the pedal sole of P. vulgata during both
undisturbed locomotion and simulated predatory attacks.
Based on the pressure recordings and behavioural obser-
vations, we conclude that limpet pedal mucus is a bio-
adhesive that provides a strong bond to the attachment
surface. Our detailed analysis of the limpet pedal mucus
has revealed novel insights into the molecular components
of limpet bio-adhesive: (i) lectin staining assays confirmed
the presence of several glycans specific to pedal sole glands
and highlighted secretory granules; (ii) transcriptome-
guided proteomics identified 171 adhesive protein candidates
present in three types of limpet pedal mucus; (iii) in situ
hybridization localized the expression of a selection of these
proteins, four of which were present only at the pedal sole.
Our annotation of pedal mucus protein sequences identified
numerous domains often found in known temporary
adhesives, along with multiple predicted sites for glycosyla-
tion. Furthermore, these proteins are capable of protein–
ligand interactions and are likely to oligomerize and cross-
link to form a strong bio-adhesive. We also identified two
protein architectures that have not been previously described
in marine adhesive secretions: first is an SCO-spondin-like
protein P-vulgata_3, which can potentially form fibres and
raises interesting questions about the re-purposing of a
highly conserved protein during evolution; second is a poten-
tially potent defensive protein P-vulgata_14, with multiple
domains for recognition and degradation of proteins and
glycans. Although we have yet to identify key molecular
differences between temporary and tidal transitory
adhesives, our study is the first in-depth molecular character-
ization of a model organism for tidal transitory adhesion and
provides a solid foundation for future work.
Data accessibility. Encoding cDNA sequences of the 14 annotated pro-
teins (P-vulgata_1 to 14) have been included in the electronic
supplementary material. Raw sequencing reads and the assembled
transcriptome has been deposited to the NCBI BioProject database
under accession number PRJNA613775.
Authors’contributions. V.K., B.L. and P.F. conceived the study. V.K. col-
lected the data for all experiments and drafted the manuscript. B.L.
helped perform in situ hybridization, lectin assays and histochemical
stainings. P.F. helped coordinate and provided guidance throughout
the study. R.W. collected the proteomics data and helped interpret
the results. All authors revised and approved the final manuscript.
Competing interests. The authors declare that they have no competing
interests.
Funding. V.K. was funded by the European Union’s Horizon 2020
research and innovation programme under the Marie Skłodowska-
Curie grant agreement no. 642861. B.L. is funded by a Schrödinger
Fellowship of the Austrian Science Fund (FWF): [J-4071]. P.F. is
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
15
Research Director of the Fund for Scientific Research of Belgium
(FRS-FNRS). Work was partially supported by a Malacological
Society of London Research Grant (awarded to V.K.), FNRS CDR
Grant no J.0013.18, by the ‘Communauté française de Belgique—
Actions de Recherche Concertées’[ARC-17/21 UMONS 3], and by
the European Cooperation in Science and Technology (COST)
Action CA15216 (STSM no. 38594 and 41591). B.L., V.K. and P.F.
are members of the COST Action ‘European Network of Bioadhesion
Expertise’(CA15216).
Acknowledgements. V.K. would like to thank Dr Walter Federle for his
helpful feedback throughout the study. V.K. is grateful to Marie
Bonneel and Morgane Algrain for their help with some of the mol-
ecular biology experiments, and to Jérôme Delroisse for his help
with transcriptomic analyses. We thank Thomas Ostermann and
Peter Ladurner (University of Innsbruck) for access to their BLAST
server and the UMONS Bioprofiling platform, Cyril-Terence Mascolo
and Corentin Decroo for their advice on mass spectrometry
experiments.
References
1. Nakano T, Ozawa T. 2007 Worldwide
phylogeography of limpets of the order
Patellogastropoda: molecular, morphological and
palaeontological evidence. J. Molluscan Stud. 73,
79–99. (doi:10.1093/mollus/eym001)
2. Denny MW. 1988 Biology and the mechanics of the
wave-swept environment. Princeton, NJ: Princeton
University Press.
3. Hahn T, Denny M. 1989 Tenacity-mediated selective
predation by oystercatchers on intertidal limpets
and its role in maintaining habitat partitioning by
‘Collisella’scabra and Lottia digitalis.Mar. Ecol.
Prog. Ser. 53,1–10. (doi:10.3354/meps053001)
4. Grenon JF, Walker G. 1981 The tenacity of the
limpet, Patella vulgata L.: an experimental
approach. J. Exp. Mar. Biol. Ecol. 54, 277–308.
(doi:10.1016/0022-0981(81)90162-3)
5. Smith AM. 1991 The role of suction in the adhesion
of limpets. J. Exp. Biol. 161, 151–169.
6. Branch GM, Marsh AC. 1978 Tenacity and shell
shape in six Patella species: adaptive features.
J. Exp. Mar. Biol. Ecol. 34, 111–130. (doi:10.1016/
0022-0981(78)90035-7)
7. Denny MW, Daniel TL, Koehl MAR. 1985
Mechanical limits to size in wave-swept
organisms. Ecol. Monogr. 55,69–102. (doi:10.2307/
1942526)
8. Coleman RA, Browne MA, Theobalds T. 2004
Aggregation as a defense: limpet tenacity changes
in response to simulated predator attack. Ecology
85, 1153–1159. (doi:10.1890/03-0253)
9. Denny MW. 2000 Limits to optimization: fluid
dynamics, adhesive strength and the evolution of
shape in limpet shells. J. Exp. Biol. 203,
2603–2622.
10. Burgos-Rubio V, De la Rosa J, Altamirano M,
Espinosa F. 2015 The role of patellid limpets as
omnivorous grazers: a new insight into intertidal
ecology. Mar. Biol. 162, 2093–2106. (doi:10.1007/
s00227-015-2739-0)
11. Smith AM. 1992 Alternation between attachment
mechanisms by limpets in the field. J. Exp. Mar.
Biol. Ecol. 160, 205–220. (doi:10.1016/0022-
0981(92)90238-6)
12. Smith AM, Kier WM, Johnsen S. 1993 The effect of
depth on the attachment force of limpets. Biol. Bull.
184, 338–341. (doi:10.2307/1542452)
13. Ellem GK, Furst JE, Zimmerman KD. 2002 Shell
clamping behaviour in the limpet Cellana
tramoserica.J. Exp. Biol. 205, 539–547.
14. Grenon J-F, Walker G. 1980 Biochemical and
rheological properties of the pedal mucus of the
limpet, Patella vulgata L. Comp. Biochem. Physiol.
66, 451–458.
15. Smith AM, Quick TJ, St. Peter RL. 1999 Differences
in the composition of adhesive and non-adhesive
mucus from the limpet Lottia limatula.Biol. Bull.
196,34–44. (doi:10.2307/1543164)
16. Davies MS, Jones HD, Hawkins SJ. 1990 Seasonal
variation in the composition of pedal mucus from
Patella vulgata L. J. Exp. Mar. Biol. Ecol. 144,
101–112. (doi:10.1016/0022-0981(90)90022-5)
17. Grenon JF, Walker G. 1978 The histology and
histochemistry of the pedal glandular system of two
limpets, Patella vulgata and Acmaea tessulata
(Gastropoda: Prosobranchia). J. Mar. Biol. Assoc. U.K.
58, 803–816. (doi:10.1017/S0025315400056770)
18. Smith AM. 2016 The biochemistry and mechanics of
gastropod adhesive gels. In Biological adhesives (ed.
AM Smith), pp. 177–192. Cham, Switzerland:
Springer.
19. Waite JH, Tanzer ML. 1981 Polyphenolic substance
of Mytilus edulis: novel adhesive containing L-Dopa
and hydroxyproline. Science 212, 1038–1040.
(doi:10.1126/science.212.4498.1038)
20. Smith AM (ed.). 2016 Biological adhesives, 2nd edn.
Cham, Switzerland: Springer.
21. Hennebert E, Maldonado B, Ladurner P, Flammang
P, Santos R. 2015 Experimental strategies for the
identification and characterization of adhesive
proteins in animals: a review. Interface Focus 5,
20140064. (doi:10.1098/rsfs.2014.0064)
22. Lengerer B, Ladurner P. 2018 Properties of
temporary adhesion systems of marine and
freshwater organisms. J. Exp. Biol. 221, jeb182717.
(doi:10.1242/jeb.182717)
23. Liang C, Strickland J, Ye Z, Wu W, Hu B, Rittschof D.
2019 Biochemistry of barnacle adhesion: an updated
review. Front. Mar. Sci. 6,1–20. (doi:10.3389/fmars.
2019.00565)
24. Hennebert E, Wattiez R, Demeuldre M, Ladurner P,
Hwang DS, Waite JH, Flammang P. 2014 Sea star
tenacity mediated by a protein that fragments, then
aggregates. Proc. Natl Acad. Sci. USA 111,
6317–6322. (doi:10.1073/pnas.1400089111)
25. Campbell KP, Maclennantll DH, Jorgensen A. 1983
Staining of the Ca
2+
-binding proteins, calsequestrin,
calmodulin, troponin C, and S-100, with the cationic
carbocyanine dye ‘Stains-all’.J. Biol. Chem. 258,
11 267–11 273.
26. Goldberg HA, Warner KJ. 1997 The staining of
acidic proteins on polyacrylamide gels:
enhanced sensitivity and stability of ‘Stains-all’
staining in combination with silver nitrate.
Anal. Biochem. 251, 227–233. (doi:10.1006/abio.
1997.2252)
27. Hennebert E, Leroy B, Wattiez R, Ladurner P.
2015 An integrated transcriptomic and proteomic
analysis of sea star epidermal secretions identifies
proteins involved in defense and adhesion.
J. Proteomics 128,83–91. (doi:10.1016/j.jprot.2015.
07.002)
28. Grabherr MG et al. 2011 Full-length transcriptome
assembly from RNA-Seq data without a reference
genome. Nat. Biotechnol. 29, 644–652. (doi:10.
1038/nbt.1883)
29. Pertea G et al. 2003 TIGR gene indices clustering
tools (TGICL): a software system for fast clustering
of large EST datasets. Bioinformatics 19, 651–652.
(doi:10.1093/bioinformatics/btg034)
30. Langmead B, Salzberg SL. 2012 Fast gapped-read
alignment with Bowtie 2. Nat. Methods 9,
357–359. (doi:10.1038/nmeth.1923)
31. Li B, Dewey CN. 2011 RSEM: accurate transcript
quantification from RNA-seq data with or without a
reference genome. BMC Bioinformatics 12, 323.
(doi:10.1186/1471-2105-12-323)
32. Simão FA, Waterhouse RM, Ioannidis P, Kriventseva
EV, Zdobnov EM. 2015 BUSCO: assessing genome
assembly and annotation completeness with single-
copy orthologs. Bioinformatics 31, 3210–3212.
(doi:10.1093/bioinformatics/btv351)
33. Afgan E et al. 2018 The Galaxy platform for
accessible, reproducible and collaborative biomedical
analyses: 2018 update. Nucleic Acids Res. 46,
W537–W544. (doi:10.1093/nar/gky379)
34. The UniProt Consortium. 2017 UniProt: the universal
protein knowledgebase. Nucleic Acids Res. 45,
D158–D169. (doi:10.1093/nar/gkw1099)
35. Mitchell AL et al. 2019 InterPro in 2019: Improving
coverage, classification and access to protein
sequence annotations. Nucleic Acids Res. 47,
D351–D360. (doi:10.1093/nar/gky1100)
36. Goujon M, McWilliam H, Li W, Valentin F,
Squizzato S, Paern J, Lopez R. 2010 A new
bioinformatics analysis tools framework at EMBL–
EBI. Nucleic Acids Res. 38, W695–W699. (doi:10.
1093/nar/gkq313)
37. Waterhouse AM, Procter JB, Martin DMA, Clamp M,
Barton GJ. 2009 Jalview Version 2—a multiple
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
16
sequence alignment editor and analysis workbench.
Bioinformatics 25, 1189–1191. (doi:10.1093/
bioinformatics/btp033)
38. Almagro Armenteros JJ, Sønderby CK, Sønderby SK,
Nielsen H, Hancock J. 2017 DeepLoc: prediction of
protein subcellular localization using deep learning.
Bioinformatics 33, 3387–3395. (doi:10.1093/
bioinformatics/btx431)
39. Julenius K. 2007 NetCGlyc 1.0: prediction of
mammalian C-mannosylation sites. Glycobiology 17,
868–876. (doi:10.1093/glycob/cwm050)
40. Gupta R, Brunak S. 2002 Prediction of glycosylation
across the human proteome and the correlation to
protein function. In Pac Symp Biocomput, Lihue,
Hawaii, pp. 310–322. Singapore: World Scientific
Publishing.
41. Steentoft C et al. 2013 Precision mapping of the
human O-GalNAc glycoproteome through SimpleCell
technology. EMBO J. 32, 1478–1488. (doi:10.1038/
emboj.2013.79)
42. Blom N, Gammeltoft S, Brunak S. 1999 Sequence
and structure-based prediction of eukaryotic protein
phosphorylation sites. J. Mol. Biol. 294, 1351–1362.
(doi:10.1006/jmbi.1999.3310)
43. Chang W-C, Lee T-Y, Shien D-M, Hsu JB-K, Horng
J-T, Hsu P-C, Wang T-Y, Huang H-D, Pan R-L. 2009
Incorporating support vector machine for identifying
protein tyrosine sulfation sites. J. Comput. Chem. 30,
2526–2537. (doi:10.1002/jcc.21258)
44. Lengerer B, Algrain M, Lefevre M, Delroisse J,
Hennebert E, Flammang P. 2019 Interspecies
comparison of sea star adhesive proteins. Phil.
Trans. R. Soc. B 374, 20190195. (doi:10.1098/rstb.
2019.0195)
45. Lengerer B, Bonneel M, Lefevre M, Hennebert E,
Leclère P, Gosselin E, Ladurner P, Flammang P. 2018
The structural and chemical basis of temporary
adhesion in the sea star Asterina gibbosa.Beilstein
J. Nanotechnol. 9, 2071–2086. (doi:10.3762/bjnano.
9.196)
46. Schindelin J et al. 2012 Fiji: an open-source
platform for biological-image analysis. Nat. Methods
9, 676–682. (doi:10.1038/nmeth.2019)
47. Federle W, Labonte D. 2019 Dynamic biological
adhesion: mechanisms for controlling attachment
during locomotion. Phil. Trans. R. Soc. B 374,
20190199. (doi:10.1098/rstb.2019.0199)
48. Hennebert E, Gregorowicz E, Flammang P. 2018
Involvement of sulfated biopolymers in adhesive
secretions produced by marine invertebrates. Biol.
Open 7, bio.037358. (doi:10.1242/bio.037358)
49. Lea IA, Sivashanmugam P, Rand MGO. 2001
Zonadhesin: characterization, localization, and zona
pellucida binding. Biol. Reprod. 65, 1691–1700.
(doi:10.1095/biolreprod65.6.1691)
50. Handford PA. 2000 Fibrillin-1, a calcium binding
protein of extracellular matrix. Biochim. Biophys.
Acta Mol. Cell Res. 1498,84–90. (doi:10.1016/
S0167-4889(00)00085-9)
51. Pjeta R et al. 2019 Temporary adhesion of the
proseriate flatworm Minona ileanae.Phil.
Trans. R. Soc. B 374, 20190194. (doi:10.1098/rstb.
2019.0194)
52. Gobron S, Monnerie H, Meiniel R, Creveaux I,
Lehmann W, Lamalle D, Dastugue B, Meiniel A.
1996 SCO-spondin: a new member of the
thrombospondin family secreted by the
subcommissural organ is a candidate in the
modulation of neuronal aggregation. J. Cell Sci. 109,
1053–1061.
53. Vera A, Recabal A, Saldivia N, Stanic K, Torrejón M,
Montecinos H, Caprile T. 2015 Interaction between
SCO-spondin and low density lipoproteins from
embryonic cerebrospinal fluid modulates their roles
in early neurogenesis. Front. Neuroanat. 9,1–12.
(doi:10.3389/fnana.2015.00072)
54. Wunderer J et al. 2019 A mechanism for temporary
bioadhesion. Proc. Natl Acad. Sci. USA 116,
201814230. (doi:10.1073/pnas.1814230116)
55. Marchler-Bauer A et al. 2017 CDD/SPARCLE:
functional classification of proteins via subfamily
domain architectures. Nucleic Acids Res. 45,
D200–D203. (doi:10.1093/nar/gkw1129)
56. Davey PA, Rodrigues M, Clarke JL, Aldred N. 2019
Transcriptional characterisation of the Exaiptasia
pallida pedal disc. BMC Genomics 20,1–15. (doi:10.
1186/s12864-019-5917-5)
57. Castañeda O, Sotolongo V, Amor AM, Stöcklin R,
Anderson AJ, Harvey AL, Engström Å, Wernstedt C,
Karlsson E. 1995 Characterization of a
potassium channel toxin from the Caribbean
sea anemone Stichodactyla helianthus.
Toxicon 33, 603–613. (doi:10.1016/0041-
0101(95)00013-C)
58. Loukas A, Hintz M, Linder D, Mullin NP, Parkinson J,
Tetteh KKA, Maizels RM. 2000 A family of
secreted mucins from the parasitic nematode
Toxocara canis bears diverse mucin domains but
shares similar flanking six-cysteine repeat motifs.
J. Biol. Chem. 275, 39 600–39 607. (doi:10.1074/
jbc.M005632200)
59. Jones HD, Trueman ER. 1970 Locomotion of the
limpet, Patella vulgata L. J. Exp. Biol. 52, 201–216.
60. Langowski JKA et al. 2019 Comparative and
functional analysis of the digital mucus glands and
secretions of tree frogs. Front. Zool. 16, 19. (doi:10.
1186/s12983-019-0315-z)
61. Lengerer B et al. 2018 Organ specific gene
expression in the regenerating tail of Macrostomum
lignano.Dev. Biol. 433, 448–460. (doi:10.1016/j.
ydbio.2017.07.021)
62. Smith AM. 2006 The biochemistry and mechanics of
gastropod adhesive gels. In Biological adhesives (eds
AM Smith, JA Callow), pp. 167–182. Berlin,
Germany: Springer.
63. Wilks AM, Rabice SR, Garbacz HS, Harro CC, Smith
AM. 2015 Double-network gels and the toughness
of terrestrial slug glue. J. Exp. Biol. 218,
3128–3137. (doi:10.1242/jeb.128991)
64. Hennebert E, Wattiez R, Waite JH, Flammang P.
2012 Characterization of the protein fraction of the
temporary adhesive secreted by the tube feet of the
sea star Asterias rubens.Biofouling 28, 289–303.
(doi:10.1080/08927014.2012.672645)
65. Kamino K, Inoue K, Maruyama T, Takamatsu N,
Harayama S, Shizuri Y. 2000 Barnacle cement
proteins: importance of disulfide bonds in their
insolubility. J. Biol. Chem. 275, 27 360–27 365.
66. Lachnit M, Buhmann MT, Klemm J, Kröger N,
Poulsen N. 2019 Identification of proteins in the
adhesive trails of the diatom Amphora
coffeaeformis.Phil. Trans. R. Soc. B 374, 20190196.
(doi:10.1098/rstb.2019.0196)
67. Smith AM, Papaleo C, Reid CW, Bliss JM. 2017
RNA-Seq reveals a central role for lectin,
C1q and von Willebrand factor A domains in
the defensive glue of a terrestrial slug. Biofouling
33, 741–754. (doi:10.1080/08927014.2017.
1361413)
68. Kamino K. 2016 Barnacle underwater attachment. In
Biological adhesives (ed. AM Smith), pp. 153–176.
Cham, Switzerland: Springer International
Publishing.
69. Amparyup P, Donpudsa S, Tassanakajon A. 2008
Shrimp single WAP domain (SWD)-containing
protein exhibits proteinase inhibitory and
antimicrobial activities. Dev. Comp. Immunol. 32,
1497–1509. (doi:10.1016/j.dci.2008.06.005)
70. Smith VJ, Fernandes JMO, Kemp GD, Hauton C.
2008 Crustins: enigmatic WAP domain-
containing antibacterial proteins from crustaceans.
Dev. Comp. Immunol. 32, 758–772. (doi:10.1016/j.
dci.2007.12.002)
71. Mason TA, McIlroy PJ, Shain DH. 2004 A cysteine-
rich protein in the Theromyzon (Annelida:
Hirudinea) cocoon membrane. FEBS Lett. 561,
167–172. (doi:10.1016/S0014-5793(04)00167-X)
72. Huang G et al. 2014 Two apextrin-like proteins
mediate extracellular and intracellular bacterial
recognition in amphioxus. Proc. Natl Acad. Sci. USA
111, 13 469–13 474. (doi:10.1073/pnas.
1405414111)
73. Gerdol M, Venier P. 2015 An updated molecular
basis for mussel immunity. Fish Shellfish Immunol.
46,17–38. (doi:10.1016/j.fsi.2015.02.013)
74. Zelensky AN, Gready JE. 2005 The C-type lectin-like
domain superfamily. FEBS J. 272, 6179–6217.
(doi:10.1111/j.1742-4658.2005.05031.x)
75. Mayadas TN, Wagner DD. 1992 Vicinal cysteines in
the prosequence play a role in von Willebrand factor
multimer assembly. Proc. Natl Acad. Sci. USA 89,
3531–3535. (doi:10.1073/pnas.89.8.3531)
76. Meiniel O, Meiniel A. 2007 The complex
multidomain organization of SCO-spondin protein is
highly conserved in mammals. Brain Res. Rev. 53,
321–327. (doi:10.1016/j.brainresrev.2006.09.007)
77. Lang T, Hansson GC, Samuelsson T. 2007 Gel-
forming mucins appeared early in metazoan
evolution. Proc. Natl Acad. Sci. USA 104, 16 209–
16 214. (doi:10.1073/pnas.0705984104)
78. Sherratt MJ, Baldock C, Louise Haston J, Holmes DF,
Jones CJP, Adrian Shuttleworth C, Wess TJ, Kielty
CM. 2003 Fibrillin microfibrils are stiff reinforcing
fibres in compliant tissues. J. Mol. Biol. 332,
183–193. (doi:10.1016/S0022-2836(03)00829-5)
79. Hellberg ME, Dennis AB, Arbour-Reily P, Aagaard JE,
Swanson WJ. 2012 The tegula tango: a
coevolutionary dance of interacting, positively
selected sperm and egg proteins. Evolution (N.Y.)
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
17
66, 1681–1694. (doi:10.1111/j.1558-5646.2011.
01530.x)
80. Kim D, Kim JA, Park J, Niazi A, Almishaal A, Park S.
2019 The release of surface-anchored alpha-tectorin,
an apical extracellular matrix protein, mediates
tectorial membrane organization. Sci. Adv. 5,1–10.
81. Harada N, Iijima S, Kobayashi K, Yoshida T, Brown
WR, Hibi T, Oshima A, Morikawa M. 1997 Human
IgGFc binding protein (FcγBP) in colonic epithelial
cells exhibits mucin-like structure. J. Biol. Chem.
272, 15 232–15 241. (doi:10.1074/jbc.272.24.
15232)
82. Gobron S, Creveaux I, Meiniel R, Didier R, Herbet A,
Bamdad M, El Bitar F, Dastugue B, Meiniel A. 2000
Subcommissural organ/Reissner’s fiber complex:
characterization of SCO-spondin, a glycoprotein with
potent activity on neurite outgrowth. Glia 32,
177–191. (doi:10.1002/1098-1136(200011)32:2<
177::AID-GLIA70>3.0.CO;2-V)
83. Sakka L, Delétage N, Lalloué F, Duval A, Chazal J,
Lemaire JJ, Meiniel A, Monnerie H, Gobron S. 2014
SCO-spondin derived peptide NX210 induces
neuroprotection in vitro and promotes fiber
regrowth and functional recovery after spinal cord
injury. PLoS ONE 9, e93179. (doi:10.1371/journal.
pone.0093179)
84. Meiniel O, Meiniel R, Lalloué F, Didier R, Jauberteau
M-O, Meiniel A, Petit D. 2008 The lengthening of a
giant protein: when, how, and why? J. Mol. Evol.
66,1–10. (doi:10.1007/s00239-007-9055-3)
85. Zhou YF, Eng ET, Zhu J, Lu C, Walz T, Springer TA.
2012 Sequence and structure relationships within
von Willebrand factor. Blood 120, 449–458. (doi:10.
1182/blood-2012-01-405134)
86. Gohad NV, Aldred N, Hartshorn CM, Jong Lee Y,
Cicerone MT, Orihuela B, Clare AS, Rittschof D, Mount
AS. 2014 Synergistic roles for lipids and proteins in the
permanent adhesive of barnacle larvae. Nat. Commun.
5,1–9. (doi:10.1038/ncomms5414)
87. He Y, Sun C, Jiang F, Yang B, Li J, Zhong C, Zheng L,
Ding H. 2018 Lipids as integral components in
mussel adhesion. Soft Matter 14, 7145–7154.
(doi:10.1039/C8SM00509E)
88. Flammang P, Michel A, Cauwenberge A, Alexandre
H, Jangoux M. 1998 A study of the temporary
adhesion of the podia in the sea star Asterias
rubens (Echinodermata, Asteroidea) through their
footprints. J. Exp. Biol. 201, 2383–2395.
89. Stewart RJ, Ransom TC, Hlady V. 2011 Natural
underwater adhesives. J. Polym. Sci. B Polym. Phys.
49, 757–771. (doi:10.1002/polb.22256)
90. Li S, Huang X, Chen Y, Li X, Zhan A. 2019
Identification and characterization of proteins
involved in stolon adhesion in the highly invasive
fouling ascidian Ciona robusta.Biochem. Biophys.
Res. Commun. 510,91–96. (doi:10.1016/j.bbrc.
2019.01.053)
91. Petrone L. 2013 Molecular surface chemistry in
marine bioadhesion. Adv. Colloid Interface Sci.
195–196,1–18. (doi:10.1016/j.cis.2013.03.006)
92. Dreanno C, Matsumura K, Dohmae N, Takio K,
Hirota H, Kirby RR, Clare AS. 2006 An
α
2
-macroglobulin-like protein is the cue to
gregarious settlement of the barnacle Balanus
amphitrite.Proc. Natl Acad. Sci. USA 103,14
396–14 401. (doi:10.1073/pnas.0602763103)
93. Flammang P. 2006 Adhesive secretions in
echinoderms: an overview. In Biological adhesives
(eds AM Smith, JA Callow), pp. 183–206. Berlin,
Germany: Springer.
94. Ottaway JR. 1979 Population ecology of the
intertidal anemone Actinia tenebrosa Farquhar
(Cnidaria: Anthozoa). Thesis, University of
Canterbury, New Zealand.
95. Cowles D. 1977 Locomotion by Epiactis prolifera
(Coelenterata: Actiniaria). Mar. Biol. 39,67–70.
(doi:10.1007/BF00395595)
96. Bowdish DME, Gordon S. 2009 Conserved domains
of the class A scavenger receptors: evolution and
function. Immunol. Rev. 227,19–31. (doi:10.1111/j.
1600-065X.2008.00728.x)
97. Yap NVL, Whelan FJ, Bowdish DME, Golding GB.
2015 The evolution of the scavenger receptor
cysteine-rich domain of the class A scavenger
receptors. Front. Immunol. 6,1–9.
98. Chaw RC, Correa-Garhwal SM, Clarke TH, Ayoub NA,
Hayashi CY. 2015 Proteomic evidence for
components of spider silk synthesis from black
widow silk glands and fibers. J. Proteome. Res. 14,
4223–4231. (doi:10.1021/acs.jproteome.5b00353)
royalsocietypublishing.org/journal/rsob Open Biol. 10: 200019
18
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