Frontiers - Biochemistry of Barnacle Adhesion: An Updated Review - Marine Science

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Front. Mar. Sci., 10 September 2019 | https://doi.org/10.3389/fmars.2019.00565
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Biochemistry of Barnacle
11,250
Adhesion: An Updated Review TOTAL VIEWS
(https://www.altmetric.com/de
Chao Liang (https://www.frontiersin.org/people/u/726994)1, domain=www.frontiersin.org&citation
$ View Article Impact (http://loop-imp
Jack Strickland2, Zonghuang Ye1, Wenjian Wu1, Biru Hu1* and
Dan Rittschof (https://www.frontiersin.org/people/u/287396)3
1
Department of Biology and Chemistry, College of Liberal Arts and Sciences, National
University of Defense Technology, Changsha, China
2
Department of Physics, North Carolina State University, Raleigh, NC, United States
3
Duke University Marine Laboratory, Beaufort, NC, United States
Barnacles are notorious marine fouling organisms, whose life cycle (https://spotlight.frontiersin.org/subm
utm_source=fweb&utm_medium=!o
initiates with the planktonic larva, followed by the free-swimming
Suggest a
cyprid that voluntarily explores, and searches for an appropriate site Research Topic >
to settle and metamorphoses into a sessile adult. Within this life (https://spotlight.frontiersin.org/subm
utm_source=fweb&utm_medium=!o
cycle, both the cyprid and the adult barnacle deposit multi-protein
adhesives for temporary or permanent underwater adhesion. Here,
we present a comprehensive review of the biochemistries behind SHARE ON
these different adhesion events in the life cycle of a barnacle. First, we

introduce the multiple adhesion events and their corresponding 0 0 0 New
adhesives from two complementary aspects: the in vivo synthesis,

storage, and secretion as well as the in vitro morphology and
Open Supplemental Data
biochemistry. The amino acid compositions, sequences, and
structures of adult barnacle adhesive proteins are specifically PEOPLE ALSO
highlighted. Second, we discuss the molecular mechanisms of adult LOOKED AT
barnacle underwater attachment in detail by analyzing the possible Injectable

Hydrogel Based on
adhesive and cohesive roles of different adhesive proteins, and based
Modified Gelatin
on these analyses, we propose an update to the original barnacle and Sodium
Alginate for Soft-
underwater adhesion molecular model. We believe that this review
Tissue Adhesive
can greatly promote the general understanding of the molecular (/articles/10.3389/f
chem.2021.744099
mechanisms underlying the reversible and irreversible underwater
/full)
adhesion of barnacles and their larvae. Such an understanding is the Yuhang Xing
(https://loop.frontiersin.o
basis for the prevention of barnacle fouling on target surfaces as well rg/people/1413960/overv
iew), Xueqin Qing, Hao
as designing conceptually new barnacle-inspired artificial underwater Xia, Shiqi Hao, Haofang
Zhu, Yiyan He, Hongli
adhesives. Mao
rg/people/1356760/overv
Introduction iew) and Zhongwei Gu
Many marine creatures inhabiting the wave-swept seashores, such as mussels Recent Advances in
(Waite, 2017), tubeworms (Stewart et al., 2011b), and barnacles (Kamino, Nanostructured
Biomimetic Dry
2006), have evolved the capability of synthesizing, secreting, and curing Adhesives
biological adhesives for temporary or permanent underwater adhesion. These (/articles/10.3389/f
bioe.2013.00022/f
organisms are fascinating, primarily because their adhesives are able to stick
ull)
a wide range of materials together quickly and firmly in water, whereas, Andras Pattantyus-
Abraham, Je!rey Krahn
almost all currently available chemical adhesives fail to do so. Conversely, (https://loop.frontiersin.o
rg/people/114537/overvie
being major fouling organisms, these hard-shell marine animals are
w) and Carlo Menon
frustrating as their adhesion on manmade architectures often causes a severe (https://loop.frontiersin.o
marine biofouling issue, which refers to the accumulation of marine w)
microbes, plants, and animals on target structures leading to millions of

The Rhythm of
dollars spent annually on antifouling (Callow and Callow, 2002; Schultz et Many: Biological
al., 2011). Therefore, understanding their underwater adhesion mechanisms Rhythms in the
Marine
is beneficial for both the design of biomimetic adhesives that can function in
Environment, from
aqueous environments (Lee et al., 2011; Zhong et al., 2014; Zhao et al., 2016) Macro-scale
and the development of new antifouling technologies (Del Grosso et al., 2016; Planktonic
Ecosystems to
Amini et al., 2017). Micro-scale
Holobionts
Owing to their wide distribution in the world’s oceans, and robust, gregarious
(/articles/10.3389/f
underwater attachment, barnacles are among the most dominant marine mars.2021.744169/
fouling organisms. They rely on a multi-protein holdfast with unique full)
Olivia Hannah Hewitt
chemistry and morphology for surface colonization (Kamino, 2010b). (https://loop.frontiersin.o
rg/people/1400087/over
Consequently, barnacles have served as model species for both marine view) and Hisham
Mohammed Shaikh
antifouling tests (Holm, 2012) and developing novel bio-inspired wet (https://loop.frontiersin.o
adhesives (Kamino, 2008). Barnacles belong to the phylum Arthropoda, rg/people/1269895/overv
iew)
differing greatly from intensively studied mussels (Mollusca), and tubeworms
(Annelida). They can be simply categorized into parasitic and non-parasitic Proteomics
insights: proteins
groups; the latter includes the two most studied clades in underwater
related to larval
adhesion research, acorn and stalked barnacles, i.e., orders Sessilia and attachment and
Pedunculata, respectively (Perez-losada et al., 2009; Power et al., 2010). metamorphosis of
marine
The life cycle of barnacles consists of four stages: nauplius, cyprid, juvenile, invertebrates
(/articles/10.3389/f
and adult (Figure 1). After six phases (I–VI), the planktonic nauplius mars.2014.00052/f
transforms into a pre-settling cyprid, which then actively explores external ull)
Kondethimmanahalli H.
substrates in search of suitable sites and settles down to metamorphose and Chandramouli
develop into benthic juvenile and adult. Thereafter, the adult barnacle is (https://loop.frontiersin.o
permanently anchored to the substrate, even after its death (Anderson, 1994; w), Pei-Yuan Qian
Khandeparker and Anil, 2007; Aldred and Clare, 2008). Within the entire life rg/people/88941/overvie
w) and Timothy Ravasi
cycle, three different adhesion events, either reversible or irreversible, are (https://loop.frontiersin.o
involved (Figure 1). First, the cyprid releases a temporary adhesive rg/people/187222/overvi
ew)
(footprint) for reversible adhesion during surface exploration. Second, the
cyprid produces a permanent adhesive (cyprid cement) for colonization on a DNA
Metabarcoding
suitable site. Third, the adult barnacle secretes barnacle cement to maintain
Methods for the
firm attachment. Consequently, the underwater adhesion studies of barnacles Study of Marine
usually differentiate into independent studies focusing on one of the three Benthic Meiofauna:
A Review
adhesion events and the corresponding adhesive. (/articles/10.3389/f
mars.2021.730063/
full)
Romy Gielings
FIGURE 1
iew), Maria Fais
(https://www.frontiersin.org/files/Articles/468324/fmars-06- rg/people/320136/overvi
ew), Diego Fontaneto
00565-HTML/image_m/fmars-06-00565-g001.jpg)
Figure 1. Schematic of the life cycle of a barnacle. The life cycle rg/people/1417487/overvi
of a barnacle consists of four stages: nauplius (I–VI), cyprid, ew), Simon Creer
juvenile, and adult. Within this life cycle, there are three (https://loop.frontiersin.o
rg/people/383964/overvi
reversible or irreversible adhesion events as summarized in (A–C). The figure was modified ew), Filipe Oliveira Costa
from Khandeparker and Anil (2007). Copyright (2007) Elsevier. (https://loop.frontiersin.o
ew), Willem Renema
iew) and Jan-Niklas
Macher
Although research on barnacle underwater adhesion has accelerated in the (https://loop.frontiersin.o
past decade, likely driven by the urgent need for novel, environmentally ew)
benign antifouling technologies (Yebra et al., 2004; Callow and Callow, 2011)
and introduction of advanced analytical tools (Hennebert et al., 2015; Aldred
and Petrone, 2016), our understanding of the mechanism behind these
processes remains incomplete. In marine environments, any submerged
objects will be covered instantly by interfacial water layers and later by
biofilms comprising microbes and their extracellular secretions (Yebra et al.,
2004). These boundary layers pose a big challenge for adhesive bonding as
they prevent adhesives from wetting and binding with underlying substrates
and greatly compromise the interfacial adhesion strength (Waite, 1987; Lee
et al., 2011). In addition, the surrounding bulk seawater, oxidative species,
and microbial communities, may also diminish underwater adhesion by
diluting, oxidizing, and degrading biological holdfasts. What adaptations
have barnacles made to overcome these challenges and achieve tenacious
underwater attachment? The question largely remains open.
To update and improve our understanding of the mechanisms behind these

reversible and irreversible adhesion events of barnacles and their larvae, a
comprehensive review is necessary given the many recent advances in this
field. Therefore, this review aims to summarize the past and present studies
of barnacle underwater adhesion. Firstly, these three different adhesion
events will be sequentially introduced. Their corresponding adhesives will be
emphasized from two complementary aspects: the in vivo synthesis, storage,
and secretion as well as the in vitro morphology and biochemistry.
Subsequently, the adult barnacle underwater attachment mechanism will be
elaborated by analyzing the possible adhesive and cohesive interactions of
different cement proteins, which have not been discussed in previous reviews
(Kamino, 2006, 2013; Power et al., 2010; Zhang et al., 2017).
Surface Exploration: Cyprid Temporary Adhesion
Surface Exploration: Why and How?

Underwater adhesion of the cyprid contains two successive stages, temporary
surface exploration, and permanent surface settlement. Prior to permanent
settlement, the cyprid must explore the substrate to decide whether to
colonize on it; the cyprid settles permanently if the substrate is satisfactory,
otherwise it returns to the water column (Aldred and Clare, 2008; Rosen-
hahn and Sendra, 2012). It was found that cyprids prefer to settle on
substrates where the temporary adhesion is strong, and thus it requires a
larger force to detach them from these preferable substrates (Aldred et al.,
2010). Moreover, cyprids have a strong tendency to settle on surfaces already
fouled by adult barnacles, especially conspecific ones, because the benthic life
style, and cross fertilization of adult barnacles require that cyprids must
settle near conspecific barnacles (Knight-Jones and Crisp, 1953).
The cyprid uses its attachment organ – the two attachment discs (Figure 2A)
on the third segment of the paired antennules – to adhere to the substrate
(Nott, 1969; Nott and Foster, 1969). Cyprid attachment discs are flat and
covered by a carpet of cuticular villi (Figures 2B,C). Among species, they vary
markedly in outlines (e.g., circular or elliptical), perimeter structures (e.g.,
velum or skirt), tilting angles relative to the long axis of the antennule, and
microvilli density (Bielecki et al., 2009; Brickner and Hoeg, 2010; Al-Yahya
et al., 2016; Yorisue et al., 2016). During surface exploration, the two
attachment discs attach and detach from the surface alternatively, allowing
the cyprid to “walk” bipedally on the surface. Simultaneously, the cyprid is
capable of precisely sensing the biochemical, physicochemical, and
topological characteristics of the substrate using an array of antennular setae
and chooses to either settle or leave. Consequently, all these factors influence
the surface choice of settling cyprids. For instance, simple cyprid settlement
assays have confirmed that adult barnacle pheromones present on the
substrate can greatly promote surface colonization of conspecific cyprids,
even when the clear substratum is not satisfactory (Crisp and Meadows,
1962; Larman and Gabbott, 1975; Matsumura et al., 1998; Dreanno et al.,
2006b). Many surface topological and physicochemical properties, e.g.,
surface textures (Berntsson et al., 2000; Aldred et al., 2010; Chaw et al.,
2011), hydrophobicity (Chaw and Birch, 2009; Phang et al., 2009; Guo et al.,
2014), surface energy, and charge (Petrone et al., 2011; Di Fino et al., 2014)
were also found to influence cyprid surface exploration. Using more complex
video-/tracking-based approaches, the dynamic cyprid surface exploration
EDITED BY process under different conditions was also monitored, offering a real-time
and quantitative method of understanding the settling behaviors of a cyprid
Andrew S. Mount
by measuring its swimming velocity, step length and duration, body
(https://loop.frontiersin.org/people/136438/overview)
Clemson University, movements, footprint deposition,
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(https://www.frontiersin.org) son et al., 2009; Chaw and Birch, 2009; Maleschlijski et al., 2012; Aldred et
REVIEWED BY
al., 2013b, 2018; Maleshlijski et al., 2016).
Nick Aldred
University of Essex, United Kingdom
FIGURE 2
Wong Yue Him
Institute for Advanced Study, Shenzhen (https://www.frontiersin.org/files/Articles/468324/fmars-06-
University, China 00565-HTML/image_m/fmars-06-00565-g002.jpg)
Figure 2. Cyprid underwater adhesion. (A) Schematic of
The editor and reviewers' a"liations are
cyprid temporary and permanent attachment organs. The
the latest provided on their Loop research
profiles and may not reflect their situation
cyprid uses unicellular antennal glands to synthesize the
at the time of review. temporary adhesive for surface exploration and kidney-shaped
cyprid glands to produce cyprid cement for permanent settlement. The cyprid gland consists
of the α and β cells. In the Amphibalanus amphitrite cyprid, Aacp20k-1 is detected in the α
TABLE OF CONTENTS
cells while Aacp20k-2, Aacp100k, and lipids are present in the β cells. (B,C) Scanning
electron microscopy photographs showing the Semibalanus balanoides cyprid attachment
Abstract disc and the covered microvilli. (D) An atomic force microscopy image showing the remnant
elliptical footprint of the A. amphitrite cyprid on the substrate. (E) Schematic of the cyprid
Introduction
permanent cement plaque. a.d., attachment disc; a.g., antennal gland; c.d., cement duct; c.e.,
Surface Exploration: Cyprid compound eye; c.g., cyprid gland; m.s., muscular sac; o., oil; l.p., lipid phase; p.p., protein
Temporary Adhesion phase. (A,E) Modified from Walker (1981). Copyright (1981) Gordon and Breach Publishers,
Inc. (B,C) Modified from Aldred et al. (2013b) under the Creative Commons CC-BY license.
Surface Settlement: Cyprid (D) Modified from Guo et al. (2014). Copyright (2014) American Chemical Society.
Permanent Adhesion
Adult Barnacle Underwater

Adhesion
Molecular Mechanism of Adult

Cyprid Temporary Adhesion
Barnacle Underwater Attachment
Final Remarks
Cyprid temporary adhesion exhibits three primary properties: tenacity,
Author Contributions speed, and reversibility. Regarding tenacity, cyprid adhesion strength varies
Funding between approximately 100–300 kPa on different substrates as measured by
a microbalance (Yule and Crisp, 1983; Yule and Walker, 1984; Maki et al.,
Conflict of Interest Statement
1994). In terms of speed, it is estimated to take only several seconds to form a
Acknowledgments
secure bond between cyprid adhesive disks and a substrate by measuring the
References
time gap between two adjacent paces (Chaw and Birch, 2009). As for
reversibility, a cyprid can not only walk on the substrate by alternatively
attaching each of its two adhesive disks, but also leave the substrate entirely
by detaching both disks. So, how does the cyprid achieve such a tenacious,
rapid, and reversible adhesion process employing a pair of attachment discs?
As stated earlier, scanning electron microscopy (SEM) observations revealed

that cyprid attachment discs are covered by microvilli and setae. This mat of
microvilli and setae are not beneficial to create the temporary seal between
attachment discs and the substrate. Moreover, cyprids do not have the
antennal musculature required for creating a closed low-pressure
environment. These observations collectively deny the assumption that
cyprids use the attachment discs as “suction cups” to adhere to surfaces.
Currently, it is widely accepted that cyprids secrete bioadhesives to assist in
temporary adhesion. In acorn barnacle cyprids, the temporary adhesive was
thought to be synthesized in unicellular antennal glands (Figure 2A) located
in the second segment of the antennules (Nott, 1969; Nott and Foster, 1969).
In the cyprid of the stalked barnacle, Octolasmis angulata, this adhesive is
produced in the unicellular glands clustered in the main body (Yap et al.,
2017). Synthesized temporary adhesives are then delivered to the attachment
discs through the ducts in the antennules. After surface exploration, the
residual footprints on the substrate are believed to be cyprid temporary 
adhesives which were first detected by Walker and Yule using a protein dye (https://www
(Walker and Yule, 1984). Over two decades later, cyprid footprints deposited
on various substrates were directly observed with atomic force microscopy
(AFM) (Figure 2D; Phang et al., 2008, 2009, 2010; Guo et al., 2014) and
imaging surface plasmon resonance (Andersson et al., 2009), thus adding
conclusive evidence to the idea that cyprids secrete adhesives for temporary
adhesion. By using AFM-based force spectroscopy, Phang et al. (2008)
detected the adhesion strength of cyprid footprints, which was only about 1/3
that of the measured adhesion strength of the cyprid. Accordingly, they
speculated that the microvilli on cyprid attachment discs might play a similar
physical adhesion role to that of fly pulvilli and gecko spatula to promote
temporary cyprid adhesion when boundary water was displaced by
temporary adhesives (Phang et al., 2008); but, this requires further
confirmation. By employing high-speed photography, Aldred et al. (2013b)
discovered that cyprid temporary adhesion was also regulated by its
behaviors. For example, during rapid walking, cyprid adhesion is
compromised by minimizing contact time and contact area of the attachment
discs, while, during careful inspection, cyprid adhesion is enhanced by
behaviorally pushing the attachment discs toward the substrate. To achieve
reversible adhesion, the cyprid tugs the antennules parallel to the surface and
peels the attachment discs from the substrate. To summarize, cyprid
temporary adhesion is biochemically, physically, and behaviorally co-
mediated.
Settlement-Inducing Protein Complex (SIPC)

The cyprid temporary adhesive mainly comprises proteins as the residual
footprints on substrates can be stained by protein dyes (Walker and Yule,
1984; Clare et al., 1994; Matsumura et al., 1998). Guo et al. (2014) discovered
that both the size and adhesion strength of cyprid footprints increase on
more hydrophobic substrates, probably implying that cyprid footprints are
abundant in hydrophobic proteins and they experience conformational
changes leading to exposure of hydrophobic domains. Cyprid footprints are
also rich in basic proteins that have an average isoelectric point (pI) of 9.6–
9.7 (Guo et al., 2016), consistent with the observation that more cyprids
choose to settle on negatively charged carboxyl self-assembled monolayers
(SAMs) (Petrone et al., 2011).
Through immunoblotting, a glycoprotein called the settlement-inducing

protein complex (SIPC) originally isolated from adult barnacles is also
identified within cyprid footprints (Matsumura et al., 1998; Dreanno et al.,
2006a). The SIPC in Amphibalanus amphitrite (GenBank accession no.
AY423545) has three subunits with apparent molecular weights (MW) of
approximately 98 kDa, 88 kDa, and 76 kDa. Each of them is glycosylated and
can be recognized by lentil lectin. Eliminating glycosylation and signal
peptides, A. amphitrite SIPC has a theoretical MW of approximately 169 kDa
and a calculated pI of approximately 5.0 (Matsumura et al., 1998; Dreanno et
al., 2006b). In a further study, the pI of isolated A. amphitrite SIPC has
consistently been determined to be 4.6–4.7 by isoelectric focusing (Petrone et
al., 2015). Based on this, the acidic pI of A. amphitrite SIPC is not in
agreement with the latest discovery that A. amphitrite cyprid footprints
mainly comprise basic proteins, which probably suggests that the SIPC is
only one among many unknown footprint proteins. Besides, sequence
alignment reveals that A. amphitrite SIPC shares approximately 30% identity
with members of the α2-macroglobulin (A2M) family (Dreanno et al.,
2006b).
So far, the SIPC is the only characterized protein in cyprid footprints and it
plays multiple roles. First, it acts as a conspecific biochemical cue to mediate
larvae-adult and larvae-larvae interactions to induce the gregarious
settlement of cyprids (Rittschof and Cohen, 2004). Settlement assays found
that even a low density of surface-bound SIPC can attract significantly more
conspecific cyprids to colonize while the homologous A2M protein fails (Mat-
sumura et al., 1998; Dreanno et al., 2006b, 2007). An SIPC homolog named
MULTIFUNCin (GenBank accession no. KC152471) has also been isolated
from Balanus glandula. Interestingly, it not only plays the same role as SIPC
in promoting gregarious attachment of cyprids, but also functions as a
feeding stimulus that induces barnacle predators hunting for barnacles (Fer-
rier et al., 2016). Another main purpose of the SIPC is functioning as an
adhesive protein to facilitate the temporary adhesion of cyprids. By using
surface plasmon resonance, Petrone et al. (2015) found that the SIPC of A.
amphitrite shows comparable adsorption on various SAMs to that of
fibrinogen, a well-known adhesive protein in the extracellular matrix. In
contrast, homologous A2M only has weak to no adsorption on the same
substrates. It has also been suggested that A. amphitrite SIPC may be able to
direct biomineralization, indicating its possible role in the formation of the
barnacle calcite basal plate during cyprid metamorphosis (Zhang et al.,
2016).
Surface Settlement: Cyprid Permanent Adhesion
Cyprid Cement Apparatus

When the cyprid finds a suitable site during surface exploration, it
immediately secretes a permanent adhesive to settle down and initiate
metamorphosis. The cyprid permanent adhesive, also called cyprid cement, is
synthesized and secreted by the cyprid cement apparatus which includes
cement glands and accessory ducts. Notably, the glands used to produce
cyprid permanent and temporary adhesives are different. In acorn barnacle
cyprids, the permanent cement gland is kidney-shaped, distributed at the
posterior of the compound eyes, and contains two types of cells (Figure 2A;
Walker, 1971; Gohad et al., 2014), while the unicellular temporary adhesive
gland is located in the second segment of the antennules (Nott, 1969; Nott
and Foster, 1969). In the cyprid of the stalked barnacle, O. angulata, the
permanent adhesive gland is rod-shaped and located at the back of the
compound eyes, while the temporary adhesive gland is oval and buried in the
mantle of the body (Yap et al., 2017).
In the 1970s, Walker first examined the permanent adhesive gland of the
Balanus balanoides cyprid (Walker, 1971). He categorized these gland cells
into columnar α and round β cells based on their morphological differences.
The two types of cells were located at the apex and the basal area of the gland,
respectively (Figure 2A). In addition, he detected phenols and poly phenolase
in the α cells but neither of these were found in the β cells. Later, Gohad et al.
(2014) discovered that the permanent adhesive of the A. amphitrite cyprid is
a dual-phase bioadhesive comprising a protein and a lipid phase (Figure 2E)
which are separately stored in the α and β cells before secretion. An in vitro
study using isolated permanent adhesive glands of the Megabalanus rosa
cyprid found that cyprid cement is secreted via exocytosis under the control
of catecholaminergic neurons (Okano et al., 1996). Briefly, when cyprid
glands are stimulated by neurotransmitters such as dopamine and
noradrenaline, the cytoplasmic vesicles containing cyprid cement migrate to
specific regions of the cell to release the glue into the extracellular ducts
through exocytosis (Okano et al., 1996). Post secretion, a significant number
of densely packed vesicles in gland cells turn into empty vacuoles (Odling et
al., 2006). By combining fluorescent labeling and laser scanning confocal
microscopy, Gohad et al. (2012) detected adrenergic-like receptors on cyprid
gland cells and antennal setae. Moreover, they found that exogenous
noradrenaline, an agonist of the adrenergic receptor can induce cyprids to
metamorphose without pre-settling on the substrate. After secretion from
these glands, cyprid cement is transported via extracellular ducts to the
muscular sac for temporary storage. When needed, it is delivered by long
cement ducts in the antennules to the attachment discs where it is released
through the openings (Figure 2A).
Biochemical Composition of Cyprid Cement

Through early histochemical studies, Hillman and Nace (1970) detected
proteins and trace lipids in the permanent adhesive of the Balanus eburneus
cyprid and Walker (1971) revealed phenols and poly phenolase, two
important components in quinone protein cross-linking in the adhesive
plaque of the B. balanoides cyprid. In recent years, with the introduction of
advanced microscale and nanoscale analytical techniques, new insights
regarding the biochemical compositions of cyprid cement have been obtained
(Aldred and Petrone, 2016). For instance, by employing AFM-based force
spectroscopy to stretch cyprid cement proteins adhered on the substrate,
Phang et al. (2006) observed gradually decreasing stretching events and
maximum extension length. This particular result was attributed to the
curing of cyprid cement and allowed researchers to estimate curing time to be
within several hours (Phang et al., 2006). Via synchrotron radiation based µ-
X-ray fluorescence analysis, Senkbeil et al. (2016) detected a large amount of
halogen elements as well as low-content metal ions (e.g., Ca2+, Mg2+, and
Fe3+) in the permanent adhesive of A. amphitrite and Balanus improvisus
cyprids. However, their possible roles have not been discussed. By using laser
scanning confocal microscopy to observe cyprid cement deposited on
different SAMs, Aldred et al. (2013a) found that a layer of non-protein
substance enclosed the proteinaceous bulk glue. Via a combination of
chemistry-specific fluorescent probes and Raman microscopy, Gohad et al.
(2014) further demonstrated that the non-protein interfacial layer is
lipidaceous and the internal bulk glue is phospho proteinaceous (Figure 2E).
The protein and lipid phases are separately stored in the α and β cells and the
lipid phase is released ahead of the protein phase upon secretion. The
interfacial location and prior secretion of the lipid phase probably indicate
that it plays the key role in removing boundary water layers and keeping
microbes away from the proteinaceous bulk glue (Gohad et al., 2014). This
discovery shed light on the important roles of non-protein components in
underwater adhesion for the first time. Likewise, He et al. (2018) noted that
lipids, as integral components, also assist in mussel underwater adhesion and
function similarly as they do in cyprid permanent adhesion. Therefore,
secreting water-repelling lipids to prepare the substrate and create an
adhesion-friendly local environment ahead of depositing multi-protein
holdfasts may be a general strategy for underwater adhesion.
As of date, no glue protein has been isolated from cyprid permanent

adhesives, but surprisingly, some barnacle cement proteins (BCPs) originally
identified from adult barnacles were found to be also present in cyprid
cement glands by immunofluorescence. As shown in Figure 2A, Aacp20k-1
(A. amphitrite cement protein-20 kDa-1) and Aacp20k-2 were separately
stored in the α and β cells (He et al., 2013). Aacp100k was detected in the β
cells as well (He et al., 2017). Whether other reported BCPs in A. amphitrite,
including Aacp19k, Aacp52k, and AaCP43 also exist in cyprid cement glands
is unclear at this moment. Nevertheless, these results suggest that adult BCPs
may play a role in cyprid permanent adhesion. To confirm this, further
studies are required to determine whether they are also present in the cured
cyprid adhesive plaque.
Adult Barnacle Underwater Adhesion

On a suitable substrate, the cyprid settles, metamorphoses into a juvenile,
and then develops into an adult. During growth, adult barnacles periodically
secrete primary cement to achieve firm attachment (Fyhn and Costlow,
1976). Adhered adult barnacles can neither move freely on the surface nor
actively detach from the substrate, because in nature, the detachment of a
barnacle generally leads to its death. Yet, laboratory studies have revealed
that barnacles dislodged from substrates (e.g., silicones) where they have
weak adhesion can also secrete secondary cement and attach to a new
substrate if their bases are intact. Accordingly, adult barnacle cement is
classified into primary and secondary cement. Generally, primary cement
refers to the cement used for natural attachment while secondary cement is
only employed during reattachment (Saroyan et al., 1970). However,
comparative studies confirm that the two types of barnacle cement are
identical because they have similar micro- and nano-morphologies,
biochemical compositions, and secondary structures (Kamino, 2006; Barlow
et al., 2010).
So far, only a few studies have discussed the relationships between adult
barnacle cement and cyprid adhesives. Kamino argues that they are different
based on the discovery that adult BCP genes of M. rosa are not expressed in
the cyprid (Kamino, 2006). While this opinion was well received by most
researchers, in A. amphitrite some adult BCPs (Figure 2A) have since been
successfully detected in cyprid permanent adhesive glands (He et al., 2013,
2017). Therefore, additional studies are required to elucidate their exact
relationships.
Synthesis, Storage, and Secretion of Barnacle

Cement
According to histological studies carried out on acorn barnacles in the 1970s
(Lacombe and Liguori, 1969; Lacombe, 1970; Walker, 1970; Fyhn and Cost-
low, 1976) and on stalked barnacles in recent years (Jonker et al., 2012; Zhe-
den et al., 2012; Lobo-da-Cunha et al., 2017), it is generally accepted that
barnacle cement is synthesized and stored in unicellular glands, and secreted
through a series of extracellular canals. Together, gland cells and drainage
canals constitute the entire cement apparatus of adult barnacles.
In most acorn barnacles, cement glands cluster in the mantle chamber next
to the basal portion of the body (Figure 3A). They often intermingle with
ovarian tissues but can easily be distinguished by Azan staining. A gland cell
is globular or oval and its diameter varies with developmental progress.
Usually, the cytoplasm of acorn barnacle gland cells is heterogeneous and
shows two distinct regions: the synthetic region that is rich in RNA and the
storage or secretory region which contains a dense mixture of proteins (Fig-
ure 3B; Lacombe and Liguori, 1969; Lacombe, 1970; Walker, 1970; Fyhn and
Costlow, 1976). BCPs are synthesized within the synthetic regions and then
transported to the storage regions where they are packed in large vacuoles
that probably have an acidic internal environment (Power et al., 2010). It is
hypothesized that the acidic environment might be beneficial for protein
condensation to form high-density secretory granules as well as self-
assembled nanofiber structures (Power et al., 2010; Liang et al., 2018). Upon
secretion, barnacle cement is delivered via an extracellular canal system
comprising collecting canals (CCs), secondary canals (SCs), and principle
canals (PCs) to the adhesive joint, where the cement is then released (Figure
3B; Power et al., 2010). The cement apparatus of stalked barnacles is largely
similar to that of acorn barnacles with only a few minor differences owing to
different life styles and body structures (Jonker et al., 2012; Zheden et al.,
2012; Lobo-da-Cunha et al., 2017). First, the unicellular glands of stalked
barnacles are gregariously located at the proximal end of the long peduncle
adjacent to the capitulum (Figure 3C) rather than the basal portion close to
the base. Second, the cytoplasm of gland cells in stalked barnacles does not
show the same distinct synthesis and storage regions as seen in acorn
barnacles. Instead, stalked barnacle glands contain many uniformly
distributed vacuoles. Third, the gland cells of stalked barnacles possess a
unique set of intracellular canals (ICCs). Consequently, in stalked barnacles
the cement is secreted via the ICCs to the extracellular CCs, and then
delivered by the SCs and PCs to the adhesive joint (Figure 3D). More details
regarding the cement apparatus of both stalked and acorn barnacles are
presented in the review by Power et al. (2010).
FIGURE 3
(https://www.frontiersin.org/files/Articles/468324/fmars-06-
Figure 3. Adult barnacles and their cement apparatuses. (A,B)
Schematics of the acorn barnacle and its cement apparatus. In
acorn barnacles, cement glands are clustered at the basal region
of their bodies and close to the substrate. The cement is
synthesized in gland cells, whose cytoplasm is separated into
synthetic and secretory regions, and conveyed downward by
collecting canals (CCs), secondary canals (SCs), and principle
canals (PCs) to the adhesion joint. (C,D) Schematics of the stalked barnacle and its cement
apparatus. Cement glands of stalked barnacles are gregariously located at the proximal end
of the peduncle and next to the capitulum. In stalked barnacles, the cement is produced in
gland cells with homogeneous cytoplasm and transported upward by intracellular canals
(ICCs), CCs, SCs, and PCs to the adhesion joint. (C,D) Modified from Jonker et al. (2012).
Copyright (2012) Wiley Periodicals, Inc.
Saroyan et al. (1970) noted that the Mallory’s Trichrome stain result of acorn
barnacle cement changes when it travels from the SCs to the PCs.
Coincidently, a hilum structure was observed at their junction in some acorn
barnacles (Lacombe, 1970). In the stalked barnacle, Lepas anatifera, Jonker
et al. (2012) failed to detect any histochemical changes in the cement during
its transportation in the canals. However, they discovered that the cement
changes from a loose, diffused substance to a denser, more clumped
substance when it passes through the SCs to the PCs (Jonker et al., 2012).
These histochemical and ultrastructural changes possibly imply that barnacle
cement has initiated polymerization during transportation in the canals. It is
likely that barnacle cement cures slowly in the canals, and therefore, has low-
extent cross-linking during secretion so that it does not block the canals
entirely. Besides, Burden et al. (2012) proposed that barnacles might employ
a step-wise cementing strategy to protect the canals from being blocked.
Briefly, a weak adhesive, barnacle cement secretion 1 (BCS1) is first released
by barnacles, and subsequently BCS2 is secreted to flush out the canals and
cooperate with BCS1 to increase adhesion strength (Burden et al., 2012).
While insightful, these in vivo histological and histochemical studies of

barnacle cement apparatuses have yet to be correlated with the in vitro
biochemical studies of BCPs that will be discussed in the next section. Thus,
the in vivo localizations of different BCPs are not currently understood.
Furthermore, this gap has led to an uncertainty whether these isolated BCPs
are truly synthesized and secreted via the above discussed pathways. Most
notably, a recent study by Fears et al. (2018) noted that in acorn barnacles,
the cement deposits at the periphery of barnacle base are transferred to the
substrate via the cuticular slip rather than the canal system (Fears et al.,
2018). Therefore, the synthesis, storage, and secretion of BCPs are far from
being fully understood.
Fibrillar Morphology of Barnacle Cement

Both morphology and adhesion ability of barnacle cement show great
variance on different substrates. On stiff substrates that barnacles can easily
attach to, such as natural objects, metals, and some polymers, barnacle
cement forms a flat, thin, and transparent layer which can duplicate the
texture of external substrates and allow for a high adhesion strength
(Dougherty, 1990; Raman et al., 2013). Conversely, on soft and elastic
substrates like hydrogels and silicones, barnacles deposit thick, opaque, and
rubber-like cement which exhibits much lower adhesion strength (Berglin
and Gratenholm, 2003; Wiegemann and Watermann, 2003; Holm et al.,
2005; Ahmed et al., 2014). This form of barnacle cement occurs probably
owing to its high water uptake, as seawater may penetrate into the loosely
sealed adhesive joint.
By using microscale and nanoscale imaging tools, the fine structures of

barnacle cement on different substrata have been intensively studied. Wiege-
mann and Watermann (2003) first discovered that the cured cement of B.
improvisus mainly comprises of nanoscale globules, which further assemble
into different microscale structures, e.g., sponge-like structures or dense
sheets on certain metals and fibril meshes on soft surfaces like
polydimethylsilane (PDMS). Similarly, the cement of Balanus reticulatus
also displays diverse morphologies including fibril meshes, broad bands, and
sponge-like structures on different surfaces (Raman and Kumar, 2011). By
using AFM imaging, Berglin and Gratenholm (2003) discovered that B.
improvisus cement forms dense sheets on polymethylmethacrylate and
dispersed nanosized granules on PDMS. Barlow et al. (2010) conducted in
vivo AFM observations on the base of A. amphitrite removed from PDMS
and detected fibrillar and globular structures in different areas. Likewise,
AFM images collected from different regions of A. amphitrite cement
deposited on a CaF2 substrate also show different morphologies (Burden et
al., 2012). These results probably suggest the spatial and temporal regulation
of barnacle cement morphologies. Sullan et al. (2009) observed the same
fibrillar structures in A. amphitrite cement remained on glass cover slips, yet,
they did not note where the images were captured with respect to the
transferred cement.
It was noted that remnants of fibrillar barnacle cement on several substrates

could be stained by amyloid fiber dyes – Thioflavin T and Congo Red –
indicating that barnacle cement fibers probably contain amyloid fibers (Sul-
lan et al., 2009; Barlow et al., 2010). Amyloid fibers are nanofibers that self-
assemble through non-covalent interactions of building blocks rich in β-sheet
secondary structures. Both circular dichroism and Fourier transform infrared
spectroscopy consistently revealed that around 40% of the total secondary
structures of A. amphitrite cement consists of β sheets (Barlow et al., 2009,
2010). Amyloid fibers have exceptional mechanical stability and show
resistance to enzymatic degradation (Fukuma et al., 2006; Smith et al., 2006;
Fowler et al., 2007; Knowles and Buehler, 2011). Thus, apart from barnacle
cement, amyloid fibers have also been detected in some other natural
underwater adhesives (Mostaert et al., 2006, 2009). These discoveries have
revealed a previously unknown function of amyloid fibers as they were
generally only associated with neural degenerative diseases in the past.
Therefore, amyloid fibers in bioadhesives are considered as functional
amyloid fibers.
Oxidative Chemistry of Barnacle Adhesive Interface

Like other arthropods, the barnacle has an outer cuticle that periodically
molts and remolds during growth. In situ observation of the dynamic process
of barnacle base expansion and cement secretion throughout the whole
molting cycle has found that at the edge of the barnacle base, where newly
synthesized cement is released, the outer cuticle of a barnacle is intermixed
with the underlying cement layers. Furthermore, the molting of these cuticles
coincides with the secretion of barnacle cement (Burden et al., 2014).
Consequently, ecdysis-related oxidases (e.g., catechol oxidase and lysyl
oxidase) and reactive oxygen species (ROS) build up at the barnacle adhesive
interface and lead to a strong oxidative stress (Golden et al., 2016), which is
speculated to play a role in catalyzing the cross-linking of barnacle cement
fibrils (So et al., 2016, 2017). The detailed cross-linking pathways will be
discussed later in this review.
Recently, Fears et al. (2018) found that A. amphitrite releases a lipid- and
ROS-abundant oxidative fluid ahead of depositing fibrous barnacle cement.
This fluid, whose relationship with ecdysis-related fluid is currently not
known, can oxidize surface-adsorbed organic matter, peel off microbial
biofilms, and affect the hydration state of the interface (Fears et al., 2018).
Similarly, it was observed that the post-settlement A. amphitrite cyprid also
secretes a ROS-containing fluid to kill bacteria and clear bacterial biofilms on
the substrate during metamorphosis (Essock-Burns et al., 2017). Taken
together, the oxidative chemistry of the barnacle adhesive interface can also
contribute to remove surface-bound organic layers in order to clear and
condition the substrate for strong underwater adhesion.
Isolation and Characterization of BCPs

It is believed that the best way to identify BCPs is to conduct comparative
studies on unsolidified liquid cement and cured cement. However, only few
studies have examined the biochemical compositions of liquid cement (Es-
sock-Burns et al., 2019), as it can be easily contaminated by other barnacle
body liquids leading to controversial conclusions (Kamino, 2010a). On
natural substrates, the solidified barnacle cement forms a thin layer with a
thickness of only several micrometers underneath the calcified base, making
it quite difficult to be collected. Moreover, it is hard to completely dissolve
the cured cement. Owing to these challenges, early research on the isolation
and characterization of BCPs advanced slowly, although it had already been
shown that the protein content of barnacle cement was over 90% (Walker,
1972; Kamino et al., 1996). Later studies noted that barnacles adhering on
soft manmade surfaces (e.g., silicones) with thick, opaque, and gummy
cement can be dislodged and successfully reattached to specially designed
substrates, providing an ideal alternative for the collection of barnacle
cement (Rittschof et al., 2008).
Until now, several approaches have been tested to dissolve cured barnacle
cement (Barnes and Blackstock, 1976; Naldrett, 1993; Kamino et al., 1996,
2000; Naldrett and Kaplan, 1997; Wiegemann et al., 2006; So et al., 2016),
two of which show promising results. The first employs high concentrations
of denaturants and reductants that are paired with a heat treatment. This
method can render more than 90% of M. rosa cement soluble and release
barnacle individuals directly from the substrate (Kamino et al., 2000). With
this method, Kamino and his colleagues isolated more than ten protein
components from M. rosa cement which were named after their apparent
MW estimated by SDS-PAGE. For example, one isolated protein was named
Mrcp100k, which denotes a 100-kDa cement protein in M. rosa. The cDNA
sequences of Mrcp100k, Mrcp52k, Mrcp20k, and Mrcp19k have been
acquired and submitted to the GenBank database (Kamino, 2006, 2008). The
second method relies on only one polar solvent, hexafluoroisopropanol, to
break down hydrogen bonds important for the stabilization of amyloid fibers.
Using this reagent, So et al. (2016) dissolved almost the entire fibrous cement
of A. amphitrite and then isolated a new protein named AaCP43, which has a
theoretical MW of about 43 kDa but migrates unusually to around 63 kDa
during electrophoresis. AaCP43, whose cDNA sequence has also been
indexed in the GenBank database, may be the homolog of Mrcp68k owing to
their similar amino acid compositions and peptide fragments. However, their
sequence identity is not known now as the sequence of Mrcp68k has not been
published. So et al. (2016) also identified some proteins homologous to
AaCP43 and Aacp19k; yet, no other new types of BCPs were found, probably
indicating that barnacles across different taxa share a core set of homologous
BCPs.
Many cDNA sequences of homologous BCPs have also been isolated from
different barnacle species through rapid amplification of cDNA ends and
RNA sequencing. Table 1 summarizes the reported cement proteins with
published amino acid sequences from various barnacles. It is worth noting
that all the cement proteins in the table are from acorn barnacles except for
the recently identified three in the stalked barnacle Pollicipes pollicipes
(Rocha et al., 2018). In the next section, the amino acid compositions and the
primary and secondary structures of different BCPs will be discussed in
detail.
TABLE 1
00565-HTML/image_m/fmars-06-00565-t001.jpg)
Table 1. Summary of BCPs with known amino acid sequences
from different barnacle species.
Amino Acid Compositions
The cp100k and cp52k contain a lot of hydrophobic amino acids and their
average GRAVY (grand average of hydropathicity) values are 0.089 and
−0.081, respectively. In the two proteins, the total content of basic Arg and
Lys (11.5% in cp100k and 15.7% in cp52k) as well as that of aromatic Phe and
Tyr (9.3% in cp100k and 13.7% in cp52k) is relatively high, while the
percentage of Cys is very low (Table 1). In contrast, cp20k lacks hydrophobic
amino acids but has an extremely high percentage of Cys. Taking full-length
Mrcp20k and Balcp20k as an example, their GRAVY values are −1.082 and
−1.188 while their Cys contents are 17.5 and 17.1%, respectively. Moreover,
there is an abundance of charged amino acids, such as His (10.4%), Asp
(11.5%), and Glu (10.4%) in Mrcp20k as well as His (20.0%) and Lys (9.5%)
in Balcp20k (Table 1). The cp19k shows a strong bias to Ser, Thr, Gly, Ala,
Lys, and Val, which collectively occupy approximately 70% of the total
residues. Notably, cp19k has an extremely low percentage of aromatic amino
acids. It can be seen from Table 1 that all these cp19k homologs do not have
Tyr or Trp and the average content of Phe is only 1.8%. The amino acid
composition of cp68k is quite similar to that of cp19k.
Based on their distinct amino acid compositions, the above five BCPs were
classified into three categories by Kamino (2006, 2008) as cement proteins
with a high percentage of hydrophobic amino acids (cp100k and cp52k),
cement proteins showing a bias to six amino acids (cp68k and cp19k), and
cement proteins rich in charged amino acids and Cys (cp20k). Alternatively,
So et al. (2016) proposed to divide these BCPs into two groups: the Leu-rich
cement protein (LrCP) group which is abundant in Leu, Ile, and Val, and the
Gly/Ser-rich cement protein (GSrCP) group that has high proportions of Gly,
Ser, Ala, and Thr. Following this classification, cp100k and cp52k belong to
the LrCP group, whereas, cp68k and cp19k fall into the GSrCP group.
Notably, cp20k falls within neither of these groups.
The post-translationally modified amino acids, 3,4-dihydroxyphenylalanine
(DOPA) and phosphorylated serine, play critical roles in mussel and
sandcastle worm underwater adhesion (Lee et al., 2011; Stewart et al., 2011a;
Waite, 2017). Similarly, could any modified amino acids also function in
barnacle underwater attachment systems? Kamino et al. (2012) insist that
BCPs do not have any post-translational modifications except for the
glycosylation of Mrcp52k. Combining chemistry-specific staining and
immunoblotting, Dickinson et al. (2016) detected phosphorylated proteins in
the adhesive interface of A. amphitrite, however, these modified proteins
have not been isolated. Recently, So et al. (2017) and Fears et al. (2018)
confirmed that these phosphorylated proteins are actually cuticular proteins
of A. amphitrite rather than its cement proteins.
Primary Structures
The sequence of Mrcp100k is relatively simple. It does not contain any

repetitive fragments but instead displays alternating hydrophilic and
hydrophobic domains (Kamino et al., 2000), which may be important for its
assembly via hydrophobic interactions (Sullan et al., 2009). The primary
structure of cp52k, including Mrcp52k and Aacp52k, is made up of four large
repeats (Figure 4). Each repeat contains about 150 amino acids and can be
further divided into different peptide segments with varying amino acid
compositions (Kamino et al., 2012; Nakano and Kamino, 2015). As shown in
Figure 4, the primary structure of cp20k can also be divided into multiple
repeats based on the regular alignment of Cys (Kamino, 2001; Mori et al.,
2007; He et al., 2013). The cp19k and cp68k possess a block copolymer-like
sequence property, whose primary structures contain two alternating blocks.
One, a STGA-rich segment, showing homology to silk proteins is dominated
by Ser, Thr, Gly and Ala, and the other, has an abundance of charged and
hydrophobic amino acids (Kamino, 2006; So et al., 2016). The homology of
BCPs to silk proteins is attractive but not very surprising given that both
barnacles and spiders belong to the phylum Arthropoda (So et al., 2016).
Obviously, both animals are capable of fabricating proteinaceous
biomaterials with superior mechanical properties. Revealing the sequence
characteristics of different BCPs further can enhance our understanding of
their structural and functional properties and may inspire the design of novel
peptide- or protein-based materials using minimal peptide motifs (Yang et
al., 2014).
FIGURE 4
(https://www.frontiersin.org/files/Articles/468324/fmars-06-00565-
HTML/image_m/fmars-06-00565-g004.jpg)
Figure 4. Amino acid sequence alignment of different BCP homologs.
The sequence of cp52k (including Mrcp52k and Aacp52k) consists of
four large repeats and each of the repeats can be further divided into
different fragments based on the distinct amino acid compositions. The
primary structure of cp20k also shows repetitive segments according to
the regular alignment of Cys. The cp19k exhibits a block copolymer-like
sequence characteristic and its primary structure comprises two
alternating blocks. One is rich in Ser, Thr, Gly and Ala (STGA-rich fragment), and the other is
dominated by hydrophobic and charged amino acids.
Secondary Structures
Owing to the great challenge of isolating natural BCPs, the secondary
structures of different BCPs were either theoretically predicted or
experimentally examined using peptide analogs or recombinant proteins. It
was predicted that approximately 87% of the Mrcp100k sequence forms β
sheets (Kamino et al., 2000). The secondary structure of cp52k has not yet
been examined, but it was found that Mrcp52k-inspired peptides could form
β sheets and self-assemble into amyloid-like fibrils when solution pH and
ionic strength increased to a certain threshold (Nakano and Kamino, 2015).
Similarly, Nakano et al. (2007) found that Mrcp20k-inspired peptides could
also transform to form β sheets and assemble into fibril networks under basic
conditions. Although intriguing, these results obtained from peptide studies
can not accurately reflect the secondary structures of full-length BCPs
because of the significantly different MW, pI, steric hindrance, and so on.
Therefore, it is uncertain whether Mrcp20k will change its secondary
structures from random coils in a 10 mM sodium phosphate buffer (pH 6.8)
to β sheets at higher pH and ionic strength just like Mrcp20k-inspired
peptides (Mori et al., 2007). Recently, Wang et al. (2018) and Liang et al.
(2018) examined the secondary structures of bacterial recombinant Balcp19k
and consistently found that it is dominated by random coils and β sheets. The
secondary structure of cp68k is currently not clear.
Molecular Mechanism of Adult Barnacle

Underwater Attachment
Underwater adhesion is a multifunctional process that primarily involves
four different steps: removing weak boundary layers, wetting, establishing
interfacial adhesion, and curing (Waite, 1987). These subfunctions were
classified into surface and bulk functions by Kamino (2008), who further
claimed that these functions are fulfilled by different components of
bioadhesives. In other words, the components of natural adhesives can be
functionally classified into their surface and bulk components. Typically,
surface components that directly couple with the substrate are located at the
interface between the bulk glue and external object. Bulk components, in
contrast, are within the glue itself and are responsible for the cross-linking of
different glue components. To achieve successful underwater bonding, not
only should surface components establish stable interfacial adhesion, but also
bulk components must develop strong bulk cohesion. Moreover, surface and
bulk components must strongly interact with each other as well. To our
knowledge, the extensively studied mussel adhesion system perfectly matches
the above assumption. In mussel adhesive plaque, foot protein-3 (fp-3) and
fp-5 that bind to the substrate directly are located at the plaque-substrate
interface, whereas fp-2 and fp-4 that form foam-like internal structures
linking interfacial adhesive proteins and byssus thread structural proteins are
distributed within the plaque (Waite, 2017). Based on these principles, the
molecular mechanism of adult barnacle underwater adhesion can be
understood by addressing the following three critical questions. First, which
BCPs are surface proteins and which ones are bulk proteins? Second, how do
surface and bulk BCPs achieve interfacial adhesion and bulk cohesion,
respectively? Third, how do surface and bulk BCPs interplay?
Based on the pioneering studies on M. rosa cement proteins, Kamino built an

original molecular model of adult barnacle underwater attachment (Kamino,
2006). In this model, cp20k, cp19k, and cp68k are speculated to be surface
proteins that adhere to substrates via non-covalent interactions, whereas
cp100k and cp52k are considered as bulk proteins that play cohesive roles via
self-assembling into amyloid fibrils (Kamino, 2006). For the first time, the
model discussed the structures and functions of different BCPs and assigned
them with surface or bulk subfunctions. In recent years, So et al. (2016, 2017)
performed parallel biochemical studies on A. amphitrite cement and
discovered that A. amphitrite and M. rosa have different cement protein
compositions. In A. amphitrite cement, Aacp19k, AaCP43 (a putative cp68k
homolog), and proteins with homology to them are major proteins, whereas
Aacp100k and Aacp52k have unexpectedly low percentages. They also noted
that underneath the A. amphitrite calcite base there exists a chitinous cuticle
layer, and multiple oxidases related to the shedding of this cuticle accumulate
in the cement (Burden et al., 2014; So et al., 2017). Based on these findings,
So et al. (2017) proposed a different enzyme-catalyzed barnacle cement
curing hypothesis, wherein cp19k and cp68k are assumed to play both
surface binding and bulk cohesion roles.
In this section, by combining the earlier model and the latest hypothesis, we
first discuss the curing mechanism of barnacle cement from three aspects:
the interfacial adhesion of surface BCPs, the bulk cohesion of bulk BCPs, and
the various interactions between surface and bulk BCPs. Based on these
discussions, we then present an updated molecular model of barnacle
underwater adhesion.
Interfacial Adhesion of Surface BCPs

Functional characterization of bacterial recombinant BCPs discovered that
Mrcp20k specifically adheres to calcite (Mori et al., 2007) while Mrcp19k
non-specifically binds to a wide variety of substrates with different surface
properties (Urushida et al., 2007). Accordingly, cp20k and cp19k are
regarded as surface BCPs playing the key surface coupling role in Kamino’s
model. Moreover, based on the empirical principle that the function of a
protein is tightly related to its location, it was postulated that cp20k is located
at the interface between the barnacle calcite base and bulk cement, while
cp19k is distributed at the junction of bulk cement and external substrate
(Kamino, 2006; Raman et al., 2016).
Cp20k: Coordination
The selective adhesion of cp20k to specific types of substrates probably

implies that chemical bonding rather than physical adhesion is responsible
for its interfacial adhesion. It is unlikely that cp20k interacts with the
barnacle calcite basal plate via covalent bonds owing to the absence of
reactive moieties on this type of inorganic surface. By carefully inspecting the
sequence of cp20k, it can be noted that some of its charged amino acids are
arranged into small clusters, which may facilitate cp20k to coordinate with
metal ions. For example, the EED, EEDDGD, and DHHDDD sequences in
Mrcp20k are conducive to its coordination with Ca2+ via side-chain carboxyl
groups (Kamino, 2001). Consistently, all the substrates to which Mrcp20k
selectively binds normally contain metal ions. Furthermore, the strong
molecule-level coordination between cp20k and the barnacle calcite base is
also in accord with the macroscopically tenacious adhesion of barnacle
cement.
However, the original speculation that cp20k serves as a barnacle calcite

base-specific coupling protein is still debated. This is partially because
functional studies on bacterial recombinant Mrcp20k reveal its ability to
directly mineralize calcite (So et al., 2015). In accordance with this, a
transcriptome study of the barnacle Tetraclita japonica formosana which
does not have a fully calcified base did not find any cp20k homolog (Lin et
al., 2014). Taken together, these results indicate that the exact role of cp20k
needs further examination.
Furthermore, in situ observation found a cuticle layer underneath the A.

amphitrite calcite base (Burden et al., 2014). Most probably, this structure is
also present at the M. rosa adhesive interface, despite it not being mentioned
in Kamino’s original model. Its existence greatly complicates our
understanding of barnacle underwater adhesion. Briefly, if this organic layer
is permeable to barnacle cement, it may not show much influence on
Kamino’s model; otherwise, the two interfaces that barnacle cement directly
contacts are cuticle and external objects. In this case, the model is quite
similar to the adhesion system of barnacles with membranous bases, but
unfortunately, such adhesion mechanisms have rarely been studied.
Cp19k and cp68k: Physical Interactions
Barnacles can tenaciously adhere to a wide range of substrates, indicating

that proteins assumed to be located at the barnacle cement-substrate
boundary, namely cp19k and cp68k, are capable of adaptively and firmly
binding to different substrates. That cp19k and cp68k contain numerous
amino acids with side-chain amine or hydroxyl groups was believed to be
favorable for the removal of surface-bound water layers (Waite, 1987; Maier
et al., 2015), a vital step for successful underwater adhesion. Furthermore, it
was found that the positively charged Lys, which is abundant in both cp19k
and cp68k, can displace the adsorbed cations on mineral substrates to
promote the surface binding of interfacial proteins (Maier et al., 2015).
From the amino acid composition of cp19k, it may be inferred that

electrostatic interactions, hydrogen bonding, hydrophobic interactions, and
van der Waals forces each play a role in its surface binding. It is well known
that the neighboring amino acids of active sites always play cooperative roles.
For example, in mussel interfacial adhesive proteins, the adjacent Lys of
DOPA cooperates with surrounding DOPA to enhance its underwater
adhesion ability (Maier et al., 2015; Li et al., 2017a, b). In the mussel foot
protein-3s, the proximal hydrophobic amino acids can increase the oxidation
potential of DOPA and decrease its self-oxidation tendency (Wei et al., 2013).
In cp19k, it is frequent that one side of the Lys region is filled with
conformation-flexible amino acids while the other side is made up of
hydrophobic amino acids. Based on this, it can be speculated that this
alignment of Lys not only endows it with conformational freedom, but also
promotes its interfacial adhesion by the synergy between positively charged
Lys and hydrophobic amino acids (Ma et al., 2015). It was recently found that
recombinant Balcp19k can self-assemble into nanofibers under suitable
conditions (Liu et al., 2017; Liang et al., 2018). More importantly, the
adhesive ability of self-assembled cp19k nanofibers becomes stronger while
also showing a resistance to the adverse influences of basic and high-salinity
seawater (Liang et al., 2018). Thus, the unique molecular design of cp19k also
enables it to self-assemble into nanofibers, which in turn boosts its overall
adhesive ability.
The cp68k has similar amino acid compositions and sequence properties to
cp19k, and thus, it is hypothesized to play an interfacial adhesion role too.
Yet, its self-assembly property and adhesive ability that have not been
examined so far must be somewhat different from cp19k, owing to their
different proportions in barnacle cement and distinctive molecular weight,
which is known to influence the adhesive ability (Jenkins et al., 2013; Kim et
al., 2018).
Internal Cohesion of Bulk BCPs
During dissolution of cured M. rosa cement, it was found that Mrcp100k and
Mrcp52k are two major protein components and only after they are dissolved
can the surface BCPs be fully released (Kamino et al., 2000). Accordingly,
cp100k and cp52k are considered as bulk BCPs. They constitute the insoluble
bulk cement of M. rosa and play a cohesive role internally as well as with
surface BCPs (Kamino, 2006; Raman et al., 2016). In contrast, So et al.
(2016) discovered that cp19k, cp68k, and their homologs, rather than cp100k
and cp52k, form the bulk cement of A. amphitrite. Therefore, in the
hypothesis proposed by So et al. (2016) these proteins are both surface and
bulk proteins.
Self-Assembled Amyloid Fibers
Kamino et al. (2000) first noticed the correlation between barnacle cement
and amyloid fibers/plaques, inspired by their similar insolubility, and high β-
sheet content. To confirm this correlation, several studies have been
conducted to examine the self-assembly properties of different BCPs. Nakano
and Kamino (2015) explored the self-assembly property of Mrcp52k-inspired
peptides owing to the unavailability of full-length cp52k. They found that
these Mrcp52k-inspired peptides can self-assemble into amyloid-like fibrils
at increased pH and ionic strengths. Later, our group verified the
amyloidogenic self-assembly ability of bacterial recombinant full-length
Mrcp52k (Zeng, 2016). Thus, cp52k, and cp100k that has a similar amino
acid composition to cp52k, are thought to play cohesive roles via self-
assembling into amyloid fibers. It has been proved that hydrogen bonds
between protein backbones as well as π-π stacking between aromatic amino
acids are important for the assembly and stabilization of amyloid fibers (Gaz-
it, 2002). Consistently, both cp100k and cp52k contain a large number of
aromatic amino acids. Besides, hydrophobic interactions may also assist in
the self-assembly of cp100k (Sullan et al., 2009).
So et al. (2016) speculate that cp19k and cp68k may also be able to self-
assemble into ordered nanofibers, as sequence alignment finds that they
contain some randomly distributed silk protein-homologous fragments.
Through a series of studies, the self-assembly property of cp19k under
different conditions has already been confirmed (Liu et al., 2017; Liang et al.,
2018), but whether its self-assembly is enabled by these silk protein-
homologous peptide fragments is currently unknown. The self-assembly
ability of cp68k has not yet been examined.
Enzyme-Catalyzed Cross-Linking
While investigating the protein components of A. amphitrite cement, So et al.

(2017) noted that ecdysis-related oxidative chemistries, including different
enzymes and ROS, would accumulate in barnacle cement. Based on this, they
hypothesized different enzyme-catalyzed BCP polymerization pathways. As
shown in Figure 5A, the peroxidase oxidizes non-peptidyl catechol precursors
present in cement layers into an active semi-quinone in a peroxide-
dependent way. Subsequently, the semi-quinone reacts with free amine
groups in BCPs, forming stable quinone protein cross-linking. This process is
similar to the cuticular sclerotization of insects. Compared with the earlier
hypothesis that BCPs were cross-linked via quinones oxidized from Tyr under
the catalysis of phenol oxidase (Lindner and Dooley, 1973), the current one is
more reasonable. This is primarily because the Tyr content in barnacle
cement is very low and phenol oxidase inhibitors do not inhibit the curing of
barnacle cement. Furthermore, free catechol precursors have also been
shown to exist in barnacle cement layers (So et al., 2017). From the
perspective of So et al. (2017) the resultant catechol groups in the peroxidase
catalyzed cross-linking pathway probably play the important role of
interfacial adhesion. In the meanwhile, under the catalysis of lysyl oxidase,
barnacle cement fibrils can also be cross-linked via lysine protein cross-
linking (Figure 5B), which is commonly observed in the cross-linking of
collagen and elastin fibrils (So et al., 2017).
FIGURE 5
Figure 5. Hypothesized cross-linking mechanisms of bulk
BCPs. (A) Quinone protein cross-linking. Non-peptidyl
catechol precursors in barnacle cement layers are first oxidized
by peroxidase in a peroxide-dependent manner into quinones that then react with amine
groups of BCPs to form stable quinone protein cross-links. (B) Lysine (Lys) protein cross-
linking. Relying on peroxide, lysyl oxidase converts Lys of BCPs into allysine that further
reacts with Lys to form multivalent cross-links. (C) Schematic of the hypothesized cohesion
mechanism of bulk BCPs. The bulk BCPs self-assemble into amyloid fibrils, which are then
cross-linked through pathways (A,B) to form interwoven fibers. (D) Schematic of typical
cross β-sheet structures of amyloid fibers. The figure was modified from So et al. (2017).
Copyright (2017) American Chemical Society.
Originally, the above cross-linking reactions were thought to occur on A.

amphitrite bulk cement proteins (cp19k and cp68k), but apparently, they
could also induce the cross-linking of cp52k and cp100k as long as they were
exposed to those oxidative chemistries. In a word, these hypothesized
enzyme-catalyzed polymerization reactions shed light on how bulk BCPs are
further cross-linked from self-assembled nanofibers to generate fibrous bulk
cement (Figures 5C,D). Notably, although the existence of peroxidase and
lysyl oxidase has been verified by proteomics (So et al., 2016), the cross-
linked products have yet to be identified. It remains inconclusive whether
bulk BCPs are cross-linked under their catalysis. Future in vitro studies could
substantiate these assumed cross-linking pathways using recombinant BCPs
and different enzymes.
Interplays Between Surface and Bulk BCPs
Covalent Cross-Linking
The indispensability of a high concentration of reductant to dissolve cured M.

rosa cement, together with the fact of all BCPs containing Cys, may easily
lead to the assumption that intermolecular disulfide bonds contribute to the
polymerization of barnacle cement. However, in the interfacial Mrcp20k
(Kamino, 2001) and the bulk Mrcp52k (Kamino et al., 2012), all Cys residues
were believed to form intramolecular disulfide bonds. Raman spectroscopy
also failed to detect any intermolecular disulfide bonds in Balanus crenatus
cement (Wiegemann et al., 2006). Furthermore, A. amphitrite cement was
fully dissolved without supplying any reductant (So et al., 2016). Collectively,
it is unlikely that surface and bulk BCPs interact with each other by
intermolecular disulfide bonds. As depicted in Figure 5, So et al. (2017)
proposed that bulk BCPs could be internally cross-linked under the catalysis
of multiple oxidases. Similarly, these enzymes may also be able to catalyze
the cross-links between surface and bulk BCPs as all of them can offer lysine
to satisfy the requirements of those reactions. It is worth noting that some
researchers assume that barnacle cement polymerization is akin to blood
coagulation based on the identification of relevant enzymes in unsolidified
liquid cement, however, these authors have not discussed how they work on
different BCPs yet (Essock-Burns et al., 2019).
Coordination
As bulk BCPs self-assemble into amyloid fibers, the associations between

surface and bulk BCPs may be similar to the interactions between amyloid
fibers and amyloid fiber binding proteins. It has been reported that a
glycoprotein named the serum amyloid P component could bind to the sugar
chains of some pathogenic amyloid fibers via coordinate bonds that rely on
Ca2+, in order to protect them from being degraded (Pepys et al., 1997). It is a
member of the pentraxin family which exists in a wide variety of vertebrates.
A pentraxin family member was discovered in the marine arthropod
horseshoe crab (Shrive et al., 2009), indicating that homologous pentraxin
family members might exist in barnacles too. From current data, it is unlikely
that these hypothesized interfacial proteins (cp20k, cp19k, and cp68k) are
members of the pentraxin family due to the low sequence homology.
However, amino acid composition and sequence analysis suggest that there is
still a possibility that cp20k coordinates with sugar chains in barnacle cement
amyloid fibers under the mediation of Ca2+. Consistently, amyloidogenic bulk
Mrcp52k is glycosylated (Kamino et al., 2012). While the function of
glycosylation of Mrcp52k is not clear, the postulation that Mrcp52k uses its
sugar chains to associate with cp20k is reasonable. Intermolecular cross-
linking based on protein-sugar interactions was also discovered in other
marine bioadhesive systems. For example, Apfp-1, a protein that links the
byssus and soft foot tissues in the fan shell, Atrina pectinata, employs DOPA-
Fe3+ coordination to interact with other foot proteins and uses a lectin
binding domain to cross-link with mannose on the cell membrane in a Ca2+
dependent manner (Yoo et al., 2016).
Non-covalent Interactions
When using a surface force apparatus (SFA) to examine the cohesion force of
peptide analogs of Mefp-5, Gebbie et al. (2017) discovered that non-covalent
cation-π interactions between benzene rings of aromatic amino acids and
side-chain amine groups of positively charged amino acids can establish
strong intermolecular interactions, especially when no hydroxyl group is on
the benzene ring (Phe > Tyr > DOPA). This finding suggests that in barnacle
cement, the lysine-rich interfacial cp19k and cp68k may be able to strongly
interplay with the internal cp100k and cp52k, which have abundant Phe and
Tyr, through cation-π interactions. Importantly, cation-π interactions can
also effectively conquer the electrostatic repulsion among likely charged
BCPs in seawater (Kim et al., 2016, 2017).
In another study, Raman et al. (2016) designed two cp19k-inspired

decapeptides to better understand the adhesion behaviors of full-length
cp19k. One (GSGSVPPPCD, Bp1) contains only hydrophobic amino acids
while the other (GSKLDLLTDG, Bp2) has both hydrophobic and charged
amino acids. Using SFA to characterize their adhesion strength between mica
and gold (or SAMs), they found that Bp2 can firmly bridge the two surfaces
via electrostatic and hydrophobic interactions, whereas Bp1 fails.
Accordingly, they proposed that the heterogeneity of hydrophobic and
charged amino acids in cp19k can promote its ability to asymmetrically
bridge hydrophobic bulk cement and charged external substrates (Raman et
al., 2016).
Updated Molecular Model of Adult Barnacle

Underwater Adhesion
According to the above analysis, we now present an updated molecular model
of adult barnacle underwater adhesion by highlighting various hypothesized
adhesive protein-substrate and cohesive protein-protein interactions.
Acorn barnacles first release an oxidative fluid to clear and condition the
substratum, and then deposit fibrous cement for adhesion (Fears et al.,
2018). In barnacles with calcified bases, their cement is secreted into the
space between the outer cuticle and the external substrate, rather than the
calcite base and the external substrate as previously described in Kamino’s
model (Kamino, 2006). The cp20k was initially suggested to be a calcite-
specific binding protein playing a role in bridging the barnacle basal plate
and bulk cement, possibly through coordination. Owing to the presence of
this cuticle layer, this speculation now sounds not so reasonable, unless the
chitinous cuticle is torn during barnacle growth, and is permeable to barnacle
cement. Kamino’s model is based solely on biochemical studies of M. rosa
and therefore, cannot be broadly applied to different barnacle species, as the
physiological structures and cement protein compositions vary considerably
among species. This model postulates that cp19k and cp68k of M. rosa play
an interfacial adhesion role via non-covalent interactions. Although physical
interactions are relatively weak, their amino acid compositions, sequences,
and structures have all been evolutionally optimized to enhance the surface
binding ability. In comparison, in A. amphitrite cement, these two types of
proteins play both interfacial adhesion and bulk cohesion roles. They rely on
pendent catechol groups to establish surface coupling, as well as self-
assembled nanofibers and enzyme-catalyzed polymerization to achieve
curing (So et al., 2017). As for M. rosa, their bulk cement proteins (cp100k
and cp52k) can cure in the same way. In this barnacle, those same enzyme-
catalyzed reactions may also promote the interplay between surface and bulk
BCPs. Besides, interfacial cp19k and cp68k may be able to use cation-π and
hydrophobic interactions to interact with hydrophobic bulk BCPs too.
Given our poor knowledge of the cement protein compositions of stalked

barnacles, their underwater adhesion molecular mechanism has not been
discussed by any studies so far. Looking forward, conducting more accurate
studies regarding these hypothesized molecular interactions is essential and
may soon be possible by taking advantage of enhanced nanoscale and
microscale surface force characterization tools, such as AFM and SFA, as well
as microbial recombinant BCPs and their peptide analogs. Furthermore,
barnacle underwater adhesion is a challenging multidisciplinary research
field, to fully resolve the underlying molecular mechanism and develop
barnacle-inspired technologies also necessitate strengthened collaborations
from scientists and engineers with different research backgrounds.
Final Remarks
All the three reversible or irreversible underwater adhesion events involved
in a barnacle’s life cycle are accomplished by depositing and curing multi-
protein natural adhesives.
During surface exploration, the cyprid uses a pair of attachment discs that are
fully covered by cuticular villi to rapidly, tenaciously, and reversibly adhere to
the substrate. This adhesion process is chemically, physically, and
behaviorally co-mediated. The cyprid footprint is the temporary adhesive,
which mainly comprises basic proteins (Guo et al., 2016). The SIPC is the
only protein identified so far in cyprid temporary adhesive and plays multiple
roles including surface adhesion, biochemical signaling, and
biomineralization. Upon finding a suitable site, the cyprid instantly deposits
cyprid cement comprising lipids and phosphoproteins for irreversible
colonization. The two chemical phases are synthesized and stored in different
gland cells. When secreted, lipids are first released to create a niche
conducive to protein adhesion. After curing, lipids form the outer shell while
phosphoproteins constitute the internal core of the cyprid adhesive plaque
(Gohad et al., 2014). A few BCPs have been unexpectedly detected in cyprid
permanent adhesive glands, but whether they are cyprid cement components
and whether they assist in cyprid irreversible adhesion is currently not clear.
The adult barnacle periodically secretes barnacle cement during growth to
secure its underwater attachment. It first releases a fluid rich in lipids and
oxidants to prepare the substrate and then deposits proteinaceous cement
(Fears et al., 2018). Barnacle cement shows fibrillar morphologies and
comprises multiple proteins with distinctive amino acid compositions and
structural properties. Kamino suggests that different BCPs have different
spatial location in the cement layer, but they all rely on non-covalent
interactions to achieve adhesion or cohesion (Kamino, 2006). In parallel, the
Wahl group revealed the ecdysis-related oxidative stress at the barnacle
adhesive interface and proposed thereof an enzyme-catalyzed BCP cross-
linking hypothesis (So et al., 2017). Unfortunately, both these hypotheses
lack experimental evidences.
The biochemical compositions of cyprid adhesives and their relationships

with barnacle cement should be further investigated in detail. To gain a
better understanding of adult barnacle underwater adhesion, the secretion
sequence of different BCPs and their spatial distribution in the cement layer,
which have not yet been examined, also need to be addressed. In addition,
further studies can be conducted to substantiate these hypothesized
molecular interactions involved in the curing of barnacle cement.
Author Contributions
CL conceived this review and wrote the manuscript with help from all other
authors. JS, ZY, WW, BH, and DR provided the helpful suggestions and
revised the manuscript.
Funding
This work was supported by an award from the National University of
Defense Technology (ZK17-03-43).
Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any
commercial or financial relationships that could be construed as a potential
conflict of interest.
Acknowledgments
The authors thank the editor and reviewers for their constructive comments
on the manuscript.
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Keywords: underwater adhesion, barnacles, cyprid, surface exploration and settlement, cement
proteins, self-assembly, curing mechanism
Citation: Liang C, Strickland J, Ye Z, Wu W, Hu B and Rittschof D (2019) Biochemistry of Barnacle

Adhesion: An Updated Review. Front. Mar. Sci. 6:565. doi: 10.3389/fmars.2019.00565
Received: 27 April 2019; Accepted: 27 August 2019;

Published: 10 September 2019.
Edited by:
Andrew Stanley Mount (https://loop.frontiersin.org/people/136438/overview), Clemson University,
United States
Reviewed by:
Nick Aldred (https://loop.frontiersin.org/people/282855/overview), Newcastle University, United
Kingdom
Wong Yue Him (https://loop.frontiersin.org/people/771316/overview), Shenzhen University, China
Copyright © 2019 Liang, Strickland, Ye, Wu, Hu and Rittschof. This is an open-access article distributed
under the terms of the Creative Commons Attribution License (CC BY) (http://creativecommon-
s.org/licenses/by/4.0/). The use, distribution or reproduction in other forums is permitted, provided the
original author(s) and the copyright owner(s) are credited and that the original publication in this journal
is cited, in accordance with accepted academic practice. No use, distribution or reproduction is
permitted which does not comply with these terms.
*Correspondence: Biru Hu, hubiru08@nudt.edu.cn (mailto:hubiru08@nudt.edu.cn)
Disclaimer: All claims expressed in this article are solely those of the authors and do not necessarily
represent those of their a"liated organizations, or those of the publisher, the editors and the reviewers.
Any product that may be evaluated in this article or claim that may be made by its manufacturer is not
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(https://www.frontiersin.org/about/impact?utm_source=fweb&utm_medium=!

in Marine Science (https://www.frontiersin.org/journa

these different adhesion events in the life cycle of a barnacle. First, we

adhesives from two complementary aspects: the in vivo synthesis,

barnacle underwater attachment in detail by analyzing the possible Injectable

microbes, plants, and animals on target structures leading to millions of

To update and improve our understanding of the mechanisms behind these

Surface Exploration: Cyprid Temporary Adhesion

Surface Exploration: Why and How?

Adult Barnacle Underwater

Molecular Mechanism of Adult

As stated earlier, scanning electron microscopy (SEM) observations revealed

Settlement-Inducing Protein Complex (SIPC)

Through immunoblotting, a glycoprotein called the settlement-inducing

Surface Settlement: Cyprid Permanent Adhesion

Cyprid Cement Apparatus

Biochemical Composition of Cyprid Cement

As of date, no glue protein has been isolated from cyprid permanent

Adult Barnacle Underwater Adhesion

Synthesis, Storage, and Secretion of Barnacle

While insightful, these in vivo histological and histochemical studies of

Fibrillar Morphology of Barnacle Cement

By using microscale and nanoscale imaging tools, the fine structures of

It was noted that remnants of fibrillar barnacle cement on several substrates

Oxidative Chemistry of Barnacle Adhesive Interface

Isolation and Characterization of BCPs

Amino Acid Compositions

The sequence of Mrcp100k is relatively simple. It does not contain any

Molecular Mechanism of Adult Barnacle

Based on the pioneering studies on M. rosa cement proteins, Kamino built an

Interfacial Adhesion of Surface BCPs

The selective adhesion of cp20k to specific types of substrates probably

However, the original speculation that cp20k serves as a barnacle calcite

Furthermore, in situ observation found a cuticle layer underneath the A.

Cp19k and cp68k: Physical Interactions

Barnacles can tenaciously adhere to a wide range of substrates, indicating

From the amino acid composition of cp19k, it may be inferred that

Self-Assembled Amyloid Fibers

While investigating the protein components of A. amphitrite cement, So et al.

Originally, the above cross-linking reactions were thought to occur on A.

Interplays Between Surface and Bulk BCPs

The indispensability of a high concentration of reductant to dissolve cured M.

As bulk BCPs self-assemble into amyloid fibers, the associations between

In another study, Raman et al. (2016) designed two cp19k-inspired

Updated Molecular Model of Adult Barnacle

Given our poor knowledge of the cement protein compositions of stalked

The biochemical compositions of cyprid adhesives and their relationships

Conflict of Interest Statement

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