Biol. Rev. (2020), 95, pp. 1812–1837.
doi: 10.1111/brv.12640
1812
Deciphering mollusc shell production: the roles
of genetic mechanisms through to ecology,
aquaculture and biomimetics
Melody S. Clark1* , Lloyd S. Peck1, Jaison Arivalagan2,3†, Thierry Backeljau4,5,
Sophie Berland6, Joao C. R. Cardoso7, Carlos Caurcel8 , Gauthier Chapelle4,
Michele De Noia9,10†, Sam Dupont11, Karim Gharbi8, Joseph I. Hoffman9, Kim S. Last12,
Arul Marie2, Frank Melzner13, Kati Michalek12, James Morris4, Deborah M. Power7,
Kirti Ramesh13, Trystan Sanders13, Kirsikka Sillanpää14, Victoria A. Sleight15,
Phoebe J. Stewart-Sinclair12 , Kristina Sundell14, Luca Telesca16,
David L. J. Vendrami9, Alexander Ventura11, Thomas A. Wilding12, Tejaswi Yarra1,8 and
Elizabeth M. Harper16
1
British Antarctic Survey, Natural Environment Research Council, High Cross, Madingley Road, Cambridge, CB3 0ET, U.K.
UMR 7245 CNRS/MNHN Molécules de Communications et Adaptations des Micro-organismes, Sorbonne Universités, Muséum National
d’Histoire Naturelle, Paris, France
3
Proteomics Center of Excellence, Northwestern University, 710 N Fairbanks Ct, Chicago, IL, U.S.A.
4
Royal Belgian Institute of Natural Sciences, Rue Vautier 29, Brussels, B-1000, Belgium
5
Evolutionary Ecology Group, University of Antwerp, Universiteitsplein 1, Antwerp, B-2610, Belgium
6
UMR 7208 CNRS/MNHN/UPMC/IRD Biologie des Organismes Aquatiques et Ecosystèmes, Sorbonne Universités, Muséum National
d’Histoire Naturelle, Paris, France
7
Centro de Ciencias do Mar, Universidade do Algarve, Campus de Gambelas, Faro, 8005-139, Portugal
8
Ashworth Laboratories, Institute of Evolutionary Biology, University of Edinburgh, Charlotte Auerbach Road, Edinburgh, EH9 3FL, U.K.
9
Department of Animal Behavior, University of Bielefeld, Postfach 100131, Bielefeld, 33615, Germany
10
Institute of Biodiversity Animal Health and Comparative Medicine, University of Glasgow, Glasgow, G12 8QQ, U.K.
11
Department of Biological and Environmental Sciences, University of Göteburg, Box 463, Göteburg, SE405 30, Sweden
12
Scottish Association for Marine Science, Scottish Marine Institute, Oban, Argyll, PA37 1QA, U.K.
13
GEOMAR Helmholtz Centre for Ocean Research, Kiel, 24105, Germany
14
Swemarc, Department of Biological and Environmental Science, University of Gothenburg, Box 463, Gothenburg, SE405 30, Sweden
15
School of Biological Sciences, University of Aberdeen, Zoology Building, Tillydrone Avenue, Aberdeen, AB24 2TZ, U.K.
16
Department of Earth Sciences, University of Cambridge, Cambridge, CB2 3EQ, U.K.
2
ABSTRACT
Most molluscs possess shells, constructed from a vast array of microstructures and architectures. The fully formed shell is
composed of calcite or aragonite. These CaCO3 crystals form complex biocomposites with proteins, which although typically less than 5% of total shell mass, play significant roles in determining shell microstructure. Despite much research
effort, large knowledge gaps remain in how molluscs construct and maintain their shells, and how they produce such a
great diversity of forms. Here we synthesize results on how shell shape, microstructure, composition and organic content
vary among, and within, species in response to numerous biotic and abiotic factors. At the local level, temperature, food
supply and predation cues significantly affect shell morphology, whilst salinity has a much stronger influence across latitudes. Moreover, we emphasize how advances in genomic technologies [e.g. restriction site-associated DNA sequencing
* Address for correspondence (Tel: +44 1223 221371; E-mail: mscl@bas.ac.uk).
†
Current address.
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
Society.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium,
provided the original work is properly cited.
Mollusc shell production
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(RAD-Seq) and epigenetics] allow detailed examinations of whether morphological changes result from phenotypic plasticity or genetic adaptation, or a combination of these. RAD-Seq has already identified single nucleotide polymorphisms
associated with temperature and aquaculture practices, whilst epigenetic processes have been shown significantly to modify shell construction to local conditions in, for example, Antarctica and New Zealand. We also synthesize results on the
costs of shell construction and explore how these affect energetic trade-offs in animal metabolism. The cellular costs are
still debated, with CaCO3 precipitation estimates ranging from 1–2 J/mg to 17–55 J/mg depending on experimental
and environmental conditions. However, organic components are more expensive (29 J/mg) and recent data indicate
transmembrane calcium ion transporters can involve considerable costs. This review emphasizes the role that molecular
analyses have played in demonstrating multiple evolutionary origins of biomineralization genes. Although these are characterized by lineage-specific proteins and unique combinations of co-opted genes, a small set of protein domains have
been identified as a conserved biomineralization tool box. We further highlight the use of sequence data sets in providing
candidate genes for in situ localization and protein function studies. The former has elucidated gene expression modularity in mantle tissue, improving understanding of the diversity of shell morphology synthesis. RNA interference (RNAi)
and clustered regularly interspersed short palindromic repeats - CRISPR-associated protein 9 (CRISPR-Cas9) experiments have provided proof of concept for use in the functional investigation of mollusc gene sequences, showing for
example that Pif (aragonite-binding) protein plays a significant role in structured nacre crystal growth and that the Lsdia1
gene sets shell chirality in Lymnaea stagnalis. Much research has focused on the impacts of ocean acidification on molluscs.
Initial studies were predominantly pessimistic for future molluscan biodiversity. However, more sophisticated experiments incorporating selective breeding and multiple generations are identifying subtle effects and that variability within
mollusc genomes has potential for adaption to future conditions. Furthermore, we highlight recent historical studies
based on museum collections that demonstrate a greater resilience of molluscs to climate change compared with experimental data. The future of mollusc research lies not solely with ecological investigations into biodiversity, and this review
synthesizes knowledge across disciplines to understand biomineralization. It spans research ranging from evolution and
development, through predictions of biodiversity prospects and future-proofing of aquaculture to identifying new biomimetic opportunities and societal benefits from recycling shell products.
Key words: integrative biomineralization, calcification, calcium, skeleton, adaptation, phenotypic plasticity, ion channels,
Crassostrea, Pinctada, Mytilus
CONTENTS
I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1814
II. Shell morphology: genetic adaptation and phenotypic plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1814
(1) Shell structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1814
(2) Shell phenotypic variability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1816
(3) Phenotypic plasticity, adaptation and epigenetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1817
(4) Genetic background and gene flow among populations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1819
III. The cost of making a shell and energetic trade-offs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1820
(1) Costs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1820
(2) Trade-offs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1821
IV. Cellular calcium transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1821
(1) Mantle tissue and calcium turnover . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1821
(2) Identification of ion channels involved in calcium transport across membranes . . . . . . . . . . . . . . . .1823
V. Mollusc genes and proteins associated with shell production . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1823
(1) Gene transcripts and proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1823
(2) Mollusc genomes and comparative genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1824
(3) The evolution of shell production in molluscs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1824
(4) A conserved biomineralization tool box . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1825
(5) Further levels of complexity: alternative splicing, isoforms and development . . . . . . . . . . . . . . . . . .1825
VI. Biomineralization genes and proteins: functionality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1826
(1) Cellular localisation of gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1826
(2) Protein activity associated with biomineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1827
(3) Disruption of biomineralization to identify gene function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1828
VII. Producing the three-dimensional structure of the shell . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1828
VIII. Future prospects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1829
(1) Molluscs in a changing world . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1829
(2) Mollusc shells for future innovation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .1830
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
Society.
Melody S. Clark et al.
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IX.
X.
XI.
XII.
Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1831
Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1831
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1832
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1832
I. INTRODUCTION
Molluscs are amongst the most successful phyla with
70000–76000 currently described species and calculated estimates of up to 200000 species (Rosenberg, 2014). A major
part of the success of the Mollusca is often claimed to be their
biomineralized shells, although some groups, including most
cephalopods (e.g. squid and octopus) and some gastropods
(e.g. nudibranchs) have secondarily lost an external shell.
Their biomineralized shells have also produced a long and
rich evolutionary history in the fossil record. Yet, the heavily
calcified shells in bivalves and gastropods are now seen as a
potential vulnerability under future environmental change,
which has fuelled research into molluscan biomineralization.
In recent years, the main focus on the consequences for molluscs under changing conditions has been on ocean acidification. The continuing reduction in seawater pH and
associated changes in carbonate chemistry mean that skeletons
of marine species are more energetically expensive to produce
with consequences for the ability of molluscs to construct and
maintain their calcium carbonate skeletons (Wittmann &
Pörtner, 2013). However, numerous studies demonstrate that
temperature, salinity and hypoxia can have greater impacts
than acidification on animal energetics: altering metabolic
rates, increasing nutritional requirements and affecting the
food supply itself (e.g. Dickinson et al., 2012; Clark
et al., 2013; Hiebenthal et al., 2013; Telesca et al., 2018,
2019). The majority of these observations are based on laboratory experiments, examining the effects of one or two (at most)
stressors on molluscs. In reality, the marine environment is far
more complex with multiple interacting biotic and abiotic
drivers that can significantly impact animal homeostasis. It is
difficult, if not impossible, to incorporate the wide range of
environmental heterogeneity, natural fluctuations and dispersal opportunities over different spatial scales into laboratory experiments (Sanford & Kelly, 2011; Urban et al., 2016).
Thus, it is perhaps not surprising that as experimental protocols become more complicated, using, for example, long
experimental timescales and multiple generations, significant
resilience is being found within populations to altered environmental conditions, much more than was predicted 10 years
ago (Clark, 2020). Whilst such experimental approaches have
limitations for predicting future biodiversity patterns at assemblage and ecosystem levels, they have two major advantages.
Namely, they can indicate differences in resilience among species and aid in uncovering the mechanisms underpinning molluscan responses to changing environments. The latter may
include evaluations of cellular energetics, transport of calcium
and the genes and proteins involved in shell production, thus
contributing to fundamental knowledge on how molluscs build
and maintain their shells.
It should be also emphasized that whilst ecological studies
evaluating molluscs’ resilience to climate change have taken
centre stage over recent years, there are many other areas
of research where a greater understanding of biomineralization is important. These include evolution of species and particular proteins, global carbon cycling and more applied
areas including optimization of aquaculture practices, development of novel bio-inspired materials (biomimicry) and reuse of shells for societal sustainability (Clark, 2020). Irrespective of the research field, what the recent ecological and
molecular-related studies highlight is that a full mechanistic
understanding of shell production, even in a single species is
an enormous task. Large knowledge gaps still exist in the fundamental understanding of how molluscs construct and
maintain their shells, and indeed in how they produce so
many diverse forms.
The aim of this review is to take a multidisciplinary
approach synthesizing current knowledge on how molluscs
produce shells. Starting with shell structures, it encompasses
mineralogy, physiology, ecology and molecular biology as
all these underpin successful mollusc shell production. Discussions centre on state-of-the-art knowledge of the molluscan genetic landscape (genes, proteins, population genetics
and epigenetics) and methodologies to increase the understanding of gene functions associated with biomineralization. Examples concentrate on model species for which
most knowledge exists, such as the commercially important
oysters Crassostrea spp., pearl oysters Pinctada spp. and blue
mussels Mytilus spp., with examples drawn from other
bivalves and gastropods as appropriate. The final
section briefly reviews socio-economic aspects with regard
to future prospects for molluscs in adapting to environmental change, their sustainable exploitation for aquaculture,
and provision of novel products for society, including biomimetic applications and how waste shell material contributes to the circular economy.
II. SHELL MORPHOLOGY: GENETIC
ADAPTATION AND PHENOTYPIC PLASTICITY
(1) Shell structure
Molluscan shells comprise layers of hierarchically arranged
biocomposite materials in which a stiff mineralized phase is
embedded in a softer organic matrix. Although there is great
diversity in shell microstructure and architecture, the basic
shell production process is shared among shelled molluscs
wherein a fleshy mantle secretes the calcareous shell onto a
proteinaceous sheet, the periostracum. Most shell production
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
Society.
Mollusc shell production
research has been on bivalves, so the following account uses
this group to illustrate the processes involved.
The two laterally disposed bivalve shells enclose the viscera, and in the post-larval stages are secreted by the mantle
lobes, which are thin layers of tissue underlying both valves
(Fig. 1). The distal edge of each mantle lobe is divided into
folds, typically three in most bivalves, although there are
exceptions where the outer fold is duplicated (e.g. Waller, 1980; Harper & Morton, 1994) [the arrangement is similar in the shelled cephalopod Nautilus but for most
gastropods the typical arrangement is only two marginal
folds – see Stasek & McWilliams (1973)]. While the innermost and middle folds are chiefly concerned with water
inflow and sensory functions, respectively, the outermost fold
secretes the shell (Yonge, 1957, 1982; Ponder &
Lindberg, 2020). The first shell material formed is the periostracum, a thin (from submicrons to tens of microns thick)
layer of quinone-tanned proteins, mucopolysaccharides,
and lipids, secreted by specialized cells in the periostracal
groove which lies between the outermost and middle mantle
folds (Saleuddin & Petit, 1983; Harper, 1997). The forming
sheet, secreted in the periostracal groove, extends and
thickens by secretion from the inner surface of the outer mantle lobe, then matures by tanning once the thickening is complete (Saleuddin & Petit, 1983) (Fig. 1). Ultimately, the
periostracal sheet reflects dorsally, forming the shell edge.
The periostracum’s primary function is to define and enclose
the space in which the shell is secreted. In taxa with a thin
periostracum, it often decays or abrades and may not persist
over older parts of the shell. However, in taxa with a thicker
periostracum it may remain on outer shell surfaces, where it
plays important secondary roles including protection from
corrosion (Tevesz & Carter, 1980). Although some bivalves
initially mineralize within the periostracum layer (Checa &
Harper, 2010), calcareous shell is predominantly laid down
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onto the periostracum by the epithelium of the outer mantle
surface. Typically, figures in scientific articles about shell formation show an apparently capacious void, the extrapallial
space, between the outer mantle surface and the most
recently secreted shell (e.g. Harper, 1997; Checa, 2000).
However, the actual gap is very narrow, around only
100 nm in the bivalve Neotrigonia spp. (Checa et al., 2014),
housing a thin film of extrapallial fluid (Fig. 1).
Molluscs display an extraordinary diversity of shell microstructure, each shell comprising two or more layers with different microstructural (and sometimes mineralogical)
arrangements (Fig. 2). The precise microstructural detail
has considerable phylogenetic and functional significance
(Taylor, Kennedy, & Hall, 1969, 1973). The mineralized
component of any particular microstructural layer in fully
formed shell is always either calcite or aragonite [although
the unstable polymorph vaterite may occur pathologically
in patches or repairs, e.g. Nehrke et al. (2012) and patches
of calcite have been identified in the aragonitic shell layers
of some individuals of chamid bivalves (Harper, 1998)].
There is also good palaeontological and phylogenetic evidence that aragonite is the ancestral molluscan state
(Taylor, 1973; Vendrasco, Checa, & Kouchinsky, 2011;
Wood & Zhuravlev, 2012). Three-dimensional arrangements of mineral units vary widely, and also occur over a
wide range of scales (Fig. 2). These units may be multiple
crystals (Taylor, 1973), with each crystal composed of
nano-units or particles as revealed by atomic force microscopy (Dauphin, 2001). The mineralized units of calcite and
aragonite microstructures fall into two broad categories:
fibres (including prisms, gastropod nacre and granules of various types) and sheets (including bivalve and cephalopod
nacre, foliae and crossed-lamellar structures) (Checa &
Salas, 2017). Despite the apparent diversity of shell microstructures, they appear to share common controls of patterns
Fig 1. Schematic of bivalve morphology. (A) Generalized bivalve morphology showing the main internal features. (B) Close-up of
growing shell edge showing different mantle folds and their relationship with the shell, periostracum and extrapallial space.
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
Society.
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Melody S. Clark et al.
Fig 2. Scanning electron micrographs showing the diversity of bivalve shell microstructures. (A) Aragonite prisms and nacre in
Aspatharia pfeifferiana. Scale bar = 50 μm. (B) Nacre (aragonite) in A. pfeifferiana. Scale bar = 10 μm. (C) Crossed-lamellar aragonite
in Ctenioides scabra. Scale bar = 5 μm. (D) ‘Homogeneous’ aragonite in Entodesma navicula. Scale bar = 5 μm. (E) Calcite prisms overetched showing thick organic envelopes in Isognomon legumen. Scale bar = 10 μm. (F) Foliated calcite in Crassostrea gigas. Scale
bar = 2 μm. (G) Chalk (calcite) in Ostrea edulis. Scale bar = 5 μm. (H) Lamellar vaterite with acicular aragonite in Corbicula fluminea.
Scale bar = 2 μm. All are fractured surfaces, except E and H, which are acid etched. Micrograph H taken by Max Frenzel.
of nucleation and growth. Surprisingly, many of the microstructures have evolved independently across or within different molluscan classes. For example, nacre has been
separately acquired in bivalves, gastropods and cephalopods,
and there are clear differences in the crystallographic detail
among groups (Vendrasco et al., 2011). Shell organic matrix
is composed largely of proteins, acidic polysaccharides and
chitin (Weiner & Dove, 2003). It is usually only a relatively
small percentage of the shell (<5%, although it is 16% of
the dry mass in certain unusual microstructures in Fan shells,
Pinna spp.), but it appears to have extremely important roles
in pre-determining the form of many shell microstructures.
For example, in shell microstructures where there is a clear
organic matrix the constituent crystalline units show strict
morphological control and orientation whereas in those
where there is little or no organic material, the growth of
crystals is more reminiscent of simple inorganic spherulitic
growths (Checa et al., 2016), as discussed in Sections V and
VI). Thus, shells are complex biocomposite materials, usually
with their structure and composition under tight taxonspecific control, but which are further modified by habitat
conditions.
(2) Shell phenotypic variability
Mollusc shell structure can be considerably influenced by a
variety of abiotic and biotic factors. The intertidal zone can
be particularly challenging, with the rigours of periodic
emersion, desiccation and increased temperatures due to
solar radiation significantly affecting shell morphologies,
but also animal distributions and metabolic rates
(Davies, 1966, 1969). Intertidal limpets generally exhibit taller, more ridged shells (providing more surface area for convective heat loss) compared with sub-tidal conspecifics
(Harley et al., 2009). Wave exposure also affects shell morphology: the nodulose form of the intertidal periwinkle Tectarius striatus (previously Littorina striata) inhabits sheltered
areas, whilst the smooth form is common in wave-exposed
areas (De Wolf, Backeljau, & Verhagen, 1998). Different
shell morphologies have likewise been described in a related
intertidal periwinkle Littorina saxatilis (Johannesson, 1986) and
the flat tree oyster Isognomon alatus (Wilk & Bieler, 2009). In
Antarctica, ice is an added environmental stress. In response
to frequent brash ice impact, intertidal limpets Nacella concinna
produce thicker, taller shells and the infaunal clam Laternula
elliptica produces more robust shells in regions with high iceberg scour compared to protected sites (Hoffman
et al., 2010; Harper et al., 2012).
Thicker shells are sometimes produced in response to predation threat, often sensed via chemical cues. The classic
examples include intertidal snails and mussels that thicken
shells in habitats where predatory crabs are present
(e.g. Appleton & Palmer, 1988; Trussell & Smith, 2000;
Trussell & Nicklin, 2002; Freeman & Byers, 2006). Chemical
cues inducing thickening can be either from the predator
and/or from damaged conspecifics (Appleton &
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
Society.
Mollusc shell production
Palmer, 1988; Freeman, Meszaros, & Byers, 2009). The
invasive mussel Xenostrobus securis produces thicker shells,
stronger byssal attachments and heavier adductor muscles
when drilling predators are present (Babarro, Vazquez, &
Olabarria, 2016). Similarly, the blue mussel Mytilus edulis
produces smaller, thicker shells with significantly larger
adductor muscles in the presence of the predatory starfish
Asterias rubens (Freeman & Byers, 2006). Interestingly, when
exposed singularly to crabs (crushing predators) or to starfish
(which prise apart the shell) M. edulis develops either a thicker
shell or larger adductor muscles, respectively. When both
cues are introduced at the same time, M. edulis did not express
either predator-specific response, indicating that such
responses are poorly integrated (Freeman et al., 2009).
The altered shell morphologies produced in response to
environmental factors are not necessarily uniform across all
the component microstructures. There may be selective deposition of different shell layers according to the stimulus. For
example, shell thickening in the periwinkle Littorina obtusata in
response to predation was due to a 91% increase in the irregular prismatic calcite layer in new shell (Brookes &
Rochette, 2007). In Pecten maximus scanning electron microscopy and atomic force microscopy revealed that weaker shells
produced in suspended aquaculture systems compared with
wild-caught animals were due to the development of modifications and abnormalities in different microstructure components
over time (Grefsrud et al., 2008). In fact, detailed monitoring
found that changes in water chemistry, food and nutrient supply operating at very small spatial scales allowed microenvironments to be detected along single mussel ropes that impacted
shell production (Michalek, 2019). At larger spatial scales Mytilus species studied across a 30 latitudinal range (3980 km spanning from the North Atlantic to the Arctic) identified
temperature and food supply as the main drivers of mussel
shape heterogeneity (Telesca et al., 2018, 2019). Salinity, however, had the strongest effect on latitudinal patterns of shell
shape. Salinity was also the major driver of shell deposition,
organic content and microstructures (Fig. 3A, B). Mussels in
low-salinity environments had thin shells and higher proportions of prismatic calcite (compared to aragonite) and organic
matrix and a thicker periostracum. Mussels in high-salinity
environments produced thicker shells with an increased proportion of the aragonitic nacreous layer, potentially providing
enhanced mechanical protection against predators (Telesca
et al., 2019) (Fig. 3A, C, D). In addition, there was a strong interaction between decreasing salinity and increasing food supply,
resulting in thicker periostraca, as a potential protection mechanism against the corrosive effects of low salinity (Telesca
et al., 2019) (Fig. 3D). Thus, many environmental factors significantly affect molluscan shell morphologies. These variant shell
morphologies may be underpinned by genetic mechanisms.
(3) Phenotypic plasticity, adaptation and
epigenetics
Variation in shell characteristics can often be attributed to
multiple environmental factors (as detailed above). However,
1817
the relative contribution of each factor to altered shell morphology and the underlying genetic mechanisms is often
unknown (e.g. Johannesson, 1986; Solas et al., 2015). Two
main genetic mechanisms may bring about this variation.
The first is genetic determination of shell characteristics, with
selection acting on standing genetic variation within populations and driving selection for characteristics more suited to
the local conditions. The second is phenotypic plasticity,
where a given genotype can produce different phenotypes
in different environments. These mechanisms are not mutually exclusive and many studies have shown them acting in
concert. Furthermore, epigenetic effects may fix favourable
traits, at least temporarily, across generations. Potential
genetic mechanisms have been investigated using several
approaches, including controlled breeding, reciprocal transplant and common garden experiments.
Genetic crosses between either half-sib pairs or different
ecotypes have demonstrated significant genetic underpinning
of shell characteristics in, for example Littorina spp., Bembicum
vittatum and Nucella lapillus (Boulding & Hay, 1993;
Parsons, 1997; Guerra-Varela et al., 2009), with the Littorina
experiments also indicating significant genotype–
environment interactions (Boulding & Hay, 1993). Adaptation of shell characteristics to altered conditions may be
rapid. For example, M. edulis occur all along the North American New England coastline. In northern New England,
M. edulis had been exposed to predation by the common crab
Carcinus maenas for >50 years, but had never encountered the
invasive predatory crab Hemigrapsus sanguineus. By contrast,
southern New England M. edulis had been exposed to
C. maenas for over 100 years and H. sanguineus for around
15 years (Freeman & Byers, 2006). When southern and
northern New England M. edulis populations were experimentally exposed to H. sanguineus, shell thickening was weaker
in northern populations that never been exposed to
H. sanguineus. This difference was attributed to rapid adaptation (over 15 years) of the southern populations (Freeman &
Byers, 2006). In recent laboratory experiments, adaptation
of M. galloprovincialis to low pH was associated with changes
in allele frequency (Bitter et al., 2019). It was suggested that
polygenic characters governed evolution to low pH and
genotype–environment interactions released ‘cryptic’ genetic
variation of fitness-related traits. Furthermore, three M. edulis
generations grown in CO2-enriched conditions revealed heritable components of calcification performance in early
development (Thomsen et al., 2017) and rapid adaptation
in shell thickness to this corrosive environment occurred in
M. chilensis (Guiñez et al., 2017).
Reciprocal transplant experiments have been performed
to test the prediction that shell shape and thickness are plastic
traits, with transplanted animals adjusting their morphologies to those of native animals. Whilst experiments analysing shell shape characteristics in Lymnaea stagnalis indicated
strong environmental control (Arthur, 1982), similar transplant experiments revealed both genetic control and phenotypic plasticity in the intertidal gastropods Nodilittorina
australis, Nucella lapillus and Acanthina monodon (Yeap, Black, &
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
Society.
Melody S. Clark et al.
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(B)
High salinity
-0.5
0.0
(C)
0.5
Shell thickness (µm)
Temperate
Polar
***
1.0
NS
2.0
1500
1250
NS
1.5
1.0
1000
0.5
750
0.0
500
FR
250
Periostracum thickness (µm)
(D)125
850
Layer thickness (µm)
Low salinity
-1.0
Organics %
Shell shape variance
(A)
Prismatic layer (calcite)
Nacreous layer (aragonite)
700
550
400
250
UK
NO
Location
GL
Low salinity
Avarage salinity
High salinity
100
75
50
25
100
5
10
15
20
25
Salinity (PSU)
30
35
30
40
50
60
Shell length (mm)
70
80
Fig 3. Influence of water salinity on Mytilus spp. shell shape, deposition and microstructure. (A) Top panel: shell shape variation of
lateral and ventral shell views with salinity indicating formation of more elongated, narrower shells and more parallel dorsoventral
margins with decreasing salinities. Middle panel: positive exponential increase of shell deposition (total thickness) with increasing
salinity. Bottom panel: relationships between the thickness of prismatic (solid line) and nacreous (dashed line) layers, and salinity
indicating a decreased proportion of prismatic calcite with increasing salinity and the deposition of relatively thicker aragonitic
nacre at salinities >27.67 psu. Mean values (lines) and confidence intervals (shaded areas) are predicted while controlling for shell
size (47.42 mm). (B) Deformation grids for both lateral and ventral Mytilus shell depicting the outline regions subject to different
degrees of change and the bindings required to pass from the average shape under low and high salinity regimes. (C) Latitudinal
variation of shell organic content within prismatic layers among shells from temperate [Brest, France (FR) and St. Andrews,
United Kingdom (UK); open bars] and polar [Tromsø, Norway (NO) and Upernavik, Greenland (GR); solid bars] regions.
Pairwise contrasts indicate significantly (***) higher proportions of organics (+29%) in high-latitude than low-latitude specimens
with non-significant (NS) variation within climatic regions. Error bars indicate 95% CI. (D) Interacting effects of salinity and shell
length on Mytilus periostracum. Periostracum thickness is modelled as a function of shell length for low (18.92 psu; solid line),
average (25.52 psu; dashed line) and high (33.13 psu; dotted line) salinities. Predicted values (lines) and confidence intervals
(shaded areas) indicate higher rates of exponential periostracal thickening with decreasing salinity. Redrawn from Telesca
et al. (2018, 2019).
Johnson, 2001; Pascoal et al., 2012; Solas, Sepulveda, &
Brante, 2013). With common garden experiments, animals
from different environments are cultivated in common conditions and expected to become more similar and lose the
characteristics of their original environments (e.g. Pascoal
et al., 2012; Clark et al., 2018). Again, a mix of responses
occurred with these experiments in different species. Shell
characteristics were genetically determined in Littorina saxatilis (Johannesson & Johannesson, 1996), but more likely
resulted from phenotypic plasticity in the freshwater snail
Semisulcospira reiniana (Urabe, 1998). Interestingly in transgenerational reciprocal transplant and common garden
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Mollusc shell production
experiments in Nucella lapillus, shell shape converged, suggesting synergy between maternal effects, genetic and plastic
responses (Pascoal et al., 2012). Shell shape also converged
in freshwater snails in a multiple-generation experiment;
however, there was a lag in convergence suggesting epigenetic effects (Gustafson et al., 2014). This last experiment
highlights one issue of using single-generation experiments.
Complete roles of genetic factors involved in shell morphology may be masked by epigenetic effects, which can take several generations to diminish or change. Epigenetics can
significantly modify responses, and the extent of epigenetic
regulation in invertebrates is seriously underestimated
(Suarez-Ulloa, Gonzalez-Romero, & Eirin-Lopez, 2015).
An early epigenetic study in molluscs using methylationsensitive polymerase chain reaction (PCR) and bisulphite
sequencing revealed extensive CpG methylation in Crassostrea
gigas (Gavery & Roberts, 2010). To date, epigenetic studies in
molluscs have predominantly involved methylation analyses,
often studying developmental effects (Fallet et al., 2020),
although other mechanisms include remodelling of chromatin structure through chemical changes to histone proteins
and regulation by small RNA molecules. Thus, epigenetic
effects may result from a mix of mechanisms in natural environments (Suarez-Ulloa et al., 2015). Epigenetic data on shell
production in molluscs are limited. In the Antarctic limpet
Nacella concinna, intertidal and subtidal individuals have significantly different shell masses, yet exhibit no significant population genetic differentiation (Hoffman et al., 2010).
Transplant and common garden experiments alongside
measurements of methylation sensitive amplification polymorphisms ((MSAPs) or methylation-sensitive amplified fragment length polymorphisms (AFLPs)) revealed that these
cohorts (intertidal and subtidal) had significantly different
methylation patterns. However, these disappeared after
9 months in a common garden experiment, suggesting that
at least some of their environmental acclimation was associated with reversible epigenetic effects through DNA methylation (Clark et al., 2018). Similarly, methylation patterns
correlated with adaptation to local habitats in the
New Zealand mud snail (Thorson et al., 2017).
The multiple factors involved in producing different shell
characteristics and the contribution of each will almost certainly be species- and population-specific. Many experiments, whilst useful for demonstrating trait heritability, did
not necessarily identify the genes responsible for morphological variation. Studies of morphological variation should consider the organism’s genetic background along with gene flow
between populations, as these analyses help to determine
whether responses are mediated by phenotypic plasticity or
adaptation (Michalek, 2019).
(4) Genetic background and gene flow among
populations
For many years, marine environments were thought to be
demographically open, especially for broadcast-spawning
species with planktonic larvae (Hellberg, 2009). The genetic
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background and gene flow between morphologically conspecific mollusc populations has been evaluated using a variety
of methods, including allozymes, random amplified polymorphic DNA (RAPD), AFLPs and microsatellites
(De Wolf et al., 1998; Hoffman et al., 2010; Zieritz
et al., 2010; Pascoal et al., 2012). These studies demonstrated
that heterogeneity in shell shape and thickness in Antarctic
limpets and freshwater mussels persist while no population
differentiation could be demonstrated in neutral markers,
suggesting that these characteristics may have a phenotypic
plasticity component (Hoffman et al., 2010; Zieritz
et al., 2010). The current low-cost sequencing techniques,
such as restriction site-associated DNA sequencing (RADseq), which allow fine-scale population structure analysis,
deliver resolutions not previously possible. As a result, significant population structure is being demonstrated, even in
broadcast-spawning molluscs (Van Wyngaarden et al.,
2018; Bernatchez et al., 2019; Vendrami et al., 2019a,
2019b). Understanding population structure is essential for
studying traits that likely result from phenotypic plasticity,
local adaptation or mixtures of both.
This approach was exemplified in a recent shell morphology study in the great scallop Pecten maximus along the Northern Irish coast (Vendrami et al., 2017). Nine populations were
genotyped using microsatellite markers and single nucleotide
polymorphisms (SNPs) (Vendrami et al., 2017). Microsatellites revealed little genetic differentiation among populations,
but using just five animals per site with RAD-Seq, Mulroy
Bay (which has long been used for commercial farming of
scallops) was separated from the eight other sites which contain wild unfarmed populations (Fig. 4A, B). Significant shell
shape and colour differences were evident in the eight genetically undifferentiated populations, suggesting these traits
have plastic components (Vendrami et al., 2017) (Fig. 4C).
However, in spite of these detailed SNP analyses, this study
could not discount local adaptation or that shell shape and
pigmentation were controlled by one or a small number
of loci.
Furthermore, whilst highly sensitive at identifying population structure, RAD-Seq is limited with regards to identifying
mechanisms underpinning traits because it analyses anonymous markers (short stretches of typically unannotated
DNA). Although SNPs associated with environmental variables can be identified, for example temperature (Van Wyngaarden et al., 2018; Vendrami et al., 2019b), it is usually not
possible to determine the causal underlying polymorphisms,
limiting our understanding of mechanisms and functions
(Van Wyngaarden et al., 2018). Mapping SNPs produced
by RAD-Seq onto reference genomes may enable the identification of genomic regions potentially under selection
(Bernatchez et al., 2019). However, for the vast majority of
molluscs, reference genomes are not available. In this respect,
the real advantage of RAD-Seq is in allowing fine-scale
genome-wide mapping of genetic backgrounds and gene flow
among populations, which can significantly impact experimental design when studying divergent shell shapes in conspecifics or closely related congenerics.
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
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Melody S. Clark et al.
Fig 4. Genetic structure and morphological variation in scallops (Pecten maximus) from Northern Ireland. (A) Map of sites produced
courtesy of Laura Gerrish, Esri, Garmin, GEBCO, NOAA NGDC and other contributors. (B) Panels (a) and (b) show scatterplots
of individual variation in the first two principal components (PCs) derived from principal component analysis conducted on
180 individuals genotyped at 13 microsatellites and 45 individuals genotyped at 10539 single nucleotide polymorphisms (SNPs),
respectively. Inertia ellipses are reported and colour-coded differently for each population. The outlier population in panel (b) is
from Mulroy Bay (site 1 in A): site of the commercial scallop farm. Panel (c) shows phenotypic shell variation among three scallop
shells from locations 1, 8 and 9, respectively. Data as reported by Vendrami et al. (2017).
III. THE COST OF MAKING A SHELL AND
ENERGETIC TRADE-OFFS
(1) Costs
Shells comprise a major proportion of marine molluscan biomass. Typically >95% of a shell comprises calcium carbonate crystals and the energetic costs during construction
derive from the metabolic costs of accumulating, transporting and precipitating these crystals. Separating these costs
from routine metabolism is non-trivial due to the overlapping
contributions of physiological processes in metabolic maintenance, soft tissue growth and calcification (Palmer, 1992). In
the first experiments to quantify these costs, Palmer (1983,
1992) measured how much extra shell material was produced
by groups of marine snails under particular sets of environmental conditions and the associated amount of food (energy)
ingested in those conditions. The costs of calcification for
Nucella lamellosa and N. lapillus were estimated by calculating
the extra energy assimilated per unit of extra shell produced
at a common rate of tissue growth after extracting the cost of
the organic matrix. Using this approach Palmer (1992) estimated that producing 1 mg of calcium carbonate cost 1–2 J
with the equivalent mass of proteinaceous organic matrix
costing 29 J/mg. Similar calcification costs were estimated
for oyster larvae (Waldbusser et al., 2013) based on calculations of the energy demand of transmembrane ion transporters, thought to be important in molluscan calcification.
These 1–2 J/mg costs were queried by Sanders et al. (2018)
using energy budget calculations derived from scope for
growth experiments of Baltic Mytilus spp. living at low salinity. The Sanders et al. (2018) calculations derived from experiments combining data from three different salinities, three
feeding regimes and two temperatures. Their data indicated
that shell production required much more energy than previously suggested. They calculated a cost of 10–55 J/mg calcium carbonate, which was 31–60% of available energy.
However, they emphasized that these calculations were for
low-salinity experiments and energetic costs decreased when
salinity increased (17–55 J/mg for 6 psu and 10–14 J/mg for
11 and 16 psu). Therefore, estimating these costs for Mytilus
mussels at full salinities (33 psu) could produce figures close
to those of Palmer (1992), especially as there are extra costs
experienced in low-salinity conditions. Solubility of calcification substrates decreases with salinity and therefore HCO3−
extraction costs are higher at lower salinities, which would
increase the costs of cellular transport of calcium ions via
transmembrane ion transporters (Waldbusser et al., 2013;
Maar et al., 2015; Sanders et al., 2018) (see Section IV). In
addition, shells of mussels in low salinities have different layer
thicknesses and microstructures (increased prismatic layer)
and thicker periostraca to protect against corrosive conditions (Telesca et al., 2019). Since the periostracum is proteinaceous, such thickening adds an energetic burden
(Palmer, 1992).
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Mollusc shell production
Although the organic matrix is only a small proportion of
the shell, it is estimated that it is 22% of the cost of producing
shells with 1.5% organic content, which increases to 50%
with 5% organic material (Palmer, 1992). These costs confirmed previous shell regeneration experiments, in which
rates of repair were inversely related to shell organic content
(Palmer, 1983). Further examples of these costs include the
organic content of new shell produced in Littorina obtusata
when responding to predation threat, which was 21% lower
compared with control animals as the priority was to produce
a thicker shell to counteract the predation threat, irrespective
of shell microstructure (Brookes & Rochette, 2007). Also limpet shells suffer erosion from being grazed by herbivores
(often by conspecifics) feeding on their encrusting corallines
and endolithic parasites (Day, Branch, & Viljoen, 2000).
Although erosion rates were greater in Cymbulla granatina (previously Patella granatina) compared with Scutellastra argenvillei
(previously Patella argenvillei), the latter produced more total
shell mass annually. Day et al. (2000) suggested this was probably due to the higher organic content of C. granatina shells
(3.58% compared with 1.75% for S. argenvillei). These observations highlight the potential long-term costs of erosion and
parasite infestation that vary among species and infestation
type. This has been estimated at 8–20% of total energy
devoted to growth and reproduction in C. granatina and S.
argenvillei, whilst infected Haliotis tuberculata produce 2–3 times
thicker shells in response to parasitic infections of worms of
the genus Polydora and the sponge Cliona celata (Peck, 1983).
Shell production costs also vary considerably with lifehistory stages. In mytilid mussels, larval skeletons have higher
organic fractions (>10%) than adult shells (<5%), with higher
relative calcification rates. This suggests higher costs of shell
growth in early stages (Thomsen et al., 2013). In addition,
Mytilus spp. larval maintenance costs in low pH reflect
enhanced calcification to compensate for increased dissolution rates (Ventura, Schulz, & Dupont, 2016). It is still
unclear how much biotic and abiotic factors impact the
energy invested into shell growth, and how this affects relative proportions of crystals and protein. Also, little is known
about the consequential energetic trade-offs with regard to
somatic and gonad growth when shell production costs
increase (Trussell & Smith, 2000).
(2) Trade-offs
It is recognized that calcareous shell secretion and maintenance are more costly in colder waters, due to the increased
solubility of calcium carbonate, making it more energetically
expensive for the animal to extract (Palmer, 1983;
Clarke, 1993; Watson et al., 2012). However, a recent study
demonstrated that high-latitude Mytilus spp. had thinner
shells, but a higher shell organic content including production of a thicker periostracum (Telesca et al., 2019). This
increase in organic material should increase energetic costs
associated with shell manufacture, but presumably the mussels benefit from reduced shell corrosion. Such costs are further exemplified in the invasive mussel Xenostrobus securis
1821
when exposed to predatory dog whelks, in which there is a
trade-off between shell growth and tenacity. The presence
of dog whelks induces X. securis to produce more proteinaceous byssus threads to increase tenacity, to the detriment
of shell growth (Babarro et al., 2016). The production of byssus threads can be very costly in some species requiring up to
44% of total carbon and 21% of total nitrogen uptake
(Hawkins & Bayne, 1985). A further example is the Antarctic
clam Laternula elliptica, which produces thicker shells in
response to damage (most likely iceberg impact; Fig. 5). In
this example, trade-offs were apparent, with the Antarctic
species having higher proportions of soft tissue mass per unit
shell, compared with lower latitude species despite the
thicker shells. This was achieved by the Antarctic Laternulas
spp. becoming more inflated in shape (Watson et al., 2012).
This trade-off of body size with shell thickness is also recognized in non-Antarctic species, the classic examples being littorinids. In these gastropods reduced body mass correlates
with increased shell thickness but reduced linear shell growth
(Trussell & Smith, 2000; Trussell & Nicklin, 2002; Brookes &
Rochette, 2007). This results in a change in shape of the shell,
which imposes additional constraints, as available living
space for tissues is constrained by the shell architecture: the
‘skeleton limitation’ hypothesis (Palmer, 1981). Gastropod
fecundity correlates with body size, and a possible consequence of size limitation is reduced fecundity (Trussell &
Smith, 2000). Furthermore, predator cues may impact gastropod feeding. In the presence of predators, littorinids are
more refuge-seeking and feed less, reducing energy available
for shell growth (Trussell & Nicklin, 2002; Brookes &
Rochette, 2007; Bourdeau, 2010). Thus, changes in shell
characteristics need to be considered both in relation to environmental factors and the consequences for future sustainability (e.g. reduced fecundity).
IV. CELLULAR CALCIUM TRANSPORT
(1) Mantle tissue and calcium turnover
The basic mechanism for shell secretion in bivalve molluscs is
regarded as common to all shell-bearing Mollusca, except the
chitons, which build their multi-part shells, consisting of eight
separate plates and girdle of spicules, within a cuticle
(Kniprath, 1980; Haas, 1981). Final shell structure involves
interactions between periostracum, specialized mantle epithelial cells, crystal nucleation events and proteinaceous scaffolds within the nanometre thick extrapallial region
(Checa, 2000; Joubert et al., 2010; Nudelman, 2015). Recent
advances in molecular tools for molluscs now make it possible
to examine these processes in more detail, and to begin identifying specific proteins involved in calcium transport.
Mantle tissue is responsible for calcium turnover and
deposition in molluscan shells (Jodrey, 1953). Furthermore,
using radioactively labelled sea water (45Ca) and excised
mantle strips, Jodrey (1953) demonstrated that calcium from
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Melody S. Clark et al.
Fig 5. Variation in inorganic content and shell thickness with habitat temperature. (A) Exterior shell view of the Antarctic bivalve
Laternula elliptica showing damage caused by iceberg impact. (B) Same shell interior showing damage extent and depth. (C) Graph
showing total animal inorganic content (ash, % dry mass) of bivalves, genus Laternula (triangles) and gastropods, family Buccinidae
(circles) with habitat temperature. Open symbols denote Northern hemisphere species and filled symbols are Southern hemisphere
species. (D) Variation in shell thickness of standard-sized animals with temperature (symbols as described in C). Shell images by
Pete Bucktrout, British Antarctic Survey, from Harper et al. (2012), reproduced under Creative Commons Attribution (https://
creativecommons.org/licenses/by/3.0/us/). Data for graphs from Watson et al. (2012).
sea water was used for shell synthesis. Most calcium uptake
for shell production in marine molluscs occurs via gill and
mantle tissues, with the haemolymph used for transportation
(Jodrey, 1953; Fan et al., 2007; Li et al., 2016; Sillanpää
et al., 2016). A small amount of calcium is rapidly turned over
and it is thought that this small fraction is used to synthesize
shell (Sillanpää et al., 2016). This calcium primarily appears
to be transported to the outer mantle epithelia in the ionic
form, with small amounts bound to proteins and smaller
ligands (Sillanpää et al., 2016; Sillanpää, Sundh, &
Sundell, 2018).
There is still considerable debate around how calcium is
transported throughout the animal and transferred across
outer mantle epithelia into the extrapallial space
(Nudelman, 2015; Ramesh et al., 2017; Sillanpää
et al., 2018). Several studies have shown haemocyte involvement in mineralization processes in Crassostrea spp., Pinctada
fucata and Haliotis tuberculata (Mount et al., 2004; Li
et al., 2016; Ivanina et al., 2018). However, haemocyte contribution may be species specific. In Crassostrea spp., for example
haemocytes are more involved in immune processes in
C. gigas than in C. virginica where ion regulation and calcium
transport appeared more prominent (Ivanina et al., 2018).
In epithelia, calcium ions might be transferred passively
between cells through paracellular pathways (including septate junctions between the epithelial cells) and/or actively
via transcellular routes (Sillanpää et al., 2018). The latter
mechanisms include calcium bound to specialized transport
proteins, potentially involving haemocytes and intracellular
vesicles, which may or may not contain amorphous calcium
carbonate (Nair & Robinson, 1998; Addadi et al., 2006; Li
et al., 2016; Ramesh et al., 2017). Furthermore, contact
between mantle cells and the growing edge might play a significant role in crystal nucleation, more so than the extrapallial fluid (Rousseau et al., 2009; Marie et al., 2012). There may
also be differences in shell formation between larvae and
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Mollusc shell production
adults. No persistent amorphous calcium carbonate (ACC)
phase was found in larval shell formation in six marine species (Kudo et al., 2010; Yokoo et al., 2011; Ramesh
et al., 2017, 2018), but roles for ACC in adult shell production
are still debated. Much of this research relied on histochemical and ultra-high-powered microscopy techniques
(Nudelman, 2015), but physiological studies can also provide
important molecular evidence for functionality of the outer
mantle epithelium, and intracellular calcium transport
processes.
(2) Identification of ion channels involved in calcium
transport across membranes
Ussing chamber methods (Ussing & Zerahn, 1951) developed to measure electrochemical properties of membranes
and epithelia are now providing detailed insights into cellular
calcium transport in molluscs. Electrophysiology of mantle
epithelia in Ussing chambers has demonstrated both their
calcium permeability, and their polarity (Coimbra
et al., 1988). Radioactive calcium ion (45Ca2+) experiments
on C. gigas outer mantle epithelium allowed studies of both
calcium transport kinetics and the identification of potential
membrane proteins involved in that process. Passive transport across septate junctions occurs at rates expected from
leaky epithelia, but the majority of transport occurs transcellularly. 45Ca ion transfer kinetics across outer mantle epithelia indicate that 60% of calcium mobilization occurs via
transcellular routes and at least two active transporters are
involved, the other 40% occurs via paracellular routes,
(Sillanpää et al., 2018).
Selective use of 45Ca2+ ions across C. gigas outer mantle
epithelia in tandem with pharmacological inhibitors
(Verapamil) indicates that calcium can enter outer mantle
epithelia through L-type and T-type voltage-gated calcium
channels located in the basolateral membrane (inner mantle
and haemolymph side) (Sillanpää et al., 2018). Similar inhibitory effects of Verapamil on calcium channels are present in
other bivalve species (Roesijadi & Unger, 1993; Wang &
Fisher, 1999). However, other membrane proteins also are
involved in this process as Verapamil only reduced calcium
transport by 20% via this route. In addition, due to Verapamil’s general action, the particular genes involved could not
be definitively identified. Calcium ion secretion from cells
into extrapallial spaces across the apical membrane is mainly
via calcium ATPases and Na+/Ca2+ exchangers, as demonstrated by caloxin and ouabain experiments (Sillanpää
et al., 2018). The primary active membrane protein here
was a plasma membrane calcium ATPase (PMCA), accounting for >50% of active calcium transport across epithelia,
with indirect evidence for a secondarily active Na+/Ca2+
exchanger (NCX) involved in this process (Sillanpää et
al., 2018). These experiments led to the development of a
Ca2+ transfer model across outer mantle epithelia in C. gigas
(Fig. 6). The activity of calcium ATPase proteins is energetically expensive in cells (McConnaughey & Whelan, 1997).
Given the major contribution of calcium ATPases to calcium
1823
intracellular transport, the increased activity of these proteins
under low-salinity environments is expected to contribute
significantly to calcification costs in Baltic Sea blue mussels,
limiting their distribution in this region (Sanders
et al., 2018; Thomsen et al., 2018). Transcriptomic studies
from carbonate manipulation experiments are providing a
more complete list of the genes involved in cellular ion transport including Na+/K+ ATPases (NKAs) (De Wit et al., 2018;
Ramesh et al., 2019). These new sequence data will facilitate
the design and testing of tailor-made pharmacological inhibitors, the development of gene knockouts and potentially
enable more accurate calculations to be made of the energetic costs associated with calcium transport.
V. MOLLUSC GENES AND PROTEINS
ASSOCIATED WITH SHELL PRODUCTION
Early work on understanding the molecular structure of
shells in the 1950s involved dissolving shells and separating
proteins by chromatography, but it was not until 1996 that
the first shell protein (nacrein-a) was finally characterized
(Miyamoto et al., 1996). For the next decade molecular data
were limited and research largely focussed on cloning and
characterization of individual genes and proteins. This changed dramatically with the development of molluscan
expressed sequence tag (EST) libraries (e.g. Jackson
et al., 2006) and subsequently, from pyrosequencing, the first
next generation sequencing (NGS) technology.
(1) Gene transcripts and proteins
In 2010, the first pyrosequencing results were published for
molluscs (Clark et al., 2010; Craft et al., 2010). The latter study
was the first specifically to describe the application of NGS to
investigate molluscan calcification processes, using comparative genomics approaches on a mantle transcriptome. Transcriptomes catalogue expressed sequences and can be
applied from single cells to whole organisms. In molluscs,
research has largely concentrated on sequencing mantle tissue,
because it secretes the proteins necessary for shell construction.
To enhance discovery of biomineralization genes, mantle
transcriptomes can be generated from shell damage-repair
experiments (Clark et al., 2013; Sleight et al., 2015; Hüning
et al., 2016; Yarra, 2018). Responses vary with the site of damage compared to the tissue sampling site (Sleight et al., 2015)
and also whether mantle edge or inner mantle regions were
targeted (Yarra, 2018). This approach, along with developmental studies on larvae raised in low-calcification-substrate
conditions (e.g. De Wit et al., 2018; Ramesh et al., 2019) is significantly increasing knowledge of putative biomineralization
genes. These approaches are particularly powerful when combined with analyses of expression modules and gene networks,
such as correlative weighted gene co-expression network analysis
(WGCNA) and programs such as the algorithm for the reconstruction of accurate cellular networks (ARACNe) (e.g. De
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
Society.
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Melody S. Clark et al.
Fig 6. Proposed model of Ca2+ transfer across outer mantle epithelia of Crassostrea gigas. Ca2+ passively flows into outer mantle
epithelium cells through basolateral voltage-gated Ca-channels (VGCCs). The Ca2+ is further actively secreted into the
extrapallial space by apical ion channels, plasma membrane calcium ATPases (PMCAs) and Na+/Ca2+ exchangers (NCXs), the
latter driven by the trans-membrane sodium-gradient created by basal Na+ K+ ATPase (NKAs). Apical L-type voltage-gated
calcium channels and basal plasma membrane calcium ATPases are also present, which suggests Ca2+ transfer in the opposite
direction, when needed. Based on data from Sillanpää et al. (2018).
Wit et al., 2018; Sleight et al., 2020). These in silico techniques
visualize gene interactions and provide major progress
towards defining biochemical pathways in non-model species.
They are particularly useful for identifying upstream control
genes and incorporation of species-specific transcripts that
have little or no annotation, into biochemical pathways
(De Wit et al., 2018; Yarra, 2018; Sleight et al., 2020; Ramsøe,
Clark, & Sleight, 2020).
(2) Mollusc genomes and comparative genomics
In 2012, a landmark paper in molluscan genomics described
the first draft of the C. gigas genome (Zhang et al., 2012). Since
then, despite molluscs being amongst the most diverse and
speciose phyla, they remain poorly represented in genome
databases [e.g. the Genomes OnLine Database (GOLD;
https://gold.jgi.doe.gov/index) and EnsemblMetazoa database (http://metazoa.ensembl.org/index.html)]. However,
this situation should change soon with the Earth Biogenome
Project (https://www.earthbiogenome.org/), which has the
ambitious aim to sequence all eukaryotic species in the next
decade and also programmes such as the Darwin Tree of Life
(https://www.darwintreeoflife.org/). Until then mollusc
genomes can be accessed far more easily than previously via
transcriptomes and there is now a specific mollusc database
of genomes and transcriptomes in ‘ensembl’ easy access format (https://molluscdb.org/).
(3) The evolution of shell production in molluscs
Comparison of these comprehensive gene data sets across
bivalve and gastropod molluscs has revealed huge diversity
in the mechanisms of shell production between even closely
related species (Kocot et al., 2016). This diversity became
apparent in an early large-scale mollusc EST study in Haliotis
asinina, where almost 85% of the secreted proteins were
encoded by novel genes (Jackson et al., 2006). Even transcriptomes from cells performing identical functions in different
species (nacre-forming cells in the bivalve Pinctada maxima
and the gastropod H. asinina) varied greatly in expressed gene
sets. After removing housekeeping sequences, only 10% of
genes from shell secretory cells were shared between the species (Jackson et al., 2010). These large differences support the
hypothesis of rapid and convergent evolution of nacre gene
pathways in diverse molluscan lineages (Jackson et al., 2010).
Rapid evolution of biomineralization pathways in molluscs
was validated by the identification of species-specific large
expansions in some gene families. Examples include tyrosinase,
shematrin, chitinase-like protein, repetitive low complexity
domain proteins (RLCDs), lysine (K)-rich mantle proteins
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Mollusc shell production
(KRMPs), carbonic anhydrase and calcitonin-like G-protein
coupled receptors (GPCRs), all of which play roles in biomineralization (McDougall, Aguilera, & Degnan, 2013; Aguilera,
Mcdougall, & Degnan, 2014; Le Roy et al., 2014; Takeuchi
et al., 2016; Cardoso et al., 2019). It is unclear why these gene
families evolved in certain species and what functions the individual duplicated genes perform. Furthermore, the genes
involved in shell production also comprise lineage-restricted
proteins and unique combinations of co-opted ancient genes.
In some cases, these genes result from domain shuffling, with
expansion and loss of particular domains in genes such as
RLCDs (Kocot et al., 2016; Aguilera, Mcdougall, &
Degnan, 2017). With such complexity, the question is whether
conserved sets of genes or proteins involved in shell secretion
exist in molluscs, that is a biomineralization toolbox.
(4) A conserved biomineralization tool box
In spite of the rapid evolution of many biomineralization
genes, some genes, including carbonic anhydrase and
RLCDs have long evolutionary histories within calcifying
metazoans. Many of these ‘ancient’ genes have multiple functions for example carbonic anhydrase is important for acid–
base physiology alongside biomineralization (Le Roy
et al., 2014; Murdock, 2020). Furthermore, recent proteomic
studies on dissected shell microstructures in a single species
showed that whilst there were different shell matrix proteins
in each layer, common proteins were present throughout
the shell (Marie et al., 2012; Gao et al., 2015).
Identification of a conserved biomineralization tool box
within the Mollusca is impeded due to the lack of comprehensive data sets across species and most data originating
from transcriptomes, where a gene is only represented if it
is expressed. In addition, transcriptome and proteome content depend highly on the methods used to generate them
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(Arivalagan Immanuel, 2017). Two recent studies addressed
some of these methodology issues by generating shell proteomes and mantle transcriptomes from the same individuals
in species with differing mineralogies (Arivalagan et al., 2017;
Yarra, 2018) (Fig. 7). These investigations revealed several
domains categorized as an evolutionarily conserved toolbox
for shell biomineralization [tyrosinase, carbonic anhydrase
(CA), chitin-binding (CB) and von Willebrand Factor A
(VWA)]. Other domains were mineralogy specific [calcite:
epidermal growth factor (EGF), fibronectin (FN3) and whey
acidic protein (WAP); aragonite: laminin (Lam-G)]
(Arivalagan et al., 2017; Yarra, 2018) (Fig. 7). These data
were substantiated by analyses comparing larval and adult
biomineralization genes, which revealed substantial differences between life-history stages, albeit with conservation of
CA, CB and VWA domains (Zhao et al., 2018).
Transcriptome and proteome data sets are highly complementary. Transcriptomes have proven more effective than
proteomes at identifying transcripts containing intrinsically
disordered domains, including those for RLCD proteins,
but proteomes proved that immune-related proteins were
incorporated into shell materials. Previously their presence
in transcriptomes was assumed to be an immune response
or contamination from haemocyctes (Arivalagan
et al., 2017; Yarra, 2018). Transcriptome studies also
highlighted signalling molecules and especially transmembrane transporters, as important (Yarra, 2018; Ramesh
et al., 2019), some of which have been independently validated using physiological studies (Sillanpää et al., 2018).
(5) Further levels of complexity: alternative splicing,
isoforms and development
Beyond the tremendous complexity already revealed in molluscan biomineralization genes described above, there are
Fig 7. Venn diagram showing conserved protein domains in four bivalve species with different shell microstructures. Data from
Arivalagan et al. (2017) and Yarra (2018) with annotation from Yarra (2018) (https://doi.org/10/cz2w). EGF, epidermal growth
factor; FN3, fibronectin 3; Lam-G, laminin G; VWA, Von Willebrand factor A; WAP, whey acidic protein.
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
Society.
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additional complications. Algorithms underpinning NGS
transcriptome assembly programs enable gene isoform or
alternatively spliced gene product discovery. Alternative
splicing could produce isoforms with different functions or
cellular locations, as recently shown with glycoprotein precursors in Pinctada imbricata fucata (Zhao et al., 2016a). Comprehensive analysis of the shell proteome and tissue-specific
transcriptomes of the snail Lymnaea stagnalis revealed several
shell-forming genes associated with the crossed-lamellar
microstructure. A significant number were alternatively
spliced, depending on tissue origin and location (Herlitze
et al., 2018). A few Lymnaea stagnalis isoforms were solely found
in either adults or larvae, suggesting a further level of biomineralization pathway sophistication (Herlitze et al., 2018).
Developmental delimitation in gene expression patterns has
also been demonstrated in various bivalves (Kakoi
et al., 2008; Miyazaki et al., 2010; Zhao et al., 2018), suggesting this phenomenon is likely widespread. This is not surprising, as early biomineralization originates in different cells
from adults. During early embryogenesis, biomineralization
of the first shell (prodissoconch in bivalves and protoconch
in gastropods) initiates in the shell gland as the mantle has
yet to develop. The subsequent shell stage (prodissoconch II
or protoconch II), is secreted by the juvenile mantle
(Verdonk & Van Den Biggelaar, 1983). Larval shells of many
species have similar aragonitic microstructures, whilst adults
display a wide variety of species-specific crystal microstructures (Eyster, 1986). There also appears to be a speciesspecific element to this developmental delimitation
(Herlitze et al., 2018; Yarra, 2018; Zhao et al., 2018; Carini
et al., 2019). However, since prodissoconch or protoconch
production is an essential precursor to adult shell, understanding how one leads to the other and the interactions
between different biomineralization pathways within the
same individual is essential [the evo-devo approach
(Jackson & Degnan, 2016)]. Studying larval developmental
programs can provide deep insight into adult tissue morphology and structure, and how their functions differ among species (Jackson & Degnan, 2016). However, achieving a
comprehensive understanding of biomineralization in adult
molluscs is still a distant goal, particularly with regard to
functional validation of genes involved in shell development.
VI. BIOMINERALIZATION GENES AND
PROTEINS: FUNCTIONALITY
Most knowledge concerning the proteins essential for shell
production has come from proteomic studies, where whole
shells or specific shell layers have been powdered and proteins extracted (Joubert et al., 2010; Marie et al., 2012; Gao
et al., 2015; Arivalagan et al., 2017). Any protein present
within the shell probably has some role in producing the relevant microstructures. Indeed functional assays (Section
VI.2) have demonstrated direct roles for many proteins in
the calcification process. By contrast, transcriptomic data,
Melody S. Clark et al.
largely obtained from mantle tissue, have demonstrated a
particular gene’s involvement through elevated expression
levels using transcript counts or reverse transcription polymerase chain reaction (RT-PCR) (Miyazaki et al., 2010; Fang
et al., 2012; Sleight et al., 2015). The mantle secretes the shell,
but it has many other functions; some proteins might be
involved in CaCO3 transport rather than in the shell structure itself. Thus, functional studies are key to deciphering
the true role of these candidate biomineralization genes. Several techniques are available to provide greater functional
characterization and evidence for participation in biomineralization pathways.
(1) Cellular localisation of gene expression
The mantle is a complex tissue (Harper, 1997) (Fig. 1). Whilst
shell damage-repair experiments often take tissue for RNA
extraction across all the mantle folds (e.g. Sleight
et al., 2015; Yarra, 2018), some have sampled with greater
precision and shown discrete expression patterns, which were
validated by in situ localization mapping (Gardner
et al., 2011). At the cellular level, similar investigations started
in 1997, when the protein MSI30 was extracted from the
nacreous layer of P. imbricata fucata and localized to the outer
mantle epithelia (Sudo et al., 1997). This was probably the
first demonstration of mantle modularity using gene expression, although previous studies had identified regional
differences in histochemical properties of mantle tissue
(Timmermans, 1968). However, the generation of EST
libraries and NGS data revealed the true extent of mantle
gene expression modularity (e.g. Jackson et al., 2006; Jackson,
Worheide, & Degnan, 2007; Gardner et al., 2011; Sleight
et al., 2016; Herlitze et al., 2018). The most recent example
was in Lymnaea stagnalis, in which 34 gene transcripts were
localized by in situ hybridization across different developmental stages (trochophore larvae and juveniles) and in adult tissue. These data revealed six distinct expression zones in adult
mantle and developmental stage-specific expression (Herlitze
et al., 2018). It is likely that even greater complexity and partitioning of mantle function will be revealed as more highthroughput in situ hybridization studies are conducted in different species in future. Such studies will play essential roles
in assigning putative biomineralization functions to genes
with no associated annotation, only mantle-specific expression (e.g. Sleight et al., 2016).
Localization studies carried out at the proteomic level
have also shown distinct partitioning of proteins between
prismatic and nacre layers using mass spectrometry (Marie
et al., 2012). Use of antibodies raised to specific shell proteins
has demonstrated that extracellular matrix proteins are associated with different crystal structures (calcite prisms or
nacre) in Pinctada (Kong et al., 2009; Fang et al., 2012; Marie
et al., 2012). Some proteins (e.g. pearlin) have been localized
to the interlamellar matrix of nacre aragonite tablets
(Montagnani et al., 2011), whilst others (e.g. caspartin) are
present in continuous films at interfaces between prisms
and surrounding insoluble sheets (Marin et al., 2005).
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Mollusc shell production
The composition and location of extracellular matrices are
being refined beyond the mass extraction of proteins from
shells. This provides greater insight into crystal–protein
matrix associations, and raises questions about how molluscs
selectively secrete proteins to distinct microstructures. This
clearly relates to the mantle zonation described above and
the models discussed in Section VII.
(2) Protein activity associated with
biomineralization
Despite many attempts over decades, only one immortalized
molluscan cell line exists, from embryos of the snail Biomphalaria glabrata, but short-term cultures are still useful (Yoshino,
Bickham, & Bayne, 2013). Calcium carbonate microcrystals
have been produced during cell culture derived from mantle
tissue of marine and freshwater pearl oysters (Samata
et al., 1994; Barik, Jena, & Ram, 2004; Gong et al., 2008). Furthermore, during in vitro primary cell line culture proteoglycans and collagen have been synthesized in Haliotis
tuberculata and nacrein protein in P. imbricata fucata (Poncet
et al., 2000; Gong et al., 2008). Nacre is well known for inducing mineralization in human and mammalian bone-forming
cells (Atlan et al., 1997), but the osteoinductive compounds in
nacre remain unidentified. The water-soluble matrix from
nacre induced mineralization in mouse pre-osteoblast cell
lines (Rousseau et al., 2003, 2008). However, current evaluations centre on ethanol-soluble matrix fractions, which have
demonstrated greater osteogenic activity, with mineralization observed in human osteoarthritis osteoblast and mouse
preosteoblast cells after adding ethanol-soluble fractions
from oysters (Zhang et al., 2016). In a different functional
assay, the chitinase-specific pharmacological inhibitor Allosamidin demonstrated a potentially important role for chitinase in shell production and shell remodelling. Larvae
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cultured with this inhibitor produced thinner shells and
applying a chitinase solution to adult shells resulted in shell
dissolution (Yonezawa et al., 2016).
In a few examples, isolated shell proteins have induced calcification activity in cell cultures. For example, when a shell
protein isolated from P. imbricata fucata, p10, was added to
mammalian mineralogenic cell lines, alkaline phosphatase
(an early biomarker of osteoblast differentiation) activity
increased. Thus, p10 has a potential biomineralizing function (Zhang et al., 2006). Mollusc genes can be inserted into
expression vectors and transfected into human cell lines,
enabling them to be assessed with heterologous ligands to test
for functional similarity in molluscs and vertebrates. For
example, the ligands, calcitonin (CALC) and parathyroid
hormone-related protein (PTHrP), in vertebrates regulate
calcium uptake and resorption, and osteoblast and osteoclast
function. Recently, homologues of the vertebrate calcitonin
system have been demonstrated in molluscs, with both multiple ligands and receptors (CALCRs) identified in
M. galloprovincialis and C. gigas (Fig. 8). Relatively few mollusc
CALCRs have been studied, but in both M. galloprovincialis
and C. gigas these receptors respond to reduced salinity
(Schwartz et al., 2019; Cardoso et al., 2020). Demonstrating
that a specific CALCR with calcitonin mediates local regulation of Ca2+ transport in the mantle in M. galloprovincialis suggests that molluscs have a regulatory system triggered from
sensory inputs that likely modulate biomineralization
(Fig. 8) (Cardoso et al., 2020). Using a different approach, a
luciferase reporter assay for the putative biomineralization
transcription factor Pf-Sp8/9 demonstrated interactions
between different shell proteins (Zheng et al., 2016). Increasing concentrations of this transcription factor were added to
human embryonic kidney cell lines containing constructs
for pearlin, prisilkin-39 and KRMP promotors (known biomineralization genes). Activity of all three genes increased
Fig 8. The bivalve calcitonin-like G-protein coupled receptor (GPCR) system. (A) Gene numbers of calcitonin-like receptors
(CALCRs) and mature calcitonin-like peptide (CALC) in human, Pacific oyster (Crassostrea gigas) and blue mussel (Mytilus
galloprovincialis). (B) Predicted structure of mussel CALCRs and activated intracellular signalling pathways in the presence of
vertebrate (human and salmon) calcitonins and mussel calcitonin-like peptide. (C) Relative abundance (fragments per kilobase of
transcript per million mapped reads; fpkm) of putative CALCR transcripts in the mussel posterior mantle edge transcriptome.
Bars show mean data + standard error of the mean (SEM). Data from Cardoso et al. (2020).
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Society.
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linearly, indicating their control by Pf-Sp8/9 (Zheng
et al., 2016). Thus, either homologous or heterologous in vitro
assays using cell culture systems are providing potentially
important functional information on shell proteins.
An additional approach examining in vitro crystallization
does not use cell cultures, but aims to identify which proteins
interact with CaCO3 to form crystals and specific crystal
microstructures. In some of the above studies, calcium precipitation assays used in tandem with cell culture experiments
demonstrated the calcium binding properties of isolated proteins. For example, caspartin isolated from the Pinna nobilis
prismatic layer strongly interacted in vitro with growing calcite
crystals (Marin et al., 2005). Its activity suggested that it was
probably a calcium carbonate nucleator, but also that it constrained crystal growth and determined crystallographic orientation in the prism layer (Marin et al., 2005). Prismalin-14
also exhibited calcium-binding properties, inhibiting calcium
carbonate activity (Kong et al., 2009; Suzuki et al., 2009).
Both p10 and a Pinctada fucata novel basic protein (PfN23)
accelerated calcium carbonate nucleation in vitro and induced
aragonite formation (Zhang et al., 2006; Fang et al., 2011).
These in vitro studies usefully demonstrate calcium binding
and uncover potential interactions between proteins and
CaCO3 precipitation and/or crystallization. This is especially useful for proteins with little sequence similarity to
known proteins and with disordered structures rather than
precise three-dimensional shapes (Amos & Evans, 2009).
However, these in vitro crystallization reactions are outside
the complex intracellular milieu and such experiments are
predominantly employed by materials scientists aiming to
engineer novel biomaterials and crystal surfaces with unique
properties (e.g. Amos & Evans, 2009; Chang & Evans, 2015).
(3) Disruption of biomineralization to identify gene
function
Gene knockout (or knock down) or gene editing techniques
are very useful for demonstrating gene functions and interactions. To date, although there have been several successful
examples in molluscs, the use of such technologies is not routine in this phylum. The earliest experiments concentrated
on P. imbricata fucata and generally used double-stranded
RNA interference (ds RNAi) to knock down target genes.
The first such experiment partially knocked down Pif (aragonite-binding protein) messenger RNA (mRNA) and resulted
in disordered growth of nacre crystals (Suzuki et al., 2009).
A subsequent experiment involving knockout of the Pinctada
fucata homeobox containing transcription factor msx gene
(PfMSX) also resulted in disordered nacre crystal growth
(Zhao et al., 2014). Furthermore, splice variants of the glycoprotein precursor (granulin/epithelin precursor) interact
with the msx PfMSX to disrupt nacre formation, and the
bone morphogenetic protein-2 (BMP-2) pathway, which is
required to maintain normal vertebrate bone homeostasis,
is involved in nacre formation (Zhao et al., 2016a, 2016b).
These studies therefore, established wider gene signalling
and network connectivity involved in nacre formation in
Melody S. Clark et al.
P. imbricata fucata. RNAi experiments also validated some
unannotated gene effects on nacre formation (Fang
et al., 2011; Funabara et al., 2018), expanding knowledge of
biomineralization beyond ‘the usual suspects’, for example
Pif, nacrein, p10. In a similar approach, using antibodies to
disrupt the function of prisilkin-39 in P. imbricata fucata, led
to dramatic morphological deformation of the inner shell
layer (Kong et al., 2009). Clustered regularly interspersed
short palindromic repeats (CRISPR)-associated protein
9 (CRISPR/Cas9) was first used in Crepidula fornicata in
2014, demonstrating the knock-in of β-catenin in embryos
(Perry & Henry, 2015), but its first use for knockout in molluscs was in 2019. This pioneering study in Lymnaea stagnalis
reported that the Lsdia1 gene set shell chirality from the
one-cell stage in (Abe & Kuroda, 2019). This research demonstrated the utility of gene-editing technologies and how
genes play key roles in three-dimensional shell structures.
Given the popularity of CRISPR/Cas9 gene editing in
non-mollusc species, this first use will very likely inspire more
shell biomineralization functional studies.
VII. PRODUCING THE THREE-DIMENSIONAL
STRUCTURE OF THE SHELL
Much evidence indicates that shell synthesis results from both
biologically controlled (genetic and cellular activity, with proteins determining mineral phases, crystal shape and nucleation) and physical mechanisms (crystal competition,
growth in confined spaces and self-organization) with interactions between the two (Checa, 2018). The balance between
these mechanisms varies with microstructure, resulting in the
wide variety of shell shapes and designs observed
(Checa, 2018). From the biological side, shell protein content
is important in crystal formation as are developmental pathways, such as that determined by the Lsdia1 gene, which dictates dextral/sinistral coiling in snails (Abe & Kuroda, 2019).
The modularity of mantle gene expression can also explain
how such complexity in shell design could be developed
(Herlitze et al., 2018).
Understanding of such biological problems can be aided
by theoretical studies embedded in mathematics and materials science (Gerber, 2017). In one mechanical model, shell
growth was described as a function of local geometry and
the mechanics of the aperture and mantle. Shell ornamentation, such as ribs on ammonite shells, could be explained
from stresses and strains on the flexible mantle tissue during
growth on the rigid shell aperture template (Moulton, Goriely, & Chirat, 2015). This was similar to a model using differential growth patterns, where varying proliferation rates and
changing the mantle edge–cell division axis bias contributed
to the production of different shell morphologies (Johnson,
Fogel, & Lambert, 2019). Another mechanical model
explained spine morphogenesis by the mechanical interaction of the secreting mantle edge and the calcified shell edge,
which the mantle adheres to during shell growth (Chirat,
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Mollusc shell production
Moulton, & Goriely, 2013). This latter model accounted for
the large diversity in spine structure by relatively small variations in the control parameters of the process (Chirat
et al., 2013). Thus, modelling mechanical processes can
potentially explain shell morphology variation, although
the underlying molecular mechanisms producing such forces
remain elusive.
Other proposed models, including activator–effector and
neural net models, are based on mathematical models developed by Turing (1952). The activator–effector model uses
shell-surface pigment patterns to code for shell design
(Fowler, Meinhardt, & Prusinkiewicz, 1992) and was recently
used to investigate pigmentation in Haliotis asinina (Budd
et al., 2014). In this species, shell pigmentation occurs via prismatic layer secretory tubules likely underpinned by coordination of signals to individual cells. These are transmitted through the entire tubule, synchronizing pigment production and secretion within the shell (Budd et al., 2014).
Neural net models assume that neural-stimulated secretion
controls cell growth, but neural nets can also encode information required for shell growth and pigmentation
(Boettiger, Ermentrout, & Oster, 2009; Gong et al., 2012).
In these models, sensory cells read the pigmentation history
and send this to a neural net, which uses this information to
predict the next pigmentation increment required and
instruct secretory cells accordingly, with direct feedback from
output to input (Boettiger et al., 2009; Gong et al., 2012).
These intra- and intercellular messages may include bioelectrical signalling, for which there is increasing evidence for an
important role in cell-to-cell communication (Adams &
Levin, 2013).
Another model provides potential links to materials
science to explain crystal microstructure self-assembly. Zlotnikov & Schoeppler (2017) suggested that shell microstructures are formed via biologically controlled extracellular
biomineralization, whereby cells create specific biochemical
and physical settings that subsequently regulate mineral
self-assembly without directly involving cells. Spontaneous
chemical interaction then occurs between CaCO3, the
organic precursors and physical properties within the
extracellular pallial space. This mechanism is similar to mineral deposition processes from colloidal chemistry
(Zlotnikov & Schoeppler, 2017). Whether any of these
models operate in vivo is not resolved and will be difficult
to demonstrate, however, they provide theoretical frameworks to describe shell structure and a basis for further
experimental design.
VIII. FUTURE PROSPECTS
(1) Molluscs in a changing world
Many factors are important in dictating species success or
failure when environments change. These include generation
times, population sizes, ability to adapt including genetics
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and phenotypic plasticity, number of propagules produced,
dispersal capability, rate of environmental change and
predator–prey interactions, amongst others (Peck, 2011;
Urban et al., 2016). In few molluscs are all, or even many,
of these factors known. Many laboratory manipulation
experiments suggest poor prospects for molluscan biodiversity, but more recent studies involving natural evaluations,
longer-term experiments and multiple generations are producing more positive results. Molluscs may be more robust
to changing environments than previously anticipated.
Indeed molluscs fared comparatively well in previous extinction events compared with other phyla (Ros &
Echevarria, 2011).
There is considerable genetic variability within many mollusc populations, which provides the material for natural
selection and rapid adaptation and many traits have plastic
characteristics (Section II.3). Certainly molluscs can thrive
in some extreme situations, including at 20–30% undersaturation of calcium carbonate in the deep sea (Allen, 1978),
around hydrothermal vents (Jollivet, 1996) and in shallower
water CO2 vents, such as at Ischia in the Tyrrhenian Sea
(Langer et al., 2014). There is also increasing recognition in
a range of taxa including molluscs, that larvae can survive
even fairly severe experimental conditions (albeit in depleted
numbers) which provides the potential for the next generation (Ventura et al., 2016; Thomsen et al., 2017). These larvae, on maturation are potentially more resilient than their
parents to the altered conditions, due to transgenerational
plasticity (Ross, Parker, & Byrne, 2016). Parental genotypes
are also critical, as very different outcomes for larval performance are produced depending on parental source, supporting the premise that resilient lines can be bred for
aquaculture (e.g. Goncalves et al., 2016). Laboratory experiments provide valuable evidence about mechanisms underlying responses to altered conditions, but results need context
from evaluations in the natural environment, as their complexity is far greater than in experimental systems. Time
series analyses of museum collections can provide valuable
insights into responses to changing environments over historical timescales (relevant for the Anthropocene).
Evaluations of Mytilus shells from the Belgian coast have
demonstrated increasing shell thickness over the past
112 years, whereas thinner shells would be predicted from
experimental data (L. Telesca, L.S. Peck, T. Backeljau,
M. Henig & E.M. Harper, in preparation). In this historical
study, shell microstructure also varied with predator pressure
from crabs, seagulls and the dog whelk Nucella lapillus. When
the latter disappeared from local habitats in 1981, mussel
shells developed with 13% less organic matrix and a 29%
thinner periostracum (L. Telesca, L.S. Peck, T. Backeljau,
M. Henig & E.M. Harper, in preparation). This exemplifies
the complexity of mollusc shell responses to changing conditions and the need to understand biotic interactions, and
responses to local conditions, including both triggers and
compensatory mechanisms. This unexpected response to historical changes is supported by another historical study in a
heavily calcified organism, the brachiopod Calloria inconspicua
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(Cross, Harper, & Peck, 2018). Calloria inconspicua collected
over the past 120 years showed no shell dissolution, but had
a 3.4% increase in shell density, despite sea surface temperature increasing by 0.6 C (1953–2016) and pCO2 increasing
by 35.7 μatm (1 standard atm = 101325 Pascal) (1988–
2016) (Cross et al., 2018). Making accurate predictions of
future mollusc biodiversity is difficult because the resilience
of the mollusc itself is not the only factor to consider, as species responses are variable and each species exists within a
complex ecosystem. There are indirect effects of changing
conditions on feeding behaviour and food supplies (Sanders
et al., 2013; Mackenzie et al., 2014), behaviour (Gunderson,
Armstrong, & Stillman, 2016) and predator–prey dynamics
(Beaty et al., 2016; Donelan & Trussell, 2019). There are also
altered disease risks and host–parasite interactions
(Marcogliese, 2008).
Most of these factors also apply to aquaculture and predicting how aquaculture should, and can, develop globally
is complex. Across the globe, molluscan aquaculture will
likely suffer from environmental change impacts, the drivers
of which will differ among nations (Fig. 9) (Stewart-Sinclair
et al., 2020). Aquaculture practices will need to evolve as conditions alter. Environmental change, for example temperature and pH, will not only have direct effects (Allison,
Badjeck, & Meinhold, 2011; Frost et al., 2012), but there will
be indirect effects of emerging diseases, invasive species, etc.
(Burge et al., 2014). Monitoring environments over large spatial and temporal scales will be key for aquaculture practices
Melody S. Clark et al.
to be modified accordingly (Allison et al., 2011; Callaway
et al., 2012). This has already been particularly successful in
the Pacific north-west shellfish industry. Mitigation measures
including buffering sea water in header tanks and spawning
stocks at key times has been very effective in a region that
has been badly affected by periodic, and unpredictable, corrosive upwellings (Barton et al., 2015). Furthermore, changing conditions may provide new opportunities for culturing
warmer water aquaculture species in traditionally colder
areas, such as around the UK and Ireland (Callaway
et al., 2012). Warmer waters can improve scallop recruitment
and would expand the current range for culturing C. gigas
oysters and potentially open up new areas for Haliotis abalone
culture (Allison et al., 2011; Callaway et al., 2012;
Goulden, 2018). Given current data, regional assessments
that account for local conditions will likely be important for
planning new aquaculture facilities (Michalek, 2019). Global
assessments are more likely to indicate new aquaculture
opportunities developing as species’ ranges change
(Shumway, 2011; Gjedrem, Robinson, & Rye, 2012).
(2) Mollusc shells for future innovation
Irrespective of future food security concerns, mollusc shells
are increasingly recognized as valuable in themselves, as
models for biomimetics or exploited in applied projects. Molluscs constitute one of the most diverse and widespread phyla
that use calcium carbonate when manufacturing their
Fig 9. Overall predicted vulnerability to climate change for shellfish mariculture in coastal nations 2090–2100 using a vulnerability
assessment model. Vulnerability (V) was measured as nation-specific mean for Exposure (E), Sensitivity (S), and Adaptive capacity (AC)
sub-layers under the IPCC RCP8.5 ‘business as usual’ scenario. These sub-layers comprised the following factors: exposure (E) was
based on changes in sea surface temperature, depth of aragonite saturation horizon, primary production and occurrence of
extreme weather events; sensitivity (S) evaluated species habitat range, production value as a normalized percentage of national
gross domestic product, and nutritional dependence based on fish protein as a percentage of animal protein in the diet of each
nation; adaptive capacity (AC) applied the Shannon index to industry diversity and governance using the World Bank world-wide
governance indicator. The high sensitivity of developing nations was based largely on the narrow habitat tolerance of the
cultivated species, whilst that of developing countries was due to the relatively high economic value of shellfish production with
limited adaptive capacity due to the small number of species cultivated. Colours represent overall V score (1 = very low, 2 = low,
3 = moderate, 4 = high, 5 = very high), while white indicates absence of data. Adapted from Stewart-Sinclair et al. (2020).
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
Society.
Mollusc shell production
protective shells and thus present great potential for novel
bio-inspired materials (Currey, 1999). One particular example is in using nacre construction as a framework for body
armour (Yadav et al., 2016). Moreover, shells are produced
at ambient temperature and pressure with water as a solvent.
Hence, they offer very different, more sustainable ways (less
energy usage, lower toxicity, etc.) to produce innovative
CaCO3 and related mineral-based materials (Nishimura,
2015; Xia, 2016). The production of hierarchical structures
from nano- to metre-scales is, however, beyond current technologies and scalability is an issue (Mann, 2000). Thus, whilst
understanding of biomineralization processes is incomplete,
the self-assembly of nanocomposites remains a major challenge for bulk biomimetic materials, but one that offers great
future promise (Morris et al., 2016).
Alongside the inspiration given by shells in biomimetic
industrial applications is the use of shells themselves. Sustainable expansion, or Blue Growth, is required and waste management in targeted sectors such as aquaculture is a key
consideration. Shells comprise a significant proportion of
the animal biomass and are usually discarded in aquaculture
practices. There are increasing pressures for recycling and
the promotion of circular economy principles (Hughes,
2017; Domenech & Bahn-Walkowiak, 2019). Much research
highlights the importance of shells in marine environments,
for example for reef restoration, which encourages mollusc
cultivation and ecosystem development (e.g. Gutierrez
et al., 2003). Such studies could also be key for environmental
change adaptation in facilitating natural ecosystem
approaches for the protection of some low-lying coasts, under
long-term sea-level rise (Bamber et al., 2019; Oppenheimer
et al., 2019). Shells have been exploited in a wide variety of
applications, including as a calcium source for laying birds,
liming agents, in wastewater biofiltration and incorporation
into construction aggregates (Morris, Backeljau, &
Chapelle, 2019). Many more innovative shell uses could be
exploited if various technical, regulatory and logistical bottlenecks can be overcome (Morris et al., 2019).
IX. FUTURE DIRECTIONS
Future research will require interdisciplinary teams with
broad knowledge bases fully to understand the complexities
of shell design and production from the molecular level to
physical shell performance. Greater genome sequencing
will be required to enable the wider identification of the
gene networks that control biomineralization. There is also
the need to understand the role that unannotated genes play
in this process, as many are often ignored in current studies
but may be critical members of biomineralization pathways
and fundamental drivers of skeletal structural diversity.
Genomes also underpin the development of advanced
molecular tools, for example in gene editing and probes
for high-resolution microscopy. The latter requires
resources including cell lines, which are virtually absent
1831
for molluscs, yet they are vital for gene manipulation studies
and for single molecule particle tracking in live cells, which
will enable comprehensive evaluations of calcium transport
through cells. The development of model mollusc species is
required to exploit the latest biophysical and biological
techniques, more often used in medical research. Given
molluscan diversity levels, a single species is unlikely to suffice and questions and requirements will dictate the mollusc
models needed.
X. CONCLUSIONS
(1) Bivalves and gastropods demonstrate considerable
inter- and intraspecific shell morphological variation,
both inherent, and in response to environmental
conditions.
(2) Variation in intraspecific shell characteristics can
result from genetic adaptation with selection acting
on standing population genetic variation and/or phenotypic plasticity. In some instances, adaptation can
be rapid, taking place within a few generations.
(3) Epigenetics may modify phenotypic traits across
generations.
(4) Shell characteristics and animal physiology are
strongly defined by adaptation to local conditions,
particularly temperature and food supply. Salinity
exerts a stronger influence across latitudes,
particularly on shell organic content and
microstructures.
(5) Energetic trade-offs can occur when accommodating
intraspecific variations in shell composition to different environmental conditions. Abiotic and biotic factors trigger regulatory processes that impact ongoing
processes such as somatic growth, energy use and
shell growth.
(6) Transport of CaCO3 within cells is mediated by epithelial processes, which often involves ion channels.
However, considerable debate continues as to how
calcium is transported in cells.
(7) Current knowledge of biomineralization molecular
pathways is largely restricted to the final proteins;
there is much less information on upstream control
sequences and gene networks associated with biomineralization and protein–protein interactions.
(8) At least some genes involved in biomineralization
pathways have evolved rapidly. These comprise a
mix of lineage-restricted proteins and unique combinations of co-opted ancient genes, which have gained
additional functions through the acquisition of new
domains.
(9) Alternative splice forms of genes provide added layers
of complexity to biomineralization pathways. Alternative splice forms of the same genes associated with
larvae and adults of the same species can differ
dramatically.
Biological Reviews 95 (2020) 1812–1837 © 2020 The Authors. Biological Reviews published by John Wiley & Sons Ltd on behalf of Cambridge Philosophical
Society.
Melody S. Clark et al.
1832
(10) Recent gene editing using CRISPR/Cas9 in molluscs
provides an important tool for advancing understanding of gene function and gene interactions.
(11) Despite mathematical model developments and the
demonstration of transcript compartmentalization
in mantle tissues, the complex variety of threedimensional shell morphologies is largely unexplained to date.
(12) Prediction of future environmental change impacts
on aquaculture is complex and needs to include indirect effects (parasites, emerging diseases, invasive species, etc.) alongside direct effects (temperature, ocean
acidification, salinity, etc.) along with species interactions and assemblage/ecosystem-level effects.
(13) Historical studies indicate a greater resilience to
chronic incremental climate change than has been
predicted using short-term experimental evaluations.
(14) Regional assessments, accounting for local habitat
conditions, will be more accurate for planning new
aquaculture facilities, whilst global assessments will
better indicate where new aquaculture opportunities
may emerge as species’ ranges alter under climate
change.
(15) Selection experiments indicate that sufficient standing genetic variation exists in molluscs to enable
breeding of lines more resilient to future conditions.
(16) Exploitation via biomimetics of shell microstructures
and properties in materials science and recycling shell
waste for a circular economy represent important
areas where mollusc research could potentially realize societal gains.
XI. ACKNOWLEDGEMENTS
This manuscript was partly funded by the European Union
Seventh Framework Programme [FP7] ITN project
‘CACHE: Calcium in a Changing Environment’ (www.
cache-itn.eu) under REA grant agreement 605051. The
authors thank everyone involved in this project including
Mark Blaxter, Xushuai Zhang, Yan Wang-Duffort, Nina
Fox, Rita Pereira, Nicola Munro, Elaina Ford, Lucy Gonzalez, Christina Chatzela, Rachel Ramirez, our EU Project
Officer Giuliana Donini and EU Mid-Term Review Expert
Guy Duke. Also, many thanks to our Project Partners: the
Association of Scottish Shellfish Growers, specifically Nick
Lake, Janet Brown and Walter Speirs, Coastal Research
and Management, Kiel especially Peter Krost, and our
External Experts Professor Catherine Boyen, Station Biologique de Roscoff and Professor Mike Thorndyke. We also
thank Laura Gerrish and Jamie Oliver (British Antarctic Survey) for drawing figures. Additional funds were provided by
Fundaç~ao para a Ciência e a Tecnologia (FCT) through project UID/Multi/04326/2019 and the European Marine
Biological Research Infrastructure Cluster-EMBRIC
(EU H2020 research and innovation program, agreement
n 654008) and a Natural Environment Research Council
Studentship (Project Reference: NE/J500173/1) to V.A.S.
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(Received 30 March 2020; revised and accepted 17 July 2020; published online 31 July 2020 )
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Gauthier Chapelle