Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyrosequencing
© Clark et al; licensee BioMed Central Ltd. 2010
Received: 22 February 2010
Accepted: 8 June 2010
Published: 8 June 2010
The Antarctic clam, Laternula elliptica, is an infaunal stenothermal bivalve mollusc with a circumpolar distribution. It plays a significant role in bentho-pelagic coupling and hence has been proposed as a sentinel species for climate change monitoring. Previous studies have shown that this mollusc displays a high level of plasticity with regard to shell deposition and damage repair against a background of genetic homogeneity. The Southern Ocean has amongst the lowest present-day CaCO3 saturation rate of any ocean region, and is predicted to be among the first to become undersaturated under current ocean acidification scenarios. Hence, this species presents as an ideal candidate for studies into the processes of calcium regulation and shell deposition in our changing ocean environments.
454 sequencing of L. elliptica mantle tissue generated 18,290 contigs with an average size of 535 bp (ranging between 142 bp-5.591 kb). BLAST sequence similarity searching assigned putative function to 17% of the data set, with a significant proportion of these transcripts being involved in binding and potentially of a secretory nature, as defined by GO molecular function and biological process classifications. These results indicated that the mantle is a transcriptionally active tissue which is actively proliferating. All transcripts were screened against an in-house database of genes shown to be involved in extracellular matrix formation and calcium homeostasis in metazoans. Putative identifications were made for a number of classical shell deposition genes, such as tyrosinase, carbonic anhydrase and metalloprotease 1, along with novel members of the family 2 G-Protein Coupled Receptors (GPCRs). A membrane transport protein (SEC61) was also characterised and this demonstrated the utility of the clam sequence data as a resource for examining cold adapted amino acid substitutions. The sequence data contained 46,235 microsatellites and 13,084 Single Nucleotide Polymorphisms(SNPs/INDELS), providing a resource for population and also gene function studies.
This is the first 454 data from an Antarctic marine invertebrate. Sequencing of mantle tissue from this non-model species has considerably increased resources for the investigation of the processes of shell deposition and repair in molluscs in a changing environment. A number of promising candidate genes were identified for functional analyses, which will be the subject of further investigation in this species and also used in model-hopping experiments in more tractable and economically important model aquaculture species, such as Crassostrea gigas and Mytilus edulis.
Laternulids are infaunal bivalve molluscs, which morphologically resemble the soft-shelled clam Mya arenaria, the major ingredient of clam chowder. In spite of a widespread latitudinal distribution ranging from the tropics, through temperate Australasia to Antarctica , research on this genus is dominated by work on the Antarctic species (Laternula elliptica). This clam has been studied for a number of years and is one of the best characterised Antarctic marine invertebrates. Studies initially focused on its ecology , and general physiology: reproduction [3, 4], development [5, 6], growth [7, 8] and seasonal energetics [9, 10]. However more recent research has focused on the longevity of this species in relation to reactive oxygen species production, antioxidant defences and cellular ageing , as this species often lives 25 years or more . It has also been the subject of significant investigation of its thermal tolerance and the expected impact of climate change [13–15].
Antarctic marine invertebrates are stenothermal  and L. elliptica is one of the more sensitive species [13–16]. These animals suffer significant mortalities at 4-5°C, but lose essential biological functions, such as the ability to bury in sediment, much earlier, at only 1-2°C over current summer maximum sea water temperatures [13–15]. This thermal response is viewed against predictions that globally oceanic sea surface temperatures are predicted to rise on average by 2°C over the next 100 years [17, 18]. However, regional differences are apparent and climate change along the Antarctic Peninsula has been particularly rapid with a temperature increase in the surface layers of the Bellingshausen Sea of 1°C in 50 years . The predictions of the effect of these thermal changes on Antarctic marine biodiversity are complex  and further complicated by reductions in ocean pH.
Antarctic species, in general, have been proposed as excellent candidates for the development of climate change molecular biomarkers , whilst L. elliptica in particular has strong support as a sentinel species . It has a circumpolar distribution and is highly abundant . It is the largest individual mollusc in terms of live weight  and one of the highest in terms of total ecological biomass . Being an infaunal filter-feeder, it also plays a significant role in benthopelagic coupling [25, 26] and therefore is a keystone species of the Antarctic marine ecosystem.
In the 250 years since the onset of the industrial revolution, ocean pH has fallen from an average of 8.16 to 8.05 and is predicted to decrease by a further 0.3-0.4 pH units by the end of this century. These predicted changes in ocean pH are greater, and far more rapid, than any experienced in the past 300 million years [27–30]. The Southern Ocean will be particularly affected as it has amongst the lowest present-day CaCO3 saturation state of any ocean region, and will therefore be among the first to become undersaturated . The ability of marine organisms to adapt to this unprecedented environmental modification of increased temperature and reduced pH is largely unknown, with particular concern expressed over calcifying animals such as echinoderms and molluscs .
Laternula species are conservative in shell form and habitat suggesting they share morphological constraints at different latitudes . Recent data suggests however, that at least in the Southern Ocean, L. elliptica exhibits a high degree of plasticity in the thickness of shell deposition. This physical characteristic varies markedly between locations along the Antarctic Peninsula. Specimens at Rothera base, Adelaide Island (67° 4' 07" S, 68° 07' 30" W) have shells at least 2-3× thicker than those at Jubany base King George Island (62.23°S, 58.67°W) (Harper, pers comm). The Rothera animals also have damage repair rates 5-12× higher than Jubany animals, possibly due to increased frequency of iceberg impact (Harper, pers comm). This plasticity in shell thickness is superimposed against a homogeneous population structure across all sites (Hoffman, pers comm). Such plasticity in shell deposition has also been observed in the Antarctic limpet, Nacella concinna .
Shells of L. elliptica were recently subjected to an end of century scenario pH 7.4 trial and showed extensive damage over 56 days . However, these were isolated shells and such studies do not take into account the ability of the animal to ameliorate shell loss via increased deposition or the effects of having an infaunal life habit. To understand the dynamics of shell turnover and the response of this process to both biotic and abiotic factors a better understanding of the molecular basis and regulation of shell formation is required. The studies which do exist have revealed that the molluscan shell is composed largely of calcium carbonate and organic macromolecules which are secreted by the mantle [35–40]. Hence, whole animal studies are essential not only to understand the shell deposition process in relation to altered temperature and pH, but also the more subtle effects of altered environmental conditions on calcium regulation in cellular processes and the energetic trade-offs of responses to climate change.
The aragonitic shell of L.elliptica comprises two layers: a very thin prismatic layer on the outside covering layers of sheet nacre on the inner side (with sheet nacre on both sides of the pallial myostracum); and a periostracum of around 10 μm surrounds the outer shell where it has not been removed by abrasion [[41, 42]; Harper, pers comm.]. The mantle secretes the shell and forms a large thin sheet of tissue between the shell and the internal organs and extends beyond the rest of the body, so that part of the edge is exposed to external conditions . It thus serves two purposes, secretion of the shell matrix and also protection from the external environment, either via sealing the edge of the shell or from damage to the shell itself (for example, after being impacted by an iceberg (Peck, pers comm)). The edge of the mantle comprises three folds, of which, only the outer fold is involved in laying down the actual shell material. The periostracum emerges from the groove between the middle and outer folds and bends back to cover the shell. The primary role of the perisotracum is believed to be shell secretion and maintenance, but there are other roles such as protection from the external environment, infestation and predatory borers . The mantle tissue for this analysis was taken as a cross section of all mantle folds and included periostracum material.
Results and Discussion
Most commonly expressed sequences with associated BLAST matches.
No of reads
Map kinase interacting serine threonine protein kinase.
California sea hare
Collagen pro-α chain
ATP synthase sub-unit α
Collagen type IV α6
B cell translocation gene
Poly adenylate binding protein
Voltage gated potassium channel complex
Stress associated endoplasmic reticulum protein (SERP2)
Transport protein SEC1 subunit α
Mitochondrial carrier protein, putative ADP/ATP translocase
Copepod sea louse
Chagas insect disease vector
Adipose differentiation-related protein
40 s ribosomal protein S2
60 s ribosomal protein L15
Y-box factor homolog
California sea hare
Vacuolar ATP synthase
Myosin heavy chain
Ribosomal protein L3
ATP synthase sub unit β
Ribosomal protein L28
Brown dog tick
Giant river prawn
Mannan-binding lectin-associated serine protease
40 s ribosomal protein S11
GABA (A) receptor associated protein
Sequence similarity searching of the GenBank non-redundant database with BLAST produced matches against only 3,098 of the contigs using a < 1e-10 cut off value. This poor level of sequence similarity matching (17%) has also been noted in previous work on this species (Truebano et al, submitted) and is due to a lack of sequences from a closely related mollusc species in the databases. This is reflected in the number of different species that show sequence matches against our data; Table 1 comprises 39 BLAST sequence similarity results with the best matches originating from 33 species ranging from hydrozoans and arthropods through to vertebrates. To date (25/01/10) there are only 25,032 nucleotide sequences, 195,275 ESTs, 14,507 proteins and 356 genes from the class Bivalvia in the public databases http://www.ncbi.nlm.nih.gov and these are dominated by entries from Mytilus and Crassostrea species. At the sub-class level, the number of nucleotide and protein entries are 86 and 19 respectively, which is further reduced to 24 and 16 at the family level. The genbank non-redundant database  is one of the best annotated sources for comparative in silico gene analyses. However, of potential use, in terms of EST verification and gene mining are other less well annotated sources of molluscan sequence data, such as the sequenced genome of the gastropod snail (Lottia gigantea) and 454 data from Mytilus species . These comprise larger molluscan datasets than found in genbank, but BLAST sequence similarity searches using a <1e-10 cut off value merely emphasized the evolutionary distance between the molluscs studied. For example, just over 2% of the Laternula contigs matched the ESTs and EST clusters produced from Lottia, although this increased to 17.5% against the Lottia filtered gene set. Less than 1% of the Laternula contigs matched the Mytilus mantle-specific 454 libraries and the 42,364 ESTs from M. californianus in GenBank. Hence there are no species closely related to L. elliptica with large amounts of sequence data in the public domain and therefore our data significantly increases resources in this area and provides an important source of comparative data for other Molluscan species.
Highly expressed sequences
The mantle, whilst not a muscle per se, is contractile and hence many of the highly expressed sequences consist of structural or muscle-related genes, such as actin, collagen, troponin, calponin, adipose differentiation-related protein and myosin , although some e.g. collagen, may also be involved in shell synthesis . Interestingly, the most commonly expressed sequence is that of a MAP kinase interacting serine threonine protein kinase (Mnk1). This gene is a transcriptional and translational regulator of mRNA, in particular acting via the phosphorylation of the elongation initiation factor (EIF4E), which is an important modulator of cell growth and proliferation . Studies in Aplysia have shown Mnk1 to be a negative regulator of cap-dependant translation in neurons , whilst in other species it has also been shown to bind stress activated p38 and may play a role in response to environmental stress . The role of this gene in cell growth links with the identification of the B cell translocation gene (also involved in cell differentiation) and the Y-box factor homologue (a transcriptional and translational regulator of mRNA) , indicating that the mantle is an area of continual growth.
From the above, the mantle is clearly a metabolically and transcriptionally active tissue. This is further exemplified by the presence of ATP synthases, an ADP/ATP translocase, NADH-ubiquinone oxidase, genes from the glycolysis pathway, ribosomal RNAs and arginine kinase. The latter is a phosphagen kinase and these enzymes are prevalent in systems with fluctuating energy demands, acting as an energy buffering system  and also as an energy shuttle delivering ATP generated by mitochondria to high energy requiring processes, such as membrane turnover and potentially shell deposition . Whilst the phosphagen kinases are a multigene family, arginine kinase is the only form of this gene in arthropods and molluscs . It also has other functions such as buffering intracellular pH which would be important in the extrapallial space with the supersaturation of shell matrix components, including calcium ions.
Animals living in constant cold temperatures could initially be thought to be more vulnerable to damage by reactive oxygen species, due to slow cell and protein turnover rates and the consequent accumulation of oxidised proteins [12, 65]. However, L. elliptica is being used as a model for ageing studies as this organism appears to have uncoupled the ageing process and antioxidant production. It has a higher antioxidant capacity compared to shorter lived temperate clams, with a constant requirement for stable antioxidant status until old age [11, 12]. Enzyme assays show that catalase, in particular, remains at a constant level throughout its lifetime and this is exemplified by the expression of catalase in the genome of adults of this species. The stress associated endoplasmic reticulum protein (SERP2) is also highly expressed and this may be linked to antioxidant capacity or it may protect unfolded target proteins against degradation and facilitate correct glycosylation. As a member of the RAMP4 family (ribosome associated membrane proteins) it may also be involved in the stabilisation of membranes in response to stress. It is known that there are problems folding proteins at low temperatures  and to date, of the few Antarctic marine species investigated, the majority do not exhibit the classical heat shock protein (HSP) stress response. Indeed, several species express the inducible form of HSP70 permanently, possibly as a measure towards more efficient folding of proteins at low temperatures  and the expression of SERP2 may contribute towards this "extra" required function and form part of a "preparative defence" strategy against the cold .
The final category of highly expressed genes comprises those involved in immune function, e.g. thymosin β , α macroglobulin and a mannan-binding lectin associated serine protease, which is a complement control module. The reason for this up-regulation may be slightly more complex than it initially appears. The mantle edge is in constant contact with the external environment and hence there will be permanent challenges to the immune system. This is compounded in the Rothera population of L. elliptica by a significant amount of physical damage (Harper, pers comm). These almost certainly will require the enhanced expression of immune-related genes, as the external protection of the shell is compromised, along with matrix deposition for shell repair. However, physical damage is a stressor in its own right which along with the effect of the cold environment (another potential stressor) may induce changes in the immune system as stress and the immune system have been found in many species to be inextricably linked .
Putative shell deposition transcripts
The ten most commonly expressed sequences (in order of abundance) in mantle tissue from 4 bivalves.
map kinase interacting serine threonine protein kinase.
collagen pro-α chain
cytochrome c oxidase 1
16 s ribosomal protein
KRMP-8, glycine rich structural protein
NADH dehydrogenase subunit 4
ATP synthase sub-unit α
cytochrome c oxidase 1
KRMP-8, glycine rich structural protein
collagen type IV α6
N14 matrix protein
NADH dehydrogenase subunit 4
NADH dehydrogenase subunit 4
NADH dehydrogenase subunit 4
Several of the most highly expressed genes in our dataset are almost certainly involved in shell deposition, including tyrosinase. The periostracum is secreted as a soluble precursor (the periostracin) and this is then cross-linked by o-diphenols and tyrosinase (or phenoloxidases) to form an insoluble periostracum [74, 75]. Tyrosinase can also be involved in pigment formation in the prismatic layer and evidence from the pearl oyster demonstrates several different paralogues of tyrosinase which are involved in these different functions [76, 77]. However, in order to discover genes within our dataset that are likely to play a role in shell deposition and calcium regulation, we searched the literature to generate an in-house database of proteins involved in extracellular matrix (ECM) formation and calcium homeostasis in metazoans (Supplemental Tables 1 and 2). Numerous transcripts were identified; hence the following section will give only a brief outline of the putative role of the more abundant transcripts.
The presence of putative transcripts for carbonic anhydrase in L. elliptica mantle is unsurprising as this protein was first identified in the shell in 1948  and it has subsequently been implicated in matrix mineralisation by generating an acidic environment through the conversion of respiratory CO2 into HCO3 in the presence of water . Putative transcripts for the matricellular glycoprotein, secreted protein acidic rich in cystein (SPARC, a basal membrane component) were also identified. This trimodular protein promotes proper assembly and maturation of the matrix scaffold and is highly conserved in animal phyla . In vertebrates the latter is achieved in part through the interaction of SPARC with fibril forming collagens (I, II, III and V) [80, 81] and although it is necessary to conduct further work to better characterize these transcripts, orthologues of collagen I, II and V were identified.
Additional transcripts identified in the L. elliptica mantle transcriptome potentially implicated in ECM formation/turnover in metazoans include the thrombospondins, which are a family of large, secreted, multi-modular, calcium-binding glycoproteins which appear to interact with collagens and integrins and have been implicated in skeletal disorders in mammals . Transcripts for tenascin, a large glycoprotein containing several fibronectin III type repeats and implicated in cell adhesion in chordates was identified in L. Elliptica . Interestingly, several transcripts for metalloproteinase 1 (collagenase 1) which are important in extracellular matrix turnover were also identified. It is apparent even from this brief consideration that numerous homologues of genes identified in the ECM of the vertebrate skeleton are also present in the mantle transcriptome. Future work will permit a more precise characterization of the localization and function of these mantle transcripts and hence provide a better understanding of shell formation. This will be essential for ecophysiological studies.
The clam orphan calcium regulatory receptors
Best database matches of the Laternul a sequences to the family 2 GPCRs.
PREDICTED: similar to parathyroid hormone receptor [Nasonia vitripennis] 4e-17 calcitonin receptor [Culex quinquefasciatus] 2e-14
PREDICTED: similar to calcitonin receptor, partial [Acyrthosiphon pisum] 9e-10
Comprehensive 454 pyrosequencing of mantle tissue from the Antarctic clam ( Laternula elliptica) has produced a transcriptome of 18,290 contigs. In spite of a low level of putative gene identifications (17%, produced via database sequence similarity searching), it was possible to identify a considerable number of transcripts putatively related to shell deposition (e.g. tyrosinase and SPARC). This was via similarity to annotated sequences from other molluscs in the databases or to genes known to be involved in skeletal formation in vertebrates. In particular, 2 putative members of family 2 GPCRs were identified which share the highest sequence similarity to the metazoan parathyroid hormone (PTH) and calcitonin/calcitonin Gene-related peptide (CLR/CGRPR) receptors. In vertebrates these genes are important mediators of the action of the calcitropic factors, calcitonin (CT) and parathyroid hormone (PTH). This dataset, whilst being the first 454 sequence to be generated from an Antarctic invertebrate, also provides a significant resource for comparative studies into protein cold adaptation and the processes of shell deposition, in particular the reaction of the shell secretome to environmental stress in the form of climate change effects. Both avenues will be explored in the future within our laboratory, both in Laternula and economically important model temperate mollusc species, such as Crassostrea gigas and Mytilus galloprovincialis.
All animals used in experimental work were collected at Rothera Research Station, Adelaide Island, Antarctic Peninsula (67° 4' 07" S, 68° 07' 30" W) by SCUBA divers during the austral summer at depths of 10-15 m. The animals were immediately returned to the laboratory where they were maintained in a through-flow aquarium with a temperature of 0.6 ± 0.3°C, under a simulated natural light:dark cycle. All animals were mature adults, with a range of shell sizes between 50.1-83.5 mm. As shell length is related to animal age: surface aging estimates using growth rings produced an mean age of 8.3 years (SE mean 0.207) with a range from 6-14 years and a median of 8 years (S. Morley pers comm). Mantle tissue was dissected from the animals and cross sections comprising all 3 folds and the periostracum were immediately flash frozen in liquid nitrogen for later RNA extraction.
RNA isolation and cDNA production
Mantle RNA was extracted from 24 animals using a modified TRI reagent protocol. After homogenization in Tri Reagent (Sigma) and chloroform extraction, the samples were subjected to a lithium chloride precipitation step. RNA was precipitated using a 1:1 isopropanol:saline solution (0.8 M sodium citrate and 1.2 M NaCl) and after resuspension, the RNA was subjected to a further precipitation using 250 μl 7.5 M LiCl. The extractions were further cleaned using RNeasy mini kit columns (Qiagen, Crawley, Sussex, UK) following manufacturer instructions in order to eliminate LiCl and salt residues. 5 μg of RNA was PCR amplified using the protocol described in  prior to preparation for the 454 run. Samples were nebulised at 30psi for one minute and subsequently purified with Ampure (Agencourt) to produce fragments 300 bp and above. The ends were polished and the 454 titananium adapters containing specific MID sequences were attached. Fragments containing both a and b adapters were selected and quantified. Libraries were amplified by emulsion PCR, beads recovered and enriched and placed on a picotiter plate for sequencing by the 454 procedure.
454 Assembly and Analysis
The raw data comprised 1,034,155 reads. Crossmatch (P. Green, unpublished) was then applied to screen for adaptor sequences and other artifacts of the pyrosequencing procedure and also vector sequences using the UniVec database http://www.ncbi.nlm.nih.gov/VecScreen/UniVec.html. Stripping the masked sequence from the ends and removing reads with masked sequence in the middle resulted in 778,629 sequences that were entered into the Newbler program  for assembly. This resulted in 18,290 contigs. All singletons were discarded. Files containing the reads have been submitted to the National Center for Biotechnology Information Short Read Archive (accession number SRA011054). The mapping facility of Newbler was applied to the assembly to determine the number of SNPs, and Phobos  was used for microsatellite discovery. The contigs were then searched for sequence similarity using BLAST  against the genbank non-redundant database  and unannotated data from other bivalve species: the gastropod snail: Lottia gigantea http://genome.jgi-psf.org/Lotgi1/Lotgi1.home.html and the Mytilus 454 mantle-specific datasets (4442949.3: M. galloprovincialis mantle unassembled and 4442954.3 M. edulis mantle unassembled)  lodged under the MG-RAST database: Meta Genome Rapid Annotation using Sub-system Technology http://metagenomics.nmpdr.org/) . The Gene Ontology (GO)  mappings were determined by an in-house database on all Swissprot and Trembl  BLAST scores below a threshold of 1e-10. Sequence manipulation was carried out using the EMBOSS suite of programmes . Sequences were clustered using ClustalW  and the alignments displayed using BoxShade v3.21 .
This paper was produced within the BAS Q4 BIOREACH/BIOFLAME core programmes. The authors would like to thank Guy Hillyard for performing the RNA extractions, Gavin Burns for the PCR amplifications, Simon Morley for obtaining sample material and all members of the Rothera Dive Team for collecting animals. Overall diving support was provided by the NERC National Facility for Scientific Diving at Oban. The sequencing was performed at the NERC Biomolecular Analysis Facility, Liverpool and we would particularly like to thank Margaret Hughes and Kevin Ashelford for their technical support. We would also like to thank Bela Tiwari of the NERC environmental Bioinformatics Centre (NEBC), CEH, Wallingford for valuable discussions on 454 software, analysis pipelines and processes and Elizabeth Harper at the University of Cambridge for advice on Laternula shell and mantle structure. Our analyses including searching against the Lottia gigantea sequence data v1.0 (July 24 2007) which was produced by the US Department of Energy Joint Genome Institute http://www.jgi.doe.gov/ in collaboration with the user community and made publicly available via their web site: http://genome.jgi-psf.org/Lotgi1/Lotgi1.home.html.
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