Insights into shell deposition in the Antarctic bivalve Laternula elliptica: gene discovery in the mantle transcriptome using 454 pyrosequencing

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Figure 1

Annotated longitudinal dissection of L. elliptica with one of the shells removed. Photograph copyright permission obtained from Erwan Amice.

Figure 2

GO categories of genes identified in L. elliptica data by BLAST sequence similarity searching.

Figure 3

Amino acid alignment of the region between transmembrane (TM) regions 7 and 9 of the α sub-unit of SEC61. Putative cold-adapted amino acid substitutions are indicated at positions 327, 328 and 339. Invertebrate-specific substitutions are labelled in red on the consensus line, insect-specific substitutions labelled in blue and the single potential bivalve-specific substitution labelled in green. Species abbreviations and accession numbers: Nan: Notothenia angustrata (Q8AY35); Pbo: Pagothenia borchgrevinki (Q8AY36); Dma: Dissostichus mawsoni (AY113841); Han: Harpagifer antarcticus (Q7T278); Ham: Hemitripterus americanus (Q8AY34); Bsa: Boreogadus saida (Q8AY33); Gog: Gadus ogac (Q8AY32) (all cold-adapted); Omy: Onchorhynchus mykiss (Q98SN9); Dre: Danio rerio (Q90ZM2); Mmu: Mus musculus (P61620); Cqu: Culex quinquefasciatus (B0WNA0); Aae: Aedes aegypti (Q17CM3); Dme: Drosophila melanogaster (Q8STG9). Lgi: Lottia gigantea (cluster: >jgi|Lotgi1|194715|estExt_Genewise1.C_sca_610223 from sequences: 4236761:1772, 4236761:2059 and 4236761:4476 extracted from http://genome.jgi-psf.org/Lotgi1/Lotgi1.home.html; Lel: Laternula elliptica, contig00029.

Figure 4

Multiple sequence alignment of the putative PTH/CALR receptor in L. elliptica contig 11573 (Lel_11573) with the N-terminal region of the putative metazoan homologues. The sequence alignment starts from the beginning of the Laternula fragment. Conserved cysteine residues are indicated by dots "•" and the Aspartic acid (D) residue within the N-terminal sequence motif C-x(4)-D-x(3,4)-C-Wx(11,12)-C-P involved in CLR/RAMP/ligand interactions indicates by a cross "+". The beginning of receptor TM1 region is indicated by an arrow and the localisation of putative glycosylation sites (NXT/S) indicated by blue dashed boxes. Amino acid conservation in the alignment is colour coded and black shaded columns mean total residue conservation. Accession numbers of the sequences used in the alignment are: Human (Hsa, PTHR1 NP_000307; CALR NP_001158209; CGRP NP_005786); Chicken (Gga, XP_418507); Zebrafish (Dre, AAI62580); Xenopus laevis (Xla, NP_001080206); Takifugu rubripes (Tru, NP_001098689); Crassostrea virginica (Cvi, JC8022 (est)); Ciona intestinalis (Cin, BAI63096); Crassostrea gigas (Cgi, AM858508); Culex quinquefasciatus (Cqu, XP_001864896); Anopheles gambiae str. PEST (Aga, XP_321982); Ixodes scapularis (Isc, XP_002414039); Apis mellifera (Ame, XP_001122670); Nasonia vitripennis (Nvi, XP_001605780). The predicted invertebrate proteins are marked in italics and were included for comparison with the bivalve (L. Elliptica; Lel) deduced amino acid sequence of contig 11573 which is highlighted in bold.

Figure 5

Multiple sequence alignment of the putative PTH/CALR receptor in L. elliptica contig 14182 (Lel_14182) with the TM domain region of putative metazoan homologues. The localization of TM1, TM2 and TM3 are indicated by lines and ICL 1 and 2 (intracellular loop) and ECL1 (extracellular loop) are named. Conserved cysteines are indicated by dots "•" and amino acid residues involved in Gs coupling are marked with a cross "+". Amino acid conservation in the alignment is colour coded and black shaded columns mean total residue conservation. Accession numbers of sequences used in the alignment are the same as for Figure 3 and predicted invertebrate proteins are marked in italics and the deduced amino acid sequence from L. elliptica (Lel) is highlighted in bold.