Transcriptomic analysis of Ruditapes decussatus oocytes

The European clam Ruditapes decussatus is native to the Mediterranean Sea and Atlantic coasts and, while its production is still relatively low in Europe (8 200 tons/year; FAO, 2006), it is a high value seafood product and economically important in many Mediterranean countries (mainly Portugal, Italy and Spain). R. decussatus culture relies mainly on natural recruitment of seed, therefore is limited by its availability, which is subject to high annual fluctuations. Over the last years, drops in natural recruitment affected the demand of hatchery seed, increasing the interest in artificial spawning and larval rearing. However little is known about the best hatching practices and the biological processes of R. decussatus influencing gamete maturation and competency. In order to address these questions and improve hatchery production as an alternative source of seed, a transcriptomic study of different larval stages and oocytes was performed following a multiplexing strategy on the Illumina platform HiSeq2000. cDNA tagged libraries for stripped oocytes, spawned eggs, larvae 48 hpf, larvae 17, 21 and 30 dpf were constructed and a 6-plex paired-end sequencing lane (2x100bp) was performed, obtaining more than 290 million reads. In order to obtain a most complete representation of the clam transcriptome, all Illumina data were merged with approximately 5,000 publicly available Sanger sequences and nearly 500,000 reads previously obtained with 454 sequencing of larvae, gonads and several adult tissues. Several strategies were tested in order to find the most effective assembly. A mixed strategy was preferred by using two different assembler software (MIRA3 and CLC Genomic Workbench) obtaining a total of 366,433 contigs. A merge was carried out through CAP3 and the amount of metacontigs was 48,329. The contigs not merged in CAP3 were joined to all the metacontigs obtaining a final transcriptome scaffold of 236,646 contigs. A preliminary annotation of 60,986 contigs (25,8%) was attained through Batch Blast similarity searches locally conducted against Danio rerio and Drosophila melanogaster Ensembl protein database. To obtain gene expression values of each contig, Illumina reads of oocytes and larval stages were mapped against the assembled reference transcriptome and RPKM (Reads Per Kilobase of exon model per Million mapped reads) were calculated. A RPKM threshold value of 1 was arbitrarily set to detect the presence of a transcript. Of 2,886 expressed contigs in spawned oocytes, 709 were annotated (against D. rerio) and characterized through the Gene Ontology (GO) analysis and the KEGG pathways identification. It was highlighted that most of the eggs expressed genes play an important role in the main biological processes for energy production, translation and cell cycle. In addition several genes with a role in oocyte maturation were identified. Finally, a comparison of sequence reads obtained from mature oocytes with those from other developmental stages identified a large set of transcripts (2,393) that appear to be exclusively expressed in released egg cells, suggesting that the oocyte transcriptome is very specific.