Sequence analysis of a normalized cDNA library of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32 strain

Abstract

In the past decades, reports on bivalves' pathogens and associated mortalities have steadily increased. To face pathogenic micro-organisms, bivalves rely on innate defenses established in hemocytes which are essentially based on phagocytosis and cytotoxic reactions. As a step towards a better understanding of the molecular mechanisms involved in the mussel Mytilus edulis innate immune system, we constructed and sequenced a normalized cDNA library specific to M. edulis hemocytes unchallenged (control) and challenged with Vibrio splendidus LGP32 strain for 2, 4 and 6 h. A total of 1,024,708 nucleotide reads have been generated using 454 pyrosequencing. These reads have been assembled and annotated into 19,622 sequences which we believe cover most of the M. edulis hemocytes transcriptome. These sequences were successfully assigned to biological process, cellular component, and molecular function Gene Ontology (GO) categories. Several transcripts related to immunity and stress such as some fibrinogen related proteins and Toll-like receptors, the complement C1qDC, some antioxidant enzymes and antimicrobial peptides have already been identified. In addition, Toll-like receptors signaling pathways and the lysosome and apoptosis mechanisms were compared to KEGG reference pathways. As an attempt for large scale RNA sequencing, this study focuses on identifying and annotating transcripts from M. edulis hemocytes regulated during an in vitro experimental challenge with V. splendidus. The bioinformatic analysis provided a reference transcriptome, which could be used in studies aiming to quantify the level of transcripts using high-throughput analysis such as RNA-Seq.

Keywords: 454 Pyrosequencing; Hemocyte; Mytilus edulis; Transcriptome; Vibrio splendidus.

Fig. 1

(A) cDNA profile before and after normalization; (B) PCR Control after normalization. Following extraction and quality control, RNA from the control and challenged hemocytes (2, 4 and 6 h) were pooled before conducting cDNA first strand synthesis. cDNA normalization was perform by Clontech Inc. CA, US using duplex-Specific Nuclease (DSN) treatment. Electrophoresis profiles were compared before (A, before) and after cDNA normalization (A, after). Following normalization, all major bands disappeared. To further control normalization, cDNAs to Elongation Factor alpha (EF) and tubulin were PCR-amplified with different cycle numbers and amplification products were compared. M: molecular size markers.

Fig. 2

(A) lengths distribution of all reads generated and (B) lengths distribution of the assembled sequences. A total of 1,024,708 reads were generated by 454 sequencing with a mean sequence length of 256 pb (histogram A plotting number of reads per read length). Following Newbler assembly, 19,622 sequences with an average length of 925 bp were generated, most of them (44%) ranged between 500 and 1000 bp in length (histogram B plotting percent of sequence in function of transcripts size ranges).

Fig. 3

Organisms that match to the assembled sequences of Mytilus edulis hemocytes exposed to Vibrio splendidus LGP32. Percentage of transcripts finding similarity with various species in the non-redundant sequence databases (Megablast program against nucleic acids).

Fig. 4

Schematic comparison of Mytilus and KEGG reference TLR pathway members. Shaded boxes indicate proteins identified in our 454 results and white boxes the absent ones.

Fig. 5

Schematic comparison of Mytilus and KEGG reference lysosome mechanisms. Shaded boxes indicate proteins identified in our 454 results and white boxes the absent ones.

Fig. 6

Schematic comparison of Mytilus and KEGG reference activation mechanism of NADPH oxidase. Shaded boxes indicate proteins identified in our 454 results and white boxes the absent ones.

Fig. 7

Schematic comparison of Mytilus and KEGG reference apoptosis pathways. Shaded boxes indicate proteins identified in our 454 results and white boxes the absent ones.