Data are representative of at least three independent experiments. Significance of the represented data was calculated using ANOVA (analysis of variance). Data are expressed as mean 6 standard errors (S.E.). Figure S1 Listeria-induced IFN-gamma production by spleen cells 9 days after infection (i.v.). Mice were infected with 10 3 CFU of Lm, 10 7 CFU wild type L.inn, or with 10 7 CFU of L.inn::vgc strain. On day 9 after infection, mice were killed and spleens removed. Single cell suspensions were stimulated in vitro with secreted soluble Listeria antigen to produce IFN-gamma. After 48 hours, culture supernatants were tested for presence of IFNgamma by ELISA. *P,0.05 (EGD-e vs L.inn::vgc). (TIF) Figure S2 Intraperitoneal infection with the L.inn::vgc strain induces protective immunity. Mice were infected intraperitoneally with Lm, L.inn and the L.inn::vgc strain as described in figure 4A . After 2 months all mice were challenged i.v. with a lethal dose (206LD 50 ) of the wild type Lm. As a control, a group of uninfected normal mice was included. Survival was monitored up to 8 days after challenge. (TIF) Figure S3 Quantification of CD44 expression on CD8 + splenocytes following primary and recall infection with Lm, L.inn and the L.inn::vgc strain. Flow cytometry was performed on spleen cells, isolated from mice on day 60 after the primary infection or day 5 after the challenge. Cells were stained with FITC-labelled anti-Lyt-2 and PE-labelled anti-CD44. Numbers shown are gated CD8 + CD44 hi T cells and analyzed with CELLQuest software. (TIF) Transcriptomics of In Vitro Immune-Stimulated Hemocytes from the Manila Clam Ruditapes philippinarum Using High-Throughput Sequencing BACKGROUND: The Manila clam (Ruditapes philippinarum) is a worldwide cultured bivalve species with important commercial value. Diseases affecting this species can result in large economic losses. Because knowledge of the molecular mechanisms of the immune response in bivalves, especially clams, is scarce and fragmentary, we sequenced RNA from immune-stimulated R. philippinarum hemocytes by 454-pyrosequencing to identify genes involved in their immune defense against infectious diseases. METHODOLOGY AND PRINCIPAL FINDINGS: High-throughput deep sequencing of R. philippinarum using 454 pyrosequencing technology yielded 974,976 high-quality reads with an average read length of 250 bp. The reads were assembled into 51,265 contigs and the 44.7% of the translated nucleotide sequences into protein were annotated successfully. The 35 most frequently found contigs included a large number of immune-related genes, and a more detailed analysis showed the presence of putative members of several immune pathways and processes like the apoptosis, the toll like signaling pathway and the complement cascade. We have found sequences from molecules never described in bivalves before, especially in the complement pathway where almost all the components are present. CONCLUSIONS: This study represents the first transcriptome analysis using 454-pyrosequencing conducted on R. philippinarum focused on its immune system. Our results will provide a rich source of data to discover and identify new genes, which will serve as a basis for microarray construction and the study of gene expression as well as for the identification of genetic markers. The discovery of new immune sequences was very productive and resulted in a large variety of contigs that may play a role in the defense mechanisms of Ruditapes philippinarum. The Manila clam (Ruditapes philippinarum) is a cultured bivalve species with important commercial value in Europe and Asia, and its culture has expanded in recent years. Nevertheless, diseases produced by a wide range of microorganisms, from viruses to metazoan parasites, can result in large economical losses. Among clam diseases, the majority of pathologies are associated with the Vibrio and Perkinsus genera [1] [2] [3] . Although molluscs lack a specific immune system, the innate response involving circulating hemocytes and a large variety of molecular effectors seems to be an efficient defense method to respond to external aggressions by detecting the molecular signatures of infection [4] [5] [6] [7] [8] ; however, not many immune pathways have been identified in these animals.