High-Throughput Sequence Analysis of Turbot (Scophthalmus maximus) Transcriptome Using 454-Pyrosequencing for the Discovery of Antiviral Immune Genes

Abstract

BackgroundTurbot (Scophthalmus maximus L.) is an important aquacultural resource both in Europe and Asia. However, there is little information on gene sequences available in public databases. Currently, one of the main problems affecting the culture of this flatfish is mortality due to several pathogens, especially viral diseases which are not treatable. In order to identify new genes involved in immune defense, we conducted 454-pyrosequencing of the turbot transcriptome after different immune stimulations.Methodology/Principal FindingsTurbot were injected with viral stimuli to increase the expression level of immune-related genes. High-throughput deep sequencing using 454-pyrosequencing technology yielded 915,256 high-quality reads. These sequences were assembled into 55,404 contigs that were subjected to annotation steps. Intriguingly, 55.16% of the deduced protein was not significantly similar to any sequences in the databases used for the annotation and only 0.85% of the BLASTx top-hits matched S. maximus protein sequences. This relatively low level of annotation is possibly due to the limited information for this specie and other flatfish in the database. These results suggest the identification of a large number of new genes in turbot and in fish in general. A more detailed analysis showed the presence of putative members of several innate and specific immune pathways.Conclusions/SignificanceTo our knowledge, this study is the first transcriptome analysis using 454-pyrosequencing for turbot. Previously, there were only 12,471 EST and less of 1,500 nucleotide sequences for S. maximus in NCBI database. Our results provide a rich source of data (55,404 contigs and 181,845 singletons) for discovering and identifying new genes, which will serve as a basis for microarray construction, gene expression characterization and for identification of genetic markers to be used in several applications. Immune stimulation in turbot was very effective, obtaining an enormous variety of sequences belonging to genes involved in the defense mechanisms.

De novo assembly and characterization of the spleen transcriptome of common carp (Cyprinus carpio) using Illumina paired-end sequencing

“…In recent years, high-throughput transcriptome sequencing has facilitated the functional genomic study of fish and has provided insights into the immunogenetics of fish. For example, previous studies have discovered the immune-relevant genes following exposure to pathogenic microorganisms through de novo transcriptome sequencing in several aquaculture fish species, including orangespotted grouper (E. coioides) [6], large yellow croaker (P. crocea) [7,8], turbot (Scophthalmus maximus) [17], Japanese sea bass (Lateolabrax japonicus) [18] and rohu carp (Labeo rohita, Hamilton) [19]. However, to our knowledge, the de novo sequencing of the spleen transcriptome of fish has been limited to only a few species, and this has hindered the progress of immunological research.…”

Transcriptome analysis of soiny mullet (Liza haematocheila) spleen in response to Streptococcus dysgalactiae

“…BLAST results revealed that 38,241 (53.25%), 42,570 (59.28%), and 33,190 (46.22%) unigenes significantly matched with the annotated sequences in NR, NT, and Swiss-Prot databases, respectively. The annotation of soiny mullet spleen transcriptome was comparable to turbot (Scophthalmus maximus) (44.84%) [24] and mud loach (Misgurnus anguillicaudatus) (43.76%) [25], while higher than that in crucian carp (17.44%) [26] and Japanese flounder 100-500 500-1000 1000-1500 1500-2000 >=2000…”

Proliferation and resistance difference of a liver-parasitized myxosporean in two different gynogenetic clones of gibel carp

“…As an orthologous to mammalian apoA-II, apo-14 is a fish-specific fundamental constituent of high-density lipoprotein (Choudhury et al 2009), and is up-regulated in liver of European seabass (Dicentrarchus labrax) infected with Vibrio anguillarum (Sarropoulou et al 2009), in skin mucus of Atlantic cod (Gadus morhua) naturally infected with V. anguillarum (Rajan et al 2013), and in liver and gill of grass carp infected with parasitic copepod Sinergasilus major (Chang et al 2005). In a high-throughput transcriptome analysis of turbot (Scophthalmus maximus) with viral infection stimulus, apo-14 was also revealed to be the second highly expressed gene (Pereiro et al 2012). Combining with the above findings, we thought that the high enhanced induction expression of apo-14 around the infected sporoplasms and plasmodia observed in clone A + might be related to defense role, and the highly inducing expression might inhibit further proliferation of the myxosporean.…”