Comparative transcriptome analysis of the Cyclina sinensis hepatopancreas provides evidence that Toll-like receptors protect against Vibrio anguillarum iYnfection

The Venus clam, Cyclina sinensis, is one of the most popular and nutritious shellfish species facing several pathogenic microbial invasions that have caused severe economic losses in China. Toll-like receptors (TLRs) play important roles in the innate immunity of molluscan species, but the available information of the C. sinensis hepatopancreas transcriptome and the results from a large-scale identification of TLRs remain limited. In this study, de novo transcriptome assembly and differential gene expression profiling of untreated (termed H) and Vibrio anguillarum-challenged (termed HV) groups of C. sinensis hepatopancreas were performed using the RNASeq platform. A total of 141,881,862 bp of high-quality clean reads were assembled into 163,129 unigenes from 206,495 transcripts with an N50 length of 1186 bp. All of these unigenes were then annotated and classified using the Swiss-Prot, Nr, Nt, KO, GO, KOG and KEGG databases. To understand and characterize the innate immune responses of C. sinensis challenged with V. anguillarum, a total of 4458 differentially expressed unigenes, including 2276 upregulated and 2182 downregulated genes, were identified, and some of these genes were associated with PRR signaling pathways in C. sinensis, including the TLR, the NOD-like receptor, the RIG-I-like receptor, NF-kappa B and the MAPK signaling pathways. From this transcriptome library, we then identified a total of 13 new TLRs that showed increased expression levels in the C. sinensis hepatopancreas at different time points after V. anguillarum infection, and these consisted of one P-TLR, one V-TLR, one Twin-TIR TLR, three Ls-TLRs and seven sP-TLRs. In summary, this study may significantly contribute to the knowledge of the innate immunity and defense mechanisms of C. sinensis in response to V. anguillarum stimulation and promote future research investigating the evolution and functions of TLRs in invertebrates.