Molecular and expression characterization of Toll-like receptor family genes from the Anadara sativa (Bivalvia, Arcidae) transcriptome

Innate immunity plays an important role in invertebrates because it provides the first line of protection by recognizing invading microbial pathogens and then activating downstream signaling pathways. However, until now, increasing reports of clam diseases did not include those of Anadara sativa, which are widely distributed and economically important maritime clams. In the present study, transcriptome libraries of untreated (termed H) and Vibrio anguillarum-challenged (termed HV) A. sativa hepatopancreases were constructed and sequenced using the Illumina HiSeq4000 platform. In total, we obtained 78,012,510 and 84,937,516 clean reads from 80,006,030 to 86,871,742 raw data reads, respectively, assembled by different software programs. Furthermore, 150,274 unigenes were generated from 196,003 transcripts, with an N50 length of 1088 bp, and then annotated with the SwissProt, NR, NT, PFAM, KO, GO, KOG and KEGG databases. Moreover, 3982 differentially expressed unigenes (H vs HV) were determined, with 3583 upregulated and 399 downregulated genes. Among these differentially expressed unigenes, 207 unigenes were found using KEGG annotation in 16 immune-related signaling pathways, such as Toll-like receptor (TLR), NOD-like receptor (NLR), and RIG-I-like receptor (RLR) signaling pathways. Finally, we selected 11 full-length TLRs and classified them into 3 groups, namely, one V-TLR, four Ls-TLR and six sP-TLR; furthermore, we validated the increased expression patterns of the 11 TLRs in response to LPS injection. In summary, these results revealed multiple findings on potential immune-related genes, such as the differential expression analysis and annotation based on the A. sativa transcriptome in response to V. anguillarum stimulation, and explored the molecular and expression characterization of A. sativa TLRs, which provide new insights into the innate immune responses and defense mechanisms in shellfish.