MiRNA-mRNA integration analysis of three continuous embryonic stages of Chinese horseshoe crab Tachypleus tridentatus

Horseshoe crabs are marine chelicerates which have existed for about 450 million years. Their long coexistence with microbial pathogens presumably is due to their efficient immune system. Herein, the first large-scale transcriptome and microRNA (miRNA) dataset of embryonic stages of the horseshoe crab Tachypleus tridentatus were presented. It provides a general overview of global gene expression changes during horseshoe crab’s early development. This study focuses on the stages 18–20 of the embryonic development of T. tridentatus, which are the first three major embryonic molt stages and involve the formation of several vital organs. We identified 138,260 unigenes, including 34 immune-related gene families such as pattern recognition receptors, signaling and transduction pathway genes, antimicrobial peptides, and coagulation cascade genes. Additionally, 1522 miRNAs (1046 known miRNAs and 476 novel miRNAs) were predicted to 29,584 target genes. Numerous mRNA-miRNA interactions were also identified. Results of Quantitative PCR proved that the immune genes such as Spaetzle, ankyrin-3, peptidoglycan-recognition protein, coagulogen, TNF-receptor-associated factor, and CD-9 antigen were negatively regulated by miR-5735, miR-200, miR-145, miR-199, miR-144, and miR-322, respectively. Futhermore, we confirmed the target regulatory relationship between miR-200 and ankyrin-3 by dual luciferase reporter gene assay. To better understand the complex developmental gene regulatory networks in T. tridentatus, the time-course gene expression data analysis is the first step. Our results provide a valuable resource for analyzing the functions of gene repertoires in embryonic developmental stages （Stage 18 to Stage 20）of horseshoe crab.