Study on the Regulatory Mechanisms of Carapace Marking Formation in Marsupenaeus japonicus

There are two phenotypes in the natural populations of Marsupenaeus japonicus, which is an ideal model for studying the formation of markings and body color in crustaceans. In a previous study, we used comparative transcriptome technology to screen some functional genes related to body color regulation. Here, high-throughput sequencing technology was used to perform microRNA (miRNA) sequencing analysis on the exoskeleton of M. japonicus with two types of carapace markings, and functional studies of related genes were performed. A total of 687 mature miRNAs belonging to 135 miRNA families were identified in this study, and 111 novel miRNAs were found. Through stringent screening conditions, a total of 18 differentially expressed miRNAs were identified, including 14 with upregulated expression and 4 with downregulated expression. Multiple target genes were predicted for almost all of the differentially expressed miRNAs. The expression levels of several target genes, such as those related to cytoplasmic microtubule organization, transmembrane transportation, and signal transduction, were confirmed using qRT-PCR. This study revealed that both the CRCN A2 and CRCN C1 genes were highly expressed in type I individuals, while the expression levels of their related miRNAs in type I individuals were lower than those in type II individuals, which is consistent with the mechanism of miRNAs negatively regulating mRNA expression. Through interference with the CRCN A2 and CRCN C1 genes, a clear regulatory relationship was found between the two genes, and the dendritic xanthophores in the carapace of M. japonicus gradually changed from bright yellow to dark black, with obvious shrinkage. In summary, our studies provide references for the regulatory mechanisms of marking formation in M. japonicus.