The use of physiological and transcriptional analyses to examine molting regulatory mechanisms in juvenile horseshoe crab Tachypleus tridentatus

Molting is a necessary developmental process in crustaceans, and is holistically regulated by a range of hormones and associated genes. In order to investigate physiological changes occurring at the molecular level during the molting cycle of juvenile Tachypleus tridentatus, chitinase and N-acetyl-β-D-glucosaminidase (β-NAGase) activities were firstly measured by absorbance spectrophotometry. In addition, ecdysone content was quantified by ELISA, and changes in expression levels of genes relevant to ecdysis were examined based on quantitative real-time PCR (qRT-PCR) and RNA-sequencing (RNA-seq). Results showed that chitinase, β-NAGase activity, and ecdysone content increased at the intermolt stages, peaked at premolt stages, and decreased to a lower level at molt and postmolt stages. Chitinase and β-NAGase were positively correlated with ecdysone. RNA-seq revealed that a total of 6482 differentially expressed genes (DEGs) in the four different molting stages, and 5763 DEGs had higher expression levels in the premolt stage than in the intermolt stage. A total of 59 shared DEGs were identified in all molting stages, and the highly expressed DEGs were mainly related to chitin binding, stratum corneum structural composition and neuropeptide hormone activity signaling pathways. KEGG pathway analysis showed that at the premolt stage, digestion, transport, and catabolic signaling pathways were significantly enriched and innate immune response pathways were significantly enriched as molting progressed. These results suggested that the molting of juvenile horseshoe crabs may be directly regulated by ecdysone, and chitinase and β-NAGase play a synergistic role with ecdysone during the molting cycle. The key biological processes and signaling pathways involved in hormone regulation, epidermal structural proteins, catabolism, apoptosis, and immune response potentially regulate the molting process of horseshoe crab juveniles. This study provides a theoretical basis for enhancing understanding of the molting and growth processes in marine arthropods, and a strong theoretical support for the developmenting of captive rearing technologies for Chinese horseshoe crabs.