Development of digestive system in the horseshoe crab Tachypleus tridentatus during the transition from endogenous to exogenous nutrition

The Chinese horseshoe crab (Tachypleus tridentatus), valued for scientific and medicinal purposes, is endangered due to pollution, habitat loss, and overharvesting. Listed as endangered by the IUCN in 2019 and protected in China since 2021, its population recovery is critical. The studies of the digestive tract are fundamental to metabolism and directly related to growth and development of aquatic animal. However, morphological and histological studies on the digestive tract of horseshoe crabs are lacking at present. To advance conservation and aquaculture strategies, this study combined histology, enzymology, molecular biology and metabolomics to investigate developmental changes in its digestive system during the transition from endogenous to exogenous nutrition. Key findings revealed reduced lipid particles and nutrient density in yellow connective tissue during ecdysis, alongside increased periostracum thickness and mucosal folds in the esophagus and proventriculus with growth. Midgut walls exhibited significantly thickened mucosal folds, submucosa, and muscle layers during ecdysis, coinciding with substantial depletion of proteins and lipids-while carbohydrates supplemented energy metabolism. Enzymatic analysis highlighted elevated cathepsin B synthesis in the first instar, likely preparing for vitellogenin hydrolysis and cathepsin L activation during molting, critical for metabolic adaptation. Metabolic data revealed distinct patterns across ecdysis and second instar. During molting, glycerophospholipid metabolism, autophagy, and mTOR/FoxO pathways supported energy recovery, lipid restructuring, and stress responses, aiding calcium ion metabolism and chitin synthesis. In contrast, second instar involved linoleic and arachidonic acid metabolism, neuroactive interactions, and the pentose phosphate pathway, likely linked to nervous and immune system maturation, antioxidant defense, and tissue repair. Molting prioritized energy by suppressing glycolysis, while second instar accumulated amino acids and lipid precursors for organ development and reproduction. These findings reveal adaptive metabolic strategies during molting and development, providing insights to optimize breeding practices, enhance aquaculture sustainability, and conserve this ecologically and economically vital endangered species.