Proteins and microorganisms reveal that feed and substrate exert different effects on the growth and development of juvenile Chinese horseshoe crabs.

In recent years, the population of Chinese horseshoe crabs (Tachypleus tridentatus) has declined sharply. As a living fossil of the ocean, they have an extremely slow growth cycle. To protect this endangered species, research has focused on identifying the optimal conditions required for its growth and development. Previous studies have focused on analyzing the optimal feed for T. tridentatus at various life stages. However, the optimal conditions required for the growth and development of first instar juveniles remain unclear. Furthermore, there is a lack of accurate and effective knowledge regarding the cultivation of first instar juveniles in aquaculture. Therefore, this study investigates the effects of feed and substrate on juvenile growth and development. Four experimental groups were established: a no-substrate, no-feeding group (WK group), a no-substrate, oyster feeding group (WS group), a group with sandy-mud substrate but no feeding (SNK group), and a group with sandy-mud substrate and oyster feeding (SNS group). The results were analyzed based on experimental records, proteomics, and microbiomics data. The results indicated that the SNS group exhibited the highest molting rate, as well as the highest weights for both first- and second instar juvenile horseshoe crabs. This indicates that the combined effect of oysters and the substrate is far greater in molting rates than the effect of either alone. Based on the proteomic results, under sandy-mud substrate conditions, oyster feeding induced substantial accumulation of mannosyl-oligosaccharide 1,2-alpha-mannosidase IA-like and Spartin, while reducing creatinase accumulation. With oyster feeding, the addition of sandy-mud substrate led to substantial accumulation of juvenile hormone acid O-methyltransferase-like isoform X1 and thromboxane-A synthase-like. Consistent with the microbiome results, juvenile horseshoe crabs in the SNS group exhibited higher average relative abundances of intestinal bacteria including Vibrionaceae, Thalassobius, and Defluviimonas.