In the pond culture of juvenile Eriocheir sinensis, a high limb-impairment rate seriously affects the culture success. Therefore, it is particularly important to artificially promote limb regeneration. This study evaluated the effects of melatonin on cheliped regeneration, digestive ability, and immunity, as well as its relationship with the eyestalk. It was found that the injection of melatonin significantly increased the limb regeneration rate compared with the saline group (P < 0.05). The qRT-PCR results of growth-related genes showed that the level of EcR-mRNA (ecdysteroid receptor) and Chi-mRNA (chitinase) expression was significantly increased following the melatonin injection, while the expression of MIH-mRNA (molt-inhibiting hormone) was significantly decreased (P < 0.05). Melatonin significantly increased lipase activity (P < 0.05). We observed that the survival rates of limb-impaired and unilateral eyestalk-ablated crabs were substantially improved following melatonin treatment, whereas the survival of the unilateral eyestalk-ablated crabs was significantly decreased compared with the control group (P < 0.05). Furthermore, the results of serum immune and antioxidant capacity revealed that melatonin significantly increased the total hemocyte counts (THC), hemocyanin content, total antioxidant capacity (T-AOC), acid phosphatase (ACP), and glutathione peroxidase activity (GSH-Px), whereas the immune-related parameters were significantly decreased in eyestalk-ablated crabs (P < 0.05). Therefore, these findings indicate that melatonin exerts a protective effect on organism injury, which could promote limb regeneration by up-regulating the expression of growth-related genes, improve digestive enzyme activity, and strengthen the immune response, particularly antioxidant capacity.

Biological mineralization is a highly programmed process in which inorganic minerals reassociate under the strict control of the extracellular matrix to form minerals with special functions and patterns. Shells are biominerals, and their synthesis is finely regulated by organic matrix including matrix proteins, polysaccharides, lipids, pigments, free amino acids, and small peptides. In this study, two matrix protein genes, hic14 and hic19, were isolated from the mantle of the mussel Hyriopsis cumingii. Tissue expression analysis showed that both proteins were expressed mainly in the mantle, and in situ hybridization of mantle tissues showed that they were specifically expressed in the dorsal epithelial cells of mantle pallial. Therefore, hic14 and hic19 were both nacreous layer matrix proteins. In the pearl insertion experiment, hic14 and hic19 kept low expression during pearl sac formation and disordered calcium carbonate deposition, and increased significantly during pearl nacre accumulation, which showed that both proteins participated in the mineralization of pearl nacre. In the RNA interference experiment, shell nacre tablet growth was inhibited after crystal nucleation due to the decreased expression of hic14, and crystal morphology and arrangement of nacre were highly modified after expression of hic19 was inhibited. These results provided further evidence that both hic14 and hic19 participated in nacreous layer biomineralization.