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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.

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Melatonin has been identified in a variety of invertebrate species, but its function is not as well understood as in crustaceans. The effects of melatonin on hemolymph glucose levels and tissue carbohydrate metabolism in the Chinese mitten crab, Eriocheir sinensis, were fully investigated in this study. Moreover, whether the eyestalk (an important endocrine center in invertebrate species) involves in this process or not, also were clarified. Analysis revealed that eyestalk ablation, especially bilateral, caused a significant decrease in the hemolymph glucose level. Moreover, injection of melatonin induced hyperglycemia in a dose-dependent manner both in intact and ablated crabs. Based on the expression of CHH mRNA in the 10 different tissues, eyestalk, thoracic ganglion, intestinal tract and hemolymph were selected to estimate the effect of melatonin on the expression of CHH mRNA. Bilateral eyestalk ablation caused a significant increase in the expression of CHH mRNA in the thoracic ganglion, intestinal tract and hemolymph compared with the controls. In addition, injection of melatonin into intact or ablated crabs elevated the CHH mRNA level in the eyestalk, thoracic ganglion and intestinal tract tissues compared with controls. The hemolymph CHH mRNA after melatonin injection was elevated only in ablated crabs. Administration of melatonin resulted in a significant decrease in total carbohydrates and glycogen levels with an increase in phosphorylase activity levels in the hepatopancreas and muscle in intact and ablated crabs. Our findings demonstrated that melatonin can induce hyperglycemic effects in both intact and ablated crabs, suggesting that this effect is probably not mediated solely via eyestalk.