Effects of miR-143 and its target receptor 5-HT2B on agonistic behavior in the Chinese mitten crab (Eriocheir sinensis).

Chinese mitten crab (Eriocheir sinensis) as a commercially important species is widely cultured in China. However, E. sinensis is prone to agonistic behavior, which causes physical damage and wastes energy resources, negatively impacting their growth and survival. Therefore, understanding the regulatory mechanisms that underlie the switching of such behavior is essential for ensuring the efficient and cost-effective aquaculture of E. sinensis. The 5-HT2B receptor is a key downstream target of serotonin (5-HT), which is involved in regulating animal behavior. In this study, the full-length sequence of 5-HT2B gene was cloned. The total length of the 5-HT2B gene was found to be 3127 bp with a 236 bp 5'-UTR (untranslated region), a 779 bp 3'-UTR, and a 2112 bp open reading frame encoding 703 amino acids. Phylogenetic tree analysis revealed that the 5-HT2B amino acid sequence of E. sinensis is highly conserved with that of Cancer borealis. Using in vitro co-culture and luciferase assays, the miR-143 targets the 5-HT2B 3'-UTR and inhibits 5-HT2B expression was confirmed. Furthermore, RT-qPCR and Western blotting analyses revealed that the miR-143 mimic significantly inhibits 5-HT2B mRNA and protein expression. However, injection of miR-143 did not decrease agonistic behavior, indicating that 5-HT2B is not involved in the regulation of such behavior in E. sinensis.

The serotonin or dopamine by cyclic adenosine monophosphate-protein kinase A pathway involved in the agonistic behaviour of Chinese mitten crab, Eriocheir sinensis.

Agonistic behaviour is common in an encounter between two crustaceans. It often causes limb disability and consumes a lot of energy, which is harmful for the growth and survival of commercially important crustaceans. In the present study, we mainly focused on the agonistic behaviour of the Chinese mitten crab, Eriocheir sinensis, which is an important species of the aquaculture industry in China. We recorded agnostic behaviour with a high-definition camera and preliminarily evaluated the role of serotonin (5-HT) or dopamine (DA)-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway and eyestalk in the behaviour. The results showed that agonistic behaviour in E. sinensis consisted of three stages: approach, contact and fight. We found that the number of fights and cumulative time of fight were significantly higher in the male vs. male group than in the female vs. female and female vs. male groups (P < 0.05). After 1 h of agonistic behaviour, 5-HT concentration showed a significant increase and DA concentration showed a significant decrease when compared with the control group (no encounter; P < 0.05). 5-HT1B and 5-HT2B mRNA levels showed a significant increase in the eyestalk (P < 0.05). 5-HT7 mRNA levels showed significant downregulation in the thoracic ganglia and DA1A mRNA levels showed upregulation in the intestine (P < 0.05). DA2 mRNA levels showed a significant decrease in the eyestalk (P < 0.05). These changes were accompanied by a significant increase in cAMP level and significant decrease in PKA level in the haemolymph (P < 0.05). In addition, a significant decrease in glucose levels was detected after the agonistic behaviour. Crustacean hyperglycemic hormone (CHH) mRNA levels showed significant upregulation in the eyestalk and significant downregulation in the intestine (P < 0.05). The number of fights and cumulative time of fight in the left eyestalk ablation (L-X vs. L-X) group were more and longer than those in the intact eyestalk (C vs. C), right eyestalk ablation (R-X vs. R-X) and bilateral eyestalk ablation (D-X vs. D-X) groups. In short, E. sinensis shows special agonistic behaviour modulated by 5-HT or DA-cAMP-PKA pathway and eyestalk, especially the left eyestalk.

The transcriptome sequencing and functional analysis of eyestalk ganglions in Chinese mitten crab (Eriocheir sinensis) treated with different photoperiods.

Photoperiod plays an important role in individual growth, development, and metabolism in crustaceans. The growth and reproduction of crabs are closely related to the photoperiod. However, as of yet, there are still no transcriptomic reports of eyestalk ganglions treated under different photoperiods in the Chinese mitten crab (Eriocheir sinensis), which is a benthonic crab with high commercial value in Asia. In this study, we collected the eyestalk ganglions of crabs that were reared under different photoperiods, including a control group (L: D = 12 h: 12 h, named CC), a constant light group (L: D = 24 h: 0 h, named LL) and a constant darkness group (L: D = 0 h: 24 h, named DD). RNA sequencing was performed on these tissues in order to examine the effects of different photoperiods. The total numbers of clean reads from the CC, LL and DD groups were 48,772,584 bp, 53,943,281 bp and 53,815,178 bp, respectively. After de novo assembly, 161,380 unigenes were obtained and were matched with different databases. The DEGs were significantly enriched in phototransduction and energy metabolism pathways. Results from RT-qPCR showed that TRP channel protein (TRP) in the phototransduction pathway had a significantly higher level of expression in LL and DD groups than in the CC group. We found that the downregulation of the pyruvate dehydrogenase complex (PDC) gene and the upregulation phosphoenolpyruvate carboxykinase (PPC) gene were involved in energy metabolism processes in LL or DD. In addition, we also found that the upregulation of the expression level of the genes Gαq, pyruvate kinase (PK), NADH peroxidase (NADH) and ATPase is involved in phototransduction and energy metabolism. These results may shed some light on the molecular mechanism underlying the effect of photoperiod in physiological activity of E. sinensis.

Melatonin Promotes Cheliped Regeneration, Digestive Enzyme Function, and Immunity Following Autotomy in the Chinese Mitten Crab, Eriocheir sinensis.

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.

Cellular and biochemical parameters following autotomy and ablation-mediated cheliped loss in the Chinese mitten crab, Eriocheir sinensis.

In the pond culture of juvenile Eriocheir sinensis, various factors have frequently led to a high rate of autotomy and limb impairments. This study evaluated the differential effects of cheliped loss with autotomy and ablation on the short-term cellular and biochemical parameters of juvenile E. sinensis. In this study, compared with the crabs before treatment, the total hemocyte counts (THC), granulocyte counts (GC), hemocyte agglutination, phosphatase activity and glucose metabolism levels were significantly increased, while hyalinocyte counts (HC) and total antioxidant capacity (T-AOC) were significantly decreased within 3 h. However, the THC levels, hemocyte agglutination and hemocyte proliferation were significantly higher in the ablation group within 3 h compared to the autotomy group. Moreover, the changes of glucose metabolism and immune-related enzymes activities in ablation groups were later than autotomy groups. The bacterial challenge showed that the mortality rate of the ablation group was significantly higher than that of the autotomy group. Therefore, the observations in this study indicate that compared with the passive trauma response of ablation, autotomy is a congenital, efficient, and active trauma response mechanism, which is of great significance to the survival and growth of E. sinensis.