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.

Establishing a deeper understanding of the osteogenic differentiation of monolayer cultured human pluripotent stem cells using novel and detailed analyses.

BACKGROUND: Derivation of osteoblast-like cells from human pluripotent stem cells (hPSCs) is a popular topic in bone tissue engineering. Although many improvements have been achieved, the low induction efficiency because of spontaneous differentiation hampers their applications. To solve this problem, a detailed understanding of the osteogenic differentiation process of hPSCs is urgently needed. METHODS: Monolayer cultured human embryonic stem cells and human-induced pluripotent stem cells were differentiated in commonly applied serum-containing osteogenic medium for 35 days. In addition to traditional assays such as cell viability detection, reverse transcription-polymerase chain reaction, immunofluorescence, and alizarin red staining, we also applied studies of cell counting, cell telomerase activity, and flow cytometry as essential indicators to analyse the cell type changes in each week. RESULTS: The population of differentiated cells was quite heterogeneous throughout the 35 days of induction. Then, cell telomerase activity and cell cycle analyses have value in evaluating the cell type and tumourigenicity of the obtained cells. Finally, a dynamic map was made to integrate the analysis of these results during osteogenic differentiation of hPSCs, and the cell types at defined stages were concluded. CONCLUSIONS: Our results lay the foundation to improve the in vitro osteogenic differentiation efficiency of hPSCs by supplementing with functional compounds at the desired stage, and then establishing a stepwise induction system in the future.

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.

5-HT2B, 5-HT7, and DA2 Receptors Mediate the Effects of 5-HT and DA on Agonistic Behavior of the Chinese Mitten Crab (Eriocheir sinensis).

The Chinese mitten crab (Eriocheir sinensis) is a commercially important crab in China and is usually managed at high stocking densities. Agonistic behavior directly impacts crab integrity, survival, and growth and results in economic losses. In the present study, we evaluated the modulatory effects of serotonin (5-HT) and dopamine (DA) though the 5-HT2 and DA2 receptor-cyclic adenosine monophosphate (cAMP)-protein kinase A (PKA) pathway on agonistic behavior. The results showed that injection of either 10-6 mol/crab 5-HT or DA reduced the agonistic behavior of E. sinensis (P < 0.05), as did 10-10 mol/crab DA and 10-8 mol/crab 5-HT and DA (P < 0.05); however, a dose of 10-10 mol/crab 5-HT promoted agonistic behavior. 5-HT significantly increased the mRNA expression level of 5-HT7 receptor and reduced that of the DA2 receptor in the cerebral ganglion (P < 0.05). In contrast to 5-HT, DA significantly decreased 5-HT2B mRNA levels and increased 5-HT7 and DA2 receptor levels in the thoracic ganglia (P < 0.05). In addition, injections of either 5-HT or DA increased the cAMP and PKA levels in hemolymph (P < 0.05). By using in vitro culture of the thoracic ganglia, the current study showed that ketanserin (5-HT2 antagonist) and [R(-)-TNPA] (DA2 agonist) had obvious effects on the expression levels of the two receptors (P < 0.05). In vivo experiments further demonstrated that ketanserin and [R(-)-TNPA] could both significantly reduce the agonistic behavior of the crabs (P < 0.05). Furthermore, both ketanserin and [R(-)-TNPA] promoted the cAMP and PKA levels (P < 0.05). The injection of CPT-cAMP (cAMP analogue) elevated the PKA levels and inhibited agonistic behavior. In summary, this study showed that 5HT-2B and DA2 receptors were involved in the agonistic behavior that 5-HT/DA induced through the cAMP-PKA pathway in E. sinensis.