Effect of dietary L-tryptophan on the survival, immune response and gut microbiota of the Chinese mitten crab, Eriocheir sinensis.

This study investigated the influence of L-tryptophan (L-trp) on the survival, immune response and gut microbiota of the Chinese mitten crab, Eriocheir sinensis (with an average weight of 16.58 ±â€¯2.20 g). After 30 days of feeding with diets supplemented with L-trp at 0.36%, 0.47%, 0.73% and 1.05% (groups 1, 2, 3 and 4, respectively), the survival rate and bacterial challenge (Aeromonas hydrophila) were evaluated, the activities of antioxidant and phosphatase enzymes in the serum were assessed, and the gut microbiota were measured via high-throughput Illumina sequencing. The results showed that the supplementation of L-trp significantly improved the survival rate of crabs (P < 0.05). After feeding for 7 days, it was observed that a high L-trp diet significantly increase the survival rate relative to a basal diet after a 96-h post-challenge with A. hydrophila (P < 0.05). The activity of CAT and AKP in the serum were increased by the addition of L-trp. The activity of CAT and AKP in the serum in group 4 were higher than those in group 1 (P < 0.05). Furthermore, we observed that adjunction of the L-trp can significantly increase the richness and diversity of the gut microbiota. The dominant phylum in the intestine of the Chinese mitten crab were Tenericutes, Proteobacteria, Firmicutes, Chloroflexi and Actinobacteria. The L-trp in the diets increased the richness of Proteobacteria, Firmicutes and Actinobacteria in the intestine significantly. These bacteria were all dominant bacteria and had a specific role in promoting the immunity of E. sinensis. Therefore, it could be inferred that L-trp supplementation is beneficial in the diet of E. sinensis. Based in these results, the dietary 0.47% or 0.73%L-trp supplemented is found to be optimum to improve E. sinensis survival.

Hemolymph transcriptome analysis of Chinese mitten crab (Eriocheir sinensis) with intact, left cheliped autotomy and bilateral eyestalk ablation.

In the pond culture of Eriocheir sinensis, high limb-autotomy seriously affects the quality and culture's economic efficiency. Based on our previous studies, limb autotomy can induce the changes of hematological immune response in E. sinensis hemolymph. Eyestalk ablation can accelerate the regeneration of limbs after autotomy. To detect the important functional genes related to the hematological molecular immunity of E. sinensis, we compared and analyzed the hemolymph transcriptome data of the intact crab, left cheliped autotomized crabs and bilateral eyestalk ablation crabs with high-throughput sequencing techniques. The results showed that the three groups obtained 62 172 414, 68 143 682, and 67 811 618 clean reads, respectively. A total of 9567 differentially expressed genes were obtained by multiple comparison of the three groups' libraries. Gene ontology (GO) functional classification analysis shows that the differential genes belong to 42 categories of biological process, cellular components and molecular function. The differentially expressed genes in the three libraries were enriched to 344 specific KEGG metabolic pathways by KEGG enrichment analysis, such as the up-regulated gene (dual oxidase (Duox), tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein (YWHAQ)) in MAPK signaling pathway, the up-regulated gene (aldehyde dehydrogenase 1 (ALDH 1)) and down-regulated gene (UDP-glucuronosyltransferase 2 (UGT 2)) in metabolism of the xenobiotics by cytochrome P450 pathway, the down-regulated gene (actin gene (AG), heat shock protein 90 (HSP 90)) in fluid shear stress and atherosclerosis pathway. To verify the expression levels of DEGs identified by RNA-Seq, the above six hematological immune-related genes were selected for qRT-PCR validation, the qRT-PCR results were consistent with the DEGs results. Our research obtained abundant E. sinensis hemolymph transcriptome information by RNA-Seq, which provides multi-level information for the cloning of novel genes and the study of hemolymph molecular immunology mechanisms of E. sinensis.

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 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.

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.

Molecular cloning and functional expression of the 5-HT7 receptor in Chinese mitten crab (Eriocheir sinensis).

Serotonin (5-HT) regulates numerous physiological functions and processes, such as light adaptation, food intake and ovarian maturation, and plays the role through 5-HT receptors. To our knowledge, this is the first study to isolate and characterize the serotonin receptor 7 (5-HT7 receptor) cDNA encoded in Eriocheir sinensis, an economically important aquaculture species in China, by performing rapid-amplification of cDNA ends. The full-length of 5-HT7 receptor gene cDNA is 2328 bp and encodes a polypeptide with 590 amino acids that are highly homologous with other crustaceans 5-HT7 receptor genes. Analysis of the deduced amino acid sequence of the 5-HT7, including 7 transmembrane domains and some common features of G protein-coupled receptors (GPCRs), indicated that 5-HT7 receptor was a member of GPCRs family. A gene expression analysis of the 5-HT7 receptor by RT-PCR revealed that the 5-HT7 receptor transcripts were widely distributed in various tissues, in which high expression levels were observed in the cranial ganglia, thoracic ganglia and intestines. Further study about the effects of photoperiods on the 5-HT7 expression in the tissues showed that a significantly increasing expression of the 5-HT7 receptor was observed in the thoracic ganglia induced by constant light. In addition, in the eyestalks, the expression levels of 5-HT7 mRNA in constant darkness and constant light were lower than control treatment. Then, the expression levels of the 5-HT7 receptor in three feeding statuses displayed that there were significantly increasing expressions in the hepatopancreas and intestines after feeding, compared with before feeding and during the feeding period. Finally, the 5-HT7 mRNA expression levels in stage III and stage IV were higher than the levels in stage I of ovarian development. Our experimental results showed that the 5-HT7 receptor structurally belongs to GPCRs, and the thoracic ganglia and eyestalks are the important tissues of the 5-HT7 receptor for light adaptation. The 5-HT7 receptor may also be involved in the physiological regulation of the hepatopancreas and intestines after ingestion in E. sinensis. In addition, the 5-HT7 receptor is involved in the process of ovarian maturation. The study provided a foundation for further research of light adaptation, digestive functions and ovarian maturation of the 5-HT7 receptor in Decapoda.

Cloning and functional characterization of the DA2 receptor gene in Chinese mitten crab (Eriocheir sinensis).

Dopamine (DA) plays a modulatory role in numerous physiological processes such as light adaptation and food intake, and exerts these functions through DA receptors (DARs). This study presents, for the first time, isolation and characterization of the dopamine receptor 2(DA2 receptor) cDNA from the intestinal tissue of Eriocheir sinensis, an economically important freshwater aquaculture species in China. The DA2 receptor cDNA sequence, which was obtained by rapid amplification of cDNA ends, is 2369bp long, encode peptide of 589 amino acid, and is highly homologous to related sequences in crustaceans. Analysis of the deduced amino acid sequence and the structure of the DA2 indicated that this receptor is a member of the family of G protein-coupled receptors (GPCRs), as it contains seven transmembrane domains and other common signatures of GPCRs. RT-PCR showed that the expression of the DA2 receptor gene was distributed in various tissues, and high expression levels were observed in the cranial ganglia and the thoracic ganglia. Further study of the effect of photoperiod on DA2 expression showed that constant darkness induced a significant increase in DA2 expression in the cranial ganglia. Finally, analysis of DA2 receptor expression under different feeding statuses showed that there was significantly greater expression in the hepatopancreas and intestines after feeding than before feeding, but there were no differences in expression between the before feeding and during feeding periods in either tissue. Our results indicate that the DA2 receptor structurally belongs to the family of G protein-coupled receptors, and that the cranial ganglia are the main tissues in which the DA2 receptor participates in light adaptation during dark hours. In addition, the DA2 receptor in E. sinensis may be involved in the physiological regulation of the hepatopancreas and digestive tract after the ingestion of food. This study provides a foundation for further exploration of the light adaptation and digestive functions of the DA2 receptor in decapods.

The Hyperglycemic Effect of Melatonin in the Chinese Mitten Crab, Eriocheir sinensis.

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.

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.

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.