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As a fat-soluble vitamin, vitamin D 3 relies on fat to perform its biological function, affecting lipid metabolism and innate immunity. This study used different percentages of lipid and vitamin D 3 diets to evaluate the synergistic effects on the growth, lipid metabolism and immunity of juvenile Eriocheir sinensis (5.83 ± 0.01 g) for 56 days, including low lipid (LL, 1.5%) and normal lipid (NL, 7.5%) and three levels of vitamin D 3 : low (LVD, 0 IU/kg), medium (MVD, 9000 IU/kg) and high (HVD, 27,000, IU/kg). The synergistic effect of lipid and vitamin D 3 was not significant on growth but significant on ash content, total protein, hepatopancreas lipid content, hemolymph 1α,25-hydroxy vitamin D 3 [1α,25(OH) 2 D 3 ] content, hepatopancreas lipolysis and synthesis genes. Crabs fed normal lipid (7.5%) and medium vitamin D 3 (9000 IU/kg) had the highest hepatopancreas index, hemolymph 1α,25(OH) 2 D 3 content, antibacterial ability, immune-related genes and hepatopancreatic lipid synthesis genes expression, but down-regulated the lipolysis genes expression. In contrast, crabs fed diets with low lipid percentage (1.5%) had low growth performance, hemolymph 1α,25(OH) 2 D 3 , mRNA levels of lipid synthesis genes, antibacterial ability and immune-related gene expression. At the 1.5% lipid level, excessive or insufficient vitamin D 3 supplementation led to the obstruction of ash and protein deposition, reduced growth and molting, aggravated the reduction in antioxidant capacity, hindered antimicrobial peptide gene expression and reduced innate immunity, and resulted in abnormal lipid accumulation and the risk of oxidative stress. This study suggests that diets' lipid and vitamin D 3 percentage can enhance antioxidant capacity, lipid metabolism and innate immunity in E. sinensis. A low lipid diet can cause growth retardation, reduce antioxidant capacity and innate immunity, and enhance lipid metabolism disorder., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Hypoxia is one of the serious stress challenges that aquatic animals face throughout their life. Our previous study found that hypoxia stress could induce neural excitotoxicity and neuronal apoptosis in Eriocheir sinensis, and observed that gamma-aminobutyric acid (GABA) has a positive neuroprotective effect on juvenile crabs under hypoxia. To reveal the neuroprotective pathway and metabolic regulatory mechanism of GABA in E. sinensis exposed to hypoxia stress, an 8-week feeding trial and acute hypoxia challenge were performed. Subsequently, we performed a comprehensive transcriptomic and metabolomic analysis of the thoracic ganglia of juvenile crabs. Differential genes and differential metabolites were co-annotated to 11 KEGG pathways, and further significant analysis showed that only the sphingolipid signaling pathway and the arachidonic acid metabolism pathway were significantly enriched. In the sphingolipid signaling pathway, GABA treatment significantly increased long-chain ceramide content in thoracic ganglia, which exerted neuroprotective effects by activating downstream signals to inhibit hypoxia-induced apoptosis. Moreover, in the arachidonic acid metabolism pathway, GABA could increase the content of neuroprotective active substances and reduce the content of harmful metabolites by regulating the metabolism of arachidonic acid for inflammatory regulation and neuroprotection. Furthermore, the decrease of glucose and lactate levels in the hemolymph suggests the positive role of GABA in metabolic regulation. This study reveals the neuroprotective pathways and possible mechanisms of GABA in juvenile E. sinensis exposed to hypoxia stress and inspires the discovery of new targets for improving hypoxia tolerance in aquatic animals., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is an immunomodulator to inhibit immune-mediated pro-inflammatory response and has been used to treat various immune-related diseases in mammals. However, the immunoregulatory effect of GABA in crustaceans has not been reported. This study evaluates the regulatory effect of dietary GABA supplementation on the innate immune status and immunoregulatory potential in lipopolysaccharide (LPS)-induced immune response in juvenile Eriocheir sinensis. Juvenile crabs were fed with six diets supplemented with graded GABA levels (0, 40, 80, 160, 320 and 640 mg/kg dry matter) for 8 weeks and then 24 h LPS challenge test was carried out. The results showed that dietary GABA supplementation significantly decreased mortality at 4 and 8 weeks. Moreover, the hemocyanin content, acid phosphatase, and alkaline phosphatase activities significantly increased in the crabs fed GABA supplementation compared with the control. On the contrary, the alanine aminotransferase and alanine aminotransferase activities in serum decreased significantly in the GABA supplementation groups compared with the control. Similarly, superoxide dismutase activity, glutathione content, and the transcriptional expression of the antioxidant-related genes and immune-related genes were significantly higher in the GABA supplementation groups than in the control. In addition, the mRNA expressions of anti-lipopolysaccharide factors (ALF 1, ALF 2, ALF 3) and inflammatory signaling pathways related genes (TLR, Myd88, Relish, LITAF, P38-MAPK, ADAM17) were significantly up-regulated in LPS stimulation groups compared with PBS treatment. Meanwhile, pro-apoptosis-related genes' mRNA expressions were significantly up-regulated, and anti-apoptosis-related genes were significantly down-regulated under LPS stimulation compared with PBS treatment. However, GABA pretreatment effectively alleviated LPS-induced immune overresponse and apoptosis. Therefore, this study demonstrates that dietary GABA supplementation could be used as an immunomodulator to improve the non-specific immunity and antioxidant capacity and alleviate the immune-mediated immune overresponse of juvenile E. sinensis., (Copyright © 2022. Published by Elsevier Ltd.)

In this study, a clip-domain serine proteinase homolog designated as MnSPH was cloned and characterized from a freshwater prawn Macrobrachium nipponense. The full-length cDNA of MnSPH was 1897 bp and contained a 1701 bp open reading frame (ORF) encoding a protein of 566 amino acids, a 103 bp 5'-untranslated region, and a 93 bp 3'-untranslated region. Sequence comparison showed that the deduced amino acids of MnSPH shared 30-59% identity with sequences reported in other animals. Tissue distribution analysis indicated that the MnSPH transcripts were present in all detected tissues with highest in the hepatopancreas and ovary. The MnSPH mRNA levels in the developing ovary were stable at the initial three developmental stages, then increased gradually from stage IV (later vitellogenesis), and reached a maximum at stage VI (paracmasis). Furthermore, the expression of MnSPH mRNA in hemocytes was significantly up-regulated at 1.5 h, 6 h, 12 h and 48 h post Aeromonas hydrophila injection. The increased phenoloxidase activity also demonstrated a clear time-dependent pattern after A. hydrophila challenge. These results suggest that MnSPH participates in resisting to pathogenic microorganisms and plays a pivotal role in host defense against microbe invasion in M. nipponense., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

An 8-week feeding trial was conducted to determine the dietary copper (Cu) on growth performance and immune responses of juvenile Chinese mitten crab Eriocheir sinensis. Six semi-purified diets with six copper levels (1.88, 11.85, 20.78, 40.34, 79.56 and 381.2 mg kg(-1) diet) of CuSO4·5H2O were fed to E. sinensis (0.45 ± 0.01 g). Each diet was fed to the crab in five replicates. The crab fed diets with 20.78 and 40.34 mg Cu kg(-1) diet had significantly greater weight gain and hemolymph oxyhemocyanin content than those fed diets with 1.88 and 381.2 mg Cu kg(-1) diet. Survival rates of crab were not significantly different between all treatment groups. The activities of copper-zinc superoxide dismutase (Cu-Zn SOD), phenoloxidase (PO), and total hemocyte count (THC) significantly increased when the supplementation of dietary copper reached 20.78-40.34 mg Cu kg(-1) diets. In the bacteria challenge experiment with Aeromonas hydrophila, survival rates significantly increased and reached a plateau when the dietary copper increased from 1.88 to 40.34 mg kg(-1), whereas significantly decreased when the dietary copper increased from 40.34 to 381.2 mg kg(-1). This study indicates that the level of dietary copper is important in regulating growth and immune response in crab., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Hemocyanin is a copper-binding protein and plays a crucial role in the physiological processes in crustacean. In this study, the cDNA encoding hemocyanin subunit from Chinese mitten crab Eriocheir sinensis (EsHc) was cloned by using EST analysis and rapid amplification of cDNA ends (RACE) approach. The full-length cDNA of EsHc was 2573 bp, consisting of a 5' untranslated region of 51 bp, a 3' untranslated region of 458 bp, and an open reading frame of 2064 bp. The deduced protein had 688 amino acid residues with molecular mass of 77,997.31 Da. Quantitative real-time RT-PCR analysis showed that the EsHc gene was expressed in haemocytes, hepatopancreas, muscles, gills, and intestines with the highest level of expression in the hepatopancreas and the lowest in the muscles. After Aeromonas hydrophila challenge, the relative expression level of EsHc in hemolymph was up-regulated at 3 h post-injection of bacteria followed by a gradual recovery from 12 to 24 h. In the second set of transcriptional studies, the mRNA expression patterns of EsHc in haemocytes and hepatopancreas were measured by quantitative real-time RT-PCR after the Chinese mitten crab were fed six diets containing different levels of copper (0, 10, 20, 40, 80 and 400 mg kg(-1)) for 8 weeks, respectively. The feeding trial showed that the expression levels of EsHc mRNA significantly increased at the copper levels of 20-40 mg kg(-1). This study implies that the expression levels of EsHc could be affected by dietary copper in the hepatopancreas and haemocytes, and hemocyanin may be potentially involved in the immune responses of the Chinese mitten crab., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

C-type lectins are pattern-recognition proteins which are functionally important for pathogen recognition and immune regulation in vertebrates and invertebrates. In this study, a lectin cDNA named as Es-Lectin was cloned and characterized from the Chinese mitten crab, Eriocheir sinensis. The full-length sequence of this Es-Lectin cDNA was 651 bp, including an open reading frame of 483 bp encoding 160 amino acids. The predicted molecular weight of the Es-Lectin was 11.8 kDa. A typical signal peptide of 21 amino acids was deduced at the N-terminus of the predicted protein. This Es-Lectin belongs to a C-type lectin and contains six cysteines, a conserved EPN motif (Glu-Pro-Asn) and an imperfect WND (Trp-Asn-Asp) motif (FND, Phe-Asn-Asp). This Es-Lectin had 55% and 32% identity with other two C-type lectins in E. sinensis, and 29-36% homology with decapods. Although the Es-Lectin was also expressed in gill, hepatopancreas, intestine, muscle and stomach, its expression in haemocytes was the greatest. The expression of Es-Lectins in haemocytes increased at 1.5 h after the Aeromonas hydrophila challenge. After a slight decrease, the Es-Lectin expression in haemocytes significantly increased at 48 h post-challenge. The diverse distribution of Es-Lectin and its enhancement by bacterial challenge indicate that C-type lectins are important in the innate immune response to bacterial infection, and can be activated for innate immune response in crab at the initial stage after pathogen infection., (Copyright © 2011 Elsevier Ltd. All rights reserved.)