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The Forkhead Box O (FOXO) gene plays a key role in various biological processes, such as growth, metabolism, development, immunity and longevity. Molting is an essential process for crustacean growth, which is mainly regulated by 20-hydroxyecdysone (20E) and molt-inhibiting hormone (MIH). Although the role of FOXO in regulating the immune response of crustaceans is well documented, its involvement in controlling crustacean molting remains unclear. In this study, a FOXO-like gene (designed as EsFOXO-like) was identified in Eriocheir sinensis, and the regulation of the 20E pathway by EsFOXO-like was also investigated. The coding sequence of EsFOXO-like was 852 bp, which consisted of 283 amino acids including a conserved Forkhead (FH) domain. EsFOXO-like shared high similarity with FOXO genes from other crustaceans, and the mRNA expression levels of the EsFOXO-like gene were highest in the hepatopancreas and lowest in the hemocytes. However, transcription and protein expression of the EsFOXO-like gene were found to be up-regulated only during the pre-molt stage in the hepatopancreas, with lower expression levels observed at the post-molt stage. To explore the role of EsFOXO-like in the 20E pathway, EsFOXO-like was firstly inhibited by a specific FOXO inhibitor (AS1842856) and then through an EsFOXO-like dsRNA injection, respectively, and the results showed that the relative expression levels of EsFOXO-like were notably decreased in the hepatopancreas after both the inhibitor and dsRNA treatments. The 20E concentration, the mRNA expression levels of the 20E receptors including the ecdysone receptor (EcR) and the retinoid-X receptor (RXR) and EsmTOR transcription in the AS1842856 group or the EsFOXO-RNAi group were all significantly higher than that in the control group, while the mRNA expression level of EsMIH was significantly decreased after EsFOXO-like inhibition. To further investigate whether the EsFOXO-like acts through mTOR or not, Rapamycin was administered to inhibit mTOR activity in EsFOXO-like inhibited crabs. The results revealed a significant reduction in the concentration of 20E and the expression level of EsMIH in the AS1842856 + Rapamycin group compared to the AS1842856 + DMSO group, accompanied by an increase in EsEcR and EsRXR expression. These findings collectively suggest that EsFOXO-like regulates the 20E pathway through mTOR, which offered valuable insights into the understanding of the molting process in crustaceans. [ABSTRACT FROM AUTHOR]

A 56‐day feeding experiment was performed to investigate the effects of the dietary lipid level on the survival, growth performance, digestive enzyme activities and antioxidant capacity of Eriocheir sinensis reared under three salinities. Six feeds with graded levels of lipids (5%, 7%, 9%, 11%, 13% and 15%) were formulated by including fish oil as the main lipid source. Sixty crab individuals (initial weight: 0.21 ± 0.02 g) were assigned to each aquarium. In total, 18 aquariums were distributed under each salinity condition. Each feed was randomly allocated to three parallel tanks of crabs. The feeding experiment lasted for 56 days. Results showed that the survival rate of E. sinensis fed diets with 7%–15% lipids just showed a decreasing tendency as the salinity increased. The highest weight gain rate (WGR) was found in the treatment with 11%, 9% and 5% lipid at the salinity of 0.5‰, 2‰ and 6‰ respectively. At the salinity of 0.5‰, 2‰ and 6‰, the highest activities of antioxidant enzymes (superoxide dismutase, catalase, glutathione peroxidase) were detected in the treatments with 11%, 13% and 7% lipid respectively. As the salinity increased, the protein and lipid contents showed an increasing trend, while the moisture content showed an opposite trend. At each salinity, arachidonic acid and docosahexaenoic acid increased, while saturated fatty acids and monounsaturated fatty acids decreased with the increase of dietary lipid level. These results indicated that increasing salinity resulted in decreased antioxidant enzyme activities and increased malondialdehyde content in the juvenile E. sinensis, especially when they were fed diets with relatively higher lipid level. Based on WGR, digestive enzyme activity and antioxidant capacity, the optimal lipid level was estimated to be 8.28–13%, 7.54–13% and 5–8.89% for juvenile E. sinensis reared at the salinity of 0.5‰, 2‰ and 6‰ respectively. [ABSTRACT FROM AUTHOR]

A 60‐day feeding experiment was performed to investigate the survival, growth, body composition, antioxidant capacity and related gene expression of Eriocheirsinensis which were fed diets with different lipid sources under three salinities. Five feeds were formulated with one of the candidate oils (fish oil, soybean oil, linseed oil, colza oil or palm oil) and were named FO, SO, LO, CO or PO respectively. Results showed that the survival rate (SR) of E. sinensis showed a decreasing tendency as the salinity increased. But SR was not significantly affected by dietary lipid sources at each salinity. Weight gain rate (WGR) were highest in the treatment of FO/PO at a salinity of 0.5‰, CO/FO/SO at a salinity of 2‰ and LO at a salinity of 6‰ respectively. At salinities of 0.5‰ and 2‰, the transcription of pro‐inflammatory and antioxidant genes in LO was significantly higher than that in FO or PO. While at a salinity of 6‰, the transcription of detected genes in FO was markedly increased compared to that in the vegetable oil treatments. At each salinity, the eicosapentaenoic acid (EPA) contents showed comparable values among different dietary treatments. However, the docosahexaenoic acid (DHA) contents in E. sinensis fed the diets with FO were still significantly higher than those with vegetable oils. These results showed that increasing salinity decreased the antioxidant capacity and therefore negatively affected the survival and growth rates of experimental animals. The beneficial effects of FO on survival and growth performance were eliminated by increasing the salinity to 6‰, which could be due to the increased lipid peroxidation and inflammatory response caused by salinity stress. It was concluded that at the salinity of 0.5‰ and 2‰, the recommended lipid sources were FO/PO and CO/FO/SO respectively. While at the salinity of 6‰, LO was the most ideal lipid source for juvenile E. sinensis. [ABSTRACT FROM AUTHOR]

A 60‐day feeding experiment was conducted to evaluate the survival, growth, antioxidant capacity and body composition of the juvenile Chinese mitten crab (Eriocheir sinensis) reared at three salinities (0.5‰, 2‰ and 6‰) in response to the different substitution levels (0%, 24%, 39%, 57% and 100%) of fish meal (FM) by soybean meal (SBM). The five diets were named as S0, S24, S39, S57 and S100 respectively. Results showed that survival rate (SR) was only significantly affected by the substitution level at the salinity of 0.5‰, with significantly higher SR observed in S39 (p < 0.05). At the salinity of 0.5‰ and 2‰, the significantly higher weight gain rate (WGR) was observed in S39 and S57 respectively (p < 0.05). At the salinity of 6‰, WGR was significantly improved by partial or complete substitution of FM by SBM. At each salinity, the highest GSH‐Px activity and lowest MDA content were observed in the treatments of S57, which were comparable to that fed S0. At the salinity of 6‰, the activities of SOD and CAT were significantly decreased in S100 than that in the other treatments (p < 0.05). The proline content significantly increased as the substitution level increased at the salinity of 2‰. At the salinity of 6‰, the contents of proline, glutamine, cysteine, methionine and lysine, first significantly increased and then decreased (p < 0.05), with the highest value observed in the treatment of S39 or S57. These results indicated that the optimal substitution level of FM by SBM was estimated to be 39% and 57% in the diets of E. sinensis at the salinity of 0.5‰ and 2‰ respectively. Although the FM can be completely replaced by SBM without negatively affecting the survival and growth performance at the salinity of 6‰, the antioxidant capacity was reduced to a large extent. [ABSTRACT FROM AUTHOR]

A 50‐day feeding experiment was conducted to investigate the effects of dietary protein level (26%, 34%, 43% and 52%) on the survival, growth performance, digestive enzyme activities, antioxidant capacity and body composition in the Chinese mitten crab (Eriocheir sinensis) reared at three salinities (0.5‰, 2‰ and 6‰). Survival rate (SR) showed a clearly decreasing tendency as the salinity elevated. The highest weight gain rate (WGR) was achieved in the treatment of 43% protein at the salinity of 0.5‰ and 2‰, and 26% protein at the salinity of 6‰ (p > 0.05). However, there was no significant interactive effect between dietary protein level and salinity on the SR and WGR of E. sinensis (p > 0.05). As salinity increased, the amylase activity and lipid deposition increased but the Na+/K+‐ATPase activity decreased. The MDA content of E. sinensis was increased at the salinity of 2‰ than the other two salinities by low or moderate dietary protein (26–43% dry diet), but was significantly decreased by high protein (52% dry diet). Correspondingly, the activities of GSH‐Px were increased at the relatively higher protein (43–52% dry diet). The significant interactive effect was only obtained between dietary protein level and salinity on the activities of CAT, GSH‐Px and MDA content of E. sinensis. Dietary protein level had a more obvious positive effects on the EAA deposition at the salinity of 2‰ and 6‰, with the highest contents of essential amino acids (EAAs) achieved by 43% dietary protein. In conclusion, the relatively higher protein could benefit the E. sinensis by inhibiting the lipid peroxidation and increasing the deposition of EAAs at the salinity of 2‰. The elevated activities of amylase could account for the low protein (26%) requirement for E. sinensis reared at the salinity of 6‰. [ABSTRACT FROM AUTHOR]

CpG oligodeoxynucleotides (ODN), as an effective adjuvant or immunopotentiator, activate the immune system and induce various immune responses. Recently, it has also been reported that high dose of CpG ODN can lead to immunosuppression. However, the underlying mechanism of CpG ODN-mediated immune response remains largely unknown in invertebrates. In the present study, the role of ERK in regulating expression levels of anti-lipopolysaccharide factors (ALFs) induced by different doses of CpG ODN 2395 was analyzed in Chinese mitten crab, Eriocheir sinensis. The mRNA expression levels of Es ALFs (Es ALF1, Es ALF2 and Es ALF3) and Es ERK in haemocytes were observed to increase from 6 h to 48 h post low doses of CpG ODN 2395 (0.5 μg and 2.5 μg) stimulation, while they were suppressed after high dose of CpG ODN 2395 (12.5 μg) injection. Meanwhile, the phosphorylation levels of ERK in haemocytes were significantly promoted after low doses of CpG ODN 2395 injection, and a reduce level of ERK phosphorylation was observed after high dose of CpG ODN 2395 injection. Further investigation showed that the expression levels of Es ALFs induced by CpG ODN 2395 were markedly down-regulated after knocking down the expression of Es ERK. Similarly, the Es ALFs mRNA expression were also inhibited post different doses of CpG ODN 2395 stimulation in PD98059 (ERK inhibitor) injection crabs. These results collectively suggest that ERK is involved in regulating the expression level of Es ALFs induced by different dose of CpG ODN 2395 in Chinese mitten crab, which contribute to the understanding of the regulation of CpG ODN involving in immune response in crustaceans. • Low dose of CpG ODN 2395 markedly enhanced Es ALFs and Es ERK expression. • High dose of CpG ODN 2395 significantly reduced Es ALFs and Es ERK expression. • Es ERK acts as a regulator involving in the Es ALFs expression induced by CpG ODN 2395. [ABSTRACT FROM AUTHOR]

In crustaceans, the steroid hormone 20-hydroxyecdysone (20E) initiates molting, and the molting process is also regulated by energy metabolism. AMPK is an energy sensor and plays a critical role in systemic energy balance. Here, the regulatory mechanism in the interaction between 20E and AMPK was investigated in Chinese mitten crab, Eriocheir sinensis. The results showed that the 20E concentration and the mRNA expression levels of 20E receptors in hepatopancreas were down-regulated post AMPK activator (AICAR) treatment, and were up-regulated after AMPK inhibitor (Compound C) injection in crabs. Besides, the molt-inhibiting hormone (MIH) gene expression in eyestalk showed the opposite patterns in response to the AICAR and Compound C treatment, respectively. Further investigation found that there was a significant reduction in 20E concentration post PI3K inhibitor (LY294002) treatment, and the phosphorylation level of PI3K was increased in hepatopancreas after AMPK inhibitor injection. On the other hand, the positive regulation of PI3K-mediated activation of AMPK was also observed, the phosphorylation levels of AMPKα, AMPKβ and PI3K in hepatopancreas were significantly increased post 20E injection. In addition, the phosphorylation levels of AMPKα and AMPKβ induced by 20E were decreased after the injection of PI3K inhibitor. Taken together, these results suggest that the regulatory cross-talk between 20E and AMPK is likely to act through PI3K pathway in E. sinensis , which appeared to be helpful for a better understanding in molting regulation. • AMPK could negatively regulate the concentration of 20E. • AMPK inhibited 20E concentration by decreasing the phosphorylation of PI3K. • The phosphorylation levels of AMPK and PI3K were increased after 20E injection. • The phosphorylation level of AMPK induced by 20E was decreased after inhibiting PI3K. [ABSTRACT FROM AUTHOR]

Hypoxia triggers diverse cell physiological processes, and the hypoxia inducible factors (HIFs) are a family of heterodimeric transcription factors that function as master regulators to respond to hypoxia in different cells. However, the knowledge about the hypoxic responses especially cell alteration mediated by HIFs under hypoxia stress is still limited in crustaceans. In the present study, a hypoxia-inducible factor-1α (HIF-1α) gene was identified (designed as Es HIF-1α). The relative mRNA expression level of Es HIF-1α was highest in hyalinocytes and lowest in granulocytes among three types of haemocytes in crabs. Hypoxia could significantly increase the Es HIF-1α protein expression level in haemocytes. Meanwhile, the proportion of hyalinocytes began to increase from 3 h post hypoxia treatment, and reached the highest level at 24 h. However, the opposite variation in proportion of granulocytes was observed under hypoxia stress. Further investigation showed that the inhibition of Es HIF-1α induced by KC7F2 (HIF-1α inhibitor) could lead to the significant decrease in the proportion of hyalinocytes under hypoxia stress, and also resulted in an increase of granulocytes proportion. While, after Es HIF-1α was activated by IOX4 (HIF-1α activator), the proportion of hyalinocytes was significantly up-regulated and the proportion of granulocytes was significantly down-regulated under post hypoxia treatment. These results collectively suggested that Es HIF-1α was involved in the regulation of proportion of three types of haemocytes induced by hypoxia stress, which provided vital insight into the understanding of the crosstalk between hypoxia and cell development in invertebrates. • The Es HIF-1α mRNA expression was highest in hyalinocytes and lowest in granulocytes. • Es HIF-1α protein expression in haemocytes is up-regulated post hypoxia treatment. • The proportion of hyalinocytes and granulocytes was differential respond to hypoxia. • Inhibition of Es HIF-1α decreases the proportion of hyalinocytes under hypoxia stress. • Activation of Es HIF-1α leads to increased hyalinocytes proportion under hypoxia stress. [ABSTRACT FROM AUTHOR]