The aim of this study was to investigate the effects of immersion on immune enzyme activity, haemolymph index, intestinal microbiome and metabolome of E. sinensis after low temperature air exposure. The results showed that low temperature air exposure induced stress response, which led to hepatopancreas injury and increased membrane permeability, but this situation was reversible and alleviated after immersion. In addition, after exposure to low temperature air, haemolymph metabolism-related substances such as glucose and total cholesterol were significantly different from the initial value (P < 0.05), and gradually returned to the initial level after immersion. The changes of intestinal flora and hepatopancreas metabolism caused by low temperature air exposure did not fully recover after immersion, and its negative effects did not completely disappear. The sequencing results showed that the species composition and diversity of intestinal microorganisms of Chinese mitten crabs were changed after low temperature air exposure and immersion treatment. The relative abundance of Bacteroidetes and Proteobacteria were increased, while the relative abundance of Firmicutes was decreased (P < 0.05). Metabolomics analysis showed that lysine levels increased significantly, taurocholic acid levels decreased significantly, and amino acid metabolism and lipid metabolism balance were disturbed in hepatopancreas of E. sinensis after exposure to low temperature air and immersion (P < 0.05). This study will provide new insights into the recovery mechanism of water immersion on Chinese mitten crabs after exposure to air.

As a key planar cell polarity protein, Van Gogh-like 2 (Vangl2) is essential for mammalian spermatogenesis. As a decapod crustacean, Eriocheir sinensis exhibits distinct spermatogenic processes due to its unique seminiferous tubule morphology and hemolymph-testis barrier (HTB). To determine whether Vangl2 performs analogous functions in E. sinensis, we identified the Es-Vangl2. Es-Vangl2 exhibited high expression and wide distribution in the testes, indicating its crucial involvement in spermatogenesis. Following targeted knockdown of Es-Vangl2in vivo, the structure of seminiferous tubules was disrupted, characterized by vacuolization of the germinal zone and obstruction of spermatozoon release. Concurrently, the integrity of the HTB was compromised, accompanied by reduced expression and aberrant localization of junction proteins. More importantly, the regulatory influence of Es-Vangl2 was manifested through modulating the organization of microfilaments, a process mediated by epidermal growth factor receptor pathway substrate 8 (Eps8). Further studies demonstrated that these phenotypes resulting from Es-Vangl2 knockdown were attributed to the inhibition of Rock signaling pathway activity, which was verified by the Es-Rock interference and Y27632 inhibition assays. In summary, the findings highlight the pivotal role of Es-Vangl2 in stabilizing HTB integrity by regulating Eps8-mediated actin remodeling through the Rock signaling pathway in the spermatogenesis of E. sinensis.

Shewanella putrefaciens has been recognized as an emerging important pathogen in aquaculture. However, scarce information is available on the characterization and microbial control of S. putrefaciens as a causal agent of hepatopancreas necrosis disease in Chinese mitten crab Eriocheir sinensis. In this study, a multi-resistant S. putrefaciens isolate (DZ-A) was identified as a causal pathogen of hepatopancreas necrosis disease in Chinese mitten crabs. It showed a lethal dose (LD50) of 2.20 × 105 CFU ml-1 in Chinese mitten crabs, and multiple resistance to aminoglycoside, chloramphenicol, macrolide, penicillin, peptide, and tetracycline antimicrobials. In addition, Bdellovibrio powder exhibited a significant antibacterial effect against the pathogenic S. putrefaciens, and conferred significant protection to challenged Chinese mitten crabs with relative percentage survivals of 80.00% to 93.33% via significant improvement in their immune response and antioxidant capability. The findings of this study provide valuable insights into the phenotypic characterization and biological control of pathogenic S. putrefaciens in Chinese mitten crabs.

The cerebral ganglion and muscle are important regulatory tissues in Eriocheir sinensis. Therefore, it is of great significance to explore their synergistic roles in this organism\'s anti-stress response. In this study, proteomics, metabolomics, and combination analyses of the cerebral ganglion and muscle of E. sinensis under alkalinity stress were performed. The cerebral ganglion and muscle played a significant synergistic regulatory role in alkalinity adaptation. The key regulatory pathways involved were amino acid metabolism, energy metabolism, signal transduction, and the organismal system. They also played a modulatory role in the TCA cycle, nerve signal transduction, immune response, homeostasis maintenance, and ion channel function. In conclusion, the present study provides a theoretical reference for further research on the mechanisms regulating the growth and development of E. sinensis in saline-alkaline environments. In addition, it provides theoretical guidelines for promoting the vigorous development of the E. sinensis breeding industry in saline-alkaline environments in the future.

(1) The development and utilization of the vast saline-alkali land worldwide is an important way to solve the worsening food crisis. Eriocheir sinensis, due to its strong osmotic regulation capability and its characteristics of being suitable for culturing in alkaline water, has become a potential aquaculture species in saline-alkali water. The brain and heart are the key tissues for signal transduction and energy supply under environmental stress. (2) This study is the first to explore the synergistic regulatory molecular mechanism by integrated analysis on cerebral ganglion proteomics and heart metabolomics of Eriocheir sinensis under alkalinity stress. (3) The results indicate that the cerebral ganglion and heart of E. sinensis were closely related in response to acute alkalinity stress. The differential regulatory pathways mainly involved regulation of energy metabolism, amino acid metabolism, and homeostasis maintenance. Importantly, alkalinity stress induced the regulation of antioxidants and further adjusted longevity and rhythm in the cerebral ganglion and heart, reflecting that the cerebral ganglion and heart may be the key tissues for the survival of Eriocheir sinensis under an alkalinity environment. (4) This study provides a theoretical reference for research on the regulation mechanism of E. sinensis under alkalinity condition and contributes to the development of aquaculture in saline-alkali water.

Molting is a crucial biological process of crustaceans. Crustaceans go through three separate stages throughout their molting process, including pre-molt, post-molt and inter-molt. However, the exact mechanism of immunological modulation during molting remains unclear. Tumor necrosis factor receptor-associated factor 6 (TRAF6) has been extensively documented to participate in immune defense. In the present study, a TRAF6 gene with two TRAF-type zinc finger domains was identified from Eriocheir sinensis (designed as EsTRAF6), and its role in regulating immune response during molting process was explored. The mRNA expression level of EsTRAF6 at pre-molt stage was higher than that at post-molt stage and inter-molt stage. After Aeromonas hydrophila stimulation, the expression levels of EsTRAF6, EsRelish and anti-lipopolysaccharide factors (ALFs) genes exhibited a considerable increase at three molting stages. Subsequently, the expression patterns of EsTRAF6 and EsRelish in response to the treatment with 20-hydroxyecdysone (20E) were examined. The mRNA expression of EsTRAF6 and EsRelish were significantly increased at 12 h after 20E injection. Additionally, the protein expression level of TRAF6 was also up-regulated in 20E group compared to control group. Furthermore, the role of EsTRAF6 in regulating the anti- ALFs expression at pre-molt stage post A. hydrophila stimulation was investigated. Following the inhibition of the EsTRAF6 transcript using RNAi or the injection of inhibitor (TMBPS), there was a notable decrease of the EsALF1, EsALF2 and EsALF3 transcripts. Moreover, a significant reduction in the phosphorylation level of NF-κB at pre-molt stage was observed after A. hydrophila stimulation in TRAF6-inhibited crabs. Collectively, our results suggest that EsTRAF6 could be induced by 20E and promoted the EsALFs expression by activating NF-κB at pre-molt stage, which provides a novel insight into the research of immune regulatory mechanism during the process of molting of crustaceans.

Cuticle proteins (CPs) are the vital components of the cuticle and chitin lining covering the digestive tract of crustaceans. In this study, four new CP genes (designated as EsCP3, EsCP4, EsCP5, and EsCP8) were initially cloned and identified from the Chinese mitten crab Eriocheir sinensis. EsCP3/4/5/8 included 375, 411, 381, and 570 bp open reading frame encoding 124, 136, 126, and 189 amino acid proteins, respectively. Except for EsCP8, EsCP3/4/5 all contained a Chitin_bind_4 domain. EsCP3/4/5/8 were clustered into different groups in the phylogenetic tree. Quantitative real-time PCR results indicated that four EsCP genes have different patterns of tissue distribution. Changes in the expression levels of these four EsCP genes were observed in the intestine of crabs under Vibrio parahaemolyticus challenge. RNA interference assay showed that the knockdown of EsCPs in the intestine could inhibit the expression of antimicrobial peptides (AMPs), including crustins and anti-lipopolysaccharide factors. In addition, the knockdown of EsRelish in the intestine decreased the expression levels of these four EsCP genes. These results indicated that EsCPs were involved in regulating the expression of AMPs, and EsCPs were regulated by EsRelish.

Molting is a key biological process of crustaceans, which is mainly regulated by 20-hydroxyecdyone (20E). The molting cycle could be divided into three main stages including pre-molt, post-molt and inter-molt stages. The mechanism of immune regulation during molting process still requires further exploration. Yorkie (Yki) is a pivotal transcription factor in the Hippo signaling pathway, and it plays an essential role in regulating cell growth and immune response. In the present study, a Yki gene was identified from Eriocheir sinensis (designed as EsYki), and the regulatory role of EsYki in controlling the expression of antimicrobial peptide genes throughout the molting process was investigated. The mRNA expression level of EsYki was higher at the pre-molt stage compared to the post-molt stage and inter-molt stage. Following the injection of 20E, there was a notable and consistent rise in the EsYki mRNA expression in haemocytes. The increase was observed from 3 h to 48 h with the maximum level at 12 h. And the phosphorylation of Yki in the haemocytes was also significantly up-regulated at 3 h post 20E injection. Moreover, the levels of EsYki mRNA expression at three molting stages were significantly increased post Aeromonas hydrophila stimulation. The maximum level was detected at post-molt stage following A. hydrophila stimulation, while the lowest level was observed at inter-molt stage. The expression pattern of EsCrus was in contrast to EsCrus. After EsYki mRNA transcripts were inhibited by Yki inhibitor (CA3), the mRNA expression levels of EsCrus1 and EsCrus2 following A. hydrophila stimulation were significantly elevated. Furthermore, the phosphorylation level of NF-κB was also increased following the inhibition of Yki. Collectively, our findings indicated that EsYki could be induced by 20E and has a suppressive effect on the expression of EsCrus via inhibiting NF-κB during molting process. This research contributes to the understanding of the immunological regulation mechanism during molting process in crustaceans.

LRR-only protein (LRRop) is an important class of immune molecules that function as pattern recognition receptor in invertebrates, however, the bacterial inhibitory activity of this proteins remain largely unknown. Herein, a novel LRRop was cloned from Eriocheir sinensis and named as EsLRRop2. The EsLRRop2 consists of six LRR motifs and formed a horseshoe shape three-dimension structure. EsLRRop2 was mainly expressed in intestine and hepatopancreas. The transcripts of EsLRRop2 in the intestine and hepatopancreas were induced by Vibrio parahaemolyticus and Staphylococcus aureus, and displayed similar transcriptional profiles. The expression levels of EsLRRop2 responded more rapidly and highly to V. parahaemolyticus than S. aureus in the intestine and hepatopancreas. Although the basal expression level of EsLRRop2 in hemocytes was relatively low, its transcripts in hemocytes were significantly induced by V. parahaemolyticus and S. aureus. The recombinant proteins of EsLRRop2 (rEsLRRop2) displayed a wide range of binding spectrum against vibrios, including V. parahaemolyticus, V. alginolyticus, and V. harveryi. The rEsLRRop2 showed dose- and time-dependent inhibitory activity against V. parahaemolyticus and S. aureus, and it could agglutinate the two bacteria. Furthermore, the inhibitory activities of rEsLRRop2 against V. parahaemolyticus, V. alginolyticus, V. harveryi and S. aureus was slightly affected by pH and salinity, and the rEsLRRop2 displayed the strongest inhibitory activity against all the three vibrios when the salinity was 20 ‰ and pH was 8.0. Collectively, these results elucidate the bacterial binding and inhibitory activities of EsLRRop2, and provide theoretical foundations for the application of rEsLRRop2 in prevention and control of vibrio diseases in aquaculture.

Eriocheir sinensis is an economically important aquatic animal. Its regulatory mechanisms underlying many biological processes are still vague due to the lack of systematic analysis tools. The protein-protein interaction network (PIN) is an important tool for the systematic analysis of regulatory mechanisms. In this work, a novel machine learning method, DGO-SVM, was applied to predict the protein-protein interaction (PPI) in E. sinensis, and its PIN was reconstructed. With the domain, biological process, molecular functions and subcellular locations of proteins as the features, DGO-SVM showed excellent performance in Bombyx mori, humans and five aquatic crustaceans, with 92-96% accuracy. With DGO-SVM, the PIN of E. sinensis was reconstructed, containing 14,703 proteins and 7,243,597 interactions, in which 35,604 interactions were associated with 566 novel proteins mainly involved in the response to exogenous stimuli, cellular macromolecular metabolism and regulation. The DGO-SVM demonstrated that the biological process, molecular functions and subcellular locations of proteins are significant factors for the precise prediction of PPIs. We reconstructed the largest PIN for E. sinensis, which provides a systematic tool for the regulatory mechanism analysis. Furthermore, the novel-protein-related PPIs in the PIN may provide important clues for the mechanism analysis of the underlying specific physiological processes in E. sinensis.