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Mud crab, Scylla paramamosain, is an important aquaculture species. In recent decades, with the development of mud crab farming, seed production has become more and more important. Therefore, a precise understanding of embryonic and larval development is required for future study or application. In this study, we described the complete embryonic and larval development of S. paramamosain under the following conditions: temperature 28°C–31°C, salinity 26,000–30,000 mg/L, pH 7.9–8.2, and dissolved oxygen 4–5 mg/L. The embryonic development was divided into five stages: cleavage, blastula, appendage, eyespot‐heartbeat, and prehatching. The entire embryonic period spanned 9–11 days. The larval development consisted of six stages: zoea I (Z1), zoea II (Z2), zoea III (Z3), zoea IV (Z4), zoea V (Z5), and the megalopal stage. The duration of the larval stage was about 22–24 days, and it took about 3 days for each stage of Z1–Z4, about 4–5 days for Z5, and about 5–6 days for the megalopal stage. In general, in this study, we presented a comprehensive description of both embryonic and larval development with massive image information, which would help diagnose different developmental stages and provide an important guidance for future study or application. [ABSTRACT FROM AUTHOR]

Mud crabs (Scylla spp.) are considered luxury seafood and high market value; hence, mud crab farming is well known in Southeast Asia. However, the prevalence of cannibalistic behavior in crabs thus requires shelter in the culture system to reduce conspecific cannibalism and enhance the survival of crabs. This study aims to determine the suitability of various seaweed species as shelter to improve the survival and production of mud crab Scylla paramamosain crablets. The experiment consisted of seven treatments and was randomly designed in triplicate tanks. The control treatment had no shelter (i.e., without seaweed in the culture tank), and in the other six treatments, red seaweed (Gracilaria tenuistipitata) or green seaweed (Enteromorpha intestinalis) was placed in the rearing tanks at three density levels: 0.5, 1, and 2 kg/m2. Instar 2 crablets were reared at a density of 300 ind/m2, at salinity of 15 g/L, and with continuous aeration. Crablets were fed frozen Artemia biomass to satiation for 4 weeks. Results showed that the survival of crabs in all treatments sharply declined from Week 3 to Week 4 of culture, where the 1 and 2 kg Gracilaria groups showed less reduction than the other treatments. At the end of the experiment, the average survival of crabs in the control group was lowest (17.4%), while the survival of crabs was significantly improved, varying within the ranges of 53.3–70.7% and 27.8–35.9% in the Gracilaria and Enteromorpha groups, respectively. Notably, higher survival resulted in lower growth rates in the seaweed treatments, but enhanced biomass and production of crab juveniles. Moreover, concentrations of TAN, NO2−, NO3−, and PO43− in the Gracilaria treatments were much lower than in both the control and the Enteromorpha treatments that improved water quality in the rearing tanks. The present findings prove that red seaweed G. tenuistipitata could be considered a suitable shelter for the reliable production of mud crab juveniles in the nursery phase. [ABSTRACT FROM AUTHOR]

Tropomyosin (TM) is an important crustacean (Scylla paramamosain) allergen. This study aimed to assess Maillard-reacted TM (TM-G) induction of allergenic responses with cell and mouse models. We analyzed the difference of sensitization and the ability to induce immune tolerance between TM and TM-G by in vitro and in vivo models, then we compared the relationship between glycation sites of TM-G and epitopes of TM. In the in vitro assay, we discovered that the sensitization of TM-G was lower than TM, and the ability to stimulate mast cell degranulation decreased from 55.07 ± 4.23% to 27.86 ± 3.21%. In the serum of sensitized Balb/c mice, the level of specific IgE produced by TM-G sensitized mice was significantly lower than TM, and the levels of interleukins 4 and interleukins 13 produced by Th2 cells in spleen lymphocytes decreased by 82.35 ± 5.88% and 83.64 ± 9.09%, respectively. In the oral tolerance model, the ratio of Th2/Th1 decreased from 4.05 ± 0.38 to 1.69 ± 0.19. Maillard reaction masked the B cell epitopes of TM and retained some T cell epitopes. Potentially, Maillard reaction products (MRPs) can be used as tolerance inducers for allergen-specific immunotherapy. [ABSTRACT FROM AUTHOR]

p53, the guardian of the genome, is a short-lived protein that is tightly controlled at low levels by constant ubiquitination and proteasomal degradation in higher organisms. p53 stabilization and activation are early crucial events to cope with external stimuli in cells. However, the role of p53 ubiquitination and its relevant molecular mechanisms have not been addressed in invertebrates. In this study, our findings revealed that both HUWE1 (HECT, UBA, and WWE domain-containing E3 ubiquitin-protein ligase 1) and TRAF6 (tumor necrosis factor receptor-associated factor 6) could serve as E3 ubiquitin ligases for p53 in mud crabs (Scylla paramamosain). Moreover, the expression of HUWE1 and TRAF6 was significantly downregulated during white spot syndrome virus (WSSV) infection, and therefore the ubiquitination of p53 was interrupted, leading to the activation of apoptosis and reactive oxygen species (ROS) signals through p53 accumulation, which eventually suppressed viral invasion in the mud crabs. To the best of our knowledge, this is the first study to reveal the p53 ubiquitination simultaneously induced by two E3 ligases in arthropods, which provides a novel molecular mechanism of invertebrates for resistance to viral infection. [ABSTRACT FROM AUTHOR]

Previously, we reported the identification, tissue distribution and confirmed the roles of Spfoxl-2 in regulating vitellogenin (vtg) expression in Scylla paramamosain. Here, we primally analyzed its potential target genes in the ovary with RNAi and RNA-Seq technology. By comparing the transcriptome data of two groups (ovaries that injected with EGFP and Foxl-2 siRNA, respectively), we found 645 DEGs (differentially expressed genes), including several conserved crucial genes involved in ovarian development, such as vtg, vitellogenin receptor (vtgR), adenylate cyclase (AC), cyclinB, and cell division cycle 2 (cdc2). In addition, these DEGs were also enriched in pathways related to ovary development, including relaxin signaling pathway, ovarian steroidogenesis, and progesterone-mediated oocyte maturation. Moreover, several genes were selected for qRT-PCR to validate the accuracy of the bioinformatic result. To the best of our knowledge, the current study was the first report about foxl-2 function through comparative transcriptome analysis in crustacean species, which identified not only relevant genes and pathways involved in ovarian development of S. paramamosain, but also provided new insights into the regulatory mechanisms of foxl-2 at the molecular level in crustacean. [ABSTRACT FROM AUTHOR]

Programmed cell death 6 (PDCD6) is a well-known apoptosis regulator that is involved in the immunity of mammals. However, the effects of miRNA-mediated regulation of PDCD6 expression on apoptosis and virus infection in organisms, especially in marine invertebrates, have not been extensively explored. In this study, PDCD6 of mud crab (Scylla paramamosain) (Sp-PDCD6) was characterized. The results showed that Sp-PDCD6 contains five EF-hands domains and could suppress virus infection via apoptosis promotion. It also presented that Sp-PDCD6 was directly targeted by miR-9875 in vitro and in vivo, miR-9875 served as a positive regulator during the virus invasion. The findings indicated that the miR-9875-PDCD6 pathway possessed fundamental effects on the immune response to virus infection in mud crab. Therefore, our research provided a novel insight into the roles of both miR-9875 and PDCD6 in the regulation of apoptosis and virus defense in mud crab.

This study was the first to evaluate multiple hormonal manipulations to hepatopancreas over the ovarian development stages of the mud crab, Scylla paramamosain. A total of 1258 metabolites in 75 hepatopancreas explants from five female crabs were induced by juvenile hormone III (JH III), methyl farnesoate (MF), farnesoic acid (FA) and methoprene (Met), as identified from combined metabolomics and lipidomics (LC-MS/MS). 101 significant metabolites and 47 significant pathways were selected and compared for their comprehensive effects to ovarian maturation. While MF played an extensive role in lipid accumulation, JH III and Met shared similar effects, especially in the commonly and significantly elevated triglycerides and lysophospholipids (fold change≥2 and ≤0.5, VIP≥1). The significant upregulation of β-oxidation and key regulators in lipid degradation by FA (P ≤ 0.05) resulted in less lipid accumulation from this treatment, with a shift toward lipid export and energy consumption, unlike the effects of MF, JH III and Met. It was possible that MF and FA played their own unique roles and acted in synergy to modulate lipid metabolism during crab ovarian maturation. Our study yielded insights into the MF-related lipid metabolism in crustacean hepatopancreas for the overall regulation of ovarian maturation, and harbored the potential use of juvenoids to induce reproductive maturity of this economic crab species.

Chromium (Cr) is an essential micronutrient for all animals, which is also an important component of glucose tolerance factor. The present study was conducted to evaluate the effect of dietary Cr levels on growth, Cr accumulation in tissues, glucose and lipid metabolism and insulin signal pathway of juvenile mud crab. Six isonitrogenous and isolipidic diets were formulated to contain 1.4 (basal diet), 1.7, 2.0, 2.2, 2.6 and 3.7 mg kg−1 Cr, respectively. The results indicated that crabs fed with 2.2 and 2.6 mg kg−1 Cr diets had the highest percent weight gain (PWG) and specific growth rate (SGR) among all treatments. Cr concentration in hepatopancreas significantly increased with increase of dietary Cr levels. Crabs fed with 3.7 mg kg−1 Cr diet exhibited lower antioxidant enzyme activities and higher malondialdehyde (MDA) in hepatopancreas and hemolymph than those fed the other diets. Crabs fed the diets with 2.2 and 2.6 mg kg−1 Cr showed higher activities of hexokinase (HK), phosphofructokinase (PFK) and pyruvate kinase (PK). The expression levels of genes related to insulin signaling pathway, glycolysis and fat synthesis, glycolipid metabolism (such as phosphatidylinositol 3-kinase (pi3k), hexokinase (hk), and fatty acid synthase (fas)) in hepatopancreas were significantly up-regulated. The expression levels of genes related to gluconeogenesis and lipolysis such as fructose-1,6-bisphosphatase (fbp) and carnitine palmitoyltransferase 1 (cpt1) were significantly down-regulated in crabs fed with optimal Cr diets. In conclusion, the optimal dietary Cr requirement was estimated to be 2.34 mg kg−1 for juvenile mud crab based on two slope broken-line regression analysis of PWG against dietary Cr levels, the results of present study indicated that optimal dietary Cr promoted the growth and Cr accumulation in tissues, and modulated insulin signaling pathway to trigger glycolysis and glycogenesis to maintain glucose homeostasis.

Crustacean cardioactive peptide (CCAP) is a pleiotropic neuropeptide, but its immunomodulatory role is not clear. Herein, the mud crab Scylla paramamosain provides a primitive model to study crosstalk between the neuroendocrine and immune systems. In this study, in situ hybridization showed that Sp-CCAP positive signal localized in multiple cells in the nervous tissue, while its conjugate receptor (Sp-CCAPR) positive signal mainly localized in the semigranular cells of hemocytes. The Sp-CCAP mRNA expression level in the thoracic ganglion was significantly up-regulated after lipopolysaccharide (LPS) stimulation, but the Sp-CCAP mRNA expression level was up-regulated firstly and then down-regulated after the stimulation of polyriboinosinic polyribocytidylic acid [Poly (I:C)]. After the injection of Sp- CCAP synthesis peptide, the phagocytosis ability of hemocytes was significantly higher than that of synchronous control group. Simultaneously, the mRNA expression of phagocytosis related gene (Sp-Rab5), nuclear transcription factor NF-κB homologues (Sp-Relish), C-type lectin (Sp-CTL-B), prophenoloxidase (Sp-proPO), pro-inflammatory cytokines factor (Sp-TNFSF , Sp-IL16) and antimicrobial peptides (Sp-ALF1 and Sp-ALF5) in the hemocytes were also significantly up-regulated at different time points after the injection of Sp -CCAP synthetic peptide, but Sp-TNFSF , Sp-ALF1 and Sp-ALF5 were down-regulated significantly at 24h. In addition, RNA interference of Sp - CCAP suppressed the phagocytic activity of hemocytes and inhibited the mRNA expression of Sp-Rab5 , Sp-Relish , Sp-CTL-B , Sp-TNFSF, Sp-IL16 and Sp-ALF5 in the hemocytes, and ultimately weakened the ability of hemolymph bacteria clearance of mud crab. Taken together, these results revealed that CCAP induced innate immune and increased the anti-infection ability in the mud crab. ● Sp - CCAP mRNA in the thoracic ganglion changed during LPS and Poly (I:C) stimulation. ● Sp-CCAP mRNA was localized in various types of cells on the nervous system, while Sp-CCAPR was localized in the semigranular cells of hemocytes. ● Sp -CCAP modulated the phagocytosis and immune effector molecules of hemocytes. ● Sp -CCAP enhanced the resistance of mud crab to bacterial infections. [ABSTRACT FROM AUTHOR]

This study investigated the effects of long-term low-salinity on the growth performance, proximate composition, amino acid and fatty acid profiles of Scylla paramamosain. The salinity of the low-salinity-culture group (LC) and maintained-salinity-culture group (MC) were set at 5 and 25 respectively. After rearing for 30 d, the survival rate (SR) of the LC group was significantly lower, whereas the average daily molting frequency (ADMF) was significantly higher than those in MC group (P <0.05). There were no significant difference for weight gain rate (WGR) and specific growth rate (SGR) between the two groups. The moisture content of the LC group was slightly higher than the MC group without significant difference. The contents of ash, crude lipid and crude protein in the LC group were lower than those in the MC group, but no significant differences were found except for ash content. A total of 17 free amino acids and 23 fatty acids were identified from the muscle of S. paramamosain. The total amino acids (TAA), essential amino acids (EAA) and essential amino acid index (EAAI) had no significant difference between the two groups. The crabs from LC group had a significantly higher HUFA, including EPA, DHA, ARA. The percentage of n-3 PUFA and the ratio of n-3/n-6 PUFA were significantly higher in the LC group. These results indicated that S. paramamosain rearing at low salinity had significantly decreased SR; however, the low-salinity environment significantly promoted the accumulation of unsaturated fatty acids (UFAs), especially the n-3 PUFA, which might be related to the osmoregulation mechanism. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are important signaling regulators that are involved in regulating the innate immunity of crustacean. However, few studies focus on the role of crustacean miRNAs in the cellular immunity have been reported. In this study, we showed that the expression of miR-133 was significantly up-regulated in the mud crab Scylla paramamosain after infection by white spot syndrome virus (WSSV) or Vibrio parahaemolyticus. The anti-miRNA oligonucleotide AMO-miR-133 was used to knock down miR-133 expression in S. paramamosain. The number of WSSV copies increased significantly in WSSV-infected crabs after miR-133 knockdown. Knockdown of miR-133 also enhanced the mortality rates of WSSV-infected and V. parahaemolyticus- infected mud crabs, and it significantly enhanced the expression of the astakine, which was confirmed by real-time quantitative PCR and western blot analysis. The data also indicate that miR-133 may affect hemocyte proliferation in S. paramamosain by regulating astaki ne expression. miR-133 Knockdown enhanced the apoptosis or phagocytosis of crab hemocytes, and increased the mortality of mud crabs after WSSV or V. parahaemolyticus infection. These results indicate that miR-133 is involved in the host immune response to WSSV and V. parahaemolyticus infection in mud crabs. Taken together, our research provides new insights for the control of viral or vibrio diseases in S. paramamosain. [ABSTRACT FROM AUTHOR]

• Sp TIA-1 is overexpressed during WSSV infection in mud crab. • Sp TIA-1 suppresses WSSV infection via apoptosis promotion. • Sp TIA-1 can activate caspase signaling in mud crab. • Sp TIA-1 can modulate MMP (⊿ψm) by regulating ROS levels. TIA-1 (T cell restricted intracellular antigen-1) is a kind of RNA-binding protein which serves as the downstream of CED-9 (a BCL2 homolog) and induces apoptosis under stress conditions. So far, the function of apoptosis mediated by TIA-1 has been extensively studied in higher animals, and apoptosis happens to be related to biological immune defense. However, the involvement of TIA-1 in the study of immune function during viral infection has not been clearly studied, especially in marine invertebrates. In the study, Sp TIA-1 in mud crab (Scylla paramamosain) was specifically identified. The Open Reading Frame (ORF) of Sp TIA-1 was consisted of 1116 nucleotide bases and encoded 372 amino acids. Besides, the results showed that the expression of Sp TIA-1 was obviously up-regulated during WSSV (White Spot Syndrome Virus) infection in hemocytes of mud crab. Furthermore, through RNAi approach, we found that Sp TIA-1 could activate Caspase-3 signaling and increase ROS levels to reduce mitochondrial membrane potential, resulting in the increase of apoptosis rate in hemocytes, which eventually suppressed WSSV multiplication in mud crab. The current study therefore improves the knowledge of antiviral immunity in mud crab and provides new insights into the innate immunity of marine crustaceans. [ABSTRACT FROM AUTHOR]

The doublesex and mab-3 related transcription factor (DMRT) gene family is conserved from invertebrates to humans. The functions of DMRT are mainly involved in sex development and the formation of many tissues and organs. In this study, a DM (Doublesex/Mab-3)-domain gene was identified in the mud crab Scylla paramamosain, and was named SpDMRT99B because of its many similarities to arthropod DMRT99B and phylogenetically close relationship with arthropod DMRT99B. The cDNA of SpDMRT99B gene is 1249 bp in length, encoding 224 amino acids. From 254 bp to 928 bp there is a conserved DM domain. No transmembrane domain was identified. Through multiple amino acid alignment and phylogenetic tree analysis, the closest gene to SpDMRT99B is Eriocheir sinensis DMRT99B, followed by Macrobrachium rosenbergii and Drosophila melanogaster DMRT99B. The expressions of the gene were characterized in different tissues of female and male crabs during early development period of crab individuals, as well as in different development periods of gonads. The results showed that SpDMRT99B gene is significantly highly expressed in testis than in ovary and other tissues. The expression level of SpDMRT99B in testis at different stages is significantly higher than that in ovary, and it is particularly highly expressed in immature testis. In early developmental stages of larvae, the expressions of SpDMRT99B remain at a low level and reach a peak at zoea stage I when the body segments shape up. It is speculated that SpDMRT99B gene might be involved in the gonadal development process and somitogenesis of S. paramamosain. [ABSTRACT FROM AUTHOR]

Abstract Background Scylla paramamosain is one of the commercially crucial marine crustaceans belonging to the genus Scylla, which is commonly distributed along the coasts of China, Vietnam, and Japan. Genomic and transcriptomic data are scarce for the mud crab. Light intensity is one of the ecological factors that affect S. paramamosain during indoor overwintering. To understand the energy metabolism mechanism adapted to light intensity, we analyzed the transcriptome of S. paramamosain hepatopancreas in response to different light intensities (0, 1.43, 40.31 μmol·m− 2·s− 1). Results A total of 5052 differentially expressed genes were identified in low light group (LL group, 3104 genes were up-regulated and 1948 genes were down-regulated). A total of 7403 differentially expressed genes were identified in high light group (HL group, 5262 genes were up-regulated and 2141 genes were down-regulated). S. paramamosain adapts to different light intensity environments through the regulation of amino acids, fatty acids, carbon and energy metabolism. Different light intensities had a strong impact on the energy generation of S. paramamosain by influencing oxygen consumption rate, aerobic respiration, glycolysis/gluconeogenesis pathway, the citrate cycle (TCA cycle) and fatty acid degradation. Conclusion Low light is more conducive to the survival of S. paramamosain, which needs to produce and consume relatively less energy to sustain physiological activities. In contrast, S. paramamosain produced more energy to adapt to the pressure of high light intensities. The findings of the study add to the knowledge of regulatory mechanisms related to S. paramamosain metabolism under different light intensities.

Abstract Background Mud crab, Scylla paramamosain, a euryhaline crustacean species, mainly inhabits the Indo-Western Pacific region. Wild mud crab spawn in high-salt condition and the salinity reduced with the growth of the hatching larvae. When the larvae grow up to megalopa, they migrate back to estuaries and coasts in virtue of the flood tide, settle and recruit adult habitats and metamorphose into the crablet stage. Adult crab can even survive in a wide salinity of 0–35 ppt. To investigate the mRNA profile after salinity stress, S. paramamosain megalopa were exposed to different salinity seawater (low, 14 ppt; control, 25 ppt; high, 39 ppt). Results Firstly, from the expression profiles of Na+/K+/2Cl- cotransporter, chloride channel protein 2, and ABC transporter, it turned out that the 24 h might be the most influenced duration in the short-term stress. We collected megalopa under different salinity for 24 h and then submitted to mRNA profiling. Totally, 57.87 Gb Clean Data were obtained. The comparative genomic analysis detected 342 differentially expressed genes (DEGs). The most significantly DEGs include gamma-butyrobetaine dioxygenase-like, facilitated trehalose transporter Tret1, sodium/potassium-transporting ATPase subunit alpha, rhodanese 1-like protein, etc. And the significantly enriched pathways were lysine degradation, choline metabolism in cancer, phospholipase D signaling pathway, Fc gamma R-mediated phagocytosis, and sphingolipid signaling pathway. The results indicate that in the short-term salinity stress, the megalopa might regulate some mechanism such as metabolism, immunity responses, osmoregulation to adapt to the alteration of the environment. Conclusions This study represents the first genome-wide transcriptome analysis of S. paramamosain megalopa for studying its stress adaption mechanisms under different salinity. The results reveal numbers of genes modified by salinity stress and some important pathways, which will provide valuable resources for discovering the molecular basis of salinity stress adaptation of S. paramamosain larvae and further boost the understanding of the potential molecular mechanisms of salinity stress adaptation for crustacean species.

Molting behavior in insects is controlled by the ecdysis triggering hormone (ETH), eclosion hormone (EH) and the crustacean cardioactive peptide (CCAP). At present, the regulation of molting behavior in crustaceans remains unclear. Here, we studied the roles of ETH, EH, and CCAP in the molt regulation of the crab, Scylla paramamosain from their expression pattern and in vivo assays. The results showed that transcripts of ETH, EH, and CCAP were mainly localized in thoracic ganglia and fluctuated periodically with the molting cycle. When ETH or CCAP was knockdown at early premolt stage (D0), molting of crabs was interrupted and all animals died at late premolt stage (D2). While the EH gene was knock-down, most crabs were dead before D2. Injection of synthetic peptide for ETH or CCAP rescued ETH- or CCAP-gene knock-down crabs separately. However, none of peptides could rescue dsEH-injected crabs. At D0 stage, knockdown of ETH down-regulated the transcriptions of EH and CCAP; while ETH was up-regulated when EH was knockdown. At D2 stage, ETH transcripts levels were reduced with the injection of dsEH but increased with the same dose of dsETH as crabs at D0 stage. Co-injection of dsETH and dsEH down-regulated ETH at D2 stage. Results showed that ETH, EH, and CCAP play essential but different roles in molt regulation in mud crab. In summary, the result of this study contributes to the discovery of different molecular mechanisms between Insecta and Crustacea and may provide insight to develop fishery drugs that helps aquacultured crustaceans to molt successfully.

MicroRNAs (miRNAs) are important signaling regulators that are involved in regulating the innate immunity of crustacean. However, few studies focus on the role of crustacean miRNAs in the cellular immunity have been reported. In this study, we showed that the expression of miR-133 was significantly up-regulated in the mud crab Scylla paramamosain after infection by white spot syndrome virus (WSSV) or Vibrio parahaemolyticus. The anti-miRNA oligonucleotide AMO-miR-133 was used to knock down miR-133 expression in S. paramamosain. The number of WSSV copies increased significantly in WSSV-infected crabs after miR-133 knockdown. Knockdown of miR-133 also enhanced the mortality rates of WSSV-infected and V. parahaemolyticus-infected mud crabs, and it significantly enhanced the expression of the astakine, which was confirmed by real-time quantitative PCR and western blot analysis. The data also indicate that miR-133 may affect hemocyte proliferation in S. paramamosain by regulating astakine expression. miR-133 Knockdown enhanced the apoptosis or phagocytosis of crab hemocytes, and increased the mortality of mud crabs after WSSV or V. parahaemolyticus infection. These results indicate that miR-133 is involved in the host immune response to WSSV and V. parahaemolyticus infection in mud crabs. Taken together, our research provides new insights for the control of viral or vibrio diseases in S. paramamosain.

Biogenic amines (BAs) play an important role in the aggressive behavior of crustaceans. In mammals and birds, 5-HT and its receptor genes (5-HTRs) are characterized as essential regulators involved in neural signaling pathways during aggressive behavior. However, only one 5-HTR transcript has been reported in crabs. In this study, the full-length cDNA of the 5-HTR1 gene, named Sp5-HTR1, was first isolated from the muscle of the mud crab Scylla paramamosain using the reverse-transcription polymerase chain reaction (RT-PCR) and rapid-amplification of cDNA ends (RACE) methods. The transcript encoded a peptide of 587 amino acid residues with a molecular mass of 63.36 kDa. Western blot results indicate that the 5-HTR1 protein was expressed at the highest level in the thoracic ganglion. Furthermore, the results of quantitative real-time PCR show that the expression levels of Sp5-HTR1 in the ganglion at 0.5, 1, 2, and 4 h after 5-HT injection were significantly upregulated compared with the control group (p < 0.05). Meanwhile, the behavioral changes in 5-HT-injected crabs were analyzed with EthoVision. After 0.5 h of injection, the speed and movement distance of the crab, the duration of aggressive behavior, and the intensity of aggressiveness in the low-5-HT-concentration injection group were significantly higher than those in the saline-injection and control groups (p < 0.05). In this study, we found that the Sp5-HTR1 gene plays a role in the regulation of aggressive behavior by BAs, including 5-HT in the mud crab. The results provide reference data for the analysis of the genetic mechanism of aggressive behaviors in crabs.

Soft-shell crabs have attracted consumers’ attention due to their unique taste and nutritional value. To evaluate the feasibility of harvest optimisation of soft-shell mud crabs, the proximate composition, mineral composition, and total carotenoid, amino acid, and fatty acid contents of edible parts of male and female soft-shell mud crabs at different moulting stages were determined and compared from a nutritional value perspective. The results showed that the sex and moulting stages could significantly affect the nutritional values of the edible portions of soft-shell crabs. The female or male soft-shell crabs in the postmoult Ⅰ stage had a much richer mineral element content than that in other moulting stages. The total carotenoid content in female soft-shell crabs was significantly higher than that in male crabs in all moulting stages, while male soft-shell crabs had better performance in amino acid nutrition than female soft-shell crabs. Moreover, it was found that soft-shell crabs in the postmoult Ⅱ stage had significantly higher contents of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), while significantly lower contents of saturated fatty acids (SFA) than those in other stages. The present study will provide a reference basis for the diversified cultivation of soft-shell crabs, and further promote the development of the mud crab industry.

The mud crab (Scylla paramamosain) is a commercially significant marine decapod crustacean. Due to its obvious sexual dimorphism, the mechanism of sex differentiation and gonadal development has attracted significant research interest. The Dmrt (double-sex and mab-3 related transcription factor) genes are vital in animal gonadal development and sex differentiation. In the present study, miR-34 was predicted to target the 3' end of Dmrt-1, idmrt-2, Dmrt-3, Dsx and Dmrt-like genes by prediction software, and the interactions between miR-34 and these Dmrt genes were validated by in vivo and in vitro experiments. Dual luciferase assay results indicated that miR-34 mimics/inhibitors co-transfected with plasmid vectors with 3' end of Dmrt-1, idmrt-2, Dmrt-3, Dsx and Dmrt-like, respectively, led to a significant decrease/increase of fluorescence activity in HEK293T cells. In vivo experiments showed that injection of agomir-34 significantly inhibited Dmrt-1, idmrt-2, Dsx and Dmrt-like expression, while injection of antagomir-34 caused the opposite result. However, Dmrt-3 expression was not affected by injection of miR-34 reagents. Meanwhile, the expression of spermatogenesis and testicular development-related molecular marker genes (IAG, foxl2 and vasa) in mud crabs was significantly changed after injecting the miR-34 reagent in vivo. Furthermore, the result of immunoblotting proved that the expression level of Dmrt-like protein can be regulated by miR-34. These results imply that miR-34 is indirectly involved in sex differentiation and testicular development of S. paramamosain by regulating Dmrt-1, idmrt-2, Dsx and Dmrt-like genes., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Inc.)

Hypoxia is a major environmental stressor that can damage the oxidation metabolism of crustaceans. Glutaredoxin (Grx) is a key member of the thioredoxin superfamily and plays an important role in the host’s defense against oxidative stress. At present, the role of Grx in response to hypoxia in crustaceans remains unclear. In this study, the full-length cDNA of Grx3 (SpGrx3) was obtained from the mud crab Scylla paramamosain, which contains a 129-bp 5′ untranslated region, a 981-bp open reading frame, and a 1,183-bp 3′ untranslated region. The putative SpGrx3 protein contains an N-terminal thioredoxin domain and two C-terminal Grx domains. SpGrx3 was expressed in all tissues examined, with the highest expression in the anterior gills. After hypoxia, SpGrx3 expression was significantly up-regulated in the anterior gills of mud crabs. The expression of Grx2 and glutathione S-transferases was decreased, while the expression of glutathione peroxidases was increased following hypoxia when SpGrx3 was silenced in vivo. In addition, the total antioxidant capacity of SpGrx3-interfered mud crabs was significantly decreased, and the malondialdehyde content was significantly increased during hypoxia. The subcellular localization data indicated that SpGrx3 was predominantly localized in the nucleus when expressed in Drosophila Schneider 2 (S2) cells. Moreover, overexpression of SpGrx3 reduced the content of reactive oxygen species in S2 cells during hypoxia. To further investigate the transactivation mechanism of SpGrx3 during hypoxia, the promoter region of the SpGrx3 was obtained by Genome Walking and three hypoxia response elements (HREs) were predicted. Dual-luciferase reporter assay results demonstrated that SpGrx3 was likely involved in the hypoxia-inducible factor-1 (HIF-1) pathway during hypoxia, which could be mediated through HREs. The results indicated that SpGrx3 is involved in regulating the antioxidant system of mud crabs and plays a critical role in the response to hypoxia.

Salinity acclimatization refers to the physiological and behavioral adjustments made by crustaceans to adapt to varying salinity environments. The eyestalk, a neuroendocrine organ in crustaceans, plays a crucial role in salinity acclimatization. To elucidate the molecular mechanisms underlying eyestalk involvement in mud crab (Scylla paramamosain) acclimatization, we employed RNA-seq technology to analyze transcriptomic changes in the eyestalk under low (5 ppt) and standard (23 ppt) salinity conditions. This analysis revealed 5431 differentially expressed genes (DEGs), with 2372 upregulated and 3059 downregulated. Notably, these DEGs were enriched in crucial biological pathways like metabolism, osmoregulation, and signal transduction. To validate the RNA-seq data, we further analyzed 15 DEGs of interest using qRT-PCR. Our results suggest a multifaceted role for the eyestalk: maintaining energy homeostasis, regulating hormone synthesis and release, PKA activity, and downstream signaling, and ensuring proper ion and osmotic balance. Furthermore, our findings indicate that the crustacean hyperglycemic hormone (CHH) may function as a key regulator, modulating carbonic anhydrase expression through the activation of the PKA signaling pathway, thereby influencing cellular osmoregulation, and associated metabolic processes. Overall, our study provides valuable insights into unraveling the molecular mechanisms of mud crab acclimatization to low salinity environments., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Apoptosis plays essential roles in the immune defense mechanism against pathogen infection. Caspase 3 is a family of cysteine proteases involved in apoptosis and the immune response. In this study, the full-length of mud crab (Scylla paramamosain) caspase 3 (designated as Sp-caspase 3) was cloned and characterized. The open reading frame of Sp-caspase 3 was comprised a 1035 bp, which encoded a putative protein of 344 amino acids. Sp-caspase 3 was ubiquitously expressed in various tissues with a high-level expression in hemocytes. Cellular localization analysis revealed that Sp-caspase 3 was located in the cytoplasm and nucleus. Over-expression of Sp-caspase 3 could induce cell apoptosis. In addition, V. Parahaemolyticus infection induced the relative expression of caspase-3 mRNA and increased caspase-3 activity. Knocking down Sp-caspase 3 in vivo significantly reduced cell apoptosis and increased mortality of mud crab after V. parahaemolyticus infection. These results indicated that Sp-caspase 3 played important roles in the immune response and apoptosis against bacterial infection. • Identified a caspase 3 gene in mud crab. • Over-expression of Sp-caspase 3 induced cell apoptosis. • Knocking down Sp-caspase 3 increased mortality of mud crab after V. parahaemolyticus infection. [ABSTRACT FROM AUTHOR]

The mud crab Scylla paramamosain is an important euryhaline mariculture species. However, acute decreases in salinity seriously impact its survival and can result in large production losses. In this study, we evaluated metabolic changes in S. paramamosain exposed to an acute salinity reduction from 23 psu to 3 psu. After the salinity decrease, hemolymph osmolality declined from 726.75 to 642.38 mOsm/kg H2O, which was close to the physiological equilibrium state. Activities of osmolality regulation-related enzymes in the gills, including Na+-K+-ATPase, CA, and V-ATPase all increased. Using LC-MS analysis, we identified 519 metabolites (mainly lipids). Additionally, 13 significant metabolic pathways (P < 0.05) were identified via enrichment analysis, which were mainly related to signal pathways, lipids, and transportation. Our correlation analysis, which combined LC-MS and previous GC-MS data, yielded 28 significant metabolic pathways. Amino acids and energy metabolism accounted for most of these pathways, and lipid metabolism pathways were insignificant. Our results showed that amino acids and energy metabolism were the dominant factors involved in the adaptation of S. paramamosain to acute salinity decrease, and lipid metabolites played a supporting role.

The HIF-1 alpha subunit (HIF-1α) is a master regulator of oxygen sensitivity. This study investigated the role of HIF-1α in the energy metabolism and immune responses of hypoxic Scylla paramamosain. After hypoxia treatment for 12 h, the expression levels of HIF-1α in the hemocytes, gills and hepatopancreas were significantly upregulated. After hypoxia and dsHIF-1α treatment for 12 h, the expression level of HIF-1α in hemocytes was successfully inhibited by specific double-strand RNAs for HIF-1α (dsHIF-1α). After inhibition of HIF-1α expression, the expression levels of prophenol oxidase, ATP synthase subunit beta, and Astakine were significantly upregulated, and those of C-type-lectin and lactate dehydrogenase (LDH) were significantly downregulated; the activities of catalase, pyruvate kinase and LDH as well as the contents of pyruvic acid and lactic acid and the levels of apoptosis and phagocytosis in hypoxic S. paramamosain were all significantly decreased. After challenge by Vibrio alginolyticus, the survival rate was significantly lower in hypoxia and dsHIF-1α treated S. paramamosain. Since cell proliferation is an energy-dependent process, the impaired glycolysis after inhibition of HIF-1α expression might be the main reason for the significant decrease of V. alginolyticus burden in hypoxic S. paramamosain. These results indicated that activated HIF-1α enhanced the resistance of S. paramamosain to V. alginolyticus under hypoxic conditions, and the enhanced resistance may be related to the changes in important immune- and metabolism-related gene expression, CAT and PO activities, total hemocyte count, apoptosis, phagocytosis, and anaerobic glycolysis.

In order to uncover the sexual difference in morphology and how early they appear during the development stage of mud crab Scylla paramamosain, we measured, observed, and biostatistically analyzed morphological traits related to sex. For unveiling the morphological differences between sexes, morphological traits involving abdomen width (AW), carapace length (CL), and carapace width (CW) were first measured during the crablet development stage of S. paramamosain in the present study. The correlation analyses and path analyses exhibited that sexual dimorphism in the third abdomen width (AW3) and fourth abdomen width (AW4) could be used for sex identification from stage C VI (stage VI of crablet). Based on the stepwise discriminant analysis and standardized traits, a sex discriminant equation was constructed, which is capable for sex identification in crablets from stage C VI. Observations for secondary sexual traits and abdomen morphology (shape and pleopods) using a dissecting microscope or scanning electron microscope indicated that sexes are easily identified at stage C VIII according to the abdomen shape; meanwhile, at stage C II based on pleopod difference, and at stage C I by the presence or absence of gonopores. The findings in this study contribute greatly to the accuracy of sex identification of S. paramamosain during the early development stage, which promotes the understanding of the morphological differentiation mechanism of sex.

This study was carried out to analyse the growth performance, feed utilization, and antioxidant capacity of juvenile mud crab (Scylla paramamosain) reared in two conditions. Here, a new indoor polypropylene tank culture system and a traditional outdoor pond cage facility were used to perform feeding experiments on juvenile crabs. After eight weeks, the survival rate, weight gain, moulting rate and feed efficiency of crabs cultured in polypropylene tanks were markedly higher than those cultured in pond cages. Moreover, the activities of serum acid phosphatase (ACP) and alkaline phosphatase (ALP), and the antioxidant indices including glutathione peroxidase (GSH-PX), glutathione transferase (GST), superoxide dismutase (SOD) and total antioxidant capacity (T-AOC) in hepatopancreas were higher in crabs cultured in the polypropylene tank system than those in pond cages. Based on these results, compared with the traditional pond cages, the polypropylene tank culture system is more suitable for the short-term culture of mud crabs. Résumé: Cette étude avait pour but d'analyser la performance de croissance, l'utilisation de la nourriture et les capacités antioxydantes des juvéniles du crabe de vase (Scylla paramamosain) élevé dans deux conditions. Un nouveau système d'élevage intérieur dans un bassin en polypropylène et la traditionnelle cage dans un étang à l'extérieur ont été utilisés pour les expériences d'alimentation sur les crabes juvéniles. Après 8 semaines, le taux de survie, le gain de poids, le rythme de mues et l'efficacité alimentaire des crabes élevés dans les bassins en polypropylène ont été plus élevés que ceux des crabes élevés dans les cages en étang. De plus les activités sériques de la phosphatase acide (ACP), de la phosphatase alcaline (ALP), les valeurs d'antioxydants dont la glutathion peroxydase (GSH-PX), la glutathion transférase (GST), la superoxyde dismutase (SOD) et les capacités antioxydantes totales (T-AOC) dans l'hépatopancréas étaient plus élevées chez les crabes des bassins en polypropylène que ceux des cages en étang. Sur la base de ces résultats, pour un élevage à court terme des crabes de vase les bassins en polypropylène conviennent mieux que les traditionnelles cages en étang. [ABSTRACT FROM AUTHOR]

The mud crab Scylla paramamosain is one of the economically important aquaculture species in China. The larval development of the mud crab is characterized by two significant morphological changes, from the 5th zoea (Z5) to the megalopa (M) stage and from the M to the first juvenile crab (C1) stage. In this study, we found that methyl farnesoate (MF) could prohibit the Z5 to M metamorphosis in a concentration-dependent manner, and that a concentration of 10 μ M MF could completely prohibit the Z5 metamorphosis. Farnesoic acid (FA) could also prohibit the Z5 metamorphosis, but its effects seemed to be concentration-independent. In addition, MF could delay rather than prohibit the M to C1 metamorphosis, while FA had no effect on the M to C1 metamorphosis at all. To summarize, it is hypothesized that either absence of MF and FA, or at least very low levels of these substances, might be necessary for a successful Z5 to M metamorphosis. Résumé: Le crabe de boue Scylla paramamosain est l'une des espèces d'aquaculture économiquement importante en Chine. Le développement larvaire de ce crabe est caractérisé par deux changements morphologiques significatifs, de la 5ème zoé (Z5) au stade mégalope (M) et de M au premier stade juvénile crabe (C1). Dans cette étude nous avons trouvé que le méthyl-farnésoate (MF) pouvait inhiber la métamorphose de Z5 d'une manière dose-dépendante, et qu'une concentration de 10 μ M de MF pouvait complètement empêcher la métamorphose de la Z5. L'acide farnésoïque (FA) peut aussi inhiber la métamorphose de la Z5, mais ses effets semblent indépendants de la dose. De plus, MF peut retarder plutôt qu'inhiber la métamorphose de M à C1, tandis que FA n'a aucun effet sur la métamorphose de M à C1. En résumé, l'hypothèse est émise que, soit l'absence de MF et FA, soit au moins un faible taux de ces substances, serait nécessaire pour une métamorphose réussie de Z5 à M. [ABSTRACT FROM AUTHOR]

Scylla paramamosain is one of the commercially crucial marine crustaceans belonging to the genus Scylla, which is commonly distributed along the coasts of China. Proteomic data are scarce for the mud crab. Salinity is one of the ecological factors that affect S. paramamosain during overwintering. To understand the energy metabolism mechanism adapted to salinity, we analysed the proteomics of S. paramamosain hepatopancreas in response to two different salinities (4‰ and 25‰). A total of 1283 proteins were detected, of which 426 down‐regulated proteins and 83 up‐regulated proteins were associated with metabolism (p < 0.05). Among these proteins related to metabolic pathways (16 pathways), we identified angiotensin‐converting enzyme, trypsin, glutamate dehydrogenase, aminomethyl transferase, hexokinase, pyruvate kinase, glucose‐6‐phosphate dehydrogenase, L‐lactate dehydrogenase, superoxide dismutase, alkaline phosphatase, catalase and xanthine dehydrogenases. Additionally, real‐time PCR showed mRNA expression of differentially expressed genes correlated positively with their protein levels. Compared with 25‰ salinity group, 4‰ salinity group was involved in amino acid metabolism, carbohydrate metabolism, energy metabolism, lipid metabolism and inorganic ion metabolism by regulating related proteins, which made the metabolism of the overwintering S. paramamosain more vigorous in the low salt environment, and hepatopancreas consumed more energy to maintain life activities. These data are the first to reveal the molecular mechanism of overwintering S. paramamosain in response to different salinities at the protein level. The study provides important information for the aquaculture production of S. paramamosain during indoor overwintering. [ABSTRACT FROM AUTHOR]

Oxidative stress is considered as the toxicity mechanism of environmental stressors on aquatic organisms. This study aims to explore the effects of oxidative stress on physiological responses, DNA damage and transcriptional profiles of the mud crabs Scylla paramamosain. In the present study, mud crabs were injected with 0.1% and 1% hydrogen peroxide (H 2 O 2) for 72 h. The results showed that superoxide dismutase and catalase activities significantly decreased after H 2 O 2 injection. Malondialdehyde content, H 2 O 2 content, aspartate aminotransferase, alanine aminotransferase and lactate dehydrogenase activity significantly increased after H 2 O 2 injection. Moreover, DNA damage occurred after H 2 O 2 injection. Transcriptome analysis showed that 531 and 372 differentially expressed genes (DEGs) were identified after 0.1% and 1% H 2 O 2 injection, respectively. These DEGs were mainly involved in the oxidative stress response and immune functions. All these results indicated that oxidative stress could impair both antioxidant defense systems and immune systems. Transcriptome analysis provided valuable information on gene functions associated with the response to oxidative stress in the mud crab. • First transcriptome of the mud crab response to hydrogen peroxide stress. • H 2 O 2 activated FOXO signaling pathway. • DNA damage response and apoptosis signaling pathways played important roles in response to oxidative stress. [ABSTRACT FROM AUTHOR]

Overwintering is an important part of the conservation of Scylla paramamosain, and salinity is a primary parameter affecting this species' metabolism at this particular time. We compared and analysed gene sequences responsible for the metabolism of S. paramamosain at 4‰ versus 25‰ salinities using transcriptome analysis, in order to find out the optimum salinity for S. paramamosain during overwintering. A total of 5895 genes were detected, of which 105 down‐regulated genes and 131 up‐regulated genes were associated with metabolism (p < 0.05). Among these genes related to metabolic pathways (117 pathways), we identified D‐aspartate oxidase, D‐amino acid oxidase, creatine kinase, sarcosine oxidase, citrate synthase, transferase, glycogen synthase, hexokinase, carbonic anhydrase, glutamine synthetase, V‐type H+‐transporting ATPase, 3′‐phosphoadenosine 5′‐phosphosulphate synthase, fatty acid synthase and others. Additionally, the consistency between qRT‐PCR results and transcriptome data reflected the reliability of transcriptome results in the present study. Compared with the 25‰ salinity group, the 4‰ salinity group promoted energy metabolism, amino acid metabolism, lipid metabolism and carbohydrate metabolism by regulating metabolism‐related genes, which made the overwintering S. paramamosain more active in the low‐salt environment, and hepatopancreas consumed more energy to maintain life activities. These results improve our understanding on molecular mechanisms and metabolic pathways associated with salinity changes in S. paramamosain during overwintering. The data presented in this study are also the first to reveal the molecular mechanisms of S. paramamosain during overwintering in response to different salinities at the gene level. [ABSTRACT FROM AUTHOR]

Overwintering is an important part of the life cycle of Scylla paramamosain. Salinity has a significant effect on the growth and metabolism of overwintering S. paramamosain. 4‰ and 25‰ are typical salinities in different regions where S. paramamosain overwinters. In order to find the optimum salinity for overwintering, the metabolites in overwintering S. paramamosain at salinities of 4‰ and 25‰ were analysed and compared using GC–MS technology. In this study, the weight gain rate of S. paramamosain was 5.51% at 4‰ salinity and 10.69% at 25‰ salinity after overwintering. A total of 1254 valid peaks and 373 metabolites were detected, including 160 significantly different metabolites (16 down‐regulated metabolites and 144 up‐regulated metabolites). In the Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis, 22 pathways were associated with metabolic changes; they have or identified L‐isoleucine, L‐lysine, urea, D‐ribose‐5‐phosphate, glucose‐6‐phosphate, fumaric acid, L‐lactic acid, docosahexaenoic acid and palmitic acid. Compared with the 25‰ salinity group, 4‰ salinity group promoted amino acid metabolism, carbohydrate metabolism, membrane transport and lipid metabolism through these differential metabolites, so that overwintering S. paramamosain needs to participate in more metabolic activities at 4‰ salinity, and hepatopancreas consumes more energy to maintain life activities, which indicated that 4‰ salinity was not conducive to the overwintering of S. paramamosain. [ABSTRACT FROM AUTHOR]

Scylla paramamosain is a kind of large euryhaline marine crab. As an important physicochemical parameter of seawater, salinity has a great impact on the survival, growth and quality of Scylla paramamosain. This research tested the content of non-volatile flavor substance, lactic acid and taurine on the 0, 1st, 3rd, 7th and 15th day in three salinity gradients (3, 13, 23) with HPLC (High-performance Liquid Chromatography) technology. Results have shown that in low salinity stress, the cumulative amount of free amino acids in muscle of Scylla paramamosain is more than that in hepatopancreas, while the cumulative amount of essential amino acids in hepatopancreas is more than that in muscle. In muscle, contents of three flavor amino acids are ranked as follows: sweet, bitter and delicious amino acid, and in hepatopancreas, it is bitter, sweet and delicious amino acid. The fluctuation rule of free amino acid, essential amino acid and lactic acid in Scylla paramamosain in the low salinity group is similar to that of other salinity control group, while the content of sweet amino acid, bitter amino acid, nucleotide, EUC, taurine is different from that of other salinity control groups. [ABSTRACT FROM AUTHOR]

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As a conditionally essential amino acid (CEAA), taurine plays an important role in the growth, development, and physiological metabolism of aquatic animals; however, its demand level in the mud crab Scylla paramamosain is still poorly understood. A 60-day growth trial was conducted to investigate the effects of different taurine levels (0% (T0.0), 0.2% (T0.2), 0.4% (T0.4), 0.8% (T0.8), and 1.6% (T1.6)) added to diet on the survival, growth, body composition, immunity, antioxidant and osmoregulatory capacity of early juvenile S. paramamosain (initial body weight 8.69 ± 0.19 mg). The results showed that dietary taurine levels >0.4% (inclusive) significantly increased the survival of early juvenile mud crabs. The growth performance (final body weight (FBW), weight gain (WG), specific growth rate (SGR)) of early juvenile mud crabs increased significantly with increasing dietary taurine levels, reaching a maximum in the T0.8 group, and the target of rapamycin (tor) gene transcription levels also peaked in the T0.8 group. The body composition of mud crabs was not affected by dietary treatments. Further, dietary taurine significantly increased digestive enzyme (trypsin, lipase, and amylase) activities and transcript levels of appetite-related genes (neuropeptide F1 (npf1) and neuropeptide F2 (npf2)) in early juvenile mud crabs. In addition, dietary taurine increased the expression levels of immune-related genes (prophenoloxidase (proPO), prophenoloxidase activating factor 1 (ppaf1), prophenoloxidase activating factor 2 (ppaf2), crustin antimicrobial peptide (crustin), lysozyme (lzm)) and antioxidant capacity (total antioxidant capacity (T-AOC), superoxide dismutase (SOD) and catalase (CAT) activities) in early juvenile mud crabs. Finally, the results of the acute salinity challenge showed that the early juvenile mud crabs' survival in the T0.8 group was significantly higher than that of the other groups, while significantly higher Na+/K+-ATPase (NKA) activity and Na+/K+-ATPase α-subunit (nka-α) and Na+/K+/2Cl−-cotransporter (nkcc) gene expression levels were also detected in the T0.8 group. Together with growth performance, immunity, antioxidant capacity and osmoregulatory capacity, the present study concluded that the appropriate dietary taurine level for early juvenile mud crabs was 1.08%–1.33%. [Display omitted] • The appropriate dietary taurine level for juvenile mud crabs was 1.08%–1.33%. • Dietary taurine above 0.4% significantly improved the survival of juvenile mud crabs. • Antioxidant capacity of mud crabs was significantly improved by 0.8% dietary taurine. • 0.8% dietary taurine significantly improved osmoregulation in juvenile mud crabs. [ABSTRACT FROM AUTHOR]

The mud crab Scylla paramamosain is a major marine crab species for aquaculture in the southern coastal regions of China. This study investigated the differences in the quality of low-salt cultured and wild mud crabs and the the salinity tolerance and growth performance of their offspring. Ovarian development and fatty acid and amino acid compositions in the hepatopancreas and ovaries of mature low-salt and wild mud crabs were compared. The gonadosomatic index was significantly lower, but the hepatopancreatic index was significantly higher in the low-salt mud crab than in the wild mud crab. The contents of C18:1, C22:6n3 (DHA), DHA/EPA, and n-3/n-6 fatty acids in the ovaries and hepatopancreas of the low-salt mud crab were significantly higher than those of the wild mud crab. Except for proline in the hepatopancreas of the low-salt group, there were no significant differences in amino acids in the ovaries and hepatopancreas between the two groups. Although the survival rate of larval mud crabs in the low-salt mud crab was significantly higher than that in the wild mud crab when the salinity was 24‰, there was no significant difference in the juvenile survival between the two groups. However, the survival rate of larval and juvenile mud crabs in the low-salt group was significantly higher than in the wild group when the salinity was 5 ‰ and 3 ‰, respectively. The final body weight, average weight, weight gain, and specific weight gain rate of male and female offspring in the low-salt group were significantly higher than in the wild group. However, the weight gain and specific growth rate of male offspring in the low-salt group in September–October were significantly lower than those of male mud crabs in the wild group. This study indicates that the mud crab can be domesticated for culture and breeding at low salinity. • The fatty acid accumulation in the ovaries and hepatopancreas of Scylla paramamosain was superior at low salinity. • The offspring of Scylla paramamosain at low salinity could adapt to sudden salinity drops and had better growth performance. • Scylla paramamosain adapted to low salinity showed a stable inheritance in offspring. [ABSTRACT FROM AUTHOR]

[Display omitted] • The main DLMs between crabs fed with different diets was PC molecules. • Lard oil reduced SFA content binding at the sn-1/3 position of PE/PS molecules. • Lard oil decreased the content of ether PLs containing DHA/EPA. • Lard oil increased the content of ether PE containing 18:0, 18:1n-9. • Lard oil decreased the content of DHA/EPA combined at sn-2 of lipid molecules. Aiming to assess the effects of lard oil (LO) and fish oil (FO) on the nutritional value of mud crabs (Scylla paramamosain), non-targeted lipidomics analysis was performed on the muscle of crabs after eight weeks of feeding trail. Compared to FO, dietary LO reduced the content of phosphatidylethanolamine (PE) and phosphatidylserine (PS) with 18:0 bound at sn-1/3 site, the content of ether phospholipids containing 20:5n-3 (EPA) and 22:6n-3 (DHA) combined at sn-2 site, and increased the content of ether PE containing 18:0 and 18:1n-9. Furthermore, the deposition of 16:0, 16:1n-7, 18:2n-6, 18:3n-3, 20:4n-6, EPA and DHA at each site of PE, PS, phosphatidylcholine and/or triacylglycerols were reduced by dietary LO, while the DHA content at the sn-2 position of PE was increased. In conclusion, the nutritional value of mud crabs was reduced by dietary LO with the manifestation of variation in FA composition and positional distribution on phospholipids. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factors −1 (HIF-1) is a crucial transcription factor that regulates the expression of glycolytic genes. Our previous study proved that the Mud crab dicistrovirus-1 (MCDV-1) can induce aerobic glycolysis that favors viral replication in mud crab Scylla paramamosain. However, the role of HIF-1 on key glycolytic genes during the MCDV-1 infection has not been examined. In this study, the intricate interplay between HIF-1 and the key glycolysis enzyme, lactate dehydrogenase (LDH), was investigated after MCDV-1 infection. The expression of LDH was significant increased after MCDV-1 infection. Additionally, the expression of HIF-1α was upregulated following MCDV-1 infection, potentially attributed to the downregulation of prolyl hydroxylase domains 2 expression. Subsequent examination of the SpLDH promoter identified the presence of hypoxia response elements (HREs), serving as binding sites for HIF-1α. Intriguingly, experimental evidence demonstrated that SpHIF-1α actively promotes SpLDH transcription through these HREs. To further elucidate the functional significance of SpHIF-1α , targeted silencing was employed, resulting in a substantial reduction in SpLDH expression, activity, and lactate concentrations in MCDV-1-infected mud crabs. Notably, SpHIF-1α -silenced mud crabs exhibited higher survival rates and lower viral loads in hepatopancreas tissues following MCDV-1 infection. These results highlight the critical role of SpHIF-1α in MCDV-1 pathogenesis by regulating LDH gene dynamics, providing valuable insights into the molecular mechanisms underlying the virus-host interaction. • The expression of SpHIF-1α and SpLDH is upregulated during MCDV-1 infection. • SpHIF-1α actively promotes SpLDH transcription through HREs. • SpHIF-1α plays a critical role in MCDV-1 pathogenesis by regulating LDH gene dynamics. [ABSTRACT FROM AUTHOR]

The innate immunity is widely regulated by microRNAs (miRNAs) and that miRNA-125 (miR-125) is involved in many diseases and host immune response. This research was aimed to explore how miR-125 regulates innate immunity in Scylla paramamosain when response to pathogen infection. AMO-miR-125 (anti-miRNA-125 oligonucleotide) was applied to specifically inhibit miR-125 in mud crabs. We found that miR-125 induced hemocyte proliferation, enhanced total hemocyte count in the crabs and protected crabs from challenged with white spot syndrome virus (WSSV) and Vibrio parahaemolyticus. Astakine regulates hemocyte proliferation, and the expression of Astakine increased after miR-125 knockdown, the results indicated that miR-125 may regulate hemocyte proliferation and total hemocyte count by regulating the expression of Astakine. Moreover, miR-125 knockdown also reduced phagocytosis and apoptosis, showing that miR-125 could affect the two processes in crab hemocytes. And the cumulative mortality of WSSV or V. parahaemolyticus infected crabs increased after AMO-miR-125 treatment, which indicated that miR-125 could reduce the death of crab caused by the pathogens and significantly affect the crab's immune response to pathogen. In summary, our data confirmed that miR-125 participates in hemocyte proliferation, the innate immunity, and the resistance to microbial disease in Scylla paramamosain. • miR-125 induced hemocyte proliferation, total hemocyte count, host phagocytosis. • miR-125 can regulate hemocyte proliferation and total hemocyte count by regulating the expression of Astakine. • miR-125 plays an important role in the crab's immune response to pathogen infection. [ABSTRACT FROM AUTHOR]

• RAS juveniles had a higher viability than WES ones. • RAS had an improved water quality and more stable water bacterial community structure. • The changed water quality indicators were closely related to the alterations in water bacterial community. • Water bacterial community of RAS had an enhanced nitrogen cycling functional potential. • The enriched heterotrophic bacteria in RAS were vital in removing nitrogenous pollutants. The structure and function of the water microbial community can change dramatically between different rearing modes. Yet investigations into the relationships between microbial community and water quality remain obscure. We provide the first evidence that rearing modes alter bacterial community and water quality in the rearing water of the mud crab (Scylla paramamosain) larvae. The juveniles in the recirculating aquaculture system (RAS) had a higher viability than those in the water exchange system (WES). RAS had the significantly lower levels of total ammonia nitrogen (TAN), NH 3 , NO 2 −-N, total nitrogen (TN), total dissolved solids (TDS), and chemical oxygen demand than those of WES. The number of significantly different amplicon sequence variants between rearing modes increased as the larvae developed. NH 3 , TAN, TDS, NO 2 −-N, and TN were closely related to the late alterations in water bacterial community. Both the FAPROTAX tool and quantitative PCR analysis showed enhanced nitrogen cycling functional potential of water bacterial community of RAS. Random forest analysis identified the enriched water bacteria especially heterotrophic bacteria such as Phaeodactylibacter, Tenacibaculum , and Hydrogenophaga , which were vital in removing nitrogenous compounds via simultaneous nitrification and denitrification. Notably, RAS could save 18.5 m3 of seawater relative to WES in larviculture on the scale of 2.5 m3. Together, these data indicate that RAS could function as microbial community and water quality management strategy in the larviculture of crab. [Display omitted] [ABSTRACT FROM AUTHOR]

The mud crab (Scylla paramamosain) possesses extensive regenerative abilities, enabling it to replace missing body parts, including claws, legs, and even eyes. Studying the genetic and molecular mechanisms underlying regenerative ability in diverse animal phyla has the potential to provide new insights into regenerative medicine in humans. In the present study, we performed mRNA sequencing to reveal the genetic mechanisms underlying the claw regeneration in mud crab. Several differentially expressed genes (DEGs) were expressed in biological pathways associated with cuticle synthase, collagen synthase, tissue regeneration, blastema formation, wound healing, cell cycle, cell division, and cell migration. The top GO enrichment terms were microtubule-based process, collagen trimer, cell cycle process, and extracellular matrix structural constituent. The most enriched KEGG pathways were ECM-receptor interaction and focal adhesion. The genes encoding key functional proteins, such as collagen alpha, cuticle protein, early cuticle protein, arthrodial cuticle protein, dentin sialophosphoprotein (DSPP), epidermal growth factor receptor (EGFR), kinesin family member C1 (KIFC1), and DNA replication licensing factor mcm2-like (MCM2) were the most significant and important DEGs suspected to participate in claw regeneration. The findings of this research offer a comprehensive and insightful understanding of the genetic and molecular mechanisms underlying claw regeneration in S. paramamosain. By elucidating the specific genes and molecular pathways implicated in this process, our study contributes significantly to the broader field of regenerative biology and offers potential avenues for further exploration in crustacean limb regeneration. [Display omitted] • Several DEGs and biological pathways were identified. • Chitin and collagen-related genes plays important role in the claw regeneration. • Key genes were collagen alpha, DSPP, FRAS1, cuticle protein, EGFR, HAUS5, and KIFC1. • Top GO terms were extracellular matrix, microtubule-based, and collagen trimer. • The most enriched KEGG pathways were ECM-receptor interaction and focal adhesion. [ABSTRACT FROM AUTHOR]

In the present study, BGISEQ-500 RNA-Seq technology was adopted to investigate how Scylla paramamosain adapts to salinity tolerance at the molecular level and explores changes in gene expression linked to salinity adaptation following exposure to both low salinity (5 ‰) and standard salinity (23 ‰) conditions. A total of 1100 and 520 differentially expressed genes (DEGs) were identified in the anterior and posterior gills, respectively, and their corresponding expression patterns were visualized in volcano plots and a heatmap. Further analysis highlighted significant enrichment of well-established gene functional categories and signaling pathways, including those what associated with cellular stress response, ion transport, energy metabolism, amino acid metabolism, H 2 O transport, and physiological stress compensation. We also selected key DEGs within the anterior and posterior gills that encode pivotal stress adaptation and tolerance modulators, including AQP, ABCA1, HSP 10, A35, CAg, NKA, VPA, CAc, and SPS. Interestingly, A35 in the gills might regulate osmolality by binding CHH in response to low salinity stress or serve as a mechanism for energy compensation. Taken together, our findings elucidated the intricate molecular mechanism employed by S. paramamosain for salinity adaptation, which involved distinct gene expression patterns in the anterior and posterior gills. These findings provide the foothold for subsequent investigations into salinity-responsive candidate genes and contribute to a deeper understanding of S. paramamosain 's adaptation mechanisms in low-salinity surroundings, which is crucial for the development of low-salinity species cultivation and the establishment of a robust culture model. [Display omitted] • Compared the transcriptomes of the anterior and posterior gills of the mud crab. • Nine DEGs were selected and their expression was validated by RT-qPCR. • The 5 ‰ group owned a better osmoregulation than 23 ‰ group. • Different functions of anterior and posterior gills in salinity adaptation. [ABSTRACT FROM AUTHOR]

Abstract Background Scylla paramamosain is a commercially important mud crab. The microbiota is a community that inhabits the crab intestine, and is important for physiological functional and host health. Results Proteobacteria, Firmicutes, Bacteroidetes, Tenericutes, Spirochaetae and Fusobacteria were the dominant phyla of the 36 representative phyla. Eleven genera of the 820 representative genera were considered as core gut microbiota and were distributed in the five dominant phyla. The core genus of the Proteobacteria included Arcobacter, Photobacterium, Vibrio, Shewanella and Desulfovibrio. The other four phyla contained one or two genera. Male and female crab samples had two different core genera, (male samples: Psychrilyobacter & Lactococcus; female samples: Clostridium_sensu_stricto_11 and Candidatus_Bacilloplasma). Conclusions This is the first time core intestinal microbiota have been identified in crab from nine coastal regions of southern China. This study provides sequencing data related to the gut microbiota of S. paramamosain, and may contribute to probiotic development for S. paramamosain aquaculture industries.

Abstract Increasing evidence has revealed a close association between intestinal bacterial community and hosts health. However, it is unclear whether and what extend Baimang disease alters the intestinal microbiota in mud crab (Scylla paramamosain). Here, we conducted intestinal contents Illumina sequencing of healthy and Baimang diseased mud crab (S. paramamosain) to understand bacterial community variations among health status. In addition, bacterial functional predication was used to investigate whether and how the bacteria variations further change their functions? The phyla of Proteobacteria, Fusobacteria, Cyanobacteria, Tenericutes, Firmicutes, Bacteroidetes, and Spirochaetae constituted over 96.44% of the total intestinal bacteria, with being the dominant taxa. The 7 most significantly different orders, including the increased four orders of Clostridiales, Entomoplasmatales, Bacteroidales, and Mycoplasmatales and the decreased three orders of Vibrionales, Campylobacterales, and Fusobacteriales, accounted for 61.14% dissimilarity, probably being the indicator taxa of Baimang disease. Accordingly, 12 Kyoto Encyclopedia of Genes and Genomes orthologies in level 3 shifted significantly at the diseased crabs. Especially, bacterial secretion system, secretion system, lipopolysaccharide biosynthesis proteins and Vibrio cholerae pathogenic cycle, being related to bacterial virulence, were reduced. In addition, the reduced butanoate metabolism, and induced methane metabolism and one carbon pool by folate were important metabolic processes of probiotic, such as Bacteroides spp. and Clostridium spp., with playing critical roles in host health. This study suggests that Baimang disease coupled altered the intestinal bacterial communities and functions, providing timely information for further analysis the influencing mechanism of Baimang disease in mud crab (S. paramamosain).

Nghiên cứu nhằm xác định quy trình ương thích hợp cho tăng trưởng, tỷ lệ sống và biến thái của ấu trùng cua biển. Thí nghiệm gồm 3 nghiệm thức: (1) ương ấu trùng theo quy trình sử dụng hóa chất; (2) ương ấu trùng theo quy trình sử dụng ozone; (3) ương ấu trùng theo quy trình sử dụng kháng sinh. Ấu trùng được bố trí trong bể 1,6 m3 với mật độ 200 con/L và độ mặn 30 ‰. Kết quả thí nghiệm cho thấy mật độ vi khuẩn tổng, Vibrio spp và tỷ lệ nhiễm ký sinh trùng trên ấu trùng thấp nhất ở nghiệm thức sử dụng ozone lần lượt là 0,86 x 104 cfu/mL, 0,16 x 104 cfu/mL và 6,40% khác biệt có ý nghĩa (p0,05) với nghiệm thức sử dụng kháng sinh (7,23%), nhưng khác biệt có ý nghĩa (p0,05) với nghiệm thức sử dụng kháng sinh (0,85),...

The dissolved oxygen (DO) level is particularly important for marine benthic crustaceans. In Scylla paramamosain, the DO level plays an important role in regulating immune and metabolic responses. The present study assessed whether the effects of hypoxia on the resistance of S. paramamosain to Vibrio alginolyticus are related to the changes in energy metabolism. In hypoxic S. paramamosain, the immune-related genes, Janus kinase, signal transducer and activator of transcription, Relish, and Astakine were significantly downregulated in hemocytes. In contrast, hypoxia inducible transcription factor-1α, lactate dehydrogenase (LDH), and prophenol oxidase mRNA levels were significantly upregulated in hypoxic S. paramamosain. The levels of pyruvic acid and lactic acid were significantly increased in hypoxic S. paramamosain, this may be closely related to the increases in pyruvate kinase and LDH activities. A significant decrease in hepatic glycogen was observed in hypoxic S. paramamosain, and the breakdown of hepatic glycogen might be the reason why no significant differences in glucose levels were found between the normoxic and hypoxic groups. In addition to the changes in metabolism-related enzyme activities, the levels of phenol oxidase (PO) activity and apoptosis increased significantly, while total hemocyte counts (THC) were significantly decreased in hypoxic S. paramamosain. After challenge with V. alginolyticus, the bacterial count was significantly lower in the hypoxic group than in the normoxic group, while the mortality rate was higher in hypoxic S. paramamosain. The decrease bacterial count might be associated with the changes in the metabolism of hypoxic S. paramamosain. These results suggested that the resistance of S. paramamosain to V. alginolyticus could be weakened when the host was under hypoxic conditions, and the weakened resistance might be related to changes in immune-related gene expression, THC, PO activity, hemocyte apoptosis, and energy metabolism.

An six-week feeding trial was conducted to evaluate the effects of dietary n-3 polyunsaturated fatty acid (n-3 PUFA) lipid sources on growth performance, antioxidant capacity, tissue fatty acid profiles, and expression levels of genes involved in lipid metabolism of juvenile green mud crab (Scylla paramamosain) (initial weight 45.3 ± 0.07 g). Three isonitrogenous and isoenergetic experimental diets were formulated to contain fish oil (FO), krill oil (KO) and linseed oil (LO), respectively. The results indicated that crabs fed the KO diet had significantly higher percent weight gain (PWG), specific growth rate (SGR) and feed efficiency (FE) than those fed the LO diet, while no statistic differences were observed between KO and FO diets. Dietary FO could significantly increase the concentrations of malondialdehyde (MDA) and lipid hydroperoxide (LPO) in hemolymph and hepatopancreas compared to dietary KO and LO, similar results were found in the levels of hepatopancreas 8-hydroxy-2-deoxyguanosine (8-OHDG). Crabs fed the KO diet could significantly increase the activities of total superoxide dismutase (T-SOD) and catalase (CAT) in comparison with FO and LO diets. Crabs fed with FO and KO had significantly higher contents of 20:5n-3 (EPA) and 22:6n-3 (DHA) in hepatopancreas and muscle than those fed with LO. While dietary LO could significantly promote the accumulation of C18:3n-3 (ALA) in hepatopancreas and muscle. The anabolic pathway relevant genes: fas and 6 pg d were up-regulated in KO diet. The catabolic pathway relevant genes, hsl, was up-regulated in FO and KO diets, cpt1, was up-regulated in KO diet. Moreover, crabs fed diet supplemented with KO could significantly increase the expression levels of srb2, srebp1 and hnf4α. These findings will provide the reference for the development of the diet for the growth stage of S. paramamosain, and could contribute to deepen the understanding of the physiological metabolism of dietary fatty acids for S. paramamosain.

The mud crab Scylla paramamosain is a species with significant sexual dimorphism in growth rate and body size, of which the females are of higher economic and nutritional values than the males. Accordingly, there is an urgent need to explore the molecular mechanism underlying sex determination and gonadal differentiation. The single-molecule long-read technology combining with RNA sequencing was employed to construct a full-length transcriptome for gonads of S. paramamosain. In total, 1,562,819 FLNC reads were obtained from 1,813,758 reads of inserts (ROIs). Among them, the 10,739 fusion isoforms corresponded to 23,634 reads and were involved in 5,369 genes in the reference annotation. According to the criteria for new transcripts, a total of 213,809 isoforms were recognized as novel transcripts and then matched against Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), NR, Swissprot, and KOG databases. We also identified 22,313 SSRs, 169,559 lncRNAs, and 25,451 SNPs. Additionally, 349,854 alternative splicing (AS) events from 8,430 gene models were detected, and 5,129 polyadenylation sites were profiled from 3,090 genes. GO and KEGG annotation indicated that AS and APA probably play important roles in the gonadal development and maturation. Besides, the DEGs associated with gonadal development and maturation were identified and analyzed based on the RNA-Seq data.

Mud crabs, found throughout the Indo‐Pacific region, are coastal species that are important fisheries resources in many tropical and subtropical Asian countries. Here, we present a chromosome‐level genome assembly of a mud crab (Scylla paramamosain). The genome is 1.55 Gb (contig N50 191 kb) in length and encodes 17,821 proteins. The heterozygosity of the assembled genome was estimated to be 0.47%. Effective population size analysis suggested that an initial large population size of this species was maintained until 200 thousand years ago. The contraction of cuticle protein and opsin genes compared with Litopenaeus vannamei is assumed to be correlated with shell hardness and light perception ability, respectively. Furthermore, the analysis of three chemoreceptor gene families, the odorant receptor (OR), gustatory receptor (GR) and ionotropic receptor (IR) families, suggested that the mud crab has no OR genes and shows a contraction of GR genes and expansion of IR genes. The numbers of the three gene families were similar to those in three other decapods but different from those in two nondecapods and insects. In addition, IRs were more diversified in decapods than in nondecapod crustaceans, and most of the expanded IRs in the mud crab genome were clustered with the antennal IR clades. These findings suggested that IRs might exhibit more diverse functions in decapods than in nondecapods, which may compensate for the smaller number of GR genes. Decoding the S. paramamosain genome not only provides insight into the genetic changes underpinning ecological traits but also provides valuable information for improving the breeding and aquaculture of this species. [ABSTRACT FROM AUTHOR]

An 8-week feeding trial was conducted to evaluate the effects of dietary n-3 LC-PUFA levels on growth performance, tissue fatty acid profiles and relative expression of genes involved in the lipid metabolism of mud crab (Scylla paramamosain). Ten isonitrogenous diets were formulated to contain five n-3 LC-PUFA levels at 7 and 12 % dietary lipid levels. The highest weight gain and specific growth rate were observed in crabs fed the diets with 19·8 and 13·2 mg/g n-3 LC-PUFA at 7 and 12 % lipid, respectively. Moisture and lipid contents in hepatopancreas and muscle were significantly influenced by dietary n-3 LC-PUFA at the two lipid levels. The DHA, EPA, n-3 LC-PUFA contents and n-3:n-6 PUFA ratio in hepatopancreas and muscle significantly increased as dietary n-3 LC-PUFA levels increased at both lipid levels. The expression levels of -6 fatty acyl desaturase and acyl-CoA oxidase in hepatopancreas increased significantly, and expression levels of fatty acid synthase, carnitine palmitoyltransferase I and hormone-sensitive TAG lipase were down-regulated, with increased dietary n-3 LC-PUFA regardless of lipid level. Based on weight gain, n-3 LC-PUFA requirements of S. paramamosain were estimated to be 20·1 and 12·7 mg/g of diet at 7 and 12 % dietary lipid, respectively. Overall, dietary lipid level influenced lipid metabolism, and purified, high-lipid diets rich in palmitic acid reduced the n-3 LC-PUFA requirement of juvenile mud crab. [ABSTRACT FROM AUTHOR]